Effects And Potential Mechanisms Of Lactiplantibacillus Plantarum NCUH001046 And Its Fermented Tomato On Obesity Alleviation

In recent decades,overweight and obesity have become a worldwide pandemic as well as a serious global public health problem.Anti-obesity pharmaceuticals are an intervention method,but their widespread use is limited by variable efficacy,adverse effects,and high price.With this in mind,it is urgent to find new safe,and effective methods to alleviate obesity.Numerous studies have shown that probiotics could play a role in alleviating obesity by modulating gut microbiota and maintaining intestinal function.In addition,with the increasing recognition of dietary therapy,the functionality of probiotic-fermented plant-based foods has been gradually explored,and they have exhibited promising alleviating effects on obesity.Therefore,this thesis screened probiotics from feces with obesity-alleviating effects,and fermented tomatoes with them to develop probiotic-fermented tomato products,and further explored the alleviating effects and potential mechanisms of probiotics and fermented tomato on obesity by combining biochemical,pathological,molecular biological,microbiological,and metabolomics technologies.Besides,the effects of probiotic fermentation on the physicochemical properties and bioactivities of tomato were also investigated,as well as the bioactive compounds in probiotic-fermented tomato with obesity-alleviating potential.The relevant outcomes of this thesis furnished safe and effective interventions and theoretical support for the prevention and alleviation of obesity and also offered high-quality strain resources and theoretical guidance for the development of functional probiotic-fermented fruit and vegetable products.The main contents and results of this thesis are listed as follows:(1)Probiotics were isolated and screened from feces and evaluated for their obesity alleviation potential in terms of lipid-lowering,enzyme activity,and microbial growth.In this thesis,strains were isolated from the feces of healthy adults and infants,and their cholesterol-lowering ability,triglyceride-lowering ability,bile salt hydrolase activity,α-glucosidase activity inhibition ability,and growth inhibition ability of Enterobacter cloacae were determined.Subsequently,the comprehensive score of probiotics was calculated by principal component analysis and the top three scoring probiotics were selected as probiotics with obesity alleviation potential,namely Limosilactobacillus reuteri NCUH064003(LR),Lactiplantibacillus plantarum NCUH001046(LP),Limosilactobacillus fermentum NCUH003068(LF).The results of in vitro properties and safety evaluation showed that the three probiotics strains exhibited favorable tolerance to simulated gastrointestinal fluids,adherence to Caco-2cells,and antioxidant activity,and were sensitive or moderately sensitive to nine common antibiotics,without hemolytic activity.(2)The alleviating effects of three probiotics with obesity-alleviating potential on obesity in mice were validated and compared.The mice were induced by a high-fat diet(HFD)for 9 weeks to establish the obese mouse model and intervened by gavage of different potential probiotics(LP,LR,and LF)at the same time,respectively.Biochemical and histopathological analyses of the mice were performed after 9 weeks of intervention.The results showed that none of the three probiotics had a significant effect on food and energy intake in mice,but all of them reduced body weight gain,liver and fat mass,adipocyte hypertrophy,and hepatic lipid accumulation to a certain degree.LP and LF remarkably alleviated dyslipidemia,systemic low-grade inflammation,lactate metabolism,and cytokine abnormalities in mice.In addition,LP markedly increased glucose tolerance and ameliorated impaired glucose tolerance in mice.By comparison,LP was found to exhibit enhanced effects than LF and LR in reducing body weight gain,epididymal white adipose tissue mass,and IL-6 level,as well as increasing fecal lipids,fecal lactate,and IL-10 level,and was therefore selected for follow-up investigations.(3)The effects of probiotic fermentation on the physicochemical properties and bioactivities of tomato were analyzed.The physicochemical properties and bioactivities of probiotic-fermented tomato were determined by HPLC,GC-QTOF/MS,amino acid analyzer,and biochemical analysis.The results showed that the viable bacterial counts reached 9.11 log CFU/m L,p H was 3.61,and acidity was 11.95g/L in live probiotic-fermented tomato(LFT)after 24 h.LP fermentation resulted in almost depletion of the sugar content of tomato,and a significant increase in the organic acid content,especially lactic acid,which was the highest content of organic acid in both LFT and sterilized fermented tomato(SFT).Compared with unfermented tomato(UFT),the contents of fresh amino acids,total phenols,and total flavonoids were significantly increased in LFT and slightly decreased after sterilization.LP fermentation markedly altered the structure and content of volatile compounds in tomato,with increased contents of ketones and alcohols,and decreased contents of aldehydes and furans(especially 2-pentyl furan)in LFT as compared with UFT,resulting in an improvement in flavor and food safety.Biochemical analysis indicated that LP fermentation remarkably improved tomato bioactivities,including antioxidant properties,α-glucosidase and α-amylase activity inhibition capacities,and Enterobacter cloacae growth inhibition capacity,leading to enhanced potential for obesity alleviation.