SOP: Determination of Total Flavonoid Content — Davis Value Test

GIFSQ — Global Institute for Food Safety & Quality

Principle

The total flavonoid content of citrus juices is measured by the Davis value test: the flavonoids develop a yellow colour with sodium hydroxide in diethylene glycol, measured at 420 nm. In grapefruit juice the value is an index of the bitterness characteristics (naringin is the main bitter constituent, along with other flavonoids); in orange juice it is an index of hesperidin content. Naringin may be converted into less bitter or non-bitter forms as the fruit matures, and hesperidin may deposit in cans or on the evaporating surface during concentrate preparation.

Apparatus required

• Colorimeter or spectrophotometer (set at 420 nm)
• Colorimeter tubes, 18 mm × 150 mm (recommended)
• Stoppered flasks
• Measuring cylinder
• Centrifuge

Reagents

a. 90% diethylene glycol: use crystal-clear diethylene glycol, diluted with water (v/v).

b. 4 N sodium hydroxide solution.

c. Purified naringin (for the grapefruit standard): recrystallize from ethyl alcohol, dry at 90 °C, and store in a desiccator. For orange juice use hesperidin instead of naringin.

Procedure

Standard curve

Prepare a stock solution containing 100 mg of purified naringin in 100 ml of distilled water, warming if necessary (70 °C) to dissolve

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Prepare standard solutions containing 10, 25, 50, 75 and 100 mg of naringin per 100 ml of distilled water

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Measure 25 ml of 90% diethylene glycol with a measuring cylinder into a stoppered flask, add 0.5 ml of the standard naringin solution and 0.5 ml of 4 N NaOH, stopper, mix, and let stand at room temperature for 10 minutes for the yellow colour to develop

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Measure the colour at 420 nm against 90% diethylene glycol as blank, and plot absorbance against concentration

Sample

Centrifuge 50 ml of the juice (or of concentrate diluted to single-strength juice, as for the determination of suspended solids)

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Decant the supernatant free from suspended matter and strain it through a wad of cotton

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Develop the colour exactly as for the standards and measure it; read the naringin concentration from the standard curve

Calculation

Davis value = milligrams of naringin per 100 ml of juice (for orange juice, use the hesperidin standard curve)

The Florida citrus industry considers 55 mg of naringin per 100 ml of juice as the maximum permissible limit in grapefruit juice products.

Precautions

• The juice must be freed from suspended matter before colour development, otherwise the reading is falsified.
• Allow the full 10 minutes for colour development at room temperature before measuring.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

Davis, W. B., Anal. Chem., 19, 476 (1947) — as cited in the book.

Hendrickson, R., Kesterson, J. W. and Edwards, G. J., Proc. Florida State Hort. Soc., 71, 194 (1958) — as cited in the book.