Structure Of Polysaccharides From Glycyrrhiza Residues And Its Regulation And Control Mechanism Of Quality And Function Of Fermented Foods

Glycyrrhiza residues are the solid waste residues after extraction of licorice root（extract）concrete by traditional decoctions,and still contains part of Glycyrrhiza polysaccharides,which can be extracted by acidic or alkaline solution.Those polysaccharides may also have various physiological activities,however,its structures have not been studied.Therefore,it is necessary to characterize the structures of polysaccharides obtained by acid and alkali extraction from Glycyrrhiza residues.Most natural polysaccharides cannot be directly digested and absorbed by the human body in stomach and small intestine,and those need to enter the gut and be decomposed by some hydralases secrected by gut microbiota.Polysaccharides can exert physiological activities by regulation and control of intestinal microbiota through its metabolites.However,the fermentation characteristics and the function of Glycyrrhiza polysaccharides on regulating intestinal microbiota have not been deeply studied.As people’s risk awareness of various chronic diseases（high blood pressure,hyperglycemia,hyperlipidemia,and etc.）has been enhanced,and the awareness of healthcare has been increasing unprecedentedly.More and more people start to consume functional food,and the outbreak of COVID-19 has further promoted this trend.There will be a greater development of functional foods in China.The Glycyrrhiza polysaccharides are applied to the functional food field to adapt to the current needs of human health.Therefore,in this study,Glycyrrhiza polysaccharides（GP）were extracted from Glycyrrhiza residues by acid and alkali extraction,and the structure and physicochemical properties of Glycyrrhiza polysaccharides were stematically elucidated.The in vitro fermentation characteristics of Glycyrrhiza polysaccharides and its effect on intestinal microbiota using human fecal microbial fermentation model in vitro were studied.The effect and regulation mechanism of Glycyrrhiza polysaccharide on the quality of fermented foods（yogurt and Chinese steamed bread）were investigated.The intestinal microbiota of normal mice was used as a medium to explore the regulation of the functional characteristics of fermented foods supplemented with Glycyrrhiza polysaccharides.The major research conclusions were summarized as follows:（1）Isolation,purification,structure and physicochemical properties of Glycyrrhiza polysaccharidesFour natural polysaccharides were prepared from Glycyrrhiza residues through acid or alkali extraction,ethanol precipitation and a series of purification methods.The molecular weights of Glycyrrhiza polysaccharides extracted by high temperature alkali-extraction（GP-HB）,low temperature alkali-extraction（GP-LB）,high temperature acid-extraction（GP-HA）and low temperature acid-extraction（GP-LA）were 3.490×10⁵g/mol,3.558×10⁵g/mol,3.519×10⁴g/mol and 2.612×10⁵g/mol,respectively.All of these four Glycyrrhiza polysaccharides were heteropolysaccharide,consisting of different molar percentages of Fuc,Rha,Ara,Gal,Glc,Man,Xyl,and Gal A,among which D-glucose（Glc）was the predominant monosaccharide.Comprehensive analysis showed that the composition of glycosidic bonds of the four Glycyrrhiza polysaccharides were different,and the→4)-Glcp-（1→linkage was the most abundant.The backbone of GP-HA and GP-HB may be composed of→4）-β-D-Glcp-(1→.The alkal-extracted Glycyrrhiza polysaccharides（GP-LB and GP-HB）had more complex branched chain structures.The acid-extracted polysaccharides（GP-HA and GP-LA）may have a triple helix conformation.Atomic force microscopy（AFM）confirmed that GP-HA possessed single branched structure,GP-LA possessed ring structures,GP-LB possessed multi-branched structure and GP-HB possessed cross-linked network structure;The GPs had numerous irregular spherical particles with smooth surface.Alkali-extracted Glycyrrhiza polysaccharides showed higher thermal stability than those of acid-extracted Glycyrrhiza polysaccharides.The GPs solution were typical pseudoplastic fluids,and the GPs extracted by alkali have higher apparent viscosity than those extracted by acid.GP-LB had high oil holding ability,emulsification activity and emulsification stability.