Proximate analysis of a food sample determines the total protein, total fat, total carbohydrate, total solid (ash) and moisture reported as the percentage composition of the product. There are food composition tables that contain proximate analysis for a large number of established foods and as new food items are added to our shopping baskets their proximate compositions are added to the database, periodically, in supplements.
Total Protein Analysis:
The total protein content of a food sample is estimated as total nitrogen (e.g., the Kjeldahl method) after digestion, salt neutralization and titration of the ammonia released against standard acid. A conversion factor is applied to calculate the total protein. Some functional groups, -NO2 and -N=N-, do not react and need further treatment if their omission will make a significant difference.
Methods used to Estimate Total Protein Content in Food:
The methods depend upon the combustion or digestion of the organic matter of the sample to release N for chemical reaction and volumetric estimation.
There are four methods are widely used to estimate the total protein content:
The Lassaigne Test is used for the qualitative measurement of N, S and the halogens. Nitrogen detection is based on the digestion/ reaction of the sample with heated Na. If N is present, NaCN is formed. This is reacted with Fe (OH) 2 to form ferrocynide. This later heated with acidified FeCl3 to form a prussian blue colored complex.
The Kjeldahl N determination, developed in 1883, depends upon the fact that most organic N compounds are converted into (NH4)2SO4 when heated with concentrated H2SO4; the exceptions are –NO2 and -N=N- groups, which if present in any quantity should be previously reduced to the amine. Digestion is carried out slowly over a microburner in a loosely stoppered digestion flask to avoid losses by splashing. Figure 7.1 shows the specially shaped Kjeldahl digestion flask and the steam distillation apparatus. The digestion products are then quantitatively rinsed into flask C, treated with excess alkali added from funnel B and the ammonia generated is distilled over into flask D.
From there it is steam distilled and condensed via the water-cooled condenser E into standard acid in flask F and in “U” traps G. The combined acid from F and G is titrated to the yield of ammonia from which the %N2 (Equation 1) and hence, the % protein (Equation 2) can be calculated, where x = ml of standard acid and w = the weight of sample.
(i) Equation – 1 %N = X/W x Molarity
(ii) Equation – 2 % Protein = X x6. 25/w x Molarity
3. Duma’s Method:
Duma’s method is based on the decomposition of compounds to CO2, H2O and gaseous N by heated CuO and a bright Cu spiral, the nitrogen being collected over a solution of KOH. The organic sample is mixed with an excess of fine CuO and inserted into the long combustion tube, together with a packing of coarse CuO and a spiral of right metallic Cu gauze- to decompose oxides of N.
The nitrometer contained a 40% solution of KOH. CO2 was used to purge the tube of air while the furnace was heated. Water and CO2 produced by the combustion of the organic sample were adsorbed or condensed and the N gas was collected in the manometer and the volume recorded and corrected for NTP.
Biuret is formed from substances containing two or more -NH- CO groups. Proteins are treated with CuSO4 in high alkaline condition to form biuret. It is a pink colored complex which can be quantified.
Total carbohydrate consists of sugars (mono and oligosaccharides) and polysaccharides (starch and the non-starch polysaccharides; pectin, soluble and insoluble dietary fibre, e.g., cellulose and hemicellulose).
Total starch (TS) is sub-divided into the following:
2 M KOH was used to dissolve the RS and the total starch was hydrolyzed with amyloglucosidase. The released glucose was determined and the TS calculated as glucose x 0.9. Total starch was measured on bread, rice, biscuits, lentils, chickpeas, beans, frozen peas, boiled potatoes and crisps.
The food sample for analysis was then buffered (pH 1.5), digested with pepsin under constant shaking to remove protein, re-buffered to pH 6.9, digested with a-amylase to hydrolyze DS, centrifuged and washed repeatedly and RS pellet was dispersed with KOH, adjusted to pH 4.75 and enzymically hydrolyzed to glucose with amyloglucosidase, centrifuged and the supernatants collected, made up to volume, washed and re-buffered. The glucose released was quantified from a standard curve.
DS was calculated as the difference between TS and RS.
Total Lipid Analysis:
Total lipid (fat) content may be calculated simply as the material extracted into diethyl ether. However, there are concerns over the availability of the many chemically different forms of fat and at least a digestion of the protein and carbohydrate would ensure the efficient release of fat from the tissue.
The important methods for total lipid content estimations are:
(i) Acid Digestion:
A simple method of measuring the total fat content of food is to digest a sample in concentrated H2SO4 and measure the remaining lipid layer in a graduated tube. The fat content of tree nuts, peanuts, sunflower seeds and olives was determined using this method, which is conveniently performed using the Gerber tube method developed for milk lipids.
(ii) Solvent Extraction Method:
This method involves the extraction of fat from food by suitable solvents.
Water is the major component of most fresh food and, therefore, the determination of moisture content defines the dry matter composition, a more quantitative starting point in the measurement of the nutritional value of the food.
There are two methods:
(i) Direct Methods:
Evaporative heating (dehydration) – It is a simple method of measuring moisture content is to heat the food to dryness by evaporating the water into the atmosphere and measure the loss in mass.
Another simple method is to place the wet sample in a desiccator with a strong desiccant, such as P2O5 and weigh the sample at intervals until equilibrium is reached.
Azeotropic Solvent Distillation:
In this method the water is distilled by the solvent, e.g., toluene and condensed into a side arm of a tube and titrate volumetrically.
(ii) Indirect Methods:
The indirect method for moisture analysis of food includes rapid and remote sensors. These techniques include the use of NMR, NIR Spectroscopy and microwave spectroscopy for the proximate analysis of water content.
This method used to calculate total amount of ash content present in the food. The prominent methods are called evaporative methods. In this method the food items are evaporated to remove the water content and the remaining residues are collected. This is known as total solids.
In the food industry, in-line sensor methods have been developed for the measurement of total solids or moisture content, e.g., in continuous fruit juice processing. The important types of inline sensor are Abbe refractometer and Maselli refractometer.