Department of Food Science, Federal University of Technology, Owerri, Nigeria.
Int J Food Microbiol. 2013 Mar 1;162(1):95-104. doi: 10.1016/j.ijfoodmicro.2013.01.001. Epub 2013 Jan 11.
Molecular identification of Bacillus spp. involved in the fermentation of African oil bean seeds for production of Ugba, as well as ability of the Bacillus spp. isolated to produce toxins, were investigated. Forty-nine bacteria were isolated from Ugba produced in different areas of South Eastern Nigeria and identified by phenotyping and sequencing of 16S rRNA, gyrB and rpoB genes. Genotypic diversities at interspecies and intraspecies level of the isolates were screened by PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence-based PCR (rep-PCR). The ability of the bacteria to produce toxins was also investigated by detection of genes encoding production of haemolysin BL (HblA, HblC, HblD), non-haemolytic enterotoxin (NheA, NheB, NheC), cytotoxin K (CytK) and emetic toxin (EM1) using PCR with specific primers. Moreover, a Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) was used to screen ability of the isolates to produce haemolysin in broth and during fermentation of African oil bean seeds. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. They were identified as Bacillus cereus sensu lato (42), Lysinibacillus xylanilyticus (3), Bacillus clausii (1), Bacillus licheniformis (1), Bacillus subtilis (1), and Bacillus safensis (1). B. cereus was the predominant Bacillus species and was present in all samples studied. Using ITS-PCR, interspecies diversity was observed among isolates, with six clusters representing each of the pre-cited species. Rep-PCR was more discriminatory (eight clusters) and allowed further differentiation at intraspecies level for the B. cereus and L. xylanilyticus isolates with two genotypes for each species. Genes encoding production of non-haemolytic enterotoxin (NheA, NheB, NheC) and cytotoxin K (CytK) genes were detected in all B. cereus isolates, while Hbl genes (HblA, HblC, HblD) were detected in only one isolate. The emetic-specific gene fragment was not detected in any of the isolates studied. None of the toxin genes screened was detected in isolates belonging to other Bacillus species. Using RPLA, haemolysin production was detected in one isolate of B. cereus, which showed positive amplicons for Hbl genes, both during cultivation in broth and during fermentation of oil bean seeds.
对参与非洲油豆种子发酵生产乌嘎巴的芽孢杆菌的分子鉴定，以及分离的芽孢杆菌产生毒素的能力进行了研究。从尼日利亚东南部不同地区生产的乌嘎巴中分离出 49 株细菌，通过 16S rRNA、gyrB 和 rpoB 基因的表型和测序进行鉴定。通过 PCR 扩增 16S-23S rDNA 基因间转录间隔区（ITS-PCR）和基于重复序列的 PCR（rep-PCR）筛选分离株的种间和种内基因型多样性。还使用针对产血红蛋白 BL（HblA、HblC、HblD）、非溶血肠毒素（NheA、NheB、NheC）、细胞毒素 K（CytK）和呕吐毒素（EM1）的基因的特异性引物的 PCR 检测来检测细菌产生毒素的能力。此外，还使用蜡状芽孢杆菌肠毒素反向被动乳胶凝集试验试剂盒（BCET-RPLA）来筛选分离株在肉汤和非洲油豆种子发酵过程中产生溶血素的能力。分离株的特征为运动、杆状、内生孢子形成、过氧化氢酶阳性、革兰氏阳性细菌。它们被鉴定为芽孢杆菌属（42）、解木聚糖芽孢杆菌（3）、克劳氏芽孢杆菌（1）、地衣芽孢杆菌（1）、枯草芽孢杆菌（1）和芽孢杆菌属 safensis（1）。芽孢杆菌是主要的芽孢杆菌属，存在于所有研究的样品中。使用 ITS-PCR，观察到分离株之间的种间多样性，每个预提到的物种都有六个聚类代表。rep-PCR 更具区分性（八个聚类），并允许对 B. cereus 和 L. xylanilyticus 分离株进行种内水平的进一步分化，每个物种都有两种基因型。在所有 B. cereus 分离株中均检测到编码非溶血肠毒素（NheA、NheB、NheC）和细胞毒素 K（CytK）基因的基因，而仅在一个分离株中检测到 Hbl 基因（HblA、HblC、HblD）。在研究的任何分离株中均未检测到呕吐特异性基因片段。在属于其他芽孢杆菌属的分离株中均未检测到筛选的任何毒素基因。使用 RPLA，在一株 B. cereus 分离株中检测到溶血素的产生，该分离株在肉汤培养和油豆种子发酵过程中均显示 Hbl 基因的阳性扩增子。
