Molecular fingerprinting of beneficial and harmful bacteria in breadmaking

The breadmaking process includes a variety of microorganisms, but only restricted species cause quality deterioration and spoilage of final products. Bacterial starter cultures and yeasts are intentionally added to the process, but microbial contaminants may enter the breadmaking processes via ingredients, bakery equipment and environment. Moulds are the most common spoilage organisms in bakery products, but also bacterial contaminants may occur. Lactic acid bacteria (LAB), mainly homo- and heterofermentative species of Lactobacillus, have an important role in the sourdough baking process. The use of sourdough seeds or starter cultures is state-of-the-art in bakeries working at an industrial scale, but LAB can also be found as contaminants. The tendency to avoid chemical preservatives has raised a problem in wheat breadmaking. Heat resistant Bacillus spores may survive throughout the baking process and cause rope spoilage of final bread. Ropiness is noticed as an unpleasant sweet odour similar to that of rotting melons, followed by a discoloration of the breadcrumb. Finally, the crumb looses its structure, becoming sticky and soft. The most frequently isolated rope-producing bacteria are B. subtilis and B. licheniformis. Bacillus spores are commonly found in soil. From this source they may enter the grains and dust, and finally the flour. Poor process hygiene is known to increase the number of rope-producing Bacillus spores. Effective process control and management should be able to identify beneficial bacteria and to trace spoilage organisms. Conventional identification methods are often time-consuming and not specific enough for this purpose. We applied the automated RiboPrinter® System for characterisation of LAB and rope-producing Bacillus strains in breadmaking processes. Ribotyping is a DNA fingerprinting method for analysing the relationship of the rRNA genes within the genomic DNA of the organism studied. In ribotyping, the whole bacterial genome is cut with a restriction enzyme and the resulting fragments are separated by agarose gel electrophoresis, transferred to a nylon membrane, and hybridised with a rDNA probe. The most important property of ribotyping is to characterise strains below the species level. We have successfully applied ribotyping to trace contamination routes and to detect troublesome "house types" in industrial processes. The advantages of the automated system are that the analysis is rapid and simple to carry out, effective, standardised, labour-saving, and suitable for different restriction enzymes. It allows an objective interpretation of results and the data can be stored for later use. The commercial database provided by the manufacturer now includes 4847 fingerprint patterns for bacteria. VTT Biotechnology has created its own database, in which LAB and Bacillus species have been one of the target groups. Considering the good discrimination power of ribotyping, often even better than that of partial 16S rDNA sequencing, the database created is extremely useful for reliable and rapid identification of starter cultures as well as of contaminants isolated from different stages of the breadmaking process or final products