南方亚麻微生物脱胶技术及其机理研究
摘要
亚麻是我国重要的纺织工业原料。亚麻原茎脱胶是亚麻初加工的第一道工序，对亚麻的纤维产量、长麻率及经济效益产生重大影响。为了建立南方亚麻新型高效脱胶技术体系，进一步推动南方亚麻产业的稳步发展，研究了南方亚麻天然水沤法脱胶过程微生物种类与数量、脱胶酶等各参数的动态变化规律，外界因子、麻茎特性等影响因子与亚麻脱胶的相互关系，并从麻茎上筛选高效脱胶菌种进行了亚麻酶法脱胶的初步研究，试验结果如下：
(1) 果胶分解菌是亚麻天然水沤麻中的重要脱胶菌群，果胶酶与亚麻脱胶效果密切相关。水源水质、浴比、起始pH、温度、添加剂等对脱胶液中的果胶分解菌数量、果胶酶活性与脱胶速度产生重要影响。果胶分解菌含量丰富的塘水有利于亚麻脱胶，浴比1：20～25、pH 7.0～7.5、温度30～35℃有利于果胶分解菌的生长繁殖；加入适量(1.5～2.0％)的含氮物质(如尿素)作为脱胶助剂，能够改善亚麻的沤麻环境，提高果胶酶的活性，加快亚麻的沤麻速度。麻茎的部位、粗细、麻龄不同，其纤维及胶质的组成和分布亦不同，对亚麻的脱胶速度均产生不同程度的影响；选茎关系到脱胶速度的快慢与脱胶质量的优劣，是亚麻脱胶工艺中必不可少的重要环节。
(2) 从亚麻茎上筛选得到了一株产果胶酶和木聚糖酶活性较高的脱胶菌株A_6，初步鉴定为环状芽孢杆菌。在摇瓶发酵条件下，A_6菌的最佳产酶条件为：亚麻茎粉7％，硫酸铵1％，磷酸氢二钾0.15％，pH 7.5，温度35℃，菌龄24h，接种量5％，250 mL三角瓶装液量70 mL，摇瓶转速200r／min，产酶时间为20-24h，果胶酶酶活高达7800 U／mL。
(3) 在摇瓶发酵条件下，A_6菌12h即能完成亚麻脱胶，比天然水沤法缩短脱胶时间84h；A_6菌以亚麻茎粉为碳源产生的酶制剂主要为胞外酶，包含果胶酶、木聚糖酶、CMC酶、淀粉酶和蛋白酶等组分。浴比1：15～20，1g麻茎添加5mL酶液，脱胶24h，纤维分散良好，脱胶后的纤维色泽、亮度和强力均优于水沤麻。
Flax (Linum usitatissimum L.) is the important raw material for textile fiber in China. The initial step for converting flax to linen is called retting, which is the major problem in processing flax. After introduction of flax to southern China, in order to establish the technical system of new pattern retting of flax in south China, major factors of natural water retting of flax, such as the type and number of retting bacteria, retting-enzymes, external retting conditions and stem characteristics were all studied deeply in the paper. And throuth screening from flax, a bacteria strain with high retting ability was obtained.
The main results are as follows:
(l)The pectinolytic bacteria played an important role in the retting. The activity of pectinase in retting water had an osculatory correlation with the retting. Water quality, ratio of flax stem to water, pH, water temperature and so on had an important effect on the growth of pectinolytic bacteria and the activity of pectinase in retting water. The optimum external retting conditions were as follows : pond water abundant in pectinolytic bacteria, water temperature 30℃-35℃, ratio of flax stem to water 1:20-25 and pH 7.0-7.5, Urea added into retting water by 2% of flax stem'mass. The different part, diameter and ripeness of flax had a different effect on flax retting. Therefore, it is necessary that the flax stems in same characeristic are selected together to ret in processing flax.
