具有katA基因的乳酸菌株对肉制品的发色机理研究

One of the most important roles of nitrite in food industry is to improve the pink color of cured meat products. However, the nitrite has acute and chronic toxicity and potential carcinogenicity. Exploring substitute is one of the best ways to solve the safety hazards. KatA gene of the strain was detected with PCR technology and good Lactobacillus strain with katA gene and good coloring effect was selected. The strain was identified by 16s rDNA sequence analysis. The new coloring product was purified with DEAE-Sepharose FF ion exchange chromatography and SephadexG-75 Gel chromatography.The amino acid composition, the amino acid sequence of C-terminal and N-terminal were analyzed. The molecular mass and the peptide mass fingerprinting of the coloring product were confirmed with MALDI-TOF-MS. Then the molecular and amino acid sequence were speculated the structure was determined in combination with Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, circular dichroism (CD), X-ray crystal diffraction analysis and nuclear magnetic resonance (NMR). Enzymes produced by the strain were purified and classified. The key enzyme was confirmed in combination with the structure of the coloring product. Enzymatic properties and secretion regularity of the key enzyme were studied. The reaction mechanism of enzymatic product and myoglobin were analyzed and the coloring mechanism of Lactobacillus strain in meat products was elucidated.

亚硝酸盐在食品工业中的重要作用之一是促进肉制品发色，但亚硝酸盐急慢性毒性较大，具有强致癌性；探寻亚硝酸盐替代品是解决这一安全隐患最好的方法之一。本项目拟用PCR技术进行katA检测，筛选具有katA基因的优良发色乳酸菌菌株，并对其进行16S rDNA 序列分析鉴定。利用DEAE-sepharose FF离子交换层析和sephadexG-75凝胶柱层析纯化发色产物。分析其氨基酸组成、C端和N端氨基酸序列、利用MALDI-TOF-MS测定发色产物的分子质量及肽质量指纹图谱并推测其分子式、氨基酸序列，结合傅里叶变换红外光谱(FTIR)、拉曼光谱、圆二色谱(CD)、X-射线晶体衍射分析、核磁共振（NMR）明确发色产物的结构。纯化、分类菌株发酵产生的酶，结合发色产物的结构，确定乳酸菌发色的关键酶。测定关键酶的酶学性质并研究其分泌规律；分析酶促产物与肌红蛋白的反应机制，阐明乳酸菌对肉制品的发色机理。

结题摘要.本项目主要是针对肉制品中常用的发色剂及防腐剂亚硝酸盐存在的急慢性毒性及强致癌性安全隐患，从发酵食品中筛选乳酸菌作为发色剂和防腐剂替代亚硝酸盐；并研究引起发色的关键酶，分析酶促产物与肌红蛋白的反应机制，阐明乳酸菌对肉制品的发色机理。.主要研究结果如下：.（1）本项目首先从发酵食品筛选出两株对肉制品具有发色与防腐作用的优良乳酸菌株，并对其进行16S rDNA 序列分析鉴定，最终确定这两株菌均为植物乳杆菌，其序列分别以登录号MH357373和MH357374保藏在GenBank数据库中。.（2）纯化后的发色产物利用紫外扫描、电子自旋振动光谱（ESR）、傅里叶变换红外光谱(FTIR)、拉曼光谱及等确定发色产物为亚硝基肌红蛋白。.（3）明确了产生该产物的关键酶为一氧化氮合酶、高铁肌红蛋白还原酶。.（4）阐明了乳酸菌对肉制品的发色机理：乳酸菌的发色机制主要是通过这两个关键酶的协调作用实现的。一氧化氮合酶是第一个关键酶，催化合成的一氧化氮与肉中的肌红蛋白发生反应生成亚硝基肌红蛋白，从而使肉制品呈现粉红色。高铁肌红蛋白还原酶作为第二关键酶首先还原呈红褐色的高铁肌红蛋白为肌红蛋白和氧合肌红蛋白，还原产物最终进一步生成亚硝基肌红蛋白，使肉制品恢复粉红色。.（5）本研究成果对于乳酸菌在肉制品中替代亚硝酸盐的发色及防腐作用研究提供了坚实的理论基础，所筛选的优良菌株对于保障肉制品的质量安全具有潜在应用前景。.（6）在本项目的资助下已正式发表高质量学术论文4篇，其中SCI期刊3篇，另外已录用2篇，大修1篇，申请专利1项“一株对肉制品具有发色及防腐功能的乳酸株（审请号201810913462.0）”，培养本科毕业生4人，硕士研究生1人，学生通过研究本项目荣获青岛农业大学校级优秀学士毕业论文1项，山东省优秀学士学位论文1项。