Summary of citrus processing and by-products research projects.
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Title: Summary of citrus processing and by-products research projects.
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Creation Date: 1957
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Year Ending June 30, 1957
Florida Citrus Experiment Station and Florida Citrus Commission
Lake Alfred, Florida

State Project 550 R. Patrick and E. C. Hill

Identification of Microorganisms in Citrus Concentrates. Streptococcus-
like organisms are associated with juice from fruit that is overmature or that
has been damaged, mechanically or by freezing. Such organisms were sometimes
found in citrus concentrates and showed rapid decline in viability when the con-
centrates were stored at OOF. These organisms have presented a diversified re-
lationship in their environmental factors and consequently are very difficult to
classify as to species. The fact that all of the unidentified cultures, that
were isolated from citrus concentrates, showed a strong reducing action in
methylene blue-milk before curdling is affected, suggests the possibility that
these organisms are closely related to the streptococcus lactis-type. Also in
favor of this relationship are the facts that some of the cultures did not ferment
glycerol or sorbitol and did tolerate 0.3 percent methylene blue. At this time
it is believed that these streptococcus-like organisms are common contaminants of
plants and soil and will not be of any public health significance, even though
they grew in 6.5 percent NaC1 medium and at 4500., which are characteristic of

Examination of Freeze-Damaged Oranges. Juices were extracted from both
Pineapple and Valencia oranges that showed evidence of different degrees of
freeze injury; juices from fruit with no or slight damage had total plate counts,
using three media, ranging from 63,500 to 120,000 per milliliter; with moderate
freeze damage from 72,500 to 456,000 per milliliter; and with extreme injury from
647,500 to 768,400 per milliliter. Valencia oranges, that were less mature than
the Pineapple oranges at the time of the freeze but hung on the trees longer
before harvesting, had lower counts than those in the juices from the damaged
Pineapple oranges. Streptococcus-like organisms were found in the juice extracted
from cold-damaged oranges. The coli-aerogenes contamination also present in a
few of the samples was of the aerobacter-type.

This fruit was obtained by Mr. George F. Westbrook and used for the prep-
aration of experimental packs of frozen orange concentrate.

Chilled Orange Juice. Chilled orange juices were packed in waxed cartons
to determine the effects of temperature of storage and stabilization temperature
on the rate of growth of microorganisms in this product. Other packs of chilled
juice were prepared from both 420 and 590 Brix frozen orange concentrates after
storage of these products for two months; these chilled juices were also stored
at different temperatures.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801 9/26/57 FW

Freshly extracted Valencia orange juice was chilled and packed in quart
cartons. The juice was not subjected to any heat treatment. Cartons were held
at 320, 400, 500, and 600F. The initial plate count for the juice was 60,000
colonies per milliliter, the diacetyl value was 3.1 parts per million and the pH
was 3.9. After two days the 600F. sample had a plate count of 2,960,000 per
milliliter and a diacetyl value of 4.9 parts per million. On the fifth day the
plate count was 11,000,000 per milliliter and the diacetyl value of the first
25 milliliter distillation fraction had reached 30.7 parts per million. A third
distillation fraction of this juice had a diacetyl value of 29.0 indicating that
this high diacetyl value was due to acetylmethyl-carbinol and the spoilage due
primarily to yeast. The cartons stored at 500F. also spoiled rapidly. In seven
days at 500F. the juice had a diacetyl value of 7.8 parts per million and a plate
count of 19,000,000 per milliliter. Chilled juice stored at 40CF. showed no in-
crease in diacetyl value after 26 days. The microbial population increased to
600,000 colonies per milliliter but there was no detectable off-flavor character-
istic of microbiological spoilage. The juice stored at 320F. showed no increase
in diacetyl value and a decrease in plate count from 60,000 to 11,000 colonies
per milliliter.

Stabilized chilled juices were prepared from freshly extracted Valencia
orange juice. Heat treatment of the juice, pH 3.8, was at 1550, 1750, and 1950F.
with the exposure time constant; one pack was not heated. The four packs of
juice were stored only at 400F. The initial count of the unheated juice was
56,500 per milliliter and this increased in 24 days to 141,000 per milliliter.
The three packs of heat-treated juices had less than 1,000 organisms per milli-
liter and there was no increase after 24 days at 400F. The diacetyl value was
initially 3.6 parts per million and there was no significant change in this
value in any of the juices throughout the storage period; also, no off-flavors
developed during storage that could be attributed to microorganisms.

Chilled juices were also prepared from frozen orange concentrates, that had
been previously made from Valencia oranges and stored at -80F. for two months.
The concentrates were mixed with cold water and the reconstituted juices were
immediately packed in cartons and stored at 400, 500, and 600F. Both 420 Brix
and 590 Brix concentrates were used and when they were processed from the same
batch of orange juice, pH 3.8, the evaporator feed juice was heated at 1750F. The
420 Brix concentrate contained unheated cut-back juice; the 590 Brix product con-
tained added cold-pressed orange oil but no cut-back juice. Since the juices
were prepared from concentrates the diacetyl values were at the very low level of
0.1 and 0.4 parts per million and the initial plate counts were about 20,000 per
milliliter. The juices stored at 600F. fermented by the third day. The products
stored at 500F. were spoiled on or before the eighth day. After 22 days, there
was no increase in diacetyl values or numbers of microorganisms in the juices
stored at 400F.