(2)A bacteria strain with high activity of pectinase and xylanase was screened from flax stem. It was preliminarily identified and named Bacillus circulans Ae. Pectinase producing conditions by Bacillus circulans A& were studied in shake flask culture. The result showed that the optimum pectinase producing conditions were as follows: flax stem power 7 %, (NH4)2SO4 1 %, K2HPO4 0.15 %, pH 7.5, inoculum age 24 hours, inoculum amount 5 %, aeration 70 mL medium / 250 mL flask, agitation speed 200r/min. After cultivating for 20-24 hours, the highest pectinase activity was 7 800 U / mL.
(3) Under the optimum growth condition, retting time of flax by Bacillus circulans A6 was 12 hours, which was shorter 84 hours than that of nature
water retting. Pectinase, xylanase, CMCase, proteinase and amylase were produced by Bacillus circulans Ae cultured with medium containing flax stem power as the sole source of Carbon. The culture filtrate of Bacillus circulans A6 at 25% (v/v) and ratios 1:15~20, were able to produce the flax fiber in 24 hours. The color, gloss and strength of fiber were better than that of nature water retting.
(1) 果胶分解菌是亚麻天然水沤麻中的重要脱胶菌群，果胶酶与亚麻脱胶效果密切相关。水源水质、浴比、起始pH、温度、添加剂等对脱胶液中的果胶分解菌数量、果胶酶活性与脱胶速度产生重要影响。果胶分解菌含量丰富的塘水有利于亚麻脱胶，浴比1：20～25、pH 7.0～7.5、温度30～35℃有利于果胶分解菌的生长繁殖；加入适量(1.5～2.0％)的含氮物质(如尿素)作为脱胶助剂，能够改善亚麻的沤麻环境，提高果胶酶的活性，加快亚麻的沤麻速度。麻茎的部位、粗细、麻龄不同，其纤维及胶质的组成和分布亦不同，对亚麻的脱胶速度均产生不同程度的影响；选茎关系到脱胶速度的快慢与脱胶质量的优劣，是亚麻脱胶工艺中必不可少的重要环节。
(2) 从亚麻茎上筛选得到了一株产果胶酶和木聚糖酶活性较高的脱胶菌株A_6，初步鉴定为环状芽孢杆菌。在摇瓶发酵条件下，A_6菌的最佳产酶条件为：亚麻茎粉7％，硫酸铵1％，磷酸氢二钾0.15％，pH 7.5，温度35℃，菌龄24h，接种量5％，250 mL三角瓶装液量70 mL，摇瓶转速200r／min，产酶时间为20-24h，果胶酶酶活高达7800 U／mL。
(3) 在摇瓶发酵条件下，A_6菌12h即能完成亚麻脱胶，比天然水沤法缩短脱胶时间84h；A_6菌以亚麻茎粉为碳源产生的酶制剂主要为胞外酶，包含果胶酶、木聚糖酶、CMC酶、淀粉酶和蛋白酶等组分。浴比1：15～20，1g麻茎添加5mL酶液，脱胶24h，纤维分散良好，脱胶后的纤维色泽、亮度和强力均优于水沤麻。
Flax (Linum usitatissimum L.) is the important raw material for textile fiber in China. The initial step for converting flax to linen is called retting, which is the major problem in processing flax. After introduction of flax to southern China, in order to establish the technical system of new pattern retting of flax in south China, major factors of natural water retting of flax, such as the type and number of retting bacteria, retting-enzymes, external retting conditions and stem characteristics were all studied deeply in the paper. And throuth screening from flax, a bacteria strain with high retting ability was obtained.
The main results are as follows:
(l)The pectinolytic bacteria played an important role in the retting. The activity of pectinase in retting water had an osculatory correlation with the retting. Water quality, ratio of flax stem to water, pH, water temperature and so on had an important effect on the growth of pectinolytic bacteria and the activity of pectinase in retting water. The optimum external retting conditions were as follows : pond water abundant in pectinolytic bacteria, water temperature 30℃-35℃, ratio of flax stem to water 1:20-25 and pH 7.0-7.5, Urea added into retting water by 2% of flax stem'mass. The different part, diameter and ripeness of flax had a different effect on flax retting. Therefore, it is necessary that the flax stems in same characeristic are selected together to ret in processing flax.
