CN115322911B - 一种异常威克汉姆酵母菌及其应用 - Google Patents
一种异常威克汉姆酵母菌及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域，尤其涉及一种异常威克汉姆酵母菌及其应用。本发明从白酒生产中的大曲分离得到一株高产4‑乙烯基愈创木酚的菌株，经过鉴定其理化性质和异常威克汉姆酵母菌相符合，将其命名为异常威克汉姆酵母Wickerhamomyces anomalus WL‑DQ‑2。本发明随后对其进行保藏，保藏编号为CGMCC NO.25203。本发明提供的异常威克汉姆酵母具有有高产4‑乙烯基愈创木酚的特性，可应用于酿造酒、蒸馏酒及其它食品领域，提高原料利用率，改善发酵食品风味品质。
Description
技术领域
本发明涉及微生物技术领域，尤其涉及一种异常威克汉姆酵母菌及其应用。
背景技术
4-乙烯基愈创木酚，分子式为C9H10O2，密度1.11×103kg/m3，熔点26～29℃，沸点100℃/667Pa，是一种挥发性酚类化合物，具有强烈香辛料、丁香和发酵香味，其感官阈值低，为0.3mg/L，是决定白酒、啤酒、葡萄酒和酱油等产品的重要风味物质，也是这些发酵产品全面风味评价必不可缺的特色成分。同时，4-乙烯基愈创木酚在日化、医药以及香精等行业也是不可多得的高档香料，其市场价值约为阿魏酸的40倍左右，并且可以进一步转化为4-乙基愈创木酚、香草醛、香草醇和香草酸等各种高附加值的化合物。天然4-乙烯基愈创木酚主要存在于玉米酒精发酵的挥发物中，为无色或淡黄色油状液体，有发酵气息、微带甜味和独特的酚感，不溶于水，但溶于甲醇和油类。
4-乙烯基愈创木酚对白酒风味有重要作用。随着白酒风味的研究的深入，调控风味化合物的策略越来越受到重视。如何提高酿造过程中高粱利用率，同时提高风味化合物的含量，利用酒醅中营养成分进行深加工，对提高谷物类发酵食品利用率，促进产业发展、提高产业效益有重要意义。除了具有作为香料和添加剂的商业价值外，4-乙烯基愈创木酚由于其抗氧化活性和抗癌活性，在医药制品领域，如用于眼科领域，其有着作为隐形眼镜溶液和滴眼剂也存在潜在价值。
目前，市场上的4-乙烯基愈创木酚多采用化学法合成，需要较多的前体物质。有些还需要较为剧烈的条件，如高温高压，微波加热诱导脱羧等，但化学合成法生产出的产品都不属于天然物质。由于4-乙烯基愈创木酚很大一部分用于香精、医药及食品工业，而化学合成品用于这些行业有一定的安全隐患。所以，寻找快速安全生产4-乙烯基愈创木酚的生物合成途径是很有必要的。
发明内容
为了解决现有的技术问题，本发明提供一种异常威克汉姆酵母菌及其应用。
第一方面，本发明提供一种异常威克汉姆酵母菌(Wickerhamom yces anomalus)，本发明从白酒生产中的大曲分离得到一株高产4-乙烯基愈创木酚的菌株，经过鉴定其理化性质和异常威克汉姆酵母菌相符合，进而对其进行生物保藏，保藏信息如下：
保藏编号：CGMCC NO.25203；分类命名：Wickerhamomyces anomalus；保藏单位：中国微生物菌种保藏管理委员会普通微生物中心；保藏地址：北京市朝阳区北辰西路1号院3号；保藏日期：2022年6月27日。
本发明进一步包括所述异常威克汉姆酵母菌(Wickerhamomyces anomalus)的微生物菌剂。
进一步地，所述微生物菌剂包括如下任意一种或多种：
i)异常威克汉姆酵母菌(Wickerhamomyces anomalus)菌体的活细胞；
ii)冷冻干燥得到的异常威克汉姆酵母菌(Wickerhamomyces anomalus)干菌体；
iii)固定化的异常威克汉姆酵母菌(Wickerhamomyces anomalus)细胞；
iv)异常威克汉姆酵母菌(Wickerhamomyces anomalus)的液体菌剂；
v)异常威克汉姆酵母菌(Wickerhamomyces anomalus)的固体菌剂。
