BIOCHEMISTRY AND HUMAN NUTRITION

Module 4. Human nutrition

Lesson 26
EVALUATION OF NUTRITIVE VALUE OF FOOD

26.1 Nutritive value

It is the indication of the contribution of a food to the nutrient content of the diet. This value depends on the quantity of a food which is digested and absorbed and the amounts of the essential nutrients (protein, fat, carbohydrate, minerals, vitamins) which it contains. This value can be affected by soil and growing conditions, handling and storage, and processing.

26.2 Calorie Content of Food (Bomb Calorimeter)

A bomb calorimeter is a type of constant-volume calorimeter used in measuring the heat of combustion of a particular reaction. Bomb calorimeters have to withstand the large pressure within the calorimeter as the reaction is being measured. Electrical energy is used to ignite the fuel; as the fuel is burning, it will heat up the surrounding air, which expands and escapes through a tube that leads the air out of the calorimeter. When the air is escaping through the copper tube it will also heat up the water outside the tube. The temperature of the water allows for calculating calorie content of the fuel.

Basically, a bomb calorimeter consists of a small cup to contain the sample, oxygen, a stainless steel bomb, water, a stirrer, a thermometer, the dewar (to prevent heat flow from the calorimeter to the surroundings) and ignition circuit connected to the bomb (Fig. 26.1)

Fig. 26.1 Bomb calorimeter

Unit—Kilocalorie (kcal)
The amount of energy which in the form of heat is required to raise the temperature of 1.0 Kg of water by 1.0 from 15 to 16^oC.

26.3 Crude Protein

The classic methods for protein concentration in food are the Kjeldahl method and the Dumas method. These tests determine the total nitrogen in a sample. The only major component of most food which contains nitrogen is protein (fat, carbohydrate and dietary fibre do not contain nitrogen). If the amount of nitrogen is multiplied by a factor depending on the kinds of protein expected in the food, the total protein can be determined. This value is known as the "crude protein" content. On food labels the protein is given by the nitrogen multiplied by 6.25, because the average nitrogen content of proteins is about 16%. The Kjeldahl test is typically used because it is the method the AOAC International has adopted and is therefore used by many food standards agencies around the world.

• Kjeldahl method

The method consists of digestion of the substance with sulfuric acid in presence of potassium sulphate that increases the boiling point of the medium (from 337°F to 373°F / 169°C to 189°C). Digestion decomposes the organic substance by oxidation to liberate the reduced nitrogen as ammonium sulfate. Chemical decomposition of the sample is complete when the medium has become clear and colorless (initially very dark).

The solution is then distilled with sodium hydroxide (added in small quantities) which converts the ammonium salt to ammonia. The amount of ammonia present (hence the amount of nitrogen present in the sample) is determined by back titration. The end of the condenser is dipped into a solution of standard boric acid solution. The ammonia reacts with the acid and the remainder of the acid is then titrated with a standard sodium carbonate solution with a methyl orange pH indicator.

26.4 Carbohydrate

The carbohydrate content of a food can be determined by calculating the percent remaining after all the other components have been measured: % carbohydrates = 100 – (% moisture + %protein + %lipid + % mineral). Nevertheless, this method can lead to erroneous results due to experimental errors in any of the other methods, and so it is usually better to directly measure the carbohydrate content for accurate measurements.

The amount of preparation needed to prepare a sample for carbohydrate analysis depends on the nature of the food being analyzed. Aqueous solutions, such as fruit juices, syrups and honey, usually require very little preparation prior to analysis. On the other hand, many foods contain carbohydrates that are physically associated or chemically bound to other components, e.g.,seeds, nuts, cereals, fruit, woody material breads and vegetables. In these it is usually necessary to isolate the carbohydrate from the rest of the food before it can be analyzed. The precise method of carbohydrate isolation depends on the carbohydrate type, the food matrix type and the purpose of analysis, however, there are some procedures that are common to many isolation techniques. For example, foods are usually dried under vacuum (to prevent thermal degradation), ground to a fine powder (to enhance solvent extraction) and then defatted by solvent extraction.

One of the most commonly used methods of extracting low molecular weight carbohydrates is to boil a defatted sample with an 80% alcohol solution. Monosaccharides and oligosaccharides are soluble in alcoholic solutions, whereas proteins, polysaccharides and dietary fiber are insoluble. The soluble components can be separated from the insoluble components by filtering the boiled solution and collecting the filtrate (the part which passes through the filter) and the retentate (the part retained by the filter). These two fractions can then be dried and weighed to determine their concentrations. In addition, to monosaccharides and oligosaccharides various other small molecules may also be present in the alcoholic extract that could interfere with the subsequent analysis e.g., amino acids, organic acids, pigments, vitamins, minerals etc. It is usually necessary to remove these components prior to carrying out a carbohydrate analysis. This is commonly achieved by treating the solution with clarifying agents or by passing it through one or more ion-exchange resins.

