CN112029673A - 一种发酵乳杆菌cicc 6278及其在新疆特色辣椒发酵中的应用 - Google Patents
一种发酵乳杆菌cicc 6278及其在新疆特色辣椒发酵中的应用 Download PDFInfo
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Abstract
本发明涉及食品微生物领域，特别涉及到一种高效快速降解亚硝酸盐，低产生物胺且具有抑制致病微生物的发酵乳杆菌(Lactobacillus fermentum)CICC 6278及其在新疆特色辣椒发酵中的应用。该菌株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 17811。本发明所述的菌株够抑制大肠埃希氏菌、金黄色葡萄球菌、单增李斯特菌和沙门氏菌生长；氨基酸脱羧酶为阴性；在改良MRS液体培养基中培养24h，亚硝酸盐降解率能够达到100％。采用微生物强化技术将该菌株应用于新疆特色辣椒发酵，亚硝酸盐的含量0.53mg/kg，生物胺的含量为2.81mg/kg，均低于对照组，发酵乳杆菌(L.fermentum)CICC 6278的强化提高了发酵辣椒的食用安全性。
Description
技术领域
本发明涉及食品微生物领域，特别涉及到一种具有快速降解亚硝酸盐，低产生物胺且具有抑制致病微生物的发酵乳杆菌CICC 6278及其在新疆特色辣椒发酵中的应用。
背景技术
乳酸菌是指革兰氏染色呈阳性、无运动性或很少有运动性、厌氧或兼性厌氧、以乳酸作为发酵代谢中主要或唯一产物等一类微生物的总称。乳酸菌广泛分布于自然界中，包括乳杆菌属，乳球菌属，链球菌属，片球菌属等属的菌株。大部分乳酸菌都具有益生功能，在发酵过程中能够产生多种有机酸，具有耐酸和耐盐特性，亚硝酸盐降解能力，抑制致病微生物特性以及低产生物胺等特性，广泛应用于果蔬发酵生产中。
果蔬发酵是利用有益微生物，控制生产条件对果蔬进行加工的一种方式，能够最大程度的保持蔬菜的营养和色香味，又具有良好的感官品质，深受消费者喜爱。果菜发酵体系实质上是多种微生物相互作用的微生态环境，其中乳酸菌发挥着至关重要的作用。本发明筛选得到的发酵乳杆菌CICC 6278，够抑制大肠埃希氏菌、金黄色葡萄球菌、单增李斯特菌和沙门氏菌等致病微生物的生长，能够快速降解亚硝酸盐，且具有低产生物胺的特性，通过微生物强化技术将该菌种应用于新疆特色辣椒发酵生产中，能够抑制发酵辣椒中致病微生物的生长，降低产品中亚硝酸盐和生物胺的含量，提高发酵辣椒的食用安全性。
发明内容
本发明的目的是提供一株发酵乳杆菌CICC 6278，其特征在于抑制致病微生物生长，快速高效的降解亚硝酸盐，低产生物胺。
本发明的另一个目的是提供一种接种上述发酵乳杆菌CICC 6278的亚硝酸盐和生物胺含量低的新疆特色发酵辣椒。
本发明在于克服现有技术的不足，通过接种能够抑制致病微生物生长，快速高效的降解亚硝酸盐，低产生物胺的发酵乳杆菌CICC 6278，即可生产出亚硝酸盐和生物胺含量低的新疆特色发酵辣椒。
本发明的具体技术方案如下：
发酵乳杆菌CICC 6278为实验室从新疆特色辣椒中分离得到的乳酸菌，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 17811，保藏日期为2019年5月15日，保藏单位地址为北京市朝阳区北辰西路1号院3号，保藏分类命名为发酵乳杆菌Lactobacillus fermentum。该菌株菌落乳白色，圆形，表面光滑，不透明，边缘整齐。菌体呈短杆状，0.3-0.4μm×1.1-2.6μm，单个或成对排列，革兰氏染色呈阳性，过氧化氢酶阴性。
发酵乳杆菌CICC 6278具有快速亚硝酸盐降解能力，在改良MRS液体培养基中培养24h，亚硝酸盐降解率能够达到100％，亚硝酸盐降解效率和速率均优于以往报道的菌株。发酵乳杆菌CICC 6278具有较强的抑制致病微生物的能力，能够抑制大肠埃希氏菌、金黄色葡萄球菌、单增李斯特菌和沙门氏菌四种常见致病菌的生长。发酵乳杆菌CICC 6278氨基酸脱羧酶为阴性，表明该菌株在生长代谢过程中产生生物胺的能力较弱。
将发酵乳杆菌CICC 6278应用于新疆特色辣椒的发酵生产过程中，所得到的发酵辣椒亚硝酸盐的含量0.53mg/kg,生物胺的含量为2.81mg/kg，降低了发酵辣椒中亚硝酸盐和生物胺，在一定程度上解决了亚硝酸盐和生物胺带来的食品安全问题。此外，接种该菌株的发酵辣椒的总酸含量达到2.84％。
附图说明
图1示发明所述发酵乳杆菌CICC 6278的扫描电镜形态
图2示发明所述发酵乳杆菌CICC 6278的16S rRNA基因系统发育树
图3示发明所述发酵乳杆菌CICC 6278亚硝酸盐降解特性
具体实施方案
下面结合实施例对本发明进一步说明，下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。
实施例1菌株CICC 6278的鉴定
本发明中的菌株CICC 6278是从新疆传统发酵辣椒中分离筛选得到，利用多相鉴定学技术，从表型和基因型水平将该菌种鉴定为发酵乳杆菌。
