耐酸石榴酒酵母的筛选及其耐酸性初步研究
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摘要
石榴品种资源丰富、分布广、营养价值高、保健功能强,开发石榴酒不仅利于石榴的消费、增加产业值,而且符合发展果酒的产业政策。目前缺乏对酸石榴酒的研发。针对高酸、低pH特点,筛选出既能耐受高酸环境、又能高效降解其中有机酸的酵母具有重要意义。本文从酸、甜石榴汁成分差异,不同酵母在酸石榴汁中发酵特性,SO2对酿酒酵母代谢有机酸的影响,酿酒酵母对柠檬酸的耐性四方面进行了研究,对了解酵母的耐酸性能、解决高酸石榴及其它高酸水果的加工具有重要意义。主要结果如下:
1.对酸、甜石榴汁的理化分析表明,酸、甜石榴汁的滴定酸分别为39.2g/L和4.4g/L,pH值分别为1.92和2.45。酸石榴汁除了高酸、低pH特点外,其它成分含量均满足酵母生长所需的营养要求。
2.用反相高效液相(RP-HPLC)测定石榴汁中的有机酸组分。发现石榴汁中含有草酸、乳酸、柠檬酸、苹果酸、α-酮戊二酸、乙酸、琥珀酸、葡萄糖酸、酒石酸、富马酸。甜石榴中的有机酸以乳酸、草酸和柠檬酸为主,分别占总有机酸含量的51.2%、26.8%和16.8%;酸石榴中的有机酸以柠檬酸、乳酸为主,分别占总有机酸含量的84.4%与14.0%。
3.酿酒酵母(Saccharomyces cerevisiae)WY-1、WY-2、WY-3和拜耳结合酵母(Zygosaccharomyces bailii)WY-4在酸石榴汁中耐50mg/L的SO2,前三株酿酒酵母的发酵性能均优于拜耳结合酵母WY-4,其发酵速度快,产酒率高,但发酵结束时滴定酸、总糖、还原糖、挥发酸含量在两种酵母之间没有差别。4株酵母代谢酸石榴汁中柠檬酸的能力没有显著差异(P>0.05),发酵结束后与柠檬酸含量下降了8.7%～13.3%相对应,总有机酸含量下降了8.1%～12.8%。
4.奇异酵母(Saccharomyces paradoxus)WY-5、柠檬形克勒克酵母(Klockera apiculata)WY-6、WY-7和马科斯克鲁维酵母(Kluyveromyces marxianus)WY-8在酸石榴汁中添加50mg/L SO2的情况下不能发酵。榨汁后不进行SO2处理,WY-5和WY-6的发酵性能优于WY-8优于WY-7。酒精度差别较大,WY-5、WY-6产酒精最高为5.96%,其次为WY-8的5.79%,WY-7的最低为5.34%;WY-6产挥发酸最多(0.5g/L),其余3株在0.2g/L～0.3g/L之间;滴定酸、pH值、总糖、干浸出物差别不大。4株酵母代谢酸石榴汁中柠檬酸、乳酸的能力没有显著差异(P>0.05),发酵结束后总有机酸含量变化幅度在0.69%～3.81%之间。
5.对8株酵母发酵的酸石榴酒进行感官品评发现,酒液均澄清透明、有光泽、酸味重;在颜色、香气浓郁程度和果香突出程度上,WY-6优于WY-1、WY-5。结合菌株的发酵特性与耐SO2能力,酿酒酵母WY-1更适于发酵酸石榴汁。
6.添加60mg/L SO2对酿酒酵母WY-1生成琥珀酸有显著的促进作用(P<0.05);对酒石酸、苹果酸、α-酮戊二酸、柠檬酸和富马酸的含量则没有显著影响(P>0.05);对草酸、乙酸和乳酸的影响依石榴成分不同而有差异。
7.在苹果汁中分别添加柠檬酸2.6、5.4、9.9、19.0、32.8、47.1、61.5g/L,对应苹果汁的pH值分别为3.20、2.97、2.74、2.51、2.31、2.17、2.03,研究了酿酒酵母WY-1在其中的生长、酒精发酵情况。柠檬酸浓度为2.6g/L～61.5g/L时,WY-1均能起发,发酵周期随柠檬酸浓度增大而延长。柠檬酸浓度2.6、5.4、9.9、19.0g/L时,对WY-1的细胞的总数、死亡率、残糖量、酒精与挥发酸含量没有显著影响(P>0.05);柠檬酸浓度为32.8g/L、47.1g/L时,WY-1细胞总数显著减少(P<0.05),死亡率极显著提高、残糖量极显著增多、挥发酸生成极显著升高(P<0.01),对酒精生成没有显著影响(P>0.05)。柠檬酸浓度2.6g/L、5.4g/L时,酵母细胞体积无显著变化(P>0.05);柠檬酸浓度大于等于9.9g/L时,细胞体积极显著减小(P<0.01)。比较47.1g/L和61.5g/L两个柠檬酸浓度,发酵苹果汁中WY-1细胞总数、死亡率没有显著差异(P>0.05),细胞体积显著减小(P<0.05),后者发酵不彻底。柠檬酸浓度为0、2.6、5.4、9.9、19.0、32.8g/L时,发酵后滴定酸升高;柠檬酸浓度为47.1g/L、61.5g/L时,发酵后滴定酸降低。
Punica granarum L. is rich in cultivar resources and widely distributed. Its fruit is featured by high nutritional value and health function. Pomegranate wine products could be fit for the development of liquor all over the world. It is important to select the wine yeast with high titratable acidity resistance and its characteristic of deacidification for the sour pomegranate juice fermentation. In this research, components of sour and sweet pomegranate juices, fermentation characteristics of 8 strains of yeasts in sour pomegranate juice, effect of sulfur dioxide (SO2) on organic acid metabolism by Saccharomyces cerevisiae, and resistance of Saccharomyces cerevisiae to citric acid were studied. The main research results were as follows:
