High pressure sensitization of heat-resistant and pathogenic foodborne spores to nisin.

Today, there is no effective non-thermal method to inactivate unwanted bacterial spores in foods. High-Pressure (HP) process has been shown to act synergistically with moderate heating and the bacteriocin nisin to inactivate spores but the mechanisms have not been elucidated. The purpose of the present work was to investigate in depth the synergy of HP and nisin on various foodborne spore species and to bring new elements of understandings. For this purpose, spores of Bacillus pumilus, B. sporothermodurans, B. licheniformis, B. weihenstephanensis, and Clostridium sp. were suspended in MES buffer, in skim milk or in a liquid medium simulating cooked ham brine and treated by HP at 500 MPa for 10 min at 50 °C or 20 °C. Nisin (20 or 50 IU/mL) was added at three different points during treatment: during HP, during and or in the plating medium of enumeration. In the latter two cases, a high synergy was observed with the inhibition of the spores of Bacillus spp. The evaluation of the germinated fraction of Bacillus spp. spores after HP revealed that this synergy was likely due to the action of nisin on HP-sensitized spores, rather than on HP-germinated spores. Thus, the combination of nisin and HP can lead to Bacillus spp. spore inhibition at 20 °C. And Nisin can act on HP-treated spores, even if they are not germinated. This paper provides new information about the inhibition of spores by the combination of HP and nisin. The high synergy observed at low temperature has not been reported yet and could allow food preservation without the use of any thermal process.

Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.

The inhibitory activities of nisin or/and potassium sorbate on spores and vegetative cells of Bacillus sporothermodurans LTIS27, which are known to be a contaminant of dairy products and to be extremely heat-resistant, were investigated. First, the tested concentrations of nisin or potassium sorbate inhibited vegetative cell growth; with the minimum inhibitory concentrations were 5 × 10(3) IU/ml and 2% (w/v), respectively. Then, the behaviour of vegetative cells and spores in presence of sub-lethal concentrations of nisin (50 UI/ml) or/and potassium sorbate (0.2%), in milk at 37 °C for 5 days, were evaluated. In the absence of inhibitors, strain grew and sporulated at the end of the exponential phase. Nisin (50 UI/ml) was able to inhibit spore outgrowth but didn't affect their germination. It induced an immediate and transitory reduction (1.6log(10) after 1 h and 2.8log(10) after 6 h of incubation) of vegetative cell growth which reappeared between 10 h and 24 h. Potassium sorbate (0.2%) had a durable bacteriostatic effect (1.1log(10) after 6 h), on vegetative cells, followed by a slower regrowth. It was able to inhibit both germination and outgrowth of spores. Association of nisin and potassium sorbate, at sub-lethal concentrations, showed a synergistic effect and resulted in a total inhibition of cells growth after 5 days. The results illustrate the efficacy of nisin and potassium sorbate in combination, and the commercial potential of applying such treatment to decontaminate any product that has a problem with persistence of bacterial spores.

Inactivation of Bacillus sporothermodurans spores by nisin and temperature studied by design of experiments in water and milk.

Spores of Bacillus sporothermodurans are known to be a contaminant of dairy products and to be extremely heat-resistant. A central composite experimental design with three factors using response surface methodology was used to evaluate the effect of nisin (50-150 UI/mL), temperature (80-100 °C), and temperature-holding time (10-20 min) on the inactivation of B. sporothermodurans LTIS27 spores in distilled water, in skim milk and in chocolate milk. The experimental values were shown to be significantly in good agreement with the values predicted by the quadratic equation since the adjusted determination coefficients (Radj(2)) were around 0.97. By analyzing the response surfaces plots, the inactivation was shown to be higher in distilled water than in skim milk under all the conditions tested. Five-log cycle reductions of B. sporothermodurans spores were obtained after a treatment at 95 °C for 12 min in presence of 125 UI of nisin/mL in distilled water or at 100 °C for 13 min in presence of 134 UI of nisin/mL in skim milk or at 100 °C for 15 min in presence of 135 UI of nisin/mL in chocolate milk. This study showed the efficiency of nisin (15-184 UI/mL) in combination with temperature (73-106 °C) to inactivate spores of B. sporothermodurans in milk.

Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology.

Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only D-glucose, L-alanine, and inosine were considered as strong independent germinants. Both inosine and L-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (L-alanine, D-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 °C in presence of 9 and 60 mM of D-glucose and L-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors.