Study On The Co-production Of γ-poly-glutamic Acid And Nattokinase By Solid-state Fermentation Of Bacillus Subtilis

γ-polyglutamic acid（γ-PGA）is an anionic polymer that can be synthesized by microorganisms.Its excellent properties have attracted wide attention in the fields of food,medicine,agriculture and cosmetics.Nattokinase（NK）is a serine protease rich in natto.It has the functions of improving blood circulation,anti-atherosclerosis,anti-hypertension and thrombolysis.There are literatures on the co-production of the two,but due to the slightly different requirements for the medium composition of theγ-PGA and NK-producing strains,the yield and activity are easily limited by the composition of the medium.The co-production ofγ-PGA and NK industrial production is difficult to develop.In this study,the co-production medium was optimized through single factor experiment and response surface method to achieve the yield and activity when the two were synthesized separately.And molecular imprinting technology was used to prepare molecular imprints for high efficiency and specificity extraction ofγ-PGA.The structure,adsorption performance,swelling performance and reusability of the prepared molecular imprints were explored.The genes expression levels of co-production strains at different stages of fermentation were analyzed by RNA-seq technology to analyze and explain the co-production mechanism.Finally,the biological control and fertilizer synergism of the soybean cake fermented product by solid state co-production and fermentation combined withγ-PGA and NK and the antibacterial activity of the soybean cake fermented product and its components were explored,which could be used as a kind of bacterial fertilizer potential strategy.The results of the study are as follows:（1）Single factor experiments and response surface methodology were used to optimize the co-production fermentation medium.A medium-based formula with highγ-PGA yield and NK activity was obtained.The result was as follows:sucrose,sodium glutamate,Mg^2+,K₂HPO₄and calcium ion concentration were optimized for the medium and then 3 factors（inoculation amount,sodium glutamate and calcium ion）that had a significant impact on the yield were selected.Response surface test determined the optimal combination of various factors,using comprehensive indicators（γ-PGA yield accounted for 60%,NK activity accounted for 40%）to carry out the Box-Behnken design test,the optimal condition is:the inoculation amount is 4.11%,The mass concentration of sodium glutamate is 35.90 g/L,and the concentration of calcium chloride is 2.10×10^-6mol·L^-1.After the optimization of the model,the yield ofγ-PGA was 16.24 g/kg（wet base）,which increased by 12.14%and the activity of NK increased by 7.38%,indicating that the obtained model had certain practical guiding significance.（2）Molecularly imprints were prepared that specifically extractedγ-PGA by molecular imprinting technology.CS（2.5 g）and PEG 2000（0.6 g）were dissolved in 100 m L of 2%glacial acetic acid solution.The mixture was heated in a water bath and 2.5%chitosan and1.5 m L glutaraldehyde were reacted at 40℃for 12 h.The optimal process determined by the orthogonal test is 100 m L 2.5%CS solution,0.55 g PEG 2000,1.5 m L glutaraldehyde solution,the temperature is 43℃and the polymerization time is 14 h.The structure of the molecularly imprinted membrane was characterized.The infrared spectrum and SEM of the membrane proved that the preparation of the molecularly imprinted membrane was successful.The adsorption performance of the molecularly imprinted membrane was studied.In the Langmuir equation,the regression coefficients R²of MIP and NIP are both higher than 0.97,which indicated that the adsorption was a single layer.The correlation coefficient of the quasi-second-order adsorption kinetics of theγ-PGA molecularly imprinted membrane is0.9725.The result showed that the adsorption of MIP toγ-PGA was chemical adsorption.The swelling rate for MIP was 213%and for NIP was 140%.It showed that the swelling performance of MIP was better.The MIP calculated by nitrogen adsorption and desorption experiment and imprinting factor（IF）was 4.76,indicating that MIP has good template recognition ability and adsorption selectivity.The adsorption capacity of MIP dropped by about 17%,while the adsorption capacity of NIP dropped by about 26%after five times of adsorption-desorption.The results showed that the ratio ofγ-PGA in ethanol extracted by precipitation method was 60.58%and the ratio ofγ-PGA extracted by MIP was increased by10.42%.This proved that the application of MIP greatly improved the extraction efficiency ofγ-PGA.