1. In the sour pomegranate juice, titratable acidity (TA) was 39.2g citric acid /L with pH value 1.92; whereas in the sweet juice, TA was 4.4g/L with pH value 2.45. Other components in sour juice, such as total sugar,α-AN and so on, were enough for wine yeasts growth except for the high acidity and low pH value.
2. A RP-HPLC method for simultaneous determination of organic acids in pomegranate juices and wines was developed. Oxalic, lactic, citric, malic,α-ketoglutaric, acetic, succinic, fumaric, tartaric and gluconic acids were found in pomegranate juices. The main organic acids were lactic acid (51.2%), oxalic acid (26.8%), and citric acid (16.8%) in sweet pomegranate juice; while citric acid (84.4%) and lactic acid (14%) were the main organic acids in sour juice.
3. Saccharomyces cerevisiae (WY-1, WY-2, WY-3) and Zygosaccharomyces bailii (WY-4) were able to endure 50mg/L SO2 in sour pomegranate juice. The wine yeast WY-1, WY-2, WY-3 had better fermentation performance with more rapid and steady fermentation speed and higher alcohol production; while WY-4 had a longer lag phase and slower fermentation speed. In all the finished wines, TA, total sugar, residual sugar and volatile acid content had no significant difference. Among the 4 wine yeast, no significant difference (P>0.05) was found in the ability of citric acid degradation, which was the main organic acid component in sour megranate juice. The total organic acid content in the finished wines decreased by 8.1~12.8% coinciding with citric acid decreasing by 8.7~13.3%.
4. Saccharomyces paradoxus (WY-5), Klockera apiculata (WY-6, WY-7), Kluyveromyces marxianus (WY-8) couldn’t ferment the sour pomegranate juice with adding 50mg/L SO2. The wine yeast WY-5, WY-6 had the best fermentation performance both with alcoholic content 5.96% in the finished wines, while WY-6 produced the most volatile acid that was 0.5g/L. The TA, pH value, total sugar, and dry extractive content had almost no difference in all finished wines. The ability of citric acid degradation for the 4 yeast had no significant difference (P>0.05) and the variation range of total organic acid content in finished wines was between 8.1% and 12.8%.
