CN110205255A - 一株高温敏感型啤酒酵母及其应用 - Google Patents
一株高温敏感型啤酒酵母及其应用 Download PDFInfo
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Abstract
本发明涉及一株高温敏感型啤酒酵母及其应用，属于微生物技术领域。本发明通过ARTP诱变啤酒酵母Pilsner，再利用酵母胞内碱性磷酸酶与BCIP反应生成蓝色沉淀这一原理，可根据蓝色出现时间与深浅进行温度敏感型突变菌的筛选，最终筛选得到一株高温敏感型啤酒酵母P‑510，在37℃下快速自溶。P‑510在37℃自溶能够促进自溶物的溶出，得到高呈味核苷酸、葡聚糖和抗氧化活性物质，可用于具有较高营养价值的啤酒酵母自溶物的生产。
Description
技术领域
本发明涉及一株高温敏感型啤酒酵母及其应用，属于微生物技术领域。
背景技术
啤酒酵母含有丰富的蛋白质(45％-60％)、氨基酸、B族维生素、多糖、矿物质等，是营养生物活性物质的天然来源。酵母抽提物是利用酵母自溶的特性将酵母菌体内的蛋白质、核酸类物质进行降解，并与其他有效成分一起被浓缩成可溶性营养物质的产品，可用于调味品、化妆品、保健品、医药等领域。其主要成分为核苷酸、氨基酸、多肽、B族维生素和微量元素。由于氨基酸、鸟苷酸(5’-GMP)和肌苷酸(5’-IMP)等成分具有独特的鲜味，酵母抽提物成为重要的天然风味增强剂，以取代加工食品中的谷氨酸钠和核苷酸。酵母自溶物的制备与酵母抽提物相同，但后续不进行高温浓缩，有利于维持多肽、氨基酸等物质的天然结构，获得具有生物活性的酵母自溶物。酵母自溶物中含有的生物活性物质有β-葡聚糖、甘露聚糖、寡糖、矿物离子和多肽等，可用于功能性食品和膳食添加剂。这是目前啤酒酵母应用的新方向之一。因此，提供一种提高酵母自溶物的溶出和营养价值的方法，具有重要的应用价值。
发明内容
本发明的第一个目的是提供一株啤酒酵母(Saccharomyces pastorianus)，分类命名为Saccharomyces pastorianus,已于2019年4月3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.17520，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
本发明的第二个目的是提供含有上述啤酒酵母的微生物制剂。
在本发明的一种实施方式中，所述微生物制剂中含有活菌数≥107CFU/g的啤酒酵母细胞。
在本发明的一种实施方式中，所述啤酒酵母细胞包括啤酒酵母干菌体或湿菌体。
本发明的第三个目的是提供一种增加酵母抽提物中呈味氨基酸含量的方法，使上述的啤酒酵母在37℃下进行自溶。
本发明的第四个目的是提供一种增加酵母抽提物中葡聚糖含量的方法，使上述的啤酒酵母在37℃-50℃下进行自溶。
本发明的第五个目的是提供上述的啤酒酵母在生产功能性食品或膳食添加剂中的应用。
本发明的第六个目的是提供上述的啤酒酵母在调味品、化妆品、保健品或制药领域中的应用。
本发明的第七个目的是提供上述的微生物制剂在生产功能性食品或膳食添加剂中的应用。
本发明的第八个目的是提供上述的啤酒酵母在调味品、化妆品、保健品或制药领域中的应用。
本发明通过ARTP诱变啤酒酵母Pilsner，再利用酵母胞内碱性磷酸酶与BCIP反应生成蓝色沉淀这一原理，可根据蓝色出现时间与深浅进行菌株的初筛。最后比较诱变菌株在37℃自溶时RNA溶出含量，筛选得到一株高温敏感型啤酒酵母P-510，在37℃下快速自溶。最终筛选得到一株高温敏感型啤酒酵母P-510，在37℃下快速自溶。P-510在37℃下自溶144h得到的自溶物呈味核苷酸含量(GMP+IMP)是原始菌的3倍，P-510自溶120h后，P-510的葡聚糖含量比Pilsner的葡聚糖含量高40.3％，可用于具有较高营养价值的啤酒酵母自溶物的生产。生物材料保藏
一株啤酒酵母(Saccharomyces pastorianus)，分类命名为Saccharomycespastorianus，已于2019年4月3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.17520，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
附图说明
图1为ARTP诱变致死率曲线。
图2为突变菌的筛选。
图3为P-510在28℃和37℃的生长情况。
图4为P-510在37℃的死亡率曲线。
图5为P-510在37℃自溶的核酸渗透率(a)和蛋白质渗透率(b)曲线。
