CN114908114A - 一种酵母细胞基因改造方法和一种重组酵母及其应用 - Google Patents
一种酵母细胞基因改造方法和一种重组酵母及其应用 Download PDFInfo
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Abstract
本发明公开一种酵母细胞基因改造方法和一种重组酵母及其应用,乳酸链球菌素Nisin是一种高效,安全,无毒的天然抑菌活性多肽,已被FDA批准用作多种食品的防腐剂。本发明采用合成生物学的方法构建酿酒酵母菌株进行发酵,使得该酿酒酵母菌株在发酵的同时能够合成外源多肽Nisin,对面包、糕点、馒头等发酵食品的防腐可做到“一步到位”,不仅能大大降低食品中添加化学防腐剂带来的成本问题和安全隐患,还简化了食品防腐步骤,在很大程度上降低了天然防腐剂Nisin的使用成本,扩展了天然防腐剂Nisin的应用方式。
Description
技术领域
本发明涉及基因工程技术领域,特别是一种酵母细胞基因改造方法和一种重组酵母及其应用。
背景技术
随着社会的发展与人民生活水平的提高,人们越来越追求更加健康、安全的饮食方式。鉴于化学合成食品防腐剂的安全性和其他缺陷,人们正在探索更安全、更方便使用的天然食品防腐剂。
乳酸链球菌素(Nisin)是一种高效、无毒的纯天然生物防腐剂,是FDA公认安全的食品添加剂,在食品工业中得到了广泛的应用。目前主要通过乳酸乳球菌亚种发酵法提取具有一定纯度的Nisin,添加到相应的食品中以抑制食品腐败菌的生长,延长食品保质期。虽然Nisin是一种高效的食品防腐剂,通常皮摩尔到纳摩尔数量级的Nisin即可发挥其抑菌作用。但目前市场上销售的都是纯度较低的乳酸链球菌素盐制剂,杂质较多且含盐量高,对食品的风味有一定的影响。另外,较低产量和较高的生产成本成为制约Nisin商业应用的瓶颈。
发明内容
本发明的主要目的是提出一种酵母细胞基因改造方法和一种重组酵母及其应用,旨在改造酵母菌使得其在食品发酵过程中同时合成天然的防腐剂Nisin,达到对食品原位生物防腐的目的。
为实现上述目的,本发明提出一种酵母细胞基因改造方法,所述酵母细胞基因改造方法的步骤包括:
将合成乳酸链球菌素的前体肽结构基因nisA和水解酶基因nisP整合至亮氨酸缺陷型载体质粒pESC-Leu,构建①号质粒pESC-Leu-His6-nisA-nisP;
将合成乳酸链球菌素的基因簇中的脱水酶基因nisB,环化酶基因nisC整合至尿嘧啶缺陷型载体质粒pESC-Ura,构建②号质粒pESC-Ura-nisB-Flag-nisC-Myc;
将表达谷氨酰-tRNA的基因tRNAGlu和GltX整合至组氨酸缺陷型载体质粒pESC-His,构建③号质粒pESC-His-pSNR52tRNAGlu-GltX;
将①号、②号和③号质粒转化至所述酵母细胞。
本发明还提出一种重组酵母,所述重组酵母由如上述实施例中的酵母细胞基因改造方法得到,所述重组酵母可胞内表达乳酸链球菌素前体肽NisA、水解酶NisP、脱水酶NisB、环化酶NisC以及谷氨酰-tRNA。
本发明还提出一种乳酸链球菌素Nisin的制备方法,所述乳酸链球菌素的制备方法的步骤包括:
S1、将权利要求2所述的重组酵母单克隆转化子在含1%-3%葡萄糖的亮氨酸、尿嘧啶和组氨酸缺陷型培养基中扩大培养,收集菌体;
S2、将收集得到的菌体置换至含1%-3%半乳糖的新鲜亮氨酸、尿嘧啶和组氨酸缺陷型培养基中诱导表达,离心收集菌体;
S3、将制备得到的菌体沉淀重悬后,破碎取上清液,从上清液中提取制备得到乳酸链球菌素Nisin。
在一实施例中,步骤S1的培养条件为30℃,摇床转速200rpm,培养24小时,测得培养菌液在600nm波长处的吸光值为2-3,收集菌体。
