Effects Of Different Substrates On Fermentation Characteristics Of Bacillus Subtilis Natto

Bacillus subtilis natto（B.natto）,a commonly used probiotic,can produce a variety of substances including alkaline protease,nattokinase（NK）,vitamin K₂,and polyglutamic acid,and has widely been used in food,medicine,and health care.It has been shown that B.natto is usually used to degrade high protein substances such as soybean,but seldom used in the hydrolysis of other substrates such as sugar and lipid content,limiting the extensive research and application of B.natto.Different from soybean（SB）in the component,Lycium barbarum（LB）,Sunflower seed kernel（SF）and bamboo shoot shell（BSS）were chosen as substrates to study their correlationship with the metabolites of B.natto in this work.This provides a theoretical basis to expand the substrate range of B.natto.The contents were detailed as the following:（1）Study of solid state fermentation-submerged fermentation（SSF-Sm F）of LB.B.natto is used as the fermentation strain with LB as the substrate,and protease yield,protein content,total sugar content,peptide content and antioxidant activity were investigated to optimize the SSF-Sm F process.The results showed that under the optimal conditions,the maximum value of protease is 11.22 IU/m L,higher than that under SSF（8.51 IU/m L）and Sm F（4.8396 IU/m L）.A positive correlation could exist between metabolite protease and protein content as well as between peptide content and antioxidant activity.（2）Investigation of the effect of SB,SF,and LB on NK synthesis of B.natto based on the transcriptomics.SB,SF,and LB were separately used as the substrate for B.natto to produce NK.The variations in reducing sugar content,protein content,NK activity,protease activity,andγ-PGA yield in the fermented substrate were determined.Transcriptomic analysis is done to study signal transduction,central metabolism,energy metabolism,and substance transport in B.natto cultured in SB,SF,and LB for 20 h,and the correlation between the medium and metabolites production was also analyzed.The results show that the highest NK yield of 9582 FU/m L was achieved in SB.The activities of NK in SF and LB were 7466 FU/m L and 0 FU/m L,respectively,indicating that protein can promote protein synthesis through glycolysis and tricarboxylic acid cycle,and sugar formation through gluconeogenesis,enhancing cell density so as to trigger the transcription of apr E.However,the polysaccharides in LB cannot provide sufficient nitrogen,leading to amino acid starvation to repress NK synthesis.（3）B.natto and LAB as strains to ferment BSS substrates and investigate the effect of Na OH or HCl solutions in concert with microbial fermentation on the extraction of SDF and IDF.The yield,physical and chemical properties,GAC and structural characteristics of SDF and IDF were compared.The results showed that compared to HCl solution,Na OH solution in cooperation with microorganisms has significant effect in WHC and WSC.In terms of yield,the highest values for IDF and SDF were for A-BN（the cooperation between HCl solution and B.natto fermentation）and B-BN（the cooperation between Na OH solution and B.natto fermentation）,respectively.IDF is superior to SDF in WHC and OHC.For WSC,the highest IDF and SDF values were A-LABN（HCl in cooperation with LAB and B.natto）and B-BN（Na OH in cooperation with B.natto）,respectively.The GAC of SDF extracted by B-LABN（Na OH in cooperation with LAB and B.natto）is as high as 9.16 mmol/L.FT-IR showed that all samples have similar typical polysaccharide spectral characteristics.The above research provides a theoretical basis for promoting the application research of B.natto in different substrates.By analyzing the mechanism of different fermentation substrates on B.natto,it provides a new idea for further improving the yield of metabolites,which is conducive to broadening the research and development of new health products and their applications in food,health care products or dietary supplements.