紫外诱变克鲁维酵母原生质体选育发酵乳清高产酒精菌株
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摘要
本论文研究了菌龄、酶解温度、酶解时间、酶浓度四个单因素对马克斯克鲁维酵母原生质体形成率和再生率的影响，采用菌龄、酶解温度、酶解时间、酶浓度、稳渗剂进行五因素四水平的正交试验，分析各因素的极差值，评价各因素的影响程度，结果表明：酶浓度的极差值最高，其次为酶解时间，然后依次为菌龄、稳渗剂、酶解温度，酶浓度为影响原生质体形成的主要因素，原生质体形成和再生的最佳条件是：菌龄为12h、酶浓度为1.5％、酶解时间为1h、酶解温度为28℃、稳渗剂为KCl，在此条件下，原生质体形成率为92.3％，再生率为29.6％。
研究了马克斯克鲁维酵母原生质体紫外线照射剂量和致死率的关系，得到了紫外线对原生质体的致死率曲线，当紫外线照射剂量为：紫外灯功率20W，照射距离30cm，照射时间为50s时，原生质体的致死率为80.2％，因此，选择该剂量为马克斯克鲁维酵母原生质体紫外线诱变的适宜剂量。
紫外线诱变马克斯克鲁维酵母原生质体后，采用TTC双层平板法进行一级筛选，通过比较各菌株的颜色深浅来衡量其产酒精能力的强弱，从平板上筛选出了颜色较深的24株菌进行二级筛选。
以乳清为培养基采用杜氏小管发酵法进行二级筛选，发酵时间为48h_，以产气速度和杜氏管产气量为指标，从中选出产气速度较快和产气量较大的8株菌进行三级筛选。
用300ml摇瓶发酵法，对二级筛选得到的8株乳酒酵母进行三级筛选，测定其酒精产率和残糖量，从中筛选出一株酒精产率较高且发酵速度较快的菌株，其96h发酵乳清酒精产率为3.8％，残糖量为0.1％，而出发菌株酒精产率为2.5％，残糖量为2.7％，诱变后的菌株较出发菌株酒精产率提高52％，该菌株定名为ZR-20。
采用连续传代的方法研究了ZR-20菌株的遗传稳定性，结果表明：连续传代10次后，ZR-20菌株发酵性能与初代菌株相比无明显变化。
对ZR-20菌株的生长和发酵特性进行了研究，分别测定了ZR-20菌株生长和发酵的最适温度、pH。结果表明：ZR-20菌株的最适生长温度为34℃、生长的最适pH为4.9，最适发酵温度为28℃，发酵的最适pH为4.9；研究了不同温度、pH、接种量对生长曲线和发酵曲线的影响。结果表明：接种量对生长曲线影响很明显，接种量越大，达到稳定期的时间越短。但是最终生物量并不受接种量的影响。在不同pH值下，生长有一定的差异，但是并不明显，在pH为5.9时达到稳定期所用时间最短：对于发酵曲线，接种量对发酵力影响不明显。在pH3.9-5.9范围内，pH值对发酵曲线的影响亦不明显。
采用发酵温度、pH、接种量进行三因素三水平的正交试验对发酵条件进行优化，结果表明：ZR-20菌株发酵乳清的最佳条件是：温度为28℃、pH值为4.9、接种量为2×10~7个/ml，在此条件下发酵96h酒精产率达到4.2％，比优化前酒精产率提高了10.5％，较出发菌株提高了68％。
The conditions that affected the protoplast generation quotiety and regeneration quotiety of K.Marxianus were studied in this paper. Five-factor and four- level orthogonal experiments were designed to study the single factor, including hypha age, enzymes, temperature and osmotants, analyze the range of each factor and assess affecting degree of each factor. Finally the optimum conditions for KMarxianus protoplast generation and regeneration were determined: hypha age was 12h, enzyme concentration was 1.5%, enzymatic hydrolysis time was In, enzymatic temperature was 28C and potassium chloride was selected as osmotants. Under the optimum condition determined, the generation quotiety and regeneration quotiety were 92.3% and 29.6% respectively.
