原生质体融合构建增香型猕猴桃酒酵母的研究
摘要
我国是世界上猕猴桃生产大国，但鲜果加工业相对落后，造成农民卖果难。猕猴桃
酒营养丰富，对人体具有多种保健作用。充分利用猕猴桃资源，发展猕猴桃酒，可以增
加猕猴桃产业的附加值，推动猕猴桃产业健康发展。但由于缺乏猕猴桃酒发酵专用酵母
菌种，导致生产的猕猴桃酒香气不足、果香不突出、典型性欠佳，严重影响了猕猴桃酒
的质量。应用现代生物技术，构建增香型猕猴桃酒酵母将对改善猕猴桃酒质量起到关键
作用。原生质体融合技术作为一种最为有效的工业微生物育种手段之一，能够实现基因
的大量、高频、多位点的重组和整合，适合用于增香酵母的构建。本研究以一株发酵力
强的酵母和一株产香性能好的酵母作为亲株，通过单亲灭活原生质体融合技术，构建出
增香型猕猴桃干酒酵母，用以增强猕猴桃酒香气，较大程度改善猕猴桃酒品质。
研究结果表明：
1．通过对Y1、Y2、Y3、Y4等4株酵母的发酵力、发酵过程中糖度变化趋势、原酒
主要理化指标和成品酒感官品评结果综合分析，选取Yl和Y3适合作为融合亲本。
2．紫外线诱变方法制备Y1单倍体遗传标记，得到一株组氨酸缺陷型突变株。
3．亲株原生质体制备的适宜条件
①菌龄：斜面菌种转接于5ml YEPD液体培养基，振荡培养12h，按1％接种量转接
于50ml YEPD液体培养基中，振荡培养5h，菌浓度达107个／m1。
②预处理：菌体108。个，预处理液3mi，30℃恒温水浴，振荡处理15min。
③酶解条件：菌体lO8个，蜗牛酶浓度20mg／ml，酶用量60mg，酶解温度37℃恒温水浴，间歇微振荡。Y1(hi s一)酶解45min，原生质体形成率98．2％，再生率20．3％；Y3酶解60min，原生质体形成率95．3％，再生率21．3％。
4．Y3原生质体灭活条件：80"C恒温水浴处理30min，灭活率达100％。
5．融合条件：两亲株原生质体各108个， 1：1比例混合，30℃恒温水浴中预培养15min，促融剂2ml．，30℃融合30min，融合率达10-8。
6．检出的69株融合子经初筛，得到10株发酵力强的融合子。进一步的猕猴桃酒发酵试验结果证明，与亲株相比，融合子F25和F41与Y1具有同样强的发酵力；发酵的猕猴桃酒理化指标和感官品评结果表明，融合子整合了亲株的优良性状。
7．GC—MS香气成分分析结果表明，亲株和融合株发酵酒的香气成分在种类上和含量上均有差异。进一步的主成分分析结果表明，各菌株香气成分的差异主要在于2-丙酸丁酯、乙酸异戊酯、己酸、2．苯乙醇、琥珀酸单乙酯、4一羟基-苯乙醇、2-甲基琥珀酸二乙酯、3．羟基．4．甲氧基．苯甲酸、肉桂酸乙酯等10种成分的含量和配比。
8．亲株和融合株发酵酒的香气成分聚类分析结果表明，各融合子在香气上与亲株
：Y3相近，Y3真正起到了香气供体的作用。
9．融合子鉴定结果表明，得到的10株融合子是真正的融合子，且具有遗传稳定性。
China is a major country producing kiwi fruit, but only a small fraction of kiwi is processed, which causes farmers to gain little benefit from kiwifruit. Kiwi wine, as a fine processed product, proved to be nutritious and healthy, can provide the kiwi industry high extra value. Therefore, making good use of kiwi resource to produce kiwi wine can alleviate the crisis between supply and demand and so help the kiwi industry lead a healthy way. However, as a result of absence of good yeasts, kiwi wine was evaluated as poor banquet, less fruity aroma, weak typicality, which gav.j kiwi wine bad quality. So, construct good yeasts became a key step to solve the kiwi wine problems. Protoplast fusion had long been proved to be an effective way to industry yeasts breeding because of its high frequency genes integration and recombination. In this paper, protoplast fusion was carried out between a yeast with strong fermentation vigor and a high aroma productive yeast to construct good yeasts for quality kiwi wine. heresults showed that,1.Y1 and Y3 were selected as parental strains in term of ermentation vigor, sugar utilization velocity during fermentation, ysio-chemical analysis of fresh wine and organleiptical evaluation of end product.2.The genetic maker (his") of Yl was obtained by UV-induced mutation. 3.Protoplast eparation procedure:?Culture: grown in liquid YEPD medium (50ml) for 5h at 28 °C on a rotary shaker (150 rpm) according to the growth curve.②Pretreatment: 108 cells were suspended in 3ml pretreatment buffer at 30°C for 15min.