以中药为基质的黑木耳多糖发酵工艺条件的研究
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摘要
黑木耳是一种营养丰富的药、食两用真菌。黑木耳中含有的黑木耳多糖因具有降血糖、降血脂、抗肿瘤以及抗凝血等生理活性而成为研究热点。本论文通过添加适当的贵州特色中药成分,以促进深层发酵黑木耳多糖的产量。研究了黑木耳多糖的摇瓶发酵工艺和胞外多糖提取工艺,并在15L发酵罐上进行发酵工艺试验。
首先考察了六种贵州特色中药的水提取液和乙醇提取液对黑木耳深层发酵的影响。结果表明所有中药的水提取液均可以在不同程度上促进黑木耳胞外多糖的产量。但是乙醇提取液对黑木耳胞外多糖产量的促抑效果不一。最终综合生物量与胞外多糖产量两项指标,选择天麻乙醇提取液作为后续试验的添加成分。
单因素试验研究了各种营养因子对黑木耳深层发酵的影响,结果表明葡萄糖、酵母膏、天麻乙醇提取液和无机盐,在一定浓度下有利于黑木耳菌体生长和胞外多糖的形成。进一步用响应面法优化了培养基配方,确定了黑木耳深层发酵培养基为:葡萄糖37.81g/L、酵母膏10.23g/L、天麻乙醇提取液30.00g/L、KH_2PO_43.94g/L、MgSO_41.97g/L。采用此培养基后,黑木耳胞外多糖产量达到了2.65g/L。
通过试验确定了黑木耳深层发酵的适宜工艺条件为:初始pH5.66(自然)、接种量10%、种龄4-5d,培养温度28℃、250mL摇瓶装液量80 mL。
通过试验得出,黑木耳发酵液胞外多糖最佳提取工艺为:第一步沉淀时采用30%的乙醇浓度,第二步沉淀时采用75%的乙醇浓度;静置温度为4℃;静置时间为24h。此工艺条件提取黑木耳胞外多糖较为合适。
在15L发酵罐上进行发酵工艺条件小试研究。通气量试验结果表明较高通气量有利于黑木耳深层发酵的发酵结果,综合考虑生产实际选择6v/v/m为宜。两段温度控制试验结果表明28℃适合菌丝体生长,25℃适宜胞外多糖的生成。所以0-84h宜采用28℃培养,84-144h宜采用25℃培养。两段pH控制试验结果表明,pH5.1适合黑木耳菌丝体生长,pH5.7有利于胞外多糖的生成。所以,0-84h宜采用pH5.1培养,84-144h宜采用pH5.7培养。最后综合以上优化结果进行的验证实验表明,采用优化后的工艺条件,胞外多糖产量最高达到了3.42g/L。
Auricularia Auricula is a kind of fungi which is both edible and medicinal.The extro-polysaccharide of Auricularia Auricula has various biological activities,such as anticogulation,antilipidemic,antiplatelet,antitumor,etc.In this paper,the primary purpose of the research was to produce better and more extro-polysaccharides by submerged fermentation from Auricularia Auricula.After examined six kinds of Chinese traditional herb medicines from Guizhou province,shaking flask fermentation, extraction,and the techniques in 15L fermentor were well studied in this paper.
The effects of water and ethanol extracts of six kinds of Chinese traditional herb medicines,which were from Guizhou province,on the submerged culture of Auricularia Auricula were firstly studied.The results showed that the yields of extro-polysaccharide were increased in different levels when added any water extracts of the six medicines. But the results varied when added ethanol extracts.Take both biomass and extro-polysaccharide into consideration,we chose the ethanol extract of Gastrodia elata.
The effects of some nutritional factors on the submerged fermentation of Auricularia Auricula were studied.And the results showed that glucose,yeast extract, salt and ethanol extract of Gastrodia elata in suitable levels could promote the yield extro-polysaccharide.An optimum medium was obtained by responding surface method experiment.The medium composition was as follows:glucose37.81 g/L,yeast extract 10.23g/L,ethanol extract of Gastrodia elata 30.00g/L,KH_2PO_43.94g/L,MgSO_41.97g/L.The maximum extro-polysaccharide yield reached 2.65g/L when used the medium above.
The optimum fermentation conditions were as follows:initial pH5.66(natural), culture temperature28℃,inoculation age 4-5d,inoculation quantity 10%,80 mL medium in 250mL shaking flask.
The extraction techniques of Auricularia Auricula extro-polysaccharide had been studied as well.The results showed that after the concentration of ethanol reached 30%in the first step,the concentration of ethanol should reach 75%in the second step. And then,deposit at temperature 4℃for 24h.
The tests in 15L fermentor showed that higher ventilatory volume was good for the submerged fermentation of Auricularia Auricula.And considering the practice, 6v/v/m was better.The result of temperature controlled strategy study showed that form 0 to 84h,it should be cultured under 28℃,and form 84 to 144h it should be cultured under 25℃.The result of pH controlled strategy showed that form 0 to 84h, it should be cultured under pH5.1,and form 84 to 144h it should be cultured under pH 5.7.Under the optimum conditions,the yield of extro-polysaccharide reached 3.42g/L.
