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Title: Pectinesterase and pectin in commercial citrus juices as determined by methods used at the Citrus Experiment Station
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Title: Pectinesterase and pectin in commercial citrus juices as determined by methods used at the Citrus Experiment Station
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Publication Date: 1955
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ji Cy o

Bulletin 570 December 1955



UNIVERSITY OF FLORIDA
AGRICULTURAL EXPERIMENT STATIONS
J. R. BECKENBACH, Director
GAINESVILLE, FLORIDA

(A contribution from the Citrus Experiment Station in cooperation
with the Florida Citrus Commission)









Pectinesterase and Pectin in Commercial Citrus
Juices as Determined by Methods Used
At the Citrus Experiment Station


A. H. ROUSE and C. D. ATKINS









TECHNICAL BULLETIN









Single copies free to Florida residents upon request to
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA






















CONTENTS
Page

INTRODUCTION .. ........... ................. 3

LITERATURE SURVEY .........- --....................-.. ......-.- 3

Pcctinesterase ..--.............- ...-..................... . 3

Pectin ....... .. ......--- ...... .-..- -............ ... 4

PECTINESTERASE ....................................... .... 6

Reagents for Pectinesterase ..........--.... ........- ...- ...- ....... 6

Analytical Method for Pectinesterase ..-.--....--........-..........-.. 7

PECTIC SUBSTANCES .......-............- .. .. .... ..- ..-........ 8

Reagents for Pectic Substances -............ ............. .. ..... .. 8

Extraction of Pectic Substances ................ ........ ..... ....... 8

Colorimetric Method for Pectic Substances .....................-........... 10

PREPARATION OF STANDARD CURVE WITH AGA ...............-.......... ....-...-. 10

INTERFERING MATERIALS ............... .....---.....- ...... 11

TECHNIQUES ....................---------..........-- ---..------ .........------- ... 12

RESULTS AND DISCUSSION .....-........--.-..-..-- ... ........ .... 13

SUMMARY .....-----.....--.........----- -..-.-............ ... 17

LITERATURE CITED ...................--- ..... ... ------ ------------.................. 17










Pectinesterase and Pectin in Commercial Citrus
Juices as Determined by Methods Used
At the Citrus Experiment Station

A. H. ROUSE and C. D. ATKINS

INTRODUCTION
The spectacular development and production of frozen con-
centrated orange and grapefruit juices during the past 10 years
has brought about increased interest in the chemical composi-
tion of citrus juices. Prevention of gelatin and clarification in
concentrated citrus juices is of major importance to processors,
since product quality is affected and economic loss may occur.
The desirable "cloud" is retained and the undesirable formation
of gel lumps is prevented by protecting the natural pectin found
in the extracted product from alteration by the enzyme, pec-
tinesterase (PE). Pectin is the naturally occurring colloidal
stabilizer that gives citrus juices a viscosity or consistency
termed "body" by the fruit juice industry. When the pectin
colloid is altered by the activity of pectinesterase, the juice
clarifies and becomes "watery", resulting in settling of the
colloidal suspended material as well as rapid separation of the
suspended water-insoluble solids called pulp.
For some time the authors have given much attention to the
study of PE activity and the pectic substances in citrus juices
and concentrates. The importance of PE activity and of pectin
stability to the quality of frozen concentrated citrus juices has
made it desirable to develop and apply procedures for determin-
ing PE activity and the kind and quantity of pectic substances
in citrus products. Thus, the purpose of this bulletin is to
present the methods found to be best suited for measurement
of PE activity, for the extraction of pectic substances, and for
estimating the extracted pectic substances.

LITERATURE SURVEY
Pectinesterase.-Though the chemistry of enzymic demethy-
lation had been known for many years, no reports dealing with
a quantitative study appeared in the literature until 1937 when
Kertesz (10)1 proposed a method for the determination of PE

Italic figures in parentheses refer to Literature Cited.







