中国主要产地葡萄酒酵母菌种构成研究
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摘要
本研究以中国主要酿酒葡萄产区(新疆、甘肃、陕西、宁夏、山东)的葡萄果粒及压榨汁为分离源,对附生的天然酵母进行分离,共得到258株酵母菌。根据形态学特征挑取部分代表性菌株,运用26S rDNA Dl/D2序列分析结合生理生化特性鉴定对这些菌株进行了分类学研究,探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布。共鉴定出13属26个种,其中优势属为葡萄酒汉逊氏酵母属Hanseniaspora(5种),分别为H. uvarum,H. clermontiae,H. vineae,H. guilliermondii和H. opuntiae;毕赤酵母属Pichia(4种),分别为P. guilliermondii,P. galeiformi,P. fermentans和P. kluyveri;假丝酵母属Candida(4种),分别为C. tropicalis,C. stellata,C. atmosphaerica和C. ethanolica;伊萨酵母属Issatchenkia(2种),I. orientalis和I.terricola。广布种为Hanseniaspora uvarum和Issatchenki orientalis。结果表明葡萄果粒表皮上具有丰富的酵母资源。对分离自不同地域的Hanseniaspora uvarum种内不同菌株进行了D1/D2序列分析比较,探讨不同地理起源地酵母种内序列稳定性及其变异。
经过杜氏管初筛、耐性实验复筛及模拟发酵试验,获得了3株性能优良的酵母菌,分别为从烟台张裕酒厂的葡萄汁分离的菌株ZYFJQ,从祁连酒业葡萄汁分离的菌株QM,宁夏玉泉营农场梅麓辄(葡萄品种)分离的菌株YQY。通过形态、培养特征和分子鉴定,菌株ZYFJQ和QM被鉴定为酿酒酵母(Saccharomyces cerevisiae),菌株YQY为葡萄酒汉逊氏酵母(Hanseniaspora uvarum)。3个菌株的GenBank登录号分别为EF550180、EU376056、EF585434。3个菌株均能耐受16%(Vol)的酒精度,400 mg/L的二氧化硫,60%的葡萄糖发酵液。模拟发酵试验表明菌株QM发酵酒度为8.8%(Vol)。
香气成分是葡萄酒的主要物化指标和风味品质构成要素,对产地葡萄酒的风格形成有重要贡献,其中酵母菌种起着重要的作用。因此,本试验以筛选的3株天然葡萄酵母菌株ZYFJQ、QM和YQY进行单菌株及组合发酵生产干红葡萄酒。其中,葡萄品种为甘肃武威及山东烟台的赤霞珠和蛇龙珠,商品菌株RC212作为对照。根据单菌株及菌株配伍酿造甘肃赤霞珠葡萄酒的理化分析及香气成分的分析结果表明,各项指标均接近商品菌株,符合葡萄酒国家标准。各酒样残糖含量在2.96～3.90 g/L之间,可滴定酸含量在5.28～6.98 g/L之间,挥发酸含量在0.18～0.36 g/L之间。不同菌株配伍后,酒度与单菌株发酵类似,但发酵香成分的种类增加。葡萄成熟果实的挥发性物质主要是酯类化合物,经过发酵原酒中挥发性物质主要有醇类、酯类、羧酸类及杂环类化合物,表明供试菌株具有一定的产香能力。所有原酒的发酵香成分主要有38种,单菌株QM和ZYFJQ发酵酒样分别为15和9种,与葡萄酒汉逊酵母菌株YQY混合发酵后分别为19和14种,主体香气物质是2-苯乙醇,特有的香气物质是去水卫矛醇及呋喃妥因。在酿造过程中不同的酵母菌株对葡萄酒香气成分的种类和含量的贡献值明显不同,这表明不同酵母菌株对特色葡萄酒的风格形成有重要影响。
258 natural yeast strains were isolated from the surface of grapes and grape juice, collected from the major wine grape cultivation areas in Xinjiang, Gansu, Shaanxi, Ningxia and Shandong Provinces in China. Then parts of the strains were selected according to morphologic characteristics, and were identified based on the 26S rDNA D1/D2 domain sequence analysis and physiological characterization, in order to clarify the diversity of yeast species and their distribution. Among them, 26 species belonging to 13 genera were recognized. The dominant genera identified were Hanseniaspora (5 species): H. uvarum, H. clermontiae, H. vineae, H. guilliermondii, H. opuntiae, respectively; Pichia (4 species): P. guilliermondii, P. galeiformi, P. fermentans, P. kluyveri, respectively; Candida (4 species): C. tropicalis, C. stellata, C. atmosphaerica, C. ethanolica, respectively; and Issatchenkia (2 species): I. orientalis, I. terricola, respectively. Hanseniaspora uvarum and Issatchenki orientalis were the most widely distributed species. The results showed that there were abundant yeast resources on the surface of the grapes. D1/D2 sequence variations were compared among the strains of Hanseniaspora uvarum from different areas.
