Group Title: Citrus Station mimeo report - Florida Citrus Experiment ; 56-9
Title: Application of enzymic hydrolysis in the analysis of naringin in grapefruit products
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 Material Information
Title: Application of enzymic hydrolysis in the analysis of naringin in grapefruit products
Series Title: Citrus Station mimeo report
Physical Description: 3 leaves : ; 28 cm.
Language: English
Creator: Ting, S. V., 1918-
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: 1955
 Subjects
Subject: Grapefruit juice -- Composition -- Florida   ( lcsh )
Grapefruit juice -- Quality -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references (leaf 3).
Statement of Responsibility: S.V. Ting.
General Note: Caption title.
General Note: "October 4, 1955."
 Record Information
Bibliographic ID: UF00072361
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 74323351

Full Text

Citrus Station Mimeo Report 56-9
October 4, 1955


Application of Enzymic Hydrolysis in the Analysis of Naringin in
Grapefruit Products

S. V. Ting

A slight bitterness is characteristic of grapefruit juice, but extreme bitter-
ness in this or other grapefruit products is undesirable because such products are
less acceptable to consumers. The substance that is largely responsible for
bitterness in grapefruit juice is naringin. Therefore, the development of any
procedure for the removal of naringin from grapefruit products is of commercial
importance since it would provide a means of improving their quality.

Investigations begun in August, 1954, have resulted in providing a method
for the removal of naringin from grapefruit juice and other grapefruit products.
The purpose of this report is to discuss this procedure and also show how it has
been applied as an analytical method for the determination of naringin in grape-
fruit products. This method has been used extensively for determining the narin-
gin content in grapefruit juices since much interest has recently been developed
concerning this glycoside, not only because of its effect on the quality of grape-
fruit products, but also of its being a possible criterion of grapefruit maturity
in as much as it is generally believed that the bitterness of grapefruit decreases
toward the latter part of the season.

Enzymic Hydrolysis of Naringin in Grapefruit Juice. Naringin is a 7-
rhamnoglucoside of the flavanone, naringenin. A specific quantitative method for
the estimation of naringin has been lacking, but the Davis (1) alkaline-diethylene-
glycol procedure for flavonoids as modified by Kesterson and Hendrickson (4) has
generally been used. This method is simple and, since naringin is the only known
major glycoside reported in grapefruit, is quite satisfactory for the purpose.

The possible occurrence of substances other than naringin in grapefruit juice
forming color with alkaline-diethyleneglycol was made evident when spectral
studies of this color revealed absorption maxima quite different from that for
naringin reported by Davis (1) to be at 420 mu. The absorption curves of the color
formed by the Davis reagents with the glycosides of rag and peel of grapefruit
showed that these glycosides have absorption peaks very close to that of naringin.

When naringin is hydrolyzed it forms rhamnose, glucose, and naringenin. This
process can be carried out by boiling the glycoside with dilute mineral acids (5),
or by treatment with an enzyme reported to occur in the leaves of grapefruit trees
(2). Attempts to obtain the enzyme from grapefruit leaves were not very successful
and the amount of enzyme isolated was too small to be of any value. However, it
was discovered that commercial concentrated pectic enzyme products, Pectinol 10-M
and Pectinol 100-D, contained, as an impurity, an enzyme that will hydrolyze
naringin. This enzyme is undoubtedly of the betaglycosidase type since similar
hydrolytic action on anthocyanins have been recently reported (3) in extracts of
fungal enzymes to which group Pectinols belong. i

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida
647 10/1/55 SVT \






When the absorbance curves of colors, formed by alkaline-diethyleneglycol
and grapefruit juice before and after hydrolysis by Pectinol 10-M were integrated,
the resultant curve showed maximum absorption at 420 mu. which is similar to that
of naringin. Chromatograms for glycosides in grapefruit juice before and after
enzymic hydrolysis showed that naringin was removed. Thus, by determining the
total glycosides before and after hydrolysis using the Davis technique, the
naringin, which is the hydrolyzed portion. may then be calculated by difference.

Optimum conditions for and various factors influencing the action of the en-
zyme capable of hydrolyzing naringin were studied. It was found that this enzyme
can act satisfactorily at a temperature as high as 600C. (l400F.) but at 700C.
(1580F.) some inactivation occurred. Complete inactivation was obtained in 5
min. at 800C. (1760F.) or 1 min. at 900C. (1940F.). On the other hand the hy-
drolytic action could take place at temperatures lower than 100C. (500F.); how-
ever, at these lower temperatures the rate of reaction dropped considerably. Like
all enzymes it has an optimum pH range which was found to be between 3.5-4.5 in a
citrate buffer. With an enzyme concentration corresponding to 1.5% Pectinol 10-M
by weight, 0.2-.08% naringin may be quantitatively (94-961%) hydrolyzed within 5
hr. These findings have been used in the development of an analytical quantita-
tive method specific for naringin in grapefruit juice, using the Davis (1) re-
agents. A copy of this method is attached to this report.

