【作者】 孙悦；

【导师】 白凤翎； 郑乾坤；

【作者基本信息】 渤海大学 ， 农业硕士（食品加工与安全）（专业学位）， 2020， 硕士

【摘要】 目前由于抗生素的滥用,细菌耐药性问题普遍存在。耐药菌极易以食物为载体,对人类健康造成威胁,耐药菌的出现已衍生为全球公共卫生的焦点问题。控制食品中的耐药菌主要应用物理法、化学法及生物法,与物理化学法相比生物抗菌剂具有安全高效、不易产生耐药等优点,是控制食品中耐药菌的优良选择。乳酸菌作为一种新型的生物抗菌剂,对食品中的有害微生物有广谱抑菌作用。本文从80株乳酸菌筛选出对耐药性埃希氏大肠杆菌(Escherichia coli)拮抗活性较强的乳酸菌菌株,对其抑菌机制进行分析,并以新鲜鸡肉为载体,分析其在鸡肉中对耐药性E.coli的影响,旨在开发一种针对耐药性E.coli的乳酸菌生物抗菌剂。主要结果如下:1.从80株乳酸菌中筛选出对耐药性E.coli拮抗活性较强的菌株XCT1-1,抑菌直径达20.31 mm。通过生理生化鉴定和16S rDNA序列分析,菌株XCT1-1为植物乳杆菌。经pH、温度、蛋白酶试验,初步判断乳酸菌无细胞上清液(cell-free supernatants,CFS)粗提物中的抑菌活性物质为细菌素类。2.菌株XCT1-1粗提物对耐药性E.coli抑菌机制的研究。菌株XCT1-1对耐药性E.coli最小抑菌浓度为6.0 mg/mL。经菌株XCT1-1粗提物、菌株XCT1-1粗提物和阿奇霉素共同作用耐药性E.coli后,导致其胞外电导率增加,胞内蛋白合成受到影响,胞内核酸发生泄漏,菌株XCT1-1粗提物和阿奇霉素共同作用效果更好。扫描电镜结果进一步表明,菌株XCT1-1粗提物、菌株XCT1-1粗提物和阿奇霉素共同处理耐药性E.coli后,可破坏大肠杆菌的细胞壁和细胞膜,前者菌体表面出现褶皱,后者菌体表面褶皱严重,菌体细胞整体结构坍塌,推测是乳酸菌粗提物提高了阿奇霉素的敏感性。3.菌株XCT1-1粗提物、菌株XCT1-1粗提物和阿奇霉素共同作用对新鲜鸡肉的抗菌效果。经菌株XCT1-1粗提物、菌株XCT1-1粗提物和阿奇霉素共同处理后的鸡肉,可有效控制细菌的生长繁殖,抑制鸡肉中E.coli数量的增长,有效延缓鸡肉中pH、TVB-N值的上升,且菌株XCT1-1粗提物和阿奇霉素共同处理后对鸡肉的抗菌效果更佳。各项指标表明添加菌株XCT1-1粗提物可延长鸡肉货架期至少2 d,菌株XCT1-1粗提物与阿奇霉素共同处理能有效延长鸡肉货架期至少4 d。表明菌株XCT1-1粗提物可提高阿奇霉素的敏感性,且具有良好的抗菌效果。更多还原

【Abstract】 Due to the improper use of antibiotics,the problem of bacterial resistance is widely presence.Drug-resistant bacteria easily threaten human health through food used as a carrier.The emergence of drug-resistant bacteria has become a focus of global public health.Physical,chemical and biological methods are mainly used to control drug-resistant bacteria in food.Compared with physical and chemical methods,biopreservatives,used as an excellent choice for controlling drug-resistant bacteria in food,have the advantages of safety,high efficiency,and resistance to drug resistance.Lactic acid bacteria(LAB),as a new biopreservative,have a broad-spectrum antibacterial effect on harmful microorganisms in food.In the study,LAB with strong antagonistic activity against drug-resistant Escherichia coli were screened from 80 strains LAB,and the antibacterial mechanism was analyzed.The effects of LAB on resistant E.coli in chicken meat was studied by using fresh chicken as a carrier.It aimed to develop a LAB biopreservative against drug-resistant E.coli.The main findings were as follows:1.Strain XCT1-1 with strong antagonistic activity against drug-resistant E.coli was screened from 80 strains LAB,and the inhibition diameter was 20.31 mm.The strain XCT1-1 was identified as Lactobacillus plantarum by physiological and biochemical identification and 16 S rDNA sequence analysis.According to the pH,temperature and protease test,the active substances in crude extract of strain XCT1-1 were preliminarily determined to be bacteriocins.2.The antibacterial mechanism of the crude extract of strain XCT1-1 against drug-resistant E.coli.The minimum inhibitory concentration of strain XCT1-1against drug-resistant E.coli was 6.0 mg/mL.After exposed to the crude extract of strain XCT1-1,crude extract of strain XCT1-1 contained with azithromycin,the extracellular conductivity of E.coli increased,intracellular protein synthesis was inhibited,and intracellular nucleic acid leaked from cells.The effects of crude extract of strain XCT1-1 combined with azithromycin was better than crude extract alone.Scanning electron microscopy images showed that the cell wall and cell membrane of drug-resistant E.coli were damaged by treatment with crude extract of strain XCT1-1,crude extract contained with azithromycin.When E.coli treated with crude extract of strain XCT1-1,the surface of the cell appeared wrinkled,and the latter overall structure of the cell collapsed..3.The antibacterial effects of crude extract of strain XCT1-1,the crude extract of strain XCT1-1 contained with azithromycin on fresh chicken.The number of microorganism and E.coli in chicken meat were effectively inhibited by treatment with the crude extract of strain XCT1-1,crude extract of strain XCT1-1 contained with azithromycin.Additionally,the increase of pH and TVB-N in chicken were effectively delayed,and the antibacterial effects of the crude extract of strain XCT1-1 contained with azithromycin were better than crude extract alone.The crude extract of strain XCT1-1 could prolong the shelf life of chicken at least 2 d,and the treatment of crude extract of strain XCT1-1contained with azithromycin could effectively extend the shelf life of chicken at least 4 d.The results showed that the crude extract of strain XCT1-1 could increase the sensitivity of azithromycin,and have good antibacterial effect on drug-resistant E.coli.更多还原