Preparation By Co-culture And Prebiotic Activity Of Schizophyllum Commune Oligosaccharide

Schizophyllan（SPG）is a β-D-glucan with a branched structure,whose repeating unit consists of threeβ-（1→3）glycosidically linked D-glucans as the main chain,connected by aβ-（1→6）glycosidically linked D-glucans as the side chain.SPG has been shown to have various physiological activities such as tumor suppression,antibacterial and anti-inflammatory,anti-radiation and immunity enhancement.Recent studies have found that SPG is effective in improving the dysbiosis of intestinal flora caused by low dietary fiber diet.However,the human intestinal microbiota cannot use polysaccharides directly;instead,they must secrete matching enzymes to breakdown them,which will reduce the utilization efficiency of polysaccharides by probiotics.Moreover,the solubility of SPG is poor and tends to form a stable triple helix structure,and the direct enzymatic hydrolysis efficiency is low.This study prepared the branched chain Schizophyllum commune oligosaccharides（Sc OG）by fungal co-culture,and used sulfur trioxide-pyridine method to obtain sulfated derivatives of oligosaccharides,then the probiotic activities of oligosaccharides and its sulfated derivatives in vitro were investigated.The main research results are as follows:（1）The initial glucose concentration,T.harzianum inoculation time,T.harzianum inoculation volume and the co-culture time in S.commune-T.harzianum co-culture system were optimized at the shake flask level.The optimal fermentation conditions were 1.83 g·L^-1for Sc OG and 22.59 U·m L^-1 forβ-1,3-glucan endo-glucanase activity.（2）Sc OG was purified by combining purification steps such as graded alcohol precipitation and column chromatography,and it was verified that Sc OG was aβ-gluco-oligosaccharide containing only glucose and with polymerization degree of 3～11.Using the linear curdlan oligosaccharides（COS）as the control,the sulfated derivatives of Sc OG and COS were prepared by the sulfur trioxide-pyridine process,named as S-Sc OG and S-COS respectively.Infrared spectrum analysis showed that the sulfate base group successfully linked to Sc OG and COS.（3）The anti-digestion ability of two oligosaccharides and their sulfated derivatives was studied by simulating saliva and gastroenteric digestion in vitro.Also,their antioxidant properties were explored.The results showed that both oligosaccharides and their sulfated derivatives had the ability to resist salivary and gastroenteric digestion without degrading to reach the large intestine.In vitro antioxidant activity experiments showed that the antioxidant capacity of Sc OG was better than COS,and the antioxidant capacity of S-Sc OG and S-COS was better than Sc OG and COS.（4）Fecal bacteria from four healthy volunteers were fermented in vitro.The results showed that S-Sc OG could change the composition of intestinal microbes and maintain the diversity of human intestinal microbes.S-Sc OG and S-COS can be selectively utilized by probiotics such as Lactobacillus and Bifidobacterium and improve their relative abundance.S-Sc OG can also improve the abundance of potential probiotics such as Clostridium and Lachnospira.In addition,intestinal microbes can use Sc OG and S-Sc OG to produce SCFAs to maintain intestinal health.（5）To further study the ability of Sc OG and its sulfated derivatives to regulate intestinal microflora and metabolites in two ulcerative colitis patients.The results showed that Sc OG was less affected by differences in individual intestinal flora.Sc OG and S-Sc OG group can regulate the intestinal microbes and its metabolites in ulcerative colitis patients in a certain extent,including increasing the abundance of Bifidobacterium and butyric-producing bacteria such as Lachnospira and Faecalibacterium.