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Rennin-like Enzyme from Mucor pusillus for Cheese Manufacture
Rennin-like Enzyme from Mucor pusillus for Cheese Manufacture G. N. RICHARDSON, J. H. NELSON, R. E. LUBNOW, and R. L. SCHWARBERG Dairyland Food Laboratories, Inc., Waukesha, Wisconsin Abstract
A fungal rennet produced h'om 3[ucor pusillus is satisfactory for cheese manufacture. Enzyme stability in powder and in saline solution is excellent. However, severe activity losses occur when blended and stored with veal rennet in liquid form. Coagulation by fungal rennet is more sensitive to high pH than veal remwt, but less sensitive than swine pepsin. Coagulation by fungal rennet is more affected by Cathan veal rennet. Fungal rennet produces a greater nonprotein-nitrogen increase in casein solution and in cheese than pepsin or rennet. Higher lipase activity is also present, suggesting advantages in acceleration of curing. Normal clotting time and curd tension can be obtained through adjustment of: rennin units in coagulant, amount of Ca ++ in milk, temperature or time of setting, or blending with veal rennet. Normal Brick, Cheddar, Pizza, and Parmesan cheeses have been made with fungal rennet. Solids losses in whey suggest careful control of factors affecting curd tension and use of a coagulant blend containing predominantly veal rennet. Durhlg the decade ending in 1965, United States cheese production increased 25% while calf slaughter decreased 40% (17, 1S). The supply of rennet extract prepared from the fourth stomach of the calf has, therefore, become critical. Swine pepsin is being used extensively and recommended as a partial replacement for veal rennet (11). Pepsin is not an ideal coagulant, because of its sensitivity to milk p H changes (11). Other coagulants, preferably of microbial origin, are continually being investigated. Knight (15) has screened numerous molds for rennin-like enzyme production and B.~/~sochlamys /'~dva was selected for continued research. The Food and Drug Administration has been petitioned (12) for clearance to use an enzyme produced by the fungus E~d(~tl~i(~ ])arasitic(~. The F D A has also been petitioned for
a change in the cheese standards (13) to include additional safe milk clotting enzymes. Meito Sangyo Kabushiki Kaisha, Nagoya, Japan, has produced a fungal rennet patented by Arima and hvasaki (4, 5). The enzyme is produced by Mucor p~silh~s vat. Liudt~ cultured by a wheat bran koji process. Pilot quantities of improved product have become available. This paper summarizes research conducted over several years in our laboratories during development of this product. Experimental Procedures
F~l~g~d r e ~ e t sol~ltio~s. A 17.8 B6 brine containing 2% propylene glycol, 0.1% sodium 1)cnzoate, and 0.25% potassium sorbate was used to dissolve fungal rennet powder. This provided an extract analogous to rennet extract, except coh)r and flavor were omitted. I~e~nd~ actirity. Rennin activity was determined as described by Ernstrom (9), except that a) Dairyland Food Laboratories, Inc.'s liquid rennet standard was selected as containing 100 remfin milts (RU) per milliliter. b) lfl0g n(mfat dry milk (ND~[) was dissolved in 500 nfl of distilled water containing 0.5 mi of 33% CaCI~" 2 H~O solution, and e) 50-ml pnrtions of substrate were used in 125-ml bottlea. pH. pH of fungal powders was measured on a l g 30 ml water dilution. pr,)tcolysis. Enzyme proteolysis was determined by measuring nonprotein nitrogen (NPN) increase during incubation in casein substrate (9). A 2~
solution of soluble casein (Sheftene
C-2) ~ was adjusted to p H 6.5 with 1.0 _x NaOH. Enzyme dilutions of rennet, pepsin, and fungal rmmet were standardized to contain 75 R U / milliliter. Trypsin solution was also prepared; however, it did not forln a clot that could be used to standardize RU. One milliliter of pH 6.5 enzyme solution was injected into a tube containing 10 ml of casein substrate. The mixture was incubated at 30 C. The reaction mixtures were added to 11 nd 25% triehloroacetic acid (TCA) at zero time and at intervals up to 60 rain, filtered, and analyzed for NPN.
Received for publication Fel)rm~ry 2, 1967. Present address: Topco Associates, Inc., Skokie, Illinois. ~ Sheffield Chemical, Norwich, New York. 1066
FUNGAL
Miero-Kjeldaht (14) was used for NPN analysis. The assay was modified for interIaboratmscomparison (10). A 6.75% casein solution was prepared by adding 6.75 g casein to 75 ml water. Casein solution was affected by adding 3.4 nfl 1.0 5r NaOH and mixing. The solution was adjusted to pH 6.6 and made up to 100 ml. The substrate comprised 20 ml of casein solution and 20 ml of 0.1 5i phosphate buffer at p H 6.6. After the substrate was tempered to 30 C, a 3-ml aliquot was removed for the blank determination and injected into a tube containing 3 ml of 24% TCA. After filtration through Whatman no. 42 filter paper, the filtrate was analyzed for NPN content. The remaining 37 ml of substrate was treated with enzyme. The volume of enzyme inocula was varied. Four milliliters of 75 RU/milliliter were inoculated in one series. This was the level used in the 2% casein study and amounted to 8.1 R U / ml of substrate. I n another series, 0.1 ml of 1 7 R U / m l solution was inoculated which resulted in 0.046 RU/nfl of substrate. Three milliliters of substrate-enzyme blend were removed, treated with TCA, and filtered at 10-, 30-, and 60-rain intervals. Results are expressed as increase in per cent NPN. Lipase ~wtivity. Lipase activity was measured with 5 ml of' tributyrhl substrate (16) in a Sargent recording p H stat. The instrmnent was charged with 0.025 ~- NaOH and set at pH 6.20, 42 C, stirrer at 8, and 0.1-inch per minute chart speed. One milliliter of a 0.01 nfl dilution of enzyme preparation is inoculated into the substrate. One lipase unit is defined as 0.025 of chart width, or 0.01 ml base. Storage stability. Blends of fungal and veal rennet solutions were held at various pH values and at 4, 38, and 60 C to determine inactivation during storage. Keeping quality of dLw powder and unmixed solutions was also assessed. Curd tensioJ~. A rennet curd tension test (6, 7) was modified to measm'e quality of fung'al rennet clots. The Cheno--Burrell 1)rocedure (7) was followed, except 15% NDS[ substratea were used. p i t and Ca ++ content of the snhstrate was modified by adding NaOH, HC1, or CaCl~ solutions. Controls were adiustcd t() equal volumes with water. Microbiological co,~ditio~, a~d to.,'icit]j. Outside laboratories evaluated products for standard plate count, yeast and uu)ld count, and coliform count with standard procedures (1). Sahnonella (2), aflatoxin (3), and acute oral LD:~ (8) tests also were run.
