Download Proline Content in Florida Frozen Concentrated Orange Juice and

Literature Cited
9. Lu, P. and J. E. Kinsella. 1972. Extractability and properties of
1. Boyd, C. E. Leaf protein from aquatic plants. Leaf Protein IBP
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and B. Roe. 1979. Protein extraction from aquatic
plants. (In preparation).
4. Delory, G. E. and E. J. King. 1945. Sodium carbonate-bicarbonate
buffer for alkaline phosphatases. Biochem. J. 39:245-247.
5. Edie, H. H. and B. W. C. Ho. 1969. Ipomoea aquatica as a veget
able in Hong Kong. Econ. Bot. 23(l):32-36.
6. Edwards, R. H., Miller, R. E., deFremery, D., Knuckles, B. E.,
Bickoff, E. M. and G. O. Kohler. 1975. Pilot plant production of an
edible white fraction leaf protein concentrate from alfalfa. /. Agric.
Food Chem. 23:620-626.
7. Gornall, A. G., Bardawill, C. S. and M. M. David. 1949. Determina
tion of serum proteins. J. Biol. Chem. 177:751-766.
8. Kohler, G. O. and E. M. Bickoff. 1971. Commercial production from
alfalfa in USA. Leaf protein IBP Handbook No. 20. Ed. by N. W.
Pirie Blackwell Scientific Publications, Oxford, p 69.
protein from alfalfa leaf meal. /. Food Sci. 37:94-99.
Company, Ithaca, New York, 21st Ed., p 1086.
11. Morton, J. F. and G. H. Snyder. 1976. Aquatic crops vs organic soil
subsidence. Proc. Fla. State Hortic. Soc. 89:125-129.
12. Morton, R. K. 1955. Extraction of enzymes from animal tissues.
Methods in Enzymology, Ed. by S. P. Colowick and N. O. Kaplan.
Vol. 1, p 25.
13. Potty, V. H. 1969. Determination of proteins in the presence of
phenols and pectins. Anal. Biochem. 29:535-539.
14. Singer, S. J., Eggman, L., Campbell, J. M. and S. G. Wildman. 1952.
The proteins of green leaves. IV. A high molecular weight protein
comprising a large part of the cytoplasmic proteins. /. Biol. Chem.
197:233-239.
F
15. Spies, J. R. and D. C. Chambers. 1951. Determination of tryptophan
using photochemical development of color. /. Biol. Chem. 191:787-
789.
16. Snyder, G. H. 1977. Personal communication.
17. Takeda, K. Y. 1975. Personal communication.
18. Watt, B. K. and A. L. Merrill. 1963. Composition of Foods, Agri
cultural Handbook No. 8, USDA, Washington, D.C. p 61.
Proc. Fla. State Hort. Soc. 92:143-145. 1979.
PROLINE CONTENT IN FLORIDA FROZEN CONCENTRATED
ORANGE JUICE AND CANNED GRAPEFRUIT JUICE1
S. V. Ting and R. L. Rouseff
Florida Department of Citrus,
AREC, P. O. Box 1088,
Lake Alfred, FL 33850
juices, its use as an index has been suggested by several
Abstract. Proline is the most abundant amino acid in
citrus juices, and has been suggested as one of the indices
of orange juice purity by some European drink manufacturers.
Using a modified photometric method of acidic ninhydrin re
action on proline developed by Chinard, the proline concn of
Florida frozen concnd orange juice (FCOJ) and canned sinale
strength grapefruit juice were determined. The proline
content in reconstituted FCOJ varied as much as two and half
fold from a low of around 60 mg/100 ml to as high as
150 mg/100 ml. Canned grapefruit juice was much lower in
its proline content than orange juice, varying from about 20
mg-60 mg/100 ml. With the exception of ornithine, other
amino acids found in citrus juices did not interfere in this
column chromatography. However, the same reagent pro
analysis.
Proline, 2, pyrollidine carboxylic acid, is the most
abundant of all amino acids in citrus juices (2, 14, 16).
Vandercook and Price (14) found an average of 169 mg
of proline in 100 ml of California Valencia orange juice
but only 94 mg in the same amount of Florida orange juice.
