No abstract

The apparent instability of ,B-galactosidase in toluene-treated cells or cell-free extracts of lactic streptococci is explained by the fact that these organisms do not contain the expected enzyme. Instead, various strains of Streptococcus lactis, S. cremoris, and S. diacetilactis were shown to hydrolyze o-nitrophenyl-f3-D-galactoside-6-phosphate (ONPG-6-P), indicating the presence of a different enzyme. In addition, lactose metabolism in S. lactis C2F was found to involve enzyme I (El), enzyme II (EII), factor III (FIII), and a heat-stable protein (HPr) of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system analogous to that of Staphylococcus aureus. Mutants of S. lactis C2F, defective in lactose metabolism, possessed the phenotype lac-gal-. These strains were unable to accumulate 4{Cthiomethyl-,3-D-galactoside, to hydrolyze ONPG, or to utilize lactose -when grown in lactose or galactose broth. In addition, these mutants contained EI and HPr, but lacked EII, FIII, and the ability to hydrolyze ONPG-6-P. This suggested that the defect was in the phosphorylation step. Lactose-negative mutants of S. lactis 7962, a strain containing j3-galactosidase, could be separated into several classes, which indicated that this organism is not dependent upon the PEP-phosphotransferase system for lactose metabolism.on August 4, 2020 by guest

The effect of sodium fluoride on lactose metabolism and o-nitrophenyl-,B-Dgalactopyranoside (ONPG) hydrolysis by Streptococcus lactis strains 7962 and C2F suggested that different mechanisms of lactose utilization existed in the two strains. Sodium fluoride prevented lactose utilization and ONPG hydrolysis by whole cells of S. lactis C2F but had no effect on S. lactis 7962. Although hydrolysis of ONPG by toluene-treated cells of S. lactis 7962 occurred without addition of phosphoenolpyruvate (PEP), toluene-treated cells of S. lactis C2F required the presence of this cofactor. Concentrated cell extracts of S. lactis C2F hydrolyzed ONPG; this hydrolysis was inhibited by NaF, but the addition of PEP, in the presence of NaF, restored maximal activity. Addition of acetyl-phosphate, carbamyl-phosphate, adenosine-5'-triphosphate, guanosine-5'-triphosphate, or uridine-5'-triphosphate did not stimulate activity. The presence of cofactors did not stimulate and NaF did not inhibit the hydrolysis in extracts of S. lactis 7962. To confirm the operation of two mechanisms, S. lactis 7962 was shown to hydrolyze lactose to glucose and galactose, whereas S. lactis C2F was unable to split the disaccharide. In addition, whole cells of S. lactis C2F rapidly accumulated a phosphorylated derivative of thiomethyl-3-D-galactoside (TMG) which behaved chromatographically and electrophoretically like TMG-PO4. Unexpectedly, S. lactis 7962 also accumulated a TMG derivative, although the rate was extremely low. These data indicate that different mechanisms of lactose utilization exist in the two strains, with a phosphorylation step dependent on PEP involved in S. lactis C2F.

3-galactosidase by several strains of Streptococcus lactis was induced by lactose. The rate of hydrolysis of o-nitrophenyl-,3-D-galactopyranoside was used to measure enzyme activity. The enzyme of all but one strain was unstable when whole cells were soIniCtreated or treated with toluene; the enzyme of one strain of S. lactis was stable to these treatments, which resulted in at least a fivefold increase in activity over that found in whole cells. The optimal assay conditions for toluene-treated cells of this strain involved incubation at 37 C in pH 7.0 sodium phosphate buffer. Lactose was the most effective inducer of enzyme synthesis. Methyl-3-D-thiogalactopyranoside, isopropyl-fl-D-thiogalactopyranoside, and galactose were also inducers of the enzyme, but were not as effective as lactose. Melibiose, maltose, and calcium lactobionate were poor inducers of enzyme synthesis. Exogenously supplied glucose repressed enzyme synthesis. The means of control of induced ,B-galactosidase synthesis in S. lactis was similar to that in Escherichia coli. added to a concentration of 0.002 g/ml. Buffer solutions. Cells were washed, suspended, and assayed in one of the following buffer solutions: 0.05 M sodium phosphate (pH 6.0 to 8.0), 0.05 M potassium phosphate (pH 7.0), 0.05 M tris (hydroxymethyl)aminomethane (Tris) (pH 7.0), 0.05 M Tris + 0.05 sodium chloride (pH 7.0), or 0.05 M Tris + 0.05 M sodium phosphate (pH 7.0). When indicated, MnCl2*4H20 was added to a concentration of 0.0004 M. Harvesting of cells. Culture samples were immediately chilled and centrifuged at 3,000 X g 937

