CN114517157A - 一株产乙酸苯乙酯的库德里阿兹威毕赤酵母x-8的筛选鉴定及其在白酒酿造中的应用 - Google Patents
一株产乙酸苯乙酯的库德里阿兹威毕赤酵母x-8的筛选鉴定及其在白酒酿造中的应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域,具体涉及一种库德里阿兹威毕赤酵母X‑8的筛选鉴定方法及其在白酒酿造过程中的应用。本发明公开的库德里阿兹威毕赤酵母X‑8,乙酸苯乙酯产量为1128.96μg/L,最适生长温度为25℃,最适pH为7,最适糖浓度为12%。库德里阿兹威毕赤酵母X‑8在模拟白酒固态发酵中应用时,酒醅中的乙酸苯乙酯含量提升约2倍。此外,微生物及风味物质的多样性也显著高于对照组。本发明在分离培养白酒酿造过程中高产白酒风味物质的功能微生物方面具有重要意义,为提升白酒的风味和品质奠定理论基础。
Description
技术领域
本发明属于微生物技术领域,尤其涉及一株库德里阿兹威毕赤酵母X-8的筛选鉴定及其在调控白酒酿造过程中乙酸苯乙酯产量中的应用。
背景技术
白酒中98%-99%成分是水和乙醇,剩余的为微量成分主要有酸、醇、酯等,是构成酒体的主要呈香、呈味物质,决定着白酒香型特点。白酒的挥发性风味物质中酯类物质的含量最多,在各香型白酒的挥发性风味物质中的含量达到了60%,是判断白酒质量的重要依据,除此之外酯类物质的气味阈值低,具有协同作用,是白酒香气的主要贡献者,其浓度的微小变化会对白酒风味产生显著影响。乙酸苯乙酯在酱香型白酒中的香气活性值大于1,是酱香型白酒的重要呈味物质。它具有玫瑰花香,蜂蜜样香气,苹果香,可以增强酱香型白酒醇厚的口感。然而,由于乙酸苯乙酯在酱香型白酒酿造过程中合成效率低,导致优质酒出酒率低,成为制约酱香型白酒品质和优质酒出酒率低的重要原因。微生物的生长代谢可以促进白酒中的有机酸和醇生成酯,因此,筛选具有合成乙酸苯乙酯能力的微生物,理性调控发酵过程,促进风味酯的原位合成,有利于从根本上保障发酵白酒的品质。
因此,本发明的目的是提供一株优质的微生物资源库德里阿兹威毕赤酵母X-8的筛选鉴定方法及其在白酒酿造中的应用,具有促进白酒中有益风味酯乙酸苯乙酯的产生,为白酒发酵过程酯的合成提供优选的微生物资源的作用。
发明内容:
本发明的目的在于提供一株产乙酸苯乙酯的库德里阿兹威毕赤酵母X-8的筛选鉴定方法及其在白酒酿造过程中的应用。
本发明通过以下技术方案来实现:
本发明公开了一株源自酱香型白酒酿造酒醅中的库德里阿兹威毕赤酵母X-8的筛选鉴定方法。
本发明还公开了一株库德里阿兹威毕赤酵母X-8在白酒酿造过程中合成乙酸苯乙酯的应用。
与现有技术相比,本发明具有以下有益的技术效果:
本发明利用微生物技术,首次从酱香型白酒酿造过程中筛选出库德里阿兹威毕赤酵母X-8,通过传统平板分离方法从酱香型白酒酿造过程的酒醅样品中筛选出1株乙酸苯乙酯产量相对较高的菌株X-8,利用HS-SPME-GC-MS技术对其发酵液中的风味物质进行测定,其乙酸苯乙酯产量为1128.96μg/L。经形态学观察、生理生化实验及分子生物学鉴定确定X-8为库德里阿兹威毕赤酵母(Pichia kudriavzevii),其最适生长温度为25℃,最适pH为7,最适糖浓度为12%。
本发明将库德里阿兹威毕赤酵母X-8在模拟白酒固态发酵中进行应用,发酵结束时,库德里阿兹威毕赤酵母X-8添加组中的酯类、醇类及其他挥发性风味物质的种类及含量均高于对照组。其中,乙酸苯乙酯含量达0.214μg/g,约为对照组的2倍。此外,苯乙醇、对乙烯基愈创木酚等特殊风味物质的含量也均有不同程度的提高。微生物群落结构分析表明,实验组中微生物多样性显著高于对照组,酒醅中细菌属分别有154个和125个,真菌属分别有113个和88个。此外,实验组中挥发性风味物质与真菌微生物的相关性增强。
