Study On Breeding Of Phytase-producing Bacillus Subtilis And Its Fermentation Products From Cottonseed Meal Source

Object:The research based on the induction of Bacillus subtilis to breed a phytase-producing ones, and determine the best conditions of mutation. Orthogonal design4factors and5levels and2factors and5levels were used to optimize fermentation parameters, substrate composition and inoculum concentration of fermented feed with Bacillus subtilis (B. subtilis), which was evaluated via quantity of B. subtilis and the activity of phytase in fermented substrates, further fermentation process was determined and lay foundation for the production of high quality fermented feed. Explore the degradation level of phytase on phytate in cottonseed meal, at the same time, by comprehensive evaluation of nutrition value and biological characteristics of source of cotton pulp fermented product to evaluate the biological characteristics and nutritional value of the fermented feed. The main objective of this research was to develop B. subtilis fermented feed from cotton meal, so as to exploit the rich cotton meal resource of Xinjiang and apply the biological characteristics of B. subtilis culture, to develop fermented feed of B. subtilis from cotton pulp source, and put it into animal production, promoting the animal growth, protecting animal health, and improving feed conversion rate.Methods:The dissertation consists of three experiments. Experiment1:Screening of phytase-producing B. subtilis, then use ultraviolet (UV), NTG, UV-NTG, NTG-UV four methods to mutagenic them, then to determine the best induced way and conditions of mutation. Experiment2:the4factors and5levels orthogonal design was used, from incubation temperature (29℃、33℃、37℃、41℃、45℃), period (12h、24h、48h、72h、96h), initial substrate moisture (30%、40%、50%、60%、70%), and pH value (6.0、6.5、7.0、7.5、8.0) four factors five levels, in accordance with the uniform design table U5(54).25experiment deals in total, to design experiment and to measure phytase activity from different treatments. Then, through analyzing the phytase activity of different combinations of fermented product, by orthogonal analysis of variance and extreme difference analysis, obtaining optimal fermentation condition parameters such as the incubation temperature, period, initial substrate moisture and pH value. Next, the design of two factors orthogonal design, from the substrate composition (cottonseed meal and maize in different proportions) and different vaccination levels (1mL、3mL、5ml、7mL、mL), two factors and five levels, according to the design table of orthogonal design52, altogether designing25experiment deals, by measuring the phytase activity and B. subtilis number, by the two factors have repeat observation data analysis of variance and multiple comparisons to confirm the composition of fermentation bottom material and proportion of inoculation. The purpose of Experiment2was to confirm the fermentation parameters of biological feed, the composition of fermentation bottom material and proportion of inoculation. The method of experiment3was that, based on the experiment2results above, the optimal fermentation process parameters was composed, and then the high-quality B. subtilis culture could be produced. After fermentation, the fermented product recovery, product B. subtilis number, phytate phosphorus, phytase activity, protease activity, and nutrients content (DM、CP、EE、NDF、Ash、Ca、P), available phosphorous were determined. So as to assess the biological characteristics and nutritional value of fermented product.Results:(1) B. subtilis was bred through four methods of ultraviolet (UV), NTG (NTG), UV-NTG, NTG-UV. The results showed that B. subtilis after100s of UV had highest phytase activity, phytase-producing reached71.96U/mL and higher38.46%than the initial strain. So UV was the best way and100s was the best conditions of mutation.(2) The optimized fermentation parameters of biological feed for phytase produced with B. subtilis were incubation temperature at37℃, incubation period of72h, moisture content of solid substrate40%, inoculum level7.0mL/100g, initial pH in7, and the substrate consisted of90%of cottonseed meal,10%of corn. (3) The study found that, the effective phosphorus content in the fermented feed from cotton meal increased by45.5%, significant difference (P<0.01), PP/TP decreased13.2%, significant difference (P<0.05), the activity of phytase increased103.72U/g, amylase activity increased24.43U/g, protease activity increased23.21U/g, and all nutritional value of biological protein feed from cotton pulp source have different degrees increase by B. subtilis fermentation. This showed that phytase-producing B. subtilis could degradation the anti-nutritional factor phytic acid in cottonseed meal source and has certain practical significance for improving the utilization rate of phosphorus in feed.