利用白地霉提高低发芽率大麦溶解性能的研究
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摘要
大麦是啤酒酿造的重要原料之一,占据啤酒原料成本相当重要的一部分。本论文针对部分国产低发芽率大麦溶解性能较差的缺点,利用白地霉提高其溶解性能。
在制麦过程中分别接种5株具有高水解酶活的白地霉,均能提高麦芽的溶解,特别是胚乳细胞壁的降解;接种G. candidum JN-4其成品麦芽的溶解指标最佳, G. candidum JN-4液态培养表明:以5oP麦汁为培养基,初始pH值为5.5,接种孢子浓度为2 % ,装液量为50 mL/250 mL,摇床转速为200 r/min,30℃培养40 h,菌体浓度可以达到5×108个/mL的最大值。
研究了制麦过程中真菌的变化。在浸麦和发芽阶段,所有的真菌微生物的总量都有明显的增加,发芽最后一天达到最大值。在焙燥阶段,真菌菌群水平下降,但并没有完全消失;制麦过程中添加白地霉能明显改变真菌群落的构成。初步探讨了微生物制麦的作用机制:浸麦初期,白地霉菌株利用接种时的菌体浓度优势,自身吸附到麦芽表面;随着制麦进行,白地霉菌株利用自身的生长优势,占据大麦表面,从而抑制其他微生物的生长代谢。发芽期间,利用自身菌丝体穿透大麦表皮,增强大麦的穿透性,并协助大麦胚乳细胞壁的降解。
以G. candidum JN-4为接种微生物,对甘肃省高海拔种植区的低发芽率大麦甘啤3号进行了微生物制麦工艺的研究。结果表明:第1次浸麦时接种104个/g大麦的菌体,提高浸麦水温度至20℃,浸麦方式采用浸5断10浸3断6浸1,发芽采用升温发芽(14℃24 h,16℃24 h,18℃24 h,20℃24 h),焙焦条件为30℃5 h,45℃5 h,50℃4 h ,60℃6 h,70℃2 h,83℃3 h工艺明显促进微生物的作用,微生物麦芽中能反应大麦胚乳细胞壁降解的粗细粉差、粘度和β-葡聚糖3个指标得到明显改善,与对照相比分别下降:10.0 %,22.4 %,36.4 %。
Barley was the major raw material used for beer brewing and it was also quite a most important part of the cost. Domestic barley had inferior characterwastics in both germination rate and malt modification. Improving the solubility of low germination rate malt by using Geotrichum candidau microorganism-malting was studied in this paper.
Five strains of Geotrichum candida with high extracellular hydrolase were inoculated in the malting process. The results showed that all the strains mentioned above were beneficial to the solubility of malt, especially the degradation of endosperm cell wall. G. candidum JN-4 was chosen as the best for microorganwasm-malting. The optimiziation of condition for spore production was studied. The optimum condition were: the medium was 5 oP wort, the initial pH was 5.5, the spores of inoculation concentration was 2 %, the volume of medium was 50 mL in 250 mL flask, the shaking speed was 200 r/min at 30℃and the culture time was 40 h. The spore yield has been reached 5×108/mL.
The changes of fungi contamination during mlating processe were studied. The amount of all fungi increased during the steeping and germination stage, with its maximum on the fianl day of germination, and partly decreased to a certain level at baking stage. Adding Geotrichum candida in malting could change the composition of fungi communities. The mechanism of microorganism-malting was preliminary studied as follows: Geotrichum candida was adsorbed to barley surface firstly due to its high inoculation concentration. Then Geotrichum candida increased rapidly and occupied the barley surface. As a consequence, the growth of other microorganisms was inhibited. During germinating stage, Geotrichum candida penetrated barley surface with its mycelium, which increased penetrability of barley and helped degradation of endosperm cell wall.
