<%BANNER%>

Guava (Psidium guajava L.) Fruit Phytochemicals, Antioxidant Properties and Overall Quality as Influenced by Postharvest...

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20110318_AAAAKS INGEST_TIME 2011-03-18T17:32:26Z PACKAGE UFE0011873_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 9088 DFID F20110318_AABNSV ORIGIN DEPOSITOR PATH nunez_f_Page_094thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
7f37104d1f085ae3dfd1a212a2664bf7
SHA-1
57dd29ef290933ee71f9596511c1f92bebfd7630
8281 F20110318_AABNSW nunez_f_Page_098thm.jpg
70d302911af2a533c24711b00571d75a
0e49349b3281713b08f0fad2a35ba512eac53d2d
1989 F20110318_AABMSA nunez_f_Page_024.txt
fe67a42519645d0b5e17ee5a071e9aa0
4b9afd275f43921b09a4fb909321732417ed8158
50429 F20110318_AABMSB nunez_f_Page_066.pro
baf03fa11cdf3b418f6220eb2003f42c
6b057e903c0ffa7c3d4afabf5231a4dcc593adde
105364 F20110318_AABMSC nunez_f_Page_061.jp2
e9eacf9fc83c6d80813b75d776027587
0303aef1aa99849790cbb676c2688000a5ea673d
8423998 F20110318_AABMRO nunez_f_Page_083.tif
530f379530f0786eda0de4ca886886f2
7908a7161d1ba925a983c45e394c0e9b96136f9e
6871 F20110318_AABMSD nunez_f_Page_088thm.jpg
0da669157f1f952b3e65917cc41b7ec9
92ef0518b3817ef89f17ea884632c03a392245b2
103329 F20110318_AABMRP nunez_f_Page_025.jp2
acea57dd0cf51f03bd87adb1734a6dee
feceabddd0bac5ba36e56fa9811e9bb045981a00
52946 F20110318_AABMSE nunez_f_Page_074.pro
7e340b56ebbdcf0af6dc7454e3a9377f
485b43ad938f064e871f033503af6991c1a95ef3
50934 F20110318_AABMRQ nunez_f_Page_031.pro
22706135c27212fe995b8f5b2a6424f0
09f7a6b4b9602d82cc0ffbfe0363ad25bd3b04c2
1051961 F20110318_AABMSF nunez_f_Page_009.jp2
ff73f2e842f7d092acc4419070544cce
8bb328b62db447b95ac48bd2d31f87bd4c8eb93e
3447 F20110318_AABMRR nunez_f_Page_101thm.jpg
41112d4a2da729b268f80278e7f7ce3a
b6df246678ab5ea495d218c54869886b64c2cc62
83405 F20110318_AABMSG nunez_f_Page_037.jpg
75389a5334440b99c3081da75c49e9b7
e3ab98bbb4a8aa96d360dd0eea7414a4e8f92e53
4731 F20110318_AABMRS nunez_f_Page_006.txt
733b66b219cfc537d030827efc0f4788
3b4ee6e3a4f7e8a4dac9ca1ee21a7a04470658a3
28032 F20110318_AABMSH nunez_f_Page_016.QC.jpg
cb8a387dae29a4f2995902caea97bd5b
39adf1869474a892c97a2130bb8df182b7979d16
76947 F20110318_AABMRT nunez_f_Page_088.jpg
eb79a9cdc7bc2da22bdb1cb0b8ed939a
5d08e6089288e7c9782f37c6d5f978d38604fc10
1759 F20110318_AABMSI nunez_f_Page_065.txt
98ae61158f405f8ea86315b919f40097
62dd919bf9a0e7d82cea7c2b2701f494a027ff84
98023 F20110318_AABMRU nunez_f_Page_005.jpg
7b32bda477f82aa11130ac21da747579
3100ba914f76551227cee740a327795cbd518a48
17220 F20110318_AABMSJ nunez_f_Page_089.QC.jpg
4a272b6101a986519ce848e88dafe9f3
8a78c1a1e876460cd5b03c286ad7219094d2d132
1704 F20110318_AABMSK nunez_f_Page_081.txt
a8ebc7402bf10bb4b977554a15e8d8a2
5681c529edbf3e7030c361bf2e0bc83db172c6cd
7095 F20110318_AABMRV nunez_f_Page_073thm.jpg
8f67f281f652bbf4c363201fcf22f444
0a278a73973e663dd36d8bd8626f363283577ccd
8564 F20110318_AABMSL nunez_f_Page_077thm.jpg
8b62b21260a0c9755a53c04a84d4d90e
bd6bcc8ad2833b54d0b0737514e6f8a90e563426
106106 F20110318_AABMRW nunez_f_Page_059.jp2
8b51d3336bf800b8c6a49f1172c02b06
48b40754fe35b62062cb4113cc05f828a9fba919
811970 F20110318_AABMTA nunez_f_Page_081.jp2
f87aac052df6d29b0727d6dca391ac74
42bb277b77fb1ff09a1574728c8e2e630269f3be
F20110318_AABMSM nunez_f_Page_081.tif
aa2c567752a196678ca12c2d4c21d722
a273457230185cabf319756f93fdb9b84e40a34e
F20110318_AABMRX nunez_f_Page_080.tif
86e51764aa0e2801945aa14caffd234f
d78ec72f449167fe1808828131a9f2a60f296396
9399 F20110318_AABMTB nunez_f_Page_064.jpg
ddbe3a972d3a9963b75963a10fd9d6fb
689ddf40863fd1a3e5a50371a5098680d9912566
1051981 F20110318_AABMSN nunez_f_Page_097.jp2
18ddbdeda74a97e189174c4cdc30da55
eb9828d9fccc6eb89e422bef9364bfac378e86ec
100624 F20110318_AABMRY nunez_f_Page_030.jpg
231b11cf7bbf076c243a3ee6aa85ff0d
14d5239e2e354be23bb6f6c4afe97b14409f2c40
80109 F20110318_AABMSO nunez_f_Page_073.jp2
f6a1110c455e865e51c679abf4258737
9ae1c93694e3a05b0b4784a3395f437b51931f84
8516 F20110318_AABMRZ nunez_f_Page_019thm.jpg
7c6e643822cefd585bbb6bbefd6091e2
5531b7d8e655678722f37eea957dad00545b28d2
849801 F20110318_AABMTC nunez_f_Page_052.jp2
f2f453be4bc9fe412089ebb2c549bf5b
b2c0ce55fab5f57c2d79b8c6348f2eda24f20e66
1943 F20110318_AABMSP nunez_f_Page_058.txt
8d6a2dc41db008cdcbff51ed708d12cd
6deda1f0224e7234f21ce60254e383429e43bc6f
105725 F20110318_AABMTD nunez_f_Page_035.jpg
0d3651614f657c305c7e344ca4b501f1
d990ff3267c90c58617a4bafd22cd43342fa1de2
104689 F20110318_AABMSQ nunez_f_Page_012.jpg
406d8e223cf923409128511d43b527b6
e944af676d95aeb67984fc1121261e29d7433df4
98777 F20110318_AABMTE nunez_f_Page_025.jpg
faba37efb483d05c2889b3fd8d968c38
d352d7a20e2bb21e52ea5ed12c5f0644538dc031
7512 F20110318_AABMSR nunez_f_Page_090thm.jpg
e994a9e5bb752bd10dec8ec484c0ec11
fcb634f15e9b9898238e02ae8869baaa4630b9f9
35872 F20110318_AABMTF nunez_f_Page_046.pro
a8383d03e56d56e8c04a0fa83ad4d038
c84296690d691a94cb960e619e259305434e7956
104724 F20110318_AABMSS nunez_f_Page_071.jpg
c837ca3fbb36ae45fd41e56a5d15e377
66a90d41d95b1be0419af7ee24acdc2734bc9a8a
1846 F20110318_AABMTG nunez_f_Page_056.txt
9c534c248647e066cd55251176686a7d
acd8af9547d42d5a51abcf50b0464c13c43df896
21462 F20110318_AABMST nunez_f_Page_008.QC.jpg
af096b6612baebdf5218ff51c916b184
e973ed7a777080b52bc4adc0dc5db6aed6d522c1
5128 F20110318_AABMTH nunez_f_Page_078thm.jpg
3945ff4d0942f6791059853ff05b6825
b1275d676c6e163c88b89f090673ce8ed2c1b14e
1053954 F20110318_AABMSU nunez_f_Page_065.tif
197b1670590d399c5108260dba4de408
fb04a2393dfa163ef85df08505ea7c9cf906ff4d
F20110318_AABMTI nunez_f_Page_014.tif
206426fa649c1b3924d815ee2b8bd933
c1133f73b548ab6201879e2a6ec5a1707b3599e9
32280 F20110318_AABMSV nunez_f_Page_068.QC.jpg
c642a5b5f3209eab739afcc29fd20515
68d49f01f702ef2e93f0f6efa164a374a16db015
8287 F20110318_AABMTJ nunez_f_Page_053thm.jpg
1381cc8b953fdaf1726c4763897c7a0a
dec2f400229c80845130c19b3562fb13d14dd8c7
6254 F20110318_AABMTK nunez_f_Page_002.jp2
902a45c913fbedfebc3750b9ba807792
9abe7fe1c8fab0e65b55f527412672b2ce61d790
52149 F20110318_AABMSW nunez_f_Page_035.pro
31b3441c0c2b586ad174c49627b541f3
e79898946f09c3e0c3cda52b62716e7c7cb9f966
35568 F20110318_AABMTL nunez_f_Page_087.pro
1ee80b6f4346b66967f9fee93fa6d46d
7bddf747249b6f530d2fcccaeefe8638eda28944
26204 F20110318_AABMSX nunez_f_Page_073.QC.jpg
a0b2d578038a32327051c4fcf52862dc
4dbe5b1fa4a55a5f8d7728695b9c3972c9e7a385
36143 F20110318_AABMUA nunez_f_Page_073.pro
584d44efc74bb6c0d9e4bb84500922ea
9b045f96a1b192c7a63d441d5f9f66ade3504cf3
8447 F20110318_AABMTM nunez_f_Page_047thm.jpg
339d26a5ca368a5479c4ea99349f4620
d35ffeb2f10b28e446a401ba1ffac8125d4e9f21
1775 F20110318_AABMSY nunez_f_Page_007.txt
a9ccd3a04bdd81b8630561228ac85402
75447c5017603fd9696f83a5e9451e3dfe3427fc
7869 F20110318_AABMUB nunez_f_Page_014thm.jpg
b307f88a67e6a8c321a593413dca5261
8a0974fe6865efbe4d9e9e66a970d2557ded3569
2029 F20110318_AABMTN nunez_f_Page_046.txt
a007f349245b17b1232a76a3a58c2acc
aa44fd0c32509e4d77c58c375054af6a21e536c2
111057 F20110318_AABMSZ nunez_f_Page_012.jp2
1e0e3061ae4c0e13dc15652377078a49
e9707a6e9935a0430b0c82eebe3faa4f60b5dcf0
25098 F20110318_AABMUC nunez_f_Page_088.QC.jpg
e90983ebbd7464717bb02b363654be24
fc311b77fc7527ed477d29768df727818680c7a0
1596 F20110318_AABMTO nunez_f_Page_008.txt
558ba6d045a0ad50874bfd5afc9de982
2c58ac020cfdda92ad6347f8a16378963bf60e35
F20110318_AABMUD nunez_f_Page_019.tif
058813722f3b6cc1bbf3f7d0eb27f002
035370992161bdedabba4527aa41634a83ca3957
F20110318_AABMTP nunez_f_Page_072.tif
0bbd853672014545b2e8e41fc792d03d
2a06c5f9604618322b7dc18f5044015c96f80504
8528 F20110318_AABMUE nunez_f_Page_038thm.jpg
805b7b3ceeb9ff38db61f176d28eb639
bf832149b8ba7a8c2f5c15707f495a8f18db7e5a
50845 F20110318_AABMTQ nunez_f_Page_063.pro
412aac55913676396eacdc55c00780bd
c91a455ce56191861e32389695a553a00b9682d6
25271604 F20110318_AABMUF nunez_f_Page_099.tif
83fc6f34f092c7cc9df430801dae5d69
5001f43c0752534469182c4454c8e2360e14c293
49181 F20110318_AABMTR nunez_f_Page_058.pro
107fefa5aafa3ae26f5b2df578a463b7
bf6bf0ea864d97e1b49e356609ff0c2153634b36
2042 F20110318_AABNAA nunez_f_Page_035.txt
fabb9341ac5175cf37639e569c39fad9
896fb05c8144214596641d862dcb2f4dfb867b3d
29247 F20110318_AABMUG nunez_f_Page_004.QC.jpg
50b824567a89302f4504132a4d82d7ef
13c91718bbcef3a9f50fccfe86941353c13e255a
11053 F20110318_AABMTS nunez_f_Page_064.jp2
053077f0d3f7326c7cfb6c40c0c0ed45
d78ad21e5d89d22f571ccfb15b5969534e837c04
397 F20110318_AABNAB nunez_f_Page_036.txt
89127098ece0ba62151ab1c0e562ff18
12a3cf690bc19f200d8375997109c314480140a3
48812 F20110318_AABMUH nunez_f_Page_023.pro
dceb7ba3288fb5c4c3ead7083a091739
79aa50d6ee5464a4f32babe2d5ac5425bef560d8
F20110318_AABMTT nunez_f_Page_037.tif
53d2950e3e084d2d1d33acdf69ab9abf
91a2423c9445085002578380d7f2519d43c6936c
1979 F20110318_AABNAC nunez_f_Page_038.txt
87668825bb01fcd9066d57c959bac1c6
1f69f730556cb7d294181dc1459761f9b385a3db
F20110318_AABMUI nunez_f_Page_084.tif
798a53a1db87132574c5a339734cbbca
d08056af9b17b2d5ac343b52e7f9ca659679176b
32252 F20110318_AABMTU nunez_f_Page_025.QC.jpg
78195bd857faed3e26ce4b442f9e6b53
f8675168a44d45e8bbbd8d38813f31a7df1bfe2c
100542 F20110318_AABMUJ nunez_f_Page_092.jpg
3c4c6fcbad33a704cbf6c620e15e18ec
0aad573029e2561a73e25f9e64904ca2a2d443dc
51940 F20110318_AABMTV nunez_f_Page_071.pro
00c298be38675a0302549a4d8a390f40
3333662898febc9bc366a05ff7271edc7f636697
1698 F20110318_AABNAD nunez_f_Page_039.txt
fca4b66c6aea327dc5ee8f6aba35aab5
ba25c67851c05abbcb9df6e81a5808eee03f0b32
F20110318_AABMUK nunez_f_Page_059.tif
03f5e68aa9ed49d730e256f0d3a057ad
523cdda8d331553bcd75e53a1595c076dd630b66
1689 F20110318_AABMTW nunez_f_Page_060.txt
46a2fc63a2e081997a82a9846c9bc5fe
cb7f3ca435b7d918a7668099b17fa2801fda3167
1745 F20110318_AABNAE nunez_f_Page_040.txt
77a9bc48c3bd22e293cacdaabe424964
98584e9d8943fa19d276a9f75d5ded7145b8ba6a
99232 F20110318_AABMUL nunez_f_Page_058.jpg
c182805c524cbd01dabd2f51aec6189a
5c672602c50b288269e715e5a98f7bba41771750
1998 F20110318_AABNAF nunez_f_Page_041.txt
b17aea1c836b490961fd2df2aead9e61
6cf38e3612b2c3dccfa75eea20639fcdddf32299
8036 F20110318_AABMVA nunez_f_Page_062thm.jpg
0ed9d1404f3063f8b144a58e04991c63
e434ac4b9c1725171224542507ee5942992332ab
4504 F20110318_AABMUM nunez_f_Page_057thm.jpg
787565a4736cb3b9fe0fc10a93a17acf
d1c4773a5e82d6a0e9671df33fc4f632d7f71b3a
95133 F20110318_AABMTX nunez_f_Page_039.jp2
6ed959f340be8e094a9561a0841fc90e
74e35e2ca2ec4abb5115d989492de792ca6636a8
1929 F20110318_AABNAG nunez_f_Page_042.txt
1b7fb1a8501265f6681d87d0b3a9cadd
c44264382914977a0c0ef9daf6fd9d336f26db16
8320 F20110318_AABMVB nunez_f_Page_091.pro
82c2fb73f50d19fd2aac328cb571351a
6f336d345cd6af6c8f0afc681b75a24a4be80963
34339 F20110318_AABMUN nunez_f_Page_019.QC.jpg
06093581399199eb0fac2a626fd95fa6
68e75f5e63d02285101b438854c74bdbd5d463f8
F20110318_AABMTY nunez_f_Page_040.tif
db339169857abb16006c70a71537bc5d
7c6ed21808a76692f183e7371acea0bd58e857c9
1910 F20110318_AABNAH nunez_f_Page_043.txt
76f9b9426137e88c002d9f6da2bfc66f
15374d74e3f0d1578d076150f8749cafae5b997b
1051965 F20110318_AABMVC nunez_f_Page_042.jp2
8bd515a576e3f8316cebb87e646c7896
d1e0a1d1447d1011c4b8e7b67db9a0353cbaec4e
34492 F20110318_AABMUO nunez_f_Page_026.QC.jpg
c73b709db44b7e05cf8461561e64e602
314016bec99b6e8f5fb233e5129cc802d25f14bf
7205 F20110318_AABMTZ nunez_f_Page_085thm.jpg
6aa93784269483c0c4b38aaa421ff32d
4e209ce9c1eb11fb5cdebe40794db0ec9f4226dd
1967 F20110318_AABNAI nunez_f_Page_044.txt
5b075feb3eca9a2fb57d770d7b96f72d
027e320ab3b9d633baa52eddcbb761153213ed75
1757 F20110318_AABMVD nunez_f_Page_004.txt
691101f5d0ba0b6d237c58c2ccc707dc
b888dcd0506bb196bb928f96aff4e2fd07b208f4
75441 F20110318_AABMUP nunez_f_Page_087.jpg
9326ddc0c50df8ba392af3b322520cae
88784ab879ebceaec49602c0b72679fac9971dbd
1743 F20110318_AABNAJ nunez_f_Page_045.txt
7086ec8a8686083c111bed2effda9776
e41eb390d09321cd03b3b8cc178ede2b154ba418
2097 F20110318_AABMVE nunez_f_Page_028.txt
d5f0b9cd28661eecde011d5c2c876dfc
2da4de12925a7bd2ad27262f1d41058ff33845cc
26838 F20110318_AABMUQ nunez_f_Page_046.QC.jpg
1ccffd02dfe5813568fe51eaa9e6ec05
e00e1778dca3ff8f29e66f9afe083369b1bf54a0
2019 F20110318_AABNAK nunez_f_Page_047.txt
202d405bed6af2ad2975d87f0c488e5c
19bd83b1ae1ff6747923a0c531547c79b6044019
99188 F20110318_AABMVF nunez_f_Page_017.jpg
a0097ad1fd614557b75d7f53a9d14d9f
dbe81e306da387eb17d7a42a0bfb5df93ea4b6f1
46477 F20110318_AABMUR nunez_f_Page_034.pro
c9522163f6a98e029843a99460b9d00a
5fb5566100a855336c774e64a6b02472777be95f
1856 F20110318_AABNBA nunez_f_Page_067.txt
b3562fbe7ff20c9b553a282abe21b593
3039a2de83d49a462d2782b9262d7d08a30d8618
1741 F20110318_AABNAL nunez_f_Page_048.txt
7093110327dbe2d278d59b26aa5da080
9bd4b4afd278d2befea6fcbfb5ebb3fac3b2fca8
101302 F20110318_AABMVG nunez_f_Page_090.jp2
3b7f6e080346d8577f9d2ce1dc362880
dbd5426a2635e7f5728e336cee28caaae702c437
1064 F20110318_AABMUS nunez_f_Page_075.txt
185225c3a4e00d44e66f9764eeb093b6
68cd7a03bbbe1725014bfe4c5dba6c3af67d2436
1878 F20110318_AABNBB nunez_f_Page_068.txt
e774a48fcffb442688f2cef669e07229
7d1c355c855ebd1793b3391265649abc98f44fc8
1930 F20110318_AABNAM nunez_f_Page_049.txt
ea5f67196202f9a6a77d7a1ac237bf6c
e5a027a073237ebace4891498b8ad48c7bb0e441
1054428 F20110318_AABMVH nunez_f_Page_057.tif
361172f1926a26e0331425d7d4bccef9
257ffb3a145ebcfa5b5514a4e5a2fe49a14e5bcb
F20110318_AABMUT nunez_f_Page_076.tif
ba27f2037c60b3990daa9d281a87bc11
68c3cf661f32617fa6fe45654397198a63a470a1
1589 F20110318_AABNBC nunez_f_Page_069.txt
fdcb808cd34cdbd77822eb542c93f22c
8bf8d1d355fcd34c123998342a241d501a02bcc9
2032 F20110318_AABNAN nunez_f_Page_050.txt
a79db57cbb2e5088c6d863e253abcb88
7126fb635f98b8a6f5983e136e3a163f64272dac
107896 F20110318_AABMVI nunez_f_Page_023.jp2
08e06fef510b96db9136b13d5eb45ec6
9f38bb44ec9440c6db1ff83477385e873082d101
106810 F20110318_AABMUU nunez_f_Page_019.jpg
aa8287cb54b515a4306d995f07f481bf
5222e712c2331001b63e1ed8c828de67e11c02cc
1924 F20110318_AABNBD nunez_f_Page_070.txt
e2aad2d78feb4e984c83f64b75bf1775
8fc86c804e95cff48b94e93931c8c59011713592
1909 F20110318_AABNAO nunez_f_Page_051.txt
d3144e92d0dd6946d239acb96f3b60cd
142fc928713d8ce303d6a195d03e11bc4e5b1153
67545 F20110318_AABMVJ nunez_f_Page_100.jp2
bb88a333a3d458c01ee707ccaae3a937
1a7251e7822e52f43f3a6afab5d1f9d135e8adb1
32282 F20110318_AABMUV nunez_f_Page_021.QC.jpg
e1ea8597704ee2b5f9913750d73c3ec1
d00b7e1c3f472208233ee94b02d218082a92b0fd
1690 F20110318_AABNAP nunez_f_Page_052.txt
9e48a63d62555bcd88fbcdd1509672ea
ee68d5a4e4c869ff3199127378fb548a70e82217
111937 F20110318_AABMVK nunez_f_Page_041.jp2
faccc6374ee9ebd1a4782db8f266dc8c
aee8e8cbff48a132b7e77cb9a923a21b8254d1f4
22377 F20110318_AABMUW nunez_f_Page_005.QC.jpg
822e8b24cb712e1af2b468fc939cda3b
12628b95873f538c396b0a815aa5cd2058fb4f99
2387 F20110318_AABNBE nunez_f_Page_071.txt
8287218af3cb7b9019882bc6a6bc04e7
34edbc25023135af638405c3020248bd8e9154f7
1970 F20110318_AABNAQ nunez_f_Page_053.txt
3a8b8c013f08f7d077b4fafdfed1aea3
74e2c8aa54c0b71869b59546aa0de560afccce7d
2290 F20110318_AABMVL nunez_f_Page_001thm.jpg
e53063c057c0eaf6131cc39fa4e7708f
ce0becf47a0709c8b5294cdc11f645626d67e8ff
29729 F20110318_AABMUX nunez_f_Page_092.QC.jpg
092a308b3880bf41e0bb7ae0eb910bb9
3c59ec7e32987d9f1426e10a8ccd60ea00583af3
1847 F20110318_AABNBF nunez_f_Page_072.txt
471cf44ac04c534abb3e13ce1766c0a6
9d329e14b0fd292d408a449b24b3125ec36dee44
1936 F20110318_AABNAR nunez_f_Page_054.txt
c6898a79b548987b3e627740a9dff693
5485b651c30b858562ceb930ff115a3c39fd1668
1160 F20110318_AABMVM nunez_f_Page_015.txt
0f5c312feb1bf23c2c0fe47b301e9a97
315ea13a02d42175e80b861a9ff6487eff86dab0
1750 F20110318_AABNBG nunez_f_Page_073.txt
132b99226a2969ced5fe7f0619a2dab1
e525b089913d21e17f69e8cd881ad8e6db961cae
F20110318_AABMWA nunez_f_Page_009.tif
99da3d88096350e61a9077258505a67b
c517b3ec31192c5d8ce48050d7c0f31266f46406
2515 F20110318_AABNAS nunez_f_Page_055.txt
fb89ddf31a74fadbae64089e5e7c1730
a9b57cf503f0d541d0341370563f2ff536135e65
F20110318_AABMVN nunez_f_Page_063.tif
c756ded9173ed8346bc672bf5eee81c4
db90e1d8f959a3a437e3d69afd7543e9a60046b0
7870 F20110318_AABMUY nunez_f_Page_021thm.jpg
69c583b8b63fc8ea5ab0a8b1ad18ce6b
b6d2e4a43e8e3a00ff520b08bd61cedfa9d2e98f
2074 F20110318_AABNBH nunez_f_Page_074.txt
c118f9d6838125f0e3512cf92e40cb50
98b91836e31cb9feeea3baf68bbb3eebd632f8e6
F20110318_AABMWB nunez_f_Page_010.tif
14dee12c6db8f80b8c83df9aa3f3c83a
fa1542455fc83d59d5094f260f360eb7cd1399b1
2718 F20110318_AABNAT nunez_f_Page_057.txt
7d69fc5091174c6838b82c24f0d4c07e
e1234c6573087ff43ab4bafc107c3df638902589
1653 F20110318_AABMVO nunez_f_Page_037.txt
9e197082047704d4d67fcebb680323b5
8c804d84f7b5412eb428e9a325a3ea00b9d5bc67
8671 F20110318_AABMUZ nunez_f_Page_093thm.jpg
0dccb9d649c110aa52568a98c5b914ea
e86023bd4241310cc6ac68ed9f7a0e9ca0f55c1a
1630 F20110318_AABNBI nunez_f_Page_076.txt
5e53ed0e39e565d6ee25b75ca407466a
9874ff0dfd45c3a49c7f1617d6a1bf61e97f61d1
F20110318_AABMWC nunez_f_Page_011.tif
085e5d5ccc10b5528667a54009458c7b
993d520cbaaec8258aa51cf4af285e89ebfb9dbc
1915 F20110318_AABNAU nunez_f_Page_059.txt
c15c5b1729921441c76b032732a9ddf2
733a63650f2bca627a1a6fc9f7015e185004c181
118967 F20110318_AABMVP UFE0011873_00001.mets FULL
a6d6ad84fc31353d83f9a5fcf99ccebe
d6d138d94c40c68840c3008dc816039b58d1058f
BROKEN_LINK
nunez_f_Page_052.tif
2082 F20110318_AABNBJ nunez_f_Page_077.txt
c0204a44f66b3103af1d5351ec1bdc11
83ded17907d176019b2ca64e8a7c25bcc02ca771
F20110318_AABMWD nunez_f_Page_012.tif
07e2928138035c2bc83766f4e4b2c580
1e72cd025de3312d321885486c753bbc0277e8a4
2311 F20110318_AABNAV nunez_f_Page_061.txt
eb4453443290e11d2117faf26f1c5e3e
8888c467b00b0e5a9e0e6c68aebae8ea160e894e
1022 F20110318_AABNBK nunez_f_Page_078.txt
b756333fb2ace84b9910f50383a57de8
42692d2eddd90dbe9cf96b5891a4416cad3e22f5
F20110318_AABMWE nunez_f_Page_013.tif
5bea8aaf8d9595750e1e87b0102d6b44
02e61070c88d6501f273fbc042aba9398a9245cd
1901 F20110318_AABNAW nunez_f_Page_062.txt
c27aaf2bba5963d33d382d268abc085c
54b094618a8c5956f9c0103fcd61550cf497f542
2520 F20110318_AABNCA nunez_f_Page_095.txt
3534401a865d73752f79ad2956b1b240
22933fbf20696f02d17845a398d88ff23aa05966
2061 F20110318_AABNBL nunez_f_Page_079.txt
1f483d17e398180a999fc1f3794aca32
21560d6344a4fc14316f77f04fd1dc6bffcde07a
F20110318_AABMWF nunez_f_Page_015.tif
775a2736f3d682bf7b947808094ce7d7
2d02c4845065cfaafeb7e6f455280fd3a161627f
2217 F20110318_AABNAX nunez_f_Page_063.txt
d9d8457bce166b9b6506d2ffa135ba0a
cbedd55b0525aae5f336d2f89f19ce73367acddb
F20110318_AABMVS nunez_f_Page_001.tif
9e6f3c66d03e9f4e5af7d0c50958b8fd
994df898c2920f91668903635a5075dce10ee816
2511 F20110318_AABNCB nunez_f_Page_096.txt
8bb0888b934a32f40bbd375c5d8ce5a8
79970e942cd1cdf8983b491fe52607a65f209f90
1720 F20110318_AABNBM nunez_f_Page_080.txt
5fb730a8c8ae349ffb2f01b1ecee36a7
c5a827ba837c75e3c7f7cde5fcb0198ed8eedb85
F20110318_AABMWG nunez_f_Page_016.tif
69cdd11d98ea9bba07735962c75e4fea
5b54264e9bff9fa7086b4249a112763560d94ead
191 F20110318_AABNAY nunez_f_Page_064.txt
7ec2d826d68fa0a2781278ebe29f4e24
80d6703a8de05bd03d3fe7f86497c39a62ff7bd6
F20110318_AABMVT nunez_f_Page_002.tif
1e52a1907345a25740092163b5cc10f6
5d78272a131c7abe1320110e29e7e32508452ca0
2366 F20110318_AABNCC nunez_f_Page_097.txt
e088fa1205e6fe0ba94ce61726350c81
4f0f0dba439990e7debb0f06b38e95c38acfdb0a
1900 F20110318_AABNBN nunez_f_Page_082.txt
b56398816fde724fbd515c1af540f5aa
c7f56a47256ca11f1d063eef882eafe745612abb
F20110318_AABMWH nunez_f_Page_017.tif
482469b62dabfcc0d60b398d269e8f68
644a37793b34b9fde39377c075ecfa984b065c5c
2010 F20110318_AABNAZ nunez_f_Page_066.txt
27b3c970eab2be5a4f2d3e15954af526
f7509ac8c3a359432a547c746fbcf2e05050603f
F20110318_AABMVU nunez_f_Page_003.tif
9f5fda28f20951bdbde0ea47adde983a
2b6b05258281bcbbf841bad21728ae365b3a79af
2348 F20110318_AABNCD nunez_f_Page_098.txt
2a2c7c86e4b8d358aacdd300fea8a302
cfe9339d85865f6540fb387dce21461c277b2e6c
1411 F20110318_AABNBO nunez_f_Page_083.txt
c4d452bc4c4c56c15f4bdaf015b5744c
c6ea48d65b6c7840626f6192f27611c4dd14442f
F20110318_AABMWI nunez_f_Page_018.tif
b8f453811efec1506eec4ffc07b61621
064af9e42fe2398ea739b40b3f824173ed96e83c
F20110318_AABMVV nunez_f_Page_004.tif
3ef950ed269695ec4ec03336f44b30ef
7a7dc2736cc89970c79bdac826d089c1f9c80db2
2466 F20110318_AABNCE nunez_f_Page_099.txt
bce6e991e34c66b53b8c751bc82eefa0
c90dc5c1e9164b0bccf7201fc0af918a4e734089
431 F20110318_AABNBP nunez_f_Page_084.txt
6a421dadbf109d43b669710919405ea0
4132f8dc62d7b3305b2b8f3b658f5e1fad861d8b
F20110318_AABMWJ nunez_f_Page_020.tif
e5d82393c003ae04564ea50bfdd87158
1f8374ebca61fa6b7a3295f11171e032d0e9d410
F20110318_AABMVW nunez_f_Page_005.tif
448e1671cee3a659ce98fb546dd0a8c2
b9b7651c52be18c748c88e7f75c6d58e1b3b1d2d
1753 F20110318_AABNBQ nunez_f_Page_085.txt
0a44685bf19cab16365109aec2e248b1
16395332507b6d87313e9a02c2d25b6fde8d07cc
F20110318_AABMWK nunez_f_Page_021.tif
f1a02ebb1af91934799a26278cafea39
07a2608926287b4015e282edf9aa864aad24655d
F20110318_AABMVX nunez_f_Page_006.tif
a9399784c414c1a1b31dcf4fac9799dd
f82d1d6880fc0cdd61bd5c4dcc1964dda162926a
1241 F20110318_AABNCF nunez_f_Page_100.txt
243c1c117d3b528d7125d9103b3db527
0f27981bd73cc104f272d06c6a62131b587ca570
1593 F20110318_AABNBR nunez_f_Page_086.txt
cb39e3799494ac459b0d45e915f58d0b
ea9b3844bf9118626584387cc07b8aea75624daa
F20110318_AABMWL nunez_f_Page_022.tif
10dc6fe66c70d3555403c3f7aa78f317
56b452a926971590c3b0c4fcf55931bea60fd3fa
F20110318_AABMVY nunez_f_Page_007.tif
11680f5596567918fcdfd08def87f0b2
f05130e7e73e9e6870188a4c3e1e03ac813df837
709 F20110318_AABNCG nunez_f_Page_101.txt
a87f5f7490305fbd81aeb66add13fdde
9ef257f4f06e3587f49d67aafddc92adee5982b9
F20110318_AABMXA nunez_f_Page_038.tif
d649a5a9475559c4e5e7d20c0a2a5dbe
7c23d0a3c41b67fa23270e81699766de52bf0619
1626 F20110318_AABNBS nunez_f_Page_087.txt
9bb221c3d88b037580711f61d7484427
2d5ac041cbbe9073c596a7b21a1521442ec0801b
F20110318_AABMWM nunez_f_Page_023.tif
7c7dbc71800d512f4175228afeb11f81
f0a56faf6684f43bbd8b9302a9da015e8715c6af
9715 F20110318_AABNCH nunez_f_Page_001.pro
6b6940938496a5c15420f2ace975b326
64fb355b15bdb0be424c0696a5ce7aab1d97c262
F20110318_AABMXB nunez_f_Page_039.tif
dec402e836e5ffda5a77c2e3e9f3da6d
85d66ee2bb505787fb8c7d363e23d91ca2b1083d
1674 F20110318_AABNBT nunez_f_Page_088.txt
6812e3f65012aaefc1afb5ece30df2e9
8ea6cc5fcd92c9f687d8816314a245283e6b542d
F20110318_AABMWN nunez_f_Page_024.tif
b9b23e951db29165d12e4bdd66753add
8361afc7d097cde66300a718ce52d81f9e67cb70
F20110318_AABMVZ nunez_f_Page_008.tif
16b87c5cca81b7cc8bce5385593f2e96
350c1db61cbc353daa1468db87ed7d3f0f136b0c
1433 F20110318_AABNCI nunez_f_Page_002.pro
2df4f827c775ef38099a10c0b7ce3fad
ef52d4b0a415dbe60ad23ed6666e6a58bdfafdc4
F20110318_AABMXC nunez_f_Page_041.tif
3850b9fb263ad029b105f565c2e74b80
c72127165ea100f7929ed97497631836da6cb14b
963 F20110318_AABNBU nunez_f_Page_089.txt
923d38ed9801ec9ac83ce04ad5a52cf8
a90129a2393d3e25db3ca91f26bcf4eaaae06780
F20110318_AABMWO nunez_f_Page_025.tif
18d7ff52bdb2c69fcaed45c66111e258
00b91d8138d783f6ab1333fe96a269e6cc905843
6908 F20110318_AABNCJ nunez_f_Page_003.pro
0126ce7128462db08488901785d6d612
8af892e8d7ad85fdb6969518cb70da5db7ff501b
F20110318_AABMXD nunez_f_Page_042.tif
2c81f9f23bd5e1dbe07f12fe914ea6de
2376bc95732eaf712db4d2e95f9b4b36d896e8a7
1891 F20110318_AABNBV nunez_f_Page_090.txt
8efcf398ac3f1731e7ef0556f3fe407d
6e23290c47e5e2d8068e616793f4d719c0504e46
F20110318_AABMWP nunez_f_Page_026.tif
d201f1ff65b96f78b67a5f3994b72fe7
1fe149f07d88378f5134cfad62c2002a369e0c0c
43411 F20110318_AABNCK nunez_f_Page_004.pro
18d7b010916b4618edcc25a630c69443
bf313b7afd8531f01522313c1a9c3dfd7fa822f2
F20110318_AABMXE nunez_f_Page_043.tif
9c4708401045849f5a156d88bec70948
18900d5c602646329e433914ec96f1504a664511
372 F20110318_AABNBW nunez_f_Page_091.txt
f3ece2767c32a5bfd445ca5d96bc7df4
4df0715c22a14fea47162fcc3a2bc2436b81ec0f
F20110318_AABMWQ nunez_f_Page_027.tif
7a261028dc6be4636df685d7cc849db5
1fd3cffc744c7edea834e9cdde37716c08af0de8
65063 F20110318_AABNCL nunez_f_Page_005.pro
0ce2eb2937e48cd00675dc9c7ce5a3bf
68c761a58d93dfa7bf6fdc5e250d15ec9e69775c
F20110318_AABMXF nunez_f_Page_044.tif
24d1eebd8d969a4c3dc0e6e8d4a4eca9
8eafb30895b68f2df8bb34857ac4d7b0fa2df57b
2164 F20110318_AABNBX nunez_f_Page_092.txt
ca93f938486dc8a864b25dd13c717342
1c690850dd27c9482032c3c065219878ce8c8b44
F20110318_AABMWR nunez_f_Page_028.tif
8181f7344a8d3f524e497cba843abe23
17075b6b33a9db37fd69db93b290ed4ccca218af
44400 F20110318_AABNDA nunez_f_Page_020.pro
901b73e5ce40c1e231175726d3442625
39630f9f7e1398f661db376e12fb57ec279ba055
102615 F20110318_AABNCM nunez_f_Page_006.pro
174ce91526e45755dabd5e9b014f87ff
a8d0793dfee544cabbd34ef402a277361d47c8e4
F20110318_AABMXG nunez_f_Page_045.tif
67387f4dedfcaf1c8c8b0c37560f4c27
d76c06bfa6807528f9f58ad6f572f7880f3d061b
F20110318_AABNBY nunez_f_Page_093.txt
37916e9a42d3282bac00bd88b68d8332
c2845bdbe445fb7088162f762810a362342ffced
F20110318_AABMWS nunez_f_Page_029.tif
987b54d772567b645ff3482fd80daeff
8521c746d97f1dc32bb43a8104387eb693c4acea
48255 F20110318_AABNDB nunez_f_Page_021.pro
3630f3505b6c7bd99759a2b594ce0e24
3cdc28e1a9f8d78500b9d82a67abca87340d5ca1
35235 F20110318_AABNCN nunez_f_Page_007.pro
8b3a0f5ed7e5384182e62370371c6c1f
a2b4125e3216a6e7557f7e76015584254559f09b
F20110318_AABMXH nunez_f_Page_046.tif
78c769ca115247fc093a5ed9d4f536a4
f84a7ccbca7a8a80bcea605c2311cb0c1e1d2a7c
2465 F20110318_AABNBZ nunez_f_Page_094.txt
a12a74fa1b5dbcebbe2f7ce5aaac2fef
a1c5d3e098852afafa19d0a358df69c1d49fcde4
F20110318_AABMWT nunez_f_Page_030.tif
e7ffb5baed60514a34b0c9d50e4fe09f
fd6f8e39942825bd6c44c8e814a08bfba6e59cfc
43394 F20110318_AABNDC nunez_f_Page_022.pro
d1c5304539c8e70c863650a1db703d5a
bcd81c017cade92bad26175508e56673b2d50082
36567 F20110318_AABNCO nunez_f_Page_008.pro
6533f64b7bf19cf54c22021a6a6e1af5
bcf475758ba982eb3c9d9385c3ca3b5a000d620e
F20110318_AABMXI nunez_f_Page_047.tif
48e0ce8bd037b4494df153515ddaca3c
43a64ee75e6b6c8bce12495ac31827a82554eddd
F20110318_AABMWU nunez_f_Page_031.tif
6a312fcf07cdd5d59feaaca0022b6a73
77aa6a27c6917d7dfa373ba271d14c645eb0bf9c
50694 F20110318_AABNDD nunez_f_Page_024.pro
1ab98e86bb293f89926c799622f1d37e
c4a04cc0e9fe7a1be9055e83f9a8ed12181dfada
66404 F20110318_AABNCP nunez_f_Page_009.pro
af41498df24dd556706651b2ca422abe
49dfb135889ed6f79383bb3468af3f491fedf917
F20110318_AABMXJ nunez_f_Page_048.tif
5609f2ce9d6c31db7b197fc21acd7d79
34b9d69a21fed7ca9897bb1ed012b836eaf58341
F20110318_AABMWV nunez_f_Page_032.tif
77946df6e0a052c40617f77294a83871
31fcbd30b1b97c29faf9f60e686e02cc68d1c606
47840 F20110318_AABNDE nunez_f_Page_025.pro
a990eccd86c28246883833e5236cb840
0bb95a78e67f3263c3353933058e5dc2215b47a5
72022 F20110318_AABNCQ nunez_f_Page_010.pro
4377826e2c30428d08dca1a2c013e76e
fcd331154d22df9fa02ee7708565ec40b1091836
F20110318_AABMXK nunez_f_Page_049.tif
006190a25da4c68503f7aa36d724bbc7
e45f3597e6b952d193c337713118028ec1aec370
F20110318_AABMWW nunez_f_Page_033.tif
d47fe8bc786d3fcf2a16ce09e2f1814e
7de58077bdf8348c0ad52fe1142e0b74b35b8b48
52451 F20110318_AABNDF nunez_f_Page_026.pro
3bf3f51da6f2519223d169c596ac2baf
01ab3b59ea580cc723eb1743affee3434c5a3045
40575 F20110318_AABNCR nunez_f_Page_011.pro
07d0ec1b24e80eaccf2d8f05cc33f282
453cf2c6e6b39431d122afc62f508ddebb69edbd
F20110318_AABMXL nunez_f_Page_050.tif
7896dc05a79c7eadd0501ac596cdf4c4
1f3cfd04a6a318e2554ab80f0f403036954870cb
F20110318_AABMWX nunez_f_Page_034.tif
0127b9272d4ba0a051768e3035f41958
2b9c5025c54a583f2d9d0b709a71a2c2eece905d
50188 F20110318_AABNCS nunez_f_Page_012.pro
8ea2800f595f73017c387a8d39efc294
4313b724044fc2953b7f7865376dad003a0771f9
F20110318_AABMXM nunez_f_Page_051.tif
ae981418c03516aa1f6885e7dfb0b9ba
498cb4bbb09932b4c36ab53f2b038ed74e8a1e77
F20110318_AABMWY nunez_f_Page_035.tif
da612f82733a0e4dd6feda87f52aa5e2
0296655e2224d07e68afcecd392161e0d6ec26d7
48957 F20110318_AABNDG nunez_f_Page_027.pro
c17363b68361b6e64bc29c2988359743
75dc60aeeb33c2a73cb29cdb56a1db0969a25135
F20110318_AABMYA nunez_f_Page_070.tif
a59a4c000b1dc8245b29fa20f19a2beb
651b103a04d018393b5103f0dd450eea77c5eb62
45049 F20110318_AABNCT nunez_f_Page_013.pro
822b3c04868d0c6c31b9e78c618b1674
fdcb23bccbb5f4c03a6a652388c6e02c5408da34
F20110318_AABMXN nunez_f_Page_053.tif
0b868d2ff0c0f1d4f615531542512142
bf84d035af66067d93ef15beb49a753d68f75c52
F20110318_AABMWZ nunez_f_Page_036.tif
8a0cf25263fc450f8271e6be3ac87973
309e0086ea435e5bca2c1ffbd4b1f9dab990f6af
53482 F20110318_AABNDH nunez_f_Page_028.pro
a598e68297e56038cf69a1bd3c3dbc74
4868e1fef79a437fd19f60fb79a98075218cdfd1
F20110318_AABMYB nunez_f_Page_071.tif
fb07e56c6a4d33d944d33512587d6905
f628bed916ca85bb16261c50f404db5f46dab3f0
48289 F20110318_AABNCU nunez_f_Page_014.pro
3dcf39423a1075860d30c43bfe28ff96
756a0ea64f2653bdf36aea8e0165516e894dceb1
F20110318_AABMXO nunez_f_Page_054.tif
751015a1c03b71b7fe718153f6c9621b
46a031a1736a0936a545b40ce1d7d26bc4677041
52987 F20110318_AABNDI nunez_f_Page_029.pro
9def28cca34864277da4fc7ddc96e0ce
955e54b1a40f5209fded87c303a4fc3dabac9735
F20110318_AABMYC nunez_f_Page_073.tif
fed59d67bbf9f7282b4ec4c8204ef73d
1004aa6cdb927eaf77af24cb69ead413ec236541
27847 F20110318_AABNCV nunez_f_Page_015.pro
6d206867af8836ad718d84107c4bacf2
69d376ab2fd77cd78d900d7162e732fcee44f5ea
F20110318_AABMXP nunez_f_Page_055.tif
62c81011216f949ae88f089ac754edd8
a0ac0f1d400f09ed783f338f434a3367df409f70
49333 F20110318_AABNDJ nunez_f_Page_030.pro
b43fc6e5e484faf78efc05f14b94feed
595e9d8256bd9cae797c95fe72c054cb88707f2c
F20110318_AABMYD nunez_f_Page_074.tif
ef6293cae92f7816f2e2678251e171b1
b3c105c1d73d40f67022c2b7590a3c719e9f7b76
42055 F20110318_AABNCW nunez_f_Page_016.pro
4c2320f9d4dfd8b3965e645e668774db
863ebc8da2bcd42053b21c43d753f405c54f71d4
F20110318_AABMXQ nunez_f_Page_056.tif
8a46c1352017fff7dc3d3e38306d059f
fe3e3169a1fa944a3420fa973caab0cf5f77da85
48753 F20110318_AABNDK nunez_f_Page_032.pro
2a4e00b415f137caaec5f98b8d17e83f
51872112db223e946685db7dccf62d29d1bb8a22
F20110318_AABMYE nunez_f_Page_075.tif
54fd818a9f94dc3d7942c4a97f7748e2
5649c85032ff7c99337e4149d5e8a7acac79c8ff
49383 F20110318_AABNCX nunez_f_Page_017.pro
3a73ec7aed5bd10a6b15bb73dd8121e2
bd182868d8380632c6fd8281e93e025bea5b7d1f
F20110318_AABMXR nunez_f_Page_058.tif
8193f795f029312747df8767819a48e3
20dd6584742f7dd78a30b0fe6ef6da41e948bee9
40434 F20110318_AABNEA nunez_f_Page_051.pro
e0c13c04d23c6fa9a13bdc3addc0f9e9
70f57dad203bf55f7d666c765a0657db83372df9
41324 F20110318_AABNDL nunez_f_Page_033.pro
6303badcd83df6a626574b854ef2bffd
8e828362b61ca8f6d9b342fc33c18fb0df8be408
F20110318_AABMYF nunez_f_Page_077.tif
62faef25b101fb438659f7baaf66ed19
18b5a63bbf887de92bf396978df6735b3d321ace
F20110318_AABMXS nunez_f_Page_060.tif
489004fcd52c2d014d0dfd851e45655f
60fb05303824cd84ccdce19599f7e0d55e758ee0
36345 F20110318_AABNEB nunez_f_Page_052.pro
c36ce452e072b6217ba06aa99e0966d1
c2e2ac374ff7108bced9a1b53364f94dcee2d4d5
8996 F20110318_AABNDM nunez_f_Page_036.pro
bf6c25996b771f8791e59683b74440ce
d84a20c4043e8817959e1f5a1b2e8d67e365e3b8
F20110318_AABMYG nunez_f_Page_078.tif
fa2cd4cd4a8904cbdc29bf44e20a3d21
9ef8c3dea814f34bb5945d5a5f2649e8458438ef
50993 F20110318_AABNCY nunez_f_Page_018.pro
e09464fb4e11afee257202c05c7012ad
c954ffbb7f46660907f65620edfe9099f244dbaf
F20110318_AABMXT nunez_f_Page_061.tif
394fe6759452df78a0ec37aea32214bb
879ac459590adc8cc9d50e91b23c993714c5b37c
49966 F20110318_AABNEC nunez_f_Page_053.pro
04978674094d518657c7fb12337f5096
966d383814b02aac911de6d629cb68079b69daf4
39651 F20110318_AABNDN nunez_f_Page_037.pro
6ba1b22bd7c88f864e6a82ea71f1ca74
f86faaecc375ed2c4bfac889dc581959fb4c5551
F20110318_AABMYH nunez_f_Page_079.tif
b4685702a18a895e605e96571e687944
8b46bf34d8685428914b89f1f114c1e6fb93eca3
52654 F20110318_AABNCZ nunez_f_Page_019.pro
9e331c94fb8d7c0a334917398ab0ad85
0e7b537abed9f2b0425226e41612a4a266c87da4
F20110318_AABMXU nunez_f_Page_062.tif
9d568a497b5f8e2e866988ba4859a057
48e57c5452508c0b4080b641d646dc8e1136bd16
38131 F20110318_AABNED nunez_f_Page_054.pro
f31051aaab8109232d93ad433dea9da2
8164586b76c35a9f0e0defaa8a2f9179a561057c
49507 F20110318_AABNDO nunez_f_Page_038.pro
8a25f3ccdfa168c2c320b0cbb4cb7e28
4cd71db07c81a149e4e9cc6bcdfd766a5450e188
F20110318_AABMYI nunez_f_Page_082.tif
1e6037ae2c81ad5c542606a1e47323b9
9f587678b418582e57481bec638b63b96ababddf
F20110318_AABMXV nunez_f_Page_064.tif
b702877d7547169eeb1acd2d803855ce
59a267ae2f571cd932ed4ecc86ac31ec73bc7fd9
53376 F20110318_AABNEE nunez_f_Page_055.pro
c15cb381e63d3284063d20646fde3c0d
ef80a17f3c9e562a34fc6ac89909ed23eec8a321
42408 F20110318_AABNDP nunez_f_Page_039.pro
4c7333556a3011385617c0f212356e09
9fb661cdccada14677f05391e69da0625f3168e1
F20110318_AABMYJ nunez_f_Page_085.tif
929904a1bff6c3496a284322f2d7f016
8e24e37c55b561cf40c672fba00d47fd24f59405
F20110318_AABMXW nunez_f_Page_066.tif
6742ed6b8f6a9815f768c04c2010b4d1
86bf3191a17bed21366033b03d8840d0bb180097
46687 F20110318_AABNEF nunez_f_Page_056.pro
3dd5bcd03b3cb91ed0adc81776e2b9e1
34bb33e61fc18aba9858bcc336dfe785dd96f87d
43895 F20110318_AABNDQ nunez_f_Page_040.pro
356f0ce31ffc3ff3ca07ac1afaab8537
9295895f7404e71cac61af4d155f4d86ab1cef8a
F20110318_AABMYK nunez_f_Page_086.tif
2c655e389312c46ebdf06f021ea6a297
12bceba00b6b8a957212a7c47adabfabd2bf88e5
F20110318_AABMXX nunez_f_Page_067.tif
81d1cf13d1e684d8c835fb2395e680e7
0850a320f42d96244d2a2097a322431961e257db
54405 F20110318_AABNEG nunez_f_Page_057.pro
17d47658d07066e4abee6de19c2b5a46
44f8c8dbba7f379d385eb9bc2326e6bcf038806a
50861 F20110318_AABNDR nunez_f_Page_041.pro
0f2424e24ebd9662e96025d2f973a3ef
14f2eabf140581cd588bdf0cbc1244318694883d
F20110318_AABMYL nunez_f_Page_087.tif
dcdbad73da08efe786ba8b21b82070be
d82074ae25c010376a82e9d9b410c40fe1c80c2f
F20110318_AABMXY nunez_f_Page_068.tif
c270123339b52345205c47009c59b159
48c03155006227fbda6c01c179dbc56fee3aae6e
129 F20110318_AABMZA nunez_f_Page_002.txt
7826687395761acaa2f9c579f596cb5d
f4869ca58ba64c11da449725de32ea63082992e4
48964 F20110318_AABNDS nunez_f_Page_042.pro
31a5658beae3e5299c8309dcd7bb2d8b
5b9d81c35f33aaacb46cc12af3de330e19278070
F20110318_AABMYM nunez_f_Page_088.tif
f657cdcea8585a95ecc70e600d3c784e
4b3b345e896d4d5b06105f3e1018b9c506251f58
F20110318_AABMXZ nunez_f_Page_069.tif
b181b4e99409837fd2da20842ec36817
746217338bf5bdd1a59b80678e5ddcbb136a0c43
48605 F20110318_AABNEH nunez_f_Page_059.pro
c4e51b9b7ba75b689105dfc53ed9a059
eeb875699e9697462f78e5253f98b18374e3ecfb
323 F20110318_AABMZB nunez_f_Page_003.txt
6b45e376c60d00a3aec570de28c388e6
e8389da101e08ef1f6df875106e24fe13448f1dd
42233 F20110318_AABNDT nunez_f_Page_043.pro
94983d3d1083871bc34016e10c5fd356
f0215ce0981ed8e65edbd4bfa9ecebeb4d4be1ef
F20110318_AABMYN nunez_f_Page_089.tif
306289998fb0b37beb3297fd556f5701
ff3b1b4b23fb2fd497b8d3c614907d20fe14c86a
42458 F20110318_AABNEI nunez_f_Page_060.pro
2086602fe612476fba66075b0bcc6bcd
5715441500bdcdf272e5c1034f6eba505064fe6a
3249 F20110318_AABMZC nunez_f_Page_005.txt
8337146adbf93afd85636d67ebdc73b4
f70c7caf34c30036ed3004ac5a7d23228ddeed01
49395 F20110318_AABNDU nunez_f_Page_044.pro
91585898a67f16983bc3e202c2e3772a
8857df42309f6719339f4ac8f9a3f91960a42520
F20110318_AABMYO nunez_f_Page_090.tif
b52f317b10e08e4c0c146fbb530bfa5e
386e0f339ad9b8e977ffaae65a0459bf51501180
52052 F20110318_AABNEJ nunez_f_Page_061.pro
854cfb93720eb86377fef91b543fb018
bdb3e1f209f53ea1e0b3ee62da08f4cab47bf2b6
2733 F20110318_AABMZD nunez_f_Page_009.txt
afe27a6a00ae28611743b7c781c3b7bc
13721bb337d518eb0c17a313b2fef034c0dc5509
37491 F20110318_AABNDV nunez_f_Page_045.pro
62f3a0ab92f84730f47d8e6280db5fa1
954d39ba6413f2e622726919663ce88e4dee20db
F20110318_AABMYP nunez_f_Page_091.tif
c828dd8192c6279ace6cd3a9b80e83a2
a88a529486d74c8637c3f329a04d897f836908d2
48162 F20110318_AABNEK nunez_f_Page_062.pro
74b290b4860256c8f47d5294d91c6b70
54e83be9c15e7e14e143bb73f7edffe5ac802685
2964 F20110318_AABMZE nunez_f_Page_010.txt
3f4a18c0a67c28e4f24573d3ca03703e
453c4463eb6b8c65b7814313ef02848664f91477
51164 F20110318_AABNDW nunez_f_Page_047.pro
30bf3ab06f5cb5c12241b571d106102e
a0fa7ed8763a6b784c5a38c811c72536b4cbb372
F20110318_AABMYQ nunez_f_Page_092.tif
1387736ab0d069ca67c11afd460414ef
75f44355cab060822ef7b50fc7fb73f6f6c3000f
3662 F20110318_AABNEL nunez_f_Page_064.pro
ed9c888983185a5bd77af638df4a1efc
1446a312779295a0936118dcc474788fafd1c132
1793 F20110318_AABMZF nunez_f_Page_011.txt
09c914994c4ff1d6282de1b8c9a3b6c1
387830eba3bf3980a471be7b666b6e9880833f77
37315 F20110318_AABNDX nunez_f_Page_048.pro
375f2f867181878c56d271e98593db67
f8239da3ff1e15cc9d92c279f1accba9dda8d9d9
F20110318_AABMYR nunez_f_Page_093.tif
25439c27a1eb18195b2aa169bd5485a4
f8ff0462ac0f8f64cd16307b40fce62084184639
26906 F20110318_AABNFA nunez_f_Page_083.pro
c86c8d9221ecbd0360e0341d23be1499
9fc9ba64a4ac7c80d5eca81e6b8e727f2610b16b
42206 F20110318_AABNEM nunez_f_Page_065.pro
d1c1478db0d8087385b7112007e70afa
426a9d5651d359cfdce3f043a3869be01140c09e
F20110318_AABMZG nunez_f_Page_012.txt
b1ab87a5dd257c4219d2fd7e1cdb1654
73f89b897b7e1c334f34109092f2c451837b159e
39215 F20110318_AABNDY nunez_f_Page_049.pro
0f2e9fa659b192486fbaab09297c65d7
f6f0b9ab325f755bba02bb93693e4677ecb64e19
F20110318_AABMYS nunez_f_Page_094.tif
ea3a63da180df8768fbe700c0f64db6d
bb3c8b9ecc328f306a2d89deee0fc44b73a9b0ef
9218 F20110318_AABNFB nunez_f_Page_084.pro
1395dc265ec4e0ce75c33e890089cae9
9b16131d3a3760efbd7442964b8b1c4c4c1c65f6
46902 F20110318_AABNEN nunez_f_Page_067.pro
29343d52988ab332f76c9223ee11e52b
953f69695def34912b3fd5269c6eaec85adc7a38
1849 F20110318_AABMZH nunez_f_Page_013.txt
95e8894d9e39f8d82f454fe397d66ab0
887b85650f33363ef3b910d6953670ba902b4754
51750 F20110318_AABNDZ nunez_f_Page_050.pro
73cfd06cf64805319a95742a4d72a86e
36ac9c09ba6c7af98e2b9a7cb038983e22b4bc86
F20110318_AABMYT nunez_f_Page_095.tif
4a70f54b882e49ea8bcdb162595d77cc
9ca12c3e304aad38b989817b0db6502de2a28899
39649 F20110318_AABNFC nunez_f_Page_085.pro
427387e175f330394e52056c06f58de7
10479f661642d49ac1d89236d914b1aca89c56ca
47344 F20110318_AABNEO nunez_f_Page_068.pro
981ccc3755fd8a83412099fd4901cc28
cd1410109484765264064e3139f8ce9ad153fee6
F20110318_AABMZI nunez_f_Page_014.txt
7b9854e4abead9f892124e2a57b0695a
660b6adf23b6cfa048f8ca2853e330ab53217cc8
F20110318_AABMYU nunez_f_Page_096.tif
fb52e6d07a4a339f32d4c571e4833b7d
3aa97148192e47b52302f65c67f2ebdb7fc0713c
34130 F20110318_AABNFD nunez_f_Page_086.pro
f8371cb9f19f383a6590cf38c00befeb
3bf059e21e18b4e547b1d39035d60cac9a5a3ea0
38872 F20110318_AABNEP nunez_f_Page_069.pro
c95ae1280eca3fdaca4e26b9d09093a0
e74afe1cccd366049e874aaa8303eb8b20987e13
1755 F20110318_AABMZJ nunez_f_Page_016.txt
ca5867cccaf6670e370969ca71c2436b
aae558f6d7df3eb636a80febd12fe8640c102f7c
F20110318_AABMYV nunez_f_Page_097.tif
c151578e98ce5bd1e5fb0043e9dedd8f
8c1a048d48874dc7a4143f9fb77b376bdb911a54
35042 F20110318_AABNFE nunez_f_Page_088.pro
2795403b95d3f9fc5af79473d295730f
70d8e783c7278ea82f0e42db937f95471aae1e84
48679 F20110318_AABNEQ nunez_f_Page_070.pro
8578889531014cb21d0eea1fd4ea4b3a
bfa12e8b5583edfd89cadd0ad95243d10d872332
1982 F20110318_AABMZK nunez_f_Page_017.txt
11c7f487bbe340b5e9e3d5f6f73620f8
0a4c80cd41973cf1353b673b3128402bc0651cdf
F20110318_AABMYW nunez_f_Page_098.tif
538a777a446b30def6d9175923c0ca26
e72fbbdab7b377003482a13d7adec125cacb3b3e
24252 F20110318_AABNFF nunez_f_Page_089.pro
40b671f929b4294de311a5f95bc5dd14
60be79d5334979c2974625bcd9e1b7492a721a69
46631 F20110318_AABNER nunez_f_Page_072.pro
5a92f19ffa35dba4824a08b4ce14ce89
fd85b217a7e048c6f4e8bfbcfcc129bd90857b64
F20110318_AABMZL nunez_f_Page_018.txt
576684e9097c65239d70bfd6876785cb
a70fd2ebd2a5bb03a2de75a3ef72edf8d963c0b5
F20110318_AABMYX nunez_f_Page_100.tif
8e304f1e4cfa5e1aab551cde525c4ad5
b43a71cee46125fc649ada4ac6676f5324a1e309
46469 F20110318_AABNFG nunez_f_Page_090.pro
e05deeba3b64bd0f6fafe18dd59a61fe
558e4e9e629618694b9fd120b291c6ab514f041d
24031 F20110318_AABNES nunez_f_Page_075.pro
b1bf24b4ee3b7e3d659bb8bf3ee60bb9
29272b5693ceae88548ebadfbb599c0ee668188c
2066 F20110318_AABMZM nunez_f_Page_019.txt
a0f4ad51a219cf7c029e7bf3a2edab00
313234247846d1db72649e0203e3e57d143ed555
F20110318_AABMYY nunez_f_Page_101.tif
4f6059f5fd3f0cd8feda05cc98f4953d
cf2a96aefd413ba37117963b465dec798ad2569d
51185 F20110318_AABNFH nunez_f_Page_092.pro
7ef448e9da9f9223433d8add38140fa4
22002962235d40854ae577da980a7896397e7e3d
34675 F20110318_AABNET nunez_f_Page_076.pro
cfca3912a90a0ff48f5c30dc8ff9129b
ae6bd01b8439cc63be44b66c716c242597e55eb0
1789 F20110318_AABMZN nunez_f_Page_020.txt
6ea0c13a2155cbc7bc84455516bad721
49f4b15bf287678edfef2eac17d028da5908308f
543 F20110318_AABMYZ nunez_f_Page_001.txt
786e5dff645425f0e92f245632cefee5
2899db823b8f584c7aca33f667f2ad8a48a3c5b8
53202 F20110318_AABNEU nunez_f_Page_077.pro
5fd2473ebabeab20b0610bee73709d0e
6484e28beaa8fd1124230e8a6912c5ff30cd64fb
1906 F20110318_AABMZO nunez_f_Page_021.txt
9760abb44c10fef32b0cd4ce46f57f66
0c9551d40c3c8525d74330bc8dabaf5e6f8f4607
60747 F20110318_AABNFI nunez_f_Page_093.pro
f4f168f7ecdf677ef308e6263e0838db
4b0a3650af0fc2121feae25edf54a025f1a7dca1
23359 F20110318_AABNEV nunez_f_Page_078.pro
3d0b56babc3b0691bd754fa8e1f31ebe
a4c36af6fccd79fb0e962d53c586b264d2e3e4ec
F20110318_AABMZP nunez_f_Page_022.txt
2edb27065e4f2886cbffb75b5af6159d
874cea3f790b7bcb917683901d16a6eac40755b0
60141 F20110318_AABNFJ nunez_f_Page_094.pro
dc60807c908d5434637e36275c557ada
bb3e547012bbb577cea5efbdd7c3509a031a9d54
52491 F20110318_AABNEW nunez_f_Page_079.pro
69444debfdccc8631491f4a1468ee14f
24617e7f2a8e93575a2a2eef5bbd494c1b905ac2
1923 F20110318_AABMZQ nunez_f_Page_023.txt
341a97b63d04ed4321eb46b7b4d3dee9
a06eee84fea5901129a08af0cbe9afc0c5f8b5d3
62608 F20110318_AABNFK nunez_f_Page_095.pro
70939487cf265ed3822c7d3bb1e3e494
50ff6a09897f01414ef84defc8a789e1731c2e93
38215 F20110318_AABNEX nunez_f_Page_080.pro
a4bd514ce3a7113e66a8d8bf040e8053
f8fa2f5ea4bc2d69e0baf950375bf56fb8996a54
1920 F20110318_AABMZR nunez_f_Page_025.txt
c3361630578678843734b5d8b8ad89cf
1d1fb85d378b71d70fd12aa6f1e5d68bfe28bdce
87224 F20110318_AABNGA nunez_f_Page_011.jpg
4110ab946f6d46e8ffb18b03ae2352ba
c9845fdce16059f25e17893d9a2507a4c25bdf09
62147 F20110318_AABNFL nunez_f_Page_096.pro
88c351e284ed018a45398b27f0bcd0da
f73e86fe5251431046f3d3a67f46c72711138b2f
36675 F20110318_AABNEY nunez_f_Page_081.pro
febc8a02b6eca9ad0b87d534be773215
ea317184e56567b98385b4ab0970a43ca01df2e2
2063 F20110318_AABMZS nunez_f_Page_026.txt
ee578ea6054c2629540ef0c1db3194f0
9e8142067b0db473dfe588a5c07ccc325bbd4181
92929 F20110318_AABNGB nunez_f_Page_013.jpg
7ca9c77bf761c7f08bece1a037535cfa
7651354a01af55d15c8b42d152d306806c0fac2b
58142 F20110318_AABNFM nunez_f_Page_097.pro
1bd50c6d400acdc961898b74c4f122e1
ce1f1086ad33c671f7c039f77ef1cb081b07452d
48113 F20110318_AABNEZ nunez_f_Page_082.pro
