Microsoft Word - 8——黄莺莺_news_.doc

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1 紫菜纳豆菌发酵物中醇提物和多糖生物活性的初步研究黄莺莺, 董焕焕, 杨苗苗, 矫丽萍, 邵霜, 岳琪, 魏玉西 (, ) 摘要 : 纳豆菌 (Bacillus natto) 发酵后的条斑紫菜经稀释离心后上清液用一定浓度的乙醇处理获得醇沉物和醇溶液将醇沉物去蛋白得到粗多糖, 进行抗氧化活性的研究 ; 将醇溶液旋转蒸发得到浸膏, 将浸膏按极性大小分相萃取分别得到石油醚相乙酸乙酯相和正丁醇相浸膏, 探讨分相浸膏对金黄色葡萄球菌枯草芽孢杆菌四联微球菌的抑菌效果通过测定多糖和分相浸膏对羟自由基 ( OH) 和 DPPH 自由基的清除能力评价其抗氧化能力结果表明 : 醇溶物对金黄色葡萄球菌具有抑制作用, 且随着浓度的升高各相浸膏对羟基自由基的清除率随之升高, 其中, 正丁醇相浸膏在质量浓度为 0.5 mg/ml 时对羟自由基的清除率达到 91.6%, 说明条斑紫菜经纳豆菌发酵可能产生了活性化合物 ; 与对照组相比, 利用纳豆菌发酵紫菜发酵物中多糖提取率增加了 0.24%, 发酵得到的多糖在质量浓度为 5.0 mg/ml 时对羟自由基的清除率达到了 95.7% 关键词 : 条斑紫菜 (Porphyra yezoensis); 纳豆菌 (Bacillus natto); 多糖 ; 醇提物 ; 抗氧化性 ; 抑菌活性中图分类号 : TS 文献标识码 : A 文章编号 : (2016) doi: /hykx , (Bacillus natto),,, [1-2], [3-4] 2000,, [5],, [6],, [7-8],, [9],,, [10],,,, 1 材料与方法 1.1 材料 ( ), (Bacillus natto) ; (Bacillus subtilis) (Staphylococcus aureus) (Micrococcus tetragenus), : ; : : (ZR2012DM014) [Foundation: Natural Science Foundation of Shandong Province, No. ZR2012DM014] : (1994-),,,, :, : , jnhyy0209@126.com; (1964-),,,,,,, :, : , yuxiw729@163.com 56 / 2016 / 40 / 6

果通过测定多糖和分相浸膏对羟自由基 ( OH) 和 DPPH 自由基的清除能力评价其抗氧化能力结果表明 : 醇溶物对金黄色葡萄球菌具有抑制作用, 且随着浓度的升高各相浸膏对羟基自由基的清除率随之升高, 其中, 正丁醇相浸膏在质量

2 1.2 主要仪器与设备 DXF-10A ( ); YXQ-LS-50SII ( ); CR21G ( ); RE-52A ( ); SW-OJ-IFD ( ); 721G ( ); ( ) 1.3 方法 [11] ( ) h,,,, 1..16, 20 min : 43, 24 h, 3 g 0.01 g,, 1..40, 5 h, (8 000 r/min)10 min 95% 80%, 4,, 12 h, ( A, A ) ( B, B ) ( C, C ), (w/w) 80 1 h,, (8 000 r/min) 20 min, ( ), 95% 80%, 4, (5 000 r/min) 10 min, :,, 490 nm [12] (%)= / 100 % : [13-15], 20 g/l, 15 ml,,, 7, 20 μl, 6, 3, 37 16~24 h,,, 20, 10 5 g/l, 7, 20 μl, 37 16~24 h, 3 [16], [17], Fenton,, 0.15 mol/l, ph ml, 6 mmol/l EDTA 0.5 ml, 520 μg/ml 0.1 ml, 3.5 ml, 0.3%(V/V) H 2 O ml, H 2 O min, 520 nm 3, Vc : (%)=(A A )/ (A A ) 100% : A ; A ; A DPPH [18], Marine Sciences / Vol. 40, No. 6 /

3.3 : 1.3.4 [13-15], 20 g/l, 15 ml,,, 7, 20 μl, 6, 3, 37 16~24 h,,, 20, 10 5 g/l, 7, 20 μl, 37 16~24 h, 3 [16], 1.3.5 1.3.5.1 [17], Fenton,, 0.15 mol/l, ph7.4 0.5 ml, 6 mmol/l EDTA 0.

