干巴菌菌丝体多糖的制备及其水解特性
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摘要
培养基优化实验表明,马铃薯葡萄糖培养基是干巴菌的最适产糖培养基,菌丝体产量和菌丝体多糖产量分别可达7.56 g/L和0.42 g/L。采用Plackett-Burman试验及响应面试验优化干巴菌多糖的提取工艺,结果表明,极显著影响因素及其最优条件为超声功率400 W、超声时间10 min、醇沉倍数3倍,优化后多糖得率可达6.98%。体外抗氧化实验证明,干巴菌多糖具有良好的抗氧化活性,并且酶水解(纤维素酶、蜗牛酶)和酸水解(硫酸)均可使多糖的抗氧化能力显著增强。采用逐级酸水解结合柱前衍生高效液相色谱法分析多糖结构,其单糖残基分布规律为:支链末端残基由半乳糖和少量甘露糖构成;半乳糖大多分布于支链外侧及支链末端;葡萄糖是主要单糖组分,主要分布于主链及支链内侧;甘露糖主要分布于支链内侧。本研究为酶水解及酸水解方法在多糖领域中的应用及干巴菌多糖的资源开发提供理论基础。
Optimization experiments showed that potato glucose medium was the optimal medium for sugar production by Thelephora ganbajun Zang, and using this medium, the mycelial yield and mycelial polysaccharide yield were 7.56 and 0.42 g/L, respectively. Plackett-Burman design and response surface methodology were used to optimize the ultrasonicassisted extraction of T. ganbajun Zang polysaccharides. Ultrasonic power, irradiation time and alcohol precipitation fold were found to be significant factors affecting the polysaccharide yield and their optimal levels were 400 W, 10 min and 3,respectively, which resulted in maximum polysaccharide yield of 6.98%. The results of in vitro antioxidant tests showed that the polysaccharides had a strong antioxidant capacity, and hydrolysis with enzymes(cellulase and snailase) or acid(sulfuric acid) could significantly enhance the antioxidant capacity of the polysaccharides. Monosaccharide composition analysis by stepwise acid hydrolysis combined with pre-column derivatization high performance liquid chromatography showed that the terminal residues of branches of the polysaccharides were composed of galactose as well as a small amount of mannose;galactose was mostly distributed on the outer side and at the end of the branches; glucose was the main monosaccharide component, and it was mainly distributed in the main chain and the inside of the branches; mannose was mainly distributed inside the branches. This study lays a theoretical foundation for the application of enzymatic hydrolysis and acid hydrolysis on polysaccharides and the exploitation and utilization of T. ganbajun Zang polysaccharides.
Optimization experiments showed that potato glucose medium was the optimal medium for sugar production by Thelephora ganbajun Zang, and using this medium, the mycelial yield and mycelial polysaccharide yield were 7.56 and 0.42 g/L, respectively. Plackett-Burman design and response surface methodology were used to optimize the ultrasonicassisted extraction of T. ganbajun Zang polysaccharides. Ultrasonic power, irradiation time and alcohol precipitation fold were found to be significant factors affecting the polysaccharide yield and their optimal levels were 400 W, 10 min and 3,respectively, which resulted in maximum polysaccharide yield of 6.98%. The results of in vitro antioxidant tests showed that the polysaccharides had a strong antioxidant capacity, and hydrolysis with enzymes(cellulase and snailase) or acid(sulfuric acid) could significantly enhance the antioxidant capacity of the polysaccharides. Monosaccharide composition analysis by stepwise acid hydrolysis combined with pre-column derivatization high performance liquid chromatography showed that the terminal residues of branches of the polysaccharides were composed of galactose as well as a small amount of mannose;galactose was mostly distributed on the outer side and at the end of the branches; glucose was the main monosaccharide component, and it was mainly distributed in the main chain and the inside of the branches; mannose was mainly distributed inside the branches. This study lays a theoretical foundation for the application of enzymatic hydrolysis and acid hydrolysis on polysaccharides and the exploitation and utilization of T. ganbajun Zang polysaccharides.
引文
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[22]MENG Q,LI Y,XIAO T,et al.Antioxidant and antibacterial activities of polysaccharides isolated and purified from Diaphragma juglandis fructus[J].International Journal of Biological Macromolecules,2017,105:43 1-437.DOI:10.1016/j.ij biomac.2017.07.062.
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[28]HAO L,SHENG Z,LU J,et al.Characterization and antioxidant activities of extracellular and intracellular polysaccharides from Fomitopsis pinicola[J].Carbohydrate Polymers,2016,141(6):54-59.DOI:10.101 6/j.carbpol.2015.1 1.048.