65534235b209159272b3bbf08ea9f06e
b472d4f2c503d66d36849afca6d8bbbfc9f05b5f
1957 F20110318_AABMZT nunez_f_Page_027.txt
3951879d6ed9b62e57ae3bd48baa11a9
1cba4de0135a4aa4b6d368b951d8572324da23fe
98951 F20110318_AABNGC nunez_f_Page_014.jpg
363b84c6abd995ba6654052a01835cfd
67b539f95c2a86d35744eab6184ef6eb2894c248
57791 F20110318_AABNFN nunez_f_Page_098.pro
8990215ee07c5ce4e802d3634b350ab0
b2ea197639f3cdc6c7b701ffc80a35ea0492154e
F20110318_AABMZU nunez_f_Page_029.txt
42090fcdd01db28e74817bfffeb4c01b
8826ee57f31420469de4a507c000ddc38d37bf5a
58502 F20110318_AABNGD nunez_f_Page_015.jpg
fb68ba3c50652928aaebb124fc551a20
71fe2d07eb282ec20f1b54915c9b1bc68813ea48
60877 F20110318_AABNFO nunez_f_Page_099.pro
22024bd54b46cedb29aed3c390ccb2cb
9dabf3ffe7d1622ee88b2473e49b4134fba21304
F20110318_AABMZV nunez_f_Page_030.txt
ee7125ee7ac874a785bc72eff24144be
943434db2447832a31245a981db15147d6f98176
87402 F20110318_AABNGE nunez_f_Page_016.jpg
6c8d04bab951ce9a7ebbf6a1a2169d8c
904ed49c62929996db675a83030183057dbe5d26
30248 F20110318_AABNFP nunez_f_Page_100.pro
0efd2915af6bf56bb0c1088962f3524e
e620807194ccc905e9832a5f1766d4521caf7ba8
2006 F20110318_AABMZW nunez_f_Page_031.txt
ec69480b0eb27dfd8b63a63caff58041
6b361d479c4552add68b188f94572fd0371595f2
102412 F20110318_AABNGF nunez_f_Page_018.jpg
93e906712ca7a4231b72dcc3579debf6
3dd81f0f62429c530d3b9f4fdb15b1bf4935bc3a
16629 F20110318_AABNFQ nunez_f_Page_101.pro
7affb515ca58c0ca19a676b3345d0cec
6d2a68b3d4acd6055b60b67020fefc635ce14bb4
1921 F20110318_AABMZX nunez_f_Page_032.txt
9413ddebbf2059d16c76c5d1872b3e4f
4a71901e33bd37281c9a3d9aaaab240e8d7569db
91850 F20110318_AABNGG nunez_f_Page_020.jpg
0f8d0e077e0cdc7f0829e4032b6e5b67
c83f5f0517af9cdd960d1078667c39982dbaac6b
30483 F20110318_AABNFR nunez_f_Page_001.jpg
ccf545a6316c0154773345e63ed54448
859153f4370cbe2dfe192965fb4da0222f4a4cef
2064 F20110318_AABMZY nunez_f_Page_033.txt
9d337494e6a565250bfa55f2e63bd8c1
e0751ee12bec38946afafaf88212fa4a7257232c
98620 F20110318_AABNGH nunez_f_Page_021.jpg
77d3de20cb8d460d3dc4ad436ca2ed25
36e25bc77edec582a322749e75d3260c9a458d12
5394 F20110318_AABNFS nunez_f_Page_002.jpg
af42d666cbb0f40dab093a924ec06bc4
b491b4830a549c87a056f07131d704251c0766db
F20110318_AABMZZ nunez_f_Page_034.txt
a9c28974315b20f01ca504844a891484
5851048cbee6d91c68c060272d40690aa56a69a9
94040 F20110318_AABNGI nunez_f_Page_022.jpg
4202abdf375566818879cb273b3135eb
abe5b1f8968126470418c8aede58ffd82f10ee96
17752 F20110318_AABNFT nunez_f_Page_003.jpg
70c380c4e12c44e5c3c0e8083d653ce8
aef8fc837eb9cf6ea644ce70194af1698f24848b
89068 F20110318_AABNFU nunez_f_Page_004.jpg
786d5b2a989a0215cf1860b5b56ab81a
d0ffa613e8cb249d6efd3cccc40d9729c7bda699
99526 F20110318_AABNGJ nunez_f_Page_023.jpg
c4233ab47d02965348d7afff8356fea1
836ec8caa862752da35a3730fbadaa26ff7d30c9
149722 F20110318_AABNFV nunez_f_Page_006.jpg
c6f058854e1954afa6d16a5384532170
5bf9e753265b7e73799508042e895860b378041b
104139 F20110318_AABNGK nunez_f_Page_024.jpg
9e3e34e0168642d9bba083d75f90d807
72fef6734820300ee1cf10b9b4c08d0343b8d5b4
57684 F20110318_AABNFW nunez_f_Page_007.jpg
87e95094f8b03d470da12cc5006d0367
0ad5aa04d3b6a0490f7ff8ebaa902db568eab7c9
100446 F20110318_AABNHA nunez_f_Page_044.jpg
56e3e95ad48b9074dfaeba381748301c
1231fa5c656ed8e8326a192fd98a93c97e380f67
106098 F20110318_AABNGL nunez_f_Page_026.jpg
946f8e3c6a9a965b9554ec2ba2b60bb2
c56bff2ba37808a8cb9afb9c0399a0939721fe48
75770 F20110318_AABNFX nunez_f_Page_008.jpg
995e3a499f4544739728b02b9f94ab85
25668ef1bb899d863fc860001f8351ede510f33c
81470 F20110318_AABNHB nunez_f_Page_045.jpg
9660589f536a75c4517bc11f06fc28d9
dec19e077e61d7d1b1355d70059538c2d095a164
100582 F20110318_AABNGM nunez_f_Page_027.jpg
d023af3cc8a5317ad9647f7d3e422fc2
36076d47d60bfd13a0ff28d54955c54bef072dc0
142545 F20110318_AABNFY nunez_f_Page_009.jpg
9e7f2bc9c8cbbcacd1bd8bb2a15e9521
b9d045f30a5c08430de40c79117a03661a42f5b0
81255 F20110318_AABNHC nunez_f_Page_046.jpg
96be9a08a838266b2773cae1247c0ffb
abd89559b117426d11eff028533355b0f2afd78b
110840 F20110318_AABNGN nunez_f_Page_028.jpg
0b26bbbae64a8d770c1a01df52f7ae90
d01c5dd378296509b47dc598284519db44e606d8
149404 F20110318_AABNFZ nunez_f_Page_010.jpg
0291d22cc534e48e85d9c008fdc62be4
5cac30b45515a0b6a5f7b98d3a61e6ee8fbaf51b
103884 F20110318_AABNHD nunez_f_Page_047.jpg
6f1e6d3163c33df7044e8fad0c19efe3
02c1c828247413a724862c6b790ca9cb64339873
107776 F20110318_AABNGO nunez_f_Page_029.jpg
fc35b08977115dfa4409466835e7ddcc
adbb818bfef9bc075554cb797d3e688733961226
81037 F20110318_AABNHE nunez_f_Page_048.jpg
e5c7bed0f34091ce64a343b7de336580
2756f3ada275aee45e82d7dfad8825948181088f
104825 F20110318_AABNGP nunez_f_Page_031.jpg
3c7227308010e9c8ecc956f7fb2f4b1a
e28d682314c6dfdac229614a6a8a7852ec9dc8b3
86009 F20110318_AABNHF nunez_f_Page_049.jpg
712510e8249e20e5fe7169accc3a86ec
3153207492d8e5cd970e937243ceacd45265d743
100344 F20110318_AABNGQ nunez_f_Page_032.jpg
60583b8d5ef6bc57bd10580b4a07e1d1
e694a2c38766df06c39176f78ff7c8d7e28e7413
105756 F20110318_AABNHG nunez_f_Page_050.jpg
cac4b60118ecca16d90999f7836da782
57ef56413abe172d31a2b333cab8317aef3f3ea4
93652 F20110318_AABNGR nunez_f_Page_033.jpg
382634055ff8d553a4030adc7d3a3f0b
fa58d92a248a04976c93efde2e6c3b440d564d61
85887 F20110318_AABNHH nunez_f_Page_051.jpg
a8d2f2d14f8c7f4d8f6b82673590ea49
78f2b01fddc83ae423ae69c2bed699f97da920b4
92867 F20110318_AABNGS nunez_f_Page_034.jpg
3fae57302290196aa3a9cf9857418806
d5a3370b3cb60cff5dff826c035c33a2204e9eaf
82889 F20110318_AABNHI nunez_f_Page_052.jpg
25f56ea889036cadbf5f0a040b71be7a
b824bee70dbad9fb89c256e37248fd977bbc3990
22236 F20110318_AABNGT nunez_f_Page_036.jpg
5a30ac499ecb0a31549e2e17870ab52d
3d632162fafa9c8963ea6b106e184a36f7bda97c
102621 F20110318_AABNHJ nunez_f_Page_053.jpg
c3047e3f865d4014cbfdb4f162394612
9944a77ff72a25882adc937c4794420079960234
103580 F20110318_AABNGU nunez_f_Page_038.jpg
fa0bda1625fd5f8d7e3a0b44c973214b
9b61dd862bc6498527563f9175316b6894b95c61
89298 F20110318_AABNGV nunez_f_Page_039.jpg
affd975a1f4089986fd646ca30b73cc3
99b5bd37c325a0726415a00cad4545cf12ddfb64
79735 F20110318_AABNHK nunez_f_Page_054.jpg
2417a42e21a46db54bc12bff6b5b9a50
12be3932984a968e1d30f36eedc70d5479cbde42
93247 F20110318_AABNGW nunez_f_Page_040.jpg
11a04ba02e1165c21f888daa6b8466a9
51eb80f6c97d876a7d5e81c8613bb94a2d131cc3
94695 F20110318_AABNHL nunez_f_Page_055.jpg
804fd47b36fe77c7ec3dde0d94d087c5
c444ebdd2cfae62f770aad767f3542a072c35c15
104679 F20110318_AABNGX nunez_f_Page_041.jpg
b292bdc1a51f14a56f7ad1e16b9eb949
c5399a41e98e388257ccbfe7a0cd98de8101b0c3
78255 F20110318_AABNIA nunez_f_Page_073.jpg
a2edb52fdaf68e7befbda7adcdb3f181
cd7fc7ab37cabb23179a82b853e408c75586b743
95935 F20110318_AABNHM nunez_f_Page_056.jpg
22d5bbcb40fa2e13726ac91110a921a7
7e5114d9da02daee5da263c23101052d455075b6
106066 F20110318_AABNGY nunez_f_Page_042.jpg
2105a082b02758d93f571154dda044fb
df7cba45eb87c09f5c33b0d8a46439849d97e7c6
110193 F20110318_AABNIB nunez_f_Page_074.jpg
61343d1e4dd11102fc5e78fa62697a4c
f06bf9a72d5e0236b6c821199f0c93afe0acaed1
59990 F20110318_AABNHN nunez_f_Page_057.jpg
056f30c671ad177254b5dcec77276744
c4fb683a790fe73d415d7181a600b36c630e48d4
83848 F20110318_AABNGZ nunez_f_Page_043.jpg
e8aec750e4e2fd851bde601bf7bc97c0
9871de00b6060449ae22d87299eb364477a06bdd
56849 F20110318_AABNIC nunez_f_Page_075.jpg
c60d20f4b3202179aabefadb6fcbc342
a023edfe0b35362626f62960b2cef7d2c5d5e87d
97930 F20110318_AABNHO nunez_f_Page_059.jpg
3af0775788d7eedcfcb63865f816d006
eea38bff16c08d835ba94dfd58b9b5d010c0b9b0
73001 F20110318_AABNID nunez_f_Page_076.jpg
95a5a1e42d1c8da9854c28796307e07a
360a44a10a238cc437c9d21979d37b381634770c
87807 F20110318_AABNHP nunez_f_Page_060.jpg
3ceaf6a415e5b37ea5e00d24328ce964
625c78043a2e13182af7f5f1aa3af3beacf2775e
107916 F20110318_AABNIE nunez_f_Page_077.jpg
f52c59f98faec8195fec2e6c0f6628c3
d15dbfdddb54620b884afce7174278fa47f8436b
102049 F20110318_AABNHQ nunez_f_Page_061.jpg
884d0612175b73c3b437b7255c589f2d
f0e00d0644af297f861aec588fa37b72805d7e02
56599 F20110318_AABNIF nunez_f_Page_078.jpg
489e6a958c2a8e07459aeaddc1cd3469
cf86fe2e3001399a95ab304521a909d9473585cc
99576 F20110318_AABNHR nunez_f_Page_062.jpg
e10dfcc49d1729dfafff1395ba4d50a2
bcb79eaf5123a6449c593bfbe9eb3eb01ceefac9
106991 F20110318_AABNIG nunez_f_Page_079.jpg
6dd60785b15b0b97a4710ce61b6a1925
cc39d96545c53a00b4f6d792332785d932874a7f
100441 F20110318_AABNHS nunez_f_Page_063.jpg
c5c2fa78a49df8c94f6254958349ce26
0b55b2664488a25a9386d75da41bde24de6b40f9
80810 F20110318_AABNIH nunez_f_Page_080.jpg
904a47bffb4abc8d8722502bb982cd79
a9411a38fd10a46d84dac555e9ff0443214c5af9
90523 F20110318_AABNHT nunez_f_Page_065.jpg
95b5e17ea580afde6bcba6d8c3f572d9
dd058e1738eefb3f7236a9cd4383c5c6f731b917
78295 F20110318_AABNII nunez_f_Page_081.jpg
bad845f3f7f0d131d5ccb6f5e9659767
9be02d51afa2d3f5ca5039d305b20c291d6ef41c
104399 F20110318_AABNHU nunez_f_Page_066.jpg
9e2558952e57e634666bc4b0651e164a
d0944f559ee1aa52bec923517fe4dafb31d98c8f
99185 F20110318_AABNIJ nunez_f_Page_082.jpg
bf3b9ffda89e3e9e68a1c065a3ad43e3
ee95ec4f9d1db57d7b72eae4f7053751ac4e3121
60239 F20110318_AABNIK nunez_f_Page_083.jpg
1acca0acebd631e752adccd640674bd8
7b8186d7628553d457f36393414f6c6dae2d6c0a
96763 F20110318_AABNHV nunez_f_Page_067.jpg
899fdb7dd4000803f15f31440777680a
6002438404b34e9c0cb73f0034c61584a739a93e
98031 F20110318_AABNHW nunez_f_Page_068.jpg
9fbfeb9ab14353c72d7ebbb322a64b3a
7f09b208e49f67e8e8912b94f079dce0892aa404
28859 F20110318_AABNJA nunez_f_Page_001.jp2
d78a5237e9108bf484052befbb158df1
fd5794f5281a7c375f9bb419bd6e2999d0c92f8b
34252 F20110318_AABNIL nunez_f_Page_084.jpg
57d571d7891f2d895faee487d632e31e
b844dbcbef614d8e53fc52546159fba212fabfaa
83129 F20110318_AABNHX nunez_f_Page_069.jpg
04f88194b554beffe7e9a587e8e56516
fd9cfac31e181bbbcbbfba990ad34e4504f24620
18576 F20110318_AABNJB nunez_f_Page_003.jp2
707268fbe39b9f3f80ea1e383005581d
7a889890e62f56dbb70e7454e6f712116c03644b
83080 F20110318_AABNIM nunez_f_Page_085.jpg
2cc4257599326449f3b8e33cb745a5ff
69b257c4fc0bc9e8e75c3c38d51fb06004e2af0e
99971 F20110318_AABNHY nunez_f_Page_070.jpg
d294d038eb834872bf32c2d82be09a91
5a3b88eb8dc1746b9a155d7d8d4b2e19ea284ff1
93071 F20110318_AABNJC nunez_f_Page_004.jp2
f08657428d2b7c0fa15671e25e8ef98c
0294095d0090c7976a05cb82786d154196c2cea0
75155 F20110318_AABNIN nunez_f_Page_086.jpg
7541167b39757652bd78b2884be59979
075e2692af14d1bc149adc535eda6c79e19b13ca
94394 F20110318_AABNHZ nunez_f_Page_072.jpg
26085068c0604fbddde15a47c78b385d
4b5b5a20d28a12495f048b44cc5caf4e01373ff5
1051979 F20110318_AABNJD nunez_f_Page_005.jp2
1b59d06eccf9c8dc2832e6540ce4c7aa
30089d8d408f2a8dde687b76f1d121eac07d890e
52767 F20110318_AABNIO nunez_f_Page_089.jpg
324349728a446a7ec0f33933589542e4
f2dfbd92b3fe344785d9b67cde5815fdf42aaabd
F20110318_AABNJE nunez_f_Page_006.jp2
c3b629c1c8f961131148ee871f1c993b
3b04424f1ba5774c902ab261bd774f5eebf90029
94613 F20110318_AABNIP nunez_f_Page_090.jpg
fcfdce97a9bf5798d308ca40cbdb532b
8f65c27a0cec65a5653958873afdc11c8b2ffa58
1051986 F20110318_AABNJF nunez_f_Page_007.jp2
c46eb98c070c569dfc5ce2e20e0b5ecf
d5c594be639b3459d95470befca999e365b4d534
20401 F20110318_AABNIQ nunez_f_Page_091.jpg
9e435ab28a434c6490b9054d69c178d3
f091720aaf984a8fe5d1d3eef094fb9aea9383f5
1051984 F20110318_AABNJG nunez_f_Page_008.jp2
5266d12339bd8af1b0fc5050c53a39f2
187cfd9278201af60179286e5b0898c07923b11e
119907 F20110318_AABNIR nunez_f_Page_093.jpg
9516e454576ae1ac6aa64a8122748853
027ab0ccafa168059234fc440303046bf2e787f2
1051973 F20110318_AABNJH nunez_f_Page_010.jp2
8a35b795ecdd2514a8c7a2353d06221c
1f182e6ad2d3ffe03286f74a170f9b42e3576ad3
118585 F20110318_AABNIS nunez_f_Page_094.jpg
77fa5f549ed429bea86d95513c6960de
3646e0255b1a29eb0a55368f9d3423c7a6759ea4
90386 F20110318_AABNJI nunez_f_Page_011.jp2
fb1548e9f8de1ce25d6e1b798ceb81cb
fcbff80e5188bd18cc2acd9ceabf838dda412c1c
125351 F20110318_AABNIT nunez_f_Page_095.jpg
d81dacdba4922f077d54af2864950432
4b21b10c3048fd6224617eb75c15b4d4076057b9
98522 F20110318_AABNJJ nunez_f_Page_013.jp2
a3356cd457db37e642fa5ba3cad6f419
270abcd0f73ae0bcfe766ed03dbabaa649ed6529
122923 F20110318_AABNIU nunez_f_Page_096.jpg
853972538574fb8e581020b350f077bc
627e6418b3dd5dad01a229aefb566e6c90586278
103836 F20110318_AABNJK nunez_f_Page_014.jp2
58a20426e64653ffed6b8c9da3b901cd
222df080f9309b1057641b38c96e0329001235e1
115308 F20110318_AABNIV nunez_f_Page_097.jpg
db1643ab9a3b9d5a179bc2cc8ecd2953
a4d03bbe10d25591b6e3b0780df7cc4499726f21
61844 F20110318_AABNJL nunez_f_Page_015.jp2
4229ebf3470fb064a9079f7761fb57f3
68e16d138f712f1947d89b42e4296249adaed282
116765 F20110318_AABNIW nunez_f_Page_098.jpg
fbaeba97c03417d359fe45e3e685ffec
bc5db617d6d2fe5f53a92b00500bec04210d00cf
127379 F20110318_AABNIX nunez_f_Page_099.jpg
dec246397684347b2267ab4cd5e16564
924426f6c2b1d3a4a83751ce5548dd439d8f89da
107851 F20110318_AABNKA nunez_f_Page_032.jp2
b80e6b056c33579307aa2a4ee090ca3e
798e8435692921620a7a9f0137b8d4e9a6f89dfb
91668 F20110318_AABNJM nunez_f_Page_016.jp2
cc2b045bd8a73befa516f119824f99b5
081913566d6dda12d9e8ad4033683943786fe8b3
67274 F20110318_AABNIY nunez_f_Page_100.jpg
b7e1901e9ce91f0105e1474b02ee5278
b3cd0d13dc6dee7385dc317998aff559192c7e4d
992061 F20110318_AABNKB nunez_f_Page_033.jp2
b2ebc0a90d8b67185882bad4c068d902
228f1512584883e3165a4ad474a8982a04636f8a
106074 F20110318_AABNJN nunez_f_Page_017.jp2
b8061232c487459d005d0971b641976c
09c60495c83fd96e5566a3d4a02f53b7fd7e0364
39503 F20110318_AABNIZ nunez_f_Page_101.jpg
6098fda2f828d3e835466a5017f72af4
80a7693c5f5d372bec849267a564899866072b74
1006784 F20110318_AABNKC nunez_f_Page_034.jp2
938b65687f24f3242f47c26382e7d2b6
0f0765bb71f7921b0c9ff8fae89f51a1fc5bd7b2
110161 F20110318_AABNJO nunez_f_Page_018.jp2
d35454df5673a861a0d8df6b23727118
d0177bdeefb8877f3a55bd6bc5555692d4fa1b82
113602 F20110318_AABNKD nunez_f_Page_035.jp2
812f1af99f80350f0fc470aeb7860753
3b7f14521beb9297095390fe26b85f3dee23761d
114534 F20110318_AABNJP nunez_f_Page_019.jp2
b60d0ffaf9cce82874f2a92628b51e43
5fe099f4f0e76b2e5a2fc5e0fbcb3d5f99b6f7a3
23810 F20110318_AABNKE nunez_f_Page_036.jp2
a52813ac160406b8b7b7594f8f7f7dc7
2a944192c8eee9f1c82941a5dc3fd36e98e3c653
97831 F20110318_AABNJQ nunez_f_Page_020.jp2
d12c1d42b1920446297aa27b13e7abb0
79fc052c1280786cd6e59763a31d04cae4fa8668
87525 F20110318_AABNKF nunez_f_Page_037.jp2
45012bcf1b3e0730c1a7d30452f63165
5cde142c8eb8683f929ba8e34205ac41087922f2
104929 F20110318_AABNJR nunez_f_Page_021.jp2
b1cf97356ff0c073b27c7a4b6051c8d1
b969b47cd4a551f329a69dfb0e41c81d007795f5
1051957 F20110318_AABNKG nunez_f_Page_038.jp2
cd4941d2ca3b43dcc296d692a001132e
c447a0560372bac17e70923a5732378c95ab2a74
1015628 F20110318_AABNJS nunez_f_Page_022.jp2
8ea06f0922c6d867d1c22ea5d4c50532
7f639e211ea4ab2f61db2716a8159a277a8acd68
1009888 F20110318_AABNKH nunez_f_Page_040.jp2
255cd5cfa04e45be19034e38b0ce3579
5bb1bd5cb6a025892b6ec8e45ff4f1ac4f757eb2
1051949 F20110318_AABNJT nunez_f_Page_024.jp2
70537bedbcf2ac1a6c13a7bf1581c97d
9ade5311895c3131760ab9612fa0d9899ad61971
87427 F20110318_AABNKI nunez_f_Page_043.jp2
5c8efb449ca937e96a8c632b9c758938
f7bcffc8c8b1f1cd16104e8af7318dc7f43574b2
111770 F20110318_AABNJU nunez_f_Page_026.jp2
54ba414075d9f12da903043fe4958fe0
f0d4ece085409c9c07b228433699e2f6b207455f
104919 F20110318_AABNKJ nunez_f_Page_044.jp2
4d274e3d572e398b4df54e65ec911ff7
663e3a19f0f038e2a0c9010e06c1e978797d214b
106600 F20110318_AABNJV nunez_f_Page_027.jp2
825564802e9055d833f5c7c30aa25f80
c165d1ef79ad36ef15994b3b4e846140d9e57450
821261 F20110318_AABNKK nunez_f_Page_045.jp2
4ccc9589a2e21e9c064069aab0c95c50
05f3d7f19ae12b109766f6d9e195e5299dbee498
116598 F20110318_AABNJW nunez_f_Page_028.jp2
53546d8e83ce4bf45ecdca3b8139356a
f8572e3e60fef13bb04df3a0d34ec7119a685d2e
802566 F20110318_AABNKL nunez_f_Page_046.jp2
5f0eb9e16db2bd08dd0f31954b5003c2
99479f9834cbdea2156da496d266e74d801a80c7
114504 F20110318_AABNJX nunez_f_Page_029.jp2
7f8f7066877f70ee2ffcd916063f19f9
591f4c2c14d910b4b242a67969f1ad71a54fed2b
94909 F20110318_AABNLA nunez_f_Page_065.jp2
7d2842197376cf1d3c4d43e7c22fe19d
a1743f1ff633ffb76e4f74085716b5b392bfb891
111559 F20110318_AABNKM nunez_f_Page_047.jp2
980623bfdcc898e4e89f356c3f1e1180
1cd0f90d2b49483d76495582b0ad30d1906b1dcb
107224 F20110318_AABNJY nunez_f_Page_030.jp2
fa5bf35d2f5f27cbaf8c5b539f705c24
95eae05dc8e3a836b622c8285c941666e6a1f1e7
108914 F20110318_AABNLB nunez_f_Page_066.jp2
3dda3580a19fe9d1b7a6ea172c751d4d
75cc18427350369d5e9aeed5f4e93f523d2fd087
111419 F20110318_AABNJZ nunez_f_Page_031.jp2
ab727713e83ca6615a4db054875ea2e3
a40fc88491634e4e50115e3d7893463052d0615c
102629 F20110318_AABNLC nunez_f_Page_067.jp2
56c1753578c4e377857336564e0701db
a235a1dc916e5a1f36daf6953bbe2e184b490bb5
843162 F20110318_AABNKN nunez_f_Page_048.jp2
2e94857e608129f312f502e6daeb30b1
032ec1c9088fa170e8e9d5c3c5b9d03d94148f3b
103321 F20110318_AABNLD nunez_f_Page_068.jp2
c7cfba3b6e1f0e32cb6029cbcc9e6fd6
87842653441dfe60e3dcfb70f7d2d8baf18b4dce
879969 F20110318_AABNKO nunez_f_Page_049.jp2
dcc3775ac40698a4f6ad5f5a6a259b18
faf8bea0ab1643ec0899bda78cb639250b31664a
88477 F20110318_AABNLE nunez_f_Page_069.jp2
90780bf1671551c77d343c25ce75619c
fa5c1d016036e6bdc538b0376f61f6f0a8f37324
113425 F20110318_AABNKP nunez_f_Page_050.jp2
8f6242da46455d71d8147c38c1cc3400
f4f5065a9193eb61e0b9769ae4dc01aa1269df84
106694 F20110318_AABNLF nunez_f_Page_070.jp2
ec2ab7707595ce9b033eae03c9480db7
d163c3546f02737ff0493edea235cf1c5c32db6c
899722 F20110318_AABNKQ nunez_f_Page_051.jp2
3302464385f4753969a6f922753c0d6c
9240a6559c331ecc9c089974fe53060c6d709ec7
1051974 F20110318_AABNLG nunez_f_Page_071.jp2
04a80ddd20995c4f50f07301c732e14e
e547da1deea82552c0dfea31dea71f7983de8ab2
108434 F20110318_AABNKR nunez_f_Page_053.jp2
856305a1aa9ddd768372eebce7f119e0
693a4ad440c3b596156f946e2700a79635743966
101773 F20110318_AABNLH nunez_f_Page_072.jp2
7fe07bc992693ea9bfb271e4286cc767
2ab6ea40f18dfdb267810116a842086ddd0ca043
835229 F20110318_AABNKS nunez_f_Page_054.jp2
5aaeae55dbdb8b5c20f5d3b3e3dd1906
3c8d498169a26fa83351966f4bda919e5418895f
113919 F20110318_AABNLI nunez_f_Page_074.jp2
465c4e5e955c88a1c8b43771c30d18cc
3743c24eeab8b8d369f97455b2b0246ec8983919
1025220 F20110318_AABNKT nunez_f_Page_055.jp2
1dc6b7047f9468e555bf20d4fa28beba
6ca55a166cce1102f33a3f56324319cdf0c72067
57323 F20110318_AABNLJ nunez_f_Page_075.jp2
5c17602b4e05f418f239e6cc52ead3e2
c435359410729e0d62690d5d213d8ad71f702bdb
101110 F20110318_AABNKU nunez_f_Page_056.jp2
345ffdc814741d75874469416ce783e5
80bb5accdd94898c6612b415e2624248fae4d683
74758 F20110318_AABNLK nunez_f_Page_076.jp2
a34147a886019fbadeb68d39685d6e89
754980af8c3a9db41ee1767b24d4b357d9dd8ba7
90079 F20110318_AABNKV nunez_f_Page_057.jp2
c363f2be29bab825a774f3e4e483c05a
dff2964ab1b63f0c83a641b36db5c897728f532c
114585 F20110318_AABNLL nunez_f_Page_077.jp2
867d5f65026332b8179857e2f8f1fb5c
2459d34d3a3123b8822e8c146ea879ff0b5050e1
105944 F20110318_AABNKW nunez_f_Page_058.jp2
d5ebe7613314d72ebcea981eb985a598
491426a45f27cbb7e10254b5da7ec298da795f40
1051978 F20110318_AABNMA nunez_f_Page_094.jp2
d036048d7aed5cfdc19775030a230ce0
4786ee97251e64125bf685fdf8aa0362d4fccdc6
510498 F20110318_AABNLM nunez_f_Page_078.jp2
04f4949ea7450e3e63327c45b4e40c40
0ff86564a99695166608c2ed1fbcb020e55700fc
93394 F20110318_AABNKX nunez_f_Page_060.jp2
b9d2e3304571a20cb48c98a35a19776b
c25293cd3c6067498ae3b715757dbff9dabb3cb8
F20110318_AABNMB nunez_f_Page_095.jp2
28a03396aeaa1e6fc13f46017a7f5d9c
91b9b80d94edc8ea641e351747379a7fab22367f
113745 F20110318_AABNLN nunez_f_Page_079.jp2
f86b69431bb43acecd3a627a3e005cbc
ba24fd0be004907cf7bcb00748b36385ce894f61
105313 F20110318_AABNKY nunez_f_Page_062.jp2
5c109d1a0f8dabcc1930cb51bf3f67a2
dc592f99af095965e360b771b5b69b1ac3a795b5
129101 F20110318_AABNMC nunez_f_Page_096.jp2
e11737b44b89ed0c1e695d1d6978bfe4
7fface161853c22f26edcb7256208d92f398f243
104914 F20110318_AABNKZ nunez_f_Page_063.jp2
847d033192ec92572a06a8345015c7dd
40429d274ec4efb62c7d3bb08ba3fe7dcfbba824
122927 F20110318_AABNMD nunez_f_Page_098.jp2
7745f14e283f1cb15861d3a4ac9f179b
30856ace2f572810ee663c6137262cb60ee91420
836259 F20110318_AABNLO nunez_f_Page_080.jp2
44bc65dd6f8382e6f57dcdcb5d33f258
5f10683664d5738e7b72da96cdb1d3794e3a60df
1051985 F20110318_AABNME nunez_f_Page_099.jp2
6985a091eaff932436c49ae11779bddd
6290387f82f9d2021a69397a82aaf073a44f494c
106243 F20110318_AABNLP nunez_f_Page_082.jp2
3633dacf99cab048824ec8553d412b07
0c380668f6df39f4ffe6543157981cbad3b6099f
40628 F20110318_AABNMF nunez_f_Page_101.jp2
2dfa3184d62a6c3c4620e78ec70d72ab
2f01291a69e73016800a7574803a2ee819b667f6
610336 F20110318_AABNLQ nunez_f_Page_083.jp2
5aa71572afc20e85b16eeeb3e2047e20
955e0af6e8d3a506deb9232d5c4d5bd67eb68fcd
34958 F20110318_AABNMG nunez_f_Page_050.QC.jpg
4085bad0053d6ec29e5af07867e82163
7738771736282180dd62cb6419380f2e7a4cb59f
279786 F20110318_AABNLR nunez_f_Page_084.jp2
41814c4789114f227f72630b99a1d803
f83d325be1020af5385cb6fbb14bf16ed496e96b
40120 F20110318_AABNMH nunez_f_Page_010.QC.jpg
f57db69e63aab093b2cd0a89b5c0f06a
8dbd2b1cdedf788d101fadb171ced687865d76c7
872439 F20110318_AABNLS nunez_f_Page_085.jp2
1c59eef68de5dfa1ddf446c9b3e603e9
ef11d8eff1052f3def404c300a9bd5f09c049ae2
33329 F20110318_AABNMI nunez_f_Page_097.QC.jpg
13b8e71f018ef8264816bc8be21f276f
5f58ea71e962e0036e5697ccb83e9043693b7923
784553 F20110318_AABNLT nunez_f_Page_086.jp2
3897b2eabdf99a3cdd7f800e23304648
c971bdb349c9e1721614899535e784b5249d6ed4
34658 F20110318_AABNMJ nunez_f_Page_096.QC.jpg
92ea32ef4a88932f385ddce7106c3017
17881d714e161421f668431c6aed5db57463d636
79534 F20110318_AABNLU nunez_f_Page_087.jp2
7021c2ccf6377cae3e3500bff58ec3df
76ecbee4a4d9abbd0abde7a779bf90843f0949a9
1684 F20110318_AABNMK nunez_f_Page_003thm.jpg
5b4a12a76037b4b5fd22dce725469d95
a31025a260abf3b7badc399197b88681a06f8208
773077 F20110318_AABNLV nunez_f_Page_088.jp2
11c37ed0b808d2313b370181cc93f395
14576ca45929ef2947e4a41e139862173f1a05db
28887 F20110318_AABNML nunez_f_Page_039.QC.jpg
6233d17a5f2a17c293fa6f0545f04759
e4ecbbeaa0263ad64a27f6667fd5ad56bab9e981
54964 F20110318_AABNLW nunez_f_Page_089.jp2
476f03111bd21131db9a91fe0b1ea46d
7ad93112a13d12da48642329623b22a7542c7215
7495 F20110318_AABNMM nunez_f_Page_049thm.jpg
6412420711d08b2792000bb84a8c1ad3
e38a7222b0b9ca3f09ae526f3c8f83b670ac78c3
21617 F20110318_AABNLX nunez_f_Page_091.jp2
775ef148f08c1682208511208785496d
c5b5f4b5092b3aa39af66169889a8a4aac2d22ce
32268 F20110318_AABNNA nunez_f_Page_067.QC.jpg
ccaeaa3ac1487e2b6fb69c10168fb8d8
b78a343d5a0354d6d6b0b8f6bb8d2d224478acd1
32273 F20110318_AABNMN nunez_f_Page_082.QC.jpg
72283b45c0db68cb1a353aca96ee6cf6
cc11db578e77c133b9c855fbfebbca8bc4b8a889
109173 F20110318_AABNLY nunez_f_Page_092.jp2
c39e6676bba3916c0701360d2d742309
b378007b54ead8420821bb17a450fe99ddc06727
8235 F20110318_AABNNB nunez_f_Page_058thm.jpg
d19e524ec87cb57b5f18c5cb8c839f1b
144a1c7bbd95e208bfd440e4b74cb84882ed56dc
3360 F20110318_AABNMO nunez_f_Page_007thm.jpg
bbff21123c9792c7f8eec5103d635094
4fac1a13386e41891e558a144c9d67380efc3ce1
127047 F20110318_AABNLZ nunez_f_Page_093.jp2
132233f3153454dbba879447b6048985
107264ebee49441eb552dbd11be106951ce0b143
29435 F20110318_AABNNC nunez_f_Page_013.QC.jpg
b3e2469afa083da6c23ad98ffe81df87
9c51cbcdf6d40777d462deddf23ff77c9d7b478c
8490 F20110318_AABNND nunez_f_Page_029thm.jpg
4d674841d5278aa743c39d9757f1a6a4
1f99dcc97104da4610891bab5a0d4d2a83656a55
29886 F20110318_AABNMP nunez_f_Page_020.QC.jpg
4e18e05d93613eb3d4968a238abd635b
818a60e620e637b5a7f17bdd57985db1c25d5226
28365 F20110318_AABNNE nunez_f_Page_051.QC.jpg
ac79664a46bf2a40e4eab124ff8fe876
73e09eafef791c25d104e7a4844ec9e4417d69a3
27220 F20110318_AABNMQ nunez_f_Page_085.QC.jpg
4049666fff6bb321ac68b3af06d48b4b
006dc998582c2378a6105c64fea14587de5db4b1
26449 F20110318_AABNNF nunez_f_Page_043.QC.jpg
f376a1d836eefc3aa5e230c7fa153b74
0b953ac679c2cdb23336b1b5f10c49c43442d551
34713 F20110318_AABNMR nunez_f_Page_093.QC.jpg
9f8084d3028ed78ddf22dd13fda6d587
84d60f7d9bc3b60410037e74143076684cff4a24
7826 F20110318_AABNNG nunez_f_Page_082thm.jpg
86db3f4c6580932d68118ed1763a3913
fe2058bd624b57afac8e5d76cbc22d448508cfe3
3515 F20110318_AABNNH nunez_f_Page_084thm.jpg
f0da23e6a35fb87e9a1a3fd9f6e07513
0a74df793a773e67b2e5a686283e1127003c1fe1
8643 F20110318_AABNMS nunez_f_Page_079thm.jpg
664232ebc86414182481d345d8260238
5ea4a6f983a6f6595a5c3d5b8e333f1a28d60981
31517 F20110318_AABNNI nunez_f_Page_059.QC.jpg
da87e60b15c69b3b066a15c8459bde68
1353b8faaa8665a734e846148720f0e865016355
32925 F20110318_AABNMT nunez_f_Page_006.QC.jpg
8b14b655fd57968f5706b94a284ad06b
211feeec44d6e013d21ce1cf61269b9c503c15ed
4845 F20110318_AABNNJ nunez_f_Page_100thm.jpg
dad90635d5376410a700b5be3cf19f7a
3c07cbd3c66063adc7085ba60731c5163d2101e4
3599 F20110318_AABNMU nunez_f_Page_064.QC.jpg
e2b8efe6f41ffff767371f1bf5ebe5c4
ac178d0fbca613646eb4fc7edcec5603413bc310
13518 F20110318_AABNNK nunez_f_Page_007.QC.jpg
ee7aa5a16bf1b535ea1084f55033559c
c3584376ea5a749e3c81ce345c3e4e92157bbcb2
6472 F20110318_AABNMV nunez_f_Page_087thm.jpg
681c26121efe7b34cb3690fe3e3f3fee
eb2405fd50b2c16669076842da64a42690ec6408
F20110318_AABNNL nunez_f_Page_002.QC.jpg
6d0af1f9b04dd2f2925ddf0e4f7945ec
bb6a62b415e2b683990b64ce04eea4f4a8cb40cf
34946 F20110318_AABNMW nunez_f_Page_029.QC.jpg
e6f0ed962364afa5054111f3e7af4e01
e24fd60ef96fae372de283d0fa59199116ed2f0b
7598 F20110318_AABNOA nunez_f_Page_033thm.jpg
fc71eaeca882d58025d9c284130926a5
5d9c2bade4a2998e7579c450e0898787081905dc
8101 F20110318_AABNNM nunez_f_Page_032thm.jpg
63ba06a5fb9e8d3cdc593a8257ee6fb7
e73c7d745081adf625ad0e5970e242567d89a79a
26781 F20110318_AABNMX nunez_f_Page_011.QC.jpg
a715e36addb84822dd9efae7935ea77a
4b41bbaa0a07d7291c2fb98c17285c26a6330fc6
36329 F20110318_AABNOB nunez_f_Page_009.QC.jpg
f0e4819d7fd4edc55cac9deb5c893da2
022c7bbca835c5d58f6153f8cb1f1566b39ef802
8437 F20110318_AABNNN nunez_f_Page_012thm.jpg
de5db2fdb75e564d9d4178048c159186
ac5ed04dbd1d532c261c9974856274397e2d8147
9274 F20110318_AABNMY nunez_f_Page_071thm.jpg
ea72ca7e7fc6be6c8806eba1aadb3ef5
dff3acebc4b7a8c2664ac6ef206e61eb1f8fc3ec
8422 F20110318_AABNOC nunez_f_Page_096thm.jpg
b11cf9f0d6778258727c4776239e2247
20465e45c1e443465c40b60b8bd83e45a430f0dd
7420 F20110318_AABNNO nunez_f_Page_004thm.jpg
0d5bf5ff2b064abc070e4b497e4e01d9
0045ddfafee4e68a72e88b45e14107963e7ae6aa
26014 F20110318_AABNMZ nunez_f_Page_086.QC.jpg
7d1d0041b4528bac2f984ea61fddd6f4
18dbc2fd027c157be8c9401e4308270325acc41a
33993 F20110318_AABNOD nunez_f_Page_030.QC.jpg
a79a61df9529fd5d7a0d0e71e51d102b
fe1e668868ce5486e0a403b1f87773829925b6c0
4916 F20110318_AABNNP nunez_f_Page_015thm.jpg
53109705ad38b285f5cf0c0c7f59daad
24f9217c79e9b7d85f1ace2876f218c29c15d0f1
9042 F20110318_AABNOE nunez_f_Page_095thm.jpg
d90b3102708aaaf6d4b558b861b4d881
622a3d060a77e4dd52f40968d0e3fcd6f7b21399
34407 F20110318_AABNOF nunez_f_Page_041.QC.jpg
453cdc8dd3812ebb15e4f22d6862ea13
856bf505104276595cf7de32dd170b8334af1dcd
6855 F20110318_AABNNQ nunez_f_Page_091.QC.jpg
2722f6f7127c09cfa561a641eb434585
556dec4f3ae98ca1b4cc86f4a0e7001be8a231d5
26809 F20110318_AABNOG nunez_f_Page_048.QC.jpg
69349ef1ed74b79b460dfebb85d58d56
04ad93c5e974f8525cc800bd6bab28d6cb9aa182
6852 F20110318_AABNNR nunez_f_Page_069thm.jpg
6b77ac3bafbf2616dda1ec73019be2b2
77acb9b7867566c2f281fdea5a9123e571d4dfc0
7813 F20110318_AABNOH nunez_f_Page_072thm.jpg
4fe752ab9eab6a0bbebd5f0f1c51d97b
78b4229981aa8ea6e2c7e4971c8182172d20ec7a
8354 F20110318_AABNNS nunez_f_Page_041thm.jpg
f18f127e5bcd5c3e7e3844c72d933ccf
ee8884f047ea500f82582006cec688cbd5d3e82c
7240 F20110318_AABNOI nunez_f_Page_013thm.jpg
5881e8c431637cffb80d524b819c758d
b490cff593bf298b38a9387c1399b199cc55f784
7444 F20110318_AABNNT nunez_f_Page_039thm.jpg
e23bd2d29379102b5f0548ec99a2d02d
d0c49e655dc596de60a01b7800ac1064d8698578
20173 F20110318_AABNOJ nunez_f_Page_083.QC.jpg
4ec90d6015b13f54e5d12603a090c124
148bfd734e54032576339fe54132c892795799ad
660 F20110318_AABNNU nunez_f_Page_002thm.jpg
227abf6dc7496475558901555e6d0d2d
e28cdd26d97d841f02673e3263c7039b155482f1
7306 F20110318_AABNOK nunez_f_Page_036.QC.jpg
36f1be1c5da2499a7db5af8442469687
86d4cb3f1ffc14682e7f99853832523d36cd08e8
33966 F20110318_AABNNV nunez_f_Page_038.QC.jpg
42effc45d026c922dedb57452d97f209
4532d0c6a585516394175c6dcf5fefdfb0312902
7725 F20110318_AABNOL nunez_f_Page_067thm.jpg
e59ff6666e20f4dd05f08e9e968c822a
b27298190482ff5ee0db1332eb22ff92e06a1629
7302 F20110318_AABNNW nunez_f_Page_052thm.jpg
eaf50292b88a0733816dab67ef32e092
7788da4d6ef4b45d7b00bd529296e864d40bad73
30831 F20110318_AABNOM nunez_f_Page_090.QC.jpg
5423bb639659c25cc0c5351a6917c81e
708be3535c569980e4084bcdadd8269890a1fa83
7066 F20110318_AABNNX nunez_f_Page_034thm.jpg
1573b36495ed91010a813a02a3250fd0
07bc7b56c0da6b2fe152986ad342645d7ac8a327
33204 F20110318_AABNPA nunez_f_Page_053.QC.jpg
da447f443776892d604fdd03ce8a7949
b1c3c695af0cc8ddf017dfac032ed43111c79131
7525 F20110318_AABNON nunez_f_Page_040thm.jpg
c4237014b488383cc1f6717c5b4d1acd
a77febd4d4e9a373417e97578a2e00829c0c452d
7816 F20110318_AABNNY nunez_f_Page_068thm.jpg
5fc9f9f095143896d35b0185ccbf8bb9
9f0448d924d392e35f127a14579e7ba2bec8e341
30292 F20110318_AABNPB nunez_f_Page_022.QC.jpg
57483de3185c5922ce9ef541273b355a
043b068522d757214a6910a8638eeb1fc53a97be
33509 F20110318_AABNOO nunez_f_Page_024.QC.jpg
24fe98b38412f38885ad7c7ef5d4ca11
657deb6f57e203e88064b55acaf723b8e5b6e1e3
7018 F20110318_AABNNZ nunez_f_Page_046thm.jpg
e043b61cdd0cad42e6e19b5d4d8bf4d0
a6f838e364bc1c22b4faed733ba4e11082dbcb0f
32439 F20110318_AABNPC nunez_f_Page_058.QC.jpg
3604c25e0db12e8ca3264a6e767ec127
744a00b9c8ca436b4733e1fff1c2b22d5fbe50ab
8434 F20110318_AABNOP nunez_f_Page_066thm.jpg
dfda97bb75232528de882114a2a25949
12915d2e685dfad85dad3cfa53fb0f99201c88af
34300 F20110318_AABNPD nunez_f_Page_031.QC.jpg
9e6ddccfdfce5cbc1c745dc820d52d69
467da03dd3a18d40aac31d2cf65ad72623c89f89
23097 F20110318_AABNOQ nunez_f_Page_076.QC.jpg
d34487214eeac2f687eb5716f7013525
4ca98c4d7a859229f515836a777d877aee581f0e
28656 F20110318_AABNPE nunez_f_Page_060.QC.jpg
942ae94ee4447a20edf91e3973695b04
d7b86f00656c6d7453981579ba5f1ff005810198
19007 F20110318_AABNPF nunez_f_Page_100.QC.jpg
9fad52aa5baf0aec9241c136e3d4589a
b0434214719de5d437376078fc36ae30bc5ca05e
29900 F20110318_AABNOR nunez_f_Page_034.QC.jpg
b377eaf23675d09e6e4c2b15ff8c3191
ea7a6fde1e81f9ddddd4d730857c35f7eaccd264
5453 F20110318_AABNPG nunez_f_Page_008thm.jpg
26d65e2001d64fe8219777ecbbfd8b24
6ecf3f957f3807ddda1d865ce23d9ea71857cc23
8747 F20110318_AABNOS nunez_f_Page_097thm.jpg
22d050589b494c80fe837a91fbb5df74
96d47b5b5046c0ce71e8e85ff35a719cc7564814
8572 F20110318_AABNPH nunez_f_Page_050thm.jpg
59d9e158038fb97bd483630b86457b2f
15af8c25a39ea35df05b9144f82cad2fa433fb10
7332 F20110318_AABNOT nunez_f_Page_048thm.jpg
a552c61234abbcbb997adbb153f01c39
5f1429fbb81343a7959b9adcb0bd5c89c8142759
7289 F20110318_AABNPI nunez_f_Page_065thm.jpg
35bbb1c8a531f4f571bf956fbf6e9f66
8a468bf8862b765b78eb55d62e0dac90149718e0
6689 F20110318_AABNOU nunez_f_Page_011thm.jpg
439c4eb429b899ad6f6cccc29dc6ee84
c37d6f6f7b0c2c96f0f29c733439fe365b8072c6
32770 F20110318_AABNPJ nunez_f_Page_027.QC.jpg
91a650048fb67a4547df996eda2b7d84
5fa06c9b7f433e312d8d1131465351655d6d2644
8247 F20110318_AABNOV nunez_f_Page_063thm.jpg
b6b5df0757495e75a4e883130527a48c
c8a254cb59d5e04a14699911205e533340f0b103
2070 F20110318_AABNPK nunez_f_Page_036thm.jpg
829b1cb5005c48cf4f5fb54ac8ffb371
e0a046d2c0e812e27ffd3dea175c7e6c1dd42c20
36545 F20110318_AABNOW nunez_f_Page_028.QC.jpg
cfeaf7718de86b3e750b67878599c76b
7f940240cdca6e94d72f4dc9f91b2a212caa2127
8253 F20110318_AABNPL nunez_f_Page_009thm.jpg
81fcda6944114e9479755cc927f6faf6
9d959ea695130880134c75cd3b9de37ae80084ee
557113 F20110318_AABNOX nunez_f.pdf
83bf9af50dbb18e121a13810b857e0d9
cbaf60d3ae17b15a3ca3d127cba7e0709171563a
8231 F20110318_AABNQA nunez_f_Page_024thm.jpg
e42424cb1eafc8bbfc2e0ed0e2e7282d
cc3ebe09dc1912fced7ec9c274d7ba8fbac9adb9
6868 F20110318_AABNPM nunez_f_Page_081thm.jpg
dc49f7aeb6cee7d0e56e38628983a524
425519a0881da05acd1ac7c2253ea0654db2ddd9
7016 F20110318_AABNOY nunez_f_Page_043thm.jpg
f4cbaa12917d36349564ec0796a9994a
346da8c56b933551b7d06d412b93142371cd318c
35620 F20110318_AABNQB nunez_f_Page_094.QC.jpg
f63771009e25829728ea92690ac04851
bcd2a7232ae989cac0563ea2ffad87816f6b9495
7376 F20110318_AABNPN nunez_f_Page_055thm.jpg
52f367e47ec7eb2da76112321631c56a
40709e0781454d2a8f3952f7bbef5573659a42ce
1889 F20110318_AABNOZ nunez_f_Page_091thm.jpg
9ca2da0590190cb1111d8f6363d57827
19be8f6abc4b69689049fe04cbdcd14c5f7fc1e1
31813 F20110318_AABNQC nunez_f_Page_063.QC.jpg
30db2cdebbbfd4efdb63bfad6f23d236
23bd07948fc8566c30c0a9c2c9f1f132b26d76a2
35700 F20110318_AABNPO nunez_f_Page_095.QC.jpg
c9e5c6f9acbcb229f5591977630fab1d
36b4407d37f273a5ee5505012c0b59cfad01524e
19135 F20110318_AABNQD nunez_f_Page_015.QC.jpg
18ecd6666145e8a58cd21cb63a8d5445
14d07cdf89899f7c3f845c84c057ad77b1ffe8f8
7917 F20110318_AABNPP nunez_f_Page_027thm.jpg
ddccdde5e74ccf9abe96d6fbc9d19dae
e371b5e6d6640e729a706f207678a87adb2ab685
26337 F20110318_AABNQE nunez_f_Page_045.QC.jpg
b05e5d255eda97a05b9ff18c68403bd2
f389824d8e24ef896a10d9e6f09da01f604b45f2
6917 F20110318_AABNPQ nunez_f_Page_080thm.jpg
9a51d0352aec322fbf0543db3efbaa14
908fac4cf2c07bcc7c6cbf6650684e624b71b0aa
7469 F20110318_AABNQF nunez_f_Page_051thm.jpg
4664fa8fde09336867d57825fe8b3206
9fafbe9c63e5c2e2fa84ad21bcc0917b392d5fcc
29798 F20110318_AABNPR nunez_f_Page_033.QC.jpg
fc0512ca6f6d35770089780782926713
ba7b4f522585f2807478eefc7206f547ee3417af
7272 F20110318_AABNQG nunez_f_Page_060thm.jpg
70d140a4bbda14835ca7a49628dddb85
061628b8515623c5a1e868979db80a515ac0e88d
6573 F20110318_AABNQH nunez_f_Page_086thm.jpg
25827f9ef5a6cd77959fce90deb5a527
0bacb85237902f749b1da7708c2c6ec132ad4a15
6912 F20110318_AABNPS nunez_f_Page_054thm.jpg
da36e9c2530fdb32f5371f88849e09cb
163ff7488ed4e00836fdf81dba17142c4965f640
27761 F20110318_AABNQI nunez_f_Page_069.QC.jpg
0c07afc257826a580807663601f6d3a5
419963230e0b9e7647cb544043d2b38e341f353a
34309 F20110318_AABNPT nunez_f_Page_047.QC.jpg
90b0cd6a100e4979f21a4a576a3c7631
458c9c958dbb298b4c2020158dc22b10bf670523
8353 F20110318_AABNQJ nunez_f_Page_025thm.jpg
383fc7a34632e27595e05d0c7ecab14e
20bd365cf84d457bc2353871b0d7fc5ddf420e23
8735 F20110318_AABNPU nunez_f_Page_099thm.jpg
75db228577655fa853973e854ebd83ea
f5926ef0ef2fe9d966cdfe74341b2b6a5b38a95f
27752 F20110318_AABNQK nunez_f_Page_049.QC.jpg
4ceb57fa5064d0d00a60c0f6893f8c99
d942b5c792334626c756d757a1fcb959d487ca06
32054 F20110318_AABNPV nunez_f_Page_032.QC.jpg
e3f87ffb75dbb7b64f3e518c43455b42
54e29c8433cba2ca26965c8cad93540723b7a5bf
34403 F20110318_AABNQL nunez_f_Page_066.QC.jpg
de1dc919609807400e2074dcdddecf5a
00d164b7a410bfe1681457824582c78b13e02e66
34453 F20110318_AABNPW nunez_f_Page_035.QC.jpg
0ff6e59b9e97e64308b36f25c33437b1
5bb45bf8f0798935d969f39c3647db2338eecaec
27591 F20110318_AABNRA nunez_f_Page_052.QC.jpg
c6491e53ec6948c2bd2215ecc5003aaf
cc73429a341bddd665437983a1c75eec4753f061
35975 F20110318_AABNQM nunez_f_Page_099.QC.jpg
bd2be447255c23477623d6e9302d4e2a
19181918ea222af4338a8fb2e0641d0078446690
34060 F20110318_AABNPX nunez_f_Page_071.QC.jpg
ad89c3a5e1029ba801402618efcfb298
4dfe678ad2da37821d1c971b56f5b5acaf15f809
25946 F20110318_AABNRB nunez_f_Page_054.QC.jpg
503e6846e732a8b14041bc6f07994f30
46aaee7d9c64fbcc9c5de975a7b8f030b729742e
32767 F20110318_AABNQN nunez_f_Page_061.QC.jpg
89d1e5299927e1e2b2ecdd306d8cb85f
dd14220c717a582a0a8d25400a19751bc54350d3
26145 F20110318_AABNPY nunez_f_Page_081.QC.jpg
d7aa6b49ee3170827df9e00602644465
09f3dea107a4b06ebe7372e8b89ee2f4a6ea29cc
30332 F20110318_AABNRC nunez_f_Page_055.QC.jpg
97122bbb10f00ae56a71d3fcb4aada8e
6da633852e8ed5b3182b629746ad3c91c94db00a
163200 F20110318_AABNQO UFE0011873_00001.xml
b1a7e3ff1df0238c050b5689b9d86e68
7ba939ff36efa48b9be4e701182975f22644c7a2
nunez_f_Page_052.tif
6646 F20110318_AABNPZ nunez_f_Page_076thm.jpg
74b83f6b3f17eeb957df534fd31d7085
54f07e5ea95d3b8daa9b474240518f80e9114172
30903 F20110318_AABNRD nunez_f_Page_056.QC.jpg
524a2f3aa7c6851410da29f1ce426545
53788a70794b7e9581436b6a8a899673e36494a1
9428 F20110318_AABNQP nunez_f_Page_001.QC.jpg
f9a885a045c00daf08f1fff01db703be
ca6418e563776dee46aefc27974ded6e978d2893
18119 F20110318_AABNRE nunez_f_Page_057.QC.jpg
7a1c37cbafec0b64b60e928f51197343
f030d74874129ae55d3a31d1b0c4de1d2f53bc13
5702 F20110318_AABNQQ nunez_f_Page_003.QC.jpg
6bfc3038e8d520186ce830ddf8f05b49
514ae786d82a99be4bf7cf1c6cc1260ac53fbbb5
33318 F20110318_AABNRF nunez_f_Page_062.QC.jpg
7ff1640d218297b41aad8df41c5028b6
065a7833acf2a6c9fb09890f9f94070a0750367c
35352 F20110318_AABNQR nunez_f_Page_012.QC.jpg
8ffaf8ef80e44d5b54ee243632ac3684
76fec7991909af15d89951274ec059c32928e4da
29016 F20110318_AABNRG nunez_f_Page_065.QC.jpg
8cede556549f024bb86cb059ee323d35
420a8422bc07bb8bce4c172887fa71a249c53223
31722 F20110318_AABNQS nunez_f_Page_014.QC.jpg
73e7a11b15c56f41c8efc1c019e72bc8
9ff411be99b994cdca5f8429e18a2f8d74bdb4ad
32386 F20110318_AABNRH nunez_f_Page_070.QC.jpg
2ddc8a99a8eb6d0b3869c864d90510c7
a5d12a6e906a49222be6489194cea3befbf1aaf5
30794 F20110318_AABNRI nunez_f_Page_072.QC.jpg
15bf1faf3ef6bbe50da8520314bd71e1
4c36261a5db50197c9204e8192d2f5a2d552e85a
33403 F20110318_AABNQT nunez_f_Page_017.QC.jpg
ce9a99e6f318f6850dc8d44713eeb888
e3e15badbdca8190cadae18f144674327eea5cda
36228 F20110318_AABNRJ nunez_f_Page_074.QC.jpg
876a70625a6fa7c2b4a7044e59689f8f
8754acd2a1b6dee434eb535a01e5dd06043806d7
34333 F20110318_AABNQU nunez_f_Page_018.QC.jpg
a0c555bbe1897ef81f2e5494054565e4
abe59d06e22aaf77c454e476db04e5f3623ebf39
19583 F20110318_AABNRK nunez_f_Page_075.QC.jpg
2fb984072f85873a182171eae2441068
f8deb7598752a5a78cafca66ad3a018eb65701f5
32757 F20110318_AABNQV nunez_f_Page_023.QC.jpg
4035ddb2dc1c1f3082c2676ed4027d49
b97638d1ac3b5642ac5cd53ebc0703c07492ce0e
34985 F20110318_AABNRL nunez_f_Page_077.QC.jpg
808135b366f0bdae0a15ebb66b7533cb
d45c90df5cbc6b4f36b8cfb08fcdeff454946355
27413 F20110318_AABNQW nunez_f_Page_037.QC.jpg
84e04f0bed00fb5c2b1f7c9d1b54916c
887962bd39590c896ff7871fcd87103193a16246
18424 F20110318_AABNRM nunez_f_Page_078.QC.jpg
74b7c5c0382efd4f8b08810ce8886292
6c4e5fb873152b553f75feb6ec5eaab06414d182
30161 F20110318_AABNQX nunez_f_Page_040.QC.jpg
602ae653272484bd57707bb0c11f0c33
2ff8b70595d4f09f8cd0dee73ed7b18c73e3eb89
7952 F20110318_AABNSA nunez_f_Page_022thm.jpg
10932d44490c8c347dcd730021f99d79
3a92748898ea99a6c8cd51e2dd944f4a18c470ba
34883 F20110318_AABNRN nunez_f_Page_079.QC.jpg
c8d3b543d1e4c4a3108fefac3823ff1a
3e5a9b4516f5a25c83b4c8422fc293f36e404f40
34153 F20110318_AABNQY nunez_f_Page_042.QC.jpg
e68192b39d07f77264c09ddd29f5c7a6
32f3e84ca004ed75bb782f9f06f98715bc33f5a4
8108 F20110318_AABNSB nunez_f_Page_023thm.jpg
1a2d143f1b181251d61ffab9f440bf49
3bf653977ae097103863520113a4e0b4bf9c2490
26153 F20110318_AABNRO nunez_f_Page_080.QC.jpg
d46447a3e81be9410e7b01141dae0035
3f85aaf1af45c894d958ba0e7402cd04ddb04cdf
31869 F20110318_AABNQZ nunez_f_Page_044.QC.jpg
2d4d67d0f86128be3839c0285e2d4d54
59b1bdab2519c166d88a908e3b2b7cc44260b24f
8587 F20110318_AABNSC nunez_f_Page_026thm.jpg
13ca7b802c473adb494acf6ae95e490c
be5e633bd75d04905a960b129f1641a8df9977f3
11717 F20110318_AABNRP nunez_f_Page_084.QC.jpg
efa66a34fcaffbc12b0dd9cc90e70924
c23fbd2cf56cb4014336dd1130cb293a966c4276
8502 F20110318_AABNSD nunez_f_Page_028thm.jpg
7472bd59257d5af976f805ec5ed110f9
7856ee0ed4906fd8774962cf46fab4553baf431b
8029 F20110318_AABNSE nunez_f_Page_030thm.jpg
9fe2049b0b3155d6772331826a0c8f71
97f0c124534d1defe02ca8484fde260a422bffce
25058 F20110318_AABNRQ nunez_f_Page_087.QC.jpg
a58235fa40a5594c4f2189fc94f345f5
0ce51e1143df8b598bc234a2b71f8eb314a49c8c
8166 F20110318_AABNSF nunez_f_Page_031thm.jpg
48c284af63ed1de1d0b9ea09ca2443eb
36a2e03b5bd58f5e614177a1a5ba8e502f2d0c4a
33113 F20110318_AABNRR nunez_f_Page_098.QC.jpg
d9c5fa3c91088182ba1b56b7ece56a54
d990871e20a9110e382148ef8c238e3480ea5a4c
8322 F20110318_AABNSG nunez_f_Page_035thm.jpg
1142d84e4237e85a1de9a22ec1e8141b
8358c1b1af105e635f0ae7563c04aad73c727c98
12796 F20110318_AABNRS nunez_f_Page_101.QC.jpg
c8cdd9d9dcb09a0a6089a94e17dc623c
cec0dbcda0455934924cff151fe487bda1006416
6776 F20110318_AABNSH nunez_f_Page_037thm.jpg
a10c9f1675230496a4605cff8612719c
e9f5950924ecd3615ea9ecd13c4f42fa08dddb48
5260 F20110318_AABNRT nunez_f_Page_005thm.jpg
632ddc0f0f45c1cfabd873325265b7c6
6d526d3bdd35a7fab1cf7495aea4170a3dd5708e
8583 F20110318_AABNSI nunez_f_Page_042thm.jpg
87d5d0f3213f84215920fc2b5876d998
6f87148c322060d3cb1dff518815b83e603447fd
8119 F20110318_AABNSJ nunez_f_Page_044thm.jpg
b6db89f7b43a4f66b1f744f3d7fabf3b
f1cf8bcdd5bc41b9e9b6db220863a640e66d901d
7323 F20110318_AABNRU nunez_f_Page_006thm.jpg
2013c95c8da59bcbc9f3a0f15584ac9d
b85eacc0aefa05701539e91d3e2a218ffa754a62
7247 F20110318_AABNSK nunez_f_Page_045thm.jpg
f03bb1d90d2efabff847c3094db9696b
e3758d852d531b92c478b489320f5f1462117f24
9509 F20110318_AABNRV nunez_f_Page_010thm.jpg
e3510fbb36fc756163e94cbd977b190b
83f1bea067f9e933a0b76fec8643869cf21a0c01
7811 F20110318_AABNSL nunez_f_Page_056thm.jpg
07b2bd1b0afbd6e4188a615a98adb685
566a3c2e610362007568ba421a34d4ed89db29ea
6939 F20110318_AABNRW nunez_f_Page_016thm.jpg
4bec38bd5f78ab0e560e10cbfb264883
cfba2c39ca04c7afad0b2a2eab5b9233a86804f1
8116 F20110318_AABNSM nunez_f_Page_059thm.jpg
1f228d8b02c7c758b90a5fd359abe77f
193706cf27c9ddfca0943801eed3c36bf66ee24c
8177 F20110318_AABNRX nunez_f_Page_017thm.jpg
1c5be02805c807e406cde5ceec890e68
12273f4c3c869afda48ebc51ff7f559dabca5d2a
8484 F20110318_AABNSN nunez_f_Page_061thm.jpg
804a8b03c34f8d14e7595582c522a160
0d56ec0902e552ea1a0f3925c53fb0ac7e8de2d7
8200 F20110318_AABNRY nunez_f_Page_018thm.jpg
7fde8a8b7fd928e1990b54c0e47b5d5c
97b806cf9e23578c59b819998d6049ca17906ec8
1032 F20110318_AABNSO nunez_f_Page_064thm.jpg
c0a65225d4670fa74f6b0e88d6127282
0ac7d2a78ae68d0517b96b168b7f9a19c2ee43a7
7533 F20110318_AABNRZ nunez_f_Page_020thm.jpg
015f8788ba085169c5ff54ce2609c71b
99f70635ed50143b5f98cc68a67880b6ecb768ff
8217 F20110318_AABNSP nunez_f_Page_070thm.jpg
8aeac71017cd09d456b2c9505f7d2ae9
ce00e993a8a8e11ccc52e57feae4c92f1ebf9a7b
9017 F20110318_AABNSQ nunez_f_Page_074thm.jpg
4fc9af23bc0d05da85d6960c315cd09f
68b354023a626e89891ee64dd0a40cc70095c7e6
5566 F20110318_AABNSR nunez_f_Page_075thm.jpg
6295ceaa124e42141008e8615f3be6c3
efb65a692a1d7f69611ab2392fcb2775929bfbbf
5399 F20110318_AABNSS nunez_f_Page_083thm.jpg
71c77cbadb2c859a7d4d2cfe5c3da179
813e40ef3d48e206ce158a8d7260e780f6638b63
4269 F20110318_AABNST nunez_f_Page_089thm.jpg
2ff94378ab23042d122c3a3b252b4695
d8b39601109ab0b5fea4f91b541244c3d7e741fe
7607 F20110318_AABNSU nunez_f_Page_092thm.jpg
fc205f49933211e9c7fa8cfa10a9e9f2
172541906500168e843ac3809ae6ea98ec08b99b



