CN1944657A - 提高酿酒酵母酒精耐性的方法 - Google Patents
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Abstract
提高酿酒酵母酒精耐性的方法。本发明将大豆粉作为发酵促进剂，应用于酒精浓醪发酵中，一方面，大豆中富含蛋白质和脂肪酸，能使酵母菌在高浓度酒精环境中有效抵抗酒精引发的膜流动性升高，并维持细胞膜的稳定性，从而帮助酵母抵抗酒精毒性，保持细胞活力，提高酒精发酵的浓度；另一方面，大豆含有多种钙、磷、铁等矿物质和微量元素，以及植酸、维生素B、胡萝卜素等物质，为酵母菌的发酵提供充足的营养，维持酵母细胞的正常形态和发酵能力，从而提高发酵后期的酒精发酵能力，使最终发酵醪的酒精浓度提高1～2°，而酒精浓度的提高将大大降低酒精蒸馏和DDGS的加工成本，使每吨酒精的生产成本可下降50～100元。
Description
【技术领域】：本发明属于酒精发酵技术领域，涉及一种提高酿酒酵母酒精耐性的方法，特别是以大豆粉为促进剂提高酿酒酵母浓醪发酵酒精度的方法。
【背景技术】：由于酒精对酵母菌的生长、发酵和存活都具有毒害作用，因此发酵终点的酒精浓度与酵母菌本身的耐酒精性能有关。酵母菌在酒精发酵过程中随着发酵液酒精浓度的升高，发酵速率逐渐下降，导致发酵周期延长，并使发酵终点酒精浓度降低。
酒精的毒性表现在：增加质子向酵母细胞的迁移速度，从而降低ATP酶活性，抑制氨基酸的吸收和细胞的繁殖；酒精还能破坏细胞膜中的磷脂双分子层结构，导致细胞内组分的流失；酒精对酒精脱氢酶的活力也有抑制作用，增加了乙醛的毒性。
研究者报道，在培养基中添加不饱和脂肪酸、氨基酸和磷脂均能在厌氧条件下促进酵母菌的生长和酒精生成。脂肪酸的存在可以弱化乙醇对细胞膜透性功能的影响，减少细胞内溶物渗出。细胞膜含有较多的长链脂肪酸可以增加疏水区的表面积和范德华力作用，降低极性，细胞含有较多长链饱和脂肪酸有助于酵母更快地从细胞中除去乙醇，恢复细胞膜的渗透功能。有报道指出，菌体的耐酒精能力随细胞膜脂肪酸不饱和度的增加而提高。蛋白质和磷脂是构成细胞膜的两大主要成分，所添加的外源氨基酸通过组入酵母菌细胞膜，使菌体在高浓度酒精冲击下有效抵抗酒精引起的膜流动性升高，从而维持细胞膜的稳定性。研究表明，磷脂酰胆碱是提高菌体耐酒精能力的关键成分。目前对磷脂影响酵母菌耐酒精能力的确切机制尚不清楚。在酒精浓醪发酵过程中，随着发酵时间的延续，除碳源外，其他营养物供应的匮乏，影响了酵母菌的正常的新陈代谢，使其发酵能力下降，细胞活力降低，发酵不彻底，最终难以达到理想的酒精浓度。据报道，改变营养条件及时补充营养物可增加酵母在高浓度酒精中的活性，并提高酒精浓度。
大豆中富含蛋白质(37％)和油脂(20％)，其中不饱和脂肪酸主要为亚油酸，占大豆油脂的50％。其所含磷脂(2％～3％)以卵磷脂、脑磷脂及磷脂肌醇为主，其中磷脂酰胆碱含量最高，占大豆磷脂的36.2％。此外，大豆还含有多种钙、磷、铁、硒、钼等矿物质和微量元素，以及生物素、维生素B、唾液酸、叶酸、泛酸、植酸和胡萝卜素等营养成分。大豆中上述成分的存在可以帮助酵母抵抗酒精毒性，保持细胞活力，从而可提高酒精发酵的浓度。
到目前为止，酒精是惟一可代替石油的一种可再生能源。而我国发酵酒精的生产成本大大高于汽油价格，成为制约燃料酒精发展的主要障碍。与传统的酒精发酵工艺相比，酒精浓醪发酵可以明显地提高单位设备的生产率和利用率并大幅度降低蒸煮、发酵、蒸馏和DDGS(干酒糟饲料)浓缩干燥过程中的能量消耗。另外，提高发酵终点的酒精浓度，可以显著降低后续酒精精馏操作的能耗及其产生的废糟液总量。虽然添加某些发酵辅助因子(如氨基酸、维生素、酵母膏、蛋白胨等)可以提高发酵速度和酒精发酵浓度，但其成本过高，不符合生产实际。我国年产酒精约300万吨，占世界第三位，但发酵浓度低、生产成本高，特别是与世界先进水平相比，发酵醪浓度低25～35％，能源消耗高30％左右。因此，提高酒精浓醪发酵的工艺技术水平，最大限度地降低生产成本，意义重大。
【发明内容】：本发明的目的是解决酒精浓醪发酵过程中酿酒酵母的酒精耐性问题，开发一种廉价而又适合于大生产应用的发酵促进剂，降低高浓度酒精对酿酒酵母的毒害作用，从而提供一种提高酿酒酵母酒精耐性的方法。
本发明为解决公知技术中存在的主要问题所采取的技术方案是：在酒精发酵中，添加一定量的大豆粉作为促进剂，其作用在于：一方面，大豆中富含蛋白质和脂肪酸，能使酵母菌在高浓度酒精环境中有效抵抗酒精引发的膜流动性升高，并维持细胞膜的稳定性，从而帮助酵母抵抗酒精毒性，保持细胞活力，提高酒精发酵的浓度；另一方面，大豆中含有多种钙、磷、铁等矿物质和微量元素，以及植酸、维生素B、胡萝卜素等物质，为酵母菌的发酵提供充足的营养，维持酵母细胞的正常形态和发酵能力，从而提高发酵后期的酒精发酵能力。