Storage temperature is the most important factor in preventing microbiologi-
cal spoilage of chilled orange juice during storage; the types and number of
microorganisms present and the pH of the juice are also important. Products
stored at 500 and 600F. spoiled very rapidly, whereas there was no evidence of

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801a 9/26/57 FWW


fermentation in chilled juices held at 320 and 400F. for three weeks. Heat treat-
ment of juice at 1550, 1750 or 1950F. before chilling reduced greatly the total
number of microorganisms, but the initial plate counts and those after the
samples had been stored at 400F. for 24 days were about the same, regardless of
the stabilization temperature. Chilled juices prepared from stabilized frozen
420 or 590 Brix orange concentrates had similar microbiological characteristics
and showed little difference in the rate of microbiological spoilage from those
prepared from freshly extracted juice which was stabilized by heat treatment
before chilling and packaging.

State Project 611 E. L. Moore, A. H. Rouse
and C. D. Atkins

Packs of 420 Brix Pineapple orange and Valencia orange concentrates were
prepared for storage to investigate further the relative effect of heating either
the single-strength juice or heating the partially concentrated juice at some
stage of the concentration process. This additional work is being done because
various research agencies have differed in their findings on this subject. Pre-
sent-day methods of juice extracting and finishing were used in preparation of
these packs to insure that sufficient water-soluble pectin would be incorporated
into the juice to establish a good turbidity or cloud. Heat treatment was
accomplished using a plate type heat exchanger.

Ten 420 Brix Pineapple orange concentrates were made from either single-
strength juice heated to 1650F. prior to evaporation, or from 2-, 3-, and 4-fold
concentrates withdrawn during the evaporation of unheated juice and subsequently
heat treated at this temperature. The heated juice and concentrates were then
individually concentrated to 550 Brix; each of the four resulting products was
divided into two portions which were cut back to 420 Brix with either 0.030
inch finisher juice or 0.040 inch Inline extractor juice. One unheated control
550 Brix concentrate was also prepared and cut back to 420 Brix with the above
two cut-back juices.

Five 420 Brix Valencia orange concentrates were prepared similarly with
three exceptions: (a) the extracted juice before finishing was passed through a
pump that would cut the pulp particles to uniform size and should liberate more
water-soluble pectin, (b) because of a difference in pH, the single-strength
juice and 2-, 3-, and 4-fold concentrates were heated to 1750F. to obtain about
the same percentage inactivation of pectinesterase as was obtained for the
Pineapple orange juice and concentrates and (c) the 550 Brix concentrates were
cut back to 420 Brix with the 0.040 inch Inline extractor juice only. The amount
of pectinesterase inactivation for the heated juices and concentrates, made from
the two orange varieties, fell within the narrow range of 94.2 to 95.8 percent.

Both the Pineapple orange and Valencia orange concentrates are being stored
at -80, 100, 200, 300, and 400F., and are being examined at periodic intervals.
In general, based on analyses to date, results are in agreement with previous

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801b 9/26/57 FWW


work which showed that concentrates prepared from juices heated at 2-, 3-, and
4-fold lost cloud more rapidly at the higher storage temperatures and did so in
direct relation to the degree of concentration before heat treatment.

State Project 649 C. D. Atkins, A. H. Rouse,
E. L. Moore and R. W. Olsen

Examination of Commercial Frozen Grapefruit Concentrates. Twenty-eight
samples of commercial frozen concentrated grapefruit juice were examined for
some characteristics that determine their physical and chemical stability.
Pectinesterase activity in these samples was restricted to a narrow range of 0.3
to 4.1 units with 78.6 percent of the samples being within the range of 0.3 to
1.9 units. These low pectinesterase values indicated that effective heat treat-
ment was used in preparation of these grapefruit concentrates. The pulp content,
determined by volume, was distributed over the range of 4.0 to 11.5 percent. The
pectic content of the samples was determined on both the reconstituted juice and
a centrifuged portion of the juice. Of the reconstituted juices, 78.6 percent
contained 20 to 39 milligrams of water-soluble pectin per 100 grams, 85.7 percent
contained 4.5 to 9 milligrams of ammonium oxalate-soluble pectin per 100 grams,
and 71.4 percent contained 5 to 14 milligrams of sodium hydroxide-soluble pectin
per 100 grams. In the reconstituted centrifuged juices the pectin content varied
from 20 to 39 milligrams per 100 grams in 85.8 percent of the samples. Water-
insoluble solids in the reconstituted juices ranged from 50 to 99 milligrams per
100 grams in 75 percent of the products, the lowest value being 40.8 and the
highest value 151.6.

None of these samples showed any initial clarification or gelation and after
storage at 800F. for 24 hours, slight gelation was noted in two concentrates and
a semi-gel formed in only one sample; under the same conditions of storage,
definite clarification occurred in two products and extreme clarification in six
samples. Neither clarification nor gelation occurred to any significant degree
in any of the 28 grapefruit concentrates after storage at 400F. for 48 hours.

Factors to Consider in Determining Clarification in Frozen Orange Concen-
trate. A study was made to determine the effect of (a) procedure for thawing
frozen orange concentrate prior to analysis, (b) conditions for centrifugation of
the reconstituted juice on the amount of turbidity or cloud in the product. Four
commercial packs of 420 Brix frozen orange concentrate were selected that con-
tained various levels of pectinesterase and pectin. A composite sample was made
of each pack to insure uniformity. Variations used for thawing the frozen samples
were (a) storage at either 400F. or 800F., and (b) in running tap water without
agitation for 30 minutes, and (c) rotation of cans in water at either 400 or
800F. After thawing, one set of the samples was examined immediately and other
sets stored at either 400 or 800F. for varying periods of time. All samples,
either initially or after storage, were reconstituted with distilled water and
then thoroughly mixed for the same period of time using an electric stirrer.
Each of the reconstituted juices was divided into two portions, one of which was

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801c 9/26/57 FWW


centrifuged in an International Centrifuge, size 1, Type SB, for 10 minutes
at 1300 revolutions per minute and the other portion for 15 minutes at 1700
revolutions per minute. Thus the percentages of pulp by volume in the
reconstituted juices were determined. Light transmittance values for the serum
were obtained using both Lurntron 401 and 402-E colorimeters; the pectin content
of the serum was also determined. As expected, centrifugation for 10 minutes at
1300 revolutions per minute resulted in greater pulp values for the reconstituted
juices than those obtained when 15 minutes at 1700 revolutions per minute were
used; however, such differences in conditions for centrifugation did not result
in any significant differences in the amount of cloud or pectin in the serum.

When the frozen orange concentrates were thawed and then examined immediately,
the various methods of thawing had no effect cn the light transmittance values or
the pectin content of the reconstituted centrifuged juices. However, agitation
during thawing of frozen samples prior to storage did affect the amount of cloud
that resulted in the reconstituted juices, especially in the concentrate having
a low pectin content and a high pectinesterase activity. Higher light trans-
mittance values, indicating less cloud, were found in products that were stored
at either 400 or 800F. in the frozen state or thawed without agitation and then
placed in storage at the elevated temperatures, than in those samples thawed
prior to storage by rotation of the cans in water. These results indicated that
it was preferable to thaw samples of frozen orange concentrates in water at 400F.
by rotating for one hour when they are to be stored at 400F.; samples to be
stored at 800F. should be rotated in water at 800F. for 15 minutes. If such pre-
cautions are not taken, variable data m.y be obtained for the same sample of
concentrate concerning the degree of clarification that occurs during storage at
either 400 or 800F.


Relationship of Heat Treatment to Quality of Processed Citrus Products. -
A procedure by which complete inactivation of the pectinesterase in grapefruit
sections packed in 211 x 400 cans with 420 Brix sugar syrup consisted of exhaust-
ing for 15 minutes in a hot water bath at 1950F. (temperature at center of can
about 1750F.), processing in a water bath for 40 minutes at 1950F. (temperature
at center of can about 1900F.), and cooling 15 minutes in running tap water.

When canned sections were experimentally heated in a water bath at a temp-
erature of 2080F. for 60 minutes or more, they turned soft in texture and tan
in color.

Grapefruit used in this study was selected so that the effect of the use of
arsenated or unarsenated fruit on the quality of canned grapefruit sections
could also be investigated. Silver Cluster grapefruit from both arsenated and
unarsenated trees were picked and processed in December of 1956, January, Feb-
ruary, and March of 1957. The arsenated trees were sprayed in May, 1956, with
1.25 pounds lead arsenate per 100 gallons of water plus 10 pounds of wettable
sulfur. The canned grapefruit sections were stored at 800F. and periodic exami-
nations for physical appearance, as well as chemical changes in water-insoluble
solids, water-soluble pectin, ammonium oxalate-soluble pectin, and sodium hydrox-
ide-soluble pectin are being continued. (A. H. Rouse, C. D. Atkins and E. L.
Florida Citrus Experiment Station and Florida Citrus Commission,
Tn-e Alfred. Florida. 801d 9/26/57 FWW


Flavor of Processed Citrus Products. Further studies on oxidized flavors,
usually described as similar to cardboard, tallow or castor oil, in frozen citrus
concentrates showed that the addition to Hamlin orange concentrate of beta-
carotene and lycopene, with or without the addition of ascorbic acid, eliminated
or reduced the occurrence of these off-flavors. (R. W. Olsen and E. L. Moore.)

The flavor of samples from 28 packs of commercial frozen grapefruit concen-
trates was evaluated by a taste panel; the samples were collected from 11 plants
during the 1955-56 season. The flavor of all of these samples was of acceptable
quality; 14 of the concentrates were graded "good" and the other 14 samples were
graded "fair". A greater variation was noted in the flavor grades designated by
each of the panel members for many of the grapefruit juices, than that usually
found when reconstituted frozen orange concentrates had been graded by the same
taste panel. Defects most frequently mentioned were bitterness and the oxidized
off-flavors. (F. W. Wenzel and R. W. Olsen.)

An investigation was started to determine the effect of various procedures
on the flavor of frozen concentrated orange juice. Packs of frozen Valencia
orange concentrate were made which included processing variables, such as yield
of juice, stabilization temperature, degree of concentration, quantity of cut-
back juice used, and pulp and recoverable oil content of the 420 Brix concentrate.
Flavor evaluation of these products has not been completed. (F. W. Wenzel and
R. W. Olsen.)

