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A linear diffusion test in sedimentation tubes filled with caseinagar gel successfully measured milk clotting enzymes at concentrations of 10-4 to 1 X 10-l rennin units/ml with 95% accuracy. Diffusion rates were unaffected by diluting enzyme samples with whey, 3% NaCl, and water, The distribution of rennet, porcine pepsin, mucor pusillus var Lindt (MP) protease, and rennet-pepsin mixtures between curd and whey was determined on milk coagulated at pH 5.2, 6.0, 6.4, and 6.6. The procedure accounted for 100 + 7% of the added enzymes. The distribution of rennet was pH dependent with 31% and 72% in curd and whey respectively at pH 6.6, and 864 and 174 respectively at pH 5.2. The distribution of MP protease was independent of pH with approximately 154 and 85% in the curd and whey at all pH values. Pepsin behaved similar to rennet but was unstable above pH 6.0. During Cheddar cheese making, 7% and 58% of the original rennet, 6% and 93% of the original MP protease, and 5% and 17% of the original rennet-pepsin mix was active in the curd and whey respectively at dipping. After overnight pressing, 6% of the rennet, 3% of MP protease, and 4% of the rennet-pepsin mix remained active in the cheese. At dipping only 9% of the original pepsin was detected in the whey. Pepsin was unstable at pH values used to release the enzyme from the curd and could not be quantitated.

Residual rennin in cheese whey and curd was measured by using a special sensitive substrate. The substrate was made by reconstituting 6 g NDM in 500 ml of buffer containing 0.2 M CaCl2, 0.5 M cacodylic acid and 0.2 M triethanolamine at pH 5.7. Cheese curd was blended into a 1:7 slurry (1 part curd, 7 parts water), and 1.67% sodium chloride was added to the why and slurry to liberate residual rennin from casein. The residual rennin in cheese whey and slurry were determined simultaneously with an identical sample containing known rennin activity. Samples with known activity were prepared by destroying the residual rennin in unknown samples after which a known amount of rennin was added back to a standard. The examine the effectiveness of this method for measuring rennin activity in whey or slurry, a recovery test was developed to measure rennin activities in the whey and curd made by centrifuging rennet-coagulated milk. The average total recovery from 15 replications was 101.6 �� 2.4%. It was found that pH was a main factor affecting rennin distribution between whey and curd. The amount in the curd increased with decreasing pH at setting. Adding 0.02% CaCl2 to milk was of little effect. The results showed that 68.1 6 �� 6.6% of the residual rennin was found in Cheddar cheese whey after dipping and 17.2 6 �� 2.6% of the residual rennin was in the curd after milling.

Two factors that are important in the production of the typical body and texture of cheddar cheese are acid development and milk fat content. When either of these is absent or present in less-than-normal amounts, the resulting cheese shows marked defects of curdiness and firmness. When acid development in a vat of cheese is arrested, it is referred to as a "dead" vat. The cheese from this vat may be salvaged by use of the hot brine method of treatment. Hot brine treated cheese, although lacking the desired acid development, is a reasonably acceptable product, having the desired characteristics of body and texture to a degree. When cheese from a "dead" vat is packed without hot brine treatment, it is inferior to the hot brine cheese. The fact that hot brine treatment improves the body and texture of non-acid or low acid cheese suggests the possibility that it might have the same effect upon cheese made from partially skimmed milk. If this is possible, it might open the way for the commercial manufacture of a cheddar-like cheese from low-fat milk using a hot brine method. This possibility forms the basis of this study.

The manufacture of cheddar cheese is greatly dependent on bacterial growth for acid production. The quality of cheese depends upon the type and extent of microbial activity. An important function of the lactic fermenting bacteria is the production of acid resulting from cellular metabolism. If little or no acid is production of acid resulting from cellular metabolism. If little or no acid is produced the resulting cheese will have an inferior body, flavor, and texture and may even cause the cheese to be used as grinders. The major causes for inhibited lactic bacterial growth are poor starter handling procedures, antibiotic in mild coming from cows treated for mastitis, quaternary ammonium compounds used in plant sanitation, and bacteriophage. The occurrence of bacteriophage contamination and the increased use of quaternary ammonium compounds and antibiotics have caused a serious problem in cheese manufacturing. Purpose of project. The purpose of this project is to determine if cheddar cheese, comparable to normal cheese, can be made from slow or non-acid milk with the use of added lactic acid. Lactic acid will be used for the purpose of substituting for the acid that is normally produced by bacterial metabolism in the manufacture of cheddar cheese.

Attempts were made to adapt the microtiter hemagglutination inhibition assay technique for the assay of enterotoxin A. The presence of a potent hemagglutinin in crude and partially purified preparations and the instability of sensitized erythrocytes prevented its use for routine analysis of enterotoxin from culture media and foods. A capillary tube immunological assay was developed in which 1 �� g of enterotoxin/ml was detected in less than 1 hr . Interfacial reaction of antisera and enterotoxin solutions in a 1 mm internal diameter capillary tube allowed rapid detection and serological typing of enterotoxins. Staphylococcus aureus growth and enterotoxin A development in Cheddar cheese slurry was evaluated. S. aureus growth and enterotoxin production occurred at 32 C. in 45 and 60% moisture cheese slurries following inoculation with 10 3 to 10 5 bacteria/gram. Hydrogen peroxide (0. 5%) treatment of slurry at 37 C did not inhibit S. aureus and enterotoxin A development. Heating slurry at 72 C for 30 min eliminated staphylococci but reinoculation with ripening organisms was essential. Addition of sorbic acid (0. 2 to 0. 3%) to a slurry adjusted to pH 5. 0 with lactic acid, inhibited staphylococci. in milk and slurry. Cheese flavor development was retarded due to inhibition of micrococci and lipolysis. Non-protein nitrogen increases paralleled that of sorbate-free controls. Sorbate treatment was preferred over other treatments .