水溶性酵母β-葡聚糖的制备及免疫活性评价

doi:10.7503/cjcu20190195

高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (9): 1873.doi: 10.7503/cjcu20190195

水溶性酵母β-葡聚糖的制备及免疫活性评价

王珊珊¹,蔡超¹,郝杰杰¹,李国云¹,王学良¹,胡明华²,范罗嫡²,于广利^1,^*()

^1. 山东省糖科学与糖工程重点实验室, 中国海洋大学医药学院, 青岛 266003
^2. 无限极(中国)有限公司, 广州 510600

收稿日期:2019-03-29 出版日期:2019-09-10 发布日期:2019-07-19
通讯作者: 于广利 E-mail:glyu@ouc.edu.cn
基金资助:
国家新药创制科技重大专项(2018ZX09305-004);山东省重大科技创新工程专项(2018SDKJ0404)

Preparation and Immunological Activity Evaluation of Water-soluble β-Glucan from Saccharomyces cerevisiae ^†

WANG Shanshan¹,CAI Chao¹,HAO Jiejie¹,LI Guoyun¹,WANG Xueliang¹,HU Minghua²,FAN Luodi²,YU Guangli^1,^*()

^1. Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, School of Medicine and Pharmacy,Ocean University of China, Qingdao 266003, China
^2. Infinitus(China) Company Ltd., Guangzhou 510600, China

Received:2019-03-29 Online:2019-09-10 Published:2019-07-19
Contact: YU Guangli E-mail:glyu@ouc.edu.cn
Supported by:
? Supported by the National Science and Technology Major Project for Significant New Drugs Development, China(2018ZX09305-004);the Major Projects of Science and Technology in Shandong Province, China(2018SDKJ0404)

摘要/Abstract

摘要：

以废啤酒酵母(Saccharomyces cerevisiae)为原料, 通过稀酸与稀碱处理获得碱不溶性酵母β-葡聚糖(SCBG), 进而在低温强碱/脲水溶液中氧化降解得到水溶性酵母β-葡聚糖(SCBGs), 再经层析柱分离得到4个纯化组分(SCBGs-0-1, SCBGs-1-1, SCBGs-1-2和SCBGs-1-3). 利用高效液相色谱、高效凝胶渗透色谱与十八角激光光散射联用技术、核磁共振波谱及与刚果红结合实验等对4个纯化组分的单糖组成、分子量及化学结构进行了分析, 并通过其对巨噬细胞RAW 264.7吞噬能力及NO和TNF-α释放量的影响评价其免疫活性. 研究结果表明, 各纯化组分均由单一的葡萄糖构成, 是一类以β-1,3-D-葡聚糖为主链且在主链C₆位羟基上具有分支的β-1,3/1,6-葡聚糖,其分子量依次为198000, 960000, 270000和18700, 除SCBGs-1-3外, 其余3个组分均具有超螺旋结构, 且4个组分均能显著增强RAW 264.7的免疫活性.

关键词: 废啤酒酵母, 水溶性β-葡聚糖;, 超螺旋结构, 免疫活性

Abstract:

The wasted beer yeast(Saccharomyces cerevisiae) was used as raw material to obtain insoluble β-glucan(SCBG) through acid and alkaline treatment. The water-soluble β-glucan(SCBGs) was prepared by oxidative degradation in the NaOH/urea aqueous solution at low temperature. Four purified components(SCBGs-0-1, SCBGs-1-1, SCBGs-1-2 and SCBGs-1-3) were further obtained by column chromatography. The monosaccharide compositions, molecular weights and chemical structures of four purified components were analyzed by high performance liquid chromatography(HPLC), high performance gel permeation chromatography-multiple angle laser light scattering(HPGPC-MALLS), nuclear magnetic resonance spectroscopy(NMR) and Congo-red test. Their immunological activities, including the effects of phagocytosis and the release of NO and TNF-α, were studied on macrophage RAW 264.7. The results showed that all purified component mainly consisted of glucose. They were kinds of β-1,3/1,6-glucans composed of β-1,3-glucan as backbone with branches at the site of C6 hydroxyl groups. The molecular weights were determined to be 198000, 960000, 270000 and 18700, respectively. All the components still retained their original supercoil structure except SCBGs-1-3. Four purified components could significantly promote the immunological activity of RAW 264.7.