Packs of 420 Brix and 530 and 590 Brix high-density frozen orange concen-
trates were made from the same batch of Valencia orange juice so that a comparison
of the flavor and other characteristics of these products could be made, both
initially and after storage. The products were prepared both with and without
the addition of cold-pressed Valencia orange oil, and one pack of 590 Brix high-
density concentrate was made using, for cut-back juice, a 240 Brix concentrate
that had been obtained by freeze concentration of Valencia orange juice. (R. W.
Olsen, C. D. Atkins and F. W. Wenzel.)

Factors Affecting Quality of Processed Grapefruit Products. Characteris-
tics of 11 experimental packs of unsweetened 380 Brix frozen concentrated
grapefruit juices were determined. These products were processed in March,
April, May, and August, 1956, using grapefruit from different localities.
Analyses of the raw juices indicated a range in soluble solids of 9.10 to 13.90
Brix; total acid, as citric, from 0.90 to 1.73 percent; Brix-to-acid ratio from
7.2 to 10.4; and pulp from 5 to 22 percent by volume. Examination of the frozen
grapefruit concentrates prepared from the raw juices showed that the soluble
solids varied only slightly from 37.90 to 38.60 Brix; total acid, as citric,
ranged from 3.82 to 5.33; Brix-to-acid ratio from 7.1 to 10.1; recoverable oil
from a trace to 0.020 milligram per 100 grams; Hunter Color Difference Meter "a"
values from -5.7 to -2.6, "b" values from 17.5 to 20.0, and "Rd" values from
15.2 to 18.7. The pulp content, by volume, in the reconstituted juices varied
from 4.0 to 12.0 percent; the total glycosides, expressed as naringin, ranged
from 56 to 74 milligrams per 100 milliliters and naringin varied from 39 to 51
milligrams per 100 milliliters, when determined by the difference in total

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.


glycosides before and after hydrolysis of the naringin by a glycosidase. Ascor-
bic acid in the reconstituted grapefruit juices ranged from 35 to 53 milligrams
per 100 milliliters. Other characteristics of 10 of these frozen grapefruit con-
centrates, together with the processing procedures used, were previously reported
(Ann. Rpt., 1956, p. 201).

The flavor of these unsweetened 380 Brix grapefruit concentrates was evalu-
ated, as well as that of 19 packs containing added sugar that were prepared from
the same 500 Brix concentrates used in making the unsweetened products. The
sweetened concentrates were made by adding either or both the minimum and maximum
amounts of sugar so that the finished products would contain 36 percent juice
solids, Brix from 380 to 480 and Brix-to-acid ratio from 10 to 13. Coldpressed
grapefruit oil was added to the sweetened products on the basis of 0.03 milliliters
per 100 grams, but none was added to the unsweetened concentrates. The reconsti-
tuted juices from each of the 30 packs were tasted by from 31 to 42 persons,
including the Citrus Station taste panel and quality control personnel of five
commercial plants. The results indicated a definite preference for the sweetened
concentrates; also a very slight preference for the sweetened products that con-
tained a minimum amount of added sugar. Flavor defects most frequently indicated
as reasons for poor flavor quality in some of the products were sourness, bitter-
ness, astringency and oxidized off-flavors. (R. W. Olsen, E. L. Moore, R. L.
Huggart, S. V. Ting, R. W. Barron, and F. W. Wenzel.)

Experimental packs of frozen grapefruit concentrates processed in 1956 were
examined to determine the effect of variety and maturity of fruit and other
factors on the quality of this product. Duncan and Marsh grapefruit were used
and the fruit was obtained from the same four plots at intervals of about six
weeks. Arsenated and unarsenated fruit of both varieties were used. Packs were
first processed on January 4 and the last packs prepared on June 29. The soluble
solids in the raw juices ranged from 9.2 to 12.90 Brix; total acid, as citric,
from 0.78 to 1.99 percent; and Brix-to-acid ratio from 6.1 to 12.8. The juices
were stabilized by heating to 1950F. in a plate-type heat exchanger, prior to
concentrating to approximately 500 Brix. The concentrated juices were cut back
to 380 Brix, using both heated and unheated cut-back juices. All of the products
were canned, frozen and stored at -80F.

The ranges in values found for various characteristics of the 20 packs of
380 Brix frozen grapefruit concentrates that contained unheated cut-back juices
follow: soluble solids 37.8 to 39.50 Brix; total acid, as citric, 3.04 to 6.62
percent; Brix-to-acid ratio, 5.9 to 12.8; recoverable oil, trace to 0.081 milli-
liters per 100 grams; Hunter Color Difference Meter "a" values, -6.5 to -2.1,
"b" values, 18.2 to 20.3, and "Rd" values, 15.6 to 20.1; in the reconstituted
juices, total glycosides, 66 to 102 milligrams per 100 milliliters; naringin, 42
to 62 milligrams per 100 milliliters; ascorbic acid, 28 to 45 milligrams per 100
milliliters; and diacetyl values, none to 1.5 parts per million.