Ting and Deszyck (13) using the procedure of Moore and
Stein (7) reported a range of about 45 to 75 mg proline in
100 ml frozen concentrated orange juice (FCOJ) reconsti
tuted to 11.8° Brix. Niedermann (8) and Koch (6) both
found that proline values in orange juice might vary from
2.5 to 3-fold among the samples. In grapefruit juice, Brenoe
(2) reported values varying from 35 to 292 mg per 100 ml.
The use of concentrations of various chemical con
stituents of fruit juices as indices of juice authenticity or as
a measure of juice content in a fruit drink has been widely
accepted. For citrus juice, formol numbers, poly phenols,
some individual amino acids, and several minerals have
been proposed either singly or in combinations for this
purpose (10). Since proline is a major amino acid of citrus
iFlorida Agricultural Experiment Stations Journal Series No. 1995.
Proc. Fla. State Hort. Soc. 92: 1979.
authors (1, 5, 6, 8, 15).
Proline produces a yellow color with ninhydrin buffered
at pH 5, and this color reaction was used by Moore and
Stein (7) in the determination of proline separated by
duces a blue or bluish red color with other amino acids.
Ting and Deszyck (13) using a mixed standard of several
major amino acids of orange juice determined proline along
with the other amino acids. Chinard (3) developed a photo
metric procedure for the estimation of proline using nin
hydrin reagent with concentrated formic acid. Under the
acidic condition with ninhydrin reagent proline first forms a
yellow color, followed by a red color having an absorption
maximum at 515 nm. Onlv orinthine and hydroxyproline
interfere in this reaction. None of the other amino acids
tested by Chinard produced any significant color with this
method.
Ouch (9) used this procedure to analyze proline in
grape juice and wine. He found no significant interference
from other grace amino acids in amounts normally present
in that fruit. He also demonstrated that the red color de
veloped need not be extracted with a water immiscible
solvent as previously proposed by Chinard (3), but instead
that the color may be diluted with isopropanol and
measured directly. Wallrauch (15) found that the use of
ethyl acetate to extract the color produced by the acidic
ninhydrin reaction was necessary for highly colored juices
with low proline content.
The purpose of this paper is to report the use of this
acidic ninhydrin method with modification and simplifica
tion to study the range of proline content in Florida FCOJ
and canned grapefruit juice as produced commercially.
Variations in proline content of orange juice due to fruit
cultivars and maturity were also studied. The simplified
method could be easily used in most quality control labora
tories equipped with an inexpensive colorimeter.
Materials and Methods
Samples
One hundred samples of FCOJ and 80 samples of canned
143
single-strength grapefruit juice used in this study were from
various processing plants in Florida. The juice samples used
to study variations of proline content with fruit maturity
in different orange cultivars were from another experiment.
These juices were extracted using an FMC Model 091 InLine Juice Extractor from fruits harvested at different times
of the season, and were pasteurized and sealed in cans. All
juice samples were stored frozen until ready for analysis.
Analytical procedure
A.
Reagents. Only two reagents are needed in this
simplified method for the determination of proline.
1. Ninhydrin reagent. The ninhydrin reagent is
prepared by dissolving 3 g of reagent grade nin
hydrin (Nutritional Biochemical Co., Cleveland,
OH) in 100 ml of diethyleneglycol
(Eastman
Organic Chemicals, Rochester, NY). The mix
ture is stirred on a magnetic stirrer until dis
solved and is stable for at least 2 weeks when
2.
B.
stored in a refrigerator.
Formic acid. The concentrated formic acid
(Fisher reagent grade 90%) is used without
dilution.
Procedure. Reconstituted FCOJ or the singlestrength canned grapefruit juice was centrifuged,
and the clarified juice diluted with water. For
orange juice a dilution of 1 to 100 is necessary in
order that the proline content in the test solution
falls within the range of linear response of the colorimetric analysis. For grapefruit juice the dilution is
made 1 to 50.
To 1 ml of the diluted juice in a 13 x 100 mm
screw cap culture tube (Corning #9826) is added
1 ml of concn formic acid. After thorough mixing, 2
ml of the ninhydrin reagent are added. The screw
caped tube is immersed in a boiling water bath for
15 min and cooled in tap water for 10 min. The
absorbance of the sample is determined at 515 nm
on a colorimeter or spectrophotometer and the concn
calculated from the standard curve of proline.