4 Florida Agricultural Experiment Stations

in tomato juice. At that time a reaction mixture of pH 6.2
was chosen and maintained by using methyl red indicator instead
of the present optimum pH 7.5 with the use of a glass electrode
pH meter.
Lineweaver and Ballou (13), in their study of the effect of
cations on the activity of alfalfa PE, and Hills and Mottern (8),
in their investigation of properties of tomato PE, improved on
Kertesz's method by showing that the enzyme from different
sources had an optimum pH and needed an optimum quantity
of electrolyte present in order to obtain the maximum activity.
The specificity of PE was further shown by MacDonnell, Jang,
Jansen and Lineweaver (15) in a study of the enzyme from
three plant sources-alfalfa, orange and tomato-as well as from
one fungal source.
In 1945 MacDonnell, Jansen and Lineweaver (14) published
a method used in studying the properties of orange PE which
is similar to that used for alfalfa PE by Lineweaver and Ballou
(13). Two years later McColloch and Kertesz (17), in their study
of tomato PE, improved the original Kertesz's method (10). The
method adopted by this laboratory for the measurement of PE
in citrus juices includes features from the procedures of both
the above methods (14, 17).
The word pectinesterase was proposed by Lineweaver and
Ballou (13) and has been accepted by the citrus processing
industry for the enzyme that hydrolyzes the ester bonds of
pectin present in citrus juices. Synonyms of pectinesterase
found in the literature are pectase, pectin-methoxylase and
pectin-methylesterase.
Pectic substances became of vital interest to processors of
frozen concentrated citrus juices when the problem of clarifi-
cation and gelation was pointed out by Rouse (23) as being
associated with the formation of low-methoxyl pectin caused by
the PE activity in citrus juice.
Pectin.-Pectin was first isolated by precipitation in alcohol,
which resulted in a crude means of estimating the quantity
present in juices or extracts. Also, the cationic precipitation of
pectin with calcium, iron, magnesium, aluminum, copper, nickel
and other metallic polyvalent ions resulted in either a coagula-
tion of impurities along with the pectin or incomplete precipita-
tion because of insufficient free carboxyl groups present in the
pectin molecule. Pectin precipitates estimated by these two
methods may have impurities ranging from 10 to 90 percent.







Pectinesterase and Pectin in Commercial Citrus Juices 5

From these procedures for estimating pectic content there were
developed the more accurate calcium pectate method of Carr6
and Haynes (4) and the pectic acid method of Wichman, which
is the method used by the A.O.A.C. (1). Improvements were
made in these two procedures by Poore (22), Hinton (9) and
Fellers and Rice (7). However, these two procedures are long
and tedious and are not applicable for routine laboratory control
in processing plants.
The pectic substances as a class are one of the most important
of the naturally occurring polyuronides in plants. The basic
structure of the pectin molecule is a mixture of polygalacturonic
acids; and, as has been shown by Myers and Baker (20), the un-
hydrolyzed pectin contains 83.2 percent galacturonic acid. Be-
cause of the heterogeneous nature of pectic substances, it has
been difficult to isolate and determine, as well as designate, the
various pectic fractions which are responsible for clarification
and gelation in citrus juices. In 1941 a committee on nomencla-
ture was appointed by the Agricultural and Food Chemistry
Division of the American Chemical Society and the proposed
nomenclature is as follows (12):
Pectic Substances: "Pectic substances" is a group desig-
nation for those complex colloidal carbohydrate derivatives which
occur in or are prepared from plants and contain a large pro-
portion of anhydrogalacturonic acid units which are thought to
exist in a chainlike combination. The carboxyl groups of poly-
galacturonic acids may be partly esterified by methyl groups and
partly or completely neutralized by one or more bases. They
always represent mixtures of polygalacturonic acids containing
variable numbers of associated units. With the exception of
pectic acid, they may also contain different proportions of methyl
ester groups in the individual polygalacturonic acids. These
may be unevenly distributed on the polygalacturonic acid. Acid
salts, again, may contain polymer units with different propor-
tions of metallic ions.
Protopectin: The term "protopectin" is applied to the water-
insoluble parent pectic substances which occurs in plants and
which upon restricted hydrolysis yields pectin or pectinic acids.
Pectinic Acids: The term "pectinic acids" is used for colloidal
polygalacturonic acids containing more than a negligible propor-
tion of methyl ester groups. Pectinic acids, under suitable con-
ditions, are capable of forming gels with sugar and acid or, if
suitably low in methoxyl content, with certain metallic ions.







