Proteolysis Index Research Articles

Monitoring Cheddar cheese ripening by chemical indices of proteolysis 1. Determination of free glutamic acid, soluble nitrogen, and liberated amino groups

Freel-glutamic acid (Glu), soluble nitrogen (N), and liberated amino groups (amino-N) were evaluated as indicators of proteolysis and maturation in a study of Cheddar cheese ripening. The analytical methods involved standardized extraction and fractionation procedures, enzymatic determination of Glu, the measurement of N by Kjeldahl and amino-N by a rapid spectrophotometric assay witho-phthaldialdehyde. Cheddar cheeses from two different production plants were stored at both 4° C and 10° C for nine months. All cheeses were periodically analyzed for Glu in an aqueous extract and for N and amino-N in citrate extracts soluble at pH 4.6 and in 12% trichloroacetic acid. All indices correlated well with each other and with the age of the cheese at a highly significant level. It was demonstrated that these parameters are well suited to determine the extent of proteolysis as an indication of the degree of maturity of ripening cheese, thus providing an objective measurement for age classification and raw material control. The convenient assays for Glu ando-phthaldialdehyde-reactive amino-N were proved suitable for routine applications, enabling a fast and sensitive check on the degree of proteolysis in cheese without sophisticated equipment.

Glucose and amino acid metabolism in chronic renal failure: effect of insulin and amino acids.

The effects of hyperinsulinemia and hyperaminoacidemia on glucose and amino acid metabolism were examined in 16 control and 13 chronic renal failure (CRF) patients under two conditions: 1) euglycemic hyperinsulinemia and 2) amino acid infusion. All studies were performed with continuous indirect calorimetry and [1-14C]leucine infusion. In CRF patients insulin-mediated whole body glucose metabolism was reduced by 35% (4.41 +/- 0.50 vs. 6.76 +/- 0.73 mg.kg-1.min-1, P less than 0.01), primarily due to a decrease in nonoxidative glucose disposal (1.70 +/- 0.70 vs. 4.32 +/- 0.60 mg.kg-1.min-1, P less than 0.01); glucose oxidation was similar in both groups. In the postabsorptive state total leucine turnover (1.56 +/- 0.06 vs. 1.75 +/- 0.06), leucine oxidation (0.25 +/- 0.01 vs. 0.30 +/- 0.01), and nonoxidative leucine disposal (1.29 +/- 0.06 vs. 1.40 +/- 0.07 mumol.kg-1.min-1) were reduced in CRF vs. control subjects (all P less than 0.05). In response to hyperinsulinemia, endogenous leucine flux (index of proteolysis), leucine oxidation, nonoxidative leucine disposal (NOLD) (index of protein synthesis), and net leucine flux into protein were similar in CRF and control subjects. In contrast, the ability of hyperaminoacidemia to enhance NOLD (1.54 +/- 0.11 vs. 2.10 +/- 0.10 mumol.kg-1.min-1, P less than 0.01) and net leucine balance (0.27 +/- 0.05 vs. 0.41 +/- 0.05, P less than 0.05) was reduced in CRF patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin and amino acid infusion on leucine and phenylalanine kinetics in type 1 diabetes.

To evaluate the anabolic effects of hyperinsulinemia and hyperaminoacidemia on amino acid (and protein) metabolism in type 1 (insulin-dependent) diabetes mellitus (IDDM), we studied leucine and phenylalanine kinetics in nine IDDM and seven control subjects, both at basal euglycemic conditions and during a euglycemic hyperinsulinemic clamp (approximately 60-80 microU/ml of plasma free insulin), combined with an intravenous infusion of amino acids (AA), which doubled plasma concentrations of most AA. In the basal state, euglycemia was maintained in IDDM subjects at the expense of a peripheral free insulin level (16 +/- 2 microU/ml) greater (P less than 0.05) than controls (9 +/- 1 microU/ml). Despite that, leucine rate of appearance (Ra), alpha-ketoisocaproate oxidation (approximating leucine-carbon oxidation), and nonoxidative leucine disposal, were greater (P less than 0.05) in IDDM than in control subjects. Phenylalanine Ra was slightly but not significantly greater in IDDM vs. control subjects. During the clamp, at comparable plasma free insulin and amino acid concentrations, oxidation was similar in the two groups, endogenous leucine and phenylalanine Ra remained significantly greater (P less than 0.05) in IDDM than in normal subjects, and leucine disposal tended also to be greater in IDDM subjects. Thus, in IDDM subjects maintained at euglycemia, endogenous Ra of essential amino acid(s) (index of endogenous proteolysis) is increased, both in the postabsorptive state and after hyperinsulinemia combined with hyperaminoacidemia, while leucine utilization for protein synthesis is not impaired.