The 40 packs of 380 Brix grapefruit concentrates, that contained both heated
and unheated cut-back juices were examined for factors affecting physical and
chemical stability. Data on the reconstituted juices varied within the following
ranges: pulp from 7 to 22 percent, water-insoluble solids from 76 to 212 milli-
grams per 100 grams, pectinesterase activity from none to 2.1 units, and water-

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801f 9/26/57 FWW

soluble pectin from 24 to 56 milligrams per 100 grams. The water-soluble pectin
was found to be significantly higher in the Duncan than in the Marsh seedless
reconstituted juices. The results from the analyses showed that concentrates
produced from arsenated fruit were just as stable as products made from unarsenated
fruit. There was neither gelation nor loss of cloud during storage of the concen-
trates at 40OF. for 48 hours. Transmittance values on the reconstituted juices
before and after storage of the concentrate varied on a Lumetron 402-E colori-
meter from 47 to 56 percent, indicating no clarification. (R. W. Olsen, A. H.
Rouse, E. L. Moore, R. L. Huggart, C. D. Atkins, S. V. Ting, R. W. Barron, E. C.
Hill, and E. J. Deszyck.)

Standardization of Processed Citrus Products. Analyses were made to deter-
mine some of the characteristics of 28 samples of commercial frozen concentrated
grapefruit juices that were packed during the 1955-56 season and collected from
11 plants with the help of personnel of the Agricultural Marketing Service, USDA,
Winter Haven, Florida. Soluble solids in the concentrates ranged from 39.9 to
45.60 Brix; total acid, as citric, from 3.89 to 4.66 percent; Brix-to-acid ratio
from 8.9 to 11.2; and ascorbic acid from 96 to 149 milligrams per 100 grams, which
corresponded to a range of 29 to 44 milligrams per 100 milliliters in the recon-
stituted juices. Recoverable oil in the reconstituted juices varied from 0.002
to 0.013 percent by volume, corresponding to 0.006 to 0.044 milligrams per 100
grams in the concentrates. Measurement of the color differences in the grapefruit
concentrates were made with the Hunter Color Difference Meter. Hunter "a" values
varied from -6.7 to -3.3; "b" values from 17.5 to 22.4; and "Rd" values from 15.9
to 22.0. Total glycosides, expressed as naringin, ranged from 36 to 64 milligrams
per 100 milliliters in the reconstituted juices; naringin varied from 20 to 41
milligrams per 100 milliliters, as determined by the difference in total glyco-
sides before and after hydrolysis of the naringin by a glycosidase. (R. L.
Huggart, R. W. Barron and S. V. Ting.)

Over 200 samples of commercial frozen orange concentrate have been collected
from Florida plants during the 1956-57 season. These samples are being examined
for flavor and various physical and chemical characteristics so that a comparison
may be made with results obtained from similar surveys made during the 1953-54 and
1954-55 citrus seasons. (F. W. Wenzel, R. L. Huggart and R. W. Barron.)

To determine individual preferences for canned grapefruit juice sweetened
with the artificial sweetener, Sucaryl, three packs of canned juice were processed.
The three canned products obtained were made from the same batch of grapefruit
juice; one pack consisted of the unsweetened juice, sugar was added to the juice
for the second pack and the third pack was prepared from juice that was sweetened
with Sucaryl. These canned grapefruit juices will be sent to the Agricultural
Marketing Service, USDA, Washington, D. C., for their evaluation prior to prep-
aration of larger packs of these products for a consumer marketing test.
(R. W. Olsen and R. W. Barron.)

Gelation in Orange Marmalade Bases. Pregelation sometimes occurs in barrels
of commercial bitter orange marmalade bases during storage in warehouses at
ambient temperatures prior to its processing into orange marmalade. Methods for
the prevention of this pregelation in such products were investigated. Three
different lots (A, B, and C) of commercial marmalade base were obtained, two of
which were expected to gel, according to initial information from the manufacturer.
Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801g 9/26/57 FWW

Four packs were made from each of the three lots in the following manner, (a) un-
treated control, (b) addition of sodium carbonate to raise the products A, B, and
C from pH 2.9, 3.3, and 2.9, respectively, to pH 4.0, (c) addition of Pectinol M
to destroy the water-soluble pectin at the original pH of the bases and (d) addi-
tion of Pectinol M to the bases after raising them to pH 4.0. Although the
marmalade bases were originally heated, residual enzymic action was present,
therefore all packs, including those treated with Pectinol M for two hours, were
reheated. Samples of all packs prepared from the three different lots of com-
mercial marmalade were stored at 100, 400, and 900F.

Control samples of lots A and C formed gels when stored at 400 and 900F. in
eight and four weeks, respectively; samples stored at 100F. showed no tendency to
form gels. Samples of lot B did not gel under any condition. Treatment with
Pectinol M, either at the original pH of the bases or after adjusting bases to
pH 4.0, prevented gelation when samples were stored for 24 weeks at the above
temperatures. At 900F. storage, sample A raised to pH 4.0, formed semi-gels in
four weeks, but the degree of gelation did not increase with continued storage;
whereas, sample C at pH 4.0 showed no tendency to form even semi-gels during
storage. Samples from the three lots receiving no added sodium carbonate in-
creased in pH, while the citric acid content decreased when stored at either 400
or 90OF.; there were no significant changes at 100F. Water-insoluble solids in
the control samples of lots A and C increased with storage, while in control
sample B and samples A and C treated with sodium carbonate and Pectinol M there
was a general trend to a decrease in water-insoluble solids. All samples re-
ceiving Pectinol M, without raising the pH, almost doubled in water-soluble pectin
by conversion of and at the expense of the sodium hydroxide-soluble pectin; very
little differences were found in the amount of oxalate-soluble pectin present
because of the reheating of the samples. (A. H. Rouse and C. D. Atkins.)