PAGE 1

GUAVA ( Psidium guajava L.) FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND OVERALL QUALITY AS INFLUENCED BY POSTHARVEST TREATMENTS By FLOR DE MARIA NUNEZ RUEDA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

PAGE 2

Copyright 2005 by Flor de Maria Nunez Rueda

PAGE 3

DEDICATION To the Lord Jesus, for giving me the stre ngth and motivation to keep pursuing my dreams. To Sabina, my grandmother, backbone of my family and shimmering light in our lives. To my nephew Diego, with lots of love from your always spoiling aunt.

PAGE 4

iv ACKNOWLEDGMENTS I wish to extend my special thanks to my major advisor, Dr. Stephen Talcott, for his support, advice, friendship and for bei ng a leading example of motivation and hard work to us. I could not have asked for mo re. I thank my advising committee, Dr. Susan Percival and Dr. Donald Huber, for their inva luable assistance and time for my project. I thank my lab partners and friends, Kim, Kristine, Jorge, Lanier, Chris, Lisbeth, and Stacy, for their never-ending assistance in my project and for making the work in the lab such an enjoyable and nurturing experien ce. Special thanks go to Dr. Joonhee Lee and soon Dr. to be David del Pozo, for investing their valuable time, sharing their knowledge and providing guidance, as we ll as a friendly shoulder to lean on. Graduate school would definitely not have been as much fun w ithout Davids friendship and craziness. I would have not reached where I am wit hout the unconditional love, support, and advice of my mother Nora, the biggest example of strength I have seen in a person. I love her so much. I thank my sister and best fr iend Tania, for her advice and big sis support during all these years. Big thanks go to my br other Jesus and my sister-in-law Yalile, for their warm support and example of perseverance and love, as well as to my little brother Guille, for having patience with me. Special thanks go to my family in Honduras, Nunez, Rueda, and Maier, for all their support and be st wishes through the distance. Last but not least, big thanks go to Gary, for sharing his love, patience, jokes and for just adding much happiness to my graduate school days.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 2.1 Guava Market and Industrial Applications.............................................................4 2.2 Guava Fruit.............................................................................................................5 2.2.1 Origin............................................................................................................5 2.2.2 Morphology..................................................................................................5 2.2.3 Postharvest Physiology.................................................................................6 2.3 Guava Phytochemicals............................................................................................8 2.3.1 Phytochemicals.............................................................................................8 2.3.3 Dietary Fiber.................................................................................................9 2.3.4 Carotenoids and Lycopene.........................................................................10 2.3.5 Guava Polyphenolics..................................................................................11 2.4 Postharvest Treatments.........................................................................................13 2.4.1 Guava Postharvest Handling and Storage..................................................13 2.4.2 Quarantine Heat Treatments.......................................................................13 2.4.3 Shelf-life Extension Treatments.................................................................15 2.5 1-Methylcyclopropene..........................................................................................15 2.5.1 1-Methylcyclopropene................................................................................15 2.5.2 1-MCP Application Conditions..................................................................16 2.5.3 1-MCP on Climacteric Fruits.....................................................................17 2.5.4 Guava and 1-MCP......................................................................................18 2.6 Polyphenolics........................................................................................................18 2.6.1 Polyphenolics.............................................................................................18 2.6.2 Polyphenolic Classification........................................................................19 2.6.3 Polyphenolics as Antioxidants...................................................................23

PAGE 6

vi 3 EFFECTS OF HOT WATER IMMERS ION TREATMENT ON GUAVA FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY.............25 3.1 Introduction...........................................................................................................25 3.2 Materials and Methods.........................................................................................26 3.2.1 Materials and Processing............................................................................26 3.2.1.1 Fruit preparation and HW treatment................................................26 3.2.1.2 Guava fruit processing.....................................................................27 3.2.2 Chemical Analysis......................................................................................27 3.2.2.1 Moisture content determination.......................................................27 3.2.2.2 Quantification of total soluble phenolics.........................................28 3.2.2.3 Analysis of ascorbic acid by HPLC.................................................28 3.2.2.4 Quantification of antioxidant capacity.............................................29 3.2.2.5 Analysis of lycopene by HPLC........................................................29 3.2.2.6 Quantification of nonlycopene carotenoids....................................30 3.2.2.7 Analysis of polyphenolics by HPLC................................................30 3.2.3 Quality Analysis.........................................................................................31 3.2.4 Statistical Analysis.....................................................................................31 3.3 Results and Discussion.........................................................................................32 3.3.1 Chemical Analysis......................................................................................32 3.3.1.1 Moisture content...............................................................................32 3.3.1.2 Total soluble phenolics.....................................................................33 3.3.1.3 Ascorbic acid....................................................................................35 3.3.1.4 Antioxidant capacity........................................................................36 3.3.1.5 Lycopene and yellow carotenoids....................................................38 3.3.1.6 Polyphenolics by HPLC...................................................................41 3.3.2. Quality Analysis........................................................................................49 3.3.2.1 pH and soluble solids.......................................................................49 3.3.2.2 Overall fruit quality..........................................................................49 3.3.3 Stage III Fruit.............................................................................................50 3.4 Conclusions...........................................................................................................51 4 EFFECTS OF 1-METHYLCYCLOPROPENE ON GUAVA FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY.............53 4.1 Introduction...........................................................................................................53 4.2 Materials and Methods.........................................................................................54 4.2.1 Materials and Processing............................................................................54 4.2.1.1 Fruit preparation and 1-MCP treatment...........................................54 4.2.1.2 Guava fruit processing.....................................................................56 4.2.2 Quality Analysis.........................................................................................56 4.2.2.1 Aesthetic fruit quality a ssessment during storage............................56 4.2.2.2 Firmness determination during storage............................................56 4.2.2.3 Titratable acidity, so luble solids and pH..........................................56 4.2.3 Chemical Analysis......................................................................................57 4.2.4 Statistical Analysis.....................................................................................57 4.3 Results and Discussion.........................................................................................57

PAGE 7

vii 4.3.1 Quality Analysis.........................................................................................57 4.3.1.1 Aesthetic fruit quality during storage...............................................57 4.3.1.2 Firmness during storage...................................................................60 4.3.1.3 Titratable acidity, soluble so lids, and pH during storage.................62 4.3.2 Chemical Analysis......................................................................................64 4.3.2.1 Moisture content...............................................................................64 4.3.2.2 Total soluble phenolics.....................................................................65 4.3.2.3 Antioxidant capacity........................................................................66 4.3.2.3 Ascorbic acid....................................................................................67 4.3.2.4 Lycopene..........................................................................................68 4.3.2.5 Polyphenolics by HPLC...................................................................70 4.3.3 1-MCP Treatment to Boxed Guavas..........................................................73 4.4 Conclusions...........................................................................................................76 5 SUMMARY AND CONCLUSIONS.........................................................................78 LIST OF REFERENCES...................................................................................................80 BIOGRAPHICAL SKETCH.............................................................................................89

PAGE 8

viii LIST OF TABLES Table page 3-1. Gradient elution runn ing program for HPLC analysis of polyphenolics.................31 3-2. Tentative identificati on of guava polyphenolics at 280 nm by HPLC based on retention time, spectral properties, a nd comparison to authentic standards.............43 3-3. Guava gallic acid (GA), gallic acid deri vatives, and an ellagic acid derivative as affected by a hot water quarantin e treatment and ripening stage.............................45 3-4. Guava procyanidins, other charact eristic unknown compounds, and total polyphenolics by HPLC as affected by a hot water quarantine treatment and ripening stage...........................................................................................................45 3-5. Quality parameters, soluble solids and pH, in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and Stage II). Data are expr essed as fresh weight basi s (mean standard error), n = 4..........................................................................................................................49 3-6. Phytochemical content and quality parame ters in Stage III guavas as affected by a hot water quarantine treatment at 46 C (mean standard error). n = 4...............51 4-1. Changes in skin coloration in non-tr eated (control) and 1-MCP-treated guavas during 1-MCP application and storage at 15 C......................................................59

PAGE 9

ix LIST OF FIGURES Figure page 2-1. Chemical structure of lyc opene, a 40-C open hydrocarbon chain...........................10 2-2. Chemical structures of a condensed ta nnin (A) and hydrolysable tannin (B). A is a typical condensed tannin composed of catechin and epicatechin; B is a polygalloyl glucose composed of a glucos e core esterified with gallic acid residues.....................................................................................................................21 2-3. Chemical structures of flavonoids apigenin and myricetin, which have previously reported in guava. They are composed of three pyrane rings.................22 3-1. Moisture content (%) of ripe guavas as affected by a hot water immersion treatment (0,15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represen t standard error of the mean, n =4..............................................33 3-2. Total soluble phenolics (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represent sta ndard error of the mean, n = 4................................34 3-3. Ascorbic acid content (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represent sta ndard error of the mean, n = 4................................36 3-4. Antioxidant capacity ( mol Trolox Equivalents/g DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars repres ent standard error of the mean, n = 4.........37 3-5. Lycopene content (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represen t standard error of the mean, n = 4.............................................39 3-6. Non-lycopene carotenoids (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represent sta ndard error of the mean, n = 4................................40 3-7. HPLC chromatogram of polyphenolic co mpounds found in guava juice-A) gallic acid, B) gallic acid derivatives, C) unknown-characteristic guava polyphenolics, D) procyanidins, and E) ellagic acid derivative. Identification (280 nm) was done by comparison to authentic sta ndards and spectral properties........................42

PAGE 10

x 4-1. Firmness (kg) of guavas treated with 1-MCP (1000 nL/L ,10 C, 24 h) during storage at 15 C. Error bars represent the sta ndard error of the mean, n = 5...........61 4-2. Titratable acidity (% citr ic acid) of guavas treated w ith 1-MCP during storage at 15 C. Error bars represent the standard error of the mean, n = 5............................63 4-3. Effect of a 1-MCP treatment (1000 nL/L ,10 C, 24 h) on guava pH during storage at 15 C. Error bars represent the sta ndard error of the mean, n = 5...........63 4-4. Effect of 1-MCP treatment (1000 nL/L at10 C, 24 h) on guava soluble solids ( Brix) during storage at 15 C. Error bars represent th e standard error of the mean, n = 5...............................................................................................................64 4-5. Effect of 1-MCP (1000 nL/L, 10 C, 24 h) on total soluble phenolics (mg/kg DW) in guava. Error bars represent the standard error of the mean, n = 23............66 4-6. Effect of 1-MCP treatment (1000 nL/L, 10 C, 24 h) on guava antioxidant capacity ( M Trolox equivalents/g DW). Error ba rs represent the standard error of the mean, n = 23...................................................................................................66 4-7. Guava ascorbic acid content (mg/kg DW) as affected by 1-MCP (1000 nL/L, 10 C, 24 h). Error bars represent the st andard error of the mean, n = 23................68 4-8. Effect of 1-MCP (1000 nL/L ,10 C, 24 h) on guava lycopene content (mg/kg DW). Error bars represent the standard error of the mean, n = 23...........................69 4-9. Guava procyanidin content (mg/kg GAE ) as affected by 1-MCP. Error bars represent the standard er ror of the mean, n = 23......................................................71 4-10. Guava ellagic acid derivative conten t (A) and character istic polyphenolics content (B) (mg/kg GAE) as affected by 1-MCP. Error bars represent the standard error of the mean, n = 23...........................................................................72 4-11. Guava gallic acid content (mg/kg GAE ) as affected by 1-MCP. Error bars represent the standard er ror of the mean, n = 23......................................................73 4-12. Firmness (kg) of boxed guavas treated with 1-MCP (1000 nL/L ,10 C, 24 h) during storage at 25 C. Error bars represent the sta ndard error of the mean, n=3..74 4-13. Titratable acidity (% citric acid) of boxed guavas treated with 1-MCP during storage at 15 C. Error bars represent the sta ndard error of the mean, n = 3...........75 4-14. Effect of 1-MCP treatment (1000 nL/L at10 C, 24 h) on boxed guavas soluble solids ( Brix) during storage at 15 C. Error bars represent the standard error of the mean, n = 3.........................................................................................................76

PAGE 11

xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science GUAVA ( Psidium guajava L.) FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND OVERALL QUALITY AS INFLUENCED BY POSTHARVEST TREATMENTS By Flor de Maria Nunez Rueda August 2005 Chair: Stephen T. Talcott Major Department: Food Science and Human Nutrition Guava ( Psidium guajava L.) fruit, appealing for its unique tropical flavors, is considered an excellent sour ce of nutrients and antioxidant phytochemicals, especially ascorbic acid. Guavas extremely perishable nature and quarantin e issues surrounding its importation somewhat limit its fresh fruit marketability within the US. Additionally, limited studies have reported resultant cha nges of postharvest pr ocesses on guavas phytochemicals and antioxidant capacity. The ob jectives of this study were to evaluate the effects of two postharvest treatments on phytochemical content, antioxidant capacity, and overall quality of pink guavas. A hot wa ter (HW) immersion technique was applied as a potential quarantine treatment, whereas 1-methylcyclopropene (1-MCP), an ethylene blocker, was applied as a treatment to extend the shelf-life of guavas. For the HW immersion study, guavas were segregated into three groups according to their ripeness level-Stage I (yellowish-g reen skin, firm texture), Stage II (25-50% yellow skin, semi-firm texture), and Stage III (>75% yellow skin, soft texture)-and

PAGE 12

xii subjected to four HW immersion times (0 (c ontrol), 15, 30, and 60 mi n) at 46C. Fruits were held at 15C until fully ripe and collected for analysis. For the 1-MCP study, unripe guava fruit were separated into two groups : Control and 1-MCP (1000 nL/L); both were held for 24 hours at 10 C during treatment applicati on. They were stored at 15 C until full ripeness and collected for analysis. Ch emical analyses for both studies included moisture, total soluble phenolics (TSP), asco rbic acid (AA), antioxi dant capacity (AOX), lycopene, yellow carotenoids, and polyphenol ics by HPLC. Quality analysis included soluble solids, pH, titratable acidity (TA) (1-MCP), and firmness (1-MCP). A HW treatment up until 30 min at 46 C insignificantly affected moisture (92.2%), TSP (20,600 mg/kg, dry weight (DW)), lycopene (501 mg/kg DW), ascorbic acid (10,800 mg/kg DW), soluble solids (7.7 Brix), pH (4.1), yellow ca rotenoids (46.7 mg/kg DW) and AOX (133 M TE/g DW) within each ripening stage. Stage I fruits treated for 60 min presented an enhancement of certain polyphenolics and a decrease in lycopene content as a response to heat stress. 1-MCP application effectively extended guava shelflife for at least 5 days at 15 C, delaying skin yellowing and retaining firmness. Moisture content (86.4%), TSP (19,700 mg/kg DW), AOX (133 M Trolox equivalents/g DW), pH (4.1), soluble solids (7.8 Brix), and TA (0.29 % citric acid) were unaffected by 1MCP. Although ascorbic acid (6,145 mg/kg DW ) and lycopene content (419 mg/kg DW) were significantly higher in 1-MCP treated fr uit, similarly to other phytochemicals, effects were independent of ethylene inhibition. A 1-MCP and HW quarantine treatment up to 30 min can be applied to fresh guava without detrimental effects on phytochemical content, antioxidant capacit y, and fruit quality, th erefore extending its fresh fruit market window.

PAGE 13

1 CHAPTER 1 INTRODUCTION The consumption trend of fresh tropical fr uits and their products is increasing steadily due to consumers education on thei r exotic flavors, nutritive value, and phytochemical content with pot ential health effects (Food and Agriculture Organization [FAO], 2004). Guava fruit ( Psidium guajava L.), an exotic from the tropics characterized by its appealing flavor and aroma, has been cat alogued as one of the most nutritious fruits due to its high content of phytochemicals, specially ascorbic acid (United States Department of Agriculture [USDA], 2004). Guavas importation as a fresh fruit is somewhat limited within the US for two ma in reasons: quarantine issues surrounding its importation and its highly perishable nature. Guavas are considered excellent sources of antioxidant phytochemicals, which include ascorbic acid, carotenoids, antioxi dant dietary fiber, and polyphenolics. After acerola cherries, guava has reported the sec ond highest concentration of ascorbic acid (ranging from 60-1000 mg/100 g) of all fru its (Mitra, 1997). Carotenoids, which are yellow, red, and orange pigments, have dem onstrated many beneficial health effects related to their antio xidant properties (Wilberg and Rodriguez-Amaya, 1995). Guavas major carotenoid, lycopene, is responsible fo r the pink coloration in pink guavas flesh (Mercadante et al., 1999). Polyphenolics fr om fruits and vegetables are widely investigated because of their role as ch emoprotective agents against degenerative diseases, antimutagenic effects, and antivir al effects, among othe rs (De Bruyne et al., 1999; Gorinstein et al., 1999; Robbins, 2003). Currently, rese arch on identif ication and

PAGE 14

2 quantification of ripe guava polyphenolics is very limited, and information is still unclear as to the type and concentration of i ndividual compounds pres ent in the fruit. Various postharvest chemical and heat appl ications exist as quarantine treatment for fresh fruit importation into the US, whic h may also preserve appearance and table quality of various fruits (Lurie, 1998). Ther mal applications are gaining more popularity due to consumers demand to ameliorate the us e of chemicals. In the case of guava, an established quarantine treatment still does not exist for importing it into the US (Gould and Sharp, 1992; USDA-Animal and Plant H ealth Inspection Service [USDA-APHIS], 2004), potentially due to the la ck of studies that demons trate a beneficial effect. Additionally, few studies repor t resultant changes that th ese processes may have on phytochemical content, nutrient st ability, and antioxidant capacity of the fruit, either in a beneficial or detrimental way. Perishability is one the main issues in postharvest handling and marketing of fresh fruits and vegetables. In the case of guava fru it, its short shelf life (7 to 10 days) limits somewhat its marketability. Numerous techno logies have been developed as means to extend their shelf-life and ea ting quality, some of which include modified atmospheres, polymeric films, irradiation, or chemical tr eatments (Mitra, 1997) A recently developed shelf-life extension tool is the app lication of a gaseous organic compound, 1methylcyclopropene (1-MCP), as an ethylene blocker, delaying or inhibiting ripening on ethylene-sensitive commodities (Blankenship and Dole, 2003). Currently, limited studies exist on the impact of 1-MCP on phytochemical content of fruits and vegetables in general, and their relationship w ith ethylene inhibition responses.

PAGE 15

3 This study evaluated the effects of tw o post-harvest treatments, a thermal quarantine hot water treatment and a 1-1-MC P application, on phytochemical content, antioxidant properties, and fr uit quality. It was hypothesized that HW treatment at 46 C for short periods would not stress guava fruit or affect phytochemical content, antioxidant properties, and quality. A 1-MCP treatme nt would affect guava ripening and characteristics associated with it, howe ver phytochemicals and antioxidant properties would remain unaffected. The specifi c objectives of this study were Post-harvest treatment-Hot Water Immersion: To evaluate the effects of a thermal quarantine treatment using a hot water im mersion technique on the phytochemical content, antioxidant capacity, and overall quality of guava fruits. Post-harvest treatment-1-MCP: To evaluate the effects of 1-MCP as a postharvest treatment for extention of fresh guava shel f-life and to determine these effects on the phytochemical content, antioxidant capacity, and overall quality of guava fruits.

PAGE 16

4 CHAPTER 2 LITERATURE REVIEW 2.1 Guava Market and Industrial Applications Exotic or minor tropical fruits, which include guava, carambola, durian, lychee, mangosteen, passionfruit and rambuttan have und ergone a significant increase in both volume and value in recent years. Their produ ction continues to steadily increase and is estimated to have reached 14.9 million metric tons (23% of total global output of tropical fruits) in 2002 and US total import vol umes were 176,000 tons for 2003 (FAO, 2004). Fresh fruit market in general is growing in the US chiefly due to an increase in consumption demand and the development of t echnologies to preserve fruit eating quality and prolong shelf-life (Kipe, 2004). Guava as an import is divided into f our categories according to the National Agriculture and Statistics Service [NASS]: pr eserved or prepared, paste and puree, jam, and dried. Brazil was the leader for guava imports into the United States in 2003, followed by Dominican Republic, Mexico, In dia, and Costa Rica. Within the US, commercial producers are Hawaii, southe rn Florida (Gould and Sharp, 1992), and southern California. Hawaii is the main grow er, with 530 harvested acres and a utilized production volume of 6.7 million pounds in 2003. The local production in Florida and Hawaii is hampered by the Caribbean fruit fly, causing serious economic losses if not controlled adequately (NASS, 2004). Currently, external or internal import of fresh guava fruit is not possible; this is mainly attributed to the tropical fruit fly and guavas very short shelf life.

PAGE 17

5 For industrial applications, guava is one of the easiest fruits to process, since the whole fruit may be fed into a pulper for m acerating into puree (Boyle, 1957). It is physically and biochemically stable in re lation to texture or pulp browning during processing (Brasil et al., 1995). It can be processed into a variety of forms, like puree, paste, jam, jelly, nectar, syr up, ice cream or juice. Within the United States processing industry, it is gaining popularity in juice blends due to it s exotic flavor and aroma. 2.2 Guava Fruit 2.2.1 Origin Guava (Psidium guajava) is an exotic fruit member of the fruit family Myrtacea. Guava, goiaba or guayaba are some of the names given to the apple of the tropics, popular for its penetrating aroma and flavor Its place of origin is quite uncertain, extending in an area from southern Mexi co through Central and South America. Currently, its cultivation has been extended to many tropical and subtropical parts of the world, where it also thrives well in the w ild (Morton, 1987;Yadava, 1996; Mitra, 1997). 2.2.2 Morphology Guava shape ranges from round, ovoid, to pear-shaped, and with an average diameter and weight ranging from 410cm and 100-400g respectively (Mitra, 1997). Classified as a berry, guava is composed by a fleshy mesocarp of varying thickness and a softer endocarp with numerous small, hard yellowish-cream seed s embedded throughout it (Malo and Campbell, 1994; Marcelin et al ., 1993). Guava pulp contains two types of cell-wall tissues: stone cells and parenchyma ce lls. Stone cells are highly lignified woody material responsible for a char acteristic sandy or gritty f eeling in the mouth when the fruit is consumed; due to their nature, they are resistant to enzymatic degradation. They account for 74% of the mesocarp tissue, while the endocarp is rich in parenchyma cells,

PAGE 18

6 which give it a softer textur e. (Marcelin et al., 1993). Exterior skin color ranges from light green to yellow when ri pe and its pulp may be white, yellow, pink, or light red. Unripe guava fruit are hard in texture, star chy, acidic in taste and astringent, due to its low sugar and high polyphenol content. Once it ri pens, the fruit becomes very soft, sweet, non-acidic, and its skin becomes thin and edible (Malo and Campbell, 2004; Mitra, 1997). Many guava cultivars exist today, however they can be broadly classified as pink or white. Seedless cultivars ar e available in many countries, which have a great potential to become popular in the US in the future (Yadava, 1996). 2.2.3 Postharvest Physiology Ripening and factors associated with it in c limacteric fruits is regulated by ethylene synthesis. Ethylene (C2H4) is a naturally-produ ced, gaseous growth regulator associated with numerous metabolic processes in plants (Mullins et al., 2000). It is produced from L-methionine via 1-aminocyclopropane-1-car boxylic acid (ACC) synthase in a complex signal transduction path way, which is still widely researched today (Salveit, 1998; Mullins et al., 2000). All plants produce ethy lene, but only climacteric fruits and wounded or stressed tissue produce suffici ent amounts to affect other tissues. In climacteric fruits, ethylene stimulates its own biosynthesis at the start of ripening, enhancing its production until reaching saturatio n levels (Salveit, 1999). Stresses such as chill injury, heat shock (Cis neros-Zevallos, 2003) or diseas e (Mullins et al., 2000), can induce ethylene production and therefore e nhance fruit ripening, and the factors associated with it. Studies evaluating respirat ory patterns of guava dem onstrated a climacteric response as increased carbon dioxide corre sponded to increased ethylene production (Akamine and Goo, 1979; Mercado-Silva et al., 1998; Bashir and Abu-Goukh, 2002).

PAGE 19

7 Guavas have a rapid rate of ripening, theref ore a relatively short sh elf life ranging from 3 to 8 days depending on the variety, harvest time, and environmental conditions (Reyes and Paull, 1995; Basseto et al., 2005) Ethylene production a nd respiration (CO2 production) increases after the first day of harv est, at the start of ripening. Guava reaches its climacteric peak between day 4 and 5 post-harvest (mature-green harvested fruits) and then declines (Akamine and G oo, 1979; Bashir and Abu-Goukh, 2002). As a guava ripens, total soluble solids and to tal sugars increase in both the peel and pulp, whereas titratable acidity declines after reaching its clim acteric peak of respiration. In general, climacteric fruits undergo cons iderable changes in sugar content during ripening, where starch and sucrose are br oken down into gluc ose (Bashir and AbuGoukh, 2002). Moisture loss in guava, especially in tropical climates, can also be substantial resulting in up to 35% weight lo ss (Mitra, 1997) that co rresponds to loss of postharvest quality and consumer acceptability. Ascorbic acid content is at its maximum level at the mature-green stage and declines as the fruit ripens in both white and pink guavas (reviewed by Bashir and Abu-Goukh, 2002), and may also be a function of postharvest handling. Lycopene synthesis in pink guavas is enhanced during ripening. In the case of tomatoes, once lycopene is accu mulated, the respiration rate decreases (Thimann, 1980). Total fiber content decrease s significantly during ripening, from 12 to 2g/100g, and it is hypothesized that is closely be related to th e activity of certain enzymes (El-Zoghbi, 1994). Abu-Goukh and Bashir (2003) studied the activitie s of some cell wall degrading enzymes in both pink and white guava and showed that pectinesterase (PE) activity increased until reach ing its climacteric and latter decreased, whereas polygaracturonase (PG) and cellulase increased as the fruit ripened in correspondence to

PAGE 20

8 fruit softening. Increase in polyphenoloxidase (PPO) activity was also reported with ripening and a decrease in polyphenolics, whic h be the responsible for the reduction of astringency (Mowlah and Itoo, 1982). Visually, the ripeness level of guava can be characteri zed by its skin color ranging from a dark green when unripe to a bright yellow or yellow-green at full ripeness. However, determination of ripeness can be misleading for some varieties and may be combined with a simple test for specific gravity, by placing fruit in water to determine if it sinks (unripe) or floats (ripe ) to obtain a clearer picture of the degree of fruit ripeness (Reyes and Paull, 1995). Objec tive determination of skin co lor has also been used to predict ripeness, with L*, a* and hue angles of 65.93, 15.92, and 110.92 respectively indicating a mature, yellow fruit (Mercado-Silv a et al., 1998). In co mbination with fruit texture, these simple assays can provide an adequate estimation of the stage of fruit ripeness. 2.3 Guava Phytochemicals 2.3.1 Phytochemicals Phytochemicals may be defined as biologi cally active compounds present in foods, nutritive or non-nutritive, whic h prevent or delay chronic diseases in humans and animals. They may also be defined as food ingredients which provide health benefits beyond their nutritional value (reviewed by Ho et al., 1992). The importance of phytochemicals has grown in recent years due to consumers increased awareness of health beneficial effects. The main phyt ochemicals found in guava are ascorbic acid, antioxidant-containing dietary fiber, carotenoids, and polyphenolics. 2.3.2 Ascorbic Acid and Other Antioxidant Vitamins

PAGE 21

9 Guavas are considered an outstanding sour ce of ascorbic acid (AA), three to six times higher than the content of an orange and after acerola cherries it has the second highest concentration among all fruits. The AA content in guava varies from 60 to 100 mg/100 g in some cultivars, and from 200 to 300 mg/100g in others, while higher reports range from 800 to 1000 mg/100g. Mitra (1997) mentions that AA content is more influenced by the fruits variety than by its ripening stage and storage conditions. Within the fruit, AA is concentrated in the sk in, followed by the mesocarp and the endocarp (Malo and Campbell, 1994). As a water-soluble vitamin, it is highly susceptible to oxidative degradation and is often used as an index for nutrient stability during processing or storage (Fennema, 1996). Guava was also found to contain alphatocopherol (vitamin E) at nearly 1.7 mg/ 100g (Ching and Mohamed, 2001), which is an important fat-soluble dietary antioxidant. 2.3.3 Dietary Fiber Dietary fiber in fruits and vegetables has been associated with a reduction in colon and other cancer risks. Soluble fiber content is generally associ ated with a reduced risk of cardiovascular disease. In a study done to a number of tropical fruits guava showed the highest content of total and soluble diet ary fibers with values of 5.60 and 2.70g/100g respectively (Gorinstein et al., 1999). Total and soluble fiber present in guava is extraordinarily high in concentration as compar ed not only to tropicals, but all fruits and vegetables. Fiber from guava pulp and peel was tested for antioxidant properties and found to be a potent source of radical-scav enging compounds, presumably from the high content of cell-wall bound polyphenolics (2.627.79% w/w basis) present in each fiber isolate (Jimenez-Escrig et al., 2001).

