Efficacy of Chicken Pepsin as a Milk Clotting Enzyme 1

Transcription

1 684 Jornal o{food Protection Vol. 41, No.9. Pages (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 of Food Technology Agricltral Research Organization, The Volcani Center P.O. Box 6, Bet Dagan, (2-5), Israel (Received for pblication December 19, 1977) ABSTRACT Comparative laboratory tests of cheesemaking show similarity between chicken pepsin and calf rennet. Sitability of chicken pepsin for large-scale prodction of Emmental (Swiss) and Kashkaval-type cheeses was tested. Use of calf rennet as a milk clotting enzyme in the manfactre of cheese has been predominant in the indstry for centries. Lately, a worldwide shortage of this enzyme has been predicted de to the increase in prodction and consmption of cheeses and the simltaneos decrease in the general availability of sckling calves' stomachs. Conseqently, a great deal of interest has been generated in research for other effective and competitive rennets. However, only a few other animal proteases - sch as pig and bovine pepsins - and some microbial rennet preparations have been fond sitable as a rennin sbstitte and are presently sed in cheesemaking (4, 8). In Israel this problem is aggravated by religios reqirements for the rital slaghtering of calves, and the prohibition of certain clotting agents from animals sch as pig pepsin. These reasons prompted local research efforts toward ftnding rennin sbstittes, which in trn have led to prodction, development, and sbseqent employment by the cheese indstry of a pepsin of avian origin. i.e.. chicken pepsin (1, 5). This paper describes a stdy of the varios properties of this enzyme, sch as milk clotting abilities and inflence on ripening of cheeses. MATERIALS AND METHODS Commercial preparations (1:5) of chicken pepsin (6.4 mg of protein!ml) (Enzyme Indstries. Emek Heter Israel) and calf rennet (1:1,} (8.1 mg of protein/ml) (Frankental and Sons Ltd., Bene Beraq, Isreal) were employed. The strengths of the enzymes were determined by comparison with Hansen Standard Rennet powder (Chr. Hansen's Lab. A/S-Copenhagen, Denmark). For laboratory experiments prified enzymes were comparatively tested, with similar reslts. 'Contribtion _from the Agricltral Research Organization, The Volcani Center, Bet Dagan, Israel series. No 278-E. Enzyme activity assays Enzyme activity was estimated by the milk clotting test done in test tbes periodically rotated in a thermostatic bath at 3 C, nless othernise specified. To ensre reprodcible reslts, a reconstitted skimmed milk known as "Berridge sbstrate" was employed. This sbstrate consists of 12 g of low-heat spray-dried skim milk powder (6 mg of whey protein/g index) dissolved in 1 ml of,1 M CaC1 2 soltion. After adding the skim milk powder to the CaC1 2 soltion, the mixtre was stirred for 2 min and left to stand at room temperatre for an additional hor. This preparation procedre was adopted since we noted that the time reqired for coaglation increased with the age of "Berridge sbstrate", particlarly for freshly prepared milk soltions. Immediately afterwards, 1 ml of milk was heated at the assay temperatre, 1 ml of enzyme soltion was added, and the clotting time was determined. lhe milk clotting time test was sed to stdy the effects of the following parameters on the activity of both enzymes. Enzyme diltions. Both commercial enzymes were dilted to the range of 1 :SO to 1:4 and their clotting times were tested. Sbstrate concentration. Soltions containing 1, 11, 12, 13, or 14% low-heat, spray-dried skim milk powder in.1 M CaCI 2 were placed in tbes each containing 1 ml of soltion. They were preincbated in a 3 C water bath for 5 min, after which 1 ml of enzyme soltion was added. It is noted that the ph (6.36) was the same for all soltions employed in this test. Sbstrate ph. In identical samples of reconstitted milk ("Berridge sbstrate"), the ph was adjsted over the range with soltions of HCI or NaOH,.2 N. The final ph was measred after 2 min of stirring and 1 h of incbation at room temperatre. Calcit<m ion concentration. Twelve grams of low-heat spray-dried skim milk was dissolved in 1 ml of distilled water containing.