CN108949595B - 一种产香酵母菌及其在红曲黄酒酿造中的应用 - Google Patents
一种产香酵母菌及其在红曲黄酒酿造中的应用 Download PDFInfo
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- CN108949595B CN108949595B CN201810898674.6A CN201810898674A CN108949595B CN 108949595 B CN108949595 B CN 108949595B CN 201810898674 A CN201810898674 A CN 201810898674A CN 108949595 B CN108949595 B CN 108949595B
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- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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Abstract
本发明公开了一种产香酵母菌及其在红曲黄酒酿造中的应用，所述产香酵母菌的分类命名为酿酒酵母JJ4（Saccharomyces cerevisiae JJ4)，已保藏于中国典型培养物保藏中心，其保藏编号为CCTCC NO：M 2018219，保藏日期为2018年4月19日。该酿酒酵母JJ4具有较好的发酵和产香能力。本发明在红曲黄酒发酵过程中添加酿酒酵母JJ4强化发酵，可丰富红曲黄酒的风味组成，显著提高酯类及酸类挥发性风味物质的总量，研制出风味独特的红曲黄酒新产品，具有很好的经济和社会效益。
Description
技术领域
本发明涉及微生物技术领域，具体涉及一种产香酵母菌及其在黄酒酿造中的应用。
背景技术
酿造红曲米是红曲黄酒酿造的主要发酵剂，其微生物来源于人工接种及制备环境的开放式自由落菌，含有曲霉、酵母菌和乳酸菌等多种微生物。酵母是黄酒发酵的灵魂和命脉，其品种好坏直接决定所酿黄酒品质的优劣。酿酒酵母能够合成多种酶，将原料中的前体物质转换成风味物质，如酯、酸、醇和醛等产物，对黄酒风味的形成有着重要的作用。醇是风味物质中的主要化合物，含量差别不大，但却是造成各种酒不同风味的原因之一，主要由酵母菌产生；酯是重要的一类风味成分，主要是酵母的生物合成，贮藏中，酸与醇的酯化作用使酯类含量明显增加，所以酒会越陈越香：黄酒中的有机酸较多，一部分来源于原料，一部分由微生物发酵生成，酸类化合物本身对酒香的直接贡献不大，但却有维持酯的风味、调整酒的风味的作用；酚类物质含量很少，但呈香作用很大；羰基化合物包括醛类和酮类，是酒中较为重要的风味成分，大多数由微生物酵解而成。优质产香酵母可代谢产生多种酯类、酸类、醇类挥发性风味物质，赋予红曲黄酒浓郁的芳香，对提高红曲黄酒品质具有重要的意义。目前针对产香酵母的研究多集中于白酒和麦曲黄酒，鲜有针对红曲黄酒酿造特性选育产香酵母的研究报道。
发明内容
本发明的目的在于提供一种产香酵母菌及其在红曲黄酒酿造中的应用。
为了是实现上述目的，本发明采用以下技术方案：
本发明提供了一种产香酵母菌，该菌株结合形态特征和基于细菌26S rDNA基因序列的系统发育分析，鉴定为酿酒酵母JJ4(Saccharomyces cerevisiae JJ4)，已保藏于中国典型培养物保藏中心，其保藏编号为CCTCC NO：M 2018219，保藏日期为2018年4月19日。
本发明提供的酿酒酵母JJ4可应用于酿造红曲黄酒，与传统发酵红曲黄酒产品CK相比，104、105、106cfu/mL接种的酿酒酵母JJ4可使酯类物质总量提高69.91％、93.09％、81.26％，使酸类物质总量提高44.60％、90.18％、137.57％，差异均达极显著水平(P<0.01)；并不会提高醇类物质的总量(P>0.05)。添加酿酒酵母JJ4可新增3-羟基己酸乙酯、硬脂酸乙酯两种物质，使壬醇、庚醇、丁二醇、二异丁基甲醇和1-壬醇等5种醇类的含量提高36.75％、89.65％、168.90％、113.79％和38.92％；使乙酸乙酯、丁酸乙酯、乙酸异戊酯、己酸乙酯、乳酸乙酯、辛酸乙酯、壬二酸二乙酯、棕榈酸乙酯等8种酯类的含量提高89.80％、442.19％、129.32％、238.08％、132.54％、230.00％、58.50％和20.34％；使己酸、辛酸、壬酸、正癸酸、月桂酸等5种酸类物质含量提高103.17％、178.51％、61.14％、136.25％和64.60％，差异均达极显著水平(P<0.01)。
