CN114134085B - 一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌及其应用 - Google Patents
一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域，尤其涉及一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌及其应用。本发明所提供的普罗旺斯类芽孢杆菌PP‑2，分类学名称为普罗旺斯类芽孢杆菌PP‑2(Paenibacillus provencensis PP‑2)，保藏编号为CCTCC NO：M20211109，现保藏于中国典型培养物保藏中心。地址：中国武汉市武汉大学。本发明分离得到一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌，可在与食品无任何直接接触的前提下可以抑制食品中亚硝胺生成，可将其制作成保鲜剂，提升熟食贮藏品质，显著抑制有害物质亚硝胺的生成，延长保质期。
Description
技术领域
本发明涉及微生物技术领域，尤其涉及一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌及其应用。
背景技术
N-亚硝胺类化合物是一种具有很强毒性的化学物质，也是最重要的化学致癌物之一，长期低剂量接触可以引起癌症。加工肉制品中的N-亚硝胺是亚硝化剂与仲胺的反应产物，亚硝化剂主要来源于添加到肉制品中的亚硝酸盐的还原产物，仲胺来源于加工过程中产生的包含仲胺基团的生物胺、蛋白质降解和蛋白质与脂质氧化形成的羰基产物。此外，N-亚硝胺的形成还受pH值、加工时间和环境温度等因素影响，而肉制品所经过的发酵、烟熏、干制和热处理等加工过程，是形成N-亚硝胺的最佳条件。然而，由于亚硝酸盐对肉制品至关重要的作用，如增强成品肉制品的颜色稳定性、促进有益微生物生长并抑制有害微生物生长繁殖，它成为肉制品加工过程中频繁使用的添加剂。因此，N-亚硝胺的产生不可避免。当前，随着食品行业的快速发展，政府和人民愈加重视食品安全，抑制食品中尤其是肉制品中亚硝胺的生成刻不容缓。
专利CN112840030A通过在烟草植物或其植物部分或植物细胞中表达去调控的阳离子外排蛋白，以减少烟草中的至少一种烟草特有亚硝胺(TSNA)或 TSNA前体的含量，但该专利针对性较强，且在实际生产中实现难度较大。专利 CN111972696A发明了一种降低烟草贮藏过程中亚硝胺的方法，具体步骤是先将烟梗和叶片喷施Na2CO3-NaHCO3缓冲液，再在此基础上喷施海藻酸溶液后进行真空处理来抑制亚硝胺的生成，显著降低吸烟危害。专利CN108077934B根据降解亚硝胺能力强、耐酸和耐胆盐的要求，将筛选得到的戊糖乳杆菌和短乳杆菌分别进行冷冻干燥、均匀粉碎后复配，后与果葡糖浆、大豆油均匀混合喷雾干燥，研发了一种泡菜乳酸菌胶囊，人服用后可降低体内亚硝胺的含量，但该专利并未进行毒理学实验，对人体安全存在威胁。专利CN104824119B将栗单宁提取物制成溶液喷洒在人类和动物的食物原料(例如油、油菜籽、谷类)或食物产品中，以抑制这些材料的氧化降解现象，同时降低微生物污染和亚硝胺和真菌毒素的形成，而且当使用合适的粟单宁浓度，还与其他多酚混合时，没有改变食物原料原本的味道和香味。但该专利中所用多酚包括绿茶提取物，朝鲜蓟提取物，橄榄树叶，橄榄酱提取物，葡萄籽提取物，迷迭香提取物，甜菜根提取物，产业化成本过高。
专利CN109679868A通过体外发酵培养乳酸菌的方式，分别在对亚硝酸盐的降解和抑制N-二甲基亚硝胺形成两个方面进行逐步筛选，得到PRO-MIX5乳酸菌发酵剂，提取其中有效起作用的靶向物质，制成微生物亚硝化抑制剂添加入产品中，抑制了N-亚硝胺形成。专利CN109329884 A将抗坏血酸钠、葡萄籽提取物、烟酰胺和辅酶Q10混合制备了一种N-亚硝胺抑制剂，添加至食品原料中，在后续加工、储藏过程中抑制亚硝胺生成。以上两篇专利CN109679868A、专利CN109329884 A等现有专利主要集中在加工前或加工过程中对食品原料亚硝胺生成的抑制，以及均需要与食品原料直接接触且进入人体，对食品原有风味、口感产生不良影响。
因此，提供一种在与食品无任何直接接触的前提下可以抑制食品中亚硝胺生成的菌株，对于抑制食品中尤其是肉制品中亚硝胺的生成具有重要的应用价值。
发明内容
本发明的目的在于克服现有技术的不足，提供一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌及其应用。本发明为解决食品冷藏过程中生成亚硝胺对人体造成危害的问题，从鱼子酱中分离得到一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌。
为实现上述目的，本发明采取的技术方案为：提供一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌(Paenibacillus provencensis)PP-2，其保藏编号为CCTCC NO：M20211109。
本发明同时提供所述普罗旺斯类芽孢杆菌的菌株发酵液冻干粉。
