Institute of Nutritional Science, Chair of Food Science, and Interdisciplinary Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, Giessen 35392, Germany.
Adisseo France S.A.S, Immeuble Anthony Parc 2, 10 Place du Général de Gaulle, Antony 92160, France.
J Chromatogr A. 2022 Aug 30;1679:463393. doi: 10.1016/j.chroma.2022.463393. Epub 2022 Jul 31.
The European Union has banned the use of antibiotic growth promoters in animal production, which has led to increased use of probiotic microorganisms. These feed additives result in higher costs for farmers, which is why the demand for a quality control system to quantify probiotics in feeds has increased in recent years. Imaging high-performance thin-layer chromatography (HPTLC) was proven to be a robust method for determining the probiotic Bacillus subtilis DSM 29784 strain based on the production of selective bacterial metabolites and thus characteristic metabolite pattern. However, to quantify the specific probiotic strain in the feed, identification of a strain-specific metabolite not produced by genetically very similar bacteria is necessary. Compared to five bacteria with high genetic similarity, a strain-specific metabolite was formed in the probiotic bacteria by a two-step cultivation procedure. Among others, antimicrobial properties were found for this metabolite, which indicated probiotic activity. The hyphenation of normal-phase HPTLC with reversed-phase high-performance liquid chromatography diode array detection and high-resolution mass spectrometry allowed the preliminary assignment of this strain-specific metabolite to the molecular formula CHNO (580.3527 Da). This metabolite, produced each time via an upstream cultivation process to generate the standard levels, was used for the quantification of probiotic active cells in the feed. Data on selectivity, linearity, detection limit, recovery, and precision have shown the good performance of the method.
欧盟已禁止在动物生产中使用抗生素生长促进剂，这导致了益生菌微生物的使用增加。这些饲料添加剂增加了农民的成本，这就是为什么近年来对一种用于定量饲料中益生菌的质量控制系统的需求增加了。成像高性能薄层色谱（HPTLC）已被证明是一种基于选择性细菌代谢产物的产生来确定益生菌枯草芽孢杆菌 DSM 29784 菌株的强大方法，从而具有特征代谢物模式。然而，要在饲料中定量特定的益生菌菌株，有必要鉴定不被遗传上非常相似的细菌产生的菌株特异性代谢物。与 5 种遗传相似度高的细菌相比，通过两步培养程序在益生菌细菌中形成了一种菌株特异性代谢物。该代谢物具有抗菌特性，表明具有益生菌活性。正相 HPTLC 与反相高效液相色谱二极管阵列检测和高分辨率质谱的联用允许对这种菌株特异性代谢物进行初步分配，其分子式为 CHNO（580.3527 Da）。该代谢物每次通过上游培养过程产生标准水平，用于定量饲料中的益生菌活细胞。选择性、线性、检测限、回收率和精密度的数据表明该方法具有良好的性能。
