田间施药对自然发酵葡萄酒酵母菌群落结构的影响
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摘要
【背景】酵母菌是葡萄酒发酵过程中一类非常重要的微生物,其多样性及群体组成对葡萄酒的质量有重要贡献。影响葡萄酒中酵母菌组成的因素有很多,但目前尚未见葡萄园田管理对葡萄酒酵母菌群落结构影响方面的报道。【目的】探索田间施药对自然发酵葡萄酒酵母菌群落结构的影响。【方法】采用分离培养、常规分子生物学鉴定和Illumina MiSeq宏基因组测序结合的方法分析不同样品中的酵母菌群落结构情况。【结果】从不使用内吸收型化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Saccharomyces、Zygoascus、Issatchenkia等7个属8个种的酵母菌,宏基因组测序结果表明有Pichia(29.42%)、Saccharomyces(21.91%)、Issatchenkia(17.99%)、 Hanseniaspora(12.10%)、 Candida(7.47%)、 Zygosaccharomyces(5.32%)、Schizosaccharomyces (3.07%)、Aureobasidium (0.29%)等属的酵母菌参与发酵;使用常规化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Cryptococcus等5个属6个种的酵母菌,宏基因组测序结果表明有Pichia (41.66%)、Hanseniaspora (21.54%)、Candida(19.11%)、 Zygosaccharomyces(7.78%)、 Schizosaccharomyces(4.04%)、 Cryptococcus(3.21%)、Saccharomyces (1.12%)、Aureobasidium (0.49%)等属的酵母菌参与发酵。【结论】两样品中酵母菌比例有显著差异,表明在酿酒葡萄的园田管理中化学农药的使用对自然发酵葡萄酒的酵母菌群落结构有较大影响。
[Background] Yeast is a very important microorganism in wine fermentation. Its diversity and population composition play an important role in wine quality. There are many factors that affect the distribution and presence of yeast flora in wine. However, the influence of grape field management, for example the use of pesticides, on wine yeast community structure has not been reported yet. [Objective] We studied the effect of pesticide application on the community structure of wine yeasts during spontaneous fermentation. [Methods] Methods of pure culture, molecular biology identification and Illumina MiSeq metagenomic sequencing were used. [Results] In the spontaneous fermentation broth of grape samples without internal absorption chemical pesticides, 7 genuses(8 species) of yeast strains including Pichia,Hanseniaspora, Schizosaccharomyces, Candida, Saccharomyces, Zygoascus, and Issatchenkia were isolated and identified, while the result of metagenomic sequencing confirmed the dominance of Pichia(29.42%),Saccharomyces(21.91%), Issatchenkia(17.99%), Hanseniaspora(12.10%), Candida(7.47%),Zygosaccharomyces(5.32%), Schizosaccharomyces(3.07%), and Aureobasidium(0.29%). In the spontaneous fermentation broth of grape samples with conventional chemical pesticides, 5 genuses(6 species)of yeast strains including Pichia, Hanseniaspora, Schizosaccharomyces, Candida, and Cryptococcus were isolated and identified, while the result of Illumina Mi Seq sequencing showed that Pichia(41.66%),Hanseniaspora(21.54%), Candida(19.11%), Zygosaccharomyces(7.78%), Schizosaccharomyces(4.04%),Cryptococcus(3.21%), Saccharomyces(1.12%), and Aureobasidium(0.49%) were involved in the fermentation. [Conclusion] Yeast community compositions in the two samples were significantly different,indicating that pesticide application on grapes had great influence on the community structure of wine yeasts during spontaneous fermentation.