RENNET
1067
E.,-t)toratory cheese making trials. Parmesan~ Cheddar, and Brick cheese were made in our labm'atorics, in 45.4-kg pilot plant cheese vats, using hig'h quality whole milk and accepted industry procedures. The cheeses were examined organoleptically by a trained flavor panel. They were also evaluated for gross composition and NPN differences. Pasta Filata cheese was produced in larger quantities and evaluated for curd handling properties. Results and Discussion
I. (;~'~cr(d characteristics of .f~gal renuet. Fungal rennet, as received, is a light grey to 1)urple-grey powder. Solutions of fungal rennet are brown, bland in taste, and have a slight, 1)leasant vegetable-like aroma. The solutions are darker than commercial rennet extract which, generally, has caramel coloring added during standardization. Absorbance readings were made at 570 m/x on equal RU/milliliter ~olutions of commercial strength veal rennet, fungal rennet, and mixtures of the two. Using a 10-ram cuvette, readings were 0.23, 0.41, 0.88, and 1 . $ 2 0 D for remmt, 10% fungal rennet, 45~ fungaI rennet, and 100% fungal rennet sample~, respectively. This severe color differential is not a problem in cheese making. Upon dilutiom the color intensity is decreased sufficiently to prevent color modification of the finished cheese. Early samples of fungal rennet had a p H of 6.6 to 6.8 and tested 330-380 RU/g. Recent shipments range in p t I from 5.0 to 5.6 and exceed 1,()00 R U,/g. ,~ol~lbility. Fungal rennet powders have improved significantly in solubility, wettability, and sedimentation characteristics. Initial powders were slow to hydrate, were gummy, viscous, and contained large particles of sediment. Cellulase treatment significantly reduced viscosity and ~edimentation; however, refinements of the bast(" enzyme production process have precluded need t'~r such treatment. It is practical to prepare fungal rennet extract with 100% market strength (75-80 RU/%l). Solution over 2~)0(~c strength is not practical in extract analogous to rennet solution because of solubility limitations. D~gr,~e o./I proteolysis. Any substitute for remwt nmst not only clot milk but must have low proteolytic activity. The high proteolytie activity of ttTpsin, papain, and ninny other coagulants prevents their use in cheese making. Fungal rennet solution is compared with t13"psin, pepsin, and rennet in Figure 1. Ten milli.T. ])AIRY SCIENCE ~'OL. 50, NO. 7
]068
R I C H A R D S O N , NELSON, LUBNO~,V, AND S C H W A R B E R G
liters of a 2% casein solution substrate was injected with 1 ml of 75 RU/ml coagulant, except that trypsin activity was only estimated. Lack of adequate clot formation and rapid proteolysis prevented standardization.
I
50
e- TRYPSIN •
A-FUNGAL
,.<, ¢y-40 /
o-RENNET
z
z rl Z
RENNET
"-PEPSIN
3o
/ 2O
I0
/
I0
20
30 4 TIME (MIN.)
SO
60
Fro. 1. Per cent NPN increase due to casein breakdown by various coagulants. The trypsin curve is typical of those enzymes which have undesirable proteolytic activity. Pepsin and rennet curves were below 3% NPN after 60 rain. Fungal rennet curves on an early sample (1963) and a recent cmnmercial shipment (1966) were above 10% NPN. Fungal rennet is thus more proteolytic under these conditions. The procedure was modified to allow comparison with another laboratoKv (10. When 0.046 R U / m l substrate was inoculated instead of the 8.1 RU/ml as used above, the fungal had a slightly lower % NPN than rennet. After 60 rain incubation, the NPN was 0.93% for rennet and 0.78% for fungal rennet. Simultaneous runs made using 8.1 RU/ml substrate had 0.37 and 11.94% NPN values for rennet and fungal, respectively. Differences in the degree of proteolysis thus do not become apparent until higher levels of iuoculum are used. This finding may be of value in determining the presence of fmlgal rennet in certain coagulant blends. The significance of' this difference in NPN increase will be discussed in a subsequent paper. Lipase activity. Lipolytic activity in fungal rennet powder was compared to rennet powder and kid pregastric esterase powder (CAPALASE Lipase Powder K~). The latter repreDairyland Food Laboratories, Inc., Waukesha, Wisconsin. J. DAIRY SCIENCE VOL. 50, NO. 7
sents a family of products used to produce lipolytic flavors in Italian cheese varieties. The rennet powders tested 1,000 RU/g. No rennin aeti~4ty was present in the pregastric esterase preparation. One gram of each powder was rehydrated in 75 ml water, mixed for 20 rain, and made up to 100 ml. One milliliter was inoculated into 5 ml tributyrin substrate and the sh)pe on the recorder indicated the reaction rate. Lipase activity was 0.23, 1.80, and 8.90 units per rain for rennet, fungal rennet, and standardized pregastric esterase preparation, respectively. There is some evidence of gastric esterase being present in rennet extract. The higher lipase activity in fungal rennet may be potentially advantageous in accelerating cheese curing. Organoleptic evaluation of cheese has not revealed any off flavor attributable to lipolysis. Pasta Filata cheese aged for three months showed no significant difference in amount of extractable free fatty acids. Thus, additional work is necessary to establish the significance of the higher lipase activity present. Storage stability. Activity retention of 1,000 RU/g fungal rennet powder is excellent. No losses have occmTed in eight months of storage at 21 C Keeping quality data on liquid products at 4, 22, and 38 C are included in Table 1. Liquid fungal rennet solutions at p H 5.75 were nmre stable than rennet or pepsin extracts. Liquid blends were not as stable as the unmixed enzyme solutions. Blends of fungal rennet and rennet lost more activity than pepsin-rennet blends. The blend containing the higher percentage of rennet lost the most activity. At 4 C the blend with the higher level of fungal rennet experienced a greater activity loss than the lower level. This was not found at higher temperatures. These results suggest that fungal rennet destroys veal rennet activity. Solutions of coagulants were heated to 60 C for three hours at different pH values to determine optimum p i t range for storage stability. Rennet has been found most stable at pH 5.5 to 6.2 (11). Comparisons were run as summaTABLE 1 Storage stability of liquid rennet, fungaJ rennet, pepsin, and blends after 4 wk at pH 5.75. Enzyme Rennet Pepsin (pH 4.3) 50/50 Rennet-pepsin blend Funga] rennet 55/45 t/ennet-fungal blend 90/10 Rennet-fungal blend
5~ Activity loss at 4C oo C 38 C < 1 < 1 ] ~ 1 22 6
5 6 1 47 54
14 44 49 13 60 80
FUNGAL RENNET
rized in Table 2-A. Fungal remwt stahility is better than rennet in the p H range evaluated. The coagulants were most stable at p H 5.25. The range of p H from 5.25 to 5.75 was optimmu dm'ing storage (Table 2-A). However, the magnitude of the losses preclude liquid blending of these enzymes. TABLE 2 Activity losses of coagulants at various pH values
1069
II. Actirily o.f fungal rennet upon milk. E!Dx't o]" milk pH. Pepsin coagulant is ]ess active in milk with high p H (11). This is generally overcome by blending with rennet, to restore control over clotting time. Fungal rennet was compared to rennet and pepsin in milks at various p H levels. Figure 2 compares clotting time from pH 6.0 to 6.8. Fungal rennet is less affected by high p H than is pepsin.
