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...

This study examined the effect of potential probiotic bacteria on growth and survival of the tilapia Oreochromis niloticus, under high density and suboptimum temperature. Presumptive Bacillus and lactic acid bacteria (LAB) were isolated from tilapia and from their culture system and were characterized for haemolytic and enzymatic activity, and antagonism against Vibrio. Selected strains were included in the diet of juvenile tilapia and evaluated during a 134‐day assay. The experiment was conducted with four treatments: (1) fish fed with commercial feed plus Dry Oil®; (2) fish fed with commercial feed plus LAB; (3) fish with bacilli in water; (4) fish with a mixture of treatments 2 and 3. Tilapias in all treatments, including bacteria, grew significantly better than fish fed with commercial feed plus Dry Oil® (control group). Survival was similar in all treatments. The physicochemical parameters of the culture system were maintained within the optimal ranges for the species, with the exception of temperature (19.9–24.82 °C). Animals fed diet supplemented with bacilli and LAB had good survival and the best growth performance, suggesting that bacteria are appropriate growth‐stimulating additives in tilapia cultivation.

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Aims:  To identify and characterize adhesion‐associated proteins in the potential probiotic Lactobacillus fermentum BCS87. Methods and Results:  Protein suspensions obtained from the treatment of Lact. fermentum BCS87 with 1 mol 1−1 LiCl were analysed by Western blotting using HRP‐labelled porcine mucus and mucin. Two adhesion‐associated proteins with relative molecular weight of 29 and 32 kDa were identified. The N‐terminal and internal peptides of the 32 kDa protein (32‐Mmubp) were sequenced, and the corresponding gene (32‐mmub) was found by inverse polymerase chain reaction. The complete nucleotide sequence of 32‐mmub revealed an open reading frame of 903 bp encoding a primary protein of 300 amino acids and a mature protein of 272 residues. A basic local alignment search showed 47–99% identity to solute‐binding components of ATP binding cassette transporter proteins in Lactobacillus, Streptococcus and Clostridium. An OpuAC‐conserved domain was identified and phylogenetic relationship analysis confirmed that 32‐Mmubp belongs to the OpuAC family. Conclusions:  Adhesion of Lact. fermentum BCS87 appeared to be mediated by two surface‐associated proteins. 32‐Mmubp is a component of ABC transporter system that also functions as an adhesin. Significance and Impact of the Study:  Characterization of 32‐Mmubp and 32‐mmub will contribute to understanding the host–bacteria interactions of Lact. fermentum with the intestinal tract of pigs.

The colonization potential of lactobacilli was investigated using small intestinal mucus extracts from 35-d-old pigs. Mucus-secreting tissue from the small intestine of piglets was gently rinsed to remove contents and then shaken in buffer to release mucus from the surface. Numbers of lactobacilli in different portions of the small intestine of 35-d-old pigs were enumerated. Also, mucus isolated from the small intestine of pigs was investigated for its capacity to support the growth of lactobacilli. Results indicated that Lactobacillus spp. inhabit the mucus layer of the small intestine and can grow and adhere to ileal mucus. From adhesion studies of Lactobacillus fermentum 104R to mucus analysed by Scatchard plot, it is suggested that an associating system showing positive cooperativity is involved. Proteinaceous compounds(s) involved in the adhesion to mucus were detected in the spent culture fluid from the growth of strain 104R. Studies are continuing in order to identify and characterize the adhesion-promoting protein(s). From the data, it is proposed that lactobacilli colonize the mucus layer of the small intestine of pigs.

The colonization potential of lactobacilli was investigated using small intestinal mucus extracts from 35‐d‐old pigs. Mucus‐secreting tissue from the small intestine of piglets was gently rinsed to remove contents and then shaken in buffer to release mucus from the surface. Numbers of lactobacilli in different portions of the small intestine of 35‐d‐old pigs were enumerated. Also, mucus isolated from the small intestine of pigs was investigated for its capacity to support the growth of lactobacilli. Results indicated that Lactobacillus spp. inhabit the mucus layer of the small intestine and can grow and adhere to ileal mucus. From adhesion studies of Lactobacillus fermentum 104R to mucus analysed by Scatchard plot, it is suggested that an associating system showing positive cooperativity is involved. Proteinaceous compound(s) involved in the adhesion to mucus were detected in the spent culture fluid from the growth of strain 104R. Studies are continuing in order to identify and characterize the adhesion‐promoting protein(s). From the data, it is proposed that lactobacilli colonize the mucus layer of the small intestine of pigs.

