| With the rapid development of social economy and the gradual improvement of people’s living standard,obesity caused by the change of living habits,eating habits and working habits has become a major threat for human health.The imbalance between energy intake and energy consumption is the primary reason causing obesity,which can be prevented or treated through adjusting the diet structure.Dietary fiber（DF）is an essential component of human diet,which plays important roles in stabilizing intestinal barrier status and maintaining intestinal microbial community structure to balance intestinal nutrient absorption and waste excretion.Increasing DF intake has become an important research topic to prevent the occurrence of obesity or improve obesity status and its complications.The two types of DFs that have attracted people’s attentions are soluble DF（SDF）and insoluble DF（IDF）.SDF is of great fermentation property to produce several short chain fatty acids（SCFAs）associated with obesity regulation,including acetate,propionate,butyrate,lactate and succinate.Unfortunately,an over-fermentation of SCFAs in animal body shows potential carcinogenic risks.For use in obesity intervention,IDFs can work as efficient as that of SDFs without any known untoward effects.However,the intervention of IDFs in obesity is still lack of long-term comparative experiments before and after obesity,especially for the work on the time-based succession rules of intestinal microbes and its relationships with obesity.It is also still unknown whether such intestinal microbes could influence obesity course by secreting certain metabolites.To answer the above questions,the program presented here use bacterial cellulose（BC）,given its advantages of high purity and strong water absorption capacity,its shortages of time-consumed production and low productivity and the little research of DF function,as an IDF material to raise the following research outlines.（1）Isolate the potential strains with high productivity of BC and analyze the BC synthesis mechanisms by whole genome sequencing and gene annotation.（2）Test BC synthesis ability and the associated metabolic pathways based on the analysis of fermentation kinetic characteristics and carbon source transformation,followed by a batch preparation of BC.（3）Prepare the diets with different components,fully investigate the physiological-intestinal microbe coupled mechanisms in mice obesity occurrence and DF regulation by physiological and biochemical analysis,histological observation,q PCR analysis of obesity-and inflammation-associated genes and intestinal microbiota analysis.（4）Construct the food-induced fat model,fully investigate the roles of BC intervention in obesity alleviation and the associated mechanisms based on physiological and biochemical analysis,histological observation,gene amplification,intestinal microbiota analysis and feces metabolite analysis.The detailed work and findings can be concluded as follows:（1）According to the microbiological characteristics of BC production,a new bacterial strain capable of BC production was isolated by using HS medium,the physicochemical properties and synthesis mechanism of bacterial cellulose were analyzed.The obtained BC-producing strain W1 showed the highest relationship with K.europaeus.The BC produced by strain W1 was of great 3D structure and stacked longitudinally by pellicles.Most observed nanocelluloses were in range of 40-60 nm of the diameter.Strain W1-produced BC had 3 typical diffraction peaks at 2θof 14.5°,16.6°and 22.7°,corresponding to the lattice planes（110）,（110）and（200）.Moreover,several infrared absorption bands were found 2900,2300,1426,1335,1314,1160,1108,1054,1030 and 900 cm-1,which were probably associated with the functional groups such as O-H,C-H,H-O-H,C-O-C,C-O-H,C-C,C-O andβ-glycosidic bond,implying that the BC produced by strain W1was mainly composed of type I cellulose.Whole genome sequencing and gene annotation showed that strain W1 had 2 bcs operons for BC synthesis and several free genes involved in glucose transformation and BC synthesis regulation.Specifically,the genes glk,pgm and UPG2 were associated with glucose metabolisms,bcs A,bcs B,bcs C,bcs D,bcs X and bcx Y were responsible for BC synthesis,while cmcax,ccp Ax and bglx A worked as BC synthesis regulators.