Group Title: Citrus Station mimeo report - University of Florida Citrus Experiment Station ; CES 65-3
Title: A Natural antioxidant in citrus fruits
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 Material Information
Title: A Natural antioxidant in citrus fruits
Series Title: Citrus Station mimeo report
Physical Description: 4 leaves : ; 28 cm.
Language: English
Creator: Ting, S. V., 1918-
Newhall, W. F
Citrus Experiment Station (Lake Alfred, Fla.)
Florida Citrus Commission
Publisher: Florida Citrus Experiment Station :
Florida Citrus Commission
Place of Publication: Lake Alfred FL
Publication Date: 1964
Subject: Citrus fruits -- Microbiology -- Florida   ( lcsh )
Orange products -- Quality -- Florida   ( lcsh )
Antioxidants   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
Bibliography: Includes bibliographical references (leaves 3-4).
Statement of Responsibility: S.V. Ting, W.F. Newhall.
General Note: Caption title.
General Note: "October 6, 1964."
 Record Information
Bibliographic ID: UF00072411
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 75959427

Full Text

Citrus Station Mimeo Report CES 65-3
October 6, 1964

A Natural Antioxidant in Citrus Fruits

S. V. Ting
Florida Citrus Commission
W. F. Newhall
University of Florida
Citrus Experiment Station
Lake Alfred, Florida

Autoxidation is generally considered to be one of the processes causing off-
flavor in citrus juices and instability of citrus oils. The most easily oxidized
substances in these citrus products are undoubtedly terpenes, and some "terpeney"
off-flavors appearing in citrus oil and citrus products have been attributed to
carvone and carveol formed by the oxidation of d-limonene (13). Commercial anti-
oxidants found (5, 7) to be effective in prolonging the storage life of oils are
butylated hydroxytoleune (BHT), butylated hydroxyanisole (BHA), nordihydro-
guaiaretic acid (NDGA), alpha-tocopherol, and the esters of gallic acid. The
search for antioxidants occurring naturally in food crops is a logical one since
they are less likely to be toxic. Various natural antioxidants from vegetable
sources have been reported (2, 8, 12) to prevent rancidity of fats. Wheat germ
oil has been used (9) to stabilize lemon and orange oil.

Several evidences have indicated that the peel of citrus fruit has anti-
oxidant activity.

1. A patent (11) was obtained in 1949 for using citrus pulp to stabilize
lard from rancidity but they did not indicate where the antioxygenic property
was located in the pulp nor did they attempt to isolate and concentrate the
substance responsible for the activity.

2. It has been observed (6) that cold-pressed orange oil has better
keeping quality than oil recovered from pulp by steam distillation.

3. A high-yielding extraction method (6) gave orange oils with high
evaporation residue and also with better stability.

4. The peroxides of citrus oil deteriorated by air oxidation were
completely removed (10) if the oil was first allowed to mix with press
liquor prior to separation.

5. In making an orange drink using comminuted whole orange, it was
found (1) that the comminuted base of the whole fruit had remarkable flavor
stability as compared to juice base containing no peel.

Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/6/64 SVT

The purpose of this work was to ascertain the location of this antioxidant
material in fruit and to estimate the relative antioxidant activity in the
component parts of the fruit.

Antioxidant activity in citrus fruits. The inhibition action exerted on
oxygen absorption of d-limonene and the lengthening of the induction period was
used to determine the antioxidant activity. To accomplish this determination of
oxygen absorption, an apparatus similar to that reported in the literature (4)
but with some modifications was made. The material to be analyzed was placed in
a specially made flask together with 25 ml. of d-limonene. Oxygen was passed
into the flask to flush out the air, after which the flask is immersed in a con-
stant temperature bath. The volume of oxygen absorbed was measured at constant
time intervals.

Temperature greatly affects the oxygen absorption rate and the induction
period of d-limonene. The purity of the d-limonene used is also very critical.
If not stored carefully, a trace of peroxide will form and the induction period
is greatly decreased. At 500C., limonene that has been stored has a shorter
induction period than a freshly distilled sample. The higher the temperature at
which the oxygen absorption test is made, the shorter the induction period. Some
batches of limonene may have very short induction periods at 600C., while others
may have induction periods as long as 3 hours at 700C. For this reason, a blank
run was made with each batch of limonene and sometimes on the same batch peri-
odically. In all tests reported, 700C. was used to minimize the time needed for
each test.

In locating the active principle in the component parts, Hamlin and Valencia
oranges were carefully separated into flavedo, albedo, and juice. Each part was
extracted with 95% ethanol and the alcohol was reduced to about 50%, using a
rotating evaporator. It then was extracted with n-hexane. An aliquot of the
n-hexane extract was used in the test.

The flavedo extract was capable in inhibiting oxidation of limonene. The
region between the flavedo and albedo containing some pigments and parts of
flavedo was termed the "inner flavedo". It showed an acceleration effect on
oxidation. These flavedo extracts represented 0.5 grams of the fresh weight.
The extracts of 10 grams of albedo showed no effect. The juice of Hamlin and
Pineapple oranges contained no antioxidant activity. However, juice of Valencia
oranges had some activity when 20 grams of the juice was extracted and used in a

Among some of the citrus fruits tested, the lime and lemon had no antioxi-
dant activity. Grapefruit contained sufficient activity to increase the
induction period of limonene.