Key words: Wasted beer yeast, Water-soluble β-glucan;, Supercoil structure, Immunological activity

中图分类号:

O629.1

TrendMD:

王珊珊, 蔡超, 郝杰杰, 李国云, 王学良, 胡明华, 范罗嫡, 于广利. 水溶性酵母β-葡聚糖的制备及免疫活性评价. 高等学校化学学报, 2019, 40(9): 1873.

WANG Shanshan, CAI Chao, HAO Jiejie, LI Guoyun, WANG Xueliang, HU Minghua, FAN Luodi, YU Guangli. Preparation and Immunological Activity Evaluation of Water-soluble β-Glucan from Saccharomyces cerevisiae ^†. Chem. J. Chinese Universities, 2019, 40(9): 1873.

图/表 8

Fig.1 Thesolubility of SCBG in H2O(A), 6%NaOH(B) and NaOH-Urea-H2O(mass ratio 6∶18∶76) with(C) or without refrigeration(D)

Fig.2 Separation of water soluble β-glucan from the wasted beer yeast by QFF(A) and Sephacryl S-300HR column chromatography(B and C)

Table 1 Physicochemical properties of β-glucan purified from the wasted beer yeast

Sample	Total sugar content(%)	Protein content(%)	Molecular weight	M_w/M_n
SCBGs-0-1	95.36	1.88	198000	1.05
SCBGs-1-1	92.73	3.50	960000	1.32
SCBGs-1-2	96.69	1.93	270000	1.11
SCBGs-1-3	85.32	8.95	18700	1.15

Fig.3 HPGPC distribution of SCBGs-0-1(A), SCBGs-1-1(B), SCBGs-1-2(C) and SCBGs-1-3(D)

Fig.4 Absorption spectra of Congo-red without(a) or with(b) SCBGs-0-1(A), SCBGs-1-1(B), SCBGs-1-2(C) and SCBGs-1-3(D)

Fig.5 1H NMR(A), 13C NMR(B) and 1H-13C HSQC(C) of SCBGs at 25 ℃ and 1H NMR of SCBGs-0-1(a), SCBGs-1-1(b), SCBGs-1-2(c) and SCBGs-1-3(d) at 50 ℃(D)

Table 2 Branching degree of four purified β-glucans

Purified β-glucans	Integral area of A/B(H1)	Integral area of B(H6)	Branching degree(%)
SCBGs-0-1	1	0.11	11
SCBGs-1-1	1	0.12	12
SCBGs-1-2	1	0.13	13
SCBGs-1-3	1	0.23	23

Fig.6 Effects of different doses of samples on the cell viability(A), pinocytotic uptake(B), NO release(C) and TNF-α release(D) of the macrophage RAW 264.7 Results are expressed as means±S.D., *P<0.05, ** P<0.01, *** P<0.001, # P<0.0001 compared with control, as determined by unpaired student’s t-test. a. Control; b. LPS; c. LNT; d. SCBGs-0-1; e. SCBGs-1-1; f. SCBGs-1-2; g. SCBGs-1-3.

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水溶性酵母β-葡聚糖的制备及免疫活性评价

Preparation and Immunological Activity Evaluation of Water-soluble β-Glucan from Saccharomyces cerevisiae ^†

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水溶性酵母β-葡聚糖的制备及免疫活性评价

Preparation and Immunological Activity Evaluation of Water-soluble β-Glucan from Saccharomyces cerevisiae †

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Preparation and Immunological Activity Evaluation of Water-soluble β-Glucan from Saccharomyces cerevisiae ^†