在一实施例中,步骤S2中培养基的体积以调节OD600值到0.1为准,培养条件为30℃,摇床转速200rpm,诱导表达4-5天,测得最终菌液在600nm波长处的吸光值为5-6,离心收集菌体。
在一实施例中,步骤S3具体包括:将制备得到的菌体沉淀用20mLddH2O重悬清洗3次,离心除去上清,加入2mL50mM的HEPES缓冲液(pH=7.5)重悬;分装到5个2mLEP管中,0.7mL/管,加入700μL0.5mm的玻璃珠,用高压均质机69Hz破碎3min×4次,15000g离心15min,收集离心后上清液。
本发明还提供一种重组酵母在发酵食品中的应用,所述应用包括上述实施例中的重组酵母在发酵食品中的应用。
本发明聚焦于微生物天然防腐剂乳酸链球菌素(Nisin)。Nisin是一种高效,安全,无毒的天然抑菌活性多肽,已被FDA批准用作多种食品的防腐剂。目前,市场上普遍通过乳酸乳球菌发酵法,提取其代谢产物Nisin。但往往工艺复杂,纯度较低。另外生产成本偏高也限制了Nisin的广泛应用。因此,本发明采用合成生物学的方法构建酿酒酵母菌株进行发酵,使得该酿酒酵母菌株在发酵的同时能够合成外源多肽Nisin。若将构建的酿酒酵母菌株应用于面包、糕点、馒头等发酵食品的生产当中,则酿酒酵母在发酵的同时可以合成活性抑菌肽Nisin,Nisin可用作食品防腐剂抑制面包、糕点、馒头等发酵食品中腐败菌的生长。本发明专利不需要将Nisin分离纯化出来,直接在面包、糕点、馒头等发酵类食品制作的过程中使用可以合成活性Nisin的酿酒酵母菌株发酵,Nisin合成产量足够发挥食品防腐剂的作用,并且纯度为100%,不需要额外添加商业化的Nisin,同时可减少此类食品中化学类防腐剂的使用,延长食品保质期。本发明对面包、糕点、馒头等发酵食品的防腐可做到“一步到位”,不仅能满足现代人们对食品健康的要求,做到食品绿色、安全、无添加,同时也能大大降低食品中添加化学防腐剂带来的成本问题和安全隐患,保障了发酵食品面包、糕点和馒头等的食品安全,简化了食品防腐步骤,将会在很大程度上降低天然防腐剂Nisin的使用成本,扩展了天然防腐剂Nisin的应用方式。可以在促进食品防腐剂技术进步的同时,带来巨大的经济效益。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图示出的结构获得其他的附图。
图1A为乳酸链球菌素(Nisin)基因簇示意图;
图1B为乳酸链球菌素(Nisin)的合成路径示意图;
图2为酿酒酵母细胞内表达乳酸链球菌素(Nisin)所需质粒的基因路线示意图;
图3为PCR鉴定酿酒酵母工程菌内转化入的各部分表达活性乳酸链球菌素(Nisin)相关基因的DNA凝胶电泳图,泳道从左至右依次是来自1号,2号酿酒酵母工程菌;
图4为酿酒酵母工程菌表达乳酸链球菌素(Nisin)抑菌实验结果图:
A)敏感菌为乳酸链球菌亚种(L.lactis subsp.cremoris strainHP);
B)敏感菌为黄色微球菌(Micrococcus flavus,M.f)
Buffer:50mM HEPES+2.5mM DTT+1μL LicP;
Nisin:商业购买的乳酸链球菌素(Nisin),不小于1000IU/mg,溶液浓度1mg/mL;
C,1和2分别代表酿酒酵母工程菌CEN-PK2-1C-0,CEN-PK2-1C-nisin-1和CEN-PK2-1C-nisin-2所表达的目标产物样品;
所有样品孔的上样量均为50μL。
本发明目的的实现、功能特点及优点将结合实施例,参照附图做进一步说明。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
需要说明,若本发明实施例中有涉及方向性指示(诸如上、下、左、右、前、后……),则该方向性指示仅用于解释在某一特定姿态(如附图所示)下各部件之间的相对位置关系、运动情况等,如果该特定姿态发生改变时,则该方向性指示也相应地随之改变。
