Effect Of Lactobacillus Plantarum NCU116 Fermentation On The Physicochemical Properties And Immunoregulatory Activity Of Asparagus Officinalis Polysaccharides

Vegetables and fruits are rich sources of dietary fiber and phytochemicals.Epidemiological studies show that the intake of vegetables and fruits is negatively correlated with various chronic diseases such as cancer,cardiovascular disease,and stroke.However,the daily intake of vegetables and fruits is lower than recommendation of World Health Organization(WHO)and Food and Agricultural Organization(FAO).As an adaptable processing technic to the changing consumption trend,lactic acid bacteria(LAB)fermentation is considered as a simple and effective biological processing technology,contributing to maintain or improve the safety,nutrition,sensory and shelf life of vegetables and fruits.LAB fermented products are frequently proven to have probiotic function,which is not only due to the addition of probiotics in the fermentation process,but also attributed to the altered matrix during fermentation.Asparagus officinalis L.(asparagus)is a famous health vegetable in China,known as "the king of fruits and vegetables" for its rich active components.Asparagus is also used as a drug to treat inflammation,fungal infections,coughs,and cancer.As a main active chemical,asparagus polysaccharide is reportedly to have anti-tumor immunoregulatory property.Thus,a green asparagus named ‘Jinggang 701',cultivated by Jiangxi Academy of Agricultural Sciences,was selected and subjected to Lactobacillus plantarum NUC116(NCU116)fermentation.Two polysaccharides were isolated from natural and fermented asparagus pulps,named Asparagus officinalis polysaccharides(AOP)and fermented-AOP(FAOP).The effects of NCU116 fermentation on the chemical properties and immunomodulatory activity of asparagus polysaccharides were studied.The main conclusions were summarized as follows:1.Effect of NCU116 fermentation on the nutritional compositions of "Jinggang 701" green asparagusThe asparagus was blanched over boiling water and crushed with a juicer to obtain asparagus pulp.The sterilized asparagus pulps after autoclave were inoculated with NCU116,following the second-sterilization to obtain fermented-asparagus pulps.The raw(RAP),blanched(BAP),and fermented asparagus powders(FAP)were obtained from the raw,blanched,and fermented pulps after drying.Based on spectrophotometry and high-speed amino acid analyser,etc.,the nutritional composition of three powders were analysed.The results showed that dry matter accounted for 7.35 ± 0.36% in the RAP.In the dry matter,the total sugar containing reducing sugar(11.03 ± 0.35%),accounted for 20.78 ± 0.76%,crude fat for 1.46 ± 0.12%,protein for 28.42 ± 2.59,mineral elements and antioxidants for a small amount.Among the antioxidant chemicals,the content of total saponins reach up to 35.31 mg/g.Compared to raw asparaguspowder,the nutritional compositions of the water-blanching asparagus powder had few alterations,but those of fermented asparagus powder had changes,which was due to the utilization of bacteria to the matrix during the fermentation and the addition of fructose syrup in the fermentation process.Consequently,the dry weight ratio increased greatly,leading to the huge increase of carbohydrate,and reducing sugar content,and further resulting in the decrease of protein,crude fat,mineral elements,amino acids,total phenols,total flavonoids and vitamin C,except saponins.2.Effects of NCU116 fermentation on physicochemical properties of AOPAOP and FAOP were separated from BAP and FAP by water extraction and alcohol precipitation.Two asparagus polysaccharides,named AOP and FAOP,were characterized by spectrophotometry,high performance anion exchange chromatography,high performance volume exclusion chromatography and scanning electron microscopy,etc.The results showed that NCU116 changed the chemical composition of asparagus polysaccharide,decreased the content of neutral sugar,but increased the content of uronic acid.In addition,the molecular weight and the particle size of AOP's solution conformation were decreased by NCU116 fermentation,and the rheological behaviour also changed.In vitro activity,NCU116 fermentation enhanced the antioxidant capacity of AOP,mainly manifested in increasing the scavenging ability