[Background] Yeast is a very important microorganism in wine fermentation. Its diversity and population composition play an important role in wine quality. There are many factors that affect the distribution and presence of yeast flora in wine. However, the influence of grape field management, for example the use of pesticides, on wine yeast community structure has not been reported yet. [Objective] We studied the effect of pesticide application on the community structure of wine yeasts during spontaneous fermentation. [Methods] Methods of pure culture, molecular biology identification and Illumina MiSeq metagenomic sequencing were used. [Results] In the spontaneous fermentation broth of grape samples without internal absorption chemical pesticides, 7 genuses(8 species) of yeast strains including Pichia,Hanseniaspora, Schizosaccharomyces, Candida, Saccharomyces, Zygoascus, and Issatchenkia were isolated and identified, while the result of metagenomic sequencing confirmed the dominance of Pichia(29.42%),Saccharomyces(21.91%), Issatchenkia(17.99%), Hanseniaspora(12.10%), Candida(7.47%),Zygosaccharomyces(5.32%), Schizosaccharomyces(3.07%), and Aureobasidium(0.29%). In the spontaneous fermentation broth of grape samples with conventional chemical pesticides, 5 genuses(6 species)of yeast strains including Pichia, Hanseniaspora, Schizosaccharomyces, Candida, and Cryptococcus were isolated and identified, while the result of Illumina Mi Seq sequencing showed that Pichia(41.66%),Hanseniaspora(21.54%), Candida(19.11%), Zygosaccharomyces(7.78%), Schizosaccharomyces(4.04%),Cryptococcus(3.21%), Saccharomyces(1.12%), and Aureobasidium(0.49%) were involved in the fermentation. [Conclusion] Yeast community compositions in the two samples were significantly different,indicating that pesticide application on grapes had great influence on the community structure of wine yeasts during spontaneous fermentation.
引文
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[6]Yan HJ,Shi Y,Liu C,et al.Screening,identification and potential use of indigenous yeasts from muscat wine spontaneous fermentation[J].Food Science,2017,38(22):117-124(in Chinese)阎贺静,时月,刘畅,等.玫瑰香干红葡萄酒自然发酵过程中优势酵母分离鉴定及其应用潜力分析[J].食品科学,2017,38(22):117-124
[7]Wang ZJ.Study on identification and diversity of wine-related yeast from Xinjiang and Gansu[D].Yangling:Master’s Thesis of Northwest A&F University,2008(in Chinese)王泽举.新疆、甘肃地区葡萄酒相关酵母菌的鉴定及多样性研究[D].杨凌:西北农林科技大学硕士学位论文,2008
[8]Yang MJ,Chen XB,Zhao JJ,et al.Identification of yeasts isolated from spontaneous fermentation of Cabernet sauvignon[J].Food and Fermentation Industries,2011,37(7):22-27(in Chinese)杨美景,陈小波,赵静静,等.赤霞珠葡萄自然发酵过程中酵母菌的分离与鉴定[J].食品与发酵工业,2011,37(7):22-27
[9]Su L,Liu SW,He L,et al.Diversity of wine related yeasts during spontaneous fermentation of Vitis.amurensis rupr wine terrior in northeastern regions of China[J].Journal of Food Science and Biotechnology,2007,26(3):110-115(in Chinese)苏龙,刘树文,何玲,等.东北山葡萄酒自然发酵酵母菌群的研究[J].食品与生物技术学报,2007,26(3):110-115
