CN111254085B - 一种降解乳酸的微生物复合菌剂 - Google Patents
一种降解乳酸的微生物复合菌剂 Download PDFInfo
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- CN111254085B CN111254085B CN202010129643.1A CN202010129643A CN111254085B CN 111254085 B CN111254085 B CN 111254085B CN 202010129643 A CN202010129643 A CN 202010129643A CN 111254085 B CN111254085 B CN 111254085B
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- lactic acid
- pichia kudriavzevii
- fermentation
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种降解乳酸的微生物复合菌剂，属于微生物技术领域。本发明的库德里阿兹威毕赤酵母，对乳酸的降解能力高达12.69g·L‑1，是模式菌株的2.04倍。同时该菌株还能够代谢乙醇，其于高粱汁培养基、30℃、200rpm发酵3d后OD600可达4.48。发酵60h，其可将高粱汁培养基中58g·L‑1的葡萄糖全部耗尽，并产生13.06g·L‑1乙醇。该库德里阿兹威毕赤酵母降解乳酸，可以缓解发酵体系的乳酸压力，使酿酒酵母得以生长以及代谢产酒。
Description
技术领域
本发明涉及一种降解乳酸的微生物复合菌剂，属于微生物技术领域。
背景技术
白酒是中国传统发酵食品的典型例子，在其厌氧发酵过程中，乳酸菌等产酸微生物代谢产生大量的酸类物质，使酒醅中难挥发性酸类物质不断积累。乳酸作为酒醅中主要的有机酸，在酱香型白酒酒醅中含量达到20～40g·kg-1酒醅，在浓香型白酒酒醅中含量达到20～30g·kg-1酒醅。乳酸的积累导致白酒发酵酒醅的酸度增加，使微生物细胞停止生长，甚至死亡。乳酸的未解离形式是亲脂性的，因此乳酸可以通过简单的扩散穿过细胞膜并流入细胞质。在接近中性的细胞质中，乳酸解离，释放出质子(H+)和乳酸根离子。这些离子带有电荷，因此它们无法穿过疏水性磷脂双分子层并积聚在细胞内部，从而导致细胞内pH(pHi)降低以及细胞质阴离子池破坏。质子和阴离子对细胞膜、核糖体核糖核酸(ribonucleic acid，RNA)和脱氧核糖核酸(deoxyribonucleic acid，DNA)、活性酶类产生破坏作用，进而损害细胞活力，影响微生物的生长代谢。
酵母菌是白酒酿造过程中重要的一类微生物，不仅是推动发酵进程的主导力量之一，同时代谢产生的挥发性风味化合物也是影响白酒风格和品质的重要因素。在酱香型白酒酿造过程中，酵母菌经堆积发酵过程得到大量的富集与繁殖，然后进入窖内参与厌氧发酵过程。采用MiSeq测序技术和Real-time qPCR方法研究酱香型白酒发酵中酵母菌群落结构组成及变化规律。研究表明除了酿酒酵母(Saccharomyces cerevisiae)外，库德里阿兹威毕赤酵母(Pichia kudriavzevii)也是白酒发酵过程中的优势酵母。库德里阿兹威毕赤酵母对中国白酒的风味做出了相当大的贡献，其可以产生酯类、高级醇和挥发性酸。然而，针对国内对白酒中库德里阿兹威毕赤酵母的研究还仅限于分离鉴定到单菌种、单菌种纯培养条件下的发酵特性研究等，并未有关于库德里阿兹威毕赤酵母在发酵过程中对生长抑制因子作用的更深入研究。