另外,若本发明实施例中有涉及“第一”、“第二”等的描述,则该“第一”、“第二”等的描述仅用于描述目的,而不能理解为指示或暗示其相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。另外,若全文中出现的“和/或”的含义为,包括三个并列的方案,以“A和/或B”为例,包括A方案,或B方案,或A和B同时满足的方案。另外,各个实施例之间的技术方案可以相互结合,但是必须是以本领域普通技术人员能够实现为基础,当技术方案的结合出现相互矛盾或无法实现时应当认为这种技术方案的结合不存在,也不在本发明要求的保护范围之内。
本发明提出一种酵母细胞基因改造方法,所述酵母细胞基因改造方法的步骤包括:
将合成乳酸链球菌素的前体肽结构基因nisA和水解酶基因nisP整合至亮氨酸缺陷型载体质粒pESC-Leu,构建①号质粒pESC-Leu-His6-nisA-nisP;
将合成乳酸链球菌素的基因簇中的脱水酶基因nisB,环化酶基因nisC整合至尿嘧啶缺陷型载体质粒pESC-Ura,构建②号质粒pESC-Ura-nisB-Flag-nisC-Myc;
将表达谷氨酰-tRNA的基因tRNAGlu和GltX整合至组氨酸缺陷型载体质粒pESC-His,构建③号质粒pESC-His-pSNR52tRNAGlu-GltX;
将①号、②号和③号质粒转化至所述酵母细胞。
本发明还提出一种重组酵母,所述重组酵母由如上述实施例中的酵母细胞基因改造方法得到,所述重组酵母可胞内表达乳酸链球菌素前体肽NisA、水解酶NisP、脱水酶NisB、环化酶NisC以及谷氨酰-tRNA。
乳酸链球菌素又称乳球菌肽或乳链菌肽,英文名为Nisin,是由乳酸乳球菌乳酸亚种(Lactococcus lactissubsp.Lactis)在代谢过程中合成和分泌的具有很强杀菌作用的小分子肽,是一种天然食品防腐剂。它对绝大多数食物腐败菌和病原菌在内的革兰氏阳性菌尤其是芽孢杆菌具有强烈的抑制作用,而对革兰氏阴性菌、酵母和霉菌无抑制作用。另外,Nisin与EDTA共同作用可抑制沙门氏菌和其他革兰氏阴性菌的生长。Nisin对热稳定,具有耐酸性,食用后在消化道内很快被α-胰凝乳蛋白酶有效降解,不会产生抗性和过敏反应,对人体无任何的毒副作用。早在1988年,nisin就被美国食品与药物管理局(FDA)批准在食品中使用。目前已被60多个国家批准作为一种无毒的天然食品防腐剂,广泛应用于乳制品、肉制品、罐头制品、果汁和酒精饮料等多种食品的防腐保鲜。
目前,Nisin的工业生产是采用Nisin产生菌乳酸乳球菌亚种,以巴氏灭菌奶添加酵母膏经蛋白酶处理后为培养基,在pH值6.0和温度30℃条件下进行发酵、提取和加工而得。虽然Nisin是一种高效的食品防腐剂,通常皮摩尔到纳摩尔数量级的Nisin即可发挥其抑菌作用。但目前市场上销售的都是纯度较低的乳酸链球菌素盐制剂,杂质较多且含盐量高,对食品的风味有一定的影响。
另一方面,酵母是安全的食品原料,在中国,酵母被广泛用来制作馒头、酿制白酒,其应用也已经有几千年的历史。目前全球酵母的产量超过100万吨,系人类年利用量唯一超过百万吨的微生物。酵母容易在实验室操作和培养;另外有一些酵母已经被开发为异源蛋白表达系统使用,利用基因技术可在酵母细胞内表达外源蛋白质。
