摘要
:本研究立足于国内和国际研究现状，利用原生质体融合技术构建增香型苹果酒酿造酵母。虽然原生质体融合技术已经是一个成熟、系统的实验体系，利用该技术进行微生物遗传育种的研究报道国内外都很多，但用于构建增香型酿造酵母的研究并不多见。
本试验以5株优良的酵母菌株为试验材料，通过对其各自发酵苹果酒的理化指标和感官指标的综合分析，得出5#酵母菌株较适合酿造优良的苹果酒；将5#酵母菌株作为诱变出发菌株，利用甲基磺酸乙酯（EMS）对其进行诱变，从突变株中筛选出1株遗传标记稳定的突变株E2，然后利用原生质体融合技术将其和发酵能力较强的3#酵母的灭活原生质体进行了融合，通过GC-MS对优良融合子及亲本发酵的苹果酒香气成分进行了鉴定分析，并结合感官品评，筛选得到3株优良的增香型苹果酒酿造菌株。主要研究结果如下：
(1) 通过对5株试验菌株发酵苹果酒理化指标和感官指标的比较分析，筛选出1株发酵苹果酒口感、风味较好，具有苹果酒典型风格的优良酵母菌株（5#菌株），然后利用EMS对其进行诱变，筛选出1株赖氨酸缺陷型突变株E2，将其确定为原生质体融合时产香能力好的亲本菌株Y。
(2) 通过对5株试验菌株的发酵速率的测定和发酵醪液理化指标的测定，发现3#菌株的发酵能力较强，将其确定为原生质体融合时发酵能力强的亲本菌株X。
(3) 根据两亲本的生长曲线图，得到两亲本的对数生长期均约为第2～10h，其对数生长早期均约为第2～4h。因此确定两亲本在制备原生质体时的前培养时间为4h。
(4) 在酶解温度35℃，蜗牛酶终浓度为1%的条件下，酶解80min时，亲本菌株Y的原生质体形成率和再生率分别可达93.5%和30.7%,亲本菌株X的原生质体形成率和再生率分别可达92.4%和29.5%,适合进行原生质体融合。
(5) 在60℃下，对亲本菌株X的原生质体进行水浴灭活，发现灭活15min，即可100%的抑制或钝化亲本菌株X原生质体活性。
(6) 将筛选的融合子及亲本酿造的苹果酒香气成分进行GC-MS鉴定分析，并结合感官品评，得到3株产香能力、发酵能力均较强的优良增香型苹果酒酵母菌株。
This study was based upon the internal and external research situation, constructed cider yeast strain having aroma-improving ability and intense fermentability by protoplast fusion. Though protoplast fusion was already a ripe systematic technique, and many researchers in home and abroad wildly used it to breed new microorganism strains, there were a few research reports of constructing aroma-improving yeast strain.
Five yeast strains were selected as experimental material in this study. According to the synthetic analysis of physic-chemical characteristics and organoleptic evaluation on ciders, strain 5# was chosen as optimal cider yeast. Then strain 5# was treated by ethyl methanesulfonate(EMS), strain E2, which has a stable hereditary marker, was isolated from mutants.Protoplasts of strain E2 were fused with inactivated protoplasts of strain 3# having intense fermentability, many stable fusants were obtained. A brewing trail with the excellent fusants and the parental strains was carried out, and the aroma components of different ciders were analyzed and identified by gas chromatography-mass spectrometry. Three fusants having beneficial characters of parental strains and aroma-improving ability were selected out. The main results in this study showed:
(1) strain 5# was chosen as optimal cider yeast according to the synthetic analysis of different ciders brewed by the five yeast strains, and treated by EMS, strain E2, which is a lysine auxotrophic strain, was selected out from mutants for one parental stain in protoplast fusion.
(2) strain 3#, which has intense fermentability, was chosen as another parental stain in protoplast fusion according to fermentative velocities of different strains and characteristics of different fermented apple juices.
(3) The logarithmic growth phase of parental stains was from 2nd hour to 10th hour and the forepart phase was from 2nd hour to 4th hour according to the growth curve, therefore we used the parental cells incubated 4 hour to make protoplast.
(4) The protoplast formation rate was 93.5% for parental stain of E2 and 92.4% for parental stain of 3# in the optimal reaction condition of at 35℃, 80min of reaction time, 1% final concentration of snailenzyme. and the protoplast regeneration
rate was 30.7% and 29.5%,respectively.
(5) The protoplast of strain 3# could be completely inactivated by 60℃ of water bath treatment for 15min.