其具体方法如下：
(1)将大豆原料粉碎，粉碎粒度在40目以上；
(2)向发酵培养液中加入上述(1)中的大豆粉，其中：对于糖质原料，向经处理好的糖液中加入10～40g/L大豆粉；对于淀粉质原料，向制好的糖化醪中加入5～30g/L大豆粉；
(3)接入培养好的酒母或活性干酵母活化液，酵母细胞接种量为1.5×107～4.5×107/mL，30～40℃发酵，发酵周期48～72h。
上述糖质原料可以是甜菜糖蜜、甘蔗糖蜜、淀粉水解糖、葡萄糖母液等。
上述淀粉质原料可以是玉米、薯干、木薯、高粱、大米、小麦等。
本发明的优点和积极效果：本发明将大豆粉作为发酵促进剂应用到酒精浓醪发酵生产中，大豆中含有的蛋白质、脂肪酸、钙、磷、铁等矿物质和微量元素，以及植酸、维生素B、胡萝卜素等物质，能使酵母菌在高浓度酒精环境中维持细胞膜的稳定性，从而帮助酵母抵抗酒精毒性，保持正常的细胞形态和细胞活力，提高酒精发酵的浓度，使最终发酵醪的酒精浓度提高1～2°，而酒精浓度的提高将大大降低酒精蒸馏和DDGS的加工成本，使每吨酒精的生产成本可下降50～100元。
【附图说明】：
图1是发酵开始时显微观察到的酵母细胞形态图(稀释倍数n＝10)；
图2是空白样发酵72h后显微观察到的酵母细胞形态图(稀释倍数n＝10)；
图3是添加大豆粉发酵72h后显微观察到的酵母细胞形态图(稀释倍数n＝10)。
【具体实施方式】：
实施例1：杜氏管发酵试验
10Bx玉米水解糖，酒精含量16％(V)～26％(V)，每一酒精梯度分别添加20g/L的大豆粉。液体酵母种子接种量为10％，置于30℃恒温箱静置培养，每个酒精浓度做三个平行试验。发酵12h后，随时观察有无气泡产生，实验结果见表1。从结果看，添加大豆粉后，酿酒酵母的耐酒精能力明显提高，达24％(V/V)，与空白比较，耐酒精能力提高了6度。
表1添加大豆粉对酿酒酵母耐酒精能力的影响
| 酒精含量％，V/V | 16 | 18 | 20 | 22 | 24 | 26 |
| 空白大豆粉 | ++++ | +++ | -++ | -+ | -+ | -- |
注：“++”表示发酵良好，“+”表示发酵，“-”表示不发酵。
实施例2：淀粉质原料酒精发酵试验
(1)将玉米原料经粉碎后，用50℃至70℃的温水按1∶2.0的加水比调浆。
(2)向(1)中加入耐高温α-淀粉酶10U/g原料，混匀后升温至85～90℃，糊化液化90min。
(3)糊化醪冷却到55～60℃，加糖化酶120U/g原料，糖化40min。
(4)糖化醪冷却至30℃，加大豆粉促进剂分别为0、5、10、20、30g/L。
(5)接入培养的酒母，酵母细胞接种量为2.35×107/mL，30℃恒温静置发酵72h。
取发酵开始和发酵72h发酵液显微观察酵母细胞的形态(镜检稀释倍数为10)，结果见图1至图3。从图1至图3看，正常酿酒酵母细胞(发酵开始时的细胞)为卵圆形；发酵72h后，不加大豆粉的样本，由于酒精的伤害作用使酵母细胞变长变瘦，而添加大豆粉的样本，其酵母细胞形态与发酵开始时基本相同，且细胞数明显增多。可见大豆粉对维持酵母细胞的正常形态和发酵能力有明显促进作用。
发酵结束后分别测定各样品的残糖和酒精浓度，结果见表2。从结果看，添加大豆粉后，发酵醪酒精浓度提高，残糖降低，发酵更加彻底。这种作用随大豆粉添加量的增大而更加显著。其中，大豆粉添加量为30g/L时，酒精度提高幅度最大，残糖也最低，但与添加量为20g/L时相比，提高幅度已不明显，考虑成本因素，大豆粉添加量以20g/L为宜。与未加大豆粉的空白相比，发酵醪酒精浓度提高1.9％(V/V)，相对出酒率提高14.3％。
表2玉米原料添加不同用量大豆粉酒精发酵效果比较(三次试验平均值)
| 大豆粉添加量(g/L) | 0 | 5 | 10 | 20 | 30 |
| 残糖(g/100mL)酒精浓度(％， v/v)相对出酒率(％) | 3.4513.3100 | 1.3614.3107.5 | 1.214.7110.5 | 0.6615.2114.3 | 0.4815.3115.0 |
实施例3：糖蜜原料酒精发酵试验
糖蜜经适当处理后加大豆粉促进剂分别为0、10、20、30和40g/L，酵母细胞接种量为2.50×107/mL，30℃恒温静置发酵72h，实验结果见表3。从结果看，添加大豆粉后，发酵醪酒精浓度提高，残糖降低，发酵更加彻底。这种作用随大豆粉添加量的增大而更加显著。当大豆粉添加量为40g/L时，与未加大豆粉的空白相比，发酵醪酒精浓度提高1.4％(V/V)，相对出酒率提高14.1％。
表3糖蜜原料添加不同用量大豆粉酒精发酵效果比较(三次试验平均值)
| 大豆粉用量(g/L) | 0 | 10 | 20 | 30 | 40 |
