摘要
黄浆水经酸性蛋白酶处理可以提高氨基氮含量,处理过的黄浆水作为木薯原料酒精发酵时的氮源营养物,促进了酵母生长,提高了原料出酒率。适量添加Mg~(2+)、Zn~(2+)对酵母酒精发酵有促进作用。麸曲可以作为酒精发酵促进剂。
实验结果表明:
(1)通过酸性蛋白酶酶解黄浆水的单因素实验,分别确定最佳的酶添加量为40u/mL,最佳pH为3.0、最佳酶解时间为10h、最佳反应温度为40℃。正交实验确定最佳的酶解方案为35u/mL添加酸性蛋白酶,在40℃,pH3.0条件下酶解12h。按此方案进行实验,黄浆水中氨基氮含量提高156.6%。
(2)添加黄浆水作为酒母培养的氮源优于尿素和硫酸铵。黄浆水做氮源时成熟酒母发酵液中细胞浓度比尿素做氮源时提高18%,比硫酸铵做氮源时提高14%。黄浆水做氮源培养的酒母进行酒精发酵时原料出酒率较尿素和硫酸铵分别提高2.2%和1.7%。黄浆水作为氮源营养物减轻了白酒厂废水处理的压力,又可提高酒精发酵得率,达到了变废为宝的目的。
(3)在实验室条件下,Mg~(2+)、Zn~(2+)对酵母酒精发酵有促进作用,最佳添加量分别为:250mg和500mg/L。K~+,Fe~(2+)对酒精发酵有抑制作用。
(4)添加麸曲后,酿酒酵母的耐酒精能力提高了3°。添加麸曲可使酒精缩短发酵时间6h。麸曲添加量为2%时发酵醪酒精浓度比对照(硫酸铵)提高1.1°,相对出酒率提高3.1%。
The content of amino nitrogen of yellow serofluid was improved with the action of acid protease, the processed yellow serofluid is beneficial in improving yield of ethanol when it is utilized as nutrient of nitrogen source of ethanol fermentation with cassava as the raw material. The proper addition of Mg2+、Zn2+ can promote ethanol fermentation with yeast. brankoji can be used as the promoter of ethanol fermentation.
The experimental results showed that:
(1) By single factor experiment of hydrolyzing yellow serofluid with acid protease, the optimal conditions was confirmed as follows: adding volume of enzyme was 40u/mL, pH3.0,time of enzyme hydrolysis was 10h,reaction temperature was 40℃. The orthogonal experiment showed the optimal conditions was confirmed as follows: adding volume of enzyme was 35u/mL, pH3.0,time of enzyme hydrolysis was 12h,reaction temperature was 40℃. The content of amino nitrogen in yellow serofluid was improved to 156.6% according to the experimental scheme confirmed above.
(2) The yellow serofluid used as nitrogen source of seeding yeast cultivation outmatch carbamide and ammonium bisulfite. The cell concentration in fermentation broth improved by 18% and 14% when yellow serofluid was used as nitrogen source compared with that of carbamide and ammonium bisulfite respectively. The field of ethanol improved by 2.2% and 1.7% when yellow serofluid was used as nitrogen source compared with that of carbamide and ammonium bisulfite respectively. The yellow serofluid which is a kind of waste water in distilled spirit plants can be recycleld, which not only lessen the pressure of waste water treatment in distilled spirit plants, but improve the ethanol production.
(3) Under the condition of laboratory, Mg2+and Zn2 can promote the fermentation of ethanol by yeast, and the optimal adding volume is 250 and 500mg/L respectively.enen a little bit of K+ or Fe2+ can inhibit ethanol fermentation.
(4) The ability of Saccharomyces cerevisiae tolerant to ethanol improved by 3 degree when brankoji was added. The ethanol fermentation time reduced 6h when brankoji was added. The concentration of ethanol in fermentation broth increased obviously , the concentration of ethanol improved by 1.1 degree and the relative ethanol production rate increased by 3.1% when the adding volume was 2%.
