UV tolerance of Lactococcus lactis 936-type phages: Impact of wavelength, matrix, and pH.

Ultraviolet C (UVC) radiation is a widely used technology for the disinfection of surfaces, air flows, water and other liquids. Although extensive research has been conducted on the UV tolerance of bacteriophages used as surrogates for waterborne viruses, limited information is available on phages relevant to food processing. Phages of dairy starters may reach high numbers in dairy facilities and cause fermentation failure with great economic losses for the dairy industry. Here, the UV tolerance of virulent phages, belonging to the 936-group (Skunavirus) of Lactococcus lactis subsp. diacetylactis F7/2, was assessed, employing both host infectivity loss and qPCR assays. A highly heat-tolerant phage (P680) and a less heat-tolerant phage (P008) were exposed to UV radiation at 265 nm (UVC), 285 nm (UVB) and 365 nm (UVA), respectively, in an aqueous suspension, using UV Light-Emitting-Diodes (LEDs) in a static set-up. UVC at 265 nm achieved the highest total inactivation, leading to a 4 log10 reduction of the phage titer at a UV dose of 327 and 164 mJ/cm2 for P680 and P008, respectively. UVB at 285 nm achieved similar inactivation levels, while UVA at 365 nm did not cause major reductions. Phages were also suspended in yoghurt serum of pH 5.5 and pH 7.0 and exposed to UVC radiation at 265 nm. The heat-tolerant phage P680 was more UV tolerant for all wavelengths, matrices and pH values tested. A higher aggregation degree together with less DNA damage was observed for both phages at pH 5.5, especially for phage P680, indicating a UV light-shielding effect. Interestingly, there were indications of some phage survivors exhibiting higher UV tolerance on re-exposure, pointing out a need for further investigation. Our results show that UV LEDs emitting at 265 nm and 285 nm are efficient in reducing the phage population significantly, but also underline that 936-type phages are relatively UV resistant. A further understanding of the main factors influencing UV efficiency could enable future use of the UV technology as an alternative or complement to thermal treatment for phage inactivation.

The impact of bacterial cell aggregation on UV inactivation kinetics.

Reconditioning of food processing water streams for reuse is an increasingly common water management practice in the food industry and UV disinfection is often employed as part of the water treatment. Several factors may impact the effect of UV radiation. Here, we aim to assess the impact of cell aggregation on UV inactivation kinetics and investigate if UV exposure induces aggregation. Three strains, isolated from food processing water reuse lines (Raoultella ornithinolytica, Pseudomonas brenneri, Rothia mucilaginosa) and both an aggregating and a non-aggregating strain of Staphylococcus aureus were exposed to UVC light at 255 nm using UV LED equipment. Total Viable Count and phase-contrast microscopy, coupled with image analysis, were used to compare the UV inactivation kinetics with the average particle size for a range of UV doses. Tailing effect, seen as a strong reduction in inactivation rate, was observed for all strains at higher UV doses (industrial strains ≥ 50 or 120 mJ/cm2, S. aureus strains  ≥ 40 or 60 mJ/cm2). The naturally aggregating strains were more UV tolerant, both within and between species. When aggregates of S. aureus were broken, UV tolerance decreased. For the processing water isolates, the lowest applied UV dose (25 mJ/cm2) significantly increased the average particle size. Application of higher UV doses obtained with longer exposure times did not further increase the particle size compared with untreated samples. For the S. aureus strains, however, no consistent change in average particle size was observed due to UV. Our results demonstrate that aggregating strains have a higher degree of protection and that UV radiation induces aggregation in some, but not all bacteria. A better understanding of the mechanisms governing microbial aggregation and survival during UV treatment could help to improve UV applications and predictions of microbial inactivation.

Stress Tolerance of Yeasts Dominating Reverse Osmosis Membranes for Whey Water Treatment.

