酒酒球菌(Oenococcus oeni)耐酸突变株苹果酸-乳酸发酵能力分析
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摘要
目的:研究酒酒球菌(Oenococcus oeni)耐酸突变菌株的抗胁迫能力和苹果酸-乳酸发酵能力,为耐酸突变菌株开发为商业发酵剂提供参考。方法:以采用离子注入诱变,分离纯化后筛选出耐酸突变菌株b1为研究对象,酒酒球菌SX-1b和商业菌株31-DH为对照,探究单因素胁迫环境、复合因素胁迫条件及模拟酒环境对菌株b1生长能力、L-苹果酸降解速率和β-葡萄糖苷酶活性的影响,评价菌株b1的苹果酸-乳酸发酵能力。结果:单因素试验结果显示,当pH 3.0、乙醇体积分数14%、L-苹果酸质量浓度3 g/L时,菌株b1的L-苹果酸降解速率和β-葡萄糖苷酶活性均高于其余菌株;正交试验进一步确定各因素对菌株生长能力、L-苹果酸降解速率和β-葡萄糖苷酶活性的影响程度为:pH值>乙醇体积分数>L-苹果酸质量浓度;当模拟酒的乙醇体积分数为14%时,菌株b1的累积L-苹果酸降解量为1.493 2 g/L,分别为SX-1b与31-DH的1.41倍和1.26倍,且菌株b1的β-葡萄糖苷酶活性最高。结论:耐酸突变菌株b1表现出良好抗胁迫能力和苹果酸-乳酸发酵能力。因此,菌株?b1具有成为商业发酵剂的潜能。
Objective: This study aimed to evaluate the stress resistance and malolactic fermentation ability of an acidtolerance mutant strain of Oenococcus oeni for the purpose of providing a basis for the development of commercial wine fermentation starters. Methods: A acid-tolerant mutant b1, obtained by N~+ ion implantation mutagenesis, was tested for the effect of various environmental stress factors and their combinations as well as wine-model conditions on its growth, L-malic acid degradation rate and β-glycosidase activity. Furthermore, we assessed the malolactic fermentation (MLF) ability of mutant b1. Results: The L-malic acid degradation rate and β-glycosidase activity of b1 were higher than those of the other strains under the conditions: pH 3.0, alcohol concentration of 14% and malic acid concentration of 3 g/L. pH had the greatest influence on the growth, L-malic acid degradation rate and β-glycosidase activity of b1, alcohol concentration was in the middle, and L-malic acid concentration was least effective. When the alcohol concentration of the model wine was 14%, the L-malic acid consumption of strain b1 was 1.493 2 g/L, which was 1.41 and 1.26 times as high as that of O. oeni SX-1b and 31-DH, respectively, and it had the highest β-glucosidase activity. Conclusion: Acid-tolerant mutant b1 has good stress resistance and malolactic fermentation ability. Therefore, it has the potential to be used as a commercial starter.
Objective: This study aimed to evaluate the stress resistance and malolactic fermentation ability of an acidtolerance mutant strain of Oenococcus oeni for the purpose of providing a basis for the development of commercial wine fermentation starters. Methods: A acid-tolerant mutant b1, obtained by N~+ ion implantation mutagenesis, was tested for the effect of various environmental stress factors and their combinations as well as wine-model conditions on its growth, L-malic acid degradation rate and β-glycosidase activity. Furthermore, we assessed the malolactic fermentation (MLF) ability of mutant b1. Results: The L-malic acid degradation rate and β-glycosidase activity of b1 were higher than those of the other strains under the conditions: pH 3.0, alcohol concentration of 14% and malic acid concentration of 3 g/L. pH had the greatest influence on the growth, L-malic acid degradation rate and β-glycosidase activity of b1, alcohol concentration was in the middle, and L-malic acid concentration was least effective. When the alcohol concentration of the model wine was 14%, the L-malic acid consumption of strain b1 was 1.493 2 g/L, which was 1.41 and 1.26 times as high as that of O. oeni SX-1b and 31-DH, respectively, and it had the highest β-glucosidase activity. Conclusion: Acid-tolerant mutant b1 has good stress resistance and malolactic fermentation ability. Therefore, it has the potential to be used as a commercial starter.
引文
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[5]PAPAGIANNI M.Metabolic engineering of lactic acid bacteria for the production of industrially important compounds[J].Computational and Structural Biotechnology Journal,2012,3(4):1-7.DOI:10.5936/csbj.201210003.
[6]MICHLMAYR H,NAUER S,BRANDES W,et al.Release of wine monoterpenes from natural precursors by glycosidases from Oenococcus oeni[J].Food Chemistry,2012,135(1):80-87.DOI:10.1016/j.foodchem.2012.04.099.
