556 THE INDIAN MEDICAL GAZETTE [Oct., 1933

A COLORIMETRIC METHOD FOR THE DETERMINATION OF MILK PROTEINS

By A. D. STEWART, m.b., f.r.c.s.e., d.p.h., d.t.m.&h.

LIEUTENANT-COLONEL, I.M.S., DIRECTOR

and

DURGA DAS MITRA, b.sc., m.b., d.p.h.

(From the All-India Institute of Hygiene and Public

Health, Calcutta)

The method is based on comparing the blue colours developed by adding Fofin's (1922) phenol reagent to different samples of milk and to a standard tyrosin solution. Of the different amino acids, only tyrosin and tryptophane (Folin and Looney, 1922) have been shown to

give rise to a blue colour with this reagent; and tryptophane has been found to give rise to 58 per cent of the colour developed by an equal amount of tyrosin. We have determined

colorimetrically the tyrosin equivalents of milk proteins for cow and buffalo milk, and these factors enable one to find out quickly the protein percentage of any sample of cow or buffalo milk. The method is that followed by Greenberg

(1929) for estimating the blood serum proteins. In the case of milk however, a turbidity and slight precipitation always result after the addition of the phenol reagent; this leads to

slight modifications of technique. By a series of colorimetric experiments and

parallel determinations of the protein contents by Kjeldahl's method, we find that, on an

average, 12 milligrammes of cow milk proteins and 1*5.2 milligrammes of buffalo milk proteins correspond respectively to 1 milligramme of

tyrosin. The following notes may be useful. Reagents required :?

(1) 5 N. NaOH.

(2) Standard solution (0.02 per cent) of pure dry tyrosin in 0.1 N (approximately) HC1.

(3) Folin's phenol reagent (Folin and Ciocal- teau, 1927).

(4) Whatman's filter paper, no. 5, size 15 centimetres.

For a series of milk samples take the corres- ponding number of marked measuring flasks.

Arrange a similar number of marked filter

funnels and beakers.

Rinse the pipettes twice with the sample after thoroughly shaking and mixing it, and pipette off 0.2 c.cm. of each sample in the case of cow's and buffalo's milk into the correspond- ing measuring flask and 4 c.cm. tyrosin solution into a separately marked flask. Add about 25 c.cm. of water to each, washing down all the traces of milk or tyrosin solution. Add 2 c.cm. of 5 N. NaOH to each, gently shake, add more water and again shake. Add 3 c.cm. of the

phenol reagent and fill up to the mark as

quickly as possible with water. Shake and keep for 10 minutes. Filter, and after 5 minutes of filtration begin to take colorimetric readings of different samples and complete all the readings within 15 minutes, i.e., from 15 minutes to

within half an hour after the addition of the

phenol reagent. This time factor is important, as the maximum

colour develops in about 15 minutes and after some time begins to fade. When taking successive samples in the same

colorimeter cup, rinse it twice with the solution under examination and wipe the colorimeter tube dry with a filter paper before immersing it into a fresh sample in the cup. The tyrosin standard is taken in the right-hand cup and fixed at 20, and the left-hand cup with the

sample is adjusted till the colour matches, when the reading is taken. Most of the experiments have been done in

duplicate. Samples should be fresh and examined colori-

metrically preferably within 4 hours. Sour milk

gives unreliable readings. The protein content is calculated from the

formula :?

Protein percentage ^ X T X ^ X j^qq where R the arbitrary reading of the standard,

i.e., 20. X the reading of the unknown. T tyrosin content in milligrammes of the

standard, i.e., 0.8 in 4 c.cm. which we take.

S = the aliquot part of the milk used in

c.cm.'s (0.2 c.cm. in this case). F = the factor of tyrosin equivalence of the

particular milk proteins (cow = 12, buffalo = 15.2).

Substituting actuals the formula reduces :?

8 X F Protein percentage = ?^?

Taking the value of F for cows to be 12, and for buffaloes to be 15.2, this formula simplifies into :?

96 121*6 Protein percentage = ^or cow's) anc*

(for buffalo's). The determination of

' F' for cow's milk.

J

Oct., 1933] MILK PROTEINS : STEWART & MITRA 557^ Table I

EXnumbent Rttadin9 1S Avercfntageeby F (calculated) number the sample Kjcidahl's 1 30.9 2.87 110

2 28 4 3.15 11-2

3 25.0 3.8 12-06 25.0

'1 29.1 3.6 13.0

28.2 3.6 12-69

6 30.1 3.06 11-5

7 27.4 3.6 12 33

83.78

Average = = 12-

7

Table II _L ADLiEJ

Showing comparison of results (1) colorimetri- cally taking F = 12 and (2) by Kjeldahls method

Cow's milk e, . . ,_?s Protein Experiment Reading of Protein (96; percentage by number the sample percentage ?? Kjeldahl's

(x) method

1 31.0 3.1 2 26.5 3.6

3 2

3.5

3 32.0 3.0 3.2

4 25.5 3.76 5 29.1 3.29 6 23.46

' 4.09 7 30.16 3.18 8 25.7 3.7 9 25.76 3.7

10 23.0 4.2 11 29.8 32

3.8 3.3 4.02 3.38 3.6 3.9 4.0 3.1

Table III

Buffalo's milk

Determination of F for buffalo s milk

Experiment Reading of vScentage by F (calculated) number the sample Kjcidahl's

1 26.45 4.5 14-85

2 27.55 4.53 3 23.86 4.78 150

4 24.6 5.13 Ijj-?, 5 26.5 4.66 6 23.0 52

15.43 14.95

7 27.3 4.4 150. 14.64 8 24.4 4.8

Taking an average F =15.2.

