灵芝在牛蒡固体培养基中发酵工艺优化及菌丝多糖的体外抗氧化活性
|
摘要
为提升牛蒡废弃物的利用价值,以低品质牛蒡作为营养基质,精选优良灵芝菌种作为发酵菌株,研究牛蒡固体发酵灵芝产多糖工艺及其体外抗氧化活性。采用单因素试验和响应面优化法确定了装瓶量、粉碎程度、液固比等工艺条件;利用水提醇沉法提取牛蒡固体发酵灵芝菌丝中多糖,并用苯酚-硫酸法测定其总多糖含量;多糖经脱蛋白、透析和脱色后,经红外光谱分析多糖特征,高效液相色谱测定其单糖组成;体外实验检测多糖对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-trinitrophenylhydrazine,DPPH)自由基、超氧阴离子自由基和羟自由基的清除能力。结果表明:最佳发酵条件为液固比0.4 mL/g、装瓶量0.2 g/mL、粉碎程度过6目筛,在此条件下实际获得的多糖含量为24.85 mg/g;红外光谱分析表明所获得的菌丝多糖具有糖类物质的特征吸收峰;高效液相色谱检测其单糖组成为甘露糖、核糖、鼠李糖、葡萄糖、半乳糖等,其中甘露糖含量最高,是葡萄糖的7.6倍;抗氧化性检测表明随着牛蒡固体发酵灵芝菌丝多糖质量浓度的增加,其对DPPH自由基、超氧阴离子自由基和羟自由基清除能力逐渐增强,且清除率均大于70%,说明多糖具有较好的体外抗氧化活性。
In order to achieve value-added utilization of burdock root waste, the solid-state fermentation of low-quality burdock root by a high-yield polysaccharide-producing strain of Ganoderma lucidum(Leyss. ex. Fr.) Karst was optimized for improved production of mycelial polysaccharide and the in vitro antioxidant activity of the polysaccharide was assessed.The fermentation conditions including amount of medium contained in culture ?asks, degree of pulverization and liquid-tosolid ratio were optimized by combined use of one-factor-at-a-time method and response surface methodology. The mycelial polysaccharide from G. lucidum was extracted by water extraction and ethanol precipitation and was quantified by the phenol-sulfuric acid method. After being deproteinized and decolorized by dialysis, the polysaccharide was characterized by infrared spectroscopy and was determined for its monosaccharide composition by high performance liquid chromatography(HPLC). Besides, the in vitro antioxidant activity was evaluated by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH),superoxide anion and hydroxyl radical scavenging assays. The results showed that the optimal fermentation conditions were as follows: liquid-to-solid ratio 0.4 mL/g, medium-to-culture ?ask ratio 0.2 g/mL, and use of particles passing through a 6-mesh sieve. The polysaccharide content of the mycelia harvested under the optimized conditions was 24.85 mg/g.Fourier-transform infrared spectroscopy(FTIR) analysis showed that the product had the characteristic absorption peaks of polysaccharide. The monosaccharide composition analysis showed that the polysaccharide was composed of mannose,ribose, rhamnose, glucose and galactose, with mannose being the most abundant monosaccharide, which was 7.6 times as high as glucose. Each of the free radical was over 70% scavenged by the polysaccharide, and the effect increased with increasing its concentration. Hence, the polysaccharide had good antioxidant activity in vitro.
