684 Journal o{Food Protection Vol. 41, No.9. Pages 684-688 (September. 1978) Copyright 1978, International Association of Milk, Food, and Environmental Sanitarians Efficacy of Chicken Pepsin as a Milk Clotting Enzyme 1 S. GORDIN and I. ROSENTHAL* Dairy Laboratory, Division ofFood Technology Agricultural Research Organization, The Volcani Center Downloaded from http://meridian.allenpress.com/jfp/article-pdf/41/9/684/1650145/0362-028x-41_9_684.pdf by guest on 26 September 2021 P.O. Box 6, Bet Dagan, (20-500), Israel (Received for publication December 19, 1977) ABSTRACT Enzyme activity assays Comparative laboratory tests of cheesemaking show similarity Enzyme activity was estimated by the milk clotting test done in test tubes periodically rotated in a thermostatic bath at 30 C, unless between chicken pepsin and calf rennet. Suitability of chicken pepsin othernise specified. To ensure reproducible results, a reconstituted for large-scale production of Emmental (Swiss) and Kashkaval-type skimmed milk known as "Berridge substrate" was employed. This cheeses was tested. substrate consists of 12 g of low-heat spray-dried skim milk powder (6 mg of whey protein/g index) dissolved in 100 ml of 0,01 M CaC1 2 solution. After adding the skim milk powder to the CaC12 solution, the mixture was stirred for 20 min and left to stand at room temperature Use of calf rennet as a milk clotting enzyme in the for an additional hour. This preparation procedure was adopted since manufacture of cheese has been predominant in the we noted that the time required for coagulation increased with the age of "Berridge substrate", particularly for freshly prepared milk industry for centuries. Lately, a worldwide shortage of solutions. Immediately afterwards, 10 ml of milk was heated at the this enzyme has been predicted due to the increase in assay temperature, 1 ml of enzyme solution was added, and the clotting production and consumption of cheeses and the time was determined. lhe milk clotting time test was used to study the effects of the simultaneous decrease in the general availability of following parameters on the activity of both enzymes. suckling calves' stomachs. Consequently, a great deal of Enzyme dilutions. Both commercial enzymes were diluted to the interest has been generated in research for other effective range of 1:SO to 1:400 and their clotting times were tested. Substrate concentration. Solutions containing 10, 11, 12, 13, or 14% and competitive rennets. However, only a few other low-heat, spray-dried skim milk powder in 0.01 M CaCI2 were placed in animal proteases - such as pig and bovine pepsins - tubes each containing 10 ml of solution. They were preincubated in a 30 and some microbial rennet preparations have been found C water bath for 5 min, after which 1 ml of enzyme solution was added. It is noted that the pH (6.36) was the same for all solutions employed in suitable as a rennin substitute and are presently used in this test. cheesemaking (4, 8). Substrate pH. In identical samples of reconstituted milk ("Berridge In Israel this problem is aggravated by religious substrate"), the pH was adjusted over the range 5.1-6.7 with solutions of HCI or NaOH, 0.2 N. The final pH was measured after 20 min of requirements for the ritual slaughtering of calves, and stirring and 1 h of incubation at room temperature. the prohibition of certain clotting agents from animals Calcit<m ion concentration. Twelve grams of low-heat spray-dried such as pig pepsin. These reasons prompted local skim milk was dissolved in 100 ml of distilled water containing 0.0-0.1M CaCI2 and was placed in tubes each containing 10 ml of research efforts toward ftnding rennin substitutes, which solution in which the milk clotting tests were done. Although addition in turn have led to production, development, and ofCaC12 changed the pH of the milk from 6.60 (0 M CaCI2} to 5.61 (0.1 subsequent employment by the cheese industry of a M CaC12), no pH correction was made. Attempts to correct the pH value by addition of acid or base yielded erratic results, most probably pepsin of avian origin. i.e .. chicken pepsin (1, 5). This due to irreversible modifications of the micellar structure of the milk paper describes a study of the various properties of this protein. enzyme, such as milk clotting abilities and influence on Reaction temperature. Tubes containing 10 ml of "Berridge substrate" were incubated in water baths, the temperatures of which ripening of cheeses. were adjusted in the range of 25 to 55 C. After 5 min of incubation, 1 ml MATERIALS AND METHODS of each of the enzymes was added and the clotting time measured. Proteolytic activity. The proteolytic activity of both enzymes was Commercial preparations (1:5000) of chicken pepsin (6.4 mg of estimated by two sets of