3 excellent yeast strains were obtained by the first screening of Durham tube method, second screening of endurance experiments and then the test of simulation fermentation. The 3 yeast strains used in this study were named ZYFJQ (GenBank accession number EF550180), isolated from the grape juice of Cabernet Gernischt(Vitis vinifera)in Changyu winery, Shandong; QM (EU376056) from the grape juice of Merlot in Qilian winery, Gansu, and YQY (EF585434) from the grape of Yuquanying farm in Ningxia, respectively. All the three strains have been identified by morphological, physiological and 26S rDNA D1/D2 sequence analysis, strains ZYFJQ and QM were Saccharomyces cerevisiae and YQY belonged to Hanseniaspora uvarum. Three strains can endure 16% (Vol) of alcohol, 400 mg/L of sulphur dioxide, 60 % of the glucose in medium. The test of simulation fermentation showed that the alcoholicity produced by yeast strain QM was 8% (Vol).
Flavor substances are the important typical factors to determine the wine quality and style, and play a significant impact on the formation of the original wine type. Therefore, Three excellencent natural yeast strains ( QM, ZYFJQ, YQY) were used in pure and mixed inoculated fermentations for original dry red wine with two grape cultivar Cabernet sauvignon and Cabemet gemischt harvested from WuWei and Yantai, meanwhile the commercial yeast Saccharomyces cerevisiae RC212 was used as control. Flavor compounds produced in wines and in must were identified by GC-MS. The results of the chemical index and aroma-component analysis showed that the chemical index of the wines with natural selected strains were close to strain RC212, which could satisfy the demand of Chinese National Wine Standard, while the reducing sugar determined varied from 2.96 to 3.90 g/L, the titratable acid between 5.30 to 7.00 g/L, and the volatile acid in the 0.20 to 0.40 g/L. The alcohol produced by the mixed fermentation were similar to the pure fermentation, while the kind and content of aroma components increased by the mixed fermentation. The GC-MS results showed that the compounds in grape juice were mainly esters, and the volatiles of the fermented wine samples were found to be mainly alcohols, esters, acids and heterocyclic compounds, which could account for that screened strains had certain ability to produce aroma compounds. The test identified 38 kinds of fermented aroma, there were 15 and 9 kinds of compounds by strain QM and ZYFJQ pure fermentation, and when mixed with strain YQY, the type of compounds added to 19 and 14, respectively. 2-benzene ethanol constituted the main characteristics of wine aroma components, and the particular compounds were dianhydrodulcitol and furans.
In the course of brewing, different yeast strains have apparently different contribution to the type and content of the wine aroma components. This showed that different yeast strains play an important part in the characteristics of the original wine style.
经过杜氏管初筛、耐性实验复筛及模拟发酵试验,获得了3株性能优良的酵母菌,分别为从烟台张裕酒厂的葡萄汁分离的菌株ZYFJQ,从祁连酒业葡萄汁分离的菌株QM,宁夏玉泉营农场梅麓辄(葡萄品种)分离的菌株YQY。通过形态、培养特征和分子鉴定,菌株ZYFJQ和QM被鉴定为酿酒酵母(Saccharomyces cerevisiae),菌株YQY为葡萄酒汉逊氏酵母(Hanseniaspora uvarum)。3个菌株的GenBank登录号分别为EF550180、EU376056、EF585434。3个菌株均能耐受16%(Vol)的酒精度,400 mg/L的二氧化硫,60%的葡萄糖发酵液。模拟发酵试验表明菌株QM发酵酒度为8.8%(Vol)。
香气成分是葡萄酒的主要物化指标和风味品质构成要素,对产地葡萄酒的风格形成有重要贡献,其中酵母菌种起着重要的作用。因此,本试验以筛选的3株天然葡萄酵母菌株ZYFJQ、QM和YQY进行单菌株及组合发酵生产干红葡萄酒。其中,葡萄品种为甘肃武威及山东烟台的赤霞珠和蛇龙珠,商品菌株RC212作为对照。根据单菌株及菌株配伍酿造甘肃赤霞珠葡萄酒的理化分析及香气成分的分析结果表明,各项指标均接近商品菌株,符合葡萄酒国家标准。各酒样残糖含量在2.96～3.90 g/L之间,可滴定酸含量在5.28～6.98 g/L之间,挥发酸含量在0.18～0.36 g/L之间。不同菌株配伍后,酒度与单菌株发酵类似,但发酵香成分的种类增加。葡萄成熟果实的挥发性物质主要是酯类化合物,经过发酵原酒中挥发性物质主要有醇类、酯类、羧酸类及杂环类化合物,表明供试菌株具有一定的产香能力。所有原酒的发酵香成分主要有38种,单菌株QM和ZYFJQ发酵酒样分别为15和9种,与葡萄酒汉逊酵母菌株YQY混合发酵后分别为19和14种,主体香气物质是2-苯乙醇,特有的香气物质是去水卫矛醇及呋喃妥因。在酿造过程中不同的酵母菌株对葡萄酒香气成分的种类和含量的贡献值明显不同,这表明不同酵母菌株对特色葡萄酒的风格形成有重要影响。
