CN109988721B - 一株能够增加酱类食品香气的鲁氏酵母菌株 - Google Patents
一株能够增加酱类食品香气的鲁氏酵母菌株 Download PDFInfo
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Images
Classifications
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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Abstract
本发明公开了一株能够增加酱类食品香气的鲁氏酵母菌株,属于生物工程技术领域。该鲁氏酵母(Zygosaccharomyces rouxii)JNBZR01已于2018年9月28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.16547。该鲁氏酵母菌株可以耐受58%的葡萄糖、60%的蔗糖、60%的果糖、65%的麦芽糖、10%的淀粉、12%的NaCl,具有淀粉酶活力,在麦汁发酵中产生的醇酯类含量较高,其可以直接添加到酱类产品中,增加酱类产品中的香气。
Description
技术领域
本发明涉及一株能够增加酱类食品香气的鲁氏酵母菌株,属于生物工程技术领域。
背景技术
鲁氏酵母(Zygosaccharomyces rouxii)在分类学上属于真核生物原界,真菌界,子囊菌门,酵母亚门,酵母纲,酵母目,酵母科,鲁氏接合酵母属。菌落呈圆形,有光泽,平坦,边缘整齐。麦芽汁中培养3天,细胞呈小圆形或卵圆形。鲁氏酵母能耐高渗透压,在食盐含量5%~8%的培养基中生长良好;能在一定浓度的果糖、葡萄糖、甘油为碳源的培养基上生长;其淀粉酶活力较强,适宜培养温度为28℃~30℃,常用于生产酱油、甜酱、味增等发酵制品。
鲁氏酵母在产品生产中有利于香气的形成。在酱油发酵中的主要作用为产生乙醇、高级醇,如异戊醇、异丁醇等醇类物质、芳香杂醇类物质(如乙醇、3-甲硫基丙醇、异戊醇(3-甲基-1-丁醇)、异丁醇(2-甲基-1-丙醇),而在高盐状态下可使糖类生成多元醇,比如丙三醇、阿拉伯糖醇、赤藓糖醇等,除此之外,还会产生呋喃酮类风味物质。
对于耐高盐的鲁氏酵母菌株研究较多,而对于耐高糖的鲁氏酵母菌株研究较少。鲁氏酵母常和其他微生物一起制作甜酱、酱油等食品,加入鲁氏酵母发酵可促进各种香气成分酯类的合成、甘油以及多元醇的生成。此外,鲁氏酵母可以增加酱油中的琥珀酸含量,使酱油的滋味得到改进;可以促进糠醛生成糠醇;还可以提高酱中的总氨基酸含量,增加甜酱的鲜味,增加酱的香味,并且提高总抗氧化能力。因此,提供一株耐高盐高糖的鲁氏酵母菌株,对于其酱类食品的制备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鲁氏酵母菌株(Zygosaccharomyces rouxii)JNBZR01,已于2018年9月28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.16547,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鲁氏酵母菌株的微生物制剂。
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的鲁氏酵母菌株干菌体。
本发明的第三个目的是提供一种增加酱类食品香气的方法,是将上述的鲁氏酵母或上述的微生物制剂添加到酱类食品中。
本发明的第四个目的是提供上述鲁氏酵母菌株在麦汁发酵中的应用。
本发明的第五个目的是提供上述鲁氏酵母菌株在生产发酵食品中的应用。
本发明的第六个目的是提供上述鲁氏酵母菌株在生产酱油、甜酱或味增中的应用。
本发明的第七个目的是提供上述微生物制剂在麦汁发酵中的应用。
本发明的第八个目的是提供上述微生物制剂在生产发酵食品中的应用。
本发明的第九个目的是提供上述的鲁氏酵母菌株在生产酱油、甜酱或味增中的应用。
本发明公开了一株耐高糖的鲁氏酵母菌株JNBZR01,其可以直接添加到酱类食品中,增加酱类食品的香气,且可以耐受酱中的高糖高盐环境,可以耐受58%的葡萄糖、60%的蔗糖、60%的果糖、65%的麦芽糖、10%的淀粉、12%的NaCl,具有淀粉酶活力,在麦汁发酵中产生的醇酯类含量较高,其中的挥发性物质增添了产物的花香味和果香味。
