CHEMISTRY OF MILK

Module 3. Milk proteins

Lesson 13
QUANTIFICATION OF PROTEINS IN MILK

13.1 Introduction

The proteins in milk are estimated by using either their component amino acids or by their chemical reactions or by their physical forms. Accordingly the methods available for estimation of proteins in milk are discussed in this lesson. The determination of the protein content of materials in which pro­teins occur in mixtures with other biological materials is not simple.

13.2 Physical Methods

13.2.1 Direct weighing

Since the ultimate objective of protein analyses is to determine the weight of protein in a given quantity of material, it might seem that separation of the protein and direct weighing would be the simplest method to use. Since removing lipids, salts, other solutes, and water completely is difficult it is not generally employed for routine analyses. Obviously,however, direct weighing is the ultimate standard for all other methods. For example, the factor for converting nitro­gen to protein was arrived at originally by preparing a sample of pure protein and determining the ratio between nitrogen and dry weight.

13.2.2 Volume measurements
Methods based on precipitating protein, centrifuging in a calibrated tube, and measuring its volume, have often been suggested. The Hart method for casein is of this type.Fat is extracted with chloroform and the casein precipitated with acetic acid,centrifuged, and measured. Methods based on volume measurements of proteins are not used to any extent in the dairy industry at present except for assessing the amount of in­soluble material in milk powders (solubility index).

13.2.3 Turbidimetric methods

It is sometimes convenient to form a suspension of insoluble protein and to estimate the protein content by optical methods. Either light transmittance or light scattering can be measured. Methods employing the former principle are called turbidimetric, those using the latter, nephelometric. In recent years a turbidimetric method devised by Harland and Ashworth has been used to a considerable extent for determining the concentration of undenatured serum proteins in heated and dry skim milks. In this method the casein and denatured serum proteins are precipitated by saturation with sodium chloride. The resulting filtrate is then diluted and acidified and its light transmittance determined. The protein con­tent is determined from a calibration curve in which per cent transmit­tance is plotted against protein nitrogen content of the filtrate (de­termined by Kjeldahl). Methods of this kind are rapid, require little sample, and are conveniently adaptable to photometers and colorime­ters found in most laboratories. Nevertheless, turbidimetric methods are not of the highest accuracy and precision because light absorption in turbid systems depends not only on the amount of dispersed ma­terial present but also on its degree of dispersion. The degree of dispersion depends on time of standing after development of turbidity, pH, salt concentration, method and rate of precipitation, and concentration of material. Only by the most rigid standardization of all of the details of such methods can they be employed at all successfully.

13.2.4 Refractive index measurements

As previously pointed out most proteins have a refractive index increment of about 0.0018, meaning that one g. of protein dissolved in 100 ml. of solution increases the refractive index by that amount. With an accurate refractometer such as the Zeiss dipping refractometer, which has a sensitivity of ±0.00003, it is possible to determine protein concentrations satisfac­torily. Of course, the refractive index of the solvent must be deter­mined as well as that of the protein solution. Temperature must be carefully controlled since most proteins exhibit a considerable tempera­ture coefficient for refractive index increment. The casein is isolated by acid pre­cipitation, washed and dissolved in alkali,and the refractive index is determined.

13.2.5 Absorption of ultraviolet radiation

The absorption of ultra­violet radiation at wavelengths in the neighborhood of 280 nm, due to tyrosine, tryptophan, and phenylalanine residues in the protein, can be used as a method for determining protein content. This method is particularly valuable for a known pure protein whose extinction coefficient (E₁^1%_cm) is known.It may also be useful as an approximate measure of the total protein content of a mixture if an average extinction coefficient can be assumed to apply. Obviously the ex­tinction coefficients of proteins vary with their contents oftyrosine, tryptophan, and phenylalanine. The presence of materials other than proteins that absorb at this wavelength would seriously limit the method, but the common salts and other solutes do not absorb radiation at 280 nm. Methods based on this principle have not been used to any extent for routine determinations of milk proteins.

13.3 Chemical Methods

13.3.1 Determination of nitrogen

By all odds the most widely used method for determining protein content is the Kjeldahl procedure for nitrogen. It is natural that this should be so since nitrogen is a characteristic element in proteins.

This method involves the oxidation of the sample with sulfuric acid and a catalyst. Carbon and hydrogen are oxidized to CO₂ and H₂O, and reduced forms of nitrogen (such as –NH₂ and >NH) are retained in the digest as ammonium ions. The digest may be made alkaline and the ammonia distilled, and titrated, or it may be determined colorimetrically directly in the digest by means of Nessler’s reagent.

Although it is widely accepted and used, the Kjeldahl method suffers from some rather serious difficulties. In the first place, there is the problem of separating protein from other nitrogenous materials. In milk about 5% of the total nitrogen is in the form of low molecular weight non-protein nitrogenous materials. The protein can be sepa­rated from these by precipitation with trichloroacetic acid. This pre­cipitation, however, does not separate protein from nitrogen-containing lipids .Since the amount of lipid nitrogen is small, comprising only about 0.270 of the total milk nitrogen, it is usually neglected (i.e., in­cluded as protein nitrogen).

A second difficulty with nitrogen methods for determining protein arises from variation in the nitrogen content of various proteins. Pro­teins vary in nitrogen content from 14 to 19% and thus a single universal conversion factor cannot be used. Fortunately the principal milk pro­teins exhibit much less variation ranging from 15.3 to 16.0%. An average factor of 6.38 (corresponding to 15.65% nitrogen) is commonly used for milk proteins to convert nitrogen to protein. In precise work on an individual milk protein the correct factor for that protein should be used.

A third group of difficulties concerned with the Kjeldahl method is the problem of proper digestion and no loss of nitrogen. A great many different modifications of the original Kjeldahl procedure have been suggested to accelerate the digestion while still attaining complete digestion. Copper, mercury, or selenium are used as catalysts and frequently Na₂S0₄ or K₂S0₄is added to elevate the boiling point during digestion..

The use of boric acid to receive the ammonia as it is distilled off is an advantage in that only one reagent, the standard acid for titrating, need to be standardized and measured accu­rately.

13.3.2 Formol titration

Considerable use has been made of titration with formaldehyde as a means of determining protein content in milk and milk fractions. Such titrations have been of particular interest as rapid methods of determining the casein content of milk for cheese making. As per Bureau of Indian Standards, for the estimation of total milk protein a factor of 1.7 is used and while for casein the factor 1.38 is used.

All such methods involve titration of a sample of milk to the end point of an indicator such as phenolphthalein, or adding a solution of formaldehyde, and titrating the acid liberated to the same end point. The amount of alkali used in the second titration is a meas­ure of the amino groups that were originally present and combined with the formaldehyde.

13.4 Instrumental Methods

Several instrumental methods have been developed for the estimation of protein content in milk and milk products. A major advantages of these instrumental methods over the other techniques mentioned earlier are that these are nondestructive, require little or no sample preparation, and measurements are rapid and precise. For quality control purposes, it is often more useful to have rapid and simple measurements of protein content and therefore IR techniques are most suitable. For fundamental studies in the laboratory, where pure proteins are often analyzed, UV-visible spectroscopic techniques are often preferred because they give rapid and reliable measurements, and are sensitive to low concentrations of protein.