猕猴桃酒生香嗜杀酵母的选育
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摘要
近十来年，我国猕猴桃酒生产得到了长足发展。但由于缺乏适宜的猕猴桃酒专用菌种，猕猴桃酒的果香不突出，典型性欠佳；此外，发酵初期的杂菌污染也是导致猕猴桃酒品质不高的重要原因。鉴于此，本研究的目的是选育出既具有优良生香性能、又具有较强嗜杀活性的猕猴桃酒酵母，使其在发酵过程中既能产生更多的香气物质，又能杀灭野生杂菌，保证纯种发酵的顺利进行，生产出高品质的猕猴桃酒。试验采用原生质体电融合方法，对野生优良酵母的分离、生香酵母的筛选、亲本原生质体制备、电融合条件、融合子的筛选与鉴定等内容进行了研究，试验取得以下主要结果。
（1）猕猴桃果实上有野生酵母存在。经过分离、培养和小型酿酒试验，从7个不同品种猕猴桃上筛选出1株发酵性能优良的猕猴桃酒用天然野生酵母。
（2）将筛选的野生酵母和12株酿酒酵母进行猕猴桃酒酿造试验，通过对各菌株发酵速度测定、酒样理化指标分析和感官评价比较，表明酵母KD起酵时间短，发酵速度快而平稳，酒精产率高，挥发酸含量低，Vc含量高；酵母KD发酵的猕猴桃酒色泽浅黄，滋味醇厚爽口，果香突出，酒香纯正，典型性强。
（3）通过GC-MS分析比较，与其他6株酵母相比，酵母KD酿造的猕猴桃酒中，香气物质种类多且含量高，共检出48种香气成分，相对含量97.33%。其中酯类物质19种，相对含量达到34.65%；高级醇7种，相对含量15.85%；有机酸6种，相对含量13.3%。KD菌株产香能力强，酒中酯类物质丰富且含量高，具有浓郁的果香和酒香，是优良的猕猴桃酒生香酵母。
（4）生香酵母KD为非营养缺陷型，不具有嗜杀活性。嗜杀酵母5045为组氨酸缺陷和核融合缺陷。以KD和5045为亲本，通过原生质体电融合将5045的嗜杀质粒导入KD中，选育出猕猴桃酒生香嗜杀酵母。
（5）研究了影响原生质体形成和再生的影响因素，如预处理液和稳渗液的选择、酶解条件和再生方法等。确定制备5045原生质体的适宜条件为：培养至4h的酵母细胞先用0.8mol/L甘露醇稳渗液洗涤，再用1:1的0.1%EDTA-Na_2+0.1%巯基乙醇复合液对酵母细胞进行预处理，并用0.8mol/L甘露醇稳渗液洗净预处理液，然后加入2.5%的蜗牛酶液，在35℃水浴酶解处理60min。在此条件下，原生质体形成率为80.97%，再生率为26.47%，形成率再生率为21.43%，原生质体制备效果最好。
（6）制备酵母KD原生质体的适宜条件为：将培养至3h的酵母细胞先用0.8mol/L甘露醇稳渗液洗涤，再用0.1%EDTA-Na_2+0.1%巯基乙醇复合液对酵母细胞进行预处理，然后加入2.5%的蜗牛酶液，在35℃酶解30min。在此条件下，原生质体形成率为81.15%，再生率为10.4%，形成率×再生率为8.44%，原生质体制备效果最好，
（7）酵母KD和5045原生质体按11比例混合进行电融合，在2KV/cm脉冲场强下，脉冲时间为40μs，脉冲间隔为600ms，脉冲3次，原生质体融合率为6.93×10~(-5)。经过初筛、复筛及酿造试验，筛选出1株性能优良的融合子F16。
（8）经对融合子F16进行细胞形态及大小测定、营养缺陷型鉴定、嗜杀活性鉴定、酿造过程中对野生酵母的抑制作用及发酵酒的感官评定和香气分析，确认F16为具有良好嗜杀活性和优良生香性能的猕猴桃酒酵母，遗传了KD酵母的发酵特性和5045的嗜杀性能，是1株应用前景广阔的猕猴桃酒酵母。
In recent ten years, the kiwifruit winemaking industry has made a rapid development inChina. But the quality of kiwifruit wine is not so perfect，the fruity aroma is not enoughstrong, and the typicality is absent. And the contamination by other yeast in the early stage offermentation also leads the flavor of kiwifruit wine deteriorative. So, the purpose of this studyis to breed kiwifruit wine yeast with not only good aroma-producing performance but alsostrong killer activity. The yeast can produce more aromas and kill the wild yeasts duringfermentation, ensure pure-bred fermentation carry out smoothly and produce high qualitykiwifruit wine. The technique of protoplast electrofusion was used in the study. Theseparation of wild yeast from kiwifruits, sifting the aroma-producing yeast, preparing theparental protoplasts, determining the parameters of electrofusion, and screening andidentifying fusants were studied. The results showed as follows.
