Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time.

Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH.IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.

Effect of incubation temperature and pH on the recovery of Bacillus weihenstephanensis spores after exposure to a peracetic acid-based disinfectant or to pulsed light.

The recovery at a range of incubation temperatures and pH of spores of Bacillus weihenstephanensis KBAB4 exposed to a peracetic acid-based disinfectant (PABD) or to pulsed light was estimated. Spores of B. weihenstephanensis were produced at 30 °C and pH 7.00, at 30 °C and pH 5.50, or at 12 °C and pH 7.00. The spores were treated with a commercial peracetic acid-based disinfectant at 80 mg·mL-1 for 0 to 200 min at 18 °C or by pulsed light at fluences ranging between 0.4 and 2.3 J·cm-2 for pulsed light treatment. After each treatment, the spores were incubated on nutrient agar at 12 °C, 30 °C or 37 °C, or at pH 5.10, 6.00 or 7.40. Incubation temperature during recovery had a significant impact only near the recovery limits, beyond which surviving spores previously exposed to a PABD or to pulsed light were not able to form colonies. In contrast, a decrease in pH of the recovery nutrient agar had a progressive impact on the ability of spores to form colonies. The time to first log reduction after PABD treatment was 29.5 ±â€¯0.7 min with recovery at pH 7.40, and was tremendously shortened 5.1 ±â€¯0.2 min with recovery at pH 5.10. Concerning the fluence necessary for the first log reduction, it was 1.5 times higher when the spores were recovered at pH 6.00 compared to a recovery at pH 5.10. The impact of recovery temperature and pH can be described with a mathematical model using cardinal temperature and pH as parameters. These effects of temperature and pH on recovery of Bacillus weihenstephanensis spores exposed to a disinfectant combining peracetic acid and hydrogen peroxide, or pulsed light are similar, although these treatments are of different natures. Sporulation temperature or pH did not impact resistance to the peracetic acid-based disinfectant or pulsed light.

Modeling the recovery of heat-treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal temperature and pH using growth limits.

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

Antibacterial activity of lactophoricin, a synthetic 23-residues peptide derived from the sequence of bovine milk component-3 of proteose peptone.

A synthetic peptide of 23 residues corresponding to the carboxyterminal 113 to 135 region of component-3 of proteose peptone (PP3) has been investigated with regard to its antibacterial properties. This cationic amphipathic peptide that we refer to as lactophoricin, displayed a growth-inhibitory activity against both gram-positive and gram-negative bacteria. For most of the strains tested, bacterial growth was observed in the presence of lactophoricin except for Streptococcus thermophilus. In that case, lactophoricin exhibited a minimum inhibitory concentration of 10 microM and a minimum lethal concentration of 20 microM. No hemolysis of human red blood cells was detected for peptide concentrations between 2 to 200 microM, indicating that lactophoricin would be noncytotoxic when used in this concentration range.

Streptococcus thermophilus 580 produces a bacteriocin potentially suitable for inhibition of Clostridium tyrobutyricum in hard cheese.

A strain of Streptococcus thermophilus that inhibits Clostridium tyrobutyricum has been isolated from raw milk. The active compound produced disappears after a treatment with protease. However, unlike most bacteriocins, it is not thermoresistant, and the activity is completely lost after 1 h at 60 degrees C. Its inhibitory spectrum is limited to other thermophilic streptococci, Brochothrix, and sporulated gram-positive rods. So this bacteriocin could be different from those already described. This bacteriocin-producing strain could be used in thermophilic starter for hard cheese making because the bacteriocin is not active against thermophilic lactobacilli. It is produced in M17 medium during the decreasing temperature phase of the hard cheese-making process temperature cycle and is also produced in milk. Moreover, when Streptococcus thermophilus was cocultured with a Lactobacillus delbrueckii subsp. lactis starter strain, it seems to enhance the bacteriocin production. However the level of activity always decreases drastically during the stationary phase. But inhibition of Clostridium tyrobutyricum spores can be obtained in small-scale curds.