5. Sensory evaluations showed that pomegranate wines fermented by 8 strains of yeasts were all luster-transparent but have a strong acid taste. The wine fermented by WY-6 was better in natural coulor and moderate flavor than that fermented by WY-1 and WY-5. Simultaneously, considering the fermentation characteristics and SO2 resistance, the Saccharomyces cerevisiae WY-1 was the optimal strain to ferment sour pomegranate juice.
6. Adding 60mg/L SO2 could significantly enhance the succinic acid production of Saccharomyces cerevisiae WY-1 (P<0.05), while decrease the production of pyruvic acid (P<0.05). SO2 addition had no significant effect on the formation of tartaric, malic,α-ketoglutaric, citric, and fumaric acids during pomegranate wine fermentation (P>0.05). Moreover, effect of adding 60mg/L SO2 on oxalic, acetic and lactic acids was different between the sweet and sour fermented pomegranate juice.
7. The Saccharomyces cerevisiae WY-1 could ferment the apple juice with citric acid addition of 0g/L to 61.5g/L, but the fermentation period was prolonged in turn. Total cell number, size, and mortality of the yeast and residual sugar, alcohol and volatile acid of the fermented apple juice were not influenced significantly (P>0.05) by citric acid at level of 0, 2.6, 5.4, 9.9, and 19.0g/L. When the citric acid added to the apple juice was up to 32.8g/L and 47.1g/L, the total cell number of the yeast was decreased significantly (P<0.05), the cell size of it was decreased extreme significantly (P<0.01), and the cell mortality of it, residual sugar content and production of volatile acid of the fermented apple juice were increased extreme significantly (P<0.01), however the production of alcohol of the yeast was not effected significantly (P>0.05). At addition of 61.5g/L citric acid, the total cell number and cell mortality were not remarkably different (P>0.05) compared to that of 47.1 g/L citric acid addition, while cell size was reduced significantly (P<0.05) and the alcohol fermentation could not finished thoroughly. When citric acid was added at level of 0, 2.6, 5.4, 9.9, 19.0, and 32.8g/L, titration acidity of the fermented apple juice was increased after fermentation, however it was decreased at level of 47.1g/L and 61.5g/L.
1.对酸、甜石榴汁的理化分析表明,酸、甜石榴汁的滴定酸分别为39.2g/L和4.4g/L,pH值分别为1.92和2.45。酸石榴汁除了高酸、低pH特点外,其它成分含量均满足酵母生长所需的营养要求。
2.用反相高效液相(RP-HPLC)测定石榴汁中的有机酸组分。发现石榴汁中含有草酸、乳酸、柠檬酸、苹果酸、α-酮戊二酸、乙酸、琥珀酸、葡萄糖酸、酒石酸、富马酸。甜石榴中的有机酸以乳酸、草酸和柠檬酸为主,分别占总有机酸含量的51.2%、26.8%和16.8%;酸石榴中的有机酸以柠檬酸、乳酸为主,分别占总有机酸含量的84.4%与14.0%。