引文
[1] 黄宇彤.世界燃料酒精生产形势[J].酿酒, 2001, 28(5): 24-26
[2] 高寿清.燃料酒精发展的国际情况与分析[J].食品与发酵工业, 2001, 27(12): 59-62
[3] 黄宇彤, 杜连祥.美国的燃料酒精工业[J].酿酒科技, 2001, (5): 99-101
[4] 傅其军, 苏毅.美国酒精工业的政策、生产和收益性[J].广西轻工业, 2005, 88(3): 29-30
[5] 朱志刚.生物质能源产业发展正当其时[N].经济日报, 2006-7-5(6)
[6] 张肖克, 黄平.落实科学发展观,加快酒精产业发展步伐[J].酿酒, 2006, 33(3): 11-14
[7] 韩德奇, 李伟.燃料乙醇的生产进展和应用探讨[J].化工技术经济, 2002, 20(6): 9-15
[8] Sheehan J., Himmel M. Enzymes, energy, and environment: A strategic perspective on the U.S Department of Energy’s research and development activities for bioethanol[J]. Biotechnol Prog, 1999, 15: 817-827
[9] 陈艳萍, 勇强, 刘朝刚, 等.戊糖发酵微生物及其选育[J].纤维素科学与技术, 2001, 9(3): 57-61
[10] Olsson L., Hahn-Hagerdal B. Fermentation of lignocellulosic hydrolysates for ethanol production[J]. Enzyme and Microbiol Technol. 1996, 18: 312-331
[11] Zaldivar J., Nielsen J., Olsson L. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration[J]. Appl Microbiol Biotechnol. 2001, (56): 17-34
[12] 陈丽杰, 白凤武, WA Anderson, et al. 超高浓度培养基条件下酵母细胞生长及酒精生成的准稳态动力学研究[J].生物加工过程, 2004, 2(2): 25-29
[13] 吕欣, 毛忠贵.高浓度酒精发酵研究进展[J].酿酒科技, 2003, 5: 58-59
[14] Jones R. Specific and nonspecific inhibitory effects of ethanol on yeast growth[J]. Enzyme Microb Technol, 1986(9): 334-338
[15] Ohta K. Hamada S. Saccharomyces cerevisiae membrane sterol modificarions in response to growth in the presence of ethanol[J]. Applied and Environmental Microbiology, 1990, 56(9): 2853-2857
[16] Fukuda, Hisashi, Par, et al. Sake brewing characteristics and multidrug resistance of trichothecin-resistant yeast mutants[J]. World Journal of Microbiology and Biotechnoloy, 1999, 15(5): 629-631
[17] Luo xinpeng. Continuous ethanol fermentation using very high gravity media andbioreactor engineering strategies[J]. Dalian University of Technology, 2005: 22-33
[18] Boulton R. B. The prediction of fermentation behavior by a kinetic model[J]. Appl Environ Microbiol, 1980, 31: 40-45
[19] Boulton R. B., Singleton V. L., Bisson L. F., et al. Principles and Practices of Winemaking[J]. Appl Biochem Biotechnol, 2001, 42: 53-55
[20] Egbosimba, E. E., Linus E., Okafor., et al. Control of ammonia uptake from malt extract medium by Saccharomyces cervisiae[J]. Inst Brew. 1988, 94: 249-252
[21] Egbosimba, E. E., and j C.Slaughter. The influence of ammonium permease activity and carbon source onthe uptake of ammonium from simple defined media by saccharomyces cervisiae[J]. Gen Micro. 1987, 133: 375-379
[22] Thomas K. C., Ingledew W. M. Fuel alcohol production: effects of free amino nitrogen of fermentation of very high gravity wheat mashes[J]. Appl Environ Microbiol, 1990, 56: 2046-2050
[23] Thomas K. C., Ingledew W. M. Production of 21%(v/v) ethanol by fermentation of very high gravity (VHG) wheat mashes[J]. Inc. Microbiol, 1992, 10: 61-68
[24] 黄宇彤, 杜连祥.玉米原料高浓度酒精发酵中试的研究[J].天津轻工业学院学报, 2002, (1): 56-58
[25] Ingeldew W. M. Kunkee R. E. Factors influence sluggish fermentation of grape juice[J].Enol Viticult, 1985, 36: 65-76
[26] McCaig R., Mckee J., Pifisterer, E. A.,et al. Very high gravity brewing-laboratory and pilot plant trials[J]. Am Soc Brew Chem, 1992, 50: 18-26
[27] Casey G. P., Magnus C. A., Ingledew, W.M. High gravity brewing: effects of nutrition on yeast composition fermentative ability and alcohol production[J]. Appl Environ Microbiol, 1984, 48: 639-46
