| According to the definition that brought forward by 'the father of prebiotics'--- Dr.Glenn Gibson in 1995, prebiotics is Non digestible food ingredients, that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improve host health. Hence, the information of gastrointestinal microflora should be taken as gist to assess the prebiotics product scientifically. This study took the fecal samples of HFA (Human Flora-associated) piglet as subject to analysis the influence of certain prebiotics on intestinal microflora of HFA piglet.1. Survey and molecular identification of hemolytic bacteria isolated from fecal samples of healthy human subjects.Because some pathogenic bacteria can produce toxin, endotoxin or hemolytic element to destory the red cell membrane, and produce the hemolytic phenomenon. To examine whether pathogenic bacteria existed in intestinal microflora of those donors, a dynamic investigation and molecular characterization was performed on the occurrence of hemolytic bacteria in 17 individuals without clinical symptoms over a three weeks period. Hemolytic bacteria were isolated in five individuals. Two types of mapping patterns were generated with ARDRA analysis of 16S rDNA of those hemolytic bacteria isolated from five individuals. Type I showed 99% homology with Bacillus cereus ATCC 10987. Type II showed 99% homology with E. coli CFT073. At last, ERIC-PCR fingerprinting was also performed to further differentiate these hemolytic bacteria on a strain level. This study showed that hemolytic bacteria were also present in healthy human fecal samples, and its presence may be relevant to the wellness the individual. So we shoud select the donor which was not existed in intestinal microflora.2. Analysis the effect of prebiotics on intestinal microflora of HFA piglet by molecular methods.It includes both in vitro and in vivo methods to study the prebiotics. The in vitro methods, that is, applies the simple batch culture or three-stage chemostat culture system to study the zymotic characteristics of oligosaccharide in different bacteria. Lots of results indicated that multiple prebiotics promoted the amount of Bifidobacteria and Lactobacillus but decreased that of Bacteroides and Clostridium. While the in vivo methods is to take the prebiotics directly by human being or to design animal models. The former is direct but demanding of a strict control, and what's more, both the difference of intestinal microflora among different human being and the sensitivity to different dosage of prebiotics would affect the results.In this study, DGGE-PCR, ERIC-PCR and Southern-blotting were adopted to analysis the fecal samples of different four stage which were collected from the HFA piglets models fed by prebiotics. On the ERIC-PCR pattern of the fecal samples of 12th, there was a band on control group pattern while disappeared on the oligosaccharide-treated group pattern. A library was constructed based on the specific band and the clones were classified into 16 types by enzyme digestion. Select the two dominant clones type to sequence and the result showed that one of them the similarity to Escherichia coli K-12 MG1655 was 97%;The other one, the similarity to Enterococcus faecalis V583 was 99%. On the other hand, it is possible that there was no significant difference on pattern because of the turbulence of intestinal microflora caused by ablactating;however, the prebiotics could affect the 12th piglet's intestinal microflora distinctly before ablactating. Furthermore, this study indicated that the prebiotics could suppress the Enterococcus faecalis V583 and Escherichia coli K-12 MG1655 probably but need to study farther.
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