The relationship between UV-light irradiated dose and lethality of K.Marxianus protoplast was also examined in this study. We got the UV-light to lethality curve. The results suggested that when UV-light irradiated dose was 20 watt, distance was 30cm and irradiated time was 50 second, the lethality of protoplast was 80.2%. The regenerated protoplast was primarily screened under the condition above.
After K.Marxianus protoplast mutagenization with UV, TTC double-plate method was chosen for primary screening. The thick degree of mutant strain color was selected as criterion to evaluated the mutant strain's ability of producing alcohol. 24 mutant strains of thick color from plates were selected for secondary screening.
Mutant strains from primary screening were incubated in whey medium and fermented in small tubes for 48h. The rate and amount of producing gas were chose as criterions for secondary screening. Finally we got 8 mutant strains of higher gas-producing rate and amount.
8 mutant strains of milk koumiss from secondary screening were fermented in 250ml shaking flakes for third screening. We got one strain of high alcohol yield and ferment rate. The alcohol concentration of this strain was 3.8%, which was increased by 52% compared with original strain, and the residue of sugar was 0.1%. We named the strain obtained ZR-20.
The effect of serial passage on the genetic stability was studied in this paper and the results demonstrated that compared with original strain, the ferment ability of ZR-20 from 1st to 10 th passage did not vary significantly.
In addition, the growth and ferment characteristics of ZR-20 were examined in this paper. We obtained series of curves including temperature, pH, ferment temperature and ferment pH for ZR-20 and studied their effection on growth and ferment. The optimum combination for ZR-20 growth was 34C, 4.9( pH), which was as same as that of original strain. The effect of grow curve and ferment curve was studied by different
temperature ,pH, incubation amount .The results indicate: growth curve was effected significantly by incubation amount ,more incubation amount ,shorter time to reach stability phase .But the final product amount was not effected by incubation amount .Growth vary but not significantly at different pH. Time to reach stability phase at pH5.9 was the shortest. Incubation amount did not effected ferment significantly to ferment curve .Ferment curve was not effected significantly by pH(3.9~5.9)
A three-factor and three-level orthogonal experiment was designed to study the effection of ferment temperature, pH and incubation amount on final ferment results, analyze the range of each factors and optimize the ferment condition of ZR-20. The optimum ferment condition for ZR-20 was determined: 28 C, pH 4.9, incubation amount 2 X 107. Under the optimum ferment condition, the alcohol concentration post final ferment of ZR-20 was 4.2%, which was increased by 10.5% and 68% respectovely compared with those of preoptimized mutant strain and original strain.
研究了马克斯克鲁维酵母原生质体紫外线照射剂量和致死率的关系，得到了紫外线对原生质体的致死率曲线，当紫外线照射剂量为：紫外灯功率20W，照射距离30cm，照射时间为50s时，原生质体的致死率为80.2％，因此，选择该剂量为马克斯克鲁维酵母原生质体紫外线诱变的适宜剂量。
紫外线诱变马克斯克鲁维酵母原生质体后，采用TTC双层平板法进行一级筛选，通过比较各菌株的颜色深浅来衡量其产酒精能力的强弱，从平板上筛选出了颜色较深的24株菌进行二级筛选。
以乳清为培养基采用杜氏小管发酵法进行二级筛选，发酵时间为48h_，以产气速度和杜氏管产气量为指标，从中选出产气速度较快和产气量较大的8株菌进行三级筛选。
用300ml摇瓶发酵法，对二级筛选得到的8株乳酒酵母进行三级筛选，测定其酒精产率和残糖量，从中筛选出一株酒精产率较高且发酵速度较快的菌株，其96h发酵乳清酒精产率为3.8％，残糖量为0.1％，而出发菌株酒精产率为2.5％，残糖量为2.7％，诱变后的菌株较出发菌株酒精产率提高52％，该菌株定名为ZR-20。
采用连续传代的方法研究了ZR-20菌株的遗传稳定性，结果表明：连续传代10次后，ZR-20菌株发酵性能与初代菌株相比无明显变化。
对ZR-20菌株的生长和发酵特性进行了研究，分别测定了ZR-20菌株生长和发酵的最适温度、pH。结果表明：ZR-20菌株的最适生长温度为34℃、生长的最适pH为4.9，最适发酵温度为28℃，发酵的最适pH为4.9；研究了不同温度、pH、接种量对生长曲线和发酵曲线的影响。结果表明：接种量对生长曲线影响很明显，接种量越大，达到稳定期的时间越短。但是最终生物量并不受接种量的影响。在不同pH值下，生长有一定的差异，但是并不明显，在pH为5.9时达到稳定期所用时间最短：对于发酵曲线，接种量对发酵力影响不明显。在pH3.9-5.9范围内，pH值对发酵曲线的影响亦不明显。
