Diffuse Reflectance Profiles of Eight Milk-Clotting Enzyme Preparations Z. USTUNOL, C. L. HICKS, and F. A. PAYNE ABSTRACT Eight milk-clotting enzyme prepartations were standardized to equal clot time and used to coagulate pasteurizedwhole milk. Diffuse re- flectance profiles were monitored for 60-min using a fiber optic sensor sensitive to infrared light at 950 nm. Modified M. miehei and M. pusillus protease, recombinant chymosin and calf rennet produced similar profiles. Rates of increasein diffuse reflectance were E. par- asitica > recombinant chymosin > calf rennet > modified M. miehei, M. pu.siI1u.s var. Lindt > 50~50blend of calf rennet and bovine pepsin > unmodified hf. miehei > pepsin. Monitoring milk coagulation as described may be useful during cheesemaking and allow setting op- timal conditions for milk-clotting enzyme preparations. INTRODUCTION USE OF MILK-CLOTTING enzymesother than calf rennet in manufacture of cheese has increasedduring the past two dec- ades. Calf rennet, a crude extract containing 85 to 95% chy- mosin and 10 to 15% bovine pepsin (Lim and Dinesen, 1973) has been the traditional and preferred milk-clotting enzyme preparation for cheese making. However world cheese pro- duction has more than doubled and calf stomachs from which calf rennet is extracted are in short supply to meet cheese- making demands. Patents have been issued for production of milk-clotting enzymes from Endothia parasitica, Mucor pus- illus var. Lindt and Mucor miehei. In the United States these enzymes have been approved for manufacturing all standard varieties of cheese (FDA, 1989). Of animal proteases, only bovine and porcine pepsin and chymosin have been of interest in cheese making (Ernstrom and Wong, 1974). Recent devel- opments in genetic engineering technology have provided re- combinant chymosin as another alternative to the shortage of calf rennet. Although these alternative milk-clotting enzyme preparations perform satisfactorily in a wide range of cheese manufacturing conditions, their use has generated important questions and considerable research. Numerous methods and instruments have been developed to monitor milk coagulation and to determine adequate firmness for cutting the curd. A curd firmness tester designedby Van- derheiden (1976) has been used sucessfully by Olson and co- workers to measure milk coagulation parameters in cheese vats (Bynum and Olson, 1982; Garnot et al., 1982; Kowalckyk and Olson, 1978). An instrument, tentatively called the “Vati- mer,” which measures resistanceto an oscillating probe was describedby Richardson et al. (1985) for continuous measure- ment of coagulation parameters. Ustunol and Hicks (1990b) reported on a Coagulation Monitoring Device (CMD), a mod- ified version of the instrument described by Richardson et al. (1985) which could monitor coagulation in six vats simulta- neously. Hori (1985) monitored coagulation using an electri- cally heated platinum wire. Kinematic viscosity of the coagulating milk was correlated with temperature change in Author Ustunol is with the Dept. of Food Science & Human Nutrition, Michigan State Univ., East Lansing, Ml 48824- 1224. Author Hicks is with the food Science Section, Dept. of Animal Sciences, Univ. of Kentucky, Lexington, KY 40546-0215. Author Payne is with the Dept. of Agricultural Engineering, Univ. of Kentucky, Lexington, KY40546. the wire. McMahon et al. (1984) reported that turbidity of undiluted samples of milk increased the absorptivity of light at 600 nm. Hardy and Fanni (1981) measured diffuse reflec- tancewith a color difference meter. Spectroscopicmethods for monitoring milk coagulation, however, have been thought lim- ited to scientific usage (Hori, 1985). With recent advancesin fiber optics and integrated circuits, many spectroscopicmeth- ods previously used only for scientific researchwill become economically viable for production plants. Previous laboratory tests have shown that diffuse reflectance profiles of coagulating milk can be determined with a fiber optic sensor which monitors the change in diffuse reflectivity of infrared radiation at a wavelength of 950&5 nm (Payne et al., 1990). Change in reflectivity of energy at 950 nm may be due to change in bound water held by casein during coagula- tion. Hicks et al. (1990) reported that diffuse reflectance pro- files followed coagulation parameters accuratelywhen changes due to pH, temperature and enzyme concentration were mon- itored by diffuse reflectance. The objective of our study was to monitor the diffuse reflectance profiles of eight commer- cially available milk-clotting enzyme preparationsover 60 min and compare several coagulation parameters. MATERIALS & METHODS Standardization of enzymes Purified and standardized single-strength calf rennet, modified and unmodified M. miehei protease, bovine pepsin (EC 3.4.23.1), and protease from M. pus& var. Lindt were provided by Marschall Products Division of Miles Laboratories Inc. (Madison. WI). Triule strength protease from E. parasitica was provided by Pfizer Inc. (Mil- waukee, WI). Recombinant chymosin (EC 3.4.23.4, isozyme B) was supplied by Chr. Hansen’s Laboratory Inc. (Milwaukee, WI). The apparatusdescribed by Sommer and Matsen (1935) was used to de- termine activity of each enzyme. Concentrationsof all enzymes were adjusted to give an equal clotting time of 5.30t0.2 min using this apparatus.Enzyme solutions were added to 25 mL pasteurizedcheese milk in 125mL wide-mouth bottles. Analysis was conducted at 35°C. Enzymes were diluted with 0.01 M sodium citrate buffer at pH 5.2 and maintained below 2°C for the duration of each experiment. Monitoring diffuse reflectance profiles Raw whole milk (3.7% fat) was pasteurized (62.K for 30 min) and cooled to 4°C. Milk was weiehed (9OO?O.lrr) into a lOOO-mL beaker and allowed to equilibrate70 35’C. One ii of standardized enzyme preparation was added to milk with stirring for 30 sec. The diffuse reflectance of coagulating milk at 35°C was measured by a fiber optic probe described by Payne et al. (1990). The probe func- tioned by transmitting light from an incandescent bulb to a submerged probe tip through a fiber optic bundle. The light reflected from the milk was received by fibers randomly mixed with the transmitting fibers that carried the reflected light to a photodetector. The detector had a built-in optical filter with a center wavelength of 950 nm and bandwidth 10 nm. The output voltage of the senor was proportional to the light reflected from the milk. A personal computer-based data acquisition system was used to monitor the diffuse reflectance during milk coagulation. The sensor output voltage was read by an analog-to-digital board (DASCON-1, Metrabyte Corp., Stoughton, MA). A BASIC program was written to managedata acquisition, filing, and plotting in real time. A subroutine was written in BASIC to calculate the first derivative of the reflectance Volume 56, No. 2, 1991-JOURNAL OF FOOD SCIENCE-411