The microorganism-malting technique had been applied to domestic barley Ganpi-3 which had low germination rate. It was showed that the optimal inoculation concentration of Geotrichum candida spore was 104/g dry barley in the first steeping water at 20℃. Long steeping technology was as follows: 5 hours of water steeping, 10 hours of air rest, 3 hours of water steeping, 6 hours of air rest and 1 hour of water steeping.
Calefactive germinationg technology: 14℃24 h,16℃24 h,18℃24 h,20℃24 h and kilning technology: 30℃5 h,45℃5 h,50℃4 h ,60℃6 h,70℃2 h,83℃3 h were adopt. The process could obviously promote effect of Geotrichum candida on barley and especially f/c extract difference, viscosity and 36.4 % which were the index reflecting the degradation of barley endosperm cell wall.Compared with the control,they decreased by 10.0 % , 22.4 % and 36.4 % respectively.
在制麦过程中分别接种5株具有高水解酶活的白地霉,均能提高麦芽的溶解,特别是胚乳细胞壁的降解;接种G. candidum JN-4其成品麦芽的溶解指标最佳, G. candidum JN-4液态培养表明:以5oP麦汁为培养基,初始pH值为5.5,接种孢子浓度为2 % ,装液量为50 mL/250 mL,摇床转速为200 r/min,30℃培养40 h,菌体浓度可以达到5×108个/mL的最大值。
研究了制麦过程中真菌的变化。在浸麦和发芽阶段,所有的真菌微生物的总量都有明显的增加,发芽最后一天达到最大值。在焙燥阶段,真菌菌群水平下降,但并没有完全消失;制麦过程中添加白地霉能明显改变真菌群落的构成。初步探讨了微生物制麦的作用机制:浸麦初期,白地霉菌株利用接种时的菌体浓度优势,自身吸附到麦芽表面;随着制麦进行,白地霉菌株利用自身的生长优势,占据大麦表面,从而抑制其他微生物的生长代谢。发芽期间,利用自身菌丝体穿透大麦表皮,增强大麦的穿透性,并协助大麦胚乳细胞壁的降解。
以G. candidum JN-4为接种微生物,对甘肃省高海拔种植区的低发芽率大麦甘啤3号进行了微生物制麦工艺的研究。结果表明:第1次浸麦时接种104个/g大麦的菌体,提高浸麦水温度至20℃,浸麦方式采用浸5断10浸3断6浸1,发芽采用升温发芽(14℃24 h,16℃24 h,18℃24 h,20℃24 h),焙焦条件为30℃5 h,45℃5 h,50℃4 h ,60℃6 h,70℃2 h,83℃3 h工艺明显促进微生物的作用,微生物麦芽中能反应大麦胚乳细胞壁降解的粗细粉差、粘度和β-葡聚糖3个指标得到明显改善,与对照相比分别下降:10.0 %,22.4 %,36.4 %。
Barley was the major raw material used for beer brewing and it was also quite a most important part of the cost. Domestic barley had inferior characterwastics in both germination rate and malt modification. Improving the solubility of low germination rate malt by using Geotrichum candidau microorganism-malting was studied in this paper.
Five strains of Geotrichum candida with high extracellular hydrolase were inoculated in the malting process. The results showed that all the strains mentioned above were beneficial to the solubility of malt, especially the degradation of endosperm cell wall. G. candidum JN-4 was chosen as the best for microorganwasm-malting. The optimiziation of condition for spore production was studied. The optimum condition were: the medium was 5 oP wort, the initial pH was 5.5, the spores of inoculation concentration was 2 %, the volume of medium was 50 mL in 250 mL flask, the shaking speed was 200 r/min at 30℃and the culture time was 40 h. The spore yield has been reached 5×108/mL.
The changes of fungi contamination during mlating processe were studied. The amount of all fungi increased during the steeping and germination stage, with its maximum on the fianl day of germination, and partly decreased to a certain level at baking stage. Adding Geotrichum candida in malting could change the composition of fungi communities. The mechanism of microorganism-malting was preliminary studied as follows: Geotrichum candida was adsorbed to barley surface firstly due to its high inoculation concentration. Then Geotrichum candida increased rapidly and occupied the barley surface. As a consequence, the growth of other microorganisms was inhibited. During germinating stage, Geotrichum candida penetrated barley surface with its mycelium, which increased penetrability of barley and helped degradation of endosperm cell wall.
