Study On Solubilization Of Candida Utilis β-Glucan And Evaluation Of Its Activity

β-glucan exists in many bacteria,fungi and higher plants.For more than 50 years,researchers have isolated,characterized and tested countless types ofβ-glucans.Saccharomyces cerevisiae has become one of the important sources of yeastβ-glucan based on its availability and low cost.Yeastβ-glucan accounts for 30-60%of the dry weight of the yeast cell wall.It is made up of 1500 glucose residues.About 60%of linear molecules are long chains connected byβ-（1→3）glycosidic bonds by 1500 glucose residues,with a molecular weight of about 240KDa.According to a large number of clinical trials,β-glucan has great potential as a medicine for the treatment or prevention of various diseases.In addition,yeastβ-D-glucan is widely used in the food industry as a thickener,emulsification stabilizer and fat substitute.It has good water retention,heat preservation,film formation and non-irritation.However,due to its dense three-strand helical structure,it is insoluble in organic solvents such as water and alcohol,which restricts its application in the fields of food,medicine and cosmetics,and might also have an important impact on its physiological functions in the body.Furthermore,the differences ofβ-glucan source,dosage,separation and purification,and physical and chemical properties would hinder the discovery of its mechanism of action.In this study Candida utilisβ-glucan was used as raw material.To obtain a green,efficient and biologically active extraction process,the effects of high-pressure steam synergistic enzymatic method and autolysis pretreatment to extract Candida utilisβ-glucan were compared.Ultrasound-assisted enzymatic was carried out to modify and improve the solubility of Candida utilisβ-glucan.The physical and chemical indexes,structural characterization and rheological properties of Candida utilisβ-glucan before and after solubilization were studied,which provided a theoretical basis and research foundation for the development of yeast cell wall polysaccharide products.The main research contents and conclusions are as follows:1.The effect of high-pressure steam and enzymatic pretreatment on the extraction ofβ-glucan from Candida utilis was investigated.In this condition,the material-to-liquid ratio was 1:10,at the temperature of 115℃,high-pressure steam treated for 2 h,then 2%papain and neutral protease were added respectively,ultrasonic extraction for 1h.The purity of Candida utilisβ-glucan could result in 89.81%,its extraction rate was twice of traditional autolysis pretreatment,reaching 18.75%.2.Comparing the in vitro antioxidant activity,physicochemical properties and solution conformation characteristics of Candida utilisβ-glucan prepared by different extraction processes of high-pressure steam synergistic enzymatic pretreatment and autolysis pretreatment,the results show that the triple helix structure and low molecular weight structural characteristics of Candida utilisβ-glucan help to improve the in vitro antioxidant capacity.At 2.0 mg/m L,AYG1（the sample treated autolysis pretreatment ultrasonic extraction once）with a Mw of 4.992×10⁶g/mol had a clearance rate of 42.28%for DPPH.At5 mg/m L,AYG2（the sample treated autolysis pretreatment and ultrasonic extraction twice）with Mw reduced to 3.554×10⁶g/mol and 1.987×10⁶g/mol HYG1（the samples pretreaed high-pressure steam enzymatic and extracted once by ultrasonic）had 78.35%and 76.45%clearance rates for DPPH.The main component of HYG1 wasβ-（1→3）-glucan connected byβ-bonds and has aβ-type configuration ofβ-（1→6）glucan branched chain.Monosaccharide composition of HYG1 wass mainly composed of 5.19mg/g glucose（Glc）and 0.52mg/g mannose（Man）,AYG1 contained 2.72mg/g glucose（Glc）and 1.24mg/g mannose（Man）.Congo red experiment and TME observation showed that there were certain differences in the solution conformation of the three yeastβ-glucans.All had a triple helix conformation in the solution.The triple helix structure of HYG1 was partially destroyed,while there was also a small amount of damage to the triple helix structure of AYG2.According to AFM,HYG1,AYG1 and AYG2 were all composed of slender worm-like chains.The chain diameter of HYG1 was much smaller than that of AYG1 and AYG2.The aggregation and entanglement phenomenon of AYG1 was the most obvious..3.To determin the technological conditions of solubilizing modified Candida utilisβ-glucan by ultrasonic enzymatic method,the ultrasonic intensity was 18W/m L,at temperature 30℃,and treated for 20 min,the solubility of Candida utilisβ-glucan reached the maximum value of 75.35%.The solubility has increased to about 3 times,compared with the traditional enzymatic hydrolysis for 20 minutes,the solubility was 28.28%.After solubilization and modification,the antioxidant activity of yeastβ-glucan is significantly improved.The clearance rate of DPPH was 12%at 300μg/m L,the clearance rate of ABTS⁺was 45.38%when the mass concentration is 2.0 mg/m L and the scavenging rate of hydroxyl radicals on 5.0 mg/m L reaches 56.76%.The zeta potential analysis of the yeastβ-glucan modified by ultrasonic synergistic enzymatic solubilization showed that the ultrasonic enzymatic solubilization and modification greatly improved the stability of the yeastβ-glucan aqueous solution.The primary structure of UEYG was analyzed by infrared spectroscopy,the primary structure was basically the same HYG1.The results of electron microscopy,AFM and SEM showed that theβ-glucan became significantly smaller and sparsely dispersed after being solubilized and modified.XRD results show that the UEYG crystal area might be destroyed,and the triple helix structure ofβ-glucan was destroyed.The thermal stability of yeastβ-glucan after solubilization modification was reduced,the thermal transition temperature of UEYG was 118℃and 95℃,respectively.4.The rheological properties of HYG1 and UEYGwas studied.The aqueous solutions of HYG1 and UEYG showed typical shear thinning behavior.In the linear viscoelastic region,the 10 mg/m L HYG1 aqueous solution exhibits liquid properties in the frequency range.In the low-frequency region,the high-concentration HYG1（40,30,20 mg/m L）solution failed to form a gel and showed liquid behavior,in the high-frequency region,it changed from a sol to a weak gel structure.Under different p H conditions,the viscosity of UEYG decreased with the increase of shear rate,and the viscosity changed more drastically as the p H value rises.In acidic and alkaline conditions,the fluidity increased,gradually turning to an ideal fluid.UEYG was very sensitive to temperature,the apparent activation energy is 0.3295 kcal.mol-1when the concentration was 30 mg/m L,20 mg/m L,and 10 mg/m L.As the ion concentration increases from 1%to 15%,the viscosity of the same concentration of Ca²⁺decreased more significantly,and the salt-tolerant effect of UEYG was poor.UEYG could be dissolved in anhydrous dimethyl sulfoxide,2%dimethyl sulfoxide and aqueous solutions.