| Pectin is a complex,colloidal heteropolysaccharide composed of structurally distinct regions or domains which include homogalacturonan(HG),rhamnogalacturonan(RG-?),rhamnogalacturonan(RG-?I).Conventional commercial pectin extraction uniformly aims at maintaining more HG content for best quality control,therefore harsh extraction condition is used,during which RG-? region was largely destroyed.However,RG-? enriched pectin has diverse bioactivity including in vitro prebiotic,immunodulatory,anti-metastasis,metabolic regulation and anti-galectin-3 activity,is gaining attention increasingly.Considering the remarkable non-digestibility of RG-?-enriched pectin in the upper gastrointestinal tract,the characteristics of RG-?-enriched fermentation by the gut microbiota in vivo and the way of exerting its biological activity are awaiting further exploration.Besides,the influence of RG-? content,neutral sugar content,molecular weight(Mw),length of side chains and variable linking types on the biological activity still remain largely enigmatic and needed to be further studied.In the previous research of our group,a RG-?-enriched pectin(WRP)was obtained from citrus canning processing water using green recovery.Therefore,in this paper,we used the WRP as the research object,and investigated its chemical structures and biological activity.Its intestinal prebiotic activity and anti-obesity effects were investigated based on the modulation of gut microbiota,changes of SCFAs and energy expenditure using healthy and obese animal model respectively.The present research can not only increase the added value of citrus canning processing water and benefit protecting environment,but also provide scientific basis for exploring the fermentation characteristics of RG-?-enriched pectin in vivo and its regulation of intestinal microbiota.The main contents of the present research are summarized as follows:(1)Preparation and structure characterization of RG-?-enriched pectinWRP recovered from citrus canning processing water was used as a raw material,a free-radical degradation system was used to degrade WRP,followed by purification to obtain DWRP-a smaller molecular weight RG-?-enriched pectin.Besides,the HG-enriched commercial pectin was used as a comparison.Through monosaccharide composition analysis,molecular weight determination,FTIR and ~1H NMR analysis,the primary structures of the three pectins were characterized;through SEC-MALLS-RI analysis and atomic force Microscopic analysis,the advanced structures(chain morphology and chain conformation)of the three pectins were characterized.The results showed that the structural characteristics of the three pectins is respectively present below:CP is a HG-dominant pectin with long linear chain and large molecular weight(Mw:492.6 k Da;RG-? molar percentage:35.77%);WRP is a RG-?-dominant pectin with highly branched chain structure and large molecular weight(Mw:531.5 k Da;RG-? molar percentage:70.44%);DWRP is a RG-?-enriched pectin with less branched chain structure and small molecular weight(Mw:12.1 k Da;RG-? molar percent content:56.29%).(2)In vivo prebiotic effect of RG-?-enriched pectin based on healthy mouse modelCP,WRP and DWRP with different structural characteristics were used for dietary intervention in healthy C57BL/6J mice fed with normal chow diet.After 9 weeks of intervention,changes of short-chain fatty acids(SCFAs)in the cecum and colon of the mice was detected by gas chromatography(GC);the Illumina Miseq PE250 high-throughput sequencing platform was employed for 16S r DNA sequencing of flora in mice cecum contents.The results showed that higher RG-? content and lower molecular weight can better promote the in vivo probiotic effect of pectin.DWRP significantly increased the probiotic abundance of Bifidobacterium and Lacobacillus in the cecum flora of mice.DWRP also significantly increased the abundance of Bacteroides thetaiotaomicron and Faecalibaculum,the former one is RG-? degrading bacteria,and the latter one is butyric acid-producing bacteria,both of them can positively affect the composition of intestinal healthy microorganisms.While the dominant caecum bacteria of mice in HFD-WRP were mainly producers of short-chain fatty acid(SCFAs),such as Ruminococcus and Butyricicoccus,blonging to the Ruminococcaceae family.In addition,by comparing SCFAs content in the cecum and colon of mice in different groups,we tentatively put forward that the HG-enriched pectin is mainly fermentated in the cecum,while the RG-?-enriched pectin cannot be completely fermented by cecum microorganisms.(3)Anti-obesity effect of RG-?-enriched pectin based on HFD induced obese mouse modelCP,WRP and DWRP were treated with HFD-induced obesity C57BL/6J mice at a dose of100 mg/kg·d for 8 weeks.Obesity-related biochemical indicators,tissue sections,immunohistochemistry,q RT-PCR and high-throughput sequencing technology were used to investigate the anti-obesity effect of the three pectin.The results showed that WRP with higher RG-? content and larger Mw has the most significant anti-obesity effect.WRP prevented obesity through combined effect of gut microbiota modification and browning of beige adipocytes promotion in HFD induced obese mice.Highly branched chain structure and large Mw enabled WRP as a more complex substrate to be utilized by a variety of microorganisms,therefore promoting the mutualism between microorganisms negatively related to obesity.On the one hand,WRP improved the gut microbiota dysbiosis caused by HFD diet:significantly(p<0.05)increased the diversity and richness of the gut microbiota;reduced the F/B ratio to normal levels;promoted microorganisms including Barnesiella,Butyrivibrio,Roseburia,Clostridium IV,etc.,which are negatively correlated to the obesity of host;reduced harmful microorganisms enriched by HFD such as Acetatifactor,Olsenella,etc.On the other hand,WRP up-regulated the expression of UCP1 protein and some related thermogenic and browning marker genes,promoted iWAT browning and heat production,increased energy consumption and reduced fat accumulation in iWAT. |
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