tests. (a) Aqueous solutions of casein (1.5%) protein!ml) (Enzyme Industries. Emek Heter Israel) and calf rennet adjusted to pH value of 5.49 were incubated at 30 C with chicken (1:10,000} (8.1 mg of protein/ml) (Frankental and Sons Ltd., Bene pepsin or calf rennet. At certain intervals samples were drawn from Beraq, Isreal) were employed. The strengths of the enzymes were the solutions and TCA solution (6%) was added in the ratio 1:1. determined by comparison with Hansen Standard Rennet powder (Chr. Samples were kept at 5 C for 30 min and then centrifuged. The Hansen's Lab. A/S-Copenhagen, Denmark). For laboratory experi­ nitrogen content of the supernatant fluid of each of the samples was ments purified enzymes were comparatively tested, with similar results. determined. (b) To solutions of "Berridge substrate" incubated at 30 C, 1 ml of calf rennet of chicken pepsin (diluted 1:100) was added. The 'Contribution _from the Agricultural Research Organization, The coagulum was knife-cut. dipped, and the nitrogen concentration in the Volcani Center, Bet Dagan, Israel. 1977 series. No 278-E. whey was determined. A similar experimental procedure served to CHICKEN PEPSIN FOR COAGULATING MILK 685 estimate the amount of enzyme transferred to the whey. Thus the fully collected whey was lyophilized, the dry material left was dissolved x-x Calf Rennet _. Chicken Pepsin in distilled water and the recovered activity of the coagulating enzyme 1000 was estimated by the milk clotting test, done in parallel to control tests with known amounts of enzyme which underwent the same treatment. u Cheese production ~"' Emmental (Swiss) and Kashkaval-type cheeses were produced at the "'E "Tnuva" Tel YosefDairy. Milk of identical origin, fat content and total 1- solids was placed in two 5000-liter vats and coagulated with the calf rennin or chicken pepsin, respectively. :§"" 500 The Emmental-type cheese was produced from 3.1 o/o fat standard­ 0 ized cows milk, HTST pasteurized (72 C for 16 sec) and cooled to 30 C. u Starter, including thermoduric bacteria. 0.3 kgofCaC12/1000 liters and either water-diluted calf or chicken pepsin (adjusted to pH 4.0 with NaHC03), was added. After ca. 30 min the coagulum was ready for cutting. The cutting, cooking, whey expulsion, molding, brining and L___ ripening were done as usual for Emmental cheese (6). O·L_ __ L-~--~--0----~------~1.-~400 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/41/9/684/1650145/0362-028x-41_9_684.pdf by guest on 26 September 2021 The Kashkaval-type cheeses was produced from 4.3-4.5% fat 1:50 1:100 1:150 1:20 standardized sheep milk, preheated to 32 C. Starter and the renneting Enzyme Dilution enzymes were added. Atter coagulation, the coagulum was cut into Fig. 1. Effect ofenzyme concentration on clotting time. pea-size grains, heated gradually to 38 C followed by cutting and cooking at 85 C under kneading. The cheese was salted in a brine solution (24o/oNaCI) for 3 days and preripened in a curing room at 10 C for 3 weeks. The cheese blocks were waxed, packed in Saran sheets, and 300 transferred to a cool (8 C) ripening room for 2 months. This parallel production was repeated three times. The cheeses produced were sampled and analysed at two-week intervals durin~~: the ripening period, starting on the fif'Jt day after production. Nitrogen (.) analyses were made with the cheese solutions in sodium citrate (9). -cu Total nitrogen and soluble nitrogen (unprecipitated at pH 4.7) were ~ determined by Kjeldahl analysis. Free amino acids were determined by cu titration of the soluble nitrogen fraction with 0.1 N NaOH after E coupling with formaldehyde. Ammonia was distilled from the cheese ..... 200 solution in the presence of BaCI2, into 0.1 N HCI and the acid that C' remained was determined by back titration with standard NaOH c: solution. The moisture content was determined gravimetrically and fat - content by the Gerber method (6). Organoleptic -and texture tests were 0 conducted by a taste panel. u RESULTS AND DISCUSSION Enzyme characterization To determine the suitability of chicken pepsin for 100 replacing calf rennet, we compared their properties 14 relevant to cheese production. The comparisons were 10 II 12 13 made under identical experimental conditions in Milk Powder Conc.(gr/100 ml 0.01 M several laboratory tests. CaCL2l Milk clotting activity. According to Holter (2), the Fig. 2. Effect of milk powder concentration on clottinl( time. relationship between clotting time (T) and enzyme concentration (C) is as follows: clotting time became. The response of both enzymes to K T +t changes in pH was the same up to pH 6.2. Above this c value the activity of chicken pepsin decreased drastically where K and tare constants, depending upon the enzyme as compared with that of the calf rennet.