CN112940953A - 一株高产3-甲基丁醇的汉逊德巴利酵母 - Google Patents
一株高产3-甲基丁醇的汉逊德巴利酵母 Download PDFInfo
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Abstract
本发明涉及利用微生物进行发酵食品生产的领域，具体涉及一株从四川传统家庭自制腊肉中分离筛选出的能够高产3‑甲基丁醇的汉逊德巴利酵母菌。能够高产3‑甲基丁醇的汉逊德巴利酵母(Debaryomyceshansenii)菌株号为LY090，在中国微生物菌种保藏管理委员会普通微生物中心登记保藏；保藏号为CGMCC NO.21374，保藏日期为2020年12月14日。该菌株不仅能高产3‑甲基丁醇，还能减少脂肪醛的积累，抑制脂肪氧化。本发明提供的汉逊德巴利酵母LY090可开发肉制品发酵剂，用于四川腊肉和发酵香肠的生产。
Description
技术领域
本发明涉及利用微生物进行发酵食品生产的领域，具体涉及一株从四川传统家庭自制腊肉中分离筛选出的能够高产3-甲基丁醇的汉逊德巴利酵母菌。
背景技术
以香肠和腊肉为代表的中式传统肉制品因其特殊的风味极受消费市场的欢迎，目前国内相关的中小型生产企业众多且产量巨大。然而，与农家自制产品相比，工厂化产品生产时间缩短，生产工艺也有微调，如替代盐和亚硝酸盐的使用、成熟温度升高，从而导致产品特征香味和风格被弱化，消费者接受度降低。采用人工发酵剂应用于工业化生产是改善产品风味、质构、色泽、安全最有效的策略，乳酸菌和凝固酶阴性葡萄球菌是最常用的人工发酵剂。乳酸菌能快速降低肉基质pH值，确保后续一系列生化反应正常进行和产品的微生物安全；凝固酶阴性葡萄球菌则在蛋白质水解、脂肪分解和颜色形成等重要生化反应中发挥作用。
除了以上两类微生物，酵母菌在肉制品成熟过程中也扮演着重要的角色。研究发现肉制品中酵母菌展现了广泛的胞外脂肪酶和蛋白酶活性，可促进肉中脂肪和蛋白质降解，其初级和次生代谢产物有助于产品特征香气和味道的形成。
酵母菌可以减少肉制品中脂肪氧化相关醛类的积累，代谢支链氨基酸促进香气成分如支链醛/醇和乙基酯的形成。近年来，国外研究者对酵母菌在西式传统肉制品中的多样性，对风味、质构及产品可接受度影响等方面进行了广泛的研究，目前意大利、西班牙、土耳其等欧洲国家已普遍将汉逊德巴利酵母作为肉品发酵剂用于改良工厂化肉制品品质。然而中国传统肉制品相关研究却很少，还没有适用于本地肉制品生产的优良酵母菌株。因此，有必要从本地传统肉制品中筛选获得优良的产香酵母菌用于中式传统肉制品的工业化生产。
发明内容
针对现有技术存在的问题，本发明目的在于提供一株高产3-甲基丁醇的汉逊德巴利酵母(Debaryomyceshansenii)LY090。
本发明所提供的菌株为汉逊德巴利酵母(Debaryomyceshansenii)，菌株号LY090，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地点为北京市朝阳区北辰西路1号院，保藏号为CGMCC NO. 21374，保藏日期为2020年12月14日。
本发明提供高产3-甲基丁醇酵母菌株的分离、筛选、鉴定。
准确称取搅碎四川腊肉样品25g，与225 mL无菌水混合于无菌均质袋中匀浆，取1mL匀浆用无菌生理盐水进行10倍梯度稀释，取适宜稀释度样液100 mL涂布于含氯霉素的PDA培养基平板，28℃培养3~5d。挑取具有酵母菌典型菌落特征的菌落进行多次平板划线纯化，保存备用。
将上述获得的酵母菌进行环境耐受性及胞外脂肪酶和蛋白酶活性的筛选，取斜面菌种一环接入5 mL的YPD液体培养基中，于28℃培养3 d，将菌液于8000 r/min、4℃离心10min，弃去上清液，收集菌体，用无菌水洗涤离心3次并重悬，调整菌体浓度为1.0×108 CFU/mL。
将上述菌悬液按1% (v/v)接种于YPD液体培养基试管中，随后在15、30和37 ℃培养3 d ，以不接种为对照，用目测法测定酵母的生长情况。同理，分别准备pH为1.5、3.0和4.5的YPD液体培养基试管（用3 mol/L HCl调节），NaCl浓度为5%、10%和15%的YPD液体培养基试管和NaNO2浓度分别为50、100和150 mg/kg的YPD液体培养基试管，将前述菌悬液按1%(v/v)接种于以上各类YPD液体培养基试管中，在28 ℃培养3 d，以不接种为对照，用目测法测定酵母的生长情况。
取上述菌悬液10μL以点种法点接在蛋白酶和脂肪酶检测平板上，25 ℃ 培养7 d，脂肪酶检测平板直接观察菌落周围透明圈的大小和清晰度，蛋白酶检测平板先滴加10% 三氯乙酸后再观察，用游标卡尺记录下透明圈和菌落大小。
将上述筛选后的酵母菌进行产香模拟发酵试验。模拟肉汤培养基分装于50 mL三角瓶，每瓶20 mL，将上述菌悬液按1% (v/v)接种于三角瓶，120 r/min，25 ℃摇床培养5 d，发酵液用GC-MS检测。