采用发酵温度、pH、接种量进行三因素三水平的正交试验对发酵条件进行优化，结果表明：ZR-20菌株发酵乳清的最佳条件是：温度为28℃、pH值为4.9、接种量为2×10~7个/ml，在此条件下发酵96h酒精产率达到4.2％，比优化前酒精产率提高了10.5％，较出发菌株提高了68％。
The conditions that affected the protoplast generation quotiety and regeneration quotiety of K.Marxianus were studied in this paper. Five-factor and four- level orthogonal experiments were designed to study the single factor, including hypha age, enzymes, temperature and osmotants, analyze the range of each factor and assess affecting degree of each factor. Finally the optimum conditions for KMarxianus protoplast generation and regeneration were determined: hypha age was 12h, enzyme concentration was 1.5%, enzymatic hydrolysis time was In, enzymatic temperature was 28C and potassium chloride was selected as osmotants. Under the optimum condition determined, the generation quotiety and regeneration quotiety were 92.3% and 29.6% respectively.
The relationship between UV-light irradiated dose and lethality of K.Marxianus protoplast was also examined in this study. We got the UV-light to lethality curve. The results suggested that when UV-light irradiated dose was 20 watt, distance was 30cm and irradiated time was 50 second, the lethality of protoplast was 80.2%. The regenerated protoplast was primarily screened under the condition above.
After K.Marxianus protoplast mutagenization with UV, TTC double-plate method was chosen for primary screening. The thick degree of mutant strain color was selected as criterion to evaluated the mutant strain's ability of producing alcohol. 24 mutant strains of thick color from plates were selected for secondary screening.
Mutant strains from primary screening were incubated in whey medium and fermented in small tubes for 48h. The rate and amount of producing gas were chose as criterions for secondary screening. Finally we got 8 mutant strains of higher gas-producing rate and amount.
8 mutant strains of milk koumiss from secondary screening were fermented in 250ml shaking flakes for third screening. We got one strain of high alcohol yield and ferment rate. The alcohol concentration of this strain was 3.8%, which was increased by 52% compared with original strain, and the residue of sugar was 0.1%. We named the strain obtained ZR-20.
The effect of serial passage on the genetic stability was studied in this paper and the results demonstrated that compared with original strain, the ferment ability of ZR-20 from 1st to 10 th passage did not vary significantly.
In addition, the growth and ferment characteristics of ZR-20 were examined in this paper. We obtained series of curves including temperature, pH, ferment temperature and ferment pH for ZR-20 and studied their effection on growth and ferment. The optimum combination for ZR-20 growth was 34C, 4.9( pH), which was as same as that of original strain. The effect of grow curve and ferment curve was studied by different
temperature ,pH, incubation amount .The results indicate: growth curve was effected significantly by incubation amount ,more incubation amount ,shorter time to reach stability phase .But the final product amount was not effected by incubation amount .Growth vary but not significantly at different pH. Time to reach stability phase at pH5.9 was the shortest. Incubation amount did not effected ferment significantly to ferment curve .Ferment curve was not effected significantly by pH(3.9~5.9)
A three-factor and three-level orthogonal experiment was designed to study the effection of ferment temperature, pH and incubation amount on final ferment results, analyze the range of each factors and optimize the ferment condition of ZR-20. The optimum ferment condition for ZR-20 was determined: 28 C, pH 4.9, incubation amount 2 X 107. Under the optimum ferment condition, the alcohol concentration post final ferment of ZR-20 was 4.2%, which was increased by 10.5% and 68% respectovely compared with those of preoptimized mutant strain and original strain.
引文
[1]林金资．乳的深度加工[J]．中国乳品工业，1995，23(4)：199～201．
[2]马丽珍．混合乳干酪的工艺研究[J]．山西农业大学，1998，03：75～77．
[3]全国乳品工业信息中心．1999年世界乳业状况[J]，中国乳品工业，1999：51．
[4]王文智．世界乳清产品贸易概况[J]．食品工业(上海)，1997(01)：27～28．
[5]汪家琴，李克杰．乳清酿酒的研究[J]．中国乳品工业，1992，20(06)：243～245．
[6]王建龙，周定．乳清的开发及综合利用[J]．中国乳品工业，1993(01)：43～48．
[7]Doultani S, Turhan KN, Etzel MR. Whey protein isolate and glycomacropeptide recovery from whey using ionexchange chromatography[J]. Journal of Food Science, 2003, 68: 1389～1395.
[8]赵敏，张敬荣．合理开发利用乳清资源[J]．中国乳品工业，1996(03)：36～37．
[9]张敬荣，张英德．乳清的开发与利用[J]．中国乳品工业，1996(06)：38～39．
[10]LAGRANGE．功能、营养、经济兼备的乳清制品[J]．中国食品工业，1998，6：32．
[11]包怡红，张兰威．乳清酿酒工艺条件的研究[J]．酿酒，2002，29(06)：23～26．
[12]张和平，郑宏旺，德力格尔桑．酶法乳清多肽饮料的研制[J]．食品科学，1994，(2)：27～31．
[13]胡萍．发酵乳清饮料的研制[J]．国外医学：卫生学分册，2000(06)：49～50．
[14]蒋福虎，马俪珍，陈建明．低度乳清酒的研制[J]，山西农业大学学报，1998，18(01)：54～57．
[15]谢继志，杨文，葛庆丰．乳清酒发酵条件及发酵动态的研究[J]，中国乳品工业，1999，27：3～6．
[16]曾志明．乳清粉中乳糖含量分析[J]．中国饲料，1998(09)：21～22．
[17]马俪珍，张秀红，蒋福虎．乳清的营养价值及乳清饮料的研究现状[J]．中国乳品工业，1999，27(05)：47～50．
[18]张秀红，马俪珍．发酵型乳清饮料的加工工艺[J]．中国食物与营养，2000(04)：32～33．
[19]Jack W, O'Brien. Dairy products as functional foods[J]. European Dairy Magazine, 2004, 12: 14～15.
[20]Hans, A Mehrens. whey: a valuable ingredient Food Technology International[J]. 2003: 35.
[21]赵敏．合理开发利用乳清资源[J]．中国乳品工业，1996，6(24)：36～37．
[22]章练红．世界单细胞蛋白开发利用概况[J]．饲料博览，1999，11(2)：24～25．
[23]李淑敏．国外乳清废液在饲料中的应用[J]．饲料研究，1990(11)：11～13．
[24]Zayed, Glet. Utilization of whey from reconstituted Skim milk for sop production[J]. Cultl Dairy Prod, 1991, 26(1): 22～28.
[25]柳萍，王建龙．乳清生物转化与废物资源化[J]．中国乳品工业，1996(02)：16～19．
[26]韩德奇，李伟，张冬捧．等．燃料乙醇的生产进展和应用探讨[J]．化工技术经济，2002(06)：9～15．
[27]Nova. J. Ehtylalcohol and yeast biomass from whey and milk Serum Khranit promst 1998, 39(6): 22～25.
[28]Marwaha J. alcohol production from whey permeate by immobilized and free cell of kluycesmarxi an usNCYC 179 Process Biochemistry, 1984(4): 70～80.
