侧孢短芽孢杆菌抗菌肽brevilaterin对细菌具有广谱抑菌活力,但是对革兰氏阴性菌的最小抑菌浓度显著高于革兰氏阳性菌,只有增大用量才能发挥广谱抑菌活性。该研究以食源性致病菌金黄色葡萄球菌、单核细胞增生李斯特菌、大肠杆菌、鼠伤寒沙门氏菌为指示菌,首先测定了食品中常用抑菌剂的最小抑菌浓度,然后分别考察了抗菌肽brevilaterin与各添加剂的联合抑菌效应,筛选了与brevilaterin具有协同作用的添加剂,进一步通过复配形成brevilaterin广谱高效抑菌配方。结果显示,天然防腐剂的抑菌效果优于化学防腐剂,且天然防腐剂对革兰氏阳性菌的抑菌效果普遍优于革兰氏阴性菌;brevilaterin分别与ε-聚赖氨酸、nisin联用对革兰氏阳性致病菌具有协同抑菌效应,与EDTA-Na2、柠檬酸、Na3PO4联用对革兰氏阴性菌具有协同抑菌效应;选取brevilaterin、nisin和柠檬酸进行复配,时间杀菌曲线结果表明该组合能够发挥协同广谱抑菌效应,brevilaterin添加量降低为原用量的1/8。说明协同抑菌物质复配可有效降低brevilaterin使用剂量,且对食源性致病菌具有广谱抑菌活性。
Brevilaterin, an antimicrobial peptide produced by Brevibacillus laterosporus, has broad antibacterial range. However, the minimum inhibitory concentration (MIC) of brevilaterin against gram-negative bacteria are much higher than that against gram-positive bacteria. Thus, a large amount of brevilaterin is required for effective and broad range antimicrobial activity. To overcome this problem, the synergistic antibacterial agents were screened, and the combined antibacterial effects of brevilaterin with other agents were evaluated. Subsequently, the antibacterial combination that had broad and high-efficient antibacterial activity were selected. In this study, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella were used as indicators for determining the minimum inhibitory concentrations (MICs). Results suggested that the antibacterial effect of natural preservatives was higher than that of the chemical compounds. Also, natural preservatives showed stronger antibacterial activities against gram-positive bacteria than gram-negative. Brevilaterin combining with ε-polylysine, or nisin showed synergistic antibacterial activity against gram-positive pathogenic bacteria, and combining with EDTA-Na2, citrate acid, or Na3PO4 showed activity against gram-negative bacteria. Finally, the time-kill curves of brevilaterin, nisin, and citrate acid combination showed broad antibacterial activity, and the MIC of brevilaterin reduced to the 1/8 of the original usage. This investigation provided theoretical foundation for the application of brevilaterin in food preservation.
[1] PILAR G, LORENA R, ANA R, et al. Food biopreservation: promising strategies using bacteriocins, bacteriophages and endolysins [J]. Trends in Food Science and Technology, 2010, 21(8): 373-382.
[2] TIWARI B K, VALDRAMIDIS V P, O'DONNELL C P. Application of natural antimicrobials for food preservation [J]. Journal of Agricultural and Food Chemistry, 2009, 57(14): 5 987-6 000.
[3] WANG Jingyi, BIE Meng, ZHOU Weijing, et al. Interaction between carboxymethyl pachyman and lotus seedpod oligomeric procyanidins with superior synergistic antibacterial activity [J]. Carbohydrate Polymers, 2019, 212: 11-20.
[4] REQUENA R, VARGAS M, CHIRALT A. Study of the potential synergistic antibacterial activity of essential oil components using the thiazolyl blue tetrazolium bromide (MTT) assay [J]. LWT-Food Science and Technology, 2019, 101: 183-190.
[5] CHAK-LUN C, REN-YOU G, NAGENDRA P S, et al. Polyphenols from selected dietary spices and medicinal herbs differentially affect common food-borne pathogenic bacteria and lactic acid bacteria [J]. Food Control, 2018, 92: 437-443.
