以青藏高原狭果茶藨子为原料,探究其对蜡样芽孢杆菌的抑菌作用及作用机制,利用纸片扩散法和二倍稀释法评价抑菌活性,通过测定膜蛋白荧光光谱、碱性磷酸酶、Na＋K＋-ATP酶、呼吸链脱氢酶活性等探究抑菌机制。结果表明,狭果茶藨子提取物对蜡样芽孢杆菌有明显的抑制作用,最低抑菌浓度和最低杀菌浓度分别为3.13和6.25 mg/mL;狭果茶藨子提取物诱导了蜡样芽孢杆菌核酸、蛋白质及碱性磷酸酶泄露,破坏了细胞壁膜的完整性,并且改变了细胞膜膜蛋白构象,Na＋K＋-ATP酶、Ca2+Mg2+-ATP酶和T-ATP 酶活性测定结果表明提取物激活了膜结合离子通道,改变细胞膜内外离子浓度以抵抗不利环境的影响;此外提取物抑制呼吸链脱氢酶活性,影响了呼吸作用和能量代谢,扫描电镜结果表明提取物导致蜡样芽孢杆菌细胞严重变形,表面有异常凸起和凹陷,说明狭果茶藨子可以有效抑制蜡样芽孢杆菌生长繁殖,具有成为天然食品保鲜剂的潜力。
Taking Ribes stenocarpum Maxim as raw material, the antimicrobial activity and mechanism of R. stenocarpum against Bacillus cereus were investigated. The antibacterial activities were evaluated by agar diffusion assay and double dilution method, the inhibition mechanism was investigated by measuring the fluorescence spectra of membrane protein, the activity of alkaline phosphatase, Na+K+-ATPase, respiratory chain dehydrogenase etc. Results showed that R. stenocarpum had significant inhibitory effect on B. cereus, the minimum inhibitory concentration and minimum bactericidal concentration were 3.13 and 6.25 mg/mL respectively. The extract of R. stenocarpum induced the leakage of alkaline phosphatase, protein and nucleotides into the culture medium, indicating damage to the integrity of cell wall and membrane, also changed the conformation of membrane protein. The activities of Na+K+-ATPase, Ca2+Mg2+-ATPase and T-ATPase were increased, the membrane bound ion channels were activated, the ion concentration inside and outside the cell membrane was changed to resist the influence of adverse environment. The activity of respiratory chain dehydrogenase was inhibited by the extract, and respiration and energy metabolism were affected. The results of scanning electron microscope showed that the extract caused serious deformation of B. cereus cells, and the surface of the cells had abnormal bulge and depression, all of which indicated that R. stenocarpum could effectively inhibit the growth and reproduction of B. cereus, it has the potential to be a natural food preservative.
[1] LAURENCE D, NADINE B, MIRJANA A, et al.An emetic Bacillus cereus outbreak in a kindergarten:Detection and quantification of critical levels of cereulide toxin[J].Foodborne Pathog Dis, 2015, 12(1):84-87.
[2] TALAHMEH N, ABU-RUMEILEH S, AL-RAZEM F.Development of a selective and differential media for the isolation and enumeration of Bacillus cereus from food samples[J].J Appl Microbiol, 2020, 128(5):1 440-1 447.
[3] 陆玲娣. 中国茶藨子属的研究[J].植物分类学报, 1995,33(1):58-75.
LU L T. A study on the genus Ribes L.in China[J].Acta Phytotaxonomica Sinica, 1995, 33(1):58-75.
[4] 王占林, 樊光辉, 贺永远.青海高原茶藨子属树种资源与育苗技术[J].北方园艺, 2012(19):43-45.
WANG Z L, FAN G H, HE Y Y.Ribes species resources and afforestation technique in Qinghai Plateau[J].Northern Horticulture, 2012(19):43-45.
[5] 金志民, 柴军红, 何婷婷,等.正交设计优化东北茶藨子的活性成分提取工艺[J].中国林副特产, 2018(6):14-16.
JIN Z M, CHAI J H, HE T T, et al.Optimization of extraction process for active ingredient from Ribes mandshuricum[J].Forest By-Product and Speciality in China, 2018(6):14-16.
[6] 叶英, 李宗仁, 曹效海,等.青藏高原狭果茶藨子果实提取物成分、抑菌活性及其抗疲劳活性[J].食品工业科技, 2019,40(13):7-13.
YE Y, LI Z R, CAO X H, et al.Components and antibacterial activities of extracts from the fruits of Ribes stenocarpum Maxim in Qinghai-Tibet Plateau and their anti-fatigue activity[J].Science and Technology of Food Industry, 2019,40(13):7-13.