本发明从酱香型白酒酿造过程中筛选出一株高产乙酸苯乙酯的库德里阿兹威毕赤酵母X-8,并将其应用到模拟白酒固态发酵体系中,为改善酱香型白酒风味、提高白酒品质提供重要的酿造工艺参考。
附图说明
图1为筛自酱香型白酒酿造酒醅中的酵母菌产乙酸苯乙酯能力的对比
图2为对照组及实验组酒醅湿度、酸度、淀粉、还原糖等理化指标的变化
图3为对照组及实验组酒醅总酯、总醇、总酸及其它风味物质含量变化
图4为对照组及实验组酒醅样品发酵过程中细菌微生物群落在属水平上的分布
图5为对照组及实验组酒醅样品发酵过程中真菌微生物群落在属水平上的分布
具体实施方式
下面结合具体的附图和实例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。下述实施例中未详细述及的操作步骤或条件,均按照本领域常规技术、条件实现,
实施例1 库德里阿兹威毕赤酵母X-8的分离鉴定方法
菌株分离:称取5g混匀的酒醅,加入45mL无菌生理盐水,常温震荡30min,吸取上清液稀释至10-5,10-6倍,取200μL稀释菌液涂布于YPD固体培养基表面,30℃培养48h,挑取具有酵母菌落特征的单菌落,转接到新的YPD固体培养基上,再次划线培养直至完全纯化,然后转入斜面培养基中低温保存。
产乙酸苯乙酯酵母的筛选:将酵母菌株种子液接种于液体高粱培养基中,在30℃,180r/min的条件下培养48h。取适量发酵液,在4℃下1000r/min离心10min,吸取5mL发酵液于进样瓶,加入2g NaCl,加入5μL内标(4-辛醇:0.5g/L),加盖密封备用。恒温(80℃)水浴30min平衡样品,将老化后的萃取头插入顶空瓶中吸附30min,将萃取头移入气相色谱的高温汽化室中解吸5min,通过GC-MS分析各挥发性物质的含量。
产乙酸苯乙酯酵母菌的鉴定:将纯化的酵母划线转接于YPD培养基上,30℃倒置培养48h,观察其在YPD培养基上的菌落形态;将一滴0.1%美蓝染液滴在洁净的载玻片上,用接种环挑取单菌落与其混匀,盖上盖玻片,用高倍镜观察酵母菌的形态。通过一系列生理生化实验包括:碳源同化实验、糖发酵实验、氮源同化实验、淀粉水解实验、尿素实验对酵母的生理特性进行分析。采用真菌DNA试剂盒方法提取酵母基因组DNA,酵母菌26S rDNA的PCR扩增,引物见表1,PCR体系见表2,PCR程序条件见表3。
表1 PCR引物
Table1 PCR primer
表2 PCR体系
Table 2 PCR system
表3 PCR程序条件
Table3 PCR program conditions
酵母菌26S rDNA基因扩增选用的是NL1和NL4,按照上述反应体系逐一添加,轻弹混匀在PCR扩增仪上进行PCR反应,PCR扩增程序参数见表。反应完成后将PCR产物进行1%琼脂糖凝胶电泳检测,确认PCR扩增片是否正确。采用试剂盒回收目的DNA片段。在北京诺赛基因组研究中心有限公司进行PCR产物的测序。测序完成后,BLAST比对获取相似序列(TXT),将测序的得到的26s rDNA序列在NCBI进行BLAST比对,将相似度较高的菌株与测序序列在MEGA7中构建菌株系统进化树。