Orange oil, distilled under vacuum to remove volatile components, was found
to contain some antioxidant activity. The activity of 1 gram of oil, calculated
from the original weight, was about equal to that extracted from 1/2 gram of

Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/6/64 SVT

Commercial antioxidants have unequal effectiveness in inhibiting oxidation
of d-limonene. The two commercial antioxidants used in such comparisons were
BHT and alpha-tocopherol. One ppm of alpha-tocopherol inhibited the oxidation
of d-limonene as much as 5 ppm of BHT. At 10 ppm alpha-tocopherol completely
inhibited autoxidation of limonene at 700C.

Isolation and examination of active principle. The active principle in
the flavedo extracts was separated on a column of alumina using as eluants mix-
tures of petroleum ether and ether. The activity was found in the 50% ether
fraction. Thin layer chromatography was also used to elucidate further the
nature of the active principle. When developed with chloroform on silica gel
plates, the activity was found in the top 25% of the area. Spraying with 0.2%
ferric chloride followed by 0.5% 2-2'dipyridyl revealed the presence of a
tocopherol-like substance in that area.

Large scale extraction using 1,400 grams of oven-dried flavedo yielded
30.1 grams of dark viscous oil. After dewaxing (5.35 g.) the oil was chro-
matographed on an alumina column (2" x 24"). Eluting with petroleum ether
containing various amounts of ethyl ether and alcohol, 100 ml. fractions were
collected. Fractions giving positive dipyridyl tests were combined and re-
chromatographed (1.52 g.) on a 1" x 8" silicic acid column. The column was
eluted with mixtures of ether and petroleum ether. Again fractions with
positive dipyridyl tests were combined and evaporated to dryness. A light-
yellow viscous oil was obtained (0.365 g.). Further purification on thin
layers of silicic acid recovered 96 mg. of pale-yellow viscous oil. Infrared
spectrum of this isolated material was found to be identical with that of an
authentic sample of d-alpha-tocopherol. There was no indication that any
tocopherol other than the alpha form was present in citrus. This was verified
by thin layer chromatography of samples of beta, gamma and delta-tocopherols.

Rakieten (14) using the dipyridyl test of Emmerie and Engel (3) reported
the apparent tocopherol content of orange juice to be not more than 99 mg. per
100 ml. Our results would indicate that the alpha-tocopherol content of orange
flavedo is less than 1 mg. per 100 g. fresh weight. While no isolation and
identification of alpha-tocopherol from orange juice was attempted in this study,
the authors were unable to detect the presence of any tocopherol-like substance
in the juice using the procedure of Emmerie and Engel (3). The juice of orange
contained little or no antioxidant activity, and it is doubtful that the alpha-
tocopherol content of the juice would be greater than that of the flavedo.


1. Charley, Vernon L. S. 1963. Some technical aspects of British comminuted
drink preparation. Food Technol. 17: 987.

2. Chipault, J. R. 1962. Antioxidant for use in foods. Chapter 12, Autoxi-
dation and antioxidants, Volume II edited by W. 0. Lunderberg. Inter-
science Publisher, New York.

Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/6/64 SVT

3. Emmerie, A. and C. Engel. 1938. Nature 142: 873.

4. French, R. B., H. S. Alcott, and H. A. Mattill. 1935. Antioxidation and
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5. Kenyon, E. M. and B. E. Proctor. 1951. Effect of antioxidants on orange
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6. Kesterson, J. W. and R. Hendrickson. 1953. Essential oils from Florida
citrus. Fla. Agri. Expt. Sta. Tech. Bul. 521.

7. Kesterson, J. W. and 0. R. McDuff. 1949. Antioxidant studies. The
American Perfumer and Essential Oil Review. October 1949.

8. Kurth, E. F. and F. L. Chan. 1951. Dihydroquercetin as antioxidant.
Jour. Amer. Oil Chemists' Soc. 28: 433.

9. Lakritz, W. 1943. Lemon and orange oil preservation. Manufacturing
Confectioner 23: 18.

10. Muncie, Fred W. 1963. Process for purification of citrus oil. U. S.
Patent No. 3,093,630.

11. Piskur, Michael M. and James W. Higgins. 1949. Antioxidants for fats.
U. S. Patent No. 2,461,080.

12. Pratt, D. E. and B. M. Watts. 1964. Antioxidant activity of vegetable
extracts. 1. Flavone aglycones. Jour. Food Sci. 29: 27.

13. Proctor, B. E. and E. M. Kenyon. 1949. Objective evaluation of odor
deterioration in orange oil. Food Technol. 2: 387.

14. Rakieten, M. L., B. Newman, K. G. Falk, and I. J. Miller. 1951. Comparison
of some constituents in fresh-frozen and freshly squeezed orange juice.
Jour. Amer. Dietet. Assn. 27: 864.

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