Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark.
Food Technology Department (DTA/IRSAT/CNRST), Ouagadougou 03 BP 7047, Burkina Faso; Université Polytechnique de Bobo-Dioulasso, 01 BP 1091 Bobo-Dioulasso, Burkina Faso.
Int J Food Microbiol. 2015 Mar 2;196:70-8. doi: 10.1016/j.ijfoodmicro.2014.11.026. Epub 2014 Nov 29.
Maari is a spontaneously fermented food condiment made from baobab tree seeds in West African countries. This type of product is considered to be safe, being consumed by millions of people on a daily basis. However, due to the spontaneous nature of the fermentation the human pathogen Bacillus cereus occasionally occurs in Maari. This study characterizes succession patterns and pathogenic potential of B. cereus isolated from the raw materials (ash, water from a drilled well (DW) and potash), seed mash throughout fermentation (0-96h), after steam cooking and sun drying (final product) from two production sites of Maari. Aerobic mesophilic bacterial (AMB) counts in raw materials were of 10(5)cfu/ml in DW, and ranged between 6.5×10(3) and 1.2×10(4)cfu/g in potash, 10(9)-10(10)cfu/g in seed mash during fermentation and 10(7) - 10(9) after sun drying. Fifty three out of total 290 AMB isolates were identified as B. cereus sensu lato by use of ITS-PCR and grouped into 3 groups using PCR fingerprinting based on Escherichia coli phage-M13 primer (M13-PCR). As determined by panC gene sequencing, the isolates of B. cereus belonged to PanC types III and IV with potential for high cytotoxicity. Phylogenetic analysis of concatenated sequences of glpF, gmk, ilvD, pta, pur, pycA and tpi revealed that the M13-PCR group 1 isolates were related to B. cereus biovar anthracis CI, while the M13-PCR group 2 isolates were identical to cereulide (emetic toxin) producing B. cereus strains. The M13-PCR group 1 isolates harboured poly-γ-D-glutamic acid capsule biosynthesis genes capA, capB and capC showing 99-100% identity with the environmental B. cereus isolate 03BB108. Presence of cesB of the cereulide synthetase gene cluster was confirmed by PCR in M13-PCR group 2 isolates. The B. cereus harbouring the cap genes were found in potash, DW, cooking water and at 8h fermentation. The "emetic" type B. cereus were present in DW, the seed mash at 48-72h of fermentation and in the final product, while the remaining isolates (PanC type IV) were detected in ash, at 48-72h fermentation and in the final product. This work sheds light on the succession and pathogenic potential of B. cereus species in traditional West African food condiment and clarifies their phylogenetic relatedness to B. cereus biovar anthracis. Future implementation of GMP and HACCP and development of starter cultures for controlled Maari fermentations will help to ensure a safe product.
玛阿里是一种由西非国家猴面包树种子制成的发酵食品调味料。这种产品被认为是安全的，每天都有数百万人食用。然而，由于发酵的自发性，人类病原体蜡样芽孢杆菌偶尔会出现在玛阿里中。本研究对来自两个玛阿里生产现场的原料（灰、钻井水（DW）和碳酸钾）、种子糊整个发酵过程（0-96 小时）、蒸汽蒸煮和晒干（最终产品）中的蜡样芽孢杆菌的分离物的演替模式和致病潜力进行了特征描述。原料中好氧嗜温细菌（AMB）的计数为 DW 中的 10(5)cfu/ml，在碳酸钾中为 6.5×10(3)到 1.2×10(4)cfu/g，在发酵过程中的种子糊中为 10(9)-10(10)cfu/g，在晒干后为 10(7) - 10(9)cfu/g。使用 ITS-PCR 从总共 290 个 AMB 分离株中鉴定出 53 株为蜡样芽孢杆菌属细菌，并用基于大肠杆菌噬菌体-M13 引物（M13-PCR）的 PCR 指纹图谱将其分为 3 组。根据 panC 基因测序，蜡样芽孢杆菌的分离株属于 PanC 型 III 和 IV，具有潜在的高细胞毒性。glpF、gmk、ilvD、pta、pur、pycA 和 tpi 基因串联序列的系统发育分析表明，M13-PCR 组 1 分离株与蜡样芽孢杆菌炭疽生物型 CI 有关，而 M13-PCR 组 2 分离株与产呕吐毒素的蜡样芽孢杆菌菌株相同。M13-PCR 组 1 分离株携带多-γ-D-谷氨酸胶囊生物合成基因 capA、capB 和 capC，与环境蜡样芽孢杆菌分离株 03BB108 具有 99-100%的同一性。在 M13-PCR 组 2 分离株中通过 PCR 证实了呕吐毒素合成酶基因簇的 cesB 的存在。在 M13-PCR 组 2 中发现了携带 cap 基因的蜡样芽孢杆菌在碳酸钾、DW、蒸煮水中和发酵 8 小时时存在。“呕吐型”蜡样芽孢杆菌存在于 DW、48-72 小时发酵的种子糊和最终产品中，而其余分离株（PanC 型 IV）则存在于灰分、48-72 小时发酵和最终产品中。这项工作阐明了传统西非食品调味料中蜡样芽孢杆菌属的演替和致病潜力，并阐明了它们与蜡样芽孢杆菌炭疽生物型的系统发育关系。未来实施 GMP 和 HACCP 以及开发用于控制玛阿里发酵的起始培养物将有助于确保产品安全。
