Study On Feed Additive Of Bacillus Intestinalis From Yellow Croaker

Isolation and screening of animal intestinal bacillus and accurately develop specific intestinal probiotics can promote intestinal awareness more effectively,make it grow rapidly and form colonies in the animal's intestinal environment.It can also maintaining the balance between the intestinal microflora and the host greatly improves the feed utilization rate,has significant economic and ecological benefits.In this study,a strain of Bacillus was isolated and identified from the intestine of wild large yellow croaker,and its culture conditions and culture medium were optimized.Finally,the feasibility of fermenting cultivate intestinal bacillus with different edible fungi strains as fermentation substrate was discussed.The specific findings are as follows:In this study,a strain of bacillus LYC-1 was first isolated from the healthy large yellow croaker's intestine using gradient dilution method of intestinal fluid;It was identified by combination of colony and morphology and sequence analysis based on 16S rDNA;The qualitative analysis of Protease,Amylase,Cellulase and Superoxide Dismutase(SOD)in this strain by hydrolysis ring;The tolerance of artificial intestinal and gastric juices and osmotic pressure tolerance of this strain were investigated.The results show:The strain was clustered with Bacillus(Genus),combined with morphological features,confirm that LYC-1 belongs Bacillus.Production enzyme analysis display,The strain has the ability to secrete proteases,amylase and SOD.Moreover,it is able to tolerate gastrointestinal fluids and high concentrations of osmotic pressure.Single-factor experiments were used to study the carbon and nitrogen sources,inorganic salts and fermentation conditions suitable for protease production by LYC-1.The conditions of the liquid fermentation and the medium were determined by the Placke-Burman design,the steepest climbing test,and the Box-Behnken method in the response surface,and It's been validated.And scale-up culture in a 25 L fermenter.The results show.The suitable culture medium and fermentation conditions of the bacterium are:Glucose 2.0%,peptone 1.8%,KH2PO4 0.2%,MgSO4 0.05%,Cacl2 0.02%,pH 8.8,temperature 25 ?,rotation speed 180 r/min,liquid volume 20 mL.The verification experiment results in a protease activity of(418.62±0.74)U/mL,a 1.92-fold increase over the original culture.In addition,the protease enzyme activity of the fermentation tank was up to 693.51 U/mL,which was 1.57 times that of the shake:flask fermentation and 3.18 times that before the optimization.The solid fermentation was used to study the culture of Flammulina velutipes,Hericium erinaceus and Pleurotus eryngii of Bacillus fermentation characteristics.To investigate the spore production of a total of 7 different culture mediums;Single factor tests were used to analyze the amount of carbon source,nitrogen source,and inorganic salt added;Design 5 factors 4 levels of orthogonal experiment.The results show:Flammulina velutipes is the most suitable fermentation substrate;The most suitable for orthogonal optimization is:Corn flour 3%,soybean meal 3%,bran 4%,KH2PO4 0.2%,CaCO3 0.2%.The number of spores can reach 7.32×109cfu/g.