PAGE 22

10 2.3.4 Carotenoids and Lycopene Carotenoids are yellow, red, and orange pi gments abundant in a wide variety of fruits and vegetables. Due to their antioxi dant properties, carotenoids have shown beneficial health effects in cancer inhibition, immuno-enhacement, and prevention of cardiovascular diseases (Wilberg and Rodr iguez-Amaya, 1995). The most important carotenoids which provide oxidative protection are -carotene, -carotene, lutein, lycopene, zeaxanthin, and -cryptoxanthin (VERIS, 2000). A well-established function is the vitamin A antioxidant activity of some of carotenoids, including -carotene, carotene, -cryptoxanthin. Carotenoids are a clas s of structurally related 40-carbon compounds (two 20-carbon tails) which consist of eight repeating isoprene units (Van de Berg et al., 2000). Lycopene, the major caroteno id present in guava (Mercadante et al., 1999), is a 40-C open chain hydrocarbon cont aining 11 conjugated and 2 non-conjugated double bonds arranged linearly (Figure 21). Currently, High Performance Liquid Chromatography (HPLC) is the preferred procedure for carotenoid analysis. Figure 2-1. Chemical structure of lycopene, a 40-C open hydrocarbon chain. Lycopene has received considerable atte ntion recently due to diverse in-vivo and in-vitro studies reporting the effect of dietary lycopene in reduction in the risk of prostate cancer and coronary heart disease (Rao a nd Agarwal, 1999). Lycopene has reported a superior antioxidant activity in relation to lutein or -carotene, due to its conjugated double bonds (reviewed by Lin and Chen, 2003). Currently, tomatoes and tomato-based

PAGE 23

11 products are the main source of dietary lycope ne. Ripe fresh tomato es have a lycopene content ranging from 4 to 8 mg/100g (Abus hita et al., 2000; Leonardi et al., 2001; Seybold et al., 2004). During tomato processing, some authors have reported lycopene and other carotenoid reduction (Takeoka et al., 2001; Sahlin et al., 2004), while others report an enhancenment, increased bioavail ability and antioxidant capacity of these compounds (Dewanto et al., 2002; Seybold et al., 2004). Lycopene content in guav a Beaumont variety has been found to be about 5-7 mg/100g fruit. Mercadante and partners (1999 ) isolated sixteen carotenoids from guava, of which thirteen were reported as guava carot enoids for the first time. In another study made to Brazilian guavas, the -carotene concentration in ripe fruits ranged from 0.3 mg/100g to 0.5 mg/100g; while the lycopene concentration ranged from 4.8 mg/100g to 5.4 mg/100g (Wilberg and Rodriguez-Amaya, 1995). 2.3.5 Guava Polyphenolics Polyphenols are the most abundant phytochemi cals in our diets, and fruits are the main contributors (Jimenez-Escrig et al., 2001). Currently, limited studies exist on the identification and quantification of guava polyphenolics. Gorinstein et al. (1999) conducted a comparative study between several tropical and subtropical fruits and found guava to be among the top three investigat ed for concentrations of gallic acid (.374 mg/100g), total phenolics (4.95 mg/100g), and th e highest total and soluble dietary fiber of the fruits investigated. Guava are so mewhat unusual in their flavonoid polyphenolic content as well, with significant levels of myricetin (55 mg/100g) and apigenin (58 mg/100g) present in edible tissues, bu t do not contain the more commonly found flavonoids quercetin and kaempferol (Miean and Mohamed, 2001) th at are abundant in

PAGE 24

12 other fruits and vegetables. Misra and Se shadri (1967) identified procyanidins, or condensed tannins in both white and pink cultiv ars, concentrated in the skin and seeds, but very little in the pulp. Also, free ellagic acid was isolated in both varieties (0.2 mg/100g in pink, 0.05 mg/100g in white). In the whole guava, total phenolics are concentrated on the peel, followed by the pulp (Bashir and Goukh, 2002). For processed products, though, location of polyphenolics does not matter since the entire fruit with peel is fed into a pulper. Although limited information is existent it has been confirmed that guava polyphenolics decrease and undergo consider able changes during maturation and subsequent ripening (Mowlah and Itoo, 1982; Itoo et al., 1987; Bashir and Goukh, 2002). According to work conducted by Itoo et al. (1987) immature, underdeveloped guava contains approximately 65% condensed tannins of its total polyphenols, which decrease dramatically as the fruit grows and devel ops. According to Mowlah and Itoo (1982) in both pink and white varieties both non tannin phenolics (simple phenolics, monomeric anthocyanins, catechins, and leucoanthocyanins) and tanni n phenolics (hydrolysable and condensed tannins) decrease during ripe ning. However, at full-ripeness non-tannin phenolics (76 and 80% of total phenolics for pink and white respectively) contents are higher than tannin phenolics (24 and 20%). Th e decrease in astringency during guava ripening has been attributed to an increase in polymerization of condensed tannins to form an insoluble polymer and hydrolysis of a soluble/astringent arabinose ester of hexahydroxydiphenic acid, a precursor of ella gic acid (Goldstein and Swain, 1963; Misra and Seshadri, 1967; Mowlah and Itoo, 1982; It oo et al., 1987). Confirming these results, an increase in free ellagic acid during ripeni ng has been reported (Goldstein and Swain,

PAGE 25

13 1963; Misra and Seshadri, 1967). Currentl y, limited information on individual polyphenolic compounds found in ri pe fruits is existent. 2.4 Postharvest Treatments 2.4.1 Guava Postharvest Handling and Storage Depending on its further use (fresh or pro cessed) postharvest conditions for guava may vary; however its short shelf life is a r ecurring pressure for growers, packers, and processors. Due to its delicate nature, it is carefully hand-harvested while still green, and immediately stored at cool temperatures. In Florida, guavas are usually stored at temperatures between 9 to 12 C (personal communication, Sardinia, 2004) due to their sensivity to chill injury. Th ey are typically shipped from packing houses in a maturegreen stage (yellowish-green skin, firm), af ter harvesting at optimum fruit size. Reyes and Paull (1995) reported less disease incidence in mature green guavas stored at 15 C as compared with fruit that were quarterand half-yellow under the same conditions. Additionally, 15 C was determined to be an opt imum holding temperature prior to processing, since it allowed gr adual ripening of mature-green fruit while delaying deterioration of quarter-yellow and ha lf-yellow fruit. Fruit stored at 5 C did not ripen and developed skin bronzing after two weeks in storage, as a consequence of chill injury. 2.4.2 Quarantine Heat Treatments Various thermal and chemical quarantine trea tments exist for fresh tropical fruits entering the US established by US Department of Agriculture-Animal and Plant Health Inspection Service-Plant Protec tion and Quarantine (USDA-APHIS-PPQ). They are set to ensure disinfectations from pests, insect s, larvae, eggs or fungus for fresh produce importation from other countries and other US st ates or territories. Du ring the past years,

PAGE 26

14 there has been an increasing interest in the use of thermal treatments as a measure of control, due to consumer demand to ameliorate the use of chemicals. Currently, there are three methods to heat commodities: hot water, vapor heat, and hot air (reviewed by Lurie, 1998). Hot water dips are effective for both fungal pathogen control and for disinfestations of insects, need ing a longer time for the latter one, since the internal core of the fruit and not just the surface needs to be brought up to the required temperature. Procedures have been develope d to disinfest a number of tr opical and subtropical fruits from various species of fruit fly (rev iewed by Paull, 1994). The USDA-APHIS-PPQ treatment manual includes treatment schedules that must be followed to import fruit into the US. In the case of mango, this includes a 46 C hot water dip th at disinfects mangoes with possible fruit fly contamination. Current ly, no established treatment schedule exists for guava by the US government (USDA-APHIS, 2004). Guava is major host for many tephritid fr uit fly species, including the Caribbean Fruit fly, Anastrepa suspensa which has been present in Florida for several years. Local guavas therefore, cannot be exported from Florida to ot her citrus-pr oducing states, somewhat limiting their market as fresh fr uit (Gould and Sharp, 1992). Gould and Sharp (1992) conducted studies to determine the suita bility of hot-water (HW) immersion as a quarantine treatment to disinfest pink guavas of Caribbean fruit flies and to asses its effect on overall fruit quality. As compared to other tropicals, such as mangos, a shorter immersion time was required to kill larvae in guava due to the size of the fruits used (approx. 90g). The storage temperature was apparently more important than a HW treatment to retain fruit quality. Guavas held at 24 C ripened within 7 days and guavas held at 10 C ripened within 11 to 18 days regardless of the length of the HW treatment.

PAGE 27

15 Probit statistical analysis estimated a probit 9 (99.9968%) mortality at 31 min at 46.1 + 0.5 C for quarantine security, which did not aff ect fruit quality. This has been one of few studies done on guava HW treatment applicati on. Further investigations are needed in order to obtain a quarantine schedule for guava. 2.4.3 Shelf-life Extension Treatments Various treatments exist to extend the shelf-life of horticultural commodities. Storage under modified atmosphere (MA), packaging (MAP) or coating in polymeric films (cellulose or carnouba -based emulsions) have been shown to be effective on many commodities, including guava. In most cas es, respiration and ethylene production are reduced, delayed or inhibited, inhibiting ripening and characte ristics associated with it (Mitra, 1997). Other shelf-life extensors whic h act directly on ethyl ene binding sites are called ethylene inhibitors or ethylene blockers. Some co mpounds employed as ethylene inhibitors for both floricultural and hortic ultural commodities include: carbon dioxide, silver thiosulfate (STS), aminoethoxyvinylglycine (AVG). 2,5-norbornadiene (2,5-NBD), and diazocyclopentadiene (DACP) (Blankenship and Dole, 2003). 1Methylcyclopropene is an ethylene blocker which is gaining popularity because of its action in a broad range of produce and its practicality of use. 2.5 1-Methylcyclopropene 2.5.1 1-Methylcyclopropene 1-Methylcyclopropene (1-MCP) is a recently developed tool used to extend the shelf life and quality of ethylene-sensitive plan t produce and research the role of ethylene responses. It is an active organic compound (C4H6) which is thought to interact with ethylene (C2H4) receptors so that ethylene cannot bind and take action. Its affinity for the receptor site, ethylene binding protein (EBP) (Mullins et al., 2000), is about ten times

PAGE 28

16 greater than that of ethyle ne. Its origin comes from b ackground work done by Sisler and Blankenship on cyclopropenes, breakdown pr oducts of diazocyclopentadiene (DACP), a known ethylene inhibitor. 1-MCP development resulted in good pract ical use because it is less volatile than cyclopropene itself and is able to act lower concentrations (ppb range). Commercialization of 1-MCP for ornamental s is sold under the trade name EthylBloc by Floralife, Inc., whereas for edible crops it is sold under the tr ade name SmartFresh by AgroFresh, Inc. Both products are gene rally regarded as safe, non-toxic, and environmentally friendly by the Environmenta l Protection Agency [EPA]. In 2000 it was approved for use in edible cr ops, while in 2002 it was exempted from the requirement from tolerance from residues (EPA, 2004).1-MC P is usually employed as a powder that forms a gas when mixed with water (reviewed by Blankenship and Dole, 2003). 2.5.2 1-MCP Application Conditions Temperature, treatment duration, concentr ation, and type of commodity are key variables affecting the efficacy of a 1-MCP tr eatment. Many studies have demonstrated a direct relationship between them. At sta ndard pressure and temperature, 1-MCP is released in approximately 20 to 30 min; how ever, at lower temperatures release might take longer (reviewed by Blankenship and Dole, 2003). DeEll et al. (2002) demonstrated that treatment applied at higher temperatures in apples required le ss exposure time; it has been hypothesized that lower temperatures might lower the affinity for the binding site of 1-MCP in apples (Mir et al ., 2001). Effective concentra tions vary widely, depending primarily on the commodity. Concentrations of between 1 and 12 L/L have been effective in blocking ethylene in broccoli. For green tomatoes, highe r concentrations for short durations have been effective. In mo st studies, treatment duration has ranged from 12 to 24 h, in order to achieve full respons e (reviewed by Blankenship and Dole, 2003).

PAGE 29

17 Multiple or single applications during a might be experimentally significant or not, depending on the commodity. Multiple applicat ions on Red Chief apples were more beneficial (Mir et al., 2001). Plant maturity and time of harv est must be also considered, whereas the more perishable the crop, the more quickly after harvest 1-MCP should be applied (reviewed by Blankenship and Dole, 2003). 2.5.3 1-MCP on Climacteric Fruits Various studies have been conducted on the effects of 1-MCP on climacteric fruits, including commodities such as apples, pears, stonefruits, bananas, melons, citrus, and mangos. Reports are variable, depending on th e commodity or even on the species. In general, as a response on ethylene inhibition, increases in respiration rates have been reduced or delayed. In avocado, a highly perishable commodity, 1-MCP treatment reduced significantly the rate of softeni ng by suppressing enzyme activities and helped retain green coloration at full ripeness stag e (Jeong et al., 2002). Soluble solids content (SSC) has been reported highe r in 1-MCPtreated pineappl es, papaya, and apples; while in mangos, oranges, apricots, and plums it was unaffected. Reports on the effect of 1MCP on titratable acidity, have been very mixed (reviewed by Blankenship and Dole, 2003). In experiments with apples, peaches, a nd nectarines an inhibition in ethylene production, softening, and titrata ble acidity was reported (Fan et al., 1999; Liguori et al., 2004). Jiang et al. (2001) found that 1-MCP a pplied preharvest to strawberries, a nonclimacteric commodity, lowered ethylene production and maintained fruit color, but it lowered anthocyanin production. In greenhouse tomatoes, 1-MCP delayed the onset of ripening-associated changes but it did not al ter significantly final values of lycopene, firmness, color, and PG activity (Mostofi et al., 2003).The effects of 1-MCP on fruit disorders and diseases has been varied, depe nding on the species. In some, cases, it has

PAGE 30

18 alleviated disorders, like reducing superficia l scald in apples (Fan et al., 1999) or decreasing internal flesh browning in apricots and pineapples (Dong et al., 2002, Blankenship and Dole, 2003). In other instan ces, a lower phenolic content in 1-MCP treated strawberries accounted for increased disease inci dence (Jiang at al., 2001). In papaya and custard apple, 1-MCP has been related to a higher incidence of external blemishes (reviewed by Blankenship and Dole 2003). Limited studies, however, exist on the impact of 1-MCP on phytochemical content of fruits and vegetables in general. 2.5.4 Guava and 1-MCP Literature on guava and 1-MCP is curre ntly very limited. Basseto and partners (2005) demonstrated the effectiv eness of application of 1-MCP to Pedro Sato guavas as well as a direct relation between concentration and exposure tim e. Fruit were subjected to different concentrations (100, 300, 900 nL/L) of 1-MCP and exposure times (3, 6, 12h) at 25 C, to improve the shelf-life of guavas ma rketed at room temperature. In general, treated fruit had a storage life twice as long as non-treated fruit (5 vs. 9 days respectively). Positive effects on skin color retention and respirati on rates were observed. Quality parameters such as SSC, ascorbic acid, and firmness were not influenced by 1MCP in all treatments. However, fruit treat ed with 900 nL/L for more than 6h did not ripen at all and treatments at 100 nL/L were ineffective. Treatments at 300 nL/L for 6 or 12 h and at 900 nL/L for 3 showed the best results, and were equally effective. 2.6 Polyphenolics 2.6.1 Polyphenolics Phenolic compounds are bioactive substan ces synthesized as secondary metabolites by all plants connected to diverse functions such as nutrient uptake, protein synthesis, enzyme activity, photosynthesis, and as st ructural components (reviewed by Robbins,

PAGE 31

19 2003). They are considered very important in foods not only because of their influence in sensory properties, but also for their potential health benefits related to their antioxidant activity (Fennema, 1996). Recent studies have shown that polyphenolics of fruits and vegetables improve lipid metabolism and preven t the oxidation of low-density lipoprotein cholesterol (LDL-C), which hinders the deve lopment of artherosclerosis (reviewed by Gorinstein et al., 1999). The term phenolic or polyphenol may be identified chemically as a substance which possesses an aromatic ring attached to one or more hydroxy substituents, and may include functional derivatives such as esters, methyl esters, glycosides or others (reviewed by Ho et al., 1991). Approxima tely 8,000 naturally occurring phenolic compounds have been identified. Phenolic plant compounds, including all aromatic molecules from phenolic acids to condensed ta nnins, are products of a plant aromatic pathway, which consists of three main s ections: the shikimic acid pathway which produces the aromatic amino acids phenylalan ine, tyrosine and tryptophan that are precursors of phenolic acids; the phenylpropa noid pathway which yields cinnamic acid derivatives that are precursors of fla vonoids and lignans; and the flavonoid pathway which produces various flavonoid compounds (reviewed by De Bruyne et al., 1999). Phenolic acids like caffeic, gallic, coumaric, chlorogenic and ferulic acids occur widely in the shikimic acid pathway of plant tissu es, which begins with the condensation of phosphoenolpyruvate and erythrose 4-phospha te (reviewed by Fennema, 1996). 2.6.2 Polyphenolic Classification Phenolics can be broadly classified in simple phenols and polyphenols, based on the number of phenol subunits present. Simple phenols, known as phenolic acids, may be classified according to their carbon frame works into two groups: 1) Hydroxylated

PAGE 32

20 derivatives of benzoic acid (C6-C1), which are very common in free state, as well as combined as esters or glycosides. This gr oup includes gallic acid, the main phenolic unit of hydrolysable tannins. 2) Hydroxylated acids derived from cinnamic acid (C6-C3), which occur mainly sterified and are very rare in free state. This group includes coumaric, caffeic, and ferulic acid (reviewed by Robbins, 2003; reviewed by Skerget, 2005). Both hydroxybenzoic and hydroxycinnamic acids are derived primarily from the phenylpropanoid pathway (Brecht et al., 2004). Polyphenols possessing at least two phenol-phenol subunits include the flavonoi ds, whereas compounds possessing three or more subunits are referred to as tannins (Robbins, 2003). Plant polyphenolics are commonly referred to as vegetable tannins (Fennema, 1996). Tannins are high molecular weight (Mr > 500) compounds containing many phenolic groups (Hagerman et al., 1998), and ar e classified according to their chemical structure into condensed and hydrolysable tannins (Fennema, 1996). Condensed tannins are oligomers or polymers composed of flav an-3-ol-nuclei, and have a lower molecular weight than hydrolysable tannins, which are polyesters of gallic and hexahydroxydiphenic acid (gallo tannins and ellagitannins, respectively). There is an additional class of polyphenols called complex tannins, in which a flavan-3-ol unit is connected to a galloor ella gitannin through a C-C linkage (reviewed by De Bruyne et al., 1999). Condensed tannins are commonly known as procyanidins or polyflavonols. Procyanidins are widespread in nature and more researched than hydrolysable tannins. They consist of chains of flavan-3-ol-units, which are commonly sterified, mainly with gallic acid units (ex: epigallocatechin gallate in tea). Specifically, the flavan-3-ols which

PAGE 33

21 are condensed tannin building blocks are (+)catechin (2,3-trans) and (-)-epicatechin (2,3cis). Flavan-3-ols are derived from a bran ch of the anthocyanin and other flavonoids pathway, of which elucidation is still unclear (reviewed by Xie and Dixon, 2005). Structural variability among proantho cyanidins depends on hydroxylation, stereochemistry at the three chiral centers, the location and type of interflavan linkage, and terminal unit structure. A classical assa y for proanthocyanidins consists of an acid hydrolysis, where the terminal units of the mo lecules convert to colored anthocyanidins. Condensed tannins can be classified into many subgroups, of which the procyanidins is the most common one (reviewed by De Bruyne et al., 1999). In guava, it has been found procyanidins to compose the major portion of guava polyphenolics (Mowlah and Itoo, 1982), however further identific ations have been limited. Figure 2-2. Chemical structures of a condensed tannin (A) an d hydrolysable tannin (B). A is a typical condensed tannin composed of catechin and epicatechin; B is a polygalloyl glucose composed of a glucos e core esterified with gallic acid residues Flavonoids are a diverse group of polyphe nolics which can polymerize to form condensed tannins. They are low-molecular we ight compounds, with the characteristic flavan nucleus and composed of three phe nolic (pyrane) rings. The major flavonoid classes include flavones, flav anones, flavonols, catechins (flavanols), anthocyanidins, A B

PAGE 34

22 isoflavones, and chalcones. Most flavonoids occur naturally as flavonoid glycosides. Quercetin, rutin, and robinin ar e the most common glycosides in the diet, which are then hydrolyzed by intestinal flora to produce th e biologically active aglycone (sugar-free flavonoid) (reviewed by Cook and Samman, 1996 ). In guava, considerable amounts of the flavonoids apigenin and myricetin ha ve been found (Miean and Mohamed, 2001). Figure 2-3. Chemical structures of flavonoi ds apigenin and myricetin, which have previously reported in guava. They are composed of three pyrane rings. Hydrolysable tannins are characterized mainly by containing a varied number of gallic acids, their major phe nolic unit (Grundhofer et al, 2001). Structural variation among them is caused by oxidative coupling of neighboring gallic acid units or by oxidation of aromatic rings. Some species of hydrolyzable ta nnins produce either gallotannins or ellagitannins, while others produce mixtur es of gallo-, ellagiand condensed tannins. Pentagalloylglucose has b een identified as the precursor for many complex tannin structures. Gallotannins consis t of a central polyol, such as glucose, surrounded by several gallic acids units. The el lagitannins are a more complex group of tannins also derived from pentagalloylglucose by oxidative reactions between gallic acid units (reviewed by Mueller-Harvey, 2001). Th e biosynthetic pathway to hydrolysable tannins may be divided in three smaller sect ions: The initial route encompases reactions that start from free gallic acid unit, which esterifies with a glucose and undergoes further

PAGE 35

23 esterification to form the end product pentag alloylglucose. Pentag alloylglucose is the starting point for the two subsequent routes. The gallotannin route is characterized by the addition of galloyl residues to pentagalloylglucose. The ellagitannin route are oxidation processes that yield C-C linkages between galloyl groups of pentagalloylglucose (reviewed by Grundhofer et al., 2001) 2.6.3 Polyphenolics as Antioxidants Polyphenols have shown potential health be nefits, chiefly rela ting to antioxidant capacity. Antioxidants prevent free radicals from harming host tissues and thus are thought to reduce the risk of certain de generative diseases such as cancer or cardiovascular disorders. Polyphenolics beha ve as antioxidants, mainly due to the reactivity of the hydroxyl substituents in the aromatic ring. There are several mechanisms, but the predominant role of antio xidant activity in polyphenols is believed to be radical scavenging via hydrogen at om donation or singlet oxygen quenching (reviewed by Robbins, 2003). In order for a poly phenol to be defined as an antioxidant it must satisfy two basic conditions: first, when present in low concentration relative to the substrate to be oxidized it can delay, retard, or prevent au toxidation or free radicalmediated oxidation; second, the resulting radi cal formed must be stable (reviewed by Shahidi and Wanasundara, 1992). Structurally, on monomeric phenolics, the ability to act as antioxidants depends on ex tended conjugation, number a nd arrangement of phenolics substituents, and molecular weight. Tannins which are highly polymerized with many phenolic hydroxyl groups, may be 15-30 tim es more effective in quenching peroxyl radicals than simple phenolics (Hagerman et al., 1998). Various in-vitro studies have demonstrated the helpful antioxidant effect s of polyphenolics in fruits and vegetables, however evidence in in-vivo studies remains unclear. Flavon oids have shown to inhibit

PAGE 36

24 LPO in-vitro by acting as scavengers as supero xide anions and hydroxyl radicals, however in-vivo, the evidence is quite unc lear (reviewed by Cook and Saman, 1996). The analytical procedures most widely used t oday to separate and id entify polyphenolics are HPLC, Gas Chromatography (GC), and HP LC-Mass Spectrometry (Robbins, 2003).

PAGE 37

25 CHAPTER 3 EFFECTS OF HOT WATER IMMERS ION TREATMENT ON GUAVA FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY 3.1 Introduction Guava ( Psidium guajava L .) the apple of the tropics, is an exotic fruit from tropical and subtropical regions Since its introduction in Florida in the late 1960s a market for fresh guava fruit and its products ha s been slowly growing in the US (personal communication, Sardinia, 2004). However, guava fresh fruit marketability is somewhat limited, mainly due to quarantine issues and the highly perishable na ture of the fruit. Unlike other tropicals, a quarantine schedul e for guava has not been approved by the USDA (USDA-APHIS, 2004), probably due to the prevalence of Caribbean fruit fly contamination (Gould and Sharp, 1992) and lack of studies demonstrating beneficial effects. Thermal quarantine treatments in particular are increasing in i ndustrial use due to consumer demand and governmental regula tions concerning the use of chemical treatments (Lurie, 1998). Currently, lim ited studies exist on thermal quarantine treatments for guava. Additionally, information on resultant changes of postharvest handling on guavas phytochemicals and antio xidant properties are very limited. The objective of this study was to evaluate the eff ects of a thermal quarantine treatment, using a simulated hot water immersion technique, on the phytochemical content, antioxidant capacity, and overall quality of guava fr uits at three stages of ripeness.

PAGE 38

26 3.2 Materials and Methods 3.2.1 Materials and Processing 3.2.1.1 Fruit preparation and HW treatment Mature (pink) guavas at va rious stages of ripeness from a single harvest were procured from C-Brand Tropi cals, Homestead, FL in Augus t 2003. Fruit were transported overnight via a courier service to the Food Science and Human Nutrition Department of the University of Florida. Upon arrival, fruit were washed and stored at 15 C for 18 hrs. Approximately 200 fruit were selected for ri peness uniformity and freedom from surface damage. Based on differences in skin color, firmness, and whether the fruit floated or sank in water (Reyes and Paull, 1995) three gr oups were segregated. Stage I fruit were mature, green fruit with a yellowish-green skin, firm texture, and floated in water Stage II fruit contained 25-50% yellow skin, semi-f irm texture, and sank in water. Stage III fruit were 75-100% yellow, soft texture, and sank in water. A hot water (HW) immersion treatment was applied according to the condi tions set by Gould and Sharp (1992). Within each ripening stage, fruit were randomly separated into four groups for each treatment time, placed into nylon bags and completely immersed in 46C water for 0 (control), 15, 30, and 60 min. Control guavas were immersed in water at 23 C, in order to have water immersion conditions. Due to the limited availa bility of Stage III fruits, only a control and 30 min immersion times were evaluated. After each respective immersion time, fruit were cooled by immersion in 23C water for 60 min. Stage I and II fruit were held in a 15 C storage room and held until fully ripe base d on color and texture as observed for the State III fruit. Stage III fruit, since they were already at a full ripe stage, were held at 15C for an additional two days to determine sh ort-term treatment effects. Therefore, this

PAGE 39

27 study focused on Stage I and II guavas where th e effects of HW treatment and changes during ripening could be evaluated. 3.2.1.2 Guava fruit processing Ripe guava fruit were collected from storage and immediately processed into a puree. After crown and peduncle were remove d, whole fruits were manually chopped into smaller cubes and processed into a puree us ing a kitchen-scale ju ice extractor (Braun, MP80) which removed excess skin and seeds. Guava composites or replicates were formed by joining 4 random fruits, making up 4 replicates for each treatment. Puree was packed in 0.1 mm thick sample bags and held frozen at -20 C until analysis. Samples were taken for moisture determin ation, soluble solids c ontent, pH, and for chemical extraction of polyphenolics, ascorbic acid, carotenoids, and antioxidant capacity (AOX). For all of the analyses except moisture, pH, and car otenoids a clarified guava juice was evaluated. To obtain this isolate, 10 g of guava puree was treated with 5 L of pectinase (Pectinex Ultra, SP-L, Novozymes), incubated at 32 C for 30 min, and centrifuged until a clear supernatant was obtaine d. The clarified juice was then filtered through cheesecloth, treated with sodium azide (0.01% w/v) to prevent microbial spoilage, and held frozen at -20 C until further analysis. 3.2.2 Chemical Analysis 3.2.2.1 Moisture content determination Moisture content was determined on th e guava puree by placing 3 g into a preweighed aluminum pan and drying to a consta nt weight in a conv ection oven (Precision Economy) at 135 C for 2 hrs (AOAC Method 920.149149(c)).

PAGE 40

28 3.2.2.2 Quantification of total soluble phenolics Total soluble phenolics were determined by the Folin-Ciocalteu assay (Swain and Hillis, 1959). Briefly, guava juice diluted 10-fold was pippeted into a test tube and 1 mL of 0.25 N Folin-Ciocalteu reagen t added. The mixture was allowed to react for and letting stand 3 minutes to allow for the reduction of phosphomolybdic-phosphotungstic acid by phenolic compounds, ascorbic acid, and other reducing agents in the juice. Subsequently, 1 mL of 1N sodium carbonate was added as an alkali to form a blue chromophore. After 7 minutes, 5 mL of dis tilled water was added and thoroughly mixed. Absorbance was read using a microplate r eader (Molecular Devices Spectra Max 190, Sunnyvale, CA) at 726 nm. Concentration of to tal soluble phenolics was quantified based on a linear regression against a standard of gall ic acid with data was expressed in gallic acid equivalents (GAE). 3.2.2.3 Analysis of ascorbic acid by HPLC Ascorbic acid was determined by revers e-phase HPLC using a Waters Alliance 2695 HPLC system equipped with a Waters 996 PDA detector (Waters Corp, Milford, MA), using a Supelcosil LC-18 column (Supelco, Belle fonte, PA) with detection at 254 nm. An isocratic running program was esta blished using a 0.2 M potassium phosphate (K2H2PO4) buffer solution at pH 2.4 (adjusted with phosphoric acid) as the mobile phase run at 1 mL/min. Ascorbic acid was iden tified by comparison to a standard (Sigma Chemical Co., St. Louis, MO) and based on UV spectroscopic properties. Samples for analysis were prepared by diluting the guava juice 10-fold with a 3% citric acid solution prior to HPLC analysis.

PAGE 41

29 3.2.2.4 Quantification of antioxidant capacity Antioxidant capacity was determined by the oxygen radical absorbance capacity (ORAC) assay as modified by Ou et al. (2001). The assay monitors the decay of fluorescein as the fluorescent probe in the pr esence of the peroxyl ra dical generator 2,2azobis (2-methylpropionamidine dihychlorid e) and is evaluated against Trolox, a synthetic, water-soluble vitamin E analog. A ssay conditions were de scribed by Talcott et al. (2003) for the use a Molecular Devices fmax 96-well fluorescent microplate reader (485 nm excitation and 538 nm emission). Fo r analysis, GJ samples were diluted 100fold in pH 7.0 phosphate buffer prior to pi petting into a microplate. Additionally, a Trolox standard curve (0, 6.25, 12.5, 25, 50 M Trolox) and a phosphate buffer blank were prepared. Fluorescence readings were taken every 2 min over a 70 min period at 37C. The rate of fluorescence decay over ti me was determined by calculating the area under the fluorescent decay curve and the antioxidant capacity quantified by linear regression based on the Trolox standard curve. Final ORAC values were expressed in M of Trolox equivalents per gram (M TE/g). 3.2.2.5 Analysis of lycopene by HPLC Lycopene was quantified by HPLC using a Dionex HPLC system equipped with a PDA 100 detector and separations made us ing a YMC Carotenoid column (250mm x 4.6 mm). An isocratic solvent delivery of 70% methyl-tert-butyl-ether and 30% methanol was run at 2 mL/min with detection at 470 nm A standard of lycope ne was isolated from from guava puree by extracting 5 g of guava puree with 20 mL of acetone and ethanol (1:1) and filtering through #4 Whatman paper. This isolate contai ned a mixture of nonlycopene carotenoids. The extraction process was repeated until the filtrate lost nearly all of its non-lycopene yellow color, whereby the addition of 100% acetone was added to

PAGE 42

30 extract lycopene. The lycopene was then part itioned into hexane that contained 100 mg/L BHT as an antioxidant Lycopene (MW 536.9) was quantified using an extinction coefficient of 3,450 at 470 nm in hexane. Purity was estimated at >98% as determined by the presence of extraneous carot enoid compounds by HPLC at 470 nm. Lycopene was extracted from guava puree using modified conditions of MartinezValverde et al. (2002) used for tomato lyc opene extraction. Approximately 0.5 g of puree was extracted with 5 mL of 100% acetone a nd vortexed vigorously. Quantitatively, 3 mL of hexane was added to the mixture, mixed, a nd water added to ensure adequate bi-layer separation. Aliquots of hexane extr acts were filtered through 0.45 m filter prior to HPLC injection. 3.2.2.6 Quantification of non-lycopene carotenoids Non-lycopene or yellow carotenoids were quantified in total using a spectrophotometer at 470 nm. Yellow carotenoids were extracted from 5g of guava puree with a known volume of acetone :ethanol (1:1) and subsequently filtered through Whatman #4 filter paper. Absorbance was recorded using a Beckman DU 60 spectrophotometer (Beckman, Fullerton, CA ) between 350 and 500 nm. Carotenoid concentration was calculated based on the extinction coefficient for -carotene. 3.2.2.7 Analysis of polyphenolics by HPLC Individual polyphenolic compounds were an alyzed by reverse-phase HPLC using a Waters Alliance 2695 HPLC system (Waters Co rp, Milford, MA) equipped with a Waters 996 PDA detector and a 5 m Waters Spherisorb ODS2 column (250 x 4.6 mm) using modified HPLC conditions described by Talcott et al. (2002). Mobile phases for gradient elution (Table 3-1) consiste d of 98:2 water and acetic acid (mobile phase A) and 68:30:2 water, acetonitrile, and acetic acid (mobile phase B) accordingly, at a flow rate of 0.8

PAGE 43

31 mL/min, and detected at 280 nm. Major pol yphenolic compounds we re characterized by spectroscopic interpretation, re tention time, and comparison to authentic standards (Sigma Chemical Co., St. Louis, MO). Follo wing filtration through at 0.45 M filter, the guava juice was injected into the HPLC without further modification. Table 3-1. Gradient elution running program for HPLC analysis of polyphenolics. Running time (min) % Mobile phase A % Mobile phase B 0.00 100-20.00 7030 30.00 5050 50.00 3070 70.00 --100 72.00 100-3.2.3 Quality Analysis The quality parameters soluble solids a nd pH were measured on the guava puree using a digital Leica Abbe Mark II re fractometer (Model 10480, Buffalo, NY) and Corning pH meter (Model 140, NY) respectively. Overall fruit quality were subjectively evaluated during storage to detect any effect s related to HW treatment or fruit decay. 3.2.4 Statistical Analysis For Stage I and Stage II fruits, the experime nt consisted of a 4 x 2 x 4 full-factorial design. The factors studied were HW im mersion time (0, 15, 30, and 60 min) and ripening stage (I and II), with a mean of 4 re plications represented in each data point. Stage III fruits were analyzed (2 x 1 x 4 design) and discusse d separately, since they were only subjected to two HW treatment times (0 and 30 min). Statisti cal analysis were conducted in JMP (SAS, Cary, NC) and consis ted of analysis of variance, Pearson Correlation, and mean separation by LSD test (P< 0.05).

PAGE 44

32 3.3 Results and Discussion 3.3.1 Chemical Analysis For chemical analyses performed, the effect of HW immersion time as compared to control within each ripening stage was evaluate d, as well as the effect of ripening stage within each HW immersion time (0, 15, 30, and 60 min), and the interaction of both factors. Results for chemical analysis, excepting polyphenolics by HPLC, were reported in dry weight basis (DW), in order to e liminate difference between samples due to varying water loss fruits e xperienced during ripening. 3.3.1.1 Moisture content Moisture content within each ripening st age was insignificantly affected by HW treatment time duration up until 60 min. Averag e moisture content for Stage I and Stage II fruit was 91.8 and 92.6% respectively (Fig ure 3-1). Total solids, determined by difference, was also insignifi cantly affected by HW treatment duration. Uniformity in total solids was an indicator of uniformity in the pool of guavas, since fruit were not affected by HW treatment duration. Due to di fferences in initial degree of ripeness, duration of HW treatment, and duration of stor age as the fruit ripened, some change in moisture content was expected. However, no significant differences in final moisture contents and subsequently total solids due to ripening stage were observed from 0 to 30 min. At 60 min in HW, Stage II fruit presente d significantly higher moisture content (1.3 %) than Stage I fruit, which is more attributed to fruit variation, since HW treatment at 60 min did not differ from control within both stag es. It is concluded that fruits at either ripening stage could be heated up to 60 min without affectin g their moisture content or total solids. To report values for fresh fru it and have more uniformity, moisture content was used to calculate chemical analys is results in dry weight basis (DW).

PAGE 45

33 Hot Water Immersion Time (min) at 46 C Moisture (%) 85 86 87 88 89 90 91 92 93 94 95 96 97 Stage I Stage II 0 15 30 60 Figure 3-1. Moisture content (%) of ripe guava s as affected by a hot water immersion treatment (0,15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n =4. 3.3.1.2 Total soluble phenolics HW treatments up to 30 min did not affect concentrations of to tal soluble phenolics (TSP) in relation to the untreated fruit within each ripening stage (Figure 3-2). At 60 min in HW, however, Stage I fruit presented lowe r TSP content than unt reated fruits, as opposed to Stage II fruit, which remained unaff ected. This lower content might be related to other metal-reducing compounds, which may c ontribute to the TSP value, due to the nature of the Folins assay. Within each HW immersion time, control fruit presented differences due to ripening stage; However within HW treatments (15 to 60 min) no significant differences in TSP were found due to ripening stage, c onfirming no significant effects attributed to ripening stage and its interaction with HW treatment. Differences found between stages in the unt reated control were probably due to fruit variability and contribution of other metal-reducing compounds, including ascorbic ac id. Stage I fruit,

PAGE 46

34 therefore, can be subjected to up until 30 mi n in HW without affec ting its TSP content, while Stage II fruit can be held up until 60 min. Hot Water Immersion Time (min) at 46 C Total Soluble Phenolics (mg/kg dwb) 5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 Stage I Stage II 0 15 30 60 Figure 3-2. Total soluble phenolics (mg/kg DW ) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represen t standard error of the mean, n = 4. Studies have demonstrated guava TSP c ontent decreases duri ng ripening (Bulk et al., 1997; Bashir and Goukh, 2002). Pink guavas in particular, have a less-marked reduction in TSP from mature green stage (Sta ge I) until full ripeness as compared to white guavas (Bashir and Goukh, 2002). For the present study, guavas from both ripening stages, treated and not treated, were able to ripen and attain TSP levels comparable to other reports in ripe guavas. Average TSP valu es for the different ri pening stages (I-III) ranged from 19,588 to 21,502 mg/kg GAE, DW (1,360 to 1,760 mg/kg GAE, FW). Bashir and Abu-Goukh (2002) reported simila r values of TSP (1200-1800 mg/kg FW) for white pulp guavas, but a lower content in pi nk pulp guavas. Kondo et al. (2005) reported

PAGE 47

35 a TSP content of 1852 mol/kg FW. Variety, origin, and even harvest season play an important make an impact in differences in TSP. 3.3.1.3 Ascorbic acid Ascorbic acid is an effective nutrient stability index during food processing and storage operations. It has been generally observed that if AA is well retained, the other nutrients are also well retained (Fenema, 1996). Ascorbic acid was insignificantly affected by HW immersion time within Stage I fruits (Figure 3-3). In a HW treatment at 38 C for 30 min applied to guavas and subseque nt exposure to chill injury conditions (5 C), these processes did not affect its ascorbic acid c ontent, although chilling injury symptoms were present. (Regalado-Contreras and Mercado-Silva, 1998). Stage II fruit at 30 min however, presented lower values as comp ared to control, which does not seem to be a treatment effect, but rather fruit varia tion effect, since fruit treated at 60 min was not different from untreated fru it. Fruit variation may be obs erved by the large error rates presented in Figure 3-3 (statist ical analysis, however, for all parameters was done using a pooled standard error). Even within the same variety, large variabili ty in ascorbic acid contents has been reported (Mitra, 1997). There were no differences in AA due to ripening stage at each treatment time. The fact that guava was unaffected by increasing HW times may not only indicat e its stability during a postharvest treatment, but the stability of other guava phytochemicals. A dditionally, since ascorbic was unaffected by most HW treatments, it may also indicate a uniformity of the pool of guavas used, most of them achieving similar contents. The average AA content for Stage I a nd Stage II fruit was 10,846 mg/kg DW (843.5 mg/kg FW) (Figure 3-3), comparable to other studies on ripe guavas. Bashir and Abu-Goukh (2002) reported an ascorbic acid content of 800 mg/kg and 670 mg/kg FW

PAGE 48

36 for white and pink guavas respectively, while Leong and Shui (2002) reported 1,310 mg/kg FW and Bulk et al. (1997) repor ted values between 882 and 1113 mg/kg FW. Hot Water Immersion Time (min) at 46 C Ascorbic Acid (mg/kg dwb) 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 16000 Stage I Stage II 0 15 30 60 Figure 3-3. Ascorbic acid content (mg/kg DW ) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represen t standard error of the mean, n = 4. Fruit variety is one of the main factors a ffecting AA content in guava, influencing more than ripening stage or stor age conditions (Mitra, 1997). 3.3.1.4 Antioxidant capacity Antioxidant capacity in fresh guava wa s insignificantly affected by increasing times of HW treatment within Stage I and St age II fruits (Figure 3-4), which presented values of 146 and 126 M TE/g DW respectively. Additiona lly, ripening stage at the time of treatment application did not affect antioxidant capacity within each HW treatment. In a study conducted in Mexico, guavas were s ubjected to chill injury conditions (5 C), where no changes in antioxidant capacity (ferric reducing antioxidant power; FRAP) and TSP were reported as compared to control, ev en when the fruit presented external chilling

PAGE 49

37 injury symptoms (Edmundo et al., 2002). The biosynthesis of certain antioxidant compounds in guava might not be affected by certain temperature changes, even though they generate stress and injuries on the commodities. However, this is extremely dependent on the degree of stress, which la rgely depends on the temperature differential, atmospheric conditions, and exposure durat ion (Lurie, 1998; Paull and Chen, 2000). Hot Water Immersion Time (min) at 46 C Antioxidant Capacity (umol Trolox Eq./g dwb) 0 20 40 60 80 100 120 140 160 180 200 Stage I Stage II 0 15 30 60 Figure 3-4. Antioxidant capacity ( mol Trolox Equivalents/g DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n = 4. The major contributors to water-soluble an tioxidant activity in guava are ascorbic acid (AA) and polyphenolics. Ascorbic acid re portedly contributes to approximately 50% guavas antioxidant activity (Leong and Shui 2002). The correlation between antioxidant capacity and ascorbic acid was low (r = 0.30, P< 0.05), probably due to the high degree of variability in ascorbic acid values. There was also a low correlation with TSP (r = 0.27, P< 0.05), since TSP content presented sign ificant differences with increasing HW treatment time. Due to the use of GJ, however overall antioxidant capacity in guava was

PAGE 50

38 probably underestimated, since lycopene, car otenoids, and other lypophilic compounds were not taken into consideration. 3.3.1.5 Lycopene and yellow carotenoids Average lycopene values for Stage I a nd Stage II guavas were 558 and 472 mg/kg DW (45.1 and 34.8 mg/kg FW) respectively (Fig ure 3-5). Studies conducted on Brazilian guavas reported similar values, ranging from 47 to 53 mg/kg FW (Padula and RodriguezAmaya, 1986; Wilberg and Rodriguez-Amaya, 1995). Lycopene content in ripe tomatoes, which ranges from 30 to 80 mg/kg FW (Abus hita et al., 2000; Thompson et al.,2000; Leonardi et al. 2000; Martinez-Va lverde et al., 2002; Seybold et al., 2004), is comparable to ripe guavas. Subjective determination of guava ripeness, based on skin color observations and texture, may have created some uncertainty about the uniformity of ripening stage. These effects were evident wh en guava fruit were cut and variation in pulp color intensity was observe d within the same ripeness stage. However, fruits were grouped in composites, joining fruits with sim ilar pulp colorations, in order to have more uniformity and reduce varia tion within treatments. Guava lycopene was unaffected by HW treatment up until 30 min within both ripening stages (Figure 3-5). At 60 min, Stage I fruits exhibite d lower lycopene content as compared to untreated fruits, whereas Stage II fruit remained unaffected. This reduction might be related to an inhibiti on in lycopene biosynthesis due to a longer exposure to heat stress. Studies of HW treatments on tomatoes ha ve reported this inhibition mainly due to an inhibition of the transcription gene for lycopene synthesis, which recovers after the removal of heat (Cheng at al., 1988; Lurie et al., 1996; Paull and Chen, 2000). Following the removal of the heat stress for 60 min, Stage I fruit recovered and synthesized lycopene, but not to the extent of the other treatments. However, this difference between

PAGE 51

39 untreated fruit and 60 min, might also be due to the significantly higher content of Stage I fruit at both 0 and 15 min, as compared to St age II. These differences might be attributed to pre-treatment storage conditions, were fru its of the different ri pening stages ripened different. Within the HW treatments, there we re no differences due to ripening stage from 15 to 60 min; however, Stage I fruit presen ted a higher content in untreated fruit. Hot Water Immersion Time (min) at 46 C Lycopene (mg/kg dwb) 100 200 300 400 500 600 700 800 Stage I Stage II 0 15 30 60 Figure 3-5. Lycopene content (mg/kg DW) in gua va as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n = 4. Non-lycopene, or yellow, carotenoids are re ferred to guava carotenoids other than lycopene. Yellow carotenoids were unaffect ed by increasing HW treatment time and by ripening stage within each HW treatment, incl uded untreated fruits (Figure 3-6). A hot water treatment applied to papaya (Carica papaya L.) did not cause any detrimental effects on the content of -carotene and lycopene (Perez and Yahia, 2004). Lycopene and other carotenoids stability in tomatoes has associated to the influence of the tomato matrix itself and its adhesion to membranes (Seybold et al., 2004). A whole guava at its early stages of ripening cont ains appreciable amounts of cellulose, hemicellulose, lignin

PAGE 52

40 (stone cells) and insoluble pectin in its cel l walls, which creates a strong matrix that may protect well guava carotenoids and other phyt ochemicals. Yellow carotenoids content of these carotenoids ranged from 42.9 to 49.9 mg/kg DW (3.27 to 4.11 mg/kg FW), which are comparable to -carotene contents of 3.7 and 5.5 mg/kg FW found in Brazilian guavas (Padula and Rodriguez-Amaya, 1986; Wilberg and Rodriguez-Amaya, 1995). Stability of carotenoids, es pecially lycopene, in toma toes during food processing operations has been widely discussed by vari ous authors, and sometimes inconsistent results are found, where lycopene content might be enhanced or reduced (Abushita et al., 2000; Leonardi et al. 2001, Seybold et al., 2004 ; Sahlin et al., 2004). A HW immersion treatment, at a lower temperature than c ooking or other processing operations, and for a shorter period of time, might be milder, in order to cause significant effects in the biosynthetic pathways of lycopene and other carotenoids. Hot Water Immersion Time ( min ) at 46 C Non-lycopene Carotenoids (mg/kg dwb) 10 20 30 40 50 60 Stage I Stage II 0 15 30 60 Figure 3-6. Non-lycopene carotenoids (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II). Error bars represent st andard error of the mean, n = 4.

PAGE 53

41 Heat treatments and other postharvest pr ocesses can delay, inhibit or accelerate ripening, as part a commoditys response to abiotic (environmental ) stress (Lurie, 1998; Paull and Chen, 2000; Jacobi et al., 2001; Basseto et al., 2004). This may also be closely associated with respiration rate, ethylene production, fruit softeni ng, enzyme activities (cell-wall degrading, ethylene-related), car otenoid development, and other components related to ripening (Paull and Chen, 2000; J acobi et al., 2001). These processes can actually be used as tools to enhance their marketability and added value of produce, for example, uniformity of skin color devel opment in mangoes (Jacobi et al., 2001) or anthocyanin accumulation in strawberries by HW treatments (Civello et al., 1997). In other cases, they can bring detrimental effect s, affecting flavor, aesthetic qualities, or inhibiting the synthesis of certain anti oxidant compounds (Cisneros-Cevallos, 2003; Sahit, 2004). Stress responses were observed in Stage I fruit heated for 60 min, where there was an enhancement in total soluble phenolics and a decrease in lycopene. Especially for lycopene, its inhibition might be detrimental to the fruits phytochemical content. However, results indicated that a HW treatment up until 30 min at 46 C insignificant major phytochemicals in guava. 3.3.1.6 Polyphenolics by HPLC Studies identifying ripe gua va polyphenolics by HPLC ar e limited. Earlier studies have been done with less precise analytical procedures in which the major classes of compounds present have been identified (Misra and Seshad ri, 1967; Mowlah and Itoo, 1982; Itoo et al., 1987). Immature, still deve loping guava are composed mainly of condensed tannins, which decrease markedly during its development and ripening, along with the rest of guava pol yphenolics (Misra and Seshadri 1967; Itoo et al., 1987). Kondo

PAGE 54

42 et al. (2005) identified galli c acid, catechin, epicatechin, and chlorogenic acid in guava skin, as well as catechin at lower concentrations in its pulp. In ripe fr uits, the presence of procyanidins, or condensed tann ins, and free ellagic acid ha s been confirmed (Misra and Seshadri, 1967). Various solvent extraction and fractionation procedures on puree and juice were attempted for HPLC analysis of guava polyphenolics with the enzyme-clarified guava juice producing the most reproducible HPLC chromatograms with maximal peak separation (Figure 3-7). In th is study, HPLC analysis of polyphenolics was used to idenfity overall data trends as affected by ripeness stage and HW treatment. Among the multitude of polyphenolic compounds present, 14 were selected based on adequate separation and abundance for treatmen t differentiation (Table 3-2). Figure 3-7. HPLC chromatogram of polyphenol ic compounds found in guava juice-A) gallic acid, B) gallic acid deriva tives, C) unknown-characteristic guava polyphenolics, D) procyanidi ns, and E) ellagic acid de rivative. Identification (280 nm) was done by comparison to authentic standards and spectral properties. AU 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 Minutes 10.00 20.00 30.00 40.00 50.00 60.00 70.00 A B B C C D D C C C C E C C 280 nm

PAGE 55

43 Peaks were tentatively iden tified and/or grouped into a common class of polyphenolics based on their spectroscopic pr operties and comparison to authentic standards (Table 32). Gallic acid (Peak 1) and an ellagic acid de rivative (Peak 12) were clearly identified by comparison to standards. Gallic acid deriva tives (Peaks 2 and 3) were tentatively identified, as they shared similar spectral properties with gallic acid. Procyanidin compounds (Peaks 6 and 7), or condensed tann ins, were identified based on their spectroscopic similarities to (+)-catechin and (-)-epicatachin, which are the bulding blocks of condensed tannins. The remaining compounds were characterized based on retention time and spectro scopic properties but were dissimilar from any known polyphenolic compounds. Further work will be needed to isolate and identify these individual polyphenolic com pounds in ripe guavas. Since gallic acid was the most prevalent compound in guava, and is found in abundance in many fruits, all compounds were quantified in gallic acid equivalents (GAE). Table 3-2. Tentative identification of gua va polyphenolics at 280 nm by HPLC based on retention time, spectral pr operties, and comparison to authentic standards. Peak No. Retention Time (min) Spectral Properties Tentative Identification 1 8.12 229.3, 271.6 Gallic Acid 2 8.90 229.3, 276.3 Gallic Acid Derivative 3 9.71 233.9, 290.5 / 233.9, 276.3 Gallic Acid Derivative 4 15.8 224.5, 262.2 Unknown 5 18.5 233.9, 266.9 Unknown 6 26.3 233.9, 281.1 Procyanidin 7 28.4 233.9, 281.1 Procyanidin 8 31.1 281.1 / 219.9, 281.1 Unknown 9 35.5 215.2, 266.9 Unknown 10 45.7 224.6, 281.1 Unknown 11 47.0 219.9, 262.2 Unknown 12 59.5 224.6, 252.8, 369.9 Ellagic Acid Derivative 13 64.6 262.2 Unknown 14 68.2 224.6, 281.1 Unknown

PAGE 56

44 Gallic acid (Peak 1) Gallic acid (GA) (Peak 1) has been reported as an effective antioxidant due to its structure and positioning of hydroxyl groups Within each ripening stage, GA was insignificantly affected by HW treatment up until 30 min (Table 3-3); however in Stage I fruit GA content was significantly higher than control at 60 min (52% increase). Stage II, however, remained unaffected. It has been disc ussed that the chronological age of a tissue is an important factor in response to HW treatment (reviewed by Paull and Chen, 2000). The presence of the heat stress for a longe r period might have re sulted in increased biosynthesis of gallic acid through the phenylpropanoid pathway, as a response to stress. Additionally, Stage I fruit probably had highe r polyphenolic content than other ripening stages, especially in its peel, most prone to be affected by abioti c stress. As shown in Table 3-3, within all HW treatment times, in cluding control, an effect of ripening stage was found, in which Stage I fruit exhibited a significantly higher GA content. The fact that Stage I fruit content was higher in all HW times, including control, indicates that it might not be attributed to a HW treatment effect, but rather pre-treatment storage conditions and post-treatment storage duration. Gallic acid derivatives (Peaks 2 and 3) GA derivatives, Peaks 2 and 3, eluted in the HPLC column immediately after GA. Due to their closeness to GA in spectral pr operties, these compounds were tentatively classified as GA derivatives (Table 3-3), and their cont ent was summed. There was no effect due to HW treatment (15 to 60 min) as compared to control within Stage II (Table 3-3). In Stage I, GA derivatives content wa s lower than control at 15 min, which

PAGE 57

45Table 3-3. Guava gallic acid (GA), gallic acid derivatives, and an ella gic acid derivative as affe cted by a hot water quarantin e treatment and ripening stage. Pk 1: Gallic Acid (GA) (mg/kg GAE2) Pks 2 and 3 : GA Derivatives (mg/kg GAE) Pk 12: Ellagic Derivative (mg/kg GAE) HW treatment (min) Stage I Stage II Stage I Stage II Stage I Stage II 0 16.2 b1 7.70 c 12.8 a 10.5 bc 1.04 c 1.71 ab 15 16.1 b 9.16 c 9.20 c 12.0 abc 1.43 bc 1.55 b 30 15.8 b 5.64 c 11.9 abc 10.3 bc 1.35 bc 2.10 a 60 24.7 a 5.71 c 13.8 a 11.8 abc 1.61 b 1.50 b 1Values with different letters w ithin columns of the same ripeness level and with in rows for each hot wa ter treatment are signif icantly different and indicate the effect of either hot water treatment or ripening stage, and their interaction (LSD test, P<0.05). 2GAE = Gallic Acid Equivalents Table 3-4. Guava procyanidins, other characteristic unknown com pounds, and total polyphenolics by HPLC as affected by a hot wat er quarantine treatment and ripening stage. Pks 6 and 7: Procyanidins (mg/kg GAE2) Unknown Guava Compounds (mg/kg GAE) Total Polyphenolics by HPLC (mg/kg GAE) HW treatment (min) Stage I Stage II Stage I Stage II Stage I Stage II 0 6.33 a1 3.93 bc 36.3 a 24.7 d 72.7 a 48.5 c 15 4.99 abc 3.60 c 36.5 a 27.0 bcd 67.7 ab 53.3 c 30 5.42 ab 4.60 bc 28.6 bc 25.2 cd 62.2 b 47.8 c 60 4.20 bc 3.53 c 29.6 b 27.7 bcd 73.9 a 50.2 c 1Values with different letters w ithin columns of the same ripeness level and with in rows for each hot wa ter treatment are signif icantly different and indicate the effect of either hot water treatment or ripening stage, and their interaction (LSD test, P<0.05). 2GAE = Gallic Acid Equivalents.

PAGE 58

probably was due to variation, since at 30 and 60 min there was no HW treatment effect.Within all HW treatments and contro l, no ripening stage effect was observed. Procyanidin compounds (Peaks 6 and 7) Peaks 6 and 7 were identified as belonging to procya nidin compounds. The presence of procyanidin compounds has been confirmed in both white and pink guavas (Mowlah and Itto, 1982), and they have been reported to be com posed mainly of (+) catechin and (+) gallocatechin (Itoo et al., 1987). Procyanidins presented no HW treatment effect in Stage II fruit; however Stage I fruit presented lower content at 60 min as compared to control (Table 3-4). This difference between control and 60 min might not at tributed to a treatment effect, rather to variation within control fruit, since an incr ease in heat stress w ould probably increase the content of procyanidin compounds rather to decrease it. A dditionally, within untreated controls, Stage I fruit presented higher procya nidin content than Stage II fruit, but no differences between ripening stages were f ound at 15, 30, and 60 min. This confirms that Stage I control fruit procyanidin content wa s significantly higher probably due to a variation in values of this compound in control fruit. It can be concluded that procyanidin content in HW treated fruit up to 30 min did not differ significantly from untreated fruits. Ellagic acid derivative (Peak 12) Peak 12 was identified as an ellagic acid de rivative, most likely a glycoside (Lee et al., 2005), due to its closeness in spectral prope rties to ellagic acid. Free ellagic acid in ripe guava was isolated and identified by Mi sra and Seshadri (1967). Similarly to other compounds, Stage II fruits ellagic acid c ontent was unaffected by increasing HW treatment up until 60 min (Table 3-3). Stage I fruit ellagic acid content at 60 min HW

PAGE 59

47 treatment, however, was signifi cantly higher than control. This trend in Stage I is comparable to gallic acid results; it must be noted though that concentrations of this compound are much lower. Very low concentrations might not give an accurate measure of a treatment effect. Within HW treatment time, Stage I fruit was significantly lower than Stage II fruit at time 0 and 30, but at time 15 and 60 min, there were no significant differences due to ripening stage. Guava characteristic unknown co mpounds (Peaks, 4, 5, 8-11, 13, 14) This group is composed of guava compounds that had characteristic and sometimes repeating spectroscopic propertie s (Table 3-4), but were not identifiable as any known polyphenolic compounds. Many of them shared si milar spectrocospic properties to gallic acid and its derivatives or pr ocyanidin compounds, which might relate them closer in further research. They were characterized by their consistency in sp ectroscopic properties among most samples, in contrast with other compounds, whose spectral properties differed mainly due to small peak areas or interference of extr aneous compounds during elution. Stage II fruit unknown compounds content presented no effect due to HW treatment. Stage I fruit content, however, decr eased with an increase in HW treatment at 30 and 60 min. This HW treatment effect wa s particular for two compounds represented by Peaks 13 and 14, which constituted 41% of the overall unknown compounds content. Peaks 13 and 14, along with gallic acid, contri buted with the larges t peak areas of all guava polyphenolics. The decrease in these co mpounds in Stage I fruits may be due to a loss of polyphenolic compounds in response to stress. Within HW treatment, significant differences were observed due to ripening stages at control and at 15 min, but not at 30

PAGE 60

48 and 60 min, which does not relate it to a HW treatment related response but rather to variation in some of the compounds. Total guava polyphenolics by HPLC (Peaks 1 to 14) Briefly, the overall trend of all 14 guava polyphenolic compounds will be discussed. Stage II fruit presented no signifi cant differences in tota l polyphenolics due to HW treatments as compared to control (Table 3-4). Stage I fruit content at 15 and 60 min was no different from control, which accounts that insignifican tly of a lower content at 30 min, in general, Stage I fruit presented no HW treatment effect. Within untreated fruit and HW treated fruit (15 to 60 min), significan t differences due to ripening stage were observed. Many postharvest stresses, in cluding heat treatments, have shown to affect the levels of polyphenolics in plant commodities, either by inducing or inhibiting their biosynthesis. When fruits or vegetables undergo stress, cinnamic acid and benzoic acid derivatives are among the first polyphenolic s to be synthesized (Sahit, 2004). The phenylpropanoid pathway, regulated by PAL, is responsible for the synthesis of hydroxycinnamic and hydroxybenzoic acids. An in crease in PAL activity due to stress may result in the accumulation of many polyphenolic compounds (Cisneros-Zevallos, 2003). In the present study, gallic acid, a hydroxycinamic acid, was significantly enhanced in Stage I fruit after 60 min, which may be closely related to a response due to a longer exposure to heat stress. Howe ver, most polyphenolics were unaffected by increasing HW treatment times, especially in Stage II fruits.