(6) The results obtained from analysis of different ciders by gas chromatography -mass spectrometry and sensory taste suggest that three fusants have better aroma-improving ability.
引文
[1]陈代杰,朱宝泉.工业微生物菌种选育与发酵控制技术[M].上海：上海科学技术文献出版社,1994
[2]陈海昌,刘 波,张苓花,等.原生质体融合技术构件糖化型啤酒酵母的研究[J].微生物学通报,1997,24(3):159-16
[3]陈庆源.苹果酒及其制造方法[J].浙江食品工业，1991,(2),12-17
[4]陈陶声,王大琛.微生物工程[M].北京：化学工业出版社,1994:23-27
[5]陈五岭,张芳琳,景建洲,等.灭活原生质体融合技术选育苏云杆菌新菌种—原生质体融合条件的研究[J].1998,28(2):147-149
[6]程东升,李国兰.电诱导细胞融合仪的研制及在真菌原生质体融合中的应用[J].微生物学通报,1995,22(3):14-17
[7]程树培,更小六,汪蓉,等.光合细菌种间原生质体融合及融合子鉴定[J].南京大学学报,1993,29(3):407-12
[8]丁友昉,陈宁.普通微生物遗传学[M].天津：南开大学出版社,1990
[9]杜金华,张开利,张锦丽,等.嗜杀苹果酒酵母的构建[J].食品与发酵工业,28(2):20-23
[10]杜连祥,王昌禄,鲁梅芳.利用细胞质导入法选育嗜杀啤酒酵母[J].生物工程学报,1996,12(1):71-77
[11]方霭其,李绍兰,陈有为,等.耐热酵母与酿酒酵母原生质体融合的研究[J].生物工程学报,1990,6(23):224-229
[12]甘志波,赵学慧.灭活黑曲霉与中期啤酒酵母原生质体融合的研究[J].华中农业大学学报,1993,12(5):502-509
[13]高年发,王淑豪,李小刚,等.酿酒酵母与粟酒裂殖酵母属间原生质体融合选育降解苹果酸强的葡萄酒酵母[J].生物工程学报,2000,16(6):718-722
[14]宫英振,王颉,李长文,等.苹果酒的香气[J].酿酒科技,2003(3),68-70
[15]郝林.食品微生物学实验技术[M].北京：中国农业出版社,2001
[16]何方.北京棒杆菌和黄色短杆菌属间原生质体融合研究.武汉大学学报，1991(4):101-06
[17]何秀良,鞠京丽,蔡崇光,等. 用原生质体融合构建高产酒精酵母株. 生物工程学报 (增刊),1996,12:163-167
[18]洪益国,郑幼霞,杨颐康,等.通过热灭活棒状杆菌原生质体种间融合选育谷氨酸高产菌株[J].生物工程学报,1988,4(4):327-330
[19]洪益国,郑幼霞,杨颐康.棒状杆菌灭活原生质体种间融合[J].遗传.1988,10(3):24-27
[20]江慧修,张金玲,周 坚,等.啤酒酿造用凝集性酵母融合育种的研究[J].微生物学
报,1993,33(1):22-31
[21]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(自然科版),1998,19(5):120-124
[22]李华,涂正顺,王华,等.猕猴桃果酒香气的气相色谱／质谱分析[J].分析化学研究简报.2002,30(6):695-698
[23]李华.现代葡萄酒工艺学[M].西安:陕西人民出版社,1994
[24]李华.葡萄酒品尝学[M].北京:中国青年出版社,1992
[25]李桃生,陈士怡.酿酒酵母与糖化酵母的种间原生质体融合及其融合子的鉴定[J].生物工程学报,1987,3(2):121-129
[26]李桃生,职慧军,罗玉萍,等.通过原生质体融合改建酵母细胞的研究[J].遗传学报,1992,19(5):467-474
[27]梁黎明.苹果酒[J].酿酒，1999,(5),43-44
[28] 彭帮柱,岳田利，袁亚宏，等.酵母菌原生质体融合技术[J].西北农业学报.2004,13(1):101-103
[29]彭帮柱,岳田利，袁亚宏，等.优良苹果酒酵母的选育[J].西北农林科技大学学报(自然科学版).2004,32(5)：81-84
[30]彭志英,食品生物技术[M].北京:中国轻工业出版社,1999
[31]阮仕立,王西锐,刘得兵,等.苹果酒的开发研究进展[J].食品与发酵工业,2000,27(4):75-77
[32]施庆珊，邱玉堂，李良秋，等.原生质体融合选育肌苷产生菌产氨短杆菌[J].微生物学杂志，2000,20(4):27-29
[33]宋安东,吴云汉,李艳梅,等.产苹果酒优良酵母菌的选育研究[J].河南农业大学学报,2002(6),(36)2:183-186
[34]孙爱东,葛毅强,张欣,等.水果制品增香技术研究进展[J].食品与发酵工业,1998, 25(1):44-49
[35]孙君社,李 雪,李军席.原生质体融合构建耐高温酵母菌株[J].食品与发酵工业,2002,28(5):1-5
[36]孙启玲,胡永松,王忠彦,等.灭活酵母细胞原生质体融合技术的研究[J].四川大学学报(自然科学版),1991,28(3):366-373
[37]涂正顺,李华,李嘉瑞,等.猕猴桃品种间果香成分的GC/MS分析[J].西北农林科技大学（自然科学版）.2004,30(2):96-100
[38]汪和睦,鲁玉瓦,武延宽,等.电场诱导营养缺陷型酵母原生质体的融合[J].生物工程学报，1986,2(3):44-51
[39]王昌禄,杜连祥,张 辉,等.利用紫外线致死原生质体融合技术选育嗜杀啤酒酵母[J].食品与发酵工业,1993,6:1-7
[40]王建华,赵学慧.螯合剂对原生质体融合的影响[J].生物工程学报,1998,14(1):112-115
[41]王立平，徐岩,王栋,等.苹果酒香气研究进展[J].食品与发酵工业,2002,28(7): 59-60
[42]王岳五,宋林生,周与良.电融合技术选育能利用木糖和纤维二糖生产乙醇的菌株[J].生物工程学报，1992,8(1):82-86
[43]王岳五,宇学锋,汪和睦,等.电诱导酵母属与短梗霉属属间融合的研究[J],真菌学报,1990,9(1):56-63
[44]王正祥,诸葛键.杀伤啤酒酵母的构建及其发酵性能研究[J].食品与发酵工业,1991,(1):1-6
[45]文铁桥,赵学慧.酵母菌属间原生质体融合构建高温酵母菌株[J].微生物学报，1999,39(2):141-147
[46]杨汝德,朱文生.双灭活原生质体融合法选育耐高温高产酒精酵母的研究[J].工业微生物,1993,23(1):9-13
[47]亚当斯 A，戈特施林 D E，凯泽 C A．刘子铎 译．遗传学实验方法指南[M].北京：科学出版社，2000
[48]袁志发.模糊数学在农林上的应用[M].陕西杨凌:天则出版社,1990
