| Litchi(Litchi chinensis Sonn.)as a typical fruit in South China,which possesses higher nutritive value.Although litchi can be used as a traditional Chinese medicine to promote blood metabolism,anemia,spleen asthma,soothe nerves,relieve insomnia and prevent amnesia in folk remedies,its material basic and interaction mechanism have been less reported.In our prophase research,we have confirmed that polysaccharide was the main bioactive compounds in litchi pulp,which has the significantly immunomodulatory activity in vitro and in vivo.However,As a representative of the complex carbohydrates,it's hardly for our organism to degrade polysaccharide directly without carbohydrate active enzymes(CAZymes)due to its complex structure,furthermore,its physiological activity mainly relays on the the digestion and fermentation of gastrointestinal system.So,the present work aims at systematically studying the characteristics of gastrointestinal digestion and fermentation of litchi polysaccharide in vivo and in vitro.In detail,studied the digestion and fermentation process of the litchi polysaccharide by using simulation experiment in vitro.And then,studied the fermentation behavior and the changes of gut microbiota in vivo by using the animal model.Finally,point out its material basis through the separation,purification and structure identification.The main methods,results and conclusions in this study are presented as follows.1.Digestion and fermentation of litchi polysaccharide in vitroThe gastric and intestinal digestion of litchi polysaccharide was investigated in vitro.It was found that the polysaccharides was influenced in gastric digestion,however,there have no effect in later intestinal digestion.In gastric digestion,a steady decrease in molecular weight(Mw)of the polysaccharide from 161.24±2.73 k Da to 102.48±1.34 k Da was observed as digestion time increased.Meanwhile,the reducing ends were increased from 15.61±0.09 mg to 26.68±0.12 mg,indicating the decrease of Mw may due to the breakdown of glyosidic bonds.In addition,there was no monosaccharide released throughout the whole digestion period.Further,in vitro fermentation of litchi polysaccharide were investigated in this study.After fermentation in vitro for 24 h,it was found that neutral sugars and p H in faecal inoculums significant decreased,with the levels of CR and bacteria remarkably increased,additionally,total SCFAs,acetic and n-butyric all significantly improved,as well as arabinosidase,galactosidase and glucosidase were both nearly increased by 1.63,1.83 and1.49–fold of the initial time.In sum,the simulated gastric juice can changed the structure of litchi polysaccharide,while the intestinal juice had no influenced on it.Results also showed that litchi polysaccharides could be utilized by human gut microbiota and produce of acetic and butyric acid.2.Fermentation of litchi polysaccharide in vivoThe immunosuppressive mice model was construct by using cyclophosphamide(Cy).Mice were given 28 days oral administration of litchi polysaccharide at the difference concentration of 100 mg/ml and 200 mg/ml to investigate the behavior of fermentation in vivo and the influence of colon microbiota.It was found that the immune organism index,the concentrations of carbohydrate enzyme,short chain fatty acid in the colon and its related receptor protein decreased significantly during treatments with cyclophosphamide.But,after the intervention of litchi polysaccharide,the contents of arabinosidase,galactosidase and glucosidase in colon fecal was significantly increased,with the total SCFAs,acetic acid,propionic acid and butyric acid and its specific receptor genes GPR41,GPR43 m RNA and protein expression were also remarkable enriched.Litchi polysaccharide can improve the intestinal dysbacteriosis,including restore the richness,diversity of fecal microbiota and control the changes of overall structure.In detail,in the level of phylum,litchi polysaccharide can contribute the significantly increased of Deferribacteres and the decreased of Cyanobacteria;Clostridia and Deferribacteres increased with the decreased of Cyanobacteria at the level of class;the relative abundance of Clostridiales and Deferribacterales was increased accompanying of the decreased of Gastranaerophilale in order level;Meanwhile,the increased of Lachnospiraceae along with the decreased of Rikenellaceae and Gastranaerophilales in family level;furthermore,in genus level,Lachnospiraceae?NK4A136?group and unclassified?f?Lachnospiraceae were remarkably enriched with the decrease of Alistipes and Prevotellaceae?UCG-001.In short,litchi polysaccharide can promote the increase of Firmicutes,restore the gut microbiota disorder caused by cyclophosphamide,hold the balance of F/B(Firmicutes/Bacteroidetes)ratio,and keep the dynamic equilibrium for intestinal micro ecology.These results indicated that litchi polysaccharides could be utilized by human gut microbiota in vivo and increase the produce of acetic,propionic and butyric acid;The intake of litchi polysaccharide could be beneficial for the growth of Firmicutes and the balance of colon microbiota.3.Structure Characteristics of litchi polysaccharideAfter the purification by DEAE Sepharose Fast Flow column and Hi Prep Sephacryl S-300HR column,we can obtain three fraction(LP1aa,LP2a,LP2b).Using ultra high polymer chromatography system(APC)to identification its purity and the weight average molecular weight of LP1aa,LP2a and LP2b were measured as 47,363 and 45 k Da,furthermore the polydispersity index of LP1aa,LP2a and LP2b were 1.49,3.18 and 1.97.The determination of monosaccharides by GC-MS manifested that LP1aa were mainly composed of Ara(37.96%)and Gal(62.04%);LP2a were mainly composed of Ara(58.10%),Gal(36.29%)and Rha(13.50%);LP2b were mainly composed of Ara(46.97%),Gal(36.29%)and Rha(16.74%).Combine the results of GC-MS,FTIR and NMR(1H NMR?13C NMR?dept135?COSY?HSQC?NOESY?HSQC-TOCSY?HMBC)spectra,can infer that the possible structure of litchi polysaccharide.The structure of LP1aa were established:... |
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