(1) There are wild yeasts on the peel of kiwifruit. After isolation and culture bysmall-scale winemaking trials, a wild kiwifruit wine yeast with good fermentationperformance was selected from seven different varieties of kiwifruit.
(2) The wild yeast and other12Saccharomyces cerevisiae were used to ferment kiwifruitjuice for wine. Through comparing the fermentation rate of all yeasts, physicochemicalindexes analysis and sensory evaluation of the kiwi wine, the yeast strain KD showed bettercharacteristics than others as follows: faster and smooth fermentation speed, higher alcoholproduction and content vitamin C, but lower volatile acid content. The kiwifruit winefermented by KD strain had high quality–light yellow color, mellow and refreshing taste,strong and pleasant fruit aroma, and pure alcoholic scent. The kiwifruit wine had harmoniousand typical fruit aroma.
(3) GC-MS analysis showed that there were more kinds of aroma components and higherlevels of their content in the kiwifruit wine fermented by KD yeast than the wine fermentedrespectively by other6yeast strains. The former wine had48kinds of aroma volatile material,and the relative content of them was97.33percent. Include19kinds of ester,7kinds ofhigher alcohol and6kinds of organic acid, the relative content were respectively34.65 percent,15.85percent, and13.3percent. Yeast strain KD had strong capacity to producearoma substances, especially more kinds of ester and higher content. The kiwi wine had rich,strong and harmonious aroma. KD was a good yeast strain for kiwifruit winemaking.
(4) S. cerevisiae KD, with capacity of produces more aromas, was non-autotrophic anddid not have killer activity. Killer yeast5045was autotrophic of histidine and was a mutant ofS. cerevisiae with defective for nuclear fusion. KD and killer yeast5045was parent. Based onProtoplast electrofusion, the killer plasmid from5045was leaded into the yeast strain KD tobreed yeast with killer activity and capacity to produce more aromas for kiwifruitwinemaking.
(5) We studied the factors which effect the formation and regeneration of the protoplastof yeast5045, such as choice the pretreatment solution and the hypertonic buffer solution,parameters of enzymolysis and cultural methods for regeneration. The optimal conditionswere summarized as follows. The cells of5045cultured for4hours were washed withhypertonic buffer solution of0.8mol/L Mannitol, then pretreated with mixture of0.1%EDTA-Na_2solution and0.1%mercaptoethanol solution (1:1, V/V) for10minutes at30℃.The pretreatment solution was washed up with0.8mol/L Mannitol. The cells were hydrolyzedby2.5%Helicase solution for60minutes at35℃. As a result, the formation rate (FR) andregeneration rate (RR) of protoplast were respectively80.97percent and26.47percent, theproduct of FR and RR was21.43percent. Protoplast formation was the best.
(6) The optimum conditions of protoplast formation of yeast strain KD were similar to5045. The cell of KD cultured for3hours also washed with hypertonic buffer solution of0.8mol/L Mannitol, then pretreated with mixture of0.1%EDTA solution and0.1%mercaptoethanol solution (1:1, V/V) for10minutes at30℃. The pretreatment solution waswashed up with0.8mol/L Mannitol. The cells were hydrolyzed by2.5%Helicase solution for30minutes at35℃. As a result, the formation rate (FR) and regeneration rate (RR) ofprotoplast were respectively81.15percent and10.4percent, the product of FR and RR was8.44percent.
(7) The protoplasts of KD and the protoplasts of5045, mixed with the ratio of1to1,were electro fused on the conditions as follow. The pulse field strength was2KV/cm, pulsetime was40microseconds, and pulse interval was600microseconds and pulse3times. Therate of protoplast fusion was6.93×10~(-5). After screening, rescreening and small-scalewinemaking, one fusant F16with excellent capability was obtained. The fusant had stablekiller activity in genetics.