[49]曾云中,张东妮,吴雪昌,等.有淀粉糖化酶活性的耐高温酿酒酵母的构建研究[J].生物工程学报,1992,8(40):363-370
[50]张博润,蔡金科.酵母菌属间原生质体融合获得能发酵乳糖生产酒精的融合体[J].微生物学报,1990,30(3):182-188
[51]张博润,姜书勤,徐婉学,等.电场诱导营养缺陷型酵母原生质体的融合[J].生物工程学报,1986,2(4):29-34
[52]张克旭,陈宁,张蓓,等.代谢控制发酵[M].北京：中国轻工业出版社,1998
[53]张明,王元君,潘仁瑞.酿酒酒酵母和粟酒裂殖酵母属间融合和融合子特性[J].真菌学报.1996,15(3):204-209
[54]张明.葡萄酒酵母与粟酒裂殖酵母融合试验[J].真菌学报,1996,15(3):204-209
[55]赵华,赵树欣,张维,等.运用紫外线灭活原生质体融合技术选育高产酯酒精酵母的研究[J].酿酒科技,1996,5:13-16
[56]周东坡,平文祥,孙剑秋,等.通过灭活原生质体融合选育啤酒酵母新菌株[J].微生物学报,1999,39(5):454-460
[57]朱宝成,王俊刚，成亚利,等.果胶酶生产菌原生质体再生及诱变育种[J].微生物学通报,1994,21(1):15-17
[58]朱平,李焕娄.微生物原生质体灭活及其在育种的应用[J].国外医药抗生素分册,1990,11(6):409-413
[59] GB/T15038-94—葡萄酒、果酒通用实验方法[S],1994
[60] Adela Bortol. Industrial yeast strain improvement: construction of strains having the killer character and capable of utilizing starch[J]. Applied Microbiology and Biotechnology, 1988, 28: 577-579
[61] Advances in Protoplast Research Proceedings of the 5th International Protoplast Symposium[C], Szeged Hungary Budapest 1979 July 9-14
[62] Arthur Senkei J R. Use of hazard analysis critical control point and alternative treatments in production apple cider[J]. Journal of Food Protection, 1999,62(7): 778- 785
[63] Avakyants S P, Rastyannikov E G, Chemyaga B S, et al. Khromato-mass- spektrometricheskoe issledovanie. Letuchikh vesnchestv vina[J]. Vinodel. Vinograd. SSSR.1981, 41: 50-53
[64] Beech F W, Davenport R R. The role of yeasts in cider making[J].Yeast Technology, 1970,3:73-146
[65] Beech F W, Carr J G. Cider and Perry[J]. Alcoholic Beverages. 1977, 1:139-313
[66] Beech F W. Cider making and cider research a review[J]. Journal of the Intitute of brewing, 1972 (78) 477-484
[67] Beech F W. Cider making and cider research[J]. Journal of science Food Agriculture, 1993,6(4):259-270
[68] Beech F W. English Cidermaking-Technology, Microbiology and Biochemistry[J] In Progress in Industrial Microbiology, 1972 , 133-213
[69] Beech F W. Yeasts in Cider Making[J].Yeast Technology. 1993,5:169-213
[70] Bortol A, Nudel C, Fraile E, et al. Isolation of yeast with killer activity and its breeding with an industrial baking strain by protoplast fusion[J]. Applied Microbiology and Biotechnology . 1986,24:414-416
[71] Bortol A, Nudel C, Fraile E, et al. Isolation of yeast with killer activity and its breeding with an industrial baking strain by protoplast fusion[J]. Applied Microbiology and Biotechnology .1986, 24: 414-416
[72] Burroughs L F, Sparks A H. The sulphite binding power of wines and ciders [J]. Journal of Science Food Agriculture. 1973,24: 187-217
[73] Carr J G. Microbiology of Wines and Ciders[J]. In Essays in Agricultural and Food Microbiology. 1973:291-307
[74] Carr J. G. Microbiology of Wines and Ciders[J]. In Essays in Agricultural and Food Microbiology, 1973:291-307
[75] Churdchai C, Haritchanan S. The Study of Protoplast Fusion between Leuconostoc oenos and Saccharomyces cerevisiae for Industrial Wine Production[J]. Proc. 4th Int. Conf. food