% Activity losses
After 3 hr at 60 C
pH a.* 4.75 5.25
5.75 6.25
55/45 RennetFungal fungal Rennet rennet blend (%) 32 13 25 20 12 20 33 13 28 63 16 39
After 2 wk at 38 C 55/45 ]~elmetfungal blen .d --(~)-53 51 51 54
4.75 62 58 58 5.25 36 76 51 5.75 70 94 62 6.25 93 93 95 * = Solutions containing sodium chloride, propylerie glycol, sodium benzoate, and potassimn sorbate. ** -Solutions prepared in distilled water. Solutions of 80 R U / m l coaguhmts were prepared in distilled water to determine how the results reported in Table 2-A were modified by the absence of preservatives. Results of heat treatment of these solutions are recorded in Table 2-B. Greater losses occurred. Rennet (prepared from 512 R U / g powder) was more stable than fungal rennet. This may partially be attributable to the sodium chloride and gelatin used in preparation of rennet powder. The severe losses in distilled water emphasize the value of the additives in solutions of fungal rennet and rennet. Mic~'obiologieal conditions. Early samples of fungal rennet contained hacterial contaminants; however, recent shipments have had low bacterial counts. A recent Standard Plate Count survey found commercial 100% rennet extracts less than 3,000 per nil. Recent shiplnents of 1,000 R U / g fungal rennet tested less than 3,000 per g. No sahnonella were present in 50 g (2) ; counts for yeast, molds, and eoliforms were less than 10/g. Toxicity. Fungal rennet has been found to be free from aflatoxin (3). Acute oral LD~ tests on rats indicate a value in excess of 20 g / k g of body weight. The product, therefore, qualifies as nontoxic orally within the meaning of the Federal IIazardous Substances Labeling Act (S).
O
2O
/
m-FUNGAL RENNET
Z
15
L9 I0 Z
I--
o-PEPSIN
o,._1 5 L#
/
o-RENNET
/'e
//-
.....___.~..,~/// i
6.0
I
i
6.2 6.4 6.6 SUBSTRATE pH
I
6.8
FIe.. 2. Ilennin test substrate clotting time at different pit values, using coagulants adjusted to equal RU/ml content at pit 6.4. Cm'd tension analyses were run in 15% reconstituted NDM, using each coagulant standardized to constant clotting strength at p i i 6.4. Curd tensions were rennet > fungal rennet > pepsin. These differences were most evident between milks at p I I 6.6 to 6.8. E]]'ect of adding Ca +'. Fungal rennet solutions standardized with the standard rennet assay appeared to be weaker when tested in cheese milk. This was found to be due partly to the concentration of Ca ++ present. CaCI~ is added to the test substrate (9). Fungal rennet is more affected by calcium, as shown in Figure 3. Thus, the fungal rennet test may be weaker with no added Ca*" but stronger with added Ca *+. These findings suggest the necessity to standardize fungal rennet strength higher than rennet. Under our laboratory test conditions 10-12~ higher test is warranted. Addition of 0.015% CaCI.~ to cheese milk has been found helpful in restoring normal curd tension. In a simulated cheese making trial rum'-liter aliquots of milk were set, using varied temperatures, coagulant, and CaCI~ addition. Subjective observations, such as the cheese maker uses, were made to deternfine flake time and cutting time. Results are shown in Table 3. J. DAIRY SCIENCE rOB. 50, NO.
1070
RICHARDSON,
140
NELSON,
LUBNO\V,
f u n g a l r e n n e t was used. Based u p o n interp o l a t i o n o£ this data it would take a b o u t 1 1 3 % of f u n g a l r e n n e t to produce a clot c o m p a r a b l e to 100% rennet. This value also agrees with the conclusion reached in the Ca** study. However, h i g h e r levels of f u n g a l r e n n e t have not always i m p r o v e d curd tension characteristics d u r i n g field trials. E.!~'ect of s~bstrate temperat~lre upon curd ten.,'io~L W h e n milk s e t t i n g t e m p e r a t u r e was varied f r o m 31.1. to 33.3 C, the em'd tension increased, as shown in Fig. 5. F u n g a l r e n n e t r e s p o n d s to t e m p e r a t u r e increase e o m p a r a b l y to rennet. However, as setting t e m p e r a t u r e s are increased ( u p to 38 C), the f u n g a l r e n n e t clots tend to be progressively weaker t h a n veal r e n n e t values. I t is evident t h a t curd tension can be modified with a d j u s t m e n t of setting t e m p e r a t u r e .
o FUNGAL RENNET • - RENNET 0
120
I00
•
I--
~ 8o LU 60
*
r'r"
<
0.. 0.. <
SCHWARBERG
AND
40
20.
200 '
400 ~
600 ~ PPM
800 ' CACI2
I000
FIG. 3. Apparent rennin test as affected by added ealelum. I n some cases the f u n g a t r e n n e t curd quality was i n f e r i o r to rennet, even following calcium a n d test a d j u s t m e n t . Effect of time upoJ~ curd te~sioa. C u r d tension evaluations in 1 5 % N D M were r u n over a p e r i o d o£ 20-60 rain. E n z y m e dilutions cont a i n e d equal RU, as d e t e r m i n e d on r e n n i n test substrate. F u n g a l r e n n e t trailed b e h i n d r e n n e t a t all points, as shown in Fig'. 4. Since f u n g a l r e n n e t d a t a tend to p a r a l l e l rennet, one method of overcoming curd tension differences is to allow a l o n g e r set time. Effect o]" hwreased inocuMm ~po~ c~H'd te~> sion. A n o t h e r method o£ increasing curd tension in f u n g a l r e n n e t clots is to increase the coagulant. C o a g u l a n t dilutions with equal clotting s t r e n g t h on test s u b s t r a t e were added in 1-ml inc r e m e n t s f r o m 1-5 nil; the r e s u l t a n t curd tension is recorded in Table 4. Curd tension reached a m a x i m u m of a b o u t 100 g w h e n either r e n n e t or
•- RENNET .,///o o-FUNGAL R E N N ~ o ~ / /
120
.S
© ~100 z 8O 0
C3 or"
4-o 0
20
30 40 50 TIME (MIN.)
60
Fie. 4. Curd tension as affected by time of cutting following coagulant inoculation.
TABLE 3 Curd formation during simulated clwese making trials Flake tinle
Setting temp 31 C 32 C
~(eat No calcium 0.015% CaCI~ No calcium 0.015% CaCI,
*Very fine flake evident. **Definite flake e~Sdent. ***Better than veal counterpart. g. DAIRy SCIEXCE ¥OL. 50, No. 7
Fun g al
---(n~h~;-11 14" 19 ~* 10 13 11 12 ~ 13"* 10 11
Time ready to cut
Veal
---(min)---40 30 40 30
Fu n gal 40 40 40 30 * ~
YUNGAL
RENNET
TABLE 4 Effect of increasing amount of coagulant upon curd tension
% of Usual inoculum
nfl of 2/100 Dilution
1
50
43 76 98 97 99
2 3 4 5
150 200 250
9°I
• RENNET
~
8O
o FUNGAL
/ ~
RENNET
COAGULANTBLEND o o PEPSINI RENNET Q, elI2%FUNGALI REN.
i~81
Fnngat rennet
Rennet
~
20~
tension
Curd
(g) 100
j
1071
a
AI24%
/
I O
Z
23 71 91 98 100
i
~-14-
10-
/ / / /
b
7C
2'0
4'0
6'0
8'o
60
% RENNET
Z
Fro. 6. Per cent rennet addition necessary to 1)]eJ~d with pepsin and with fungal rennet, to restore clotting time in ptI 6.5 raw whole milk substrate containing 2c~ added lactic culture. (Fungal renm,t standardized 12 and 24 RU/ml higher than rem~et on rennin test substrate.)
