An Enzymic Method for the Determination of Naringin in
Grapefruit Products
S. V. Ting

Apparatus and Reagents


Apparatus:

1. Water bath controlled at 500 + l1C.

2. 18 x 150 mm culture tubes calibrated at 25 ml.

3. 23 x 300 mm culture tubes.

4. 1 ml. Mohr pipette graduated at 0.1 ml.

5. 100-ml beakers.

Reagents:

1. Diethyleneglycol Technical grade (Carbide and Carbon Chemicals Co.).

2. Alkaline-diethyleneglycol. Place 20 ml of ,N NaOH in a 1000-ml graduated
cylinder and add diethyleneglycol to the mark. Mix well by pouring the
contents back and forth into a 2-liter Erlenmeyer flask and by rotating
the flask. This reagent contains 0.5 ml NaOH in 25 ml.

3. Enzyme preparation. Pectinol 10-M (Rohm and Haas Co.).

4. Naringin. Eastman, practical grade, naringin recrystallized from water.

5. 5N NaOH.

Procedure

Preparation of standard curve. Dilute a 0.05% solution of recrystallized narin-
gin into a series of concentrations varying from 0.01 to 0.05% in 0.0]%increments.
Pipette 0.5 ml of each of the standard solutions to a clean 18 x 150 mm calibrated
tube and add the alkaline-diethyleneglycol to mark. Invert the tube several times
to mix the contents, and read the absaorency (optical density) of the yellow
color formed in a colorimeter, using a 420 mu filter, 15 minutes after adding the
reagent. Plot the absorbance against the concentration to form a standard curve
and/or calculate the factor.

Total flavonoids or total elycosides. Pipette 0.5 ml of grapefruit juice
into a calibrated tube, make to mark with the alkaline-diethyleneglycol and mix.
Read in a colorimeter at 420 mu after standing for 15 minutes and calculate the
total glycosides as per cent naringin.

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
645 9/30/55 SVT





-2-


Total flavonoids or total glycosides after hydrolysis. To hydrolyze the
naringin in the grapefruit juice, place 25 ml of it in a 100-ml beaker and adjust
the hydrogen ion concentration to pH 4 with 5N NaOH. Add the strong base drop-
wise from a burette, and stir the sample thoroughly after the addition of each
drop. Usually 4 to 6 drops will be needed. Then add 0.375 gram of Pectinol 10-M
to the mixture and transfer the contents to a 23 x 200 mm tube. Cap the tube
lightly with a cork stopper, and place it in a water bath maintained at 500 1 100.
for 5 hours. Then determine the total glycoside content as described in the pre-
vious paragraph. The instrument in this case is nulled with an enzyme blank.

Naringin. The naringin content in the grapefruit juice is the difference
in total glycosides determined before and after enzymic hydrolysis.




































Using Davis method for determination of flavonoids in citrus fruits. Anal.
Chem., 19, 476-8 (1947).

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
645a 9/30/55 SVT










Table 1


Effect of Extraction Pressure and Pulp Content on the Total Glycosides and Naringin
in Canned Unsweetened Grapefruit Juices
m m no I ,,I


Lou ares sure extraction


High pressure extraction


Pulp Total glycosides Naringin Pulp Total glycosides N__aringin


mE./100 /ml.


mg./l00 ml. .


% vol.


mg./l00 ml.


mg./100 ml


cap '..s.i ', -- ________ I .... -- T ...
Before enzymic After enzymic Before enzymic After enzymic
hydrolysis hydrolysis hydrolysis hydrolysis

12/ 9/54 5.0 53 26 27 11.0 96 41 55

2/15/55 6.5 80 33 47 7.0 84 34 50

4/20/55 9.0 71 30 41 12.0 94 36 58

6/14/55 10.5 68 29 39 14.5 79 33 46


The seedy grapefruit used for these packs was picked from different grove
on each packing date was thoroughly mixed before being separated into two
pressures


plots; however, all of the fruit used
lots for extraction using different


Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
638 9/27/55 SVT


Date
irkAed


Svon _








Table 2


Comparison of Bitterness in Unsweetened Canned Grapefruit Juices with Total
Glycoside and Naringin Content
i I I LIII I 1 l l l I I I I ] I I ]I I I 1 I I II


Extraction
Pressure


Total glycosides


Before enzymic
hydrolysis


After enzymic
I hydrolysis


Naringin


Intensity of i
1 i
bitterness I


mg./100 ml. mg./100 ml. mg./100 ml. I
Packed 12/9/54
High 96 41 55 Extreme
Low 53 26 27 Slight
Difference 43 15 28 "
Packed 2/15/55
High 84 34 50 Moderate
Low 80 33 47 Moderate
Difference 4 i 1 3

Packed 4/20/55
High 94 36 58 Moderate
Low 71 41 Slight
Difference 23 6 17

Packed 6/14/55
High 79 33 46 Slight
Low _68 29 39 Sight


Difference


11


Significant
difference i
bitterness i
5% level


Yes




No




Yes




No


The terms, moderate or extreme, used to indicate the intensity of bitterness considered to be
of consumer significance.

Florida Citrus Experiment Station
and Florida Citrus Commission, Lake Alfred, Fla.
638a 9/28/55 SVT


I


4 i


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