所述GC-MS检测方法为：取4 mL发酵液置于20 mL顶空瓶中，加入1 g NaCl，将老化后的萃取头（75 um，CAR/PDMS，Supelco）插入样品瓶顶空部分，于50 ℃吸附30 min，吸附后的萃取头取出插入气相色谱进样口，于250 ℃解吸3 min。程序升温条件：柱初温40℃，维持3 min，然后以4 ℃/min的速率升温至80 ℃，保持1 min，以5 ℃/min升到150 ℃，保持2min，再以10 ℃/min的升温速率升至230 ℃，保持1 min。毛细管柱DB-WAX(30m×0.25mm×0.25um)，以氦气做载气，流速为1 mL/min。电离方式为El，电子能量70ev，离子源温度230℃，接口温度为280 ℃，四级杆温度150 ℃，扫描范围：30 ~ 550 m/z。
各风味组分的表示方法：各组分经谱库检索确定，仅选择匹配度大于80%的组分，相对含量以峰面积占检出气体总峰面积百分比表示。
菌株形态鉴定：肉眼观察菌落形态为圆形，凸起，边缘整齐，表面湿润的乳白色菌落。与光学显微镜下观察菌株细胞特征，其细胞呈球状，多边出芽生殖，未观察到假菌丝。对照《酵母菌的特征与鉴定手册》进行生理生化鉴定，能够发酵葡萄糖、半乳糖、麦芽糖、蔗糖和棉子糖，可以同化蛋白胨和硝酸钾，不能同化尿素，脲酶试验为阴性。初步判定菌株LY090为汉逊德巴利酵母。
菌株LY090分子遗传学鉴定。
菌株LY090经26S rDNA序列测定，获得如下序列：
GAGGAAAAGAAACCAACAGGGATTGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGCACCTTCGGTGTCCGAGTTGTAATTTGAAGAAGGTAACTTTGGAGTTGGCTCTTGTCTATGTTCCTTGGAACAGGACGTCACAGAGGGTGAGAATCCCGTGCGATGAGATGCCCAATTCTATGTAAAGTGCTTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGAGATCAGACTTGGTATTTTGCGATCCTTTCCTTCTTGGTTGGGTTCCTCGCAGCTTACTGGGCCAGCATCGGTTTGGATGGTAGGATAATGACTAAGGAATGTGGCTCTACTTCGGTGGAGTGTTATAGCCTTGGTTGATACTGCCTGTCTAGACCGAGGACTGCGTCTTTTGACTAGGATGTTGGCATAATGATCTTAAGCCACCCGTCTT
该菌株的26S rDNA 基因测序结果与 GenBank 数据库中序列进行Blast同源性对比，该菌株序列与Debaryomyceshansenii菌株基因序列高度同源，得到登录号为MT422073，结合形态学和生理生化指标，最终将菌株LY090鉴定为汉逊德巴利酵母。
本发明所提供的高产支链醇酵母菌株是从四川传统家庭自制腊肉中分离筛选得到的，该菌株具有很强的肉制品生产环境耐受性，胞外脂肪酶活性强，在模拟肉汤培养基中能显著增加3-甲基丁醇的含量。
本发明所用培养基为：PDA培养基：马铃薯浸粉3 g，葡萄糖20 g，琼脂粉15 g，氯霉素0.1 g。YPD液体培养基：蛋白胨20 g，酵母膏10 g，葡萄糖20 g，水1000 mL，121℃灭菌15min。加入2%琼脂为YPD固体培养基。模拟肉汤培养基：瘦猪肉1000 g切成小丁，加1000 mL蒸馏水，加热沸腾后继续煮制20 min，四层纱布过滤，添加3%食盐和150 mg/kg亚硝酸钠，蒸馏水补足到1000 mL，121 ℃灭菌15 min。
与现有技术相比，本发明的积极效果是：从四川传统家庭自制腊肉中筛选获得高产3-甲基丁醇的汉逊德巴利，汉逊德巴利酵母很早被用于西式肉制品生产的人工发酵剂之一，该菌株来源安全，具有环境耐受性、胞外脂肪酶活性强、高产3-甲基丁醇等优点，可为四川传统肉制品工业化生产提供良好菌源，具有良好的应用潜力。
附图说明
图1为酵母菌LY090的菌落形态和细胞形态图。
图2为LY090胞外脂肪酶和蛋白酶活性。
图3为接种LY090后挥发性风味物质GC-MS分析总离子流色谱图。
图4为接种LY090发酵后主要挥发性物质含量变化图。
具体实施方式
实例1：菌株LY090接种于模拟肉汤培养基发酵产3-甲基丁醇的效果。
将菌株LY090按1%接种于20ml模拟肉汤培养基中，以不接种菌株LY090的20 ml上述模拟肉汤培养基作为空白对照，25℃振荡培养5 d，用[0012]和[0013]所述方法测定挥发性风味物质。
菌株LY090发酵模拟肉汤培养基产生3-甲基丁醇的峰面积(AU)达到474.85×106，对照组未检测到3-甲基丁醇。