In order to achieve value-added utilization of burdock root waste, the solid-state fermentation of low-quality burdock root by a high-yield polysaccharide-producing strain of Ganoderma lucidum(Leyss. ex. Fr.) Karst was optimized for improved production of mycelial polysaccharide and the in vitro antioxidant activity of the polysaccharide was assessed.The fermentation conditions including amount of medium contained in culture ?asks, degree of pulverization and liquid-tosolid ratio were optimized by combined use of one-factor-at-a-time method and response surface methodology. The mycelial polysaccharide from G. lucidum was extracted by water extraction and ethanol precipitation and was quantified by the phenol-sulfuric acid method. After being deproteinized and decolorized by dialysis, the polysaccharide was characterized by infrared spectroscopy and was determined for its monosaccharide composition by high performance liquid chromatography(HPLC). Besides, the in vitro antioxidant activity was evaluated by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH),superoxide anion and hydroxyl radical scavenging assays. The results showed that the optimal fermentation conditions were as follows: liquid-to-solid ratio 0.4 mL/g, medium-to-culture ?ask ratio 0.2 g/mL, and use of particles passing through a 6-mesh sieve. The polysaccharide content of the mycelia harvested under the optimized conditions was 24.85 mg/g.Fourier-transform infrared spectroscopy(FTIR) analysis showed that the product had the characteristic absorption peaks of polysaccharide. The monosaccharide composition analysis showed that the polysaccharide was composed of mannose,ribose, rhamnose, glucose and galactose, with mannose being the most abundant monosaccharide, which was 7.6 times as high as glucose. Each of the free radical was over 70% scavenged by the polysaccharide, and the effect increased with increasing its concentration. Hence, the polysaccharide had good antioxidant activity in vitro.
引文
[1]何虎翼,甘秀芹,陆柳英,等.不同牛蒡品种根的营养品质分析[J].中国蔬菜,2011,12(6):73-75.
[2]蒋淑敏.牛蒡化学成分和药理作用的研究现状[J].时珍国医国药,2001,12(10):941-941.DOI:10.3969/j.issn.1008-0805.2001.10.082.
[3]郝林华,陈靠山,李光友,等.利用牛蒡渣提取高活性膳食纤维的工艺[J].食品与发酵工业,2003,29(4):41-44.DOI:10.3321/j.issn:0253-990X.2003.04.010.
[4]蒋晓文,白俊鹏,田星,等.牛蒡根中黄酮苷类化学成分及其抗氧化活性构效关系的研究[J].中草药,2016,47(5):726-731.
[5]谷绒,万国福,张嫚,等.牛蒡红枣复合养生饮料的研制[J].食品研究与开发,2017,38(1):67-72.DOI:10.3969/j.issn.1005-6521.2017.01.016.
[6]SIMONA C,CRISTINA M,OLIVIU V,et al.polyphenolic profile,anti-inflammatory and antinociceptive activity of an extract from Arctium lappa L.roots[J].Notulae Botanicae Horti Agrobotanici ClujNapoca,2017,45(1):59-64.DOI:10.15835/nbha45110549.
[7]李超.牛蒡根活性成分的高速剪切辅助提取及其对牛肉丸储藏稳定性的影响[J].中国食品添加剂,2017(9):151-159.DOI:10.3969/j.issn.1006-2513.2017.09.017.
[8]?ILERD?I?J,GALI? M,VUKOJEVI?J,et al.Potential of selected fungal species to degrade wheat straw,the most abundant plant raw material in Europe[J].BMC Plant Biology,2017,17(2):76-81.DOI:10.1186/s12870-017-1196-y.
[9]刘高强,孙虹,章春莲.赤芝胞外三萜体外抑制几种细菌生长的研究[J].食品科技,2010,35(2):156-159.DOI:10.13684/j.cnki.spkj.2010.02.066.
[10]FIERASCU R C,GE ORGIEV M I,FIERASCU I,et al.Mitodepressive,antioxidant,antifungal and anti-inflammatory effects of wild-growing Romanian native Arctium lappa L.(Asteraceae)and Veronica persica Poiret(Plantaginaceae)[J].Food and Chemical Toxicology,2018,111:44-52.DOI:10.1016/j.fct.2017.11.008.
[11]CAI Q,LI Y,PEI G.Polysaccharides from Ganoderma lucidum attenuate microglia-mediated neuroinflammation and modulate microglial phagocytosis and behavioural response[J].Journal of Neuroinflammation,2017,14:63-76.DOI:10.1186/s12974-017-0839-0.
[12]OUYANG M Z,LIM L Z,LV W J,et al.Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapy-related fatigue in mice[J].International Journal of Biological Macromolecules,2016,91:905-910.DOI:10.1016/j.ijbiomac.2016.04.084.