6 Florida Agricultural Experiment Stations

The salts of pectinic acids are either normal or acid pectinates.
Pectin: The general term "pectin" (or pectins) designates
those water-soluble pectinic acids of varying methyl ester con-
tent and degree of neutralization which are capable of forming
gels with sugar and acid under suitable conditions.
Pectic Acids: The term pecticc acids" is applied to pectic
substances mostly composed of colloidal polygalacturonic acids
and essentially free from methyl ester groups. The salts of
pectic acids are either normal or acid pectates.
Based on the present knowledge of pectin chemistry and this
accepted nomenclature, Kertesz and McColloch (11), McCready
and McComb (18), McColloch (16), and Owens, McCready, Shep-
herd, Schultz, Pippen, Swenson, Miers, Erlandsen and Maclay
(21) have developed extraction procedures for separating the
pectic substances and new methods of determination.
Pectic substances in citrus juices have been determined by
optical rotation (19) and by a colorimetric reaction with car-
bazole (3). The latter method was first applied by Stark (24)
to the pectic substances in cotton and by Averill (3) to frozen
citrus concentrates. Further modifications of the colorimetric
method by Dietz and Rouse (5) led to a new and rapid procedure
suited for routine laboratory analyses of pectin in citrus juices.
This colorimetric determination of pectin is based on Dische's (6)
carbazole-hexuronic acid-sulfuric acid reaction. Depending upon
the type of standard, results may be expressed as pectin or cal-
cium pectate in terms of anhydrogalacturonic acid, since this
is the basic structural unit of the pectin molecule.
Later Atkins and Rouse (2) revised the extraction procedure
for isolating the pectic substances in citrus juices and con-
centrates and suggested new techniques for the colorimetric de-
termination.
PECTINESTERASE
Reagents for Pectinesterase.-Pectin substrate. Mix 10 g. of
citrus pectin (undiluted powdered citrus pectin having a mini-
mum methoxyl content of 9 percent) with 11.7 g. of sodium
chloride and slowly add to 800 ml. distilled water with constant
agitation. After complete dispersion of the pectin make to 1
liter with water. This is a 1 percent substrate containing 0.2M
sodium chloride. Add 4 to 6 drops of toluene to the pectin solu-
tion and keep at refrigerated temperatures, 4.50 to 100C. to
prevent microbiological growth. The substrate is warmed to
room temperature, 260 to 300C., prior to the PE determination.








Pectinesterase and Pectin in Commercial Citrus Juices '7

Sodium hydroxide, reagent grade, 0.2N. Eight g. of sodium
hydroxide dissolved in 1,000 ml. water.
Sodium hydroxide, reagent grade, standard 0.05N. Two g. of
sodium hydroxide dissolved in 1,000 ml. water and standardize
according to A.O.A.C. procedures.
Analytical Method for Pectinesterase.-The juice sample is
comminuted for three min. in an Osterizer or Waring Blendor.
Into a 150 ml. beaker weigh accurately on a torsion balance 1 to
5 g. or pipette carefully 1 to 5 ml. of citrus juice or concentrate
and add 50 ml. of pectin substrate. Single-strength juice or
reconstituted juice is usually pipetted while a concentrated juice
is weighed. The substrate and juice sample are mechanically
stirred in the beaker and rapidly titrated to pH 7.5 with ca. 0.2N
sodium hydroxide. Either an automatic titrator or a pH meter
with extension electrodes may be used. A temperature of 30C.
and a reaction time of 30 min. are preferred. During this re-
action period the mixture is titrated with 0.05N sodium hydrox-
ide to maintain a pH 7.5. The reaction period begins as soon
as the pH is adjusted to 7.5. Blanks containing pectin sub-
strate, to which may be added sample juice that has been boiled
for five min., are titrated to pH 7.5 and maintained at this pH
for the reaction period. The ml. of 0.05N sodium hydroxide
used during the 30-min. period multiplied by the normality
(0.05N) is equivalent to ml. of N sodium hydroxide. If more
than 9.6 ml. of 0.05N sodium hydroxide (equivalent to 50 ml.
of subtrate containing 0.5 g. of citrus pectin which has a
methoxyl content of 10 percent) are required during the 30-min.
period, then a shorter reaction period should be used so that
not more than 30 percent demethylation (17) of the pectin
substrate occurs.
Pectinesterase units may be expressed by the symbols
(PE.u.)ml. or (PE.u.)g., which represents the milliequivalents
of ester hydrolyzed per min. per ml. or g. of juice or concentrate.
The equation used for computing PE units per g. of citrus con-
centrate is as follows:
ml. sodium hydroxide X normality
(PE.u.)g. concentrate -
"weight of sample X 30 min.
These are multiplied by 10 4 for easy interpretation. Another
means of expression is by the symbol (PE.u.)g. of soluble solids
(Brix) as determined by a refractometer. Degree Brix is not
only rapidly measured by refractometer, but is also a term
commonly used and understood by the citrus industry for ex-