Characteristics of Processed Citrus Products Made from Freeze-Damaged Fruit.-
A study of the characteristics of 37 experimental packs of frozen concentrated
orange juice, most of which contained juice from freeze-damaged fruit, was com-
pleted. A reduction in total acid, and resulting increase in pH and Brix-to-acid
ratio, were characteristic in the concentrates made from freeze-damaged fruit.
The pulp and flavonoid content of the reconstituted juice usually increased with
extent of freeze injury in the oranges. A very high relative viscosity was
found to be typical of products prepared from cold-damaged Pineapple-type oranges,
but this was not true when damaged Valencia oranges were used. An increase in
total pectin was characteristic of concentrates prepared from freeze-injured
fruit. Gelation and clarification were almost always worse when concentrates
were made from damaged Pineapple-type oranges than when undamaged fruit was used.
Higher temperatures than 1750F. were necessary for stabilization to prevent ge-
lation and clarification, but heat treatment of the cut-back juice was not found
necessary for that purpose when the evaporator-feed juice was heated at 1950F.
Valencia orange concentrates made from freeze-damaged fruit showed little tend-
ency to gel or clarify. Loss of flavor and/or the development of off-flavor may
occur rapidly in concentrate prepared from fruit injured during a freeze.
(George F. Westbrook and F. W. Wenzel.)

Production and Use of Activated Citrus Sludge. The production of activated
citrus sludge using the laboratory activated sludge system was concluded. Samples

Florida Citrus Experiment Station
and Florida Citrus Commission, Lake Alfred, Florida.
801h 9/26/57 FWW


of dried sludge were collected representing 15 variations of nutrient levels and
drying methods. During all 15 runs the reductions in B.O.D. and organic solids
were excellent. The samples collected have been analyzed for nitrogen, thiamine,
pantothenic acid and vitamin B12.

The nitrogen content of the sludge samples ranged from 4.3 to 7.0 percent,
thiamine from none to 9.6 micrograms per gram, vitamin B12 from 2.1 to 24.9
micrograms per gram and pantothenic acid from 5.9 to 41.5 micrograms per gram.
The amount of nutrient nitrogen used during sludge production proved to be
beneficial to the nitrogen content of the final dry product but quite detrimental
to thiamine production. Additions of cobaltous chloride appeared to be beneficial
to both thiamine and B12 production. The effect of the addition of phosphate on
the production of the above mentioned constituents was not detectable, but the
combination of nitrogen and phosphate gave a marked increase in vitamin B12 pro-

The method of drying was also found to be another factor that determined the
amount of B vitamins in the sludge. Oven drying at 1050C. for 24 hours destroyed
a large amount of the thiamine and vitamin 112. Drum drying is the method of
choice if the vitamin content of the dried product is of primary concern.

In the past during pilot plant operation, serious deterioration of the
activated sludge culture occurred at a temperature of 410C.; this high tempera-
ture was caused by overloading the system. So that a study could be made of the
effect of high temperatures on the activated sludge culture without the influence
of an overload, the laboratory aeration unit was converted from a three-stage to
a single-stage unit; heating tape was installed on the outside of the aeration

As a result of a four-month study on the effect of increased temperature on
the activated sludge culture it was found that (1) an activated sludge unit could
be operated at 360C. with no deleterious effect on the culture or the degree of
treatment obtained; (2) if, in approaching a temperature of 36C., a rapid rise
in temperature of 40 or 5C. occurred, a temporary period of unhealthy conditions
was encountered but recovery took place if no further increase in temperature
occurred; (3) as temperatures were increased above 3600., the number and species
of protozoa began to decrease; (4) at a temperature of 430C. the culture was
completely devoid of all protozoa, and (5) at a temperature of 4600C. the degree
of treatment obtained was so poor that further investigation was not deemed

The modified method (1956 Ann. Rpt., p. 206) for determining the strength
of citrus waste water by means of chemical oxygen demand analysis was changed
further. By comparison with a set of standards sealed in glass the chemical
oxygen demand of a sample may be estimated, according to the new procedure, to
the nearest 100 parts per million without the use of titration or colorimetric
instruments. The method is satisfactory when speed is more essential than
accuracy. (M. H. Dougherty, R. R. McNary and R. W. Wolford.)

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801i 9/26/57 FWW


Inositol in Citrus Fruits. In the course of these studies it was established
that inositol occurs naturally in citrus fruits as the free, water soluble, myo-
form. It was found to be distributed throughout the fruit with the largest
amount in the juice; also decreasing amounts in order in the rag, peel and pulp.

Primary emphasis has been placed on the development of an economical method
for recovery of inositol from citrus juices, peel and waste liquids from both
processing and by-products plants. An ammoniacal lead acetate precipitation of
the filter press water, obtained from the by-product recovery of hesperidin,
caused nearly complete removal of myo-inositol from solution. Since in the
hesperidin recovery process the orange peel was reacted with lime before pressing,
no prior precipitation with an alkaline hydroxide was conducted. The alkaline
press water which had been acidified to pH 5.3 with hydrochloric acid for the
crystallization of hesperidin was heated to 700C., neutralized to pH 7.0 with
ammonium hydroxide and precipitated with lead acetate. In this way the lead hy-
droxide wad formed in solution and if the proper amount of the acetate was
employed, the inositol could be readily precipitated as a lead complex. The
quantity of lead acetate used was varied between 0.32 and 1.3 grams per gram of
solids in the press water. An amount of 0.64 grams per gram of solids proved to
be sufficient for good inositol recovery if the first lead precipitate was re-
moved and the filtrate was allowed to stand for 12 to 24 hours. At the end of
that time the pH had lowered to 5.5 or 6.0 and if neutralized again with ammonium
hydroxide, more of the lead acetate still in solution was then precipitated. This
precipitate contained the remainder of the inositol.