0 ~n 6C Z ,,i I--
L) 40 3O i
Si.I
i
i
32.2 TEMR (*C.)
I
33.3
Fro. 5. Effect of substrate settil~g temperature upon curd tension. Analyzed 30 rain after setting.
Effect of blending .reJ~net sol~tio~,~ ~po~ clottbtg time. Coagulant blends containing pepsin and rennet routinely contain at least 25c~ rennet, to control clotting time (11). To evaluate what percentage of rennet would restore clotting time to a coagulant blend, mixtures of rennet with pepsin and with fungal rennet were prepared. They were blended just prior to testing, to mininfize keeping quality losses. Rennet and pepsin eoag'ulants were adjusted to 100 R U / m l on test milk substrate. Two fungal rennet solutions were adjusted to contain 112 and 124 RU/nfl, respectively. Clotting time was determined on raw whole milk containing 2% added lactic culture. This produced a p H 6.5 substrate resembling cheese milk at time of coagulant addition. Results are shown in Figure 6. None of the blends restored clotting time equal to 100~}
rennet. The blends containing the highest fungal rennet test had the shortest clotting times. An increase in the proportion of veal rennet in rennet-pepsin blends reduced the clotting times more sharply than analogous proportions of rennet in either of the rennet-fungal rennet blends. Therefore, to maintain normal clotting times, a higher proportion of rennet is required in a rennet-pepsin blend than in a remtet-fungal blend. These observations suggest the need to use a different test substrate for fungal rennet rather than the high solids ND~I, CaCI~ fortified produet. An unfortified whole or skinunilk adjusted to pH 0.5 is suggested. E,rploratory c]~eese makh~g trials. Pilot plant quantities of Cheddar, Brick, and Parmesan cheese were manufactured from high quality whole milk. An increase in whey fat when fung'al rennet was used has prompted additional yield studies under commercial conditions. Onemonth-old Brick and ten-months-old Parmesan cheese samples were of marketable quality. The minor defects observed could not be attributed to fungal rennet. Cheddar cheese appeared normal at two and five months. At 14 months, the Cheddar samples were slightly bitter. Samples made with fnngal rennet were more bitter than the control. The N P N value for Cheddar using rennet was 83.8%, compared with 42.0 J. DAIRY SCINNCE VOL. 50, NO. 7
1072
R I C H A R D S O N , NELSON, LUBNO\V, AND S C H W A R B E R G
a n d 35.5% f o r two lots made f r o m f u n g a l rennet. A t e n d e n c y f o r h i g h e r N P N values in f u n g a l r e n n e t cheese has been established in s u b s e q u e n t work. F u l l cream P a s t a F i l a t a cheese curd was p r e p a r e d w i t h s t a n d a r d procedures. To evaluate curd m o l d i n g a n d s t r e t c h i n g p r o p e r t i e s of p r o d u c t made with f u n g a l rennet, vats were set using all f u n g a l rennet, veal, a n d 10/90, 4 5 / 5 5 blends of these coagulants. E x p e r i e n c e d I t a l i a n cheese makers j u d g e d curd quality u s i n g as c r i t e r i a : m a t t i n g characteristics, hot iron test, molding characteristics, a n d f a t losses in whey. All blends behaved n o r m a l l y in evehw respect. The v a t p r e p a r e d f r o m 100% funo'al r a n 8 rain b e h i n d in total make time, due to a slower flake a n d set tinm. No a t t e m p t h a d been m a d e to a d j u s t nfilk conditions to reduce these differences. Curd w o r k i n g p r o p e r t i e s were n o r m a l in all vats. F a t loss in the all f u n g a l r e n n e t v a t was 0.42%, instead of the n o r m a l 0.30c~. A p p a r ently, this was related to the slow ~et a n d curd fragility.
Acknowledgment Tile authors express their gratitude to Dr. C. A. Ernstrom for his valuable suggestions, to Dawes Laboratories, Inc. for their cooperation and for providing the initial samples of fungal rennet, and to Mrs. H. Beikerdffe, techuieian.
References (1) American Public Health Association. 1961. Standard Methods for the Exanfination of Dairy Products. 11th ed. Public Itealth Ass., New York. (2) Angelotti, tt., Crane, A., Hunter, J. E., Klusmeyer, P. W., Huston, M. L., Lamprech, E. D., and Silliker, J. H. 1966. Recomnlendations for Sampling aa~d Laboratory Analysis of Eggs, Egg Products, and Prepared Mixes. Food Technol., 2 0 ( 4 ) : 121. (3) Anonymous. 1966. 25 Nuts and Nut Produets. J. Ass. Offie. Agr. Chemists, 49: 229.
J. DAIRY SCIENCE VOL. 50, NO. 7
(4) Arima, K., and Iwasaki, S. 1964. Milk Coagulating Enzyme "Microbial Rennet" and Method of Preparation Thereof. U. S. Pat. 3,151,039. (5) Arima, K., and Iwasaki, S. 1965. Process for Making Cheese. U. S. Pat. 3,212,905. (6) Babcock, C. J., Chambers, L. A., Flora, C. C., ttull, M. E., Mueller, W. S., Sommer, H. H., Storrs, A. B., and Doan, F. J., Chairman. 1941. Einal Report of Committee on Methods of Deternfining the Curd Tension of Milk. J. Dairy Sci., 24: 825. (7) Cherry-Burrell Corp. ]955. Bull. 1P5132-M. Chicago, Ill. (~) Code of Federal Regulations, P a r t 1 9 1 Hazardous Suhstanees: Definitions and Procedural and Interpretive Regulation. (9) Ernstrom, C. A. 1958. Heterogeneity of Cr.vstalline Remlin. J. Dairy Sci., 41: 1663. (lo) Ernstrom, C. A. 1966. Personal comnmnieation. ('11) Ernstrom, C. A., and Tittsler, R. P. ]966. Rennin Action and Cheese Chenlistry. pp. 592-610. I ~ Webb, B. It., and Johnson, A. tI., eds. Fundamentals of Dairy Science. Avi Publ. Co., Westport, Conn. (12) Federal Register. Oct. 5, 1966. 31:(193) 12967. (13) Federal Register. Oct. 6, 1966. 31: (194) 130~)5. (14) Killer, A., Plazin, J., and Van Slyke, D. D. 1948. A Study of Conditions for KjeldahI Determination of Nitrogen in Proteins. Description of Methods with Mercury Catalyst and Titrimetric and Gasometric Measurements of the Anmlonia Formed. J. Biol. Chem., 176: 1401. (15) Knight, S. G. 1966. Production of a Renninlike Enzyme by Molds. Can. J. Microbio]., 12: 420. (16) Richardson, G. H., and Nelson, J. H. 1967. Assay and Characterization of Pregastrie Esterase. J. Dairy Sei., 50:1061. (17) USDA. Statistical Reporting Service. 1965. Livestock Slaughter. }I + An 1-2-1(66). (18) USDA. Statistical Reporting Service. 1965. Milk Production and Dairy Products, Animal Statistical Smnmary. Da 3(66), pp. 5 and 12.
A fungal rennet produced h'om 3[ucor pusillus is satisfactory for cheese manufacture. Enzyme stability in powder and in saline solution is excellent. However, severe activity losses occur when blended and stored with veal rennet in liquid form. Coagulation by fungal rennet is more sensitive to high pH than veal remwt, but less sensitive than swine pepsin. Coagulation by fungal rennet is more affected by Cathan veal rennet. Fungal rennet produces a greater nonprotein-nitrogen increase in casein solution and in cheese than pepsin or rennet. Higher lipase activity is also present, suggesting advantages in acceleration of curing. Normal clotting time and curd tension can be obtained through adjustment of: rennin units in coagulant, amount of Ca ++ in milk, temperature or time of setting, or blending with veal rennet. Normal Brick, Cheddar, Pizza, and Parmesan cheeses have been made with fungal rennet. Solids losses in whey suggest careful control of factors affecting curd tension and use of a coagulant blend containing predominantly veal rennet. Durhlg the decade ending in 1965, United States cheese production increased 25% while calf slaughter decreased 40% (17, 1S). The supply of rennet extract prepared from the fourth stomach of the calf has, therefore, become critical. Swine pepsin is being used extensively and recommended as a partial replacement for veal rennet (11). Pepsin is not an ideal coagulant, because of its sensitivity to milk p H changes (11). Other coagulants, preferably of microbial origin, are continually being investigated. Knight (15) has screened numerous molds for rennin-like enzyme production and B.~/~sochlamys /'~dva was selected for continued research. The Food and Drug Administration has been petitioned (12) for clearance to use an enzyme produced by the fungus E~d(~tl~i(~ ])arasitic(~. The F D A has also been petitioned for
a change in the cheese standards (13) to include additional safe milk clotting enzymes. Meito Sangyo Kabushiki Kaisha, Nagoya, Japan, has produced a fungal rennet patented by Arima and hvasaki (4, 5). The enzyme is produced by Mucor p~silh~s vat. Liudt~ cultured by a wheat bran koji process. Pilot quantities of improved product have become available. This paper summarizes research conducted over several years in our laboratories during development of this product. Experimental Procedures
F~l~g~d r e ~ e t sol~ltio~s. A 17.8 B6 brine containing 2% propylene glycol, 0.1% sodium 1)cnzoate, and 0.25% potassium sorbate was used to dissolve fungal rennet powder. This provided an extract analogous to rennet extract, except coh)r and flavor were omitted. I~e~nd~ actirity. Rennin activity was determined as described by Ernstrom (9), except that a) Dairyland Food Laboratories, Inc.'s liquid rennet standard was selected as containing 100 remfin milts (RU) per milliliter. b) lfl0g n(mfat dry milk (ND~[) was dissolved in 500 nfl of distilled water containing 0.5 mi of 33% CaCI~" 2 H~O solution, and e) 50-ml pnrtions of substrate were used in 125-ml bottlea. pH. pH of fungal powders was measured on a l g 30 ml water dilution. pr,)tcolysis. Enzyme proteolysis was determined by measuring nonprotein nitrogen (NPN) increase during incubation in casein substrate (9). A 2~
solution of soluble casein (Sheftene
C-2) ~ was adjusted to p H 6.5 with 1.0 _x NaOH. Enzyme dilutions of rennet, pepsin, and fungal rmmet were standardized to contain 75 R U / milliliter. Trypsin solution was also prepared; however, it did not forln a clot that could be used to standardize RU. One milliliter of pH 6.5 enzyme solution was injected into a tube containing 10 ml of casein substrate. The mixture was incubated at 30 C. The reaction mixtures were added to 11 nd 25% triehloroacetic acid (TCA) at zero time and at intervals up to 60 rain, filtered, and analyzed for NPN.
Received for publication Fel)rm~ry 2, 1967. Present address: Topco Associates, Inc., Skokie, Illinois. ~ Sheffield Chemical, Norwich, New York. 1066
FUNGAL
Miero-Kjeldaht (14) was used for NPN analysis. The assay was modified for interIaboratmscomparison (10). A 6.75% casein solution was prepared by adding 6.75 g casein to 75 ml water. Casein solution was affected by adding 3.4 nfl 1.0 5r NaOH and mixing. The solution was adjusted to pH 6.6 and made up to 100 ml. The substrate comprised 20 ml of casein solution and 20 ml of 0.1 5i phosphate buffer at p H 6.6. After the substrate was tempered to 30 C, a 3-ml aliquot was removed for the blank determination and injected into a tube containing 3 ml of 24% TCA. After filtration through Whatman no. 42 filter paper, the filtrate was analyzed for NPN content. The remaining 37 ml of substrate was treated with enzyme. The volume of enzyme inocula was varied. Four milliliters of 75 RU/milliliter were inoculated in one series. This was the level used in the 2% casein study and amounted to 8.1 R U / ml of substrate. I n another series, 0.1 ml of 1 7 R U / m l solution was inoculated which resulted in 0.046 RU/nfl of substrate. Three milliliters of substrate-enzyme blend were removed, treated with TCA, and filtered at 10-, 30-, and 60-rain intervals. Results are expressed as increase in per cent NPN. Lipase ~wtivity. Lipase activity was measured with 5 ml of' tributyrhl substrate (16) in a Sargent recording p H stat. The instrmnent was charged with 0.025 ~- NaOH and set at pH 6.20, 42 C, stirrer at 8, and 0.1-inch per minute chart speed. One milliliter of a 0.01 nfl dilution of enzyme preparation is inoculated into the substrate. One lipase unit is defined as 0.025 of chart width, or 0.01 ml base. Storage stability. Blends of fungal and veal rennet solutions were held at various pH values and at 4, 38, and 60 C to determine inactivation during storage. Keeping quality of dLw powder and unmixed solutions was also assessed. Curd tensioJ~. A rennet curd tension test (6, 7) was modified to measm'e quality of fung'al rennet clots. The Cheno--Burrell 1)rocedure (7) was followed, except 15% NDS[ substratea were used. p i t and Ca ++ content of the snhstrate was modified by adding NaOH, HC1, or CaCl~ solutions. Controls were adiustcd t() equal volumes with water. Microbiological co,~ditio~, a~d to.,'icit]j. Outside laboratories evaluated products for standard plate count, yeast and uu)ld count, and coliform count with standard procedures (1). Sahnonella (2), aflatoxin (3), and acute oral LD:~ (8) tests also were run.