3.酿酒酵母(Saccharomyces cerevisiae)WY-1、WY-2、WY-3和拜耳结合酵母(Zygosaccharomyces bailii)WY-4在酸石榴汁中耐50mg/L的SO2,前三株酿酒酵母的发酵性能均优于拜耳结合酵母WY-4,其发酵速度快,产酒率高,但发酵结束时滴定酸、总糖、还原糖、挥发酸含量在两种酵母之间没有差别。4株酵母代谢酸石榴汁中柠檬酸的能力没有显著差异(P>0.05),发酵结束后与柠檬酸含量下降了8.7%～13.3%相对应,总有机酸含量下降了8.1%～12.8%。
4.奇异酵母(Saccharomyces paradoxus)WY-5、柠檬形克勒克酵母(Klockera apiculata)WY-6、WY-7和马科斯克鲁维酵母(Kluyveromyces marxianus)WY-8在酸石榴汁中添加50mg/L SO2的情况下不能发酵。榨汁后不进行SO2处理,WY-5和WY-6的发酵性能优于WY-8优于WY-7。酒精度差别较大,WY-5、WY-6产酒精最高为5.96%,其次为WY-8的5.79%,WY-7的最低为5.34%;WY-6产挥发酸最多(0.5g/L),其余3株在0.2g/L～0.3g/L之间;滴定酸、pH值、总糖、干浸出物差别不大。4株酵母代谢酸石榴汁中柠檬酸、乳酸的能力没有显著差异(P>0.05),发酵结束后总有机酸含量变化幅度在0.69%～3.81%之间。
5.对8株酵母发酵的酸石榴酒进行感官品评发现,酒液均澄清透明、有光泽、酸味重;在颜色、香气浓郁程度和果香突出程度上,WY-6优于WY-1、WY-5。结合菌株的发酵特性与耐SO2能力,酿酒酵母WY-1更适于发酵酸石榴汁。
6.添加60mg/L SO2对酿酒酵母WY-1生成琥珀酸有显著的促进作用(P<0.05);对酒石酸、苹果酸、α-酮戊二酸、柠檬酸和富马酸的含量则没有显著影响(P>0.05);对草酸、乙酸和乳酸的影响依石榴成分不同而有差异。
7.在苹果汁中分别添加柠檬酸2.6、5.4、9.9、19.0、32.8、47.1、61.5g/L,对应苹果汁的pH值分别为3.20、2.97、2.74、2.51、2.31、2.17、2.03,研究了酿酒酵母WY-1在其中的生长、酒精发酵情况。柠檬酸浓度为2.6g/L～61.5g/L时,WY-1均能起发,发酵周期随柠檬酸浓度增大而延长。柠檬酸浓度2.6、5.4、9.9、19.0g/L时,对WY-1的细胞的总数、死亡率、残糖量、酒精与挥发酸含量没有显著影响(P>0.05);柠檬酸浓度为32.8g/L、47.1g/L时,WY-1细胞总数显著减少(P<0.05),死亡率极显著提高、残糖量极显著增多、挥发酸生成极显著升高(P<0.01),对酒精生成没有显著影响(P>0.05)。柠檬酸浓度2.6g/L、5.4g/L时,酵母细胞体积无显著变化(P>0.05);柠檬酸浓度大于等于9.9g/L时,细胞体积极显著减小(P<0.01)。比较47.1g/L和61.5g/L两个柠檬酸浓度,发酵苹果汁中WY-1细胞总数、死亡率没有显著差异(P>0.05),细胞体积显著减小(P<0.05),后者发酵不彻底。柠檬酸浓度为0、2.6、5.4、9.9、19.0、32.8g/L时,发酵后滴定酸升高;柠檬酸浓度为47.1g/L、61.5g/L时,发酵后滴定酸降低。
Punica granarum L. is rich in cultivar resources and widely distributed. Its fruit is featured by high nutritional value and health function. Pomegranate wine products could be fit for the development of liquor all over the world. It is important to select the wine yeast with high titratable acidity resistance and its characteristic of deacidification for the sour pomegranate juice fermentation. In this research, components of sour and sweet pomegranate juices, fermentation characteristics of 8 strains of yeasts in sour pomegranate juice, effect of sulfur dioxide (SO2) on organic acid metabolism by Saccharomyces cerevisiae, and resistance of Saccharomyces cerevisiae to citric acid were studied. The main research results were as follows:
1. In the sour pomegranate juice, titratable acidity (TA) was 39.2g citric acid /L with pH value 1.92; whereas in the sweet juice, TA was 4.4g/L with pH value 2.45. Other components in sour juice, such as total sugar,α-AN and so on, were enough for wine yeasts growth except for the high acidity and low pH value.
