A Colorimetric Method for the Determination of Milk Proteins
Transcript of A Colorimetric Method for the Determination of Milk Proteins
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.