Changes in protein synthesis during thermal adaptation of Propionibacterium freudenreichii subsp. shermanii

Dairy propionibacteria are present in Graviera Kritis, a traditional Gruyère-type cheese made without added propionic starter. Ten isolated strains were identified by a combination of SDS-PAGE, species-specific PCR and according to their ability to ferment lactose. They were all found to belong to the Propionibacterium freudenreichii subsp. shermanii species. Because of the stressing Gruyère technology, which includes cooking at 52 to 53 degrees C their thermotolerance was investigated at 55 degrees C. Thermotolerant and thermosensitive strains were clearly discriminated. Interestingly, the reference strain CIP 103027 belongs to the sensitive subset. One sensitive strain, ACA-DC 1305 and one tolerant, ACA-DC 1451, were selected for further study and compared to CIP 103027. For the sensitive strains ACA-DC 1305 and CIP 103027, heat pre-treatment at 42 degrees C conferred thermoprotection of cells at the lethal temperature of 55 degrees C, while there was less effect on the tolerant ACA-DC 1451. No cross-protection of salt-adapted cells against heat stress was observed for none of the strains. Differential proteomic analysis revealed distinct but overlapping cell responses to heat stress between sensitive and tolerant strains. Thermal adaptation upregulated typical HSPs involved in protein repair or turnover in the sensitive one. In the tolerant one, a distinct subset of proteins was overexpressed, whatever the temperature used, in addition to HSPs. This included enzymes involved in propionic fermentation, amino acid metabolism, oxidative stress remediation and nucleotide phosphorylation. These results bring new insights into thermoprotection in propionibacteria and the occurrence of divergent phenotypes within a same subspecies.     

Effect of Lactobacillus helveticus and Propionibacterium freudenrichii ssp. shermanii combinations on propensity for split defect in Swiss cheese

One of the least controlled defects in Swiss cheese is development of splits that appear during refrigerated storage after cheese is removed from the warm room. Such fissures, or cracks, in the body of the cheese can be as short as 1 cm, or long enough to span a 90-kg block. A 2 x 2 x 2 factorial experiment was used to determine the effect of different Lactobacillus helveticus/Propionibacterium freudenreichii ssp. shermanii starter culture combinations on the occurrence of split defect in Swiss cheese. Eights vats of cheese were made in summer and eight in winter. Each 90-kg block of cheese was cut into twenty-four 4-kg blocks and graded based on the presence of splits. Only small variations were found in the composition of cheeses made during the same season. There were no correlations between moisture, pH, fat, protein, calcium, lactose contents, D/L lactate ratio, or protein degradation that could be used to predict splits after 90 d of storage. However, cheese made in the summer had 2% higher moisture content and a greater prevalence of splits. There was a sixfold increase in amount of downgraded cheese between the best and worst culture combinations used during cheese manufacture. After 90-d storage, 14 to 90% of cheese had splits in the summer, and 1 to 6% in the winter. Split formation increased with time from 60 to 120 d of storage and extent of split formation was influenced by both the lactobacilli and propionibacteria cultures used.     

Evaluating the effects of Lactobacillus animalis and Propionibacterium freudenreichii on performance and rumen and fecal measures in lactating dairy cows

