Effect of Acidulants and Milk-Clotting Enzymes on Yield, Sensory Quality, and Proteolysis of Pizza Cheese Made by Direct Acidification1, 2

Effect of Acidulants and Milk-Clotting Enzymes on Yield, Sensory Quality, and Proteolysis of Pizza Cheese Made by Direct Acidification 1, E. L. QUARNE, W. A. LARSON, AND N. F. OLSON Department of Food Science and Industries, University of Wisconsin, Madison Abstract

Various acidulants (hydrochloric, phosphoric, and lactic acids) and milk-clotting enzymes (pepsin, veal rennet, and a con> mercial fungal rennet) were used to make Pizza cheese by the direct acidificationcontinuous agitation procedure without added lactic starter cultures. Recovery of solids-not-fat was the highest in the finished cheese when phosphoric acid was used. The type of acid had no effect on recovery of fat. F a t and solids-not-fat recovery in cheese was not affected by the type of enzyme. Proteolysis, as measured by levels of nitrogen soluble at p H 4.4 and fornml nitrogen, in cheese during storage at 4 C was greatest in lots made with fungal rennet and least in those made with pepsin. Levels of nitrogen soluble in 12% trichloracetic acid were lowest in cheese made with veal rennet. Flavor grades after one week and one and two months of storage at 4 C were highest in cheese made with pepsin and lowest in cheese made with veal rennet. Cheese nmde with veal rennet was criticized for excessive bitterness after one month. Addition of acid to milk prior to cheese making was suggested in early work on manufacture of cheese from pasteurized milk and has been practiced traditionally in making Ricotta cheese (11, 13). The selection of acid has been based on the feasibility of its use, economics, and effect on the quality and composition of cheese. I n the manufacture of Cottage cheese by direct acidification, Ernstrom (9) stated that it was possible to produce satisfactory curd with various acids. Shehata et al. (21, 22) found that the type of acid used to acidify milk to p H 5.6 before coagulation with rennet in

direct acidification procedures had significant effects on the composition and characteristics of Blue and Pizza cheeses. Although the acid used in direct acidification procedures for Pizza cheese may affect the curd characteristics and rate of curd formation, coagulation of milk results from rennet action (2, 10). Veal rennet has been used irt all previous studies on this procedure. However, other milk coagulating enzymes should function satisfactorily under conditions of this system. The milk-clotting activity of pepsin has been shown to be higher than rennet at p t t levels below 6.4, if the milk-clotting activities were standardized to equal levels at the normal p H of milk (8, 11). Microbial rennet preparations have been tested and apparently are capable of coagulating milk to produce an acceptable cheese (17, 20, 23). The activity of certain of these preparations was increased by higher calcium and lower p H levels (17, 20). A n y substitute for veal rennet must not only coagulate milk but must have low proteolytic activity and produce cheese with acceptable flavor and rheological characteristics. Cheese made with pepsin has been criticized for retaining its curdiness longer during ripening than cheese made with veal rennet. This undoubtedly was caused by the lower levels of protein degradation in cheese made with pepsin (5, 14-16, 19). I t has been reported that microbial rennet preparations accelerated flavor and body development and proteolysis during ripening over that obtained with veal rennet (3, 17). I n cheese manufactured by direct acidification procedures, the agents responsible for changes in the curd during subsequent storage must be normal constituents of milk or enzymes used to coagulate the milk, since lactic starter cultures are not used and bacterial contamination during manufacture is minimal. Because of the importance of the coagulating enzyme used in direct acidification, the effects of different milk-clotting enzymes on proteolysis and sensory characteristics of cheese during ripening were determined. Effects of the acidu~ lant and enzyme on recovery of milk fat and solids-not-fat were determined also.

Received for publication January 26, 1968. 1Published with approval of the Director of the Agricultural Experiment Station, University of Wisconsin, Madison. This study was supported in part by a grant from the Cooperative State l~esearch Service, United States Department of Agriculture. 848

