Some Observations on the Hull Method for Measurement of Proteolysis in Milk1

TECHNICAL

NOTES

SOME' O B S E R V A T I O N S ON T H E H U L L M E T H O D FOI~ M E A S U R E M E N T OF P R O T E O L Y S I S IN M I L K 1 Although the widely used (2, 3) Hull method (1) for the determination of hydrolysis of milk protein is rapid and convenient, certain difficulties have been encountered in this laboratory daring its application. Highly turbid, rather than clear, solutions were often obtained dm~ing the final stage of the assay. In addition, when using autoclaved milk, bacterial proteolysis could not be accurately determined. These difficulties and their corrective measures will be described. According to the Hull procedure, a clear trichloroacetic acid filtrate is obtained from the milk sample to be assayed. The p H of the filtrate is increased by the addition of a sodium carbonate-sodium methaphosphate solution. Finally, upon the addition of Folin-Ciocalteau phenol reagent, a clear blue solution results. Using a suitable blank, the intensity

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phenol reagent was added. Probable causes of t.his turbidity, including the age and storage conditions of the milk, the age, purity, and concentration of the various reagents, and the temperature at which the assay was performed, were investigated. The last factor, temperature, proved to be the cause of the observed turbidity. As shown in Figm'e 1; temperatures of the reaction mixture below 26 C resulted in marked turbidity; whereas, above this temperature the solutions were clear. Also, once the clear solution was obtained, lowering the temperature did not cause the solution to become turbid. Since room temperatures below 26 C are common, it is suggested that special precautions be taken to conduct the Hull assay under moderately warm test conditions. F o r example, the sodium carbonate solution may be kept at about 40 C in a water bath prior to the assay. As a final note, it should be mentioned that low levels of bacterial proteolysis could not be determined accurately in samples obtained from milk autoclaved at 121 C f o r 12 rain. Autoclaving may have caused the release of amino acids sensitive to this test; the blue color which resulted was so intense that amino acid release due to bacterial proteolysis was difficult to detect colorimetrically, even with a suitable blank of the same autoclaved milk. This difficulty was corrected by the use of milk pasteurized 30 rain at 62 C. J. E. CI~TI W. E. SARDINE AlqD

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P. R. ELLIKBR Department of Microbiology Oregon State University Corvallis

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TEMPERATURE,°C FIG. 1. Effect o.f reaction mixture temperature on absorbance at 650 m/z by trichloroa~etie aeld fii~ra.te of milk obtained by t2~e method of Hull (1).

REFERENCES

(1) ttULL, M. E. Studies on Milk Proteins. II. Colorimetric Determination of the Partial Hydrolysis of the Proteins in Milk. J. Dairy Sci., 30: 881. 1947. (2) STADHOU]mRS,J., AND VXnINaA, tI. A. Some Experiments Related to the Inhibitory Action of Milk Peroxidase on Lactic Acid Streptococci. Netherlands Milk Dairy J., 16: 96. 1962. (3) WILLIAMSON, W. T., AN]) SPECK, M. L. Proteo]ysis and Curd Tension ir~ Milk Associated with Accelerated Starter Culture Growth. J. Dairy Sci., 45: 164. 1962.

of the colored solution may be measured photometrically and related to a standard curve of tyrosine equivalents. During the present study, a white precipitate frequently formed a few minutes after the Supported by NI H Grant EF-69. 337