Stock proline standards of 100 mg per liter of water
was diluted to a series of working standards containing
0, 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 ^g per ml, which were
analyzed in the same manner as described above. A linear
absorbance response with concn was obtained within the
concn range. The amount of proline in a sample was cal
culated from the standard curve. A set of standards was
run each time with the samples.
Results and Discussion
Proline in reconstituted FCOJ. The mean proline
content of 100 samples of reconstituted FCOJ (12.8° Brix)
was 107.8 mg/100 ml with a range from 60 to 153 mg per
100 ml. A distribution of these samples is shown in Fig. 1. A
large number of samples had proline contents above 100
mg/100 ml with a second peak in the distribution centered
around 130 mg/100 ml.
Variations of proline in orange juice due to maturity
and cultivar. An investigation was carried out to determine
the seasonal variations of proline in juice of the main
cultivars of Florida oranges. The results are shown in Fig.
2. The juices of early and mid-season oranges are low in
proline when the fruit is immature or just begins to reach
the Florida standards (11). Proline increased rapidly with
season. However, when the fruit reach their maturity, the
proline content was low compared to late season fruit.
'Hamlin', the early maturing orange cultivar, was found to
144
PROLINE CONTENT
mg/ 100ml
Fig. 1. Frequency distribution of proline content in 12.8° Brix
reconstituted FCOJ. Mean = 107.8 mg/100 ml; standard deviation =
21.5 mg/100 ml; n = 100.
be extremely low in proline even after the fruit had attained
maturity but continued to increase in proline as the season
advanced. 'Pineapple' oranges ripen during January and
February and its proline content was considerably higher
than 'Hamlin'. With 'Valencia' oranges the proline content
fluctuates although it is always high as compared to the
earlier maturing cultivars. The fluctuation of proline in the
late season fruit probably is caused by the climate during
that time of the year when the weather favors vegetative
growth of the tree (12, 16).
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100
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-80
PINEAPPLE I n^* *
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Fig. 2. Seasonal variations of proline content in juices of 3 Florida
orange cultivars. (1973-74 season).
The mean and range of proline content of Florida
canned grapefruit juice. The average proline content of 80
samples of canned grapefruit juice was 39 mg/100 ml, about
one-third of that of reconstituted FCOJ, and the coefficient
of variation was 37% as compared to about 20% for orange
juice. A distribution of the proline values of these samples
is shown in Fig. 3. The lowest value for grapefruit juice
was 16 mg/100 ml and the highest was 92 mg/100 ml. It is
not known whether the seasonal and varietal variation in
proline content as found in oranges also occur in grape
fruit.
Conclusion
Due to its wide variations, the use of proline concn of
citrus juices as an index of juice content suffers from the
Proc. Fla. State Hort. Soc. 92: 1979.
most citrus quality control laboratories for the Davis test
The ninhydrin reagent will also produce a red color
with hydroxyproline and orinthine. No hydroxyproline has
ever been reported to occur in citrus, and orinthine is only
about 0.5-1.5% of proline as determined in orange juice
(2. 6).
Literature Cited
15
25
35
45
55
PROLINE CONTENT
65
95
75
mg / 100ml
Fig. 3. Frequency distribution of proline content in Florida canned
grapefruit juice. Mean = 39.1 mg/100 ml; standard deviation = 14.6
mg/100 ml; n « 80.
same shortcomings of many other juice characteristics used
for this purpose. However, in the 100 samples of orange
juice analyzed, the mean was 107.8 mg/100 ml with a
standard deviation of 21.5 mg/100 ml. A confidence at the
level of 95% could be expected that the juice should not
have a value below the 2 standard deviations of the mean,
or 63.8 mg/100 ml for a 12.8° Brix orange juice. The
amount must be adjusted for juices reconstituted to lower
Brix values. The use of proline content as an index for
grapefruit juice will be even less meaningful because of its
low values and high standard deviation (14.6 mg/100 ml).
Products containing significant amount of juices derived
from less mature early and mid-season Florida oranges
may have low proline content. With proportioned amount
of the late season orange juice added to blend for Brix
to acid ratios and for color, the resulting product may
reach the minimum values for this amino acid as proposed
by Bielig et al. (1) and Koch (5, 6).