8 Florida Agricultural Experiment Stations

pressing solids. The equation used to compute PE units per g.
of soluble solids is:
ml. sodium hydroxide X mormality
(PE.u.)g. soluble solids =
weight of sample X 30 min. X Brix
100
PECTIC SUBSTANCES
Reagents for Pectic Substances.-Ethyl alcohol, reagent grade,
95 percent.
Ethyl alcohol, ca. 63 percent. Add 1 volume of distilled water
to 2 volumes of reagent grade 95 percent ethyl alcohol.
Ethyl alcohol, purified. Reflux 1 liter of 95 percent reagent
grade ethyl alcohol with 4 g. of zinc dust and 2 ml. concentrated
sulfuric acid for 24 hr. Distill, using all-glass apparatus. Redis-
till from zinc dust and potassium hydroxide, using 4 g. of each
to 1 liter of alcohol.
Ammonium oxalate, reagent grade, 0.75 percent solution.
Sodium hydroxide, reagent grade, 1N. Forty g. sodium hy-
droxide dissolved in 1,000 ml. water.
Sulfuric acid, reagent grade, concentrated. Reject lots of acid
that give a color when added to carbazole solution.
Carbazole, 0.1 percent alcoholic solution. Dissolve 0.1 g. of
reagent grade carbazole in purified ethyl alcohol and dilute to
100 ml. A blank of 1 ml. of water, 0.5 ml. of carbazole solution,
and 6 ml. of sulfuric acid carried through the entire procedure
should be water-white or nearly so.
Galacturonic acid monohydrate, reagent grade. Check the
purity by titrating 0.5 g. with 0.1N sodium hydroxide to pH
8.0. The theoretical molecular weight of galacturonic acid hy-
drate is 212.
Extraction of Pectic Substances.-The pectic substances are
divided into three fractions by progressive extractions with dis-
tilled water, 0.75 percent ammonium oxalate and 0.05N sodium
hydroxide. All separations are made by centrifugation followed
by decantation.
Purification and extraction procedures require approximately
three hr. and the colorimetric determination of anhydrogalac-
turonic acid takes about 20 min. Duplicate samples carried
through the extraction and colorimetric procedures should agree
within 5 percent.
Citrus juice or concentrate is comminuted for three min. in an
Osterizer or similar blender. Weigh 16 g. of juice or 4 g. of
concentrate into a tared 50 ml., graduated, short conical bottom,







Pectinesterase and Pectin in Commercial Citrus Juices 9

centrifuge tube. To the 4 g. of concentrate add 12 ml. of distilled
water. Hot, 75C., 95 percent ethyl alcohol is added to a volume
of 40 ml. and the mixture heated for 10 min. in a water bath at
850C. with occasional stirring, using a glass rod. The stirring
rod is then rinsed off with 95 percent alcohol and the volume
of the mixture made up to 50 ml. in the tube. Centrifuge the
tube at 2,300 r.p.m. (1,150 .\ gravity) for 15 min. and after
decanting discard the supernatant solution. Repeat the leaching
with hot 63 percent alcohol for 10 min. in a water bath at 850C.,
centrifuge and again decant and discard the supernatant solu-
tion.
Add ca. 5 ml. distilled water to the tube and disperse the pre-
cipitate with a rubber policeman. Rinse policeman with distilled
water, make contents to a volume of 35 ml. and stir vigorously
and continuously for 10 min. This is accomplished either by
a mechanical stirrer or by bubbling air through the mixture in
the tube. The bubbling device is made by connecting to a source
of air a capillary tube about six in. long which is inserted into
the centrifuge tube. The stirrer or capillary tube is rinsed with
approximately 5 ml. distilled water increasing the volume to 40
ml., the tube centrifuged at 2,300 r.p.m. for 15 min. and the
liquid decanted into a 100 ml. volumetric flask. Repeat the water
extraction and after centrifuging decant into the same volumetric
flask. Add 5 ml. IN sodium hydroxide to the water extract and
dilute to volume. Mix and let stand 15 min. before beginning
the colorimetric procedure.
To the residue in the centrifuge tube add 5 ml. of 0.75 per-
cent ammonium oxalate solution and disperse the precipitate
with a rubber policeman. Rinse policeman with oxalate solu-
tion, make contents to a volume of 35 ml., and stir vigorously
and continuously for 10 min. as described above. Make to volume
of 40 ml. with oxalate solution. Centrifuge as before and decant
into a 100 ml. volumetric flask. Repeat the oxalate extraction,
centrifuge and decant into the same 100 ml. flask. Add 5 ml.
IN sodium hydroxide to the oxalate extract and dilute to volume.
Mix and let stand for 15 min. before beginning the colorimetric
procedure.
The residue remaining in the centrifuge tube is washed into a
100 ml. volumetric flask, 5 ml. 1N sodium hydroxide is added,
and the contents made to volume with distilled water. Mix, let
stand 15 min. with occasional shaking, and filter.