（2）The kinetic processes and BC productivity and transformation pathways in W1were studied,followed-by a batch preparation of BC.In HS medium,the growth of strain W1followed a nontypical S type curve.While BC productivity increased first followed-by a stable status,the medium glucose（GLU）decreased dramatically,both of BC and residual GLU correlated well with bacterial biomass（r2=0.88,p<0.001&r2=0.96,p<0.001）.Strain W1 could efficiently utilize fructose（FRU）,GLU,glycerol（GLY）and mannitol（MAN）to produced BC,being the highest productivity and transformation rate in FRU by1.529 g L-1 and 7.65%.However,few utilization of acetic acid,ethonal,lactic acid and sucrose were observed.Interestingly,all the above carbon sources were transformed to BC with an average diameter of 40-50 nm.The BCs produced from FRU、GLU、GLY and MAN had 3 typical diffraction peaks at 2θof 14.5°,16.6°and 22.7°,with a same interplanar crystal spacing（d）,quite different apparent crystal size（ACS）,and crystallinity index（CI）by 0.64-0.89.Unlike X ray diffraction（XRD）data,several typical spectra bands obtained by Fourier transform infrared spectroscopy（FTIR）were similar for the BCs produced from different carbon sources.All BC samples were mainly composed of type I cellulose.The genome annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis revealed that the biochemical transformation pathways associated with the utilization of and BC production from FRU,GLU,GLY and MAN were found in strain W1,but which was not the case for other carbon sources.Based on the HS medium and bacterial growth characteristics,a batch preparation platform of BC with simple process,convenient operation and low cost was built.（3）The physiological-intestinal microbe coupled mechanisms in regulating mice obesity occurrence by the BC intervention were fully investigated.After 9 weeks of feed with different diet components,the high fat diet（H）induction resulted in the highest mice body weight,liver and adipose tissue weight,serum concentration of GLU,TG,TC and LDL-C,and the liver concentration of TG and TC,but the lowest serum HDL-C concentration.While high fat+high BC diet（HBC）reduced mice body weight by 14%,which could resulted in a substantial reduction of liver and epididymal fat（1.54 vs.1.87 g,p<0.01;1.63 vs.2.21 g,p<0.01）.The data obtained from high fat+high fiber diet（HHF）fell in between H and HBC treatments.The serum concentrations of GLU,TG,TC,HLD-C and LDL-C in HBC were significantly different from H by 32%（p<0.01）,20%（p<0.001）,18%（p<0.01）,14%（p<0.05）and 32%（p<0.01）.The liver TG and TC concentrations followed a similar trend to serum TG and TC.Histological slice observation showed that H treatment caused liver cells to become larger,hepatocellular steatosis,structural damage in liver lobular,liver cord and liver blood sinusoids.H treatments also shortened the ileum villi and lead to the disappearance of intestinal alkaline phosphatase complex（IAP）.In HBC and HHF treatments,the liver cells had normal structure,the liver macrophage and lipid droplet decreased significantly,so did for ileum structure and IAP content.q PCR analysis revealed that H treatment resulted in a upregulation of Srebp-1c and Fas genes for fat acid synthesis in liver by 7.5-fold and 5.9-fold,a downregulation of Occludin and ZO-1 gene for tight junction protein synthesis in intestinal by 58%and 57%,and a upregulation of TNF-αgene for tumor inflammatory factor synthesis by 8.4-fold and IL-1βand IL-6 genes for interleukin cytokines by 5.2-fold and 3.0-fold,respectively.Both BC and normal plant cellulose significantly affected the expression of above genes,while the former showed a better effect than the latter,which may be related to its higher purity and unique structural characteristics.The analysis of species diversity showed that the observed OTUs of intestinal microbes in mice decreased from 817±115 in C treatment to 675±18 in H treatment（p<0.01）.Bacterial cellulose intervention reconstructed the bacterial diversity and increased OTUs to779±19（p<0.05）,which was little better than HHF treatment.While Good’s coverage in H was the highest,Chao1 and PD whole tree indexes showed opposite.The effects of BC on Good’s coverage,Chao1 and PD whole tree indexes followed a similar trend as OTUs.The results of species annotation showed that the relative abundance of Firmicutes（F）was the highest in each group,followed by Bacteroidetes（B）and Proteobacteria（P）.H treatment could