(2)A bacteria strain with high activity of pectinase and xylanase was screened from flax stem. It was preliminarily identified and named Bacillus circulans Ae. Pectinase producing conditions by Bacillus circulans A& were studied in shake flask culture. The result showed that the optimum pectinase producing conditions were as follows: flax stem power 7 %, (NH4)2SO4 1 %, K2HPO4 0.15 %, pH 7.5, inoculum age 24 hours, inoculum amount 5 %, aeration 70 mL medium / 250 mL flask, agitation speed 200r/min. After cultivating for 20-24 hours, the highest pectinase activity was 7 800 U / mL.
(3) Under the optimum growth condition, retting time of flax by Bacillus circulans A6 was 12 hours, which was shorter 84 hours than that of nature
water retting. Pectinase, xylanase, CMCase, proteinase and amylase were produced by Bacillus circulans Ae cultured with medium containing flax stem power as the sole source of Carbon. The culture filtrate of Bacillus circulans A6 at 25% (v/v) and ratios 1:15~20, were able to produce the flax fiber in 24 hours. The color, gloss and strength of fiber were better than that of nature water retting.
引文
[1] 任鸿桥，关宏梅，徐蓬，等．浅谈亚麻纺织产品的开发与应用[J]．黑龙江纺织，2003，(3)：10-11．
[2] 许益存，韩树明，张立明，等．实施亚麻战略加快黑龙江省种植业结构调整[J]．中国农机化，2003，(4)：9-11．
[3] 孙焕良，王冬毛．湖南春季亚麻引种栽培的初步研究[J]．中国麻作，1995，17(3)：12-15．
[4] 刘东鑫，赵介仁、孙焕良等．关于发展我国南方亚麻产业化问题的初步探讨[J]．中国麻业，1998，20(1)：42-45．
[5] 吕江南，胡镇修．我国南方亚麻开发的几点思考[J]．农牧产品开发，1999，(7)：34-35．
[6] 周文新，孙焕良，郭清泉，等．我国南方亚麻产业化发展刍论[J]．农业现代研究，2001，22(1)：7-10．
[7] 张宝胜，鲁文良．浅析亚麻纤维脱胶与亚麻粗纱煮练漂白工艺[J]．黑龙江纺织，1999，(4)：1-2．
[8] 孙庆祥．麻类作物微生物脱胶综述[J]．中国麻作，1981，(1)：38-41．
[9] 彭源德，刘正初，孙焕良等．南方亚麻微生物脱胶技术研究，Ⅰ外界因子对亚麻天然水浸沤麻的影响[J]．中国麻作，1997，19(2)：37-40．
[10] 陈灿，孙焕良，彭源德等．南方亚麻微生物脱胶技术研究Ⅱ．麻茎特性对亚麻脱胶的影响[J]．中国麻作，2000，22(2)：34-37．
[11] 江洁，刘晓兰，等．化学助剂法亚麻微生物脱胶新技术研究的[J]．纺织学报，2003，24(1)：46-48．
[12] 李宗道，等著．麻类生物工程进展(M)．中国农业出版社，1999，267-268．
[13] 王立群，关凤芝，张福修等．亚麻微生物脱胶技术的研究Ⅰ．脱胶菌种的筛选[J]．东北农业大学学报，1994，25(2)：182-185．
[14] 江洁，刘晓兰，等．亚麻脱胶菌种的选育及脱胶过程的初步研究[J]．微生物学报，1998，25(3)：150-153．
[15] G FILA ,L M MANICI and F Caputo. In vitro evaluation of dew-retting of flax by fungi from southern Europe[J].Ann.appl.Biol, 2001, 138: 343-351.