③Optimal cells wall enzymatic lysis condition: suspended 20mg/ml snailase in PBS pH 6.0, incubated at 37"C (Yl for 45 min, Y3 for 60 min) with interval gentle shaking. The ratio of protoplasts formation reached to 98.2% and frequency of protoplasts regeneration 20.3% for Yl. 95.3% and 21.3% for Y3. 4. Inactivation condition of Y3 protoplasts was obtained: 80°C for SOmin. S.PEG-induced protoplast fusion was carried out between live Yl(his') protoplasts and Y3 heat-inactivated protoplasts. Fusants were recovered on YNB minimal medium. The fusion frequency was 10-5.6.Sixty-nine clones obtained were screened for the larger colony, only 10 clones: Fl, F12, F13, F17, F25, F30, F38, F41, F52 and F65 had good fermentation vigor.Further screen work was carried out by kiwi wine fermentation using these fusants. Results showed that F25 and F41 had strong vigor and good aroma producing characteristics.T.Aromatic components GC-MS determination showed that volatile compounds in kiwi wines fermented
with parental and fusants varied in kinds and amount. Further principle components analysis showed that difference lied in some esters and phenyl ethyl alcohol.8.Cluster analysis of aromatic compounds in kiwi wine showed that Y3 was donor of aroma.9.Identification results showed that 10 fusants were real hybrids between parental strains and stable during subculture.
酒营养丰富，对人体具有多种保健作用。充分利用猕猴桃资源，发展猕猴桃酒，可以增
加猕猴桃产业的附加值，推动猕猴桃产业健康发展。但由于缺乏猕猴桃酒发酵专用酵母
菌种，导致生产的猕猴桃酒香气不足、果香不突出、典型性欠佳，严重影响了猕猴桃酒
的质量。应用现代生物技术，构建增香型猕猴桃酒酵母将对改善猕猴桃酒质量起到关键
作用。原生质体融合技术作为一种最为有效的工业微生物育种手段之一，能够实现基因
的大量、高频、多位点的重组和整合，适合用于增香酵母的构建。本研究以一株发酵力
强的酵母和一株产香性能好的酵母作为亲株，通过单亲灭活原生质体融合技术，构建出
增香型猕猴桃干酒酵母，用以增强猕猴桃酒香气，较大程度改善猕猴桃酒品质。
研究结果表明：
1．通过对Y1、Y2、Y3、Y4等4株酵母的发酵力、发酵过程中糖度变化趋势、原酒
主要理化指标和成品酒感官品评结果综合分析，选取Yl和Y3适合作为融合亲本。
2．紫外线诱变方法制备Y1单倍体遗传标记，得到一株组氨酸缺陷型突变株。
3．亲株原生质体制备的适宜条件
①菌龄：斜面菌种转接于5ml YEPD液体培养基，振荡培养12h，按1％接种量转接
于50ml YEPD液体培养基中，振荡培养5h，菌浓度达107个／m1。
②预处理：菌体108。个，预处理液3mi，30℃恒温水浴，振荡处理15min。
③酶解条件：菌体lO8个，蜗牛酶浓度20mg／ml，酶用量60mg，酶解温度37℃恒温水浴，间歇微振荡。Y1(hi s一)酶解45min，原生质体形成率98．2％，再生率20．3％；Y3酶解60min，原生质体形成率95．3％，再生率21．3％。
4．Y3原生质体灭活条件：80"C恒温水浴处理30min，灭活率达100％。
5．融合条件：两亲株原生质体各108个， 1：1比例混合，30℃恒温水浴中预培养15min，促融剂2ml．，30℃融合30min，融合率达10-8。
6．检出的69株融合子经初筛，得到10株发酵力强的融合子。进一步的猕猴桃酒发酵试验结果证明，与亲株相比，融合子F25和F41与Y1具有同样强的发酵力；发酵的猕猴桃酒理化指标和感官品评结果表明，融合子整合了亲株的优良性状。
7．GC—MS香气成分分析结果表明，亲株和融合株发酵酒的香气成分在种类上和含量上均有差异。进一步的主成分分析结果表明，各菌株香气成分的差异主要在于2-丙酸丁酯、乙酸异戊酯、己酸、2．苯乙醇、琥珀酸单乙酯、4一羟基-苯乙醇、2-甲基琥珀酸二乙酯、3．羟基．4．甲氧基．苯甲酸、肉桂酸乙酯等10种成分的含量和配比。
8．亲株和融合株发酵酒的香气成分聚类分析结果表明，各融合子在香气上与亲株
：Y3相近，Y3真正起到了香气供体的作用。
9．融合子鉴定结果表明，得到的10株融合子是真正的融合子，且具有遗传稳定性。
China is a major country producing kiwi fruit, but only a small fraction of kiwi is processed, which causes farmers to gain little benefit from kiwifruit. Kiwi wine, as a fine processed product, proved to be nutritious and healthy, can provide the kiwi industry high extra value. Therefore, making good use of kiwi resource to produce kiwi wine can alleviate the crisis between supply and demand and so help the kiwi industry lead a healthy way. However, as a result of absence of good yeasts, kiwi wine was evaluated as poor banquet, less fruity aroma, weak typicality, which gav.j kiwi wine bad quality. So, construct good yeasts became a key step to solve the kiwi wine problems. Protoplast fusion had long been proved to be an effective way to industry yeasts breeding because of its high frequency genes integration and recombination. In this paper, protoplast fusion was carried out between a yeast with strong fermentation vigor and a high aroma productive yeast to construct good yeasts for quality kiwi wine. heresults showed that,1.Y1 and Y3 were selected as parental strains in term of ermentation vigor, sugar utilization velocity during fermentation, ysio-chemical analysis of fresh wine and organleiptical evaluation of end product.2.The genetic maker (his") of Yl was obtained by UV-induced mutation. 3.Protoplast eparation procedure:?Culture: grown in liquid YEPD medium (50ml) for 5h at 28 °C on a rotary shaker (150 rpm) according to the growth curve.②Pretreatment: 108 cells were suspended in 3ml pretreatment buffer at 30°C for 15min.③Optimal cells wall enzymatic lysis condition: suspended 20mg/ml snailase in PBS pH 6.0, incubated at 37"C (Yl for 45 min, Y3 for 60 min) with interval gentle shaking. The ratio of protoplasts formation reached to 98.2% and frequency of protoplasts regeneration 20.3% for Yl. 95.3% and 21.3% for Y3. 4. Inactivation condition of Y3 protoplasts was obtained: 80°C for SOmin. S.PEG-induced protoplast fusion was carried out between live Yl(his') protoplasts and Y3 heat-inactivated protoplasts. Fusants were recovered on YNB minimal medium. The fusion frequency was 10-5.6.Sixty-nine clones obtained were screened for the larger colony, only 10 clones: Fl, F12, F13, F17, F25, F30, F38, F41, F52 and F65 had good fermentation vigor.Further screen work was carried out by kiwi wine fermentation using these fusants. Results showed that F25 and F41 had strong vigor and good aroma producing characteristics.T.Aromatic components GC-MS determination showed that volatile compounds in kiwi wines fermented
with parental and fusants varied in kinds and amount. Further principle components analysis showed that difference lied in some esters and phenyl ethyl alcohol.8.Cluster analysis of aromatic compounds in kiwi wine showed that Y3 was donor of aroma.9.Identification results showed that 10 fusants were real hybrids between parental strains and stable during subculture.
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