Interaction Between Lactobacillus Fermentum LF-12 And Kudingcha Dicaffeoylquinic Acid

Kudingcha(KDC)is mainly produced from the plant Ilex kudingcha from the Ilex genus of the Aquifoliaceae family and the Ligustrum genus of Oleaceae family.It is a kind of herbal tea with bitter and sweet taste that has a wide audience in China.The main components of Kudingcha are flavonoids,polysaccharides,saponins,polyphenols and volatile oils.Among them,polyphenols are one of the main active ingredients in Kudingcha.Especially Caffeoylquinic acids(CQAs)compounds,the main componets of Kudingcha polyphenols,including caffeic acid(CA),monocaffeoquinic acid(MonoCQAs)And dicaffeoyl quinic acid(DiCQAs).DiCQAs are rich in Kudingcha with fewer investigations compared to other types of phenolic acids,that’s the reason why they are selected as research materials.Dietary polyphenols play an important role in human health However,due to its polyhydroxy structure and large molecular weight,it is difficult to be degraded by the upper digestive tract,and remain intact when entering the colon,it interacts with intestinal microorganisms,on the one hand,it affects the metabolic pathway of the intestinal flora,and activates certain polyphenolsmetabolizing related genes and express certain enzymes in microorganisms.On the other hand,the activation of specific enzymes in polyphenol-hydrolyzed strains allows polyphenols to be effectively degraded,thereby increasing the bioavailability of polyphenols in the human body.Intestinal microorganisms have been a hotspot in recent years.Based on the results of this experiment,we speculate that polyphenols interact with specific strains of intestinal microorganisms and specific enzymes to exert their biological activities.The main contents and results are as follows:1.Isolation and purification of specific esterase from DiCQAs-hydrolyzed strainIn this section,a gut-derived microorganism Lactobacillus fermentum LF-12 capable of hydrolyzing DiCQAs was isolated from the feaces of volunteers.Different carbon sources have different effects on the growth of L.fermentum LF-12.When lactose is used as the carbon source,L.fermentum LF-12 can exhibit the best DiCQAs hydrolysis ability.In the presence of DiCQAs,LF-12 can express an intracellular enzyme capable of hydrolyzing DiCQAs.The enzyme was purified by ammonium sulfate fractionation,dialysis,DEAE-Sepharose Fast Flow ion exchange and Sephacryl S-200 molecular exclusion chromatography.Followed by prokaryotic expression.After purification,its molecular weight is about 31.9 kDa and pI is about 4.71.2.Study on the characteristics of DiCQAs on L.Fermentum LF-12 and its hydrolaseStudies have shown that different concentrations of polyphenols have different effects on the growth of bacteria.Specifically,the appropriate concentration of polyphenols can promote the growth of bacteria,while high concentrations of polyphenols can inhibit the growth of bacteria.The experiments in this section investigate the growth of L.fermentum LF-12 by using different concentrations of DiCQAs.The optimal DiCQAs concentration is 1.5 mg/ml.At this concentration,the extracellular alkaline phosphatase(AKP)and extracellular soluble protein of L.fermentum LF-12 are both at a low level,indicating that this concentration of DiCQAs does not destroy bacterial integrity.The experiments in this section also explore the enzymatic properties of DiCQAs hydrolase in L.fermentum LF-12.The enzyme shows its highest hydrolyzing ability at 45℃;the optimum pH is 6.5;ethylenediaminetetraacetic acid(EDTA)，Co2+ and Cu2+ can inhibit the activity of the esterase,while Ca2+ and Mg2+ may be used as coenzymes of the enzyme to increase enzyme activity;methanol,acetonitrile,acetone,dimethylformaldehyde(DMSO)and ethanol at different concentrations can reduce enzyme activity,indicating that the enzyme is sensitive to organic solvents;When 3,4-DiCQA,3,5-DiCQA and 4,5DiCQA act as substrate,the Km value of the enzyme was significantly different(p<0.05),and the Km value was lowest when 3,4-DiCQA was used as substrate.Therefore,3,4-DiCQA has the highest affinity for the enzyme among the three substrates.Finally,the molecular docking of DiCQAs with the simulated protein 6a6o.1.A was studied.The optimal binding energies of 3,4-DiCQA,3,5-DiCQA and 4,5-DiCQA with 6a6o.1.A after simulation were-6.51 Kcal/mol,-3.46 Kcal/mol,3.76 Kcal/mol espectively.The order of binding energy is 3,4-DiCQA<4,5-DiCQA<3,5-DiCQA,which is consistent with the order of Km values in enzyme kinetics,the forces of 3,4-DiCQA with 6a6o are mainly van der Waals forces,weak hydrogen bonds,carbon-hydrogen bonds,alkylation and π-alkylation interactions.The interactions between 3,5-DiCQA and 6a6o are mainly van der Waals force,weak hydrogen bond,pi-donor,and π-alkylation.The interactions between 4,5-DiCQA and 6a6o are mainly van der Waals forces,weak hydrogen bonds and π-alkylation.3.Proteomics of DiCQAs-treated L.fermentum LF-12This section continues to discuss the effect of DiCQAs on the L.fermentum LF12 protein group at a concentration of 1.5 mg/ml.The Data independent acquisition(DIA)method was used to study the differential proteins in the experimental group and the control group.A total of 1207 proteins were quantified,of which 202 were differential proteins.There were 112 proteins with significant differences;a total of 68 proteins were up-regulated and 44 proteins were down-regulated.Of the 202 differential proteins,141 GO terms were annotated into biological processes,7 were annotated into cell components,and 54 were annotated into molecular functions.Studies on the KEGG enrichment scatter plot of differential proteins showed that metabolic pathways,antibiotic biosynthesis,secondary metabolite biosynthesis,and amino acid biosynthesis are enriched in proteins.When discussing the effect of the addition of DiCQAs on specific proteins in the glycolysis/gluconeogenesis pathway,the pentose phosphate pathway,and the galactose metabolism pathway in the metabolic pathway,it was found that the addition of DiCQAs can improve the carbon source utilization pathway and amino acids metabolism pathway.