The survival of four strains of lactic acid bacteria in human gastric juice, in vivo and in vitro, and in buffered saline, pH 1 to 5, has been investigated. The strains studied include two Lactobacillus acidophilus strains, Lactobacillus bulgaricus, and Streptococcus thermophilus. In addition, the adhesion of these strains to freshly collected human and pig small intestinal cells and to pig large intestinal cells has been studied and the effect of milk on both survival and adhesion tested. As a result of these investigations, an in vitro test system for screening potential cultures for use as human dietary adjuncts can be developed. The ability to survive in gastric juice and to adhere varied significantly for the strains tested; L. acidophilus ADH survived and adhered better than the others while S. thermophilus survived and adhered poorly. For all strains, both survival and adhesion was enhanced by milk. As all strains adhered to some extent to both human and pig intestinal cells, the adhesion mechanism is probably a nonspecific attachment as opposed to other reported specific Lactobacillus adhesion to gastric tissue. From the survival and adhesion data it seems feasible to obtain elevated levels of viable Lactobacillus sp. in human intestine by careful selection of the bacterial strains ingested. Furthermore, the in vitro methods used here should be valuable to screen potential strains. The data presented here can then be correlated with human in vivo studies monitoring the beneficial effect of ingestion of these Lactobacillus.

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidaselabeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesionpromoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesionpromoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.Proteinaceous surface appendages or coverings, e.g., fimbriae, flagella, or surface (S)-layers with affinity for mammalian extracellular matrix (55) or mucosa (17, 28), have been extensively characterized for many pathogenic bacteria. It is thought that adhesion to mammalian extracellular matrix components is a prerequisite for bacterial colonization and invasion to subepithelial tissues (55) Cell surface-adhesive proteins from nonpathogenic bacteria and their role in colonization has not been as thoroughly characterized.Lactobacillus, one of the common indigenous organisms of the gastrointestinal tracts of mammals (46,48,49) and a potential probiotic microbe that contributes to the health of the host (11,16), has the capacity to adhere to epithelial cells (5,6,9,10,16,21,23,24,33,34,44,49) and mucus gel (25,32,35,38,41,51,52) from the intestinal tracts of different species. Lactobacillus surface proteins have been proposed to be involved in colonization of gastrointestinal epithelial cells and mucosa of mammals (9,12,21,41,44,53). Collagen binding by lactobacilli (1, 47) is known, and purification of collagen binding proteins from L...

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Aims: To optimize a spray coating process for the production of encapsulated microspheres containing viable Bi®dobacterium cells and to determine whether the readily gelatinized modi®ed starch coating used in this study improved bacterial survival in foods or under acid conditions. Methods and Results: An air inlet temperature of 100°C was demonstrated to be optimal for the spray drying process, as it afforded good drying, low outlet temperatures (45°C) and resulted in less than 1 log reduction in bi®dobacteria numbers during drying. Maximum recovery yields of 30% were obtained after optimizing the air aspiration conditions. The average size of the Bi®dobacterium PL1-containing starch microparticles was determined by scanning electron microscopy to be of the order of 5 lm. The starch-coated cells did not display any enhanced viability compared with free PL1 cells when exposed to acid conditions for 6 h or in two dry food preparations over 20 d storage at ambient temperature (19±24°C). Determination of 1491 nucleotides of the 16S rRNA gene from PL1 indicated that it shared 97% homology with a previously sequenced Bi®dobacterium ruminantium strain. Conclusions: Our data demonstrated that, although spray drying is a valuable process for encapsulating bi®dobacteria, further work is required to ascertain a more appropriate coating material that will protect this strain against adverse environmental conditions. Signi®cance and Impact of the Study: The production of small, uniformly coated microspheres containing viable bi®dobacteria using an affordable and industrially convenient process, such as spray drying, has commercial implications for the production of probiotic products. Although popular for use as a coating polymer by the food industry, this study indicated that modi®ed starches might not be suitable for use as an encapsulating material for probiotic strains.