1988年,Buchaman首次提出Nisin的合成机制,指出其前体物质是由57个氨基酸组成,包括含有23个氨基酸的N端前导序列(leader peptide)和34个氨基酸的C端核心序列,经过翻译后酶修饰才可以形成具有生物活性的大分子。据报道,合成活性乳酸链球菌素(Nisin)的基因是由11种基因组成的一个基因簇,它们按转录顺序依次排列为nisABTCIP,nisRK,和nisFEG,如图1A所示。其中,nisA是合成NisinA的结构基因;nisB、nisC,nisT,和nisP分别代表Nisin的翻译后修饰酶基因,控制合成脱水酶NisB,环化酶NisC,转运酶NisT和水解酶NisP;nisR,nisK则是是参与调控Nisin基因簇表达的基因;nisI,nisFEG是对乳酸乳球菌发挥免疫作用的基因。要实现在外源微生物表达具有生物活性的Nisin,在目标基因上游插入强启动子后,至少还需要基因簇中nisA、nisB、nisC、nisP四种基因。
乳酸链球菌素Nisin的合成路径如图1B所示,乳酸链球菌素前体肽NisA,被脱水酶NisB作用脱去8分子的水,随后在环化酶NisC的作用下分子内生成的碳碳双键与邻近半胱氨酸上的巯基发生加成反应,生成带有硫醚键的分子内环状结构。最后在水解酶NisP的作用下,切除前导肽(leaderpeptide),释放出包含34个氨基酸的生物活性Nisin。从1988年发现Nisin的合成机制至今,对Nisin的分子结构以及其生物合成路径的研究更加深入。其中,除了Nisin的源产生菌乳酸乳球菌乳酸亚种外,研究者也利用外源微生物合成生物活性Nisin,以研究Nisin的生物合成路径中,各种酶的作用机制,从而提高微生物合成Nisin的产量。
本发明通过对酵母细胞的基因进行改造,以使酵母细胞内表达出活性乳酸链球菌素Nisin。酵母细胞可以是酿酒酵母细胞,也可以是其他酵母细胞如啤酒酵母细胞、毕赤酵母细胞、面包酵母细胞等,可根据实际需要进行选择,在此不做过多限定。
在本发明一实施例中,采用酿酒酵母细胞进行基因改造,具体方案如下:采用酿酒酵母表达外源蛋白的载体质粒pESC-Leu、pESC-Ura和pESC-His,该质粒为穿梭质粒,能够在酵母菌和大肠杆菌之间穿梭使用,即同时具备酿酒酵母中同源重组克隆、自我复制、重组子筛选的能力,和在大肠杆菌中稳定性复制和保持的能力,便于DNA纯化分离操作。pESC-Leu、pESC-Ura和pESC-His载体质粒上均有两个蛋白质表达盒子,以酿酒酵母GLA1,10为启动子,并在多克隆位点处设计了蛋白标签序列。将合成乳酸链球菌素前体肽的基因nisA和水解酶基因nisP整合至亮氨酸缺陷型载体质粒pESC-Leu,构建①号质粒pESC-Leu-His6-nisA-nisP;将合成Nisin基因簇中的脱水酶基因nisB和环化酶基因nisC整合至尿嘧啶缺陷型载体质粒pESC-Ura,构建②号质粒pESC-Ura-nisB-Flag-nisC-Myc;将表达glutamyl-tRNAGlu的基因tRNAGlu和GltX整合至组氨酸缺陷型载体质粒pESC-His,构建③号质粒pESC-His-pSNR52tRNAGlu-GltX。研究表明,glutamyl-tRNAGlu在脱水酶NisB催化乳酸链球菌素前体肽NisA脱水步骤中起到关键作用,使得完全脱水的产物提高近3倍。将①号、②号和③号质粒同时转化至酿酒酵母细胞,构建包含以上六种基因的酿酒酵母工程菌株。诱导各部分基因在酿酒酵母细胞内过表达,生成的活性Nisin理论上会分布在酿酒酵母细胞内,后期通过破裂酿酒酵母细胞,释放出目标抗菌肽Nisin。酿酒酵母细胞内表达活性Nisin所需质粒的基因线路图设计如图2所示。
下面通过具体实验来说明经过基因改造后的重组酵母细胞内表达出了活性乳酸链球菌素Nisin。实验条件和过程如下:
1.菌种和生长条件
野生型酿酒酵母CEN-PK2-1C来自于本实验室。大肠杆菌TOP10菌株用于质粒构建和增殖。在所有实验中,大肠杆菌生长培养基均使用含有100mg/L氨苄青霉素的LB培养基。野生型酿酒酵母的培养使用YPD培养基,转化了载体质粒的酿酒酵母工程菌株均使用对应营养缺陷的合成培养基(SC-Leu/Ura/His)。所有琼脂培养基含琼脂(agar)的浓度为1.5%。其中,用于酿酒酵母转化子扩大培养和诱导酿酒酵母表达目标基因时的培养基中添加的葡萄糖和半乳糖的浓度均为20g/L。所有大肠杆菌的培养条件均为温度37℃,摇床转速200rpm;所有酿酒酵母的培养条件均为温度30℃,摇床转速200rpm。
