Isolation,Purification,Structural Identification and Bioactivity of Polysaccharides from Bamboo Shoots(Chimonobambusa Quadrangularis)Processing By-products

Author:Chen Guang Jing

Supervisor:zuo jian quan


Degree Year:2019





Polysaccharides are widely found in plants,animals,microorganisms,and algae.Especially,plant-derived polysaccharides have received much attention because of their pharmacological properties such as antioxidant,anti-fatigue,anti-allergic,anti-tumor,anti-inflammatory,immunomodulatory,anti-diabetic anti-diabetic and prebiotic behaviors.Moreover,these natural products can be used as potential candidates for safe,highly stable and effective natural drugs due to their relatively nontoxicity and insignificant side effects,which is the main problem associated with synthetic drugs.Due to that mentioned above,discovery and evaluation of new polysaccharides from the various plants have become a hot research spot.With the largest bamboo industry worldwide,China has the largest annual output of fresh bamboo shoots(5-6 million tons).As fresh bamboo shoots easily turn brown and sequentially lignify,processing techniques such as drying,pickling,and canning are frequently employed for bamboo shoots.However,after processing,bamboo shoot residues(50%–70%of the whole bamboo shoots,fresh weight)are usually discarded or used as animal feed.Large volumes of wastes are produced by the bamboo shoot industries,creating serious environmental and disposal problems.Chimonobambusa quadrangularis,also known as“Square Bamboo”due to its square-shaped culm,is a bamboo species originally cultivated in the provinces of Southwest China,especially in Chongqing and Guizhou.Given its taste and nutritional values,this bamboo shoot is favored by consumers.Increasing consumption of bamboo shoot processing products has led to the serious discarding problem of shoot residues.Therefore,interest in the exploitation of the processing by-products to generate value-added products has increased recently.However,to the best of our knowledge,the main processing by-products of bamboo shoots(C.quadrangularis),bamboo shoot residues(culms,BSRs),have not been investigated as potential source of bioactive polysaccharides.Therefore,in this study,we aim to find a way for converting BSRs as value-added products,as well as develop bioactive polysaccharides from these residues as functional additives in the food and pharmaceutical industries.The specific objectives of this study are as follows:(1)to apply an efficient accelerated solvent extraction(ASE)technology to rapidly extract polysaccharides from the BSRs;(2)to evaluate the influences of extraction methods on the yield,physicochemical properties,in vitro prebiotic activity and antioxidant activity of polysaccharides from BSRs(CPS);(3)to isolate and purify the crude CPS as well as determine their structural characterization;(4)to examine whether the digestive system(saliva,simulated gastric and small intestinal conditions)could break down and large intestinal microbiota could utilize the purified polysaccharides from BSRs(PCPS-2);(5)to examine whether the purified polysaccharides from BSRs(PCPS-2)can treat dextran sulfate sodium(DSS)-induced ulcerative colitis in BALB/c mice.The main results of this study are summarized as follows:(1)Accelerated solvent extraction(ASE)of polysaccharides from the BSRsThe Box–Behnken design(BBD),a popular form of RSM,was introduced to optimize the extraction conditions of polysaccharides from BSRs using the ASE and to simultaneously evaluate the effects of extraction parameters on their yields.Subsequently,ASE and hot water extraction(HWE)were applied and compared in the extraction of polysaccharides from the BSRs(CPS).A maximal ASE-CPS yield was obtained by optimized extraction conditions(temperature126°C,2 cycles,and 22 min)using response surface methodology.The yield of polysaccharides from ASE(9.96%±0.39%)was significantly higher than that from HWE(7.16%±0.32%;p<0.05).The neutral sugar content of ASE-CPS was 43.35±2.13%,similar to that of HWE-CPS(45.68±1.06%;p>0.05).The apparent viscosities of all CPS solutions at each concentration obviously decreased with the increase of shear rate,which is a typical characteristic of pseudoplastic fluid showing shear-thinning flow behavior.Meanwhile,HWE-CPS displayed lower apparent viscosity at different concentrations compared with ASE-CPS.The apparent viscosities of ASE-CPS and HWE-CPS solution obviously reduced after the addition of ClCa2.But the apparent viscosity of HWE-CPS decreased much more dramatically compared with ASE-CPS.Furthermore,both ASE-CPS and HWE-CPS exhibited a gel-like behavior,with G′always being higher than G″.The addition of CaCl2 could decrease the gel strength of ASE-CPS and HWE-CPS solutions.However,the reduction of the gel strength of ASE-CPS due to the addition of CaCl2 was smaller than that of HWE-CPS at all frequencies.All the results revealed that ASE could be a promising and alternative technique to extract polysaccharides for BSRs due to its high yield and efficiency.