PAGE 61

49 3.3.2. Quality Analysis 3.3.2.1 pH and soluble solids Quality parameters for fresh guava fruit and its industrial applications include fruit diameter and weight, percentage of seeds, puree color, skin color, ac idity, flavor, soluble solids, pH, and ascorbic acid (Boyle et al., 1957). Soluble so lids and pH were unaffected by HW treatment duration (15 to 60 min) within both ripening stages (Table 3-6). Heat treatment, water or hot air (38 to 48 c for 1 h to 3 days), had no effect on tomato soluble solids or acidity (Lurie and Klein, 1991; McD onald et al., 1997). In the case of mango, soluble solids are not affected by an insect vapor heat treatment (J acobi and Giles, 1997, Jacobi et al., 2001). Differences in pH were observed due to ripening stage at 30 and 60 min, while for soluble solids Stage I fru it present higher contents at 60 min. Table 3-5. Quality parameters, soluble solids a nd pH, in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and Stage II). Data are e xpressed as fresh weight basis (mean standard error), n = 4. Soluble Solids (Brix) pH HW treatment (min) Stage I Stage II Stage I Stage II 0 7.77 0.49 ab 7.37 0.22 b 4.05 0.09 ab 4.05 0.03ab 15 8.39 0.33 a 7.68 0.18 ab 4.03 0.05 b 4.15 0.03ab 30 8.12 0.33 ab 7.25 0.19 b 4.03 0.02 b 4.18 0.05a 60 8.51 0.47 a 7.29 0.26 b 4.03 0.02 b 4.18 0.05 a 3.3.2.2 Overall fruit quality Observations performed during the storag e period following the HW treatments reported no differences in aesthetic quality of HW-treated guavas as compared to nontreated fruit. HW treated guava s of all ripening stages did no t presented visible signs of heat injury. Some damages as a consequence of heat stress on HW treated fruit include irreversible changes such as skin scalding, sk in browning and failure to soften (Paull and

PAGE 62

50 Chen, 1990; Jacobi and Giles, 1997), among ot hers. Gould and Sharp (1992) reported no signs of damage to guavas after HW treatment for 35 min at 46 C. Guavas in the present study were comparable in weight and shap e to Gould and Sharp (1992) study, which are an important factor which affects the unifo rmity of heating (Paul and Chen, 2000) and any subsequent damages present. Additiona lly, Gould and Sharp (2002) reported storage temperature after treatment was more important in maintaining fruit quality than the HW treatment itself. Although stor age temperatures were not as sessed in the present study, duration of storage at 15 C might have played an important role in differences observed between Stage I and II fruits in chemical properties, although aesth etic quality was not affected. All guavas attained full ripeness a nd showed no differences among themselves in quality attributes like firmness and skin coloration independently differences in initial fruit ripeness and storage temperatures. 3.3.3 Stage III Fruit Stage III fruit were analyzed independently, being the main interest to determine if the hot water treatment affected phytochemical pr operties of ripe guava fruit or not. Fruit were treated for 30 min at 46 and along with a control gr oup, they were allowed to reach full ripeness for approximately two days at 15 C. Moisture content, total soluble phenolics, antioxidant capacity, ascorbic ac id, lycopene, and yellow carotenoids were unaffected by HW treatment in Stage III guavas. Quality parameters such as pH and soluble solids were also unaffected. Probabl y, the fruit had alrea dy developed all its quality and phytochemical proper ties by the time of treatment application. Comparable to Stage I and II, the HW treatment caused no ch ange in guava quality and phytochemistry.

PAGE 63

51 Table 3-6. Phytochemical content and quality pa rameters in Stage III guavas as affected by a hot water quarantine treatment at 46 C (mean standard error). n = 4. Time (min) Moisture (%) Soluble Phenolics (mg/kg dwb) AOX Capacity ( mol TE/g) Ascorbic Acid (mg/kg dwb) 0 93.21 0.25 a 20070 1200 a 135.5 19 a 13120 1900 a 30 92.85 0.23 a 19110 740 a 110.7 6.4 a 10170 1000 a Lycopene (mg/kg) Yellow carotenoids (mg/kg dwb) Soluble Solids ( Brix) pH 0 458.5 81 a 45.98 6 a 6.79 0.25 a 4.225 0 a 30 437.3 76 a 41.98 3 a 7.15 0.23 a 4.100 0 b 3.4 Conclusions A quarantine hot water treatment at 46 C for up to 30 min can be applied to guavas (Stage I-III) without affecting its phytochemi cals (total soluble phenol ics, ascorbic acid, lycopene, yellow carotenoids, polyphenolic co mpounds), antioxidant capacity and quality (pH, brix, overall fruit appearance). Unifor mity of the pool of guavas was confirmed by moisture content, ascorbic acid, yellow carot enoids, and quality parameters. After 60 min in hot water, Stage I fruit presented an increa se in gallic acid, which might be attributed to an increase in biosynthesis of polypheno lic compounds as a response to heat stress. Additionally, a decrease in lycopene conten t was observed, which is also related to reversible inhibition in its biosynthesis when stress was a pplied. The chronological age of the tissue plays an important role, especially when the stress is applied for a longer period of time. Many of the differences observed in most parameters were due to ripening stage and storage conditions rather th an a hot water treatment effect Although it is not feasible to treat ripe guavas, Stage III fruit phytoc hemicals and quality parameters were not affected by the HW quarantine treatment for 30 min. Stage I fruit guavas treated for 30

PAGE 64

52 min at 46 C are preferred for HW treatment, since they could have a longer shelf life and allow more marketability to guavas.

PAGE 65

53 CHAPTER 4 EFFECTS OF 1-METHYLCYCLOPROPENE ON GUAVA FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY 4.1 Introduction Guava ( Psidium guajava L.) and more specifically guava juice and puree have increased in popularity within US markets (NAS S, 2004) due to its exotic tropical flavor and overall consumer appeal. Fresh guava is hi ghly perishable with a retail shelf life of approximately 7-10 days, creating recurrent pressure for packers and distributors to deliver a consistent product with widespread consumer acceptability. Due to the delicacy of its skin and rapid loss in firmness, special care is taken in most postharvest handling operations such as individually paper-wr apped fruit placed into specially designed packages (personal communication, Sardinia, 2005). Numerous technologies have been developed in the past years to extend shelf lif e of fruits and vegeta bles, and at the same time preserve their table quality, allowing thei r marketability to distant markets. These include storage under controlled atmosphere s (CA), in polybags or with modified atmosphere packaging (MAP), and coating wi th polymeric films have shown to prolong the shelf-life of many commodities (Mitra, 1997). In the case of Florida guavas, many of these technologies have been evaluated by packers without ap preciable success (Sardinia, 2005). A recent technology emerged from the fi eld of ethylene inhibitors is 1methylcyclopropene, or 1-MCP, a gaseous co mpound that when applied to a commodity binds to ethylene receptors causing an inhi bition in ethylene action (Sisler and Serek,

PAGE 66

54 1997). Due to ethylenes close relation to va rious ripening processe s, many beneficial effects have been attributed to 1-MCP in the extension of shelflife (Blankenship and Dole, 2003). Its practicality of use, low cost, and beneficial effects are an attractive way of increasing fresh fruit marketing competitiven ess. However, despite its vast potential for the fresh fruit industry, little is know n of 1-MCPs effect on phytochemicals and antioxidant properties, especially with guava The objective of the present study was not only to evaluate quality parameters of guava as affected by a 1-MCP treatment, but also its effects on polyphenolics and antioxidant properties. 4.2 Materials and Methods 4.2.1 Materials and Processing 4.2.1.1 Fruit preparation and 1-MCP treatment Mature, green pink fleshed guavas (a hybrid variety) procured from Sardinia Farms (Homestead, Florida) were harvested on July 7, 2004. They were transported to the Food Science and Human Nutrition Department of the University of Florida, washed, and stored at 15 C until treatmen t application. Fruit were uniform in size, shape, firmness, and skin color (green) and free from any surf ace damage. Fruit were then transported to the Horticultural Sciences Department, Univer sity of Florida, a nd approximately 320 fruit were selected and randomly separated into two groups: Control and 1-MCP treated. Both groups were arranged separa tely into two impermeable 174 L capacity chambers in a storage room held at 10 C. Calculations by regression (Huber, 2004) were performed to measure the amount of 1-methylcyclopropene (1-MCP, Smartfresh, Agrofresh, Inc.) powder, based on total fruit weight, to yiel d a final concentration of 1,000 nL/L gaseous 1-MCP inside the chamber. The powder was di ssolved in 40 mL of deionized water in a 125 mL flask, which was sealed, and vorte xed. The flask was placed in the 1-MCP

PAGE 67

55 chamber, unsealed, and the chamber door immediately sealed. The same conditions applied to the chamber contai ning the control group using a flask of water without the 1MCP. The 1-MCP treatment (1000 nL/L) was maintained for 24 hours, with a second application after the fi rst 12 hours. At the conclusion of the treatment, fruit were again transported to the Food Science and Human Nu trition Department and held at a storage room at 15 C until complete ripeness. Ripe fruit were removed from storage for physicochemical analysis when the outer skin became thin, completely yellow and the fruit presented a soft texture, characteristic s for a ready-to-eat, ripe fruit. Day 0 was established as the 24 hour 1MCP application period, while Da y 1 was established as first day of storage at 15 C, less than an hour after the fruits were removed from 1-MCP treatment. Subsequent days were 24 hours apart from the preceding day. For quality analysis over time, a group of five guavas were randomly obtained from each group every 3 to 4 days for evaluation of firm ness followed by measurements for pH, total soluble solids, and titratable acidity. Additionally, a secondary experiment wa s simultaneously conducted to evaluate the effects of 1-MCP applicati on on boxed guavas, in effort to assess applicability of the 1-MCP treatment on fruit ready for shipping. Approximately 180 fruit were selected and also separated into Control and 1-MCP treate d. Fruits for each treatment were arranged inside four small cardboard boxes (22 to 23 guavas per box), which were stacked inside their respective chambers and treated as previo usly described and allo wed to ripen at 15 C. During storage, three guava s were obtained every 3 to 4 days for quality analysis during ripening.

PAGE 68

56 4.2.1.2 Guava fruit processing Procedures for guava fruit processing were followed as outlined in Chapter 3 with composites of 5 guavas evaluated within each treatment. Fruits were processed when they achieved full ripeness, according to parameters described in Chapter 3. Collection of ripe fruits was done periodically, until the last fruits achieved full ripeness. 4.2.2 Quality Analysis 4.2.2.1 Aesthetic fruit quality assessment during storage Following treatment with 1-MCP (Day 0), fr uit were assessed daily every day for changes in aesthetic quality characterist ics (skin coloration, firmness, presence of damages/diseases) during the storage period at 15 C (Days 1 to 26). 4.2.2.2 Firmness determination during storage Firmness on fresh guava fruits, obtained ev ery 3 to 4 days during ripening, was measured using an Instron Universal Te sting Instrument (M odel 4411-C8009, Canton, Mass.), equipped with a 5 kg load cell a nd an 8-mm diameter compressive probe, adapting conditions from Bashir et al. 2002, Reyes and Paull 1995, and Ergun and Huber 2004. The probe was positioned at zero force contact with the surface of the guava. Probe penetration was set at 10 mm (1 cm) at a crosshead speed of 50 mm/min, and readings were taken at 3 equidistant poi nts on the equatorial region of the fruit. Firmness data was expressed as the maximum force ( kg) attained during penetration. 4.2.2.3 Titratable acidity, soluble solids and pH Titratable acidity analysis was performe d the guavas obtained every 3 to 4 days during storage. Approximately 3 g of puree were combined with 10 mL of deionized water and titrated with 0.1 N NaOH to an end point of pH 8.2. TA was calculated based on the volume of NaOH used and results were expressed as % citric acid, which is the

PAGE 69

57 major organic acid in guava (Wilson et al., 1982). Soluble so lids (SS) and pH measurements were performed as outlined in Chapter 3; Additionally, SS and pH were performed on all final ripe samples. 4.2.3 Chemical Analysis Chemical analysis (total soluble phenolic s, antioxidant capacity, ascorbic acid, lycopene, polyphenolics by HPLC and moisture content) were conducted according to the procedures outlined in Chapter 3. 4.2.4 Statistical Analysis The experimental design consisted of a completely randomized design with two treatments: control and 1-MCP. Statistical analysis consiste d of t-test using JMP (SAS, Cary, NC) to compare differences between treatments (P< 0.05). 4.3 Results and Discussion 4.3.1 Quality Analysis Detailed treatments comparisons of fruit quality were assessed during storage since one of the main focuses of 1-MCP is exte nding produce shelf-life and preserving many of the physicochemical and quality attributes of fresh guava. The most important aesthetic quality parameters for guava are firmness and skin coloration. In order to differentiate between stages of skin coloration during ripeni ng, 4 color criteria were used to describe the fruit that included green (mature-green stag e), yellowish-green (a brighter green color with yellow tints), turning (40 to 70 % surface yellow), and yellow (>70% surface yellow). 4.3.1.1 Aesthetic fruit quality during storage. Days 0 to 7: First identifiable differences between treatments

PAGE 70

58 Perceivable differences in surface color we re not apparent until 4 days in storage when approximately 20% of the control fr uit were classified as yellowish-green compared to green for the 1-MCP treated fr uit. On Day 5, 30% of control fruit were yellowish-green and turning, while the 1-MCP group presente d 5% of its fruits at a yellowish-green stage. By Day 7, some bruises on guava skin became apparent on control fruit, probably due to a more advanced degree of ripening. Days 9 to 15: Control guavas ripening Day 9 was characterized by the first colle ction of ripe guavas from the control fruits, which accounted for approximately 35% of control fruits. Additionally, control guavas from boxed treatment were collected. The 1-MCP group remained primarily in the green color stage, including boxed guavas. A difference in fruit texture between the treatments was apparent. On Day 13, the firs t collection of ripe 1-MCP guavas was done, close to 13% of the 1-MCP group; while 80% of the control fruits had already been collected for ripeness. Ripe 1-MCP fruits had completely yellow skin coloration and were firmer than control fruits. The last guavas from the control group were collected on Day 15, when 80% of the original 1-MCP group was still in the process of ripening. A brief summary of changes in skin coloration during storage is presented in Table 4-1. Days 16 to 26: 1-MCP guavas ripening By Day 17, only about 30% of 1-MCP ripe fruits had been collected. Most fruit remained green, while 40% of the group was turning or yellow. 1-MCP guava ripening continued until Day 26, when the last batch was collected. Approximately 70% of the 1MCP ripe guavas were collected in the period from Day 18 to Day 26. Reyes and Paull (1995) have reported that guavas stored at 15 C usually attain full ripeness in a period

PAGE 71

59 between 8 to 11 days. Although, variety, harv est time, post harvest handling and other parameters shall be considered. Comparably, most of the control guavas (82%) achieved full ripeness between Day 9 and 13 (12 to 15 days after harvest). 1-MCP was able to extend the shelf life was able to extend the shelf-life of guavas for at least 5 days. Green (%*) Yellowish/green (%) Turning (%) Yellow (%) Days in Storage at 15 C Control 1-MCP Control 1-MCP Control 1-MCP Control 1-MCP 0 100 100 5 70 100 20 10 7 2 95 73 5 25 9 90 60 10 10 30 13 80 20 20 30 50 15 60 20 5 5 100 15 18 -20 -30 -30 -20 22 --50 -20 -30 26 ----100 Table 4-1. Changes in skin coloration in non-treated (control) a nd 1-MCP-treated guavas during 1-MCP application and storage at 15 C. % of fruit from the treatment containing determined skin coloration. The influence of 1-MCP on diseases or di sorders has been specific depending on the species showing mixed results (Blanke nship et al., 2003). 1-MCP treated mangos reported twice the amount of stem rot than co ntrol fruit (Hofman et al., 2001), whereas in apples it has reduced superficial scald (Fan et al., 1999) and in papaya it has shown less incidence in decay (Ergun and Huber, 2004). Along with bruising, an incidence of brown spots around the crown and othe r parts of the fruit was observed in some fruits during ripening, which was likely due to firm rot, a common disease in guavas usually induced by bruising (Ko and Kunimoto, 1980; Reyes and Paull, 1995). As the fruit became riper, bruises and spots became more apparent, especi ally in control fruits, which started to ripen earlier. Due to a slowing down in their ripening process, 1-MCP fruits did not show

PAGE 72

60 these disorders as markedly, however by Days 20 to 26, they were more apparent. Approximately 15% of the original fruits from both groups were lost mainly due to incidence of firm rot. 1-MCP treatment to guavas did not ameliorated or induced this disorder. 4.3.1.2 Firmness during storage Firmness loss during climacteric fruit ripening is directly related to disassembly of cell wall components (Lohani et al., 2003) and modification of pectin fractions mainly, with an increase in pectin solubilization (H uber, 1983). These changes are resultant of an increase in activity of cell wall hydrolases, which have been closely associated to ethylene (Brummell and Harpster, 2001). Ce ll wall hydrolases polygalacturonase (PG), pectinesterase (PE) and cellulose in both white and pink flesh guavas have shown to increase in activity during ripe ning, with a correlation between increase in activity of PG and cellulose and loss of flesh firmness (Abu-Goukh and Bashir, 2003). A 1-MCP treatment effect was observed si nce Day 4, were 1-MCP fruits presented significantly firmer texture as compared to c ontrol (Figure 4-1). The trend continued until Day 12, before the last procurement of c ontrol guavas was performed. Comparably, Basseto et al. (2005) reported that Pedro Sato guavas treated at 900 nL/L (6 to 12 h at 25 C) retained firmness as compared to a control, while fruits treated at lower concentrations presented no differences in te xture. They reported, however, these guavas (900 nL/L) were not able to attain full ripeness, as opposed to the present study. Important factors to consider are the relationship between application time and temperature; in the present study guavas were exposed for a longer period of time at a lower temperature.

PAGE 73

61 During guava storage, there were no signi ficant differences in firmness between each sampling point, from Day 1 until Day 13 for control fruits and until Day 17 for 1MCP treated fruits, as observed in Figure 4-1. A decline in firmness was expected in control fruits during storage, since softening is directly related to an increase in days of ripening in guava (Abu-Goukh and Bashir, 2003 ).However, there were no significant differences between sampling points. This was likely attributed to the variability of the samples procured, since they were chosen based on physiological similarities to 1-MCP fruits collected each sampling day. Mercado-Silva et al. (1998) reported a large variability in firmness between different ri pening stages of guavas. 1-MCP fruits maintained their firmness throughout the entire storage period, even when they attained full ripeness. Firmness (Kg) 0 1 2 3 4 5 6 7 8 9 Control 1-MCP 1 5 9 13 17Storage Time at 15 C (Days) Figure 4-1. Firmness (kg) of guavas treated with 1-MCP (1000 nL/L ,10 C, 24 h) during storage at 15 C. Error bars represent the sta ndard error of the mean, n = 5. Various effects of ethylene during fruit ri pening are apparently associated with alterations in the properties of cellular membranes. It is thought that ethylene, along with

PAGE 74

62 other growth substances found in plants, bi nds to some components of the cellular membrane (proteins, glycoproteins, and lipid s) thereby initiating secondary responses (Noogle and Fritz, 1983). Such bindings, especi ally with proteins, may have immediate effects, including protein conformation or cell turgor changes, or tr igger other processes which might take a longer time to become vi sible. It has been discussed that when ethylene binds to proteins possessing enzyma tic activity, the act of binding may activate them and alters their rate of degradation (Noogle and Fritz, 1983). By inhibiting ethylene action, 1-MCP has been related to the delay or inhibition of the activity of cell wall hydrolases, responsible for tissue softening. 4.3.1.3 Titratable acidity, soluble solids, and pH during storage Titratable acidity (TA) and pH in 1-MC P fruits was higher than control only on Day 5 of storage, but not insignificantly diffe rent at other days (F igures 4-2 and 4-3). This difference might be attributed to va riation, as seen in the large error rate. Independently of Day 5, 1-MCP did not affect TA and pH during storage time. Basseto et al. (2005) reported that 1-MC P treated fruit (900 nL/L) ma intained higher titratable acidity levels during entire storage, attribut ing it to a ripening delay. Reports on influence of 1-MCP on titratable acidity during are mi xed, depending on the t ype of commodity or even variety (Blankenship and Dole, 2003). Additionally, pH on final ripe samples was not influenced by 1-MCP (average = 4.07) Within the 1-MCP group, TA and pH remained unaffected by increasing storage tim e. Within the control group, Day 13 TA was significantly higher than Day 5, but not differe nt from the rest of the sampling points; while pH remained unaffected. Accord ing to results reported by Reyes and Paull (1995), both TA and sol uble solids as a function of fruit ag e rather than stage of ripeness, were both quality parameters are maintained during ripening

PAGE 75

63 Figure 4-2. Titratable acidity (% citric acid) of guavas treat ed with 1-MCP during storage at 15 C. Error bars represent the stan dard error of the mean, n = 5. Figure 4-3. Effect of a 1MCP treatment (1000 nL/L ,10 C, 24 h) on guava pH during storage at 15 C. Error bars represent the standa rd error of the mean, n = 5. Soluble solids were not influenced by the 1-MCP treatment both during storage (Figure 4-4) and in final ripe guavas (average = 7.81 Brix). There has been no reported effect on soluble solids on Brazilian guava (Basseto et al., 2005), mango, custard apple pH 3.8 3.9 4.0 4.1 4.2 4.3 4.4 Control 1-MCP 1 5 9 13 17Storage Time at 15 C (Days) Titratable Acidity (% Citric Acid) 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 Control 1-MCP 1 5 9 13 17Stora g e Time at 15 C ( Da y s )

PAGE 76

64 (Hofman et al., 2001), apricots, and plums (Dong et al., 2002). Guava soluble solids maintained uniformity during the storage ti me within both the control and 1-MCP group. The role of ethylene on starch and/or sugar conversion is still not clear, with mixed reports of whether 1-MCP affects their conv ersion or not (Blankens hip and Dole, 2003). Soluble Solids ( Brix) 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 Control 1-MCP 1 59 13 17Storage Time at 15 C (Days) Figure 4-4. Effect of 1-MC P treatment (1000 nL/L at10 C, 24 h) on guava soluble solids ( Brix) during storage at 15 C. Error bars represent th e standard error of the mean, n = 5. 4.3.2 Chemical Analysis For all chemical analysis performed, except for polyphenolics by HPLC, results were reported in dry weight basis (DW), in order to eliminate difference between samples due to varying water loss fruits experience d during ripening, an impor tant factor since 1MCP fruits were stored considerab ly longer than c ontrol fruit. 4.3.2.1 Moisture content Moisture content in the final, ripe fru it was not significantly different between control and 1-MCP treated fr uit at 86.6 and 85.9% respectively. Although, moisture

PAGE 77

65 content determined at final ripe guava puree samples is not the same as a measure of individual fruit weight loss dur ing ripening, it gives a clearer picture of differences in final samples due to moisture. It was expect ed that 1-MCP treated fruits probably had a higher weight loss due to their longer time in storage. However, no differences in final moisture values were reported for the present study. 4.3.2.2 Total soluble phenolics Total soluble phenolics (TSP) content wa s unaffected by 1-MCP treatment (Figure 4-5). Although some aspects of the ripening pr ocess were delayed in 1-MCP fruits, these presented TSP values of full ripe fruit, as co mpared to control. Total phenolics from both groups probably decreased during ripening achie ving similar values, independently of the inhibition of ethylene. However, the a ddition of ethylene may affect polyphenolic increase in biosynthesis (Cisneros-Cevallos, 1997). In lettuce, phe nylalanine lyase (PAL) activity was induced by exogenous ethylene, cau sing an increase in phenolics compounds (Ke and Salveit, 1988; Dixon and Paiva, 1995; Tomas-Barberan et al., 1997). Also in lettuce, 1-MCP application (1000 nL/L) did no t affect polyphenolic content as compared to control; however, when 1-MCP was applied prior to exposure to ethylene, there was a significant reduction in ethylene-induced pol yphenolic synthesis (Campos-Vargas and Salveit, 2002). The addition of exogenous ethylene rather th an endogenous ethylene inhibition by 1-MCP seems to affect more th e levels of polyphenolic s. In apples, total phenolics exhibited an ethylene-independent regulation when ethylene was inhibited (Defilippi et al., 2004). It is co ncluded that 1-MCP did not aff ect the final levels of total soluble phenolics in guava, where probably polyphenolic regulation was not affected by inhibition of ethylene. The inhibition of et hylene itself, even at a relatively high concentration of 1-MCP, was not enough to affect polyphenolic bi osynthetic pathways.

PAGE 78

66 Guava Treatments After Ripening at 15 C Total Soluble Phenolics (mg/kg dwb) 10000 12000 14000 16000 18000 20000 22000 Control 1-MCP Figure 4-5. Effect of 1-MCP (1000 nL/L, 10 C, 24 h) on total soluble phenolics (mg/kg DW) in guava. Error bars represent the standard error of the mean, n = 23. 4.3.2.3 Antioxidant capacity Figure 4-6. Effect of 1-MC P treatment (1000 nL/L, 10 C, 24 h) on guava antioxidant capacity ( M Trolox equivalents/g DW). Error bars represent the standard error of the mean, n = 23. Antioxidant capacity of ripe guavas was unaffected by 1-MCP treatment (Figure 46). This observation supports results for TSP. Since antioxidant capac ity in guava was not Guava Treatments After Ripening at 15 C Antioxidant Capaci ty (uM TE/g dwb) 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 Control 1-MCP

PAGE 79

67 affected by 1-MCP this might explain a degree of ethylene independence in the regulation of most antioxi dant phytochemicals during climacteric ripening. 4.3.2.3 Ascorbic acid 1-MCP treated fruit presented significantly higher levels of ascorbic acid as compared to Control fruit (Figure 4-7). Differenc es in ascorbic acid might be attributed to fruit variability than the 1-MCP treatment its elf. Basseto and partners (2005) reported no differences between 1-MCP and control fruit, determined by a titration method. The use of a more precise analytical method resulted in a more accurate quant ification of ascorbic acid. However, it resulted in a high variability between samples. A variability can be attributed to the assay for running ascorbic acid, however, careful care was taken when making composites of five fruits and extrac ting guava juice and the standard for HPLC had a low standard error (1.7%). It has been reported a high variability in ascorbic acid levels in guava, even within the same variety (reviewed by Mitra, 1997). Additionally, the fact that antioxidant capacity and T SP were not influenced by 1-MCP is a good indicator that ascorbic acid probably was not affected. Apart from being an essential nutrient fo r humans, ascorbic acid within the plant has numerous roles mainly related to three biol ogical activities: as an antioxidant, as a donor/acceptor in electron transport at the pl asma membrane or chloroplasts, and as an enzyme co-substrate (Davey et al., 2000). Alt hough the biosynthetic pathway for ascorbic acid has not been completely elucidated, a pathway via hexose sugars (GDP-mannose, GDP-L galactose, L-galactose, galacto-1,4-lactone) has been proposed recently (Davey et al., 2000; Smirnoff and Wheeler, 2000; Barata-Soares, 2004). The relationship existent between ascorbic acid and ethylene lies wi thin ethylenes biosynthetic pathway, where ACC oxidase (enzyme responsible for the last step in ethylene biosynthesis), uses

PAGE 80

68 ascorbic acid as a co-substrate, apparently oxidizing it to dehydroasc orbate (Davey et al., 2000; Smith et al., 2000). Ethylene levels pr oduced by most fruits are very low (ppb range), therefore its normal production or subseq uent inhibition might be to low to result in detectable changes in ove rall ascorbic acid levels (personal communication, Huber, 2005), specially in guavas, which contain a larg e pool of ascorbic acid as compared to most fruits. Additionally other mechanisms su ch as heat, light or wounding tend to affect ascorbic acid to a greater extent. Guava Treatments After Ripening at 15 C Ascorbic Acid (mg/kg dwb) 3000 3500 4000 4500 5000 5500 6000 6500 7000 Control 1-MCP Figure 4-7. Guava ascorbic acid content (mg/kg DW) as affected by 1-MCP (1000 nL/L, 10 C, 24 h). Error bars represent the standard error of the mean, n = 23. 4.3.2.4 Lycopene Results from lycopene analysis presente d significantly higher content in 1-MCP fruits as opposed to control fruits (Figure 48). Equal or even higher lycopene values in 1MCP fruit as compared to control indicat es that although there was an alteration on ethylene production and other ripening proce sses were affected, lycopene accumulation resulted in guava during its ripening. This might suggest that lycopene accumulation pathways in guava may not be directly rela ted to ethylene pathways, although they might

PAGE 81

69 affect the rate of lycopene accumulati on during ripening. Comparably, Mostofi and partners (2003) reported final lycopene values in 1-MCP treated tomatoes as insignificantly different from control, how ever, the treatment delayed the onset of lycopene accumulation during storage at 15 C. In another study on fresh cut tomatoes using other ethylene inhibitors and exoge nous ethylene, although ethylene inhibition slowed down the rate of lycopene accumula tion probably due to other processes slowing down, it was concluded that ethylene pr oduction is not essential for lycopene biosynthesis in tomato fruit (Edwards et al., 1983). Guava Treatments After Ri p enin g at 15 C Lycopene (mg/kg dwb) 100 150 200 250 300 350 400 450 500 Control 1-MCP Figure 4-8. Effect of 1-MCP (1000 nL/L ,10 C, 24 h) on guava lycopene content (mg/kg DW). Error bars represent the st andard error of the mean, n = 23. It is known from studies on tomatoes that lycopene accumulation is mainly in the last period of the ripening process (Giova nelli et al., 2004). Although ethylene plays an important role in accumulation of tomato carotenoids during ripe ning, since it regulates phytoene synthase, the lycope ne-producing enzyme, it has be en discussed that it might not be the key regulator for lycopene accu mulation in particular. Work conducted on

PAGE 82

70 tomatoes by Alba et al. (2002) demonstrated that lycopene accumulation in pericarp tissue during tomato ripening is mainly re gulated by phytochromes or chromoproteins, and this can be independent of ethylene biosynthesis. The regulation of carotenoid biosynthesis genes in particular has also been proposed as primary mechanism that controls lycopene accumulation in to mato fruits (Ronen et al., 1999). Lycopene is the major carotenoid in gu ava and its content is comparable or sometimes higher than tomato. Unfortunately th e lack of lycopene in the skin hides its presence in the pulp, making fruit selection based on lycopene an impossible selling point in the market. Considering new evidences fo r correlation between lycopene consumption and reduced rates of prosta te cancer (Giovannuci et al., 1995; Rao and Agarwal, 1999), it is of particular importance preserving or even enhancing its cont ent during postharvest operations. The opportunity of marketing guava as an excellent source of lycopene is present. 4.3.2.5 Polyphenolics by HPLC Polyphenolics by HPLC were identified by re tention time, spectr al properties, and comparison to authentic standards. Based on the work done on the HW treatment study, approximately 16 peaks were selected from the guava chromatogram. These peaks were divided into 4 groups based on spectral prope rties and retention time: gallic acid, procyanidins, characteristic phenolics, and an ellagic acid derivative. Gallic acid and ellagic acid derivative were two indivi dual identifiable compounds. Characteristic phenolics group contained characteristic guava compounds which are unknown, with spectral properties similar to the ones described for the HW treatment study. The polyphenolic profile of guavas from the 1-MC P study was similar to the HW treatment study.

PAGE 83

71 1-MCP did not have an influence on the levels of procyanidins, ellagic acid derivative and other characteris tic polyphenolics of ripe gua va (Figures 4-9 and 4-10). The observed results confirm results observed for antioxidant capacity and TSP, which help explain an ethylene-independent s ynthesis guava polyphenolic compounds when ethylene is inhibited. In the case of gallic acid (Figure 4-11), however, 1-MCP treated fruit exhibited higher content, which might be attributed more to variability in content between samples, specially within the c ontrol group. However, due to gallic acids abundance in a wide variety of commodities and its still not completely known synthesis mechanism, the possibility of an interacti on with ethylene inhibition should also be considered. Guava Treatments After Ripening at 15 C Procyanidins (mg/kg GAE) 0 1 2 3 4 5 6 7 8 9 10 Control 1-MCP Figure 4-9. Guava procyanidin content (mg/kg GAE) as affected by 1-MCP. Error bars represent the standard er ror of the mean, n = 23.

PAGE 84

72 A Guava Treatments After Ripening at 15 C Ellagic Acid Derivative (mg/kg GAE) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Control 1-MCP B Guava Treatments After Ripening at 15 C Characteristic Polyphenolics (mg/kg GAE) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Control 1-MCP Figure 4-10. Guava ellagic acid derivative content (A) and charac teristic polyphenolics content (B) (mg/kg GAE) as affected by 1-MCP. Error bars represent the standard error of the mean, n = 23.

PAGE 85

73 Guava Treatments After Ripening at 15 C Gallic Acid (mg/kg GAE fwb) 0 5 10 15 20 25 30 35 40 45 Control 1-MCP Figure 4-11. Guava gallic acid content (mg/kg GAE) as affected by 1-MCP. Error bars represent the standard er ror of the mean, n = 23. 4.3.3 1-MCP Treatment to Boxed Guavas The present study evaluated the eff ects of 1-MCP on guava quality and phytochemical content when application was conducted on guavas arranged inside their boxes, in a simulated environment prior to shipment. Guavas treated in boxes presented similar aesthetic characteristics during st orage as loose fruit from the previous experiment. On Day 0, during 1-MCP applica tion, guavas within th e boxes presented no differences among themselves. From Days 5 to 8 in storage the first identifiable differences were detected, where 1-MCP pres ented a better retenti on of green coloration and firmness, as compared to control fruit. Similarly to the main study, Day 9 was characterized by the first collection of ri pe guavas, which accounted for 14% of the control group; while most 1-MCP guavas ma intained their firmness and green coloration. The first collection of ripe 1-MCP guavas (17% of the 1-MCP group) was conducted on Day 12, when already more than 50% of the control fruit had already ripened and been procured. Similar to the main experiment, co llection of the last gr oup of control guavas was conducted on Day 15, while collection of 1-MCP ripe guavas continued until Day

PAGE 86

74 26. Results from aesthetic quality evaluations indicated an effect due to 1-MCP treatment in color retention, where 1-MCP treated frui ts presented a delay in skin coloration development for at least 5 days, comparab ly to the main study. Chemical analysis performed resulted in no si gnificant differences in mois ture content (86.1 %), total soluble phenolics (19,100 mg/kg DW), antioxidant capacity (121 M Trolox equivalents/g DW), ascorbic acid (7,090 mg /kg DW), lycopene (853 mg/kg DW), soluble solids (8.13 Brix) and pH (4.10). Firmness measured over time, however, pr esented no significant differences during storage (Figure 4-12), as opposed to the main experiment. However, as observed by the large error rates, this statistical lack of ef fect might be due to variation in samples. A clear distinction between treatments can be observed, especially after Day 9, where 1MCP fruit presents higher firmness values. Probably, a larger number of replications Firmness (Kg) 0 1 2 3 4 5 6 7 8 Control 1-MCP 15 9 13 17 Storage time at 15 C (Days) Figure 4-12. Firmness (kg) of boxed gua vas treated with 1-MCP (1000 nL/L ,10 C, 24 h) during storage at 25 C. Error bars represent the standard error of the mean, n=3.

PAGE 87

75 might have demonstrated a clearer effect. T itratable acidity (Figure 4-13) and soluble solids (Figure 4-14) were unaffected by 1-MCP during storage, comparable to the main experiment. The nature of the packaging material used is an important factor to consider. It seems that the 1-MCP gas was able to penetrate to the walls of the cardboard box, diffusing itself among the fruit, delaying skin yellowing and retaining firmness, as described by aesthetic quality evaluations. Pr obably, if the boxes had more perforations, a better penetration and diffusion of the gas w ould have happened. In a work conducted in plums packaged similarly inside perforated boxes, 1-MCP proved be tter effectiveness than plums treated in bulk (Valero et al., 2004) Therefore, the possi bility of applying 1MCP on packaged guavas has a potential of being explored further for commercial applications, especially due to the nature of the fruit, since an easier way to handle any postharvest process might be beneficial. Titratable Acidity (% Citric Acid) 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 Control 1-MCP 1 5 9 13 17Storage time at 15 C (Days) Figure 4-13. Titratable acidit y (% citric acid) of boxed guava s treated with 1-MCP during storage at 15 C. Error bars represent the sta ndard error of the mean, n = 3.

PAGE 88

76 Soluble Solids ( Brix) 5 6 7 8 9 10 11 12 13 Control 1-MCP 1 5 9 13 17Storage time at 15 C (Days) Figure 4-14. Effect of 1-MCP treatment (1000 nL/L at10 C, 24 h) on boxed guavas soluble solids ( Brix) during storage at 15 C. Error bars represent the standard error of the mean, n = 3. 4.4 Conclusions Extension of shelf-life in guavas is of extreme value due the highly perishable nature of these exotics. A 1-MCP treatment (1000 nL/L for 24 h at 10 C) applied to pink fleshed guavas was effective in extending their shelf life and retaining quality characteristics, without detrimental effects to their phytochemicals. Guava shelf life was extended for at least five days during storage at 15 C, with a delay in skin yellowing and retention of firmness. Quality parameters such as titratable acidity, soluble solids, and pH were maintained during storage, and una ffected by a 1-MCP treatment. Additionally, there was no significant eff ect of 1-MCP in total sol uble phenolics and antioxidant capacity. Ascorbic acid and lycopene presen ted significantly higher values in 1-MCP treated fruit; however these differences were not attributed to a treatment effect, but rather to fruit variability and certain i ndependence of ethylene in their biosynthetic

PAGE 89

77 pathways. Procyanidin compounds, total polyphe nolics, and an ellagic acid derivative were not affected by a 1-MCP treatment, wh ich supports results fo r antioxidant activity and TSP. An ethylene inhibition in guava, even at a relatively high concentration, resulted in insignificant eff ect in most of its phytochemicals. Results from the boxed guavas study indicate a potential for a pplying 1-MCP on boxed guavas, and further investigation in a near future would be benefi cial for packers. Literature presents mixed results on the effects of 1-MCP on physicoche mical properties of various commodities, being species specific and very dependent on application cond itions. Results of this study conclude that 1-MCP can be applied to guava successfully, without negative impacts on its aesthetic quality, phytochemicals, and antio xidant properties. Further research is needed to determine optimal application conditions.

PAGE 90

78 CHAPTER 5 SUMMARY AND CONCLUSIONS Guava marketability as a fresh fruit is somewhat limited due to lack of an established quarantine treatment and its hi ghly perishable natu re. To provide an overview, these studies consis ted of application of two pos tharvest treatments, a hot water immersion technique as a quarantine treatment and a 1-methylcyclopropene (1MCP) application to extend guava shelf-life. The effects evaluated not only included overall quality parameters, but guava phytochemicals (polyphenolics, ascorbic acid, carotenoids, and lycopene) and antioxidant properties. A hot water immersion treatment at 46 C for up to 30 min may be applied to gua vas at three ripening stages without affecting their quality and phytochemical conten t. Stage I fruits treated longer than 30 min experienced an increase in certain polyphenolic compounds and a decrease in lycopene content. This was a response to heat stress, where bi osynthesis of certain polyphenolic compounds was enhanced and lyco pene biosynthesis might have been reversibly inhibited affecting its final concentrations. Othe r differences reported were mainly attributed to ripeni ng stage than the HW treatment itself. 1-MCP application (1000 nL/L for 24 h at 10 C) successfully extended the shelf life of guavas for at least 5 days during storage at 15 C, presenting positive effects which included skin yellowing delay and retention of firmne ss. Although shelf-life was extended, 1-MCP insignificantly affected quality parameters (titratable acid ity, soluble solids, and pH) and phytochemical content. It was observed a higher ascorbic ac id and lycopene cont ent in 1-MCP treated fruits, which was not directly related to a 1-MCP effect. It was concluded that the

PAGE 91

79 biosynthesis pathways for most antioxidant compounds in guava are independent from inhibition of ethylene action. A HW immersi on treatment and a 1-MCP treatment may be applied successfully to guavas, maintaining their quality attributes and especially not affecting detrimentally phytochemical compounds.

PAGE 92

80 LIST OF REFERENCES Abu-Goukh, A.; Bashir, H. A. Changes in pe ctic enzymes and cellu lose activity during guava fruit ripening. Food Chem 2003 83, 213-218. Abushita, A.A,; Daood, H.G.; Biacs, P.A. Changes in carotenoids and antioxidant vitamins in tomatoes as a function of varietal and tec hnological factors J. Agric. Food Chem. 2000 48, 2075-2081. Akamine, E.; Goo, T. Respiration and ethyl ene production in fruits of species and cultivars of Psidium and species of Eugenia J. Amer. Soc. Hort. Sci. 1979 104, 632-635. Alba, R.; Cordonnier-Pratt, M.; Pratt, L. Fr uit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato. Plant Physiol. 2000 123, 363-370. Association of Analytical Communities. Official methods of analysis of AOAC International, 17th Ed.; Horwitz: Gaithersburg, MD, 2000. Barata-Soares, A.D.; Gomez, M.; De Mesquita, C.; Lajolo, F. Ascorbic acid biosynthesis: A precursor study on plants. Brasilian J. Plant Physiol. 2004 3, 147-154. Bashir, H. A.; Abu-Goukh, A. Compositiona l changes during guava fruit ripening. Food Chem 2002 80, 557-563. Basseto, E.; Jacomino, A.P.; Pinheiro, A.L.; Kl uge, R.A. Delay of ripening of Pedro Sato guava with 1Methylcyclopropene. Postharvest Biol. Technol. 2005 35, 303308. Blankenship, S.; Dole, J. 1Methylcyclopropene: A Review. Postharvest Biol. Technol 2003 28, 1-25 Boyle, F.; Seagrave, H.; Sakata, S.; Sherman, D. Commercial guava processing in Hawaii, Bulletin.University of Hawaii: Honolulu, Hawaii, 1957. Brasil, I.; Arraes Maia, G.; Wilane de Fi gueiredo, R. Physical-chemical changes during extraction and clarifica tion of guava juice. Food Chem 1995 54; 383-386. Brecht,J.; Saltveit, M.; Talcott, S.; Schneid er, K.; Felkey, K.; Bartz, J. Fresh-cut vegetables and fruits. In: Janick (ed.). Horticultural reviews. Vol. 30; John Wiley and Sons: Hoboken, New Jersey, 2004.

PAGE 93

81 Brummel, D.; Harpster, M. Cell wall metabolis m in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 2001 77, 311-340. Bulk, R.; Babiker, E.; Tinay, A. Changes in chemical composition of guava fruits during development and ripening. Food Chem. 1997 59, 395-399. Campos-Vargas, R.; Saltveit, M.E. Involveme nt of putative chemi cal wound signals in the induction of phenolics meta bolism in wounded lettuce. Physiol. Plant. 2002, 114, 73-84. Cheng,T.; Floros, J.; Shewfelt, R.; Chang, C. The effect of high temperature stress on ripening of tomatoes ( Lycopersicum esculentum ). J. Plant Physiol. 1988 132, 459464. Ching, L.; Mohamed, S. Alpha-tocopherol content in 62 edible tropical plants. J. Agric. Food Chem 2001 49, 3101-2105. Cisneros-Zevallos, L. The use of controlled postharvest abiotic st resses as a tool for enhancing the nutraceutical c ontent and adding-value of fr esh fruits and vegetables. J. Food Sci 2003 68, 1560-1565. Civello, P.; Martinez, G.; Chaves, A.; A non, M. Heat treatments delay ripening and postharvest decay of strawberry fruit. J. Agric. Food Chem. 1997 45, 4589-4594. Cook, N.; Samman, S. Flavonoids: Chemistry, me tabolism, cardioprotective and dietary sources. Nutr. Biochem. 1996 7 66-76. Davey, M.; Montagu, M.; Inze, D.; Sanmartin, M. ; Kanellis, A.; Smirnoff, N.; Benzie, I.; Strain, J.; Favell, D.; Fletcher, J. Plan t L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Sci. Food Agric. 2000, 80, 825-860. DeEll, J.; Murr, D.; Porteous, M.; Rupasinghe H. Influence of temperature and duration of 1-methylcyclopropene (1-M CP) treatment on apple quality. Postharvest Biol. Technol. 2002 24, 349-353. De Bruyne, T.; Pieters, L.; Deelstra, H ., Vlietinck, Condensed vegetable tannins: Biodiversity in structure and biological activities Biochemical Systematics and Ecology. 1999 27, 445-449 Defilippi, B.G.; Dandekar, A.M.; Kader, A.A. Impact of suppresion of ethylene action or biosynthesis on flavor metabolites in apple. J. Agric. Food Chem. 2004 52, 5694-5701. Dewanto, V.; Wu, X.; Adom, K.K.; Liu, R.H. Thermal processing enhances the nutritional Value of tomatoes by in creasing total antioxidant activity. J. Agric. Food Chem. 2002 50, 3010-3014.

PAGE 94

82 Dixon, R.; Paiva, N. Stress-induced phenylpropanoid metabolism. Plant Cell. 1995. 7, 1085-1097. Dong, L.; Lurie, S.; Zhou, H. Effect of 1methylcyclopropene on ripening of Canino apricots and Royal Zee plums. Postharvest Biol. Technol. 2002, 24, 135-145. Edmundo, M.P.; Elia, N.; Edmundo, M.S. Antioxidant capacity of guava fruit and jicama roots under chilling inju ry conditions. Annual IFT Meeting and Expo.2002. Edwards, J.I.; Saltveit, M.E.; Henderson, W.R. Inhibition of lycopene synthesis in tomato pericarp tissue by inhibitors of ethylen e biosynthesis and reversal with applied ethylene. J. Amer. Soc. Hort. Sci. 1983 108, 512-514. El-Zoghbi, M. Biochemical changes in some tropical fruits during ripening. Food Chem 1994 49, 33-37. Environmental Protection Agency [EPA]. Biopesticide Federal Register Notices by Active Ingredient. 1-MCP. 2003. Available online: www.epa.gov Last accessed: April, 2005. Fallik, E. Prestorage hot water treatmen ts (immersion, rinsing and brushing). Postharvest Biol. Technol. 2004 32, 125-134. Fan, X.; Blankenship, S.; Mattheis, J. 1-Me thylcyclopropene inhi bits apple ripening. J. Am. Soc. Hort. Sci. 1999 124, 690-695. Fennema, O.R. Food Chemistry 3rd Ed.; Marcel Dekke r: New York, NY, 1996. Food and Agriculture Organization [FAO ]. Commodity Market Review 2003-2004. Available online: http://www.fao.org/documents. Last accessed: August, 2004. Giovanelli, G.; Lavelli, V.; Peri, C.; Simona, Nobili. Variation in antioxidant components of tomato during vine and post-harvest ripening. J. Sci. Food Agric. 1999 79, 1583-1588. Goldstein, J.; Swain, T. Changes in tannins in ripening fruits. Phytochemistry. 1963 2, 371-383. Gorinstein, S.; Zemser, M.; Haruenkit, R.; Chuthakorn, R.; Grauer, F.; Martin-Belloso, O.; Trakhtenberg, S. Comparative content of total polyphenols and dietary fiber in tropical fruits and persimmon. J. Nutr. Biochem 1999 10, 367-371. Gould, W.; Sharp, J. Hot-water immersion quara ntine treatment for guavas infested with Caribbean fruit fly (diptera: tephritidae). J. of Economic Entomology 1992 85, 1235-1239. Grundhofer, P., Niemetz, R., Schilling, G., Gross, G. Biosynthesis and subcellular distribution of hydrolyzable tannins. Phytochemistry 2001 57 915-927.

PAGE 95

83 Hagerman, A.; Riedl, K.; Jones, A.; Sovik, K.; Ritchard, N.; Hartzfeld, Riechel, T. High molecular weight plant polyphenolics (t annins) as biological antioxidants. J. Agric. Food Chem 1998 46, 1887-1892. Ho, C.; Lee, C.; Huang M. Phenolic compounds in food and their effects on health I. ; American Chemical Society: Washington, DC, 1992. Hofman, P.; Jobin-De cor, M.; Meiburg, G.; Macnish, A.; Joyce, D. Ripening and quality responses of avocado, custard appl e, mango and papaya fruit to 1methylclopropene. Aust. J. Exp. Agric. 2001 41, 567-572. Huber, D.J. The role of cell wa ll hydrolases in fruit softening. Hort. Rev. 1983, 5,169-219. Itoo, S.; Matsuo, T.; Ibushi, Y.; Tamari, N. S easonal changes in the levels of polyphenols in guava fruit and leaves a nd some of their properties. J. Japan. Soc. Hort. Sci. 1987 56, 107-113. Jacobi, K.; Giles, J. Quality of Kensington mango ( Mangifera indica Linn.) fruit following combined vapour heat dissinfe station and hot water disease control treatments. Postharvest Biol. Technol. 1997 12, 285-292. Jacobi, K.; MacRae, E.; Hetherington, S. Post harvest heat disinfestation treatments of mango fruit. Scientia Horticulturae. 2001, 89, 171-193. Janick, J. Horticultural Reviews: Volume 30 ; John Wiley and Sons: Hoboken, HJ, 2004. Jeong, J.; Huber, D.J.; Sargent, S. Influence of 1-methylcyclopropene (1-MCP) on ripening and cell-wall matrix polysaccharides of avocado ( Persea americana ) fruit. Postharvest Biol. Technol. 2002 25, 241-256. Jiang, Y.; Joyce, D.; Terry, L. 1-methylcyclop ropene treatment affects strawberry fruit decay. Postharvest Biol. Technol. 2001, 23, 227-232. Jimnez-Escrig, A.; Rincn, M.; Pulido, R.; Saura-Calixto, F. Guava fruit ( Psidium guajava L.) as a new source of an tioxidant dietary fiber. J. Agric. Food Chem 2001 49, 5489-5493. JMP; SAS Institute, Inc., SAS Campus Drive, Cary, NC, 1996. Ke, D.; Salveit, M.E. Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce. Plant Physiol. 1988 1136-1140. Kipe, S. The World Fresh Fruit Market USDA Foreign Agricultural Service. Horticultural and Tropical Products Division. 2004. Available online: www.fas.usda.gov/htp/ Presentations/2004 Last accessed: August, 2004. Ko, W.; Kunimoto, R. Guava fru it firm rot induced by bruising. Hort. Science 1980 15, 722-723.

PAGE 96

84 Kondo,S.; Kittikorn, M.; Kanlayanarat, S. Preh arvest antioxidant act ivities of tropical fruits and the effect of low temperatur e strage on antioxidants and jasmonates. Postharvest Biol. Technol. 2005 In Press. Lee, J.H.; Johnson, J.V.; Talcott, S.T. Identi fication of ellagic acid conjugates and other polyphenolics in mature muscadine grapes by HPLC-ESI-MS. 2005 ASAP Article. Leonardi, C.; Ambrosino, P.; Esposito, F.; Fogliano,V. Antioxidative activity and carotenoid and tomatine contents in diffe rent typologies of fresh consumption tomatoes. J. Agric. Food Chem. 2000, 48, 4723-4727. Leong, L.; Shui, G. An investigation of an tioxidant capacity of fruits in Singapore markets. Food Chem. 2002 76, 69-75. Lin, C.H.; Chen, B.H. Determination of carotenoids in tomato juice by liquid chromatography. J.of Chromatography 2003 1012, 103-109. Liguori, G.; Weksler, A.; Zutahi, Y.; Lurie, S.; Kosto, I. Effect of 1-MCP on melting flesh peaches and nectarines. Postharvest Biol. Technol 2004 31, 263-268. Lohani, S.; Trivedi, P.; Nath, P. Changes in activities of cell wall hydrolases during ethylene-induced ripening in banana: effect of 1-MCP, ABA, and IAA. Postharvest Biol. Technol 2004 31, 119-126. Lurie, S. Postharvest heat treatments. Postharvest Biol. Technol. 1998 14, 257-269. Lurie, S.; Handros, A.; Fallek, E.; Shapira, R. Reversible inhibi tion of tomato gene expression at high temperature. Plant Physiol 1996 110, 1207-1214. Lurie, S.; Klein, J. Acquisition of low-temper ature tolerance in to matoes by exposure to high-temperature stress. J. Amer. Soc. Hort. Sci. 1991 116, 1007-1012. Malo, S.E; Campbell,C.W. 1994.The Guava. University of Florida-IFAS extension. Bulletin. Available online: http://edis.ifas.ufl.edu/MG 045. Last accessed: August, 2004. Marcelin, O.; Williams, P.; Brillouet, J. Isol ation and characterization of the two main cell-wall types from guava ( Psidium guajava L.) pulp. Carbohydr. Res. 1993 240, 233-243. Martinez-Valverde, I.; Pe riago, M.; Provan, G.; Chesson, A. Phenolic compounds, lycopene and antioxidant activity in commercial varieties of tomato ( Lycopersicum esculentum ). J. Sci. Food Agric. 2002 82, 323-330. Mercadante, A.; Steck, A.; Pfander, H. Carotenoids from Guava ( Psidium guajava L. ): Isolation and Structure Elucidation. J. Agric. Food Chem 1999 47, 145-151.

PAGE 97

85 Mercado-Silva, E.; Benito-Bautista, P.; Garc ia-Velasco, M. Fruit development, harvest index and ripening changes of gua vas produced in central Mexico. Postharvest Biol. Technol. 1998 13, 143-150. Miean, K.H.; Mohamed, S. Flavonoid (myrice tin, quercetin, keampferol, luteolin, and apigenin) content of edible tropical plants. J. Agric. Food Chem 2001 49, 31062112. Mir, N.; Curell, E; Khan, N.; Whitaker, M.; Beaudry, R. Harvest maturity, storage temperature, and 1-MCP application fr equency alters firmness retention and chlorophyll fluorescence of R edchief Delicious apples. J. Am. Soc. Hort. Sci. 2001 126, 618-624. Misra, K.; Seshadri, T. Chemical components of the fruits of Psidium guajava Phytochemistry. 1967 7, 641-645. Mitra, S. Postharvest physiology and storage of tropical and subtropical fruits, CAB International: New York, New York, 1997. Morton, J.F. The Guava. Fruits of warm climates Media Incorporated: Greensboro, NC, 1987. Mostofi, Y.; Toivonen, P.; Lessani, H.; Babalar, M.; Lu, C. Effects of 1methylcyclopropene on ripening of gr eenhouse tomatoes at three storage temperatures. Postharvest Biol. Technol. 2003 27, 285-292. Mowlah, G.; Itoo, S. Quantitative ch anges in guava polyphenols and the polyphenoloxidase (PPO) at different stages of maturation, ripening, and storage. Nippon Kogyo Gakkaishi. 1982 29, 413-417. Mueller-Harvey, I. Analysis of hydrolysable tannins. Anim. Feed Sci. Technol. 2001 91, 3-20. Mullins, E.D.; McCollum, T.G.; McDonald, R.E. Consequences of ethylene metabolism of inactivating ethylene r eceptor sites in diseased non-climacteric fruit. Postharvest Biol. Technol. 2000 19, 155-164. National Agriculture Statisti cs Service [NASS]. 2004. Guava output falls for the third straight year.Bulletin: Hawa ii Guavas. Available online: www. nass .usda.gov Last accessed: February, 2005. Noogle, G. and Fritz, G. Introductory plant physiology 2nd Edition, Prentice Hall Inc.: Englewood Cliffs, New Jersey, 1983. Ou, B.; Hampsch-Woodill, M.; Prior, R.L. Development and validation of an improved oxygen radical absorbance capac ity assay using fluorescein as a fluorescent probe. J. Agric. Food Chem. 2001 49, 4619-4626.

PAGE 98

86 Padula, M.; Rodriguez-Amaya, D. Characteris ation of the caroteno ids and assesment of the vitamin A value of Brasilia n guavas (Psidium guajava L). Food Chem. 1986 20, 11-19. Paull, R. Response of tropical horticultu ral commodities to insect desinfestation treatments. Hort. Sci. 1994 29, 988-996. Paull, R.; Chen, N. Heat treatment and fruit ripening. Postharvest Biol. Technol. 2000 21-37. Rao, A.; Agarwal, S. Role of lycopene as antioxidant carotenoid in the prevention of chronic diseases: A review. Nutr. Res. 1999 19, 305-323. Regalado-Contreras, E.; Mercado-Silva, E. E ffect of hot water treatment on the ascorbic acid and reduced glutathione le vels in guava fruit during cold storage. Annual IFT Meeting and Expo. 1998. Reyes, M.U.; Paull, R.E. Effect of storag e temperature and ethylene treatment on guava (Psidium guajava L.) fruit ripening. Postharvest Biol. Technol 1995 6, 357-365. Rice-Evans, C.; Miller, N.; Paganga, G. Stru cture-antioxidant activ ity relationships of flavonoids and phenolic acids. Free Radical Biol. Med. 1995 20, 933-956. Robbins, R. Phenolic acids in foods: An overview of analytical methodology. J. Agric. Food Chem. 2003 51, 2866-2887. Ronen, G.; Cohen, M.; Zamir, D.; Hirschberg, J. Regulation of carot enoid biosynthesis during tomato fruit development expressi on of the gene for lycopene epsiloncyclase is down regulated during ripening and is elevated in the mutant Delta. Plant Journal 1999 17, 341-351. Sahlin, E; Savage; G.P.; Lister, C.E. Invest igation of the antioxi dant properties of tomatoes after processing. Journal of Food Composition and Analysis 2004 17, 635-647. Saltveit, M. Effect of ethylene on quality of fresh fruits and vegetables. Postharvest Biol. Technol. 1999 15, 279-292. Salveit, M. Effect of 1-methylcycl opropene on phenylpropanoid metabolism, the accumulation of phenolics compounds, a nd browning of whole and fresh-cut iceberg lettuce. Postharvest Biol. Technol. 2004 34, 75-80. Selvarajah, S.; Bauchot, A.D.; and John, P. Intern al browning in cold-s tored pineapples is suppressed by a postharvest applic ation of 1-methylcyclopropene. Postharvest Biol. Technol. 2001 23: 167

PAGE 99

87 Seybold, C.; Frohlich, K.; Bitsch, R.; Otto, K onrad; Bohm, V. Changes in contents of carotenoids and vitamin E during tomato processing. J. Agric. Food Chem 2004 52, 7005-7010. Shahidi, F.; Wanasundara, P. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 1992 32, 67-103. Sisler, E.C., Serek, M. Inhibitors of ethylen e responses in plants at a receptor level. Recent developments Physiol. Plant. 1997, 100, 577-582. Skerget, M.; Kotnik, P.; Hadolin, M.; Rizn er, A.; Marjana, S.; Zeljko, K. Phenols, proanthocyanidins, flavones, and flavonol s in some plant materials and their antioxidant activities. Food Chem. 2005 89, 191-198. Smirnoff, N.; Wheeler, G. Ascorbic acid in plants: Biosynthesis a nd function. Critical Reviews in Plant Sciences, 2000 19, 267-290. Swain, T. and Hillis, W.E. 1959. The phenolic constituents of Purmus domestica I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric 1959 10 63-68. Takeoka, G. R.; Dao, L.; Flessa, S.; Gillesp ie, D. M.; Jewell, W. T.; Huebner, B.; Bertow,D.; Edeler, SE. Processing effect s on lycopene content and antioxidant activity oftomatoes. J. Agric. Food Chem. 2001 49, 3713-3717. Talcott, S.T., Howard, L, Brenes, C.H. An tioxidant changes and sensory properties of carrot puree processed with a nd without periderm tissue. J. Agric. Food Chem 2000 48 1315-1321. Talcott, S.T., Percival, S.S., Pittet-Moore, J. Celoria, C. Phytochemical composition and antioxidant stability of for tified yellow passion fruit ( Passiflora edulis ). J. Agric. Food Chem 2003 48 4, 1315-1321 Thimann, K. Senescence in plants CRC Press: Boca Raton, FL, 1980. Thompson, K.; Marshall, M.; Sims, C.; Sargent, S.; Scott, J. Cultivar, maturity, and heat treatment on lycopen content in tomatoes. J. Food Sci. 2000 65, 791-793. Tomas-Barberan, F.A.; Loaiza-Velarde, J.; B onfanti, A.; Saltveit, M.E. Early woundand ethylene-induced changes in phenylpropanoid metabolism in harvested lettuce. J. Am. Soc. Hortic. Sci. 1997 122, 399-404. United States Department of Agri culture [USDA]. Available online: http://www.usda.gov Last accessed: August, 2004. United States Department of Agriculture-An imal and Plant Health Inspection Service [USDA-APHIS]. 2004. Av ailable online: http://www.aphis.usda.gov Last accessed: March, 2005.

PAGE 100

88 Van de Berg, H.; Faulks, R.; Granado, H. F. ; Hirschberg, J.; Olmedilla, B.; Sandmann,G.; Southon, S.; Stahl, W. The potential for th e improvement of carotenoid levels in foods and the likely systemic effects. J. Sci. Food. Agric. 2000 80, 880-912. Valero, D.; Martinez-Romero, J.M.; Guillen, F.; Castillo, S.; Serrano, M. Could 1-MCP treatment effectiveness in plum be affected by packaging? Postharvest Biol. Technol. 2004 295-303. VERIS Research Information Service. 2000. Carotenoid fact book. VERIS, La Grange, IL. Wilberg, V.; Rodriguez-Amaya, D. HPLC quant itation of major carotenoids of fresh and processed guava, mango, and papaya. Lebensm-Wiss. U. Technol 1995, 28, 478480. Wilson, C.; Shaw, P.; Campbell, C. Determin ation of organic acids and sugars in guava( Psidium guajava L.) cultivars by high-performance liquid chromatography. J. Sci. Food Agric. 1982 33, 777-780. Xie, D.; Dixon, R. Proanthocya nidin biosynthesis-still more questions than answers? Phytochemistry. In Press. Yadava, U. Guava production in Georgia under co ld-protection structure. In: J. Janick (ed.) Progress in new crops ; ASHS Press: Arlington, VA, 1996.

PAGE 101

89 BIOGRAPHICAL SKETCH Flor de Maria Nunez Rueda was bor n on January 4, 1981, and raised in Tegucigalpa, Honduras, Central America. Afte r graduating from high school, she entered the Escuela Agricola Panamericana (University of Zamorano), earning her Bachelor of Science in agroindustry in December 2003. During spring 2003 she performed an internship with Dr. Steve Talcott at the F ood Science and Human Nutrition Department at University of Florida and was offered an assi stantship to pursue he r graduate studies. She earned a Master of Science in food science and human nutrition at the University of Florida in August 2005.


Permanent Link: http://ufdc.ufl.edu/UFE0011873/00001

Material Information

Title: Guava (Psidium guajava L.) Fruit Phytochemicals, Antioxidant Properties and Overall Quality as Influenced by Postharvest Treatments
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011873:00001

Permanent Link: http://ufdc.ufl.edu/UFE0011873/00001

Material Information

Title: Guava (Psidium guajava L.) Fruit Phytochemicals, Antioxidant Properties and Overall Quality as Influenced by Postharvest Treatments
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011873:00001


This item has the following downloads:


Full Text


















GUAVA (Psidium guajava L.) FRUIT PHYTOCHEMICALS, ANTIOXIDANT
PROPERTIES AND OVERALL QUALITY AS INFLUENCED BY POSTHARVEST
TREATMENTS

















By

FLOOR DE MARIA NUNEZ RUEDA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Flor de Maria Nunez Rueda














DEDICATION


To the Lord Jesus, for giving me the strength and motivation to keep pursuing my dreams.

To Sabina, my grandmother, backbone of my family and shimmering light in our lives.

To my nephew Diego, with lots of love from your always spoiling aunt.















ACKNOWLEDGMENTS

I wish to extend my special thanks to my major advisor, Dr. Stephen Talcott, for

his support, advice, friendship and for being a leading example of motivation and hard

work to us. I could not have asked for more. I thank my advising committee, Dr. Susan

Percival and Dr. Donald Huber, for their invaluable assistance and time for my project.

I thank my lab partners and friends, Kim, Kristine, Jorge, Lanier, Chris, Lisbeth,

and Stacy, for their never-ending assistance in my project and for making the work in the

lab such an enjoyable and nurturing experience. Special thanks go to Dr. Joonhee Lee and

soon Dr. to be David del Pozo, for investing their valuable time, sharing their knowledge

and providing guidance, as well as a friendly shoulder to lean on. Graduate school would

definitely not have been as much fun without David's friendship and craziness.

I would have not reached where I am without the unconditional love, support, and

advice of my mother Nora, the biggest example of strength I have seen in a person. I love

her so much. I thank my sister and best friend Tania, for her advice and big sis support

during all these years. Big thanks go to my brother Jesus and my sister-in-law Yalile, for

their warm support and example of perseverance and love, as well as to my little brother

Guille, for having patience with me. Special thanks go to my family in Honduras, Nunez,

Rueda, and Maier, for all their support and best wishes through the distance. Last but not

least, big thanks go to Gary, for sharing his love, patience, jokes and for just adding much

happiness to my graduate school days.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F TA B LE S ............. ..................... ....... ........ ................. ... viii

LIST OF FIGURES ......... ............................... ........ ............ ix

ABSTRACT .............. .......................................... xi

CHAPTER

1 IN TRODU CTION ................................................. ...... .................

2 LITERATURE REVIEW ........................................................................4

2.1 Guava Market and Industrial Applications.................................... .............4
2 .2 G uava F ruit ...................... ............... ............. .. ............................... . 5
2 .2 .1 O rigin .................................................. 5
2 .2 .2 M orphology ....................................................... 5
2.2.3 Postharvest Physiology....................................................... ............... 6
2.3 G uava Phytochem icals......................................... .................................. 8
2.3.1 Phytochemicals.................. ...... ........... ..................8
2.3.3 D dietary Fiber...................................................... 9
2.3.4 Carotenoids and Lycopene ........................................ ...... ............... 10
2.3.5 G uava P olyphenolics ................................................................................ 11
2.4 Postharvest Treatm ents ............................ ............................. ..... ............... 13
2.4.1 Guava Postharvest Handling and Storage ...............................................13
2.4.2 Quarantine Heat Treatments................................. ............... 13
2.4.3 Shelf-life Extension Treatments.......................................................15
2.5 1-M ethylcyclopropene .......................................................... ............... 15
2.5.1 1-M ethylcyclopropene.................................................................... ..... 15
2.5.2 1-MCP Application Conditions.....................................................16
2.5.3 1-M CP on Clim acteric Fruits ........................................ ............... 17
2.5.4 Guava and 1-M CP ...................................... ......... ... .................. 18
2 .6 P o ly p h en o lic s .................................................................................................. 1 8
2 .6 .1 P oly p h en olics ............................. .... .................. .. .. ........ .... ............18
2.6.2 Polyphenolic Classification ............................................... .................. 19
2.6.3 Polyphenolics as Antioxidants ....................................... ............... 23



v









3 EFFECTS OF HOT WATER IMMERSION TREATMENT ON GUAVA FRUIT
PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY.............25

3 .1 In tro du ctio n ...................................... ............................ ................ 2 5
3 .2 M materials and M ethods .............................................................. .....................26
3.2.1 M materials and Processing .............................. ............................ ........ 26
3.2.1.1 Fruit preparation and HW treatment ............................. 26
3.2.1.2 Guava fruit processing .......................................... ............... 27
3.2.2 Chem ical A nalysis............................................... ............................. 27
3.2.2.1 M oisture content determination ....................................... .......... 27
3.2.2.2 Quantification of total soluble phenolics .........................................28
3.2.2.3 Analysis of ascorbic acid by HPLC .............................................28
3.2.2.4 Quantification of antioxidant capacity ..........................................29
3.2.2.5 Analysis of lycopene by HPLC ................................ ... ..................29
3.2.2.6 Quantification of non-lycopene carotenoids .............. .....................30
3.2.2.7 Analysis of polyphenolics by HPLC........................................... 30
3 .2 .3 Q u ality A n aly sis .................................................. ............ .......... .. .. ...3 1
3.2.4 Statistical A analysis ........................................ .......... .... ........ .... 31
3 .3 R esu lts an d D iscu ssion ........................................ ...................... .....................32
3.3.1 C hem ical A naly sis.......... .................................. ...... ........ .... ......... 32
3.3.1.1 M moisture content........................................ ........................... 32
3.3.1.2 Total soluble phenolics.................................. ....................... 33
3.3.1.3 A scorbic acid......................... .. ........ .. .............. .......... 35
3.3.1.4 A ntioxidant capacity ............................................. ............... 36
3.3.1.5 Lycopene and yellow carotenoids ............................... ................38
3.3.1.6 Polyphenolics by H PLC ....................................... ............... 41
3.3.2. Quality A analysis ...................................... .. ................ ............. 49
3.3.2.1 pH and soluble solids ............................................ ............... 49
3.3.2.2 O overall fruit quality ..................................... ........... ............... 49
3 .3 .3 Stag e III F ru it ............................................... ................ 50
3 .4 C o n clu sio n s.................................................. ................ 5 1

4 EFFECTS OF 1-METHYLCYCLOPROPENE ON GUAVA FRUIT
PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY............53

4.1 Introduction........................................................................ ....... ...... 53
4 .2 M materials and M ethods .............................................................. .....................54
4.2.1 M materials and Processing. ............................ ............................ ........ 54
4.2.1.1 Fruit preparation and 1-MCP treatment ............... ............ .....54
4.2.1.2 Guava fruit processing ........................................ ............... 56
4.2.2 Quality Analysis ................ ........... .................. .............. .......... ....56
4.2.2.1 Aesthetic fruit quality assessment during storage..........................56
4.2.2.2 Firmness determination during storage......................................56
4.2.2.3 Titratable acidity, soluble solids and pH......................................56
4 .2 .3 C h em ical A n aly sis........................................................... .....................57
4.2.4 Statistical A analysis ........................... ............. .................. ......57
4 .3 R results and D iscu ssion ........................................... ........................................ 57









4.3.1 Quality Analysis ................ .. .. ...... ...... ........ .. ... ... ................. 57
4.3.1.1 Aesthetic fruit quality during storage.................... ........ ........57
4.3.1.2 Firm ness during storage ........................................ ............... .... 60
4.3.1.3 Titratable acidity, soluble solids, and pH during storage ...............62
4.3.2 C hem ical A nalysis........................................................... ............... 64
4.3.2.1 M oisture content......................................... .......................... 64
4.3.2.2 Total soluble phenolics.................................. ....................... 65
4.3.2.3 A ntioxidant capacity ........................................ ..... ............... 66
4.3.2.3 A scorbic acid ........................... ..... .. ...... .............. ......67
4 .3 .2 .4 L y copene .................................................. .............. ............ 68
4.3.2.5 Polyphenolics by HPLC ....................................... ............... 70
4.3.3 1-M CP Treatment to Boxed Guavas .................................. ............... 73
4 .4 C o n clu sio n s.................................................. ................ 7 6

5 SUMMARY AND CONCLUSIONS.............. ....................................... 78

L IST O F R E F E R E N C E S ...................... .. .. ............. ....................................................80

B IO G R A PH IC A L SK E TCH ..................................................................... ..................89
















LIST OF TABLES


Table p

3-1. Gradient elution running program for HPLC analysis of polyphenolics. ................31

3-2. Tentative identification of guava polyphenolics at 280 nm by HPLC based on
retention time, spectral properties, and comparison to authentic standards.............43

3-3. Guava gallic acid (GA), gallic acid derivatives, and an ellagic acid derivative as
affected by a hot water quarantine treatment and ripening stage...........................45

3-4. Guava procyanidins, other characteristic unknown compounds, and total
polyphenolics by HPLC as affected by a hot water quarantine treatment and
ripening stage. ........................................................................45

3-5. Quality parameters, soluble solids and pH, in guava as affected by a hot water
immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I
and Stage II). Data are expressed as fresh weight basis (mean standard error),
n = 4 ................................................................................4 9

3-6. Phytochemical content and quality parameters in Stage III guavas as affected by
a hot water quarantine treatment at 46 C (mean standard error). n = 4..............51

4-1. Changes in skin coloration in non-treated (control) and 1-MCP-treated guavas
during 1-MCP application and storage at 15 C. ............................................. 59















LIST OF FIGURES


Figure page

2-1. Chemical structure of lycopene, a 40-C open hydrocarbon chain. ..........................10

2-2. Chemical structures of a condensed tannin (A) and hydrolysable tannin (B). A is
a typical condensed tannin composed of catechin and epicatechin; B is a
polygalloyl glucose composed of a glucose core esterified with gallic acid
residues .................................. .......................... .... ...... ........ 21

2-3. Chemical structures of flavonoids apigenin and myricetin, which have
previously reported in guava. They are composed of three pyrane rings ...............22

3-1. Moisture content (%) of ripe guavas as affected by a hot water immersion
treatment (0,15, 30, and 60 min at 46 C) and ripening stage (Stage I and II).
Error bars represent standard error of the mean, n =4........ ......... ............... 33

3-2. Total soluble phenolics (mg/kg DW) in guava as affected by a hot water
immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I
and II). Error bars represent standard error of the mean, n = 4.............................34

3-3. Ascorbic acid content (mg/kg DW) in guava as affected by a hot water
immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I
and II). Error bars represent standard error of the mean, n = 4.............................36

3-4. Antioxidant capacity ([tmol Trolox Equivalents/g DW) in guava as affected by a
hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening
stage (Stage I and II). Error bars represent standard error of the mean, n = 4. ........37

3-5. Lycopene content (mg/kg DW) in guava as affected by a hot water immersion
treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II).
Error bars represent standard error of the mean, n = 4.............................................39

3-6. Non-lycopene carotenoids (mg/kg DW) in guava as affected by a hot water
immersion treatment (0, 15, 30, and 60 min at 460C) and ripening stage (Stage I
and II). Error bars represent standard error of the mean, n = 4.............................40

3-7. HPLC chromatogram of polyphenolic compounds found in guava juice-A) gallic
acid, B) gallic acid derivatives, C) unknown-characteristic guava polyphenolics,
D) procyanidins, and E) ellagic acid derivative. Identification (280 nm) was
done by comparison to authentic standards and spectral properties ......................42









4-1. Firmness (kg) of guavas treated with 1-MCP (1000 nL/L ,100C, 24 h) during
storage at 15 C. Error bars represent the standard error of the mean, n = 5...........61

4-2. Titratable acidity (% citric acid) of guavas treated with 1-MCP during storage at
15 C. Error bars represent the standard error of the mean, n = 5............................63

4-3. Effect of a 1-MCP treatment (1000 nL/L ,100C, 24 h) on guava pH during
storage at 15 C. Error bars represent the standard error of the mean, n = 5 ..........63

4-4. Effect of 1-MCP treatment (1000 nL/L atl00C, 24 h) on guava soluble solids
(Brix) during storage at 15 OC. Error bars represent the standard error of the
m ean, n = 5. ...........................................................................64

4-5. Effect of 1-MCP (1000 nL/L, 100C, 24 h) on total soluble phenolics (mg/kg
DW) in guava. Error bars represent the standard error of the mean, n = 23...........66

4-6. Effect of 1-MCP treatment (1000 nL/L, 100C, 24 h) on guava antioxidant
capacity (pM Trolox equivalents/g DW). Error bars represent the standard error
of the m ean, n = 23 ...................................................... ......................66

4-7. Guava ascorbic acid content (mg/kg DW) as affected by 1-MCP (1000 nL/L,
100C, 24 h). Error bars represent the standard error of the mean, n = 23 ...............68

4-8. Effect of 1-MCP (1000 nL/L ,100C, 24 h) on guava lycopene content (mg/kg
DW). Error bars represent the standard error of the mean, n = 23...........................69

4-9. Guava procyanidin content (mg/kg GAE) as affected by 1-MCP. Error bars
represent the standard error of the mean, n = 23................................................ 71

4-10. Guava ellagic acid derivative content (A) and characteristic polyphenolics
content (B) (mg/kg GAE) as affected by 1-MCP. Error bars represent the
standard error of the m ean, n = 23 ......... .... ............................... ............... 72

4-11. Guava gallic acid content (mg/kg GAE) as affected by 1-MCP. Error bars
represent the standard error of the mean, n = 23................................................ 73

4-12. Firmness (kg) of boxed guavas treated with 1-MCP (1000 nL/L ,100C, 24 h)
during storage at 25 C. Error bars represent the standard error of the mean, n=3..74

4-13. Titratable acidity (% citric acid) of boxed guavas treated with 1-MCP during
storage at 15 C. Error bars represent the standard error of the mean, n = 3...........75

4-14. Effect of 1-MCP treatment (1000 nL/L atl0C, 24 h) on boxed guavas soluble
solids (Brix) during storage at 15 OC. Error bars represent the standard error of
the m ean, n = 3. .................. ......... ...................................... ............................. 76















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

GUAVA (Psidium guajava L.) FRUIT PHYTOCHEMICALS, ANTIOXIDANT
PROPERTIES AND OVERALL QUALITY AS INFLUENCED BY POSTHARVEST
TREATMENTS

By

Flor de Maria Nunez Rueda

August 2005

Chair: Stephen T. Talcott
Major Department: Food Science and Human Nutrition

Guava (Psidium guajava L.) fruit, appealing for its unique tropical flavors, is

considered an excellent source of nutrients and antioxidant phytochemicals, especially

ascorbic acid. Guava's extremely perishable nature and quarantine issues surrounding its

importation somewhat limit its fresh fruit marketability within the US. Additionally,

limited studies have reported resultant changes of postharvest processes on guava's

phytochemicals and antioxidant capacity. The objectives of this study were to evaluate

the effects of two postharvest treatments on phytochemical content, antioxidant capacity,

and overall quality of pink guavas. A hot water (HW) immersion technique was applied

as a potential quarantine treatment, whereas 1-methylcyclopropene (1-MCP), an ethylene

blocker, was applied as a treatment to extend the shelf-life of guavas.

For the HW immersion study, guavas were segregated into three groups according

to their ripeness level-Stage I (yellowish-green skin, firm texture), Stage II (25-50%

yellow skin, semi-firm texture), and Stage III (>75% yellow skin, soft texture)-and









subjected to four HW immersion times (0 (control), 15, 30, and 60 min) at 460C. Fruits

were held at 150C until fully ripe and collected for analysis. For the 1-MCP study, unripe

guava fruit were separated into two groups: Control and 1-MCP (1000 nL/L); both were

held for 24 hours at 10 OC during treatment application. They were stored at 15 C until

full ripeness and collected for analysis. Chemical analyses for both studies included

moisture, total soluble phenolics (TSP), ascorbic acid (AA), antioxidant capacity (AOX),

lycopene, yellow carotenoids, and polyphenolics by HPLC. Quality analysis included

soluble solids, pH, titratable acidity (TA) (1-MCP), and firmness (1-MCP).

A HW treatment up until 30 min at 46C insignificantly affected moisture (92.2%),

TSP (20,600 mg/kg, dry weight (DW)), lycopene (501 mg/kg DW), ascorbic acid (10,800

mg/kg DW), soluble solids (7.70Brix), pH (4.1), yellow carotenoids (46.7 mg/kg DW)

and AOX (133 [M TE/g DW) within each ripening stage. Stage I fruits treated for 60

min presented an enhancement of certain polyphenolics and a decrease in lycopene

content as a response to heat stress. 1-MCP application effectively extended guava shelf-

life for at least 5 days at 15 C, delaying skin yellowing and retaining firmness. Moisture

content (86.4%), TSP (19,700 mg/kg DW), AOX (133 [M Trolox equivalents/g DW),

pH (4.1), soluble solids (7.8 Brix), and TA (0.29 % citric acid) were unaffected by 1-

MCP. Although ascorbic acid (6,145 mg/kg DW) and lycopene content (419 mg/kg DW)

were significantly higher in 1-MCP treated fruit, similarly to other phytochemicals,

effects were independent of ethylene inhibition. A 1-MCP and HW quarantine treatment

up to 30 min can be applied to fresh guava without detrimental effects on phytochemical

content, antioxidant capacity, and fruit quality, therefore extending its fresh fruit market

window.














CHAPTER 1
INTRODUCTION

The consumption trend of fresh tropical fruits and their products is increasing

steadily due to consumer's education on their exotic flavors, nutritive value, and

phytochemical content with potential health effects (Food and Agriculture Organization

[FAO], 2004). Guava fruit (Psidium guajava L.), an exotic from the tropics characterized

by its appealing flavor and aroma, has been catalogued as one of the most nutritious fruits

due to its high content of phytochemicals, specially ascorbic acid (United States

Department of Agriculture [USDA], 2004). Guava's importation as a fresh fruit is

somewhat limited within the US for two main reasons: quarantine issues surrounding its

importation and its highly perishable nature.

Guavas are considered excellent sources of antioxidant phytochemicals, which

include ascorbic acid, carotenoids, antioxidant dietary fiber, and polyphenolics. After

acerola cherries, guava has reported the second highest concentration of ascorbic acid

(ranging from 60-1000 mg/100 g) of all fruits (Mitra, 1997). Carotenoids, which are

yellow, red, and orange pigments, have demonstrated many beneficial health effects

related to their antioxidant properties (Wilberg and Rodriguez-Amaya, 1995). Guava's

major carotenoid, lycopene, is responsible for the pink coloration in pink guava's flesh

(Mercadante et al., 1999). Polyphenolics from fruits and vegetables are widely

investigated because of their role as chemoprotective agents against degenerative

diseases, antimutagenic effects, and antiviral effects, among others (De Bruyne et al.,

1999; Gorinstein et al., 1999; Robbins, 2003). Currently, research on identification and









quantification of ripe guava polyphenolics is very limited, and information is still unclear

as to the type and concentration of individual compounds present in the fruit.

Various postharvest chemical and heat applications exist as quarantine treatment

for fresh fruit importation into the US, which may also preserve appearance and table

quality of various fruits (Lurie, 1998). Thermal applications are gaining more popularity

due to consumer's demand to ameliorate the use of chemicals. In the case of guava, an

established quarantine treatment still does not exist for importing it into the US (Gould

and Sharp, 1992; USDA-Animal and Plant Health Inspection Service [USDA-APHIS],

2004), potentially due to the lack of studies that demonstrate a beneficial effect.

Additionally, few studies report resultant changes that these processes may have on

phytochemical content, nutrient stability, and antioxidant capacity of the fruit, either in a

beneficial or detrimental way.

Perishability is one the main issues in postharvest handling and marketing of fresh

fruits and vegetables. In the case of guava fruit, its short shelf life (7 to 10 days) limits

somewhat its marketability. Numerous technologies have been developed as means to

extend their shelf-life and eating quality, some of which include modified atmospheres,

polymeric films, irradiation, or chemical treatments (Mitra, 1997). A recently developed

shelf-life extension tool is the application of a gaseous organic compound, 1-

methylcyclopropene (1-MCP), as an ethylene blocker, delaying or inhibiting ripening on

ethylene-sensitive commodities (Blankenship and Dole, 2003). Currently, limited studies

exist on the impact of 1-MCP on phytochemical content of fruits and vegetables in

general, and their relationship with ethylene inhibition responses.









This study evaluated the effects of two post-harvest treatments, a thermal

quarantine hot water treatment and a 1-1-MCP application, on phytochemical content,

antioxidant properties, and fruit quality. It was hypothesized that HW treatment at 46 C

for short periods would not stress guava fruit or affect phytochemical content, antioxidant

properties, and quality. A 1-MCP treatment would affect guava ripening and

characteristics associated with it, however phytochemicals and antioxidant properties

would remain unaffected. The specific objectives of this study were

Post-harvest treatment-Hot Water Immersion: To evaluate the effects of a thermal

quarantine treatment using a hot water immersion technique on the phytochemical

content, antioxidant capacity, and overall quality of guava fruits.

Post-harvest treatment-1-MCP: To evaluate the effects of 1-MCP as a postharvest

treatment for extention of fresh guava shelf-life and to determine these effects on

the phytochemical content, antioxidant capacity, and overall quality of guava

fruits.














CHAPTER 2
LITERATURE REVIEW

2.1 Guava Market and Industrial Applications

Exotic or minor tropical fruits, which include guava, carambola, durian, lychee,

mangosteen, passionfruit and rambuttan have undergone a significant increase in both

volume and value in recent years. Their production continues to steadily increase and is

estimated to have reached 14.9 million metric tons (23% of total global output of tropical

fruits) in 2002 and US total import volumes were 176,000 tons for 2003 (FAO, 2004).

Fresh fruit market in general is growing in the US chiefly due to an increase in

consumption demand and the development of technologies to preserve fruit eating quality

and prolong shelf-life (Kipe, 2004).

Guava as an import is divided into four categories according to the National

Agriculture and Statistics Service [NASS]: preserved or prepared, paste and puree, jam,

and dried. Brazil was the leader for guava imports into the United States in 2003,

followed by Dominican Republic, Mexico, India, and Costa Rica. Within the US,

commercial producers are Hawaii, southern Florida (Gould and Sharp, 1992), and

southern California. Hawaii is the main grower, with 530 harvested acres and a utilized

production volume of 6.7 million pounds in 2003. The local production in Florida and

Hawaii is hampered by the Caribbean fruit fly, causing serious economic losses if not

controlled adequately (NASS, 2004). Currently, external or internal import of fresh guava

fruit is not possible; this is mainly attributed to the tropical fruit fly and guava's very

short shelf life.









For industrial applications, guava is one of the easiest fruits to process, since the

whole fruit may be fed into a pulper for macerating into puree (Boyle, 1957). It is

physically and biochemically stable in relation to texture or pulp browning during

processing (Brasil et al., 1995). It can be processed into a variety of forms, like puree,

paste, jam, jelly, nectar, syrup, ice cream or juice. Within the United States processing

industry, it is gaining popularity in juice blends due to its exotic flavor and aroma.

2.2 Guava Fruit

2.2.1 Origin

Guava (Psidium guajava) is an exotic fruit member of the fruit family Myrtacea.

Guava, goiaba or guayaba are some of the names given to the "apple of the tropics",

popular for its penetrating aroma and flavor. Its place of origin is quite uncertain,

extending in an area from southern Mexico through Central and South America.

Currently, its cultivation has been extended to many tropical and subtropical parts of the

world, where it also thrives well in the wild (Morton, 1987;Yadava, 1996; Mitra, 1997).

2.2.2 Morphology

Guava shape ranges from round, ovoid, to pear-shaped, and with an average

diameter and weight ranging from 4-10cm and 100-400g respectively (Mitra, 1997).

Classified as a berry, guava is composed by a fleshy mesocarp of varying thickness and a

softer endocarp with numerous small, hard yellowish-cream seeds embedded throughout

it (Malo and Campbell, 1994; Marcelin et al., 1993). Guava pulp contains two types of

cell-wall tissues: stone cells and parenchyma cells. Stone cells are highly lignified woody

material responsible for a characteristic sandy or gritty feeling in the mouth when the

fruit is consumed; due to their nature, they are resistant to enzymatic degradation. They

account for 74% of the mesocarp tissue, while the endocarp is rich in parenchyma cells,









which give it a softer texture. (Marcelin et al., 1993). Exterior skin color ranges from

light green to yellow when ripe and its pulp may be white, yellow, pink, or light red.

Unripe guava fruit are hard in texture, starchy, acidic in taste and astringent, due to its

low sugar and high polyphenol content. Once it ripens, the fruit becomes very soft, sweet,

non-acidic, and its skin becomes thin and edible (Malo and Campbell, 2004; Mitra,

1997). Many guava cultivars exist today, however they can be broadly classified as pink

or white. Seedless cultivars are available in many countries, which have a great potential

to become popular in the US in the future (Yadava, 1996).

2.2.3 Postharvest Physiology

Ripening and factors associated with it in climacteric fruits is regulated by ethylene

synthesis. Ethylene (C2H4) is a naturally-produced, gaseous growth regulator associated

with numerous metabolic processes in plants (Mullins et al., 2000). It is produced from

L-methionine via 1-aminocyclopropane-l-carboxylic acid (ACC) synthase in a complex

signal transduction pathway, which is still widely researched today (Salveit, 1998;

Mullins et al., 2000). All plants produce ethylene, but only climacteric fruits and

wounded or stressed tissue produce sufficient amounts to affect other tissues. In

climacteric fruits, ethylene stimulates its own biosynthesis at the start of ripening,

enhancing its production until reaching saturation levels (Salveit, 1999). Stresses such as

chill injury, heat shock (Cisneros-Zevallos, 2003) or disease (Mullins et al., 2000), can

induce ethylene production and therefore enhance fruit ripening, and the factors

associated with it.

Studies evaluating respiratory patterns of guava demonstrated a climacteric

response as increased carbon dioxide corresponded to increased ethylene production

(Akamine and Goo, 1979; Mercado-Silva et al., 1998; Bashir and Abu-Goukh, 2002).









Guavas have a rapid rate of ripening, therefore a relatively short shelf life ranging from 3

to 8 days depending on the variety, harvest time, and environmental conditions (Reyes

and Paull, 1995; Basseto et al., 2005). Ethylene production and respiration (CO2

production) increases after the first day of harvest, at the start of ripening. Guava reaches

its climacteric peak between day 4 and 5 post-harvest (mature-green harvested fruits) and

then declines (Akamine and Goo, 1979; Bashir and Abu-Goukh, 2002).

As a guava ripens, total soluble solids and total sugars increase in both the peel and

pulp, whereas titratable acidity declines after reaching its climacteric peak of respiration.

In general, climacteric fruits undergo considerable changes in sugar content during

ripening, where starch and sucrose are broken down into glucose (Bashir and Abu-

Goukh, 2002). Moisture loss in guava, especially in tropical climates, can also be

substantial resulting in up to 35% weight loss (Mitra, 1997) that corresponds to loss of

postharvest quality and consumer acceptability. Ascorbic acid content is at its maximum

level at the mature-green stage and declines as the fruit ripens in both white and pink

guavas (reviewed by Bashir and Abu-Goukh, 2002), and may also be a function of

postharvest handling. Lycopene synthesis in pink guavas is enhanced during ripening. In

the case of tomatoes, once lycopene is accumulated, the respiration rate decreases

(Thimann, 1980). Total fiber content decreases significantly during ripening, from 12 to

2g/100g, and it is hypothesized that is closely be related to the activity of certain enzymes

(El-Zoghbi, 1994). Abu-Goukh and Bashir (2003) studied the activities of some cell wall

degrading enzymes in both pink and white guava and showed that pectinesterase (PE)

activity increased until reaching its climacteric and latter decreased, whereas

polygaracturonase (PG) and cellulase increased as the fruit ripened in correspondence to









fruit softening. Increase in polyphenoloxidase (PPO) activity was also reported with

ripening and a decrease in polyphenolics, which be the responsible for the reduction of

astringency (Mowlah and Itoo, 1982).

Visually, the ripeness level of guava can be characterized by its skin color ranging

from a dark green when unripe to a bright yellow or yellow-green at full ripeness.

However, determination of ripeness can be misleading for some varieties and may be

combined with a simple test for specific gravity, by placing fruit in water to determine if

it sinks (unripe) or floats (ripe) to obtain a clearer picture of the degree of fruit ripeness

(Reyes and Paull, 1995). Objective determination of skin color has also been used to

predict ripeness, with L*, a* and hue angles of 65.93, 15.92, and 110.920 respectively

indicating a mature, yellow fruit (Mercado-Silva et al., 1998). In combination with fruit

texture, these simple assays can provide an adequate estimation of the stage of fruit

ripeness.

2.3 Guava Phytochemicals

2.3.1 Phytochemicals

Phytochemicals may be defined as biologically active compounds present in foods,

nutritive or non-nutritive, which prevent or delay chronic diseases in humans and

animals. They may also be defined as food ingredients which provide health benefits

beyond their nutritional value (reviewed by Ho et al., 1992). The importance of

phytochemicals has grown in recent years due to consumers increased awareness of

health beneficial effects. The main phytochemicals found in guava are ascorbic acid,

antioxidant-containing dietary fiber, carotenoids, and polyphenolics.

2.3.2 Ascorbic Acid and Other Antioxidant Vitamins









Guavas are considered an outstanding source of ascorbic acid (AA), three to six

times higher than the content of an orange and after acerola cherries it has the second

highest concentration among all fruits. The AA content in guava varies from 60 to 100

mg/100 g in some cultivars, and from 200 to 300 mg/100g in others, while higher reports

range from 800 to 1000 mg/100g. Mitra (1997) mentions that AA content is more

influenced by the fruit's variety than by its ripening stage and storage conditions. Within

the fruit, AA is concentrated in the skin, followed by the mesocarp and the endocarp

(Malo and Campbell, 1994). As a water-soluble vitamin, it is highly susceptible to

oxidative degradation and is often used as an index for nutrient stability during

processing or storage (Fennema, 1996). Guava was also found to contain alpha-

tocopherol (vitamin E) at nearly 1.7 mg/100g (Ching and Mohamed, 2001), which is an

important fat-soluble dietary antioxidant.

2.3.3 Dietary Fiber

Dietary fiber in fruits and vegetables has been associated with a reduction in colon

and other cancer risks. Soluble fiber content is generally associated with a reduced risk of

cardiovascular disease. In a study done to a number of tropical fruits guava showed the

highest content of total and soluble dietary fibers with values of 5.60 and 2.70g/100g

respectively (Gorinstein et al., 1999). Total and soluble fiber present in guava is

extraordinarily high in concentration as compared not only to tropicals, but all fruits and

vegetables. Fiber from guava pulp and peel was tested for antioxidant properties and

found to be a potent source of radical-scavenging compounds, presumably from the high

content of cell-wall bound polyphenolics (2.62-7.79% w/w basis) present in each fiber

isolate (Jimenez-Escrig et al., 2001).









2.3.4 Carotenoids and Lycopene

Carotenoids are yellow, red, and orange pigments abundant in a wide variety of

fruits and vegetables. Due to their antioxidant properties, carotenoids have shown

beneficial health effects in cancer inhibition, immuno-enhacement, and prevention of

cardiovascular diseases (Wilberg and Rodriguez-Amaya, 1995). The most important

carotenoids which provide oxidative protection are a-carotene, P-carotene, lutein,

lycopene, zeaxanthin, and P-cryptoxanthin (VERIS, 2000). A well-established function is

the vitamin A antioxidant activity of some of carotenoids, including a-carotene, P-

carotene, 0-cryptoxanthin. Carotenoids are a class of structurally related 40-carbon

compounds (two 20-carbon tails) which consist of eight repeating isoprene units (Van de

Berg et al., 2000). Lycopene, the major carotenoid present in guava (Mercadante et al.,

1999), is a 40-C open chain hydrocarbon containing 11 conjugated and 2 non-conjugated

double bonds arranged linearly (Figure 2-1). Currently, High Performance Liquid

Chromatography (HPLC) is the preferred procedure for carotenoid analysis.








Figure 2-1. Chemical structure of lycopene, a 40-C open hydrocarbon chain.

Lycopene has received considerable attention recently due to diverse in-vivo and

in-vitro studies reporting the effect of dietary lycopene in reduction in the risk of prostate

cancer and coronary heart disease (Rao and Agarwal, 1999). Lycopene has reported a

superior antioxidant activity in relation to lutein or P-carotene, due to its conjugated

double bonds (reviewed by Lin and Chen, 2003). Currently, tomatoes and tomato-based









products are the main source of dietary lycopene. Ripe fresh tomatoes have a lycopene

content ranging from 4 to 8 mg/100g (Abushita et al., 2000; Leonardi et al., 2001;

Seybold et al., 2004). During tomato processing, some authors have reported lycopene

and other carotenoid reduction (Takeoka et al., 2001; Sahlin et al., 2004), while others

report an enhancement, increased bioavailability and antioxidant capacity of these

compounds (Dewanto et al., 2002; Seybold et al., 2004).

Lycopene content in guava 'Beaumont' variety has been found to be about 5-7

mg/100g fruit. Mercadante and partners (1999) isolated sixteen carotenoids from guava,

of which thirteen were reported as guava carotenoids for the first time. In another study

made to Brazilian guavas, the P-carotene concentration in ripe fruits ranged from 0.3

mg/100g to 0.5 mg/100g; while the lycopene concentration ranged from 4.8 mg/100g to

5.4 mg/100g (Wilberg and Rodriguez-Amaya, 1995).

2.3.5 Guava Polyphenolics

Polyphenols are the most abundant phytochemicals in our diets, and fruits are the

main contributors (Jimenez-Escrig et al., 2001). Currently, limited studies exist on the

identification and quantification of guava polyphenolics. Gorinstein et al. (1999)

conducted a comparative study between several tropical and subtropical fruits and found

guava to be among the top three investigated for concentrations of gallic acid (.374

mg/100g), total phenolics (4.95 mg/100g), and the highest total and soluble dietary fiber

of the fruits investigated. Guava are somewhat unusual in their flavonoid polyphenolic

content as well, with significant levels of myricetin (55 mg/100g) and apigenin (58

mg/100g) present in edible tissues, but do not contain the more commonly found

flavonoids quercetin and kaempferol (Miean and Mohamed, 2001) that are abundant in









other fruits and vegetables. Misra and Seshadri (1967) identified procyanidins, or

condensed tannins in both white and pink cultivars, concentrated in the skin and seeds,

but very little in the pulp. Also, free ellagic acid was isolated in both varieties (0.2

mg/100g in pink, 0.05 mg/100g in white). In the whole guava, total phenolics are

concentrated on the peel, followed by the pulp (Bashir and Goukh, 2002). For processed

products, though, location of polyphenolics does not matter since the entire fruit with

peel is fed into a pulper.

Although limited information is existent, it has been confirmed that guava

polyphenolics decrease and undergo considerable changes during maturation and

subsequent ripening (Mowlah and Itoo, 1982; Itoo et al., 1987; Bashir and Goukh, 2002).

According to work conducted by Itoo et al. (1987) immature, underdeveloped guava

contains approximately 65% condensed tannins of its total polyphenols, which decrease

dramatically as the fruit grows and develops. According to Mowlah and Itoo (1982) in

both pink and white varieties both "non tannin phenolics' (simple phenolics, monomeric

anthocyanins, catechins, and leucoanthocyanins) and "tannin phenolics" (hydrolysable

and condensed tannins) decrease during ripening. However, at full-ripeness non-tannin

phenolics (76 and 80% of total phenolics for pink and white respectively) contents are

higher than tannin phenolics (24 and 20%). The decrease in astringency during guava

ripening has been attributed to an increase in polymerization of condensed tannins to

form an insoluble polymer and hydrolysis of a soluble/astringent arabinose ester of

hexahydroxydiphenic acid, a precursor of ellagic acid (Goldstein and Swain, 1963; Misra

and Seshadri, 1967; Mowlah and Itoo, 1982; Itoo et al., 1987). Confirming these results,

an increase in free ellagic acid during ripening has been reported (Goldstein and Swain,









1963; Misra and Seshadri, 1967). Currently, limited information on individual

polyphenolic compounds found in ripe fruits is existent.

2.4 Postharvest Treatments

2.4.1 Guava Postharvest Handling and Storage

Depending on its further use (fresh or processed) postharvest conditions for guava

may vary; however its short shelf life is a recurring pressure for growers, packers, and

processors. Due to its delicate nature, it is carefully hand-harvested while still green, and

immediately stored at cool temperatures. In Florida, guavas are usually stored at

temperatures between 9 to 12 C (personal communication, Sardinia, 2004) due to their

sensivity to chill injury. They are typically shipped from packing houses in a mature-

green stage (yellowish-green skin, firm), after harvesting at optimum fruit size. Reyes

and Paull (1995) reported less disease incidence in mature green guavas stored at 150C as

compared with fruit that were quarter- and half-yellow under the same conditions.

Additionally, 15C was determined to be an optimum holding temperature prior to

processing, since it allowed gradual ripening of mature-green fruit while delaying

deterioration of quarter-yellow and half-yellow fruit. Fruit stored at 5C did not ripen and

developed skin bronzing after two weeks in storage, as a consequence of chill injury.

2.4.2 Quarantine Heat Treatments

Various thermal and chemical quarantine treatments exist for fresh tropical fruits

entering the US established by US Department of Agriculture-Animal and Plant Health

Inspection Service-Plant Protection and Quarantine (USDA-APHIS-PPQ). They are set to

ensure disinfectations from pests, insects, larvae, eggs or fungus for fresh produce

importation from other countries and other US states or territories. During the past years,









there has been an increasing interest in the use of thermal treatments as a measure of

control, due to consumer demand to ameliorate the use of chemicals. Currently, there are

three methods to heat commodities: hot water, vapor heat, and hot air (reviewed by Lurie,

1998). Hot water dips are effective for both fungal pathogen control and for

disinfestations of insects, needing a longer time for the latter one, since the internal core

of the fruit and not just the surface needs to be brought up to the required temperature.

Procedures have been developed to disinfest a number of tropical and subtropical fruits

from various species of fruit fly (reviewed by Paull, 1994). The USDA-APHIS-PPQ

treatment manual includes treatment schedules that must be followed to import fruit into

the US. In the case of mango, this includes a 46 C hot water dip that disinfects mangoes

with possible fruit fly contamination. Currently, no established treatment schedule exists

for guava by the US government (USDA-APHIS, 2004).

Guava is major host for many tephritid fruit fly species, including the Caribbean

Fruit fly, Anastrepa suspense, which has been present in Florida for several years. Local

guavas therefore, cannot be exported from Florida to other citrus-producing states,

somewhat limiting their market as fresh fruit (Gould and Sharp, 1992). Gould and Sharp

(1992) conducted studies to determine the suitability of hot-water (HW) immersion as a

quarantine treatment to disinfest pink guavas of Caribbean fruit flies and to asses its

effect on overall fruit quality. As compared to other tropicals, such as mangos, a shorter

immersion time was required to kill larvae in guava due to the size of the fruits used

(approx. 90g). The storage temperature was apparently more important than a HW

treatment to retain fruit quality. Guavas held at 24 C ripened within 7 days and guavas

held at 10 C ripened within 11 to 18 days regardless of the length of the HW treatment.









Probit statistical analysis estimated a probit 9 (99.9968%) mortality at 31 min at 46.1 +

0.5 C for quarantine security, which did not affect fruit quality. This has been one of few

studies done on guava HW treatment application. Further investigations are needed in

order to obtain a quarantine schedule for guava.

2.4.3 Shelf-life Extension Treatments

Various treatments exist to extend the shelf-life of horticultural commodities.

Storage under modified atmosphere (MA), packaging (MAP) or coating in polymeric

films (cellulose or camouba-based emulsions) have been shown to be effective on many

commodities, including guava. In most cases, respiration and ethylene production are

reduced, delayed or inhibited, inhibiting ripening and characteristics associated with it

(Mitra, 1997). Other shelf-life extensors which act directly on ethylene binding sites are

called ethylene inhibitors or ethylene blockers. Some compounds employed as ethylene

inhibitors for both floricultural and horticultural commodities include: carbon dioxide,

silver thiosulfate (STS), aminoethoxyvinylglycine (AVG). 2,5-norbornadiene (2,5-NBD),

and diazocyclopentadiene (DACP) (Blankenship and Dole, 2003). 1-

Methylcyclopropene is an ethylene blocker which is gaining popularity because of its

action in a broad range of produce and its practicality of use.

2.5 1-Methylcyclopropene

2.5.1 1-Methylcyclopropene

1-Methylcyclopropene (1-MCP) is a recently developed tool used to extend the

shelf life and quality of ethylene-sensitive plant produce and research the role of ethylene

responses. It is an active organic compound (C4H6) which is thought to interact with

ethylene (C2H4) receptors so that ethylene cannot bind and take action. Its affinity for the

receptor site, ethylene binding protein (EBP) (Mullins et al., 2000), is about ten times









greater than that of ethylene. Its origin comes from background work done by Sisler and

Blankenship on cyclopropenes, breakdown products of diazocyclopentadiene (DACP), a

known ethylene inhibitor. 1-MCP development resulted in good practical use because it

is less volatile than cyclopropene itself and is able to act lower concentrations (ppb range).

Commercialization of 1-MCP for ornamentals is sold under the trade name EthylBloc

by Floralife, Inc., whereas for edible crops it is sold under the trade name SmartFresh

by AgroFresh, Inc. Both products are generally regarded as safe, non-toxic, and

environmentally friendly by the Environmental Protection Agency [EPA]. In 2000 it was

approved for use in edible crops, while in 2002 it was exempted from the requirement

from tolerance from residues (EPA, 2004). 1-MCP is usually employed as a powder that

forms a gas when mixed with water (reviewed by Blankenship and Dole, 2003).

2.5.2 1-MCP Application Conditions

Temperature, treatment duration, concentration, and type of commodity are key

variables affecting the efficacy of a 1-MCP treatment. Many studies have demonstrated a

direct relationship between them. At standard pressure and temperature, 1-MCP is

released in approximately 20 to 30 min; however, at lower temperatures release might

take longer (reviewed by Blankenship and Dole, 2003). DeEll et al. (2002) demonstrated

that treatment applied at higher temperatures in apples required less exposure time; it has

been hypothesized that lower temperatures might lower the affinity for the binding site of

1-MCP in apples (Mir et al., 2001). Effective concentrations vary widely, depending

primarily on the commodity. Concentrations of between 1 and 12 iL/L have been

effective in blocking ethylene in broccoli. For green tomatoes, higher concentrations for

short durations have been effective. In most studies, treatment duration has ranged from

12 to 24 h, in order to achieve full response (reviewed by Blankenship and Dole, 2003).









Multiple or single applications during a might be experimentally significant or not,

depending on the commodity. Multiple applications on 'Red Chief apples were more

beneficial (Mir et al., 2001). Plant maturity and time of harvest must be also considered,

whereas the more perishable the crop, the more quickly after harvest 1-MCP should be

applied (reviewed by Blankenship and Dole, 2003).

2.5.3 1-MCP on Climacteric Fruits

Various studies have been conducted on the effects of 1-MCP on climacteric fruits,

including commodities such as apples, pears, stonefruits, bananas, melons, citrus, and

mangos. Reports are variable, depending on the commodity or even on the species. In

general, as a response on ethylene inhibition, increases in respiration rates have been

reduced or delayed. In avocado, a highly perishable commodity, 1-MCP treatment

reduced significantly the rate of softening by suppressing enzyme activities and helped

retain green coloration at full ripeness stage (Jeong et al., 2002). Soluble solids content

(SSC) has been reported higher in 1-MCP- treated pineapples, papaya, and apples; while

in mangos, oranges, apricots, and plums it was unaffected. Reports on the effect of 1-

MCP on titratable acidity, have been very mixed (reviewed by Blankenship and Dole,

2003). In experiments with apples, peaches, and nectarines an inhibition in ethylene

production, softening, and titratable acidity was reported (Fan et al., 1999; Liguori et al.,

2004). Jiang et al. (2001) found that 1-MCP applied preharvest to strawberries, a non-

climacteric commodity, lowered ethylene production and maintained fruit color, but it

lowered anthocyanin production. In greenhouse tomatoes, 1-MCP delayed the onset of

ripening-associated changes but it did not alter significantly final values of lycopene,

firmness, color, and PG activity (Mostofi et al., 2003).The effects of 1-MCP on fruit

disorders and diseases has been varied, depending on the species. In some, cases, it has









alleviated disorders, like reducing superficial scald in apples (Fan et al., 1999) or

decreasing internal flesh browning in apricots and pineapples (Dong et al., 2002,

Blankenship and Dole, 2003). In other instances, a lower phenolic content in 1-MCP

treated strawberries accounted for increased disease incidence (Jiang at al., 2001). In

papaya and custard apple, 1-MCP has been related to a higher incidence of external

blemishes (reviewed by Blankenship and Dole, 2003). Limited studies, however, exist on

the impact of 1-MCP on phytochemical content of fruits and vegetables in general.

2.5.4 Guava and 1-MCP

Literature on guava and 1-MCP is currently very limited. Basseto and partners

(2005) demonstrated the effectiveness of application of 1-MCP to 'Pedro Sato' guavas as

well as a direct relation between concentration and exposure time. Fruit were subjected to

different concentrations (100, 300, 900 nL/L) of 1-MCP and exposure times (3, 6, 12h) at

250 C, to improve the shelf-life of guavas marketed at room temperature. In general,

treated fruit had a storage life twice as long as non-treated fruit (5 vs. 9 days

respectively). Positive effects on skin color retention and respiration rates were observed.