[7]张明霞,吴玉文,段长青.葡萄与葡萄酒香气物质研究进展[J].中国农业科学,2008,41(7):2098-2104.DOI:10.3864/j.issn.0578-1752.2008.07.030.
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[9]张春芝,江志国.微生物对葡萄酒香气的影响综述[J].中国酿造,2013,32(9):28-31.DOI:10.3969/j.issn.0254-5071.2013.09.008.
[10]GRIMALDI A,BARTOWSKY E,JIRANEK V.Screening of Lactobacillus spp.and Pediococcus spp.for glycosidase activities that are important in oenology[J].Journal of Applied Microbiology,2005,99(5):1061-1069.DOI:10.1111/j.1365-2672.2005.02707.x.
[11]GAGNE S,LUCAS P M,PERELLO M C,et al.Variety and variability of glycosidase activities in an Oenococcus oeni strain collection tested with synthetic and natural substrates[J].Journal of Applied Microbiology,2011,110(1):218-228.DOI:10.1111/j.1365-2672.2010.04878.x.
[12]LI Y H,MA Y H,FAN M T,et al.Assessment of beta-D-glucosidase activity of intact cells of two Oenococcus oeni strains with synthetic and natural substrates[J].Czech Journal of Food Sciences,2016,34(1):16-23.DOI:10.17221/216/2015-CJFS.
[13]UGLIANO M,MOIO L.The influence of malolactic fermentation and Oenococcus oeni strain on glycosidic aroma precursors and related volatile compounds of red wine[J].Journal of the Science of Food and Agriculture,2006,86(14):2468-2476.DOI:10.1002/jsfa.2650.
[14]SOLIERI L,GENOVA F,DE PAOLA M,et al.Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations:a framework for selection of new starter cultures[J].Journal of Applied Microbiology,2010,108(1):285-298.DOI:10.1111/j.1365-2672.2009.04428.x.
[15]FIA G,MILLARINI V,GRANCHI L,et al.Beta-glucosidase and esterase activity from Oenococcus oeni:screening and evaluation during malolactic fermentation in harsh conditions[J].LWT-Food Science and Technology,2018,89:262-268.DOI:10.1016/j.lwt.2017.10.060.
[16]BRAVO-FERRADA B M,HOLLMANN A,BRIZUELA N,et al.Growth and consumption of L-malic acid in wine-like medium by acclimated and non-acclimated cultures of Patagonian Oenococcus oeni strains[J].Folia Microbiologica,2016,61(5):365-373.DOI:10.1007/s12223-016-0446-y.
[17]陈其玲,任晓宁,王玲,等.酒酒球菌β-葡萄糖苷酶活性与耐酸胁迫能力的相关性分析[J].食品科学,2017,38(2):115-120.DOI:10.7506/spkx1002-6630-201702019.
[18]JIN G,WANG H,ZHANG C H,et al.Characterization and amino acid metabolism performances of indigenous Oenococcus oeni isolated from Chinese wines[J].European Food Research and Technology,2014,238(4):597-605.DOI:10.1007/s00217-013-2112-9.
[19]杨世玲,刘叶,薛楚然,等.酒酒球菌β-葡萄糖苷酶基因克隆及生物信息学分析[J].中国食品学报,2017,17(7):199-207.DOI:10.16429/j.1009-7848.2017.07.025.
[20]BARBAGALLO R N,SPAGNA G,PALMERI R,et al.Assessment ofβ-glucosidase activity in selected wild strains of Oenococcus oeni for malolactic fermentation[J].Enzyme and Microbial Technology,2004,34(3/4):292-296.DOI:10.1016/j.enzmictec.2003.11.013.
[21]TOURDOT-MARECHAL R,FORTIER L C,GUZZO J,et al.Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni:a relationship between reduction of ATPase activity and lack of malolactic activity[J].Fems Microbiology Letters,1999,178(2):319-326.DOI:10.1111/j.1574-6968.1999.tb08694.x.
[22]BETTERIDGE A,GRBIN P,JIRANEK V.Improving Oenococcus oeni to overcome challenges of wine malolactic fermentation[J].Trends in Biotechnology,2015,33(9):547-553.DOI:10.1016/j.tibtech.2015.06.008.
[23]SUMBY K M,GRBIN P R,JIRANEK V.Implications of new research and technologies for malolactic fermentation in wine[J].Applied Microbiology&Biotechnology,2014,98(19):8111-8132.DOI:10.1007/s00253-014-5976-0.
[24]WANG P X,LI A X,DONG M,et al.Induction,purification and characterization of malolactic enzyme from Oenococcus oeni SD-2a[J].European Food Research and Technology,2014,239(5):827-835.DOI:10.1007/s00217-014-2276-y.