258 natural yeast strains were isolated from the surface of grapes and grape juice, collected from the major wine grape cultivation areas in Xinjiang, Gansu, Shaanxi, Ningxia and Shandong Provinces in China. Then parts of the strains were selected according to morphologic characteristics, and were identified based on the 26S rDNA D1/D2 domain sequence analysis and physiological characterization, in order to clarify the diversity of yeast species and their distribution. Among them, 26 species belonging to 13 genera were recognized. The dominant genera identified were Hanseniaspora (5 species): H. uvarum, H. clermontiae, H. vineae, H. guilliermondii, H. opuntiae, respectively; Pichia (4 species): P. guilliermondii, P. galeiformi, P. fermentans, P. kluyveri, respectively; Candida (4 species): C. tropicalis, C. stellata, C. atmosphaerica, C. ethanolica, respectively; and Issatchenkia (2 species): I. orientalis, I. terricola, respectively. Hanseniaspora uvarum and Issatchenki orientalis were the most widely distributed species. The results showed that there were abundant yeast resources on the surface of the grapes. D1/D2 sequence variations were compared among the strains of Hanseniaspora uvarum from different areas.
3 excellent yeast strains were obtained by the first screening of Durham tube method, second screening of endurance experiments and then the test of simulation fermentation. The 3 yeast strains used in this study were named ZYFJQ (GenBank accession number EF550180), isolated from the grape juice of Cabernet Gernischt(Vitis vinifera)in Changyu winery, Shandong; QM (EU376056) from the grape juice of Merlot in Qilian winery, Gansu, and YQY (EF585434) from the grape of Yuquanying farm in Ningxia, respectively. All the three strains have been identified by morphological, physiological and 26S rDNA D1/D2 sequence analysis, strains ZYFJQ and QM were Saccharomyces cerevisiae and YQY belonged to Hanseniaspora uvarum. Three strains can endure 16% (Vol) of alcohol, 400 mg/L of sulphur dioxide, 60 % of the glucose in medium. The test of simulation fermentation showed that the alcoholicity produced by yeast strain QM was 8% (Vol).
Flavor substances are the important typical factors to determine the wine quality and style, and play a significant impact on the formation of the original wine type. Therefore, Three excellencent natural yeast strains ( QM, ZYFJQ, YQY) were used in pure and mixed inoculated fermentations for original dry red wine with two grape cultivar Cabernet sauvignon and Cabemet gemischt harvested from WuWei and Yantai, meanwhile the commercial yeast Saccharomyces cerevisiae RC212 was used as control. Flavor compounds produced in wines and in must were identified by GC-MS. The results of the chemical index and aroma-component analysis showed that the chemical index of the wines with natural selected strains were close to strain RC212, which could satisfy the demand of Chinese National Wine Standard, while the reducing sugar determined varied from 2.96 to 3.90 g/L, the titratable acid between 5.30 to 7.00 g/L, and the volatile acid in the 0.20 to 0.40 g/L. The alcohol produced by the mixed fermentation were similar to the pure fermentation, while the kind and content of aroma components increased by the mixed fermentation. The GC-MS results showed that the compounds in grape juice were mainly esters, and the volatiles of the fermented wine samples were found to be mainly alcohols, esters, acids and heterocyclic compounds, which could account for that screened strains had certain ability to produce aroma compounds. The test identified 38 kinds of fermented aroma, there were 15 and 9 kinds of compounds by strain QM and ZYFJQ pure fermentation, and when mixed with strain YQY, the type of compounds added to 19 and 14, respectively. 2-benzene ethanol constituted the main characteristics of wine aroma components, and the particular compounds were dianhydrodulcitol and furans.
In the course of brewing, different yeast strains have apparently different contribution to the type and content of the wine aroma components. This showed that different yeast strains play an important part in the characteristics of the original wine style.
引文
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