附图说明
图1为产香酿酒酵母JJ4的菌落形态图；
图2为产香酿酒酵母JJ4的微观形态图；
图3为产香酿酒酵母JJ4与相关种的26S rDNA序列系统发育树。
具体实施方式
以下实施例中所用的实验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均可从商业途径获得。
商用酵母JAQ：宁德市黄家酒业有限公司提供。
酵母菌分离纯化培养基：改良马铃薯葡萄糖培养基PDA。马铃薯200g/L、葡萄糖20g/L、去氧胆酸钠0.5g/L、氯霉素0.1g/L、琼脂15g/L。其中去氧胆酸钠可以抑制丝状真菌菌丝的蔓延生长。
麦芽汁培养基：可溶性固形物含量10％麦芽汁，使用乳酸及氢氧化钠调节pH值至5.4，121℃灭菌20min，冷却备用。加2％琼脂可制成麦芽汁琼脂培养基。
白糖水培养基：配制140g/L的白糖水，添加5g/L谷氨酸钠或5g/L尿素，121℃灭菌20min，冷却后备用。
大米糖化液培养基：称量100g的糯米于500mL锥形瓶中，浸泡过夜后沥干，121℃灭菌20min，冷却后添加150mL无菌水，再分别加入用量为每g米饭70U、560U的α-淀粉酶和糖化酶，酶解后基质固形物含量30％，总酸0.23g/L，pH值6.0。
实施例1菌株的分离、筛选、鉴定及保存
采用平板划线法，从风味优良的蜂窝样品发酵液中分离酵母菌；通过发酵风味和酿造特性试验，筛选出具有独特发酵风味的优良酵母菌。
1.菌株的分离、纯化
以风味优良的蜂窝样品为选育菌源，梯度稀释并涂布于酵母菌分离纯化培养基平板上，经多次划线分离和镜检，得到较纯的菌株。根据TTC颜色变化试验，挑选出具有典型酵母菌菌落特征的菌株6株，编号为JJ1、JJ2、JJ3、JJ4、JJ5、JJ6，得到的菌株分别接种于麦芽汁斜面培养基上，28℃培养1d，4℃冰箱保藏，备用。
2.菌株的初筛
将分离得到的菌株分别以2％的接种量加入大米糖化液培养基中，在20℃控温发酵3d，以发酵风味为指标初筛产香酵母菌株。
发酵样品按其风味特征分为3级。一级为发酵香浓郁，标记为“+++”；；二级为发酵一般，标记为“++”；三级为发酵香不明显，标记为“-”。请24个经培训过的专业人士进行独立评价，取平均值统计分析。
6株菌株在不同培养基中的发酵风味试验结果见表1。酵母菌JJ2、JJ4和JJ6在大米糖化液中具有浓郁的发酵香，评分为“+++”，酵母菌JJ3在大米糖化液中具有发酵香，评分为“+”，酵母菌JAQ、JJ1和JJ5均不能产生发酵香，评分为“-”。结果表明，具有独特发酵风味的优良酵母菌共有3株，编号分别为JJ2、JJ4和JJ6。
表1 6株酵母菌在不同培养基中的发酵风味评价
3.菌株的复筛
按米饭：水为1:1落缸，加入总重5％的乌衣红曲米和5％的酵母菌JJ2、JJ4、JJ6，控温25±2℃发酵，发酵结束后检测各处理酒精度、总酸，并进行感官风味评价，以不添加酵母菌的处理为对照，筛选红曲黄酒酿造适宜酵母菌株，结果见表2。由表2可见，初筛酵母菌JJ2、JJ4、JJ6的总酸比对照组分别降低44.71％、46.31％、40.13％，差异均达极显著水平(P<0.01)；酒精度及残糖水平各处理间无显著差异；酵母菌JJ2、JJ4和JJ6均可显著提高红曲黄酒的风味，其中以JJ4效果最佳。结果表明，红曲黄酒酿造的适宜产香酵母为酵母菌JJ4。
表2酵母菌复筛
4.菌株的鉴定
将菌株JJ4接种于MEA培养基，28℃培养7d，观察到呈乳白色，质地粘稠，表面反光，边缘不整齐的菌落，如图1所示。如图2，显微镜下观察到细胞呈椭圆形或棒状，大小2.5-6×4-15μm，繁殖方法为出芽生殖。
表3菌株JJ4的生理生化特征
注：+表示反应为阳性，-表示反应为阴性。
从表3中可以看出，菌株JJ4可以D-葡萄糖、海藻糖、D-麦芽糖、D-棉籽糖、D-半乳糖、D-蔗糖为碳源。
对菌株JJ4进行26S rDNA检测，测序结果如SEQ ID NO.1所示。序列比对结果如下：
Alignment：酵母菌JJ4
采用MEGA5.0软件，邻位连接法显示菌株“酵母菌JJ4”与相关种的26S rDNA序列系统发育树，进行1000次的相似度重复计算，图3中发育树节点只显示Bootstrap值大于70％数值，上标“T”代表模式菌株。
