Interactions Between Infectious Foodborne Viruses and Bacterial Biofilms Formed on Different Food Contact Surfaces.

Bacterial biofilms contribute to contamination, spoilage, persistence, and hygiene failure in the food industry, but relatively little is known about the behavior of foodborne viruses evolving in the complex communities that make up biofilm. The aim of this study was to evaluate the association between enteric viruses and biofilms on food contact surfaces. Formed biofilms of mono- and multispecies cultures were prepared on glass, stainless steel, and polystyrene coupons and 105 pfu/ml of murine norovirus, rotavirus, and hepatitis A virus were added and incubated for 15 min, 90 min, and 24 h. The data obtained clearly demonstrate that the presence of biofilms generally influences the adhesion of enteric viruses to different surfaces. Many significant increases in attachment rates were observed, particularly with rotavirus whose rate of viral infectious particles increased 7000 times in the presence of Pseudomonas fluorescens on polystyrene after 24 h of incubation and with hepatitis A virus, which seems to have an affinity for the biofilms formed by lactic acid bacteria. Murine norovirus seems to be the least influenced by the presence of biofilms with few significant increases. However, the different factors surrounding this association are unknown and seem to vary according to the viruses, the environmental conditions, and the composition of the biofilm.

Interactions between spoilage bacteria in tri-species biofilms developed under simulated meat processing conditions.

The formation of biofilms in the food industry is a major issue, as they are a frequent source of contamination of products, which can result in significant economic losses for processors through spoilage of foods or pose serious health concerns for consumers when foodborne pathogens are present. In this study, experiments were carried out using CDC Biofilm Reactors to produce biofilms on two test surfaces (polystyrene and stainless steel coupons) under a regimen for simulated meat processing conditions (SMPC). This entailed a 12 day regimen of daily cycles of filling the reactors with a meat slurry and letting stand for 16 h, followed by draining and refilling with water for an 8 h period in order to mimic a possible scenario of fluctuating periods of nutrient availability and starvation in a meat processing facility. Strains of Pseudomonas fluorescens, Lactobacillus plantarum and Leuconostoc pseudomesenteroides were used for mono and mixed cultures biofilms as they are relevant spoilage bacteria in the meat processing industry. In monoculture, the viable cell densities (CFU/cm2) of the two lactic acid bacteria species tested were higher for biofilms grown on polystyrene as compared to those obtained on stainless steel, whereas viable cell numbers in P. fluorescens monoculture were surface-independent. Synergistic interactions were demonstrated during growth of multi-species biofilms. Results from experiments where one of the 3 strains was inoculated 24 h before introduction of the other two strains showed increased levels of L. plantarum within biofilms grown on both test surfaces. The model developed here serves as a baseline to study the interactions between potential spoilage bacteria during biofilm development.

The effect of bacteriophages on the acidification of a vegetable juice medium by microencapsulated Lactobacillus plantarum.

Starter cultures are increasingly being used for the production of sauerkraut, kimchi and other fermented vegetables. The goal of this study was to determine whether the microencapsulation of a bacterial culture can prevent phage infection during vegetable fermentation. Lactobacillus plantarum HER1325 was microencapsulated in alginate beads. Some beads were used without further processing, while others were freeze-dried prior to testing. Fresh beads (diameter of 2 mm) and dried cultures of the lactobacilli (particle size of 53-1000 µm) were added to a vegetable juice medium (VJM) at 1 × 107 CFU/mL. The virulent phage HER325 was added at an initial titer of 1 × 104 PFU/mL. In the absence of phages, the pH of the vegetable juice dropped to 4.2 after 40 h of fermentation at 19 °C. In the presence of phage HER325, acidification by both the non-microencapsulated and microencapsulated starter cultures stopped after 24 h. In all assays, the alginate particles dissolved during the 40 h of VJM fermentation. When 15 g/L of calcium chloride was added to the VJM, the alginate beads did not dissolve and significant phage protection was observed. The results suggest that phage-protected microencapsulated starter cultures can be used for vegetable fermentation if means are taken to prevent them from dissolving during acidification.

Evaluation of survival of murine norovirus-1 during sauerkraut fermentation and storage under standard and low-sodium conditions.