2.质粒的构建
将控制表达活性Nisin的基因簇中所必须的六种基因,分别整合至三种载体质粒pESC-Leu/pESC-Ura/pESC-His。
其中,乳酸链球菌素前体肽基因nisA和水解酶基因nisP通过吉布森組裝GibsonAssembly的方法,分别插入到载体质粒pESC-Leu的第一个和第二个基因表达盒子中,得到质粒①pESC-Leu-his6-nisA-nisP,其中his6表示在nisA基因上游连接由6个组氨酸组成的蛋白质标签基因,方便后续对酿酒酵母表达乳酸链球菌素前体肽NisA的检测。本实验设计中,将用水解酶LicP代替乳酸链球菌素合成中的原始水解酶NisP,控制最终的水解反应,释放出具有生物活性的Nisin。
脱氢酶基因nisB和环化酶基因nisC通过吉布森組裝Gibson Assembly的方法,分别插入到载体质粒pESC-Ura的第一个和第二个基因表达盒子中,得到质粒②pESC-Ura-nisB-Fla-nisC-Myc,其中,Flag和Myc分别为在基因nisB和nisC的下游加入的蛋白质标签基因,方便后续对脱水酶NisB和环化酶NisC的检测。
将表达乳酸链球菌glutamyl-tRNAGlu的基因tRNAGlu和GltX通过吉布森組裝Gibson Assembly的方法,分别插入到载体质粒pESC-His的第一个和第二个基因表达盒子中,其中,用组成型启动子pSNR52取代原始质粒pESC-His的第一个基因表达盒子上的启动子GAL10,得到质粒③pESC-His-pSNR52tRNAGlu-GltX。将③号质粒上的启动子pSNR52,用酿酒酵母自身的glutamyl-tRNA启动子promoter-tRNA代替,得到的质粒④pESC-His-ptRNAGlu-GltX。
将GibsonAssembly产物用化学转化法转化入大肠杆菌Top10感受态,在含有100mg/L氨苄青霉素的LB琼脂培养基上培养,筛选出阳性单克隆送生工生物工程(上海)股份有限公司测序。将得到的测序结果正确的表达载体质粒,用酵母转化试剂盒Frozen-EZYeast Transformation II Kit转化入酿酒酵母菌株CEN-PK2-1C中。
得到对照组酿酒酵母转化子CEN-PK2-1C-0、一号酿酒酵母工程菌株CEN-PK2-1C-Nisin-1和二号酿酒酵母工程菌株CEN-PK2-1C-Nisin-2,其中,对照组酿酒酵母转化子CEN-PK2-1C-0转化了三个空载质粒pESC-Leu、pESC-Ura和pESC-His;一号酿酒酵母工程菌株CEN-PK2-1C-Nisin-1包含有质粒①pESC-Leu-his6-LicP-nisA、质粒②pESC-Ura-NisB-Fla-NisC-My和质粒③pESC-His-pSNR52tRNAGlu-GltX;二号酿酒酵母工程菌株CEN-PK2-1C-Nisin-2包含有质粒①pESC-Leu-his6-LicP-nisA、质粒②pESC-Ura-NisB-Fla-NisC-My,以及质粒④pESC-His-ptRNAGlu-GltX。
分别用PCR鉴定的方法,鉴定出导入的合成Nisin的基因全部正确的酿酒酵母转化子,如图3所示。
3.诱导酿酒酵母工程菌表达Nisin
