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Importance of the host specificity in the selection of probiotic bacteria.

The gastrointestinal tract is a complex and dynamic ecosystem. Commensal microorganisms (C), which proliferate in the intestine from birth, are crucial for gut homeostasis while non commensal (NC) microorganisms are transient and enter the organism from the environment and foods. We studied comparatively the influence of oral administration of C and NC Lactobacillus fermentum and Lactobacilus acidophilus on the gut-associated lymphoid tissue (GALT) of conventional mice. To determine the importance of the selection of probiotic host-specificity bacteria with immunomodulating capacity, we examined the interaction with the gut by transmission electron microscopy and FITC-labelled bacteria. We compared the immunomodulation capacities of C and NC strains by studying the number of IgA secreting cells and cytokine profile. No differences were found in the number of IgA+ cells; however, the pattern of cytokine response to C and NC bacteria was different. With regard to proinflammatory cytokine (IFNgamma and TNFalpha), we found that TNFalpha was mainly produced by NC bacteria, while C bacteria were able to elicit mainly IFNgamma. The regulatory cytokines (IL-10 and IL-4) were induced with different patterns for both C and NC strains. No differences in the pathway of internalization to the gut between C and NC were found. In summary, we determined that C and NC bacteria interact with the intestine in the same way; both C and NC bacteria were able to reinforce the surveillance of the gut mucosal immune system. The cytokine profile showed that C bacteria would be involved in the regulation of intestinal homeostasis rather than in the immune activation as the NC bacteria.

Administration, Oral↗

[Evaluation of the effect of probiotic cultures on two different yogurt brands over a known population of Staphylococcus aureus and the production of thermonuclease].

The effect of probiotic cultures over known populations of Staphylococcus aureus inoculated in yogurt was studied; also the production and stability of its thermonuclease during yogurt storage was evaluated. In three different occasions, two different yogurt brands, one with additional probiotic cultures (Lactobacillus casei and L. acidophilus), were inoculated with known populations of S. aureus in high and low concentration (10(9) CFU/g and 10(7) CFU/g), respectively. These samples were stored for 28 days at 5 degrees C. Every four days the count of lactic bacteria, S. aureus and pH were evaluated, according to the methodology described in the Compendium of Methods for the Microbiological Examination of Foods, Vanderzant & Splittstoesser. The presence of thermonuclease was determined using petrifilm for S. aureus from 3M company. The pH and lactic bacteria population were constant during the testing period. Yogurt with additional probiotic cultures (high and low concentration) lowered the population of S. aureus to non detectable levels in 8 days; but, S. aureus could be cultured from yogurt without probiotics even after 24 days of incubation. Same time, the presence of thermonuclease was positive in all tests; it was not affected by probiotics. The presence of thermonuclease is related to the production of S. aureus enterotoxin. This work emphasizes again the beneficial effects of probiotic cultures in yogurt over bacteria and the importance of keeping hygienic practices in order to avoid the contamination of food with S. aureus and the eventual production of its enterotoxin, since it is not affected by probiotics.

Animals↗

Susceptibility to some antifungal drugs of vaginal lactobacilli isolated from healthy women.

Samples were taken from the lateral vaginal wall of 19 healthy women. Lactobacilli were isolated from ten of these women. Fifty-eight Lactobacillus isolates were identified as L. gasseri, L. vaginalis, L. acidophilus, L. delbrueckii spp. lactis, L. crispatus, L. plantarum, L. cellobiosus, L. jensenii, L. salivarius, L. curvatus, L. brevis and L. oris. The inhibitory effect of isoconazole and oxiconazole in 0.2, 0.1 and 0.05 microg/ml concentrations on 58 Lactobacillus spp. and Candida albicans (AJD 180) were investigated, using an agar diffusion method. Although both antifungal drugs showed a large inhibitory effect on C. albicans, oxiconazole had a very low inhibitory effect on Lactobacillus isolates. The natural resistance of lactobacilli to anti-fungal drugs may enable the development of an antifungal drug(s)/ probiotic combination for antifungal activity and lactobacilli replacement therapy.

Antifungal Agents↗

Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley.