Effect of Infused Branched-Chain Amino Acids on Muscle and Whole-Body Amino Acid Metabolism in Man

1. Using the forearm balance method, together with systemic infusions of L-[ring-2,6-3H]phenylalanine and L-[1-14C]leucine, we examined the effects of infused branched-chain amino acids on whole-body and skeletal muscle amino acid kinetics in 10 postabsorptive normal subjects; 10 control subjects received only saline. 2. Infusion of branched-chain amino acids caused a four-fold rise in arterial branched-chain amino acid levels and a two-fold rise in branched-chain keto acids; significant declines were observed in circulating levels of most other amino acids, including phenylalanine, which fell by 34%. Plasma insulin levels were unchanged from basal levels (8 +/- 1 mu-units/ml). 3. Whole-body phenylalanine flux, an index of proteolysis, was significantly suppressed by branched-chain amino acid infusion (P less than 0.002), and forearm phenylalanine production was also inhibited (P less than 0.03). With branched-chain amino acid infusion total leucine flux rose, with marked increments in both oxidative and non-oxidative leucine disposal (P less than 0.001). Proteolysis, as measured by endogenous leucine production, showed a modest 12% decrease, although this was not significant when compared with saline controls. The net forearm balance of leucine and other branched-chain amino acids changed from a basal net output to a marked net uptake (P less than 0.001) during branched-chain amino acid infusion, with significant stimulation of local leucine disposal. Despite the rise in whole-body non-oxidative leucine disposal, and in forearm leucine uptake and disposal, forearm phenylalanine disposal, an index of muscle protein synthesis, was not stimulated by infusion of branched-chain amino acids. 4. The results suggest that in normal man branched-chain amino acid infusion suppresses skeletal muscle proteolysis independently of any rise of plasma insulin. Muscle branched-chain amino acid uptake rose dramatically in the absence of any apparent increase in muscle protein synthesis, as measured by phenylalanine disposal, or in branched-chain keto acid release. Thus, an increase in muscle branched-chain amino acid concentrations and/or local branched-chain amino acid oxidation must account for the increased disposal of branched-chain amino acids.

Failure of Dehydroepiandrosterone to Influence Energy and Protein Metabolism in Humans*

It was reported recently that 4 weeks of dehydroepiandrosterone (DHEA) treatment [5.55 mmol/day (1600 mg/day), orally] reduced body fat and increased lean body mass in healthy men. The present study was performed to examine whether these effects could be explained by increased energy expenditure and muscle protein synthesis. Eight healthy men were given placebo and DHEA (1600 mg/day) for 4 weeks each in a double blind cross-over study. DHEA treatment caused a 9-fold increase in mean plasma DHEA sulfate concentrations, but had no significant effect on body weight or on two indices of lean body mass (total body water and total body potassium). DHEA had no effect on any of the parameters of energy and protein metabolism, including resting metabolic rate, total energy expenditure (estimated by the 2H2(18)O method during the final 2 weeks of each treatment period), leucine flux (an index of whole body proteolysis), the nonoxidized portion of leucine flux (an index of whole body protein synthesis), and the rate of incorporation of leucine into muscle protein. Circulating levels of cholesterol, T3, and T4 also were unaffected by DHEA. These data suggest that DHEA is not an important regulator of energy or protein metabolism in humans.