10 Florida Agricultural Experiment Stations

Analyze 1 ml. aliquots from each of the above three extracts
by the colorimetric method.
Colorimetric Method for Pectic Substances.-Pipette 1 ml. ali-
quots of the extract into each of 2 large test tubes (25 x 200 mm.)
and add 0.5 ml. of the 0.1 percent alcoholic carbazole to the
sample tube and 0.5 ml. of purified ethyl alcohol to the blank
tube. A white flocculent precipitate will form in the sample tube.
Add 6 ml. of concentrated sulfuric acid to each of the tubes
with constant agitation. An automatic acid burette is advan-
tageous for adding the acid and should be set to deliver the
6 ml. of acid in seven sec. to obtain a temperature of 85 C. (heat
of solution). The test tubes are immediately placed in a water
bath heated to 850C.; otherwise varying intensities of red color
may be produced in duplicate samples. The tubes should re-
main in the bath for five min. During this waiting period the
temperature need not be maintained. Remove tubes from water
bath, allow to cool for 15 min., and transfer the solutions to
microabsorption cells. Read immediately the percent trans-
mittance in an electrophotometer with a 525 millimicron (m[)
filter, after nulling the instrument against the blank. Refer to
the standard curve to obtain the concentration of anhydrogalac-
turonic acid (AGA) in micrograms (pg.). Calculate the per-
cent AGA in the samples using the formula:

Ag. AGA X dilution volume (ml.) X 100
Percent AGA =
1,000,000 X sample weight (g.)

PREPARATION OF STANDARD CURVE WITH AGA
Weigh accurately 120.5 mg. of galacturonic acid monohydrate,
vacuum dried five hr. at 300C. or dried over phosphorus pent-
oxide at room temperature, and transfer to a liter volumetric
flask. Add 0.5 ml. N sodium hydroxide and dilute to volume with
distilled water. Mix thoroughly and allow the solution to stand
overnight. This standard solution contains 100 Ig. of AGA per
ml. Molecular weight ratio of AGA to galacturonic acid monohy-
drate is 176/212.
Prepare working standards covering the range of 10 to 70 tg.
of AGA per ml. Develop the color standards as described for
sample extract using 1 ml. aliquots of each working standard,
and record the percentages of transmittance. For the curve
using the Fisher electrophotometer, plot the log percent trans-








Pectinesterase and Pectin in Commercial Citrus Juices 11

mittance against concentration of AGA in pg.; this should be a
straight line as shown in Fig. 1.

80-

0
z


w
J


o /
o
U,

0 -
50
-J


w

0 4o



o:30-

IJ-
w
a 20
I3
U-







0 10 20 30 40 50 60 70

MICROGRAMS/ ML. OF ANHYDROGALACTURONIC ACID

Fig. 1.-Standard curve for galacturonic acid.

INTERFERING MATERIALS

Impurities found in 95 percent ethyl alcohol may form highly
colored products when heated with carbazole. A 1 ml. sample
of distilled water carried through the procedure should have a
light transmittance greater than 98 percent when compared
against a blank in which purified alcohol was substituted for







12 Florida Agricultural Experiment Stations

the carbazole solution, using a Fisher electrophotometer with
a 525 m/ filter and a 3 ml. micro cylindrical absorption cell.
Sugars, citric acid and ascorbic acid will react with carbazole
to form colored products of varying degrees of intensity. At a
wave length of 525 mM (Beckman spectrophotometer, model B)
the maximum interference was caused by fructose (5).
The two alcoholic extractions with ethyl alcohol were sufficient
to reduce the concentration of these soluble juice constituents
(18) to a level of negligible interference. The pectic substances
were also precipitated from colloidal solution by the dehydrating
action of the ethyl alcohol. Ethyl alcohol left in the precipitate
following the second alcoholic extraction acts as a wetting agent
and aids in dispersing the pectic substances in the following
water extraction.
TECHNIQUES
Attention should be called to the following techniques that
result in more consistent data when used in the analytical pro-
cedure for determining pectic substances in citrus products.
Centrifugation of the water and oxalate extractions of pectin
from the alcohol precipitate of citrus concentrate facilitates
complete sedimentation. When the water and oxalate extractions
of pectin are separated by centrifugation from the alcohol pre-
cipitate of fresh citrus juice, inevitably some insoluble solids
remain floating. On decantation it is impossible to prevent this
floating material from passing into the volumetric flask. To
prevent the occurrence of floating material about 1/2 teaspoonful
of disintegrated, wetted paper pulp (Whatman Ashless Tablets)
is added to the 50 ml. centrifuge tube during the first water
extraction. The paper pulp aids the sedimentation during centrif-
ugation by carrying the insoluble solids to the bottom of the
centrifuge tube.
A technique that should be carefully followed in the
colorimetric procedure is regulation of the time required to add
the 6 ml. of concentrated sulfuric acid. The analyst should
have a dispenser set to deliver 6 ml. sulfuric acid in seven sec.
to obtain a temperature of 850C. (heat of solution) and should
immediately place the large test tube in a water bath heated
to 850C. Otherwise, varying intensities of red color may be
produced in duplicate samples. The tube should remain in the
bath for five min. and during this waiting period the temperature
need not be maintained.