Table IV

Showing results calculated colorimetrically and those determined by Kjeldahl's method

Experiment Reading of Protein Proteinpercentage number the sample percentage by Kjeldahls

1 24.7 4.9 4.8

2 242 5.0 4.7

3 31.0 3.9 3.8

4 25.4 4.8 ,

4.78

5 23.3 5.2 6

Human milk

0*4 c.cm. milk was used for each colorimetric experiment.

Table V

Showing the protein percentage obtained by Kjeldahl's method and 'F' calculated on

colorimetric readings and protein percentage

~? <? - <~> 1 225 1.79 10.18 2 33.26 1.4 11.6 3 23.0 2.68 15.4 4 282 2.04 14.38 5 37.5 1.4 13.1 6 27.5 1.96 13.47 7 23.1 2.5 14.4 8 20.6 2.9 14.93 9 24.4 2.4 14.6 10 26.6 2.1 13.96 11 25.4 1.8 11.43 12 24.1 2.1 12.65 13 23.8 22 12.09 14 26.4 22 14.5 15 17.8 2.6 11.57 16 20.8 1.97 11.2 17 24.0 2.5 15.0 18 28.8 1.8 12.96

237.47 Taking the average F = =132.

18

These samples came from women of various nationalities, of different ages, and different serial orders of pregnancies. They are all how- ever from parturient women during the first week of lactation. The protein shows a wide variation in content. Lowenfeld, Widdows, Bond, and Taylor (1927) showed that the

average percentage of protein in human milk varies considerably during lactation and the same authors found that the first month of lactation is a period during which the protein content falls from a high to a low percentage. The calculated value of F in column 4 shows

variations from 10.18 to 15, suggesting that the tyrosin and tryptophane content of human milk varies considerably in the first week of lacta- tion. The calculated average for F = 13.2 is

evidently not a close one and, as might be ex- pected, the values obtained colorimetrically in

table VI are not in close agreement with actual

Kjeldahl findings. The method is therefore not

suitable for determining the protein of human milk during the first week of lactation. We had

no opportunities to examine human milk after the first week of lactation.

Table VI

Human milk

Showing colorimetric findings and actual protein percentage by Kjeldahl's method

Experiment Reading of Protein Protein-percentage number the sample percentage by Kjeldahl's

1 21.0 2.5 2.1 2 26.0 2.0 16 3 26.9 2.0 1.7 4 24.2 5.0 4.7

The percentage calculated colorimetrically is that by volume, whereas Kjeldahl's determina- tion gives percentage by weight. For practical

558 THE INDIAN MEDICAL GAZETTE [Oct., 1933

I

purposes this difference may be neglected as it is a difference only in the second place of decimals. The percentage of milk proteins has been

made a standard by the Madras Food Adultera- tion Act, 1918, and the validity of such a

standard has been verified by Stewart and

Chatterjee (1931) in Calcutta samples of milk. A simple method which can be completed within half an hour, with very small samples, either

individually or in a series, may be useful. In the case of known cow and buffalo milks

reliable results can be obtained. Our thanks are due to Lieut.-Col. Boyd,

Principal of the Medical College Hospitals, and Lieut.-Col. Gow of the Eden Hospital for

supplying samples of human milk from the Eden Hospital. We thank Mr. P. C. Mitra for

supplying pure samples of cow's and buffalo's milk.

References

Folin, O. (1922). Laboratory Manual of Biological Chemistry. New York: D. Apnleton and Co.

Folin. 0., and Ciocalteau. V. (1927). Journ. Biol.

Chem., Vol. LXXIII, p. 627. Folin, O., and Looney, J. M. (1922). Ibid., Vol. LI,

p. 421.

Greenberg, D. M. (1929). Ibid.. Vol. LXXXIX, p. 545.

Lowenfeld, M. F., Widdows, S. T., Bond. M., and Tavlor, E. I. (1927). Biochem. Journ.. Vol. XXI. p. 1.

Stewart, A. D., and Chatteriee, N. K. (1931). Indian Med. Gaz., Vol. LXVI, p. 320. Widdows, S. T.. Lowenfeld, M. F.. Bond. M.. and

Tavlor, E. I. (1930). Biochem. Journ.. Vol. XXIV, p. 327.