Structural and Conformational Characteristics of Glucomannans from Different Sources and Their Intestinal Fermentation Properties in Mice

Author:Shi Xiao Dan

Supervisor:nie shao ping yin jun yi


Degree Year:2019





Glucomannans(GMs)are a kind of carbohydrate polymers,which are widely spread and have relatively simple chemical structure.Plants in Amorphophallus,Aloe,and Dendrobium genus are main sources of GMs.Even though GMs from the three kinds of plants have similar structural type,they are found to have big differences in structural features.Based on our previous studies on GMs from D.officinale(DOP)and Aloe gel,the current study mainly analyzed the structural characteristics of GMs from konjac tubers and Aloe leaves.Then,solution properties,solid microstructure,physicochemical property,and intestinal fermentation property in mice of GMs from konjac tubers,Aloe leaves,and Dendrobium stem were determined.Main contents and results of the present study were listed as follows:(1)Processing method of GMs from konjac tubers was explored and their structures were characterized.Cold water extraction,ethanol precipitation,enzymatic purification,acidic ethanol precipitation,and freeze-drying were used to extract and isolate polysaccharides from tubers of A.rivirei(konjac),A.albus,and two local types of A.bulbifer(wild type M and wild type B),which were designated as KGM,AGM,MGM,and BGM,successively.The four GMs had about 90%(w/w)neutral sugar,11%(w/w)moisture,and 1%2%(w/w)ash.The degree of acetylation was in the range of0.781.25.Mannose and glucose as the main components existed in molar ratios of1.41,1.41,1.21,and 1.58 for KGM,AGM,MGM,and BGM,respectively.Relative molecular weights of the four fractions increased in the order of:KGM<AGM<MGM<BGM.Structural analysis showed that all of the four samples hadβ-(1→4)-linked glucomannan as main chains.There were a few branches linked at O-2,6 of mannosyl residues and/or O-3,6 of glucosyl residues.The acetyl groups mainly attached to O-2and O-3 of mannosyl residues by NMR spectra analysis.(2)Polysaccharides from the leaf skin of Aloe vera were isolated,and their structures were characterized.Crude polysaccharide(AS-WE)was extracted and it was fractionated into three fractions(designated as ASP-4N,ASP-6N,and ASP-8N)using gradient ammonium sulfate precipitation.The physicochemical properties were determined.Results showed that there were 74%79%of neutral sugar and small amounts of protein and uronic acid.Result of high performance gel permeation chromatography(HPGPC)indicated that the three fractions were homogeneous.The relative molecular weights for ASP-4N,ASP-6N,and ASP-8N were 339,130,and 67.6kDa,successively.They were predominantly composed of mannose and glucose,and the ratios of mannose to glucose were 19.13,8.97,and 2.96,successively.Structural characteristics investigated by methylation analysis and 1D/2D NMR spectroscopy suggested that the three fractions was highly acetylated linear(1→4)-β-glucomannans.There were also some→3)-β-Manp-(1→residues mixed in the backbone.The acetyl groups mainly attached to O-2,O-3,and/or O-6 of mannopyranosyl residues in the backbone as mono-,di-,and/or tri-acetylated forms according to the NMR data.(3)Fine structure of polysaccharide from the gel of A.vera(AGP40)were identified.Three fractions(PAH-1,PAH-2,and PAH-3)and the other one(AGP40-EH)were collected from partial acid hydrolysates and enzymatic hydrolysates,respectively.AGP40 and the four hydrolyzed samples were subjected to methylation and NMR analysis.Results showed that AGP40 had a backbone consisting of major→4)-β-Manp-(1→residues,minor→3)-β-Manp-(1→,and→4)-β-Glcp-(1→residues.Low degree of branches distributed at O-6 of 4-linked mannose in the main chain.Similar to those GMs from Aloe leaf skin,the acetyl groups in AGP40 presented as mono-or di-substituents at O-2,O-3,and/or O-6 of→4)-β-Manp-(1→residues.