A C I D U L A N T S AND ~ I L K - C L O T T I N G

Experimental Procedures

Preparation of milk. Milk for cheese making was prepared by methods described previously (18), except that whole milk was homogenized at 35.2 kg/cm 2 for all lots. I n the study of acidulants, approximately 0.40, 0.50, and 0.24 nil, respectively, of 37.5% hydrochloric, 85% lactic, and 85% phosphoric acids were required to acidify 1.0 kg of milk to p H 5.6. I n the study of enzymes, hydrochloric acid was used to acidify the milk. Cheese making. Curd was made by the direct acidification continuous agitation method (18). Veal rennet was used in the study of acidulants, while fungal rennet (1), pepsin, and three different lots of veal rennet were used in studying milk-clotting enzymes. The levels of enzymes used in cheese making were standardized to give clotting times equivalent to that of a commercial single strength veal rennet, added at the rate of 10 ml per 45.4 kg o!~milk. Clotting times were determined in 2% fat milk at p i t 5.6, using Ernstrom's method (7). Preliminary experiments indicated that 7.5 ml of pepsin and 10 ml of fungal rennet were required per 45.4 kg milk to give clotting times equivalent to veal rennet. Lots of cheese, whey, and molding water were sampled and analyzed for fat and total solids and the data used to calculate recovery of fat and solids-not-fat (18). All lots of cheese were stored at 4 C. Sensory evaluation. Cheeses were graded at one week by one judge and at one month ~nd two months by a panel of three judges. Flavor and body were scored according to the scale of 1 to 6: 6, excellent, 5, desirable, 4, satisfactory, 3, slightly objectionable, 2, objectionable, and 1, unsalable. Proteolysis of cheese. Samples of cheese were prepared and analyzed at one week and two months for total nitrogen, nitrogen soluble at p H 4.4, and formol nitrogen according to the procedures of Vakaleris (24). An aliquot of the cheese slurry, used for determination of total nitrogen, was used for the determination of 12% trichloroacetic acid (TCA) soluble nitrogen. Results

Comparison of acidulants. Mean moisture levels in Pizza cheese made with phosphoric, hydrochloric, and lactic acids were 48.92, 50.04, and 51.21%, respectively. No attempts were made to modify manufacturing procedures to correct for these differences in moisture. Mean percentages of fat in the dry matter were 37.23,

849

ENZYMES

TABLE 1. Effect of different acidulants on recovery of milk fat and solids-not-fat with the direct acidification continuous agitation procedure using homogenized milk. ~ Acid Fraction Cheese Whey Molding water Total

Cheese Whey Molding water Total

ItC1

Lactic

H3PO~

- - ( % ]%covery of milk fat) b 89.44 89.66 89.34 6.93 6.22 5.12 4.32 5.16 4.74 100.69 101.04 99.20 (% Recovery of milk - - s o l i d s - n o t - f a t ) t,_ 31.21c 31.58c 32.10c 67.34 68.53 67.30 1.05 1.00 1.l 0 99.60 101.11 100.50

a Milk homogenized at 35.2 kg/cm :. b Means of three replicate trials. c Solids-not-fat of cheese does not include NaC1. 37.83, and 37.52%, respectively, in cheese made with phosphoric, hydrochloric, and lactic acid. The type of acid had no apparent effect on the handling properties of the curd during the stretching and molding operations. Table 1 indicates that fat recovery in cheese was not affected by the acidulant used in direct acidification procedure. However, recovery of solids-not-fat was greater in cheese made with phosphoric acid than that made with hydrochloric and lactic acids (Table 1). This difference was statistically significant (P ----0.01). The increase in solids-not-fat recovery may have been caused, in part, by increased retention of calcium in the curd (21). The differences in solids-not-fat recovery were reflected in cheese yields, since phosphoric acid also gave higher yields than other acids. Yields of 9.25, 9.34, and 9.39 kg/100 kg milk for hydrochloric, lactic, and phosphoric acids, respectively, were significantly different (P ~-- 0.05). Comparison of enzymes. Five milk-coagulating enzymes were compared, in two experimental series, for effects on fat and solids-notfat recovery, using a Randomized Complete Block design within each series (Table 2). Recovery of fat was higher in cheese made with veal rennet A or B, as compared to pepsin in Series 1, while recovery was higher in cheese made with pepsin than that made with veal rennet C or fungal rennet in Series 2. Recovery of solids-not-fat in cheese was fairly uniform between types of enzymes and was not correlated with variations in fat recovery. Differences in fat and solids-not-fat recovery in cheese were not statistically significant (P---0.05) between the types of enzymes within each J. DAIRY SCIENCE VOL. 51, NO. 6

850

QUARNE, LARSON, AND OLSON

TABLE 2. Recovery of milk f a t and solids-not-fat in the direct acidification continuous agitation procedure using different milk-clotting enzymes, a

TABLE 3. Distribution of nitrogen at one week and two months in cheese nmnufactured with pepsin, fungal rennet, and three different lots of veal rennet. ~