[13]GAO Y,GU C,WANG K,et al.The effects of hypoglycemia and weight loss of total lignans from Fructus Arctii in KKAy mice and its mechanisms of the activity[J].Phytotherapy Research,2017,1:127-139.DOI:10.1002/ptr.6003.
[14]WU H C,CHEN H M,SHIAU C Y.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Journal of Food and Nutrition Research,2003,36:949-957.DOI:10.1016/S0963-9969(03)00104-2.
[15]王雅,苗敬芝,董玉玮.超声波协同复合酶法提取杏鲍菇多糖及抗氧化活性的研究[J].食品工业,2014,35(3):134-137.
[16]YEN G C,HSIEH P P.Antioxidative activity and scavenging effects on active oxygen of xylose-lysine maillard reaction products[J].Journal of the Science of Food and Agriculture,1995,67(3):415-420.DOI:10.1002/jsfa.2740670320.
[17]盛悦,何音华,沈月,等.猴头菌液体固体发酵产物多糖抗氧化活性研究[J].农产品加工,2017,430(4):9-12.DOI:10.16693/j.cnki.1671-9646(X).2017.04.031.
[18]段金柱,曹淡君.固体发酵与液体发酵生产纤维素酶产率与催化性能比较[J].粮食与饲料工业,2000(3):24-26.DOI:10.3969/j.issn.1003-6202.2000.03.011.
[19]孙丽萍,金卫根.大型野生真菌选育种培养基研究[J].江西农业学报,2007,19(4):71-72.DOI:10.3969/j.issn.1001-8581.2007.04.026.
[20]付铭,冮洁,狄文婷,等.大米为基质灵芝菌丝体固态发酵条件的优化[J].食品工业科技,2014,35(2):186-191.DOI:10.13386/j.issn1002-0306.2014.02.001.
[21]生东明,马莺.固态发酵生产灵芝多糖培养基的优化[J].现代食品科技,2004,20(2):18-20.DOI:10.3969/j.issn.1673-9078.2004.03.006.
[22]周小苹,杨海军.灵芝菌丝体固态发酵黑茶的初步研究[J].食品工业,2018,39(8):187-190.DOI:10.13386/j.issn1002-0306.2014.02.001.
[23]于华峥,刘艳芳,周帅,等.灵芝子实体、菌丝体及孢子粉中多糖成分差异比较研究[J].菌物学报,2016,35(2):170-177.DOI:10.13346/j.mycosystema.140242.
[24]黄静涵,艾斯卡尔·艾拉提,毛健.灵芝多糖的分离纯化及结构鉴定[J].食品科学,2011,32(12):301-304.
[25]冯胜平.不同栽培品种灵芝抗氧化作用及灵芝多糖分离纯化、免疫活性研究[D].成都:成都中医药大学,2015.
[26]CARLOTTO J,DE SOUZA L M,BAGGIO C H,et al.Polysaccharides from Arctium lappa L.:chemical structure and biological activity[J].International Journal of Biological Macromolecules,2016,91:954-960.DOI:10.1016/j.ijbiomac.2016.06.033.
[27]AI-LATI A,LIU S,JI Z,et al.Structure and bioactivities of a polysaccharide isolated from Ganoderma lucidum in submerged fermentation[J].Bioengineered,2017,8(5):565-571.DOI:10.1080/21655979.2017.1283459.
[28]郑一美,温正如,宋宇鹏,等.牛蒡多糖组分的高效阴离子交换-脉冲安培分析[J].浙江大学学报(理学版),2014,41(5):537-541.DOI:10.3785/j.issn.1008-9497.2014.05.011.
[29]TIAN X,GUO L P,HU X L,et al.Protective effects of Arctium lappa L.roots against hydrogen peroxide-induced cell injury and potential mechanisms in SH-SY5Y cells[J].Cellular and Molecular Neurobiology,2015,35(3):335-344.DOI:10.1007/s10571-014-0129-7.
[30]NIE J,YANG S L,MO M H,et al.In vitro antioxidant activity of hot water extracts from 7 different sources of Ganoderma Lucidum[J].Medicinal Plant,2017,8(4):62-71.DOI:10.19600/j.cnki.issn2152-3924.2017.04.014.
[31]ZHANG T X,LIU H B,ZHANG W Z,et al.Enzymatic extraction and DPPH free radical scavenging properties of polysaccharides from Ganoderma lucidum[J].Medicinal Plant,2010,1(12):76-79.
[32]张天笑,刘红兵,张文竹.复合酶提取灵芝多糖工艺及其抗氧化能力研究[J].安徽农业科学,2011,39(8):4496-4498.DOI:10.3969/j.issn.0517-6611.2011.08.034.
[2]蒋淑敏.牛蒡化学成分和药理作用的研究现状[J].时珍国医国药,2001,12(10):941-941.DOI:10.3969/j.issn.1008-0805.2001.10.082.
[3]郝林华,陈靠山,李光友,等.利用牛蒡渣提取高活性膳食纤维的工艺[J].食品与发酵工业,2003,29(4):41-44.DOI:10.3321/j.issn:0253-990X.2003.04.010.
[4]蒋晓文,白俊鹏,田星,等.牛蒡根中黄酮苷类化学成分及其抗氧化活性构效关系的研究[J].中草药,2016,47(5):726-731.
[5]谷绒,万国福,张嫚,等.牛蒡红枣复合养生饮料的研制[J].食品研究与开发,2017,38(1):67-72.DOI:10.3969/j.issn.1005-6521.2017.01.016.
[6]SIMONA C,CRISTINA M,OLIVIU V,et al.polyphenolic profile,anti-inflammatory and antinociceptive activity of an extract from Arctium lappa L.roots[J].Notulae Botanicae Horti Agrobotanici ClujNapoca,2017,45(1):59-64.DOI:10.15835/nbha45110549.
[7]李超.牛蒡根活性成分的高速剪切辅助提取及其对牛肉丸储藏稳定性的影响[J].中国食品添加剂,2017(9):151-159.DOI:10.3969/j.issn.1006-2513.2017.09.017.
[8]?ILERD?I?J,GALI? M,VUKOJEVI?J,et al.Potential of selected fungal species to degrade wheat straw,the most abundant plant raw material in Europe[J].BMC Plant Biology,2017,17(2):76-81.DOI:10.1186/s12870-017-1196-y.
[9]刘高强,孙虹,章春莲.赤芝胞外三萜体外抑制几种细菌生长的研究[J].食品科技,2010,35(2):156-159.DOI:10.13684/j.cnki.spkj.2010.02.066.
[10]FIERASCU R C,GE ORGIEV M I,FIERASCU I,et al.Mitodepressive,antioxidant,antifungal and anti-inflammatory effects of wild-growing Romanian native Arctium lappa L.(Asteraceae)and Veronica persica Poiret(Plantaginaceae)[J].Food and Chemical Toxicology,2018,111:44-52.DOI:10.1016/j.fct.2017.11.008.
[11]CAI Q,LI Y,PEI G.Polysaccharides from Ganoderma lucidum attenuate microglia-mediated neuroinflammation and modulate microglial phagocytosis and behavioural response[J].Journal of Neuroinflammation,2017,14:63-76.DOI:10.1186/s12974-017-0839-0.
[12]OUYANG M Z,LIM L Z,LV W J,et al.Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapy-related fatigue in mice[J].International Journal of Biological Macromolecules,2016,91:905-910.DOI:10.1016/j.ijbiomac.2016.04.084.
[13]GAO Y,GU C,WANG K,et al.The effects of hypoglycemia and weight loss of total lignans from Fructus Arctii in KKAy mice and its mechanisms of the activity[J].Phytotherapy Research,2017,1:127-139.DOI:10.1002/ptr.6003.
[14]WU H C,CHEN H M,SHIAU C Y.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Journal of Food and Nutrition Research,2003,36:949-957.DOI:10.1016/S0963-9969(03)00104-2.
[15]王雅,苗敬芝,董玉玮.超声波协同复合酶法提取杏鲍菇多糖及抗氧化活性的研究[J].食品工业,2014,35(3):134-137.