RENNET
1067
E.,-t)toratory cheese making trials. Parmesan~ Cheddar, and Brick cheese were made in our labm'atorics, in 45.4-kg pilot plant cheese vats, using hig'h quality whole milk and accepted industry procedures. The cheeses were examined organoleptically by a trained flavor panel. They were also evaluated for gross composition and NPN differences. Pasta Filata cheese was produced in larger quantities and evaluated for curd handling properties. Results and Discussion
I. (;~'~cr(d characteristics of .f~gal renuet. Fungal rennet, as received, is a light grey to 1)urple-grey powder. Solutions of fungal rennet are brown, bland in taste, and have a slight, 1)leasant vegetable-like aroma. The solutions are darker than commercial rennet extract which, generally, has caramel coloring added during standardization. Absorbance readings were made at 570 m/x on equal RU/milliliter ~olutions of commercial strength veal rennet, fungal rennet, and mixtures of the two. Using a 10-ram cuvette, readings were 0.23, 0.41, 0.88, and 1 . $ 2 0 D for remmt, 10% fungal rennet, 45~ fungaI rennet, and 100% fungal rennet sample~, respectively. This severe color differential is not a problem in cheese making. Upon dilutiom the color intensity is decreased sufficiently to prevent color modification of the finished cheese. Early samples of fungal rennet had a p H of 6.6 to 6.8 and tested 330-380 RU/g. Recent shipments range in p t I from 5.0 to 5.6 and exceed 1,()00 R U,/g. ,~ol~lbility. Fungal rennet powders have improved significantly in solubility, wettability, and sedimentation characteristics. Initial powders were slow to hydrate, were gummy, viscous, and contained large particles of sediment. Cellulase treatment significantly reduced viscosity and ~edimentation; however, refinements of the bast(" enzyme production process have precluded need t'~r such treatment. It is practical to prepare fungal rennet extract with 100% market strength (75-80 RU/%l). Solution over 2~)0(~c strength is not practical in extract analogous to rennet solution because of solubility limitations. D~gr,~e o./I proteolysis. Any substitute for remwt nmst not only clot milk but must have low proteolytic activity. The high proteolytie activity of ttTpsin, papain, and ninny other coagulants prevents their use in cheese making. Fungal rennet solution is compared with t13"psin, pepsin, and rennet in Figure 1. Ten milli.T. ])AIRY SCIENCE ~'OL. 50, NO. 7
]068
R I C H A R D S O N , NELSON, LUBNO~,V, AND S C H W A R B E R G
liters of a 2% casein solution substrate was injected with 1 ml of 75 RU/ml coagulant, except that trypsin activity was only estimated. Lack of adequate clot formation and rapid proteolysis prevented standardization.
I
50
e- TRYPSIN •
A-FUNGAL
,.<, ¢y-40 /
o-RENNET
z
z rl Z
RENNET
"-PEPSIN
3o
/ 2O
I0
/
I0
20
30 4 TIME (MIN.)
SO
60
Fro. 1. Per cent NPN increase due to casein breakdown by various coagulants. The trypsin curve is typical of those enzymes which have undesirable proteolytic activity. Pepsin and rennet curves were below 3% NPN after 60 rain. Fungal rennet curves on an early sample (1963) and a recent cmnmercial shipment (1966) were above 10% NPN. Fungal rennet is thus more proteolytic under these conditions. The procedure was modified to allow comparison with another laboratoKv (10. When 0.046 R U / m l substrate was inoculated instead of the 8.1 RU/ml as used above, the fungal had a slightly lower % NPN than rennet. After 60 rain incubation, the NPN was 0.93% for rennet and 0.78% for fungal rennet. Simultaneous runs made using 8.1 RU/ml substrate had 0.37 and 11.94% NPN values for rennet and fungal, respectively. Differences in the degree of proteolysis thus do not become apparent until higher levels of iuoculum are used. This finding may be of value in determining the presence of fmlgal rennet in certain coagulant blends. The significance of' this difference in NPN increase will be discussed in a subsequent paper. Lipase activity. Lipolytic activity in fungal rennet powder was compared to rennet powder and kid pregastric esterase powder (CAPALASE Lipase Powder K~). The latter repreDairyland Food Laboratories, Inc., Waukesha, Wisconsin. J. DAIRY SCIENCE VOL. 50, NO. 7
sents a family of products used to produce lipolytic flavors in Italian cheese varieties. The rennet powders tested 1,000 RU/g. No rennin aeti~4ty was present in the pregastric esterase preparation. One gram of each powder was rehydrated in 75 ml water, mixed for 20 rain, and made up to 100 ml. One milliliter was inoculated into 5 ml tributyrin substrate and the sh)pe on the recorder indicated the reaction rate. Lipase activity was 0.23, 1.80, and 8.90 units per rain for rennet, fungal rennet, and standardized pregastric esterase preparation, respectively. There is some evidence of gastric esterase being present in rennet extract. The higher lipase activity in fungal rennet may be potentially advantageous in accelerating cheese curing. Organoleptic evaluation of cheese has not revealed any off flavor attributable to lipolysis. Pasta Filata cheese aged for three months showed no significant difference in amount of extractable free fatty acids. Thus, additional work is necessary to establish the significance of the higher lipase activity present. Storage stability. Activity retention of 1,000 RU/g fungal rennet powder is excellent. No losses have occmTed in eight months of storage at 21 C Keeping quality data on liquid products at 4, 22, and 38 C are included in Table 1. Liquid fungal rennet solutions at p H 5.75 were nmre stable than rennet or pepsin extracts. Liquid blends were not as stable as the unmixed enzyme solutions. Blends of fungal rennet and rennet lost more activity than pepsin-rennet blends. The blend containing the higher percentage of rennet lost the most activity. At 4 C the blend with the higher level of fungal rennet experienced a greater activity loss than the lower level. This was not found at higher temperatures. These results suggest that fungal rennet destroys veal rennet activity. Solutions of coagulants were heated to 60 C for three hours at different pH values to determine optimum p i t range for storage stability. Rennet has been found most stable at pH 5.5 to 6.2 (11). Comparisons were run as summaTABLE 1 Storage stability of liquid rennet, fungaJ rennet, pepsin, and blends after 4 wk at pH 5.75. Enzyme Rennet Pepsin (pH 4.3) 50/50 Rennet-pepsin blend Funga] rennet 55/45 t/ennet-fungal blend 90/10 Rennet-fungal blend
5~ Activity loss at 4C oo C 38 C < 1 < 1 ] ~ 1 22 6
5 6 1 47 54
14 44 49 13 60 80
FUNGAL RENNET
rized in Table 2-A. Fungal remwt stahility is better than rennet in the p H range evaluated. The coagulants were most stable at p H 5.25. The range of p H from 5.25 to 5.75 was optimmu dm'ing storage (Table 2-A). However, the magnitude of the losses preclude liquid blending of these enzymes. TABLE 2 Activity losses of coagulants at various pH values
1069
II. Actirily o.f fungal rennet upon milk. E!Dx't o]" milk pH. Pepsin coagulant is ]ess active in milk with high p H (11). This is generally overcome by blending with rennet, to restore control over clotting time. Fungal rennet was compared to rennet and pepsin in milks at various p H levels. Figure 2 compares clotting time from pH 6.0 to 6.8. Fungal rennet is less affected by high p H than is pepsin.