The crude myo-inositol was freed by treating the lead precipitate in aqueous
slurry with 30 percent phosphoric acid, by carbonation with carbon dioxide and by
sulfite formation with sulfurous acid, sodium meta-bisulfite, or sulfur dioxide.
The latter reaction which formed the insoluble lead sulfite allowed good release
of the inositol from the lead complex.

When difficulty was encountered in the crystallization of inositol from the
crude solution it was revealed that the interference could be removed by pyridine
extraction. In this way the myo-inositol was successfully crystallized from the
pyridine insoluble material. The presence of water soluble inorganic substances
had little or no effect on the crystallization in this procedure. The identity
of the interfering organic impurity has not yet been determined.

Another procedure for the isolation of inositol from the crude solutions
consisted of adding acetic acid to the solution to make an acid concentration of
90 percent and then precipitating with isopropyl alcohol. Two precipitations
in thit way produced an inositol solution in which crystallization could be pro-
moted by usual methods. (R. W. Wolford, R. R. McNary and M. H. Dougherty.)

Citrus Vinegar. Over 30 gallons of grapefruit vinegar were made.. Samples
of this, as well as samples of orange and tangerine vinegars, were submitted to
several food companies, various organizations, and many persons for evaluation of
flavor. Many favorable comments were received indicating that these vinegars had
distinctive and desirable flavor characteristics; also, that they could be mer-
chandised in a manner similar to wine vinegar.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801j 9/26/57 FWW


The brown sediment that appears in citrus vinegar has been traced to the
commercial pectic enzyme preparation that has been used to remove the pectin.
The sediment was resistant to hydrolysis; but when heated to 1500C. with 20 per-
cent hydrochloric acid in a sealed tube for six hours, sufficient breakdown
occurred that nine amino acids were found in the hydrolyzate. The pectic enzyme,
when similarly hydrolyzed, yielded the same amino acids. Paper chromatography
was used to indicate the amino acids.

The same pectic enzyme preparation has been useful in removing the bitter
glucoside, naringin, from grapefruit juice that was being made into vinegar. The
amount required to remove all of the naringin was found to be two grams per
liter of juice.

Several times a deterioration of the surface film of vinegar bacteria has
taken place when the slow vinegar process was being used. Investigation of the
heavy metal content of healthy versus sickly films indicated that metallic con-
tamination was not the cause of the disease. The cause has not yet been deter-
mined. (R. R. McNary, R. W. Wolford and M. H. Dougherty.)

Effect of Borax and Arsenic Sprays on Certain Characteristics of Extracted
Juices of Marsh Grapefruit. Data for degrees Brix, pulp by volume, water-
insoluble solids, water-soluble pectin, pectinesterase activity, and turbidity or
cloud were obtained on juices extracted from Marsh grapefruit picked from trees
that had received arsenic and boron sprays. Lead arsenate spray applications
consisted of 0, 0.4, 1.25 and 3.0 pounds per 100 gallons of water and borax was
sprayed at the rate of 1.0 pound per 100 gallons of water. Regardless of the
amount of arsenic spray, juice extracted from fruit which was sprayed with boron
was 23.5 and 20.9 percent lower in water-insoluble solids and pectinesterase
activity, respectively. The juices from fruit from trees receiving 1.25 and 3.0
pounds of lead arsenate per 100 gallons of water, regardless of boron spray,
contained less water-soluble pectin than grapefruit from unarsenated trees or
from those receiving only 0.4 pound of lead arsenate per 100 gallons of the
spray application. (A. H. Rouse, C. D. Atkins, E. L. Moore, and E. J. Deszyck.)

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801k 9/26/57 FWW


State Project 607 J. W. Kesterson and R. Hendrickson

The ultraviolet and infrared absorption spectra for coldpressed orange,
grapefruit, tangerine, lime, and lemon oils have been studied for an additional
season. The data obtained verifies the findings as reported last year. As a
result of this two-year study, it becomes even more apparent that these criteria
can be used as a standard of purity and identity for citrus oils.

By the use of infrared and ultraviolet absorption data, it is possible to
distinguish between citrus oils produced from color-added, packing house elimi-
nations as compared to those produced from grove run fruit. When color-added
fruit is used, lower CD and peak absorption values occur in the ultraviolet. An
absorption area at 13.1 mu in the infrared is greatest for the color-added fruit.
These differences will vary according to the quantity of color-added fruit pro-
cessed. Evaporation residues from color-added fruit give a characteristic orange
color; whereas, grove run fruit gives a greenish-orange colored residue.