A fermented probiotic maize porridge with high energy density and low viscosity was prepared, using maize flour and barley malt. The porridge was fermented with four probiotic strains (grown separately): Lactobacillus reuteri, Lb. acidophilus (LA5 and 1748) and Lb. rhamnosus GG. These strains were inoculated at two levels; to obtain approx. 7 or 6 log cfu g(-1) in the porridge at 0 h. The porridge was fermented for 24 h at 37 degrees C, and analysed for viable cell count, pH, organic acids, volatile aromatic compounds and sugar content. The inoculated cell concentration was shown to be particularly important during the first hours of the fermentation period, showing a delayed production of most metabolites in porridge inoculated with approx. 6 log cfu g(-1). Most strains reached maximum cell count after 12-h fermentation (7.2-8.2 log cfu g(-1)), with a pH below 4.0. Depending on the strain, lactic acid was produced in amounts ranging from 1360 to 4000 mg kg(-1). Lb. reuteri metabolised succinate, while pyruvate and small amounts of diacetyl were detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748. High amounts of diacetyl (6 mg kg(-1)) and acetoin (27 mg kg(-1)) were detected in porridge inoculated with Lb. rhamnosus GG. Porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, contained acetaldehyde, while both Lb. reuteri and Lb. rhamnosus GG reduced the acetaldehyde to ethanol. Lb. reuteri utilised both maltose and glucose as carbohydrate sources, while Lb. acidophilus LA5, Lb. acidophilus 1748 and Lb. rhamnosus GG utilised only glucose.

Carbohydrate Metabolism↗

Plasmid profiling and curing of Lactobacillus strains isolated from the gastrointestinal tract of chicken.

In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.

Animals↗

Effects of yeasts and bacteria on the levels of folates in rye sourdoughs.

Fermentation of rye dough is often accompanied with an increase in folate content. In this study, three sourdough yeasts, Candida milleri CBS 8195, Saccharomyces cerevisiae TS 146, and Torulaspora delbrueckii TS 207; a control, baker's yeast S. cerevisiae ALKO 743; and four Lactobacillus spp., L. acidophilus TSB 262, L. brevis TSB 307, L. plantarum TSB 304, and L. sanfranciscensis TSB 299 originally isolated from rye sourdough were examined for their abilities to produce or consume folates. The microorganisms were grown in yeast extract-peptone-d-glucose medium as well as in small-scale fermentations that modelled the sourdough fermentation step used in rye baking. Total folate contents were determined using Lactobacillus rhamnosus (ATCC 7469) as the growth indicator organism. The microorganisms studied did not excrete folates into the media in significant amounts. Yeasts increased the folate contents of sterilised rye flour-water mixtures from 6.5 microg/100 g to between 15 and 23 microg/100 g after 19-h fermentation, whereas lactic acid bacteria decreased it to between 2.9 and 4.2 microg/100 g. Strains of Lactobacillus bulgaricus, L. casei, L. curvatus, L. fermentum, L. helveticus, Pediococcus spp., and Streptococcus thermophilus that were also tested gave folate contents after fermentation that varied between 2 and 10.4 microg/100 g. Although the four Lactobacillus spp. from sourdough consumed folates their effect on folate contents in co-cultivations was minimal. It was concluded that the increase of folate content during fermentation was mainly due to folate synthesis by yeasts. Fermentation of non-sterilised flour-water mixtures as such resulted in three-fold increases in the folate contents. Two folate producing bacteria were isolated from the non-sterilised flour and identified as Enterobacter cowanii and Pantoea agglomerans.

Bread↗

Lactobacilli counts in crop, ileum and caecum of growing broiler chickens fed on practical diets containing whole or dehulled sweet lupin (Lupinus angustifolius) seed meal.

1. Four experiments with growing broiler chickens were carried out to study the effects of the inclusion in their diets of lupin (Lupinus angustifolius) seed meal of E. coli and lactobacilli counts in crop, ileum and caecca at 3 or 4 weeks of age. 2. Diets were formulated to contain the same amounts of metabolisable energy (12.55 MJ/kg) and protein (210 g/kg). Raw whole (heat-untreated) or dehulled sweet (low in alkaloids) lupin seed meal (400 and 320 g/kg respectively) were used to prepare the lupin-based diets, whose protein content was completed with either defatted soyabean meal or casein. 3. Final body weight and food intake of chickens fed on whole lupin seed meal diets were lower than controls, but gain: food ratios were not different. However, birds given the diet with dehulled lupin seed meal had similar body weight, food intake and gain: food values as those of controls. 4. While E. coli counts were not affected, lactobacilli numbers were consistently increased compared to controls in all intestinal sections of chickens fed on the whole or dehulled lupin-based diets, irrespective of the age of the birds or the presence of soyabean meal or casein in the diet. The lactobacilli species isolated were: Lactobacillus fermentum, L. acidophilus, L. salivarius and L. brevis. 5. The results suggest that the use of whole or dehulled sweet lupin seed meal in diets for growing broilers might enhance the growth of lactic acid--fermenting bacteria in the gut.