（2）Glycolysis characteristics of Glycyrrhiza polysaccharides in vitro and regulation of human intestinal microbiotaGlycyrrhiza polysaccharides（GP）could be used by intestinal microbiota in in vitro fermentation.The molecular weight and total sugar content decreased during fermentation.All of these four Glycyrrhiza polysaccharides showed relatively slower fermentation rate,and D-glucose（Glc）may be the monosaccharide most easily utilized and fermented by gut microbiota.Glycyrrhiza polysaccharides could promote the production of short chain fatty acids（SCFAs）.The effects of Glycyrrhiza polysaccharides on intestinal microbiota composition were significant,the microbiotal community diversity of low temperature acid-extraction Glycyrrhiza polysaccharides（LA）group increased significantly,compared with that of the blank control group（D）,the abundance of Actinobacteriota in each treatment group was significantly increased,while the relative abundance of other phylum was different,both Glycyrrhiza polysaccharide and fructooligosaccharide（G）could increase the abundance of some major genera such as Megamonas and Bifidobacterium,but the abundance of minor bacteria were different.The dominant microbiota among treatment groups were significant different（p<0.05）.The abundance of intestinal bacteria involved in carbohydrate transportation and metabolism was the highest in all treatment groups.（3）Effect and regulation mechanism of Glycyrrhiza polysaccharides on the quality of yogurtGlycyrrhiza polysaccharide（GP）/Casein（CS）could form a stable system.The electrostatic interaction,hydrogen bonding and hydrophobicity were involved in the formation of the composites.The incorporation of Glycyrrhiza polysaccharide at different concentrations accelerated the growth of lactic acid bacteria.GP could suitably improve the water-holding capacity and texture,educe the precipitation rate of whey of the yogurt.And the yogurt prepared with GP showed the higher viscosity and shorter transverse relaxation time of mobile water(T₂₃)value than control group（CG）.Moreover,the microstructure analysis results indicated that the casein network of yogurt with GP was more compact and cohesive than those of others.Yogurt prepared with 0.1%GPs exhibited the best sensory acceptance.Collectively,GP significantly improved the structural and texture properties of the yogurts.（4）Effect of Glycyrrhiza polysaccharide on quality and function of Chinese steamed bread and its regulation mechanismGlycyrrhiza polysaccharides exhibited certain inhibition activity onα-amylase andα-glucosidase.CD spectra and fluorescence intensity measurements confirmed that the secondary structure ofα-amylase andα-glucosidase changed and the enzyme activity was reduced after the addition of GP.The quality,starch digestion and storage properties of Chinese steamed bread were significantly affected by Glycyrrhiza polysaccharide.The addition of GP increased the specific volume of CSB in a dose-dependent manner,and the specific volume of CSB-2 was 2.55 m L/g.GP also contributed to the increase in hardness（from 1240.17 to 2539.34 g）and chewiness（893.85 to 1959.27 g）of fresh CSB.In addition,GP could maintain the integrity of the protein network within the CSB.The scores for sensory evaluation indicators of CSB-1 were relatively balanced.More importantly,the addition of GP altered starch digestive properties,and the content of the resistant starch（RS）was increased from8.62%（CSB-0）to 43.46%（CSB-2）.GP led to a significant reduction of the expected glycemic index（e GI）of CSB,and the e GI of CSB was decreased from 97.50（CSB-0）to 73.8（CSB-2）.In addition,the results of X-ray diffraction（XRD）and differential scanning calorimeter（DSC）revealed the addition of GP delayed the staling of CSB during storage.（5）The study of the regulation of Glycyrrhiza polysaccharides on fermented food based on intestinal microbiotaFermented functional foods supplemented with Glycyrrhiza polysaccharides were safe to mice and did not affect the normal growth and development of mice.Glycyrrhiza polysaccharide food could promote the production of SCFAs in the intestine of mice,reduce the p H value of colonic contents,and increase the water content of faeces.The effect of Glycyrrhiza polysaccharide fermented foods on intestinal microbiota composition of normal mice was significant,the microbiota categories among different groups were remarkably different.The relative abundance of Actinobacteriota was increased significantly in group of Chinese steamed bread supplemented with Glycyrrhiza polysaccharides,the relative abundance of Bacteroidota and Firmicutes was higher in the yogurt group.The microbiota composition at genus level was significantly different amont these groups,Chinese steamed bread or yogurt（GP series）with Glycyrrhiza polysaccharides had the potential to enrich Muribaculacea,and also promoted the relative abundance of minor potentially beneficial bacteria in the mice gut.The key microbiota among treatment groups were significant different.