Center for Research and Development in Food Cryotechnology, La Plata, Argentina.
J Appl Microbiol. 2012 Feb;112(2):363-71. doi: 10.1111/j.1365-2672.2011.05210.x. Epub 2012 Jan 3.
To set-up an experimental and analytical methodology to evaluate the feasibility of developing simple, accurate and quantitative models based on Raman spectroscopy and multivariate analysis for the quantification of metal ions adsorbed to the bacterial surface of Lactobacillus kefir.
One millilitre cultures from two strains of Lact. kefir in the stationary phase were harvested and washed twice with ultra pure water. The bacterial pellets were resuspended into 1 ml solutions of Pb(+2), Cd(+2) or Ni(+2) ranging from 0 to 0·9 mmol l(-1). The suspensions were further incubated for 1 h at 30°C at pH 5·5. After centrifugation, the pellets were kept to register the Raman spectra and the supernatants were used for the analytical determination of Pb(+2) , Cd(+2) and Ni(+2). Micro-organisms nontreated with metal ions were used as controls. Principal component analysis (PCA) was performed over the preprocessed Raman spectra to evaluate whether the clusters obtained could be correlated with the concentration of metal ions attached to the bacterial biomass. After that, partial least squares (PLS) models were calibrated with the aim of quantifying the metal ions adsorbed to the bacterial surface. According to the analytical determinations, the maximum binding capacity of all the metals (q(max)) attained values that are comparable with those observed for other lactic acid bacteria (ca. 0·200 mmol g(-1)). The spectral analysis revealed that the main functional groups involved in the bacteria/metal interaction are carboxylates, phosphates and polysaccharides. In PCA, the first two principal components explain more than 72% variance of the spectral data set contained in the data structure, allowing a clear discrimination among samples of different concentrations. Based on this information and using as reference the results obtained by analytical methods, PLS prediction models were successfully defined for the quantification of Pb(+2), Cd(+2) and Ni(+2) attached to the bacterial surface.
The calibration and validation of methods based on multivariate analysis allowed the definition of models for the quantification of Pb(+2), Cd(+2) and Ni(+2) attached to bacterial surfaces. The high percentages of explained variances in PCA gave a strong support to calibrate the prediction models, depicting very good correlations with the reference method (correlations ∼0·90 in all cases).
Lactobacillus kefir CIDCA 8348 and JCM 5818 bind Pb(+2), Cd(+2) and Ni(+2) in an efficient way. This fact gives support for their potential use as sequestrants of traces of these metals in products addressed to human and animal consume. The prediction models developed would be useful for the determination of the investigated metal ions in unknown samples giving at the same time, structural information about this interaction. This is certainly the most important contribution of this work.
建立一种实验和分析方法，以评估基于拉曼光谱和多元分析开发简单、准确和定量模型的可行性，用于定量测定乳酸克鲁维酵母细菌表面吸附的金属离子。
从处于静止期的两种乳酸克鲁维酵母菌株的 1 毫升培养物中收获并用水超纯洗涤两次。将细菌沉淀物重悬于浓度范围为 0 至 0.9 mmol l(-1)的 Pb(+2)、Cd(+2)或 Ni(+2)溶液 1 ml 中。将悬浮液在 30°C、pH 5.5 下进一步孵育 1 小时。离心后，保留沉淀物以记录拉曼光谱，并用上清液进行 Pb(+2)、Cd(+2)和 Ni(+2)的分析测定。未用金属离子处理的微生物用作对照。对预处理后的拉曼光谱进行主成分分析 (PCA)，以评估获得的聚类是否可以与附着在细菌生物量上的金属离子浓度相关联。之后，使用偏最小二乘法 (PLS) 模型进行校准，目的是定量测定吸附在细菌表面的金属离子。根据分析测定结果，所有金属的最大结合能力 (q(max)) 达到了与其他乳酸细菌观察到的值相当的水平（约 0.200 mmol g(-1))。光谱分析表明，参与细菌/金属相互作用的主要功能基团是羧酸盐、磷酸盐和多糖。在 PCA 中，前两个主成分解释了数据结构中包含的光谱数据集的 72%以上的方差，可以清楚地区分不同浓度的样品。基于这些信息并参考分析方法的结果，成功定义了用于定量测定附着在细菌表面的 Pb(+2)、Cd(+2)和 Ni(+2)的 PLS 预测模型。
基于多元分析的方法的校准和验证允许定义用于定量测定附着在细菌表面的 Pb(+2)、Cd(+2)和 Ni(+2)的模型。PCA 中解释方差的高百分比为校准预测模型提供了有力支持，与参考方法（在所有情况下相关性约为 0.90）呈现出很好的相关性。
乳酸克鲁维酵母 CIDCA 8348 和 JCM 5818 有效地结合 Pb(+2)、Cd(+2)和 Ni(+2)。这一事实支持了它们作为这些金属痕量的螯合剂在面向人类和动物消费的产品中的潜在用途。开发的预测模型将有助于在未知样品中测定研究的金属离子，同时提供有关这种相互作用的结构信息。这无疑是这项工作的最重要贡献。