[29]Athanasiadis I. whey liquid waste of the dairy industry as raw material for potable alcoholproduction by kefir granules[J]. Journal of Agricultural and Food Chemistry, 2002(50): 7231～7234.
[30]李涛，费中伟．开发乳清产品满足国内需要[J]．中国食品工业，1999(05)：28～30．
[31]滕国新，周伟明．中国干酪及乳清制品的现状与产业化进程[J]．食品工业科技，2000(01)：76～78．
[32]Madigan，M．T，Marrinko J．微生物生物学[M]．北京：科学出版社，2001：390．
[33]http://biology.kenyon.edu/courses/biol114/Chap06/Chapter_06a.html#class.
[34]曹友声，刘仲敏．现代工业微生物学[M]．长沙：湖南科学技术出版社，1998．
[35]Doc Kaiser's Microbiology Home Page http://www.cat.cc.rod.us/courses/bio141/lecguide/unit4/geneties/mutation/mutation.Html.
[36]周东坡，平文祥著．微生物原生质体融合[M]．哈尔滨：黑龙江省科学技术出版社，1990．
[37]林稚兰，黄秀梨主编．现代微生物学与实验技术[M]北京：科学出版社，2000．
[38]Nobuhiko Mukai, Chiharu Nishimori. Beer Brewing Using a Fusant between a Sake Yeast and a Brewer's Yeast[J]. Journal of Bioscience and Bioengineering, 2001, 91: 482～486.
[39]杜珠还，张泉渡，华祖尧等．紫外线和氯化锂对棒状杆菌原生质体的诱变作用[J]．生物工程学报，1985，1(4)：59～62．
[40]王弘，齐秀兰，李福德．紫外诱变原生质体选育赖氨酸高产菌株[J]．生物工程学报，1990，6(1)：32～381．
[41]方善康，王晓辉．酒化根霉原生质体的诱变育种[J]．中国酿造，1990，1：28～291．
[42]李庆余，朱宝臣，李潞滨等．维生素B2产生菌原生质体诱变选育[J]．微生物学报，1990，30(4)：312～314．
[43]李明春，邢来君．γ-亚麻酸产生菌的原生质体诱变育种[J]．微生物学通报，1998，25(1)：9～12．
[44]Lorenz MG, Wackernagel W. Bacterial genetransfer by natural genetic transformationin the environment[J]. Microbiol Rev 1994, 58: 563～603.
[45]陈春涛，王岁楼，卫军．产类胡萝卜素酵母菌原生质体的制备、再生与诱变．生物技术，2002，112：23～25．
[46]徐柳．高产胞外植酸酶酵母菌的选育[J]．四川大学学报(自然科学皈)，2002，4(139)：31～34．
[47]赵华．原生质体紫外诱变提高生香酵母产酯能力的研究[J]．天津微生物，1996(2)：13～
18．
[48]管敦仪．啤酒工业手册(中册)[M]，北京：轻工业出版社，1996．
[49]唐孝宣，肖信发，徐京宁．微生物原生质体融合技术的进展[J]．工业微生物，1987，17(5)：23～29．
[50]黎碧莲，涂桂洪．酿酒酵母与扣囊拟内孢霉属间原生质体融合的研究[J]．暨南大学学报(自然科学版)，1998，19(5)：120～124．
[51]甘志波，赵学慧．酿酒酵母RasseⅫ原生质体的形成与再生[J]．微生物学杂志．1994，19(5)：12～16．
[52]webl. tch. harvard.edu/silverman/protocols/YACS/protoplast/protoplast.Pdf.