Relationship of resting metabolic rate to body composition and protein turnover

This study examined whether variability among healthy young adults in resting metabolic rate, normalized for the amount of metabolically active tissue (assessed by total body potassium), is related to protein turnover. Resting metabolic rate was measured by indirect calorimetry for 2 h in 26 men and 21 women, 19-33 yr old, with simultaneous estimation of protein turnover during a 4-h infusion of L-[1-13C]leucine. After adjusting metabolic rate for total body potassium, the standard deviation was only 89 kcal/day, or 5.5% of the average value. There was a high correlation between leucine flux (an index of proteolysis) and metabolic rate (r = 0.84) and between the nonoxidized portion of leucine flux (an index of protein synthesis) and metabolic rate (r = 0.83). This relationship was weaker, but still significant, after adjusting leucine metabolism and metabolic rate for total body potassium (r = 0.36 for leucine flux vs. metabolic rate, r = 0.33 for nonoxidized portion of leucine flux vs. metabolic rate, P less than 0.05). The regression analysis suggested that the contribution of protein turnover to resting metabolic rate was approximately 20% in an average subject. Metabolic rate and protein turnover were highest in the subjects with the greatest amount of body fat, even after accounting for differences in whole body potassium. Neither resting metabolic rate nor protein turnover was related to total or free concentrations of thyroxine or triiodothyronine, within the euthyroid range.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of protein turnover by carbohydrate overfeeding in men.

The effect of carbohydrate overfeeding on protein metabolism was studied in 11 healthy men. Total urinary nitrogen output during 10 days of carbohydrate overfeeding (1,600 extra kcal/day) decreased 27% relative to nitrogen excretion during 10 days of weight maintenance, indicating protein accretion during over-feeding. However, postabsorptive nitrogen excretion did not change, which means that the positive nitrogen balance associated with overfeeding results from enhanced postprandial nitrogen retention. Overfeeding reduced postabsorptive glucose concentrations 4 +/- 1% and increased glucose production rate 14 +/- 2% and glucose clearance 17 +/- 4%. Overfeeding increased plasma concentrations of insulin, glucagon, and 3,5,3'-triiodothyronine approximately 20%. Alanine and branched-chain amino acid concentrations were increased after overfeeding, but serine, threonine, and asparagine concentrations were reduced. Postabsorptive leucine flux, which is an index of proteolysis, was measured using L-[1-13C]leucine as a tracer. Overfeeding increased leucine flux 13 +/- 2% compared with values after 10 days on a weight-maintenance diet. If it is assumed that overfeeding did not alter the fraction of 13CO2 not recovered in breath, there was no change in the portion of leucine flux that was oxidized. Thus the difference between flux and oxidation, which is a theoretical index of protein synthesis, increased 12 +/- 3% after overfeeding. These data suggest that excess caloric intake, without an increase in protein intake, stimulates post-absorptive proteolysis and protein synthesis.

Preservation of insulin effects on glucose production and proteolysis during fasting.

To determine whether fasting alters the ability of insulin to suppress endogenous glucose production or proteolysis, isotopic flux measurements of glucose, leucine, alpha-keto-isocaproate, and alanine were made in 13 normal volunteers after a 14-h (day 1) and an 84-h (day 4) fast. Using the "pancreatic clamp" technique, we achieved small increments in plasma insulin concentrations, as well as constant and identical plasma hormone and glucose concentrations, on both study days in seven subjects. The remaining six subjects were infused with saline and served as controls. Leucine rate of appearance (an index of proteolysis) was greater on day 4 than on day 1 (P less than 0.001), but decreased to equal values during the pancreatic clamp on each study day. During the pancreatic clamp, endogenous glucose production decreased to lesser (P less than 0.005) values on day 4 than on day 1. In conclusion, insulin suppresses proteolysis equally well before and after brief fasting, and endogenous glucose production is more completely suppressed by insulin after brief fasting than after an overnight fast.

Accelerated ripening of cheese using liposome‐encapsulated enzyme

SummaryImproved efficiency of liposome encapsulation of a cheese‐ripening enzyme and increased retention of encapsulated enzyme by cheese curd are reported. The economy of enzyme usage for accelerating the rate of ripening of Cheddar cheese has been improved about 100‐fold over previously reported methods. This has made feasible the use of microencapsulated enzymes for large‐scale cheese production. Objective measurement of cheese ripening by proteolytic indices, and subjective evaluation of flavour quality and intensity by trained taste panels, indicate that cheeses are ripened by the microencapsulated enzyme in half the normal time. Investigations aimed at further improving the efficiency and commercial feasibility of the process are reported, and the possibility of using this technology for other aspects of food processing are discussed.