作为本发明所述菌株发酵液冻干粉的优选实施方式，所述菌株发酵液冻干粉是将所述普罗旺斯类芽孢杆菌接种于LB液体培养基中，37℃，140-160rpm，培养48-72h成为液体菌剂，冻干得到菌株发酵液冻干粉。
本发明同时提供所述的普罗旺斯类芽孢杆菌或所述的菌株发酵液冻干粉在抑制食品中亚硝胺的生成中的应用。
作为本发明所述应用的优选实施方式，所述食品是肉制品。
作为本发明所述应用的优选实施方式，所述应用为将普罗旺斯类芽孢杆菌或其菌株发酵液冻干粉，与食品一同放置密闭容器中，以抑制食品中亚硝胺的生成。
作为本发明所述应用的优选实施方式，所述放置是在26～30℃下放置2～4 天。
本发明还提供所述的普罗旺斯类芽孢杆菌或所述的菌株发酵液冻干粉在制备食品保鲜剂中的应用。
作为本发明所述应用的优选实施方式，所述食品保鲜剂为冰箱保鲜盒中的食品保鲜剂。
本发明的有益效果：
本发明分离得到一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌，可在与食品无任何直接接触的前提下可以抑制食品中亚硝胺生成，可将其制作成保鲜剂，提升熟食贮藏品质，显著抑制有害物质亚硝胺的生成，延长保质期。
生物材料保藏
本发明所提供的普罗旺斯类芽孢杆菌PP-2，分类学名称为普罗旺斯类芽孢杆菌PP-2(Paenibacillus provencensis PP-2)，保藏编号为CCTCC NO： M20211109，现保藏于中国典型培养物保藏中心，地址：中国武汉市武汉大学，保藏日期2021年09月01日。
附图说明
图1为基于16S rDNA序列的菌株PP-2的系统发育树。
图2为实验中各组午餐肉罐头、卤牛肉中N-二甲基亚硝胺含量；其中，空白组即为对照组。
具体实施方式
为更清楚地表述本发明的技术方案，下面结合具体实施例进一步说明，但不能用于限制本发明，此仅是本发明的部分实施例。
实施例1：菌株的分离与鉴定
1)采样：从浙江省衢州市鲟龙科技股份有限公司取得储存时间为6个月的鱼子酱；
2)取3g鱼卵加入6mL无菌生理盐水得到菌液，在37℃下孵育2h；
3)取上述菌液100μL，加入900μL无菌生理盐水将菌液稀释至10％，按上述操作依次将菌液梯度稀释至106倍，采用涂布平板法各取100μL菌液加入LB 营养琼脂培养基中，在37℃培养24h；
4)将样品送至上海美吉生物医药科技有限公司进行高通量测序鉴定出6株菌株。
将其中的1株菌株进行菌种鉴定，提取菌株基因组，对其16S rDNA序列 PCR并测序，经过Blast比对鉴定其种属，结果显示，菌株为普罗旺斯类芽孢杆菌(Paenibacillusprovencensis)，命名为普罗旺斯类芽孢杆菌(Paenibacillus provencensis)PP-2，并在中国典型培养物保藏中心进行保藏，保藏编号为CCTCC NO：M20211109。
实施例2：菌株抑制亚硝胺生成的应用
实验对象为添加0.5％亚硝酸钠的午餐肉罐头，采用普罗旺斯类芽孢杆菌 PP-2菌株发酵液冻干粉进行实验。普罗旺斯类芽孢杆菌PP-2菌株发酵液冻干粉的制备方法为：将普罗旺斯类芽孢杆菌PP-2，接种于LB液体培养基中，37℃， 140-160rpm，培养48-72h成为液体菌剂，冻干得到菌株发酵液冻干粉。
各组添加0.5％亚硝酸钠的午餐肉罐头、卤牛肉均为100g，对照组无其它添加；实验组1添加1g普罗旺斯类芽孢杆菌PP-2菌粉发酵液冻干粉，菌粉均置于平皿中，分别与午餐肉罐头、卤牛肉放于密闭保鲜盒中，在28℃下放置3天。随后按照GB 5009.26-2016《食品安全国家标准食品中N-亚硝胺类化合物的测定》进行午餐肉罐头、卤牛肉中亚硝胺类物质测定，测定结果见图2。结果表明，午餐肉罐头和卤牛肉28℃贮藏3天后，实验组中N-二甲基亚硝胺(NDMA)含量明显低于对照组，说明PP-2发酵液冻干粉能够在贮藏过程中抑制N-亚硝胺类化合物的生成。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (8)
1.一株可抑制亚硝胺生成的普罗旺斯类芽孢杆菌（Paenibacillus provencensis）PP-2，其保藏编号为CCTCC NO：M20211109。
2.权利要求1所述普罗旺斯类芽孢杆菌的菌株发酵液冻干粉。
3.根据权利要求2所述的菌株发酵液冻干粉，其特征在于，所述菌株发酵液冻干粉是将所述普罗旺斯类芽孢杆菌接种于LB液体培养基中，37℃，140-160rpm，培养48-72h成为液体菌剂，冻干得到菌株发酵液冻干粉。
4.权利要求1所述的普罗旺斯类芽孢杆菌或权利要求2-3任一所述的菌株发酵液冻干粉在抑制肉制品中亚硝胺的生成中的应用。
5.根据权利要求4所述的应用，其特征在于，所述应用为将普罗旺斯类芽孢杆菌或其菌株发酵液冻干粉，与肉制品一同放置密闭容器中，以抑制肉制品中亚硝胺的生成。
6.根据权利要求5所述的应用，其特征在于，所述放置是在26~30℃下放置2~4天。
7.权利要求1所述的普罗旺斯类芽孢杆菌或权利要求2-3任一所述的菌株发酵液冻干粉在制备肉制品保鲜剂中的应用。
8.权利要求7所述的应用，其特征在于，所述肉制品保鲜剂为冰箱保鲜盒中的肉制品保鲜剂。
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