Screening, Optimization And Application Of Alkali-tolerant And High-temperature Pullulanase Strains

Pullulanase(EC3.2.1.1/41)specifically hydrolyzes the a-1,6-linkages in branched oligosaccharides of amylopectin,and leads to the formation of amylose.At present,more pullulanase production strains are mainly concentrated in aerobacter aerobacter,aquatic thermotoler,Bacillus cereus,Lactobacillus and so on.Most of the pullulanase produced by these strains are acidase,enzyme activity is low under alkaline conditions,which greatly limits the application of pullulanase in detergents and other industries.In this paper,a wild strain of production pullulanase was isolated from soil.According to its morphological characteristics,physiological and biochemical identification(16S rDNA),the strain was identified as Bacillus megaterium.The single factor test and response surface analysis were carried out on the conditions for pullulanase production.Through the study of the properties of crude enzyme,it was found that the optimum pH was 8.0,the optimum temperature was 50 ?,which had good alkali resistance and high temperature resistance.To broaden the application of alkaline pullulan in food,this experiment explored the application of hydrolyzate maltooligosaccharides in food preservation.The main research contents and results are as follows:1.Screening and identification of alkali-resistant high temperature resistant strains that can product pullulanaseBased on morphological observation,Gram staining,physiological and 16S rDNA biochemical tests,this bacterial strains was identified as Bacillus megaterium.2.Optimization of the enzyme-producing ability of Bacillus megateriumBased on the single factor test,the optimum conditions for producing enzyme were determined by Plackett-Burman and Box-Behnken software.The optimum conditions were as follows:glutinous rice starch 0.85%,yeast extract:tryptone(1:2)4%,KH2PO4 0.05%,MgSO4.7H2O 0.01%,NaCl 0.2%,MnSO4.7H2O 0.005%,culture medium initial pH 8.8,culture temperature 21.0 ?,fermentation time 55 h,enzyme activity value 0.77 U/mL.3.Study on the properties of crude enzymeThe fermentation broth was centrifuged to remove the cells?ammonium sulfate concentration and dialysis desalination,obtain the crude enzyme so.lution of pullulanase.The optimum pH was about 8.0,and it was stable in the range of pH 7.0?9.0.The optimum reaction temperature was about 50 ?,The heat stability is better in the range of 35?50 ?.SDS-PAGE and zymogram analysis showed that there were two pullulanase active bands in the crude enzyme solution.The crude hydrolyzate contains maltose,maltotriose and a small amount of maltotetraose by thin layer chromatography.4.Effect of maltooligosaccharides on preservation of tilapia filletsDifferent concentrations of maltooligosaccharides were used for preservation of tilapia fillets.Sensory changes,pH value,conductivity,total number of bacteria,volatile basic nitrogen(TVB-N),thiobarbituric acid(TBA)and water loss rate were determined to study the tilapia fillets changes of the freshness during storage in ice.Studies have shown that the addition of 2%maltooligosaccharides is the best for tilapia.