[10]Liu SW,Chang YW,Hu T,et al.Study on yeasts of different ages of grape in the course of spontaneous fermentation[J].Journal of Northwest A&F University(Natural Science Edition),2008,37(7):51-56,63(in Chinese)刘树文,常亚维,胡廷,等.不同树龄葡萄自然发酵过程中酵母菌的研究[J].西北农林科技大学学报:自然科学版,2008,37(7):51-56,63
[11]Marsit S,Dequin S.Diversity and adaptive evolution of Saccharomyces wine yeast:a review[J].FEMS Yeast Research,2015,15(7):fov067
[12]Santiago-Urbina JA,Arias-García JA,Ruiz-Terán F.Yeast species associated with spontaneous fermentation of taberna,a traditional palm wine from the southeast of Mexico[J].Annals of Microbiology,2015,65(1):287-296
[13]Schuller D,Cardoso F,Sousa S,et al.Genetic diversity and population structure of Saccharomyces cerevisiae strains isolated from different grape varieties and winemaking regions[J].PLoSOne,2012,7:e32507
[14]Csoma H,Zakany N,Capece A,et al.Biological diversity of Saccharomyces yeasts of spontaneously fermenting wines in four wine regions:comparative genotypic and phenotypic analysis[J].International Journal of Food Microbiology,2010,140(2/3):239-248
[15]Wang HH,Liu TM,Wang K,et al.Identification of yeasts species in the fermentation of dry red wine of Yantai[J].China Brewing,2011,30(1):33-36(in Chinese)王会会,刘天明,王可,等.烟台干红葡萄酒发酵过程中酵母种类鉴定[J].中国酿造,2011,30(1):33-36
[16]Kurtzman CP,Robnett CJ.Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit(26S)ribosomal DNA partial sequences[J].Antonie Van Leeuwenhoek,1998,73(4):331-371
[17]Zhao S,Zhou N,Zhao ZY,et al.Endophytic bacterial diversity and dynamics in root of Salicornia europaea estimated via high throughput sequencing[J].Acta Microbiologica Sinica,2016,56(6):1000-1008(in Chinese)赵帅,周娜,赵振勇,等.基于高通量测序分析盐角草根部内生细菌多样性及动态规律[J].微生物学报,2016,56(6):1000-1008
[18]Xing MY,Du H,Xu Y.Diversity and succession of lactic acid bacteria during sesame-flavor liquor fermentation[J].Microbiology China,2018,45(1):19-28(in Chinese)邢敏钰,杜海,徐岩.芝麻香型白酒发酵过程中乳酸菌多样性及其演替规律[J].微生物学通报,2018,45(1):19-28
[19]Sternes PR,Lee D,Kutyna DR,et al.A combined meta-barcoding and shotgun metagenomic analysis of spontaneous wine fermentation[J].GigaScience,2017,6(7):1-10
[20]Morgan HH,du Toit M,Setati ME.The grapevine and wine microbiome:insights from high-throughput amplicon sequencing[J].Frontiers in Microbiology,2017,8:820
[21]Yang Y,Xu YW,Xue JX,et al.Validate the identification effect of WL nutrient agar on wine yeast[J].Journal of Microbiology,2007,27(5):75-78(in Chinese)杨莹,徐艳文,薛军侠,等.WL营养琼脂对葡萄酒相关酵母的鉴定效果验证[J].微生物学杂志,2007,27(5):75-78
[22]Xu YW,Yang Y,Xue JX,et al.26S rDNA RFLP analysis as a tool to characterize non-Saccharomyces yeasts[J].Journal of Microbiology,2007,27(4):23-26(in Chinese)徐艳文,杨莹,薛军侠,等.26S r DNA-RFLP分析在非酿酒酵母菌分类研究中的应用[J].微生物学杂志,2007,27(4):23-26
[23]Li KM,Fu GM,Wu CF,et al.Dynamics of eukaryotic microbial community succession during the traditional fermentation of special-flavor liquor[J].Food Science,2017,38(22):131-136(in Chinese)李凯敏,付桂明,吴酬飞,等.特香型白酒酿造过程中真核微生物菌群演替[J].食品科学,2017,38(22):131-136
[24]Wang H.Study on the composition of wine yeast strains from main producing areas in China[D].Jinan:Master’s Thesis of Shandong Polytechnic University,2008(in Chinese)王慧.中国主要产地葡萄酒酵母菌种构成研究[D].济南:山东轻工业学院硕士学位论文,2008