Animal Feed↗

Note: lack of influence of adherent Lactobacillus isolates on the attachment of Escherichia coli to the intestinal epithelial cells of chicken in vitro.

Two Lactobacillus isolates, Lact. acidophilus I 26 and Lact. fermentum I 25, were selected, based on their poor aggregation with Escherichia coli and strong ability to adhere to ileal epithelial cells (IEC), to study in vitro interactions with E. coli O1:K1, O2:K1 and O78:K80 in an IEC radioactive-assay under the conditions of exclusion (lactobacilli and IEC, followed by the addition of E. coli), competition (lactobacilli, IEC and E. coli together) and displacement (E. coli and IEC, followed by the addition of lactobacilli). The results indicated that Lact. acidophilus I 26 and Lact. fermentum I 25 could not significantly reduce the attachment of E. coli O1:K1, O2:K1 and O78:K80 to IEC under the three conditions tested in vitro, except that the attachment of E. coli O1:K1 was slightly reduced by Lact. fermentum I 25 in the test for competition.

Animals↗

The adherence of lactic acid bacteria to the columnar epithelial cells of pigs and calves.

The adhesion of various lactobacilli and streptococci to columnar epithelial cells of pigs and calves were studied, by in vitro methods. The porcine strains isolated most frequently were Lactobacillus delbrueckii, Lact, acidophilus and Lact. fermentum. Thirteen of the 22 lactobacilli were adhesive. All the streptococci isolated belonged to Lancefield's D-group; none of them adhered to pig epithelial cells. The adhesive strains (9 of 22) of calves were identified as Lact. fermentum. Adherence was variable even between strains of the same species. Isolates from plant material, cultured milk and cheese did not adhere to the columnar epithelial cells in vitro. The adhesive porcine strains tolerated low pH and bile acids, which is important for their survival under conditions in the stomach and intestine.

Adhesiveness↗

Lack of therapeutic effect of a specially designed yogurt for the eradication of Helicobacter pylori infection.

BACKGROUND: Current antibiotic treatment for Helicobacter pylori infection is often associated with frequent adverse effects and resistance to antibiotics. Alternative treatment methods to control H. pylori infection are needed. Some specific strains of lactic acid bacteria (probiotics) in dairy products are known to inhibit the growth of H. pylori in vitro. A clinical trial was conducted to see the efficacy of a specially designed yogurt product containing specific probiotics on the eradication of H. pylori. METHOD: The yogurt was prepared using three Lactobacillus spp. (L. acidophilus and L. casei) and one commercial starter culture (L. acidophilus, L. bulgaricus and Streptococcus thermophilus). All these cultures were previously evaluated, and found to have strong in vitro inhibitory effects on the growth of H. pylori. Twenty-seven asymptomatic women positive for H. pylori on gastric biopsy and 13C urea breath test were recruited, and administered 175 ml of the yogurt three times a day for 30 days. The 13C urea breath test was administered again, one month after stopping the yogurt treatment to detect the presence of H. pylori. RESULTS: In 26 of 27 subjects, the urea breath test values remained positive, indicating that the consumption of the yogurt had no effect on the eradication of H. pylori. CONCLUSION: Although the designed fermented milk containing lactobacilli is very effective in the inhibition of H. pylori growth in vitro, eradication of this infection in humans is difficult to achieve by consuming this product.

Adult↗

Changes in the microflora and physiology of the anterior intestinal tract of pigs weaned at 2 days, with special reference to the pathogenesis of diarrhea.

The gastrointestinal microflora and gastric physiology of piglets weaned at 2 days was compared with that of piglets allowed to continue sucking the sow. Although there was a significantly higher count of Escherichia coli in the stomach, duodenum, and jejunum of the early-weaned compared with sow-reared pigs, these differences were not detectable in samples from the ileum. There were no quantitative differences in lactobacilli and in streptococci between the two treatments. Lactobacillus fermentum, L. acidophilus, Streptococcus salivarius, S. bovis, and related biotypes were isolated from both groups of pigs. L. fermentum and S. salivarius were isolated more frequently from sow-reared piglets. The weight of digesta in the stomach was greater in weaned than in sucking pigs and was even greater in scouring weaned pigs, suggesting that in scouring pigs there may be gastric stasis. The gastric pH was higher in the weaned pigs at 4 days of age, but gradually decreased up to 10 days, during which time the lactic acid concentration rose. In weaned pigs there was a highly significant negative correlation between pH and lactic acid concentration in the stomach digesta, and also a positive correlation between pH and number of E. coli. These correlations suggest that lactic acid, from bacterial fermentation, is the major component in the regulation of gastric pH in weaned pigs. Three of twenty sucking pigs, but none of the weaned pigs, were secreting HCl (chloride concentration > 3 mg/g, pH < 3.5). In sucking pigs there was an inverse relationship between the chloride and lactic acid concentrations in the digesta. In weaned scouring pigs there was a nonsignificant increase in pepsin concentration in the stomach tissue. There was a threefold increase in the total proteolytic activity of the stomach tissue.

