曲酸对冷鲜鸭肉中优势腐败菌的抑制作用及其抑菌机理
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摘要
以冷鲜鸭肉中优势腐败菌假单胞菌、气单胞菌、热杀索丝菌和具有致腐能力的病原微生物沙门氏菌为研究对象,采用抑菌圈直径、液体生长曲线和最小抑菌浓度评价曲酸的抑菌作用,分析曲酸对细胞膜和菌株胞内物质的影响,探索曲酸的抑菌机理。结果表明:曲酸对4株菌株均具有良好的抑制作用,其中热杀索丝菌对曲酸处理敏感,最小抑菌质量浓度为0.5 mg/mL,液体体系下低质量浓度曲酸溶液处理即能完全抑制其生长;随着曲酸质量浓度增加,沙门氏菌、假单胞菌和气单胞菌的抑菌圈直径增加,对假单胞菌和气单胞菌的抑制尤为明显,其抑菌圈直径增加1倍以上;2.0 mg/mL曲酸处理在液体体系中能完全抑制上述3株菌株的生长。曲酸处理能够提高菌株细胞膜疏水性1～2倍,降低细胞膜表面的选择渗透性和破坏细胞膜结构,菌株培养液中Ca~(2+)、K~+、Mg~(2+)离子浓度增加;曲酸处理还可破坏呼吸链脱氢酶、促使β-半乳糖苷酶和碱性磷酸酶等内容物质泄漏、菌体细胞膜破裂形成孔洞。同时,曲酸处理引起菌株胞外蛋白质含量显著上升(P<0.05),胞内分子质量大于66.4 kDa和小于20 kDa的蛋白质减少。整体来看,曲酸抑制4株菌株的机理基本一致,主要是通过促进疏水物质暴露、破坏菌株相关酶和改变细胞膜通透性,同时干扰菌株蛋白质新陈代谢,从而抑制菌株的增殖。
The bacteriostatic effects of kojic acid against the dominant spoilage bacteria Pseudomonas sp., Aeromonas sp. and Brochothrix thermosphacta and the pathogenic bacterium Salmonella sp. in chilled duck meat were studied by determining the diameter of inhibition zone, growth curves in LB liquid medium and minimal inhibitory concentration(MIC). The underlying mechanism was explored by analyzing the effect of kojic acid on the cell membrane and intracellular substances. The results showed that kojic acid had good bacteriostatic properties against all four strains. Brochothrix thermosphacta was sensitive to kojic acid treatment with an MIC of 0.5 mg/mL, and its growth in the liquid medium was completely low concentration of kojic acid. The diameter of zone of inhibition of Salmonella, Pseudomonas, andAeromonas increased significantly with the increase of kojic acid concentration, with an over 2-fold increase being observed for Pseudomonas and Aeromonas. Kojic acid treatment at 2.0 mg/mL could completely inhibit the growth of all above 3 strains in the liquid medium. Kojic acid treatment could increase cell surface hydrophobicity by two to three folds, reduce the selective permeability of the cell membrane, and destroy the structure of the cell membrane, thereby leading to increased levels of extracellular Ca~(2+), K~+ and Mg~(2+). Kojic acid treatment could also destroy respiratory chain dehydrogenase activity, and result in the leakage of intracellular β-galactosidase and alkaline phosphatase and cell membrane rupture to form pores. Besides, kojic acid treatment caused a significant increase in extracellular protein content, with a simultaneous decrease in intracellular proteins with molecular mass more than 66.4 kDa and less than 20 kDa. Overall, kojic acid inhibited the four strains via the same mechanism, namely, by promoting hydrophobic substance exposure, destroying related enzyme activities and changing cell membrane permeability, as well as disturbing protein metabolism.