Pectinesterase and Pectin in Commercial Citrus Juices 13

RESULTS AND DISCUSSION

The two methods just described were used in analyzing 221
commercial samples of frozen concentrated orange juice from
23 concentrate plants in Florida during the 1953-54 season.
From 16 of these manufacturers, which represented the major-
ity of the frozen concentrated orange juice production in Florida,
9 to 13 samples were obtained for testing from each processor
during the months of December 1953 through June 1954. This
accounted for 177 samples, or 80 percent of the commercial
samples received. Collection of two to eight samples from each
of the other seven plants accounted for the remaining 20 per-
cent. Data presented in Table 1 are the mean values found for
pectin and pectinesterase in concentrates from each of the 23
plants. PE activity is expressed as (PE.u.)g. of concentrate X
104.

60-


u) 50 -
-1
a-

S4-

0
30-
.-
I-
0


10-



0 20 40 60 80 100 120 140 160 180
(PE.u.)g. x 104 OF CONCENTRATE

Fig. 2.-Occurrence of pectinesterase activity in commercial samples of
frozen concentrated orange juice.

The range of PE activity varied in the commercial samples
of concentrated orange juice from a high of 173.6 to a low of 11.9
units. For reconstituted juices, these two extremes were by
calculation 50.6 and 3.4 units. Slightly over 50 percent of the
commercial samples contained between 20 and 39 units (Fig. 2).









TABLE 1.-MEAN VALUES FOUND FOR PECTIN AND PECTINESTERASE IN COMMERCIAL FROZEN CONCENTRATED ORANGE JUICES -
PROCESSED IN FLORIDA, 1953-54.

Pectin as Anhydrogalacturonic Acid-g./100g. Cone. Percentage of Total Pectin PE Activity

Plant _Soluble in Soluble in per g. cone.

HO_ (NHI)CGIO NaOH I Total HO (NH,),CO i NaOH X 104

A 0.076 0.079 0.116 0.271 28.1 29.5 42.4 106.0
B 0.096 0.056 0.103 0.255 37.7 21.1 40.2 23.5
C 0.075 0.079 0.104 0.258 28.7 31.0 40.3 72.5
D 0.138 0.068 0.107 0.313 43.8 21.8 34.4 37.1
E 0.093 0.068 0.116 0.277 33.7 24.5 41.8 89.6
F 0.120 0.058 0.116 0.294 40.7 19.9 39.4 31.6
G 0.097 0.083 0.130 0.310 31.5 26.9 41.6 104.2
H 0.135 0.073 0.138 0.346 39.1 21.1 39.8 25.3
I 0.094 0.054 0.104 0.252 37.4 21.5 41.1 33.0
J 0.121 0.062 0.121 0.304 39.9 20.5 39.6 37.4
K 0.144 0.080 0.163 0.387 37.4 20.9 41.7 23.6
L 0.112 0.055 0.106 0.273 41.1 20.1 38.8 26.6
M 0.120 0.066 0.116 0.302 39.4 22.2 38.4 42.4 n
N 0.153 0.077 0.157 0.387 39.5 20.2 40.3 47.5
O 0.154 0.072 0.140 0.366 42.2 19.7 38.1 41.1
P 0.114 0.057 0.101 0.272 41.8 20.9 37.3 25.3
Q 0.097 0.078 0.108 0.283 34.6 27.5 37.9 71.8
R 0.126 0.063 0.120 0.309 40.9 20.3 38.8 24.2
S 0.143 0.077 0.149 0.369 39.0 20.9 40.1 67.8
T 0.111 0.059 0.103 0.273 40.6 21.7 37.7 31.9
U 0.166 0.083 0.180 0.429 38.9 19.4 41.7 44.4
V 0.100 0.064 0.101 0.265 37.7 24.2 38.1 42.1
W 0.110 0.072 0.109 0.291 38.0 24.9 37.1 36.0