[28] Casey G. P, Magnus C. A., Ingledew, W. M. High gravity brewing: nutrient enhanced production of high concentration of ethanol by brewing yeast[J]. Biotechnology Lett, 1983, 5:429-434
[29] Thomas K. C., Ingledew W. M. Fuel alcohol production: effects of free amino nitrogen of fermentation of very high gravity wheat mashes[J]. Appl Environ Microbiol, 1990, 56: 2046-2050
[30] Thomas K. C., Hynes S. H., Jones A.M, et al. Production of fuel alcohol from wheat by VHG technology: effect of sugar concentration and temperature[J]. Appl Biochem Biotechnol, 1993, 43: 211-216
[31] Jones A. M., Ingledew W. M. Fuel alcohol production: optimization of temperature for efficient very high gravity fermentation[J]. Appl Environ Microbiol., 1994, 60:1048-1051
[32] Ingledew W.M., Jones A. M., Bhattry R. S, et al. Fuel alcohol production from hullless barley[J]. Cereal Chem, 1995, 72: 147-150
[33] 吕欣, 毛忠贵.高浓度酒精发酵研究进展[J].酿酒科技, 2003, 5: 58-59
[34] Alfenore S. C., Molina-Jouve S. E. Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process[J]. Appl Microbiol Biotechnol, 2002, 60: 67-72
[35] 胡纯铿, 白凤武, 安利佳.Mg2+和 Ca2+对自絮凝颗粒酵母耐酒精性能的影响及作用机制的比较研究[J].高效化学工程学报, 2004, 18(2): 179-184
[36] Jones R. P., Greenfield P. F. A review of yeast ionic nutrition: growth and fermentation requirements[J]. Process Biochem, 1984, 19: 48-60
[37] Dombek K. M., Ingraim I. O. Magnesium limitation and its role in the apparent toxicity of ethanol during yeast fermentation[J]. Appl Environ Microbiol, 1986, 52: 975-981
[38] 胡纯铿, 白凤武, 安利佳.细胞膜蛋白质氨基酸组成对自絮凝酵母耐酒精能力的影响及作用机制[J] .微生物学报, 2004, 44(5 ): 636-640
[39] Petra Bafrncova, Daniela Smogrovicova. Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae[J]. Biotechnology letters, 1999, 21: 337-341
[40] Pena A., Pardo J. P. Early metabolic effects and mechanism of ammonium transport in yeast[J]. Arch Biochem Biophys.1987, 253: 431-438
[41] Lagunas R. C., Dominguez A. Mechanisms of appearance of the Pasteur effects in Saccharomyces cerevisiae inactivation of the sugar transport systems[J]. Bacteriol, 1982, 152: 19-25
[42] Salmon J. M. Effect of sugar transport inactivation in Saccharomyces cerevisiae at low and intermediate growth temperatures[J]. Biotech, 1989, 28: 301-303
[43] 肖冬光.提高淀粉质原料酒精浓醪发酵酒精度的方法 [P].中国专利 : 200410094105.4, 2004.12.30
[44] 赵江, 赵华.木薯酒精发酵条件优化[J] .酿酒, 2003, 30(2): 76-78
[45] 徐铁军, 赵心清, 周友超, 等.自絮凝酵母 SPSC01 在组合反应器系统中酒精连续发酵的研究[J].生物工程学报, 2005, 21(1): 113-117
[46] 严正, 孜力汗, 李宁, 等.多级串联悬浮床反应器系统中自絮凝颗粒酵母乙醇连续发酵耦合废糟液直接全循环使用的研究[J].生物工程学报, 2005, 24(4): 628-632
[47] 葛旭萌, 白凤武.乙醇连续发酵过程中自絮凝酵母颗粒粒径分布的模拟[J].化工学报, 2006, 57(4): 892-897
[48] 黄宇彤, 伍松陵, 杜连祥.玉米酒精超高浓度发酵工艺条件的优化[J].食品工业 科技, 2002, 123(8): 67-69
[49] 张建华, 段作营, 李永飞, 等.酒精蒸馏废液全循环工艺研究[J].食品与发酵工业, 2006, 32(4): 31-34
[50] 吕欣, 段作营, 毛忠贵.高基质浓度酒精发酵过程中副产物的研究[J].食品科技, 2003, 8: 89-91
[51] 赵新淮, 冯新彪.蛋白质水解物水解度的测定[J].食品科学, 1994, 11: 65-67
[52] Sampermans S., Mortier J., Soares E.V. Flocculation onset in Saccharomyces cerevisiae: the role of nutrients[J]. Appl Microbiol, 2005, 98: 525-531
[53] Soares E. V., Duarte A. A. Addition of nutrients induce a fast loss of flocculation in starved ce1ls of Saccharomyces cerevisiae[J]. Biotechnology Letters. 2002, 24:1957-1960
[54] 董霞, 李崎, 顾国贤.啤酒有机酸类物质研究进展[J].酿酒, 2003, 30(6):63-65
[55] 肖冬光.酒精废液回用理论的探讨[J].酿酒科技,2001, (6) 76-78
[56] 孙建义, 许梓荣.微量元素影响酵母生长的响应曲面分析[J].中国粮油学报, 1998,6: 52-54
[57] 魏述众主编.生物化学[M].北京:中国轻工业出版社, 1996: 235
[58] 张书祥, 肖亚中, 任杰.添加营养盐对酒精酵母发酵的影响[J]生物学杂志, 1997,14(1): 23-25