生物材料保藏
一株鲁氏酵母菌株(Zygosaccharomyces rouxii)JNBZR01,分类命名为鲁氏结合酵母Zygosaccharomyces rouxii,已于2018年9月28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 16547,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为菌株1,2,3号的菌体形态。
图2为菌株1号的系统发育树。
具体实施方式
(一)GC-MS分析方法
以2-辛醇为内标作半定量分析。分析条件为:GC条件:PEG-20M弹性石英毛细管柱,30m×0.25m×0.25μm;载气为高纯氦气,恒定流量为0.8mL/min;升温程序:从180℃开始,保持2min,以3℃/min升温到230℃,保持10min;进样口温度250℃,出样口温度200℃;检测电压350V。MS条件:EI离子源,发射电流200μA,电子能量70eV,扫描范围20U~550U。
实施例1菌株的筛选和鉴定
1.菌株的初步筛选
将置于室外的柿饼上长出的酵母菌接入40%的葡萄糖平板中,置于28℃培养箱培养。
2.菌落特征及菌体形态
平板上的菌均为乳白色,表面光滑,湿润,粘稠,易挑起,菌落大小适中。在显微镜下菌体形态如图1所示。
3.菌株的分子生物学鉴定
将初筛选取的7株菌于YPD液体培养基上2%接种量活化,28℃、150r/min培养24h。取适量培养液离心1min(12000r/min,4℃),弃尽上清液,得到适量菌体(菌总数约为107cfu/mL),用天根酵母基因组DNA提取试剂盒提取基因组DNA。用酵母ITS通用引物对正向引物ITS1、反向引物ITS4扩增基因组DNA。PCR反应条件为:94℃预变性5min后进入以下循环:94℃变性45s,55℃退火40s,72℃延伸60s,35个循环;72℃延伸10min。PCR产物用1.0%琼脂糖凝胶电泳检测。产物送往苏州金唯智生物科技有限公司测序。
将7株菌的测序结果进行比对,结果表明,7株菌中包含3株不同的菌株,分别命名为1号、2号、3号菌株。
其中,1号菌株的16S rDNA序列在NCBI中使用BLAST程序进行同源性比较,与Zygosaccharomyces Rouxii AB302829.1同源性最高,相似度达98.21%。系统发育树见图2,可以确定该菌株是鲁氏酵母菌(Zygosaccharomyces Rouxii),将其送往保藏机构保藏,获得保藏编号CCTCC NO:16547,分类命名为鲁氏结合酵母Zygosaccharomyces Rouxii。
4.菌株的生理生化特征
(1)菌株的耐高浓度葡萄糖测试
将1号、2号、3号菌株在不同浓度的高浓度葡萄糖YPD平板上作扇形划线,置于28℃静置培养48小时,观察其菌落的生长情况来确定菌株的最高葡萄糖耐受浓度。由平板上生长情况可以看出1号,2号,3号的最高葡萄糖耐受浓度见表1分别为58%,55%,56%。
(2)菌株在不同碳源条件下耐受浓度测试
将初筛得到的1号、2号、3号菌株在以蔗糖、果糖或麦芽糖为唯一碳源的YPD平板上作扇形划线,置于28℃静置培养48小时,观察其菌落的生长情况来确定菌株在不同碳源条件下的耐受浓度。由平板上生长情况可以看出1号,2号,3号在不同碳源条件下的耐受浓度见表1。最高的蔗糖耐受浓度均为60%,最高果糖耐受浓度均为60%,最高麦芽糖耐受浓度均为65%。
(3)菌株的淀粉酶活力的测试
将初筛得到的1号、2号、3号菌株在以不同浓度的淀粉为唯一碳源的YPD平板上作扇形划线,置于28℃静置培养48小时,观察其菌落的生长情况来确定3株菌的淀粉酶活力。由平板上生长情况可以看出1号,2号,3号在10%的淀粉为碳源的YPD平板上均可生长,经过碘液染色可看出菌落周围有透明圈,说明其淀粉酶活力较强。
(4)菌株的耐盐性的测试
将初筛得到的1号、2号、3号菌株在不同浓度的NaCl的YPD平板上作扇形划线,置于28℃静置培养48小时,观察其菌落的生长情况来确定菌株的最高NaCl耐受浓度。由平板上生长情况可以看出1号、2号、3号菌株的最高NaCl耐受浓度见表1,分别为12%,8%,9%。
实施例2 3株菌在麦汁发酵中的应用