The bacteriostatic effects of kojic acid against the dominant spoilage bacteria Pseudomonas sp., Aeromonas sp. and Brochothrix thermosphacta and the pathogenic bacterium Salmonella sp. in chilled duck meat were studied by determining the diameter of inhibition zone, growth curves in LB liquid medium and minimal inhibitory concentration(MIC). The underlying mechanism was explored by analyzing the effect of kojic acid on the cell membrane and intracellular substances. The results showed that kojic acid had good bacteriostatic properties against all four strains. Brochothrix thermosphacta was sensitive to kojic acid treatment with an MIC of 0.5 mg/mL, and its growth in the liquid medium was completely low concentration of kojic acid. The diameter of zone of inhibition of Salmonella, Pseudomonas, andAeromonas increased significantly with the increase of kojic acid concentration, with an over 2-fold increase being observed for Pseudomonas and Aeromonas. Kojic acid treatment at 2.0 mg/mL could completely inhibit the growth of all above 3 strains in the liquid medium. Kojic acid treatment could increase cell surface hydrophobicity by two to three folds, reduce the selective permeability of the cell membrane, and destroy the structure of the cell membrane, thereby leading to increased levels of extracellular Ca~(2+), K~+ and Mg~(2+). Kojic acid treatment could also destroy respiratory chain dehydrogenase activity, and result in the leakage of intracellular β-galactosidase and alkaline phosphatase and cell membrane rupture to form pores. Besides, kojic acid treatment caused a significant increase in extracellular protein content, with a simultaneous decrease in intracellular proteins with molecular mass more than 66.4 kDa and less than 20 kDa. Overall, kojic acid inhibited the four strains via the same mechanism, namely, by promoting hydrophobic substance exposure, destroying related enzyme activities and changing cell membrane permeability, as well as disturbing protein metabolism.
引文
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[19]何世微,陶毅明,王贵平,等.曲酸对菠萝蜜多酚氧化酶的抑制作用和抑菌实验[J].食品工业科技,2015,36(11):159-161;166.DOI:10.13386/j.issn1002-0306.2015.11.023.
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[23]郝刚,乐国伟,施用晖.抗菌肽Buforin Ⅱ衍生物特异性结合金黄色葡萄球菌DNA抑菌机理的研究[J].畜牧兽医学报,2014,45(7):1154-1161.DOI:10.11843/j.issn.0366-6964.2014.07.018.
[24]WANG Chenjie,CHANG Tong,YANG Hong,et al.Surface physiological changes induced by lactic acid on pathogens in consideration of p Ka,and pH[J].Food Control,2014,46:525-531.DOI:10.1016/j.foodcont.2014.06.024.
[25]SITOHY M Z,MAHGOUB S A,OSMAN A O.In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit[J].International Journal of Food Microbiology,2012,154(1/2):19-29.DOI:10.1016/j.ijfoodmicro.2011.12.004.
[26]赵春贵,张立伟,王晖,等.肉桂酸及其衍生物抗氧化活性研究[J].食品科学,2005,26(1):218-222.DOI:10.3321/j.issn:1002-6630.2005.01.051.
[27]PRAMANIK K,GHOSH P K,RAY S,et al.An in silico,structural,functional and phylogenetic analysis with three dimensional protein modeling of alkaline phosphatase enzyme of Pseudomonas aeruginosa[J].Journal of Genetic Engineering&Biotechnology,2017,15(2):527-537.DOI:10.1016/j.jgeb.2017.05.003.
[28]王巍,牟德华,李丹丹.山楂果胶寡糖的抑菌性能及机理[J].食品科学,2018,39(3):110-116.DOI:10.7506/spkx1002-6630-201803017.
[29]黄诗琪.客家黄酒多酚抑菌及抗氧化性研究[D].广州:仲恺农业工程学院,2016:49-50.
[30]孙京新,王文娟.茶多酚对假单胞菌抑菌机理研究[J].肉类研究,2009,23(10):48-51.DOI:10.3969/j.issn.1001-8123.2009.10.015.
[31]张伟娜.ε-聚赖氨酸的抑菌特性及对猪肉保鲜效果研究[D].济南:齐鲁工业大学,2013:23.