[53]陈海昌．南阳5号酵母原生质体制备和再生条件的研究[J]．微生物学杂志，1985，5(1)：13～17．
[54]卫军．原生质体融和选育高产β—胡萝卜素酵母菌[J]．生物技术，2001，112：23～25．
[55]唐孝宣，肖信发，徐克宁．微生物原生质体融合技术的进展[J]．工业微生物，1987，17(5)：23～29．
[56]明道绪．生物统计附试验设计1989，11，357～358．
[57]李用芳．面包酵母原生质体的制备，再生及紫外诱变的初步研究[J]．生物技术，2000，10(2)：23～27．
[58]王唐义，徐开成．产酯酵母的诱变育种[J]．酿酒科技，1999(3)：26～27．
[59]张伟．原生质体紫外诱变筛选还原双乙酰能力强的啤酒酵母[J]．酿酒，2003，3：63～65．
[60]王梅，张澎湃，帅桂兰．TTC在黄酒酵母选育中的应用[J]．酿酒，2001，28(5)：62～64．
[61]胡子斌．TTC～脱氢酶活性常温萃取测定法及应用[J]．工业水处理，2001，21(10)：29～31．
[62]胡永松，王忠彦．微生物与发酵工程[M]．成都：四川大学出版社，1994．
[63]寇运同，胡永松，王忠彦等．高温酵母发酵特性及功能的研究[J]．酿酒科技．1995，(3)：70～72．
[64]大连轻工业学院等合编食品分析[M]．北京：中国轻工业出版社，1994．
[65]Maria, dasGracas, Carneiro da Cunha. Protoplast formation and regeneration from Streptomyces clavuligerus NRRL 3585 and clavulanic acid production[J]. Braz. J. Microbio-1. 2003, 33: 55～58.
[66]Hopwood, D. A Genetic studies with bacterial protoplasts. Ann. Rev. Microbiol. 1981, 35: 237～272.
[67]ling, G. T, Normansell. Protoplast isolation and regeneration in Streptomyces clavuligerus[J]. J. Gen. Microbiol, 1989, 135: 2289～2297.
[68]Wenlin Wei. Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae to produce sorbitol from Jerusalem artichokes[J]. Biotechnology Letters, 2001,
23(10)：799～803．
[69] 钟亚铃，汤岳琴，木田建次．耐温絮凝酵母Saccharomyces cerevisiae KF～7的细胞活性及发酵特性研究[J]．食品与发酵工业，1997，24(4)：1～5．
[70] 范怀德．青霉素在酒精发酵中的应用[J]．甘肃科技，1999(5)：33．
[71] 李剑芳，张灏．发酵猕猴桃汁中产香酵母的分离、鉴定及生长特性的研究[J]．食品科学，2001，22(9)：19～22．
[2]马丽珍．混合乳干酪的工艺研究[J]．山西农业大学，1998，03：75～77．
[3]全国乳品工业信息中心．1999年世界乳业状况[J]，中国乳品工业，1999：51．
[4]王文智．世界乳清产品贸易概况[J]．食品工业(上海)，1997(01)：27～28．
[5]汪家琴，李克杰．乳清酿酒的研究[J]．中国乳品工业，1992，20(06)：243～245．
[6]王建龙，周定．乳清的开发及综合利用[J]．中国乳品工业，1993(01)：43～48．
[7]Doultani S, Turhan KN, Etzel MR. Whey protein isolate and glycomacropeptide recovery from whey using ionexchange chromatography[J]. Journal of Food Science, 2003, 68: 1389～1395.
[8]赵敏，张敬荣．合理开发利用乳清资源[J]．中国乳品工业，1996(03)：36～37．
[9]张敬荣，张英德．乳清的开发与利用[J]．中国乳品工业，1996(06)：38～39．
[10]LAGRANGE．功能、营养、经济兼备的乳清制品[J]．中国食品工业，1998，6：32．
[11]包怡红，张兰威．乳清酿酒工艺条件的研究[J]．酿酒，2002，29(06)：23～26．
[12]张和平，郑宏旺，德力格尔桑．酶法乳清多肽饮料的研制[J]．食品科学，1994，(2)：27～31．
[13]胡萍．发酵乳清饮料的研制[J]．国外医学：卫生学分册，2000(06)：49～50．
[14]蒋福虎，马俪珍，陈建明．低度乳清酒的研制[J]，山西农业大学学报，1998，18(01)：54～57．
[15]谢继志，杨文，葛庆丰．乳清酒发酵条件及发酵动态的研究[J]，中国乳品工业，1999，27：3～6．
[16]曾志明．乳清粉中乳糖含量分析[J]．中国饲料，1998(09)：21～22．
[17]马俪珍，张秀红，蒋福虎．乳清的营养价值及乳清饮料的研究现状[J]．中国乳品工业，1999，27(05)：47～50．
[18]张秀红，马俪珍．发酵型乳清饮料的加工工艺[J]．中国食物与营养，2000(04)：32～33．
[19]Jack W, O'Brien. Dairy products as functional foods[J]. European Dairy Magazine, 2004, 12: 14～15.
