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DETECTION of GELATIN in CULTURED BUTTERMILK and COTTAGE CHEESE by G

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DETECTION of GELATIN in CULTURED BUTTERMILK and COTTAGE CHEESE by G 314 ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS [Vol. XVII, NoJ DETECTION OF GELATIN IN CULTURED BUTTERMILK AND COTTAGE CHEESE By G. A. RICHARDSON and N. P. TARASSUK (Division of Dairy Industry, University of California University Farm, Davis, California) Downloaded from https://academic.oup.com/jaoac/article/17/2/314/5765365 by guest on 29 September 2021 The Stokes method1 for the detection of gelatin in milk and cream was accepted as official by the Association of Official Agricultural Chemists.2 It has been recognized, however, that this method gives erroneous results in the case of sour milk, sour cream, cultured buttermilk and cottage cheese. This is presumably due to the presence in these products of sub- stances resulting from the partial hydrolysis of the proteins, such sub- stances resembling gelatin in their behavior to the reagents used in the Stokes test. The Seidenberg hot water modification3 was designed to make the Stokes test specific for gelatin, but it often fails clearly to dif- ferentiate gelatin from the hydrolytic products of the proteins, especially in cultured buttermilk and cottage cheese. For example, Mendelsohn4 reported that the modification failed in the case of cream to which rennet had been added, a positive test for gelatin being obtained on samples that were known not to contain gelatin. Preliminary studies in connection with cultured buttermilk showed that the Seidenberg modification yields fairly accurate results under well- controlled conditions. The test, however, was found to be too uncertain, especially from the standpoint of regulatory laboratories. If certain dis- criminating observations are made, the method of Stokes leads to fairly correct decisions in the detection of gelatin in cultured buttermilks whether fresh or old, with or without rennet. Thus, when picric acid is added to the filtrate resulting from the addition of mercuric nitrate, old samples and those containing gelatin or rennet will become cloudy im- mediately. On shaking and standing, precipitates will form in all samples of cultured buttermilks, more especially in old samples and those to which rennet has been added. The types of precipitates are different. Those re- sulting from compounds derived from proteolysis are flocculent, settle rapidly, are non-adhesive to the walls of the container, and are easily dis- turbed. The serum is clear. The precipitates of gelatin-picrate are more crystalline in nature, settle slowly, and adhere tenaciously to the bottom and sides of the container. The serum will remain opalescent for days. By making use of these observations during the past three years, three sepa- rate classes of upper division students were enabled to report correctly on heterogeneously arranged groups of unknown samples of cultured butter- milk. It is imperative, however, that for precise work the method be as inde- » Analyst, 22, 320 (1897). * Methods of Analysis, A.O.A.C, 1930, 223. »Ind. Eng. Chem., 5, 927 (1913;. « Analyst, 55, 567 (1930). 1984] EICHAEDSON, TAKASSUK! GELATIN IN BUTTERMILK AND CHEESE 315 pendent as possible from a procedure in which a comparison of types of precipitate is used in drawing final conclusions. In addition, the method should also be applicable to cottage cheese, with and without rennet. The procedure outlined above does not yield satisfactory results with cottage cheese. Downloaded from https://academic.oup.com/jaoac/article/17/2/314/5765365 by guest on 29 September 2021 Jacobs and Jaffe1 recently reviewed the literature on the detection of gelatin in dairy products and reported a new method. They use basic lead nitrate as the protein precipitant, and calcined charcoal as adsorbent for the "pseudo-gelatins" formed in the souring process. Picric acid or tannic acid is used as the final precipitant. This method, however, in the hands of the writers gave erroneous results with very old samples of cot- tage cheese and cultured bettermilk, and with those containing rennet. The successful use of trichloracetic acid, especially in high concentra- tions, for the precipitation of the proteins of milk suggested that this acid might aid in differentiating gelatin from the compounds resulting from the partial hydrolysis of the milk proteins of cultured buttermilk and cot- tage cheese. Sanders2 found that four parts of 10 per cent trichloracetic acid solution to one part of milk was very effective in preparing protein-free filtrates for mineral determinations. Moir3 used trichloracetic acid in his study of the distribution of the proteins of milk. Sanders4 recently reported that trichloracetic acid in sufficient concentration is al- most as efficient as tungstic acid as a precipitant for the nitrogen com- pound of milk. EXPERIMENTAL Cultured Buttermilk The procedure that was found satisfactory for cultured buttermilk is as follows: REAGENTS (a) Mercuric nitrate*—DissolveHg in twice its weight of HNO3 and dilute this solution to 25 times its volume with H2O. (It is imperative that the reagent be free from the mercurous ion.) (b) Picric acid.