ABSTRACT. In this study, five microalgal strains were isolated from Bahía de La Paz, Baja California Sur, Mexico and identified as Grammatophora sp., Navicula sp., Rhabdonema sp., Schizochytrium sp., and Nitzschia sp., and their evaluation as potential food for Artemia franciscana. The isolated strains were cultured outdoors and harvested after four days. Chaetoceros muelleri was cultured under laboratory conditions and used as control. The protein, lipid, and carbohydrate composition and the fatty acid profiles of the strains were determined by gas chromatography. To assess the effect of microalgal strains on A. franciscana, decapsulated cysts were cultured at outdoor conditions in 15 L containers. The experiment was conducted for twelve days. Samples from the five different feeding treatments were taken at the beginning and end of the experiment to assess number, size, and weight of Artemia larvae. Treatment with Rhabdonema sp. showed larvae with a lower percentage of polyunsaturated fatty acids (PUFAs) while Grammatophora sp. showed those with the greatest PUFA proportion, even more than those fed Chaetoceros muelleri (control). Larvae consuming Schizochytrium sp. had no docosahexanoic (DHA) nor eicosapentaenoic (EPA) fatty acid content. Growth and survival of A. franciscana did not show significant differences among feed treatments, except when it was fed Nitzschia sp., showing lower survival and dry weight. Treatment based on Schizochytrium sp. and Rhabdonema sp. had a greater A. franciscana size but reduced dry weight; additional tests including two or more algal species for every treatment should be carried out to determine the best yield. Keywords: brine shrimp, arid zones, nutrition, microalgae, fatty acids, aquaculture. Evaluación del potencial de microalgas endémicas para el cultivo deArtemia franciscana RESUMEN. Se aislaron cinco cepas de microalgas de la Bahía de La Paz, Baja California Sur, Mexico, identificadas como Grammatophora sp., Navicula sp., Rhabdonema sp., Schizochytrium sp., and Nitzschia sp., y su evaluación como alimento potencial para Artemia franciscana. Las cepas algales fueron aisladas, purificadas y cultivadas al exterior. Chaetoceros muelleri fue cultivada en condiciones de laboratorio y utilizada como control. El análisis bioquímico y el perfil de ácidos grasos correspondientes al cuarto día de cultivo de las microalgas, se efectuó mediante cromatografía de gases. El experimento con Artemia franciscana se realizó al exterior por doce días en tanques de 15 L. Se realizaron muestreos al inicio y al final del trabajo para determinar el incremento en talla, peso seco y sobrevivencia. El tratamiento con la microalga identificada como Rhabdonema sp. mostró larvas con menor porcentaje de ácidos grasos polinsaturados (PUFAs) mientras que aquellas alimentadas con Grammatophora sp. presentaron la mayor proporción, superando a las del control Chaetoceros muelleri. Con Schizothyrium sp. no presentaron los ácidos grasos docosahexanoico (DHA) y eicosapentanoico (EPA). La sobrevivencia obtenida...

Important centres for the pork industry have become growth in arid regions in the world and pig production needs alternatives to increase the productivity. A screening of predominant Lactobacillus strains from healthy piglets was performed in order to select specific probiotics. The ability of 164 strains to grow at different temperatures and concentrations of NaCl was evaluated. Results showed that all of them grew at 45 °C, 75% at 50 °C and 64% resisted 680 mM of salt. Adhesion to mucus and gastric mucin was evaluated showing 45% of strains isolated from faeces were able to adhere whereas 71% of strains from mucus showed mucus binding activity. Among the 164 isolates, 27 adhesive strains were identified using comparisons with 16S rDNA and intergenic 16-23S sequences. Results indicated that L. fermentum and L. reuteri were the most common species in faeces and mucus, respectively. Ability to grow in gastrointestinal mucus was evaluated showing that 92.6% of strains were able to replicate. Additionally, bacterial strain grown in 3.5% MRS with bile salts was evaluated. These results indicated that animals inhabiting isolated arid coasts are a rich source of probiotics, which resist adverse environmental conditions and can colonise the intestinal tract of pigs.