Inflammatory and neoplastic lesions of the oral cavity

To determine the oral candidal carriage (OCC), activity of virulent factors and fluconazole susceptibility in subjects with type 2 diabetes mellitus (T2DM) and investigate their association with HbA1c measurements.A cross sectional study was conducted on 100 diabetics and 100 healthy volunteers. The virulence was assessed by measuring the phospholipase activity and proteolysis index. Fluconazole susceptibility was performed using the gradient diffusion method. The OCC, virulence factors and antifungal susceptibility were correlated with patients’ HbA1c measurements.The OCC and candidal density carriage was significantly higher in diabetics. Candida albicans (C. albicans) was the most frequently isolated species followed by Candida tropicalis (C. tropicalis). Relatively uncommon species, Candida lusitaniae (C. lusitaniae) and Candida lipolytica (C. lipolytica) were isolated from the diabetics. Prevalence of virulence factor, proteinase, was greater in diabetic group (p < 0.05). Reduced fluconazole susceptibility was noted among the isolates from diabetics; however it was not statistically significant (p = 0.593). Except one, all the susceptible-dose dependent and resistant isolates were Candida no-albicans (C. non-albicans).C. albicans remains the predominant pathogen in diabetics, although other species are on the rise. Compared to control group, the isolated species from T2DM group had higher proteinase activity. Resistance to fluconazole was considerably greater among the C. non-albicans isolates from T2DM group. These findings warrant effective treatment modalities to reduce the occurrence of oropharyngeal candidiasis.

Indices of Fibrinogen Proteolysis and Platelet Activation During the Resolution of Pulmonary Embolism

Indices of fibrinogen cleavage by thrombin (fibrinopeptide A, FPA); by plasmin (BB1-42 and serum FDP); of the platelet release reaction (beta thromboglobulin and platelet factor 4); and antithrombin III (AT III) levels were measured serially in 46 patients with pulmonary emboli in whom either substantial (SR) or impaired (IR) resolution was documented. The mean FPA level in the 25 patients with IR was significantly higher than the level in the 21 patients with SR (p less than 0.01). The increased thrombin activity in the IR group was not due to AT III deficiency or increased platelet reactivity but the elevated FPA levels in the presence of therapeutic levels of anticoagulation indicated that the dose of heparin was inadequate. The higher BB1-42 and FDP levels in this group reflected the increased plasmin activity that follows increased thrombin activity. In a multivariate discriminant analysis, only the FPA data could be used to predict the degree of resolution. Increased thrombin action impairs resolution presumably by producing greater amounts of accreted fibrin on the impacted embolus.

Effect of Chilling and Forewarming Treatments on Heat Stability and Proteolysis in Milk from Individual Cows

Milk samples from 12 Brown Swiss cows were partially defatted by centrifugation at 2500×g for 30min at 37°C and then examined. Coagulation time at 140°C and tyrosine value, as an index of proteolysis, was determined for raw, chilled (2°C/48h), and forewarmed (95°C/10min) portions from each sample. Coagulation times of chilled samples were longer than those of corresponding raw milks. Coagulation times of forewarmed samples were shorter for 9 cows and longer for 3 cows than those of corresponding raw samples. Addition of β-lactoglobulin, bovine serum albumin, or sodium and potassium salts to raw milk did not affect heat stabilities of the resultant systems before or after chilling or forewarming. Tyrosine values increased both for chilled and forewarmed milks.

Proteolysis in Milk Associated with Increasing Somatic Cell Counts

Individual cow samples were collected and preserved with potassium dichromate. Somatic cells counts were determined. Tyrosine value was used as an index of proteolysis. Sixty-six samples ranged in somatic cell count from < 50,000 to > 2,000,000/ml. Initial milk tyrosine values and tyrosine values for milks incubated for 24h at 37°C showed proteolytic activity increased with increasing somatic cell count. The increase in proteolysis in preserved milk refrigerated for 72h at 6.7°C was over 1.5 times greater in milks with > 1,000,000 cells/ml than in milks with < 60,000 cells/ml. When preserved milks were laboratory pasteurized, cooled, and stored at 6.7°C for 14 d, some proteolytic activity was detected in milks at all concentrations of somatic cells, and proteolysis increased as somatic cell counts increased. Laboratory-pasteurized samples of milk with various somatic cell counts were also incubated at 30°C for 3 and 6h to duplicate the proteolysis that could occur during the ripening, coagulation, cutting, and cooking steps of cheese making. Again, the greatest increases in tyrosine were in milks with high somatic cell count. Protease(s) associated with elevated somatic cell counts will damage raw milk quality upon storage, pasteurized fluid milk over shelf-life, and milk during cheese making.