首先考察了六种贵州特色中药的水提取液和乙醇提取液对黑木耳深层发酵的影响。结果表明所有中药的水提取液均可以在不同程度上促进黑木耳胞外多糖的产量。但是乙醇提取液对黑木耳胞外多糖产量的促抑效果不一。最终综合生物量与胞外多糖产量两项指标,选择天麻乙醇提取液作为后续试验的添加成分。
单因素试验研究了各种营养因子对黑木耳深层发酵的影响,结果表明葡萄糖、酵母膏、天麻乙醇提取液和无机盐,在一定浓度下有利于黑木耳菌体生长和胞外多糖的形成。进一步用响应面法优化了培养基配方,确定了黑木耳深层发酵培养基为:葡萄糖37.81g/L、酵母膏10.23g/L、天麻乙醇提取液30.00g/L、KH_2PO_43.94g/L、MgSO_41.97g/L。采用此培养基后,黑木耳胞外多糖产量达到了2.65g/L。
通过试验确定了黑木耳深层发酵的适宜工艺条件为:初始pH5.66(自然)、接种量10%、种龄4-5d,培养温度28℃、250mL摇瓶装液量80 mL。
通过试验得出,黑木耳发酵液胞外多糖最佳提取工艺为:第一步沉淀时采用30%的乙醇浓度,第二步沉淀时采用75%的乙醇浓度;静置温度为4℃;静置时间为24h。此工艺条件提取黑木耳胞外多糖较为合适。
在15L发酵罐上进行发酵工艺条件小试研究。通气量试验结果表明较高通气量有利于黑木耳深层发酵的发酵结果,综合考虑生产实际选择6v/v/m为宜。两段温度控制试验结果表明28℃适合菌丝体生长,25℃适宜胞外多糖的生成。所以0-84h宜采用28℃培养,84-144h宜采用25℃培养。两段pH控制试验结果表明,pH5.1适合黑木耳菌丝体生长,pH5.7有利于胞外多糖的生成。所以,0-84h宜采用pH5.1培养,84-144h宜采用pH5.7培养。最后综合以上优化结果进行的验证实验表明,采用优化后的工艺条件,胞外多糖产量最高达到了3.42g/L。
Auricularia Auricula is a kind of fungi which is both edible and medicinal.The extro-polysaccharide of Auricularia Auricula has various biological activities,such as anticogulation,antilipidemic,antiplatelet,antitumor,etc.In this paper,the primary purpose of the research was to produce better and more extro-polysaccharides by submerged fermentation from Auricularia Auricula.After examined six kinds of Chinese traditional herb medicines from Guizhou province,shaking flask fermentation, extraction,and the techniques in 15L fermentor were well studied in this paper.
The effects of water and ethanol extracts of six kinds of Chinese traditional herb medicines,which were from Guizhou province,on the submerged culture of Auricularia Auricula were firstly studied.The results showed that the yields of extro-polysaccharide were increased in different levels when added any water extracts of the six medicines. But the results varied when added ethanol extracts.Take both biomass and extro-polysaccharide into consideration,we chose the ethanol extract of Gastrodia elata.
The effects of some nutritional factors on the submerged fermentation of Auricularia Auricula were studied.And the results showed that glucose,yeast extract, salt and ethanol extract of Gastrodia elata in suitable levels could promote the yield extro-polysaccharide.An optimum medium was obtained by responding surface method experiment.The medium composition was as follows:glucose37.81 g/L,yeast extract 10.23g/L,ethanol extract of Gastrodia elata 30.00g/L,KH_2PO_43.94g/L,MgSO_41.97g/L.The maximum extro-polysaccharide yield reached 2.65g/L when used the medium above.
The optimum fermentation conditions were as follows:initial pH5.66(natural), culture temperature28℃,inoculation age 4-5d,inoculation quantity 10%,80 mL medium in 250mL shaking flask.
The extraction techniques of Auricularia Auricula extro-polysaccharide had been studied as well.The results showed that after the concentration of ethanol reached 30%in the first step,the concentration of ethanol should reach 75%in the second step. And then,deposit at temperature 4℃for 24h.
The tests in 15L fermentor showed that higher ventilatory volume was good for the submerged fermentation of Auricularia Auricula.And considering the practice, 6v/v/m was better.The result of temperature controlled strategy study showed that form 0 to 84h,it should be cultured under 28℃,and form 84 to 144h it should be cultured under 25℃.The result of pH controlled strategy showed that form 0 to 84h, it should be cultured under pH5.1,and form 84 to 144h it should be cultured under pH 5.7.Under the optimum conditions,the yield of extro-polysaccharide reached 3.42g/L.
引文
[1] Akira M, Mariko K, Takuma S, et al. Studies on interrelation of structure and antitumor effects of polysaccharides: Antitumor action of periodate-modified, branched (1-3)-P-D-glucan of Auricularia Auricula-judae, and other polysaccharides containing (1-3)-glycosidic linkages[J]. Carbohydr Res, 1981,92(1):115-129.
