Enzymatic Production Of Galactomannan Oligosaccharides From Gleditsia Microphylla And Evaluation Of Their Prebiotic Activity

In the context of the national strategy of establishing the great food perspectives and building a healthy China,fully utilizing China’s rich forestry resources and using biotechnology to prepare high value-added food and feed additives is an important way to achieve the great food perspectives.This article aims to improve the economic value of Gleditsia microphylla,which has huge reserves and widespread distribution.The main research is to prepare high-value Gleditsia microphylla galactomannan oligosaccharides by enzymatic method and evaluate the effect of Gleditsia microphylla galactomannan oligosaccharides on human intestinal bacteria,providing a theoretical basis for the further development and utilization of Gleditsia microphylla galactomannan oligosaccharides.The content of galactomannan in Gleditsia microphylla seeds is 28.92%,which galactomannan is mainly distributed in endosperm,with a content as high as 62.67%,which is an excellent galactomannan resource.Using Gleditsia microphylla seed endosperm as substrate,under the optimal hot water extraction conditions（extraction temperature 70°C,solid-liquid ratio 1:200,extraction for 5 hours）,the extraction rate of Gleditsia microphylla galactomannan reached 93.98%,and the ratio of mannose to galactose in galactomannan was 2.54.The structural characteristics of Gleditsia microphylla galactomannan were analyzed,and its weight-average molecular weight was 6.36×10⁵ Da;when the concentration of Gleditsia microphylla galactomannan solution was 2.5%,its viscosity reached 16.53 Pas,with low concentration and high viscosity characteristics;infrared spectroscopy and nuclear magnetic resonance were used for analysis to verify that the structure of Gleditsia microphylla galactomannan is similar to that of other plant-derived galactomannans.The separation of Gleditsia microphylla galactomannan oligosaccharides was successfully achieved by ethanol graded alcohol precipitation.Based on this,combined with acid hydrolysis and high performance anion exchange chromatography（HPAEC）,scientific and accurate quantitative analysis of Gleditsia microphylla galactomannan oligosaccharides was achieved.Based on this,the effects of different substrate forms（Gleditsia microphylla galactomannan and Gleditsia microphylla seeds）on the enzymatic preparation of galactomannan oligosaccharides were systematically studied.The results showed that when Gleditsia microphylla galactomannan sample was used as substrate,the optimal conditions for enzymatic preparation of galactomannan oligosaccharides were 50°C,p H5,15 U/g-GM and 20 g/L polysaccharide concentration.At this time,the yield of galactomannan oligosaccharides was 52.22%and the enzyme selectivity was 92.61%.When crushed Gleditsia microphylla seeds were used as substrate,the optimal conditions for enzymatic preparation of galactomannan oligosaccharides were 50°C,p H5,20 U/g-GM and 40 g/L polysaccharide concentration.At this time,the highest yield of galactomannan oligosaccharides reached 70.78%and the enzyme selectivity was 82.26%.（3）In vitro fermentation of intestinal bacteria from human fecal,the galactomannan and galactomannan oligosaccharide was ultilized as carbon sources,and the results showed that the effect of both carbon sources on the concentration of short-chain fatty acids was insignificant;the utilization of galactomannan oligosaccharide by intestinal microbial was higher than that of galactomannan,and the proliferation effect of galactomannan oligosaccharide was higher than that of galactomannan.Their utilization rates were 98.40%and 74.61%,and their OD₆₀₀ were2.03 and 1.73,respectively.The results of 16s r RNA gene sequencing showed that both galactomannan and oligosaccharide could improve the diversity of intestinal bacteria,increase the relative abundance of phylum Bacteroides（beneficial bacteria）and significantly inhibit the phylum Fusobacteria（harmful bacteria）.Among them,galactomannan proliferated more significantly in the phyla Bacteroidetes,and galactomannan oligosaccharide inhibited phylum Fusobacteria more effectively,but galactomannan had a proliferative effect on the phylum Proteobacteria.The analysis of the results at the genus level were similar to the results at the phyla level,with galactomannan significantly proliferating the genus Parabacteroides compared to galactomannan oligosaccharide,while galactomannan oligosaccharide was more effective in inhibiting the genus Fusobacterium,while galactomannan oligosaccharide was found to have a proliferative effect on the genus Escherichia-Shigella.The changes of intestinal bacteria structure were associated with metabolism functions.The galactomannan oligosaccharides had more significant effects on several metabolic functions,including metabolic functions,carbohydrate metabolism,and cofactor and vitamin metabolism（level 2 metabolism functions）,compared to galactomannan,while lipoic acid metabolism,which is classified as a level 3 metabolism function,was significantly affected by galactomannan oligosaccharides.Finally,the correlation analysis between short-chain fatty acids and p H and genus level intestinal bacteria showed that p H and SCFAs had a significant effect on intestinal bacteria,with ambient p H of intestinal bacteria showing a significant positive correlation with Vagococcus.The Bacteroides,Megamonas and Kluyvera showed a significant positive correlation with the concentration of SCFAs.In addition,the environmental p H was negatively correlated with the Phascolarctobacterium and Parabacteroides,with Parabacteroides being the most significant.Gleditsia microphylla galactomannan and galactomannan oligosaccharides were used as carbon sources for fecal bacteria fermentation experiments to evaluate the prebiotic activity of Gleditsia microphylla galactomannan and galactomannan oligosaccharides.The results showed that both Gleditsia microphylla galactomannan and its oligosaccharides could be absorbed and utilized by intestinal bacteria,effectively proliferating intestinal bacteria.Among them,the utilization efficiency of galactomannan oligosaccharides was 98.40%,significantly higher than that of galactomannan（74.61%）,and the proliferation effect of galactomannan oligosaccharides on intestinal bacteria was better.Intestinal bacteria structure analysis showed that adding Gleditsia microphylla galactomannan and its oligosaccharides significantly increased the diversity of intestinal bacteria.Galactomannan had a significant proliferation effect on phyla Bacteroidetes,but a poor inhibition effect on phyla Fusobacteria.Galactomannan oligosaccharides were the opposite,with a poor proliferation effect on phyla Bacteroidetes and a significant inhibition effect on phyla Fusobacteria.At the genus level,Fusobacterium was more significantly inhibited by galactomannan oligosaccharides,while Parabacteroides was better proliferated by galactomannan.In addition,analysis of predicted metabolic function results found that galactomannan oligosaccharides significantly improved carbohydrate metabolism and co-factor and vitamin metabolism compared to galactomannan,and had a significant impact on thioctic acid metabolism.In summary,this article uses Gleditsia microphylla seed powder as substrate,adopts Richoderma reesei-derivedβ-mannanase restrictive hydrolysis,and efficiently prepares Gleditsia microphylla galactomannan oligosaccharides.In addition,Gleditsia microphylla galactomannan oligosaccharides have potential prebiotic activity.This study provides a basic basis for the large-scale production of Gleditsia microphylla galactomannan oligosaccharides and their impact on human intestinal bacteria.