significantly increase the harmful intestinal microbiota including Firmicutes and Proteobacteria and decreased the relative abundance of beneficial intestinal microbiota Bacteroidetes by（F+P）/B increasing from 2.09±0.34 to 5.00±0.47（p<0.0001）.Both HBC and HHF resulted in a dramatical decrease of（F+P）/B（3.32±0.21,p<0.01;2.57±0.82,p<0.001）.As a result of the full analysis based on the data at phylum,class,order,family,genus and OTUs,the OTUs 1108 and 1294 of NK4A136 group,OTU1645 of Blautia,OTU 1919 of Coprococcus 1 and OTUs 1109、1115,1168,1600,1611and 1888 of unknown group of Lachnospiraceae,OTUs 1116,1206 and 1251 of Ruminiclostridium of Hungateiclostridiaceae and OTU 1114 of E.coprostanoligenes of Eubacterium of Eubacteriaceae in Firmicutes,the OTU 1330 of Helicobacter and OTU 6of Desulfovibrio in Proteobacteria and the OTU 238 of Bacteroides and OTU 1000 of S24-7 group of unknown family in Bacteroidetes were possible members of the harmful intestinal microbiota associated with obesity occurrence.In contrast,the OTU 180 of Alloprevotella,OTU 227 of Prevotella UCG-001 group and OTUs 221,234,403 and 766of S24-7 group of unknown family in Bacteroidetes,and OTUs 1636 and 1638 of unknown group of unknown genus and OTU 1861 of NK4A136 group of unknown genus of Lachnospiraceae in Firmicutes were possible members of the beneficial intestinal microbiota associated with obesity alleviation.（4）The food-induced fat model was successfully constructed,and the intervention treatment effects of BC on obesity mice were fully evaluated based on physiological and biochemical analysis,intestinal microbiota analysis and feces metabolite analysis.In 6weeks of intervention,the mice body weight changed slightly in C treatment and continuously increased in H treatment,while HBC treatment decreased as compared to H treatment,suggesting that BC intervention could significantly alleviate the obesity process.Bacterial cellulose supplement slowed down the obesity development.Moreover,the presence of BC significantly decreased the liver weight from 2.33±0.34 to 1.79±0.22 g（p<0.01）,but which showed little difference in epididymal fat and perirenal fat.In serum,the GLU concentration of H treatment was higher than that of HBC treatment(8.8 vs.5.2mmol L-1,p<0.0001),so did for serum TG,TC and HDL-C,and liver TG and TC.In H treatment,the mice liver cells and structure were destroyed,the macrophage increased dramatically and an adipose accumulation was found.Moreover,H treatment shortened the ileum villi and decreased the IAP complex.As expected,BC intervention showed beneficial effects on liver cell and structure being back to normal status,while the macrophage and adipose tissue decreased,and the ileum villi structure and IAP complex increased.q PCR analysis showed that BC intervention inhibited the rapid upregulation of Srebp-1c and Fas genes in liver as compared with H treatment（4.5-vs.2.1-fold and 4.1-vs.1.5-fold,p<0.0001）.Similar positive effects of BC on the upregulating expression of Occludin（p<0.01）and ZO-1（p<0.05）and the downregulating expression of TNF-αand IL-1β（p<0.0001）were also observed.The analysis of species diversity showed that the observed OTUs in H were significantly lower than that in C（443±27 vs.643±74,p<0.0001）,but BC supplement had little effect on OTUs change（486±24）.H treatment could significantly increase Good’s coverage and decreased Chao1,PD whole tree,Shannon and Simpson diversity.However,only PD whole tree diversity was significantly affected by the supplement of BC（p<0.05）.Among treatments,Firmicutes,Bacteroidetes,Proteobacteria,Verrucomicrobia（V）and Actinobacteria（A）predominated,while the relative low abundant phylum such as Deferricteres（D）,Saccharibacteria（S）and Tenericutes（T）in Chapter 3 changed dramatically in this study.Specifically,H treatment lead to a significant decrease of Bacteroidetes and a significant increase of Proteobacteria,resulting in an increase of（F+P+A）/（B+V）at 4.82±1.23 to 2.93±0.77（p<0.01）.While HBC treatment significantly decreased the relative abundance of Actinobacteria（p<0.0001）,it increased Verrucomicrobia from 4.7%to 16%（p<0.01）.As a result of the full analysis based on the data at phylum,class,order,family,genus and OTUs,the OTU 443 of Coriobacteriaceae UCG-002 group in Actinobacteria,OTU 63 of unknown genus and OTU 5 of Faecalibaculum of Erysipelotrichaceae,OTUs 516,751 and 767 of unknown