| Hyaluronic acid(HA),a polysaccharide with multiple physiological effects,has been classified as a new food resource by the National Health Commission of the People’s Republic of China in early 2021 and is allowed to be added to common foods.At present,studies on HA are mainly focused on cosmetic and medical aspects,while the effects on host metabolism,intestinal microbiota,and health after consumption as a food ingredient are still in the initial stage,especially the effects on type 2 diabetes are rarely reported.In order to evaluate whether HA can exert anti-diabetic activity based on changes in metabolic profiles and intestinal flora,a mouse model of type 2 diabetes was established by high-fat feeding combined with intraperitoneal injection of streptozotocin(STZ).After 4 weeks of HA intervention,the physiological and biochemical indexes of different treatment groups were compared and analyzed,and the intestinal contents of each groups were collected.The changes of metabolic profiles and intestinal microbiota in type 2 diabetic mice after HA intervention were analyzed by non-targeted metabolomics and 16 S r RNA high-throughput sequencing to evaluate whether HA has anti-diabetic activity.Based on this,aiming at the problem of dyslipidemia in type 2 diabetes,the potential probiotics with cholesterol-lowering function were screened,and their probiotic properties were preliminarily studied.This study further explored the physiological effects of HA,broadened the application range of HA,and provided a theoretical basis for the development of HA food and HA functional food.The main results are as follows :1.The results of physiological index determination in mice showed that the mice in the model group had symptoms such as weight loss,overeating,hyperglycemia,impaired glucose tolerance,decreased insulin secretion,increased inflammatory level and liver injury.After 4 weeks of experiment,the body weight of mice in the model group was 37.84±1.44 g,the food intake was 15.32±1.87 g,the fasting blood glucose value was 24.98±2.34 mmol/L,and the glucose AUC value was significantly increased.The serum insulin concentration was 61.5±7.39 pg/m L,the serum TNF-α concentration was 18.38±1.81 μg/m L,and the liver cells were massively necrotic,the cells were severely vacuolated,and the liver was severely damaged.After HA intervention,low-dose HA reduced the food intake of diabetic mice to 12.20±2.57 g;the medium dose of HA reduced the fasting blood glucose and serum TNF-α concentration of diabetic mice to 21.80±0.58 mmol/L and 8.44±1.85 μg/m L,respectively.High dose of HA can improve the symptoms of weight loss caused by diabetes.Low,medium and high doses of HA could significantly increase the serum insulin concentration of diabetic mice,with an increase of 26.15%,20.93% and 28.11%,respectively,and the symptoms of impaired glucose tolerance were relieved.In addition,HA reduced the number of necrotic hepatocytes,reduced intracellular vacuoles,and made the cells more orderly.2.The non-targeted metabolomics results showed that HA intervention for 4 weeks altered the metabolic profile of diabetic mice,with 122,142,and 189 differential metabolites produced in the low,medium,and high dose groups,respectively.The content of free amino acids such as arginine and taurine and dipeptide and tripeptide increased significantly.The metabolic pathways significantly enriched in the differential metabolites included Glycine,serine and threonine metabolism,Arginine and proline metabolism,beta-Alanine metabolism,and ABC transporters.Among them,Glycine,serine and threonine metabolism,Arginine and proline metabolism and beta-Alanine metabolism were up-regulated.Oxidative phosphorylation activity was down-regulated.3.The sequencing results of intestinal microbiota showed that HA increased the Bacteroidetes/Firmicutes ratio,with the most significant increase of 0.15 in the medium-dose group.The intestinal microbial diversity increased,and the abundance of various bacteria increased,such as Oscillospira,Clostridium,Coprococcus,Turicibacter,Weissella,and Lactobacillus.Among them,Turicibacter,Coprococcus,Weissella,and Lactobacillus increased most significantly,and the abundance of the harmful bacteria Desulfovibrio decreased.Functional prediction analysis showed that metabolic pathways such as Bacterial chemotaxis,Flagellar assembly,Insulin signaling pathway,and Linoleic acid metabolism were upregulated,while Tryptophan metabolism and Phenylalanine metabolism were down-regulated.In addition,the intervention of HA significantly increased the contents of acetic acid,propionic acid,butyric acid,and valeric acid,with the content of 2427±12.53,747.6±5.36,451.7±2.10,and 35.06±0.49μg/g,respectively.4.There were 10 strains with morphological differences were screened,and strain F5 with the highest in vitro cholesterol clearance rate was selected for the follow-up experiments.The strain F5 was identified by molecular biology as Weissella confusa.The strain had acid and bile salt resistance,and the common logarithm values of viable bacterial density were 6.39±0.16 and 4.09±0.16 after 3 h under the condition of p H 2.0acid and 0.3% bile salt,respectively,with survival rates of 82.45% and 54.53%.The self-condensation rate of the strain at 24 h was 52.11%.In addition,the strain showed strong inhibition of Staphylococcus aureus with an inhibition circle diameter of14.91±0.12 mm.In summary,HA intervention can change the structure and composition of intestinal flora,increase the number of beneficial bacteria,and then change the intestinal flora and metabolic function of the body,and change the types and contents of metabolites in the intestine.HA may promote the intestinal and body health of type 2diabetic mice by this mechanism,so as to improve the physiological indexes of diabetic mice.In addition,Weissella confusa,a strain screened from mouse feces under HA intervention,is a potential probiotic with many excellent characteristics,which is expected to colonize in the intestine of type 2 diabetic mice and improve their lipid metabolism disorders. |
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