SOP: Fractionation of Pectic Substances in Citrus Juices
GIFSQ — Global Institute for Food Safety & Quality
Principle
The water-soluble pectic substances are sufficiently high-methoxyl pectins; in citrus juices these give the juice its body or consistency and serve as colloidal stabilizers for the insoluble suspended solids. The sodium hexametaphosphate-soluble pectic substances are the low-methoxyl pectinates of the polyvalent cations, magnesium and calcium; the sequestering effect of the polyphosphates on calcium and magnesium renders the low-methoxyl pectinates soluble, and their presence causes gelation and clarification in frozen concentrates. The sodium hydroxide-soluble pectic substances include protopectin and the calcium and magnesium pectates not removed by the sodium hexametaphosphate extractions — insoluble compounds formed as end products of chemical or enzymic demethylation of pectin. Based on solubility, the pectic substances of citrus juices are classified into three fractions by progressive extractions with distilled water, 0.4% sodium hexametaphosphate, and 0.05 N NaOH.
Apparatus required
- Blender
- Tared 50-ml graduated centrifuge tube
- 100-ml centrifuge tube
- Water bath
- Centrifuge
- 100-ml volumetric flasks
Reagents
a. 95% ethyl alcohol.
b. 60% alcohol.
c. 1 N NaOH.
d. 0.4% sodium hexametaphosphate solution.
e. 0.05 N NaOH.
Procedure — Sample preparation and alcohol precipitation
Blend the concentrate in a blender for 3 min
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Pipette 5 ml of the concentrate to a tared 50-ml graduated centrifuge tube and weigh accurately
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Add 5 ml distilled water and 30 ml of 95% ethyl alcohol — in the case of canned or fresh juice, pipette 20 ml of sample into a 100-ml centrifuge tube and precipitate the pectic material with 60 ml of 95% ethyl alcohol, giving a 70% alcoholic mixture
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Heat the alcoholic mixture for 10 min in a water bath at 85 ± 2°C, with occasional stirring; allow the solution to boil gently during the last 5 min of the heating period
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Centrifuge the tubes at 2,100 r.p.m. (1000 × gravity) for 10 min; decant and discard the supernatant alcoholic solution
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Add 40 ml of 60% alcohol to the precipitate and mix thoroughly; heat for 10 min in a water bath at 85°C, centrifuge, decant and discard the supernatant liquid as before
Procedure — Water-soluble fraction
Add 40 ml of distilled water to the centrifuge tube and stir until the mixture is evenly dispersed
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Allow the mixture to stand for 10 min at room temperature, and stir again
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Centrifuge as before and decant into a 100-ml volumetric flask; repeat the water extraction using 40 ml of distilled water, centrifuge, and decant into the same volumetric flask
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Add 5 ml of 1 N NaOH to the water extract to deesterify the pectins to sodium polygalacturonate; dilute to volume with water
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Let it stand for 30 min and estimate the pectin by the SOP-R33 Colorimetric Method
Procedure — Hexametaphosphate-soluble fraction
To the residue in the centrifuge tube, add 40 ml of 0.4% sodium hexametaphosphate solution, stir, and allow the solution to stand for 10 min at room temperature
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Centrifuge and decant the supernatant liquid to a 100-ml volumetric flask; make a second extract of the residue similarly and decant into the same volumetric flask
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Add 5 ml of 1 N NaOH; allow it to stand for 30 min and estimate the pectin colorimetrically
Procedure — Alkali-soluble fraction
Extract the residue remaining in the centrifuge tube with 40 ml of 0.05 N NaOH for 15 min at room temperature, stirring occasionally
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Centrifuge as before for 10 min and decant the supernatant liquid into a 100-ml volumetric flask; dilute to volume with water and estimate the pectin by the colorimetric procedure
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
Dietz, J.H. and A.H. Rouse, Food Res., 18, 169 (1953) — cited in the book.