Studies On Preparation Of Yeast β-glucan And Fermentation Process Of β-1,3-glucanase

Yeast b-glucan is a major component of yeast cell wall. b-Glucan with a certain molecular weight and triple helical structure has certain biological activities. Production and application of b-1,3-glucanase have a huge market potential with deeper researches on active yeast glucan. Main researches to prepare and enhance the active ingredient in water-soluble yeast glucan which has a certain molecular weight and triple helical structure in the thesis have carried on as follows.(1) A quick measure method of congo red for the yeast β-glucan with triple helix structure was established. Final concentration of dimethyl sulfoxide and the maximum absorption wavelength of the cogon red system was 2% and 523 nm, respectively. The regression equation for qulities of yeast β-glucan with triple helix structure(Y) and ΔOD523 values(x) was Y = 923.0x-5.641, R2=0.996. The linear relationship was very good. The RSD of precision and accuracy of the method which was used to test the β-glucan with triple helix structure content in yeast β-glucan samples were 2.21% and 2.32%, respectively. It was an accurate and efficient measure method.(2) Cell wall(P0) obtained after Saccharomyces cerevisiae autolysis was used as base materials. Yeast b-glucan P2 and P3 products were prepared by hot acid hydrolysis with different hydrolysis time under the condition of same high temperature and low p H with cell wall dissolution rate as a process control indicator. b-Glucan and water-soluble b-glucan with triple helical structure contents in P2 and P3 samples were 27.05% and 33.35%, 0.21% and 0.35%, respectively. Then, according to the water-soluble b-glucan with triple helical structure contents, an optimized yeast b-glucan preparation process with the hot acid hydrolysis method was obtained as follows: feeding amount of P0 was 5%, p H was 2.00 and the time in water bath at 90°C was 15 h. Thus the water-soluble b-glucan with triple helical structure contents of the obtained sample(Ps) was 0.57%, which was increased by 171% and 62.86%, respectively, compared to P2 and P3.The hot acid hydrolysis process was substituted by a b-1,3-glucanase enzymatic hydrolysis. Optimized preparation process was that feeding amount of P0 was 5%, b-1,3-glucanase was 0.50%, p H was 5.50 and the time in water bath at 50°C was 6 h. Thus the water-soluble b-glucan with triple helical structure contents of the obtained sample(Pm) was 0.94%, which was increased by 64.91%. Meanwhile, the preparation time was reduced by 60% compared to optimized hot acid hydrolysis process.The average molecular weights of water-soluble part of yeast b-glucan samples(P0, P2, P3, Ps and Pm) were 95 KD, 54 KD, 52 KD, 70 KD and 79 KD, respectively, which were tested by gel permeation chromatography(GPC). They were all in the range of bioactive yeast b-glucan molecular weight.(3) Identification plate method for b-1,3-glucanase was used to prove that Zygosaccharomyces rouxii A which was a strain with the Na Cl-tolerant ability of 24%, had the characteristics of synthesizing and secreting β-1,3-glucanase. Z. rouxii A was used to produce b-1,3-glucanase in a 10 L fermentor, and the fermentation results were obtained as follows: glucose was the optimum carbon source(YEPD) for growth and enzyme production; fermentation efficiency with YEPD batch culture was significantly higher than that with glycerol(YEPG) and ethanol(YEPE), respectively; the growth efficiency(biomass), the largest b-1,3-Glucanase enzyme activity and enzyme productivity with YEPD batch cultivation were increased by 1.89% and 29.88%, 114% and 19.65%, 188% and 33% than that with YEPG and YEPE batch culture, respectively; compared with the YEPD batch cultivation, the time of maximum biomass was shorten by 12 h and the maximum biomass was increased by 19.29% with the exponential fed-batch with YEPF medium from 15 h to 23 h; moreover, b-1,3-glucanase was nearly logarithmically synthesized to the largest enzyme activity(44.99 U/m L), the time of synthesis was shortened by 6 h and enzyme productivity was up to 2.14 U/m L/h that was increased by 76.86%. The aim of improving enzyme production was achieved.