[29]XU Y,JIANG H Y,SUN C Y,et al.Antioxidant and hepatoprotective effects of purified Rhodiola rosea polysaccharides[J].International Journal of Biological Macromolecules,2018,117:167-178.DOI:10.1016/j.ijbiomac.2018.05.168.
[30]陈春娟,朱振元,陈璐.低分子量蛹虫草多糖降血糖活性的研究[J].现代食品科技,2017(4):25-30.DOI:10.13982/j.mfst.1673-9078.2017.4.005.
[31]韩莎莎,黄臻颖,沈照鹏,等.酶法降解坛紫菜多糖及其产物分析[J].食品科学,2015,36(21):145-149.DOI:10.7506/spkx1002-6630-201521028.
[2]ZHAO Y,HOU B,TANG Z,et al.Application of ultrasonics to enhance the efficiency of cleaning Thelephora ganbajun[J].Ultrasonics Sonochemistry,2009,16(2):209-211.DOI:10.1016/j.ultsonch.2008.08.006.
[3]张岚,和劲松,王雪峰,等.响应面优化复合酶法提取干巴菌多糖工艺[J].食品研究与开发,2017,38(20):51-54.DOI:10.3969/j.issn.1005-6521.2017.20.010.
[4]LIN H,JI-KAI L.Two novel phenylacetoxylated p-terphenyls from Thelephora ganbajun Zang[J].Zeitschrift Fur Naturforschung Section C-a Journal of Biosciences,2001,56(1 1/12):983-987.DOI:10.15 15/znc-2001-11-1213.
[5]李丽娟,王涛,申元英,等.干巴菌对小鼠免疫功能影响的实验研究[J].食品研究与开发,2012,33(6):48-51.DOI:10.3969/j.issn.1005-6521.2012.06.011.
[6]徐菘阳,陆文娟,陶明煊,等.干巴菌多糖对急性酒精损伤小鼠的抗氧化作用[J].食品工业科技,2017,38(15):314-318.DOI:10.13386/j.issn 1002-0306.2017.15.059.
[7]HAO L,SHENG Z,LU J,et al.Characterization and antioxidant activities of extracellular and intracellular polysaccharides from Fomitopsis pinicola[J].Carbohydrate Polymers,2016,141(6):54-59.DOI:10.1016/j.carbpol.2015.1 1.048.
[8]WANG Y,TIAN Y,SHAO J,et al.Macrophage immunomodulatory activity of the polysaccharide isolated from Collybia radicata mushroom[J].International Journal of Biological Macromolecules,2018,108:300-306.DOI:10.1016/j.ijbiomac.2017.12.025.
[9]SINGDEVSACHAN S K,AUROSHREE P,MISHRA J,et al.Mushroom polysaccharides as potential prebiotics with their antitumor and immunomodulating properties:a review[J].Bioactive Carbohydrates&Dietary Fibre,2016,7(1):1-14.DOI:10.1016/j.bcdf.2015.1 1.001.
[10]MENG X,LIANG H,LUO L.Antitumor polysaccharides from mushrooms:a review on the structural characteristics,antitumor mechanisms and immunomodulating activities[J].Carbohydrate Research,2016,424:30-41.DOI:10.101 6/j.carres.2016.02.008.
[11]REN Z,LI J,XU N,et al.Anti-hyperlipidemic and antioxidant effects of alkali-extractable mycelia polysaccharides by Pleurotus eryngii var.tuolensis[J].Carbohydrate Polymers,2017,175:282-292.DOI:10.101 6/j.carbpol.2017.08.009.
[12]CHEN T Q,WU J G,KAN Y J,et al.Antioxidant and hepatoprotective activities of crude polysaccharide extracts from Lingzhi or Reishi medicinal mushroom,Ganoderma lucidum(Agaricomycetes),by ultrasonic-circulating extraction[J].International Journal of Medicinal Mushrooms,2018,20(6):58 1-593.DOI:10.1615/IntJMedMushrooms.201 8026536.
[13]KHAN I,HUANG G,LI X,et al.Mushroom polysaccharides from Ganoderma lucidum and Poria cocos reveal prebiotic functions[J].Journal of Functional Foods,2018,41:191-201.DOI:10.1016/j.jff.2017.12.046.
[14]LIU Y,ZHOU Y,LIU M,et al.Extraction optimization,characterization,antioxidant and immunomodulatory activities of a novel polysaccharide from the wild mushroom Paxillus involutus[J].International Journal of Biological Macromolecules,2018,112:326-332.DOI:10.1016/j.ij biomac.2018.01.132.