[7] 冯亚净, 张媛媛, 王瑞鑫,等.五味子木脂素对大肠杆菌的抑菌机理及效果[J].食品与发酵工业, 2016,42(2):72-76.
FENG Y J, ZHANG Y Y, WANG R X, et al.Antibacterial effect of Lignanoid from Fructus Schisandrae chinensis on the Escherichia coli and its mechanism[J].Food and Fermentation Industries, 2016,42(2):72-76.
[8] KANG S M, KONG F H, SHI X Y, et al.Antibacterial activity and mechanism of lactobionic acid against Pseudomonas fluorescens and Methicillin-resistant Staphylococcus aureus and its application on whole milk[J].Food Control, 2020,108:106 876.
[9] CAI X T, WANG X, CHEN Y C, et al.A natural biopreservative:Antibacterial action and mechanisms of Chinese Litsea mollis hemsl.extract against Escherichia coli dh5α and Salmonella spp.[J].Journal of Dairy Science, 2019, 102(11):9 663-9 673.
[10] WANG L H, WANG M S, ZENG X, et al.Membrane and genomic DNA dual-targeting of citrus flavonoid naringenin against Staphylococcus aureus[J].Integrative Biology (Camb), 2017, 9(10):820-829.
[11] SONG M Z, WANG X R, MAO C Q, et al.The discovery of a potential antimicrobial agent:The novel compound natural medicinal plant fermentation extracts against Candida albicans[J].IOP Conference Series:Materials Science and Engineering, 2018, 301(1):012 026.
[12] LIU X, CAI J X, CHEN H M, et al.Antibacterial activity and mechanism of linalool against Pseudomonas aeruginosa[J].Microb Pathog, 2020, 141:103 980.
[13] WANG B Y, GAO X L, LIU B G, et al.Protective effects of curcumin against chronic alcohol-induced liver injury in mice through modulating mitochondrial dysfunction and inhibiting endoplasmic reticulum stress[J].Food & Nutrition Research, 2019,63:3 597.
[14] 郑志永, 姚善泾.比色法测定大肠杆菌呼吸链脱氢酶活性[J].浙江大学学报(工学版), 2005(3):119-122.
ZHENG Z Y, YAO S J.Colorimetric assay for respiratory chain dehydrogenase activity in Escherichia coli[J].Journal of Zhejiang University(Engineering Science), 2005(3):119-122.
[15] LI Y Q, HAN Q, FENG J L, et al.Antibacterial characteristics and mechanisms of ε-poly-lysine against Escherichia coli and Staphylococcus aureus[J].Food Control, 2014, 43:22-27.
[16] 周云冬, 章漪玲, 宗红, 等.牡丹花蕾提取物对铜绿假单胞菌的抑菌活性及其机理[J].食品与发酵工业, 2019,45(13):92-97.
ZHOU Y D, ZHANG Y L, ZONG H, et al.Antibacterial activities and mechanisms of Paeonia suffruticosa Andr.buds extract against Pseudomonas aeruginosa[J].Food and Fermentation Industries, 2019,45(13):92-97.
[17] CAO J R, FU H J, GAO L H, et al.Antibacterial activity and mechanism of lactobionic acid against Staphylococcus aureus[J].Folia Microbiol (Praha), 2019, 64(6):899-906.
[18] XIANG Q S, KANG C D, NIU L Y, et al.Antibacterial activity and a membrane damage mechanism of plasma-activated water against Pseudomonas Deceptionensis CM2[J].LWT, 2018, 96:395-401.
[19] 翟艺宗, 黄昌林, 常祺, 等.中频脉冲电流经皮刺激肝区对运动性疲劳大鼠肝细胞线粒体Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性的影响[J].解放军医学杂志, 2015,40(4):327-330.
ZHAI Y Z, HUANG C L, CHANG Q, et al.Effects of percutaneous midband pulse current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats[J].Medical Journal of Chinese People's Liberation Army, 2015,40(4):327-330.
[20] LIN Y L, TANG X, XU L Z, et al.Antibacterial properties and possible action mechanism of chelating peptides-zinc nanocomposite against Escherichia coli[J].Food Control, 2019, 106:106 675.
[21] LI X C, HE C F, SONG L Y, et al.Antimicrobial activity and mechanism of Larch bark procyanidins against Staphylococcus aureus[J].Acta Biochim Biophys Sin (Shanghai), 2017, 49(12):1 058-1 066.
[22] 李志刚, 陈宝峰, 张中华, 等.辅助能量物质强化环磷酸腺苷发酵合成机制[J].中国生物工程杂志, 2020,40(Z1):102-108.
LI Z G, CHEN B F, ZHANG Z H, et al.The physiological mechanism for enhanced cyclic adenosine monophosphate biosynthesis by auxiliary energy substance[J].China Biotechnology, 2020,40(Z1):102-108.