Sci.Tech.,2001,121-136
[76] Dimick P S, Hoskin J C. CRC Critical Reviews[J]. In Food Science and Nutrition,1983,18:387-409
[77] Dixon J, Hewett E W. Exposure to hypoxia conditions alters volatile concentrations of apple caltivars[J]. Journal of the Science Food and Agriculture,2001,8:22-29
[78] Duenas M, Irastorza A. et al. Influence of apple juice treatments on the cider making process [J]. Journal of the Intitute of brewing. 1997,103(4):251-255
[79] Durr P .The flavor of cider.food flavor. Part B The flavor of Beverages[M]. Ed by Morton I D ,Macleod A G .Amsterdam Netherlands: Elsevier Science Press, 1986
[80] Gomis D B, Gutierrez M D, Moranm M J. Analytical control of cider production by two technological methods[J]. Journal of the Institute of Brewing, 1991, 97: 453-456
[81] Hopwood D A, Wright H M. Factors affecting frequency in protoplast fusions of streptomyces coelicolor[J].J. Gen. Microbiol.,1979,111:137-143
[82] Jarvls B, Forster M J, Kinsella W P. Factors affecting the development of cider flavor[J]. Journal of Applied Bacteriology Symposium Supplement,1995,79: 5-18
[83] Javadekar VS, Sivaraman H, gokhale DV. Industrial yeast strain improvement: construction of a highly flocculent yeast with a killer character by protoplast fusion[J]. Journal Of Industrial Microbiology, 1995,15: 94-102
[84] Jepsen O M. The sensory and analytical evaluation of apple juice volatiles[J]. International Federation of Fruit Juice Producers. Scientific Technical Commission, 1978, 15, 349-61
[85] Kao K N, Michayluk M R A. Method for high frequency intergeneric fusion of plant protoplasts[J]. Planta , 1974, 115: 355-367
[86] Kenji Kida, Kiyotaka Kume, Shigeru Mrimura, et al. Repeated-batch fermentation process using a thermotolerant flocculation yeast constructed by protoplast fusion[J]. Journal of Fermentation and Bioengineering. 1992,74(3): 169-173
[87] Lea A G H. Analytical techniques for the estimation of sulphite inding components in cider and wine [J]. Interional Journal of Food Science and Technology, 2000(35):105-112
[88] Lea A G H. Fermented Beverage Product[M]. Blackie. Ed by Lea A G H, Piggott J R. London: Academic and Professional Press,1995:66-67
[89] Leguerinel I, Cleret J J, Bourgeois C, et al. Yeast strain and kinetic aspects of the formation of flavor components in cider[J]. Journal of the Institute of Brewing, 1989,95:405-409
[90] Leguerinel I, Cleret J J, Bourgeois C, et al. Yeast strain and the formation of flavor components in cider[J].Journal of the Institute of Brewing,1988,96: 391-395
[91] Mangas J J，Cabranes C et al. Influence of cider-making technology on cider taste[J]. Journal Article,1994,17(6):583-586
[92] Masao kishida, Tomoaki muguruma, kazunobusakanaka,etal. Chromosomal deletion or rearrangement in chimeric hybrids of saccharomycopsis fibuligera and saccharomyces diastaticus obtained by cell fusion[J]. Journal Of Fermentation And Bioengineering, 1996, 81(4):281-285