Filamentous yeast species belonging to the closely related Saprochaete clavata and Magnusiomyces spicifer were recently found to dominate biofilm communities on the retentate and permeate surface of Reverse Osmosis (RO) membranes used in a whey water treatment system after CIP (Cleaning-In-Place). Microscopy revealed that the two filamentous yeast species can cover extensive areas due to their large cell size and long hyphae formation. Representative strains from these species were here further characterized and displayed similar physiological and biochemical characteristics. Both strains tested were able to grow in twice RO-filtrated permeate water and metabolize the urea present. Little is known about the survival characteristics of these strains. Here, their tolerance toward heat (60, 70, and 80°C) and Ultraviolet light (UV-C) treatment at 255 nm using UV-LED was assessed as well as their ability to form biofilm and withstand cleaning associated stress. According to the heat tolerance experiments, the D60°C of S. clavata and M. spicifer is 16.37 min and 7.24 min, respectively, while a reduction of 3.5 to >4.5 log (CFU/mL) was ensured within 5 min at 70°C. UV-C light at a dose level 10 mJ/cm2 had little effect, while doses of 40 mJ/cm2 and upward ensured a ≥4log reduction in a static laboratory scale set-up. The biofilm forming potential of one filamentous yeast and one budding yeast, Sporopachydermia lactativora, both isolated from the same biofilm, was compared in assays employing flat-bottomed polystyrene microwells and peg lids, respectively. In these systems, employing both nutrient rich as well as nutrient poor media, only the filamentous yeast was able to create biofilm. However, on RO membrane coupons in static systems, both the budding yeast and a filamentous yeast were capable of forming single strain biofilms and when these coupons were exposed to different simulations of CIP treatments both the filamentous and budding yeast survived these. The dominance of these yeasts in some filter systems tested, their capacity to adhere and their tolerance toward relevant stresses as demonstrated here, suggest that these slow growing yeasts are well suited to initiate microbial biofouling on surfaces in low nutrient environments.

Assessing the capacity of growth, survival, and acid adaptive response of Listeria monocytogenes during storage of various cheeses and subsequent simulated gastric digestion.

Different physicochemical and microbiological characteristics of cheeses may affect Listeria monocytogenes potential to grow, survive, or exhibit an acid adaptive response during storage and digestion. The objectives of the present study were to assess: i) the survival or growth potential of L.monocytogenes on various cheeses during storage, ii) the effect of initial indigenous microbiota on pathogen growth in comparison to expected growth curves retrieved by existing predictive models, and iii) the impact of habituation on/in cheeses surfaces on the subsequent acid resistance during simulated gastric digestion. Portions of cream (Cottage and Mascarpone), soft (Anthotyros, Camembert, Mastelo®, Manouri, Mozzarella, Ricotta), and semi-hard (Edam, Halloumi, Gouda) cheeses were inoculated with ca. 100CFU/g or cm2 of L.monocytogenes and stored under vacuum or aerobic conditions at 7°C (n=4). The impact of varying (initial) levels of starter culture or indigenous spoilage microbiota on pathogen growth was evaluated by purchasing cheese packages on different dates in relation to production and expiration date (subsequently reflecting to different batches) mimicking a potential situation of cheese contamination with L.monocytogenes during retail display. Values of pH and aw were also monitored and used to simulate growth of L. monocytogenes by existing models and compare it with the observed data of the study. Survival in simulated gastric fluid (SGF) (pH1.5; HCl; max. 120min) was assessed at three time points during storage. Mascarpone, Ricotta, Mozzarella, Camembert, and Halloumi supported L.monocytogenes growth by 0.5-0.8logCFU/g or cm2per day, since low initial levels of total viable counts (TVC) (1.8-3.8logCFU/g or cm2) and high pH/aw values (ca. 6.23-6.64/0.965-0.993) were recorded. On Cottage, Anthotyros, Manouri, Mastelo®, Edam, and Gouda, the pathogen survived at populations similar or lower than the inoculation level due to the high reported competition and/or low pH/aw during storage. L. monocytogenes growth was significantly suppressed (p<0.05) on samples purchased close to expiration date (bearing high TVC), compared to those close to production date, regardless of cheese. Cheeses which supported growth of L.monocytogenes enabled higher survival in gastric acidity along their shelf-life compared to cheeses which did not support growth. However, even in the latter cheeses (i.e., Cottage, Mastelo®, Gouda), total elimination of a persisting low initial contamination was not always achieved. Such findings may provide useful evidence for assessing the risk posed by various cheeses types in relation to their compliance with food safety regulations.