2. A RP-HPLC method for simultaneous determination of organic acids in pomegranate juices and wines was developed. Oxalic, lactic, citric, malic,α-ketoglutaric, acetic, succinic, fumaric, tartaric and gluconic acids were found in pomegranate juices. The main organic acids were lactic acid (51.2%), oxalic acid (26.8%), and citric acid (16.8%) in sweet pomegranate juice; while citric acid (84.4%) and lactic acid (14%) were the main organic acids in sour juice.
3. Saccharomyces cerevisiae (WY-1, WY-2, WY-3) and Zygosaccharomyces bailii (WY-4) were able to endure 50mg/L SO2 in sour pomegranate juice. The wine yeast WY-1, WY-2, WY-3 had better fermentation performance with more rapid and steady fermentation speed and higher alcohol production; while WY-4 had a longer lag phase and slower fermentation speed. In all the finished wines, TA, total sugar, residual sugar and volatile acid content had no significant difference. Among the 4 wine yeast, no significant difference (P>0.05) was found in the ability of citric acid degradation, which was the main organic acid component in sour megranate juice. The total organic acid content in the finished wines decreased by 8.1~12.8% coinciding with citric acid decreasing by 8.7~13.3%.
4. Saccharomyces paradoxus (WY-5), Klockera apiculata (WY-6, WY-7), Kluyveromyces marxianus (WY-8) couldn’t ferment the sour pomegranate juice with adding 50mg/L SO2. The wine yeast WY-5, WY-6 had the best fermentation performance both with alcoholic content 5.96% in the finished wines, while WY-6 produced the most volatile acid that was 0.5g/L. The TA, pH value, total sugar, and dry extractive content had almost no difference in all finished wines. The ability of citric acid degradation for the 4 yeast had no significant difference (P>0.05) and the variation range of total organic acid content in finished wines was between 8.1% and 12.8%.
5. Sensory evaluations showed that pomegranate wines fermented by 8 strains of yeasts were all luster-transparent but have a strong acid taste. The wine fermented by WY-6 was better in natural coulor and moderate flavor than that fermented by WY-1 and WY-5. Simultaneously, considering the fermentation characteristics and SO2 resistance, the Saccharomyces cerevisiae WY-1 was the optimal strain to ferment sour pomegranate juice.
6. Adding 60mg/L SO2 could significantly enhance the succinic acid production of Saccharomyces cerevisiae WY-1 (P<0.05), while decrease the production of pyruvic acid (P<0.05). SO2 addition had no significant effect on the formation of tartaric, malic,α-ketoglutaric, citric, and fumaric acids during pomegranate wine fermentation (P>0.05). Moreover, effect of adding 60mg/L SO2 on oxalic, acetic and lactic acids was different between the sweet and sour fermented pomegranate juice.
7. The Saccharomyces cerevisiae WY-1 could ferment the apple juice with citric acid addition of 0g/L to 61.5g/L, but the fermentation period was prolonged in turn. Total cell number, size, and mortality of the yeast and residual sugar, alcohol and volatile acid of the fermented apple juice were not influenced significantly (P>0.05) by citric acid at level of 0, 2.6, 5.4, 9.9, and 19.0g/L. When the citric acid added to the apple juice was up to 32.8g/L and 47.1g/L, the total cell number of the yeast was decreased significantly (P<0.05), the cell size of it was decreased extreme significantly (P<0.01), and the cell mortality of it, residual sugar content and production of volatile acid of the fermented apple juice were increased extreme significantly (P<0.01), however the production of alcohol of the yeast was not effected significantly (P>0.05). At addition of 61.5g/L citric acid, the total cell number and cell mortality were not remarkably different (P>0.05) compared to that of 47.1 g/L citric acid addition, while cell size was reduced significantly (P<0.05) and the alcohol fermentation could not finished thoroughly. When citric acid was added at level of 0, 2.6, 5.4, 9.9, 19.0, and 32.8g/L, titration acidity of the fermented apple juice was increased after fermentation, however it was decreased at level of 47.1g/L and 61.5g/L.
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