(8) Through comparing the cell shape and size, auxotrophic determination, killer activityidentification, detection the inhibition action of fusant F16to wild yeast during brewing process, aroma analysis and sensory evaluation, the results showed that fusant F16inheritedthe good fermentation characteristics of KD and the strong killer performance of5045. F16was yeast for kiwifruit winemaking with superior capacity to produce more aromas and finekiller activity, and the prospect using in kiwifruit wine industry was broad.
（1）猕猴桃果实上有野生酵母存在。经过分离、培养和小型酿酒试验，从7个不同品种猕猴桃上筛选出1株发酵性能优良的猕猴桃酒用天然野生酵母。
（2）将筛选的野生酵母和12株酿酒酵母进行猕猴桃酒酿造试验，通过对各菌株发酵速度测定、酒样理化指标分析和感官评价比较，表明酵母KD起酵时间短，发酵速度快而平稳，酒精产率高，挥发酸含量低，Vc含量高；酵母KD发酵的猕猴桃酒色泽浅黄，滋味醇厚爽口，果香突出，酒香纯正，典型性强。
（3）通过GC-MS分析比较，与其他6株酵母相比，酵母KD酿造的猕猴桃酒中，香气物质种类多且含量高，共检出48种香气成分，相对含量97.33%。其中酯类物质19种，相对含量达到34.65%；高级醇7种，相对含量15.85%；有机酸6种，相对含量13.3%。KD菌株产香能力强，酒中酯类物质丰富且含量高，具有浓郁的果香和酒香，是优良的猕猴桃酒生香酵母。
（4）生香酵母KD为非营养缺陷型，不具有嗜杀活性。嗜杀酵母5045为组氨酸缺陷和核融合缺陷。以KD和5045为亲本，通过原生质体电融合将5045的嗜杀质粒导入KD中，选育出猕猴桃酒生香嗜杀酵母。
（5）研究了影响原生质体形成和再生的影响因素，如预处理液和稳渗液的选择、酶解条件和再生方法等。确定制备5045原生质体的适宜条件为：培养至4h的酵母细胞先用0.8mol/L甘露醇稳渗液洗涤，再用1:1的0.1%EDTA-Na_2+0.1%巯基乙醇复合液对酵母细胞进行预处理，并用0.8mol/L甘露醇稳渗液洗净预处理液，然后加入2.5%的蜗牛酶液，在35℃水浴酶解处理60min。在此条件下，原生质体形成率为80.97%，再生率为26.47%，形成率再生率为21.43%，原生质体制备效果最好。
（6）制备酵母KD原生质体的适宜条件为：将培养至3h的酵母细胞先用0.8mol/L甘露醇稳渗液洗涤，再用0.1%EDTA-Na_2+0.1%巯基乙醇复合液对酵母细胞进行预处理，然后加入2.5%的蜗牛酶液，在35℃酶解30min。在此条件下，原生质体形成率为81.15%，再生率为10.4%，形成率×再生率为8.44%，原生质体制备效果最好，
（7）酵母KD和5045原生质体按11比例混合进行电融合，在2KV/cm脉冲场强下，脉冲时间为40μs，脉冲间隔为600ms，脉冲3次，原生质体融合率为6.93×10~(-5)。经过初筛、复筛及酿造试验，筛选出1株性能优良的融合子F16。
（8）经对融合子F16进行细胞形态及大小测定、营养缺陷型鉴定、嗜杀活性鉴定、酿造过程中对野生酵母的抑制作用及发酵酒的感官评定和香气分析，确认F16为具有良好嗜杀活性和优良生香性能的猕猴桃酒酵母，遗传了KD酵母的发酵特性和5045的嗜杀性能，是1株应用前景广阔的猕猴桃酒酵母。
In recent ten years, the kiwifruit winemaking industry has made a rapid development inChina. But the quality of kiwifruit wine is not so perfect，the fruity aroma is not enoughstrong, and the typicality is absent. And the contamination by other yeast in the early stage offermentation also leads the flavor of kiwifruit wine deteriorative. So, the purpose of this studyis to breed kiwifruit wine yeast with not only good aroma-producing performance but alsostrong killer activity. The yeast can produce more aromas and kill the wild yeasts duringfermentation, ensure pure-bred fermentation carry out smoothly and produce high qualitykiwifruit wine. The technique of protoplast electrofusion was used in the study. Theseparation of wild yeast from kiwifruits, sifting the aroma-producing yeast, preparing theparental protoplasts, determining the parameters of electrofusion, and screening andidentifying fusants were studied. The results showed as follows.