Development of fecal Lactobacillus and coliform in healthy newborn pigs during the first 48 h after birth was studied. Lactobacilli were detected (104 per g) in the feces of newborn pigs as early as 4 h after birth and colifroms by 8 h (105 per g). By 24 h the two types were present in near equal numbers (104 to 105/g). A frozen concentrate of a human isolate of Lactobacillus lactis was fed to piglet litters (8 to 10 animals per litter) from the time of their birth. Bottle feeding resulted in reduced fecal coliforms in nursing pigs but lactobacilli were not increased in number. After 54 days of treatment, the Lactobacillus to coliform ratio (L/C) was 1280:1; in control pigs not fed lactobacilli, the ratio was 2: 1. A continued suppression of coliforms was observed for 30 days after treatment was discontinued. The influence of Lactobacillus on the bacterial flora of the gastrointestinal tract was studied. With scouring pigs, enteropathogenic Escherichia coli (EEC) were present in larger numbers in tissue homogenates of the tract than in the lumen. The virulence of the EEC found prosent was confirmed by experimental infection in pigs. In control, nonscouring pigs only non-EEC were isolated from tissue sections. In Lactobacillus-fed pigs, E. coli was reduced to low numbers; also, the few E. coli observed were non-enteropathogenic. There were higher numbers of lactobacilli in tissue sections of Lactobacillus-fed pigs than in control and scouring pigs. The lactobacilli isolated from tissue homogenates of the treated animals resembled biochemically and serologically (fluorescent antibody staining) the Lactobacillus which was fed. Histological studies were done to observe the bacteria in frozen sections of washed intestine obtained from Lactobacillus-fed pigs; staining revealed large numbers of gram-positive bacilli. On the other hand, control pigs which died of scouring revealed many coliform types present. Pigs in groups receiving colostrum and lactobacilli did well; no evidence of diarrhea was seen and many lactobacilli were observed in tissue throughout the small intestine. Even after the challenge with EEC serotype 09:K:NM, these two groups of pigs did not show any signs of disease and few coliform types (cocco-bacillary forms) were observed. Pigs not receiving colostrum but only lactobacilli did not scour before challenge and many lactobacilli were present in tissue from the small intestine. However, 72 h after challenge these latter animals revealed symptons of diarrhea and coliforms were seen in the small intestine tissue in addition to lactobacilli.

Early and recent literature on the nomenclature and sources of lactic streptococci in nature is reviewed. Evidence is presented in favor of including Streptococcus diacetilactis as a recognized species in the group N streptococci. Despite many research efforts, the natural habitat of Streptococcus cremoris remains unknown. An appeal for more ecological studies on these important bacteria is made.

Field and laboratory observations (Frazier et al. 1935) long have indicated that the temperature of incubation of Swiss cheese starter cultures significantly influences their ability to develop following the rather severe heat exposure to which they are subjected during manufacture. If it were true that the temperature of growth had an effect on heat resistance of bacteria, this fact would be of significance in various fermentations, both commercial and natural, and would prove of general interest from the standpoint of the physiology of bacteria. Therefore, an investigation was initiated to determine the influence of incubation temperature and time on the thermal resistance of certain Swiss cheese starter cultures. For the purpose of comparison, similar studies were undertaken with a typical strain (H-52) of Escherichia coli. The results of the investigations on E. coli are presented in this paper. Because it produced less acid, was able to develop under a wider variety of environmental conditions, and could be counted fairly accurately by the plate method, E. coli gave results which were more conclusive than those obtained with the lactic starter cultures and indicated that E. coli, a favorite subject for experimentation, is an ideal organism for studies on the heat resistance of vegetative cells. Despite the apparent importance of the relationship between the growth temperature and thermal resistance of bacterial cells, iThis work has been aided by a grant from the Wisconsin Alumni Research Foundation.

The ability of Streptococcus diacetilactis to inhibit a variety of food spoilage organisms and pathogens in milk and broth cultures was demonstrated. Test organisms included Pseudomonas and Alcaligenes species, Salmonella, Staphylococcus aureus, Clostridium perfringens, and Vibrio parahaemolyticus. In general, approximately 99.0% and 99.9% inhibition was observed in milk and broth, respectively. Possible practical applications of the inhibition were examined. Addition of S. diacetilactis extended the shelf life of artificially contaminated cottage cheese and prevented proteolysis in milk at 7.5 C by Pseudomonas fluorescens. Staphylococcus aureus was inhibited greater than 99% in vanilla cream filling, ham sandwich spread, chicken gravy, soy milk, and ground beef stored at 25 C for 24 hr. Development of the gram-negative flora of ground beef was also inhibited greater than 99% after storage at 7.5 C for 7 days. Possible roles for several factors in the mechanism of inhibition by S. diacetilactis are briefly discussed. The effects of pH reached and acids produced by S. diacetilactis on the growth of S. aureus are described. A greater role for the lactic acid bacteria in fermentations in the food industry is suggested.