Safe Self-inducible Expression And High-density Fermentation Of Soybean Umami Peptide

Umami peptides,as new“green and safe”peptides flavoring agent,have great potential in the development of new flavor seasonings.The research on the industrial preparation method of umami peptide is an important step during promoting the application of umami peptide.Currently,the preparation methods reported mainly focus on the traditional enzymatic extraction and chemical synthesis.These two methods may have problems as low purity of the product or the potential toxicity of chemical reagents and so on,which make us urgently need to explore a safe production method with high purity and high yield.The microbial cloning provides a new idea for the safe and efficient preparation of umami peptides.Soybean is popular among consumers for its delicious taste,rich protein and bioactive peptides.Soybean Umami Peptide（SUP,Ser-Ser-Arg-Asp-Glu-Gln-Ser-Arg）has also received attention from researchers in the field of umami peptides.And currently,the research on safe clonal expression and fermentation production SUP with microbial system is relatively lacking,and further exploration is needed.In this study,the umami peptide was taken as the research object.Then its safe and efficient preparation process was studied by food biotechnology such as molecular docking,clone expression,Maillard reaction and high-density fermentation.The specific research contents are as follows:Firstly,PEP-FOLD3 and AutoDock vina molecular docking technology were used to analyze the three-dimensional structure difference between SUP had added 6×His tag or not,and to investigate the feasibility of adding affinity tag to C-terminus of umami peptide SUP.It was found that the addition of the affinity tag 6×His tag at the C-terminus of SUP only extended theα-helix structure of the SUP,and the overall structural did not change much.The molecular docking result with the umami receptor also showed the interaction sites of them are mostly the same and the hydrogen bonds of the one added 6×His tag were increased.It is speculated that SUP can activate the umami receptor after adding the 6×His tag to make the human body feel the umami taste.So the SUP added 6×His tag was used as the expression sequence of the SUP,B.subtilis 168 as the expression host strain,and the self-induced expression vector pMA09srfA constructed on the basis of pMA09 and srfA as the SUP expression vector to construct the umami peptide safe auto-inducible expression system B.subtilis 168/pMA09srfA-SUP.After expression and purification,SDS-PAGE electrophoresis showed that the target protein SUP was successfully expressed.The sensory verification results showed that the umami taste of SUP dialysate reached 6.5 points,and there was a certain umami enhancement effect.It indicated that B.subtilis 168/pMA09srfA-SUP may be used as an expression engineering strain of umami peptide SUP to produce umami peptide.Further the taste characteristics of SUP Maillard reaction were analyzed.Firstly,combining with the single factor experiment and orthogonal test results,the optimal Maillard reaction conditions were as follows:the reducing sugar was xylose,the reaction temperature was 120°C,the reaction time was 80 min,and the initial pH of the reaction was 7.5.Subsequently,the difference in taste between SUP and its Maillard reaction product was compared.The results showed that after the Maillard reaction,the umami and umami-enhanced effect of SUP was improved,its kokumi taste and persistent feeling were enhanced.And the bitterness and acidity were reduced.In addition,during the Maillard reaction,SUP produces nitrogen oxides,sulfides,and hydrocarbons,alcohols,aldehydes,etc.,which form meat flavor,coke flavor and roasted aroma,making the flavor of product be more full and rich.It indicated that SUP and xylose reacted at 120°C for 80 min,which could further enhance the umami taste and effectively improve the overall flavor of SUP.It can be used to prepare SUP Maillard compound flavoring agent.In order to expand the expression level of SUP,a high-density fermentation study was carried out on the recombinant expression bacteria.Firstly,the fermentation conditions were optimized at the shake flask level,and the optimal culture process was obtained:glucose was used as the fermentation carbon source,the initial glucose concentration was 20 g/L,the NH₄⁺concentration was 0.25 mol/L,and the culture temperature was at 37°C,the concentration of bacteria and SUP reached 2.773 g/L and 0.128 g/L,respectively.Based on the optimization of shake flasks,batch fermentation experiments were carried out,and the maximum accumulation of bacteria and products was 25.57 g/L and 0.97 g/L,respectively.Further use the exponential flow feeding strategy to optimize SUP high-density fermentation to increase SUP production.The results showed that the final concentration of recombinant expression strain B.subtilis 168/pMA09srfA-SUP reached 46.21 g/L,and the SUP concentration reached1.76 g/L.Its SUP yield is 1.8 times than that of batch fermentation,indicating that the exponential flow feeding strategy can effectively increase the high-density fermentation yield,which is an optional effective fermentation feeding strategy.