(1) There are wild yeasts on the peel of kiwifruit. After isolation and culture bysmall-scale winemaking trials, a wild kiwifruit wine yeast with good fermentationperformance was selected from seven different varieties of kiwifruit.
(2) The wild yeast and other12Saccharomyces cerevisiae were used to ferment kiwifruitjuice for wine. Through comparing the fermentation rate of all yeasts, physicochemicalindexes analysis and sensory evaluation of the kiwi wine, the yeast strain KD showed bettercharacteristics than others as follows: faster and smooth fermentation speed, higher alcoholproduction and content vitamin C, but lower volatile acid content. The kiwifruit winefermented by KD strain had high quality–light yellow color, mellow and refreshing taste,strong and pleasant fruit aroma, and pure alcoholic scent. The kiwifruit wine had harmoniousand typical fruit aroma.
(3) GC-MS analysis showed that there were more kinds of aroma components and higherlevels of their content in the kiwifruit wine fermented by KD yeast than the wine fermentedrespectively by other6yeast strains. The former wine had48kinds of aroma volatile material,and the relative content of them was97.33percent. Include19kinds of ester,7kinds ofhigher alcohol and6kinds of organic acid, the relative content were respectively34.65 percent,15.85percent, and13.3percent. Yeast strain KD had strong capacity to producearoma substances, especially more kinds of ester and higher content. The kiwi wine had rich,strong and harmonious aroma. KD was a good yeast strain for kiwifruit winemaking.
(4) S. cerevisiae KD, with capacity of produces more aromas, was non-autotrophic anddid not have killer activity. Killer yeast5045was autotrophic of histidine and was a mutant ofS. cerevisiae with defective for nuclear fusion. KD and killer yeast5045was parent. Based onProtoplast electrofusion, the killer plasmid from5045was leaded into the yeast strain KD tobreed yeast with killer activity and capacity to produce more aromas for kiwifruitwinemaking.
(5) We studied the factors which effect the formation and regeneration of the protoplastof yeast5045, such as choice the pretreatment solution and the hypertonic buffer solution,parameters of enzymolysis and cultural methods for regeneration. The optimal conditionswere summarized as follows. The cells of5045cultured for4hours were washed withhypertonic buffer solution of0.8mol/L Mannitol, then pretreated with mixture of0.1%EDTA-Na_2solution and0.1%mercaptoethanol solution (1:1, V/V) for10minutes at30℃.The pretreatment solution was washed up with0.8mol/L Mannitol. The cells were hydrolyzedby2.5%Helicase solution for60minutes at35℃. As a result, the formation rate (FR) andregeneration rate (RR) of protoplast were respectively80.97percent and26.47percent, theproduct of FR and RR was21.43percent. Protoplast formation was the best.
(6) The optimum conditions of protoplast formation of yeast strain KD were similar to5045. The cell of KD cultured for3hours also washed with hypertonic buffer solution of0.8mol/L Mannitol, then pretreated with mixture of0.1%EDTA solution and0.1%mercaptoethanol solution (1:1, V/V) for10minutes at30℃. The pretreatment solution waswashed up with0.8mol/L Mannitol. The cells were hydrolyzed by2.5%Helicase solution for30minutes at35℃. As a result, the formation rate (FR) and regeneration rate (RR) ofprotoplast were respectively81.15percent and10.4percent, the product of FR and RR was8.44percent.
(7) The protoplasts of KD and the protoplasts of5045, mixed with the ratio of1to1,were electro fused on the conditions as follow. The pulse field strength was2KV/cm, pulsetime was40microseconds, and pulse interval was600microseconds and pulse3times. Therate of protoplast fusion was6.93×10~(-5). After screening, rescreening and small-scalewinemaking, one fusant F16with excellent capability was obtained. The fusant had stablekiller activity in genetics.
(8) Through comparing the cell shape and size, auxotrophic determination, killer activityidentification, detection the inhibition action of fusant F16to wild yeast during brewing process, aroma analysis and sensory evaluation, the results showed that fusant F16inheritedthe good fermentation characteristics of KD and the strong killer performance of5045. F16was yeast for kiwifruit winemaking with superior capacity to produce more aromas and finekiller activity, and the prospect using in kiwifruit wine industry was broad.
引文
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