[32]赵磊,张鹤龑,郝添阳,等.蔗糖癸酸酯对沙门氏菌的抗菌作用及其机理初探[J].现代食品科技,2016,32(5):46-51.DOI:10.13982/j.mfst.1673-9078.2016.5.08.
[2]RAJAMANIKYAM M,GADE S,VADLAPUDI V,et al.Biophysical and biochemical characterization of active secondary metabolites from Aspergillus allahabadii[J].Process Biochemistry,2017,56(2):45-56.DOI:10.1016/j.procbio.2017.02.010.
[3]KAYAHARA H,SHIBATA N,TADASA K,et al.Amino acid and peptide derivatives of kojic acid and their antifungal properties[J].Journal of the Agricultural Chemical Society of Japan,2014,54(9):2441-2442.DOI:10.1080/00021369.1990.10870342.
[4]EMAMI S,GHAFOURI E,FARAMARZI M A,et al.Mannich bases of 7-piperazinylquinolones and kojic acid derivatives:synthesis in vitro antibacterial activity and in silico study[J].European Journal of Medicinal Chemistry,2013,68(7):185-191.DOI:10.1016/j.ejmech.2013.07.032.
[5]苏国成,汤凤霞,杨秋明,等.曲酸对常见食品污染菌的抑制作用[J].食品与发酵工业,2005,31(3):47-51.DOI:10.3321/j.issn:0253-990X.2005.03.013.
[6]EMAMI S,GHAFOURI E,FARAMARZI M A,et al.Mannich bases of 7-piperazinylquinolones and kojic acid derivatives:synthesis,in vitro antibacterial activity and in silico study[J].European Journal of Medicinal Chemistry,2013,68(12):185-191.DOI:10.1016/j.ejmech.2013.07.032.
[7]LIU X,JIANG Q,XIA W.One-step procedure for enhancing the antibacterial and antioxidant properties of a polysaccharide polymer:kojic acid grafted onto chitosan[J].International Journal of Biological Macromolecules,2018,113:1125-1133.DOI:10.1016/j.ijbiomac.2018.03.007.
[8]KIM J,CAMPBELL B,CHAN K,et al.Synergism of antifungal activity between mitochondrial respiration inhibitors and kojic acid[J].Molecules,2013,18(2):1564-1581.DOI:10.3390/molecules18021564.
[9]LIU X,XIA W,JIANG Q,et al.Effect of kojic acid-grafted-chitosan oligosaccharides as a novel antibacterial agent on cell membrane of Gram-positive and Gram-negative bacteria[J].Journal of Bioscience&Bioengineering,2015,120(3):335-339.DOI:10.1016/j.jbiosc.2015.01.010.
[10]HAFIZ M,SHOAIB S,AHMAD S K,et al.Pre-storage kojic acid application delays pericarp browning and maintains antioxidant activities of litchi fruit[J].Postharvest Biology and Technology,2017,132(6):154-161.DOI:10.1016/j.postharvbio.2017.06.004.
[11]陆正清,王艳.曲酸的发酵法生产及其在食品加工中的应用[J].中国调味品,2008,33(1):65-67;71.DOI:10.3969/j.issn.1000-9973.2008.01.015.
[12]GAO Z,SUR,QI W,et al.Copper nanocluster-based fluorescent sensors for sensitive and selective detection of kojic acid in food stuff[J].Sensors and Actuators B,2014,195(1):359-364.DOI:10.1016/j.snb.2014.01.051.
[13]赵新河,李枚秋,钟秋平.曲酸及其衍生物的研究进展[J].中国酿造,2007,13(10):4-7.DOI:10.3969/j.issn.0254-5071.2007.10.002.
[14]张志强,孙杨赢,潘道东,等.两种保鲜液对冷鲜鸭胸肉品质的影响[J].食品工业科技,2016,37(18):324-328;335.DOI:10.13386/j.issn1002-0306.2016.18.053.