[16]YEN G C,HSIEH P P.Antioxidative activity and scavenging effects on active oxygen of xylose-lysine maillard reaction products[J].Journal of the Science of Food and Agriculture,1995,67(3):415-420.DOI:10.1002/jsfa.2740670320.
[17]盛悦,何音华,沈月,等.猴头菌液体固体发酵产物多糖抗氧化活性研究[J].农产品加工,2017,430(4):9-12.DOI:10.16693/j.cnki.1671-9646(X).2017.04.031.
[18]段金柱,曹淡君.固体发酵与液体发酵生产纤维素酶产率与催化性能比较[J].粮食与饲料工业,2000(3):24-26.DOI:10.3969/j.issn.1003-6202.2000.03.011.
[19]孙丽萍,金卫根.大型野生真菌选育种培养基研究[J].江西农业学报,2007,19(4):71-72.DOI:10.3969/j.issn.1001-8581.2007.04.026.
[20]付铭,冮洁,狄文婷,等.大米为基质灵芝菌丝体固态发酵条件的优化[J].食品工业科技,2014,35(2):186-191.DOI:10.13386/j.issn1002-0306.2014.02.001.
[21]生东明,马莺.固态发酵生产灵芝多糖培养基的优化[J].现代食品科技,2004,20(2):18-20.DOI:10.3969/j.issn.1673-9078.2004.03.006.
[22]周小苹,杨海军.灵芝菌丝体固态发酵黑茶的初步研究[J].食品工业,2018,39(8):187-190.DOI:10.13386/j.issn1002-0306.2014.02.001.
[23]于华峥,刘艳芳,周帅,等.灵芝子实体、菌丝体及孢子粉中多糖成分差异比较研究[J].菌物学报,2016,35(2):170-177.DOI:10.13346/j.mycosystema.140242.
[24]黄静涵,艾斯卡尔·艾拉提,毛健.灵芝多糖的分离纯化及结构鉴定[J].食品科学,2011,32(12):301-304.
[25]冯胜平.不同栽培品种灵芝抗氧化作用及灵芝多糖分离纯化、免疫活性研究[D].成都:成都中医药大学,2015.
[26]CARLOTTO J,DE SOUZA L M,BAGGIO C H,et al.Polysaccharides from Arctium lappa L.:chemical structure and biological activity[J].International Journal of Biological Macromolecules,2016,91:954-960.DOI:10.1016/j.ijbiomac.2016.06.033.
[27]AI-LATI A,LIU S,JI Z,et al.Structure and bioactivities of a polysaccharide isolated from Ganoderma lucidum in submerged fermentation[J].Bioengineered,2017,8(5):565-571.DOI:10.1080/21655979.2017.1283459.
[28]郑一美,温正如,宋宇鹏,等.牛蒡多糖组分的高效阴离子交换-脉冲安培分析[J].浙江大学学报(理学版),2014,41(5):537-541.DOI:10.3785/j.issn.1008-9497.2014.05.011.
[29]TIAN X,GUO L P,HU X L,et al.Protective effects of Arctium lappa L.roots against hydrogen peroxide-induced cell injury and potential mechanisms in SH-SY5Y cells[J].Cellular and Molecular Neurobiology,2015,35(3):335-344.DOI:10.1007/s10571-014-0129-7.
[30]NIE J,YANG S L,MO M H,et al.In vitro antioxidant activity of hot water extracts from 7 different sources of Ganoderma Lucidum[J].Medicinal Plant,2017,8(4):62-71.DOI:10.19600/j.cnki.issn2152-3924.2017.04.014.
[31]ZHANG T X,LIU H B,ZHANG W Z,et al.Enzymatic extraction and DPPH free radical scavenging properties of polysaccharides from Ganoderma lucidum[J].Medicinal Plant,2010,1(12):76-79.
[32]张天笑,刘红兵,张文竹.复合酶提取灵芝多糖工艺及其抗氧化能力研究[J].安徽农业科学,2011,39(8):4496-4498.DOI:10.3969/j.issn.0517-6611.2011.08.034.