Animals↗

Gut mucosal immunostimulation by lactic acid bacteria.

The beneficial properties of lactic acid bacteria (LAB) on human health have been frequently demonstrated. The interaction of LAB with the lymphoid cells associated to the gut to activate the mucosal immune system and the mechanisms by which they can exert an adjuvant effect is still unclear, as well as if this property is common for all the LAB. We studied the influence of the oral administration of different geneous of LAB such as Lactobacillus casei, L. acidophilus, L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. plantarum, Lactococcus lactis and Streptococcus thermophilus. We determined if the LAB assayed were able to stimulate the specific, the non-specific immune response (inflammatory response), or both. We demonstrated that all the bacteria assayed were able to increase the number of IgA producing cells associated to the lamina propria of small intestine. This effect was dose dependent. The increase in IgA+ producing cells was not always correlated with an increase in the CD4+ T cell number, indicating that some LAB assayed only induced clonal expansion of B cells triggered to produce IgA. Most of them, induced an increase in the number of cells involved in the inflammatory immune response. CD8+ T cell were diminished or not affected, with exception of L. plantarum that induced an increase at low dose. This fact would mean that LAB are unable to induce cytotoxicity mechanisms. We demonstrated the importance in the selection of LAB to be used as gut mucosal adjuvant. The different behaviours observed among them on the gut mucosal immune response, specially those that induce inflammatory immune response, show that not all the LAB can be used as oral adjuvant and that the beneficial effect of them can not generalized to genous or specie. The immunoadjuvant capacity would be a property of the strain assayed.

Adjuvants, Immunologic↗

Human ileostomy glycoproteins as a model for small intestinal mucus to investigate adhesion of probiotics.

Human ileostomy glycoproteins were used as a model for small intestinal mucus to investigate the adhesion of 12 Lactobacillus strains, one Lactococcus strain and one Propionibacterium strain, Both probiotic and dairy strains were tested. Adhesive and non-adhesive Escherichia coli strains were used as controls. All the strains were also tested for their adhesion to polystyrene. Adhesion to ileostomy glycoproteins and to polystyrene varied significantly among the strains tested. Lactobacillus rhamnosus (human isolate), Lactobacillus GG, Lact. acidophilus 1 and P. freudenreichii adhered to ileostomy glycoproteins. Adhesion was concentration-dependent and the most adhesive strains were able to saturate the substratum. These results indicate that human ileostomy glycoproteins can be used as a model system to select potential probiotic strains to complement the adhesion test with intestinal cell lines.

Bacterial Adhesion↗

Influence of the oral administration of lactic acid bacteria on iga producing cells associated to bronchus.

Intestinal, respiratory and genitourinary mucosal surfaces are the most important routes of entry for microbial pathogens. The stimulus of the mucosal immunity is not easy because the trigger keys for the activation do not follow the ones of the systemic immune response. In previous works we have demonstrated that some Lactic Acid Bacteria (LAB), when orally administered, can induce an enhance of the gut immune response. Taking into account the concept of a common mucosal response, we studied the effect of orally administered mice with Lactobacillus casei, L. acidophilus, L. rhamnosus, L. delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus and Lactococcus lactis on the IgA secreting cells associated to bronchus. As shown before, oral immunostimulation with LAB induced an increase of the IgA* cells at intestinal level by a dose depending effect. In this study it is also showed that the LAB assayed, with exception of L. acidophilus, were able to enhance IgA+ cells at bronchial level, being also this effect dose dependent. The increment induced by some LAB in the number of IgA+ cells on the mucosa surface of the lower respiratory tract may be very important to prevent bronchus diseases.

Journal Article↗

Safety and efficacy of probiotic lactobacilli in promoting growth in post-weaning Swiss mice.

