青海东部野生酵母物种多样性研究
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摘要
本文以青海东部的互助、民和、门源等10个州县的果园或耕地土样为分离源,分离得到98株酵母菌株,利用26S rDNA D1/D2区域序列分析并结合形态学和生理生化特性对这些菌株进行了分类学研究,探讨了青海东部土壤中酵母的物种多样性及其分布。共鉴定出10属13种(其中有两个新种),分别为假丝酵母属Candida,隐球酵母属Cryptococcus,德巴利酵母属Debaryomyces,地霉属Geotrichum,有孢汉逊酵母属Hanseniaspora,伊萨酵母属Issatchenkia,毕赤酵母属Pichia,有孢圆酵母属Torulaspora,红酵母属Rhodotorula和Zygowilliopsis属。Geotrichum candidum和Rhodotorula mucilaginosa为该地区的优势种,这一结果表明,青海东部地区含有相对丰富的酵母种类和少数形态独特的酵母类型。
以98株野生酵母菌株为材料,通过杜氏管发酵法初筛出35株发酵力强、有香味的酵母菌株,对其抗性和发酵特性的评价结果表明:35株酵母菌对SO2具有高度的抗性,大部分菌株在浓度600mg/L时仍可正常生长并发酵,有7株耐NaCl高达120.0g/L,菌株QXN2在160.0g/L的浓度下仍可正常生长并发酵;但大多数菌株的酒精耐受力不高,有6株可耐12%(Vol)的乙醇,但已不能发酵产气;酵母菌株的耐温性较高,菌株QHL4、QXS1、QJD12可耐受55℃的高温;其pH耐受范围在2.5-13.5之间;大部分菌株都可在600g/L的葡萄糖溶液中生长,其中9株仍可发酵。对9株发酵力较好且产香的酵母菌株的酒精发酵实验结果表明:4株酵母酒精度为0,具有浓郁的果香;4株产酒度在2.1-2.4%(Vol)之间,且残糖较低,有浓郁果香。这些酵母菌株有望应用于面包生产、无醇饮料的开发、工业废水处理等领域。
最后对分离自甘肃甘南自治州的两株酵母GS9B和GS9D分别进行了形态鉴定、生理生化鉴定和分子鉴定。菌株GS9B与GS9D菌落形态略有差异,生理生化鉴定结果大多数一致,少数氮源同化实验结果有差异;基于26S rDNA D1/D2区序列分析显示:两株菌独立的聚在一枝,与Cryptococcus spp等其他属碱基差异超过10%;这一结果显示菌株GS9B和GS9D可归为一个新的酵母属,分别命名为Geotrichum brunus Xu & Liu sp. nov和Geotrichum nigrans Xu & Liu sp. nov。
In this paper, 98 natural yeast strains were isolated from the soil samples of orchard and cultivated land, collected from 10 counties (Huzhu, Minhe, Mengyuan, etc) in the east part of Qinghai Province in China. Then parts of the strains were classified 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, 13 species belonging to 10 genera (two strains might represent two new species) were recognized. The dominant genera identified were Candida.spp, Cryptococcus.spp, Debaryomyces.spp, Hanseniaspora.spp, Geotrichum.spp, Pichia.spp, Torulaspora.spp, Issatchenkia.spp, Rhodotorula.spp and Zygowilliopsis.spp. Geotrichum candidum and Rhodotorula mucilaginosa were the most widely distributed species. The results showed that there existed abundant yeast resources and new species yeasts in the soil samples in the east part of Qinghai Province in China.
98 natural yeast strains were used as the experimental samples.35 strains with high fermentation capacity and thick aroma were screened out by Durham tube method. The results of resistance evaluation and fermentation characteristics showed that 35 strains could grew in high concentration of SO2.The most strains were still normal growth and fermentation in the concentration of 600 mg/L.7 strains grew in NaCl over 120.0g/L.QXN2 strain could grow stilly normally and ferment in the concentration of 160.0g/L. Alcohol-resistance of the majority strains were low.6 strains were resist to 12%(vol) of ethanol, but did not ferment.A few yeast strains can resist high temperature, QHL4、QXS1、QJD12 strains could grow at 55℃.Their range of pH tolerance were between 2.5-12.0.Most strains could grow in 600.0g/L glucose solution and 9 of them could still ferment.9 excellent strains were screened out which fermented well and produced nice smell. The result of alcohol fermentation showed that alcoholicity of 4 yeast strains was zero, but all of them had thick aroma.4 strains alcoholicity were between 2.1-2.4%(vol),with lower residual sugar and fruit fragrant. They may be used as the ferment for the bread baking, non-alcohol beverage, waste water purification and so on.
