Fermentation Process Of Polysaccharide From Polygonatum Odoratum And Its Effect On Macrophage Immunity

Polygonatum odoratum（Mill.）Druce belongs to Liliaceae and Polygonatum Mill.It is a traditional medicine and edible homologous Chinese medicinal material.Its main active components include Polygonatum odoratum polysaccharide、flavonoids、amino acids、steroidal saponins、alkaloids and some trace elements.According to the reports in China and abroad,Polygonatum odoratum has many functional activities,such as antioxidant,antiviral,antibacterial,hypoglycemic and lipid-lowering,and regulating immunity.Among them,polysaccharides have been considered to be one of the most important active substances in Polygonatum odoratum,and have been a research hotspot for researchers.At present,the research on polysaccharides from Polygonatum odoratum mainly focuses on the traditional extraction methods and processes of polysaccharides,and less attention is paid to the production of polysaccharides by microbial fermentation with high efficiency,high yield and low energy consumption.In particular,the comparison of differences between fermented and unfermented polysaccharides from Polygonatum odoratum has not been reported,and the relationship between the structure and activity of polysaccharides has not been clarified.Further in-depth study on the structure and function of polysaccharides from Polygonatum odoratum is conducived to the rational utilization of Polygonatum odoratum resources.In this study,the screening of fermentation strains,the optimization of fermentation process of Polygonatum odoratum polysaccharide,the separation and purification of fermented and unfermented Polygonatum odoratum polysaccharide,and their biological activities were discussed for further study.The main research results are as follows:（1）Optimization of fermentation process for preparation of Polygonatum odoratum polysaccharide by microbial fermentationIn the early stage of the experiment,nine strains（Enterococcus faecalis,Lactobacillus fermentation,Pediococcus acidilactici,Lactobacillus rhamnosus,Lactobac secaliphilus,Bacillus licheniformis,Bacillus sp.PD-1,Bacillus amyloliquefaciens,Bacillus subtilis）preserved in the laboratory were used to ferment Polygonatum odoratum,and the strains with the highest water-soluble polysaccharide content were screened for subsequent experiments.Optimization of fermentation process by single factor and response surface method.Including the amount of Polygonatum odoratum powder,the type of nitrogen source,the type of inorganic salt,the quantity of inoculation,the temperature of fermentation,the rotational speed of culture and the fermentational time,seven single-factor experiments on the increase rate of Polygonatum odoratum polysaccharide were investigated.By comparison,three significant indigenous factors were screened out,namely the amount of inoculation,the fermentational temperature and the fermentational time.The Box-Behnken design methodology was used for the response surface optimization of three factors and three levels to obtain the optimal fermentation procession:the amount of Polygonatum odoratum powder 4%,the yeast extract powder 3%and Na Cl 1%.Under the conditions of inoculation amount 3%,temperature 35°C,shaking speed 160 r/min and fermentation time 48 h,the polysaccharide was increased from 4.56 mg/m L to 7.83mg/m L,and the yield of polysaccharide was increased by 71.78%.（2）Study on the biological activity of polysaccharides from unfermented Polygonatum odoratumThe unfermented polysaccharide POP₁ and fermented polysaccharide POP₂ were obtained by extracting the fermented and unfermented polysaccharide from Polygonatum odoratum,removing protein by Sevage method,removing pigment by AB-8 macroporous resin and removing salt by dialysis bag.The antioxidant activities of POP₁ and POP₂ were determined by in vitro antioxidant method.The results showed that POP₁ and POP₂ showed certain antioxidant capacity and were dependent on polysaccharide concentration.Compared with untreated Yuzhu polysaccharide（POP₁）,fermented Polygonatum odoratum polysaccharide（POP₂）had 19.81%higher DPPH·radical scavenging capacity,30.66%higher ABTS⁺·radical scavenging capacity,35.43%higher OH^-radical scavenging capacity and 21.5%higher reducing power.Immunological activity of POP₁ and POP₂ was studied by cell model.RAW264.7macrophages were induced by lipopolysaccharide（LPS）to establish an inflammatory model in vitro,and the final concentration was 1μg/m L.The results of cell toxicity test showed that polysaccharide had no toxicity at the concentration of 50-200μg/m L.POP₁and POP₂ at this concentration could significantly inhibit LPS-induced phagocytosis and NO secretion of RAW264.7 inflammatory cells,and the inhibitory effect at high concentration was better than that at low concentration,and the inhibitory effect of POP₂ was better than that of POP₁.This study found that POP₂ was significantly stronger than POP₁ in antioxidant activity and immunoregulatory activity.（3）Purification and configuration of polysaccharide from Polygonatum odoratumPOP₁ and POP₂ were separated by DEAE-52 anion exchange column chromatography and further purified by Sephadex G-50 gel column chromatography.The structure of Polygonatum odoratum polysaccharide was preliminarily studied by UV and FT-IR.The results were as follows:POP₁ was separated by DEAE-52 fiber column to obtain the dd H₂O elution fraction POP₁-I and 0.1 mol/L Na Cl solution elution fraction POP₁-II.POP₂ was used to obtain the elution fractions of dd H₂O,0.1mol/L Na Cl and 0.3 mol/L Na Cl solution,namely POP₂-I,POP₂-II and POP₂-III.The polysaccharide components POP₁-I,POP₁-II,POP₂-I,POP₂-II and POP₂-III were subjected to Sephadex G-50 gel column chromatography to obtain the purified polysaccharide components POP₁-I-a,POP₁-II-a,POP₂-I-a,POP₂-II-a and POP₂-III-a.UV spectra showed that POP₁-I-a,POP₁-II-a,POP₂-I-a,POP₂-II-a,POP₂-III-a had no protein,nucleic acid and other impurities.Fourier transform infrared spectroscopy showed that POP₁-I-a was acidic polysaccharide withα-glycosidic bond andβ-glycosidic bond,POP₁-II-a was neutral polysaccharide withβ-glycosidic bond.POP₂-I-a is a neutral polysaccharide containingα-pyranose ring andβ-pyranose ring,POP₂-II-a is a neutral polysaccharide containingα-pyranose ring andβ-pyranose ring,POP₂-III-a is a neutral polysaccharide containingβ-pyranose ring.