Enzyme Series I Fraction

Veal rennet A

Veal rennet B

Pepsin

Cheese Whey Molding water Total

- - ( % Recovery of milk f a t ) b--91.91 91.32 89.99 5.73 6.00 5.34 3.02 3.32 3.77 100.66 100.64 99.]0

Cheese Whey Molding water Total

( % Recovery of milk -solids-not-fat) b 32.90 33.79 33.41 67.20 67.41 66.29 0.88 0.96 0.94 100.98 102.16 100.64 Series 2 Veal rennet C

Fungal ~ennet

Pepsin

Cheese Whey Molding water Total

- - ( % Recovery of milk f a t ) k--85.86 86.30 88.84 8.05 8.32 6.27 5.29 5.01 4.85 99.20 99.63 99.96

Cheese Whey Molding water Total

( % Recovery of milk -solids-not-fat ) b 33.20 33.08 33.05 65.82 66.28 66.34 0.98 1.02 1.03 100.00 100.38 100.42

Milk homogenized at 35.2 k g / c m ~. b Means of three replicate trials. series. The lack of significant differences is n o t s u r p r i s i n g , since milk-clotting activities of the enzymes were standardized, time of coagulation in t h e v a t was similar, a n d the h a n d l i n g p r o cedure of the curd was the same. T h e r e were no a p p a r e n t differences in the rheological p r o p erties of curd, m a d e with the different enzymes, d u r i n g cheese making. Yields of cheese were n o t significantly different ( P = 0.05) between lots m a d e w i t h the different enzymes. Proteolysis of cheese. As shown in T a b l e 3, proteolysis was g r e a t e r in cheese m a d e with f u n g a l r e n n e t t h a n in cheese m a d e w i t h veal r e n n e t . The least proteolysis, except levels of trichloroacetic acid soluble nitrogen, occurred in cheese m a d e w i t h pepsin. R i p e n i n g f o r two m o n t h s e x a g g e r a t e d the differences between enzymes. The relative p r o t e o l y t i c activities of these c o a g u l a t i n g enzymes are in a g r e e m e n t w i t h p r e v i o u s r e p o r t s on cheese made b y t r a d i t i o n a l p r o c e d u r e s (3, 5, 16, 17, 19). Levels of soluble a n d f o r m o l n i t r o g e n ilJ. DAIRY SCIE~GE ~OL. 51, NO. 6

Enzyme

Total Nin cheese

TCA Solusoluble ble Formol N as N as N as per cent per cent per cent of total of total of total i~ N N

(%) Veal rennet A Veal rennet B Veal rennet C Pepsin Fungal rennet Veal rennet A Veal rennet B Veal rennet C Pepsin Fungal rennet

Cheese ripened one week 4.09 10.56 1.80 0.15 3.79 10.84 1.84 0.14 3.87 9.89 1.96 0.07 3.89 9.57 1.96 0.10 3.79 11.60 2.19 0.29 Cheese ripened two months 4.08 19.22 3.50 0.65 3.91 19.05 3.28 0.69 3.88 19.78 3.81 0.65 3.94 17.64 4.42 0.61 3.94 27.45 6.55 1.43

Data are means of three replicate trials, except pepsin data which are means of six replicates. l u s t r a t e effects of r e n n e t a n d lactic s t a r t e r cult u r e s on p r o t e i n breakdown. P e r c e n t a g e s of soluble n i t r o g e n a t one week a n d two m o n t h s of storage were sinfilar to those r e p o r t e d f o r C h e d d a r cheese m a d e b y the t r a d i t i o n a l m e t h od, while f o r m o l n i t r o g e n levels a n d r a t i o s of f o r m o l to soluble n i t r o g e n were m u c h lower (25, 26). The r a t i o o£ f o r m o l to soluble n i t r o gen was 0.19:1 f o r C h e d d a r cheese e x a m i n e d a t two m o n t h s of age b y V a k a l e r i s et al., b u t only 0.03:1 in P i z z a cheese stored f o r two m o n t h s i n this study. Similarly, the acid soluble n i t r o g e n was h i g h e r t h a n levels observed b y E m m o n s et el. (6) in C h e d d a r cheese, p r e s u m a b l y reflecting m o i s t u r e differences, b u t levels of trichloroacetic acid soluble a n d f o r m o l n i t r o g e n levels were m u c h lower in o u r cheese. The low trichloroacetic acid soluble a n d f o r m o l n i t r o g e n levels reflect the absence of lactic s t a r t e r cult u r e s in the e x p e r i m e n t a l P i z z a cheese, a n d suggest the i m p o r t a n c e of s t a r t e r s in d e g r a d i n g soluble n i t r o g e n f r a c t i o n s p r o d u c e d b y r e n n e t action in cheese (4, 6). Sensory evaluation. As indicated in Table 4, cheese m a d e w i t h p e p s i n or f u n g a l r e n n e t h a d h i g h e r flavor scores t h a n cheese made w i t h veal r e n n e t ; p e p s i n cheese was s u p e r i o r to f u n g a l r e n n e t cheese. Differences between flav o r scores of cheese made w i t h p e p s i n a n d t h a t m a d e w i t h r e n n e t p r e p a r a t i o n s increased w i t h r i p e n i n g , bitterness b e i n g the m a i n criticism of veal r e n n e t cheese. A second b a t c h of commercial p e p s i n also p r o d u c e d cheese which