Pectinesterase and Pectin in Commercial Citrus Juices 15

Of the remaining samples, 41 percent showed more enzymic ac-
tivity; whereas 9 percent of the samples showed less. For the
higher activities, shorter reaction times during analyses were
used so that not more than 30 percent demethylation (17) of the
pectin substrate occurred.
Pectic substances were'divided into three groups on the basis
of their solubility at room temperature in water (HO), in am-
monium oxalate ((NH4),2C2O), and in sodium hydroxide
(NaOH). Pectins are expressed as anhydrogalacturonic acid
(AGA) in g. per 100 g. of concentrate and as percentage of total
pectin soluble in each of the three extractive solvents. The water
extraction removed the water-soluble pectic substances of suf-
ficiently high methoxyl content to be termed pectins (12). In
citrus juices these high-methoxyl pectins give the juice its body
or consistency and serve as colloidal stabilizers for the insoluble
suspended particles.
Ammonium oxalate-soluble pectic substances are the insoluble
salts of pectic acids and the low-methoxyl pectinic acids, re-
sulting from the combination of these acids with polyvalent
cations, such as calcium and magnesium. These water-insoluble
salts are called pectates and pectinates and in citrus juices are
formed as end products of chemical and enzymic deesterification
of pectin. The oxalate has a sequestering effect on the calcium
and magnesium ions, thus solubilizing the low-methoxyl pectin-
ates and pectates. The presence of low-methoxyl pectins causes
the two defects, gelation and clarification, in frozen concentrated
citrus juices. When present in low concentrations the low-
methoxyl pectins form a flocculent precipitate with polyvalent
metallic ions which occludes the insoluble solids and results in
clarification of the reconstituted juice upon complete settling of
the pectic material. At higher concentrations the low-methyl-
ester pectins form a gel structure in combination with polyvalent
metallic ions. This gel formation in cans of frozen concentrated
citrus juice is the defect termed "gelation" by the citrus proces-
sor.
Sodium hydroxide-soluble pectic substance represents the
protopectin fraction, which is the precursor of pectin. Proto-
pectin is also known as the water-insoluble parent pectic sub-
stance (12) which yields pectin upon acid hydrolysis in citrus
juices. During extended storage, hydrolysis of protopectin
might increase the water-soluble pectin content of citrus juices.
When total pectin only is desired during analysis, sodium hydrox-








16 Florida Agricultural Experiment Stations


40

30-
WATER SOLUBLE
20-

10 -




80
so-


70-

60

-J
5L 50-
a
S40-
SAMMONIUM OXALATE SOLUBLE

1 30
U.


10-





30



SODIUM HYDROXIDE SOLUBLE

20

10 -


0 0.075 0.100 0.125 0.150 0.175 0200 0225 0.250
GRAMS OF PECTIN/100 GRAMS CONCENTRATE

Pig. 3.-Occurrence of the three groups of pectic substances in commercial
samples of frozen concentrated orange juice.








Pectinesterase and Pectin in Commercial Citrus Juices 17

ide can be used as the extractant to render not only protopectin
but also water-insoluble pectates and pectinates soluble simul-
taneously.
In the commercial samples, the water-, oxalate- and sodium
hydroxide-soluble pectins varied from 0.041 to 0.210, 0.043 to
0.127 and 0.076 to 0.232 g. per 100 g. of concentrate, respectively.
However, in 83 percent of the samples the water-soluble fraction
occurred between 0.075 and 0.150 g.; whereas, in 74 percent
of the samples the ammonium oxalate-soluble pectin amounted
to 0.075 g. or less. Occurrence of the three pectic fractions in
commercial samples of frozen concentrated orange juice in re-
lation to the amount of pectin in different ranges is presented
graphically in Figure 3.

SUMMARY
Pectinesterase and pectic substances have become of consider-
able importance in the production of frozen concentrated citrus
juices because of difficulties encountered in clarification and gela-
tion. Methods are presented for the measurement of PE activity
and for the extraction and colorimetric estimation of pectic
substances in citrus juices and concentrates. These methods
have been used at the Florida Citrus Experiment Station for
routine control work and for a survey of 221 commercial samples
of frozen concentrated orange juices from 23 Florida processors.

