| The yield of soluble whole-cell protein from Alicylobacillus acidoterrestris DSM 3922T cells,is dependent on the effectiveness of cell lysis post fermentation.Various cell lysis approaches were investigated,including ultrasound,boiling,lysozyme,and cell lysis reagent treatment,and the degree of cell lysis was analyzed by SEM.The different samples of soluble protein were determined by SDS protein polyacrylamide gel electrophoresis.The results showed that the cell lysis reagent treatment was the most effective in extracting soluble whole-cell protein.The optimization of the soluble whole-cell protein were carried out by one-factor-at-a-time experiments and response surface method.Verifying the repeatability of SDS-PAGE electrophoresis,and analysis the whole-cell protein of Alicyslobacillus as a method for identification;Finding out the reason for the unobvious reaction of API-50CH kits,this research modified the standard method of API system,which was applied to the rapid identification of Alicyslobacillus.The results of this study are as follows:1、The yield of soluble whole-cell protein from A.acidoterrestris DSM 3922T cells is dependent on the effectiveness of cell lysis.Various cell lysis approaches were investigated,including including ultrasound,boiling,lysozyme,and cell lysis reagent treatment.The results showed that the cell lysis reagent treatment was the most effective in extracting soluble whole-cell protein.Different methods showed various protein profiles.The background of protein bands of the soluble whole-cell protein by lysis reagent treatment is light,and the number of protein bands is the highest among the four cell lysis methods.As a result,using lysis reagent treatment to extract soluble whole-cell protein is most feasible.2、The most significant processing parameters,namely incubation temperature,reaction time,and the ratio of cell lysis reagent volume and bacteria weight(A/B ratio),were optimized by using the Box-Behnken(BB)with a quadratic regression model built by Response Surface Methodology(RSM).The influence order of factors to protein extraction rate is:A/B ratio>time>temperature.The highest soluble whole-cell protein yield at 161.18mg/g was attained under optimal time,temperature,and A/B ratio at 37.4min,18.9℃,and 27.0 ml/g,respectively.3、Through comparing culture conditions on protein profiles,the results showed incubation time,medium and incubation temperature did not affect protein profiles.Different batches of the same strain and different protein profiles of the same sample showed minor discrepancies.Different cell lysis methods significantly affect the protein profiles.4、The protein profiles of whole-cell proteins by SDS-PAGE of 20 showed different degrees of variation,which can be applied to distinguish acidophilic bacteria.The results were consistent with the analysis of the 16S rDNA sequence sequencing,indicating that this method is feasible for identification of Bacillus subtilis and Alicyclobacillus.5、API-50CHB can not be used for the identification of Alicyclobacillus had nothing to do with indicator,but was related with the amount of acid produced by glycolytic reaction.The amount of fermented acid,which did not reach the threshold of indicator,was too little to make the indicator occur change.Using a modified method(adjust the inoculum concentration to 4 MacFarland),API-50CH kits showed obvious results.6、The API 50CH results of A.acidoterrestris DSM 3922T were obtained by the modified methods.The API-50CH results showed the glycolytic fermentation profiles of Alicyclobacillus(12)and B.subtilis(8)had significant differences,but A.acidoterrestris had the same glycolytic fermentation profiles except AAT12.The cluster analysis showed that this method can separate Alicyclobacillus from B.subtilis. |