图6为Pilsner和P-510的生长曲线。
图7为37℃自溶液中葡聚糖含量。
图8为37℃下相同菌浓自溶液DPPH清除率。
具体实施方式
(一)高效液相色谱法测定自溶液中鸟苷酸、肌苷酸含量
利用高效液相色谱法测定自溶液中鸟苷酸、肌苷酸含量，色谱条件：如表1，流速0.5mL·min-1，波长254nm，色谱柱Waters XSELECTTM HSS T3 5μm 4.6×250mm Column。
表1色谱条件
(二)测定葡聚糖含量的方法
通过苯胺蓝法测定自溶液中葡聚糖含量。取1mL自溶液12000rpm离心5min，吸取上清液100μL加入1mL苯胺蓝工作液。50℃暗反应保温30min使荧光物质形成。置于室温30min,彻底混匀，吸取180μL至黑色96孔板进行检测。激发波长为405nm，发射波长为495nm，测定反应液荧光值。苯胺蓝工作液：12.7mL超纯水，3.70mL甘氨酸/氢氧化钠缓冲液(1.0M甘氨酸，1.25M氢氧化钠)和2.50mL染液(5.0g/L苯胺蓝)混合后在黑暗中过夜储存。
(三)DPPH清除率测定
以DPPH清除率为指标表征自溶液的抗氧化能力。采用分光光度计法，无水乙醇配制0.5072mM DPPH自由基醇溶液后，取1mL DPPH自由基醇溶液与1mL自溶液混合，避光反应30min测定其OD517值。DPPH清除率计算公式如下：
式中：A0，DPPH溶液与无水乙醇混合后测定的OD517值；Ai，DPPH溶液与样品反应测得OD517值；Aj，无水乙醇与样品混合反应后测得OD517值。
实施例1：高温敏感型啤酒酵母的筛选
(1)ARTP诱变时间的选择
出发菌株Pilsner培养至对数期，用生理盐水稀释至菌液细胞数达107个·mL-1左右。取上述菌悬液进行ARTP诱变处理，处理时间分别为15s、30s、45s、60s、75s。诱变结束后，将处理过的载片置于1mL无菌生理盐水中。取菌液合理稀释后涂布于YPD平板，置于28℃培养。绘制致死率曲线。见图1。由于啤酒酵母的选育需要保持菌株的成品啤酒主体风味，一般选择75％-85％的致死率，因此选择46s为诱变时间。
(2)突变菌的筛选
利用酵母胞内碱性磷酸酶与BCIP反应生成蓝色沉淀这一原理，可根据蓝色出现时间与深浅进行温度敏感型突变菌的筛选。将经过46s诱变所得到的菌液稀释10倍，涂布于含有40mg·L-1BCIP的YPD平板，28℃培养36h。通过平板影印法将菌落复制于YPD平板，并将其放置于28℃培养，同时将含有40mg·L-1BCIP的YPD平板放置于37℃培养24h。挑选菌落颜色变蓝的单菌落过夜培养于20mL YPD，将菌液OD600调至0.6-0.8，同时划线于两块YPD平板，分别放置于28℃和37℃培养24h。获得16株温度敏感型突变菌，见图2。
(3)突变菌37℃自溶能力测定
菌株过夜活化，离心收集菌体，1.0g酵母泥加入10mL柠檬酸缓冲液(pH＝4.0)中同时记录细胞个数。180r·min-1、37℃摇床自溶30h，通过NanoDrop测定上清液中核酸浓度。见表2，得到5-10为自溶能力较好的突变菌。
表2突变菌株37℃自溶物RNA相对含量
实施例2：高温敏感型啤酒酵母的生理性能
(1)突变菌37℃的生长状况和自溶能力
为了进一步分析P-510在37℃的生理性能，测定了其在37℃的生长状况(图3)和死亡率(图4)。P-510在37℃表现出明显的生长抑制。在37℃下培养12h后，Pilsner的死亡率变化不明显，84h后死亡率为6.5％，P-510的死亡率迅速增加，84h后至95.5％，说明P-510对高温敏感。同时测定P-510在37℃自溶过程中核酸(a)和蛋白质(b)的渗透率，如图5所示。在自溶过程中，自溶液核酸、蛋白质含量均增加，核酸含量增加量大于蛋白质含量增加量。120h后P-510核酸含量为232.3mg·L-1，而Pilsner的核酸含量为83.43mg·L-1，且P-510核酸含量增加率大于Pilsner和3-3。蛋白质含量增加幅度较小，在0-24h变化最大。120h后P-510蛋白质含量为7.57mg·L-1，而Pilsner的蛋白质含量为6.02mg·L-1。综上说明P-510在37℃较Pilsner更易自溶，对温度更敏感。
(2)生长曲线和发酵能力
诱变可能会对菌株的生长能力和发酵性能造成影响，因此测定了突变菌的生长曲线和发酵能力。如图6所示，P-510和Pilsner同时进入对数期，进入平台期后菌浓比原始菌略低，但菌株总体生长一致，说明经过诱变处理后P-510的生长能力没有受到影响。同时如表3所示，Pilsner和P-510在11℃发酵7天后发酵液的酒精度和实际发酵度差异不大，说明P-510能够进行正常发酵。
