High Protease Activity Of Probiotic Bacillus Screening And Corn Gluten Meal Fermentation Crafts Research

Corn gluten meal,is a byproduct of corn processed starch.The content of hydrophobic amino acids?such as Isoleucine,Leucine,Alanine and Valine,etc.?and Glutamicacid is high,tryptophan and Lysine and other essential amino acid is very small,affecting its application value in food,feed and other industries.The use of microbes to reduce the degradation of corn gluten meal into a digestion and absorption of antioxidants and other functions of corn peptides,is a potential application of processing technology.In this study,10 strains of Bacillus with relatively strong proteolytic capacity were screened from the plate culture medium of corn gluten meal,and the alkaline protease activity of the 10 strains was determined by the method of phenolic phenol reagent,re-screening to get a strong ability to hydrolyze corn gluten powder Bacillus X1-1.The strain X1-1 was identified as Bacillus subtilis by comparison of bacterial16S rDNA sequence analysis and phylogenetic tree comparison and primers YyaR-F/TetB-R.The physiological and biochemical properties of Bacillus subtilis X1-1 were identified,which was able to secrete three kinds of proteases in the basic fermentation medium of corn gluten meal,the acid protease was 35 U/mL,the alkaline protease activity was 105 U/mL,and the neutral protease activity was 120 U/mL.The Oxford Cup antagonistic test results show that X1-1 has a protective effect on E.coli..The plate count method wasused as the evaluation index,and the results were compared with the bacterial growth test,showed that X1-1 strain had obvious ability to resist sodium sulfite and could grow well in medium containing 0.2%sodium sulfite,which was similar to that of corn gluten meal has a good ability to grow and protein hydrolysis capacity.The effect of six factors including substrate concentration,seed liquid inoculation amount,shaking speed,initial pH of culture medium,fermentation temperature and fermentation time on the content of soluble protein was studied by single factor experiment.Establishing six factor three level orthogonal design to optimize the corn protein powder fermentation process,the best culture conditions for protease generation were as follows:6%inoculation,temperature 37?,shaking speed 180 rpm,culture time 36 h.Fermentation medium composition:Corn gluten meal 40 g/L,KH₂PO₄ 1.65 g/L,K₂HPO₄ 0.5 g/L,CaCl₂ 0.1 g/L,NaCl 0.5g/L,Protein Peptone 2.0 g/L,pH 7.5.After fermentation,the ratio of soluble protein to total protein was 75.2%,compared with pre-fermentationwhich,increased 4.63 fold.The ability of Bacillus subtilis X1-1 to degrade maize protein powder with acid,medium and alkaline protease preparation was compared.Its degradation ability was significantly higher than acid,neutral protease preparations,and alkaline protease degradation ability is not much difference.The corn protein powder hydrolyzed by alkaline protease has obvious bitterness,and the taste of corn protein powder hydrolyzed by crude enzyme solution of X1-1 fermented corn protein powder has been improved obviously.After fermentation,the fermentation broth was spray dried and dried to obtain a solid sample of corn peptides.Compared with the corn protein powder,the corn peptides contained 2×10⁸ cfu/g,the neutral protease activity was 2217U/g,the crude protein and the small peptides were 69.7%and 47.6%respectively,compared with the raw material,the contents of protein and soluble peptide increased 1.81 and 5.35 fold,respectively.It was found that the molecular weight of the protein in maize peptides was decreased obviously,mainly due to the small molecular protein with molecular weight below 17 kD,and its hydrophilicity was also improved.The antioxidant activity of maize peptides was determined by DPPH method.It was found that with the increase of maize peptide concentration,the scavenging ability of free radicals was higher and higher,the highest clearance rate was up to 95.57%,compared with the raw material,increased 50%.When the concentration reached 1.0mg/mL,its antioxidant capacity is higher than the same concentration of vitaminc.