(2)Effects of different extraction techniques on physicochemical properties,antioxidant and prebiotic activities in vitro of polysaccharides from BSRs(CPS)The BSRs were extracted to obtain CPS by using hot water(HWE),accelerated solvent extraction(ASE),ultrasound-assisted extraction(UAE),microwave-assisted extraction(MAE),and enzyme-assisted extraction(EAE)processes,respectively.Then the characterizations of five different CPS samples were analyzed on the base of chemical analysis,molecular weight,monosaccharide composition and preliminary structural feature.The digestibility by artificial human gastric juice andα-amylase of the resulting CPS samples were assessed.Then the antioxidant activities in vitro of the five CPS samples were compared.At last,the influence of the five CPS samples as carbon sources alternative to glucose on the proliferation of bifidobacteria and lactobacilli in liquid culture were evaluated.The effects of these CPSs on the growth and SCFA production of bifidobacteria and lactobacilli were compared.The experimental results showed that the extraction yields,uronic acid contents,monosaccharide contents,molecular weight,particle sizes,zeta potentials and conformation and antioxidant activities of the five CPS were significantly different.CPS extracted by ASE method(ASE-CPS)possessedthehighestextractionyield(9.94%),thehighest medium-high-molecular-weight value(136.07 kDa)and notable antioxidant ability.UAE-CPS had the highest uronic acid(9.42%),the lowest medium-high-molecular weight value(117.49 kDa),the largest negative charge(-16.38),and the smallest diameter(116.8 nm).Besides,UAE-CPS exhibited the best antioxidant abilities including oxygen radical absorbance capacity(ORAC),radical scavenging powers(DPPH,ABTS,hydroxyl and superoxide radical)and chelating activity on ferrous ion.All of the five CPS samples were resistant to hydrolysis by artificial gastric juice andα-amylase in vitro.Furthermore,the five polysaccharides significantly stimulated the growth of the tested probiotics and increased lactic,acetate,propionate,and butyrate acids production from fermentation for 48 h compared to the control(p<0.05).The polysaccharides prepared using UAE and EAE processes displayed better prebiotic activity than the other fractions as they induced notably more proliferation of probiotic bacteria and higher production of SCFAs.(3)Isolation,purification,and structural elucidation of polysaccharides from BSRs(CPS)The crude polysaccharides(CPS)were isolated and purified using Cellulose DEAE-52anion-exchange and Sephadex G-100 size exclusion column.Then the structure of the purified polysaccharides was characterized by periodate oxidation and smith degradation analysis,methylation analysis,UV,FT-IR,GC,HPLC,GC-MS,NMR,SEM,AFM,CD,and XRD.Two novel water-soluble polysaccharides(PCPS-1 and PCPS-2)were obtained from BSRs through the UAE extraction and purified by Cellulose DEAE-52 anion-exchange and Sephadex G-100 size exclusion column chromatography.Chemical composition analysis revealed that PCPS-1had an average molecular weight of 24.58 kDa and consisted of mannose,rhamnose,glucuronic acid,glucose,galactose and arabinose with molar percentages of 0.54,0.49,0.33,5.93,46.79,and 45.93%,respectively.PCPS-2 had an average molecular weight of 123.45 kDa and comprised mannose,rhamnose,glucuronic acid,glucose,galactose and arabinose with molar percentages of 0.46,1.57,1.40,3.36,45.82,and 47.38%,respectively.PCPS-1 and PCPS-2 were found to contain 93.26 and92.25%neutral sugar,respectably.Besides,the content of uronic acid in PCPS-1 and PCPS-2 was0.28 and 0.65%,respectably.Methylation,GC–MS and NMR analysis revealed that the main glycosidicbondsinPCPS-1comprised→5)-α-L-Araf-(1→,→3,5)-α-L-Araf-(1→,→6)-β-D-Galp-(1→,→3,6)-β-D-Galp-(1→,T-β-D-Galp,→3)-β-D-Galp-(1→and T-α-D-Glc with molar percentages of 37.16,9.01,17.38,14.22,11.19,6.08,and 4.95%,respectively.PCPS-2consisted of→5)-α-L-Araf-(1→,→3,5)-α-L-Araf-(1→,→6)-β-D-Galp-(1→,→3,6)-β-D-Galp-(1→,T-β-D-Galp,→3)-β-D-Galp-(1→and T-α-D-Glcp with molar percentages of 37.16,9.01,17.38,14.22,11.19,6.08,and 4.95%,respectively.The SEM analysis showed that PCPS-1 have laminated structure with smooth surface,while PCPS-2 exhibit a rodlike structure with smooth surface.CD and AFM results showed that the molecules of both PCPS-1 and PCPS-2 intertwined with each other and formed irregular aggregates.XRD result indicated that both PCPS-1 and PCPS-2 display a few microcrystalline structure but mainly contain amorphous portions.