本发明进一步提供所述的异常威克汉姆酵母菌(Wickerhamomyces anomalus)，或所述的微生物菌剂在生产4-乙烯基愈创木酚中的应用。
本发明进一步提供所述的异常威克汉姆酵母菌(Wickerhamomyces anomalus)，或所述的微生物菌剂在提高发酵产品中4-乙烯基愈创木酚含量中的应用。
本发明进一步提供所述的异常威克汉姆酵母菌(Wickerhamomyces anomalus)，或所述的微生物菌剂在提高发酵产品风味中的应用。
进一步地，所述发酵产品包括：白酒、啤酒、葡萄酒或酱油中的一种或多种。
进一步地，可以将所述异常威克汉姆酵母菌(Wickerhamomyces anomalus)加入至酒醅深加工中，应用于4-乙烯基愈创木酚生产。
进一步，可以采用所述异常威克汉姆酵母菌(Wickerhamomyces anomalus)所得的脱羧酶进行降解阿魏酸。
第二方面，本发明提供所述的异常威克汉姆酵母菌(Wickerhamomyces anomalus)的发酵方法，包括：
将所述异常威克汉姆酵母菌(Wickerhamomyces anomalus)接种于发酵产品中进行发酵。
进一步地，所述接种于发酵产品中进行发酵为：接种于高粱汁液态发酵培养基中进行发酵，或，添加至谷物类发酵食品生产中。
进一步地，所述接种于高粱汁液态发酵培养基中进行发酵为：
接种于高粱汁液态发酵培养基中，在28～32℃的条件下培养3～5d。
本发明具备如下有益效果：
本发明从大曲中分离得到了一株高效产生4-乙烯基愈创木酚的异常威克汉姆酵母菌(Wickerhamomyces anomalus)，其具备较高的4-乙烯基愈创木酚的生产能力。本发明提供的异常威克汉姆酵母菌可以接种高粱汁液态培养基中进行4-乙烯基愈创木酚的生产，并且产量显著高于其他菌株。基于该异常威克汉逊酵母在白酒酿造过程中高产4-乙烯基愈创木酚特性，可以将其应用于酒糟的生物再利用领域。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
本实施例提供异常威克汉姆酵母Wickerhamomyces anomalus筛选及鉴定的方法，具体流程如下：
1、取10g浓香型大曲溶于90mL无菌生理盐水中，30℃条件下摇床振荡培养30分钟后进行梯度稀释，涂布WL营养琼脂培养基，于30℃恒温条件下培养3-5天，根据平板上菌落形态特征，收集Wickerhamomyces anomalus孢子进行保菌。
2、将菌株送至北京睿博生物技术有限公司进行序列测定后在NCBI中经Blast比较分析确定为异常威克汉逊酵母菌，确定所筛选得高产4-乙烯基愈创木酚的异常威克汉逊酵母在分类学上都属于Wickerhamomyces anomalus异常威克汉逊酵母，最终所得到一株高产4-乙烯基愈创木酚的异常威克汉逊酵母菌，命名为异常威克汉逊酵母Wickerhamomycesanomalus。
本发明将异常威克汉逊酵母Wickerhamomyces anomalus进行保藏，保藏信息如下：
保藏编号：CGMCC NO.25203；分类命名：Wickerhamomyces anomalus；保藏单位：中国微生物菌种保藏管理委员会普通微生物中心；保藏地址：北京市朝阳区北辰西路1号院3号；保藏日期：2022年6月27日。
WL营养琼脂培养基(g·L-1)：酵母粉5.00，酸水解酪蛋白5.00，葡萄糖50.00，磷酸二氢钾0.55，氯化钾0.425，氯化钙0.125，硫酸镁0.125，氯化铁0.0025，硫酸锰0.0025，溴甲酚绿0.022，琼脂17.00，加1000mL蒸馏水。
实施例2
本实施例验证实施例1涉及的异常威克汉逊酵母Wickerhamomyces anomalus代谢产4-乙烯基愈创木酚的功能，具体如下：
高粱液态发酵培养基：取适量的高粱粉碎后，以高粱粉：水＝1：4的比例混合，煮沸后冷却，加入耐高温α-淀粉酶，90℃水浴液化2h，冷却至60℃加入糖化酶酶解1h。采用脱脂棉网过滤，并将滤后液离心取上清液调整糖度为10°BX，pH 6.0。之后分装于250mL锥形瓶中，每瓶50mL，115℃条件下灭菌20min。