The microorganism-malting technique had been applied to domestic barley Ganpi-3 which had low germination rate. It was showed that the optimal inoculation concentration of Geotrichum candida spore was 104/g dry barley in the first steeping water at 20℃. Long steeping technology was as follows: 5 hours of water steeping, 10 hours of air rest, 3 hours of water steeping, 6 hours of air rest and 1 hour of water steeping.
Calefactive germinationg technology: 14℃24 h,16℃24 h,18℃24 h,20℃24 h and kilning technology: 30℃5 h,45℃5 h,50℃4 h ,60℃6 h,70℃2 h,83℃3 h were adopt. The process could obviously promote effect of Geotrichum candida on barley and especially f/c extract difference, viscosity and 36.4 % which were the index reflecting the degradation of barley endosperm cell wall.Compared with the control,they decreased by 10.0 % , 22.4 % and 36.4 % respectively.
引文
[1]酿酒工业协会啤酒分会2006年中国酿酒工业协会啤酒分会工作报告. 2007.
[2]刘玉春,许如根,吕超等.增强国产啤酒大麦在国内啤麦原料市场竞争力的几点建议[J].大麦科学, 2003 11: 46～47.
[3]尚雪英.甘肃啤酒大麦产业的比较优势及竞争力分析[J]. 2007, 23(10).
[4]李崎译. Malting and Brewing Science [M].北京:中国轻工业出版社, 2005.
[5] Edney M J, Eglinton J K, Collins H M, et al. Importance of endosperm modification for malt Wort fermentability [J]. Journal of the Institute of Brewing, 2007, 113: 228-238.
[6] Henry R J. Factors Influencing the Rate of Modification of Barleys During Malting [J]. J Cereal Sci, 1989, 10(1): 51-59.
[7] Laitila A, Kotaviita E, Peltola P, et al. Indigenous microbial community of barley greatly influences grain germination and malt quality [J]. Journal of the Institute of Brewing, 2007, 113(1): 9-20.
[8] Gyllang H, Kjellen K, Haikara A, et al. Evaluation of Fungal Contaminations on Barley and Malt [J]. Journal of the Institute of Brewing, 1981, 87(4): 248-251.
[9] Linko M, Haikara A, Ritala A, et al. Recent advances in the malting and brewing industry [J]. J Biotechnol, 1998, 65(2-3): 85-98.
[10] Etchevers G C, Banasik O J and Watson C A. Microflora of barley and its effects on malt and beer properties: a review [J]. Brewers' Digest, 1977, 52(1): 46-50, 53.
[11] Vaughan A, O'Sullivan T and van Sinderen D. Enhancing the microbiological stability of malt and beer - A review [J]. Journal of the Institute of Brewing, 2005, 111(4): 355-371.
[12] Lefyedi M L and Taylor J R N. Control of the growth of coliforms and moulds in sorghum malting by bacterial and yeast cultures [J]. Journal of the Institute of Brewing, 2007, 113: 123-129.
[13] Yin X S, Macgregor A W and Clear R M. Field Fungi and Beta-Glucan Solubilase in Barley Kernels [J]. Journal of the Institute of Brewing, 1989, 95(3): 195-198.
[14] Flanniga.B. Degradation of Arabinoxylan and Carboxymethyl Cellulose by Fungi Isolated from Barley Kernels [J]. Transactions of the British Mycological Society, 1970, 55(OCT): 277-&.
[15] Flanniga.B and Sellars P N. Activities of Thermophilous Fungi from Barley Kernels against Arabinoxylan and Carboxymethyl Cellulose [J]. Transactions of the British Mycological Society, 1972, 58(APR): 338-&.
