Some probiotic properties of chicken lactobacilli

The beneficial effect of lactobacilli has been attributed to their ability to colonize human and animal gastrointestinal tracts. In this work, adhesion assays with three lactobacillus strains and intestinal fragments obtained from chickens were assessed. Lactobacillus animalis and L. fermentum were able to adhere to three kinds of epithelial cells (crop, small and large intestines) with predominance to small intestine. Among the strains considered, L. fermentum subsp. cellobiosus showed the lowest and L. animalis the highest adhesion ability. Scanning electron microphotographs showing L. animalis and L. fermentum adhering to intestinal cells were obtained. The characterization of L. animalis adhesion indicated that lectin-like structure of this strain has glucose/mannose as specific sugars of binding. However, a calcium requirement was not observed. The adhesion of L. fermentum was reduced by addition of sialic acid or mannose (P < 0.01). These carbohydrates can be involved in the interaction between adhesin and epithelial surface. In this case, the dependence on bivalent cations was demonstrated. Lactobacillus fermentum was effective in reducing the attachment of Salmonella pullorum by 77%, while L. animalis was able to inhibit (90%, 88%, and 78%) the adhesion of S. pullorum, S. enteritidis, and S. gallinarum to host-specific epithelial fragments respectively. Our results from this in vitro model suggest that these lactobacilli are able to block the binding sites for Salmonella adhesion.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains (Salmonella enteritidis and Escherichia coli). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3.0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo. It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains ( Salmonella enteritidis and Escherichia coli ). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3·0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo . It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Gel bands were excised and in-gel digested with trypsin. The resulting peptides were analysed by capillary-LC-ESI-MS/MS. The peptide sequences were used for a database search and allowed identification of a total of 29 proteins, many of which could potentially be involved in the action of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics.     

Adhesion of probiotic lactobacilli to chick intestinal mucus

In the present work, interactions between three Lactobacillus strains (Lactobacillus fermentum CRL1015, Lactobacillus animalis CRL1014, and Lactobacillus fermentum CRL1016) and chicken small intestinal mucus were determined. Three lactobacilli isolated from chicken and selected by their potentially probiotic properties were able to grow in mucus preparations. Three peaks from gel filtration chromatography of intestinal mucus were obtained. The adhesion to three mucus fractions (I, II, and III), especially fraction III, was higher (P < 0.01) in L. fermentum CRL1015 than L. animalis CRL1014. Pretreatment of this fraction with proteases and metaperiodate showed lower (P < 0.01) adhesion values than that of the control, suggesting that a glycoprotein from the mucus acts as a receptor for L. fermentum CRL1015. Highest adhesion values were obtained at pH 7 and 42 degrees C, and neither the removal of divalent cations with ethylenediaminetetraacetic acid (EDTA) nor the addition of calcium produced significant variation from the adhesion values of the control (P > 0.01). This adhesion was only inhibited by N-acetyl-glucosamine. Salmonella pullorum and Salmonella gallinarum showed high (P < 0.01) values of adhesion to chick intestinal mucus. The results obtained from assays of the inhibition of adherence of Salmonella spp. to mucus, immobilized in polystyrene tissue culture wells, indicated that the pathogen adhesion was not reduced by lactobacilli (P > 0.05) or their spent culture supernatants (P > 0.05), suggesting that these strains did not interfere with the binding sites for Salmonella spp. adhesion to the small intestinal mucus.     

Characterization of intestinal lactobacilli as putative probiotic candidates

AIMS: To use antioxidative activity and antagonistic properties of lactobacilli against selected pathogens and members of the normal microflora as a basis for screening probiotic candidates. METHODS AND RESULTS: Antagonistic activity of lactobacilli against target bacteria in both microaerobic and anaerobic environments was tested. Production of antagonistic metabolites (ethanol, hydrogen peroxide (H2O2), acetic, lactic and succinic acid) by lactobacilli as well as their total antioxidative activity were assessed. In general, the lactobacilli tested were most effective against Gram-negative bacteria and their antagonistic activity was strain-specific. However, obligately heterofermentative lactobacilli had the strongest activity when tested in a microaerobic environment. Additionally, facultatively heterofermentative lactobacilli were equally effective in either milieu and produced significant levels of acetic and lactic acid. Moreover, obligately homofermentative lactobacilli had high H2O2 production and total antioxidative activity but weak antagonistic activity. CONCLUSIONS: Antioxidative and antagonistic activity of intestinal lactobacilli is strain-specific but typically can be related to their fermentation type which may be used for rapidly screening large numbers of lactobacilli for probiotic candidates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first report on the utilization of group characteristics to screen lactobacilli intended for specific probiotic use. Such uses include the targeting of particular gut niches and pathogens as well as allowing for long-term benefits to the host.     