[25]OLGUIN N,BORDONS A,REGUANT C.Multigenic expression analysis as an approach to understanding the behaviour of Oenococcus oeni in wine-like conditions[J].International Journal of Food Microbiology,2010,144(1):88-95.DOI:10.1016/j.ijfoodmicro.2010.08.032.
[26]GRIMALDI A,BARTOWSKY E,JIRANEK V.A survey of glycosidase activities of commercial wine strains of Oenococcus oeni[J].International Journal of Food Microbiology,2005,105(2):233-244.DOI:10.1016/j.ijfoodmicro.2005.04.011.
[27]MANSFIELD A K,ZOECKLEIN B W,WHITON R S.Quantification of glycosidase activity in selected strains of Brettanomyces bruxellensis and Oenococcus oeni[J].American Journal of Enology&Viticulture,2002,53(4):303-307.
[28]杨芮,吕珍,文彦,等.酒类酒球菌中β-葡萄糖苷酶性质研究[J].食品科学,2013,34(23):206-211.DOI:10.7506/spkx1002-6630-201323043.
[29]MICHLMAYR H,SCHUMANN C,DA SILVA N M B B,et al.Isolation and basic characterization of a beta-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture[J].Journal of Applied Microbiology,2010,108(2):550-559.DOI:10.1111/j.1365-2672.2009.04461.x.
[30]MILLER B J,FRANZ C M A P,CHO G S,et al.Expression of the malolactic enzyme gene (mle) from Lactobacillus plantarum under winemaking conditions[J].Current Microbiology,2011,62(6):1682-1688.DOI:10.1007/s00284-011-9914-4.
[2]MARGALEF-CATALA M,FELIS G E,REGUANT C,et al.Identification of variable genomic regions related to stress response in Oenococcus oeni[J].Food Research International,2017,102:625-638.DOI:10.1016/j.foodres.2017.09.039.
[3]VIGENTINI I,PICOZZI C,TIRELLI A,et al.Survey on indigenous Oenococcus oeni strains isolated from red wines of Valtellina,a cold climate wine-growing Italian area[J].International Journal of Food Microbiology,2009,136(1):123-128.DOI:10.1016/j.ijfoodmicro.2009.09.009.
[4]YANAGIDA F,SRIONNUAL S,CHEN Y S.Isolation and characteristics of lactic acid bacteria from koshu vineyards in Japan[J].Letters in Applied Microbiology,2008,47(2):134-139.DOI:10.1111/j.1472-765X.2008.02398.x.
[5]PAPAGIANNI M.Metabolic engineering of lactic acid bacteria for the production of industrially important compounds[J].Computational and Structural Biotechnology Journal,2012,3(4):1-7.DOI:10.5936/csbj.201210003.
[6]MICHLMAYR H,NAUER S,BRANDES W,et al.Release of wine monoterpenes from natural precursors by glycosidases from Oenococcus oeni[J].Food Chemistry,2012,135(1):80-87.DOI:10.1016/j.foodchem.2012.04.099.
[7]张明霞,吴玉文,段长青.葡萄与葡萄酒香气物质研究进展[J].中国农业科学,2008,41(7):2098-2104.DOI:10.3864/j.issn.0578-1752.2008.07.030.
[8]MATEO J J,JIMENEZ M.Monoterpenes in grape juice and wines[J].Journal of Chromatography A,2000,881(1/2):557-567.DOI:10.1016/S0021-9673(99)01342-4.
[9]张春芝,江志国.微生物对葡萄酒香气的影响综述[J].中国酿造,2013,32(9):28-31.DOI:10.3969/j.issn.0254-5071.2013.09.008.
[10]GRIMALDI A,BARTOWSKY E,JIRANEK V.Screening of Lactobacillus spp.and Pediococcus spp.for glycosidase activities that are important in oenology[J].Journal of Applied Microbiology,2005,99(5):1061-1069.DOI:10.1111/j.1365-2672.2005.02707.x.
[11]GAGNE S,LUCAS P M,PERELLO M C,et al.Variety and variability of glycosidase activities in an Oenococcus oeni strain collection tested with synthetic and natural substrates[J].Journal of Applied Microbiology,2011,110(1):218-228.DOI:10.1111/j.1365-2672.2010.04878.x.
[12]LI Y H,MA Y H,FAN M T,et al.Assessment of beta-D-glucosidase activity of intact cells of two Oenococcus oeni strains with synthetic and natural substrates[J].Czech Journal of Food Sciences,2016,34(1):16-23.DOI:10.17221/216/2015-CJFS.
[13]UGLIANO M,MOIO L.The influence of malolactic fermentation and Oenococcus oeni strain on glycosidic aroma precursors and related volatile compounds of red wine[J].Journal of the Science of Food and Agriculture,2006,86(14):2468-2476.DOI:10.1002/jsfa.2650.
[14]SOLIERI L,GENOVA F,DE PAOLA M,et al.Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations:a framework for selection of new starter cultures[J].Journal of Applied Microbiology,2010,108(1):285-298.DOI:10.1111/j.1365-2672.2009.04428.x.
[15]FIA G,MILLARINI V,GRANCHI L,et al.Beta-glucosidase and esterase activity from Oenococcus oeni:screening and evaluation during malolactic fermentation in harsh conditions[J].LWT-Food Science and Technology,2018,89:262-268.DOI:10.1016/j.lwt.2017.10.060.
[16]BRAVO-FERRADA B M,HOLLMANN A,BRIZUELA N,et al.Growth and consumption of L-malic acid in wine-like medium by acclimated and non-acclimated cultures of Patagonian Oenococcus oeni strains[J].Folia Microbiologica,2016,61(5):365-373.DOI:10.1007/s12223-016-0446-y.
[17]陈其玲,任晓宁,王玲,等.酒酒球菌β-葡萄糖苷酶活性与耐酸胁迫能力的相关性分析[J].食品科学,2017,38(2):115-120.DOI:10.7506/spkx1002-6630-201702019.
[18]JIN G,WANG H,ZHANG C H,et al.Characterization and amino acid metabolism performances of indigenous Oenococcus oeni isolated from Chinese wines[J].European Food Research and Technology,2014,238(4):597-605.DOI:10.1007/s00217-013-2112-9.
[19]杨世玲,刘叶,薛楚然,等.酒酒球菌β-葡萄糖苷酶基因克隆及生物信息学分析[J].中国食品学报,2017,17(7):199-207.DOI:10.16429/j.1009-7848.2017.07.025.
[20]BARBAGALLO R N,SPAGNA G,PALMERI R,et al.Assessment ofβ-glucosidase activity in selected wild strains of Oenococcus oeni for malolactic fermentation[J].Enzyme and Microbial Technology,2004,34(3/4):292-296.DOI:10.1016/j.enzmictec.2003.11.013.
[21]TOURDOT-MARECHAL R,FORTIER L C,GUZZO J,et al.Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni:a relationship between reduction of ATPase activity and lack of malolactic activity[J].Fems Microbiology Letters,1999,178(2):319-326.DOI:10.1111/j.1574-6968.1999.tb08694.x.
[22]BETTERIDGE A,GRBIN P,JIRANEK V.Improving Oenococcus oeni to overcome challenges of wine malolactic fermentation[J].Trends in Biotechnology,2015,33(9):547-553.DOI:10.1016/j.tibtech.2015.06.008.
[23]SUMBY K M,GRBIN P R,JIRANEK V.Implications of new research and technologies for malolactic fermentation in wine[J].Applied Microbiology&Biotechnology,2014,98(19):8111-8132.DOI:10.1007/s00253-014-5976-0.
[24]WANG P X,LI A X,DONG M,et al.Induction,purification and characterization of malolactic enzyme from Oenococcus oeni SD-2a[J].European Food Research and Technology,2014,239(5):827-835.DOI:10.1007/s00217-014-2276-y.
[25]OLGUIN N,BORDONS A,REGUANT C.Multigenic expression analysis as an approach to understanding the behaviour of Oenococcus oeni in wine-like conditions[J].International Journal of Food Microbiology,2010,144(1):88-95.DOI:10.1016/j.ijfoodmicro.2010.08.032.
[26]GRIMALDI A,BARTOWSKY E,JIRANEK V.A survey of glycosidase activities of commercial wine strains of Oenococcus oeni[J].International Journal of Food Microbiology,2005,105(2):233-244.DOI:10.1016/j.ijfoodmicro.2005.04.011.
[27]MANSFIELD A K,ZOECKLEIN B W,WHITON R S.Quantification of glycosidase activity in selected strains of Brettanomyces bruxellensis and Oenococcus oeni[J].American Journal of Enology&Viticulture,2002,53(4):303-307.
[28]杨芮,吕珍,文彦,等.酒类酒球菌中β-葡萄糖苷酶性质研究[J].食品科学,2013,34(23):206-211.DOI:10.7506/spkx1002-6630-201323043.
[29]MICHLMAYR H,SCHUMANN C,DA SILVA N M B B,et al.Isolation and basic characterization of a beta-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture[J].Journal of Applied Microbiology,2010,108(2):550-559.DOI:10.1111/j.1365-2672.2009.04461.x.
[30]MILLER B J,FRANZ C M A P,CHO G S,et al.Expression of the malolactic enzyme gene (mle) from Lactobacillus plantarum under winemaking conditions[J].Current Microbiology,2011,62(6):1682-1688.DOI:10.1007/s00284-011-9914-4.