3 2.0 ml, 0.2 mmol/l DPPH 2.0 ml, 30 min, 517 nm A i ;, 2.0 ml DPPH 2.0 ml A 0 ; 2.0 ml 2.0 ml A j Vc : (%)=[1 (A i A j )/ A 0 ] 100% : A i - ; A j - ; A [19], 4.5 ml Tris-HCl (50 mmol/l, ph8.2) 25 ml, 4.2 ml, 37, 20 min ml 3 mmol/l ( 10 mmol/l HCl ), 0.5 ml 10 mmol/l HCl HCl,,, 325 nm 30 s (A a ) (A b ) 2 ml,,,, (A i ) 10 mmol/l HCl, (A j ), 3 Vc : (%)=[(A a A b ) (A i A j )]/(A a A b ) 紫菜多糖提取及含量测定结果, 2.40%, 1.94%, 0.46%( 2) [20],, 表 2 多糖提取率及蛋白质去除率 Tab. 2 The extraction ratio of crude polysaccharides and the rate of protein removal (%) (%) 醇提物的抑菌活性 ,, 2.3.2,,, 5.00 mm, 1 2 结果与分析 2.1 两组提取物旋蒸后各相浸膏的质量 150 g,, 1, 表 1 紫菜纳豆菌发酵乙醇提取物浸膏质量 Tab. 1 The mass of the alcohol extract from Porphyra yezoensis fermented by Bacillus natto (g) Fig. 1 The antibacterial ring diameter of the alcohol extract in the presence of Staphylococcus aureus,,,, ; 5 g/l 10 g/l, 58 / 2016 / 40 / 6

5 ml 10 mmol/l HCl HCl,,, 325 nm 30 s (A a ) (A b ) 2 ml,,,, (A i ) 10 mmol/l HCl, (A j ), 3 Vc : (%)=[(A a A b ) (A i A j )]/(A a A b ) 100 2.2 紫菜多糖提取及含量测定结果, 2.40%, 1.94%, 0.

4 :,,,,,,,,, 2.4 醇提物和多糖的抗氧化活性 ( OH) 2, Vc, 0.5 g/l, 63.04% 80.12% 91.61%, Vc 60.09% Vc,, :,,,,, 3 Fig. 3 The effect of polysaccharides on the scavenging of hydroxyl free radicals DPPH DPPH 4 Vc 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 g/l, DPPH 40.00%, 69.30%, 80.80%, 89.28%, 91.13%, 95.21%,, DPPH,, DPPH :,,,, DPPH, DPPH 2 Fig. 2 The effect of various organic extracts on the scavenging of hydroxyl free radicals 3 0.5~5.0 g/l,,, 5.0 g/l, 95.7%, Vc 0.5~5.0 g/l 100.0%, 4 DPPH Fig. 4 The effect of various organic extracts on the scavenging of DPPH free radicals Marine Sciences / Vol. 40, No. 6 /

30%, 80.80%, 89.28%, 91.13%, 95.21%,, DPPH,, DPPH :,,,, DPPH, DPPH 2 Fig.

5 ,,, DPPH ~5.0 g/l,,, 5.0 g/l, 64.0%, Vc( 80.9%) 5 Fig. 5 The effect of polysaccharides on the scavenging of superoxide anion free radicals 3 结论,,,,,,,,,, 5.0 g/l, 95.7%( Vc ), : [1] Sumi H, Hamada H, Tsushima H, et al. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto: a typical and popular soybean food in the Japanese diet[j]. Experientia, 1987, 43 (10): [2] Liang X, Zhang L, Zhong J, et al. Secretory expression of a heterologous nattokinase in Lactococcus lactis[j]. Appl Microbiol Biotechnol, 2007, 75(1): [3] Pais E, Alexy T, Holsworth RE Jr, et al. Effects of nattokinase.a pro-fibrinolytic enzyme on red blood cell aggregation and whole blood viscosity[j]. Clin Hemorheol Microcirc, 2006, 35(1-2): [4] Sumi H, Hamada H, Nakanishi K, et al. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase[j]. Acta Haematol, 1990, 84(3): [5],,. [J]., 2007, 23(10): Yang Yu, Zhang Lijing, Seigo Amachi. Selection of high Nattokinase-producing Strains and Optimization of the Fermentation Conditions[J], Modern Food Science and Technology, 2007, 23(10): [6],,,. [J]., 2009, 6(25): Ming Feiping, Zhao Peijing, Liao Chunfang, et al. Optimization of Liquid Fermentation of Bacillus natto[j]. Modern Food Science and Technology, 2009, 6(25): [7],,,. [J]., 2011, 30(6): Cong Junying, Zhang Shulian, Wang Yang, et al. The degree of proteolysis from fish protein fermentation by Bacillus natto[j]. Journal of Dalian Polytechnic University, 2011, 30(6): [8],,. [J]., 2013, 38(11): Fu Bo, Wu Ligang, Duan Shan, et al. Study on influencing factors of protease activity during the process of fermentation of shrimp head and shell with Bacillus natto[j]. China Condiment, 2013, 38(11): [9] Zhang Q, Li N, Liu X, et a1. The structure of a sulfated galactan from Porphyra haitanensis and its in vivo antioxidant activity[j]. Carbohydr Res, 2004, 339(1): [10],,,. [J] , 39(4): Zheng Wenxiang, Zheng Huibin, Wang Baozhou, et al. Characterization of protein extracted from Porphyra haitanensis by fermentation combined with enzymolysis[j]. Food and Fermentation Industries, 2013, 39(4): [11]. [J]. ( ), 2004, 3(4): Cheng Xixiong. A preliminary study on the bacteriostasis of Allium macrostemon[j]. Journal of Hangzhou Teachers College, 2004, 3(4): / 2016 / 40 / 6

