| The disorder of lipid metabolism caused by high-fat diet has become an important factor affecting people's health.The regulation of lactic acid bacteria on lipid metabolism disorder has become a hot spot in the study of lactic acid bacteria to help reduce blood lipids.In this paper,we thoroughly understand the genetic and metabolic basis of Lactobacillus rhamnosus hsryfm 1301 by whole genome sequencing,and explore its probiotic characteristics.At the same time,the effects of lactobacillus rhamnosus hsryfm 1301 fermented milk on lipid metabolism were evaluated by in vivo experiments.Furthermore,transcription and metabolomics were used to study the regulation of Lactobacillus rhamnosus hsryfm 1301 fermented milk on lipid metabolism,and to explore the mechanism of action of Lactobacillus rhamnosus hsryfm 1301 fermented milk to reduce blood lipids.The main contents and results are as follows:1 In vitro characteristics and functional gene analysis of Lactobacillus rhamnosus hsryfm 1301The ability of 10 strains of Lactobacillus rhamnosus to resist acid,bile salt,bacteriostatic,cholesterol lowering and triglyceride was studied to screen strains with excellent functional properties in vitro.The results showed that Lactobacillus rhamnosus hsryfm 1301 has strong acid and bile-tolerant ability,the survival rate in pH 3.0 environment reached 72.42%,and the survival rate in 0.1%and 0.3%bile salt concentration also reached 65.34%.and 63.54%.At the same time,it has strong inhibition ability against Escherichia coli,Salmonella and Staphylococcus aureus,and the diameter of the inhibition zone is greater than 12 mm.In addition,Lactobacillus rhamnosus hsryfm 1301 also has strong ability to degrade cholesterol and triglycerides,and the degradation rates are 63.90%and 37.25%,respectively.Whole genome sequencing of Lactobacillus rhamnosus hsryfm 1301 was performed to analyze the functional genes of the strain.The results showed that the Lactobacillus rhamnosus hsryfm 1301 genome was 3022021 bp in length and contained 3094 protein coding genes,15 rRNA genes and 59 tRNA genes.Through the KEGG pathway annotation analysis,it was found that 46 genes in the Lactobacillus rhamnosus hsryfm 1301 genome were involved in 11 pathways related to lipid metabolism,including fatty acid synthesis,fatty acid decomposition and unsaturated fatty acid synthesis.This suggests that Lactobacillus rhamnosus hsryfm 1301 may achieve its auxiliary hypolipidemic function by regulating lipid metabolism.2 Effect of fermentation time on Lactobacillus rhamnosus hsryfm 1301 fermented milkThe effects of prolonged fermentation time on the beneficial metabolites of fermented milk and their antibacterial and antioxidant properties were studied.The results showed that the number of viable cells did not change significantly after 72 h of fermentation(P>0.05).Fermented for 72 h facilitated the production and accumulation of metabolites.The 72 h fermented milk contained 39 volatile flavor substances,significantly higher than 17 h(P<0.05),and acetaldehyde,diacetyl and other characteristic flavor substances.The content was also significantly increased(P<0.05).In addition,the content of short-chain fatty acids in fermented milk for 72 h was significantly higher than 17 h(P<0.05),and the contents of acetic acid and propionic acid were 1.0170 mg/mL and 0.8012 mg/mL,respectively.The inhibition zone diameter of the 72 h fermented milk against Staphylococcus aureus,Salmonella,Pseudomonas,Escherichia coli,Bacillus cereus and Bacillus subtilis was more than 19.75 mm,which was significantly higher than 17 h(P<0.05).The total antioxidant capacity was 6.67 U/mL,which was significantly higher than 4.94 U/mL at 17 h(P<0.05).The metabolites of the 72 h fermented milk were better accumulated,the antibacterial ability and antioxidant capacity were enhanced,and the probiotic function was better3 Effect of Lactobacillus rhamnosus hsryfm 1301 fermented milk on lipid metabolism disorderA rat model of hyperlipidemia was established by feeding high-fat diet to study the regulation of Lactobacillus rhamnosus hsryfm 1301 fermented milk on lipid metabolism disorder in hyperlipidemic rat model.The results showed that the contents of triglyceride(TG),total cholesterol(TC)and low density lipoprotein(LDL-C)in the serum of rats treated with Lactobacilltts rhamnosus hsryfm 1301 were 1.04?mol/L and 2.12 ?mol,respectively./L,1.57 mmol/L,significantly lower than the model group rats(P<0.05).At the same time,serum high-density