[20]Hans, A Mehrens. whey: a valuable ingredient Food Technology International[J]. 2003: 35.
[21]赵敏．合理开发利用乳清资源[J]．中国乳品工业，1996，6(24)：36～37．
[22]章练红．世界单细胞蛋白开发利用概况[J]．饲料博览，1999，11(2)：24～25．
[23]李淑敏．国外乳清废液在饲料中的应用[J]．饲料研究，1990(11)：11～13．
[24]Zayed, Glet. Utilization of whey from reconstituted Skim milk for sop production[J]. Cultl Dairy Prod, 1991, 26(1): 22～28.
[25]柳萍，王建龙．乳清生物转化与废物资源化[J]．中国乳品工业，1996(02)：16～19．
[26]韩德奇，李伟，张冬捧．等．燃料乙醇的生产进展和应用探讨[J]．化工技术经济，2002(06)：9～15．
[27]Nova. J. Ehtylalcohol and yeast biomass from whey and milk Serum Khranit promst 1998, 39(6): 22～25.
[28]Marwaha J. alcohol production from whey permeate by immobilized and free cell of kluycesmarxi an usNCYC 179 Process Biochemistry, 1984(4): 70～80.
[29]Athanasiadis I. whey liquid waste of the dairy industry as raw material for potable alcoholproduction by kefir granules[J]. Journal of Agricultural and Food Chemistry, 2002(50): 7231～7234.
[30]李涛，费中伟．开发乳清产品满足国内需要[J]．中国食品工业，1999(05)：28～30．
[31]滕国新，周伟明．中国干酪及乳清制品的现状与产业化进程[J]．食品工业科技，2000(01)：76～78．
[32]Madigan，M．T，Marrinko J．微生物生物学[M]．北京：科学出版社，2001：390．
[33]http://biology.kenyon.edu/courses/biol114/Chap06/Chapter_06a.html#class.
[34]曹友声，刘仲敏．现代工业微生物学[M]．长沙：湖南科学技术出版社，1998．
[35]Doc Kaiser's Microbiology Home Page http://www.cat.cc.rod.us/courses/bio141/lecguide/unit4/geneties/mutation/mutation.Html.
[36]周东坡，平文祥著．微生物原生质体融合[M]．哈尔滨：黑龙江省科学技术出版社，1990．
[37]林稚兰，黄秀梨主编．现代微生物学与实验技术[M]北京：科学出版社，2000．
[38]Nobuhiko Mukai, Chiharu Nishimori. Beer Brewing Using a Fusant between a Sake Yeast and a Brewer's Yeast[J]. Journal of Bioscience and Bioengineering, 2001, 91: 482～486.
[39]杜珠还，张泉渡，华祖尧等．紫外线和氯化锂对棒状杆菌原生质体的诱变作用[J]．生物工程学报，1985，1(4)：59～62．
[40]王弘，齐秀兰，李福德．紫外诱变原生质体选育赖氨酸高产菌株[J]．生物工程学报，1990，6(1)：32～381．
[41]方善康，王晓辉．酒化根霉原生质体的诱变育种[J]．中国酿造，1990，1：28～291．
[42]李庆余，朱宝臣，李潞滨等．维生素B2产生菌原生质体诱变选育[J]．微生物学报，1990，30(4)：312～314．
[43]李明春，邢来君．γ-亚麻酸产生菌的原生质体诱变育种[J]．微生物学通报，1998，25(1)：9～12．
[44]Lorenz MG, Wackernagel W. Bacterial genetransfer by natural genetic transformationin the environment[J]. Microbiol Rev 1994, 58: 563～603.