A non‐virulent Carnobacterium sp., designated strain K1, isolated from the gastrointestinal tract of Atlantic salmon, Salmo salar L., which produced inhibitory substances against bacterial fish pathogens, was examined in vitro for characteristics important for the colonization of the fish gastrointestinal tract and in vivo for persistence in the tract after oral dosing. In vitro growth experiments showed that the cells of this strain were metabolically active in both the intestinal mucus and faeces from salmonids. The production of growth inhibitors against the two common fish pathogens Vibrio anguillarum and Aeromonas salmonicida by Carnobacterium sp. strain K1 was demonstrated in vitro in mucus and faecal extracts. Furthermore, it was demonstrated that the Carnobacterium cells remained viable in the gastrointestinal tract for several days and that no detrimental effect to the fish was observed as a result of the presence of the bacterium.

BackgroundProbiotic supplementation significantly reduces the risk of necrotising enterocolitis (NEC) and all cause mortality in preterm neonates. Independent quality assessment is important before introducing routine probiotic supplementation in this cohort.AimTo assess product quality, and confirm that Bifidobacterium breve (B. breve) M-16V supplementation will increase fecal B. breve counts without adverse effects.Methods and ParticipantsStrain identity (16S rRNA gene sequencing), viability over 2 year shelf-life were confirmed, and microbial contamination of the product was ruled out. In a controlled trial preterm neonates (Gestation <33 weeks) ready to commence or on feeds for <12 hours were randomly allocated to either B. breve M-16V (3×109 cfu/day) or placebo (dextrin) supplementation until the corrected age 37 weeks. Stool samples were collected before (S1) and after 3 weeks of supplementation (S2) for studying fecal B. breve levels using quantitative PCR (Primary outcome). Secondary outcomes included total fecal bifidobacteria and NEC≥Stage II. Categorical and continuous outcomes were analysed using Chi-square and Mann-Whitney tests, and McNemar and Wilcoxon signed-rank tests for paired comparisons.ResultsA total of 159 neonates (Probiotic: 79, Placebo: 80) were enrolled. Maternal and neonatal demographic characteristics were comparable between the groups. The proportion of neonates with detectable B. breve increased significantly post intervention: Placebo: [S1:2/66 (3%), S2: 25/66 (38%), p<0.001] Probiotic: [S1: 29/74 (40%), S2: 67/74 (91%), p<0.001].Median S1 B. breve counts in both groups were below detection (<4.7 log cells.g−1), increasing significantly in S2 for the probiotic group (log 8.6) while remaining <4.7 log in the control group (p<0.001). There were no adverse effects including probiotic sepsis and no deaths. NEC≥Stage II occurred in only 1 neonate (placebo group).Conclusion B. breve M-16V is a suitable probiotic strain for routine use in preterm neonates.Trial RegistrationAustralia New Zealand Clinical Trial Registry ACTRN 12609000374268

More than 400 isolates from the intestine and the external surface of farmed Scophtalmus maximus as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regard to their sites of colonization, especially the intestinal regions and sites within each region. Of the total number of bacterial isolates from the intestine, 28% were inhibitory against V. anguillarum HI 11345. A marine biochemical assay was used to order the inhibitory strains into different phena. Most inhibitory bacteria were found in the rinse and mucus fractions of the gastrointestinal tract. No correlations among the different phena, site of colonization, and inhibitory effect could be found; however, a biochemical div!rsity was noted in the strains with an inhibitory effect. Of the isolates with an inhibitory effect against V. anguillarum HI 11345, 60% had an inhibitory effect on five other fish-pathogenic serotypes of V. anguillarum. Inhibitory effects of the isolates were also shown against Aeromonas salmonicida and Aeromonas hydrophila.

The role of the intestinal tract in Vibrio anguillarum infection of turbot, Scophthalmus maximus (L.), fingerlings was investigated in two in vivo models and the possible mechanisms involved were studied in vitro. Viable V. anguillarum cells were detected in spleens from more than 50% of the fish administered the pathogen orally or rectally, suggesting that the intestinal tract is a portal of entry for V anguillarum. In transmission electron micrographs, V. anguillarumlike cells were seen close to the rectal epithelium, suggesting penetration of the mucus layer, but no epithelial cell penetration or endocytosis was evident. Attachment to intact turbot intestines was investigated, and 80% or mote of the bacterial cells still remained attached after serial washings. A significantly higher number of cells attached to rectal segments than to the other intestinal segments. In vitro, V. anguillarum cells did not adhere specifically to intestinal mucus, but rather accumulated close to intestinal mucus interfaces and subsequently penetrated them. It is proposed that the intestinal tract of turbot is a portal of entry for V. anguillarum and that the cells penetrate the intestinal mucus overlaying the epithelial cells.