[15]MA G,YANG W,ZHAO L,et al.A critical review on the health promoting effects of mushrooms nutraceuticals[J].Food Science and Human Wellness,2018,7:125-133.DOI:10.1016/j.fshw.2018.05.002.
[16]KOTHARI D,PATEL S,KIM S K.Anticancer and other therapeutic relevance of mushroom polysaccharides:a holistic appraisal[J].Biomedicine&Pharmacotherapy,2018,105:377-394.DOI:10.1016/j.biopha.2018.05.138.
[17]PENG Z,XUE-YUE L,YANG W,et al.Optimization of submerged fermentation of Thelephora ganbajun Zang[J].Journal of Basic Microbiology,2014,54(8):866-872.DOI:10.1002/j obm.201200757.
[18]GAN D,MA L,JIANG C,et al.Production,preliminary characterization and antitumor activity in vitro of polysaccharides from the mycelium of Pholiota dinghuensis Bi[J].Carbohydrate Polymers,201 1,84(3):997-1003.DOI:10.1016/j.carbpol.2010.12.058.
[19]DUBOIS M,GILLES K A,HAMILTON J K,et al.Colorimetric method for determination of sugars and related substances[J].Analytical Chemistry,1956,28(3):350-356.
[20]STAUB A M.Removal of proteins-Sevag method[J].Methods in Carbohydrate Chemistry,1965,5:5-6.
[21]DIVYA K,VIJAYAN S,JISHA M S.Antifungal,antioxidant and cytotoxic activities of chitosan nanoparticles and its use as an edible coating on vegetables[J].International Journal of Biological Macromolecules,2018,114:572-577.DOI:10.1016/j.ijbiomac.2018.03.130.
[22]MENG Q,LI Y,XIAO T,et al.Antioxidant and antibacterial activities of polysaccharides isolated and purified from Diaphragma juglandis fructus[J].International Journal of Biological Macromolecules,2017,105:43 1-437.DOI:10.1016/j.ij biomac.2017.07.062.
[23]LIU Y,ZHANG B,IBRAHIM S A,et al.Purification,characterization and antioxidant activity of polysaccharides from Flammulina velutipes residue[J].Carbohydrate Polymers,2016,145:71-77.DOI:10.1016/j.carbpol.2016.03.020.
[24]WANG K,LI M,WEN X,et al.Optimization of ultrasound-assisted extraction of okra(Abelmoschus esculentus(L.)Moench)polysaccharides based on response surface methodology and antioxidant activity[J].International Journal of Biological Macromolecules,2018,114:1056-1063.DOI:10.101 6/j.ij biomac.201 8.03.145.
[25]MA Y L,ZHU D Y,THAKUR K,et al.Antioxidant and antibacterial evaluation of polysaccharides sequentially extracted from onion(Allium cepa L.)[J].International Journal of Biological Macromolecules,2018,111:92-101.DOI:10.1016/j.ijbiomac.2017.12.1 54.
[26]XU Z,WANG H,WANG B,et al.Characterization and antioxidant activities of polysaccharides from the leaves of Lilium lancifolium Thunb[J].International Journal of Biological Macro molecules,2016,92:148-155.DOI:10.1016/j.ijbiomac.2016.07.028.
[27]SUN J,ZHOU B,TANG C,et al.Characterization,antioxidant activity and hepatoprotective effect of purple sweetpotato polysaccharides[J].International Journal of Biological Macromolecules,2018,1 15:69-76.DOI:10.1016/j.ij biomac.2018.04.033.
[28]HAO L,SHENG Z,LU J,et al.Characterization and antioxidant activities of extracellular and intracellular polysaccharides from Fomitopsis pinicola[J].Carbohydrate Polymers,2016,141(6):54-59.DOI:10.101 6/j.carbpol.2015.1 1.048.
[29]XU Y,JIANG H Y,SUN C Y,et al.Antioxidant and hepatoprotective effects of purified Rhodiola rosea polysaccharides[J].International Journal of Biological Macromolecules,2018,117:167-178.DOI:10.1016/j.ijbiomac.2018.05.168.
[30]陈春娟,朱振元,陈璐.低分子量蛹虫草多糖降血糖活性的研究[J].现代食品科技,2017(4):25-30.DOI:10.13982/j.mfst.1673-9078.2017.4.005.
[31]韩莎莎,黄臻颖,沈照鹏,等.酶法降解坛紫菜多糖及其产物分析[J].食品科学,2015,36(21):145-149.DOI:10.7506/spkx1002-6630-201521028.