Isolation of lactobacilli with probiotic properties from the human stomach

Aims: Recent evidence suggests that the human gastric microbiota is much more diverse than previously thought. The aim of this study was to assess the potential for isolating lactobacilli from the human stomach. Methods and Results: Lactobacilli were selectively cultured from gastric biopsies from 12 patients undergoing routine endoscopy. Lactobacilli were present in four of 12 biopsies. We isolated, in total 10 different strains representing five species (Lactobacillus gasseri, L. fermentum, L. vaginalis, L. reuteri and L. salivarius). The 10 isolates varied greatly in their ability to inhibit the growth of two Gram‐positive bacteria and two Gram‐negative bacteria. Furthermore, the acid and bile resistance profiles of the 10 isolates spanned a wide range. Conclusions: Five different Lactobacillus species were cultured from human gastric biopsies for the first time. Significance and Impact of the Study: Diverse Lactobacillus species are more prevalent in the human stomach than previously recognized, representing an untapped source of bacteria with beneficial probiotic and/or biotechnological properties.     

In vitro appraisal of the probiotic value of intestinal lactobacilli

Lactobacilli play a distinctive role in the microbial balance of the chicken gut. In experiments simulating the chicken crop, the antagonism of lactobacilli against Enterobacteriaceae and Salmonella typhimurium was demonstrated and was attributed to lactic acid production. Moreover, adhesion to the crop epithelium was a common characteristic of intestinal lactobacilli. As opposed to salmonellas, lactobacilli were sensitive to deconjugated bile salts at 2.5mm. This sensitivity could lower their chance of proliferation in the small bowel of the chicken tract.The authors are at the Laboratory of Microbial Ecology, Faculty of Agricultural Sciences, State University of Gent, Coupure L., 653, B-9000 Gent, Belgium.     

Adherence capacities of oral lactobacilli for potential probiotic purposes

The most abundantly used probiotic strains come from the genus Lactobacillus and only a few studies have investigated their role in oral health. Even if a positive correlation has been established between the saliva Lactobacillus count and dental caries, this genus is generally recognized as safe (GRAS). Moreover, lactobacilli could in some cases play a beneficial role by inhibiting the growth of some oral pathogenic bacteria. This activity could justify their use as probiotic. To establish the potential health benefit of probiotic candidates, appropriate in?vitro tests are required, particularly on their adhesive capacity. The aim of this work was to investigate the adhesive properties and surface characteristics of 70 oral lactobacilli that could be used as probiotics for oral health. For this, three methods were used: biofilm formation on a glass surface and on saliva-coated hydroxyapatite discs and the microbial adhesion to solvent method. The results of the biofilm formed on glass surface showed 13 strains with an adhesion score equal to or higher than 3. 57/70 (81%) of the tested lactobacilli did not form any biofilm on glass surfaces. All of the 13 strains formed biofilms on HA discs. Among these 13 strains, 10/13 (77%) showed low surface hydrophobicity (0-35%) and 3/13 (23%) showed medium hydrophobicity (36-70%). Some of the selected strains showed potentially useful adhesive capacity. This work paves the way for the selection of probiotics that could be used for oral health purposes with the aim to reduce carious risk.     

Cell surface appendages of lactobacilli

Abstract Seventeen vaginal isolates of lactobacillus were negatively stained using 1% phosphotungstic acid and examined by transmission electron microscopy. One or two flagella were observed in a polar or, less frequently, sub-polar position on 14 of the 17 isolates. The structures, measuring 12 nm in diameter and up to 12 μm long, were seen on cells cultured in both solid and liquid MRS medium. Fimbriae, measuring 3.5 nm in diameter and 0.1 μm to 1.0 μm in length, extended from the cell surface of 15 of the 17 lactobacillus isolates. Production of fimbriae was maximal when the lactobacilli were cultured on solid medium in an anaerobic atmosphere. Fimbriation significantly increased adhesion of lactobacilli to vaginal epithelial cells in vitro.     

