Studies On The High-yield Of Amylase And Protease Feeding Function Bacteria

Probiotics, also known as Living bacterium agent or Microbial ecological agent, as a kind of noresidue, non-toxic side effects, no drug-resistant to green additive, it is more widely used in animalhusbandry. Commonly used four main categories probiotics are lactic acid bacteria, Bacillus, yeast,photosynthetic bacteria. Which forage Bacillus become a hot topic because of strong resistance, hightemperature resistant, easy storage and other unique features. The purpose of this test is to get a strongstrain of the degradation of starch and protein by protoplast fusion technique.Test separate a high yieldof amylase and protease strains from the sample, get five high-yield enzyme through the mutationbreeding methods, and protoplast fusion, and use it to improve the yield of bacillus subtilis amylase andprotease ability, provides a theoretical basis for Bacillus subtilis probiotics study.1. This study first to high-yield amylase and protease feeding function bacteria breeding theresearch.45degradation with high amylase and protease activity were isolated from soil and fresh pigmanure samples. Through the hydrolysis of starch Medium and casein hydrolysis medium to getscreening screened10strains and measure the amylase and protease activity of these strains. Screeningout one high amylase and protease strain of JAPPS2. Starch and protease activity respectively is1.846IU/mL�'�9.848IU/mL. Determination the growth curve and enzyme curve of the starting strain.Determine the logarithmic phase of the starting strain is8-12h, after12h into stable period, enzymeactivity elevate at8-14h.2. The high yield of amylase and protease forage bacteria mutation breeding the research. Used byultraviolet radiation, Ethyl sulfate as single mutagen treated, determinate the enzyme activity of incomemutant strains. Filter out the high-yield enzyme mutants strains, these are UJ-3、UJ-5、DJ-1and DJ-2.Determination of amylase and protease activity respectively are4.457U/mLand1.509U/mL、5.092U/mLand11.728U/mL、4.513U/mLand12.306U/mL、5.124U/mLand13.031U/mL.The best conditions forsingle mutagenic treatment were ultraviolet irradiation160s; the concentration of DES is1.5mg/mL,processing40min. Using UV–DES compound mutagens, JASSB2Bacillus subtilis as original strain,on the basis of the single-factor test, used by the quadratic regression orthogonal rotation combinationtrial design. Optimize the mutagenesis conditions of composite mutagenesis JASSB2Bacillus subtilisand mutagenic treatment the JASSB2Bacillus subtilis under the optimized mutagenic conditions,determination the mutant enzyme activity of combined mutagenesis. The experimental results showthat under the conditions of ultraviolet irradiation160s; the concentration of DES is1.5mg/mL,processing40min can screening the mutant strains of U-DJ4,the amylase and protease activityrespectively is5.280U/mL�'�13.324U/mL3. Genome shuffling breeding high amylase and protease strains the research. The experimentalresults show that: the best conditions for Protoplast formation, inactivated, and fusion is processing15min, Lysozyme concentration is1.0mg/mL,37℃enzymolysis15h; Protoplast uv-inactivated is15min, heat-inactivated is60℃processing90min; Protoplast fusion conditions are the PEG6000concentration is40%, fusion temperature is34℃, fusion time is40min, at this point fusion rate can up to5.6×10^-5. To use mutagenesis proceeds strain as Original strain, After two rounds of shuffling get thereorganization bacteria of F299, It amylase and protease activity respectively is6.732U/mL and18.842U/mL。...