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The aim of this study was to screen Bacillus strains from the guts of Scophthalmus maximus, Paralichthys olivacues, Epinephelus coioides and Clupanodon punctatus, for use as probiotics in aquaculture. Eight Bacillus strains were screened, and strain M001 was selected for probiotic study based on its antagonistic activity against multiple aquatic bacterial pathogens including Vibrio anguillarum, V.campbellii, V.vulnificus, V.parahamolyticus, Streptococcus sp. and Edwardsiella tarda. M001 was identified as B.amyloliquefaciens based on the biochemical tests and 16S rRNA gene analysis. In vitro experiments revealed that M001 was able to grow at a wider range of temperature, pH and salinity and was capable to use turbot mucus as nutrient for growth. Additionally, M001 isolate greatly inhibited the growth of V.anguillarum by producing antibacterial substances and was acid tolerance, non-antibiotic resistance and non-harmful. Thereafter, the potential probiotic effect of M001 was tested in turbot by dietary administration of M001 at a dose of 10(8)CFUg(-1) diet for 42days. No significant differences of weight gain, specific growth rate and feed ratio were found in the M001-diet group of fish compared with control fish, but which increased, respectively, by 5.5%, 4.7% and 7.0% after 42days of feeding. Several digestive enzyme activities were found to increase significantly in the M001-diet group, including protease and amylase activities in hepatopancreas, protease activity in intestine and lipase activity in stomach (P

Pig weaning period is frequently associated with infectious disease, mainly caused by enterotoxigenic Escherichia coli (ETEC) K88. Plant extracts exert different beneficial effects and may represent antibiotic alternatives to reduce piglet infection. In this study, plant extracts and other natural substances (PENS) have been evaluated on the pig intestinal IPEC-1 cells, for potential protection against ETEC K88 induced membrane damage. Several PENS have been considered: yeast extract, yeast nucleotides, unsaturated oligo-mannuronic acid, ulvan, bromelain and three fractions of bovine colostrums, as anti-inflammatory and immunomodulatory compounds; daidzein and Chlorella vulgaris extract, as anti-oxidant compounds; allicin, cinnamaldehyde and carvacrol, as anti-bacterial compounds. First, possible toxic effect of PENS on cell membrane permeability was verified by assessing the transepithelial electrical resistance (TEER) and paracellular flux of the extracellular marker phenol red. The highest non-toxic PENS concentration was added to ETEC infected cells to test the protection against membrane damage. The results showed that yeast extract, daidzein, bovine colostrum, bromelain and allicin protected the cells against the increased membrane permeability caused by ETEC, whereas the other PENS did not show this ability. Allicin protection was not due to its anti-bacterial activity, since ETEC growth was unaffected by the presence of allicin.

Keywords: creep feeding, weaning, intestinal microbiota, Lactobacillus sobrius, prebiotics, daidzein, probiotics, 16S rRNA, DGGE, cloning, real-time PCR Nursing is a major critical period in the life of piglets. On one hand maternal antibodies are not able to cross the placenta, thus piglets are born without circulating antibodies and consequently lack maternal passive protection. On the other hand, creep-feeding and weaning increase susceptibility to gut disorders, infections and diarrhea. Therefore clarification of the composition and function of the normal gut microbiota of piglets is pivotal as a knowledge base for the design of innovative nutritional strategies based on pre- and probiotics to keep piglets healthy. The objectives of this study were to describe the composition and function of the intestinal microbiota of piglets during the nursing period through creep feeding and weaning, to in vivo and in vitro evaluate the effect of daidzein on composition and function of intestinal microbiota of nursing piglets in order to evaluate its prebiotic function, and to investigate the probiotic effect of Lactobacillus sobrius S1 on composition and function of intestinal microbiota of nursing piglets. The porcine intestinal microbiota development and diversity, the in vivo and in vitro evaluation of prebiotic effect of daidzein and the investigation of probiotic effect of Lactobacillus sobrius S1 on composition and function of intestinal microbiota of piglets during nursing period through creep feeding and weaning was described using real-time PCR, and PCR analysis of 16S ribosomal RNA gene by denaturing gradient gel electrophoresis (DGGE) and cloning, in combination with analysis of gas production, lactate and VFA yield. The data obtained during the course of the study indicated that 1) Early creep-feeding stabilizes the microbiota of piglets around the weaning period. 2) Lactobacillus communities follow a successional change associated with piglet growth and diet shifting. Creep feeding stabilizes the Lactobacillus community of weaning piglets. Within the Lactobacillus community, some members like L. reuteri and L. amylovorus / L. sobrius might be permanent colonizers, while L. delbruckii, L. acidophilus and L. crispatus are more likely to be transient members of the Lactobacillus communities in the piglet´s GI tract. 3) Both in vitro and in vivo evaluations indicated that daidzein has the potential for use as a prebiotic additive in animal feed. 4) Lactobacillus sobrius S1 has the potential of promoting beneficial bacteria and inhibiting pathogens.