曼氏无针乌贼缠卵腺抗菌糖蛋白提取工艺优化
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摘要
为优化曼氏无针乌贼缠卵腺抗菌糖蛋白的提取工艺,探索糖蛋白的抗菌活性。本研究在单因素试验基础上,通过响应面试验对糖蛋白的提取工艺进行优化,同时对粗糖蛋白采用阴离子交换柱(DEAE-52)层析进行分离纯化,并进行电泳鉴定。结果表明,糖蛋白的提取优化参数为:NaOH浓度为0.43 mol/L、提取时间2.8 h、提取温度27℃,液料比30∶1,在此条件下,得出抗菌粗糖蛋白实际得率为8.31%±0.18%。粗糖蛋白经阴离子交换柱(DEAE-52)层析有效分离获得组分2和组分3等2个糖蛋白组分,经SDS-PAGE电泳表明2个糖蛋白中的活性蛋白成分均为单一蛋白。结果同时表明,组分2与组分3对大肠杆菌与金黄色葡萄球菌具有非常显著的抑菌效果(P<0.01),且抗菌活性远强于同浓度山梨酸钾(P<0.01)。本研究可为曼氏无针乌贼缠卵腺糖蛋白在生物防腐剂方面的开发利用提供理论基础。
To optimize the extraction condition for the antibacterial glycoproteins from the nidamental gland of the Sepiella maindroni and explore the antibacterial activity of the glycoproteins,this paper optimized the extraction process of glycoprotein by response surface test on the basis of single factor test.At the same time,the crude glycoprotein was separated and purified by DEAE-52 Cellulose ion exchange column chromatography and identified by electrophoresis.The results showed that the optimized parameters were 0.43 mol/L of NaOH,extraction times of 2.8 h,extraction temperature of 27 ℃,and liquid to material ratio was 30∶1.In this condition,the actual antibacterial crude glycoprotein yield was 8.31%±0.18%.Two glycoprotein components,component 2 and component 3,were efficiently separated by DEAE-52 Cellulose ion exchange column chromatography.The SDS-PAGE electrophoresis showed that the active protein components of the two glycoproteins were single proteins.The results also showed that component 2 and component 3 had very significant antibacterial effect against Escherichia coli and Staphylococcus aureus(P<0.01),and the antibacterial activity was much stronger than the same concentration of potassium sorbate(P<0.01).This study can provide a theoretical basis for the development and utilization of glycoproteins from nidamental gland of the Sepiella maindroni in biological preservatives.
To optimize the extraction condition for the antibacterial glycoproteins from the nidamental gland of the Sepiella maindroni and explore the antibacterial activity of the glycoproteins,this paper optimized the extraction process of glycoprotein by response surface test on the basis of single factor test.At the same time,the crude glycoprotein was separated and purified by DEAE-52 Cellulose ion exchange column chromatography and identified by electrophoresis.The results showed that the optimized parameters were 0.43 mol/L of NaOH,extraction times of 2.8 h,extraction temperature of 27 ℃,and liquid to material ratio was 30∶1.In this condition,the actual antibacterial crude glycoprotein yield was 8.31%±0.18%.Two glycoprotein components,component 2 and component 3,were efficiently separated by DEAE-52 Cellulose ion exchange column chromatography.The SDS-PAGE electrophoresis showed that the active protein components of the two glycoproteins were single proteins.The results also showed that component 2 and component 3 had very significant antibacterial effect against Escherichia coli and Staphylococcus aureus(P<0.01),and the antibacterial activity was much stronger than the same concentration of potassium sorbate(P<0.01).This study can provide a theoretical basis for the development and utilization of glycoproteins from nidamental gland of the Sepiella maindroni in biological preservatives.
引文
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19 Ebran N,Julien S,Orange N,et al.Pore-forming properties and antibacterial activity of proteins extracted from epidermal mucus of fish[J].Comp Biochem & Physiol A Mol & Integr Physiol,1999,122:181-189.
20 Shi XD.Purification,characterization,immunoassay and function study of vitellogenin and lipovitellin from rosy barb[D].Qingdao:Ocean University of China(中国海洋大学),2004.