1、形态学鉴定
参照《常见细菌系统鉴定手册》对菌株CICC 6278进行形态特征进行鉴定，并对其进行扫描电镜观察(图1)。MRS培养基，37℃培养24h，菌株菌落乳白色，圆形，表面光滑，不透明，边缘整齐。菌体呈短杆状，0.3-0.4μm×1.1-2.6μm，单个或成对排列，革兰氏染色呈阳性，过氧化氢酶阴性。
2、生理生化特征鉴定
使用API 50CHL鉴定系统进行菌株CICC 6278的碳源利用产酸特性，具体鉴定结果见表1。
表1副干酪乳杆菌CICC 6277生理生化特征鉴定结果
3、16S rRNA基因的PCR扩增
使用细菌基因组DNA提取试剂盒提取乳酸菌的基因组。利用引27f(5’-AGAGTTTGATCCTGGCTCAG-3’)和1492r(5’-GGTTACCTTGTTACGACTT-3’)对16S rRNA基因进行扩增。PCR反应体系为：模板10×PCR buffer 5μL、dNTP(2.5mmol/L)4μL、模板2μL、 Taq DNA聚合酶1μL、引物各1μL,补充去离子水至50μL。反应条件:94℃5min，94℃ 50s，52℃50s，72℃50s，33个循环，72℃7min。
4、序列分析
将所得的16S rRNA基因序列用BLAST软件与NCBI数据库进行相似性比对，运用MEGA 5.0软件采用Neighbor Joining方法构建系统发育树。菌株CICC 6278与发酵乳杆菌处于同一个分支(图2)，16S rRNA基因序列相似度为99.86％，且与其他菌株的相似度均在98.65％以下，再结合表型特征，鉴定为发酵乳杆菌。其16S rRNA基因序列登录号为MN314273。
实施例2发酵乳杆菌CICC 6278的特性研究
1、亚硝酸盐降解能力
按照GB 5009.33-2010：《食品中亚硝酸盐与硝酸盐的测定》测定发酵乳杆菌CICC6278亚硝酸盐降解能力。1％接种量接种到含0.125mg/mL NaNO2的MRS培养基中，37℃培养24h，亚硝酸盐降解率能够达到100％(图3)。
2、氨基酸脱羧酶试验
微生物代谢过程中，在氨基酸脱羧酶作用下，生成生物胺类物质。筛选氨基酸脱羧酶为阴性的菌株作为发酵剂，会降低果蔬发酵生产中生物胺的含量。氨基酸脱羧酶试验结果表明，发酵乳杆菌CICC 6278色氨酸脱羧酶、赖氨酸脱羧酶和鸟氨酸脱羧酶活性均为阴性。
3、抑菌能力
利用滤纸片法测定发酵乳杆菌CICC 6278对肠炎沙门氏菌、大肠杆菌、金黄色葡萄球菌和单增李斯特菌的抑制程度。发酵乳杆菌CICC 6278过夜培养物对肠炎沙门氏菌的抑菌直径达到24.2mm，对大肠杆菌的抑菌直径达到16.3mm，对金黄色葡萄球菌的抑菌直径达到16.4mm，对单增李斯特菌抑菌直径达到12.2mm。
实施例3发酵乳杆菌CICC 6278在新疆特色辣椒发酵中的应用
选择新鲜的新疆辣椒，清洗，破碎后，加入3％蔗糖和3％盐，接种1％发酵乳杆菌CICC 6278(108CFU/mL)，30℃恒温发酵7天，即得到发酵辣椒。将未接种乳酸菌的辣椒为对比试验组。按照GB 5009.33-2010：《食品中亚硝酸盐与硝酸盐的测定》，测定发酵辣椒的亚硝酸盐含量。按照GB/T 5009.208-2008：《食品中生物胺含量的测定》，使用HPLC法对接种发酵乳杆菌CICC 6278的发酵辣椒和自然发酵辣椒中生物胺的种类和含量进行测定。按照GB/T 12456-2008：《食品中总酸的测定》中酸碱滴定法测定发酵辣椒的总酸含量。通过构建有机酸分析模型，使用HPLC法对接种发酵乳杆菌CICC 6278的发酵辣椒和自然发酵辣椒中的有机酸种类和含量进行测定。具体测定结果见表2。
表2发酵辣椒测定指标
发酵乳杆菌CICC 6278用于新疆特色辣椒发酵，所得到的发酵辣椒亚硝酸盐的含量 0.53mg/kg,低于对照组亚硝酸盐含量(2.63mg/kg)，远远低于国家标准20mg/kg。接种菌株CICC 6278的发酵辣椒中生物胺主要为腐胺，其含量为2.81mg/kg，与对照组相比，生物胺含量下降了85.5％。因此接种发酵乳杆菌CICC 6278，能够显著降低了发酵蔬菜中残留亚的硝酸盐和生物胺含量，解决了亚硝酸盐和生物胺带来的食品安全问题。此外，接种发酵乳杆菌CICC 6278的发酵辣椒总酸含量2.84％，高于自然发酵辣椒，并且乳酸、琥珀酸和乙酸的含量菌明显增加，这表明发酵乳杆菌CICC 6278的强化提升了发酵辣椒的品质。
Claims (3)
1.一种发酵乳杆菌 (Lactobacillus fermentum) CICC 6278，该菌株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 17811。
2.权利要求1所述的发酵乳杆菌 (Lactobacillus fermentum) CICC 6278在辣椒发酵生产中的应用。
3.权利要求2所述的应用，其中所述辣椒为新疆特色辣椒。
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