[25]Sturm J,Grossmann M,Schnell S.Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations[J].Journal of Applied Microbiology,2006,101(6):1241-1248
[26]Lopandic K,Tiefenbrunner W,Gangl H,et al.Molecular profiling of yeasts isolated during spontaneous fermentations of Austrian wines[J].FEMS Yeast Research,2008,8(7):1063-1075
[27]Comitini F,Ciani M.Influence of fungicide treatments on the occurrence of yeast flora associated with wine grapes[J].Annals of Microbiology,2008,58(3):489-493
[2]Soufleros EH,Bouloumpasi E,Tsarchopoulos C,et al.Primary amino acid profiles of Greek white wines and their use in classification according to variety,origin and vintage[J].Food Chemistry,2003,80(2):261-273
[3]Wang ZJ,Liu YL,Liu AG,et al.Survey on yeast population dynamics during wine spontaneous fermentation in Xinjiang[J].Journal of Huazhong Agricultural University,2008,27(5):664-667(in Chinese)王泽举,刘延琳,刘爱国,等.新疆葡萄酒自然发酵过程酵母菌的种类和动态变化[J].华中农业大学学报,2008,27(5):664-667
[4]Cheng L,Li Z,Wang J.Study on yeasts during the spontaneous fermentation of grapevine[J].Journal of Chinese Institute of Food Science and Technology,2010,10(2):131-137(in Chinese)程雷,李梓,王军.葡萄自然发酵过程中酵母菌的研究[J].中国食品学报,2010,10(2):131-137
[5]Cheng SW,Qu HG,Luan LY,et al.Screening and fermentation characteristics of indigenous Saccharomyce scerevisiae from spontaneous fermentation of Chardonnay grapes[J].Journal of Chinese Institute of Food Science and Technology,2017,17(8):119-125(in Chinese)程仕伟,屈慧鸽,栾丽英,等.新疆霞多丽自然发酵醪中本土酿酒酵母的选育与发酵特性[J].中国食品学报,2017,17(8):119-125
[6]Yan HJ,Shi Y,Liu C,et al.Screening,identification and potential use of indigenous yeasts from muscat wine spontaneous fermentation[J].Food Science,2017,38(22):117-124(in Chinese)阎贺静,时月,刘畅,等.玫瑰香干红葡萄酒自然发酵过程中优势酵母分离鉴定及其应用潜力分析[J].食品科学,2017,38(22):117-124
[7]Wang ZJ.Study on identification and diversity of wine-related yeast from Xinjiang and Gansu[D].Yangling:Master’s Thesis of Northwest A&F University,2008(in Chinese)王泽举.新疆、甘肃地区葡萄酒相关酵母菌的鉴定及多样性研究[D].杨凌:西北农林科技大学硕士学位论文,2008
[8]Yang MJ,Chen XB,Zhao JJ,et al.Identification of yeasts isolated from spontaneous fermentation of Cabernet sauvignon[J].Food and Fermentation Industries,2011,37(7):22-27(in Chinese)杨美景,陈小波,赵静静,等.赤霞珠葡萄自然发酵过程中酵母菌的分离与鉴定[J].食品与发酵工业,2011,37(7):22-27
[9]Su L,Liu SW,He L,et al.Diversity of wine related yeasts during spontaneous fermentation of Vitis.amurensis rupr wine terrior in northeastern regions of China[J].Journal of Food Science and Biotechnology,2007,26(3):110-115(in Chinese)苏龙,刘树文,何玲,等.东北山葡萄酒自然发酵酵母菌群的研究[J].食品与生物技术学报,2007,26(3):110-115
[10]Liu SW,Chang YW,Hu T,et al.Study on yeasts of different ages of grape in the course of spontaneous fermentation[J].Journal of Northwest A&F University(Natural Science Edition),2008,37(7):51-56,63(in Chinese)刘树文,常亚维,胡廷,等.不同树龄葡萄自然发酵过程中酵母菌的研究[J].西北农林科技大学学报:自然科学版,2008,37(7):51-56,63
[11]Marsit S,Dequin S.Diversity and adaptive evolution of Saccharomyces wine yeast:a review[J].FEMS Yeast Research,2015,15(7):fov067
[12]Santiago-Urbina JA,Arias-García JA,Ruiz-Terán F.Yeast species associated with spontaneous fermentation of taberna,a traditional palm wine from the southeast of Mexico[J].Annals of Microbiology,2015,65(1):287-296