genus of Lachnospiraceae,OTU 755 of Blautia,OTU 929 of Eubacterium,OTU 34 of Peptococcacea,OTU 957 of Romboutsia and OTU 476 of Intestinimonas in Firmicutes,OTU 93 of Bacteroides in Bacteroidetes and OTU 4 of Desulfovibrio and OTU 750 of Helicobacter in Proteobacteria were members of the harmful intestinal microbiota,possibly associated with continuous obesity.In contrast,the OTUs 137 and 165 of Bacteroides,OTU96 of S24-7 group of unknown genus,OTU 195 of Odoribacter and OTU 193 of Prevotella Ga6A1 group in Bacteroidetes,OTU 456 of NK4A136 group,OTUs 462 and 778 of unknown group,OTUs 459 and 591 of Ruminiclostridium and OTU 461of Intestinimonas of Lachnospiraceae in Firmicutes,and OTU 445 of Akkermansia in Verrucomicrobia were members of the beneficial intestinal microbiota,possibly associated with obesity resistance.Except for the unknown genus and Blautia of Lachnospiraceae,Bacteroides,Eubacteriu,Desulfovibrio,Helicobacter,S24-7 group of unknown genus,Prevotella and NK4A163group,all other groups substantially changed through community succession before and after the study.Finally,the metabonomics analysis showed that taurochenodeoxycholic acid（TA,a type of bile acid）increased in HBC treatment（negative model:2.19-fold,p=0.083）,having comparative difference from H treatment（positive model:3.13-flod,p=0.033;negative model:3.14-fold,p=0.022）.Unlike TA,another bile acid（i.e.,glycoursodeoxycholic acid,GA）was downregulated in the presence of BC（negative model:0.32-fold,p=0.012）.As for succinate,HBC treatment decreased its concentration（negative model:0.33-fold,p=0.015）,a significant difference was observed between HBC and H（0.12-fold,p=0.034）.We also found a significant increase of glutamine in H treatment（negative model:1.92-fold,p=0.048）,which was enhanced further by HBC treatment,although no difference was observed。A significant increase of L-citrulline was found in H treatment（negative:2.5-fold,p=0.026）,but BC supplement significantly decreased L-citrulline as compared to H treatment（0.44-fold,p=0.02）.Finally,a well-known anti-obesity hormone were both found in H and HBC treatments by a decrease trend（positive model:0.017-fold for H,p=0.42;0.017-fold for HBC,p=0.046）,but the two treatments showed no difference.We speculated that the change of TA may be related Coriobacteriaceae,and the change of succinate may be related to Bacteroidetes and Odoribacter.The relationship between other substances and intestinal microbes is still unclear.Conclusions:A new bacterial strain,belonging to K.europaeus,capable of BC production,was isolated and characterized.Strain W1 produced a typical BC with great 3D nanostructure,the XRD peaks and absorption peaks of FTIR functional groups are typical characteristics of BC,which was mainly consisted of type I cellulose.The BC synthesis in strain W1 was achieved by the regulation of 2 bcs operons.The bacterial growth and BC synthesis followed type I fermentation model.The BC productivity was relative higher in the medium containing GLU,FRU,GLY or MAN as the sole carbon source,all of which could be annotated in KEGG pathways.The fermentation kinetics laid the foundation for the mass production of BC by plastic tray method combined with the characteristics of carbon source utilization of strain W1.The supplement of BC displayed a good effect on both the prevention and intervention treatment of obesity.In this case,the mice had a lower body weight,liver weight and adipose tissue weight,a downregulation of fat acid synthesis in liver and inflammatory factors synthesis in epididymal fat and a upregulation of intestinal barrier function genes,a significant decrease of the relative abundance of harmful intestinal microbiota and increase of beneficial intestinal microbiota,and intestinal microbes-associated metabolites change including bile acid increase,succinate decrease,glutamine increase and L-citrulline decrease.A significant microbial community succession occurred over a long-term intervention of high fat diet.The information on some key microbial communities and their metabolites that may be related to the physiological function and intestinal microecological balance of mice were obtained in this study,which can provide theoretical reference and experimental basis for the systematic research of BC as an IDF on the occurrence,development and intervention of obesity. |
PDF Full Text Request