（3）The monitoring of the fermentation process showed the highest yield ofγ-PGA（358.5 g/kg,w/w,dry base）and the highest activity of NK（1388 U/g）and 5%was selected according to the yield ofγ-PGA and the activity of NK（w/w）sucrose and 4%（w/w）sodium glutamate are added.And the samples for RNA library construction:fermentation process and differentially expressed gene production time 6 h（NK production time）,9 h（γ-PGA production time）and 24 h（NK highest activity time）.The third-generation high-throughput technology RNA-seq transcriptome sequencing was used to sequence the three samples of the library and the gene expression levels of the three stages were compared,and the law of co-production was preliminarily explored.Through the analysis of its metabolic law to explain its co-production mechanism,while looking for potential targets to increase the production ofγ-PGA and NK activity.The specific pathways of differentially expressed genes are grouped based on the KEGG database.Among them,the proportion of genes involved in carbohydrate metabolism including PTS system and pyruvate metabolism was up-regulated.Among them,the expression of two genes encoding GDH（gud B and roc G）indicated that nattokinase did not inhibit the synthesis of glutamine.When the synthesis rate of NK was high,it might be related to the inhibition of the expression ofγ-PGA synthesis genes.And the gene expression level of apr N gradually increased,indicating that NK is not restricted by the joint production conditions during the fermentation process.Part ofγ-PGA was degraded due to the action ofγ-PGA depolymerase.Through the analysis of metabolism pathway,glt ABC（co-encoding glutamate-α-ketoglutarate amidotransferase）,glt P（glutamine）Acid transporter)and ycg MNO were all up-regulated,while gud B（encoding glutamate dehydrogenase）was down-regulated.（4）The method of co-producingγ-PGA and NK through solid-state fermentation of Bacillus subtilis natto was applied to the biological control and growth of cucumber seedlings.First,we explored the optimal fermentation conditions（the inoculum was 6%,the initial moisture was 55%,the initial p H was 7.0,the fermentation temperature was 37°C and the fermentation time was 72 h）.In the pot experiment,the biological control and fertilizer synergistic effects of fermented soybean cake were explored.The resulted showed that the use of fermented soybean cake cultured with Bacillus subtilis to conduct biological control research on cucumber fusarium wilt,the mortality rate of cucumber seedlings was reduced from 92%to 17%.And in the fertilizer efficiency experiment,the dry weight of the stem and root of cucumber seedlings and the ratio of root to shoot increased by 16.9%and 11.8%,respectively.At 1/2 nutrition,they increased by 19.6%and 13.9%,and 1/3 nutrition increased by 27.0%and 22.5%,respectively.The addition of fermented soybean cake culture significantly increased the dry weight of roots and shoots and the ratio of roots to shoots（R/S）at various nutritional levels.At lower nutrient levels,the increase was more pronounced.The stable existence of Bacillus subtilis in the soil also proves that the strain had the potential as a biocontrol bacteria.The results of antibacterial experiments on different components of fermented soybean cake（Bacillus subtilis,γ-PGA and NK crude enzyme solution）showed that the addition of crude enzyme solution increased the survival rate of seedlings by 20%,indicating that the effect of nattokinase crude enzyme solution was not as good asγ-PGA,but its components had antifungal ability.Bacillus subtilis was effective against Staphylococcus aureus and Fusarium oxysporum obvious inhibition,the inhibition rates were 29.87%and31.20%.Therefore,fermented soybean cake cultured with strain co-produceγ-PGA and NK,which could be used as a potential bacterial fertilizer utilization strategy.In this study,the medium conditions for the co-production ofγ-PGA and NK by Bacillus subtilis solid-state fermentation were optimized;the mechanism of the co-production ofγ-PGA and NK by Bacillus subtilis was summarized by RNA-seq;a specific adsorption ofγ-PGA molecularly imprinted membrane was prepared,and the performance of the molecularly imprinted membrane to adsorbγ-PGA was characterized;fermented soybean cake was added to the soil of cucumber seedlings to explore the biological control and fertilizer synergistic effect of fermented soybean cake on cucumber seedlings.This study provided a certain theoretical basis for the industrialization and application of co-production and fermentation ofγ-PGA and NK.