CN114703075A - 一株抑制发酵肉制品中n-亚硝胺类物质生成的酵母及其应用 - Google Patents
一株抑制发酵肉制品中n-亚硝胺类物质生成的酵母及其应用 Download PDFInfo
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Abstract
本发明属于生物工程领域,涉及一株酵母,具体涉及一株抑制发酵肉制品中N‑亚硝胺类物质生成的酵母及其应用。其分类名为Meyerozyma guilliermondii J1‑2,保藏编号为CCTCC NO:M 2022405,保藏单位为中国典型培养物保藏中心,保藏时间为2022年4月11日,保藏地址为中国.武汉.武汉大学。该菌株可以将N‑亚硝胺的前体物质亚硝酸盐进行降解,降解速率最高为15%,并进一步抑制N‑二乙基亚硝胺等N‑亚硝胺类物质的生成。将其应用于发酵肉制品中,对发酵肉制品中N‑亚硝胺类物质含量的抑制率为50%,具有改善肉制品口感和风味、增强肉制品品质以及提升肉制品安全性的作用。
Description
技术领域
本发明属于生物工程领域,涉及一株酵母,具体涉及一株抑制发酵肉制品中N-亚硝胺类物质生成的酵母及其应用。
背景技术
N-亚硝胺(N-nitrosamines,NAs)是一类亚硝基化合物的总称,通常由二级胺的亚硝化反应生成。食品中常见的亚硝胺有N-亚硝基二甲胺(N-Nitrosodimethylamine,NDMA)、N-亚硝基甲乙胺(N-nitrosomethylethylamine,NMEA)、N-亚硝基二乙胺(N-nitrosodiethylaminen,NDEA)、N-亚硝基吡咯烷(N-Nitrosopyrrolidine,NPYR)、N-亚硝基吗啉(N-nitrosomorpholine,NMOR)、N-亚硝基二丙胺(N-Nitrosodi-n-propylanime,NDPA)、N-亚硝基哌啶(N-Nitrosopiperidine,NPIP)、N-亚硝基二丁胺(N-Nitrosodibutylamine,NDBA)等。其中,国际癌症研究机构把NDMA和NDEA列为2A致癌物,超过10种亚硝胺被列为2B致癌物。
在肉制品加工中,亚硝酸盐最大使用量是0.15g/kg,常常用作发色剂来保障产品的外观品质,也具有良好的抑菌作用,同时可以增强肉制中脂肪的稳定性,抑制脂质氧化,延长货架期,使肉制品的风味和品质不受影响。同时,亚硝酸盐与肌肉蛋白质分解出的胺类物质的反应,生成亚硝基类化合物。因此,亚硝酸盐是亚硝胺类物质的前体物质,减少亚硝胺类物质的前提物质是控制肉制品中亚硝胺类物质含量的主要策略。
发酵肉制品是指在自然或人工控制条件下,利用微生物或酶的发酵作用,使原料肉发生一系列生物化学变化及物理变化,而形成具有特殊风味、色泽和质地以及较长保藏期的肉制品。常见的发酵菌株有乳酸菌、酵母菌、葡萄球菌和霉菌等,但是能够抑制发酵肉制品中N-亚硝胺类物质的酵母类发酵菌株还未见报道。
发明内容
针对上述技术问题,本发明提出了一株抑制发酵肉制品中N-亚硝胺类物质生成的酵母及其应用,解决了发酵肉制品加工中,以亚硝酸盐为前体物质生成NDEA等亚硝胺类物质的高风险性问题。
为了达到上述目的,本发明的技术方案是这样实现的:
一株抑制发酵肉制品中N-亚硝胺类物质生成的酵母,所述的酵母分类名为Meyerozyma guilliermondii J1-2(季也蒙毕赤酵母J1-2);保藏号为CCTCC NO: M2022405;保藏单位为中国典型培养物保藏中心;保藏时间为2022年4月11日,保藏地址为中国.武汉.武汉大学。
进一步,所述的酵母在降解N-亚硝胺前体物质亚硝酸盐中的应用。
进一步,所述的酵母在抑制发酵肉制品中N-二乙基亚硝胺等亚硝胺类物质的生成的应用。
进一步,所述的应用,步骤如下:
(1)发酵剂制备:在无菌条件下将J1-2菌株接种到培养基进行活化、培养获得季也蒙毕赤酵母菌液,经离心浓缩得发酵剂;
(2)肉制品的预腌制:将新鲜牛肉去除筋腱,整理为大小均匀的块状,在无菌容器中,将炒制后的食盐、花椒、八角和香叶混匀加入肉块中,进行初步腌制;
(3)预腌制肉制品的腌制:向步骤(2)所得的预腌制肉制品中添加50-55°白酒、白砂糖、小茴香粉、姜粉、大蒜粉、白胡椒粉和亚硝酸钠辅料,继续腌制,期间翻倒至少3次,得到腌制好的肉制品;