Quality parameters such as SSC, ascorbic acid, and firmness were not influenced by 1-

MCP in all treatments. However, fruit treated with 900 nL/L for more than 6h did not

ripen at all and treatments at 100 nL/L were ineffective. Treatments at 300 nL/L for 6 or

12 h and at 900 nL/L for 3 showed the best results, and were equally effective.

2.6 Polyphenolics

2.6.1 Polyphenolics

Phenolic compounds are bioactive substances synthesized as secondary metabolites

by all plants connected to diverse functions such as nutrient uptake, protein synthesis,

enzyme activity, photosynthesis, and as structural components (reviewed by Robbins,









2003). They are considered very important in foods not only because of their influence in

sensory properties, but also for their potential health benefits related to their antioxidant

activity (Fennema, 1996). Recent studies have shown that polyphenolics of fruits and

vegetables improve lipid metabolism and prevent the oxidation of low-density lipoprotein

cholesterol (LDL-C), which hinders the development of artherosclerosis (reviewed by

Gorinstein et al., 1999).

The term 'phenolic' or 'polyphenol' may be identified chemically as a substance

which possesses an aromatic ring attached to one or more hydroxy substituents, and may

include functional derivatives such as esters, methyl esters, glycosides or others

(reviewed by Ho et al., 1991). Approximately 8,000 naturally occurring phenolic

compounds have been identified. Phenolic plant compounds, including all aromatic

molecules from phenolic acids to condensed tannins, are products of a plant aromatic

pathway, which consists of three main sections: the shikimic acid pathway which

produces the aromatic amino acids phenylalanine, tyrosine and tryptophan that are

precursors of phenolic acids; the phenylpropanoid pathway which yields cinnamic acid

derivatives that are precursors of flavonoids and lignans; and the flavonoid pathway

which produces various flavonoid compounds (reviewed by De Bruyne et al., 1999).

Phenolic acids like caffeic, gallic, coumaric, chlorogenic and ferulic acids occur widely

in the shikimic acid pathway of plant tissues, which begins with the condensation of

phosphoenolpyruvate and erythrose 4-phosphate (reviewed by Fennema, 1996).

2.6.2 Polyphenolic Classification

Phenolics can be broadly classified in simple phenols and polyphenols, based on

the number of phenol subunits present. Simple phenols, known as phenolic acids, may be

classified according to their carbon frameworks into two groups: 1) Hydroxylated









derivatives of benzoic acid (C6-C1), which are very common in free state, as well as

combined as esters or glycosides. This group includes gallic acid, the main phenolic unit

of hydrolysable tannins. 2) Hydroxylated acids derived from cinnamic acid (C6-C3),

which occur mainly sterified and are very rare in free state. This group includes

coumaric, caffeic, and ferulic acid (reviewed by Robbins, 2003; reviewed by Skerget,

2005). Both hydroxybenzoic and hydroxycinnamic acids are derived primarily from the

phenylpropanoid pathway (Brecht et al., 2004). Polyphenols possessing at least two

phenol-phenol subunits include the flavonoids, whereas compounds possessing three or

more subunits are referred to as tannins (Robbins, 2003).

Plant polyphenolics are commonly referred to as "vegetable tannins" (Fennema,

1996). Tannins are high molecular weight (Mr > 500) compounds containing many

phenolic groups (Hagerman et al., 1998), and are classified according to their chemical

structure into condensed and hydrolysable tannins (Fennema, 1996). Condensed tannins

are oligomers or polymers composed of flavan-3-ol-nuclei, and have a lower molecular

weight than hydrolysable tannins, which are polyesters of gallic and

hexahydroxydiphenic acid (gallotannins and ellagitannins, respectively). There is an

additional class of polyphenols called "complex" tannins, in which a flavan-3-ol unit is

connected to a gallo- or ellagitannin through a C-C linkage (reviewed by De Bruyne et al.,

1999).

Condensed tannins are commonly known as procyanidins or polyflavonols.

Procyanidins are widespread in nature and more researched than hydrolysable tannins.

They consist of chains of flavan-3-ol-units, which are commonly sterified, mainly with

gallic acid units (ex: epigallocatechin gallate in tea). Specifically, the flavan-3-ols which










are condensed tannin building blocks are (+)-catechin (2,3-trans) and (-)-epicatechin (2,3-

cis). Flavan-3-ols are derived from a branch of the anthocyanin and other flavonoids

pathway, of which elucidation is still unclear (reviewed by Xie and Dixon, 2005).

Structural variability among proanthocyanidins depends on hydroxylation,

stereochemistry at the three chiral centers, the location and type of interflavan linkage,

and terminal unit structure. A classical assay for proanthocyanidins consists of an acid

hydrolysis, where the terminal units of the molecules convert to colored anthocyanidins.

Condensed tannins can be classified into many subgroups, of which the procyanidins is

the most common one (reviewed by De Bruyne et al., 1999). In guava, it has been found

procyanidins to compose the major portion of guava polyphenolics (Mowlah and Itoo,

1982), however further identifications have been limited.

oO,0 A B c B
A G G
m3 ____ =XGOG


H HO
OH G>G G




OHcondensed tannins. They are low-molecular weight compounds, with the characteristic
Hlo 4_ 0 IJI

MO OH 0


Figure 2-2. Chemical structures of a condensed tannin (A) and hydrolysable tannin (B). A
is a typical condensed tannin composed of catechin and epicatechin; B is a
polygalloyl glucose composed of a glucose core esterified with gallic acid
residues

Flavonoids are a diverse group of polyphenolics which can polymerize to form

condensed tannins. They are low-molecular weight compounds, with the characteristic

flavan nucleus and composed of three phenolic (pyrane) rings. The major flavonoid

classes include flavones, flavanones, flavonols, catechins (flavanols), anthocyanidins,












isoflavones, and chalcones. Most flavonoids occur naturally as flavonoid glycosides.

Quercetin, rutin, and robinin are the most common glycosides in the diet, which are then

hydrolyzed by intestinal flora to produce the biologically active aglycone (sugar-free

flavonoid) (reviewed by Cook and Samman, 1996). In guava, considerable amounts of

the flavonoids apigenin and myricetin have been found (Miean and Mohamed, 2001).



OH
OH OH

0nH v I) YK H OH
'OH V1YYOH
H 0 OH 0
Apigenin Myrictin


Figure 2-3. Chemical structures of flavonoids apigenin and myricetin, which have
previously reported in guava. They are composed of three pyrane rings.

Hydrolysable tannins are characterized mainly by containing a varied number of

gallic acids, their major phenolic unit (Grundhofer et al, 2001). Structural variation

among them is caused by oxidative coupling of neighboring gallic acid units or by

oxidation of aromatic rings. Some species of hydrolyzable tannins produce either

gallotannins or ellagitannins, while others produce mixtures of gallo-, ellagi- and

condensed tannins. Pentagalloylglucose has been identified as the precursor for many

complex tannin structures. Gallotannins consist of a central polyol, such as glucose,

surrounded by several gallic acids units. The ellagitannins are a more complex group of

tannins also derived from pentagalloylglucose by oxidative reactions between gallic acid

units (reviewed by Mueller-Harvey, 2001). The biosynthetic pathway to hydrolysable

tannins may be divided in three smaller sections: The initial route encompasses reactions

that start from free gallic acid unit, which esterifies with a glucose and undergoes further









esterification to form the end product pentagalloylglucose. Pentagalloylglucose is the

starting point for the two subsequent routes. The gallotannin route is characterized by the

addition of galloyl residues to pentagalloylglucose. The ellagitannin route are oxidation

processes that yield C-C linkages between galloyl groups of pentagalloylglucose

(reviewed by Grundhofer et al., 2001)

2.6.3 Polyphenolics as Antioxidants

Polyphenols have shown potential health benefits, chiefly relating to antioxidant

capacity. Antioxidants prevent free radicals from harming host tissues and thus are

thought to reduce the risk of certain degenerative diseases such as cancer or

cardiovascular disorders. Polyphenolics behave as antioxidants, mainly due to the

reactivity of the hydroxyl substituents in the aromatic ring. There are several

mechanisms, but the predominant role of antioxidant activity in polyphenols is believed

to be radical scavenging via hydrogen atom donation or singlet oxygen quenching

(reviewed by Robbins, 2003). In order for a polyphenol to be defined as an antioxidant it

must satisfy two basic conditions: first, when present in low concentration relative to the

substrate to be oxidized it can delay, retard, or prevent autoxidation or free radical-

mediated oxidation; second, the resulting radical formed must be stable (reviewed by

Shahidi and Wanasundara, 1992). Structurally, on monomeric phenolics, the ability to act

as antioxidants depends on extended conjugation, number and arrangement of phenolics

substituents, and molecular weight. Tannins, which are highly polymerized with many

phenolic hydroxyl groups, may be 15-30 times more effective in quenching peroxyl

radicals than simple phenolics (Hagerman et al., 1998). Various in-vitro studies have

demonstrated the helpful antioxidant effects of polyphenolics in fruits and vegetables,

however evidence in in-vivo studies remains unclear. Flavonoids have shown to inhibit






24


LPO in-vitro by acting as scavengers as superoxide anions and hydroxyl radicals,

however in-vivo, the evidence is quite unclear (reviewed by Cook and Saman, 1996). The

analytical procedures most widely used today to separate and identify polyphenolics are

HPLC, Gas Chromatography (GC), and HPLC-Mass Spectrometry (Robbins, 2003).














CHAPTER 3
EFFECTS OF HOT WATER IMMERSION TREATMENT ON GUAVA FRUIT
PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY

3.1 Introduction

Guava (Psidium guajava L.), the "apple of the tropics", is an exotic fruit from

tropical and subtropical regions. Since its introduction in Florida in the late 1960's a

market for fresh guava fruit and its products has been slowly growing in the US (personal

communication, Sardinia, 2004). However, guava fresh fruit marketability is somewhat

limited, mainly due to quarantine issues and the highly perishable nature of the fruit.

Unlike other tropicals, a quarantine schedule for guava has not been approved by the

USDA (USDA-APHIS, 2004), probably due to the prevalence of Caribbean fruit fly

contamination (Gould and Sharp, 1992) and lack of studies demonstrating beneficial

effects.

Thermal quarantine treatments in particular are increasing in industrial use due to

consumer demand and governmental regulations concerning the use of chemical

treatments (Lurie, 1998). Currently, limited studies exist on thermal quarantine

treatments for guava. Additionally, information on resultant changes of postharvest

handling on guava's phytochemicals and antioxidant properties are very limited. The

objective of this study was to evaluate the effects of a thermal quarantine treatment, using

a simulated hot water immersion technique, on the phytochemical content, antioxidant

capacity, and overall quality of guava fruits at three stages of ripeness.









3.2 Materials and Methods

3.2.1 Materials and Processing

3.2.1.1 Fruit preparation and HW treatment

Mature (pink) guavas at various stages of ripeness from a single harvest were

procured from C-Brand Tropicals, Homestead, FL in August 2003. Fruit were transported

overnight via a courier service to the Food Science and Human Nutrition Department of

the University of Florida. Upon arrival, fruit were washed and stored at 15 oC for 18 hrs.

Approximately 200 fruit were selected for ripeness uniformity and freedom from surface

damage. Based on differences in skin color, firmness, and whether the fruit floated or

sank in water (Reyes and Paull, 1995) three groups were segregated. Stage I fruit were

mature, green fruit with a yellowish-green skin, firm texture, and floated in water. Stage

II fruit contained 25-50% yellow skin, semi-firm texture, and sank in water. Stage III

fruit were 75-100% yellow, soft texture, and sank in water. A hot water (HW) immersion

treatment was applied according to the conditions set by Gould and Sharp (1992). Within

each ripening stage, fruit were randomly separated into four groups for each treatment

time, placed into nylon bags and completely immersed in 460C water for 0 (control), 15,

30, and 60 min. Control guavas were immersed in water at 23 C, in order to have water

immersion conditions. Due to the limited availability of Stage III fruits, only a control

and 30 min immersion times were evaluated. After each respective immersion time, fruit

were cooled by immersion in 230C water for 60 min. Stage I and II fruit were held in a 15

C storage room and held until fully ripe based on color and texture as observed for the

State III fruit. Stage III fruit, since they were already at a full ripe stage, were held at

15C for an additional two days to determine short-term treatment effects. Therefore, this









study focused on Stage I and II guavas where the effects of HW treatment and changes

during ripening could be evaluated.

3.2.1.2 Guava fruit processing

Ripe guava fruit were collected from storage and immediately processed into a

puree. After crown and peduncle were removed, whole fruits were manually chopped into

smaller cubes and processed into a puree using a kitchen-scale juice extractor (Braun,

MP80) which removed excess skin and seeds. "Guava composites" or replicates were

formed by joining 4 random fruits, making up 4 replicates for each treatment. Puree was

packed in 0.1 mm thick sample bags and held frozen at -20 oC until analysis.

Samples were taken for moisture determination, soluble solids content, pH, and for

chemical extraction of polyphenolics, ascorbic acid, carotenoids, and antioxidant capacity

(AOX). For all of the analyses except moisture, pH, and carotenoids a clarified guava

juice was evaluated. To obtain this isolate, 10 g of guava puree was treated with 5 pL of

pectinase (Pectinex Ultra, SP-L, Novozymes), incubated at 32 C for 30 min, and

centrifuged until a clear supernatant was obtained. The clarified juice was then filtered

through cheesecloth, treated with sodium azide (0.01% w/v) to prevent microbial

spoilage, and held frozen at -20 oC until further analysis.

3.2.2 Chemical Analysis

3.2.2.1 Moisture content determination

Moisture content was determined on the guava puree by placing 3 g into a pre-

weighed aluminum pan and drying to a constant weight in a convection oven (Precision

Economy) at 135 C for 2 hrs (AOAC Method 920.149149(c)).









3.2.2.2 Quantification of total soluble phenolics

Total soluble phenolics were determined by the Folin-Ciocalteu assay (Swain and

Hillis, 1959). Briefly, guava juice diluted 10-fold was pippeted into a test tube and 1 mL

of 0.25 N Folin-Ciocalteu reagent added. The mixture was allowed to react for and

letting stand 3 minutes to allow for the reduction of phosphomolybdic-phosphotungstic

acid by phenolic compounds, ascorbic acid, and other reducing agents in the juice.

Subsequently, 1 mL of IN sodium carbonate was added as an alkali to form a blue

chromophore. After 7 minutes, 5 mL of distilled water was added and thoroughly mixed.

Absorbance was read using a microplate reader (Molecular Devices Spectra Max 190,

Sunnyvale, CA) at 726 nm. Concentration of total soluble phenolics was quantified based

on a linear regression against a standard of gallic acid with data was expressed in gallic

acid equivalents (GAE).

3.2.2.3 Analysis of ascorbic acid by HPLC

Ascorbic acid was determined by reverse-phase HPLC using a Waters Alliance

2695 HPLC system equipped with a Waters 996 PDA detector (Waters Corp, Milford,

MA), using a Supelcosil LC-18 column (Supelco, Bellefonte, PA) with detection at 254

nm. An isocratic running program was established using a 0.2 M potassium phosphate

(K2H2PO4) buffer solution at pH 2.4 (adjusted with phosphoric acid) as the mobile phase

run at 1 mL/min. Ascorbic acid was identified by comparison to a standard (Sigma

Chemical Co., St. Louis, MO) and based on UV spectroscopic properties. Samples for

analysis were prepared by diluting the guava juice 10-fold with a 3% citric acid solution

prior to HPLC analysis.









3.2.2.4 Quantification of antioxidant capacity

Antioxidant capacity was determined by the oxygen radical absorbance capacity

(ORAC) assay as modified by Ou et al. (2001). The assay monitors the decay of

fluorescein as the fluorescent probe in the presence of the peroxyl radical generator 2,2'-

azobis (2-methylpropionamidine dihychloride) and is evaluated against Trolox, a

synthetic, water-soluble vitamin E analog. Assay conditions were described by Talcott et

al. (2003) for the use a Molecular Devices fmax 96-well fluorescent microplate reader

(485 nm excitation and 538 nm emission). For analysis, GJ samples were diluted 100-

fold in pH 7.0 phosphate buffer prior to pipetting into a microplate. Additionally, a

Trolox standard curve (0, 6.25, 12.5, 25, 50 [aM Trolox) and a phosphate buffer blank

were prepared. Fluorescence readings were taken every 2 min over a 70 min period at

37C. The rate of fluorescence decay over time was determined by calculating the area

under the fluorescent decay curve and the antioxidant capacity quantified by linear

regression based on the Trolox standard curve. Final ORAC values were expressed in |JM

of Trolox equivalents per gram (iM TE/g).

3.2.2.5 Analysis of lycopene by HPLC

Lycopene was quantified by HPLC using a Dionex HPLC system equipped with a

PDA 100 detector and separations made using a YMC Carotenoid column (250mm x 4.6

mm). An isocratic solvent delivery of 70% methyl-tert-butyl-ether and 30% methanol

was run at 2 mL/min with detection at 470 nm. A standard of lycopene was isolated from

from guava puree by extracting 5 g of guava puree with 20 mL of acetone and ethanol

(1:1) and filtering through #4 Whatman paper. This isolate contained a mixture of non-

lycopene carotenoids. The extraction process was repeated until the filtrate lost nearly all

of its non-lycopene yellow color, whereby the addition of 100% acetone was added to









extract lycopene. The lycopene was then partitioned into hexane that contained 100 mg/L

BHT as an antioxidant. Lycopene (MW 536.9) was quantified using an extinction

coefficient of 3,450 at 470 nm in hexane. Purity was estimated at >98% as determined by

the presence of extraneous carotenoid compounds by HPLC at 470 nm.

Lycopene was extracted from guava puree using modified conditions of Martinez-

Valverde et al. (2002) used for tomato lycopene extraction. Approximately 0.5 g of puree

was extracted with 5 mL of 100% acetone and vortexed vigorously. Quantitatively, 3 mL

of hexane was added to the mixture, mixed, and water added to ensure adequate bi-layer

separation. Aliquots of hexane extracts were filtered through 0.45 tm filter prior to

HPLC injection.

3.2.2.6 Quantification of non-lycopene carotenoids

Non-lycopene or "yellow" carotenoids were quantified in total using a

spectrophotometer at 470 nm. Yellow carotenoids were extracted from 5g of guava puree

with a known volume of acetone:ethanol (1:1) and subsequently filtered through

Whatman #4 filter paper. Absorbance was recorded using a Beckman DU 60

spectrophotometer (Beckman, Fullerton, CA) between 350 and 500 nm. Carotenoid

concentration was calculated based on the extinction coefficient for B-carotene.

3.2.2.7 Analysis of polyphenolics by HPLC

Individual polyphenolic compounds were analyzed by reverse-phase HPLC using a

Waters Alliance 2695 HPLC system (Waters Corp, Milford, MA) equipped with a Waters

996 PDA detector and a 5 [tm Waters Spherisorb ODS2 column (250 x 4.6 mm) using

modified HPLC conditions described by Talcott et al. (2002). Mobile phases for gradient

elution (Table 3-1) consisted of 98:2 water and acetic acid (mobile phase A) and 68:30:2

water, acetonitrile, and acetic acid (mobile phase B) accordingly, at a flow rate of 0.8









mL/min, and detected at 280 nm. Major polyphenolic compounds were characterized by

spectroscopic interpretation, retention time, and comparison to authentic standards

(Sigma Chemical Co., St. Louis, MO). Following filtration through at 0.45 [M filter, the

guava juice was injected into the HPLC without further modification.

Table 3-1. Gradient elution running program for HPLC analysis of polyphenolics.
Running time (min) % Mobile phase A % Mobile phase B
0.00 100 --
20.00 70 30
30.00 50 50
50.00 30 70
70.00 -- 100
72.00 100 --

3.2.3 Quality Analysis

The quality parameters soluble solids and pH were measured on the guava puree

using a digital Leica Abbe Mark II refractometer (Model 10480, Buffalo, NY) and

Corning pH meter (Model 140, NY) respectively. Overall fruit quality were subjectively

evaluated during storage to detect any effects related to HW treatment or fruit decay.

3.2.4 Statistical Analysis

For Stage I and Stage II fruits, the experiment consisted of a 4 x 2 x 4 full-factorial

design. The factors studied were HW immersion time (0, 15, 30, and 60 min) and

ripening stage (I and II), with a mean of 4 replications represented in each data point.

Stage III fruits were analyzed (2 x 1 x 4 design) and discussed separately, since they were

only subjected to two HW treatment times (0 and 30 min). Statistical analysis were

conducted in JMP (SAS, Cary, NC) and consisted of analysis of variance, Pearson

Correlation, and mean separation by LSD test (P< 0.05).









3.3 Results and Discussion

3.3.1 Chemical Analysis

For chemical analyses performed, the effect of HW immersion time as compared to

control within each ripening stage was evaluated, as well as the effect of ripening stage

within each HW immersion time (0, 15, 30, and 60 min), and the interaction of both

factors. Results for chemical analysis, excepting polyphenolics by HPLC, were reported

in dry weight basis (DW), in order to eliminate difference between samples due to

varying water loss fruits experienced during ripening.

3.3.1.1 Moisture content

Moisture content within each ripening stage was insignificantly affected by HW

treatment time duration up until 60 min. Average moisture content for Stage I and Stage

II fruit was 91.8 and 92.6% respectively (Figure 3-1). Total solids, determined by

difference, was also insignificantly affected by HW treatment duration. Uniformity in

total solids was an indicator of uniformity in the pool of guavas, since fruit were not

affected by HW treatment duration. Due to differences in initial degree of ripeness,

duration of HW treatment, and duration of storage as the fruit ripened, some change in

moisture content was expected. However, no significant differences in final moisture

contents and subsequently total solids due to ripening stage were observed from 0 to 30

min. At 60 min in HW, Stage II fruit presented significantly higher moisture content (1.3

%) than Stage I fruit, which is more attributed to fruit variation, since HW treatment at 60

min did not differ from control within both stages. It is concluded that fruits at either

ripening stage could be heated up to 60 min without affecting their moisture content or

total solids. To report values for fresh fruit and have more uniformity, moisture content

was used to calculate chemical analysis results in dry weight basis (DW).











97
96 Stage I
I- I Stage II
95
94
-- 93
92


090
89
88
87
86
85
0 15 30 60
Hot Water Immersion Time (min) at 46 C

Figure 3-1. Moisture content (%) of ripe guavas as affected by a hot water immersion
treatment (0,15, 30, and 60 min at 46 C) and ripening stage (Stage I and II).
Error bars represent standard error of the mean, n =4.

3.3.1.2 Total soluble phenolics

HW treatments up to 30 min did not affect concentrations of total soluble phenolics

(TSP) in relation to the untreated fruit within each ripening stage (Figure 3-2). At 60 min

in HW, however, Stage I fruit presented lower TSP content than untreated fruits, as

opposed to Stage II fruit, which remained unaffected. This lower content might be related

to other metal-reducing compounds, which may contribute to the TSP value, due to the

nature of the Folin's assay. Within each HW immersion time, control fruit presented

differences due to ripening stage; However within HW treatments (15 to 60 min) no

significant differences in TSP were found due to ripening stage, confirming no significant

effects attributed to ripening stage and its interaction with HW treatment. Differences

found between stages in the untreated control were probably due to fruit variability and

contribution of other metal-reducing compounds, including ascorbic acid. Stage I fruit,










therefore, can be subjected to up until 30 min in HW without affecting its TSP content,

while Stage II fruit can be held up until 60 min.


27500
Stage I
25000 Stage II
"D
S22500

S 20000

0 17500 -







7500
c-




S 5000
0 15 30 6012500




Hot Water Immersion Time (min) at 46 C


Figure 3-2. Total soluble phenolics (mg/kg DW) in guava as affected by a hot water
al., 1997; Bashir and Goukh, 2002). Pink guavas in particular, have a less-marked
l- 7500



0 15 30 60






Hotreduction in TSP from mature green stage (Stage ) until full ripeness as compared to

Figure 3-2.guavas (Bashir and Goukh, 2002). For the presenting guava as affected by a from both ripening

immstages, treated and not treatment (0, 15, 30, and 60 mn attain TSP levels comparable to
(Stage I and II). Error bars represent standard error of the mean, n = 4.

Studies have demonstrated guava TSP content decreases during ripening (Bulk et

al., 1997; Bashir and Goukh, 2002). Pink guavas in particular, have a less-marked

reduction in TSP from mature green stage (Stage I) until full ripeness as compared to

white guavas (Bashir and Goukh, 2002). For the present study, guavas from both ripening

stages, treated and not treated, were able to ripen and attain TSP levels comparable to

other reports in ripe guavas. Average TSP values for the different ripening stages (I-III)

ranged from 19,588 to 21,502 mg/kg GAE, DW (1,360 to 1,760 mg/kg GAE, FW).

Bashir and Abu-Goukh (2002) reported similar values of TSP (1200-1800 mg/kg FW) for

white pulp guavas, but a lower content in pink pulp guavas. Kondo et al. (2005) reported









a TSP content of 1852 [tmol/kg FW. Variety, origin, and even harvest season play an

important make an impact in differences in TSP.

3.3.1.3 Ascorbic acid

Ascorbic acid is an effective nutrient stability index during food processing and

storage operations. It has been generally observed that if AA is well retained, the other

nutrients are also well retained (Fenema, 1996). Ascorbic acid was insignificantly

affected by HW immersion time within Stage I fruits (Figure 3-3). In a HW treatment at

38 C for 30 min applied to guavas and subsequent exposure to chill injury conditions (5

C), these processes did not affect its ascorbic acid content, although chilling injury

symptoms were present. (Regalado-Contreras and Mercado-Silva, 1998). Stage II fruit at

30 min however, presented lower values as compared to control, which does not seem to

be a treatment effect, but rather fruit variation effect, since fruit treated at 60 min was not

different from untreated fruit. Fruit variation may be observed by the large error rates

presented in Figure 3-3 (statistical analysis, however, for all parameters was done using a

pooled standard error). Even within the same variety, large variability in ascorbic acid

contents has been reported (Mitra, 1997). There were no differences in AA due to

ripening stage at each treatment time. The fact that guava was unaffected by increasing

HW times may not only indicate its stability during a postharvest treatment, but the

stability of other guava phytochemicals. Additionally, since ascorbic was unaffected by

most HW treatments, it may also indicate a uniformity of the pool of guavas used, most

of them achieving similar contents.

The average AA content for Stage I and Stage II fruit was 10,846 mg/kg DW

(843.5 mg/kg FW) (Figure 3-3), comparable to other studies on ripe guavas. Bashir and

Abu-Goukh (2002) reported an ascorbic acid content of 800 mg/kg and 670 mg/kg FW










for white and pink guavas respectively, while Leong and Shui (2002) reported 1,310

mg/kg FW, and Bulk et al. (1997) reported values between 882 and 1113 mg/kg FW.

16000
15000 Stage I
14000 I Stage II
13000
12000
31 11000 -
E 10000
V 9000
< 8000
7000
0
O 6000-
5000
4000
3000
2000
0 15 30 60
Hot Water Immersion Time (min) at 46 C


Figure 3-3. Ascorbic acid content (mg/kg DW) in guava as affected by a hot water
immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage
(Stage I and II). Error bars represent standard error of the mean, n = 4.

Fruit variety is one of the main factors affecting AA content in guava, influencing more

than ripening stage or storage conditions (Mitra, 1997).

3.3.1.4 Antioxidant capacity

Antioxidant capacity in fresh guava was insignificantly affected by increasing

times of HW treatment within Stage I and Stage II fruits (Figure 3-4), which presented

values of 146 and 126 tM TE/g DW respectively. Additionally, ripening stage at the time

of treatment application did not affect antioxidant capacity within each HW treatment. In

a study conducted in Mexico, guavas were subjected to chill injury conditions (5 C),

where no changes in antioxidant capacity (ferric reducing antioxidant power; FRAP) and

TSP were reported as compared to control, even when the fruit presented external chilling










injury symptoms (Edmundo et al., 2002). The biosynthesis of certain antioxidant

compounds in guava might not be affected by certain temperature changes, even though

they generate stress and injuries on the commodities. However, this is extremely

dependent on the degree of stress, which largely depends on the temperature differential,

atmospheric conditions, and exposure duration (Lurie, 1998; Paull and Chen, 2000).

200

180 T I Stage I
SI Stage II
S" 160
W
X
0 140

120-
E
100

o 80

O 60

|- 40
0
20-
0
0 15 30 60
Hot Water Immersion Time (min) at 46 C

Figure 3-4. Antioxidant capacity (tmol Trolox Equivalents/g DW) in guava as affected
by a hot water immersion treatment (0, 15, 30, and 60 min at 46 C) and
ripening stage (Stage I and II). Error bars represent standard error of the mean,
n =4.

The major contributors to water-soluble antioxidant activity in guava are ascorbic

acid (AA) and polyphenolics. Ascorbic acid reportedly contributes to approximately 50%

guava's antioxidant activity (Leong and Shui, 2002). The correlation between antioxidant

capacity and ascorbic acid was low (r = 0.30, P< 0.05), probably due to the high degree

of variability in ascorbic acid values. There was also a low correlation with TSP (r =

0.27, P< 0.05), since TSP content presented significant differences with increasing HW

treatment time. Due to the use of GJ, however, overall antioxidant capacity in guava was









probably underestimated, since lycopene, carotenoids, and other lypophilic compounds

were not taken into consideration.

3.3.1.5 Lycopene and yellow carotenoids

Average lycopene values for Stage I and Stage II guavas were 558 and 472 mg/kg

DW (45.1 and 34.8 mg/kg FW) respectively (Figure 3-5). Studies conducted on Brazilian

guavas reported similar values, ranging from 47 to 53 mg/kg FW (Padula and Rodriguez-

Amaya, 1986; Wilberg and Rodriguez-Amaya, 1995). Lycopene content in ripe tomatoes,

which ranges from 30 to 80 mg/kg FW (Abushita et al., 2000; Thompson et al.,2000;

Leonardi et al. 2000; Martinez-Valverde et al., 2002; Seybold et al., 2004), is comparable

to ripe guavas. Subjective determination of guava ripeness, based on skin color

observations and texture, may have created some uncertainty about the uniformity of

ripening stage. These effects were evident when guava fruit were cut and variation in

pulp color intensity was observed within the same ripeness stage. However, fruits were

grouped in composites, joining fruits with similar pulp colorations, in order to have more

uniformity and reduce variation within treatments.

Guava lycopene was unaffected by HW treatment up until 30 min within both

ripening stages (Figure 3-5). At 60 min, Stage I fruits exhibited lower lycopene content as

compared to untreated fruits, whereas Stage II fruit remained unaffected. This reduction

might be related to an inhibition in lycopene biosynthesis due to a longer exposure to heat

stress. Studies of HW treatments on tomatoes have reported this inhibition mainly due to

an inhibition of the transcription gene for lycopene synthesis, which recovers after the

removal of heat (Cheng at al., 1988; Lurie et al., 1996; Paull and Chen, 2000). Following

the removal of the heat stress for 60 min, Stage I fruit recovered and synthesized

lycopene, but not to the extent of the other treatments. However, this difference between










untreated fruit and 60 min, might also be due to the significantly higher content of Stage I

fruit at both 0 and 15 min, as compared to Stage II. These differences might be attributed

to pre-treatment storage conditions, were fruits of the different ripening stages ripened

different. Within the HW treatments, there were no differences due to ripening stage from

15 to 60 min; however, Stage I fruit presented a higher content in untreated fruit.


800
Stage I
S Stage II
700
So
600
0)
500 -

S400

j 300

200

100
0 15 30 60
Hot Water Immersion Time (min) at 46 C

Figure 3-5. Lycopene content (mg/kg DW) in guava as affected by a hot water immersion
treatment (0, 15, 30, and 60 min at 46 C) and ripening stage (Stage I and II).
Error bars represent standard error of the mean, n = 4.

Non-lycopene, or yellow, carotenoids are referred to guava carotenoids other than

lycopene. Yellow carotenoids were unaffected by increasing HW treatment time and by

ripening stage within each HW treatment, included untreated fruits (Figure 3-6). A hot

water treatment applied to papaya (Carica papaya L.) did not cause any detrimental

effects on the content of p-carotene and lycopene (Perez and Yahia, 2004). Lycopene and

other carotenoids stability in tomatoes has associated to the influence of the tomato

matrix itself and its adhesion to membranes (Seybold et al., 2004). A whole guava at its

early stages of ripening contains appreciable amounts of cellulose, hemicellulose, lignin










(stone cells) and insoluble pectin in its cell walls, which creates a strong matrix that may

protect well guava carotenoids and other phytochemicals. Yellow carotenoids content of

these carotenoids ranged from 42.9 to 49.9 mg/kg DW (3.27 to 4.11 mg/kg FW), which

are comparable to p-carotene contents of 3.7 and 5.5 mg/kg FW found in Brazilian

guavas (Padula and Rodriguez-Amaya, 1986; Wilberg and Rodriguez-Amaya, 1995).

Stability of carotenoids, especially lycopene, in tomatoes during food processing

operations has been widely discussed by various authors, and sometimes inconsistent

results are found, where lycopene content might be enhanced or reduced (Abushita et al.,

2000; Leonardi et al. 2001, Seybold et al., 2004; Sahlin et al., 2004). A HW immersion

treatment, at a lower temperature than cooking or other processing operations, and for a

shorter period of time, might be milder, in order to cause significant effects in the

biosynthetic pathways of lycopene and other carotenoids.



E' 60 -M Stage I
I-I Stage II

450-

0
g
5 40
10
0


0






0 15 30 60
Hot Water Immersion Time (min) at 46 C

Figure 3-6. Non-lycopene carotenoids (mg/kg DW) in guava as affected by a hot water
immersion treatment (0, 15, 30, and 60 min at 460C) and ripening stage (Stage
I and II). Error bars represent standard error of the mean, n = 4.
I and II). Error bars represent standard error of the mean, n =4.









Heat treatments and other postharvest processes can delay, inhibit or accelerate

ripening, as part a commodity's response to abiotic (environmental) stress (Lurie, 1998;

Paull and Chen, 2000; Jacobi et al., 2001; Basseto et al., 2004). This may also be closely

associated with respiration rate, ethylene production, fruit softening, enzyme activities

(cell-wall degrading, ethylene-related), carotenoid development, and other components

related to ripening (Paull and Chen, 2000; Jacobi et al., 2001). These processes can

actually be used as tools to enhance their marketability and added value of produce, for

example, uniformity of skin color development in mangoes (Jacobi et al., 2001) or

anthocyanin accumulation in strawberries by HW treatments (Civello et al., 1997). In

other cases, they can bring detrimental effects, affecting flavor, aesthetic qualities, or

inhibiting the synthesis of certain antioxidant compounds (Cisneros-Cevallos, 2003;

Sahit, 2004). Stress responses were observed in Stage I fruit heated for 60 min, where

there was an enhancement in total soluble phenolics and a decrease in lycopene.

Especially for lycopene, its inhibition might be detrimental to the fruit's phytochemical

content. However, results indicated that a HW treatment up until 30 min at 46 C

insignificant major phytochemicals in guava.

3.3.1.6 Polyphenolics by HPLC

Studies identifying ripe guava polyphenolics by HPLC are limited. Earlier studies

have been done with less precise analytical procedures in which the major classes of

compounds present have been identified (Misra and Seshadri, 1967; Mowlah and Itoo,

1982; Itoo et al., 1987). Immature, still developing guava are composed mainly of

condensed tannins, which decrease markedly during its development and ripening, along

with the rest of guava polyphenolics (Misra and Seshadri, 1967; Itoo et al., 1987). Kondo










et al. (2005) identified gallic acid, catechin, epicatechin, and chlorogenic acid in guava

skin, as well as catechin at lower concentrations in its pulp. In ripe fruits, the presence of

procyanidins, or condensed tannins, and free ellagic acid has been confirmed (Misra and

Seshadri, 1967).

Various solvent extraction and fractionation procedures on puree and juice were

attempted for HPLC analysis of guava polyphenolics with the enzyme-clarified guava

juice producing the most reproducible HPLC chromatograms with maximal peak

separation (Figure 3-7). In this study, HPLC analysis of polyphenolics was used to

identity overall data trends as affected by ripeness stage and HW treatment. Among the

multitude of polyphenolic compounds present, 14 were selected based on adequate

separation and abundance for treatment differentiation (Table 3-2).

0.22

0.20 280 nm
0.18
0.16
A C
0.14
C
0.12-
0.10- B
0.08 C
0.06- C

0.04 E


0.00,
10.00 20.00 30.00 40.00 50.00 60.00 70.00
Minutes

Figure 3-7. HPLC chromatogram of polyphenolic compounds found in guava juice-A)
gallic acid, B) gallic acid derivatives, C) unknown-characteristic guava
polyphenolics, D) procyanidins, and E) ellagic acid derivative. Identification
(280 nm) was done by comparison to authentic standards and spectral
properties._










Peaks were tentatively identified and/or grouped into a common class of polyphenolics

based on their spectroscopic properties and comparison to authentic standards (Table 3-

2). Gallic acid (Peak 1) and an ellagic acid derivative (Peak 12) were clearly identified by

comparison to standards. Gallic acid derivatives (Peaks 2 and 3) were tentatively

identified, as they shared similar spectral properties with gallic acid. Procyanidin

compounds (Peaks 6 and 7), or condensed tannins, were identified based on their

spectroscopic similarities to (+)-catechin and (-)-epicatachin, which are the building

blocks of condensed tannins. The remaining compounds were characterized based on

retention time and spectroscopic properties but were dissimilar from any known

polyphenolic compounds. Further work will be needed to isolate and identify these

individual polyphenolic compounds in ripe guavas. Since gallic acid was the most

prevalent compound in guava, and is found in abundance in many fruits, all compounds

were quantified in gallic acid equivalents (GAE).

Table 3-2. Tentative identification of guava polyphenolics at 280 nm by HPLC based on
retention time, spectral properties, and comparison to authentic standards.

Peak Retention Time Spectral Properties Tentative Identification
No. (min)
1 8.12 229.3, 271.6 Gallic Acid
2 8.90 229.3, 276.3 Gallic Acid Derivative
233.9, 290.5 / 233.9,
3 9.71 276.3 Gallic Acid Derivative
4 15.8 224.5, 262.2 Unknown
5 18.5 233.9, 266.9 Unknown
6 26.3 233.9, 281.1 Procyanidin
7 28.4 233.9, 281.1 Procyanidin
8 31.1 281.1 / 219.9, 281.1 Unknown
9 35.5 215.2, 266.9 Unknown
10 45.7 224.6, 281.1 Unknown
11 47.0 219.9, 262.2 Unknown
12 59.5 224.6, 252.8, 369.9 Ellagic Acid Derivative
13 64.6 262.2 Unknown
14 68.2 224.6, 281.1 Unknown









Gallic acid (Peak 1)

Gallic acid (GA) (Peak 1) has been reported as an effective antioxidant due to its

structure and positioning of hydroxyl groups. Within each ripening stage, GA was

insignificantly affected by HW treatment up until 30 min (Table 3-3); however in Stage I

fruit GA content was significantly higher than control at 60 min (52% increase). Stage II,

however, remained unaffected. It has been discussed that the chronological age of a tissue

is an important factor in response to HW treatment (reviewed by Paull and Chen, 2000).

The presence of the heat stress for a longer period might have resulted in increased

biosynthesis of gallic acid through the phenylpropanoid pathway, as a response to stress.

Additionally, Stage I fruit probably had higher polyphenolic content than other ripening

stages, especially in its peel, most prone to be affected by abiotic stress. As shown in

Table 3-3, within all HW treatment times, including control, an effect of ripening stage

was found, in which Stage I fruit exhibited a significantly higher GA content. The fact

that Stage I fruit content was higher in all HW times, including control, indicates that it

might not be attributed to a HW treatment effect, but rather pre-treatment storage

conditions and post-treatment storage duration.

Gallic acid derivatives (Peaks 2 and 3)

GA derivatives, Peaks 2 and 3, eluted in the HPLC column immediately after GA.

Due to their closeness to GA in spectral properties, these compounds were tentatively

classified as GA derivatives (Table 3-3), and their content was summed. There was no

effect due to HW treatment (15 to 60 min) as compared to control within Stage II (Table

3-3). In Stage I, GA derivatives content was lower than control at 15 min, which












Table 3-3. Guava gallic acid (GA), gallic acid derivatives, and an ellagic acid derivative as affected by a hot water quarantine
treatment and ripening stage.
Pk 1: Gallic Acid (GA) Pks 2 and 3 : GA Derivatives Pk 12: Ellagic Derivative
HW treatment (mg/kg GAE2) (mg/kg GAE) (mg/kg GAE)
(min)
Stage I Stage II Stage I Stage II Stage I Stage II

0 16.2 b1 7.70 c 12.8 a 10.5 bc 1.04 c 1.71 ab
15 16.1 b 9.16 c 9.20 c 12.0 abc 1.43 bc 1.55 b
30 15.8 b 5.64 c 11.9 abc 10.3 bc 1.35 bc 2.10 a
60 24.7 a 5.71 c 13.8 a 11.8 abc 1.61 b 1.50 b
1Values with different letters within columns of the same ripeness level and within rows for each hot water treatment are significantly different, and indicate the
effect of either hot water treatment or ripening stage, and their interaction (LSD test, P<0.05). 2GAE = Gallic Acid Equivalents

Table 3-4. Guava procyanidins, other characteristic unknown compounds, and total polyphenolics by HPLC as affected by a hot water
quarantine treatment and ripening stage.


Pks 6 and 7: Procyanidins Unknown Guava Compounds Total Polyphenolics by HPLC
HW treatment (mg/kg GAE2) (mg/kg GAE) (mg/kg GAE)
(min)


Stage I Stage II Stage I Stage II Stage I Stage II
0 6.33 a' 3.93 bc 36.3 a 24.7 d 72.7 a 48.5 c
15 4.99 abc 3.60 c 36.5 a 27.0 bcd 67.7 ab 53.3 c
30 5.42 ab 4.60 bc 28.6 bc 25.2 cd 62.2 b 47.8 c
60 4.20 bc 3.53 c 29.6 b 27.7 bcd 73.9 a 50.2 c
'Values with different letters within columns of the same ripeness level and within rows for each hot water treatment are significantly different, and indicate the
effect of either hot water treatment or ripening stage, and their interaction (LSD test, P<0.05). 2GAE = Gallic Acid Equivalents.









probably was due to variation, since at 30 and 60 min there was no HW treatment

effect.Within all HW treatments and control, no ripening stage effect was observed.

Procyanidin compounds (Peaks 6 and 7)

Peaks 6 and 7 were identified as belonging to procyanidin compounds. The

presence of procyanidin compounds has been confirmed in both white and pink guavas

(Mowlah and Itto, 1982), and they have been reported to be composed mainly of(+)

catechin and (+) gallocatechin (Itoo et al., 1987).

Procyanidins presented no HW treatment effect in Stage II fruit; however Stage I

fruit presented lower content at 60 min as compared to control (Table 3-4). This

difference between control and 60 min might not attributed to a treatment effect, rather to

variation within control fruit, since an increase in heat stress would probably increase the

content of procyanidin compounds, rather to decrease it. Additionally, within untreated

controls, Stage I fruit presented higher procyanidin content than Stage II fruit, but no

differences between ripening stages were found at 15, 30, and 60 min. This confirms that

Stage I control fruit procyanidin content was significantly higher probably due to a

variation in values of this compound in control fruit. It can be concluded that procyanidin

content in HW treated fruit up to 30 min did not differ significantly from untreated fruits.

Ellagic acid derivative (Peak 12)

Peak 12 was identified as an ellagic acid derivative, most likely a glycoside (Lee et

al., 2005), due to its closeness in spectral properties to ellagic acid. Free ellagic acid in

ripe guava was isolated and identified by Misra and Seshadri (1967). Similarly to other

compounds, Stage II fruits ellagic acid content was unaffected by increasing HW

treatment up until 60 min (Table 3-3). Stage I fruit ellagic acid content at 60 min HW









treatment, however, was significantly higher than control. This trend in Stage I is

comparable to gallic acid results; it must be noted though that concentrations of this

compound are much lower. Very low concentrations might not give an accurate measure

of a treatment effect. Within HW treatment time, Stage I fruit was significantly lower

than Stage II fruit at time 0 and 30, but at time 15 and 60 min, there were no significant

differences due to ripening stage.

Guava characteristic unknown compounds (Peaks, 4, 5, 8-11, 13, 14)

This group is composed of guava compounds that had characteristic and sometimes

repeating spectroscopic properties (Table 3-4), but were not identifiable as any known

polyphenolic compounds. Many of them shared similar spectrocospic properties to gallic

acid and its derivatives or procyanidin compounds, which might relate them closer in

further research. They were characterized by their consistency in spectroscopic properties

among most samples, in contrast with other compounds, whose spectral properties

differed mainly due to small peak areas or interference of extraneous compounds during

elution.

Stage II fruit unknown compounds content presented no effect due to HW

treatment. Stage I fruit content, however, decreased with an increase in HW treatment at

30 and 60 min. This HW treatment effect was particular for two compounds represented

by Peaks 13 and 14, which constituted 41% of the overall unknown compounds content.

Peaks 13 and 14, along with gallic acid, contributed with the largest peak areas of all

guava polyphenolics. The decrease in these compounds in Stage I fruits may be due to a

loss of polyphenolic compounds in response to stress. Within HW treatment, significant

differences were observed due to ripening stages at control and at 15 min, but not at 30









and 60 min, which does not relate it to a HW treatment related response but rather to

variation in some of the compounds.

Total guava polyphenolics by HPLC (Peaks 1 to 14)

Briefly, the overall trend of all 14 guava polyphenolic compounds will be

discussed. Stage II fruit presented no significant differences in total polyphenolics due to

HW treatments as compared to control (Table 3-4). Stage I fruit content at 15 and 60 min

was no different from control, which accounts that insignificantly of a lower content at 30

min, in general, Stage I fruit presented no HW treatment effect. Within untreated fruit

and HW treated fruit (15 to 60 min), significant differences due to ripening stage were

observed.

Many postharvest stresses, including heat treatments, have shown to affect the

levels of polyphenolics in plant commodities, either by inducing or inhibiting their

biosynthesis. When fruits or vegetables undergo stress, cinnamic acid and benzoic acid

derivatives are among the first polyphenolics to be synthesized (Sahit, 2004). The

phenylpropanoid pathway, regulated by PAL, is responsible for the synthesis of

hydroxycinnamic and hydroxybenzoic acids. An increase in PAL activity due to stress

may result in the accumulation of many polyphenolic compounds (Cisneros-Zevallos,

2003). In the present study, gallic acid, a hydroxycinamic acid, was significantly

enhanced in Stage I fruit after 60 min, which may be closely related to a response due to

a longer exposure to heat stress. However, most polyphenolics were unaffected by

increasing HW treatment times, especially in Stage II fruits.









3.3.2. Quality Analysis

3.3.2.1 pH and soluble solids

Quality parameters for fresh guava fruit and its industrial applications include fruit

diameter and weight, percentage of seeds, puree color, skin color, acidity, flavor, soluble

solids, pH, and ascorbic acid (Boyle et al., 1957). Soluble solids and pH were unaffected

by HW treatment duration (15 to 60 min) within both ripening stages (Table 3-6). Heat

treatment, water or hot air (38 to 48 c for 1 h to 3 days), had no effect on tomato soluble

solids or acidity (Lurie and Klein, 1991; McDonald et al., 1997). In the case of mango,

soluble solids are not affected by an insect vapor heat treatment (Jacobi and Giles, 1997,

Jacobi et al., 2001). Differences in pH were observed due to ripening stage at 30 and 60

min, while for soluble solids Stage I fruit present higher contents at 60 min.

Table 3-5. Quality parameters, soluble solids and pH, in guava as affected by a hot water
immersion treatment (0, 15, 30, and 60 min at 46 C) and ripening stage
(Stage I and Stage II). Data are expressed as fresh weight basis (mean +
standard error), n = 4.
HW Soluble Solids (Brix) pH
treatment
(min) Stage I Stage II Stage I Stage II
0 7.77 0.49 ab 7.37 0.22 b 4.05 0.09 ab 4.05 0.03ab
7.68 0.18
15 8.39 0.33 a 8 0.18 4.03 0.05 b 4.15 0.03ab
ab
30 8.12 0.33 ab 7.25 0.19 b 4.03 0.02 b 4.18 0.05a
60 8.51 0.47 a 7.29 0.26 b 4.03 0.02 b 4.18 0.05 a

3.3.2.2 Overall fruit quality

Observations performed during the storage period following the HW treatments

reported no differences in aesthetic quality of HW-treated guavas as compared to non-

treated fruit. HW treated guavas of all ripening stages did not presented visible signs of

heat injury. Some damages as a consequence of heat stress on HW treated fruit include

irreversible changes such as skin scalding, skin browning and failure to soften (Paull and









Chen, 1990; Jacobi and Giles, 1997), among others. Gould and Sharp (1992) reported no

signs of damage to guavas after HW treatment for 35 min at 46 C. Guavas in the present

study were comparable in weight and shape to Gould and Sharp (1992) study, which are

an important factor which affects the uniformity of heating (Paul and Chen, 2000) and

any subsequent damages present. Additionally, Gould and Sharp (2002) reported storage

temperature after treatment was more important in maintaining fruit quality than the HW

treatment itself. Although storage temperatures were not assessed in the present study,

duration of storage at 15 C might have played an important role in differences observed

between Stage I and II fruits in chemical properties, although aesthetic quality was not

affected. All guavas attained full ripeness and showed no differences among themselves

in quality attributes like firmness and skin coloration independently differences in initial

fruit ripeness and storage temperatures.

3.3.3 Stage III Fruit

Stage III fruit were analyzed independently, being the main interest to determine if

the hot water treatment affected phytochemical properties of ripe guava fruit or not. Fruit

were treated for 30 min at 46 and along with a control group, they were allowed to

reach full ripeness for approximately two days at 15 OC. Moisture content, total soluble

phenolics, antioxidant capacity, ascorbic acid, lycopene, and yellow carotenoids were

unaffected by HW treatment in Stage III guavas. Quality parameters such as pH and

soluble solids were also unaffected. Probably, the fruit had already developed all its

quality and phytochemical properties by the time of treatment application. Comparable to

Stage I and II, the HW treatment caused no change in guava quality and phytochemistry.









Table 3-6. Phytochemical content and quality parameters in Stage III guavas as affected
by a hot water quarantine treatment at 46 C (mean standard error). n = 4.

Time Moisture Soluble Phenolics AOX Capacity Ascorbic Acid
(min) (%) (mg/kg dwb) (jtmol TE/g) (mg/kg dwb)
0 93.21 0.25 a 20070 1200 a 135.5 19 a 13120 1900 a
30 92.85 0.23 a 19110 740 a 110.7 6.4 a 10170 1000 a

Lycopene Yellow carotenoids Soluble Solids pH
(mg/kg) (mg/kg dwb) (o Brix)
0 458.5 81 a 45.98 6 a 6.79 0.25 a 4.225 0 a
30 437.3 76 a 41.98 3 a 7.15 0.23 a 4.100 0 b


3.4 Conclusions

A quarantine hot water treatment at 46 C for up to 30 min can be applied to guavas

(Stage I-III) without affecting its phytochemicals (total soluble phenolics, ascorbic acid,

lycopene, yellow carotenoids, polyphenolic compounds), antioxidant capacity and quality

(pH, brix, overall fruit appearance). Uniformity of the pool of guavas was confirmed by

moisture content, ascorbic acid, yellow carotenoids, and quality parameters. After 60 min

in hot water, Stage I fruit presented an increase in gallic acid, which might be attributed

to an increase in biosynthesis of polyphenolic compounds as a response to heat stress.

Additionally, a decrease in lycopene content was observed, which is also related to

reversible inhibition in its biosynthesis when stress was applied. The chronological age of

the tissue plays an important role, especially when the stress is applied for a longer period

of time. Many of the differences observed in most parameters were due to ripening stage

and storage conditions rather than a hot water treatment effect. Although it is not feasible

to treat ripe guavas, Stage III fruit phytochemicals and quality parameters were not

affected by the HW quarantine treatment for 30 min. Stage I fruit guavas treated for 30






52


min at 46 C are preferred for HW treatment, since they could have a longer shelf life and

allow more marketability to guavas.














CHAPTER 4
EFFECTS OF 1-METHYLCYCLOPROPENE ON GUAVA FRUIT
PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY

4.1 Introduction

Guava (Psidium guajava L.) and more specifically guava juice and puree have

increased in popularity within US markets (NASS, 2004) due to its exotic tropical flavor

and overall consumer appeal. Fresh guava is highly perishable with a retail shelf life of

approximately 7-10 days, creating recurrent pressure for packers and distributors to

deliver a consistent product with widespread consumer acceptability. Due to the delicacy

of its skin and rapid loss in firmness, special care is taken in most postharvest handling

operations such as individually paper-wrapped fruit placed into specially designed

packages (personal communication, Sardinia, 2005). Numerous technologies have been

developed in the past years to extend shelf life of fruits and vegetables, and at the same

time preserve their table quality, allowing their marketability to distant markets. These

include storage under controlled atmospheres (CA), in polybags or with modified

atmosphere packaging (MAP), and coating with polymeric films have shown to prolong

the shelf-life of many commodities (Mitra, 1997). In the case of Florida guavas, many of

these technologies have been evaluated by packers without appreciable success (Sardinia,

2005).

A recent technology emerged from the field of ethylene inhibitors is 1-

methylcyclopropene, or 1-MCP, a gaseous compound that when applied to a commodity

binds to ethylene receptors causing an inhibition in ethylene action (Sisler and Serek,









1997). Due to ethylene's close relation to various ripening processes, many beneficial

effects have been attributed to 1-MCP in the extension of shelf-life (Blankenship and

Dole, 2003). Its practicality of use, low cost, and beneficial effects are an attractive way

of increasing fresh fruit marketing competitiveness. However, despite its vast potential

for the fresh fruit industry, little is known of 1-MCP's effect on phytochemicals and

antioxidant properties, especially with guava. The objective of the present study was not

only to evaluate quality parameters of guava as affected by a 1-MCP treatment, but also

its effects on polyphenolics and antioxidant properties.