ACIDULANTS

AND ~¢IILK-CLOTTING ENZYMES

TABLE 4. Flavor evaluation of cheese during ripenlng2 Enzyme

Score

Criticism

Veal rennet A Veal rennet B Veal rennet C Pepsin Fungal rennet

Cheese ripened one week 3.67 Very slightly bitter 3.00 Slightly bitter 3.50 Very slightly bitter 4.25 Slightly unclean 4.17 Slightly rancid

Veal rennet A Veal rennet B Veal rennet C Pepsin Fungal rennet

Cheese ripened one month 3.50 Slightly bitter 2.67 Bitter 2.33 Bitter 4.58 Slightly unclean 4.00 Very slightly bitter

Veal rennet A Veal rennet B Veal rennet C Pepsin Fungal rennet

Cheese ripened two months 2.67 Bitter 2.83 Bitter 2.67 Bitter 4.20 Slightly unclean 3.30 Slightly bitter

a Scores are means of three replicates, except pepsin scores which are means of six replicates. scored higher than cheese made with veal rennet. Cheese made with both batches of pepsin was relatively bland in flavor. There were no clear-cut relationships between flavor scores and proteolysis in cheese made with the various enzymes. Levels of acid soluble nitrogen in cheese made with veal rennet, which were bitter, were intermediate between those of cheese made with pepsin and fungal rennet. ttowever, cheese made with f u n g a l rennet and pepsin contained higher levels of trichloroacetic acid soluble nitrogen than lots made with veal rennet a f t e r two months of ripening. A t this time, ratios of trichloroaeetic acid soluble to soluble nitrogen were similar f o r cheese made with pepsin and fungal rennet, and were higher than ratios for cheese made with veal rennet. The ratios of the lots made with veal rennet also fell within a narrow range. Since it has been shown that bitterness in Cheddar cheese can be caused by the inability of certain strains of lactic streptococci to hydrolyze certain peptides, it is possible that the lack of bitterness in pepsin and f u n g a l rennet cheese resulted f r o m more extensive hydrolysis of the soluble paracasein by these enzymes. However, the lack of correlation between bitterness and levels of formol nitrogen and ratios of formol to trichloroacetie acid soluble nitrogen suggests qualitative differences between the proteolytic action of these enzymes (4, 12). The possible development of bitterness in cheese made by direct acidification without added lactic starter cultures must be considered if veal rennet is