[16] Hoy J L, Macauley B J and Fincher G B. Cellulases of Plant and Microbial Origin in Germinating Barley [J]. Journal of the Institute of Brewing, 1981, 87(2): 77-80.
[17] Boivin P and Malanda M. Influence of starter cultures in malting on the microflora development and malt quality [J]. Journal of the Institute of Brewing, 1993, 99(3): 207.
[18] Noots I, Derycke V, Cornelis K, et al. Degradation of starchy endosperm cell walls in nongerminating sterilized barley by fungi [J]. J Agric Food Chem, 2001, 49(2): 975-981.
[19] Noots I, Derycke V, Jensen H E, et al. Studies on barley starchy endosperm cell wall degradation by Rhizopus VII [J]. J Cereal Sci, 2003, 37(1): 81-90.
[20] Noots I, Derycke V, Michiels C, et al. Improvement of malt modification by use of Rhizopus VII as starter culture [J]. J Agric Food Chem, 2001, 49(8): 3718-3724.
[21] Haikara A and Laitila A. Influence of lactic acid starter cultures on the quality of malt and beer [J]. Journal of the Institute of Brewing, 1995, 101(3): 163.
[22] Lowe D P, Arendt E K, Soriano A M, et al. The influence of lactic acid bacteria on the quality of malt [J]. Journal of the Institute of Brewing, 2005, 111(1): 42-50.
[23] Hang Y D and Woodams E E. Production and Characterization of Polygalacturonase from Geotrichum-Candidum [J]. World J Microbiol Biotechnol, 1992, 8(5): 480-482.
[24] Boivin P. Interest in rapid methods for the detection of mould spoilage on malting barley [J]. Stored malting barley: management of quality using an expert system, Reims, France, 28-29 May, 2001, 2005: 185-193.
[25] Boivin P and Malanda M. Improvement of malt quality and safety by adding starter culture during the malting process [J]. Technical Quarterly, Master Brewers' Association of the Americas, 1997, 34(2): 96-101.
[26] Boivin P, Malanda M and Dumoulin M. Improving the quality of malting barley by employing microbial starter cultures in the field [J]. Journal of the Institute of Brewing, 1999, 105(3): 145.
[27] Baxter E D, Slaiding I R and Kelly B. Behavior of ochratoxin A in brewing [J]. J Am Soc Brew Chem, 2001, 59(3): 98-100.
[28]中国科学院微生物研究所.中国菌种目录[M].北京:中国科学出版社, 1988.
[29] Foszczynska B, Dziuba E and Stempniewicz R. The use of Geotrichum candidum starter culture for protection of barley and its influence on biotechnological qualities of malts [J]. Electronic Journal of Polish Agricultural Universities, 2004, 7(2).
[30] Schwarz W H, Schimming S and Staudenbauer W L. Degradation of Barley Beta-Glucan by Endoglucanase-C of Clostridium-Thermocellum [J]. Applied Microbiology and Biotechnology, 1988, 29(1): 25-31.
[31]孙淑琴,邵冬梅.比色法快速测定糖化酶活力新方法[J].河北省科学院学报, 1997, (2): 35-39.
[32] Bailey M J, Biely P and Poutanen K. Interlaboratory Testing of Methods for Assay of Xylanase Activity [J]. J Biotechnol, 1992, 23(3): 257-270.
[33]诸葛健.工业微生物实验技术手册[M].中国轻工业出版社, 1994.
[34]蔡定域.酿酒工业分析手册[M].轻工业出版社, 1988.
[35] Piegza M, Witkowska D, Stempniewicz R, et al. Geotrichum hydrolytic activity in milled malt and barley medium [J]. Electronic Journal of Polish Agricultural Universities, 2005, 8(1).
[36]梁刚.啤酒大麦和麦芽微生物对啤酒质量的影响[J].酿酒科技, 1999, 91((1)): 56-57.
[37] Noots I, Delcour J A and Michiels C W. From field barley to malt: Detection and specification of microbial activity for quality aspects [J]. Crit Rev Microbiol, 1999, 25(2): 121-153.
