Chemical Analysis determining sugar, fiber and moisture Determination of sugar content

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Took 5 g of sample into a beaker and added 100 ml of tepid water. The mixture of suspension until all the soluble matters were dissolved and filtered into a 250 volumetric flask. Pipetted 100 ml of the solution get readied into a conical flask; added 10 ml diluted HCl and boiled for 5 min. On cooling, neutralize the solution to phenolphthalein with 10% NaOH and compensate to volume in a 250 volumetric flask. This solvent was utilized for titration against Fehling's solution and reading was computed as follow.

% Total sugar = Factor (4.95) x dilution (250) x 2.5

Titre x wt of sample x 10

% Reducing sugar = Factor (49.5) x dilution (250)

Titre x wt of sample x 10

The estimation of the difference between the total sugar content and reducing sugar content were non-reducing sugar.

Determination of dietary fiber

Using the AOAC enzymatic-gravimetric method measured the total dietary fiber content. The foundation of this method is the isolation of dietary fiber by enzymatic digestion of the rest of the constituents of the material. The residue is measured gravimetrically.

Starch is digested by combining amylase (pH=6.0, Ta=100°C, t=30min) and amyloglucosidase (pH=7.5, Ta=60°C, t=30min); protein is digested with protease

(pH = 4.5, T a = 60°C, t = 30 min).

Analyses are performed using a total dietary fiber assay kit (Sigma product n ° TDF 100 kit). Ethanol is added to precipitate the soluble fiber. Filtration is carried out in crucibles with 0.5 g of wet and homogeneously distributed celite. The residue is then filtered off and washed with 78 % and 95 % ethanol and acetone. Crucibles with the residue are dried to measure the weight of residue. Protein, ash and starch were measured in each residue in order to correct the values for dietary fiber. Weight of dietary fiber gave was calculated by the following formula.

Dietary Fiber % = (c-b) - (d-b) x 100


3. Determination of moisture content

One of the most important factors influencing seed quality and storability is moisture content. Consequently, the estimation during seed quality computation is important. Moisture content can be expressed either on wet weight foundation or on dry weight foundation, in grain testing, it is always expressed on a wet weight basis, and the method for its determination is given later. Moisture content can be determined either by air oven or moisture meter. However, if prescribed standard for moisture content is less than 8%, air oven method shall be used.

Air oven method: In this method, moisture is removed by drying at a specified temperature for a specified duration. The moisture content is expressed as a percentage of the wet weight. It is the most common and standard method for moisture determination.

Moisture meters: A category of moisture meters are available in the market.

These meters evaluate seed moisture quickly but the estimation is not as precise as by the air-oven method. The meters should be calibrated and standardized against the air-oven method.

Moisture content of the sample is calculated using the following equation:

W% = A-B x 100



%W = Percentage of moisture in the sample,

A = Weight of wet sample (grams), and

B = Weight of dry sample (grams)

4. Determination of ash content

Loading 10 g of each sample added in a silica crucible. The crucible was heated in a shroud furnace for about 180-300 min at 600 °C. It was cooled in desiccators and loaded to completion of ashing. To ensure completion of ashing, it was heated again in the furnace for 30-60 min more, cooled and loaded. This was repeated consequently till the weight became constant. Weight of ash content was calculated by the equations.

Ash % = Weight of ashed sample x 100

Weight of sample taken

5. Determination of protein

Protein was determined using Kjeldahl method as describe in AOAC (2000). 2 g of sample taked in a vacuum and the addition of 10 g potassium sulphate and 1 g copper sulphate added following amount of 30 ml concentrated sulfuric acid (H2SO4). The mixture was heated first gently and then strongly once the frothing had ceased. When the solution became colorless or clear, it was heated for another hour, allowed to cool, diluted with distilled water (washing the digestion flask) and transferred to 800 ml vacuum. 3 or 4 pieces of granulated zinc and 100 ml of 40% caustic soda were being an addition and the flask was connected with the splash heads of the distillation apparatus. Next 25 ml of 0.1 N sulphuric acid was taken in the receiving flask and distilled. When two-thirds of the liquid had been distilled, it was tested for completion of reaction. The vacuum was removed and titrated against 0.1 N caustic soda solution using methyl red indicator for computation of Kjeldahl nitrogen, which in turn gave the protein content. The percent of protein content was calculated by the equations.

N%= 1.4 (V2-V1) x Normality of Hcl x 250 (dilution)

Weight of Sample

Whereas, the estimation of conversion of nitrogen percentage to protein was protein content

Protein % = N% x Conversion factor (6.25)

Where conversion factor = 100/N (N% in fruit products)

6. Determination of fat

Fat content was determined gravimetrically after extraction with ethoxyethane and petroleum ether from an ammonia alcoholic solution of the sample. 10 g of sample was taken into a Mojonnier tube. Added 1 ml of 0.880 with 10 ml ethanol mixed well and cooled. Added 25 ml diethyl ether, stopper the tube, shacked vigorously and then added 25 ml petroleum ether and left the tube to be stand for 1 hr. The extraction was replaced thrice using a mixture of 5 ml petroleum ether and adding the extraction to the distillation flask. Distilled off the solvents, dried the flask for 1 hr at 100 C and reweighed. The percentage fat

Content of the sample was calculated by the following formula which gave that the difference in the weight or the original flask and the flask plus extracted fat represent the weight of fat present in the original sample.

% Fat content of sample = W2 W1 x 100



W1 = Weight of empty vacuum (g)

W2 = Weight of vacuum + fat (g)

W3 = Weight of sample taken (g)

7. Determination of plasma glucose

After collection of the whole blood containing sodium fluoride as preservative (2.5 mg/dL of blood) the blood sample was centrifuged and plasma was aspirated and deproteinized by gel filtration method. The glucose method is and an adaptation of the hexokinase-glucose-6 phosphate dehydrogenase method. Hexokinase (HK) catalyzes the phosphorylation of glucose by adenosine-5'-triphophate (ATP) to glucose-6-phophate which is oxidized to 6-phosphogluconolactone by glucose-6-phosphate dehydrogenase (G-6-PDH) with simultaneous reduction of nicotinamide-adenine dinucleotide phosphate (NADP). One mole of NADP is reduced to one mole of NADPH for each mole of glucose present. The absorbance due to NADPH (and thus the glucose concentration) is determined using a bichromatic (340 and 383 nm) endpoint technique.

Glucose + ATP → Glucose-6-phosphate+ ADP

Glucose 6 Phosphate + NADP → 6 - phosphogluconolactone + NADPH



Calculation of glycemic index (GI)

Glycemic index of food is the ratio of blood glucose area under the curve for test food compared with a reference food (bread or glucose). The GI is usually defined as the area under the glycemic response curve during a 2 hrs period after consumption of 50-g carbohydrate from a test food, and values are expressed relative to the effect of either white bread or glucose. Originally the reference was glucose contained 50 g CHO.

In the fruit, the GI was calculated as

GI = Blood glucose response area of fruit x 100

Blood glucose response area of glucose