Role of cellular proteinases in acute myocardial infarction I. proteolysis in nonischemic and ischemic rat myocardium and the effects of antipain, leupeptin, pepstatin and chymostatin administered in vivo

To test the hypothesis that cellular proteinases contribute to ischemic myocellular death, measurements were made of tyrosine release (an index of overall proteolysis) from incubated slices of nonischemic and ischemic myocardium obtained at various times after coronary artery occlusion in rats. Proteolysis failed to increase in ischemic myocardium throughout the first 24 hours of occlusion, when irreversible damage develops, indicating that cellular proteinases do not undergo generalized activation in this phase. These data represent the first assessment of myocardial proteolysis throughout the development of ischemic death, and suggest that cellular proteinases do not play a causal role in this process. However, the possibility remains that ischemia selectively accelerates the breakdown of vital proteins, a phenomenon that may not be detected by measuring overall proteolysis. To determine whether future studies on the effects of proteolytic inhibition on infarct size are feasible, the ability of the proteinase inhibitors antipain, leupeptin, pepstatin and chymostatin, given in vivo, to interfere with proteolysis in ischemic myocardium was also evaluated. Leupeptin (10 or 40 mg/kg) inhibited proteolysis in a dose-related fashion (-49 and -72%, respectively, p less than 0.001). Antipain (20 mg/kg) decreased protein breakdown by 60% (p less than 0.001). The combination of antipain (20 mg/kg), leupeptin (40 mg/kg) and pepstatin (5 mg/kg) suppressed proteolysis almost completely at both 15 minutes (-88%, p less than 0.001) and at 6 hours (-72%, p less than 0.05) of ischemia, that is, throughout the development of irreversible injury. These results demonstrate that whatever proteolysis is occurring during acute myocardial infarction is largely mediated by cathepsins A, B, D, L and H and by calcium-activated neutral protease (that is, the enzymes sensitive to the inhibitors used). Because antipain, leupeptin and pepstatin significantly suppress such proteolysis, these agents might be useful in further assessing any potential contribution of cellular proteinases to the production of ischemic myocellular death. In addition, this study provides a new experimental model that affords serial assessments of regional myocardial proteolysis during the evolution of myocardial infarction.

Application of Dialysis of Measure Protease Activity in Milk

A dialysis technique for measuring proteolysis in milk was investigated. This method consists of adding buffered enzyme substrate mixture to a dialysis bag that is immersed into a test tube containing 10ml distilled water. After incubation at 37°C for a specific time the bag is discarded. Ten milliliters of 15% sodium carbonate solution is added to contents of the test tube followed by phenol reagent. Color is developed for 15min before absorbance at 650nm is measured. This simple procedure had a sensitivity equal to the Hull method, required less glassware and reagents, and did not form precipitates.This dialysis test with 30-min incubation also may be used to determine free tyrosine as an index of proteolysis in stored milk. It is as sensitive as the Hull method.

Beneficial effects of ibuprofen in acute myocardial ischemia.

Ibuprofen, a nonsteriodal anti-inflammatory agent, was studied in the early stages of myocardial ischemia in order to determine whether it helps preserve myocardial integrity. Ibuprofen was administered intravenously at a dose of 12.5 mg/kg at the time of coronary artery occlusion and again 2.5 h later. Ibuprofen significantly prevented the loss of myocardial creatine phosphokinase (CPK) release in ischemic cardiac tissue. In addition, this drug significantly returned S-T segment elevation toward normal values, and significantly prevented the myocardial loss of compounds having free amino nitrogen groups, an index of proteolysis. Although ibuprofen moderated the increased plasma CPK activity, plasma CPK values 5 h after coronary occlusion were above control values. Thus, ibuprofen significantly prevented alterations in three of the four indices used to assess myocardial ischemic damage. The protective mechanism of ibuprofen may be via stabilization of cellular membranes (i.e., lysosomal membranes) and to a lesser extent on reduction in myocardial oxygen demand.

Pancreatic hypoperfusion and the production of a myocardial depressant factor in hemorrhagic shock.