Two experiments evaluated the effect of supplementation with a bacterial direct-fed microbial on performance and apparent total-tract nutrient digestion of dairy cows. In experiment 1, 30 multiparous cows (75 ± 32 d in milk) were randomly assigned to 1 of 2 treatments fed for 10 wk. All cows were fed a diet containing 23.8% starch. Treatments were top dressed to rations twice daily and consisted of a combination of Lactobacillus animalis (1 × 10⁹ cfu/d) and Propionibacterium freudenreichii (2 × 10⁹ cfu/d; LAPF) or carrier alone (CON). In experiment 2, 6 ruminally cannulated cows (123 ± 129 d in milk) were randomly assigned to a crossover design with two 6-wk periods. Cows received the same CON or LAPF treatment as in experiment 1. Cows were fed the same 23.8% starch diet as experiment 1 during wk 1 through 5 of each period, and then cows were abruptly switched to a 31.1% starch diet for wk 6. For both experiments, intake and milk yield were measured daily, and milk samples were collected weekly. In experiment 1, fecal grab samples were collected every 6 h on d 7 of experimental wk 1, 2, 4, 6, 8, and 10. Fecal consistency was scored, and fecal starch was measured in daily composite samples. Fecal composites from a subset of 7 cows per treatment were used to measure apparent total-tract nutrient digestion. In experiment 2, rumen pH was continuously recorded during wk 5 and 6. On d 7 of wk 5 (the final day of feeding the 23.8% starch ration), d 1 of wk 6 (the day of diet transition), and d 7 of wk 6 (the final day of feeding the 31.1% starch ration), rumen in situ digestion was determined. Samples of rumen fluid and feces were collected every 6 h on those days for measurement of fecal starch (composited by cow within day), rumen volatile fatty acids, and fecal pH. Rumen and fecal samples were collected at one time point on those days for microbiota assessment. In experiment 1, treatment did not affect intake, milk yield, milk composition, or fecal score. The LAPF treatment decreased fecal starch percentage and tended to increase starch digestion compared with CON, but the differences were very small (0.59 vs. 0.78% and 98.74 vs. 98.46%, respectively). Digestion of other nutrients was unaffected. In experiment 2, LAPF increased rumen pH following the abrupt switch to the high-starch diet, but milk yield was lower for LAPF compared with CON (35.7 vs. 33.2 kg/d). Contrary to the decrease in fecal starch with LAPF observed in experiment 1, fecal starch tended to be increased by LAPF following the abrupt ration change in experiment 2 (2.97 vs. 2.15%). Few effects of treatment on rumen and fecal microbial populations were detectable. Under the conditions used in our experiments, addition of the bacterial direct-fed microbials did not have a marked effect on animal performance, ruminal measures, or total-tract nutrient digestion.     

Inability of probiotic bacterial strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 to induce human platelet aggregation in vitro

Platelet aggregation contributes to the pathogenesis of infective endocarditis, and aggregation of platelets induced by lactobacilli is thought to be an important contributory factor in the development and progression of Lactobacillus endocarditis. The main purpose of this study was to examine the effect of immunity-enhancing probiotic strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 on the activation and aggregation of human blood platelets. Whole blood samples from healthy individuals were incubated in vitro with HN001 or HN019 and subsequently labeled with platelet-specific monoclonal antibodies, fluorescein isothiocyanate-conjugated anti-CD41a (expressed on normal platelets), and phycoerythrin-streptavidin-conjugated anti-CD62p (expressed on activated platelets) before analysis by flow cytometry. Platelet-rich plasma was used to assist the gating of the platelet cluster. ADP and epinephrine were used as the physiological platelet activation agonists. Platelet aggregation-inducing strain Streptococcus sanguis 133-79 was used as a positive control strain. The mean fluorescence intensity of phycoerythrin and the percentage of platelets expressing the CD62p marker were used to assess the degree of platelet activation. The percentage of CD62p-positive platelets and the light scatter profiles of the agonist-activated platelets were used to identify the occurrence and degree of platelet aggregation. HN001 and HN019 had no effect on spontaneous platelet activation and aggregation; they also failed to exacerbate the platelet aggregation activity induced by ADP and epinephrine. Therefore, these test probiotic strains HN001 and HN019 are less likely to participate in the pathogenesis of infective endocarditis or other thrombotic disorders with regard to platelet aggregation factors.     

Effect of clinical and probiotic Lactobacillus rhamnosus strains on intestinal permeability and bacterial translocation in healthy and colitic rats

Lactobacillus rhamnosus is one of the most widely used probiotic microorganisms. Although this microorganism is not known for its virulence some rare cases of bacteraemia and endocarditis have been observed. Thus, it is important to identify the possible risks associated with each strain. Comparison of clinical and probiotic strains may give information on properties that could be a safety concern. A possible adverse effect is bacterial translocation. Animals with induced-colitis constitute a good model to assess translocation in the case of mucosal barrier disruption. We aimed at determining the ability of five L. rhamnosus strains, from clinical or probiotic origin, showing different in vitro properties, to induce in vivo translocation in both, healthy and colitic animals. We also tested the effects on the gut mucosal lining by measuring intestinal permeability. None of the in vitro parameters used for selection of the strains included in this study appear to be a risk for translocation or mucosal barrier disruption.     