Screening for lactobacilli with probiotic properties in the infant gut microbiota

Lactobacilli are believed to be beneficial for the human hosts and are currently being evaluated as potentially probiotic bacteria. In this study, Lactobacillus strains were isolated from infant faeces and were examined in vitro for potential probiotic properties. Faecal specimens from 63 healthy, full-term infants were collected at 4, 30 and 90 days after delivery. Seventy-four Lactobacillus strains were isolated and one or more different phenotypes from each infant (n = 44) were selected for further testing. The bacterial isolates were identified mainly as L. gasseri, L. crispatus, Lactobacillus paracasei, L. salivarius, L. fermentum after amplification and sequencing of 16s rRNA gene. The strains were examined for acid and bile tolerance, adhesion to Caco-2 cells, antibiotic susceptibility and antimicrobial activity against selected enteric pathogens. The great majority of the isolated lactobacilli were susceptible to ampicillin, amoxicillin/clavulanic acid, tetracycline, erythromycin, cephalothin, chloramphenicol and rifampicin. Resistance to vancomycin or bacitracin was detected to 34% of the strains. Twenty strains out of forty-four exhibited significant tolerance to bile salts. Those strains were subsequently tested for resistance to low pH conditions (pH 2 and 3). Interestingly, 85% (17 strains) of the tested lactobacilli remained unaffected at pH 3 after 3 h of incubation, 6 strains were found resistant at pH 2 after 1.5 h and only 2 strains found resistant after 3 h of incubation. Two of the strains were able to adhere to Caco-2 cells. In conclusion, two isolates fulfilled the in vitro probiotic criteria and are good candidates for further in vivo evaluation.     

Effect of some pharmaceutical excipients on the survival of probiotic vaginal lactobacilli

Lactobacilli are the predominant microorganisms of the vaginal bacterial microbiota, and they play a major role in the maintenance of a healthy urogenital tract. In consequence, the interest in their potential use as probiotics has significantly increased during the last decade. In the present study we assessed the influence of different excipients on the survival of 4 probiotic vaginal lactobacilli incorporated into glycerinated gelatin ovules and stored at 5 degrees C for 60 d. Results showed that viability after storage was a strain-dependent characteristic, but inclusion of ascorbic acid significantly increased survival in 3 of the 4 strains tested. The best survival was observed for Lactobacillus salivarius CRL 1328 in ovules containing skimmed milk. No significant differences in viability were observed between control ovules (glycerogelatin base without excipients) and those containing lactose or Tween 80 for any of the strains tested. Lactobacillus acidophilus CRL 1259 and Lactobacillus crispatus CRL 1266 were, respectively, the most resistant and sensitive strains to the storage with the different substances. In conclusion, these results provide a basis for selecting excipients to improve the survival of lactobacilli in a probiotic product, in an attempt to ensure the delivery of an adequate number of viable cells to the urogenital tract.     

Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances

AIMS: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products. METHODS AND RESULTS: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C. Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C. Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx. 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact. salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact. rhamnosus GG. CONCLUSIONS: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage. SIGNIFICANCE AND IMPACT OF THE STUDY: High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.     

Viability of vaginal probiotic lactobacilli during refrigerated and frozen storage

The viability of six different strains of probiotic vaginal Lactobacillus was examined in two different cryoprotective media, during refrigerated versus frozen storage, and using two traditional types of stock cultures for starting the biomass production. Freezing at -20 degrees C and -70 degrees C had much less adverse effect on viability than did storage at 7 degrees C, and the reduction in viability was greater at -20 degrees C than at -70 degrees C. The strains showed variation in the extent of the viability losses during both types of storage. Milk-yeast extract (MYE) was shown to be the more suitable protective medium to maintain viability of the strains during the storage. The vaginal Lactobacillus strains are most stable in MYE at -70 degrees C with only a small decrease of the viability observed under these conditions. The viable cell counts of Lactobacillus paracasei CRL 1251 and CRL 1289, L. crispatus CRL 1266 and L. salivarius CRL 1328 remained around 1 x 10(8) CFU/mL after 24 months of storage at -70 degrees C, or up to 18 months for L. acidophilus CRL 1259.     

Usefulness of epifluorescence for quantitative analysis of lactobacilli in probiotic feed

AIMS: Enumeration of total, active or viable probiotic micro-organisms from liquid or solid commercial feedstuffs was studied during processing and storage. METHODS AND RESULTS: After sample preparation, an epifluorescence microscopy technique and a plating method were investigated comparatively. It was shown that (i) on the day of manufacture, active or viable bacteria were in equivalent amounts and that viable numbers then decreased, depending on the different processing and storage factors enhancing ABNC production, (ii) the amount of total and active lactobacilli remained close and quite stable for months at a high level (>10(8) active fluorescent units). CONCLUSIONS: Processing and storage promoted ABNC cells in the products tested. Consequently, both techniques should be used to evaluate the viable-dead-active status of bacteria for which functional properties are claimed. SIGNIFICANCE AND IMPACT OF THE STUDY: Enumeration of the whole probiotic bacterial population should be take into account for guidelines and labelling since non-viable bacteria could have a probiotic effect.     

Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens

AIM: To develop in vitro assays for comparing the antagonistic properties and anti-oxidative activity of probiotic Lactobacillus and Bifidobacterium strains against various entero- and urinary pathogens. METHODS AND RESULTS: The antagonistic activity of five probiotic lactobacilli (Lactobacillus rhamnosus GG, Lactobacillus fermentum ME-3, Lactobacillus acidophilus La5, Lactobacillus plantarum 299v and Lactobacillus paracasei 8700:2) and two bifidobacteria (Bifidobacterium lactis Bb12, Bifidobacterium longum 46) against six target pathogens was estimated using different assays (solid and liquid media, anaerobic and microaerobic cultivation) and ranked (low, intermediate and high). Bacterial fermentation products were determined by gas chromatography, and the total anti-oxidative activity of probiotics was measured using linolenic acid test. Pyelonephritic Escherichia coli was highly suppressed by GG and both bifidobacteria strains. Lactobacilli strains 8700:2, 299v and ME-3 were the most effective against Salmonella enterica ssp. enterica in microaerobic while ME-3 and both bifidobacteria expressed high activity against Shigella sonnei in anaerobic milieu. Lact. paracasei, Lact. rhamnosus and Lact. plantarum strains showed intermediate antagonistic activity against Helicobacter pylori under microaerobic conditions on solid media. The highest anti-oxidative activity was characteristic for Lact. fermentum ME-3 (P < 0.05). No efficient antagonist against Clostridium difficile was found. The positive correlations between the pH, lactic acid production and anti-microbial activity for all tested probiotics were assessed. CONCLUSIONS: Developed experimental assays enable to compare the anti-microbial and -oxidative activity of Lactobacillus and/or Bifidobacterium probiotics, which have been claimed to possess the ability of suppressing the growth of various enteric and urinary pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening Lactobacillus and Bifidobacterium sp. strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and urinary tract infections.     

Characterization of lactobacilli towards their use as probiotic adjuncts in poultry

AIMS: A total of 112 strains of lactic acid bacteria of duck origin were studied for their use as a probiotic feed supplement. METHODS AND RESULTS: In vitro studies included aggregation, co-aggregation, cell surface hydrophobicity and adhesion activities on poultry crop cells and human Hep2-cells. Additionally, growth with bile acids (chicken bile, ox gall and taurocholic acid) and tolerance to acidic pH were tested. Among all the isolates, two strains (Lactobacillus animalis TMW 1.972 and Lactobacillus salivarius TMW 1.992) were selected for a survival test in poultry. Monitoring and differentiation of these strains was achieved by selective detection as rifampicin and erythromycin double-resistant mutants. After a single feed administration, both micro-organisms were shown to persist in the crop and caecum of ducks for a period of 18 and 22 days, respectively. For identification of Lact. animalis and Lact. salivarius, two specific PCRs targeted against 16S rDNA were developed. CONCLUSIONS: Within the autochtoneous microflora of ducks, two strains of lactobacilli exhibited strong potential as probiotic adjuncts. The results indicate that the natural gut microflora of poultry serves as an excellent source for optimal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: A general strategy for the selection of probiotic strains is presented. The suggested sequence of tests allows identification of the most promising candidates within complex ecosystems or large strain collections with minimal expenditure.     

Estimation of vaginal probiotic lactobacilli growth parameters with the application of the Gompertz model

Lactobacilli are widely described as probiotic microorganisms used to restore the ecological balance of different animal or human tracts. For their use as probiotics, bacteria must show certain characteristics or properties related to the ability of adherence to mucosae or epithelia or show inhibition against pathogenic microorganisms. It is of primary interest to obtain the highest biomass and viability of the selected microorganisms. In this report, the growth of seven vaginal lactobacilli strains in four different growth media and at several inoculum percentages was compared, and the values of growth parameters (lag phase time, maximum growth rate, maximum optical density) were obtained by applying the Gompertz model to the experimental data. The application and estimation of this model is discussed, and the evaluation of the growth parameters is analyzed to compare the growth conditions of lactobacilli. Thus, these results in lab experiments provide a basis for testing different culture conditions to determine the best conditions in which to grow the probiotic lactobacilli for technological applications.     

Cross‐talk between probiotic lactobacilli and host immune system

The mechanism by which probiotic lactobacilli affect the immune system is strain specific. As the immune system is a multicompartmental system, each strain has its way to interact with it and induce a visible and quantifiable effect. This review summarizes the interplay existing between the host immune system and probiotic lactobacilli, that is, with emphasis on lactobacilli as a prototype probiotic genus. Several aspects including the bacterial‐host cross‐talk with the mucosal and systemic immune system are presented, as well as short sections on the competing effect towards pathogenic bacteria and their uses as delivery vehicle for antigens.