(4)腌制肉制品的发酵:将步骤(1)制备的发酵剂按一定的接种比例接种到步骤(3)制备的腌制肉制品中,发酵;
(5)将步骤(4)所得的发酵好的肉放入烟熏炉中,进行烘制,即为成品。
进一步,所述步骤(1)中菌液制备方法为:在无菌条件下挑取单菌落接种于含50mL液体培养基的250mL摇瓶中,28℃、200rpm、培养48h,制得菌液;再在4500rpm条件下离心,将菌液浓缩为5mL,得到发酵剂。
进一步,所述步骤(2)中肉块大小为30cm*20cm*2cm,食盐、花椒、八角和香叶添加量分别为牛腩肉质量的5%,0.02%,0.01%,0.005%,腌制时间为24h,腌制温度为4℃。
进一步,所述步骤(3)中50-55°白酒添加量为牛腩肉质量的1%、白砂糖添加量为牛腩肉质量的2%、小茴香粉添加量为牛腩肉质量的0.2%、姜粉添加量为牛腩肉质量的0.05%、大蒜粉添加量为牛腩肉质量的0.2%、白胡椒粉添加量为牛腩肉质量的0.05%、亚硝酸钠添加量为牛腩肉质量的0.05%。
进一步,所述步骤(3)中翻倒为每隔12h翻倒一次,腌制温度为4℃。
进一步,所述步骤(4)中发酵剂的接种比例为106 CFU/g,发酵温度为30℃,发酵时间为48h。
进一步,所述步骤(5)中烘制温度为40℃,烘制时间为48h。
本发明具有以下有益效果:
1、本发明的季也蒙毕赤酵母J1-2通过降低NDEA的前体物质亚硝酸盐,降解速率最高达15%,抑制发酵肉制品中的NDEA,等亚硝胺类物质的生成,抑制率为50%。
2、本发明基于对发酵过程的分析以及相关实验,证明发酵肉制品中NDEA等亚硝胺含量的减少是微生物的作用,微生物代谢降解了N-亚硝胺的前体物质亚硝酸盐。
3、本发明提出一株抑制发酵肉制品中N-亚硝胺类物质生成的酵母及其应用,解决了发酵肉制品加工中,以亚硝酸盐为前体物质生成NDEA等亚硝胺类物质的高风险性问题。本发明的季也蒙毕赤酵母J1-2菌株从地方特色腊肉中筛选出来,发酵产物食品安全性高,能够直接用于发酵肉制品的生产。发酵肉制品营养丰富,风味、色泽和质地独特,保质期长等优点。
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获得其他的附图。
图1为本发明季也蒙毕赤酵母J1-2菌株对亚硝酸盐的降解速率。
图2为本发明季也蒙毕赤酵母J1-2菌株发酵肉制品中NDEA的含量。
图3为本发明季也蒙毕赤酵母J1-2菌株发酵肉制品中N-亚硝胺类物质的色谱图。
图4为本发明季也蒙毕赤酵母J1-2菌株美兰染色显微镜检图。
图5为本发明季也蒙毕赤酵母J1-2菌株的16S rDNA基因系统发育树图谱。
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付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。
本发明所用孟加拉红固体培养基的配方为:蛋白胨0.5%,葡萄糖1%,KH2PO4 0.1%,MgSO4 0.05%,琼脂2%,孟加拉红0.0033%,氯霉素0.01%,121℃灭菌15min。
本发明所用液体培养基的配方为:蛋白胨0.5%,葡萄糖1%,KH2PO4 0.1%,MgSO40.05%,孟加拉红0.0033%,氯霉素 0.01%,121℃灭菌15min。
实施例1:季也蒙毕赤酵母J1-2的分离、筛选和鉴定
(1.1)采样:本发明涉及菌株从河南信阳地方特色手工腊肉中筛选而来。
(1.2)分离和筛选:在无菌条件下,称取25g样品,剪碎,加入225mL无菌生理盐水,放入无菌均质袋中,用均质器进行均质。用无菌生理盐水进行10倍梯度稀释涂布平板,在28℃条件下倒置培养48h。挑取平板上不同形态的菌株,观察菌落形态,将获得的单菌落进行平板划线处理,进行多次纯化,用作初筛菌株。
挑取生长较好、性状不同初筛菌株接种于孟加拉红固体培养基上,28℃条件下倒置培养48h,进行复筛。选择有30-150菌落的平板,选择菌落为单一的,大小不一的,光泽度不一的菌落,进行划线纯化培养;用美兰染色法进行镜检,直至镜检下为纯种的菌株。