[38]朱衡,翟峰,朱立煌.利用氯化苄提取适于分子生物学分析的真菌DNA [J].真菌学报, 1994, (1): 34-40.
[39] Bryan G T, Daniels M J and Osbourn A E. Comparison of Fungi within the Gaeumannomyces-Phialophora Complex by Analysis of Ribosomal DNA-Sequences [J]. Appl Environ Microbiol, 1995, 61(2): 681-689.
[40]李永仙,尹象胜,顾国贤等.刚果红法测定麦汁和啤酒中的β-葡聚糖[J].无锡轻工大学学报, 1997, 16: 8-13.
[2]刘玉春,许如根,吕超等.增强国产啤酒大麦在国内啤麦原料市场竞争力的几点建议[J].大麦科学, 2003 11: 46～47.
[3]尚雪英.甘肃啤酒大麦产业的比较优势及竞争力分析[J]. 2007, 23(10).
[4]李崎译. Malting and Brewing Science [M].北京:中国轻工业出版社, 2005.
[5] Edney M J, Eglinton J K, Collins H M, et al. Importance of endosperm modification for malt Wort fermentability [J]. Journal of the Institute of Brewing, 2007, 113: 228-238.
[6] Henry R J. Factors Influencing the Rate of Modification of Barleys During Malting [J]. J Cereal Sci, 1989, 10(1): 51-59.
[7] Laitila A, Kotaviita E, Peltola P, et al. Indigenous microbial community of barley greatly influences grain germination and malt quality [J]. Journal of the Institute of Brewing, 2007, 113(1): 9-20.
[8] Gyllang H, Kjellen K, Haikara A, et al. Evaluation of Fungal Contaminations on Barley and Malt [J]. Journal of the Institute of Brewing, 1981, 87(4): 248-251.
[9] Linko M, Haikara A, Ritala A, et al. Recent advances in the malting and brewing industry [J]. J Biotechnol, 1998, 65(2-3): 85-98.
[10] Etchevers G C, Banasik O J and Watson C A. Microflora of barley and its effects on malt and beer properties: a review [J]. Brewers' Digest, 1977, 52(1): 46-50, 53.
[11] Vaughan A, O'Sullivan T and van Sinderen D. Enhancing the microbiological stability of malt and beer - A review [J]. Journal of the Institute of Brewing, 2005, 111(4): 355-371.
[12] Lefyedi M L and Taylor J R N. Control of the growth of coliforms and moulds in sorghum malting by bacterial and yeast cultures [J]. Journal of the Institute of Brewing, 2007, 113: 123-129.
[13] Yin X S, Macgregor A W and Clear R M. Field Fungi and Beta-Glucan Solubilase in Barley Kernels [J]. Journal of the Institute of Brewing, 1989, 95(3): 195-198.
[14] Flanniga.B. Degradation of Arabinoxylan and Carboxymethyl Cellulose by Fungi Isolated from Barley Kernels [J]. Transactions of the British Mycological Society, 1970, 55(OCT): 277-&.
[15] Flanniga.B and Sellars P N. Activities of Thermophilous Fungi from Barley Kernels against Arabinoxylan and Carboxymethyl Cellulose [J]. Transactions of the British Mycological Society, 1972, 58(APR): 338-&.
[16] Hoy J L, Macauley B J and Fincher G B. Cellulases of Plant and Microbial Origin in Germinating Barley [J]. Journal of the Institute of Brewing, 1981, 87(2): 77-80.
[17] Boivin P and Malanda M. Influence of starter cultures in malting on the microflora development and malt quality [J]. Journal of the Institute of Brewing, 1993, 99(3): 207.
[18] Noots I, Derycke V, Cornelis K, et al. Degradation of starchy endosperm cell walls in nongerminating sterilized barley by fungi [J]. J Agric Food Chem, 2001, 49(2): 975-981.
[19] Noots I, Derycke V, Jensen H E, et al. Studies on barley starchy endosperm cell wall degradation by Rhizopus VII [J]. J Cereal Sci, 2003, 37(1): 81-90.
