Fermentation Optimization Of Feather-degrading By Bacillus Cereus X5

Keratins are insoluble fibrous proteins highly cross-linked with disulfide bridges,hydrogen bonds,and hydrophobic interactions.The tightly packed supercoiled polypeptide chains result in high mechanical,stability and resistance to proteolysis by common proteases such as trypsin,pepsin,and papain.Keratin is the major component of feathers,which represent up to 10%of the total,weight of mature chickens.Since feathers are generated as a waste product in commercial,poultry-processing plants,reaching millions of tons per year worldwide,keratinous wastes are increasingly accumulating and there is a demand for developing al.ternatives for their recycling.Waste feathers are currently converted to feather meal,by a hydrothermal,process;however,this process destroys certain amino acids,yielding a product with poor digestibility and variable nutrient qual.ity.Keratinolytic microorganisms may have important biotechnological,applications in the environment-friendly processing of keratinous wastes from the poultry industry.Particularly,since poultry feathers and al.so feather meal.are nexpensive,abundant,and readily available,the utilization of such byproducts as substrates by keratinolytic microorganisms represents a feasible microbial,technology for obtaining proteolytic enzymes,microbial.biomass,and protein hydrolysates with lower production costs.Bacillus cereus X5,an Feather-degrading bacterium,was isolated from the reeling mill waste water and further identified with registration number FJ763651 on Genbank,have a stable effect on degradation of feather.By using waste feather as the substrate,The fermentation medium and fermentation conditions were statistical,optimized,and then determination the content of amino acid and protein in the fermented liquid.The main results were as follows.1.10 strains of feather-degrading bacteria was used to carry out the degrading ability test,the degradation ability test medium:K2HPO4 5.59 g,KH2PO4 0.41g,Ca3(PO4)2 0.004g feather 10g,watet 1000mL.The degradation rate of more than 80%strains:X5 88.9%,S6489%,S68 88.7%;the content of amino acid was above 2000 ?g/mL strains:X5,X8,X10,S64and S68.The congtent of protein of S59 was the highest,388?g/mL,S64 and S68strains,the protein content were 249 ?g/mL and 235 ?g/mL.Combined with the stability of degradation rate,protein and amino acid content,Bacillus cereus X5 was used for fermentation optimization2.Through the cultivation in shake-flask fermentation,,the effects of fermentation medium and environmental,conditions on the titer of fermentation broth had been studied.Determine the inorganic sal.t composition of fermentation medium by one-factor-at-a-time experiments and orthogonal,design method.In the optimization of medium,the influences of KH2PO4,K2HPO4 and CaCO3 on the Feather degradation rate broth were first eval.uated using Plackett Burman design.Among of the components tested,Both KH2PO4 and K2HPO4 had significant effect on the Feather degradation rate.The path of steepest ascent was used to approach the optimal.region of the medium composition.In the last step,the optimal.concentrations of components were determined by central,composite design and response surface anal.ysis.The components of optimum medium for Feather degradation rate were:CaCO3 0.1%,KH2PO4 0.068%,K2HPO40.32%,feather 8%.Based on the optimal.medium,the inoculation volume,volume of medium,fermentation temperature,initial.pH and fermentation time were confirmed by the experiment of one-factor-at-a-time.The optimized fermentation conditions for Feather degradation rate were:inoculation volume 2%(v/v),fermentation temperature 38?,initial.pH 8.0,rotate speed 220r/m,incubation time 40h.Using the best fermentation and fermentation conditions for the shake-flask fermentation.Feather degradation rate increased to 81.3%from 41.3%,feather by adding amount to 8%from 1%.3.Through the ninhydrin and amino acid can be quantitatively happened the color reaction was to increase amino acid contents,Through the Coomassie brilliant blue G-250 and protein can be quantitatively happened the color reaction was to increase protein contents,After the examination,Amino acid content is 48.4 g/L,protein content is 8.12 g/L.4.Fermentation liquid centrifugal,filter supernatants were collected,by a rotary evaporator,frozen dried powder of amino acid type and content analysis,the results show that,the fermentation broth containing 17 amino acids,and amino acids composition of feathers,and contains a variety of animal of the essential amino acids,in which glutamic acid relative content of 10.1%,cysteine relative content6.75%,the relative content of phenylalanine proline 16.4%,relative content of 8.26%.