(2)鉴定:将纯化好的单菌落进行美兰染色处理,观察菌落形态,如图4所示。采用16S rDNA通用PCR引物ITS1(5’-TCCGTAGGTGAACCTGCGG-3’)和ITS4(5’-TCCTCCGCTTATTGATATGC-3’)作为上下游引物进行PCR扩增反应;进行扩增,扩增条件见表1,PCR产物4℃保藏。由北京六合华大基因科技有限公司(武汉分公司)完成测序(结果见表2),采用16S rDNA测序方法对菌株进行分子学生物鉴定。经BLAST比对,用NeighborJoining构建目标菌株的系统发育树,结果如图5所示。菌株J1-2与多株季也蒙毕赤酵母(Meyerozyma guilliermondii)同源性达到99%,与模式菌株季也蒙毕赤酵母(Meyerozyma guilliermondii)OK267918.1处于同一分支,可鉴定菌株J1-2为季也蒙毕赤酵母(Meyerozyma guilliermondii)。
表1 PCR反应体系和反应条件
表2 菌株测序结果
实施例2:季也蒙毕赤酵母J1-2发酵肉制品
(1)菌株初筛:利用地方特色手工腊肉,结合梯度稀释平板法,进行菌株初筛;
(2)菌株复筛:在固体培养基上对步骤(1)所得的菌株进行复筛;
(3)纯化培养:对单菌株进行纯化培养,结合美兰染色法进行镜检,并进行鉴定;
(4)发酵剂制备:在无菌条件下挑取单菌落接种于含50mL液体培养基的250mL摇瓶中,28℃、200rpm、培养48h,制得菌液;再在4500rpm条件下离心,将菌液浓缩为5mL,得到发酵剂;
(5)在液体培养基中,进行降解亚硝酸盐速率的测定,测定方法为:按照1%的接种量,将菌液接种于含有50mg/kg的NaNO2液体培养基中,28℃培养48h,每隔8h测定一次亚硝酸盐含量。图1为本实施例季也蒙毕赤酵母J1-2菌株对亚硝酸盐的降解速率,如图1所示,季也蒙毕赤酵母J1-2降解亚硝酸盐速率高达15%;
(6)肉制品的腌制、发酵和烘制:先将新鲜牛腩肉去除筋腱,整理为30cm*20cm*2cm大小均匀的块状,备用,然后,在无菌容器中将炒制后的5%牛腩肉质量的食盐、0.02%牛腩肉质量的花椒、0.01%牛腩肉质量的八角、0.005%牛腩肉质量的香叶混匀加入肉块中,在4℃条件下,进行初步腌制24h;之后,添加1%牛腩肉质量的50-55°白酒、2%牛腩肉质量的白砂糖、0.2%牛腩肉质量的小茴香粉、0.05%牛腩肉质量的姜粉、0.2%牛腩肉质量的大蒜粉、0.05%牛腩肉质量的白胡椒粉、0.05%牛腩肉质量的亚硝酸钠,每隔12h翻倒一次,进行4次翻倒后,按照106 CFU/g的接种比例将发酵剂接种到肉块中,在30℃条件下发酵48h;最后,将发酵好的肉放入烟熏炉中,在40℃条件下烘制48h,即为成品。
实施例3:季也蒙毕赤酵母J1-2抑制发酵肉制品中NDEA等亚硝胺类物质的效果
取上述样品10g样品与15mL乙腈加入50mL冷冻离心管中,充分匀浆后置于-20℃冰箱中静置30min。随后向离心管中分别加入4g硫酸镁、1g NaCl和一个磁力搅拌子。旋涡振荡3min后,混合物在3800g,4℃条件下离心10min。取6mL上清置于10mL离心管中,分别加入50mg PSA、150mg C18E和900mg无水硫酸钠。旋涡振荡1min后,于3800g,4℃条件下离心10min。取5mL上清在室温条件下氮吹浓缩至1mL体积。浓缩液过0.22µm滤膜后用于GC-MS/MS分析。如图3所示,NDMA、NMEA、NDEA、NDPA、NDBA、NPIP、NPYR、NMOR的保留时间分别为9.233、10.349、11.048、15.082、19.026、19.302、19.763、20.4181min。通过上述流程,最终获得一株抑制发酵肉制品中N-亚硝胺类物质生成的季也蒙毕赤酵母J1-2并进行应用。在上述条件下,发酵肉制品中N-亚硝胺类物质含量见如图2所示,发酵肉制品中NDEA含量为24.70μg/kg,空白的发酵肉制品中NDEA含量为49.00μg/kg,其他亚硝胺类物质未检出,季也蒙毕赤酵母对发酵肉制品中NDEA等亚硝胺的抑制率为50%。