At last, the strains GS9B and GS9D were identified in detail based on morphological, physiological and biochemical characterization .There were some strains. The other characteristics of two strains were identical. Molecular taxonomic analysis based on the 26S rDNA D1/D2 domain also shows that the two strains should be defined as a new genus because they clustered in a branch alone. These new species differed from Cryptococcus spp and other genus with about 10% nucleotide divergences in the D1/D2 regions. So they were named Geotrichum brunus Xu & Liu sp. nov and Geotrichum nigrans Xu & Liu sp. nov, considering their colony morphology similar to Geotrichum spp.
以98株野生酵母菌株为材料,通过杜氏管发酵法初筛出35株发酵力强、有香味的酵母菌株,对其抗性和发酵特性的评价结果表明:35株酵母菌对SO2具有高度的抗性,大部分菌株在浓度600mg/L时仍可正常生长并发酵,有7株耐NaCl高达120.0g/L,菌株QXN2在160.0g/L的浓度下仍可正常生长并发酵;但大多数菌株的酒精耐受力不高,有6株可耐12%(Vol)的乙醇,但已不能发酵产气;酵母菌株的耐温性较高,菌株QHL4、QXS1、QJD12可耐受55℃的高温;其pH耐受范围在2.5-13.5之间;大部分菌株都可在600g/L的葡萄糖溶液中生长,其中9株仍可发酵。对9株发酵力较好且产香的酵母菌株的酒精发酵实验结果表明:4株酵母酒精度为0,具有浓郁的果香;4株产酒度在2.1-2.4%(Vol)之间,且残糖较低,有浓郁果香。这些酵母菌株有望应用于面包生产、无醇饮料的开发、工业废水处理等领域。
最后对分离自甘肃甘南自治州的两株酵母GS9B和GS9D分别进行了形态鉴定、生理生化鉴定和分子鉴定。菌株GS9B与GS9D菌落形态略有差异,生理生化鉴定结果大多数一致,少数氮源同化实验结果有差异;基于26S rDNA D1/D2区序列分析显示:两株菌独立的聚在一枝,与Cryptococcus spp等其他属碱基差异超过10%;这一结果显示菌株GS9B和GS9D可归为一个新的酵母属,分别命名为Geotrichum brunus Xu & Liu sp. nov和Geotrichum nigrans Xu & Liu sp. nov。
In this paper, 98 natural yeast strains were isolated from the soil samples of orchard and cultivated land, collected from 10 counties (Huzhu, Minhe, Mengyuan, etc) in the east part of Qinghai Province in China. Then parts of the strains were classified 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, 13 species belonging to 10 genera (two strains might represent two new species) were recognized. The dominant genera identified were Candida.spp, Cryptococcus.spp, Debaryomyces.spp, Hanseniaspora.spp, Geotrichum.spp, Pichia.spp, Torulaspora.spp, Issatchenkia.spp, Rhodotorula.spp and Zygowilliopsis.spp. Geotrichum candidum and Rhodotorula mucilaginosa were the most widely distributed species. The results showed that there existed abundant yeast resources and new species yeasts in the soil samples in the east part of Qinghai Province in China.
98 natural yeast strains were used as the experimental samples.35 strains with high fermentation capacity and thick aroma were screened out by Durham tube method. The results of resistance evaluation and fermentation characteristics showed that 35 strains could grew in high concentration of SO2.The most strains were still normal growth and fermentation in the concentration of 600 mg/L.7 strains grew in NaCl over 120.0g/L.QXN2 strain could grow stilly normally and ferment in the concentration of 160.0g/L. Alcohol-resistance of the majority strains were low.6 strains were resist to 12%(vol) of ethanol, but did not ferment.A few yeast strains can resist high temperature, QHL4、QXS1、QJD12 strains could grow at 55℃.Their range of pH tolerance were between 2.5-12.0.Most strains could grow in 600.0g/L glucose solution and 9 of them could still ferment.9 excellent strains were screened out which fermented well and produced nice smell. The result of alcohol fermentation showed that alcoholicity of 4 yeast strains was zero, but all of them had thick aroma.4 strains alcoholicity were between 2.1-2.4%(vol),with lower residual sugar and fruit fragrant. They may be used as the ferment for the bread baking, non-alcohol beverage, waste water purification and so on.
At last, the strains GS9B and GS9D were identified in detail based on morphological, physiological and biochemical characterization .There were some strains. The other characteristics of two strains were identical. Molecular taxonomic analysis based on the 26S rDNA D1/D2 domain also shows that the two strains should be defined as a new genus because they clustered in a branch alone. These new species differed from Cryptococcus spp and other genus with about 10% nucleotide divergences in the D1/D2 regions. So they were named Geotrichum brunus Xu & Liu sp. nov and Geotrichum nigrans Xu & Liu sp. nov, considering their colony morphology similar to Geotrichum spp.
引文
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