根据菌株的基因测序结果与酿酒酵母具有高度的同源性，结合菌株的生理生化特征等鉴定结果，将菌株JJ4鉴定为Saccharomyces cerevisiae JJ4，已保藏于中国典型培养物保藏中心，其保藏编号为CCTCC NO：M 2018219，保藏日期为2018年4月19日。
实施例2酿酒酵母JJ4的应用
本实施例考察产香酵母对红曲黄酒品质和风味的影响，使用的酵母菌JJ4由福建省农业科学院农业工程技术研究所提供。
大米糖化液培养基：按米饭与水比例为1：1落缸，按淀粉酶、糖化酶用量分别为每g米饭70U、560U添加α-淀粉酶和糖化酶，60℃酶解2h，冷却，备用。酶解后基质固形物35％、总酸0.23g/L、pH值6.0。
1.酵母菌的活化
从斜面勾取1环产香酿酒酵母JJ4至50mL大米糖化液培养基，25℃恒温培养24h，得到活化后的产香酿酒酵母JJ4。
2.强化发酵工艺
按米饭：水为1:1落缸，加入总重5％的古田红曲米和起始菌量分别达104(JA)、105(JB)、106(JC)cfu/mL的酿酒酵母JJ4强化发酵，以未接种JJ4的处理(CK)为对照，发酵结束后测定发酵酒的酒度、总酸、pH值、β-苯乙醇、游离氨基酸、非糖可固及总酯含量，并进行感观评价，考察添加酿酒酵母JJ4强化发酵对红曲黄酒品质的影响。
3.优选菌株的特征挥发性风味物质
按米饭：水为1:1落缸，加入总重5％的古田红曲米，加入起始菌量达104(JA)、105(JB)、106(JC)cfu/mL的酿酒酵母JJ4强化发酵，在大米糖化液培养基中以105)cfu/mL加入酿酒酵母JJ4强化发酵，以未添加酿酒酵母JJ4的同基质发酵液(CK)为对照，以2-辛醇为内标物采用GC-MS法检测各处理挥发性风味物质组分及含量，明确酿酒酵母JJ4在酒精发酵过程中的特征挥发性风味物质。
4.特征挥发性风味物质的测定
采用HS-SPMEGC和MS联用法测定。
(1)挥发性风味物质SPME萃取
萃取头的老化：按照SUPELCO公司推荐的条件，第一次使用时将50/30μmDVB/CAR/PDMS萃取头在气相色谱进样口250℃老化1h；之后每次使用前老化0.5h，至无干犹峰出现。
样品顶空固相微萃取：在15mL顶空瓶中加入0.6mL发酵酒液，5.4mL水，2g NaCl，10μg/L的2-辛醇和磁力搅拌子的顶空瓶，密封后于45℃水浴中预热10min，插入萃取头，萃取吸附45min后取出进行GC-MS分析，GC解吸5min。
(2)GC-MS分析：
GC条件色谱柱：进口温度250℃，分流比为10:1；色谱柱：
-Wax毛细管柱(30m×0.25mm×0.25μm)；程序升温：起始温度40℃，保持5min，以5℃/min升至120℃，然后以10℃/min升至240℃，保持7min；载气：He；载气流速为1Ml/MIN；检测器为质谱检测器。
MS条件离子化方式：EI电离；电离电压：70eV；全扫描；接口温度：250℃；离子源温度：230℃；检测器电压：1050V。
5.测定方法
红曲黄酒酒精度、总酸含量的测定、风味评价：均参照GB/T 13662-2008《黄酒》进行。
6.结果与分析
6.1酿酒酵母JJ4对红曲黄酒理化指标的影响
由表4、表5可见，添加酿酒酵母JJ4强化发酵可使传统红曲黄酒的总酸含量下降35.37％(P<0.01)，总酯含量提高15.16％(P<0.01)，对酒精度、残糖含量、非糖可固水平等指标无显著影响(P>0.05)。由表6可知，添加JJ4强化发酵会使红曲黄酒苦味氨基酸含量提高13.40％、14.41％、11.39％，差异均达极显著水平(P<0.01)；使甜味氨基酸含量降低0.85％(P>0.05)、4.90％(P<0.01)、14.88％(P<0.01)。因此，添加酿酒酵母JJ4强化发酵降低黄酒的酸刺激感，使黄酒口感风味愉悦，当接种量106cfu/mL时，黄酒酸味下降但苦味凸显。
表4理化指标的变化
表5风味指标的变化
表6游离氨基酸含量的变化
6.2酿酒酵母JJ4的特征挥发性风味物质