-.1m CaCI 2 and was placed in tbes each containing 1 ml of soltion in which the milk clotting tests were done. Althogh addition ofcac1 2 changed the ph of the milk from 6.6 ( M CaCI 2 } to 5.61 (.1 M CaC1 2 ), no ph correction was made. Attempts to correct the ph vale by addition of acid or base yielded erratic reslts, most probably de to irreversible modifications of the micellar strctre of the milk protein. Reaction temperatre. Tbes containing 1 ml of "Berridge sbstrate" were incbated in water baths, the temperatres of which were adjsted in the range of 25 to 55 C. After 5 min of incbation, 1 ml of each of the enzymes was added and the clotting time measred. Proteolytic activity. The proteolytic activity of both enzymes was estimated by two sets of tests. (a) Aqeos soltions of casein (1.5%) adjsted to ph vale of 5.49 were incbated at 3 C with chicken pepsin or calf rennet. At certain intervals samples were drawn from the soltions and TCA soltion (6%) was added in the ratio 1:1. Samples were kept at 5 C for 3 min and then centrifged. The nitrogen content of the spernatant flid of each of the samples was determined. (b) To soltions of "Berridge sbstrate" incbated at 3 C, 1 ml of calf rennet of chicken pepsin (dilted 1:1) was added. The coaglm was knife-ct. dipped, and the nitrogen concentration in the whey was determined. A similar experimental procedre served to

2 CHICKEN PEPSIN FOR COAGULATING MILK 685 estimate the amont of enzyme transferred to the whey. Ths the flly collected whey was lyophilized, the dry material left was dissolved in distilled water and the recovered activity of the coaglating enzyme was estimated by the milk clotting test, done in parallel to control tests with known amonts of enzyme which nderwent the same treatment. Cheese prodction Emmental (Swiss) and Kashkaval-type cheeses were prodced at the "Tnva" Tel YosefDairy. Milk of identical origin, fat content and total solids was placed in two 5-liter vats and coaglated with the calf rennin or chicken pepsin, respectively. The Emmental-type cheese was prodced from 3.1 o/o fat standardized cows milk, HTST pasterized (72 C for 16 sec) and cooled to 3 C. Starter, inclding thermodric bacteria..3 kgofcac1 2 /1 liters and either water-dilted calf or chicken pepsin (adjsted to ph 4. with NaHC 3 ), was added. After ca. 3 min the coaglm was ready for ctting. The ctting, cooking, whey explsion, molding, brining and ripening were done as sal for Emmental cheese (6). The Kashkaval-type cheeses was prodced from % fat standardized sheep milk, preheated to 32 C. Starter and the renneting enzymes were added. Atter coaglation, the coaglm was ct into pea-size grains, heated gradally to 38 C followed by ctting and cooking at 85 C nder kneading. The cheese was salted in a brine soltion (24o/oNaCI) for 3 days and preripened in a cring room at 1 C for 3 weeks. The cheese blocks were waxed, packed in Saran sheets, and transferred to a cool (8 C) ripening room for 2 months. This parallel prodction was repeated three times. The cheeses prodced were sampled and analysed at two-week intervals drin~~: the ripening period, starting on the fif'jt day after prodction. Nitrogen analyses were made with the cheese soltions in sodim citrate (9). Total nitrogen and solble nitrogen (nprecipitated at ph 4.7) were determined by Kjeldahl analysis. Free amino acids were determined by titration of the solble nitrogen fraction with.1 N NaOH after copling with formaldehyde. Ammonia was distilled from the cheese soltion in the presence of BaCI 2, into.1 N HCI and the acid that remained was determined by back titration with standard NaOH soltion. The moistre content was determined gravimetrically and fat content by the Gerber method (6). Organoleptic -and textre tests were condcted by a taste panel. RESULTS AND DISCUSSION Enzyme characterization To determine the sitability of chicken pepsin for replacing calf rennet, we compared their properties relevant to cheese prodction. The comparisons were made nder identical experimental conditions in several laboratory tests. Milk clotting activity. According to Holter (2), the relationship between clotting time (T) and enzyme concentration (C) is as follows: K T +t c where K and tare constants, depending pon the enzyme.and milk sbstrate, respectively. The reslts of this test for chicken pepsin and calf rennet are shown in Fig. 1. A good inverse proportionality relation between the amont of enzyme and coaglation time was obtained for