A number of chemical methods used to determine monosaccharides and oligosaccharides are based on the fact that many of these substances are reducing agents that can react with other components to yield precipitates or colored complexes which can be quantified. The concentration of carbohydrate can be determined gravimetrically, spectrophotometrically or by titration. Non-reducing carbohydrates can be determined using the same methods if they are first hydrolyzed to make them reducing. It is possible to determine the concentration of both non-reducing and reducing sugars by carrying out an analysis for reducing sugars before and after hydrolysis. Many different chemical methods are available for quantifying carbohydrates. Most of these can be divided into three catagories: titration, gravimetric and colorimetric

• Colorimetric Methods

The Anthrone method is an example of a colorimetric method of determining the concentration of the total sugars in a sample. Sugars react with the anthrone reagent under acidic conditions to yield a blue-green color. The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed. The solution is then allowed to cool and its absorbance is measured at 620 nm. There is a linear relationship between the absorbance and the amount of sugar that was present in the original sample. This method determines both reducing and non-reducing sugars because of the presence of the strongly oxidizing sulfuric acid. Like the other methods it is non-stoichemetric and therefore it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration.

The Phenol - Sulfuric Acid method is an example of a colorimetric method that is widely used to determine the total concentration of carbohydrates present in foods. A clear aqueous solution of the carbohydrates to be analyzed is placed in a test-tube, then phenol and sulfuric acid are added. The solution turns into yellow-orange color as a result of the interaction between the carbohydrates and the phenol. The absorbance at 420 nm is proportional to the carbohydrate concentration initially in the sample. The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so that this method determines the total sugars present. This method is non-stoichemetric and so it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration.

• Gravimetric Method
• Crude Fiber Method

The crude fiber method gives an estimate of indigestible fiber in foods. It is determined by sequential extraction of a defatted sample with 1.25% H2SO4 and 1.25% NaOH. The insoluble residue is collected by filtration, dried, weighed and ashed to correct for mineral contamination of the fiber residue. Crude fiber measures cellulose and lignin in the sample, but does not determine hemicelluloses, pectins and hydrocolloids, because they are digested by the alkali and acid and are therefore not collected. For this reason many food scientists believe that its use should be discontinued. Nevertheless, it is a fairly simple method to carry out and is the official AOAC method for a number of different foodstuffs.

26.5 Ether Extraction for Lipid Estimation

The method described by Soxhlet in 1879 is the most commonly used example of a semi-continuous method applied to extraction of lipids from foods. According to the Soxhlet's procedure, oil and fat from solid material are extracted by repeated washing (percolation) with an organic solvent, usually hexane or petroleum ether, under reflux in a special glassware as shown in figure.

In this method the sample is dried, ground into small particles and placed in a porous cellulose thimble. The thimble is placed in an extraction chamber (2), which is suspended above a flask containing the solvent (1) and below a condenser (4). The flask is heated and the solvent evaporates and moves up into the condenser where it is converted into a liquid that trickles into the extraction chamber containing the sample. The extraction chamber is designed so that when the solvent surrounding the sample exceeds a certain level it overflows and trickles back down into the boiling flask. At the end of the extraction process, which lasts a few hours, the flask containing the solvent and lipid is removed. In some device a funnel (3) allows to recover the solvent at the end of the extraction after closing a stopcock between the funnel and the extraction chamber. The solvent in the flask (1) is then evaporated and the mass of the remaining lipid is measured. The percentage of lipid in the initial sample can then be calculated.

Fig. 26.2 Soxhlet appratus

Despite disadvantages of this procedure (poor extraction of polar lipids, long time involved, large volumes of solvents, hazards of boiling solvents, several methods involving automatic solvent extraction were described. Different automated or semi-automated extraction instruments may be found on the market.

26.6 Nutrition Value of Milk

Milk is a prefect food as it provides vital nutrients like proteins, EFA,vitamins, minerals and lactose in balanced proportions. It complements and supplements nutrients available from grains, legumes, vegetables, fruits, meat, seafoods and poultary. Milk has high nutrient density. High concentration of major nutrients at relatively low caloric value. Concentrations of nutrients and energy in milk are given in table 26.1

Table 26.1 Nutrition value of cow, buffalo and goat milk per 100g

26.7 Milk Fat

• The Good and the Bad

• Despite the fact that the cholesterol level in milk is low, milk fat is considered hypercholesterolemic. This is mainly because of its high-saturated fatty acid content (60 to 65%). Palmitic (C16:0) and C14:0 acids have been shown to be hypercholesterolemic while shorter fatty acids (C4-C10) are neutral. Stearic, C18:1 and C18:2 acids are hypocholesterolemic.
• Bovine milk fat contains significant amounts of short chain fatty acids and relatively lower concentrations of C18 fatty acids than are found in other sources of animal fat such as beef or pork. Bovine milk is also a poor source of polyunsaturated fatty acids. Milk fatty acids are derived in part from dietary long chain fatty acids, microbial synthesis of fatty acids and body stores of fat with the remainder coming from de novo synthesis in the mammary glands. Manipulating the diet of the dairy cow can substantially alters the balance between mammary de novo synthesis of short and medium chain fatty acids, and dietary long chain fatty acids presented to the mammary gland.

26.8 Biological Importance of Milk Proteins

• Milk proteins are the main source of amino acids for the newborn. Casein micelles also provide Ca and P for skeletal development. Casein micelles are highly digestible by the proteolytic enzymes of the newborn.
• Some milk proteins have intracellular functions. For instance, Beta-lactalbumin forms a part of the enzyme lactose snynthase.
• Milk contains proteins such as lactoferrin and lysozyme. The antibacterial properties of these materials, lysozyme digesting bacterial polysaccharides and lactoferrin sequestering iron required by bacteria emphasize their importance in reducing mastitis infections. Lactoferrin concentration is high in the dry bovine mammary gland.
• In many mammalian species including bovine colostrum is a vital way of transferring passive immunity from the mother to the newborn.