挑选鉴定正确的酿酒酵母转化子:对照组CEN-PK2-1C-0,一号工程菌株CEN-PK2-1C-Nisin-1和二号工程菌株CEN-PK2-1C-Nisin-2。在含2%葡萄糖的亮氨酸(Leu),尿嘧啶(Ura)和组氨酸(His)缺陷型培养基(Sc-Leu-Ura-His)中扩大培养,培养条件为30℃,摇床转速200rpm。时间为24小时。测得酿酒酵母培养菌液在600nm波长处的吸光值OD600为2-3,收集菌体,置换至含2%半乳糖的新鲜SC-Leu-Ura-HIS培养基,液体培养基的体积以调节OD600值到0.1为准,在摇床转速为200rpm,温度为30℃诱导表达4-5天。测得最终菌液的OD600值为5-6。离心收集菌体。暂存于-80℃冰箱。
4.抑菌活性实验验证酿酒酵母表达Nisin的抑菌活性
将以上对照组,1号,2号样品收集的菌体沉淀(每个样品收集100mL菌液),从-80℃冰箱取出,20mLddH2O重悬清洗3次,离心除去上清,对照组,1号,2号样品加入约2mL 50mMHEPES缓冲液(pH=7.5)重悬。分装到5个2mL的EP离心管中,每管0.7mL。加入约700μL0.5mm的玻璃珠,用高压均质机69Hz破碎3min×4次,15000g离心15min,收集离心后上清液。对照组,1号,2号样品均取出200μL酿酒酵母破碎上清液,加入2.5mM二硫苏糖醇DTT,加入4μLLicP酶,在室温下做酶切反应;另外对照组,1号,2号样品每个样品取出200μL置于室温做对照组,30小时后做抑菌实验。在抑菌实验前,每个样品中均加入终浓度为0.1%的TFA终止酶切反应。
1)以乳酸链球菌(L.lactis subsp.cremoris strain HP)为敏感菌做抑菌实验:配制M17/agar培养基,其中,取7.45gM17样品粉末,3g琼脂,用200mL去离子水重悬,121℃灭菌15min,冷却至50℃左右时,加入终浓度为0.5%的无菌葡萄糖溶液充分混匀。取出其中的20mL,待培养基温度降至40℃左右时,按照250:1的体积比,加入OD600=0.5的乳酸链球菌培养液,混匀,倒入直径约9cm的细菌培养板,室温下在无菌操作箱中吹干约20min。用打孔器在凝固的琼脂培养板上打孔,加入制备好的Nisin抗菌溶液对照组和1、2号样品组,30℃培养箱静置培养24小时后,相机拍照记录抑菌实验结果,如图4A所示。由抑菌实验结果可以看出,与酿酒酵母背景表达产物相比,酿酒酵母工程菌株CEN-PK2-1C-Nisin-1和2表达的Nisin均表现出对乳酸链球菌HP较强的抑制活性。
2)以黄色微球菌(Micrococcus flavus,M.f)为敏感菌做抑菌实验:LB/agar培养基,其中,取7g LB琼脂(不含糖)干粉,加入200mL去离子水重悬,121℃高压灭菌15min,待培养基冷至50℃左右,加入终浓度0.1%的无菌葡萄糖溶液,充分混匀。用50mL无菌离心管,取其中的20mL,待温度降至40℃左右时,按照培养基与菌液体积比500:1,加入OD600=0.5的黄色微球菌,混匀,倒入直径约9cm的细菌培养板,室温下在无菌操作箱中吹干约20min。用打孔器在凝固的琼脂培养板上打孔,加入制备好的Nisin抗菌溶液对照组和1、2号样品组,30℃培养箱静置培养24小时后,相机拍照记录抑菌实验结果,如图4B所示。由抑菌实验结果可以看出,与酿酒酵母背景表达产物相比,酿酒酵母工程菌株CEN-PK2-1C-Nisin-1和2表达的Nisin均表现出对黄色微球菌M.f较强的抑制活性。
本发明还提供一种重组酵母在发酵食品中的应用,所述应用包括上述实施例中的重组酵母在发酵食品中的应用。