发酵培养：高粱液态发酵培养基灭菌后接种异常威克汉逊酵母菌液，3mL液体培养基接种30μL菌液，于30℃培养4d，间隔12h摇晃震荡一次。采用真空吸附辅助萃取方法结合气相色谱-质谱联用仪器测定接种异常威克汉逊酵母的培养基4-乙烯基愈创木酚的含量变化。定量采用选择离子扫描模式(SIM)，并结合4-辛醇内标法定量，最终与其他微生物相比，本发明分离得到的异常威克汉逊酵母菌具有较强产生4-乙烯基愈创木酚能力，结果如下表所示，本发明进行5次重复性实验，得到如下结果：
表1异常威克汉逊酵母菌生产4-乙烯基愈创木酚的能力
| 4-乙烯基愈创木酚产量(μg·L-1) | |
| 96h发酵产物 | |
| 第一组 | 102.25 |
| 第二组 | 148.67 |
| 第三组 | 104.76 |
| 第四组 | 150.00 |
| 第五组 | 99.03 |
试验例1
本试验例进一步采用实施例1相同的方法针对现有的异常威克汉姆酵母的4-乙烯基愈创木酚生产水平进行检测(PDA-DQ-1以及WL-DQ-5均为本发明分离得到的同种不同株的异常威克汉逊酵母菌)，得到如下结果：
表2不同异常威克汉姆酵母菌
| 4-乙烯基愈创木酚产量(μg·L-1) | |
| 菌株名称 | 96h发酵产物 |
| WL-DQ-2 | 109.09 |
| PDA-DQ-1 | 29.60 |
| WL-DQ-5 | 79.25 |
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (10)
1.一种异常威克汉姆酵母菌（Wickerhamomyces anomalus）WL-DQ-2，其特征在于，保藏编号为CGMCC NO.25203。
2.一种微生物菌剂，其特征在于，包括权利要求1所述的异常威克汉姆酵母菌（Wickerhamomyces anomalus）。
3.根据权利要求2所述的微生物菌剂，其特征在于，所述微生物菌剂包括如下任意一种或多种：
i）异常威克汉姆酵母菌（Wickerhamomyces anomalus）菌体的活细胞；
ii）冷冻干燥得到的异常威克汉姆酵母菌（Wickerhamomyces anomalus）干菌体；
iii）固定化的异常威克汉姆酵母菌（Wickerhamomyces anomalus）细胞；
iv）异常威克汉姆酵母菌（Wickerhamomyces anomalus）的液体菌剂；
v）异常威克汉姆酵母菌（Wickerhamomyces anomalus）的固体菌剂。
4.权利要求1所述的异常威克汉姆酵母菌（Wickerhamomyces anomalus），或权利要求2或3所述的微生物菌剂在生产4-乙烯基愈创木酚中的应用。
5.权利要求1所述的异常威克汉姆酵母菌（Wickerhamomyces anomalus），或权利要求2或3所述的微生物菌剂在提高发酵产品中4-乙烯基愈创木酚含量中的应用。
6.权利要求1所述的异常威克汉姆酵母菌（Wickerhamomyces anomalus），或权利要求2或3所述的微生物菌剂在提高发酵产品风味中的应用。
7.根据权利要求5或6所述的应用，其特征在于，所述发酵产品包括：白酒、啤酒、葡萄酒或酱油中的一种或多种。
8.权利要求1所述的异常威克汉姆酵母菌（Wickerhamomyces anomalus）的发酵方法，其特征在于，包括：
将所述异常威克汉姆酵母菌（Wickerhamomyces anomalus）接种于发酵产品中进行发酵。
9.根据权利要求8所述的发酵方法，其特征在于，所述接种于发酵产品中进行发酵为：接种于高粱汁液态发酵培养基中进行发酵，或，添加至谷物类发酵食品生产中。
10.根据权利要求9所述的发酵方法，其特征在于，所述接种于高粱汁液态发酵培养基中进行发酵为：
接种于高粱汁液态发酵培养基中，在28~32 ℃的条件下培养3~5d。
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