both enzymes. Inflence of sbstrate concentration on coaglation time. The reslts (Fig. 2) show an increase in coaglation time with higher skim milk powder concentrations, similar for both enzymes. inflence of milk acidity on coaglation time. The acidity of milk has long been recognized as one of the critical parameters in cheese prodction. To determine the inflence of this factor, the ph of otherwise identical samples of reconstitted milk was adjsted over the range of The data obtained are in Fig. 3. In general, the greater the milk acidity, the shorter the 1 ~ "' "' E 1- "" : 5 Fig. 1. -(.) c ~ c E... C' c: - x-x Calf Rennet _. Chicken Pepsin O L L L-~--~------~------~1.-~4 1:5 1:1 1:15 1:2 Enzyme Diltion Effect of enzyme concentration on clotting time. 1 II Milk Powder Conc.(gr/1 ml.1 M CaCL2l Fig. 2. Effect of milk powder concentration on clottinl( time. clotting time became. The response of both enzymes to changes in ph was the same p to ph 6.2. Above this vale the activity of chicken pepsin decreased drastically as compared with that of the calf rennet. It is noted that porcine pepsin is also inactive at ph vales above 6 (7) Dependence of coaglation time on concentration of Ca ions. Addition of CaCI 2 to pasterized milk improves the clotting activity of the coaglating enzyme, and at the same time may reglate the water content of the cheese. The dependence of clotting time on concentration of Ca ions (Fig. 4) indeed verified the fact that increased amonts of CaC1 2 shorten the clotting time. The clotting activity of chicken pepsin at low concentrations of CaC1 2 was inhibited more than that of calf rennet. This is most probably de to the high ph vales of the sbstrate at these concentrations of CaC1 2 (see Materials and Methods) which inhibited the activity of this enzyme. Dependence of coaglation time on milk temperatre. Since the enzymatic activity of proteases is temperatredependent, this parameter is of primary importance in

3 686 GORDIN AND ROSETHAL *"-X Calf Rennet..-.Chicken Pepsin x--x Calf Rennet -Chicken Pepsin > = - Fig. 5. ~--~--L---L---~--~--~----~ Milk Temperatre( C) Dependence on coaglation time on milk temperatre. ph Fig. 3. Dependence of coaglation time on milk acidity. 1 CD 8.3 ) E 1- C7> 6 c 4 Incbation Time (min) Fig. 6. Effect of incbation time on NPN. observed, which indicated an enhanced proteolytic activity nder these experimental conditions. Nitrogen loss in whey. The proteolytic activity of a coaglant in prodction of cheese is also expressed by the nitrogen loss to the whey. This parameter is a primary indicator of the rate of tilization of milk proteins in the final prodct, i.e., cheese. Or reslts indicate that the nitrogen losses in whey which was separated from crd nder identical experimental conditions for both enzymes, were virtally identical and nchanged with the incbation time (Fig. 7). *-X Calf Rennet -e Chicken Pepsin - Chicken Pepsin x---k Calf Rennet Fig. 4. cheese manfactring. Obviosly the gradient of temperatre cold be different for different enzymes. Reslts of these measrements (Fig. 5) indicates that the chicken pepsin was more temperatre-sensitive over the range of C. Proteolytic activity. The proteolytic activity of a coaglating enzyme can be estimated by release of a small nitrogen-containing fraction (NPN) from the protein. As shown in Fig. 6. the proteolytic activity of both enzymes in the first 15 min of incbation was similar. Sbseqently, in the chicken pepsin-treated soltions. an increase in the solble nitrogen release was z ~ Fig. 7. Nitrogen loss in whey. rjr--~t Incbation Time (min} 18

4 CHICKEN PEPSIN FOR COAGULATING MILK 687 Transfer of enzyme to whey. Development of the body and flavor in cheese depends in part on the activity of the coaglant sed. The residal coaglant in crd has an inflence on the ripening process and on cheese qality. To estimate the amont of residal enzyme in crd, the amont of enzyme transferred to the whey was determined. The reslts showed that ca. SOo/o of the initial chicken pepsin and 3o/o of the initial calf rennet activity were fond in the whey. We assme that the residal enzyme was retained in the crd. Cheesemaking The flnal criterion of performance for a rennet sbstitte is the qality of cheese made with it. Following laboratory experiments, it was evident that a sbstantial