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[5] Kim SW, Hwang HJ, Xu CP. Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738[J]. J appl Microbiol,2003,94(1): 120-126.
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[8] Noda N. 2,4-Bis(4-hydroxybenzyl) phenol from Gastrodiaelata[J]. Phytochemistry, 1995,39(5):1247.
[9] Seon JY, Myeong AY, Yu RP. The nontoxic mushroom Auricularia Auricula contains a polysaccharide with anticoagulant activity mediated by antithrombin[J]. Thrombosis Res,2003,112:151-158.
[10] Taguchi H. Studies on the constituents of Gastrodia elata Blume[J]. Chem Pharm Bull. 1981,29 (1): 55-62.
[11] Wang HB, Zhang JP, Wang H. The measuring of red cell membrance CR1 molecule and its clinic meaning [J]. Chin J Microbiol Immunol,2000,20:381.
[12] Wu J, Ding ZY, Zhang KC. Improvement of exopolysaccharide production by macro-fungus Auricularia Auricula in submerged culture[J]. Enzyme Microbial Technology,2006,39:743-749.
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[2] Dubois M, Gilles KA, Hamilton JK. Colorimetric method for determination of sugars and related substances[J]. Analy Chem, 1956,28: 350-356.
[3] Hammerschmidt DE. Szechwan prupura[M]. N Engl J Med, 1980.
[4] Hao XY. Anewdi-(p-hydroxybenzyl) hydroxylamine from Gastrodiaelata Bl[J]. Chinese Chemical Letters, 1999,10(6):467.
[5] Kim SW, Hwang HJ, Xu CP. Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738[J]. J appl Microbiol,2003,94(1): 120-126.
[6] Lin JH. Parishins B and C from rhizomes of Gastrodiaelat[J]. Phytochemistry, 1996,42(2):549.
[7] Li PZ, Zhang KC. Studies on pH controlled fermentation of bioactive exo-polysaccharides by Ganoderma lucidum[J]. Bull Microbiol, 2002, 27(1):5-8.
[8] Noda N. 2,4-Bis(4-hydroxybenzyl) phenol from Gastrodiaelata[J]. Phytochemistry, 1995,39(5):1247.
[9] Seon JY, Myeong AY, Yu RP. The nontoxic mushroom Auricularia Auricula contains a polysaccharide with anticoagulant activity mediated by antithrombin[J]. Thrombosis Res,2003,112:151-158.
[10] Taguchi H. Studies on the constituents of Gastrodia elata Blume[J]. Chem Pharm Bull. 1981,29 (1): 55-62.
[11] Wang HB, Zhang JP, Wang H. The measuring of red cell membrance CR1 molecule and its clinic meaning [J]. Chin J Microbiol Immunol,2000,20:381.
[12] Wu J, Ding ZY, Zhang KC. Improvement of exopolysaccharide production by macro-fungus Auricularia Auricula in submerged culture[J]. Enzyme Microbial Technology,2006,39:743-749.
[13]Yun-Choi HS.Cissiumal dehyde,a new fruan from the tubers of Gastrodiaelata[J].Natural Product Science(Korea),1997,3(2):104.
[14]Yu XB,Luo CC,Miu J.Effects of controlled pH on submerged fermentation of Cordycepssinensis[J].Food & Ferment Ind,2002,123(13):98-100.
[15]Zhang HB,Cheng LZ,Sha Tao.The effects of a controlled pH environment on polysaccharide synthesis by Aureobasidium pullulans[J].Bull Microbiol,2001,28(1):35-38.
[16]Zhang LN,Yang LQ,Ding Q.Studies on molecular weights of polysaccharides of Auricularia auricula-judae[J].Carbohydr Res,1995,270(1):1-10.
[17]暴增海,柳焕章,李月梅.食用菌栽培原理与技术[M].北京:中国标准出版社,2000.
[18]李时珍.本草纲目[M].北京:人民卫生出版社,1982.
[19]蔡小玲,章佩芬.黑木耳多糖、红菇多糖降的胆固醇作用研究[J].深圳中西医杂志,2002,12(3):137-139.
[20]陈依军,夏尔宁.黑木耳、银耳及银耳孢子多糖延缓衰老作用[J].现代应用医学,1989,6(2):9-10.
[21]丁黎.木耳多糖的分离及其组成单糖的分折[J].特产研究,1994,1:44-45.
[22]樊黎生,龚晨睿,张声华.黑木耳多糖抗辐射效应的动物实验[J].营养学报,2005,27(6):525-526.
[23]范亚明,王朋等.黑木耳抗脂质过氧化的初步探讨[J].心肺血管病杂志,1995,14(4)236-238.
[24]韩春然,马永强,唐娟.黑木耳多糖的提取及降血糖作用[J].食品与生物技术学报,2006,25(5):111-114.
[25]胡文铎,崔乃杰,高仲阳.国家基本药物及新药临床指南[M].天津技术翻译出版社,1997.
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