(4)Digestion under saliva,simulated gastric and small intestinal conditions and fermentation in vitro by human intestinal microbiota of PCPS-2The digestibility of PCPS-2 was investigated using an in vitro digestion model.The PCPS-2samples after digestion were purified by Cellulose DEAE-52 anion-exchange column chromatography and their main glycosidic bonds were analyzed by NMR.Meanwhile,the fermentation in vitro of PCPS-2 by human gut microbiota was investigated.Results showed that the molecular weight,the main glycosidic bonds,and the reducing sugar content of PCPS-2 were not significantly changed,and no free monosaccharides released from PCPS-2 were detected after the salivary,gastric and intestinal digestion,suggesting that the backbone of PSPS-2 was resistant to be cleaved in the saliva and gastrointestinal tract environments.It was found that pH in the fermentation system significantly decreased from 8.18 to 3.1,and the production of lactic acid,acetic acid,propionic acid and total short-chain fatty acid all significantly increased from 0.64 to 8.95 mM,1.73 to 14.03 mM,0.38 to 7.16 mM,and 3.11 to 31.37 mM,respectively.Theα-L-arabinofuranosidase andβ-D-galactosidase activities were significantly increased(p<0.05).The results indicated that PCPS-2 could be broken down and utilized by gut microbiota,and could significantly enhance the production of short-chain fatty acids.(5)PCPS-2 attenuates colitis in BABL/c mice and its underlying mechanismsThe protective effects of PCPS-2 on an acute ulcerative colitis BALB/c mice model induced by3%DSS were examined.Meanwhile,the potential mechanism was investigated by ELISA,qRT-PCR and western blot technology.Our data indicated that PCPS-2 administration could improve clinical signs and symptoms,and alleviate colonic pathological damage including body weight loss,diarrhea,fecal occult blood,shortening of colon,edema,ulceration and splenomegaly.Meanwhile,histological analysis indicated that PCPS-2 could improve the signs of histological damage such as abnormal crypts,crypt loss,and inflammatory cell infiltration induced by DSS.PCPS-2 suppressed the secretion of interleukin(IL)-1β,interleukin(IL)-6,interleukin(IL)-18 and tumor necrosis factor(TNF)-α,and reestablished the balance of pro-and anti-inflammatory cytokines in DSS-induced acute ulcerative colitis mice.Besides,PCPS-2 could down-regulate key markers of oxidative stresses,including nitric oxide(NO),malondialdehyde(MDA),total superoxide dismutase(T-SOD),and myeloperoxidase(MPO).Moreover,PCPS-2 suppressed the expression of cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS)and decreased the expression of related mRNA.PCPS-2 administration could also markedly suppress the activation of NLRP3 inflammasome.At last,PCPS-2 could inhibit the expression of phosphorylated nuclear factor-κB(NF-κB)p65 and NF-κB inhibitor alpha(IκB-α)in DSS-induced acute ulcerative colitis mice.In conclusion,in this study,an efficient accelerated solvent extraction(ASE)technology was applied for rapid extraction of polysaccharides from the processing by-products(bamboo shoot residues)of C.quadrangularis.Different extraction methods could have significantly impact on the antioxidant and prebiotic activities in vitro of polysaccharides from BSRs(CPS).The polysaccharides prepared using ultrasound-assisted extraction processes displayed better antioxidant and prebiotic activities in vitro.The two-step purification technical rout depending on the Cellulose DEAE-52 anion-exchange and Sephadex G-100 size exclusion column was successfully established and applied to purify CPS.Two novel water-soluble polysaccharides(PCPS-1 and PCPS-2)were obtained.Both PCPS-1 and PCPS-2 mainly consisted of→5)-α-L-Araf-(1→,→3,5)-α-L-Araf-(1→,→6)-β-D-Galp-(1→,→3,6)-β-D-Galp-(1→,T-β-D-Galp,→3)-β-D-Galp-(1→and T-α-D-Glcp.The backbone of PSPS-2 was resistant to be cleaved in the saliva and gastrointestinal tract environments.Moreover,the main glycosidic bonds of PCPS-2 were not significantly changed after the salivary,gastric and intestinal digestion.PCPS-2 could be broken down and utilized by gut microbiota,and could significantly enhance the production of lactic acid,acetic acid,and propionic acid.PCPS exhibited appreciable protective effects on an acute ulcerative colitis BALB/c mice model induced by 3%DSS.Its mechanism could be related to suppression of the secretion of pro-inflammatory cytokines and the expression of cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(iNOS),improvement of oxidative stress,and inhibition of NLRP3 inflammasome and NF-κB pathways.