(4)The alleviating effects of probiotic-fermented tomato on obesity in mice were investigated.Mice were induced by HFD for nine weeks to establish the obese mouse model and were gavaged with UFT,LFT,and SFT,at the same time,respectively.Results indicated that UFT,LFT,and SFT had no significant effect on food and energy intake,but all reduced body weight gain,liver,and fat mass to some degree.Histopathological analysis showed that LFT and SFT also reduced adipocyte hypertrophy and hepatic lipid accumulation.In addition,biochemical analysis showed that LFT and SFT not only ameliorated impaired glucose tolerance,but also effectively alleviated dyslipidemia,systemic low-grade inflammation,lactate metabolism,and cytokine abnormalities in mice caused by high-fat diet.Notably,LFT showed enhanced effects than SFT and UFT in reducing body weight gain,liver and fat mass,TG,LPS,and TNF-α levels,which could be associated with LP and the content of bioactive compounds in LFT.(5)The potential mechanisms of probiotics and fermented tomato in alleviating obesity in mice were investigated.Molecular biology,microbiome,and metabolomics technologies were employed to investigate the alleviating effects and potential mechanisms of probiotics and fermented tomato on obesity in mice.The results of hepatic lipid metabolism-related pathway protein expression showed that LP,LFT and SFT markedly increased the phosphorylation of AMPK,resulting in markedly upregulated phosphorylation of ACC and expression of CPT-1 and PPARα,markedly down-regulated FAS expression,and enhanced fatty acids(FFAs)β-oxidation as well as inhibited the synthesis of FFAs,which led to a decrease in hepatic lipid synthesis.The results of 16 S r RNA amplicon sequencing and correlation analysis indicated that LP and LFT restored the diversity and richness of gut microbiota in mice,lowered the relative abundance of positively obesity-related genera such as Mucispirillum,Olsenella,Streptococcus,and Clostridium,while increased the relative abundance of obesity-negative genera such as Roseburia,Coprococcus,and Bacteroides,and also promoted short-chain fatty acid production.Serum metabolites were investigated using untargeted metabolomics and combined with multivariate statistical analysis,which demonstrated that the high-fat diet led to significant alterations in serum metabolite levels,whereas supplementation with LP and LFT significantly reversed the up-or down-regulation of the levels of partial metabolites,which mainly included phosphatidylcholine,phosphatidylethanolamine,sphingomyelin,and unsaturated fatty acids.Pathway enrichment analysis of the differential metabolites revealed that both LP and LFT significantly regulated four lipid metabolic pathways(glycerophospholipid metabolism,sphingolipid metabolism,alpha-linoleic acid metabolism,and arachidonic acid metabolism)and one amino acid metabolism(phenylalanine,tyrosine and tryptophan biosynthesis and glycine,serine,and threonine metabolism and tryptophan metabolism,respectively).Combined with the results of the correlation analysis,LP and LFT may modulate gut microbiota and systemic metabolic disorders by regulating the levels of key genera and metabolites associated with obesity and increase the levels of key metabolites(e.g.,adiponectin,short-chain fatty acids)to activate the AMPK and PPAR signaling pathways to reduce hepatic lipid synthesis and alleviate the development of obesity and its complications.(5)Potential bioactive compounds in LFT with obesity-alleviating effects were explored.Differential metabolites between UFT and LFT were analyzed using untargeted metabolomics,and the results showed that five phenolic compounds,chlorogenic acid,capsiate,tiliroside,irisflorentin,and homoeriodictyol were the differential phenolic compounds,and their contents were significantly increased after LP fermentation,and especially chlorogenic acid was the most abundant differential phenolic compound in LFT.Correlation analysis showed that these five phenols were negatively correlated with body weight,TG,TNF-α,and IL-6.The alleviating effects of irisflorentin and homoeriodictyol on lipid metabolism disorders were explored by establishing oleic acid-induced hyperlipidemic Hep G2 cells,and the results demonstrated that 40 μg/m L of IF and 100 μg/m L of HE significantly reduced cholesterol and triglyceride contents in hyperlipidemic Hep G2 cells,and markedly increased AMPK,PPARα and CPT-1 transcription,promoted β-oxidation of FFAs and reduced lipid synthesis.Combined with the results of existing publications and the results of cellular experiments,it could be hypothesized that the above five phenolic compounds may be potential bioactive compounds in LFT with alleviating effects on obesity and lipid metabolism disorders,with chlorogenic acid contributing the most to these effects.