Lactobacillus rhamnosus MA27/6B and L. acidophilus MA27/6R are strains used in feed as probiotics. Their safety profiles and growth-stimulating properties were investigated via in vivo studies on young Swiss mice. After repeated administrations of different probiotic preparations in drinking water, safety parameters determined from liver, spleen and total weight remained unchanged. The growth-stimulating properties of viable or dead lactobacilli were studied after supplementation in drinking water. The feed intake (FI), water intake and body weight gain (WG) of the animals were compared to those of control mice. The lactobacilli supplementation of a sub-optimal diet made of barley allow recording of measurable growth performance of mice. It significantly increased WG compared to control groups (P < 0.01), by +28.9% and +31.7% for L. rhamnosus MA27/6B and L. acidophilus MA27/6R, respectively. This WG was correlated with a decrease in the consumption index. The effect of the dose ingested was also investigated: 10(8) lactobacilli CFU/mouse/day produced greater WG than 10(2), 10(4) or 10(6) CFU/mouse/day. No significant differences in growth performance parameters were observed between mice fed with 10(8) cells of viable or nonviable preparations. The mouse assay described could be used as a preliminary criterion when screening candidate probiotics for growth performance properties.

Animals↗

Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin.

In an effort to demonstrate the potential usefulness of Bacillus subtilis (natto) as a probiotic, we examined the effect of this organism on the growth of three strains of lactobacilli co-cultured aerobically in vitro. Addition of B. subtilis (natto) to the culture medium resulted in an increase in the number of viable cells of all lactobacilli tested. Since B. subtilis (natto) can produce catalase, which has been reported to exhibit a similar growth-promoting effect on lactobacilli, we also examined the effect of bovine catalase on the growth of Lactobacillus reuteri JCM 1112 and L. acidophilus JCM 1132. Both catalase and B. subtilis (natto) enhanced the growth of L. reuteri JCM 1112, whereas B. subtilis (natto) but not catalase enhanced the growth of L. acidophilus JCM 1132. In a medium containing 0.1 mM hydrogen peroxide, its toxic effect on L. reuteri JCM 1112 was abolished by catalase or B. subtilis (natto). In addition, a serine protease from B. licheniformis, subtilisin, improved the growth and viability of L. reuteri JCM 1112 and L. acidophilus JCM 1132 in the absence of hydrogen peroxide. These results indicate that B. subtilis (natto) enhances the growth and (or) viability of lactobacilli, possibly through production of catalase and subtilisin.

Bacillus subtilis↗

Chemical composition of the cell wall of lactic acid bacteria and related species.

In order to examine the relationship between biological activities and the cell wall content, the murein type and the teichoic acid of the cell wall from five strains of bacteria were studied. Two of these Lactobacillus casei CRL 431 and L. acidophilus CRL 730, are used in a commercial fermented milk (BIO MILK), which is believed to be beneficial for health. The other strains, Lactococcus lactis CRL 526, Pediococcus pentosaceus CRL 923 and Propionibacterium acidipropionici CRL 1198 were included in order to compare the cell wall structures of active and inactive strains. A method was designed to confirm the amino acids of the peptidoglycan in impure substrates. Four of the studied strains, L. casei, L. acidophilus, L. lactis and P. acidipropionici, contained glycerol teichoic acids. L. casei, L. acidophilus, P. pentosaceus and L. lactis contained A4 alpha type murein, while P. acidipropionici contained A3 gamma type. The capacity of orally administered peptidoglycans of the studied strains to stimulate phagocytosis by mouse peritoneal macrophages was analyzed. Only the PG of L. casei showed this activity. No differences were observed between active and inactive strains with respect to the chemical composition of the peptidoglycan. Therefore the biological activity is unlikely to be due to the peptidoglycan structure.

Animals↗

Acute oral toxicity and bacterial translocation studies on potentially probiotic strains of lactic acid bacteria.

Three potentially probiotic lactic acid bacteria (LAB) strains, Lactobacillus rhamnosus HN001 (DR20(TM)), Lb. acidophilus HN017 and Bifidobacterium lactis HN019 (DR10()), have recently been identified and characterized. The present study was designed to evaluate the acute oral toxicity of these strains to mice, and also to investigate bacterial translocation and gut mucosal pathology in BALB/c mice fed HN019, HN001 or HN017 for 8 consecutive days at a high dose of 10(11)cfu/mouse/day. Results showed that these probiotic strains had no adverse effect on general health status, feed intake, body weight gain and intestinal mucosal morphology (villus height, crypt depth, epithelial cell height and mucosal thickness). No viable bacteria were recovered from blood and tissue samples (mesenteric lymph nodes, liver and spleen) of mice, and no treatment-associated illness or death was observed. According to these results, the oral LD(50) of HN019, HN001 and HN017 is more than 50g/kg/day for mice, and their acceptable daily intake (ADI) value is 35g dry bacteria per day for a 70-kg person. This suggests that the probiotic strains HN019, HN001 and HN017 are non-pathogenic and likely to be safe for human consumption.

Administration, Oral↗