Ultraviolet absorption criteria have been used to determine the quality of
coldpressed tangerine oil during the past season. Pure tangerine oil has peak
absorptions at 325 mu and 270 mu. The peak absorptions at 325 mu occur at 1.05
to 1.28 with CD values of 0.53 to 0.68. The peak absorptions at 270 mu occur at
1.27 to 1.43 with CD values of 0.20 to 0.24. One lot of tangerine oil, produced
from tangerine peel containing approximately five percent orange peel, gave peak
absorption values at 327 mu and 267 mu. At 327 mu the peak absorption occurred
at 0.98 with a CD value of 0.48. At 267 mu the peak absorption occurred at 1.21
with a CD value of 0.16. Orange oil has peak absorptions at 330 mu which range
from 0.42 to 0.68 and CD values from 0.24 to 0.42. Since orange oil is so differ-
ent from tangerine oil, it requires only a small addition of orange oil to greatly
influence the ultraviolet characteristics of tangerine oil. As shown above, the
addition of five percent orange peel to tangerine peel shifted the peak absorptions
from 325 mu and 270 mu to 327 mu and 267 mu respectively. The peak absorption and
CD value was also reduced below the minimum for pure tangerine oil.

The effect of total rainfall on the aldehyde content of coldpressed Valencia
orange oil has been studied for a period of nine years. Normal or above normal
rainfall gives an oil with a normal aldehyde content. One drought year following
a rainy period will give an oil of increased aldehyde content. The second and
third year in succession will result in an extremely low aldehyde content oil.
This phenomenon has occurred twice during the past nine years.

State Project 622 J. W. Kesterson and R. Hendrickson

This project was inactive during the year because the recovery of this bio-
flavonoid had previously been worked out in the laboratory. However, the
pharmaceutical industry has shown considerable interest in naringin, and the
necessary steps have been taken to do pilot plant work on the recovery of this
product during the coming season.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801 1 9/26/57 FWW


State Project 646 R. Hendrickson and J. W. Kesterson

Continued widespread pharmaceutical interest in citrus bio-flavonoids has
led to the successful production of hesperidin as an established new by-product,
the production of which is a direct result of recent laboratory findings of this
laboratory. The therapeutic uses of this product suggested the need for more
specific analytical procedures for hesperidin. Considerable attention was given
to the adaptation of an AOAC semi-micro alkoxyl determination. Concurrently
evaluated was a gravimetric procedure whereby hesperidin, dissolved in cold
alkaline methyl alcohol under vacuum, was reprecipitated after removal of impuri-
ties. Results obtained by these new procedures were compared with the older
standard photometric Davis test and other less used chemical procedures. Crude
hesperidin extracts analyzed by the methoxyl procedure gave erroneously high re-
sults, due probably to incompletely degraded pectinous-like impurities. Analyses
of high purity hesperidin were considerably more accurate by this procedure ex-
cept when one of the lower alcohols was used in the purification technique, which
led again to high results. The gravimetric procedure consistently showed hesperi-
din samples to be of lower purity than by Davis test, with best agreement again
occurring at highest purity. Although neither procedure consistently assured
more accurate results than the Davis test, each procedure can yield valuable
information when the history of a hesperidin sample is known.

The previously mentioned gravimetric procedure, which in reality is a puri-
fication technique having only slight commercial possibilities, was found to
yield an exceedingly pure hesperidin when analyzed by Davis test. A process
capable of economically manufacturing hesperidin of equal purity required con-
siderable investigation and entailed numerous failures. Of the numerous trials,
washing hesperidin with exceedingly large volumes of boiling slightly alkaline
water was one of the better methods of removing the last three percent of impuri-
ties. One contaminating impurity, hesperitin, was isolated, purified and its
interference in the Davis test evaluated. The analytical methoxyl procedure was
especially useful in quality-checking hesperitin.

In continued attempts to improve the recovery of hesperidin from oranges,
two detergent systems were tried with no marked improvement. Although the deter-
gent system appeared to increase hesperidin crystal size, filtration rate and
purity were not enhanced. Improved recovery of hesperidin from oranges was
possible, however, by using only a segregated portion of the peel ejected from
one particular extractor. By avoiding the separated portion containing mostly
rag and pulp, higher hesperidin yields were obtained. This procedure looks
promising where limited by-product processing facilities are available.

The previously developed technique of purifying naringin using isopropanol
and isolating a high purity alcoholate was further investigated to show the im-
portance of anhydrous conditions in the process. Solubility data for this new
product in alcohol and water mixtures at various temperatures were studied. Al-
though patent possibilities for this product were abandoned in favor of publication,
it is noted that a similar product has since been marketed nationally.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801 m 9/26/57 FW


Conversion of Citrus Terpenes To Useful Chemical Compounds

State Project 817

W. F. Newhall

This project was initiated on October 17, 1956, with the object of synthe-
sizing from citrus terpenes, particularly d-limonene, chemical compounds which
would extend the uses of these materials and bring a better monetary return to
the citrus industry.

A study is now in progress to devise means of preparing in good yield
oxygenated derivatives of d-limonene such as A' 8 (O)-p-menthenel-, 2-diol (I).
This glycol has been prepared from d-limonene by a number of authors but the
best yield obtained has been 24 percent (E. Earl Royals et al., J. Am. Chem.
Soc. 77, 3405-8, 1955).

Extensive oxidation studies of d-limonene have failed so far to furnish
the 1, 2 diol (I) in any better yield than 15-20 percent. However, an analog of
I which is a crystalline solid has been prepared from d-limonene in 40-45 per-
cent yield. This work is being continued.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
801n 9/26/57 FWW