气相色谱-质谱(GC-MS/MS)条件:GC条件如下:进样量为10µL;色谱柱为DB-WAX(60m×0.25mm×0.25µm);进样口温度为250℃;升温程序:初始40℃,保持3min,首先以10℃/min从40℃升至110℃,随后以15℃/min从110℃升至200℃,最后以40℃/min从200℃升至240℃。载气为高纯度氦气(99.999%),流速为25mL/min;质谱连接线路温度为 250℃。
MS/MS条件如下:扫描模式为多反应检测扫描(MRM);离子源为EI源,电子能量为70eV;离子源温度为230℃。
对比例:空白
本对比例空白的制备方法,步骤如下:
先将新鲜牛腩肉去除筋腱,整理为30cm*20cm*2cm大小均匀的块状,备用。然后,在无菌容器中将炒制后的5%牛腩肉质量的食盐、0.02%牛腩肉质量的花椒、0.01%牛腩肉质量的八角、0.005%牛腩肉质量的香叶混匀加入肉块中,在4℃条件下,进行初步腌制24h;之后,添加1%牛腩肉质量的50-55°白酒、2%牛腩肉质量的白砂糖、0.2%牛腩肉质量的小茴香粉、0.05%牛腩肉质量的姜粉、0.2%牛腩肉质量的大蒜粉、0.05%牛腩肉质量的白胡椒粉、0.05%牛腩肉质量的亚硝酸钠,每隔12h翻倒一次,进行4次翻倒后,腌制48h即可,不进行接种,在30℃条件下进行自然发酵48h;最后,将发酵好的肉放入烟熏炉中,在40℃条件下烘制48h,即为成品。
对空白样品的的N-亚硝胺化合物的含量测定方法同实施例3,结果如图2、图3所示。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1. 一株抑制发酵肉制品中N-亚硝胺类物质生成的酵母,其特征在于:所述的酵母分类名为Meyerozyma guilliermondii J1-2;保藏号为CCTCC NO: M 2022405;保藏单位为中国典型培养物保藏中心;保藏时间为2022年4月11日,保藏地址为中国.武汉.武汉大学。
2.权利要求1所述的酵母在降解N-亚硝胺前体物质亚硝酸盐中的应用。
3.权利要求1所述的酵母在抑制发酵肉制品中N-二乙基亚硝胺类物质生成的应用。
4.根据权利要求2或3所述的应用,其特征在于,所述的应用步骤如下:
(1)发酵剂制备:在无菌条件下将J1-2菌株接种到孟加拉红液体培养基进行活化、培养获得J1-2菌液,经离心浓缩得发酵剂;
(2)肉制品的预腌制:将新鲜牛肉去除筋腱,整理为大小均匀的块状,在无菌容器中,将炒制后的食盐、花椒、八角和香叶混匀加入肉块中,进行初步腌制;
(3)预腌制肉制品的腌制:向步骤(2)所得的预腌制肉制品中添加50-55°白酒、白砂糖、小茴香粉、姜粉、大蒜粉、白胡椒粉和亚硝酸钠辅料,继续腌制,期间翻倒至少3次,得到腌制好的肉制品;
(4)腌制肉制品的发酵:将步骤(1)制备的发酵剂按一定的接种比例接种到步骤(3)制备的腌制肉制品中,发酵;
(5)将步骤(4)所得的发酵好的肉放入烟熏炉中,进行烘制,即为成品。
5.根据权利要求4所述的应用,其特征在于,所述步骤(1)中菌液制备方法为:在无菌条件下挑取单菌落接种于含50mL液体培养基的250mL摇瓶中,28℃、200rpm、培养48h,制得菌液;再在4500rpm条件下离心,将菌液浓缩为5mL,得到发酵剂。
6.根据权利要求4所述的应用,其特征在于,所述步骤(2)中肉块大小为30cm*20cm*2cm,食盐、花椒、八角和香叶添加量分别为牛腩肉质量的5%,0.02%,0.01%,0.005%,腌制时间为24h,腌制温度为4℃。
7.根据权利要求4所述的应用,其特征在于:所述步骤(3)中50-55°白酒添加量为牛腩肉质量的1%、白砂糖添加量为牛腩肉质量的2%、小茴香粉添加量为牛腩肉质量的0.2%、姜粉添加量为牛腩肉质量的0.05%、大蒜粉添加量为牛腩肉质量的0.2%、白胡椒粉添加量为牛腩肉质量的0.05%、亚硝酸钠添加量为牛腩肉质量的0.05%。
8.根据权利要求4所述的应用,其特征在于:所述步骤(3)中翻倒为每隔12h翻倒一次,腌制温度为4℃。
9. 根据权利要求4所述的应用,其特征在于:所述步骤(4)中发酵剂的接种比例为106 CFU/g,发酵温度为30℃,发酵时间为48h。
10.根据权利要求4所述的应用,其特征在于:所述步骤(5)中烘制温度为40℃,烘制时间为48h。
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