Potential Mechanism Of Glucose Intolerance Induced By Biogenic Preservative Nisin

Gut microbiota is known as the body’s"invisible organ".It can help to digest food,synthesize vitamines and maintain intestinal function.Gut microbiota can also interact with other body systems through the circulatory,immune,endocrine,and nervous systems,combining environmental factors with genetic,immune,and metabolic signals to further impact host physiology and health.With the spread of industrial lifestyles worldwide,the resulting"industrial"gut microbiota with lower microbial diversity is considered as one of the causes of metabolic disorders and the progression of many chronic diseases.Up to now,gut microbiota has been proven to be the driver of a variety of metabolic disorders and even diseases.Therefore,it is necessary to view the gut microbiota as a central hub for exposure to chemicals.In modern society,antibacterial substances with bactericidal and bacteriostatic functions are widely used in production and life.This study focused on the impact of commonly used antibacterial substances in food on body health,namely food preservatives and antibiotics.The development of food industry and the globalization of food circulation require the use of food preservatives in food processing to achieve the purpose of food preservation and prolonging food shelf life.Up to now,some studies have reported the negative effects of food preservatives on gut microbiota and body health.These results showed that although food preservatives in the market have been evaluated,they are not totally safe owing to these evaluations mainly focus on the acute toxicity and carcinogenicity of food preservatives.In view of the key role of gut microbiota in body health regulation,it is necessary to systematically evaluate the effects of commonly used food preservatives on body health from the perspective of gut microbiota and metabolism,which will further improve the safety assessment data of food preservatives and provide references for the rational use of preservatives in the food industry.Therefore,this paper focused on food preservatives and systematically evaluated their effects on gut microbiota and metabolism.In addition,improper use of antibiotics in animal husbandry and aquatic products will lead to antibiotic residues in primary agricultural and livestock products.Antibiotics entering the body through the food chain will exert a variety of negative effects,such as gut microbiota dysbiosis,evolution and transfer of antibiotic resistance genes,and even induce pathological symptoms such as antibiotic-related diarrhea.Therefore,it is necessary to use antibiotics rationally and develop effective intervention strategies to repair the adverse effects of antibiotics.Main research contents and results were summarized as follows:1.The effects of commonly used food preservatives on gut microbiota and glucose metabolism were evaluated,including chemical synthetic sources（sodium benzoate,potassium sorbate,sodium diacetate,methyl parabens,ethyl parabens,sodium nitrate and sodium propionate）and biological sources（nisin,natamycin,lysozyme andε-polylysine）.On this basis,the dose-dependent effect of nisin on glucose metabolism,the potential mechanism of its interference with glucose metabolism and the role of gut microbiota mediating glucose metabolism dysfunction induced by nisin were further explored.The results were as follows:（1）Food preservatives perturbed gut microbiota and glucose metabolism in healthy mice:The results of glucose tolerance test showed that chronic exposure to food preservatives for eight weeks induced glucose intolerance in healthy mice,regardless of their origin,among which the biogenic nisin induced the most significant glucose intolerance in healthy mice.In addition,16S r DNA sequencing results showed that both kinds of food preservatives significantly altered the gut microbiota of healthy mice,among which nisin exerted the most significant.In addition,nisin significantly changed gut microbiota metabolism,leading to a significant downregulation of amino acid.The above results showed that compared with chemical synthetic food preservatives,biological food preservatives were not always superior to synthetic ones.It is not suitable to directly use them as substitutes for synthetic food preservatives.（2）Dose-dependent effects of nisin on glucose metabolism:The effects of nisin on glucose metabolism based on acceptable daily intake（ADI）and three times ADI level were investigated through glucose tolerance and insulin tolerance tests.The results showed that nisin at the ADI level did not induce significant glucose intolerance in mice,but nisin at three times the ADI level significantly interfered with glucose metabolism,induced glucose intolerance and insulin resistance in mice.At the same time,significantly decreased glucagon-like peptide 1（GLP-1）and increased glucagon level were found in the 3ADI group.（3）Potential mechanism of the glucose metabolism disorder induced by nisin:In view of the significant influence of nisin on amino acid metabolism,this study focused on the amino acid sensing pathway in the intestine.The contents of amino acids and metabolites in the small intestine and colon contents were analyzed through targeted metabolomics.The results showed that amino acids and metabolites showed a dose-dependent downregulation in the nisin treatment groups.Multiple physicochemical experiments further showed that nisin treatment down-regulated the protease activity in the intestinal tract,resulting in the disturbance of protein hydrolysis and general downregulation of amino acids release.The general downregulation of amino acids and hormone secretion alteration of amino acid sensing pathway suggested that nisin might interfere with glucose metabolism via amino acid nutrient sensing pathway.To test this hypothesis,free amino acid supplemented diet was designed to explore its effect on glucose metabolism in nisin-treated mice.The results showed that amino acid diet reversed the glucose intolerance induced by nisin and Ca²⁺-Sensing Receptor（Ca SR）antagonist treatment reproduced the phenotypic characteristic of nisin treatment.Therefore,nisin can perturb glucose metabolism through Ca SR amino acid nutrient sensing pathway.（4）Gut microbiota mediated nisin-induced glucose metabolism disorder:This study focused on small intestinal and colon microbiota.Firstly,metagenomic sequencing was used to explore the dose-dependent effects of nisin on microbiota.The results showed that ADI and 3 times the ADI level of nisin significantly changed the small intestinal microbiota.And only 3 times the ADI level of nisin significantly altered the colon microbiota.In view of the important regulatory role of small intestinal nutrient sensing pathway and small intestinal microbiota in digestion,absorption and metabolism,this study explored the role of small intestinal microbiota in mediating glucose metabolism disorder induced by nisin through microbiota transplantation.The microbiota of the control and the 3ADI groups were transplanted into antibiotic treated mice,and the metabolic changes were explored by glucose tolerance test,amino acid content and protease activity detection,and key glucose metabolism regulation hormone quantitative detection.The results showed that compared with the control group,increased blood glucose level,decreased amino acid and increased glucagon level were found in the 3ADI microbiota transplantation group,which reproduced the metabolic phenotype characteristics of the 3ADI group.2.Based on the common residual antibiotics in food,gut microbiota dysbiosis model was established.Then,green banana powder,a common food material in daily life,was selected to repair the perturbed intestinal microecology in order to develop a diet intervention strategy widely applicable to the public.Compared with the natural repair ability of gut microbiota after antibiotics treatment,the repair ability of green banana powder was evaluated by fluorescence in situ hybridization,intestinal permeability measurement,fluorescence quantification PCR,western blotting and 16S r DNA sequencing.The results showed that,compared with natural repair group,green banana powder reduced the intestinal permeability,reduced the gut microbiota erosion,increased the expression of mucin-2 and repaired the intestinal mucus barrier.In addition,green banana powder significantly enriched a variety of beneficial bacteria,and the enrichment level of Bacteroidales S24-7 was positively correlated with the recovery rate of gut microbiota,indicating that Bacteroidales S24-7 might mainly drive the recovery of gut microbiota.These results suggested that green banana powder can be used as a functional food ingredient to repair antibiotic-induced dysbiosis and intestinal barrier damage.