% Activity losses
After 3 hr at 60 C
pH a.* 4.75 5.25
5.75 6.25
55/45 RennetFungal fungal Rennet rennet blend (%) 32 13 25 20 12 20 33 13 28 63 16 39
After 2 wk at 38 C 55/45 ]~elmetfungal blen .d --(~)-53 51 51 54
4.75 62 58 58 5.25 36 76 51 5.75 70 94 62 6.25 93 93 95 * = Solutions containing sodium chloride, propylerie glycol, sodium benzoate, and potassimn sorbate. ** -Solutions prepared in distilled water. Solutions of 80 R U / m l coaguhmts were prepared in distilled water to determine how the results reported in Table 2-A were modified by the absence of preservatives. Results of heat treatment of these solutions are recorded in Table 2-B. Greater losses occurred. Rennet (prepared from 512 R U / g powder) was more stable than fungal rennet. This may partially be attributable to the sodium chloride and gelatin used in preparation of rennet powder. The severe losses in distilled water emphasize the value of the additives in solutions of fungal rennet and rennet. Mic~'obiologieal conditions. Early samples of fungal rennet contained hacterial contaminants; however, recent shipments have had low bacterial counts. A recent Standard Plate Count survey found commercial 100% rennet extracts less than 3,000 per nil. Recent shiplnents of 1,000 R U / g fungal rennet tested less than 3,000 per g. No sahnonella were present in 50 g (2) ; counts for yeast, molds, and eoliforms were less than 10/g. Toxicity. Fungal rennet has been found to be free from aflatoxin (3). Acute oral LD~ tests on rats indicate a value in excess of 20 g / k g of body weight. The product, therefore, qualifies as nontoxic orally within the meaning of the Federal IIazardous Substances Labeling Act (S).
O
2O
/
m-FUNGAL RENNET
Z
15
L9 I0 Z
I--
o-PEPSIN
o,._1 5 L#
/
o-RENNET
/'e
//-
.....___.~..,~/// i
6.0
I
i
6.2 6.4 6.6 SUBSTRATE pH
I
6.8
FIe.. 2. Ilennin test substrate clotting time at different pit values, using coagulants adjusted to equal RU/ml content at pit 6.4. Cm'd tension analyses were run in 15% reconstituted NDM, using each coagulant standardized to constant clotting strength at p i i 6.4. Curd tensions were rennet > fungal rennet > pepsin. These differences were most evident between milks at p I I 6.6 to 6.8. E]]'ect of adding Ca +'. Fungal rennet solutions standardized with the standard rennet assay appeared to be weaker when tested in cheese milk. This was found to be due partly to the concentration of Ca ++ present. CaCI~ is added to the test substrate (9). Fungal rennet is more affected by calcium, as shown in Figure 3. Thus, the fungal rennet test may be weaker with no added Ca*" but stronger with added Ca *+. These findings suggest the necessity to standardize fungal rennet strength higher than rennet. Under our laboratory test conditions 10-12~ higher test is warranted. Addition of 0.015% CaCI.~ to cheese milk has been found helpful in restoring normal curd tension. In a simulated cheese making trial rum'-liter aliquots of milk were set, using varied temperatures, coagulant, and CaCI~ addition. Subjective observations, such as the cheese maker uses, were made to deternfine flake time and cutting time. Results are shown in Table 3. J. DAIRY SCIENCE rOB. 50, NO.
1070
RICHARDSON,
140
NELSON,
LUBNO\V,
f u n g a l r e n n e t was used. Based u p o n interp o l a t i o n o£ this data it would take a b o u t 1 1 3 % of f u n g a l r e n n e t to produce a clot c o m p a r a b l e to 100% rennet. This value also agrees with the conclusion reached in the Ca** study. However, h i g h e r levels of f u n g a l r e n n e t have not always i m p r o v e d curd tension characteristics d u r i n g field trials. E.!~'ect of s~bstrate temperat~lre upon curd ten.,'io~L W h e n milk s e t t i n g t e m p e r a t u r e was varied f r o m 31.1. to 33.3 C, the em'd tension increased, as shown in Fig. 5. F u n g a l r e n n e t r e s p o n d s to t e m p e r a t u r e increase e o m p a r a b l y to rennet. However, as setting t e m p e r a t u r e s are increased ( u p to 38 C), the f u n g a l r e n n e t clots tend to be progressively weaker t h a n veal r e n n e t values. I t is evident t h a t curd tension can be modified with a d j u s t m e n t of setting t e m p e r a t u r e .
o FUNGAL RENNET • - RENNET 0
120
I00
•
I--
~ 8o LU 60
*
r'r"
<
0.. 0.. <
SCHWARBERG
AND
40
20.
200 '
400 ~
600 ~ PPM
800 ' CACI2
I000
FIG. 3. Apparent rennin test as affected by added ealelum. I n some cases the f u n g a t r e n n e t curd quality was i n f e r i o r to rennet, even following calcium a n d test a d j u s t m e n t . Effect of time upoJ~ curd te~sioa. C u r d tension evaluations in 1 5 % N D M were r u n over a p e r i o d o£ 20-60 rain. E n z y m e dilutions cont a i n e d equal RU, as d e t e r m i n e d on r e n n i n test substrate. F u n g a l r e n n e t trailed b e h i n d r e n n e t a t all points, as shown in Fig'. 4. Since f u n g a l r e n n e t d a t a tend to p a r a l l e l rennet, one method of overcoming curd tension differences is to allow a l o n g e r set time. Effect o]" hwreased inocuMm ~po~ c~H'd te~> sion. A n o t h e r method o£ increasing curd tension in f u n g a l r e n n e t clots is to increase the coagulant. C o a g u l a n t dilutions with equal clotting s t r e n g t h on test s u b s t r a t e were added in 1-ml inc r e m e n t s f r o m 1-5 nil; the r e s u l t a n t curd tension is recorded in Table 4. Curd tension reached a m a x i m u m of a b o u t 100 g w h e n either r e n n e t or
•- RENNET .,///o o-FUNGAL R E N N ~ o ~ / /
120
.S
© ~100 z 8O 0
C3 or"
4-o 0
20
30 40 50 TIME (MIN.)
60
Fie. 4. Curd tension as affected by time of cutting following coagulant inoculation.
TABLE 3 Curd formation during simulated clwese making trials Flake tinle
Setting temp 31 C 32 C
~(eat No calcium 0.015% CaCI~ No calcium 0.015% CaCI,
*Very fine flake evident. **Definite flake e~Sdent. ***Better than veal counterpart. g. DAIRy SCIEXCE ¥OL. 50, No. 7
Fun g al
---(n~h~;-11 14" 19 ~* 10 13 11 12 ~ 13"* 10 11
Time ready to cut
Veal
---(min)---40 30 40 30
Fu n gal 40 40 40 30 * ~
YUNGAL
RENNET
TABLE 4 Effect of increasing amount of coagulant upon curd tension
% of Usual inoculum
nfl of 2/100 Dilution
1
50
43 76 98 97 99
2 3 4 5
150 200 250
9°I
• RENNET
~
8O
o FUNGAL
/ ~
RENNET
COAGULANTBLEND o o PEPSINI RENNET Q, elI2%FUNGALI REN.
i~81
Fnngat rennet
Rennet
~
20~
tension
Curd
(g) 100
j
1071
a
AI24%
/
I O
Z
23 71 91 98 100
i
~-14-
10-
/ / / /
b
7C
2'0
4'0
6'0
8'o
60
% RENNET
Z
Fro. 6. Per cent rennet addition necessary to 1)]eJ~d with pepsin and with fungal rennet, to restore clotting time in ptI 6.5 raw whole milk substrate containing 2c~ added lactic culture. (Fungal renm,t standardized 12 and 24 RU/ml higher than rem~et on rennin test substrate.)