[93] Nedovic V A, Duriux A, Van Nedervelde L, et al. Continuous cider fermenttion with co-immobilized yeast and Leuxonostoc oenos . Cells Enzyme and Microbial Technology 2000(26): 834-839
[94] Ough C S, Bell A. Effects of nitrogen fertilization of grapevines on amino acid metabolism and higher alcohol formation during grape juice fermentation[J]. American Journal of Enology andViticulture,1980,31:110-120
[95] Ough C S, Guymon J F, Crowell E A. Formation of higher alcohols during fermentation[J]. Journal of Food Science, 1966, 31:620-625
[96] Paillard N N M.The Flavour of Apple, Pears and Quinces.In Food Flavours Part C.The Flavour of Fruits[M] :Ed by Morton I D ,Macleod A G .Amsterdam Netherlands ;Elsevier Science Press,1990:38-41
[97] Pollard A, Kieser M E, Beech E V. Factors influencing the flavor of cider: the effecter of fermentation treatments on fusel oil production[J]. Journal of Applied Bacteriology, 1966,29(2):253-259
[98] Pollard A, Margaret E K, Pauline M S，et al. Fusel oils in ciders and parries[J].Journal of Science of Food and Agriculture, 1965,16: 384-389
[99] Rajko V, Janez H. Synthesis of higher alcohols during cider pressing[J]. Food Chemistry, 1999,67:287-294
[100] Ranger R S. Van H, Luyben K C.The formation of esters and higher alcohols during brewery fermentation: the effect of carbon dioxide pressure[J]. Journal of the Institute of Brewing, 1992,98:509-513
[101] Rapp A, Mandcry N. New progress in wine and wine research[J]. Experientia, 1987,42:873-884
[102] S.Y.Chang, S.Y.Hiang, M.C.Chang, et al. Intraspecific protoplast fusion of Candida tropicalis for enhancing phenol degradation[J]. Appl. Microbiol. Biotech, 1995,43(3):534-538
[103] Sauvageot F, Vivier P. Effects of malolactic fermentation on sensory properties of four Burgundy wines[J]. American Journal of Enology and Viticulture,1997,48:187-192
[104] Schreier P, Drawert F, Schenid M C. Changes in the composition of neutral volatile
components during the production of apple brandy[J]. Journal of the Science Food and Agriculture,1978,29:728-736
[105] Sipiczki M, Ferenczy L. Protoplast fusion of Schizosaccharomyces pombe auxotrophic mutants of identical mating type[J]. Molecular and General Genetics , 1977, 151: 77-81
[106] Sulo P, Michlcakova S, Reiser V, Construction and properties of k1 type killer wine yeasts[J]. Biotechnology Letters, 1992,14(1): 55-60
[107] Williams A A, Tucknott O G. Volatile constituents of fermented cider-draught dry cider blend[J]. Journal of science Food Agriculture. 1971,22:264-269
[108] Williams A A.Flavor research and the cider industry[J].Journal of the Institute of Brewing,1974,80:455-470
[109] Williams A. A. May H. V. Examination of cider volatiles using electron impact and chemical ionisation gas chromatography mass spectrometry [J]. Journal of institute of Brewing, 1981(87):372-375
[110]Zimmermann U, Scheurich P. High frequency fusion of plants protoplasts by electric field[J]. Plantae, 1981,151: 26-32