实例2：菌株LY090应用于四川腊肉对挥发性风味物质的影响。
四川腊肉制备：取新鲜猪五花肉10kg，切分为240mm×80mm×30mm长条，将300g食盐和20g花椒混合后均匀涂抹肉条表面，肉条平均分为2组，即对照组和实验组，分别均匀码放入不锈钢桶，于温度4℃、相对湿度90%腌制7d，中间翻缸2次；腌完后将肉条用小勾挂起，在温度15℃、相对湿度75%人工气候箱中风干成熟20d即为成品。
用[0009]所述方法制备菌株LY090菌悬液，上述实验组在入桶腌制之前将制备好的LY090菌悬液接种于肉条，菌体浓度达到1×106 CFU/g肉。风干成熟完成后，分别取对照组和实验组肉条，用组织捣碎机破碎，用[0012]和[0013]所述方法测定测定挥发性风味物质。
由12名专业品评人员组成品评小组对实验组和对照组四川腊肉进行感官品评并打分，采用SPSS22.0软件对综合得分进行单因素方差分析。
接种菌株LY090四川腊肉中3-甲基丁醇峰面积比对照组提高了68.3%。经感官品评分析，实验组综合得分显著（p＜0.05）高于对照组。说明菌株LY090接种发酵可提高四川腊肉挥发性风味3-甲基丁醇含量，显著增强消费者可接受度。
以上的实例仅仅是对本发明的实施方式进行描述，而并非对本发明的范围进行限定，对于本领域的技术人员来说，可以对上述说明进行改进或变形，但所有的这些改进或变形均应落入本发明的权利要求确定的保护范围。
序列表
<110> 四川农业大学
<120> 一株高产3-甲基丁醇的汉逊德巴利酵母
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 587
<212> DNA
<213> 汉逊德巴利酵母(Debaryomyces hansenii)
<400> 1
gaggaaaaga aaccaacagg gattgcctta gtaacggcga gtgaagcggc aaaagctcaa 60
atttgaaatc tggcaccttc ggtgtccgag ttgtaatttg aagaaggtaa ctttggagtt 120
ggctcttgtc tatgttcctt ggaacaggac gtcacagagg gtgagaatcc cgtgcgatga 180
gatgcccaat tctatgtaaa gtgctttcga agagtcgagt tgtttgggaa tgcagctcta 240
agtgggtggt aaattccatc taaagctaaa tattggcgag agaccgatag cgaacaagta 300
cagtgatgga aagatgaaaa gaactttgaa aagagagtga aaaagtacgt gaaattgttg 360
aaagggaagg gcttgagatc agacttggta ttttgcgatc ctttccttct tggttgggtt 420
cctcgcagct tactgggcca gcatcggttt ggatggtagg ataatgacta aggaatgtgg 480
ctctacttcg gtggagtgtt atagccttgg ttgatactgc ctgtctagac cgaggactgc 540
gtcttttgac taggatgttg gcataatgat cttaagccac ccgtctt 587
Claims (3)
1. 一株高产3-甲基丁醇的汉逊德巴利酵母(Debaryomyceshansenii)LY090，其特征在于保藏于中国普通微生物菌种保藏管理中心，保藏地点为北京市朝阳区北辰西路1号院，保藏号为CGMCC NO. 21374，保藏日期为2020年12月14日。
2.根据权利要求1所述汉逊德巴利酵母(Debaryomyceshansenii)LY090的应用，其特征在于：接种于模拟肉汤培养基发酵高产3-甲基丁醇。
3.根据权利要求1所述汉逊德巴利酵母(Debaryomyceshansenii)LY090的应用，其特征在于：该菌株用于四川腊肉生产提高挥发性风味3-甲基丁醇含量并增强消费者接受度。
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| CN114317300A (zh) * | 2022-01-17 | 2022-04-12 | 东北农业大学 | 一株能够提升风干肠风味的汉逊德巴利酵母菌及其应用 |
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| CN114317300A (zh) * | 2022-01-17 | 2022-04-12 | 东北农业大学 | 一株能够提升风干肠风味的汉逊德巴利酵母菌及其应用 |
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