—Saturated aqueous solution. (c) Trichloracetic acid.—20% aqueous solution. PREPARATION OF SAMPLE If the cultured buttermilk is cold, warm it to approximately 25°C. and stir thor- oughly. PROCEDURE To 10 cc. of the sample, add 10 cc. of the mercuric nitrate reagent. Shake the mixture, allow to stand 5 minutes, and filter through a retentive, medium-fast filter paper (Filtrate No. 1). ilnd. Eng. Chem. Anal. Ed., 4, 418 (1932). »/. Bid. Chem., 90, 755 (1931). »Analyst, 56, 228 (1931). < This Journal, 16, 140 (1933). • Methods of Analysis, A.O.A.C, 1930, 223, 26. TABLE I.—Observations recorded in testing commercial cultured buttermilks for gelatin SAMPLE 1 2 3 4 5 6 Filtrate from mercuric nitrate Fairly clear Fairly clear Very turbid Turbid Turbid Fairly clear (No. 1) Aliquot of filtrate No. 1 plus § Small amount Small amount Colloidal tur- Turbid, no Flocculent pre- Voluminous vol. of saturated picric acid of flocculent of flocculent bidity, no floes fiocs visible cipitate. flocculent pre- precipitate. precipitate. visible Serum col- cipitate. Serum clear Serum clear loidally tubid Serum clear Prediction No gelatin No gelatin Gelatin Gelatin Gelatin and No gelatin but rennet rennet Aliquot of filtrate No. 1 chilled in Almost clear" Slight opales- Turbid Slightly turbid Colloidal tur- Turbid, (red- ice water, plus § volume of cence bidity dish, Millon's 20% trichloracetic acid reaction) After standing overnight at Small amount Small amount Dense colloid- Colloidal tur- Flocculent pre- Flocculent pre- 8-10°C, with occasional shak- of flocculent of flocculent al turbidity bidity cipitate. cipitate ing precipitate. precipitate. Serum turbid Serum clear Serum clear Serum clear (reddish) (reddish) Filtrate from trichloracetic acid Clear Clear Dense colloidal Colloidal tur- Dense colloidal Clear mixture heated to 50°C. plus \ turbidity bidity turbidity vol. of saturated picric acid (50°C.) Conclusions No gelatin No gelatin Gelatin Gelatin Gelatin and No gelatin but rennet rennet Actual content Fresh cultured Old cultured Fresh cultured Fresh cultured Fresh cultured Fresh cultured buttermilk— buttermilk— buttermilk plus buttermilk plus buttermilk plus buttermilk plus no gelatin no gelatin 2 % gelatin 0.1% gelatin 0.15% gelatin rennet plus rennet Downloaded from https://academic.oup.com/jaoac/article/17/2/314/5765365 by guest on 29 September 2021 September 29 on guest by https://academic.oup.com/jaoac/article/17/2/314/5765365 from Downloaded 1984] RICHAEDSON, TAEASSUK: GELATIN IN BUTTEKMILK AND CHEESE 317 To an aliquot of Filtrate No. 1, add § volume of saturated picric acid. Observe the mixture for clearness and type of precipitate. Chill the remainder of the filtrate (No. 1) in ice water, add £ volume of 20% trichloracetic acid, shake well, and allow to stand at 8-10°C. about 16 hours, with occasional shaking particularly during the early part of the period. Observe the filtrate-trichloracetic acid mixture for clearness and type of precipitate. Filter cold, using a medium-fast filter paper (Filtrate No. 2). Warm the filtrate to 50-55°C, hold at this temperature for at Downloaded from https://academic.oup.com/jaoac/article/17/2/314/5765365 by guest on 29 September 2021 least 5 minutes, and add \ volume of warm saturated picric acid (50°C). Observe while still warm. Table 1 illustrates what the analyst may expect to observe, and from which he may draw conclusions. It is very evident that the method of Stokes would have led to incorrect conclusions in the case of each sample. When the type of precipitate and the nature of the serum found in con- junction with the Stokes test are considered, the prediction was correct. The use of trichloracetic acid clearly substantiates the prediction. It should be mentioned that apparently no sample had undergone deteriora- tion. It is recognized that it is not the practice to add rennet to cultured buttermilk, but the possibility of protein hydrolysis occurring during the pasteurization treatment and subsequent culturing procedure is not re- mote. To be able to distinguish the reactions of rennet from those of gela- tin gives the test greater reliability. The samples referred to in Table 1 and others were tested by the tech- nic of Jacobs and Jaffe.1 The test proved to be excellent for perfectly fresh samples of cultured buttermilk, but it yielded erroneous results in the case of old samples or those to which rennet had been added. When the filtrates from the basic lead nitrate and charcoal were subjected to the trichloracetic acid treatment prior to the addition of tannic or picric acid, the results were as definite as those shown in Table 1. The test so modified offers no advantages, however, and it is more laborious than the tri- chloracetic acid modification of the Stokes method. COTTAGE CHEESE The procedure outlined for cultured buttermilk was adapted to cottage cheese by altering the method of preparing the sample, and by a double precipitation with mercuric nitrate.
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