应用1号、2号、3号菌株进行发酵液基本醇酯及发酵液酒精度测定。1号、2号、3号三株菌在以1%的接种量20mL YPD中活化24h,以107cfu/mL接入麦汁浓度12°P的麦汁中,25℃静置培养6天,每天称重。1天内减重少于0.1g视为发酵完成。发酵完成后,气相色谱测定发酵液中基本的醇酯含量。结果见表2。由表1、表2、表3可看出1号、2号、3号菌株对于不同糖的耐受浓度差别较小,1号菌株对于NaCl的耐受性最好,在麦汁发酵中酯类含量最高。
实施例3 1号酵母菌株(JNBZR01)菌体挥发性物质
根据实施例2中对1号、2号、3号菌株在麦汁中发酵所得醇酯含量的结果,选取醇酯含量最高的1号菌株进行酵母菌体挥发性物质的测定。将1号酵母菌株按1%的接种量接种到200mL YPD中过夜培养。无菌水清洗一次后,离心去上清。在20mL顶空瓶中,加入2mL酵母菌泥样品,10μL 10280μg/L 2-辛醇,在45℃预热5min,萃取60min。萃取完成后,将萃取头插入进样口,解吸附5min,进行GC-MS分析。1号酵母菌株菌体挥发性物质含量测定结果见表4,苯乙醇、苯乙醛等物质有令人愉悦的花香味,乙酸乙酯、乙酸甲酯、3-甲基丁醛有果香味。将该酵母菌体直接添加到酱类食品中会增加食品的香气。因此决定选取1号菌株进行保藏。1号菌株为鲁氏酵母菌株(Zygosaccharomyces rouxii)JNBZR01,分类命名为鲁氏结合酵母Zygosaccharomyces rouxii,于2018年9月28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 16547,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。
表1菌株1,2,3号在不同碳源条件下最高耐受浓度结果
1号 | 2号 | 3号 | |
葡萄糖耐受浓度 | 58% | 55% | 56% |
蔗糖耐受浓度 | 60% | 60% | 60% |
果糖耐受浓度 | 60% | 60% | 60% |
麦芽糖耐受浓度 | 65% | 65% | 65% |
淀粉耐受浓度 | 10% | 10% | 10% |
表2菌株1,2,3号NaCl最高耐受浓度结果
1号 | 2号 | 3号 | |
NaCl耐受浓度 | 12% | 8% | 9% |
表3菌株1,2,3号发酵液基本醇酯含量及酒精度
1号 | 2号 | 3号 | |
乙醛(mg/L) | 5.46 | 5.91 | 4.57 |
乙酸乙酯(mg/L) | 0.45 | 0.42 | 0.36 |
正丙醇(mg/L) | 3.97 | 2.39 | 3.64 |
异丁醇(mg/L) | 21.18 | 19.58 | 17.68 |
乙酸异戊酯(mg/L) | 0.01 | 0.01 | 0.01 |
异戊醇(mg/L) | 34.96 | 33.14 | 28.65 |
酒精度(%vol) | 1.38 | 1.62 | 1.62 |
表4酵母菌株1号菌体挥发性物质含量测定
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)
1.一株鲁氏酵母菌株(Zygosaccharomyces rouxii),已于2018年9月28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.16547,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所述鲁氏酵母菌株的微生物制剂。
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的鲁氏酵母菌株干菌体。
4.一种增加酱类食品香气的方法,其特征在于,将权利要求1所述的鲁氏酵母或权利要求2所述的微生物制剂添加到酱类食品中。
5.权利要求1所述的鲁氏酵母菌株在麦汁发酵中的应用。
6.权利要求1所述的鲁氏酵母菌株在生产发酵食品中的应用。
7.权利要求1所述的鲁氏酵母菌株在生产酱油、甜酱或味增中的应用。
8.权利要求2所述的微生物制剂在麦汁发酵中的应用。
9.权利要求2所述的微生物制剂在生产发酵食品中的应用。
10.权利要求2所述的鲁氏酵母菌株在生产酱油、甜酱或味增中的应用。
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