4.2 Materials and Methods

4.2.1 Materials and Processing

4.2.1.1 Fruit preparation and 1-MCP treatment

Mature, green pink fleshed guavas (a hybrid variety) procured from Sardinia Farms

(Homestead, Florida) were harvested on July 7, 2004. They were transported to the Food

Science and Human Nutrition Department of the University of Florida, washed, and

stored at 15 C until treatment application. Fruit were uniform in size, shape, firmness,

and skin color (green) and free from any surface damage. Fruit were then transported to

the Horticultural Sciences Department, University of Florida, and approximately 320 fruit

were selected and randomly separated into two groups: Control and 1-MCP treated. Both

groups were arranged separately into two impermeable 174 L capacity chambers in a

storage room held at 10 oC. Calculations by regression (Huber, 2004) were performed to

measure the amount of 1-methylcyclopropene (1-MCP, Smartfresh, Agrofresh, Inc.)

powder, based on total fruit weight, to yield a final concentration of 1,000 nL/L gaseous

1-MCP inside the chamber. The powder was dissolved in 40 mL of deionized water in a

125 mL flask, which was sealed, and vortexed. The flask was placed in the 1-MCP









chamber, unsealed, and the chamber door immediately sealed. The same conditions

applied to the chamber containing the control group using a flask of water without the 1-

MCP. The 1-MCP treatment (1000 nL/L) was maintained for 24 hours, with a second

application after the first 12 hours. At the conclusion of the treatment, fruit were again

transported to the Food Science and Human Nutrition Department and held at a storage

room at 15 C until complete ripeness. Ripe fruit were removed from storage for

physicochemical analysis when the outer skin became thin, completely yellow and the

fruit presented a soft texture, characteristics for a "ready-to-eat", ripe fruit. Day 0 was

established as the 24 hour 1-MCP application period, while Day 1 was established as first

day of storage at 15 OC, less than an hour after the fruits were removed from 1-MCP

treatment. Subsequent days were 24 hours apart from the preceding day. For quality

analysis over time, a group of five guavas were randomly obtained from each group

every 3 to 4 days for evaluation of firmness followed by measurements for pH, total

soluble solids, and titratable acidity.

Additionally, a secondary experiment was simultaneously conducted to evaluate

the effects of 1-MCP application on boxed guavas, in effort to assess applicability of the

1-MCP treatment on fruit ready for shipping. Approximately 180 fruit were selected and

also separated into Control and 1-MCP treated. Fruits for each treatment were arranged

inside four small cardboard boxes (22 to 23 guavas per box), which were stacked inside

their respective chambers and treated as previously described and allowed to ripen at 15

C. During storage, three guavas were obtained every 3 to 4 days for quality analysis

during ripening.









4.2.1.2 Guava fruit processing

Procedures for guava fruit processing were followed as outlined in Chapter 3 with

composites of 5 guavas evaluated within each treatment. Fruits were processed when they

achieved full ripeness, according to parameters described in Chapter 3. Collection of ripe

fruits was done periodically, until the last fruits achieved full ripeness.

4.2.2 Quality Analysis

4.2.2.1 Aesthetic fruit quality assessment during storage

Following treatment with 1-MCP (Day 0), fruit were assessed daily every day for

changes in aesthetic quality characteristics (skin coloration, firmness, presence of

damages/diseases) during the storage period at 15 OC (Days 1 to 26).

4.2.2.2 Firmness determination during storage

Firmness on fresh guava fruits, obtained every 3 to 4 days during ripening, was

measured using an Instron Universal Testing Instrument (Model 4411-C8009, Canton,

Mass.), equipped with a 5 kg load cell and an 8-mm diameter compressive probe,

adapting conditions from Bashir et al. 2002, Reyes and Paull 1995, and Ergun and Huber

2004. The probe was positioned at zero force contact with the surface of the guava. Probe

penetration was set at 10 mm (1 cm) at a crosshead speed of 50 mm/min, and readings

were taken at 3 equidistant points on the equatorial region of the fruit. Firmness data was

expressed as the maximum force (kg) attained during penetration.

4.2.2.3 Titratable acidity, soluble solids and pH

Titratable acidity analysis was performed the guavas obtained every 3 to 4 days

during storage. Approximately 3 g of puree were combined with 10 mL of deionized

water and titrated with 0.1 N NaOH to an end point of pH 8.2. TA was calculated based

on the volume of NaOH used and results were expressed as % citric acid, which is the









major organic acid in guava (Wilson et al., 1982). Soluble solids (SS) and pH

measurements were performed as outlined in Chapter 3; Additionally, SS and pH were

performed on all final ripe samples.

4.2.3 Chemical Analysis

Chemical analysis (total soluble phenolics, antioxidant capacity, ascorbic acid,

lycopene, polyphenolics by HPLC and moisture content) were conducted according to the

procedures outlined in Chapter 3.

4.2.4 Statistical Analysis

The experimental design consisted of a completely randomized design with two

treatments: control and 1-MCP. Statistical analysis consisted of t-test using JMP (SAS,

Cary, NC) to compare differences between treatments (P< 0.05).

4.3 Results and Discussion

4.3.1 Quality Analysis

Detailed treatments comparisons of fruit quality were assessed during storage since

one of the main focuses of 1-MCP is extending produce shelf-life and preserving many of

the physicochemical and quality attributes of fresh guava. The most important aesthetic

quality parameters for guava are firmness and skin coloration. In order to differentiate

between stages of skin coloration during ripening, 4 color criteria were used to describe

the fruit that included green (mature-green stage), yellowish-green (a brighter green color

with yellow tints), turning (40 to 70% surface yellow), and yellow (>70% surface

yellow).

4.3.1.1 Aesthetic fruit quality during storage.

Days 0 to 7: First identifiable differences between treatments









Perceivable differences in surface color were not apparent until 4 days in storage

when approximately 20% of the control fruit were classified as yellowish-green

compared to green for the 1-MCP treated fruit. On Day 5, 30% of control fruit were

yellowish-green and turning, while the 1-MCP group presented 5% of its fruits at a

yellowish-green stage. By Day 7, some bruises on guava skin became apparent on control

fruit, probably due to a more advanced degree of ripening.

Days 9 to 15: Control guavas ripening

Day 9 was characterized by the first collection of ripe guavas from the control

fruits, which accounted for approximately 35% of control fruits. Additionally, control

guavas from boxed treatment were collected. The 1-MCP group remained primarily in

the green color stage, including boxed guavas. A difference in fruit texture between the

treatments was apparent. On Day 13, the first collection of ripe 1-MCP guavas was done,

close to 13% of the 1-MCP group; while 80% of the control fruits had already been

collected for ripeness. Ripe 1-MCP fruits had completely yellow skin coloration and were

firmer than control fruits. The last guavas from the control group were collected on Day

15, when 80% of the original 1-MCP group was still in the process of ripening. A brief

summary of changes in skin coloration during storage is presented in Table 4-1.

Days 16 to 26: 1-MCP guavas ripening

By Day 17, only about 30% of 1-MCP ripe fruits had been collected. Most fruit

remained green, while 40% of the group was turning or yellow. 1-MCP guava ripening

continued until Day 26, when the last batch was collected. Approximately 70% of the 1-

MCP ripe guavas were collected in the period from Day 18 to Day 26. Reyes and Paull

(1995) have reported that guavas stored at 15 OC usually attain full ripeness in a period









between 8 to 11 days. Although, variety, harvest time, post harvest handling and other

parameters shall be considered. Comparably, most of the control guavas (82%) achieved

full ripeness between Day 9 and 13 (12 to 15 days after harvest). 1-MCP was able to

extend the shelf life was able to extend the shelf-life of guavas for at least 5 days.

Yellowish/green
Green (%*) (%) Turning (%) Yellow (%)

Days in Storage
at 15 OC

Control 1-MCP Control 1-MCP Control 1-MCP Control 1-MCP
0 100 100
5 70 100 20 10
7 2 95 73 5 25
9 90 60 10 10 30
13 80 20 20 30 50
15 60 20 5 5 100 15
18 -- 20 -- 30 -- 30 -- 20
22 -- 50 -- 20 -- 30
26 -- -- -- -- 100
Table 4-1. Changes in skin coloration in non-treated (control) and 1-MCP-treated guavas
during 1-MCP application and storage at 15 OC.
% of fruit from the treatment containing determined skin coloration.

The influence of 1-MCP on diseases or disorders has been specific depending on

the species showing mixed results (Blankenship et al., 2003). 1-MCP treated mangos

reported twice the amount of stem rot than control fruit (Hofman et al., 2001), whereas in

apples it has reduced superficial scald (Fan et al., 1999) and in papaya it has shown less

incidence in decay (Ergun and Huber, 2004). Along with bruising, an incidence of brown

spots around the crown and other parts of the fruit was observed in some fruits during

ripening, which was likely due to firm rot, a common disease in guavas usually induced

by bruising (Ko and Kunimoto, 1980; Reyes and Paull, 1995). As the fruit became riper,

bruises and spots became more apparent, especially in control fruits, which started to

ripen earlier. Due to a slowing down in their ripening process, 1-MCP fruits did not show









these disorders as markedly, however by Days 20 to 26, they were more apparent.

Approximately 15% of the original fruits from both groups were lost mainly due to

incidence of firm rot. 1-MCP treatment to guavas did not ameliorated or induced this

disorder.

4.3.1.2 Firmness during storage

Firmness loss during climacteric fruit ripening is directly related to disassembly of

cell wall components (Lohani et al., 2003) and modification of pectin fractions mainly,

with an increase in pectin solubilization (Huber, 1983). These changes are resultant of an

increase in activity of cell wall hydrolases, which have been closely associated to

ethylene (Brummell and Harpster, 2001). Cell wall hydrolases polygalacturonase (PG),

pectinesterase (PE) and cellulose in both white and pink flesh guavas have shown to

increase in activity during ripening, with a correlation between increase in activity of PG

and cellulose and loss of flesh firmness (Abu-Goukh and Bashir, 2003).

A 1-MCP treatment effect was observed since Day 4, were 1-MCP fruits presented

significantly firmer texture as compared to control (Figure 4-1). The trend continued until

Day 12, before the last procurement of control guavas was performed. Comparably,

Basseto et al. (2005) reported that 'Pedro Sato' guavas treated at 900 nL/L (6 to 12 h at

25 C) retained firmness as compared to a control, while fruits treated at lower

concentrations presented no differences in texture. They reported, however, these guavas

(900 nL/L) were not able to attain full ripeness, as opposed to the present study.

Important factors to consider are the relationship between application time and

temperature; in the present study guavas were exposed for a longer period of time at a

lower temperature.







61


During guava storage, there were no significant differences in firmness between

each sampling point, from Day 1 until Day 13 for control fruits and until Day 17 for 1-

MCP treated fruits, as observed in Figure 4-1. A decline in firmness was expected in

control fruits during storage, since softening is directly related to an increase in days of

ripening in guava (Abu-Goukh and Bashir, 2003).However, there were no significant

differences between sampling points. This was likely attributed to the variability of the

samples procured, since they were chosen based on physiological similarities to 1-MCP

fruits collected each sampling day. Mercado-Silva et al. (1998) reported a large

variability in firmness between different ripening stages of guavas. 1-MCP fruits

maintained their firmness throughout the entire storage period, even when they attained

full ripeness.

9.

8 -- Control
--- 1-MCP
7

6
7 5-5



4
3

2
1-

0
1 5 9 13 17
Storage Time at 15 C (Days)


Figure 4-1. Firmness (kg) of guavas treated with 1-MCP (1000 nL/L ,100C, 24 h) during
storage at 15 C. Error bars represent the standard error of the mean, n = 5.

Various effects of ethylene during fruit ripening are apparently associated with

alterations in the properties of cellular membranes. It is thought that ethylene, along with









other growth substances found in plants, binds to some components of the cellular

membrane (proteins, glycoproteins, and lipids) thereby initiating secondary responses

(Noogle and Fritz, 1983). Such bindings, especially with proteins, may have immediate

effects, including protein conformation or cell turgor changes, or trigger other processes

which might take a longer time to become visible. It has been discussed that when

ethylene binds to proteins possessing enzymatic activity, the act of binding may activate

them and alters their rate of degradation (Noogle and Fritz, 1983). By inhibiting ethylene

action, 1-MCP has been related to the delay or inhibition of the activity of cell wall

hydrolases, responsible for tissue softening.

4.3.1.3 Titratable acidity, soluble solids, and pH during storage

Titratable acidity (TA) and pH in 1-MCP fruits was higher than control only on

Day 5 of storage, but not insignificantly different at other days (Figures 4-2 and 4-3).

This difference might be attributed to variation, as seen in the large error rate.

Independently of Day 5, 1-MCP did not affect TA and pH during storage time. Basseto et

al. (2005) reported that 1-MCP treated fruit (900 nL/L) maintained higher titratable

acidity levels during entire storage, attributing it to a ripening delay. Reports on influence

of 1-MCP on titratable acidity during are mixed, depending on the type of commodity or

even variety (Blankenship and Dole, 2003). Additionally, pH on final ripe samples was

not influenced by 1-MCP (average = 4.07). Within the 1-MCP group, TA and pH

remained unaffected by increasing storage time. Within the control group, Day 13 TA

was significantly higher than Day 5, but not different from the rest of the sampling

points; while pH remained unaffected. According to results reported by Reyes and Paull

(1995), both TA and soluble solids as a function of fruit age rather than stage of ripeness,

were both quality parameters are maintained during ripening










0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15


0.10

0.05




Figure 4-2.



4.4


4.3


4.2


S4.1


4.0


3.9


3.8


1 5 9 13


Storage Time at 15 C (Days)
Titratable acidity (% citric acid) of guavas treated with 1-MCP during storage
at 15 OC. Error bars represent the standard error of the mean, n = 5.


1 5 9 13 17
Storage Time at 15 C (Days)


Figure 4-3. Effect of a 1-MCP treatment (1000 nL/L ,100C, 24 h) on guava pH during
storage at 15 OC. Error bars represent the standard error of the mean, n = 5.

Soluble solids were not influenced by the 1-MCP treatment both during storage


(Figure 4-4) and in final ripe guavas (average = 7.81 OBrix). There has been no reported


effect on soluble solids on Brazilian guava (Basseto et al., 2005), mango, custard apple


--- Control
-0- 1-MCP


-*- Control
-0- 1-MCP











(Hofman et al., 2001), apricots, and plums (Dong et al., 2002). Guava soluble solids

maintained uniformity during the storage time within both the control and 1-MCP group.

The role of ethylene on starch and/or sugar conversion is still not clear, with mixed

reports of whether 1-MCP affects their conversion or not (Blankenship and Dole, 2003).

11.0
10.5
10.0
9.5
3
S9.0 -
S8.5
o 8.0
CO
'U 7.5
5 7.0
6.5
6.0 -- Control
5.5- -0- 1-MCP
5.0 -
1 5 9 13 17

Storage Time at 15 C (Days)


Figure 4-4. Effect of 1-MCP treatment (1000 nL/L atl0C, 24 h) on guava soluble solids
(Brix) during storage at 15 OC. Error bars represent the standard error of the
mean, n = 5.

4.3.2 Chemical Analysis

For all chemical analysis performed, except for polyphenolics by HPLC, results

were reported in dry weight basis (DW), in order to eliminate difference between samples

due to varying water loss fruits experienced during ripening, an important factor since 1-

MCP fruits were stored considerably longer than control fruit.

4.3.2.1 Moisture content

Moisture content in the final, ripe fruit was not significantly different between

control and 1-MCP treated fruit at 86.6 and 85.9% respectively. Although, moisture









content determined at final ripe guava puree samples is not the same as a measure of

individual fruit weight loss during ripening, it gives a clearer picture of differences in

final samples due to moisture. It was expected that 1-MCP treated fruits probably had a

higher weight loss due to their longer time in storage. However, no differences in final

moisture values were reported for the present study.

4.3.2.2 Total soluble phenolics

Total soluble phenolics (TSP) content was unaffected by 1-MCP treatment (Figure

4-5). Although some aspects of the ripening process were delayed in 1-MCP fruits, these

presented TSP values of full ripe fruit, as compared to control. Total phenolics from both

groups probably decreased during ripening achieving similar values, independently of the

inhibition of ethylene. However, the addition of ethylene may affect polyphenolic

increase in biosynthesis (Cisneros-Cevallos, 1997). In lettuce, phenylalanine lyase (PAL)

activity was induced by exogenous ethylene, causing an increase in phenolics compounds

(Ke and Salveit, 1988; Dixon and Paiva, 1995; Tomas-Barberan et al., 1997). Also in

lettuce, 1-MCP application (1000 nL/L) did not affect polyphenolic content as compared

to control; however, when 1-MCP was applied prior to exposure to ethylene, there was a

significant reduction in ethylene-induced polyphenolic synthesis (Campos-Vargas and

Salveit, 2002). The addition of exogenous ethylene rather than endogenous ethylene

inhibition by 1-MCP seems to affect more the levels of polyphenolics. In apples, total

phenolics exhibited an ethylene-independent regulation when ethylene was inhibited

(Defilippi et al., 2004). It is concluded that 1-MCP did not affect the final levels of total

soluble phenolics in guava, where probably polyphenolic regulation was not affected by

inhibition of ethylene. The inhibition of ethylene itself, even at a relatively high

concentration of 1-MCP, was not enough to affect polyphenolic biosynthetic pathways.













22000


20000


18000


16000


14000


12000


S1Control
___ 1-MCP


10000 --I I
Guava Treatments After Ripening at 15 C


Figure 4-5. Effect of 1-MCP (1000 nL/L, 100C, 24 h) on total soluble phenolics (mg/kg
DW) in guava. Error bars represent the standard error of the mean, n = 23.


4.3.2.3 Antioxidant capacity


145
140
135 -
130
125
120 -
115
110
105
100
95
90
85
80


S-M Control
S1-MCP


Guava Treatments After Ripening at 15 C


Figure 4-6. Effect of 1-MCP treatment (1000 nL/L, 100C, 24 h) on guava antioxidant
capacity ([tM Trolox equivalents/g DW). Error bars represent the standard
error of the mean, n = 23.


Antioxidant capacity of ripe guavas was unaffected by 1-MCP treatment (Figure 4-


6). This observation supports results for TSP. Since antioxidant capacity in guava was not









affected by 1-MCP this might explain a degree of ethylene independence in the

regulation of most antioxidant phytochemicals during climacteric ripening.

4.3.2.3 Ascorbic acid

1-MCP treated fruit presented significantly higher levels of ascorbic acid as

compared to Control fruit (Figure 4-7). Differences in ascorbic acid might be attributed to

fruit variability than the 1-MCP treatment itself. Basseto and partners (2005) reported no

differences between 1-MCP and control fruit, determined by a titration method. The use

of a more precise analytical method resulted in a more accurate quantification of ascorbic

acid. However, it resulted in a high variability between samples. A variability can be

attributed to the assay for running ascorbic acid, however, careful care was taken when

making composites of five fruits and extracting guava juice and the standard for HPLC

had a low standard error (1.7%). It has been reported a high variability in ascorbic acid

levels in guava, even within the same variety (reviewed by Mitra, 1997). Additionally,

the fact that antioxidant capacity and TSP were not influenced by 1-MCP is a good

indicator that ascorbic acid probably was not affected.

Apart from being an essential nutrient for humans, ascorbic acid within the plant

has numerous roles mainly related to three biological activities: as an antioxidant, as a

donor/acceptor in electron transport at the plasma membrane or chloroplasts, and as an

enzyme co-substrate (Davey et al., 2000). Although the biosynthetic pathway for ascorbic

acid has not been completely elucidated, a pathway via hexose sugars (GDP-mannose,

GDP-L galactose, L-galactose, galacto-1,4-lactone) has been proposed recently (Davey et

al., 2000; Smirnoff and Wheeler, 2000; Barata-Soares, 2004). The relationship existent

between ascorbic acid and ethylene lies within ethylene's biosynthetic pathway, where

ACC oxidase (enzyme responsible for the last step in ethylene biosynthesis), uses










ascorbic acid as a co-substrate, apparently oxidizing it to dehydroascorbate (Davey et al.,

2000; Smith et al., 2000). Ethylene levels produced by most fruits are very low (ppb

range), therefore its normal production or subsequent inhibition might be to low to result

in detectable changes in overall ascorbic acid levels (personal communication, Huber,

2005), specially in guavas, which contain a large pool of ascorbic acid as compared to

most fruits. Additionally other mechanisms such as heat, light or wounding tend to affect

ascorbic acid to a greater extent.

7000

6500 Control
/ 1-MCP
-Q 6000
_0
S5500 -
E
-o 5000

4500
O
0 4000

3500

3000
Guava Treatments After Ripening at 15 C

Figure 4-7. Guava ascorbic acid content (mg/kg DW) as affected by 1-MCP (1000 nL/L,
100C, 24 h). Error bars represent the standard error of the mean, n = 23.

4.3.2.4 Lycopene

Results from lycopene analysis presented significantly higher content in 1-MCP

fruits as opposed to control fruits (Figure 4-8). Equal or even higher lycopene values in 1-

MCP fruit as compared to control indicates that although there was an alteration on

ethylene production and other ripening processes were affected, lycopene accumulation

resulted in guava during its ripening. This might suggest that lycopene accumulation

pathways in guava may not be directly related to ethylene pathways, although they might











affect the rate of lycopene accumulation during ripening. Comparably, Mostofi and

partners (2003) reported final lycopene values in 1-MCP treated tomatoes as

insignificantly different from control, however, the treatment delayed the onset of


lycopene accumulation during storage at 15 OC. In another study on fresh cut tomatoes

using other ethylene inhibitors and exogenous ethylene, although ethylene inhibition

slowed down the rate of lycopene accumulation probably due to other processes slowing

down, it was concluded that ethylene production is not essential for lycopene

biosynthesis in tomato fruit (Edwards et al., 1983).

500

450 I Control
SI -- 1-MCP
400
"0
350

300
C:
0- 250
0
o
.j
200

150

100
Guava Treatments After Ripening at 15 C

Figure 4-8. Effect of 1-MCP (1000 nL/L ,100C, 24 h) on guava lycopene content (mg/kg
DW). Error bars represent the standard error of the mean, n = 23.

It is known from studies on tomatoes that lycopene accumulation is mainly in the

last period of the ripening process (Giovanelli et al., 2004). Although ethylene plays an

important role in accumulation of tomato carotenoids during ripening, since it regulates

phytoene synthase, the lycopene-producing enzyme, it has been discussed that it might

not be the key regulator for lycopene accumulation in particular. Work conducted on









tomatoes by Alba et al. (2002) demonstrated that lycopene accumulation in pericarp

tissue during tomato ripening is mainly regulated by phytochromes or chromoproteins,

and this can be independent of ethylene biosynthesis. The regulation of carotenoid

biosynthesis genes in particular has also been proposed as primary mechanism that

controls lycopene accumulation in tomato fruits (Ronen et al., 1999).

Lycopene is the major carotenoid in guava and its content is comparable or

sometimes higher than tomato. Unfortunately the lack of lycopene in the skin hides its

presence in the pulp, making fruit selection based on lycopene an impossible selling point

in the market. Considering new evidences for correlation between lycopene consumption

and reduced rates of prostate cancer (Giovannuci et al., 1995; Rao and Agarwal, 1999), it

is of particular importance preserving or even enhancing its content during postharvest

operations. The opportunity of marketing guava as an excellent source of lycopene is

present.

4.3.2.5 Polyphenolics by HPLC

Polyphenolics by HPLC were identified by retention time, spectral properties, and

comparison to authentic standards. Based on the work done on the HW treatment study,

approximately 16 peaks were selected from the guava chromatogram. These peaks were

divided into 4 groups based on spectral properties and retention time: gallic acid,

procyanidins, characteristic phenolics, and an ellagic acid derivative. Gallic acid and

ellagic acid derivative were two individual identifiable compounds. Characteristic

phenolics group contained characteristic guava compounds which are unknown, with

spectral properties similar to the ones described for the HW treatment study. The

polyphenolic profile of guavas from the 1-MCP study was similar to the HW treatment

study.







71


1-MCP did not have an influence on the levels of procyanidins, ellagic acid


derivative and other characteristic polyphenolics of ripe guava (Figures 4-9 and 4-10).


The observed results confirm results observed for antioxidant capacity and TSP, which


help explain an ethylene-independent synthesis guava polyphenolic compounds when


ethylene is inhibited. In the case of gallic acid (Figure 4-11), however, 1-MCP treated


fruit exhibited higher content, which might be attributed more to variability in content


between samples, specially within the control group. However, due to gallic acid's


abundance in a wide variety of commodities and its still not completely known synthesis


mechanism, the possibility of an interaction with ethylene inhibition should also be


considered.

10
9 Control
8 1-MCP
8
6L





2-e
O

E


E 4
3

2


0

Guava Treatments After Ripening at 15 C


Figure 4-9. Guava procyanidin content (mg/kg GAE) as affected by 1-MCP. Error bars
represent the standard error of the mean, n = 23.














SControl
m1-MCP


~T


Guava Treatments After Ripening at 15 C


I Control
rI 1-MCP


Guava Treatments After Ripening at 15 C
B


Figure 4-10. Guava ellagic acid derivative content (A) and characteristic polyphenolics
content (B) (mg/kg GAE) as affected by 1-MCP. Error bars represent the
standard error of the mean, n = 23.











45
40 T Control
I I 1-MCP
35

< 30
r 25
5)
E 20

._o

(. 10
5
0
Guava Treatments After Ripening at 15 C


Figure 4-11. Guava gallic acid content (mg/kg GAE) as affected by 1-MCP. Error bars
represent the standard error of the mean, n = 23.

4.3.3 1-MCP Treatment to Boxed Guavas

The present study evaluated the effects of 1-MCP on guava quality and


phytochemical content when application was conducted on guavas arranged inside their


boxes, in a simulated environment prior to shipment. Guavas treated in boxes presented


similar aesthetic characteristics during storage as loose fruit from the previous


experiment. On Day 0, during 1-MCP application, guavas within the boxes presented no


differences among themselves. From Days 5 to 8 in storage the first identifiable


differences were detected, where 1-MCP presented a better retention of green coloration


and firmness, as compared to control fruit. Similarly to the main study, Day 9 was


characterized by the first collection of ripe guavas, which accounted for 14% of the

control group; while most 1-MCP guavas maintained their firmness and green coloration.


The first collection of ripe 1-MCP guavas (17% of the 1-MCP group) was conducted on


Day 12, when already more than 50% of the control fruit had already ripened and been


procured. Similar to the main experiment, collection of the last group of control guavas


was conducted on Day 15, while collection of 1-MCP ripe guavas continued until Day







74


26. Results from aesthetic quality evaluations indicated an effect due to 1-MCP treatment

in color retention, where 1-MCP treated fruits presented a delay in skin coloration

development for at least 5 days, comparably to the main study. Chemical analysis

performed resulted in no significant differences in moisture content (86.1 %), total

soluble phenolics (19,100 mg/kg DW), antioxidant capacity (121 |M Trolox

equivalents/g DW), ascorbic acid (7,090 mg/kg DW), lycopene (853 mg/kg DW), soluble

solids (8.13 Brix) and pH (4.10).

Firmness measured over time, however, presented no significant differences during

storage (Figure 4-12), as opposed to the main experiment. However, as observed by the

large error rates, this statistical lack of effect might be due to variation in samples. A

clear distinction between treatments can be observed, especially after Day 9, where 1-

MCP fruit presents higher firmness values. Probably, a larger number of replications


8

7

6


(1)
4

Ei 3

2
-*- Control
1 -0- 1-MCP

0 -
1 5 9 13 17

Storage time at 15 C (Days)

Figure 4-12. Firmness (kg) of boxed guavas treated with 1-MCP (1000 nL/L ,100C, 24 h)
during storage at 25 oC. Error bars represent the standard error of the mean,
n=3.










might have demonstrated a clearer effect. Titratable acidity (Figure 4-13) and soluble

solids (Figure 4-14) were unaffected by 1-MCP during storage, comparable to the main

experiment. The nature of the packaging material used is an important factor to consider.

It seems that the 1-MCP gas was able to penetrate to the walls of the cardboard box,

diffusing itself among the fruit, delaying skin yellowing and retaining firmness, as

described by aesthetic quality evaluations. Probably, if the boxes had more perforations, a

better penetration and diffusion of the gas would have happened. In a work conducted in

plums packaged similarly inside perforated boxes, 1-MCP proved better effectiveness

than plums treated in bulk (Valero et al., 2004). Therefore, the possibility of applying 1-

MCP on packaged guavas has a potential of being explored further for commercial

applications, especially due to the nature of the fruit, since an easier way to handle any

postharvest process might be beneficial.


0.45

0.40

0 0.35 -

F 0.30 -


S 0.2 -


I--
0.15 -*- Control
-0- 1-MCP

0.10 -0.
1 5 9 13 17
Storage time at 15 C (Days)


Figure 4-13. Titratable acidity (% citric acid) of boxed guavas treated with 1-MCP during
storage at 15 C. Error bars represent the standard error of the mean, n = 3.










13

12 -- Control
-0- 1-MCP
11

S10

9
U)
0 9

S8







1 5 9 13 17
Storage time at 15 C (Days)


Figure 4-14. Effect of 1-MCP treatment (1000 nL/L atl00C, 24 h) on boxed guavas
soluble solids (Brix) during storage at 15 OC. Error bars represent the
standard error of the mean, n = 3.

4.4 Conclusions

Extension of shelf-life in guavas is of extreme value due the highly perishable

nature of these exotics. A 1-MCP treatment (1000_nL/L for 24 h at 10 OC) applied to pink

fleshed guavas was effective in extending their shelf life and retaining quality

characteristics, without detrimental effects to their phytochemicals. Guava shelf life was

extended for at least five days during storage at 15 OC, with a delay in skin yellowing and

retention of firmness. Quality parameters such as titratable acidity, soluble solids, and pH

were maintained during storage, and unaffected by a 1-MCP treatment. Additionally,

there was no significant effect of 1-MCP in total soluble phenolics and antioxidant

capacity. Ascorbic acid and lycopene presented significantly higher values in 1-MCP

treated fruit; however these differences were not attributed to a treatment effect, but

rather to fruit variability and certain independence of ethylene in their biosynthetic









pathways. Procyanidin compounds, total polyphenolics, and an ellagic acid derivative

were not affected by a 1-MCP treatment, which supports results for antioxidant activity

and TSP. An ethylene inhibition in guava, even at a relatively high concentration,

resulted in insignificant effect in most of its phytochemicals. Results from the boxed

guavas study indicate a potential for applying 1-MCP on boxed guavas, and further

investigation in a near future would be beneficial for packers. Literature presents mixed

results on the effects of 1-MCP on physicochemical properties of various commodities,

being species specific and very dependent on application conditions. Results of this study

conclude that 1-MCP can be applied to guava successfully, without negative impacts on

its aesthetic quality, phytochemicals, and antioxidant properties. Further research is

needed to determine optimal application conditions.














CHAPTER 5
SUMMARY AND CONCLUSIONS

Guava marketability as a fresh fruit is somewhat limited due to lack of an

established quarantine treatment and its highly perishable nature. To provide an

overview, these studies consisted of application of two postharvest treatments, a hot

water immersion technique as a quarantine treatment and a 1-methylcyclopropene (1-

MCP) application to extend guava shelf-life. The effects evaluated not only included

overall quality parameters, but guava phytochemicals (polyphenolics, ascorbic acid,

carotenoids, and lycopene) and antioxidant properties. A hot water immersion treatment

at 46 C for up to 30 min may be applied to guavas at three ripening stages without

affecting their quality and phytochemical content. Stage I fruits treated longer than 30

min experienced an increase in certain polyphenolic compounds and a decrease in

lycopene content. This was a response to heat stress, where biosynthesis of certain

polyphenolic compounds was enhanced and lycopene biosynthesis might have been

reversibly inhibited affecting its final concentrations. Other differences reported were

mainly attributed to ripening stage than the HW treatment itself. 1-MCP application

(1000 nL/L for 24 h at 10 C) successfully extended the shelf life of guavas for at least 5

days during storage at 15 OC, presenting positive effects which included skin yellowing

delay and retention of firmness. Although shelf-life was extended, 1-MCP insignificantly

affected quality parameters titratablee acidity, soluble solids, and pH) and phytochemical

content. It was observed a higher ascorbic acid and lycopene content in 1-MCP treated

fruits, which was not directly related to a 1-MCP effect. It was concluded that the






79


biosynthesis pathways for most antioxidant compounds in guava are independent from

inhibition of ethylene action. A HW immersion treatment and a 1-MCP treatment may be

applied successfully to guavas, maintaining their quality attributes and especially not

affecting detrimentally phytochemical compounds.
















LIST OF REFERENCES

Abu-Goukh, A.; Bashir, H. A. Changes in pectic enzymes and cellulose activity during
guava fruit ripening. Food Chem. 2003, 83, 213-218.

Abushita, A.A,; Daood, H.G.; Biacs, P.A. Changes in carotenoids and antioxidant
vitamins in tomatoes as a function of varietal and technological factors. J. Agric.
Food Chem. 2000, 48, 2075-2081.

Akamine, E.; Goo, T. Respiration and ethylene production in fruits of species and
cultivars of Psidium and species of Eugenia. J. Amer. Soc. Hort. Sci. 1979, 104,
632-635.

Alba, R.; Cordonnier-Pratt, M.; Pratt, L. Fruit-localized phytochromes regulate lycopene
accumulation independently of ethylene production in tomato. Plant Physiol. 2000,
123, 363-370.

Association of Analytical Communities. Official methods of analysis ofAOAC
th
International, 17 Ed.; Horwitz: Gaithersburg, MD, 2000.

Barata-Soares, A.D.; Gomez, M.; De Mesquita, C.; Lajolo, F. Ascorbic acid biosynthesis:
A precursor study on plants. Brasilian J. Plant Physiol. 2004, 3, 147-154.

Bashir, H. A.; Abu-Goukh, A. Compositional changes during guava fruit ripening. Food
Chem. 2002, 80, 557-563.

Basseto, E.; Jacomino, A.P.; Pinheiro, A.L.; Kluge, R.A. Delay of ripening of 'Pedro
Sato' guava with 1-Methylcyclopropene. Po,\halt vet Biol. Technol. 2005, 35, 303-
308.

Blankenship, S.; Dole, J. 1-Methylcyclopropene: A Review. Powharl, e~tBiol. Technol.
2003, 28, 1-25.

Boyle, F.; Seagrave, H.; Sakata, S.; Sherman, D. Commercial guava processing in
Hawaii, Bulletin.University of Hawaii: Honolulu, Hawaii, 1957.

Brasil, I.; Arraes Maia, G.; Wilane de Figueiredo, R. Physical-chemical changes during
extraction and clarification of guava juice. Food Chem. 1995, 54; 383-386.

Brecht,J.; Saltveit, M.; Talcott, S.; Schneider, K.; Felkey, K.; Bartz, J. Fresh-cut
vegetables and fruits. In: Janick (ed.). Horticultural reviews. Vol. 30; John Wiley
and Sons: Hoboken, New Jersey, 2004.


80









Brummel, D.; Harpster, M. Cell wall metabolism in fruit softening and quality and its
manipulation in transgenic plants. PlantMol. Biol. 2001, 77, 311-340.

Bulk, R.; Babiker, E.; Tinay, A. Changes in chemical composition of guava fruits during
development and ripening. Food Chem. 1997, 59, 395-399.

Campos-Vargas, R.; Saltveit, M.E. Involvement of putative chemical wound signals in
the induction of phenolics metabolism in wounded lettuce. Physiol. Plant. 2002,
114, 73-84.

Cheng,T.; Floros, J.; Shewfelt, R.; Chang, C. The effect of high temperature stress on
ripening of tomatoes (Lycopersicum esculentum). J. Plant Physiol. 1988, 132, 459-
464.

Ching, L.; Mohamed, S. Alpha-tocopherol content in 62 edible tropical plants. J. Agric.
Food Chem. 2001, 49, 3101-2105.

Cisneros-Zevallos, L. The use of controlled postharvest abiotic stresses as a tool for
enhancing the nutraceutical content and adding-value of fresh fruits and vegetables.
J. FoodSci. 2003, 68, 1560-1565.

Civello, P.; Martinez, G.; Chaves, A.; Anon, M. Heat treatments delay ripening and
postharvest decay of strawberry fruit. J. Agric. Food Chem. 1997, 45, 4589-4594.

Cook, N.; Samman, S. Flavonoids: Chemistry, metabolism, cardioprotective and dietary
sources. Nutr. Biochem. 1996, 7, 66-76.

Davey, M.; Montagu, M.; Inze, D.; Sanmartin, M.; Kanellis, A.; Smimoff, N.; Benzie, I.;
Strain, J.; Favell, D.; Fletcher, J. Plant L-ascorbic acid: chemistry, function,
metabolism, bioavailability and effects of processing. JSci. FoodAgric. 2000, 80,
825-860.

DeEll, J.; Murr, D.; Porteous, M.; Rupasinghe, H. Influence of temperature and duration
of 1-methylcyclopropene (1-MCP) treatment on apple quality. Po\i1,ht ve\t Biol.
Technol. 2002, 24, 349-353.

De Bruyne, T.; Pieters, L.; Deelstra, H., Vlietinck, Condensed vegetable tannins:
Biodiversity in structure and biological activities. Biochemical Systematics and
Ecology. 1999, 27, 445-449

Defilippi, B.G.; Dandekar, A.M.; Kader, A.A. Impact of suppression of ethylene action or
biosynthesis on flavor metabolites in apple. J. Agric. Food Chem. 2004, 52, 5694-
5701.

Dewanto, V.; Wu, X.; Adom, K.K.; Liu, R.H. Thermal processing enhances the
nutritional Value of tomatoes by increasing total antioxidant activity. J. Agric.
Food Chem. 2002, 50, 3010-3014.






82


Dixon, R.; Paiva, N. Stress-induced phenylpropanoid metabolism. Plant Cell. 1995. 7,
1085-1097.

Dong, L.; Lurie, S.; Zhou, H. Effect of 1-methylcyclopropene on ripening of 'Canino'
apricots and 'Royal Zee' plums. Po,/iha, \e't Biol. Technol. 2002, 24, 135-145.

Edmundo, M.P.; Elia, N.; Edmundo, M.S. Antioxidant capacity of guava fruit and jicama
roots under chilling injury conditions. Annual IFT Meeting and Expo.2002.

Edwards, J.I.; Saltveit, M.E.; Henderson, W.R. Inhibition of lycopene synthesis in tomato
pericarp tissue by inhibitors of ethylene biosynthesis and reversal with applied
ethylene. J. Amer. Soc. Hort. Sci. 1983, 108, 512-514.

El-Zoghbi, M. Biochemical changes in some tropical fruits during ripening. Food Chem.
1994, 49, 33-37.

Environmental Protection Agency [EPA]. Biopesticide Federal Register Notices by
Active Ingredient. 1-MCP. 2003. Available online: www.epa.gov Last accessed:
April, 2005.

Fallik, E. Prestorage hot water treatments (immersion, rinsing and brushing). Postharvest
Biol. Technol. 2004, 32, 125-134.

Fan, X.; Blankenship, S.; Mattheis, J. 1-Methylcyclopropene inhibits apple ripening. J.
Am. Soc. Hort. Sci. 1999, 124, 690-695.

Fennema, O.R. Food Chemistry, 3rd Ed.; Marcel Dekker: New York, NY, 1996.

Food and Agriculture Organization [FAO]. Commodity Market Review 2003-2004.
Available online: http://www.fao.org/documents. Last accessed: August, 2004.

Giovanelli, G.; Lavelli, V.; Peri, C.; Simona, Nobili. Variation in antioxidant components
of tomato during vine and post-harvest ripening. J. Sci. FoodAgric. 1999, 79,
1583-1588.

Goldstein, J.; Swain, T. Changes in tannins in ripening fruits. Phytochemistry. 1963, 2,
371-383.

Gorinstein, S.; Zemser, M.; Haruenkit, R.; Chuthakorn, R.; Grauer, F.; Martin-Belloso,
O.; Trakhtenberg, S. Comparative content of total polyphenols and dietary fiber in
tropical fruits and persimmon. J. Nutr. Biochem. 1999, 10, 367-371.

Gould, W.; Sharp, J. Hot-water immersion quarantine treatment for guavas infested with
Caribbean fruit fly (diptera: tephritidae). J. of Economic Entomology. 1992, 85,
1235-1239.

Grundhofer, P., Niemetz, R., Schilling, G., Gross, G. Biosynthesis and subcellular
distribution of hydrolyzable tannins. Phytochemistry. 2001, 57, 915-927.









Hagerman, A.; Riedl, K.; Jones, A.; Sovik, K.; Ritchard, N.; Hartzfeld, Riechel, T. High
molecular weight plant polyphenolics (tannins) as biological antioxidants. J. Agric.
FoodChem. 1998, 46, 1887-1892.

Ho, C.; Lee, C.; Huang M. Phenolic compounds in food and their effects on health I.;
American Chemical Society: Washington, DC, 1992.

Hofman, P.; Jobin-De cor, M.; Meiburg, G.; Macnish, A.; Joyce, D. Ripening and quality
responses of avocado, custard apple, mango and papaya fruit to 1-
methylclopropene. Aust. J. Exp. Agric. 2001, 41, 567-572.

Huber, D.J. The role of cell wall hydrolases in fruit softening. Hort. Rev. 1983, 5,169-219.

Itoo, S.; Matsuo, T.; Ibushi, Y.; Tamari, N. Seasonal changes in the levels of polyphenols
in guava fruit and leaves and some of their properties. J. Japan. Soc. Hort. Sci.
1987, 56, 107-113.

Jacobi, K.; Giles, J. Quality of Kensington mango (Mangifera indica Linn.) fruit
following combined vapour heat dissinfestation and hot water disease control
treatments. Pothii \r vt Biol. Technol. 1997, 12, 285-292.

Jacobi, K.; MacRae, E.; Hetherington, S. Postharvest heat disinfestation treatments of
mango fruit. Scientia Horticulturae. 2001, 89, 171-193.

Janick, J. Horticultural Reviews: Volume 30; John Wiley and Sons: Hoboken, HJ, 2004.

Jeong, J.; Huber, D.J.; Sargent, S. Influence of 1-methylcyclopropene (1-MCP) on
ripening and cell-wall matrix polysaccharides of avocado (Persea americana) fruit.
Postharvest Biol. Technol. 2002, 25, 241-256.

Jiang, Y.; Joyce, D.; Terry, L. 1-methylcyclopropene treatment affects strawberry fruit
decay. Poithai\l 't Biol. Technol. 2001, 23, 227-232.

Jimenez-Escrig, A.; Rincon, M.; Pulido, R.; Saura-Calixto, F. Guava fruit (Psidium
guajava L.) as a new source of antioxidant dietary fiber. J. Agric. Food Chem.
2001, 49, 5489-5493.

JMP; SAS Institute, Inc., SAS Campus Drive, Cary, NC, 1996.

Ke, D.; Salveit, M.E. Plant hormone interaction and phenolic metabolism in the
regulation of russet spotting in iceberg lettuce. Plant Physiol. 1988, 1136-1140.

Kipe, S. The World Fresh Fruit Market. USDA Foreign Agricultural Service.
Horticultural and Tropical Products Division. 2004. Available online:
www.fas.usda.gov/htp/Presentations/2004 Last accessed: August, 2004.

Ko, W.; Kunimoto, R. Guava fruit firm rot induced by bruising. Hort. Science. 1980, 15,
722-723.









Kondo,S.; Kittikorn, M.; Kanlayanarat, S. Preharvest antioxidant activities of tropical
fruits and the effect of low temperature storage on antioxidants and jasmonates.
Postharvest Biol. Technol. 2005, In Press.

Lee, J.H.; Johnson, J.V.; Talcott, S.T. Identification of ellagic acid conjugates and other
polyphenolics in mature muscadine grapes by HPLC-ESI-MS. 2005, ASAP Article.

Leonardi, C.; Ambrosino, P.; Esposito, F.; Fogliano,V. Antioxidative activity and
carotenoid and tomatine contents in different typologies of fresh consumption
tomatoes. J. Agric. Food Chem. 2000, 48, 4723-4727.

Leong, L.; Shui, G. An investigation of antioxidant capacity of fruits in Singapore
markets. Food Chem. 2002, 76, 69-75.

Lin, C.H.; Chen, B.H. Determination of carotenoids in tomato juice by liquid
chromatography. J.ofChromatography. 2003, 1012, 103-109.

Liguori, G.; Weksler, A.; Zutahi, Y.; Lurie, S.; Kosto, I. Effect of 1-MCP on melting
flesh peaches and nectarines. Postharvest Biol. Technol. 2004, 31, 263-268.

Lohani, S.; Trivedi, P.; Nath, P. Changes in activities of cell wall hydrolases during
ethylene-induced ripening in banana: effect of 1-MCP, ABA, and IAA. Postharvest
Biol. Technol. 2004, 31, 119-126.

Lurie, S. Postharvest heat treatments. Postharvest Biol. Technol. 1998, 14, 257-269.

Lurie, S.; Handros, A.; Fallek, E.; Shapira, R. Reversible inhibition of tomato gene
expression at high temperature. Plant Physiol. 1996, 110, 1207-1214.

Lurie, S.; Klein, J. Acquisition of low-temperature tolerance in tomatoes by exposure to
high-temperature stress. J. Amer. Soc. Hort. Sci. 1991, 116, 1007-1012.

Malo, S.E; Campbell,C.W. 1994.The Guava. University of Florida-IFAS extension.
Bulletin. Available online: http://edis.ifas.ufl.edu/MG045. Last accessed: August,
2004.

Marcelin, O.; Williams, P.; Brillouet, J. Isolation and characterization of the two main
cell-wall types from guava (Psidium guajava L.) pulp. Carbohydr. Res. 1993, 240,
233-243.

Martinez-Valverde, I.; Periago, M.; Provan, G.; Chesson, A. Phenolic compounds,
lycopene and antioxidant activity in commercial varieties of tomato (Lycopersicum
esculentum). J. Sci. FoodAgric. 2002, 82, 323-330.

Mercadante, A.; Steck, A.; Pfander, H. Carotenoids from Guava (Psidium guajava L.):
Isolation and Structure Elucidation. J. Agric. Food Chem. 1999, 47, 145-151.









Mercado-Silva, E.; Benito-Bautista, P.; Garcia-Velasco, M. Fruit development, harvest
index and ripening changes of guavas produced in central Mexico. Po,\h/,i ue\t Biol.
Technol. 1998, 13, 143-150.

Miean, K.H.; Mohamed, S. Flavonoid (myricetin, quercetin, keampferol, luteolin, and
apigenin) content of edible tropical plants. J. Agric. Food Chem. 2001, 49, 3106-
2112.

Mir, N.; Curell, E; Khan, N.; Whitaker, M.; Beaudry, R. Harvest maturity, storage
temperature, and 1-MCP application frequency alters firmness retention and
chlorophyll fluorescence of 'Redchief Delicious' apples. J. Am. Soc. Hort. Sci.
2001, 126, 618-624.

Misra, K.; Seshadri, T. Chemical components of the fruits ofPsidium guajava.
Phytochemistry. 1967, 7, 641-645.

Mitra, S. Postharvest physiology and storage of tropical and subtropical fuits, CAB
International: New York, New York, 1997.

Morton, J.F. The Guava. Fruits of warm climates, Media Incorporated: Greensboro, NC,
1987.

Mostofi, Y.; Toivonen, P.; Lessani, H.; Babalar, M.; Lu, C. Effects of 1-
methylcyclopropene on ripening of greenhouse tomatoes at three storage
temperatures. Postharvest Biol. Technol. 2003, 27, 285-292.

Mowlah, G.; Itoo, S. Quantitative changes in guava polyphenols and the
polyphenoloxidase (PPO) at different stages of maturation, ripening, and storage.
Nippon Kogyo Gakkaishi. 1982, 29, 413-417.

Mueller-Harvey, I. Analysis of hydrolysable tannins. Anim. Feed Sci. Technol. 2001, 91,
3-20.

Mullins, E.D.; McCollum, T.G.; McDonald, R.E. Consequences of ethylene metabolism
of inactivating ethylene receptor sites in diseased non-climacteric fruit. Postharvest
Biol. Technol. 2000, 19, 155-164.

National Agriculture Statistics Service [NASS]. 2004. Guava output falls for the third
straight year.Bulletin: Hawaii Guavas. Available online: www.nass.usda.gov. Last
accessed: February, 2005.

Noogle, G. and Fritz, G. Introductory plant physiology 2nd Edition, Prentice Hall Inc.:
Englewood Cliffs, New Jersey, 1983.

Ou, B.; Hampsch-Woodill, M.; Prior, R.L. Development and validation of an improved
oxygen radical absorbance capacity assay using fluorescein as a fluorescent probe.
J. Agric. Food Chem. 2001, 49, 4619-4626.









Padula, M.; Rodriguez-Amaya, D. Characterisation of the carotenoids and assessment of
the vitamin A value of Brasilian guavas (Psidium guajava L). Food Chem. 1986, 20,
11-19.

Paull, R. Response of tropical horticultural commodities to insect desinfestation
treatments. Hort. Sci. 1994, 29, 988-996.

Paull, R.; Chen, N. Heat treatment and fruit ripening. Po(,thal ve't Biol. Technol. 2000,
21-37.

Rao, A.; Agarwal, S. Role of lycopene as antioxidant carotenoid in the prevention of
chronic diseases: A review. Nutr. Res. 1999, 19, 305-323.

Regalado-Contreras, E.; Mercado-Silva, E. Effect of hot water treatment on the ascorbic
acid and reduced glutathione levels in guava fruit during cold storage. Annual IFT
Meeting and Expo. 1998.

Reyes, M.U.; Paull, R.E. Effect of storage temperature and ethylene treatment on guava
(Psidium guajava L.) fruit ripening. Po(,tha vi't Biol. Technol. 1995, 6, 357-365.

Rice-Evans, C.; Miller, N.; Paganga, G. Structure-antioxidant activity relationships of
flavonoids and phenolic acids. Free Radical Biol. Med. 1995, 20, 933-956.

Robbins, R. Phenolic acids in foods: An overview of analytical methodology. J. Agric.
Food Chem. 2003, 51, 2866-2887.

Ronen, G.; Cohen, M.; Zamir, D.; Hirschberg, J. Regulation of carotenoid biosynthesis
during tomato fruit development expression of the gene for lycopene epsilon-
cyclase is down regulated during ripening and is elevated in the mutant Delta. Plant
Journal 1999, 17, 341-351.

Sahlin, E; Savage; G.P.; Lister, C.E. Investigation of the antioxidant properties of
tomatoes after processing. Journal of Food Composition and Analysis. 2004, 17,
635-647.

Saltveit, M. Effect of ethylene on quality of fresh fruits and vegetables. Postharvest Biol.
Technol. 1999, 15, 279-292.

Salveit, M. Effect of 1-methylcyclopropene on phenylpropanoid metabolism, the
accumulation of phenolics compounds, and browning of whole and fresh-cut
'iceberg' lettuce. P(,oilhi VetBiol. Technol. 2004, 34, 75-80.

Selvarajah, S.; Bauchot, A.D.; and John, P. Internal browning in cold-stored pineapples is
suppressed by a postharvest application of 1-methylcyclopropene. Po(,\thltii e't Biol.
Technol. 2001, 23: 167-170









Seybold, C.; Frohlich, K.; Bitsch, R.; Otto, Konrad; Bohm, V. Changes in contents of
carotenoids and vitamin E during tomato processing. J. Agric. Food Chem. 2004,
52, 7005-7010.

Shahidi, F.; Wanasundara, P. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 1992, 32,
67-103.

Sisler, E.C., Serek, M. Inhibitors of ethylene responses in plants at a receptor level.
Recent developments. Physiol. Plant. 1997, 100, 577-582.

Skerget, M.; Kotnik, P.; Hadolin, M.; Rizner, A.; Marjana, S.; Zeljko, K. Phenols,
proanthocyanidins, flavones, and flavonols in some plant materials and their
antioxidant activities. Food Chem. 2005, 89, 191-198.

Smirnoff, N.; Wheeler, G. Ascorbic acid in plants: Biosynthesis and function. Critical
Reviews in Plant Sciences, 2000, 19, 267-290.

Swain, T. and Hillis, W.E. 1959. The phenolic constituents ofPurmus domestic. I. The
quantitative analysis of phenolic constituents. J. Sci. FoodAgric. 1959, 10, 63-68.

Takeoka, G. R.; Dao, L.; Flessa, S.; Gillespie, D. M.; Jewell, W. T.; Huebner, B.;
Bertow,D.; Edeler, SE. Processing effects on lycopene content and antioxidant
activity oftomatoes. J Agric. Food Chem. 2001, 49, 3713-3717.

Talcott, S.T., Howard, L, Brenes, C.H. Antioxidant changes and sensory properties of
carrot puree processed with and without periderm tissue. J Agric. Food Chem.
2000, 48, 1315-1321.

Talcott, S.T., Percival, S.S., Pittet-Moore, J. Celoria, C. Phytochemical composition and
antioxidant stability of fortified yellow passion fruit (Passiflora edulis). J. Agric.
Food Chem. 2003, 48, 4, 1315-1321

Thimann, K. Senescence inplants, CRC Press: Boca Raton, FL, 1980.

Thompson, K.; Marshall, M.; Sims, C.; Sargent, S.; Scott, J. Cultivar, maturity, and heat
treatment on lycopen content in tomatoes. J. Food Sci. 2000, 65, 791-793.

Tomas-Barberan, F.A.; Loaiza-Velarde, J.; Bonfanti, A.; Saltveit, M.E. Early wound- and
ethylene-induced changes in phenylpropanoid metabolism in harvested lettuce. J.
Am. Soc. Hortic. Sci. 1997, 122, 399-404.

United States Department of Agriculture [USDA]. Available online:
http://www.usda.gov. Last accessed: August, 2004.

United States Department of Agriculture-Animal and Plant Health Inspection Service
[USDA-APHIS]. 2004. Available online: http://www.aphis.usda.gov. Last
accessed: March, 2005.









Van de Berg, H.; Faulks, R.; Granado, H. F.; Hirschberg, J.; Olmedilla, B.; Sandmann,G.;
Southon, S.; Stahl, W. The potential for the improvement of carotenoid levels in
foods and the likely systemic effects. J. Sci. Food. Agric. 2000, 80, 880-912.

Valero, D.; Martinez-Romero, J.M.; Guillen, F.; Castillo, S.; Serrano, M. Could 1-MCP
treatment effectiveness in plum be affected by packaging? Po/,tht ivIt Biol.
Technol. 2004, 295-303.

VERIS Research Information Service. 2000. Carotenoid fact book. VERIS, La Grange,
IL.

Wilberg, V.; Rodriguez-Amaya, D. HPLC quantitation of major carotenoids of fresh and
processed guava, mango, and papaya. Lebensm-Wiss. U. Technol. 1995, 28, 478-
480.

Wilson, C.; Shaw, P.; Campbell, C. Determination of organic acids and sugars in
guava(Psidium guajava L.) cultivars by high-performance liquid chromatography.
J. Sci. FoodAgric. 1982, 33, 777-780.

Xie, D.; Dixon, R. Proanthocyanidin biosynthesis-still more questions than answers?
Phytochemistry. In Press.

Yadava, U. Guava production in Georgia under cold-protection structure. In: J. Janick
(ed.) Progress in new crops; ASHS Press: Arlington, VA, 1996.