[15]云美林.利用生物抑菌涂膜延长冷鲜羊肉保质期的效果研究[D].呼和浩特:内蒙古农业大学,2012:1-4.
[16]曾晓房,林惠珍,邝智祥,等.冰鲜肉中腐败菌的研究现状[J].安徽农业科学,2010,38(34):19550-19552;19558.DOI:10.3969/j.issn.0517-6611.2010.34.124.
[17]林惠珍.冰鲜鸭优势腐败菌的分析及其控制技术的研究[D].福州:福建农林大学,2011:4-5.
[18]WANG C,CHEN Q,ZHANG C,et al.Characterization of a broad host-spectrum virulent Salmonella bacteriophage fmb-p1 and its application on duck meat[J].Virus Research,2017,236(5):14-23.DOI:10.1016/j.virusres.2017.05.001.
[19]何世微,陶毅明,王贵平,等.曲酸对菠萝蜜多酚氧化酶的抑制作用和抑菌实验[J].食品工业科技,2015,36(11):159-161;166.DOI:10.13386/j.issn1002-0306.2015.11.023.
[20]刘蔚,周涛.ε-聚赖氨酸抑菌机理研究[J].食品科学,2009,30(9):15-20.DOI:10.3321/j.issn:1002-6630.2009.09.002.
[21]何丽.基于微生物控制的冷鲜淡水鱼分割产品的保鲜技术研究[D].武汉:武汉轻工大学,2016:31-32.
[22]冯建岭.ε-聚赖氨酸的防腐效果及对大肠杆菌抑制机理的研究[D].济南:齐鲁工业大学,2014:25-26;29;34-35.
[23]郝刚,乐国伟,施用晖.抗菌肽Buforin Ⅱ衍生物特异性结合金黄色葡萄球菌DNA抑菌机理的研究[J].畜牧兽医学报,2014,45(7):1154-1161.DOI:10.11843/j.issn.0366-6964.2014.07.018.
[24]WANG Chenjie,CHANG Tong,YANG Hong,et al.Surface physiological changes induced by lactic acid on pathogens in consideration of p Ka,and pH[J].Food Control,2014,46:525-531.DOI:10.1016/j.foodcont.2014.06.024.
[25]SITOHY M Z,MAHGOUB S A,OSMAN A O.In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit[J].International Journal of Food Microbiology,2012,154(1/2):19-29.DOI:10.1016/j.ijfoodmicro.2011.12.004.
[26]赵春贵,张立伟,王晖,等.肉桂酸及其衍生物抗氧化活性研究[J].食品科学,2005,26(1):218-222.DOI:10.3321/j.issn:1002-6630.2005.01.051.
[27]PRAMANIK K,GHOSH P K,RAY S,et al.An in silico,structural,functional and phylogenetic analysis with three dimensional protein modeling of alkaline phosphatase enzyme of Pseudomonas aeruginosa[J].Journal of Genetic Engineering&Biotechnology,2017,15(2):527-537.DOI:10.1016/j.jgeb.2017.05.003.
[28]王巍,牟德华,李丹丹.山楂果胶寡糖的抑菌性能及机理[J].食品科学,2018,39(3):110-116.DOI:10.7506/spkx1002-6630-201803017.
[29]黄诗琪.客家黄酒多酚抑菌及抗氧化性研究[D].广州:仲恺农业工程学院,2016:49-50.
[30]孙京新,王文娟.茶多酚对假单胞菌抑菌机理研究[J].肉类研究,2009,23(10):48-51.DOI:10.3969/j.issn.1001-8123.2009.10.015.
[31]张伟娜.ε-聚赖氨酸的抑菌特性及对猪肉保鲜效果研究[D].济南:齐鲁工业大学,2013:23.
[32]赵磊,张鹤龑,郝添阳,等.蔗糖癸酸酯对沙门氏菌的抗菌作用及其机理初探[J].现代食品科技,2016,32(5):46-51.DOI:10.13982/j.mfst.1673-9078.2016.5.08.