本发明一实施例采用合成生物学的方法构建酿酒酵母菌株进行发酵,使得该酿酒酵母菌株在发酵的同时能够合成外源抗菌肽Nisin。将构建的酿酒酵母菌株应用于面包、糕点、馒头等发酵食品的生产当中,则酿酒酵母在发酵的同时可以合成活性抑菌肽Nisin,在后期高温加热加工过程中,酿酒酵母细胞破裂,释放Nisin抑制面包、糕点、馒头等发酵食品中腐败菌的生长。本发明应用不需要将Nisin分离纯化出来,直接在面包、糕点、馒头等发酵类食品制作的过程中使用可以合成活性Nisin的酿酒酵母菌株发酵,Nisin合成产量足够发挥食品防腐剂的作用,并且纯度为100%,不需要额外添加商业化的Nisin,同时可减少此类食品中化学类防腐剂的使用,延长食品保质期。本发明对面包、糕点、馒头等发酵食品的防腐可做到“一步到位”,不仅能满足现代人们对食品健康的要求,做到食品绿色、安全、无添加,同时也能大大降低食品中添加化学防腐剂带来的成本问题和安全隐患,保障了发酵食品面包、糕点、馒头等的食品安全,简化了食品防腐步骤,将会在很大程度上降低天然防腐剂Nisin的使用成本,扩展了天然防腐剂Nisin的应用方式。可以在促进食品防腐剂技术进步的同时,带来巨大的经济效益。
以上所述仅为本发明的优选实施例,并非因此限制本发明的专利范围,凡是在本发明的发明构思下,利用本发明说明书及附图内容所作的等效结构变换,或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。
Claims (7)
1.一种酵母细胞基因改造方法,其特征在于,所述酵母细胞基因改造方法的步骤包括:
将合成乳酸链球菌素Nisin的前体肽结构基因nisA和水解酶基因nisP整合至亮氨酸缺陷型载体质粒pESC-Leu,构建①号质粒pESC-Leu-His6-nisA-nisP;
将合成乳酸链球菌素Nisin的基因簇中的脱水酶基因nisB,环化酶基因nisC整合至尿嘧啶缺陷型载体质粒pESC-Ura,构建②号质粒pESC-Ura-nisB-Flag-nisC-Myc;
将表达谷氨酰-tRNA的基因tRNAGlu和GltX整合至组氨酸缺陷型载体质粒pESC-His,构建③号质粒pESC-His-pSNR52tRNAGlu-GltX;
将①号、②号和③号质粒转化至所述酵母细胞。
2.一种重组酵母,其特征在于,所述重组酵母由如权利要求1所述的酵母细胞基因改造方法得到,所述重组酵母可胞内表达乳酸链球菌素前体肽NisA、水解酶NisP、脱水酶NisB、环化酶NisC以及谷氨酰-tRNA。
3.一种乳酸链球菌素Nisin的制备方法,其特征在于,所述乳酸链球菌素的制备方法的步骤包括:
S1、将权利要求2所述的重组酵母单克隆转化子在含1%-3%葡萄糖的亮氨酸、尿嘧啶和组氨酸缺陷型培养基中扩大培养,收集菌体;
S2、将收集得到的菌体置换至含1%-3%半乳糖的新鲜亮氨酸、尿嘧啶和组氨酸缺陷型培养基中诱导表达,离心收集菌体;
S3、将制备得到的菌体沉淀重悬后,破碎取上清液,从上清液中提取制备得到乳酸链球菌素Nisin。
4.如权利要求3所述的乳酸链球菌素Nisin的制备方法,其特征在于,步骤S1的培养条件为30℃,摇床转速200rpm,培养24小时,测得培养菌液在600nm波长处的吸光值为2-3,收集菌体。
5.如权利要求4所述的乳酸链球菌素Nisin的制备方法,其特征在于,步骤S2中培养基的体积以调节OD600值到0.1为准,培养条件为30℃,摇床转速200rpm,诱导表达4-5天,测得最终菌液在600nm波长处的吸光值为5-6,离心收集菌体。
6.如权利要求5所述的乳酸链球菌素Nisin的制备方法,其特征在于,步骤S3具体包括:将制备得到的菌体沉淀用20mL dd H2O重悬清洗3次,离心除去上清,加入2mL 50mM的HEPES缓冲液(pH=7.5)重悬;分装到5个2mL EP管中,0.7mL/管,加入700μL 0.5mm的玻璃珠,用高压均质机69Hz破碎3min×4次,15000g离心15min,收集离心后上清液。
7.一种重组酵母在发酵食品中的应用,其特征在于,所述应用包括如权利要求2所述的重组酵母在发酵食品中的应用。
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