(4)The solution properties,rheological properties,and microstructure of four GMs from konjac tubers were determined.Intrinsic viscosity of four GMs from konjac tubers was detected using Ubbelohde viscometer,while other conformational parameters including weight-average molecular weight(Mw),radius of gyration,and hydrodynamic radius were tested by high performance size-exclusion chromatography connected with multi-angle laser light scattering(HPSEC-MALLS).It was found that the intrinsic viscosity increased positively with the increase of Mw.SEC data showed that all of the four GMs were in random coil conformation under the detected condition(0.1 M NaNO3).There were also a few aggregations.The consistent length of KGM,AGM,MGM,and BGM were in the ranges of 58 nm and 810 nm calculated from the random flight model and worm-like chain model,respectively.Data of rheological experiments suggested that the four GMs all presented as a kind of pseudoplastic fluids.Under the oscillation mode,obvious cross points were only found in the curves of storage modulus(G′)and loss modulus(G″)versus frequency of MGM(3.2 Hz)and BGM(2.0 Hz).Microstructure observed through scanning electronic images(SEM)showed that the four GMs mainly had sheet-like,linear,and spherical shapes.(5)The solution properties and microstructure of DOP and four GMs from A.vera were determined.Data from SEC and Ubbelohde viscometer showed that intrinsic viscosity of the three fractions(ASP-4N,ASP-6N,and ASP-8N)from Aloe leaf skin and AGP40 from Aloe gel were positively related to the Mw.However,DOP with lower Mw showed higher intrinsic viscosity,indicating that the intrinsic viscosity of GMs may be not only affected by Mw,but also other structural features such as degree of acetylation.The HPSEC-MALLS data suggested that DOP and four GMs from Aloe leaf all presented random coil conformations,and they all had flexible polymer chains.All the GMs had fibrous,ribbon-like,and spherical morphology by SEM observation.(6)The chemical components,structural characteristics,and intrinsic viscosity of KGM,DOP,and GMs from A.vera were summarized and compared.The main structural characteristics of GMs were found to be different in molecular weight,mannose to glucose ratio(M/G),and degree of acetylation(DA).The physicochemical properties including water absorption capacity(WAC),solubility,fat absorption capacity(FAC),ζ-potential,and viscosity of six GMs were also determined and compared.Results showed that molecular weight was greatly related to the WAC,solubility,and viscosity.Theζ-potential showed obviously positive link to DA to some extent,however,it may also depend on the monosaccharide composition and molecular weight.The FAC of GMs were jointly determined by several different structural factors.Based on the above data,molecular weight,DA,and M/G could jointly play roles on these physicochemical properties of GMs.(7)Fermentation property of KGM,DOP,and GMs from A.vera in the intestine of normal Kunming mice were compared.General health status,biochemical levels,and production of intestinal short chain fatty acids(SCFAs)were evaluated with or without GMs supplements.After continuous treatment for 14 days,general health status of normal Kunming mice in oral administrated GMs groups did not significantly differ from that of mice in control group(saline treated group).Only the levels of some detected serum chemicals fluctuated in normal range when comparing with those of normal mice.After fermentation in the intestine,the production of SCFAs changed obviously.In detail,KGM and DOP significantly promoted the production of SCFAs in the cecum,colon,and feces.While GMs from A.vera only markedly influenced the SCFAs production in the colon.Data analysis showed that molecular weight and DA could positively promote the production of SCFAs,on the contrary,the values of M/G showed negative effects.The increase of SCFAs after fermentation indicated that those GMs may have great potentiality in promoting intestinal health.In conclusion,GMs from konjac tubers and Aloe leaf were all identified to be linear acetylated(1→4)-β-glucomannans with small amounts of branches in the current study.The acetyl groups mainly distributed at O-2,O-3 and/or O-6 of mannopyranosyl residues in the main chain.The main structural difference could be found in molecular weight,M/G,and DA aspects.Specifically,KGM had the highest molecular weight and lowest M/G ratio,while GMs from Aloe leaf showed the highest DA.DOP contained the lowest DA,moderate molecular weight,and M/G ratio.All GMs mainly performed random coil conformations in 0.1 M NaNO3 solution.They all presented fibrous,ribbon-like,and spherical morphology in solid state.The difference in structural characteristics of GMs resulted in different physicochemical properties and productions of SCFAs.