Fermentation Preparation And Prebiotics Activity Of Extracellular Oligosaccharides Produced By Rhizobium Radiobacter

R.radiobacter is a typical nitrogen-fixing microorganism in soil.The current research on Rhizobia mainly focuses on its nitrogen-fixing symbiosis,and the surface oligosaccharides secreted by Rhizobia are closely related to the successful symbiosis of legumes.Therefore,studying the chemical structure and physical and chemical properties of oligosaccharides produced by Rhizobia,which is helpful to understand their structure activity relationship and broaden their potential application fields.In this paper,the fermentation process of R.radiobacter ATCC 13333 was optimized to improve the production of extracellular oligosaccharides,and then the synthesis mechanism of extracellular oligosaccharides under the influence of different nitrogen concentrations was studied under the transcriptomics level.This study provided a theoretical basis for the development of extracellular oligosaccharides fermentation of R.radiobacter ATCC 13333.Secondly,through the separation and purification of the fermentation broth,COGs-1 and COGs-3 were obtained and their structural properties were analyzed in detail.Finally,based on the unique macrocyclic structure of COGs-1,it was used as an ideal embedding carrier to embed curcumin and explored the in vitro prebiotic activity of the inclusion complex,which broadened the application prospect of R.radiobacter ATCC 13333 extracellular oligosaccharide.The main results are as follows:（1）To increase the fermentation yield of extracellular oligosaccharides,the single factor method and response surface method were used to optimize the medium composition and culture conditions of R.radiobacter ATCC 13333.The optimal shaking flask fermentation conditions were determined as 20 g·L^-1 mannitol,2.5 g·L^-1 glutamic acid,and an initial p H of7.0.Under this condition,the yield of extracellular oligosaccharides was 2.68 g·L^-1 at 144 h,which was 88.8%higher than before optimization.In order to solve the problem that excessive concentration of nitrogen source would lead to the increase of byproduct melanin concentration,a novel two-stage p H combined with dissolved oxygen control strategy was proposed at the 7L fermentation tank level.The p H was set to 7.0 in the growth phase,and then the p H was adjusted to 6.0 and dissolved oxygen was controlled at 40%in extracellular oligosaccharides biosynthesis phase.Under this strategy,the yield and biomass of extracellular oligosaccharides increased to 4.18 g·L^-1 and 7.84 g·L^-1 at 120 h,which were 88.3%and 115.4%higher than those of uncontrolled p H fermentation,respectively.At the same time,the concentration of melanin in fermentation broth was inhibited,which was significantly reduced to 0.11 g·L^-1.（2）In order to explore the effects of different glutamic acid concentrations on the expression of genes related to extracellular oligosaccharides and melanin production in R.radiobacter ATCC 13333,comparative transcriptomics sequencing analysis was performed.The results showed that compared with the high glutamic acid concentration,the gene expression levels of glucokinase,glucose pyrophosphorylase,UDP-glucose-4-differential isomerase and glycosyltransferase in the glycolysis pathway under low glutamic acid concentration were significantly increased,while the gene expressions of metabolic pathways related to energy such as tricarboxylic acid cycle were significantly decreased.This indicated that low concentration of glutamic acid was conducive to the conversion of glucose and contributed to the improvement of oligosaccharides production,while at higher glutamic acid concentration,more ATP,intermediate metabolites,and cofactors were generated,which enhanced cell growth and melanin biosynthesis.（3）The extracellular oligosaccharides in fermentation medium were prepared by ethanol fractional precipitation,solid phase extraction,and anion exchange chromatography.Two main oligosaccharides were obtained:COGs-1 and COGs-3.The contents of total glucans of COGs-1 and COGs-3 were 96.2±0.05%and 95.9±1.02%respectively.Based on Fourier transform infrared spectroscopy,monosaccharide composition,methylation,and nuclear magnetic resonance analyses.The COGs-1 was a family of unbranched cyclic oligosaccharides composed of onlyβ-1,2-linked D-glucopyranose residues with degree of polymerization between 17 and23,which was a typical of cyclicβ-1,2-glucans.The molecular weight and molecular formula of COGs-3 were 1584 Da and C₅₉H₉₂O₄₉,respectively.The structure of COGs-3 consisted ofβ-Glcp-（1→3）-β-Glcp-（1→3）-β-Glcp-（1→6）-β-Glcp-（1→6）-β-Glcp-（1→4）-β-Glcp-（1→4）-β-Glcp-（1→3）-α-Galp modified with two succinate and one pyruvate groups,which was a novel type of succinoglycan.The good thermal stability of COGs-1 and COGs-3 was demonstrated by differential scanning thermal analysis and thermogravimetric analyses.（4）Based on the characterization of COGs-1 with unique macrocyclic structure,it was used for embedding curcumin to explore its prebiotics activity.The stability constant of curcumin/Cys inclusion complexes（Cys/Cur）was 930 M^-1 through the phase solubility study.The Cys/Cur inclusion complex was then characterized by Fourier transform infrared spectroscopy,differential scanning calorimeter,scanning electron microscope,and nuclear magnetic resonance.The results showed that Cys could form inclusion complexes with curcumin.The results of in vitro simulated digestion experiment showed that Cys showed high digestion resistance,and the release rate of curcumin in Cys/Cur inclusion complex reached 88±1.8%.The results of typical probiotics fermentation experiments showed that the Cys and Cys/Cur inclusion complex groups could significantly promote the growth of probiotics and the generation of short-chain fatty acids compared with the Blank group.The results of the static fermentation of healthy human feces showed that the concentrations of acetic acid and lactic acid concentrations in the Cys group were significantly increased,the abundance of key bacterial species such as Bifidobacterium and Lactobacillus increased,and the relative abundance of pathogenic bacteria such as Klebsiella decreased significantly compared with the blank group.Compared with the Cys and fructooligosaccharide groups,Cys/Cur inclusion complex group significantly increased the concentrations of acetic acid,propionic acid,and lactic acid concentrations.The proliferation of beneficial genera such as Bifidobacterium and Lactobacillus was considerably promoted and the relative abundance of potential pathogenic bacteria such as Lactococcus,Streptococcus and Klebsiella was markedly inhibited.In conclusion,both Cys and Cys/Cur inclusion complex significantly increased the production of SCFAs,changed the composition of intestinal flora,promoted the proliferation of beneficial bacteria,and inhibited the growth of harmful bacteria.The Cys/Cur inclusion complex had double prebiotic function.