851

used and the cheese is stored f o r several months. However, this should not pose any p a r t i c u l a r problem with Pizza cheese made by direct acidification, since this cheese is normally used fresh or within a few weeks a f t e r manufacture. E v a l u a t i o n of body of cheese indicated more variation in body scores within each type than between types of enzymes. Curdiness at two months of age correlated with protein breakdown. Cheese made with f u n g a l rennet had the highest acid soluble nitrogen content and was least curdy, while cheese made with pepsin had the lowest level of soluble nitrogen and exhibited the most curdiness. References (1) Arima, K., and S. Iwasaki. 1964. Milk Coagulating Enzyme "Microbial Rennet" and Method of Preparation Thereof. U.S. Pa±. 3,151,039. (2) Breene, W. M., W. V. Price, and C. A. Ernstrom. 1964. Manufacture of Pizza cheese without starter. J. Dairy Sci., 47: 1173. (3) Chakravorty, S. C., R. A. Srinivasan, I. J. Babbar, A. T. Dudaa~i, S. D. Burde, and K. K. Iya. 1966. Comparative study of proteolysis during ripening of Cheddar cheese by bacterial milk clotting enzymes and rennet extract. Proc. X V I I t h Intern. Dairy Congr., D: 187. (4) Czulak, J., and P. D. Shimmin. 1961. Further notes on bitter flavor in cheese. Australian J. Dairy Technol., 16: 96. (5) Davies, W. L., J. G. Davis, D. V. Deardon, and A. T. R. Ma.ttick. 1934. Studies in Cheddar cheese. I I I . The role of rennin pepsin, and lactobacilli. J. Dairy Res., 5: 144. (6) Emmons, D. B., W. A. MeGugan, J. A. Elliot, and Pamela M. Morse. 1962. Effect of strain of starter eultm'e and of manufacturing procedure on bitterness and protein breakdown in Cheddar cheese. J. Dairy Sei., 45: 332. (7) Ernstrom, C. A. 1958. Heterogeneity of crystalline rennin. J. Dairy Sci., 41: 1663. (8) Ernstrom, C. A. 1961. Milk clotting activity of pepsin and rennet. Milk Prod. J., 52(5): 8. (9) Ernstrom, C. A. 1965. Continuous Cottage cheese manufacture without culture. Milk Ind., 56(1) • 36. (10) Ernstrom, C. A. 1965. Mechanized "Pizza cheese" making--by means of a continuous, direct acidification method. Mfd. Milk Prod. J., 57(7) : 7. (11) Ernstrom, C. A., and R.. P. Tittsler. 1966. ]~ennhl action and cheese chemistry, p. 590. I n Fundamentals of DMry Chemistry. Webb, B. It., and Johnson, A. I-I,, eds. AVI Publishing Co., Inc., Westport, Conn. (12) Foltmann, B. 1966. A review on proren5. DAIRY SCIENCE VOL. 51, NO. 6

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(13)

(14)

(15)

(16)

(17)

(18)

(19)

QUARNE, LARSON, AND OLSON nin and rennin. Compt. rend. tray. lab. Carlsberg, 35: 143. Kosikowski, F. V. 1966. Cheese and Fermented Milk Foods. Edwards Brothers, Inc., Ann Arbor, Michigan. Linklater, P. M., and C. A. Ernstrom. 1961. Crystalline rennin in the manufacture of Cheddar cheese. J. Dairy Sci., 44: 1621. Maragoudakis, M. E., J. O. Young, and R. W. Stein. 1961. Use of rennet and a rennet-like substitute in Cheddar cheese manufacture. J. Dairy Sci., 44: 2339. Melachouris, N. P., anl S. L. Tuckey. 1964. Comparison of the proteolysis produced by rennet extract and the pepsin preparation Metroclot during ripening of Cheddar cheese. J. Dairy Sci., 47:1. Melachouris, N. P., and S. L. Tuckey. 1967. Properties of a milk-clotting microbial enzyme. J. Dairy Sci., 50:943. Quarne, E. L., W. L. Larson, and N. F. Olson. 1968. Recovery of milk solids in direct acidification and traditional procedures of manufacturing Pizza cheese. J. Dairy Sei., 51: 527. Raadsveld, C. W. 1964. Use of rennet and pepsin in cheesemaking. Misset's Zuivel, 70: 51. [Dairy Sci. Abstr., 1964, Abstr. 1856.]

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(20) Richardson, G. H., J. H. Nelson, 1~. E. Lubnow, and R. L. Schworberg. 1967. Renninlike enzyme from Mucor pusillus for cheese manufacture. J. Dairy Sci., 50:1066. (21) Shehata, A. E., Meena Iyer, N. F. Olson, anl T. Richardson. 1967. Effect of type of acid used in direct acidification procedures on moisture, firmness, and calcium levels of cheese. J. Dairy Sci., 50: 824. (22) Shehata, A. E., and N. F. Olson. 1966. Manufacture of Blue cheese by direct acidification methods. J. Dairy Sci., 49: 1025. (23) Shovers, J., and V. S. Bavisotto. 1967. Fermentation-derived enzyme substitute for animal rennet. J. Dairy Sci., 50: 942. (24) Vakaleris, D. G. 1955. A study of formol titration as a measure of changes in protein during cheese ripening. M.S. thesis, University of Wisconsin, Madison. (25) Vakaleris, D. G., N. F. Olson, W. V. Price, and S. G. Knight. 1960. A study of the ripening of Dariwold and Cheddar cheese with special emphasis on proteolysis. J. Dairy Sci., 43: 1058. (26) Vakaleris, D. G., and W. V. Price. 1959. A rapid spectrophotometric method for measuring cheese ripening. J. Dairy Sci., 43 : 264.