2 Ren GY,Li BF,Zhao X,et al.Ultrasound-assisted extraction technology for the extraction of glycoprotein from jellyfish(Rhopilema esculentum) oral-arms[J].Trans Chin Soc Agric Eng(农业工程学报),2008,24:255-259.
3 Yu D,Yang YF,Wang JB,et al.Study of glycoprotein extracted and purified from Ruditapes philippinarum inhibits the growth of tumor cell in vitro[J].J Zhejiang Ocean Univ:Nat Sci(浙江海洋学院学报:自科版),2011,30:336-339.
4 Zhang JY,Liu H,Sun HQ,et al.Extraction and purification of glycoprotein from Giant salamander's mucus and its antioxidant properties[J].J Chin Inst Food Sci Tech(中国食品学报),2018,18:181-187.
5 Xu YJ,Su YT,Ge XM,et al.Purification and characterization of antioxidant glycoproteins from the yellow seahorse Hippocampus kuda Bleeker[J].Chin J Mar Drug(中国海洋药物),2017,36(6):39-46.
6 Huang LZ,Zhong M,Hu XQ,et al.Anti-Oxidant activities in vitro of glycoprotein from Crassostrea Rivularis Crould by different extraction methods[J].J Guangdong Ocean Univ(广东海洋大学学报),2011,31(3):95-98.
7 Feng XM,Han YQ,Zhao ZQ,et al.Isolation,purification and characterization of glycoprotein from Oyster[J].Nat Prod Res Dev(天然产物研究与开发),2008,20:709-712.
8 Gu QQ,Fang YC,Xin XL,et al.Tumor suppressive activity and effect on immune function of Chlamys farreri[J].Acta Nutri Sin(营养学报),2001,23:200-202.
9 Mallya S K,Partin J S,Valdizan M C,et al.Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos[J].J Cell Biol,1992,117:1211-1221.
10 Zhang JS,Lei XL.Extraction of glycoprotein from squid viscera and its immunomodulation[J].Mod Food Sci & Technol(现代食品科技),2008,24:167-169.
11 Liu SJ,Wang Q,Wang Y,et al.Salmon stalk glandular glycoprotein MGS hypolipidemic and non-specific immune function research[J].J Fisheries Res(渔业研究),2014,6:436-441.
12 Kisugi J,Ohye H,Kamiya H,et al.Biopolymers from marine invertebrates.X.Mode of action of an antibacterial glycoprotein,aplysianin E,from eggs of a sea hare,Aplysia kurodai[J].Chem & Pharmaceut Bull,1989,37:3050-3053.
13 Jiang B,Wang YY,Liu CJ,et al.Extraction,purification and content determination of polysaccharide in Gingko Leaves[J].Lishizhen Med Mater Med Res(时珍国医国药),2007,18:2729-2731.
14 Xu WL.Anti-cancer activity of the purified extract glycoprotein with salamander skin mucus[D].Hanzhong:Shanxi Institute of Technology(陕西理工学院),2015.
15 Jiao QC.Introduction to Sugar Engineering(糖工程概论)[M].Beijing:Science Press,2010.
16 Wen J,Xie XA,Li Y,et al.Technology and antioxidant activity of tea-residue protein using microwave-assisted alkaline extraction[J].J Cereals & Oils(粮食与油脂),2017,30(5):41-44.
17 Wang Q.Studied on extraction and bioactivity analysis of mucin from nidamental gland of Todarodes pacificus[D].Fuzhou:Fujian Agriculture and Forestry University(福建农林大学),2013.
18 Fu D.Studies on antibacterial activity and antibacterial mechanism of ovomucin[D].Wuhan:Huazhong Agriculture University(华中农业大学),2015.
19 Ebran N,Julien S,Orange N,et al.Pore-forming properties and antibacterial activity of proteins extracted from epidermal mucus of fish[J].Comp Biochem & Physiol A Mol & Integr Physiol,1999,122:181-189.
20 Shi XD.Purification,characterization,immunoassay and function study of vitellogenin and lipovitellin from rosy barb[D].Qingdao:Ocean University of China(中国海洋大学),2004.
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