Metabolomics Analysis And Safety Evaluation Of Biogenic Amines Pathway Of Lactic Acid Bacteria Based On

Background:Lactic acid bacteria is widely used in food industry which has aroused extensive concern about their potential biogenic amines pollution. Previous research on amino acid-amine pathways was focous on genomics, proteomics, transcriptomics and enzyme kinetics. Metabolomics is a new technology which has developed dramaticly recent years. With an nontarget nuclear magnetic resonance spectroscopy approach, it could give a comprehensive analysis of the metabolome. This research was aimed to develop a high-resolution1H NMR and a high-resolution magic angle spin1H NMR method for metabolomic analysis of lactic acid bacteria culture supernatant, cell extraction and cell pellet, and to elucidate the regulation mechanism of biogenic amine pathway based on chemomtrics and the methods above metioned. Furthermore, we discussed the perspectivesof NMR metabolomics in food safety area.Objectives:(1)Developing a HR-NMR method for lactic acid bacteria culture supernatant and cell extraction, and a HR-MAS-NMR method for cell pellet through optimizing of current protocols.(2)Discussing the difference of bioamine-producing LAB metabolme under different culture conditions and the regulation mechanism of amino acid-amine pathway, searching for relating biomarkers.(3)Discussing the perspectives of NMR metabolomics in microbiological safety evaluation based on analysis of LAB isolates under different culture conditions.Methods:(1)NMR spectroscopy from MRS culture supernatant under different pH values was collected and evaluated by PCA. Four extraction methods in publications were evaluated by extracting the same quantity of cell pellet. MAS-NMR parameters were optimized for better spectroscopy.(2)Culture supernatant, cell extraction and cell pellet from six different media in mid-exponential phase and early stationary phase was collected and data sets were analysed by HR NMR, HR MAS-NMR and PCA(principle component analysis). Typical metabolites were calculated by chemomtrics to explain their interrelations. Then the influence of high concentration of free amino acid, glucose, low pH value and chitosan oligosaccharide on metabolome of biogenic amine producing strain was evaluated and the regulation mechanism of amine pathway discussed.(3)Culture supernatant of LAB isolates was collected in mid-exponential phase and early stationary phase. Typical metabolites were calculated to numeralizing their characteristics.Results:(1)Most metabolites including glucose, lactate, acetate, alanine, glysine had stable chemical shifts between pH6.0-8.0, while histidine and histamine’s chemical shifts changed evidently. Most metabolites had a smaller chemical shift under higher pH value, but glucose and methionine appeared in the opposite way. Ultrasonic was more effective than frozen-thaw repeated for the cell pellet extraction. Extraction with organic phase had a lower efficiency than extraction without organic phase, while it also had a smaller CV.(2)Growth characteristics of ATCC33222under different culture condition was various. High concentration glucose could significantly prolong the exponential phase, increase lactate yields and restrain amine production. COS could also repress amine production while reducing proliferation. Low pH value could stimulate amine pathway, especially early production of diamine which was harmful for bacteria growth.(3)We identified6tyramine-producing strains and10non-tyramine-producing strains,4homofermentation strains and12heterofermentation strains. Main metabolites and the changes between different time points could be quantified. COS, high concentration free amino acid and low pH value decreased lactate yeilds of homofermentaion lactobacillus brevis. Lactate could be further metabolized into acetate and other metabolites during stationary phase.Conclusions:(1)The optimized pH value for1H NMR experiment of LAB culture supernatant and cell extraction was7.4. Considering yields, reproducibility, convenience and speed, acetonitrile/water (1/1) combined with ultrasonic extraction was recommended in this research.(2) amino acid-biogenic amine pathway have two major functions:providing energy and a defense mechanism to withstand an acidic environment. Putrescine, cadaverine and histamine were similar in energy supply while bacteria would prefer to produce diamine to resist acidic environment. Cell could dynamically adjust the concentration of intracellular metabolites to make a balance of osmotic pressure and pH value. High concentraion of free amino acid was a prerequisit of amine pathway. Low pH value could stimulate amine production, while glucose and COS could act the opposite way. HR-MAS-NMR has been proved to be an effective way to detect intercellular metabolites of Gram-positive strain.(3) Tyramine pathway had no competitive relation with glycolysis pathway in energy supply function. Tyramine could increase lactate yields and reduce strain growth. It also could up-regulate lactate pathway in heterofermentation bacteria. Metabolomics technoque based on NMR could not only determine amine-producing or non-amine-producing, but also homo-or heterofermentation. It could quantify main products of heterofermentaion strain and changes over time. This method could be used as a new tool for strain screening and safety evaluation.