| Sea cucumbers have been loved by people since ancient times.It is regarded as the first of the"Eight Treasures of the Sea".The processing of sea cucumber is mainly based on traditional processed products and simplified processed products.Intensive and deep processing products are still lacking and cannot meet the diversified needs of the market.Fermented sea cucumber products are still in the blank stage.In this study,the response surface methodology was used to optimize the process conditions of Bacillus subtilis Natto fermented fresh sea cucumbers,and the polysaccharides of sea cucumbers before and after fermentation were extracted.In order to provide theoretical basis for the development of new fermented sea cucumber products.Its main findings are as follows:On the basis of single factor,using the protease activity as the detection index,the response surface methodology was used to optimize the various process conditions of the fermented sea cucumber by Bacillus subtilis Natto.The main factors that have a significant effect on protease activity were obtained by Plackett-Burman experiment.Use the center combination experiment to establish a regression model to obtain the optimal process conditions for the main factors:The addition of sea cucumber,glucose and inoculation were 3.5%,7.5 g/L and 4%respectively.Under optimal conditions,the protease activity reached 535.12 U/m L,which was not significantly different from the predicted value of 522.56 U/m L,indicating that the model was reliable.The polysaccharides of Stichopus japonicus before and after fermentation were extracted by CPC method.There was no significant difference in the components of Stichopus japonicus polysaccharides before and after fermentation,and the total sugar contents were 51.91%and 49.09%respectively.The monosaccharide composition of Stichopus japonicus polysaccharide before and after fermentation was analyzed by HPLC.The results showed that they were composed of mannose,glucosamine,glucuronic acid,galactosamine,glucose,galactose and fucose,and the mass percentages were 5.82:6.52:4.53:10.13:5.93:6.10:60.99 and 5.47:4.67:4.95:9.71:1.38:4.81:69.01 respectively.High molecular weight gel exclusion chromatography was used to determine the relative molecular mass of Polysaccharides from sea cucumber before and after fermentation.The relative molecular mass of these polysaccharides was 1.07×10-6 and 1.16×10-6.Fourier transform infrared spectroscopy showed that the polysaccharides of Stichopus japonicus before and after fermentation contained fucose,acetylamino and sulfate substituents.The results of differential scanning calorimetry showed that the fermented Apostichopus japonicus polysaccharide had higher thermal stability.The antioxidant activity of Stichopus japonicus polysaccharide before and after fermentation was measured.The ABTS free radical scavenging ability of fermented Stichopus japonicus polysaccharide was higher than that of Stichopus japonicus polysaccharide,but the DPPH free radical scavenging ability and hydroxyl free radical scavenging ability were lower than that of Stichopus japonicus polysaccharide.Both polysaccharides had no iron reducing ability.The digestion process of Stichopus japonicus polysaccharide before and after fermentation was studied by simulating oral stomach small intestine digestion in vitro.In the process of simulated oral digestion,the relative molecular weight and reducing sugar content of the two polysaccharides did not change,and there was no free monosaccharide at the end of digestion,indicating that the Stichopus japonicus polysaccharide could not be digested by saliva before and after fermentation.In the process of simulated gastric digestion,the relative molecular weight of the two polysaccharides did not change,but the content of reducing sugar increased.A small amount of free glucuronic acid was detected at the digestion end point,indicating that the gastric juice could not digest the polysaccharides of Stichopus japonicus before and after fermentation,but could hydrolyze glucuronic acid.In the process of simulated small intestine digestion,the relative molecular weight of the two polysaccharides decreased,the content of reducing sugar did not change,and there was no free monosaccharide at the end of digestion,indicating that the polysaccharide of Apostichopus japonicus could be partially degraded by the small intestine before and after fermentation.The in vitro simulation experiment was conducted to study the fermentation characteristics of two polysaccharides by intestinal microorganisms.At the end of fermentation,the relative molecular weight and total sugar content of the two polysaccharides decreased,indicating that Stichopus japonicus polysaccharides can be utilized by intestinal microorganisms before and after fermentation.16S r DNA amplicon sequencing was used to study the effects of two polysaccharides on intestinal microorganisms.Before and after fermentation,Apostichopus japonicus polysaccharides did not change the species abundance and uniformity of intestinal microbiota,but could change the composition of intestinal microbiota. |
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