Modeling the combined effect of temperature, pH, acetic and lactic acid concentrations on the growth/no growth interface of acid-tolerant Bacillus spores.

The application of minimal processing technologies has led to increased spoilage incidents in low-acid pasteurized sauces due to the outgrowth of acid-tolerant spore-forming spoilage bacteria (ATSSB). Controlling the germination and subsequent growth of ATSSB spores is vital to enhance the ambient storage stability of pasteurized sauces. This study developed and validated a set of growth/no growth (G/NG) models for spores of two ATSSB strains (Bacillus velezensis and Bacillus subtilis subsp. subtilis) isolated from pasteurized sauces. The G/NG data at two levels of temperature (22 and 30 °C) were collected in Nutrient Broth (a = 0.98 adjusted with NaCl) by a full factorial design with five equidistant levels of pH (4.4-5.6), four concentrations of total acetic acid (0.0-0.3% (w/w)), and four concentrations of total lactic acid (0.00-1.00% (w/w)). The growth, starting from heat-treated (10 min 80 °C) spores, of each strain was assessed under 160 combinations by regular optical density measurements during three months. Twelve replicates were made for each combination. The developed models demonstrate that without organic acids even the lowest pH (4.4) allows a high growth possibility of the ATSSB spores, while acetic and lactic acids exhibit a significant antibacterial activity, which can be enhanced at decreased pH. The growth starting from B. subtilis spores can be inhibited for at least three months with 1.0% (w/w) total lactic acid in the water phase at both temperatures, which was not the case for B. velezensis, while 0.3% acetic acid achieves a full inhibition on both strains at 22 °C. With a combination of 0.3% acetic acid and 0.7% lactic acid, no growth should occur in the investigated range. This research is one of the first studies exploring the feasibility of ambient storage for low-acid pasteurized sauces eliminating preservatives such as benzoic and sorbic acids, and proves the synergistic effect of decreased pH and the presence of acetic and lactic acids on inhibiting bacterial growth from ATSSB spores.