[16] 彭源德，刘正初，冯湘沅等．亚麻快速生物技术研究Ⅰ亚麻快速脱胶菌株的选育[J]，中国麻业，2003，25(3)：135-138．
[17] Akin, D.E., Morrison, W.H. III, Gamble, G.R., etal., Effect of retting enzymes on the structure and composition of flax cell wall[J]. Textile Res.J. 1997, 67: 279-287.
[18] Henriksson, G.,Akin , D.E., Rigsby, L.L., etal. Influence of chelating agents and mechanical pretreatment on enzymatic retting of flax[J]. Textile Res.J,1997, 67: 829-836.
[19] Danny E. Akin, Jonn A. Foulk, Roy B. Dodd, David D. McAlister Ⅲ, Enzyme-retting of flax and characterization of processed fibers[J], Journal of Biotechnology, 2001, 89:193-203.
[20] Van Sumere,C.E, Retting of Flax with special reference to enzyme retting .In:Sharma, H.S.S.,Van Sumere ,C.F.(Eds.),The Biology and Processing of Flax. M Publications, Belfast ,Northem Ireland ,1992, pp.157-198.
[21] Henriksson, G.,Akin, D.E., Slomczynski, D., etal. Poduction of highly efficient enzymes for flax retting by Rhizomucar pusillus[J].J.Biotechnol. 1999, 68: 115-123.
[22] J.D.Evans, D.E.Akin ,etal Flax-retting by polygalacuronase-containing enzyme mixtures and effects on fiber properties[J]. Journal of Biotechnology, 2002.
[23] Seaby D A, Mercer P c. Development of a hand tool to test the degree of retting of flax-straw[J]. Annals of Applied Biology,1984,104: 567-573.
[24] Fischer K, Topf W. Entwicklung objektiver Qualitatskriterien fur Flachs. Teil2:TextiltechnologischUntersuchungen[J].MellianTextilberichte,1988, 12: 858-861.
[25] Brown A E ,Sharma H S S , Black D L R. Relationship between pectin content of stems of flax cultivars, fungal cell wall-degrading enzymes and pre-harvest retting[J]. Annals of Applied Biology,1986, 109: 345-351.
[26] Morvan C, Morvan O, Januneau A, Demarty M. Capacite d'echange cationique des fibres de lin[J].Comptes Rendus de l'Academie d'Agriculture de France,1985, 71:837-841
[27] Meijer W J M, Vertregt N, Rutgers B, van de Waart M. The pectin content as a measure of the retting and tettability of flax[J]. Industrial Crops and Products,1995, 4: 273-284.
[28] Henriksson G,Akin D E, Hanlin R T, Rodriguez C, Archibald D D ,Rigsby L L,
Eriksson K-E L. Identification and retting efficiencies of fungi isolated from dew-retted flax in the United States and Europe[J]. Applied and Environmental Microbiology, 1997, 63: 3950-3956.
[29] Dujardin, A.Vias-roten .De Westvlaamsche bokhandel s.v. Groeninghe drukkeij, Kortrijk, Belgium,1942.