Hemorrhagic shock was produced in dogs by bleeding to a systemic blood pressure of 45 mm Hg for 3 hours, followed by reinfusion of the shed blood. A rapid decrease in pancreatic blood flow occurred and pancreatic perfusion remained at 15-25% of control over the entire 3-hour oligemic period. As a consequence of this marked degree of pancreatic hypoperfusion, autolytic changes occurred in pancreatic acinar cell ultrastructure, particularly in the enlarging of lysosomes which developed many vacuoles. Plasma proteolytic indices (e.g., cathepsin D activity and amino nitrogen concentration) markedly increased during shock as well as the activity of a myocardial depressant factor (MDF). MDF was also produced in incubated pancreatic homogenates obtained from nonshocked dogs and in non-incubated homogenates from shocked dogs. MDF activity in the homogenates was closely correlated with amino nitrogen concentration. These data suggest that pancreatic hypoperfusion plays a key role in MDF formation and ultimately in the pathogenesis of circulatory shock. Moreover, MDF activity was found not to be associated either with pentobarbital concentration or the salt content of active fractions of plasma and pancreatic tissue. Ashing of active fractions was very effective in destroying MDF activity. These data are consistent with the earlier findings that indicate MDF to be a peptide having a molecular weight of 500-1,000.

Proteolysis detection in milk. I. Interpretation of tyrosine value data for raw milk supplies in relation to natural variation, bacterial counts and other factors.

SummaryA method for detection of proteolysis in milk was evaluated. Amino acids and peptides soluble in trichloroacetic acid were estimated by the Lowry–Folin procedure with expression of results in terms of colour yield equivalent to that of tyrosine (the ‘tyrosine value’ (TV)). Some variables and alternatives in the method of estimation were investigated.TVs for milk freshly drawn from individual cows were extremely varied (0·31–0·92 mg/ml). TVs for samples of refrigerated milk from daily supply farms were in the range 0·40–0·58 mg/ml; no relationships between TV and bacterial counts were evident with these samples. With bulk refrigerated raw milk supplied to Brisbane for liquid consumption from country depots and local farms equipped with bulk vats, significant positive relationships were found between TV and total bacterial count (TBC) for supplies from some sources, but in no instance was a significant relationship found between TV and psychrotroph or proteolytic psychrotroph count. Significant positive relationships were found between TV of bulk milk supplies from some sources and atmospheric temperature, and between TBC of the supplies and atmospheric temperature. The significance of these various relationships for bulk milks is discussed.Natural variation in TV imposed limitations on reliability of the method to provide an index of proteolysis; in general the results indicated that its application was restricted to bulk milk cold-stored for at least 3 days.

Effect of Some Manufacturing Variables on the Leakage Defect of Blue Cheese1,2

Effect of certain manufacturing variables on the leakage (water-exuding defect of 60-day-old Blue cheese was examined by a split-plot experimental design. The variables on a pilot-plant scale were starter culture, the treatment of cream or milk for whitening, amounts and types of nonfat dry milk fortification of vat milk, amount of salt added to the curd at hooping, temperature during overnight drainage in hoops, and type of coating used over the cheese during curing. Chemical analyses of cheeses for pH, salt, milk fat, and proteolysis index were made after 24 hr and (or) 60 days after manufacture. Flavor evaluations were made after 60 days.Statistical analyses of the data showed that the starter culture, rate of salt addition, temperature of overnight drainage, and the whitening treatment for the cream were highly significant in influencing the leakage tendency of 60-day-old Blue cheese. No statistical correlation, however, was obtained between any of the manufacturing variables and the flavor scores of cheeses.

LIPOLYTIC AND PROTEOLYTIC ACTIVITY OF ENTEROCOCCI AND LACTIC GROUP STREPTOCOCCI ISOLATED FROM YOUNG CHEDDAR CHEESE

Lipolytic and proteolytic screening techniques were applied to cultures isolated from young Cheddar cheese manufactured in 10 Iowa cheese plants. Twenty-one cultures were selected for study. These included 16 enterococci and 5 lactic group streptococci. These strains were examined for lipolytic activity when grown in skimmilk, cream, and skimmilk containing tributyrin; changes in proteolysis index, plate counts, and pH in skimmilk incubated at 7, 15, 21, and 32 C also were determined, And, combinations of enterococci and lactic streptococci were studied.One-half of the Streptococcus durans strains frequently produced as much as 10 times more acetic acid than the others; the five strains of lactic streptococci consistently produced the lowest quantities of acetic acid. Compared with enterococci, except for Streptococcus faecalis var. liquefaciens, the lactic streptococci were more proteolytic, produced lower pH values, and had less viability at 15, 21, and 32 C. Enterococci other than S. faecalis var. liquefaciens were not proteolytic. All cultures showed tributyrinase activity; enterococci were the most active. Combining enterococci and lactic streptococci produced anomalous results.