Patton Thomas, Barrett John, Brennan James, Moran Noel
Institute of Technology Sligo, IT Sligo, Ballinode, Sligo, Ireland.
J Microbiol Methods. 2006 Jan;64(1):84-95. doi: 10.1016/j.mimet.2005.04.007. Epub 2005 Jun 24.
The antimicrobial activity of manuka honey has been well documented (Molan, 1992a,b,c, 1997) [Molan, P.C., 1992. The antibacterial activity of honey. 1: the nature of the antibacterial activity. Bee World 73 (1) 5-28; Molan, P.C., 1992. The antibacterial activity of honey. 2: variation in the potency of the antibacterial activity. Bee World 73 (2) 59-76; Molan, P.C., 1992. Medicinal uses for honey. Beekeepers Quarterly 26; Molan, P.C., 1997. Finding New Zealand honeys with outstanding antibacterial and antifungal activity. New Zealand Beekeeper 4 (10) 20-26]. The current bioassays for determining this antimicrobial effect employ a well diffusion (Ahn and Stiles, 1990) [Ahn, C., Stiles, M.E., 1990. Antibacterial activity of lactic acid bacteria isolated from vacuum-packed meats. Journal of Applied Bacteriology 69, 302-310], (Weston et al., 1999) [Weston, R.J., Mitchell, K.R., Allen, K.L., 1999. Antibacterial phenolic components of New Zealand manuka honey. J. Food Chem. 64, 295-301] or disc diffusion (Taormina et al., 2001) [Taormina, Peter J., Niemira, Brendan A., Beuchat, Larry R., 2001. Inhibitory activity of honey against food borne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. Int. J. Food Microbiol. 69, 217-225] assay using zones of inhibition as indicators of bacterial susceptibility. The development of a 24-h spectrophotometric assay employing 96-well microtiter plates, that is more sensitive and more amenable to statistical analysis than the assays currently employed, was undertaken. This simple and rapid assay permits extensive kinetic studies even in the presence of low honey concentrations, and is capable of detecting inhibitory levels below those recorded for well or disc diffusion assays. In this paper, we compare the assay to both well and disc diffusion assays. The results we obtained for the spectrophotometric method MIC values show that this method has greater sensitivity than the standard well and disc diffusion assays. In addition, inter- and intra-assay variance for this method was investigated, demonstrating the methods reproducibility and repeatability.
麦卢卡蜂蜜的抗菌活性已有充分记载（莫兰，1992年a、b、c，1997年）[莫兰，P.C.，1992年。蜂蜜的抗菌活性。1：抗菌活性的本质。《蜜蜂世界》73(1) 5 - 28；莫兰，P.C.，1992年。蜂蜜的抗菌活性。2：抗菌活性效力的变化。《蜜蜂世界》73(2) 59 - 76；莫兰，P.C.，1992年。蜂蜜的药用用途。《养蜂人季刊》26；莫兰，P.C.，1997年。寻找具有出色抗菌和抗真菌活性的新西兰蜂蜜。《新西兰养蜂人》4(10) 20 - 26]。目前用于测定这种抗菌效果的生物测定法采用杯碟扩散法（安和斯泰尔斯，1990年）[安，C.，斯泰尔斯，M.E.，1990年。从真空包装肉类中分离的乳酸菌的抗菌活性。《应用细菌学杂志》69，302 - 310]，（韦斯顿等人，1999年）[韦斯顿，R.J.，米切尔，K.R.，艾伦，K.L.，1999年。新西兰麦卢卡蜂蜜的抗菌酚类成分。《食品化学杂志》64，295 - 301]或纸片扩散法（陶尔米纳等人，2001年）[陶尔米纳，彼得·J.，涅米拉，布伦丹·A.，贝查特，拉里·R.，2001年。过氧化氢的存在和抗氧化能力水平对蜂蜜抑制食源性病原体活性的影响。《国际食品微生物学杂志》69，217 - 225]，以抑菌圈作为细菌敏感性的指标。我们开展了一项使用96孔微量滴定板的24小时分光光度测定法，该方法比目前使用的测定法更灵敏且更适合进行统计分析。这种简单快速的测定法即使在蜂蜜浓度较低的情况下也能进行广泛的动力学研究，并且能够检测到低于杯碟扩散法或纸片扩散法记录的抑制水平。在本文中，我们将该测定法与杯碟扩散法和纸片扩散法进行了比较。我们通过分光光度法获得的最低抑菌浓度（MIC）值结果表明，该方法比标准的杯碟扩散法和纸片扩散法具有更高的灵敏度。此外，我们还研究了该方法的批间和批内差异，证明了该方法的可重复性和再现性。