Substances with biological activity of vitamin B12 formed during cultivation of Propionibacterium freudenreichii in the presence of precursors (short communications)

The papers of Kolhouse et al. and Cooper et al. described the occurrence of vitamin B12 analogues of unknown origin in blood serum. Some of these analogues may be derived from slaughter cattle raised on feed supplemented with vitamins and minerals, as was observed by Allen. Herbert et al. found vitamin B12 analogues in multivitamin preparations produced in U.S.A., and Kanazawa et al. in human liver, red cells and brain. It is not clear so far, if and how do vitamin B12 analogues interfere with vitamin B12 metabolism. When P. freudenreichii was cultivated in the presence of o-phenylenediamine and 5,6-dimethylbenzimidazole, which may be considered precursors as well as antimetabolites of vitamin B12, the stimulation of biosynthesis of substances with biological activity of vitamin B12 took place. Various signs show that these substances are probably vitamin B12 analogues. During stimulated and nonstimulated production of vitamin B12 by P. freudenreichii, two substances with vitamin B12 biological activity have always been obtained. Their relation was not stable and differed according to the conditions of cultivation. Every attempt to stimulate the biosynthesis of vitamin B12 resulted in the suppression of production of the substance with higher molecular weight, even if the biosynthesis of cobalamin (lower molecular weight) was increased. In our note we want to pay attention to the character of substances arising in the stimulated biosynthesis.     

The protective effect of monosodium glutamate on survival of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus E-97800 (E800) strains during spray-drying and storage in trehalose-containing powders

The use of trehalose as a means of preserving Lactobacillus rhamnosus GG (LGG) and L. rhamnosus E-97800 (E800) during spray-drying and the effects of incorporated monosodium glutamate (MSG) in the carrier medium on the survival rates during drying and storage were examined. E800 was more resistant to heat than LGG in 20%, w/w, trehalose; the d-values at 65 °C were 14 s and 5.1 s, respectively. An air outlet temperature of 65–70 °C was taken as optimal for the drying process, as the resultant moisture levels in trehalose containing these bacteria were 4.1% (w/w) and 3.79% (w/w) with corresponding viable counts of 3.65 × 10⁸ cfu mL^?1 and 1.80 × 10⁹ cfu mL^?1, respectively. The presence of MSG increased the final viable counts of LGG and E800 to 3.05 × 10⁹ cfu mL^?1 and 1.30 × 10⁹ cfu mL^?1, respectively. Survival of LGG and E800 remained constant at a minimum level of ～10⁸ cfu mL^?1 during storage at 25 °C in trehalose–MSG medium.     

The influence of vegetarian nutrition on the structure of mucosa in human colon

Structural and functional peculiarities of digestive tract's wall depending from the character of nutrition (vegetarian or mixed diets) are interpreted in morphology. Proper facts and results of investigations of other authors about morphological structure of tunica mucosa colonic according of type of nutrition are discussed.     

The effect of guar gum on microbial activity in the human colon

On feeding a human volunteer a control diet for 21 days, followed by one containing 13·9 g guar/day for a further 21 days, the major changes seen were an increase in faecal output and water content and a decrease in faecal pH and transit time. Faecal output remained high for at least 10 days after return to the control diet. Short chain fatty acid (SCFA) levels/g wet weight of faeces were unchanged during the guar diet but total excretion of SCFA increased because of increased faecal output. Bacterial output was increased although the proportion of guar fermenters remained approximately 20%. Utilization of guar was complete from the beginning of the guar diet, with no period of adaptation. Flatus production substantially increased during the first 10 days of the guar diet but was less noticeable thereafter, suggesting that changes in the pattern of metabolism were taking place in response to continued feeding of guar. Addition of guar to the diet increased α-galactosidase, mannanase and β-mannosidase activity in faeces and most of the activity was associated with the bacterial fraction. In vitro incubation of faecal slurries from the control and guar diets with guar, galactose or mannose showed that SCFA production was lowest and gas production highest when guar was substrate. No effect of diet was seen on utilization of carbohydrate, SCFA and gas production or enzyme activity. Enzyme activity was only induced by guar and was mainly cell-associated.     

The critical role of urease in yogurt fermentation with various combinations of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus

The protocooperation between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus relies on metabolite exchanges that accelerate acidification during yogurt fermentation. Conflicting results have been obtained in terms of the effect of the Strep. thermophilus urease and the NH₃ and CO₂ that it generates on the rate of acidification in yogurt fermentation. It is difficult to perform a systematic study of the effects of urease on protocooperation because it is necessary to distinguish among the direct, indirect, and strain-specific effects resulting from the combination of the strains of both species. To evaluate the direct effects of urease on protocooperation, we generated 3 urease-deficient mutants (ΔureC) of fast- and slow-acidifying Strep. thermophilus strains and observed the effects of NH₃ or CO₂ supplementation on acidification by the ΔureC strains. Further, we examined 5 combinations of 3 urease-deficient ΔureC strains with 2 CO₂-responsive or CO₂-unresponsive strains of L. bulgaricus. Urease deficiency induced a shortage of ammonia nitrogen and CO₂ for the fast- and slow-acidifying Strep. thermophilus and for the CO₂-responsive L. bulgaricus, respectively. Notably, the shortage of ammonia nitrogen had more severe effects than that of CO₂ on yogurt fermentation, even if coculture with L. bulgaricus masked the effect of urease deficiency. Our work established (1) that urease deficiency inhibits the fermentative acceleration of protocooperation regardless of the Strep. thermophilus and L. bulgaricus strain combinations, and (2) that urease is an essential factor for effective yogurt acidification.     

The effect of calcium ions on adhesion and competitive exclusion of Lactobacillus ssp. and E. coli O138

The adhesion abilities of 11 strains of Lactobacillus were determined in vitro using the IPEC-J2 cell line as a model system. Bacteria cultures included the probiotic strains L. rhamnosus GG, L. reuteri ATCC 55730, L. johnsonii NCC 533 and L. reuteri DSM 12246, and new isolates of Lactobacillus ssp. Adhesion was quantified by scintillation counting of radiolabelled bound bacteria. The highest adhesion of 38%, was determined for L. reuteri DSM 12246 followed by L. plantarum Q47 with an adhesion level of 24%. Other strains showed moderate to low binding of less than 16%. Competitive adhesion experiments on IPEC-J2 cells demonstrated that strongly adhesive strains, as L. reuteri DSM 12246 and L. plantarum Q47, significantly reduced the attachment of the less adhesive strains, such as L. rhamnosus GG and L. johnsonii NCC 533, both under condition of co-incubation and in displacement assays, indicating that bacteria may share the same binding sites for attachment to intestinal cells. Furthermore, it was revealed that calcium ions significantly increased the binding of tested lactobacilli to IPEC-J2 cells; and therefore, added calcium may be useful in enhancing the adhesion of normally weakly adhesive probiotic cultures. In contrast, no significant change in adhesion of lactobacilli was observed in the presence of Mg and Zn ions. Displacement assays performed with pathogenic E. coli O138 showed that all tested Lactobacillus strains reduced the attachment of E. coli O138 to IPEC-J2 by more than 2-fold both in the presence and the absence of calcium ions. The strains of Lactobacillus did not differ significantly in the extent of their inhibition of E. coli O138 adhesion, indicating that the reduced adhesion of E. coli O138 was due to steric hindrance of the binding sites rather than to specific interactions.     

Effect of inulin on the growth and metabolism of a probiotic strain of Lactobacillus rhamnosus in co-culture with Streptococcus thermophilus

Metabolic studies are very important to improve quality of functional dairy products. For this purpose, the behaviors of pure cultures of Streptococcus thermophilus (St) and Lactobacillus rhamnosus (Lr) as well a co-culture of them (St–Lr) were investigated during skim milk fermentation, and the inulin effect as prebiotic was assessed. Lr was able to metabolize 6 g/100 g more galactose than St and St–Lr. Final lactic acid production by Lr was higher (9.8 g/L) compared to St (9.1 g/L) and St–Lr (9.1 g/L). Acetic acid concentration varied from 0.8 g/L (St–Lr) to 1.5 g/L (Lr) and that of ethanol from only 0.2 g/L (St–Lr) to 0.4 g/L (Lr), which suggests the occurrence in Lr of a NADH oxidase activity and citrate co-metabolization via pyruvate, both dissipating a part of the reducing power. Diacetyl and acetoin accumulated at the highest levels (18.4 and 0.8 mg/L, respectively) with St–Lr, which suggests possible synergistic interactions between these microorganisms as well as the Lr capability of co-metabolizing citrate in the presence of lactose. Inulin stimulated both biomass growth and levels of all end-products, as the likely result of fructose release from its partial hydrolysis and subsequent metabolization as an additional carbon and energy source.     

Impact of a plant extract on the viability of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in nonfat yogurt

The effect of a plant extract (prepared from olive, garlic, onion and citrus with sodium acetate as a carrier) on the viability of yogurt starter cultures was studied. Nonfat yogurt was prepared with various levels of supplements: plant extract (0, 0.5 or 1.0%, w/v) or l-cysteine HCl (0.014 or 0.028%, w/w). Microbial and physicochemical analyses were conducted weekly for 50 days. Fermentation time increased for supplemented yogurts compared with the non-supplemented yogurt. Lactobacillus bulgaricus counts in supplemented yogurts were >6 log cfu mL^?1 for a longer time (7–21 days) compared with the non-supplemented yogurt. Streptococcus thermophilus counts in all yogurts were > 6 log cfu mL^?1 throughout the storage. Overall, redox potential and titratable acidity of yogurts on day 50 were greater compared with day 1, but pH and syneresis were less. Plant extract at 0.5% enhanced L. bulgaricus viability in nonfat yogurt while least affecting the physicochemical characteristics.     

Immunoadjuvant activity of oral Lactobacillus casei: influence of dose on the secretory immune response and protective capacity in intestinal infections.

Lactobacilli, often used as effectors of host functions, could play an important role in maintaining human health by controlling other intestinal microorganisms capable of producing harmful effects. Using an experimental model, we studied the effect of different oral doses of Lactobacillus casei on the secretory IgA response and the protective capacity of the microorganism in preventing intestinal infections. The optimization of the protective dose of Lb. casei by previous feeding and the use of the lactobacillus as an immunological way to control enteric infections were investigated. We found that conventional mice were protected against infection with Salmonella typhimurium and Escherichia coli by previous feeding for 2 consecutive days with a daily Lb. casei dose of 1.2 x 10(9) cfu/mouse. Previous feeding for 7 d proved less effective, and feeding for 5 d afforded no protection at all. We were also able to demonstrate that the protective effect of Lb. casei against Sal. typhimurium and Esch. coli was connected mainly with the high level of IgA antipathogen antibodies present in intestinal secretions. beta-Glucuronidase (EC 3.2.1.31) and beta-galactosidase (EC 3.2.1.23) activities, measured both in the intestinal fluid and histological samples, showed a marked increase in intestinal inflammatory response on day 5 of feeding. These results show that Lb. casei plays an important role in the prevention of enteric infections, a low dose being enough for protection against intestinal infections by increasing IgA secretion into the intestinal lumen, thus providing adequate defences for the mucosal surface. A previously administered dose of this magnitude could therefore be used as an oral adjuvant in preventing enteric infections.     

Safety assessment of potential probiotic lactic acid bacterial strains Lactobacillus rhamnosus HN001, Lb. acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice

The general safety of immune-enhancing lactic acid bacteria (LAB) strains Lactobacillus rhamnosus HN001 (DR20), Lb. acidophilus HN017, and Bifidobacterium lactis HN019 (DR10) was investigated in a feeding trial. Groups of BALB/c mice were orally administered test LAB strains or the commercial reference strain Lb. acidophilus LA-1 at 2.5 x 10(9), 5 x 10(10) or 2.5 x 10(12) colony forming units (CFU)/kg body weight/day for 4 weeks. Throughout this time, their feed intake, water intake, and live body weight were monitored. At the end of the 4 week observation period, samples of blood, liver, spleen, kidney, mesenteric lymph nodes, and gut tissues (ileum, caecum, and colon) were collected to determine: haematological parameters (red blood cell and platelet counts, haemoglobin concentration, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration); differential leukocyte counts; blood biochemistry (plasma total protein, albumin, cholesterol, and glucose); mucosal histology (epithelial cell height, mucosal thickness, and villus height); and bacterial translocation to extra-gut tissues (blood, liver, spleen, kidney and mesenteric lymph nodes). DNA finger printing techniques were used to identify any viable bacterial strains recovered from these tissues. The results demonstrated that 4 weeks consumption of these LAB strains had no adverse effects on animals' general health status, haematology, blood biochemistry, gut mucosal histology parameters, or the incidence of bacterial translocation. A few viable LAB cells were recovered from the tissues of animals in both control and test groups, but DNA fingerprinting did not identify any of these as the inoculated strains. The results obtained in this study suggest that the potentially probiotic LAB strains HN001, HN017, and HN019 are non-toxic for mice and are therefore likely to be safe for human use.     

Hair growth promoting effect of essence manufactured with products fermented by Lactobacillus rhamnosus and Backryeoncho (Opuntia ficus-indica var. sarboten) fruits in mice

Hair loss is a clinical problem that is becoming more common in the world but it is generally poorly controlled and more effective treatments are required. In this study, we evaluated the hair growth promoting effect of essence manufactured with products fermented by backryeoncho (Opuntia ficus-indica var. sarboten) fruits and Lactobacillus rhamnosus on hair growth in mice. The bacterial number after fermentation always remained higher than 6 log CFU/mL and the pH of those ranged from 4 to 6. Mice topically applied with 0.1 mL essence at once daily for 8 days after shaving furs of their back showed more significant hair growth promoting effect than that of minoxidil. Body weight and food intake of all groups didn’t show significant difference. Furthermore, histological evaluation showed that the essence containing fermented products markedly increased the depth and size of the hair follicles, as compared to the minoxidil.     

The influence of Lactobacillus plantarum culture inoculation on the fate of Staphylococcus aureus and Salmonella typhimurium in Montasio cheese.

The growth and survival of Staphylococcus aureus and Salmonella typhimurium were investigated during the manufacturing and ripening of raw milk Montasio cheese. Initial inoculated populations in the cheese milk were about 10(5) cfu/ml for S. aureus and 10(6) cfu/ml for S. typhimurium. Samples of curds and cheeses were taken during manufacturing and storage and analysed for pH and microbial populations. S. aureus increased slightly in number during the early period of ripening and attained a population of about 10(6) cfu/ml during the remaining period of storage. S. typhimurium decreased during cheesemaking and storage but persisted through 90 days. The addition of Lactobacillus plantarum culture (0.2% v/v) produced a marked reduction in populations of the test strains in 10 days of storage. Enterotoxin A was not detected in Montasio cheese even with a S. aureus population of 1.1 X 10(7) cfu/ml. L. plantarum strains were also tested by the spot method and the associative growth approach for their antagonistic activity against S. aureus and S. typhimurium. The compound excreted by L. plantarum was active only toward S. aureus. Furthermore, its activity was destroyed by protease treatment. These results indicated that while the growth of S. typhimurium is reduced by the acid production, S. aureus inhibition can be ascribed to bacteriocin production.