Experientia, 1987, 43 (10): 1110-1111. [2] Liang X, Zhang L, Zhong J, et al. Secretory expression of a heterologous nattokinase in Lactococcus lactis[j].

6 [12],. - [J]., 2004, 7(17): Zhang Qing, Zhang Tianming. Determination of polysaccharide by phenol-sulfuric acid method[j]. Shandong Food Science and Technology, 2004, 7(17): [13] STROBEL G A. Rainforest endophytes and bioactive products[j]. Critical Reviews in Biotechnology, 2002, 22(2): [14],,,. [J]., 2006, 19(6): Wang Jun, Yao Ying, Zhang, Jianpeng, et al. Preparation and biological activity of polysaccharides from Ostrea rivularis in the east China sea[j]. Journal of Medical Postgraduates, 2006, 19(6): [15]. [M]. :, 1986: Zhou Deqing. Microbiology Laboratory Manual[M]. Shanghai: Shanghai Scientific and Technical Publishers, 1986: [16]. [M]. :, 1998: Su Shiyan. Handbook of food microbiology testing[m]. Beijing: China Light Industry Press, 1998: [17]. [M]. :, 2004: 344. Ling Guanting. Antioxidant food and health[m]. Beijing: Chemical Industry Press, 2004: 344. [18],,,. (Subergorgia suberosa) [J]., 2010, 34(9): Liu Lei, Wei Yuxi, Wang Changyun, et al. Study on the Anti-oxidation and Bacteriostasis Activities of the Ethanol Extract from Subergorgia suberosa[j]. Marine Sciences, 2010, 34(9): [19],,,. [J]., 2013, 34(14): Chen Yiyong, Zhang Tianjun, Yang Jun, et al. Viscosity and Antioxidation Activity of Polysaccharides from Porphyra Haitanensis of Nantong[J]. Food Research and Development, 2013, 34(14): [20],,. [J]., 2013, 30(5): Zhang Liyan, Hao Min, Li Qiusha. Purification and Enzymological Properties of Natto Fungus Glycosidase [J]. Chinese Journal of Drug Evaluation, 2013, 30(5): Preliminary research on the biological activity of alcohol extract and polysaccharides from Porphyra yezoensis fermented by Bacillus natto HUANG Ying-ying, DONG Huan-huan, YANG Miao-miao, JIAO Li-ping, SHAO Shuang, YUE Qi, WEI Yu-xi (College of Life Sciences, Qingdao University, Qingdao , China) Received: Sep. 15, 2015 Key words: Porphyra yezoensis; Bacillus natto; polysaccharides; alcohol extract; antioxidant activity; antibacterial activity Abstract: The fermentation product of Porphyra yezoensis by Bacillus natto was diluted and centrifuged, and the supernatant was treated with alcohol to obtain an alcohol-based precipitation extract. The fermentation product was also deproteinized to extract crude polysaccharides to study the antioxidant activity, while the extract was subjected to rotary evaporation and extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to evaluate the related extract for antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Micrococcus tetragenus. The antioxidant activity was evaluated based on the scavenging rate to hydroxyl free radicals ( OH) and 1, 1-diphenyl-2-picryl-hydrazyl radicals (DPPH ). The results revealed that the alcohol soluble substances showed an inhibitory effect against S. aureus, and the scavenging rate to OH presented a dose-response relationship. Among them, the scavenging rate to OH by the n-butanol extract reached 91.6% at a concentration of 5.0 mg/ml. Compared with unfermented P. yezoensis, the extraction ratio of crude polysaccharides from fermented P. yezoensis increased by 0.24%, and the scavenging rate of polysaccharides to OH at a concentration of 5.0 mg/ml increased up to 95.7%. These results demonstrated that the fermented product possesses both antibacterial and antioxidant properties. ( 本文编辑 : 康亦兼 ) Marine Sciences / Vol. 40, No. 6 /

Wang Jun, Yao Ying, Zhang, Jianpeng, et al. Preparation and biological activity of polysaccharides from Ostrea rivularis in the east China sea[j].

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