[13]Schuller D,Cardoso F,Sousa S,et al.Genetic diversity and population structure of Saccharomyces cerevisiae strains isolated from different grape varieties and winemaking regions[J].PLoSOne,2012,7:e32507
[14]Csoma H,Zakany N,Capece A,et al.Biological diversity of Saccharomyces yeasts of spontaneously fermenting wines in four wine regions:comparative genotypic and phenotypic analysis[J].International Journal of Food Microbiology,2010,140(2/3):239-248
[15]Wang HH,Liu TM,Wang K,et al.Identification of yeasts species in the fermentation of dry red wine of Yantai[J].China Brewing,2011,30(1):33-36(in Chinese)王会会,刘天明,王可,等.烟台干红葡萄酒发酵过程中酵母种类鉴定[J].中国酿造,2011,30(1):33-36
[16]Kurtzman CP,Robnett CJ.Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit(26S)ribosomal DNA partial sequences[J].Antonie Van Leeuwenhoek,1998,73(4):331-371
[17]Zhao S,Zhou N,Zhao ZY,et al.Endophytic bacterial diversity and dynamics in root of Salicornia europaea estimated via high throughput sequencing[J].Acta Microbiologica Sinica,2016,56(6):1000-1008(in Chinese)赵帅,周娜,赵振勇,等.基于高通量测序分析盐角草根部内生细菌多样性及动态规律[J].微生物学报,2016,56(6):1000-1008
[18]Xing MY,Du H,Xu Y.Diversity and succession of lactic acid bacteria during sesame-flavor liquor fermentation[J].Microbiology China,2018,45(1):19-28(in Chinese)邢敏钰,杜海,徐岩.芝麻香型白酒发酵过程中乳酸菌多样性及其演替规律[J].微生物学通报,2018,45(1):19-28
[19]Sternes PR,Lee D,Kutyna DR,et al.A combined meta-barcoding and shotgun metagenomic analysis of spontaneous wine fermentation[J].GigaScience,2017,6(7):1-10
[20]Morgan HH,du Toit M,Setati ME.The grapevine and wine microbiome:insights from high-throughput amplicon sequencing[J].Frontiers in Microbiology,2017,8:820
[21]Yang Y,Xu YW,Xue JX,et al.Validate the identification effect of WL nutrient agar on wine yeast[J].Journal of Microbiology,2007,27(5):75-78(in Chinese)杨莹,徐艳文,薛军侠,等.WL营养琼脂对葡萄酒相关酵母的鉴定效果验证[J].微生物学杂志,2007,27(5):75-78
[22]Xu YW,Yang Y,Xue JX,et al.26S rDNA RFLP analysis as a tool to characterize non-Saccharomyces yeasts[J].Journal of Microbiology,2007,27(4):23-26(in Chinese)徐艳文,杨莹,薛军侠,等.26S r DNA-RFLP分析在非酿酒酵母菌分类研究中的应用[J].微生物学杂志,2007,27(4):23-26
[23]Li KM,Fu GM,Wu CF,et al.Dynamics of eukaryotic microbial community succession during the traditional fermentation of special-flavor liquor[J].Food Science,2017,38(22):131-136(in Chinese)李凯敏,付桂明,吴酬飞,等.特香型白酒酿造过程中真核微生物菌群演替[J].食品科学,2017,38(22):131-136
[24]Wang H.Study on the composition of wine yeast strains from main producing areas in China[D].Jinan:Master’s Thesis of Shandong Polytechnic University,2008(in Chinese)王慧.中国主要产地葡萄酒酵母菌种构成研究[D].济南:山东轻工业学院硕士学位论文,2008
[25]Sturm J,Grossmann M,Schnell S.Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations[J].Journal of Applied Microbiology,2006,101(6):1241-1248
[26]Lopandic K,Tiefenbrunner W,Gangl H,et al.Molecular profiling of yeasts isolated during spontaneous fermentations of Austrian wines[J].FEMS Yeast Research,2008,8(7):1063-1075
[27]Comitini F,Ciani M.Influence of fungicide treatments on the occurrence of yeast flora associated with wine grapes[J].Annals of Microbiology,2008,58(3):489-493
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