Organic Acid Detection And Neuroprotective Effect Of Lactobacillus Acidophilus Metabolites

The brain-gut axis is a network containing the gastrointestinal tract,enteric nervous system,and brain.Two-way information exchange between these entities affects important functions such as immunity,digestion,metabolism,and stress response.More and more animal and clinical studies have shown that the intestinal flora plays an important role in the two-way response of the intestine axis,especially with the functional development of the central nervous system and the occurrence of diseases.Lactobacillus acidophilus is the main bacteria of the intestinal flora,and is also one of the representatives of probiotics.It has a positive effect on human health,It has been widely used in the food industry and medicine.However,the reason for the interaction between probiotics and the nervous system is not yet clear.Based on the above research background,firstly,Lactobacillus acidophilus was resuscitated and activated,its growth curve was measured,and its growth period was analyzed to obtain Lactobacillus acidophilus culture supernatant.Then high performance liquid chromatography was used to establish a method for the analysis of metabolized organic acid content in the supernatant of Lactobacillus acidophilus.Finally,the effect of L.a.s on the proliferation of NSCs was studied.In this section,Lactobacillus acidophilus?ATCC 4356?purchased from the China General Microbiological Culture Collection Center was resuscitated and activated,and the microscopic morphology of Lactobacillus acidophilus was observed by scanning electron microscopy.At the same time,the growth curve was measured,the results showed that the growth stability of Lactobacillus acidophilus was between 20h and 32h.Afterwards,the content of lactic acid and acetic acid in the metabolites of Lactobacillus acidophilus was determined by high performance liquid chromatography,and the optimal chromatographic conditions were obtained:The separation was performed on C₁₈ column.The column temperature was 30°C.The mobile phase was 0.01mol/L NaH₂PO₄ solution?1mol/L phosphoric acid was adjusted to pH 2.6?.The flow rate was 1mL/min.The detection wavelength was 215nm.The whole process was completed in 10min.Under optimized chromatographic conditions,the linear range of lactic acid and acetic acid was1.0-50.0mmol/L,the recoveries of lactic acid and acetic acid were 97.5%-103.0%,96.5%-103.8%,and the relative standard deviations were 1.39%.and 3.56%.This method was used to quantify the supernatant during the stationary phase of growth.It was found that the lactic acid and acetic acid content reached a maximum for L.a.s in the culture time of 30h,indicating that the content of metabolites in L.a.s was the largest,so the choice of incubation time was 30 hours.Finally,the effects of L.a.s on the proliferation of NSCs were investigated.The primary extraction and passage culture of NSCs were performed firstly.The results of immunocytochemical identification showed that the extracted NSCs have self-renewal and multi-differentiation potential and can be used as seed cells for experiments.L.a.s cultured for30h were treated with different volume fractions of NSCs.Morphological observation and fluorescence staining showed that NSCs grew normally.The results of CCK-8 assay showed that when the volume fraction of L.a.s added was 4%,the proliferation rate of NSCs reached the highest,and the cell survival rate increased to 122.72±7.37%in the control group.At this concentration,the effect of L.a.s on the differentiation behavior of NSCs can be further investigated to explore its neuroprotective effects in more depth.