[20] Noots I, Derycke V, Michiels C, et al. Improvement of malt modification by use of Rhizopus VII as starter culture [J]. J Agric Food Chem, 2001, 49(8): 3718-3724.
[21] Haikara A and Laitila A. Influence of lactic acid starter cultures on the quality of malt and beer [J]. Journal of the Institute of Brewing, 1995, 101(3): 163.
[22] Lowe D P, Arendt E K, Soriano A M, et al. The influence of lactic acid bacteria on the quality of malt [J]. Journal of the Institute of Brewing, 2005, 111(1): 42-50.
[23] Hang Y D and Woodams E E. Production and Characterization of Polygalacturonase from Geotrichum-Candidum [J]. World J Microbiol Biotechnol, 1992, 8(5): 480-482.
[24] Boivin P. Interest in rapid methods for the detection of mould spoilage on malting barley [J]. Stored malting barley: management of quality using an expert system, Reims, France, 28-29 May, 2001, 2005: 185-193.
[25] Boivin P and Malanda M. Improvement of malt quality and safety by adding starter culture during the malting process [J]. Technical Quarterly, Master Brewers' Association of the Americas, 1997, 34(2): 96-101.
[26] Boivin P, Malanda M and Dumoulin M. Improving the quality of malting barley by employing microbial starter cultures in the field [J]. Journal of the Institute of Brewing, 1999, 105(3): 145.
[27] Baxter E D, Slaiding I R and Kelly B. Behavior of ochratoxin A in brewing [J]. J Am Soc Brew Chem, 2001, 59(3): 98-100.
[28]中国科学院微生物研究所.中国菌种目录[M].北京:中国科学出版社, 1988.
[29] Foszczynska B, Dziuba E and Stempniewicz R. The use of Geotrichum candidum starter culture for protection of barley and its influence on biotechnological qualities of malts [J]. Electronic Journal of Polish Agricultural Universities, 2004, 7(2).
[30] Schwarz W H, Schimming S and Staudenbauer W L. Degradation of Barley Beta-Glucan by Endoglucanase-C of Clostridium-Thermocellum [J]. Applied Microbiology and Biotechnology, 1988, 29(1): 25-31.
[31]孙淑琴,邵冬梅.比色法快速测定糖化酶活力新方法[J].河北省科学院学报, 1997, (2): 35-39.
[32] Bailey M J, Biely P and Poutanen K. Interlaboratory Testing of Methods for Assay of Xylanase Activity [J]. J Biotechnol, 1992, 23(3): 257-270.
[33]诸葛健.工业微生物实验技术手册[M].中国轻工业出版社, 1994.
[34]蔡定域.酿酒工业分析手册[M].轻工业出版社, 1988.
[35] Piegza M, Witkowska D, Stempniewicz R, et al. Geotrichum hydrolytic activity in milled malt and barley medium [J]. Electronic Journal of Polish Agricultural Universities, 2005, 8(1).
[36]梁刚.啤酒大麦和麦芽微生物对啤酒质量的影响[J].酿酒科技, 1999, 91((1)): 56-57.
[37] Noots I, Delcour J A and Michiels C W. From field barley to malt: Detection and specification of microbial activity for quality aspects [J]. Crit Rev Microbiol, 1999, 25(2): 121-153.
[38]朱衡,翟峰,朱立煌.利用氯化苄提取适于分子生物学分析的真菌DNA [J].真菌学报, 1994, (1): 34-40.
[39] Bryan G T, Daniels M J and Osbourn A E. Comparison of Fungi within the Gaeumannomyces-Phialophora Complex by Analysis of Ribosomal DNA-Sequences [J]. Appl Environ Microbiol, 1995, 61(2): 681-689.
[40]李永仙,尹象胜,顾国贤等.刚果红法测定麦汁和啤酒中的β-葡聚糖[J].无锡轻工大学学报, 1997, 16: 8-13.