表3 Pilsner、P-510发酵液的酒精度和实际发酵度
| 指标 | Pilsner | P-510 |
| 酒精度/％v·v<sup>-1</sup> | 3.7±0.1 | 3.62±0.1 |
| 实际发酵度/％ | 55.3±1.0 | 54.6±1.1 |
实施例3：高温敏感型突变菌37℃自溶物分析
(1)呈味核苷酸含量测定
将实施例1中筛选出的高温敏感型啤酒酵母P-510在37℃自溶，利用高效液相色谱法测定自溶液中鸟苷酸、肌苷酸含量。如表4所示，P-510在37℃下自溶144h得到的自溶物呈味核苷酸含量(GMP+IMP)是原始菌的3倍，说明高温敏感型的P-510在37℃更易自溶，有利于获得高呈味核苷酸含量的酵母抽提物及自溶物。
表4 37℃自溶时相同菌浓的Pilsner、P-510自溶液中GMP、IMP含量
(2)葡聚糖含量测定
自溶过程中细胞壁受到压力，多种水解酶作用于细胞壁，随后细胞壁组分溶出，其中包括生物活性物质β-葡聚糖。分别测定Pilsner和P-510在37℃自溶液中β-葡聚糖的含量，如图3所示。自溶时间越长，溶出葡聚糖含量越高。如图7所示，自溶120h后，P-510的葡聚糖含量比Pilsner的葡聚糖含量高40.3％，说明P-510的高温敏感突变性状使其在37℃下自溶时溶出葡聚糖含量增加。
(3)DPPH清除率测定
酵母抽提物及自溶物具有一定抗氧化性，可作为功能性食品添加剂。主要是由于酵母自溶过程中蛋白质降解成的活性肽，细胞壁组分溶出的葡聚糖和甘露聚糖等物质具有抗氧化性。分别测定Pilsner和P-510在37℃自溶液中DPPH清除率，如图8所示。37℃下Pilsner自溶120h时DPPH清除率最高为79.80％，P-510在80h时DPPH清除率最高为86.09％，比Pilsner高6.29％。可能是由于蛋白酶催化蛋白质中肽键水解时，温度会影响多肽链的长度、游离氨基酸的数量和氨基酸顺序，从而影响自溶液的抗氧化性。因此P-510在37℃自溶有利于获得抗氧化性较强的酵母抽提物及自溶物。
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)
1.一株啤酒酵母(Saccharomyces pastorianus)，已于2019年4月3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.17520，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
2.含有权利要求1所述啤酒酵母的微生物制剂。
3.如权利要求2所述的微生物制剂，其特征在于，所述微生物制剂中含有活菌数≥107CFU/g的啤酒酵母细胞。
4.如权利要求3所述的微生物制剂，其特征在于，所述啤酒酵母细胞包括啤酒酵母干菌体或湿菌体。
5.一种增加酵母抽提物中呈味氨基酸含量的方法，其特征在于，使权利要求1所述的啤酒酵母在37℃-50℃下进行自溶。
6.一种增加酵母抽提物中葡聚糖含量的方法，其特征在于，使权利要求1所述的啤酒酵母在37℃-50℃下进行自溶。
7.权利要求1所述的啤酒酵母在生产功能性食品或膳食添加剂中的应用。
8.权利要求1所述的啤酒酵母在调味品、化妆品、保健品或制药领域中的应用。
9.权利要求2或3所述的微生物制剂在生产功能性食品或膳食添加剂中的应用。
10.权利要求2或3所述的啤酒酵母在调味品、化妆品、保健品或制药领域中的应用。
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| 高玉荣等: "富硒啤酒酵母自溶条件的研究", 《黑龙江八一农垦大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020248962A1 (zh) * | 2019-06-11 | 2020-12-17 | 江南大学 | 一株高温敏感型啤酒酵母及其应用 |
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| Publication number | Publication date |
|---|---|
| US20210321654A1 (en) | 2021-10-21 |
| CN110205255B (zh) | 2020-09-04 |
| WO2020248962A1 (zh) | 2020-12-17 |
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