The simplified method of determining proline could
give the quality control laboratories an additional tool for
monitoring the characteristics of the juice. The reagents
are easy to prepare and stable. The diethyleneglycol used
to prepare the ninhydrin reagent is generally available in
1. Bielig, H. J., W. Feathe, J. Koch, S. Wallrauch and K. Wucherpfennig. 1977. Standards and variations of the determined charac
teristics for apple, grape and orange juices. Obst Gemuese-Verwert.
Ind. 62:209-219, und Fluss. Obst 44:215-225. (In German).
2. Brenoe, C. 1971. Research studies of the distribution of free amino
acids and other compounds in orange fruits picked in Greece during
the period January to March 1968 and collected during the period
January to March 1969. Husholdingsradets Tekniske Meddeleser
11:15-59.
3. Chinard, F. P. 1952. Photometric estimation of proline and
orinthine. /. Biol. Chem. 199:91-95.
4. Davis, W. B. 1947. Determination of flavanone in citrus fruits.
Anal. Chem. 19:476-478.
5. Koch, J. 1975. About the characteristics of manufactured orange
juice as basis for a uniform estimation of the product by means
of official food regulations. Fluss. Obst. 42:217-222. (In German).
6.
. 1979. The free amino acids in commercial orange
juice. Fluss. Obst. 46:212-216. (In German).
7. Moore, S. and W. H. Stein. 1948. Photometric ninhydrin method
for use in the chromatography of amino acids. /. Biol. Cheim.
176:367-387.
8. Niedmann, P. D. 1976. A contribution to the quantitative deter
mination of free amino acids and ammonia in orange juice. Dtsch.
Lebensm. Rundsch. 72:119-126. (In German).
9. Ouch, C. S. 1969. Rapid determination of proline in grapes and
wines. /. Food Sci. 34:228-230.
10. Royo Iranzo, J. 1974. Methods for the detection of adulteration in
citrus juices. Proceeding International Conference on Quality and
Detection of Adulteration in Citrus Juices. Instituto de Agroquimica y Technologia de Alimentos, Valencia, Spain. (In Spanish).
11. State of Florida, 1974 Citrus Fruit Laws. Editorially Revised. Florida
Department of Citrus, Lakeland, Florida.
12. Ting, S. V. and J. A. Attaway. 1971. Citrus Fruits. In The Bio
chemistry of Fruits and their Products, A. C. Hulme, Editor. Aca
demic Press, New York and London.
13.
and E. J. Deszyck. 1960. Total amino acid content of
chilled orange juice and frozen concentrate. Fla. State Hort. Soc.
73:252-257.
14. Vandercook, C. and E. L. Price. 1972. The application of amino
acid composition to the characterization of citrus juices. /. Food
Sci. 37:384-386.
15. Wallrauch, S. 1976. Determination of proline in fruit juices, Sig
nificance for the estimation. Fluss. Obst 45:430-437.
16. Wedding, W. T. and R. P. Horspool. 1955. Juice composition
change during orange development. Calif. Citrog. 40:106-107.
Proc. Fla. State Hort. Soc. 92:145-148. 1979.
TANGERETIN CONTENT OF FLORIDA CITRUS PEEL
AS DETERMINED BY HPLC
R. L. ROUSEFF AND S. V. TlNG
Florida Department of Citrus,
Agriculture Research and Education Center, IFAS,
P. a. Box 1088,
Lake Alfred, FL 33850
Additional
index
words,
albedo,
flavedo,
dietary
fiber,
fiavones.
Abstract, Eleven varieties of Florida citrus have been
analyzed for peel tangeretin contents using a new HPLC
method. Highest tangeretin concentrations are found in the
flavedo. Of the varieties tested, the average flavedo con-
Proc. Fla. State Hort. Soc. 92: 1979.
tributes to only 42.5% of the fresh peel weight, yet it
contains 87.5% of the total amount of tangeretin in the peel.
Mandarin peel had the greatest tangeretin concentration (183
ppm) while lemon peel had the least (.06 ppm). Orange peel
contained, on the average, slightly greater amounts of
tangeretin than grapefruit peel. The tangeretin content of
any citrus peel based product can be increased or decreased
dramatically depending on the variety and the portion of
the peel used as a starting material.
Tangeretin is one of several methoxylated fiavones found
in citrus and has been found in all of the component parts
of the Valencia orange (4). It has been reported by Robbins
145