Does the high biodiversity of lactococcal bacteriophages allow predictions about their different UV-C susceptibilities?

Fermentation processes can only succeed if intact and active starter cultures are present. Bacteriophages, which can lyse bacteria and thus bring entire fermentation processes to a standstill, therefore pose a major threat. Cheese production, for example, is often affected. The by-product whey can be highly contaminated with bacteriophages (≤109 plaque-forming units/mL) and in this state, further utilization is a quality and processing risk. Therefore, an orthogonal process consisting of membrane filtration followed by UV-C irradiation could be applied to eliminate bacteriophages and to generate "phage-free" whey. In order to define suitable process parameters, 11 lactococcal bacteriophages belonging to different families and genera and differing in their morphology, genome size, heat resistance, and other attributes, were screened for their UV-C resistance in whey. P369 was found to be the most resistant and could thus be well-suited as a biomarker. Starting from a 4 log unit bacteriophage reduction by membrane filtration, another 5 log unit decrease should be realized when applying a UV-C dose of 5 J/cm2. A clear correlation of UV-C sensitivity to the chosen attributes studied such as bacteriophage morphology and genome size was difficult and ambiguous, presumably because other yet unidentified parameters are important. Mutation experiments were performed with the representative bacteriophage P008 by multiple cycles of UV-C irradiation and propagation. A few mutational events were found, but could not be linked to an artificially generated UV-C resistance, indicating that the process used would probably not lose its effectiveness over time.

Whey powders are a rich source and excellent storage matrix for dairy bacteriophages.

Thirteen whey powders and 5 whey powder formulations were screened for the presence of dairy bacteriophages using a representative set of 8 acid-producing Lactococcus lactis and 5 Streptococcus thermophilus, and 8 flavour-producing Leuconostoc pseudomesenteroides and Leuconostoc mesenteroides strains. Lytic L. lactis phages were detected in all samples, while S. thermophilus and Leuconostoc phages were present in 50% or 40% of the samples, respectively. Maximal phage titers were 6×107 plaque-forming units (pfu)/g of whey powder for L. lactis phages, 1×107pfu/g for Leuconostoc phages and 1×105pfu/g for S. thermophilus phages. In total, 55 phages were isolated and characterized. Thirty one of the 33 lactococcal phages tested belonged to the wide-spread 936 phage group. In the course of this study, a PCR detection method for Leuconostoc phages (Ali et al., 2013) was adapted to new phage isolates. Furthermore, a remarkably high stability of phages in whey powder samples was documented during a long-term storage period of 4 years.

Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage.

Due to changes in the design of industrial food processing and increasing international trade, highly thermoresistant spore-forming bacteria are an emerging problem in food production. Minimally processed foods and products with extended shelf life, such as milk products, are at special risk for contamination and subsequent product damages, but information about origin and food quality related properties of highly heat-resistant spore-formers is still limited. Therefore, the aim of this study was to determine the biodiversity, heat resistance, and food quality and safety affecting characteristics of aerobic spore-formers in the dairy sector. Thus, a comprehensive panel of strains (n=467), which originated from dairy processing environments, raw materials and processed foods, was compiled. The set included isolates associated with recent food spoilage cases and product damages as well as isolates not linked to product spoilage. Identification of the isolates by means of Fourier-transform infrared spectroscopy and molecular methods revealed a large biodiversity of spore-formers, especially among the spoilage associated isolates. These could be assigned to 43 species, representing 11 genera, with Bacillus cereus s.l. and Bacillus licheniformis being predominant. A screening for isolates forming thermoresistant spores (TRS, surviving 100°C, 20 min) showed that about one third of the tested spore-formers was heat-resistant, with Bacillus subtilis and Geobacillus stearothermophilus being the prevalent species. Strains producing highly thermoresistant spores (HTRS, surviving 125°C, 30 min) were found among mesophilic as well as among thermophilic species. B. subtilis and Bacillus amyloliquefaciens were dominating the group of mesophilic HTRS, while Bacillus smithii and Geobacillus pallidus were dominating the group of thermophilic HTRS. Analysis of spoilage-related enzymes of the TRS isolates showed that mesophilic strains, belonging to the B. subtilis and B. cereus groups, were strongly proteolytic, whereas thermophilic strains displayed generally a low enzymatic activity and thus spoilage potential. Cytotoxicity was only detected in B. cereus, suggesting that the risk of food poisoning by aerobic, thermoresistant spore-formers outside of the B. cereus group is rather low.

Review: elimination of bacteriophages in whey and whey products.

As the cheese market faces strong international competition, the optimization of production processes becomes more important for the economic success of dairy companies. In dairy productions, whey from former cheese batches is frequently re-used to increase the yield, to improve the texture and to increase the nutrient value of the final product. Recycling of whey cream and particulated whey proteins is also routinely performed. Most bacteriophages, however, survive pasteurization and may re-enter the cheese manufacturing process. There is a risk that phages multiply to high numbers during the production. Contamination of whey samples with bacteriophages may cause problems in cheese factories because whey separation often leads to aerosol-borne phages and thus contamination of the factory environment. Furthermore, whey cream or whey proteins used for recycling into cheese matrices may contain thermo-resistant phages. Drained cheese whey can be contaminated with phages as high as 10(9) phages mL(-1). When whey batches are concentrated, phage titers can increase significantly by a factor of 10 hindering a complete elimination of phages. To eliminate the risk of fermentation failure during recycling of whey, whey treatments assuring an efficient reduction of phages are indispensable. This review focuses on inactivation of phages in whey by thermal treatment, ultraviolet (UV) light irradiation, and membrane filtration. Inactivation by heat is the most common procedure. However, application of heat for inactivation of thermo-resistant phages in whey is restricted due to negative effects on the functional properties of native whey proteins. Therefore an alternative strategy applying combined treatments should be favored - rather than heating the dairy product at extreme temperature/time combinations. By using membrane filtration or UV treatment in combination with thermal treatment, phage numbers in whey can be reduced sufficiently to prevent subsequent phage accumulations.

Classification of lytic bacteriophages attacking dairy Leuconostoc starter strains.

A set of 83 lytic dairy bacteriophages (phages) infecting flavor-producing mesophilic starter strains of the Leuconostoc genus was characterized, and the first in-depth taxonomic scheme was established for this phage group. Phages were obtained from different sources, i.e., from dairy samples originating from 11 German dairies (50 Leuconostoc pseudomesenteroides [Ln. pseudomesenteroides] phages, 4 Ln. mesenteroides phages) and from 3 external phage collections (17 Ln. pseudomesenteroides phages, 12 Ln. mesenteroides phages). All phages belonged to the Siphoviridae family of phages with isometric heads (diameter, 55 nm) and noncontractile tails (length, 140 nm). With the exception of one phage (i.e., phage ΦLN25), all Ln. mesenteroides phages lysed the same host strains and revealed characteristic globular baseplate appendages. Phage ΦLN25, with different Y-shaped appendages, had a unique host range. Apart from two phages (i.e., phages P792 and P793), all Ln. pseudomesenteroides phages shared the same host range and had plain baseplates without distinguishable appendages. They were further characterized by the presence or absence of a collar below the phage head or by unique tails with straight striations. Phages P792 and P793 with characteristic fluffy baseplate appendages could propagate only on other specific hosts. All Ln. mesenteroides and all Ln. pseudomesenteroides phages were members of two (host species-specific) distinct genotypes but shared a limited conserved DNA region specifying their structural genes. A PCR detection system was established and was shown to be reliable for the detection of all Leuconostoc phage types.