因此，获取能降解乳酸的库德里阿兹威毕赤酵母，对于以库德里阿兹威毕赤酵母作为主要功能微生物的发酵食品的生产就显得尤为重要，对于提高发酵产品的产量和质量具有重要价值。
发明内容
为了解决上述问题，本发明提供了一株能够高效降解乳酸的库德里阿兹威毕赤酵母 DC-16，已于2020年1月13日保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO.19337，保藏地址为北京市朝阳区北辰西路1号院3号。
本发明还提供了一种降解乳酸的方法，是以上述库德里阿兹威毕赤酵母DC-16为发酵菌株，在发酵过程中降解乳酸。
在本发明的一种实施方式中，所述发酵是以高粱汁培养基作为发酵培养基。
在本发明的一种实施方式中，所述高粱汁培养基为将高粱:水＝1:4(M:V)，加淀粉酶蒸煮液化，60℃加糖化酶糖化，过滤离心，调节糖度至7°Bx得到的。
在本发明的一种实施方式中，所述降解具体是：向添加了乳酸的高粱汁培养基中，按 5～10％的接种量(v:v)接种上述库德里阿兹威毕赤酵母DC-16，28～32℃，180～220rpm培养 60～100h。
本发明还提供了一种降解乳酸的方法，是以上述库德里阿兹威毕赤酵母DC-16和酿酒酵母DC-3为发酵菌株，在发酵过程中降解乳酸。所述酿酒酵母DC-3已于2020年1月13日保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO.19336，保藏地址为北京市朝阳区北辰西路1号院3号。
在本发明的一种实施方式中，所述降解具体是：将库德里阿兹威毕赤酵母DC-16和酿酒酵母DC-3接种至高粱汁培养基中，并添加乳酸，28～32℃，180～220rpm培养60～100h。
在本发明的一种实施方式中，所述库德里阿兹威毕赤酵母DC-16和酿酒酵母DC-3的初始接种浓度均为107～108个·mL-1，接种浓度比为1：1。
本发明还提供了含上述库德里阿兹威毕赤酵母DC-16的微生物制剂。
本发明还提供了上述库德里阿兹威毕赤酵母DC-16或上述微生物制剂在酿酒领域的应用。
本发明还提供了上述库德里阿兹威毕赤酵母DC-16或上述微生物制剂在食品领域的应用。
本发明的有益效果：
本发明提供了一株库德里阿兹威毕赤酵母，其对乳酸的降解能力高达12.69g·L-1，是模式菌株的2.04倍。同时该菌株还能够代谢乙醇，其于高粱汁培养基、30℃、200rpm发酵3d 后菌体浓度OD600可达4.48；发酵60h，其可将高粱汁培养基中58g·L-1的葡萄糖全部耗尽，并产生13.06g·L-1乙醇。该库德里阿兹威毕赤酵母降解乳酸，可以缓解发酵体系的乳酸压力，使酿酒酵母得以生长以及代谢产酒。
生物材料保藏
库德里阿兹威毕赤酵母(Pichia kudriavzevii)DC-16，已于2020年1月13日保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO.19337，保藏地址为北京市朝阳区北辰西路1号院3号。
酿酒酵母(Saccharomyces cerevisiae)DC-3已于2020年1月13日保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO.19336，保藏地址为北京市朝阳区北辰西路1 号院3号。
附图说明
图1：不同浓度乳酸下实验菌株和模式菌株的生长曲线，其中，A：20g L-1乳酸下实验菌株和模式菌株的生长曲线；B：30g L-1乳酸下实验菌株和模式菌株的生长曲线；C：40gL-1乳酸下实验菌株和模式菌株的生长曲线；D：不同浓度乳酸下实验菌株和模式菌株的比生长速率。