of DPPH,hydroxyl and superoxide anion these three free radical.3.The effect of NCU116 fermentation on splenic immune activity of asparagus polysaccharideCyclophosphamide(CTX)was used to modelling the immunosuppressive mice.Based on the previously results about basic structural information of AOP and FAOP,effect of NCU116 fermentation on their regulation of splenic immune activity were further investigated.The animals were randomly divided into 8 groups: normal control(NC)group,CTX model(CTX)group,AOP low/medium/high-dose(AOP-L/M/H)group,FAOP low/medium/high-dose(FAOP-L/M/H)group.The conclusions were as follows:(1)AOP-H and FAOP-H increased serum antibody level and maintained Th2 immune response.In addition,compared with AOP,FAOP can significantly promote the expression of Th1 nuclear transcription factor and the secretion of Th1 specific cytokine IFN-?,and inhibit the expression of Th17 nuclear transcription factor and the secretion of Th17 specific cytokine IL-17.FAOP administration stimulated a gradual shift from Th17-dominant acute inflammatory response to Th1-dominant defensive immune response.4.Effects of NCU116 fermentation on AOP and FAOP alleviated CTX-induced liver injury induced by CTX and regulated serum metabonomicThe effects of AOP and FAOP on CTX-induced liver injury were studied based on real-time quantitative PCR,Western blot,and liquid chromatography mass spectrometry,etc.The results showed both AOP and FAOP could elevate the liver injury induced by CTX and protect the liver,mainly manifest in inhibiting liver biochemical markers(alt,AST,AKP and LDH)and proinflammatory cytokines(TNF-? and IL-1 ?),and enhancing antioxidant system(T-AOC,SOD,CAT and MDA).Compared to AOP,FAOP also significantly promoted GSH biosynthesis and normalized the disorder of bile acid receptors(FXR and SHP)and their key enzymes(CYP7A1,CYP8B1 and CYP27A1).Specifically,the regulation of FAOP on bile acid homeostasis might be related to the up-regulation of liver SCFA receptors GPR41 and GPR109 A expression,and the promotion of intestinal SCFA production.Furthermore,serum metabolism revealed that FAOP had better liver protection than AOP,with the evidence on the distribution of bile acid pool and the construction of related metabolic pathways.5.Effect of NCU116 fermentation on AOP and FAOP regulated intestinal mucosal immunity and intestinal microbiota compositionBased on real-time quantitative PCR,Western blot,and DNA sequencing,we further investigated the effects of AOP and FAOP on intestinal mucosal immune damage induced by CTX and intestinal microbiota composition.The results showed that FAOP improved intestinal injury induced by CTX,mainly by improving the pathological structure,stimulating the differentiation of T helper cells,remodelling the homeostasis of intestinal cytokines,up-regulating the expression of tight junction protein,promoting the synthesis of short chain fatty acids and regulating the composition of intestinal microflora.CTX mainly increased the abundance of Erysipelotrichaceae,while FAOP mainly promoted the abundance of ·and AOP mainly promoted the abundance of Lactobacillus gasseri,Pneumatosis intellectualis and Pterinochilus murinus.Taken together,NCU116 was successfully applied to asparagus fermentation to isolate AOP and FAOP.NCU116 fermentation changed the structural properties of AOP,and increased its in vitro antioxidant activity.Based on the change of AOP's structural information and the increase of its in vitro activity,its immunomodulatory activity in vivo was further explored.The results showed that compared with AOP,FAOP was more efficient to regulate the intestinal microflora and promote the synthesis and production of SCFA,whose dysfunction could repair the barrier function of intestinal mucosa,inhibit splenic Th17 inflammatory response caused by intestinal bacteria translocation to spleen,promote Th1 immune response,and eliminate pathogens in vivo.Meanwhile,FAOP also regulated the bile acid circulation and liver metabolism by regulating intestinal flora,and inhibit liver inflammatory injury by promoting the systemic circulation of SCFA draining into the liver to restore the antioxidant capacity of liver,accelerate the metabolism of toxic products of CTX in the liver,restore serum metabolites,and protect the mice injured by CTX.