likelihood existed that chicken pepsin cold replace calf rennet in cheese prodction. The sitability of chicken pepsin was examined by preparing two kinds of representative cheeses, Emmental and Kashkaval-type, for which several parameters were compared dring the ripening period. Ths total solids, fat content, total nitrogen, solble nitrogen, free amino acids and ammonia were monitored at different time intervals, from the fifth day ntil the ninth month of ripening. In the Emmental-type cheese no differences cold be detected by these tests between cheese made with chicken pepsin or rennet. Most significant was the finding that no enhanced proteolytic decomposition was recorded for chicken pepsin, as reflected by comparison of solble nitrogen, free amino acids and ammonia analyses (Table 1). In Kashkaval-type cheese, the tendencies in changes of total solids (52-53 o/o), fat content (45-46 o/o), total nitrogen (8o/o), free amino acids and ammonia were also similar for the two enzymes (Table 2). However, the solble nitrogen measrements indicated a more enhanced proteolytic cleavage for chicken pepsin. Specifically, this parameter varied between.89 and 2.83 o/o, and.66 and 1. 9 o/o for cheese made with chicken pepsin or calf rennet, respectively. It is possible that this difference between the Kashkaval-type and Emmentaltype cheese was de to the lower water content of the latter. However, organoleptic tests showed no deftnite difference in taste, flavor or body textre between the cheeses. A bitter taste was not detectable in any of the samples tested. Attention to ph control and temperatre, together with a slight modification in the techniqe of cheesemaking, have enabled satisfactory prodction of many types of cheeses in Israel with chicken pepsin. All nripened soft cheeses and some of the ripened ones (Emmental, Kashkaval, Edam, Danbo-type) have been manfactred sccessflly sing this enzyme. Cheddar cheese made with chicken pepsin has been reported (3) to be of poor qality, having soft body, weak flavor and intense off-flavors which indicate extensive proteolysis. The difference between this reslt and the present stdy cold be de to the different processing conditions of the cheeses tested. Ths the processing of Emmental and Kashkaval-type cheeses reqires high temperatres of 52 and 85 C, respectively, while dring TABLE I. Compositional ana(yses ofemmental-type cheese made with di(f'erent clotting enzymesa Rennet Chicken pepsin Ripening time Solble Free Ammonia Solble Free Ammonia (days) nitrogen amino acids nitrogen amino acids , , athe percent of total solids which varied between 62-63o/oserved as the basis for calclations. The amont of fat was nchanged at 51%. TABLE 2. cheese made with Rennet Ripening time Solble Free Ammonia (days) nitrogen amino acids Chicken pepsin 5.66, as percent oftotal solids.

5 688 GORDIN AND ROSETHAL cheddaring the maximm temperatre achieved is only 38 C. (6). The heat treatment of the crd might partially inactivate the enzyme, ths affecting the degree of proteolysis dring ripening. ACKNOWLEDGMENTS The athors express their appreciation to Mr. H. Balaban and Mr. M. Frank of the Tnva Tel-Yosef Dairy for their valable help in the cheese prodciton; and to Miss Christine Navrot and Mrs. Solange Bernstein for their technical assistance. REFERENCES 1. Bohak. Z Prification and characterization of chicken pepsinogen and chicken pepsin. J. Bioi. Chern. 244: Ernstrom, C. A Milk-clottingenzymes and their action. In B. H. Webb, A. H. Johnson, J. A. Alford (eds.) Fndamentals of dairy chemistry. A vi. Pblishing Co., Westport, Connectict. 3. Green. M. L Assessment of swine, bovine and chicken pepsins as rennet sbstittes for Cheddar cheese-making. J. Dairy Res. 39: Green, M. L Review of the progress of dairy science: Milk coaglants. J. Dairy Res. 44: Gtfeld, M., and P. P. Rosenfld The soltion to Israel's rennet shortage. Dairy Indst. 4: Kosikowski, F. V Cheese and fermented milk foods, 2nd edition, Edwards Brothers, Inc., Ann Arbor, Michigan. 7. Ryle, A. P The porcine pepsins and pepsinogens. p In G. E. Perlman and L. Lorand (ed.) Methods in Enzymology, Vol. 19 Academic Press, New York. 8. Sardinas, J. L Calf rennet sbstittes. Process Biochem Vakaleris, D. G., and W. V. Price A rapid spectrophotometric method for measring cheese ripening. J. Dairy Sci. 42: