Forage Bacillus Natto Probiotics Research And Development

Natto is a kind of functional food, and Bacillus natto is one of probiotics that was permitted to be used in animal feed by State Agricultural Department. In this thesis, the isolation, identificatuion and selection of Bacillus natto strains were carried our, the producing technology of Bacillus natto contained product for feed by soy-been meal as the main raw material was researched, and the nutritional and functional properties of the Bacillus natto fermented product were evaluated. On these bases, the pilot experiment was done and the animal feeding experiment of the pilot product was carried out as well. The results obtained were as follows:1) Strain isolation and molecular identification of Bacillus natto. Natto number 1,3,4,6 were isolated and the standard natto 0 was purchased. DNA sequencing results showed that the full length of the 5 Natto 16S rDNA segments was 1417 bp. The alignment of the 5 Natto 16S rDNA with the sequence of B. subtilis subsp. natto 16S rDNA (AY608741) showed that all sequence had 99% identity.2) By testing under different culturing conditions such as temperature, pH and ventilation, the selection of Natto strain was carried out. The result showed that the influence of temperature, pH and air on Natto number 1,2,3,4,5,6,7 was different. Natto 4 was the most suitable for production use. And then some biological characteristics of Natto 4 were studied. The conclusion was that Natto 4 could endure high concentration of cholate, it was more sensitive to penicillin than to aureomycin, and could inhibit the growth of E. coli and aurococcus significantly.3) The growth of Natto bacteria reached peak density after 16 h of fermentation. The results of technology optimization showed that the suitable growing conditions for the bacteria were : the soy-bean meal marinated with water of pH 6.5 before fermentation , and the content of water, sodium, glucose, and gultin in culture medium was respectively 60 % (w/w) , 0.3 % (w/w), 5 % (w/w) and 5 % (w/w) . The suitable proportion of the fermented soy-bean meal and corn powder was 1:1 (w/w) in drying process. The effect of heating at 65 ℃ within 60 min on the live bacteria quantity was not significant. The live bacteria quantity would decline sharply when the heating temperature reached to 75 ℃ or higher.4) The acid protease content, protein content, peptide content and peptide mapping on the course of fermentation were studied. The result showed that the curves of the acid protease activity and peptide content were consistent to the growth curve of Natto. After 12 h of fermentation, the acid protease activity reached peak. After 14-16 h of fermentation, peptide content reached highest. The protein content decreased slightly during fermentation. Peptide mapping suggested that some peptides with molecular weight between 2561-4000 appeared after fermentation. The molecular weight of most of the peptides decreased from 1100 before fermentation to lower than 800.5) Pilot experiment and animal feeding experiment had been done. The live bacteria in the obtained product reached 7.5×10⁸ cfu/g, which was higher than that in the Standard of State Agricultural Department, which was 1 × 10⁸ cfu/g. The content of peptide, VB₂ and dipicolinic acid in the fermented product was greatly higher than in the raw culture. Animal experiment results showedthat adding 2kg/t of the product to the feed of wening piglets could increase the growth by 53.56 %, and decrease the feed-weight ratio by 20.82 %, and the growth of E. coli was inhibited to some extent and the growth of Bifidobacterium and lactic acid bacillus was enhanced significantly.