酵母菌生物特性研究:(1)温度耐受性测定:采用YPD培养基(自然pH),在培养基中接入待测酵母,在20℃、25℃、30℃、35℃、40℃、45℃、50℃下180rpm,培养24h。600nm波长下的OD值作为最适温度测定结果。(2)pH耐受性测定:采用YPD培养基,调节培养基的pH为1.0-12.0,在培养基中接入待测酵母,30℃,180rpm,培养24h。600nm波长下的OD值作为最适pH测定结果。(3)乙醇耐受性测定:采用YPD培养基,在培养基中加入浓度(v/v)为0%、3%、6%、9%、12%、15%、18%、21%的乙醇,在培养基中接入待测酵母,30℃,180rpm,培养24h。600nm波长下的OD值作为最大乙醇耐受测定结果。(4)高盐耐受性测定:采用YPD培养基,在培养基中加入浓度(g/L)为0%、5%、10%、15%、20%、25%、30%的氯化钠,在培养基中接入待测酵母,30℃,180rpm,培养24h。600nm波长下的OD值作为最大盐浓度耐受测定结果。(5)高糖耐受性测定。采用YPD培养基,在培养基中加入浓度(g/L)为2%、12%、22%、32%、42%、52%、62%、72%的葡萄糖,在培养基中接入待测酵母,30℃,180rpm,培养24h。600nm波长下的OD值作为最大糖浓度耐受测定结果。
实施例2 库德里阿兹威毕赤酵母X-8在模拟白酒固态发酵中应用
菌悬液的制备:无菌条件下,在YPD培养基上活化Pichia kudriavzevii X-8,然后在培养箱中28℃倒置培养48h,挑取单菌落一环移到装有50mL YPD液体培养基的三角瓶中,28℃摇床培养24h后得到107-108cfu/mL的酵母菌悬液,备用。
工艺流程:原料:高粱800g;粉碎:整粒与碎粒之比为8∶2;润粮:润粮水温控制在90℃左右,润粮总用水量约为原料的50%-56%,润粮时间24h;蒸梁:平铺200g稻壳,高粱铺好,蒸1.5h,约有70%左右的原料蒸熟,即可出甑,出甑后再泼上85℃的热水(原料量的12%);摊晾、拌曲:当温度降到32℃左右加入大曲粉(10%);堆积:堆积72h;加菌:将活化好的酵母离心,收集菌体,加生理盐水重悬混入样品中,空白以生理盐水作为对照,加菌量为2%;发酵:入坛发酵30天,设置对照组(未添加Pichia kudriavzevii X-8)及实验组(添加Pichia kudriavzevii X-8);取样:在0、6、12、18、24、30天进行取样。
模拟白酒固态发酵酒醅理化指标测定:方法参照中国酒业协会团体标准(T/CBJ004-2018)。
模拟白酒固态发酵酒醅风味物质分析方法:取5g酒醅置于50mL离心管中,加20mL无菌生理盐水,充分振荡后在冰浴中进行超声处理,在4℃下离心(1000×g)5min。取8mL上清液移于顶空瓶中,加入3g NaCl及6μL 4-辛醇作为内标物质。
Claims (3)
1.一种源自酱香型白酒的具有高产乙酸苯乙酯特性的库德里阿兹威毕赤酵母X-8的分离鉴定方法。方法包括:稀释涂布、平板划线、液体高粱培养基中培养、形态学观察、生理生化鉴定、分子生物学鉴定、生物特性(pH、乙醇、高盐、高糖耐受性)鉴定。其中,高粱培养基组成包括:250g高粱中加入1L去离子水混合;煮沸糊化,加入200μL耐高温α淀粉酶,90℃液化1h;冷却后加入0.125g糖化酶,60℃糖化2h;冷却至室温,用4层纱布过滤;将滤液10000r/min,离心10min,取上清;调糖度至10Brix,115℃灭菌20min。
2.由权利要求1所述库德里阿兹威毕赤酵母X-8,经培养后,其乙酸苯乙酯产量为1128.96μg/L,其最适生长温度为25℃,最适pH为7,最适糖浓度为12%。
3.由权利要求1所述库德里阿兹威毕赤酵母X-8在模拟白酒固态酿造中的应用,其特征在于,发酵结束时,库德里阿兹威毕赤酵母X-8添加组中的酯类、醇类及其他挥发性风味物质的种类及含量均高于对照组。其中,酒醅中乙酸苯乙酯含量达0.214μg/g,约为对照组的2倍。此外,苯乙醇、对乙烯基愈创木酚等特殊风味物质的含量也均有不同程度的提高。且实验组中微生物多样性显著高于对照组,实验组中挥发性风味物质与真菌微生物的相关性增强。
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