图2：不同浓度乳酸下实验菌株和模式菌株的乳酸消耗比较，其中，A：20g L-1乳酸下实验菌株和模式菌株的乳酸消耗比较；B：30g L-1乳酸下实验菌株和模式菌株的乳酸消耗比较；C：40g L-1乳酸下实验菌株和模式菌株的乳酸消耗比较；D：不同浓度乳酸下实验菌株和模式菌株的乳酸消耗量。
图3：不同浓度乳酸下实验菌株和模式菌株的乳酸消耗速率比较，其中，A：20g L-1乳酸下实验菌株和模式菌株的乳酸消耗速率比较；B：30g L-1乳酸下实验菌株和模式菌株的乳酸消耗速率比较；C：40g L-1乳酸下实验菌株和模式菌株的乳酸消耗速率比较。
图4：库德里阿兹威毕赤酵母DC-16生长曲线。
图5：不同时间下菌株DC-16的葡萄糖消耗量(A)和乙醇生成量(B)。
图6：乳酸胁迫下单培养(库德里阿兹威毕赤酵母DC-16)与共培养(库德里阿兹威毕赤酵母DC-16和酿酒酵母DC-3)生物量的比较。
图7：乳酸胁迫下单培养(库德里阿兹威毕赤酵母DC-16)与共培养(库德里阿兹威毕赤酵母DC-16和酿酒酵母DC-3)乙醇生成量(A)和乙醇生成速率(B)的比较。
图8：乳酸胁迫下单培养(库德里阿兹威毕赤酵母DC-16)与共培养(库德里阿兹威毕赤酵母DC-16和酿酒酵母DC-3)乳酸消耗量(A)和乳酸消耗速率(B)的比较。
具体实施方式
(一)培养基
富集培养基：乳酸40g L-1，葡萄糖50g L-1，蛋白胨20g L-1，酵母膏10g L-1，磷酸氢二钾2g L-1，氯化钠1g L-1，硫酸镁0.1g L-1，硫酸锰0.05g L-1。
筛选培养基：乳酸40g L-1，葡萄糖50g L-1，蛋白胨20g L-1，酵母膏10g L-1。
高粱汁培养基：将高粱:水＝1:4(M:V)，加淀粉酶蒸煮液化，60℃加糖化酶糖化，过滤离心，调节糖度至7°Bx。
(二)检测乳酸含量的方法
用0.22μm有机相针头式滤器对发酵液进行过滤，将滤液转移至液相小瓶。色谱柱：Bio-Rad Aminex HPX-87H Ion Exclusion Column，柱温：60℃；检测器：紫外检测器(PDA)，检测波长210nm；流动相：5mmol L-1H2SO4，流速：0.6mL min-1。
(三)检测乙醇含量的方法
用0.22μm有机相针头式滤器对发酵液进行过滤，将滤液转移至液相小瓶。色谱柱：Bio-Rad Aminex HPX-87H Ion Exclusion Column，柱温：60℃；检测器：示差折光检测器(RID)；流动相：5mmol L-1H2SO4，流速：0.6mL min-1。
实施例1菌株的筛选
酱香型白酒发酵过程中筛选得到了一株耐乳酸的库德里阿兹威毕赤酵母，过程如下。
采集某酱香型白酒发酵酒醅的样品。称取10g样品放入250mL锥形瓶，加入90mL无菌生理盐水，加入玻璃珠振荡均匀。吸取0.1mL上清液于100mL富集培养基中，在30℃，200rpm条件下培养2～4d，观察培养液是否浑浊；若培养液已明显浑浊时，吸取0.1mL富集培养液于新的100mL液态筛选培养基中，30℃，200rpm培养2d，培养3～4次后将培养液梯度稀释于YPD固体培养基上，培养3～4d后，其单菌落即为抗乳酸特性的目标菌株。
从该平板中随机挑选7个单菌落，接种到添加40g L-1乳酸的高粱汁液体培养基中，调节 pH为3.5。30℃，200rpm发酵3d。测定发酵液中的乳酸浓度，其中一株菌株降解乳酸的量最高，为12.69g L-1。将该菌株划线到斜面培养基中培养并保藏在甘油管中，命名为DC-16。
实施例2菌株的鉴定
(1)菌落特征及菌体形态
菌落呈白色，表面粗糙，质地均匀，易挑取。通过显微镜观察形态结果发现分离筛选得到的菌种细胞呈椭圆状，部分正在出芽分裂。
(2)生理生化特征
将DC-16接种于高粱汁培养基，30℃、200rpm发酵1d后，DC-16进入稳定期。如图4所示，发酵3d时，DC-16的生物量OD600可达4.48。
