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The following points highlight the top four methods used for estimation of reducing sugar. The methods are: 1. Nelson—Somogyi Method 2. Folin Wu Method 3. Dinitrosalicylic Acid Method 4. Glucose Oxidase Method.
1. Nelson—Somogyi Method:
Sugars arising out of the presence of an aldehyde or ketogroup with reducing property like glucose, maltose, lactose and galactose are known as reducing sugars.
Principle:
When a reducing sugar is heated with alkaline copper tartarate, the copper is reduced from cupric to cuprous oxide. This when treated with arsenomolybdic acid, molybdic acid is reduced to molybdenum blue. This blue colour is compared with a set of standards at 620 nm in a colorimeter.
Requirements:
Reagents:
(i) Alkaline copper tartarate:
(a) Dissolve 2.5 g anhydrous sodium carbonate plus.
2.0 g sodium bicarbonate plus.
2.5 g potassium sodium tartarate plus.
20.0 g anhydrous sodium sulphate in 80.0 ml water and make up the volume to 100 ml.
(b) Dissolve 15.0 g copper sulphate in small volume of distilled water. Add one drop of concentrated sulphuric acid and make up the volume to 100 ml. Mix 4 ml of (b) and 96 ml of (a) before use.
(ii) Arsenomolybdate reagent:
Dissolve 2.5 g of ammonium molybdate in 45 ml of distilled water. Add 2.5 ml sulphuric acid and mix well. Then add 0.3 g disodium hydrogen arsenate dissolved in 25 ml distilled water. Mix well and incubate at 37°C for 24-48 hours.
(iii) Standard glucose solution:
Stock:
100 mg of glucose made up to 100 nil in distilled water.
Working standard:
Dilute 10 ml of stock solution to 100 ml with distilled water = 100 µg/ml.
1. Sample: fermented broth.
2. Water bath.
3. 2 ml graduated pipettes.
4. Test tubes in rack.
5. Spectronic 20.
Procedure:
1. Pipette out aliquots of 0.1 or 0.2 ml and 1.0 ml in separate test tubes label.
2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml of working standard into separate tubes and label.
3. Using distilled water make up the volume to 2 ml in both tubes of sample and standard.
4. Set up a blank in another tube with 2 ml water blank.
5. Add 1.0 ml of alkaline copper tartarate to each tube and place all the tubes in boiling water bath for 10 minutes.
6. Cool the tubes and add 1.0 ml of arsenomolybdic acid to all the tubes.
7. Make up the volume to 10 ml with distilled water in all the tubes.
8. After 10 minutes, read absorbance of the blue colour developed at 620 nm.
9. Plot a graph with µg of sugar against absorbance and calculate the amount of reducing sugar present in the fermented broth.
2. Folin Wu Method:
Requirements:
Reagents:
1. Phospho Molybdic Acid (Qualigen):
Add 200 ml of 10% NaOH solution and 200 ml of water to 35 g molybdic acid and 5.0 g of sodium tungstate in a one litre beaker. Boil vigorously for 20-40 minutes. Cool, dilute to 350 ml. Add 125 ml H3PO4 (orthophosphoric acid) dilute to 500 ml.
3. Alkaline Copper Solution (Qualigen):
Dissolve 40 g Na2CO3 in 400 ml water and transfer to one litre volumetric flask. Dissolve 7.5 g tartaric acid to this solution and then 4.5 g CuS04.5H2O, mix and dilute to one litre in a volumetric flask. If sediment is formed on standing, decant and use supernatant.
4. Standard and Glucose:
Dissolve 50 mg of anhydrous glucose in 0.25% benzoic acid and make up the volume to 100 ml in a volumetric flask.
Standard glucose solution = 500 µg/ml.
5. Tubes with stand.
6. Spectronic 20.
7. Water bath (100°C).
Procedure:
1. Take tubes with 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 ml of standard glucose solution along with blank with 2 ml distilled water and 2 ml of experimental broth.
2. Add distilled water to all the tubes with standard glucose to make up the volume to 2.0 ml.
3. Add 2.0 ml of alkaline copper sulphate to all tubes including bank and experimental broth.
4. Keep all the tubes in boiling water bath (100°C) for 10 minutes or more till a precipitate appears.
5. Add 2 ml of phosphomolybdic acid to all the tubes and make up the volume to 25 ml using distilled water.
6. Read absorbance, of the blue colour developed, at 420 nm.
7. Plot a graph with µg of glucose against OD and find out the concentration of sugar in the unknown sample.
3. Dinitrosalicylic Acid Method:
This method is useful when a large number of samples are to be handled at a time.
Requirements:
Reagents:
1. Dinitrosalicylic acid (DNSA)
Dissolve 1.0 g dinitrosalicylic acid plus 200.0 mg crystalline phenol plus 50.0 mg sodium sulphite in 100 ml 1% NaOH by stirring and store at 4°C. Due to sodium sulphite, the reagent deteriorates. If long storage is required, add sodium sulphite at the time of use.
2. 40% Rochella salt solution (potassium sodium tartarate).
3. Graduated pipettes.
4. Sample: fermented broth.
5. Test tubes and rack.
6. Water bath (100°C).
7. Spectronic 20.
8. Standard glucose solution.
9. Distilled water.
Procedure:
1. Pipette out 0.1, 0.2 and 1.0 ml aliquots in test tubes and label.
2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml of standard glucose into separate tubes and label.
3. Make up the volume to 3.0 ml, with distilled water, in all the tubes and then add 3.0 ml of DNSA reagent.
4. Heat the tubes in boiling water bath for 5 minutes and when contents are still warm add 1.0 ml 40% Rochelle salt solution.
5. Cool and read the red colour developed at 510 nm.
6. Plot a graph using µg of sugar against absorbance and find out the concentration of residual sugar in the fermented broth.
4. Glucose Oxidase Method:
This method gives the exact concentration of glucose, eliminating interference by other reducing sugars.
Principle:
The oxidation of alfa-D-glucose to glucose-1,5- lactone (gluconic acid) and H2O2 is catalysed by glucose oxidase. From H2O2, O2 that is liberated will react with o-dianisidine and oxidises it to a red chromophore product.
Requirements:
Reagents:
1. Standard glucose solution
Dissolve 100 mg of anhydrous glucose in 100 ml distilled water. Dilute 10 ml of this stock solution to 100 ml with distilled water which will be the working standard.
2. Glucose-oxidase peroxidase reagent:
Dissolve 25.0 mg of o-dianisidine in 1.0 ml methanol. Add 49.0 ml of 0.1 (M) phosphate buffer (pH 6.5). Add to this 5.0 mg of peroxidase and 5.0 mg of Glucose oxidase.
3. Deproteinised fruit juice.
4. Test tubes and stand.
5. 1 ml graduated pipettes.
6. Incubator (37°C).
7. 6(N) HCl.
8. Spectronic 20.
Procedure:
1. Take 0.5 ml fruit juice and add 0.5 ml distilled water and 1.0 ml glucose-oxidase- peroxidase reagent, label.
2. Prepare a series of 0.2, 0.4, 0.6, 0.8 and 1.0 ml of working standard glucose solution and make up the volume to 1.0 ml and label.
3. Pipette out 1.0 ml of distilled water in a separate tube, as blank.
4. Add 1.0 ml of glucose-oxidase-peroxidase reagent to all the tubes and incubate at 37°C for 40 minutes.”
5. Add 2.0 ml 6(N) HCl to all the tubes to terminate the reaction.
6. Read the red colour intensity at 540 nm.
7. Plot a graph of standard sugar with concentration against absorbance.
8. Find out the concentration of glucose in the fruit juice glucosein from the graph.