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[2] 许益存，韩树明，张立明，等．实施亚麻战略加快黑龙江省种植业结构调整[J]．中国农机化，2003，(4)：9-11．
[3] 孙焕良，王冬毛．湖南春季亚麻引种栽培的初步研究[J]．中国麻作，1995，17(3)：12-15．
[4] 刘东鑫，赵介仁、孙焕良等．关于发展我国南方亚麻产业化问题的初步探讨[J]．中国麻业，1998，20(1)：42-45．
[5] 吕江南，胡镇修．我国南方亚麻开发的几点思考[J]．农牧产品开发，1999，(7)：34-35．
[6] 周文新，孙焕良，郭清泉，等．我国南方亚麻产业化发展刍论[J]．农业现代研究，2001，22(1)：7-10．
[7] 张宝胜，鲁文良．浅析亚麻纤维脱胶与亚麻粗纱煮练漂白工艺[J]．黑龙江纺织，1999，(4)：1-2．
[8] 孙庆祥．麻类作物微生物脱胶综述[J]．中国麻作，1981，(1)：38-41．
[9] 彭源德，刘正初，孙焕良等．南方亚麻微生物脱胶技术研究，Ⅰ外界因子对亚麻天然水浸沤麻的影响[J]．中国麻作，1997，19(2)：37-40．
[10] 陈灿，孙焕良，彭源德等．南方亚麻微生物脱胶技术研究Ⅱ．麻茎特性对亚麻脱胶的影响[J]．中国麻作，2000，22(2)：34-37．
[11] 江洁，刘晓兰，等．化学助剂法亚麻微生物脱胶新技术研究的[J]．纺织学报，2003，24(1)：46-48．
[12] 李宗道，等著．麻类生物工程进展(M)．中国农业出版社，1999，267-268．
[13] 王立群，关凤芝，张福修等．亚麻微生物脱胶技术的研究Ⅰ．脱胶菌种的筛选[J]．东北农业大学学报，1994，25(2)：182-185．
[14] 江洁，刘晓兰，等．亚麻脱胶菌种的选育及脱胶过程的初步研究[J]．微生物学报，1998，25(3)：150-153．
[15] G FILA ,L M MANICI and F Caputo. In vitro evaluation of dew-retting of flax by fungi from southern Europe[J].Ann.appl.Biol, 2001, 138: 343-351.
[16] 彭源德，刘正初，冯湘沅等．亚麻快速生物技术研究Ⅰ亚麻快速脱胶菌株的选育[J]，中国麻业，2003，25(3)：135-138．
[17] Akin, D.E., Morrison, W.H. III, Gamble, G.R., etal., Effect of retting enzymes on the structure and composition of flax cell wall[J]. Textile Res.J. 1997, 67: 279-287.
[18] Henriksson, G.,Akin , D.E., Rigsby, L.L., etal. Influence of chelating agents and mechanical pretreatment on enzymatic retting of flax[J]. Textile Res.J,1997, 67: 829-836.
[19] Danny E. Akin, Jonn A. Foulk, Roy B. Dodd, David D. McAlister Ⅲ, Enzyme-retting of flax and characterization of processed fibers[J], Journal of Biotechnology, 2001, 89:193-203.
[20] Van Sumere,C.E, Retting of Flax with special reference to enzyme retting .In:Sharma, H.S.S.,Van Sumere ,C.F.(Eds.),The Biology and Processing of Flax. M Publications, Belfast ,Northem Ireland ,1992, pp.157-198.
[21] Henriksson, G.,Akin, D.E., Slomczynski, D., etal. Poduction of highly efficient enzymes for flax retting by Rhizomucar pusillus[J].J.Biotechnol. 1999, 68: 115-123.
[22] J.D.Evans, D.E.Akin ,etal Flax-retting by polygalacuronase-containing enzyme mixtures and effects on fiber properties[J]. Journal of Biotechnology, 2002.
[23] Seaby D A, Mercer P c. Development of a hand tool to test the degree of retting of flax-straw[J]. Annals of Applied Biology,1984,104: 567-573.
[24] Fischer K, Topf W. Entwicklung objektiver Qualitatskriterien fur Flachs. Teil2:TextiltechnologischUntersuchungen[J].MellianTextilberichte,1988, 12: 858-861.
[25] Brown A E ,Sharma H S S , Black D L R. Relationship between pectin content of stems of flax cultivars, fungal cell wall-degrading enzymes and pre-harvest retting[J]. Annals of Applied Biology,1986, 109: 345-351.
[26] Morvan C, Morvan O, Januneau A, Demarty M. Capacite d'echange cationique des fibres de lin[J].Comptes Rendus de l'Academie d'Agriculture de France,1985, 71:837-841
[27] Meijer W J M, Vertregt N, Rutgers B, van de Waart M. The pectin content as a measure of the retting and tettability of flax[J]. Industrial Crops and Products,1995, 4: 273-284.
[28] Henriksson G,Akin D E, Hanlin R T, Rodriguez C, Archibald D D ,Rigsby L L,
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