高粱汁培养基中葡萄糖含量为58g·L-1。如图5所示，发酵60h，DC-16可将高粱汁培养基中58g·L-1的葡萄糖全部耗尽，并产生13.06g·L-1乙醇。
(3)分子生物学鉴定
接种菌株DC-16至YPD培养基中，培养1d，提取该菌的总DNA作为PCR模板。采用酵母ITS通用引物进行PCR扩增，本研究选用的通用引物为ITS1和ITS4。PCR扩增条件： 94℃5min,94℃30s,55℃30s,72℃1min,循环30次；72℃10min。PCR扩增产物经 1％琼脂糖凝胶检验合格后送往苏州金唯智生物科技有限公司进行测序。将测序结果上传至美国国立生物技术信息中心(national center for biotechnology information，NCBI)数据库中进行BLAST比对，发现该菌株为Pichia kudriavzevii。
综合菌落形态特征、生理生化特征以及ITS序列分析，将菌株DC-16初步鉴定为库德里阿兹威毕赤酵母DC-16。该菌株已经提交并保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO.19337，保藏地址为北京市朝阳区北辰西路1号院3号。
实施例3本发明的酵母菌对乳酸的降解情况
采用添加了不同乳酸浓度的高粱汁培养基作为发酵培养基，对实验菌株库德里阿兹威毕赤酵母DC-16(以下简称为P.k.DC-16)和其模式菌株库德里阿兹威毕赤酵母ATCC24210(以下简称为P.k.ATCC 24210)进行乳酸的代谢实验。根据筛选过程中酒醅中乳酸的检测结果，设计代谢实验中乳酸添加量分别为20，30，40g·L-1。
实验及对照组设定：
对照组1：向添加20g·L-1乳酸的高粱汁培养基中按7％接种量(v:v)接种P.k.ATCC24210；
实验组1：向添加20g·L-1乳酸的高粱汁培养基中按7％接种量(v:v)接种P.k.DC-16；
对照组2：向添加30g·L-1乳酸的高粱汁培养基中按7％接种量(v:v)接种P.k.ATCC24210；
实验组2：向添加30g·L-1乳酸的高粱汁培养基中按7％接种量(v:v)接种P.k.DC-16；
对照组3：向添加40g·L-1乳酸的高粱汁培养基中按7％接种量(v:v)接种P.k.ATCC24210；
实验组3：向添加40g·L-1乳酸的高粱汁培养基中按7％接种量(v:v)接种P.k.DC-16。
其中，模式菌株P.k.ATCC 24210购于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC 2.1465。添加乳酸的高粱汁培养基均用5M NaOH调节pH为3.5(真实酒醅的pH)，菌株是按7％接种量(v:v)接种到培养基中，30℃，200rpm培养72h。每隔 12h定时取样，测定发酵液中乳酸的含量以及OD600。
结果显示，相比模式菌株P.k.ATCC 24210，实验菌株P.k.DC-16的终点生物量更高，比生长速率更快，具体结果见图1。其中，模式菌株P.k.ATCC 24210在20，30，40g·L-1乳酸胁迫时终点OD600分别为3.32，2.90，2.74；实验菌株P.k.DC-16在20，30，40g·L-1乳酸胁迫时终点OD600分别为3.81，3.49，3.18，分别是模式菌株的1.14，1.20，1.16倍。模式菌株P.k.ATCC 24210在20，30，40g·L-1乳酸胁迫时的比生长速率分别是0.2074，0.1956，0.1936t-1；实验菌株P.k.DC-16在20，30，40g·L-1乳酸胁迫时的比生长速率分别是0.2207，0.2139， 0.2035t-1，分别是模式菌株的1.06，1.09，1.05倍。以上结果表明P.k.DC-16的乳酸耐受性更高，更适应高乳酸环境的生产过程。
与模式菌株P.k.ATCC 24210相比，实验菌株P.k.DC-16可以降解更多的乳酸，具体结果见图2。当高粱汁培养基分别添加20，30，40g·L-1乳酸时，模式菌株P.k.ATCC 24210可以分别降解7.62，3.80，6.21g·L-1乳酸；实验菌株P.k.DC-16可以分别降解10.48，10.73，12.69 g·L-1乳酸，分别是模式菌株的1.37，2.83，2.04倍。
与模式菌株P.k.ATCC 24210相比，实验菌株P.k.DC-16降解乳酸的速率更快，具体结果见图3。发酵12h，乳酸的消耗速率最快。当高粱汁培养基分别添加20，30，40g·L-1乳酸时，模式菌株P.k.ATCC 24210在12h的乳酸消耗速率分别是0.31，0.08，0.27g·L-1·h-1；实验菌株P.k.DC-16的乳酸消耗速率分别是0.38，0.22，0.43g·L-1·h-1，分别是模式菌株的1.24，2.70， 1.57倍。
实施例4降解乳酸的复合菌剂
采用添加了40g·L-1乳酸的高粱汁培养基作为发酵培养基，对实验菌株P.k.DC-16和酿酒酵母DC-3(以下简称为S.c.DC-3)进行独立或联合发酵实验。
对照组1：将P.k.DC-16的种子液以终浓度2×107个·mL-1的接种量接种至高粱汁培养基中，并添加40g·L-1乳酸。
对照组2：将S.c.DC-3的种子液以终浓度2×107个·mL-1的接种量接种至高粱汁培养基中，并添加40g·L-1乳酸。
实验组：P.k.DC-16和S.c.DC-3的种子液按107(个·mL-1)：107(个·mL-1)的接种量接种至高粱汁培养基中，并添加40g·L-1乳酸。
其中，发酵培养条件是30℃200rpm培养72h。每隔12h定时取样，测定发酵液中两种酵母的生物量以及乙醇、乳酸的含量。
单培养和共培养过程中P.k.DC-16和S.c.DC-3的生物量如图6所示。由于高浓度的乳酸对酵母菌的毒害作用，发酵初期P.k.DC-16和S.c.DC-3的生物量在单培养和共培养过程中都有所减少。
发酵12h时，P.k.DC-16在单培养过程中从2.0×107个·mL-1减少为3.8×106个·mL-1，而在共培养过程中从1.0×107个·mL-1减少为2.8×106个·mL-1；随后P.k.DC-16的生物量缓慢上升，在72h达到最高值，其中单培养终点的生物量为5.7×107个·mL-1，共培养终点的生物量为5.0×107个·mL-1。P.k.DC-16的生物量在单培养和共培养中没有显著变异(P<0.05)。
S.c.DC-3单培养过程中48h达到最低生物量7.1×104个·mL-1，而在共培养中S.c.DC-3 的延迟期缩短，在12h达到最低生物量5.0×105个·mL-1，随后维持稳定。48h时共培养中 S.c.DC-3(9.6×105个·mL-1)的生物量是单培养的13倍。以上结果表明，P.k.DC-16利用乳酸，可以缓解乳酸对酿造体系中产酒酵母的胁迫作用，使其更好地进行后续的酒精发酵。
在40g·L-1乳酸胁迫时，S.c.DC-3的产乙醇能力受到严重的抑制。如图7所示，发酵结束时，P.k.DC-16的乙醇产量为16.23g·L-1，而S.c.DC-3的乙醇产量仅为4.66g·L-1。将P.k. DC-16和S.c.DC-3一起培养时，乙醇产量为18.08g·L-1，与单培养相比有所增加。发酵12h 时，P.k.DC-16单培养的乙醇生成速率为0.26g·L-1·h-1，S.c.DC-3单培养的乙醇生成速率在单培养12h时为0.21g·L-1·h-1。而共培养时为0.42g·L-1·h-1，分别是P.k.DC-16单培养和S.c.DC-3 单培养的1.64和3.64倍。与单菌相比P.k.DC-16和S.c.DC-3共培养可以提高乙醇的产量和生产速率。
如图8所示，发酵结束时P.k.DC-16可以利用培养基中7.20g·L-1乳酸，S.c.DC-3可以利用4.44g·L-1乳酸。而P.k.DC-16和S.c.DC-3共培养时，培养基中乳酸的含量减少了13.71g·L-1，乳酸降解量显著增加(P＜0.01)。P.k.DC-16乳酸消耗速率的最大值为0.16g·L-1·h-1，S.c.DC-3 的最大乳酸消耗速率为0.15g·L-1·h-1。然而，共培养时乳酸的最大消耗速率显著增加(0.32 g·L-1·h-1)。说明与单培养相比，P.k.DC-16和S.c.DC-3共培养可以增强酵母对乳酸的利用能力。
基因lldD编码乳酸脱氢酶，可以将乳酸降解为丙酮酸，是参与降解乳酸的关键基因。共培养中P.k.DC-16和S.c.DC-3的lldD基因转录情况如表1所示。培养12h时只有P.k.DC-16 转录了基因lldD，而S.c.DC-3未转录基因lldD，说明当乳酸浓度较高时，只有P.k.DC-16参与乳酸的降解。当培养24h时，培养基中的乳酸浓度减少为35.46g L-1，此时P.k.DC-16和S.c.DC-3均转录基因lldD，说明低乳酸浓度时，P.k.DC-16和S.c.DC-3共同参与乳酸的降解。
表1共培养中2株酵母菌lldD基因转录情况
注：“+”表示在样品中有检出lldD基因的转录，“–”表示没有检测到该基因的转录。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一株库德里阿兹威氏毕赤酵母(Pichia kudriavzevii)DC-16，已于2020年1月12日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.19337，保藏地址为北京市朝阳区北辰西路1号院3号。
2.一种降解乳酸的方法，其特征在于，所述方法是以权利要求1所述的库德里阿兹威氏毕赤酵母DC-16为发酵菌株，在发酵过程中降解乳酸。
3.如权利要求2所述的方法，其特征在于，所述发酵是以高粱汁培养基作为发酵培养基。
4.如权利要求2或3所述的方法，其特征在于，所述降解具体是：向添加了乳酸的高粱汁培养基中按5～10％的接种量接种权利要求1所述的库德里阿兹威氏毕赤酵母DC-16，28～32℃，180～220rpm培养60～100h。
5.一种降解乳酸的方法，其特征在于，所述方法是以权利要求1所述的库德里阿兹威氏毕赤酵母DC-16和保藏编号为CGMCC NO.19336的酿酒酵母(Saccharomyces cerevisiae)DC-3为发酵菌株，在发酵过程中降解乳酸。
6.如权利要求5所述的方法，其特征在于，所述降解具体是：将库德里阿兹威氏毕赤酵母DC-16和酿酒酵母DC-3接种至高粱汁培养基中，并添加乳酸，28～32℃，180～220rpm培养60～100h。
7.如权利要求6所述的方法，其特征在于，所述库德里阿兹威氏毕赤酵母DC-16和酿酒酵母DC-3的初始接种浓度均为107～108个·mL-1。
8.含权利要求1所述的库德里阿兹威氏毕赤酵母DC-16的微生物制剂。
9.权利要求1所述的库德里阿兹威氏毕赤酵母DC-16或权利要求8所述的微生物制剂在酿酒领域的应用。
10.权利要求1所述的库德里阿兹威氏毕赤酵母DC-16或权利要求8所述的微生物制剂在食品领域的应用。
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