Identification of spore-forming bacteria contaminating ingredients for bread production: a concern for bread quality and safety?

Spore-forming bacteria are frequently isolated from raw materials of vegetable origin and they may cause spoilage and toxicity problems when these materials are used for food production. The rope spoilage of bread is associated to the presence of spores of Bacillus species in raw materials and may represent a microbiological concern relevant for bakery industries mainly under warm and humid conditions that may be frequent in Mediterranean countries. Moreover, ropy bread could also be a risk to consumer health; in fact, Bacillus species are known to cause food poisoning through the production of toxins. A relevant outbreak of rope spoilage in Southern Italy prompted this study aimed to identify spore-forming bacterial species more frequently contaminating semolina and associated to rope in bread, by using appropriate techniques for their molecular characterization and identification. A total of 176 spore-forming bacterial isolates, from 93 samples of durum wheat semolina, durum wheat grain, bread improvers, brewer's yeast and bread, were analysed by rep-PCR. Isolates representative of each of the 54 different rep-PCR profiles were identified by 16S rRNA gene sequencing and also by sequencing the gyrA or gyrB genes. Strains of the B. cereus group were further characterized by partial sequencing of the panC gene. Moreover, in order to assess the ability of all strains to cause ropy, a screening test was performed in bread slices. Our results indicated that more than 102 spores/g - amount that may cause microbial alteration in the final product - were detected in 23% of durum wheat semolina samples and in 42 % of other raw material samples. Molecular identification of isolates revealed a high bacterial diversity at the species level leading to the identification of the following species: Bacillus amyloliquefaciens, B. subtilis, B. licheniformis, B. mojavensis, B. safensis, B. oleronius, B. simplex, B. megaterium, B. pumilus, Paenibacillus peoriae, P. lautus, Paenibacillus spp., Lysinibacillus spp. and species of the B. cereus group. On the basis of partial sequencing of the panC gene, strains of the B. cereus group were included in phylogenetic groups which also include potentially dangerous strains with a cytotoxic activity and involved in food poisoning. It is also noteworthy that B. amyloliquefaciens was the species more frequently isolated and its importance for rope spoilage was recognized in this work for the first time, probably due to its previous misidentification as B. subtilis. Our results also indicated that strains of other species, mainly B. subtilis and those of the toxigenic B. cereus group, could cause rope spoilage, although different strains of the same species may behave differently. In conclusion, our results indicate that raw materials used to produce bread may represent a source of spore-forming bacteria potentially responsible for spoilage and toxicity problems, therefore more attention should be devoted to this aspect.