[6] SKARIYACHAN S, GOVINDARAJAN S. Biopreservation potential of antimicrobial protein producing Pediococcus spp. towards selected food samples in comparison with chemical preservatives [J]. International Journal of Food Microbiology, 2019, 291:189-196.
[7] LUCILLE G, JEROME M, LE S, et al. Development of antifungal ingredients for dairy products: From in vitro screening to pilot scale application [J]. Food Microbiology, 2019, 81: 97-107.
[8] YANG Xu, HUANG En, AHMED E Y. Brevibacillin, a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus [J]. Microbiological Research, 2017, 195: 18-23.
[9] GHARSALLAOUI A, OULAHAL N, JOLY C, et al. Nisin as a Food Preservative: Part 1: Physicochemical properties, antimicrobial activity, and main uses [J]. Critical Reviews in Food Science and Nutrition, 2016, 62(8): 1 262-1 274.
[10] SUN Zhilan, LI Pengpeng, LIU Fang, et al. Synergistic antibacterial mechanism of the Lactobacillus crispatus surface layer protein and nisin on Staphylococcus saprophyticus [J]. Scientific Reports, 2017, 7: 265.
[11] 马俊美,宁亚维,王志新,等.侧孢短芽孢杆菌抗菌肽的结构与性质[J].食品与生物技术学报, 2016, 345(6): 629-634.
[12] 杨倩,于宏伟,郭润芳,等.侧孢短芽孢杆菌S62-9产抗菌物质的分离纯化及部分特性的研究[J].河北农业大学学报, 2010, 33(2): 74-78.
[13] 李兴峰,刘豆,薛江超,等.天然食品防腐剂的协同抗菌作用[J].中国食品学报, 2014, 14(3): 140-144.
[14] BAG A, CHATTOPADHYAY R R. Synergistic antibacterial and antibiofilm efficacy of nisin in combination with p-coumaric acid against food-borne bacteria Bacillus cereus and Salmonella typhimurium [J]. Letter in Applied Microbiology, 2017, 65: 366-372.
[15] 宋萌,付强,时艺翡,等.ε-聚赖氨酸复配防腐剂在酱腌菜中的应用[J].食品科学, 2018, 39(10): 276-282.
[16] 王霄晔,任婧寰,王哲,等. 2017 年全国食物中毒事件流行特征分析[J].疾病监测, 2018, 33(5): 359-363.
[17] SCHARFF R L. Economic burden from health losses due to foodborne illness in the United States [J]. Journal of Food Protection, 2012, 75(1), 123-131.
[18] 贾英民,李兴峰,王志新,等. 一种从发酵液中快速提取食用抗菌肽的方法:中国[P]. ZL 201210019936.X.
[19] WIEGAND I, HILPERT K, HANCOCK R E W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances [J]. Nature Protocols, 2008, 3(2): 163-175.
[20] SEMIS R, NAHMIAS M, LEV S, et al. Evaluation of antifungal combinations of nystatin-intralipid against Aspergillus terreus using checkerboard and disk diffusion methods [J]. Journal de Mycologie Médicale, 2015, 25: 63-70.
[21] CHRISTIANE M V, EDUARDO B D O, STEPHANIA N R, et al. Evaluating strategies to control enzymatic browning of minimally processed yacon (Smallanthus sonchifolius) [J]. Food and Bioprocess Technology, 2015, 8(9): 1 982-1 994.
[22] 陈历水,刘松玲,倪军,等. 1株来源于腐败肉制品的沙雷氏菌的分离鉴定与特性研究[J].中国食品学报, 2014, 14(10): 235-240.
[23] 张莉莉,于长青.磷酸三钠对提高冷却猪肉持水性优化条件的研究[J].农产品加工, 2009(8): 71-73.
[24] 谭春明,孙通,薛勇,等.大米肽功能饮料的研制[J].食品与发酵工业, 2017, 43(3): 157-162.
[25] 林琳,张雪婧,张可星,等.柠檬酸钠与桂皮提取物对各种革兰氏阴性食源性病原菌的抑菌效果[J].中国食品添加剂, 2014(1): 77-80.
