Determination of Sugar Content: Biochemical Analysis

Determination of sugars (total sugar, reducing sugar and non-reducing sugar) were carried out though Lane and Eynon Method as described by James (1995).

Total sugar and reducing sugar: Took 5 g of sample into a beaker and added 100 ml of warm water. The solution was stired until all the soluble matters were dissolved and filtered through wattman paper into a 250 volumetric flask. Pipetted 100 ml of the solution prepared 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 make up to volume in a 250 volumetric flask. This solution was used for titration against Fehling’s solution and reading was calculated as follow.

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

Titre x wt of sample x 10

% 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

Non-reducing sugar was estimated as the difference between the total sugar content and reducing sugar content.

Determination of dietary fiber

Total dietary fibre content is measured according to the AOAC enzymatic-gravimetric method. The basis of this method is the isolation of dietary fibre 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 fibre assay kit (Sigma product n ° TDF 100 kit). Ethanol is added to precipitate the soluble fibre. 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 fibre. Weight of dietary fiber gave was calculated by the following formula.

Crud Fiber % = (c-b) – (d-b) x 100

(a)

[6].

Determination of moisture content

Moisture content is one of the most important factors influencing seed quality and storability, Therefore, its estimation during seed quality determination is important. Seed moisture content can be expressed either on wet weight basis or on dry weight basis, in seed testing, it is always expressed on a wet weight basis, and the method for its calculation is given later. Seed 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.

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

(2) Moisture meters: A variety of moisture meters are available in the market.

These meters estimate 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.

The moisture content of the sample is calculated using the following equation:

W% = A-B x 100

B

Where:

%W = Percentage of moisture in the sample,

A = Weight of wet sample (grams), and

B = Weight of dry sample (grams)

Determination of ash content

For determination of ash content, method of AOAC (2000) was followed. According to the method, 10 g of each sample was weighed in a silica crucible. The crucible was heated in a muffle furnace for about 3-5 h at 600 °C. It was cooled in desiccators and weighed to completion of ashing. To ensure completion of ashing, it was heated again in the furnace for half an hour more, cooled and weighed. This was repeated consequently till the weight became constant (ash became white or grayish white). Weight of ash gave the ash content was calculated by the following formula.

Ash % = Weight of ashed sample x 100

Weight of sample taken

Determination of protein

Protein was determined using micro Kjeldahl method as describe in AOAC (2000).

2 g of sample material was taken in a Kjeldahl flask and 30 ml concentrated sulfuric acid (H2SO4) was added followed by the addition of 10 g potassium sulphate and 1 g copper sulphate. 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 Kjeldahl flask. Three or four pieces of granulated zinc and 100 ml of 40% caustic soda were added 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 flask was removed and titrated against 0.1 N caustic soda solution using methyl red indicator for determination of Kjeldahl nitrogen, which in turn gave the protein content. The nitrogen percent was calculated by the following formula.

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

Weight of Sample

Whereas, protein content was estimated by conversion of nitrogen percentage to protein (James, 1995).

Protein % = N% x Conversion factor (6.25)

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

Determination of fat

Fat was determined by Mojonnier method (James,1995). The fat content was determined gravimetrically after extraction with diethyl ether (ethoxyethane) and petroleum ether from an ammonia alcoholic solution of the sample. About 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

W3

Where:

W1 = Weight of empty flask (g)

W2 = Weight of flask + fat (g) and

W3 = Weight of sample taken (g).