lipoprotein(HDL-C)content and aspartate aminotransferase(AST)and alanine aminotransferase(ALT)activities were significantly increased(P<0.05),the content was 0.66 mmol/L,91.90 U/L,4.53 U/L.In addition,the free fatty acid(FFA)in the serum and liver of Lactobacillus rhamnosus hsryfm 1301 fermented milk group was 1259.28 ?mol/L and 143.16?mol/L,respectively,which was significantly lower than that of the model group(P<0.05).At the same time,the contents of fatty acid synthase(FAS)and acetyl coA carboxylase(ACC),the key enzymes of fatty acid synthesis in the liver,were also significantly lower than the model group(P<0.05).Hyperlipidemia severely damaged the liver cell structure of rat liver.There were a lot of hepatocyte fat vacuoles in hepatocytes,and the milk vacuoles of rat liver cells decreased after the intervention of Lactobacillus rhamnosus hsryfm 1301 fermented milk.It indicated that Lactobacillus rhamnosus hsryfm 1301 fermented milk can reduce the production of excessive fatty acids by inhibiting fatty acid synthase,thereby reducing liver lipid deposition and body fat accumulation,regulating lipid metabolism disorder and achieving auxiliary hypolipidemic function.4 Mechanism of regulation of lipid metabolism disorder by Lactobacillus rhamnosus hsryfm 1301 fermented milkThe regulation effect of Lactobacillus rhamnosus hsryfm 1301 fermented milk on the transcriptional level of lipid metabolism related genes in rat liver was studied by RT-PCR.The results showed that the fermented milk of Lactobacillus rhamnosus hsryfm 1301 significantly up-regulated the level of AMP-dependent protein kinase(AMPK)and liver X receptors a(LXRa)in rat liver(P<0.05),but there was no significant change in peroxisome proliferators-activated receptors(PPAR?)(P>0.05).At the same time,sterol-regulatory element-binding protein-1c(SREBP-1C)which is the downstream genes of AMPK and LXRa and its regulated fatty acid synthase levels of FAS and ACC were significantly down-regulated(P<0.05).The expression levels of fatty acid transferases FAT/CD36 and ABCA1 also increased significantly(P<0.05).It indicated that Lactobacillus rhamnosus hsryfm 1301 fermented milk can also accelerate the lipid metabolism by regulating lipid transport and accelerating the decomposition of fatty acids and cholesterol in the liver.The regulation effect of Lactobacillus rhamnosus hsryfm 1301 fermented milk on the metabolites in rat serum was studied by metabolomics.The results showed that the fermented milk of Lactobacillus rhamnosus hsryfm 1301 had a regulatory effect on 50 metabolites in the serum of rats,and 33 of them had significant changes(P<0.05).The contents of 13 metabolites such as 2-Methylbenzoic acid,Estradiol valerate,Anthranilic acid(Vitamin Ll),Pelargonic acid and 1-Stearoyl-2-arachidonoyl-sn-glycerol in the serum of fermented milk intervention group increased significantly(P<0.05).20 metabolites such as 1-Myristoyl-sn-glycero-3-phosphocholine,Oleic acid,Formylanthranilic acid,D-Pipecolinic acid and Ethyl glucuronide were significantly decreased(P<0.05).Among them,2-Methylbenzoic acid has the highest up-regulation ratio,and the P/M multiple is 3.606561386.Ethyl glucuronide has the highest down-regulation ratio,and the P/M multiple is only 0.365010518.Cluster analysis of differential metabolites showed that the selected candidate metabolites were reasonable and accurate.KEGG metabolic pathway analysis of differential metabolites showed that Lactobacillus rhamnosus hsryfm 1301 fermented milk intervention to restore lipid metabolism by regulating the synthesis of unsaturated fatty acids,insulin resistance,amino acid metabolism and choline metabolism.In summary,Lactobacillus rhamnosus hsryfm 1301 fermented milk regulates key metabolic metabolites while promoting the transcription of liver AMPK and LXRa,inhibiting the transcription of SREBP-1C and its downstream fatty acid synthesis related genes FAS and ACC,thereby inhibiting fatty acid synthesis..At the same time,Lactobacillus rhamnosus hsryfm 1301 fermented milk promotes the transcription of lipid transport related genes FAT/CD36 and ABCA1 by promoting the transcription of AMPK,thereby promoting lipid transport and accelerating lipid decomposition.Inhibition of fatty acid synthesis and promotion of lipid transport is an important pathway for Lactobacillus rhamnosus hsryfm 1301 fermented milk to regulate lipid metabolism and achieve an auxiliary hypolipidemic function. 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