[45]陈春涛，王岁楼，卫军．产类胡萝卜素酵母菌原生质体的制备、再生与诱变．生物技术，2002，112：23～25．
[46]徐柳．高产胞外植酸酶酵母菌的选育[J]．四川大学学报(自然科学皈)，2002，4(139)：31～34．
[47]赵华．原生质体紫外诱变提高生香酵母产酯能力的研究[J]．天津微生物，1996(2)：13～
18．
[48]管敦仪．啤酒工业手册(中册)[M]，北京：轻工业出版社，1996．
[49]唐孝宣，肖信发，徐京宁．微生物原生质体融合技术的进展[J]．工业微生物，1987，17(5)：23～29．
[50]黎碧莲，涂桂洪．酿酒酵母与扣囊拟内孢霉属间原生质体融合的研究[J]．暨南大学学报(自然科学版)，1998，19(5)：120～124．
[51]甘志波，赵学慧．酿酒酵母RasseⅫ原生质体的形成与再生[J]．微生物学杂志．1994，19(5)：12～16．
[52]webl. tch. harvard.edu/silverman/protocols/YACS/protoplast/protoplast.Pdf.
[53]陈海昌．南阳5号酵母原生质体制备和再生条件的研究[J]．微生物学杂志，1985，5(1)：13～17．
[54]卫军．原生质体融和选育高产β—胡萝卜素酵母菌[J]．生物技术，2001，112：23～25．
[55]唐孝宣，肖信发，徐克宁．微生物原生质体融合技术的进展[J]．工业微生物，1987，17(5)：23～29．
[56]明道绪．生物统计附试验设计1989，11，357～358．
[57]李用芳．面包酵母原生质体的制备，再生及紫外诱变的初步研究[J]．生物技术，2000，10(2)：23～27．
[58]王唐义，徐开成．产酯酵母的诱变育种[J]．酿酒科技，1999(3)：26～27．
[59]张伟．原生质体紫外诱变筛选还原双乙酰能力强的啤酒酵母[J]．酿酒，2003，3：63～65．
[60]王梅，张澎湃，帅桂兰．TTC在黄酒酵母选育中的应用[J]．酿酒，2001，28(5)：62～64．
[61]胡子斌．TTC～脱氢酶活性常温萃取测定法及应用[J]．工业水处理，2001，21(10)：29～31．
[62]胡永松，王忠彦．微生物与发酵工程[M]．成都：四川大学出版社，1994．
[63]寇运同，胡永松，王忠彦等．高温酵母发酵特性及功能的研究[J]．酿酒科技．1995，(3)：70～72．
[64]大连轻工业学院等合编食品分析[M]．北京：中国轻工业出版社，1994．
[65]Maria, dasGracas, Carneiro da Cunha. Protoplast formation and regeneration from Streptomyces clavuligerus NRRL 3585 and clavulanic acid production[J]. Braz. J. Microbio-1. 2003, 33: 55～58.
[66]Hopwood, D. A Genetic studies with bacterial protoplasts. Ann. Rev. Microbiol. 1981, 35: 237～272.
[67]ling, G. T, Normansell. Protoplast isolation and regeneration in Streptomyces clavuligerus[J]. J. Gen. Microbiol, 1989, 135: 2289～2297.
[68]Wenlin Wei. Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae to produce sorbitol from Jerusalem artichokes[J]. Biotechnology Letters, 2001,
23(10)：799～803．
[69] 钟亚铃，汤岳琴，木田建次．耐温絮凝酵母Saccharomyces cerevisiae KF～7的细胞活性及发酵特性研究[J]．食品与发酵工业，1997，24(4)：1～5．
[70] 范怀德．青霉素在酒精发酵中的应用[J]．甘肃科技，1999(5)：33．
[71] 李剑芳，张灏．发酵猕猴桃汁中产香酵母的分离、鉴定及生长特性的研究[J]．食品科学，2001，22(9)：19～22．