Study On The Molecular Mechanism Of Lactic Acid Bacteria To Imprqve The Quality Of Sourdough

In recent years,the consumption of fermented food in China has been keeping rising.Fermented food with multiple varieties,high quality,and health function are increasingly expected by consumers.The microorganisms involved in its fermentation process are yeast and Lactic acid bacteria(LAB).And studies on the mechanism of yeast fermentation in sourdough have been extensively reported,but studies on improving the quality and flavor of sourdough with LAB are rarely reported.Dough,which is the living environment of LAB,interacts with the bacteria.The relationship of LAB and dough is interdependence and interaction.But in revealing the mechanism of action of the relationship is still weak.Different LAB in the dough are affected by the fermentation conditions and can initiate different metabolic pathways to produce special flavoring substances and functional ingredients,thus forming a unique product.In recent years,the consumption of fermentation foods in our country have also been increasing year by year.Fermented foods with varieties,high quality and functions are increasingly expected by consumers.Compared with the West,China still lacks systematic theoretical research on the topic of fermentation pasta.Therefore,two LAB isolated from sourdough were selected in this study.And the effect of flour on metabolism of LAB was investigated by transcriptomics sequencing.At the same time,we use traditional molecular cloning to express the relevant enzymes,in order to study the relevant genes from the molecular level.To clarify the way that lactic acid bacteria improve the quality of sourdough,and enrich the research on the molecular mechanism of fermented foods in our country.First,after the dough was fermented by L.acidophilus 0201 or L.helveticus 0103,the sourdough pH,total titratable acid content and free amino acid content were measured.The results show that they both have good fermentation characteristics.Next,metabolic pathways and related genes with significant changes before and after fermentation of L.acidophilus 0201 and L.helveticus 0103 were obtained by using transcriptome sequencing.After fermentation,87 genes of L.acidophilus 0201 were significantly up-regulate and 23 genes were significantly down-regulated.As to L.helveticus 0103,there were 577 genes up-regulated after fermentation,and 36 genes were significantly down-regulated.For L.acidophilus 0201,we screened 14 genes out of 7 metabolic pathways based on its high-producing arginine and valine and verified by RT-qPCR.As to L.helveticus 0103 high-yield of glutamate,we screened 43 genes out of 11 metabolic pathways based on transcriptome sequencing and verified by RT-qPCR.Based on the verification of RT-qPCR,we selected two genes LBA1116(pyrroline-5-carboxylate reductase,PCR)and LBA1957(prolyl aminopeptidase,PEPL)involved in the arginine and valine metabolism pathway of L.acidophilus 0201.And two genes in the glutamate and glutamate pathways of L.helveticus 0103: LHE_RS16190(UDP-N-acetylmuramoyl alanine--D-glutamate ligase,uridine diphosphate,MURD)and LHE_RS19445(orotate phosphoribosyl transferase,OPT)were cloned and expressed.Four genes expression vectors were successfully constructed and induced by IPTG in E.coli BL21.Except pyrroline-5-carboxylate reductase,other three enzymes were successfully expressed and purified by 6×His tagging to obtain the purified protein.The purified PEPL,MURD,and OPT mixed with adjuvants that were used to immunize mice to obtain anti-serums for Western Blot detection.We found that the PEPL produced by L.acidophilus 0201 at 2,4 and 8 h after fermentation was significantly up-regulated by 1.28,1.47 and 1.66 fold,respectively,compared with the control group.The MURD produced by L.helveticus 0103 at 2 h,4 h,and 8 h of fermentation was significantly up-regulated by 1.41,1.37,and 1.51 fold,respectively,compared to the control group.The OPT was significantly up-regulated by 1.24,1.47,and 1.67 times,respectively.Moreover,after the exogenous addition of PEPL,the contents of arginine and proline were 82 mg/kg and 120 mg/kg,respectively,which were significantly higher than control group(44 mg/kg and 76 mg/kg).Then,we evaluated breads that were bake by the fermented doughs with L.acidophilus 0201 or L.helveticus 0103.Image analysis,texture analysis,and sensory evaluation were carried out.In the image analysis,the bread husk prepared by LAB have better color than the control group,and the void of bread core is small and uniform.In the texture analyzer test,In the texture analyzer test,the hardness of the bread prepared by L.acidophilus 0201 was 104.0 g and the Springiness was 96.23%,which was significantly better than the hardness and springiness of the control group 170.4 g and 90.97%.In the sensory evaluation test,the bread with lactic acid bacteria had a more obvious acid odor,and the appearance and taste scores were better than the control group.This study explored the molecular mechanism of L.acidophilus 0201 and L.helveticus 0103 in improving the quality of sourdough,enriched the molecular level study of the application of lactic acid bacteria in fermentation pasta,and proposed a new research method for LAB and food matrix.At the same time,it provides a theoretical basis for optimizing the production process of sourdough,and also provides a solid foundation for industrial production.