0 ~n 6C Z ,,i I--
L) 40 3O i
Si.I
i
i
32.2 TEMR (*C.)
I
33.3
Fro. 5. Effect of substrate settil~g temperature upon curd tension. Analyzed 30 rain after setting.
Effect of blending .reJ~net sol~tio~,~ ~po~ clottbtg time. Coagulant blends containing pepsin and rennet routinely contain at least 25c~ rennet, to control clotting time (11). To evaluate what percentage of rennet would restore clotting time to a coagulant blend, mixtures of rennet with pepsin and with fungal rennet were prepared. They were blended just prior to testing, to mininfize keeping quality losses. Rennet and pepsin eoag'ulants were adjusted to 100 R U / m l on test milk substrate. Two fungal rennet solutions were adjusted to contain 112 and 124 RU/nfl, respectively. Clotting time was determined on raw whole milk containing 2% added lactic culture. This produced a p H 6.5 substrate resembling cheese milk at time of coagulant addition. Results are shown in Figure 6. None of the blends restored clotting time equal to 100~}
rennet. The blends containing the highest fungal rennet test had the shortest clotting times. An increase in the proportion of veal rennet in rennet-pepsin blends reduced the clotting times more sharply than analogous proportions of rennet in either of the rennet-fungal rennet blends. Therefore, to maintain normal clotting times, a higher proportion of rennet is required in a rennet-pepsin blend than in a remtet-fungal blend. These observations suggest the need to use a different test substrate for fungal rennet rather than the high solids ND~I, CaCI~ fortified produet. An unfortified whole or skinunilk adjusted to pH 0.5 is suggested. E,rploratory c]~eese makh~g trials. Pilot plant quantities of Cheddar, Brick, and Parmesan cheese were manufactured from high quality whole milk. An increase in whey fat when fung'al rennet was used has prompted additional yield studies under commercial conditions. Onemonth-old Brick and ten-months-old Parmesan cheese samples were of marketable quality. The minor defects observed could not be attributed to fungal rennet. Cheddar cheese appeared normal at two and five months. At 14 months, the Cheddar samples were slightly bitter. Samples made with fnngal rennet were more bitter than the control. The N P N value for Cheddar using rennet was 83.8%, compared with 42.0 J. DAIRY SCINNCE VOL. 50, NO. 7
1072
R I C H A R D S O N , NELSON, LUBNO\V, AND S C H W A R B E R G
a n d 35.5% f o r two lots made f r o m f u n g a l rennet. A t e n d e n c y f o r h i g h e r N P N values in f u n g a l r e n n e t cheese has been established in s u b s e q u e n t work. F u l l cream P a s t a F i l a t a cheese curd was p r e p a r e d w i t h s t a n d a r d procedures. To evaluate curd m o l d i n g a n d s t r e t c h i n g p r o p e r t i e s of p r o d u c t made with f u n g a l rennet, vats were set using all f u n g a l rennet, veal, a n d 10/90, 4 5 / 5 5 blends of these coagulants. E x p e r i e n c e d I t a l i a n cheese makers j u d g e d curd quality u s i n g as c r i t e r i a : m a t t i n g characteristics, hot iron test, molding characteristics, a n d f a t losses in whey. All blends behaved n o r m a l l y in evehw respect. The v a t p r e p a r e d f r o m 100% funo'al r a n 8 rain b e h i n d in total make time, due to a slower flake a n d set tinm. No a t t e m p t h a d been m a d e to a d j u s t nfilk conditions to reduce these differences. Curd w o r k i n g p r o p e r t i e s were n o r m a l in all vats. F a t loss in the all f u n g a l r e n n e t v a t was 0.42%, instead of the n o r m a l 0.30c~. A p p a r ently, this was related to the slow ~et a n d curd fragility.
Acknowledgment Tile authors express their gratitude to Dr. C. A. Ernstrom for his valuable suggestions, to Dawes Laboratories, Inc. for their cooperation and for providing the initial samples of fungal rennet, and to Mrs. H. Beikerdffe, techuieian.
References (1) American Public Health Association. 1961. Standard Methods for the Exanfination of Dairy Products. 11th ed. Public Itealth Ass., New York. (2) Angelotti, tt., Crane, A., Hunter, J. E., Klusmeyer, P. W., Huston, M. L., Lamprech, E. D., and Silliker, J. H. 1966. Recomnlendations for Sampling aa~d Laboratory Analysis of Eggs, Egg Products, and Prepared Mixes. Food Technol., 2 0 ( 4 ) : 121. (3) Anonymous. 1966. 25 Nuts and Nut Produets. J. Ass. Offie. Agr. Chemists, 49: 229.
J. DAIRY SCIENCE VOL. 50, NO. 7
(4) Arima, K., and Iwasaki, S. 1964. Milk Coagulating Enzyme "Microbial Rennet" and Method of Preparation Thereof. U. S. Pat. 3,151,039. (5) Arima, K., and Iwasaki, S. 1965. Process for Making Cheese. U. S. Pat. 3,212,905. (6) Babcock, C. J., Chambers, L. A., Flora, C. C., ttull, M. E., Mueller, W. S., Sommer, H. H., Storrs, A. B., and Doan, F. J., Chairman. 1941. Einal Report of Committee on Methods of Deternfining the Curd Tension of Milk. J. Dairy Sci., 24: 825. (7) Cherry-Burrell Corp. ]955. Bull. 1P5132-M. Chicago, Ill. (~) Code of Federal Regulations, P a r t 1 9 1 Hazardous Suhstanees: Definitions and Procedural and Interpretive Regulation. (9) Ernstrom, C. A. 1958. Heterogeneity of Cr.vstalline Remlin. J. Dairy Sci., 41: 1663. (lo) Ernstrom, C. A. 1966. Personal comnmnieation. ('11) Ernstrom, C. A., and Tittsler, R. P. ]966. Rennin Action and Cheese Chenlistry. pp. 592-610. I ~ Webb, B. It., and Johnson, A. tI., eds. Fundamentals of Dairy Science. Avi Publ. Co., Westport, Conn. (12) Federal Register. Oct. 5, 1966. 31:(193) 12967. (13) Federal Register. Oct. 6, 1966. 31: (194) 130~)5. (14) Killer, A., Plazin, J., and Van Slyke, D. D. 1948. A Study of Conditions for KjeldahI Determination of Nitrogen in Proteins. Description of Methods with Mercury Catalyst and Titrimetric and Gasometric Measurements of the Anmlonia Formed. J. Biol. Chem., 176: 1401. (15) Knight, S. G. 1966. Production of a Renninlike Enzyme by Molds. Can. J. Microbio]., 12: 420. (16) Richardson, G. H., and Nelson, J. H. 1967. Assay and Characterization of Pregastrie Esterase. J. Dairy Sei., 50:1061. (17) USDA. Statistical Reporting Service. 1965. Livestock Slaughter. }I + An 1-2-1(66). (18) USDA. Statistical Reporting Service. 1965. Milk Production and Dairy Products, Animal Statistical Smnmary. Da 3(66), pp. 5 and 12.