由表7可知，酿酒酵母JJ4可代谢葡萄糖产生β-苯乙醇等12种醇类物质、棕榈酸乙酯、乙酸乙酯等16种酯类物质、辛酸、壬酸、己酸等7种酸类物质以及醛类物质苯甲醛。将其应用于红曲黄酒酿造，结果见表8。与传统发酵红曲黄酒产品CK相比，104、105、106cfu/mL接种的酿酒酵母JJ4并可使酯类物质总量提高69.91％、93.09％、81.26％，使酸类物质总量提高44.60％、90.18％、137.57％，差异均达极显著水平(P<0.01)；并不会提高醇类物质的总量(P>0.05)。添加酿酒酵母JJ4可新增3-羟基己酸乙酯、硬脂酸乙酯两种物质，使壬醇、庚醇、丁二醇、二异丁基甲醇和1-壬醇等5种醇类的含量提高36.75％、89.65％、168.90％、113.79％和38.92％；使乙酸乙酯、丁酸乙酯、乙酸异戊酯、己酸乙酯、乳酸乙酯、辛酸乙酯、壬二酸二乙酯、棕榈酸乙酯等8种酯类的含量提高89.80％、442.19％、129.32％、238.08％、132.54％、230.00％、58.50％和20.34％；使己酸、辛酸、壬酸、正癸酸、月桂酸等5种酸类物质含量提高103.17％、178.51％、61.14％、136.25％和64.60％，差异均达极显著水平(P<0.01)。结果表明，与传统发酵工艺相比，添加酿酒酵母JJ4强化发酵可丰富红曲黄酒的风味组成，显著提高酯类及酸类挥发性风味物质的总量，从而提高红曲黄酒的风味。
表7酿酒酵母JJ4的特征挥发性风味物质
表8酿酒酵母JJ4对红曲黄酒挥发性风味物质的影响
序列表
<110> 福建省农业科学院农业工程技术研究所
<120> 一种产香酵母菌及其在红曲黄酒酿造中的应用
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<213> 人工序列(Artificial Sequence)
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ttccttgtct atgttccttg gaacaggacg tcatagaggg tgagaatccc gtgtggcgag 120
gagtgcggtt ctttgtaaag tgccttcgaa gagtcgagtt gtttgggaat gcagctctaa 180
gtgggtggta aattccatct aaagctaaat attggcgaga gaccgatagc gaacaagtac 240
agtgatggaa agatgaaaag aactttgaaa agagagtgaa aaagtacgtg aaattgttga 300
aagggaaggg catttgatca gacatggtgt tttgtgccct ctgctccttg tgggtagggg 360
aatctcgcat ttcactgggc cagcatcagt tttggtggca ggataaatcc ataggaatgt 420
agcttgcctc ggtaagtatt atagcctgtg ggaatactgc cagctgggac tgaggactgc 480
gacgtaagtc aaggatgctg gcataatggt tatatgccgc ccgtcttg 528
Claims (2)
1.一种产香酵母菌，其分类命名为酿酒酵母（Saccharomyces cerevisiae)，已保藏于中国典型培养物保藏中心，其保藏编号为CCTCC NO：M 2018219，保藏日期为2018年4月19日。
2.如权利要求1所述的产香酵母菌在红曲黄酒酿造中的应用。
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| CN105176854A (zh) * | 2015-07-14 | 2015-12-23 | 福建省农业科学院农业工程技术研究所 | 一种红曲黄酒酿造用的酿酒酵母菌株 |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105176854A (zh) * | 2015-07-14 | 2015-12-23 | 福建省农业科学院农业工程技术研究所 | 一种红曲黄酒酿造用的酿酒酵母菌株 |
Non-Patent Citations (2)
| Title |
|---|
| Chemical and sensory profiles of makgeolli, Korean commercial rice wine,;Heeyong Jung et al.;《Food Chemistry》;20141231;第624-632页 * |
| 酵母菌对红曲黄酒风味物质形成的影响;陈玉香;《中国食品学报》;20150731;第218-224页 * |
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