Studies On The Preparation, Conformation And Immunobiological Activity Of β-D-Glucans In Saccharomyces Cerevisiae

β-D-Glucans, homopolymers of glucose, was widely distributed in the cell walls of microorganisms, mushrooms and plants. One important source ofβ-D-glucans was the cell wall of yeasts, particularly of the baker's and brewer's yeast Saccharomyces cerevisiae. Since the 1940s, numerous studies have demonstrated thatβ-D-glucans could enhance innate host defenses by binding to specific macrophage receptors and activating macrophage, resulted in antitumour, antibacterial, and wound-healing activities. Moreover, yeastβ-D-glucans was widely applied in the food industry as a thickening and water-holding agent, emulsifying stabilizer and fat replacer. The mensuration way for detectingβ-D-glucan was a difficult problem and there had no a standard mensuration way in the world. Routine mensuration way for detectingβ-D-glucan was phenol-sulfated acid method, but the method was easily to be disturbed by other polysaccharides and lead to receive incorrect results. In our research, a new method for quantitative determination of yeastβ-D-glucan was proposed. Firstly, vortex-mini-bead disruption way was used to broke the yeast cell walls and the ratio of broken yeast cells reached 95.28%; Secondly, modified sulfuric acid hydrolysis method was adopted to completely release glucose and the recoveries of hydrolysis ofβ-D-glucan came to 98.76%; Based on this, using GOPOD enzyme way, the content of the yeastβ-D-glucan was obtained. The new the relative standard deviation value and the average recovery of the new method were 0.46% and 99.96%, respectively. The ratios ofβ-D-glucan in various strain Saccharomyces cerevisiae were detected with the new mensuration method. The results indicates that the ratios ofβ-D-glucan in various strain Saccharomyces cerevisiae were distinctly difference (P≤0.05). The most difference of cell wall and cell dry mass reached 138.10% and 189.52%, respectively.Theβ-D-glucans content of yeast must be influenced by culture medium and condition, so we optimized the Fermentation condition. Firstly, the influences of carbon source, nitrogen source, and enzyme activation on content ofβ-glucan were evaluated using factional factorial design. Based on this, the experiment for optimization of theβ-glucan fermentation medium was carried out with the design of the rotation-regression-orthogonal combination and received model equation: Y = 106.89+3.74X₁-8.85X₁²-5.72X₂²-7.96X₃²-7.52X₄². The optimal parameters were obtained by multinomial regression techniques as follows: glucose: 32.7 g/L, peptone: 18.9 g/L, yeast extract: 15.7 g/L;glycerin: 10.4 g/L. The optimized culture medium allowedβ-glucan to increase from 65.80 mg/100ml to 109.33 mg/100ml. Secondly, adopting orthogonal design to optimize theβ-glucan fermentation condition, the sequences of primary and secondary influence factor were quantity of culture medium, culture temperature, initial pH and quantity of inoculation. A satisfactory productivity of yeastβ-D-glucans was obtained under the condition: pH5.0, 5ml/100ml (quantity of inoculation), 32℃and 60 ml/flask (quantity of culture medium), the quantity ofβ-D-glucans increased from 109.33 mg/100ml to 128.30 mg/100ml.In the basis of optimize fermentation, the yeast biomass and quantity ofβ-D-glucans were simulated by Logistic, BoxLucas1 and SRichards2 model. The Logistic model mostly suitted to the yeast biomass, the simulation equation as follows: y = 1317. 3?1 +(1x2/0180..96)2.7, the correlation coefficient was 0.975. The best simulated model ofβ-D-glucans was SRichards2 model, the simulation equation as followed: the correlation coefficient was 0.996. The glucose consume model is founded with Exponemtial ExpDec1 model, which correlated experiment well with of R2 of 0.977, the simulation equationIn the past decades,β-D-glucans have been often isolated by an acid-alkaline method. However, using these methods had some drawbacks: the great mass ofβ-D-glucans were degraded and distributed in all levels in supernatant. This led to lowerβ-D-glucans yields compared with the original content in the cell walls and remarkably influenced on its biological function. A new mild method to extractβ-D-glucans from S.cerevisiae cells was proposed in this paper, which was composed of induced autolysis, water and organic solvent treatment, homogenization and protease hydrolysis. The broken yeast cell was key and difficulty in theβ-D-glucans isolation procedure, so yeast cells were firstly treated by hot water. Treated by hot water, the mechanical strength of the yeast cell wall decreased with the removal of the mannoproteins, which caused the yeast cell walls easy to be disrupted. As a result, the ratio of broken yeast cell slightly exceeded 95% after three passes in homogenization treatment in 70 MPa. Finally,β-D-glucans were obtained at a yield of 91% of the original ratio in the yeast cell walls and with a purity of up to 93% (w/w). Because of infusibility,β-D-glucans was limited in the industry application.β-D-glucans modification was achieved by ultrasonication and homogeneous sulfation in this paper. The optical ultrasonical condition was 400W (power), 24s (treat time), 6s (intermission time) and 20 circle times. Using ultrasonication, the average diameter ofβ-D-glucans reduced from 56.49μm to 2.49μm.β-D-glucans sulfation reaction was in Urea-DMSO homogeneous system, in which 5% sulfate acid was employed as esterifiable reagent. The reaction carried through 4 h at 100℃, the sulfated yield was approximate 87.97% (w/w) and DS was 0.43 in the end.In order to make clear basic configuration ofβ-D-glucans and its sulfated derivative, some modern analysis technology FTIR, EA and NMR were adopted. These spectra revealed thatβ-D-glucans were (1→3)-linkedβ-D-glucopyranan backbone with (1→3)-β-branches every nine or five units and the etherification ofβ-D-glucans mainly exist on C6-SO3. Based on the elemental analysis, the empirical formula of sulfatedβ-D-glucans was (C6H10O5)20·9SO3·20H2O. The solution properties of sulfatedβ-D-glucans which was essential for the further insight on the correlation of structure to bioactivities, have not been reported. So based on YFY and KP wormlike cylinder models, the molecular parameters of sulfatedβ-D-glucans in solution were obtained by laser light scattering and viscometry in this paper. The results indicated that the conformational parameters of sulfatedβ-D-glucans were calculated to be 646 nm-1 for ML, 5.1 nm for q and 16.3 for C∞by wormlike cylinder mode, indicating a relatively extended flexible chain in the aqueous solution.Further, the immunobiological activity ofβ-D-glucans and its sulfated derivative was approved by mice experimentation. Preliminary immunopharmacological tests suggested that ultrasonic and sulfatedβ-D-glucans could significantly increased the ConA-induced spleen lymphocytes proliferation, the hemolytic level and the activity of NK cells (P≤0.01).