Study On Isoiation And Characterization Of A Thermotolerant Feather-degrading Bacterium,Fermentation And Enzymatic Properties

In recent years,the production and consumption of poultry products have grown globally.Feather waste is produced after poultry slaughter and production of large,the annual output of feathers is about one million tons in China,The abolition of a large number of feathers not only caused a huge waste of resources,but also pollute the environment.Feather meal is completed in all kinds of amino acids,amino acids must be ready,especially sulfur-containing amino acids higher.So the full use of such resources will be a very important supplement to protein feed.Although keratin waste is rich in amino acids,it is a potentially large source of feed protein,but the digestibility is very low.If you can improve the utilization of keratin wastes,not only for the aquaculture industry to provide a large number of feed protein,but also to promote all the keratin waste collection and processing industry chain development,so that the use of these waste and Reduce emissions have a positive meaning,to reduce the pollution caused by the environment.To obtain high efficient thermotolerant keratin-degraded bacteria,train Y6 was isolated by feather meal as its sole source of carbon,nitrogen.The strain was identified as Bacillus licheniformis,which is based on colony morphology,morphological characteristics,physiological and biochemical characteristics,16SrRNA sequence analysis and its 16S rRNA sequence using BLAST of NCBI due to 99%homology with Bacillus licheniformis strain Xmb047 similarity,and named as Bacillus licheniformis Y6(Gen Bank ID:KY082766).The bacteria was used to degrade the whole feather,and after 72 h fermentation,the feathers were only left scapus,and the branches were completely degraded,indicating that the Y6 strain had a strong ability to degrade feather.Using ICR white mouse as the experimental animals,using the number of viable bacteria in up to 3.3 x 1014CFU/mL Bacillus licheniformis Y6 that had been cultivated for 24 h,and supernatant medium of Bacillus licheniformis Y6 feathers that had been fermented for 60h fed mice.The acute toxicity tests showed that mouse group did not die,they were alive and well.Execution of the test group and control group mouse,anatomy observed that liver,kidney,lungs,intestines and other organs of expernmental group mouse had no change.Above results showed that Bacillus licheniformis Y6 and its fermentation are non-toxic products on mouse.In order to optimize the conditions of solid State fermentation,mensurated the weight loss rate and the in vitro digestibility of the fermented product,optimized the conditions(feather meal dosage,the buffer solution dosage,Buffer pH,inoculum size,the thickness of culture medium,fermentation temperature,and fermentation time).The results showed that the best feathering dosage was 60%,the best buffer solution dosage was 150%,the best Buffer pH was 8.0,the best inoculum size was 20%,the best thickness of culture medium was 2.0 cm,the best fermentation temperature was 55 0C and the best fermentation time was 6 days.The in vitro digestibility and weight loss of solid-state fermentation products were 61.43%and 38.41%,respectively.The aim of this experiment was to isolate and investigate the protease of Bacillus licheniformis Y6.the fermented feather powder was used to isolate and purify the fermented product to obtain crude enzyme solution.The protease was purified from crude enzyme by 70%Ammonium Sulfate,Sephdex G-100 chromatography and analyzed by SDS-PAGE and zymography.The effect of protease activity on temperature and pH was detected and the characteristics of protease were investigated.It was shown that there were two proteases in crude enzyme.One protease with molecular weights of 50 KD showed an optimal activity at 70 0C and pH 8.0.Metalloproteinase inhibitors arid serine protease inhibitors can partially inhibit the activity of the enzyme,the enzyme is classified as serine metalloproteinase.This study indicated that Bacillus licheniformis Y6 is an appropriate strain for exploring microorganic fermentation system to degrade feather keratin,and had a great applied potential in producing high quality protein feed.