LITERATURE CITED
1. Association of Official Agricultural Chemists. Official methods of
analysis. 7th Ed., 327-328. 1950. Washington 4, D. C.
2. ATKINS, C. D., and A. H. RousE. The effect of different methods of
juice extraction on the pectin content of Valencia orange juice.
Proc. Fla. State Hort. Soc. 66: 289-292. 1953.
3. AVERILL, W. Enzyme studies relative to the gelatin and clarification of
concentrated citrus juices. Ph.D. thesis, Food Tech. Dept. Univ. of
Mass., Amherst, Mass. 1951.
4. CARRa, M. H., and D. HAYNES. The estimation of pectin as calcium
pectate and the application of this method to the determination of
the soluble pectin in apples. Biochem. Jour. 16: 60-69. 1922.
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Biol. Chem. 167: 189-198. 1947.
7. FELLERS, C. R., and C. C. RICE. Rapid centrifugal method for pectic
acid determination. Ind. Eng. Chem., Anal. Ed. 4: 268-271. 1932.








18 Florida Agricultural Experiment Stations

8. HILLS, C. H., and H. H. MOTTERN. Properties of tomato pectase. Jour.
Biol. Chem. 168: 651-663. 1947.

9. HINTON, C. L. Fruit pectins, their chemical behavior and jellying
properties. Chem. Publishing Co. 1940.

10. KERTESZ, Z. I. Pectic enzymes. I. The determination of pectin-
methoxylase activity. Jour. Biol. Chem. 121: 589-598. 1937.

11. KERTESZ, Z. I., and R. J. McCOLLOCH. The pectic substances of mature
John Baer tomatoes. N.Y. Agr. Exp. Sta. Bul. 745: 4-7. 1950.
12. KERTESZ, Z. I., G. L. BAKER, G. H. JOSEPH, H. H. MOTTERN and A. G.
OLSEN. Report of the committee for the revision of the nomencla-
ture of pectic substances. Chem. Eng. News. 22: 105-106. 1944.

13. LINEWEAVER, HANS, and G. A. BALLOU. The effect of cations on the
activity of alfalfa pectinesterase (pectase). Arch. Biochem. 6:
373-387. 1945.

14. MACDONNELL, L. R., E. F. JANSEN and HANS LINEWEAVER. The prop-
erties of orange pectinesterase. Arch. Biochem. 6: 389-401. 1945.

15. MACDONNELL, L. R., ROSIE JANG, E. F. JANSEN and HANS LINEWEAVER.
The specificity of pectinesterases from several sources with some
notes on purification of orange pectinesterase. Arch. Biochem. 28:
260-273. 1950.

16. McCOLLOCH, R. J. Determination of pectic substances and pectic
enzymes in citrus and tomato products. Fruit and Vegetable Chem-
istry Lab., Pasadena, Calif., Bureau of Agr. Ind. Chem., USDA.
AIC-337: 1-6. (mimeographed) 1952.

17. McCOLLOCH, R. J., and Z. I. KERTESZ. Pectic enzymes. VIII. A com-
parison of fungal pectin-methylesterase with that of higher plants,
especially tomatoes. Arch. Biochem. 13: 217-229. 1947.

18. MCCREADY, R. M., and E. A. McCOMB. Extraction and determination
of total pectic materials in fruits. Anal. Chem. 24: 1986-1988. 1952.
19. McCREADY, R. M., A. D. SHEPHERD, H. A. SWENSON, R. F. ERLANDSEN
and W. D. MACLAY. Determination of citrus pectic substances by
optical rotation. Anal. Chem. 23: 975-977. 1951.

20. MYERS, P. B., and G. L. BAKER. Fruit Jellies. VIII.-The role of
pectin. 4. The physico-chemical properties of pectin. Del. Agr.
Exp. Sta. Bul. 187: 31. 1934.

21. OWENS, H. S., R. M. MCCREADY, A. D. SHEPHERD, T. H. SCHULTZ, E. L.
PIPPEN, H. A. SWENSON, J. C. MIERS, R. F. ERLANDSEN and W. D.
MACLAY. Methods used at Western Regional Research Laboratory
for extraction and analysis of pectic materials. West. Reg. Res.
Lab. Albany, Calif., Bureau of Agr. Ind. Chem., USDA AIC-340:
1-17. (mimeographed) 1952.

22. POORE, H. D. Recovery of naringin and pectin from grapefruit residue.
Ind. Eng. Chem. 26: 637-639. 1934.








Pectinesterase and Pectin in Commercial Citrus Juices 19

23. ROUSE, A. H. Gel formation in frozen citrus concentrates thawed and
stored at 400 F. Proc. Fla. State Hort. Soc. 62: 170-173. 1949.
24. STARK, S. M., JR. Determination of pectic substances in cotton. Anal.
Chem. 22: 1158-1160. 1950.





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