Sodium reduction strategies have raised a few concerns in regards to possible outbreaks in unpasteurised raw fermented vegetables. Among potential outbreak agents, foodborne viruses are recognized as an important cause of food-borne illnesses. As most of them are acid-resistant, evaluation of the efficacy of lactic fermentation in inactivating enteric viruses must be considered to ensure the safety of these foods. In particular with the sodium reduction trend which could impair adequate fermentation in vegetables, we have challenged sauerkraut fermentation at a final concentration of 4 log TCID50/mL with the murine norovirus (MNV-1). Three sodium chloride concentrations (1.0%, 1.5%, 2.0%) were evaluated in spontaneous and starter fermentation of sauerkraut and were followed during fermentation and over a storage phase of 90 days. Detection of MNV-1 genetic material was carried out by real-time RT-PCR and the infectivity on cell culture. Real-time RT-PCR results showed that viral RNA was still detected after 90 day in sauerkraut under all the different conditions. Furthermore, MNV-1 viral particles were able to infect RAW cells after 90 days of storage with a non-significant viral charge reduction. Sodium reduction has a significant impact on the fermentation processing of sauerkraut but no influence on the destruction of norovirus particles or on their survival.

Antimicrobial action of hydrolyzed chitosan against spoilage yeasts and lactic acid bacteria of fermented vegetables.

The antimicrobial properties of various chitosan-lactate polymers (ranging from 0.5 to 1.2 MDa in molecular weight) against two yeasts isolated from fermented vegetables and against three lactic acid bacteria from a mixed starter for sauerkraut on methylene blue agar (MBA) and in vegetable juice medium (VJM) were investigated. Chitosan-lactate reduced the growth of all microorganisms in solid (MBA) as well as in liquid (VJM) medium. In MBA, a concentration of 5 g/liter was needed to inhibit the growth of Saccharomyces bayanus, while 1 g/liter was sufficient to inhibit the growth of Saccharomyces unisporus. Lactic acid bacteria were also inhibited in this range of concentrations. The low-molecular-weight chitosan-lactate DP3 (0.5 kDa) was most efficient in solid medium (MBA), and inhibitory activities decreased with increasing hydrolysate lengths. In liquid medium (VJM), 0.5 g of chitosan-lactate per liter reduced the growth rates for both yeasts, but 10 g/liter was insufficient to prevent yeast growth. Intermediate-molecular-weight chitosan-lactate (5 kDa) was more efficient than chitosan of low molecular weight. Native chitosan (1.2 MDa) showed no inhibition in either medium. Microscopic examination of S. unisporus Y-42 after treatment with chitosan-lactate DP25 showed agglutination of a refractive substance on the entire cell wall, suggesting an interaction between chitosan and the cell wall. When chitosanase was added to the culture media containing chitosan-lactate, refractive substances could not be observed.

Kefir consumption does not alter plasma lipid levels or cholesterol fractional synthesis rates relative to milk in hyperlipidemic men: a randomized controlled trial [ISRCTN10820810].

BACKGROUND: Fermented milk products have been shown to affect serum cholesterol concentrations in humans. Kefir, a fermented milk product, has been traditionally consumed for its potential health benefits but has to date not been studied for its hypocholesterolemic properties. METHODS: Thirteen healthy mildly hypercholesterolemic male subjects consumed a dairy supplement in randomized crossover trial for 2 periods of 4 wk each. Subjects were blinded to the dairy supplement consumed. Blood samples were collected at baseline and after 4 wk of supplementation for measurement of plasma total, low-density lipoprotein, and high-density lipoprotein cholesterol and triglyceride concentrations, as well as fatty acid profile and cholesterol synthesis rate. Fecal samples were collected at baseline and after 2 and 4 wk of supplementation for determination of fecal short chain fatty acid level and bacterial content. RESULTS: Kefir had no effect on total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglyceride concentrations nor on cholesterol fractional synthesis rates after 4 wk of supplementation. No significant change on plasma fatty acid levels was observed with diet. However, both kefir and milk increased (p < 0.05) fecal isobutyric, isovaleric and propionic acids as well as the total amount of fecal short chain fatty acids. Kefir supplementation resulted in increased fecal bacterial content in the majority of the subjects. CONCLUSIONS: Since kefir consumption did not result in lowered plasma lipid concentrations, the results of this study do not support consumption of kefir as a cholesterol-lowering agent.