| Obesity is a chronic metabolic disease caused by excessive accumulation of body fat due to energy metabolism imbalance.In recent years,dietary supplementation to improve obesity has become a hot research topic in the field of food and nutrition because of its broad target and low side effects.β-glucan,as a functional dietary fiber,has a positive effect on obesity treatment.Currently,commercial β-glucan is mainly extracted from cereal grains and yeast cell walls,which has the problems of cumbersome extraction and purification process and low content and,which greatly limits the large-scale industrial production.The use of bacterial fermentation to produce extracellular β-glucan has the advantages of low production cost and simple extraction process,providing a new way for the industrial production of β-glucan.Currently,a kind of β-glucan named Curdlan has been produced commercially by Agrobacterium fermentum,but its water-insoluble property at room temperature limits its application.In this study,a strain of extracellular water-soluble β-glucan-producing microorganism was developed.The production level of β-glucan was improved by mutagenesis breeding and gene knockout,and systematically researched its structural properties and biosynthetic mechanism.Subsequently,β-glucan was added to high fat food to investigate its effect on the digestion and absorption of triglyceride(TG),and its effect on TG metabolism in obese mice induced by high fat diet(HFD)was analyzed by long-term intervention.Firstly,an exopolysaccharide-producing strain(named ZB01)was obtained from an offshore saline soil and identified as Rhizobium pusense by 16 S r DNA sequencing.The polysaccharide was prepared by alcohol precipitation,protein removal,dialysis and column purification,and was identified as a(1→3)glycosidically linked straight-chain glucan with repeating units consisting of β-D-Glcp-(1→3)and a small amount of α-D-Glcp-(1→3)by monosaccharide composition analysis,molecular weight detection and structural analysis.Theβ-glucan yield was increased from 3.97 g/L to 9.52 g/L by UV-Atmospheric and Room Temperature Plasma(ARTP)compound mutagenesis breeding and optimization of medium and culture conditions.Secondly,a genome-wide analysis of strain ZB01 was performed,and the gene cluster related to β-glucan synthesis(named glu)was obtained by bioinformatics analysis,and its structural composition was analyzed to be more similar to the succinylglycan synthesis gene cluster exo.In addition,the correlation between this gene cluster and β-glucan synthesis was further verified by combining transcriptome differential analysis.Based on the results of the histological analysis,the specific roles of these genes on β-glucan synthesis were verified by knocking out 13 genes related to β-glucan synthesis in the gene cluster glu,and the phenotypes of the mutants △ glu A,△ glu D,△ glu H,and △ glu N were found to be significantly changed from the original strain.The four knocked out genes encoded β-glucan synthesis pathway blocker protein,β-1,3-glucosyltransferase,β-1,3-1,4-glucosidase and extracellular polysaccharide polymerase,respectively.The β-glucan production of the mutant strain △glu A,which knocked out the gene encoding the deterrent protein,reached 11.27 g/L,which was 7.30 g/L higher than that of the original strain.The results indicated that the negative regulatory property of glu A on β-glucan synthesis.In addition,the remaining 9 genes on the gene cluster have less effect on the amount of polysaccharide secretion.Finally,β-glucan was added to high-fat foods.In vitro simulated digestion experiments,in vitro simulated absorption experiments,and animal experiments confirmed that the addition of β-glucan could effectively reduce the digestion and absorption of TG,Besides,microstructural characterization using simulated digests revealed that it may be due to β-glucan binding to fat droplets and bridging them together,thus reducing the contact area with lipase.Furthermore,the application of β-glucan as a dietary supplement in an animal model of HFD-induced obesity effectively alleviated the weight gain of mice,reduced the tissue indices of blood glucose,epididymal fat(e WAT)and subcutaneous fat(SAT),and increased brown adipose fat(BAT)and altered TG levels in serum,liver and feces.In addition,β-glucan supplementation increased the relative abundance of beneficial bacteria such as Bacteroidaceae,Bacteroides and Alistipes in the intestine of HFD diet mice,and decreased the Firmicutes/Bacteroidete ratio(related to fat accumulation).The relative abundance of Lactobacillaceae(causing disorders of lipid metabolism),Erysipelotrichaceae(associated with metabolic diseases)and Faecalibaculum(enrichment due to high-fat diet)was reduced.A correlation analysis of the genus-level variation of intestinal flora with obesity and TG metabolism-related indicators revealed that Alistipes and Bacteroides were negatively correlated with serum and liver TG levels,and positively correlated with fecal TG levels.Subsequent,lipid omics analysis was conducted on the liver,an important metabolic organ,and 14 different shared lipids were selected for supplementation β-glucan significantly increased these different lipid levels in HFD fed mice.Based on pathway enrichment analysis,it was found that glycerophospholipid metabolic pathway may play a key role in high-fat dietinduced obesity and β-glucan regulation of high-fat diet-induced obesity.Meanwhile,it was found that the supplementation of β-glucan reduced the expression of fatty acid synthase(FAS)genes related to lipid formation in the liver of HFD mice,as well as the expression of peroxisome proliferators activate receptor γ(PPAR-γ)involved in the conversion of free fatty acid(FFA)to TG level and upregulation of the expression level of peroxisome proliferators activate receptor α(PPAR-α)involved in fatty acid oxidation through RT-q PCR. |
