Structure And Activity Analysis Of Polysaccharide From Sparassis Latifolia Fermentation

Sparassis latifolia is a new type of edible fungus with market potential,which contains polysaccharide,superoxide dismutase,ergosterol and other active ingredients.Polysaccharide had attracted widespread attention due to its good pharmacological effects.However,the current research on the activities and structure of S.latifolia polysaccharide mainly focused on the fruiting body,which was difficult to meet the needs of research,development and application.The technology of extraction,structure and activity analysis of mycelium polysaccharide is not sophisticated,which affects the indepth development and utilization of S.latifolia.In this paper,by screening and comparing strains from different sources,the extraction,isolation and purification process of the fermentation polysaccharide of S.latifolia were systematically studied,and the structure and activity of the polysaccharide were analyzed,which provided reference for the development and application of liquid fermentation polysaccharide of S.latifolia.The main research contents and results are as follows:（1）The experimental strain XQJ-1 was collected and screened.A total of 4 strains of S.latifolia were isolated and collected,and all of them were identified as S.latifolia after reexamination.Through liquid culture and solid culture,XQJ-1 was selected as the experimental strain for subsequent liquid fermentation,with the dry weight of mycelium and polysaccharide yield as indexes.At the same time,the analysis of biological characteristics showed that XQJ-1 had good mushroom growth.（2）The fermentation and extraction conditions of polysaccharide from XQJ-1 strain were optimized.The liquid culture process of the XQJ-1 strain was monitored and the optimal culture time was determined to be 17 days.The best liquid medium composition obtained by single factor experiment was:potato 20%,soluble starch 1.5%,beef extract 0.8%,KH₂PO₄0.1%,Mg SO₄0.1%,VB₁0.01%,and initial p H was 5.The hot dip extraction method of S.latifolia mycelium polysaccharide was further improved through response surface method,and the reformative conditions were:extraction temperature 89℃,extraction time 2.4 h,liquid-to-material ratio 31 m L·g^-1,and actual yield is 8.82%±0.15%,which has good fit and repeatability and can be used in subsequent experiments.（3）The homogeneous component SLP-1 was obtained by isolation and purification of the fermentation polysaccharide of S.latifolia.The impurities in crude polysaccharide were removed by enzymatic method and Sevage method combined with enzymatic method.A neutral polysaccharide component and two acidic polysaccharide components were separated by column chromatography.The main component SLP-1 was non-starch polysaccharide and did not contain protein,reducing sugar and other impurities.The weight-average molecular weight and homogeneity of SLP-1 were determined by gel permeation chromatography.The results showed that the weight-average molecular weight of SLP-1was 25641,which was a homogeneous polysaccharide component.（4）The structure analysis and activity of polysaccharide fermented by S.latifolia was studied.The structure of SLP-1 was analyzed by infrared spectroscopy,liquid chromatography and other methods.The results showed that SLP-1 wasβ-type polysaccharide,containing C-H,C=O,C-OH and other functional groups,and was composed of glucose and other six monosaccharides.Micromorphology showed that SLP-1 was composed of spherical structures of different sizes,and did not have a triple helix structure.The results of activity experiment showed that both the crude polysaccharide SLPS-1 and the purified polysaccharide SLP-1 had certain inhibitory effects on the proliferation of human colon cancer Caco-2 cells in vitro.The results of inverted microscope and confocal laser microscope showed that the polysaccharide could cause cell death and morphological changes,which had adverse effects on the growth of human colon cancer cells.In this study,the extraction,separation and purification methods of S.latifolia fermented polysaccharide were explored,purified polysaccharide were obtained,and the structure of the polysaccharide was preliminarily analyzed.Cell experiments confirmed that the S.latifolia polysaccharide had certain inhibitory effects on colon cancer Caco-2 cells in vivo,which provides a reference for the development of S.latifolia edible and medical products.