| 残糖(g/100mL)酒精浓度(％，v/v)相对出酒率(％) | 3.889.9100 | 3.0510.4105.1 | 2.5110.7108.1 | 1.8311.2113.1 | 1.6411.3114.1 |
Claims (3)
1、一种提高酿酒酵母酒精耐性的方法，其特征是本发明将大豆粉作为发酵促进剂，应用于酒精浓醪发酵中，以提高酒精发酵的浓度，其具体方法如下：
(1)将大豆原料粉碎，粉碎粒度在40目以上；
(2)向发酵培养液中加入上述(1)中的大豆粉，其中：对于糖质原料，向经处理好的糖液中加入10～40g/L大豆粉；对于淀粉质原料，向制好的糖化醪中加入5～30g/L大豆粉；
(3)接入培养好的酒母或活性干酵母活化液，酵母细胞接种量为1.5×107～4.5×107/mL，30～40℃发酵，发酵周期48～72h。
2、根据权利要求1所述的提高酿酒酵母酒精耐性的方法，其特征是糖质原料是甜菜糖蜜、甘蔗糖蜜、淀粉水解糖、葡萄糖母液。
3、根据权利要求1所述的提高酿酒酵母酒精耐性的方法，其特征是淀粉质原料是玉米、薯干、木薯、高粱、大米、小麦。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103484503A (zh) * | 2013-07-15 | 2014-01-01 | 广西轻工业科学技术研究院 | 一种酒精发酵促进剂及其制法和用法 |
| US9012190B2 (en) | 2011-06-15 | 2015-04-21 | Butamax Advanced Biofuels Llc | Use of thiamine and nicotine adenine dinucleotide for butanol production |
| CN113717918A (zh) * | 2021-07-02 | 2021-11-30 | 广西乐酵生物科技有限公司 | 一种可替代抗生素、无机盐和酸性蛋白酶的多功能酒精酵母促进剂 |
| CN116121092A (zh) * | 2022-11-01 | 2023-05-16 | 四川大学 | 多重胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用 |
| EP4273221A1 (en) | 2022-05-06 | 2023-11-08 | Uniwersytet Zielonogórski | Method of obtaining saccharomyces cerevisiae yeast biomass with increased resistance to osmotic stress and application of the biomass |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9012190B2 (en) | 2011-06-15 | 2015-04-21 | Butamax Advanced Biofuels Llc | Use of thiamine and nicotine adenine dinucleotide for butanol production |
| CN103484503A (zh) * | 2013-07-15 | 2014-01-01 | 广西轻工业科学技术研究院 | 一种酒精发酵促进剂及其制法和用法 |
| CN103484503B (zh) * | 2013-07-15 | 2015-09-02 | 广西轻工业科学技术研究院 | 一种酒精发酵促进剂及其制法和用法 |
| CN113717918A (zh) * | 2021-07-02 | 2021-11-30 | 广西乐酵生物科技有限公司 | 一种可替代抗生素、无机盐和酸性蛋白酶的多功能酒精酵母促进剂 |
| EP4273221A1 (en) | 2022-05-06 | 2023-11-08 | Uniwersytet Zielonogórski | Method of obtaining saccharomyces cerevisiae yeast biomass with increased resistance to osmotic stress and application of the biomass |
| CN116121092A (zh) * | 2022-11-01 | 2023-05-16 | 四川大学 | 多重胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用 |
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