Difference Analysis Of Folate Biosynthesis Of Lactobacillus Plantarum Close Relatives And Optimization Of Fermentation Conditions

The chemical name of folic acid is pteroyl monoglutamate,which is a water-soluble form of vitamin B9.Folic acid is an essential trace element involved in almost all biological metabolism,because it plays a key role in the synthesis and metabolism of substances,so people cannot survive without leaving folic acid.Since the vertebrate cells themselves cannot synthesize the micronutrients,they can only be obtained by food intake.Therefore,if the folic acid intake is too small,the corresponding folic acid deficiency disease will occur in the body.Folic acid is widely present in various foods,but it is extremely unstable,and its loss rate during cooking and preservation is high.In order to solve this problem,folic acid is currently synthesized mainly by supplemental chemical.However,studies have shown that chemical synthesis of folic acid is on the human body.There are certain side effects.At the same time,folic acid deficiency is still a major global nutritional problem,so it is extremely urgent and important to develop functional foods rich in folic acid.In this study,two sub-species Lb.plantarum subsp.Plantarum YM 4-3 and Lb.paraplantarum AY01 strains of Lactobacillus plantarum were used to ferment three fermentation substrates of MRS,skim milk and soybean milk,Under the fermentation of naturally fermented and maintained at pH 6.0 conditions,the growth status of Lactobacillus plantarum YM 4-3 and AY01 was quite different.L.plantarum produces a large amount of organic acid during natural fermentation,which causes the pH of the culture medium to decrease.Since microbial cells are very sensitive to changes in pH,even lactic acid bacteria inhibit their life activities at lower pH conditions.The results showed that the YM4-3 and AY01 strains had a shorter duration of stability during natural fermentation.As the fermentation time prolonged,the viable cell count was lower than that of maintaining pH 6.0;in addition,in the MRS matrix The number of viable bacteria decreased rapidly,and when skim milk and soy milk were used as the substrate,the higher activity was maintained for a long time,indicating that skim milk and soybean milk can protect the plant Lactobacillus in an acidic environment.When the pH 6.0 fermentation was maintained,the YM4-3 and AY01 strains had a longer duration of stability,and the number of viable cells remained higher as the fermentation time prolonged.Folic acid is unstable under acidic conditions,while folic acid is stable under alkaline or neutral conditions.However,the current research fails to effectively control the pH of the fermentation process,resulting in a certain degree of degradation of folic acid.In this study,the fermenter was used to automatically monitor the process in real time,and the pH of the fermentation process was maintained at 6.0 to investigate the effect of pH on folic acid synthesis.L.plantarum needs to consume folic acid during the growth process,so during the cultivation process,L.plantarum will first consume the folic acid in the medium and then self-synthesize to supply cell consumption.The folic acid assay showed that the folic acid content in the MRS matrix was higher,about 33-57 ng/mL,and the folic acid content in the soybean milk was about 3 ng/mL.No folate content was detected in the skim milk.Compared with 0h,the folic acid in MRS and soybean milk decreased significantly at 12 h,indicating that Lactobacillus plantarum first used the folic acid in the matrix to supply cell consumption.When MRS was used as the fermentation substrate,the folic acid content was higher under natural fermentation conditions,and only the AY01 strain had a higher folic acid content in the matrix after 60 h.When skim milk and soymilk were used as fermentation substrates,the folic acid content was improved under the fermentation conditions of pH 6.0.And YM4-3 had higher folic acid yield when fermented with soybean milk.In order to study the effect of pH on the genes involved in folate synthesis during the culture process,the main genes for the synthesis of 6-hydroxymethyldihydropterin pyrophosphate pathway during folate biosynthesis were quantitatively detected by real-time PCR.The results showed that there were some differences in the response of various subspecies of Lactobacillus plantarum to fermentation conditions.When maintaining pH6.0,YM4-3 strains fol B,fol K,fol E,fol P and fol Q were all down-regulated in MRS matrix.Correspondingly,the folate content decreased,while the relative expression of the gene was up-regulated at 36 h in AY01 strain;when fermented in skim milk,the folate synthesis-related gene of AY01 strain was down-regulated,and folic acid accumulated a little;but under this condition,The relative expression level of folate synthesis related genes in YM4-3 strain was significantly increased in skim milk.In this study,the growth characteristics of different subspecies of Lactobacillus plantarum,different fermentation substrates and the ability of folic acid synthesis under different pH conditions were studied to lay a theoretical foundation for the development of Lactobacillus plantarum and its metabolites as novel functional foods..