Effect of Extraction Pressure and Pulp Content on the Total Glycosides and
Naringin in Canned Unsweetened Grapefruit Juices. Single-strength unsweetened
juice of seedy grapefruit was canned in the pilot plant in December, February,
April and June during the 1954-55 season. Fruit was obtained from different grove
blocks at the Station on each of the 4 processing dates. Fruit for each run was
composite and divided into two six-box lots. One lot was extracted with low
pressure (approximately 30 p.s.i.g.) and the other with high pressure (approxi-
mately 70 p.s.i.g.) on a Citro-Mat juice extractor. The juice was finished using
a"0.030" screen, pasteurized, canned and stored at 400F. The same processing pro-
cedures were used for all packs.

Pulp determinations were made on samples from the 8 packs. Three cans from
each pack were used and four analyses were made using juice from each can, re-
sulting in a total of 12 determinations for any one pack. The pulp values listed
in Table 1 are averages of these 12 determinations for each pack.

Total glycosides of the juices were determined, before and after enzymic
hydrolysis, with the Davis alkaline-diethyleneglycol procedure. The naringin
content was calculated from the difference in glycosides of the two determinations.

The data on the glycoside and pulp content of these 8 packs of canned un-
sweetened grapefruit juices are shown in Table 1. Little seasonal trend in gly-
coside content was found probably because the sources of fruit were not the same.
The pulp content in the juices was higher toward the latter part of the season.
In the packs processed in December, April and June, high extraction pressure con-
sistantly increased the pulp content and both the total glycosides and naringin
in these juices. No explanation is available for the inconsistency in the packs
processed in February.


Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
647a 10/1/55 SVT






Comparison of Bitterness in Canned Unsweetened Grapefruit Juices with Total
Glycoside and Naringin Content. To determine whether significant differences in
bitterness existed in the 2 packs of canned grapefruit juices processed on the
same day, samples from these packs were given to a small taste panel. The tri-
angular test (two samples alike, one different) was used, and each of the 4 sets
of 2 different juices were presented to the panel 3 times. After attempting to
separate the 3 juice samples used in each test, the panel members were asked to
indicate the intensity of bitterness in the samples. The results of these tests
are reported in Table 2.

Significant differences in bitterness were found between the high and low
extraction pressure juices processed in December and April, when the differences
in naringin content were 28 mg./1OO ml. and 17 mg./100 ml., respectively. No
significant difference was found in either the February or June packs, where the
difference in naringin was 3 mg./lO ml. and 7 mg./100 ml.

It is interesting to note that 55 mg./100 ml. naringin in a juice packed in
December was considered as extremely bitter by the panel, while 50 mg./lO ml. in
a February pack and 58 mg./100 ml. in an April pack was considered as moderate.
The low extraction pressure juice in December was considerably lower in naringin
content and less intense in bitterness; also, it undoubtedly accentuated the
intensity of bitterness of the juice with which it was being compared.

In summary, this investigation has provided (a) a procedure for the removal
of naringin, and therefore the major portion of the bitterness, from grapefruit
products, (b) a quantitative method for the determination of naringin in grape-
fruit juices, and (c) data that show the effect of extraction pressure and pulp
content on the naringin content and bitterness in canned grapefruit juices.

It is also realized that various commercial applications may be made for the
improvement in the quality of grapefruit products by using the procedure of en-
zymic hydrolysis of naringin which is discussed in this report. For example this
procedure offers possibilities for "debittering" of grapefruit juice prior to or
during processing and for the production from grapefruit peel of a nonbitter bland
syrup. Investigations on these and other possible commercial applications of en-
zymic hydrolysis of either naringin in grapefruit products or hesperidin in orange
products are now being considered.

Acknowledgment
The assistance of Mr. R. W. Olsen in the packing of the canned grapefruit
juices used in this study was appreciated.

Literature Cited
1. Davis, W. B. Determination of flavonoids in citrus fruits. Anal. Chem.,
1, 476-8 (1947).
2. Hall, Donald H. A new enzyme. Chemistry and Industry, 57, 473 (1938).
3. Huang, H. T. Decolorization of anthocyanins by fungal enzymes. Agr. and
Food Chem., 2, 141-6 (1955).
4. Kesterson, J. W. and Hendrickson, R. Naringin, a bitter principle of grape-
fruit. Univ. of Fla. Agr. Expt. Sta. Bull. 511 (1953).

5. Pulley, George N. and von Loesecke, Harry W. Preparation of rhamnose from
naringin. J. Am. Chem. Soc., 6(, 175 (1939).

Florida Citrus Experiment Station Lake Alfred, Florida. 647b 10/3/55-SVT
and Florida Citrus Commission,




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