Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum – Evaluation and validation with MAP and smoked foods

Clostridium Sporogenes as Surrogate for Proteolytic C. Botulinum - Development and Validation of Extensive Growth and Growth-Boundary Model

“…The observed growth rates were not significantly different (P = 0.52). Similar results have previously been reported by several studies and also for non-proteolytic strains of C. botulinum as discussed by Koukou et al (2022). Aerobic storage or storage in modified atmospheres including oxygen does not exclude the presence of anaerobic microenvironments within products or depletion of the available oxygen by spoilage bacteria (FDA, 2021).…”

“…On the basis of the performed model evaluation (Table 6, Section 3.4) it is suggested to identify no-growth conditions with ψ-values > 2.0 for processed cheese and meat products to account for variability of data with A f -values of 1.58-1.87. The ψ-values above 2 allow variability in the environmental factors before growth occurs (Mejlholm and Dalgaard, 2009;Koukou et al, 2022). As an example, the hot filled processed cheese suggested above with 3750 ppm of water phase citric acid and a pH of 5.8 (ψ = 2.5) is predicted to prevent growth also at 30 • C (ψ = 1.6) and 35…”

“…At each sampling, 10 g of cheese were diluted 5-fold with 40 mL of sterile physiological saline (0.85%, w/v NaCl) with 0.1% Bacto Peptone (Bacto™ Peptone, 211677, BD Bioscience, San Jose, USA) and homogenized in a stomacher (Stomacher 400 Circulator, Seward Medical, London, UK) at normal speed for 60 s. Due to lack of selective culturebased media for enumeration of Clostridia (EFSA, 2005), a real-time PCR (qPCR) method was used for detection and quantification of C. sporogenes. Previous studies for non-proteolytic strains of C. botulinum in heat-treated ("sterile") fish showed non-significant statistical difference between the growth rates estimated from cell concentrations determined by qPCR or plate counts (Koukou et al, 2022). The homogenate was prepared for qPCR analysis as described below.…”

“…qPCR amplification was performed using the real-time PCR System Mx3000P™ (Agilent Technologies, Glostrup, Denmark). Species-specific primers designed by Koo et al (2019) on the 16S rRNA gene of C. sporogenes were used and their specificity was verified with DNA extracted from 23 different species of Gram positive and Gram negative bacteria as previously described (Koukou et al, 2022). Each qPCR reaction mixture had a final volume of 20 μL and contained 2 μL of template DNA, 1.6 μL of forward and 1.6 μL of reverse primers (final concentrations of 400 nM), 10 μL of DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific™, Naerum, Denmark) (final concentration 1 mM) and 4.8 μL of molecular water.…”

“…A standard curve was obtained by using purified genomic DNA from Cs-mix. DNA was extracted from six 10-fold serial dilutions (seven to one log CFU/ml) of a Cs-mix culture as previously described (Koukou et al, 2022). The linear relation between the seven bacterial concentrations, the corresponding cycle threshold (C t ) values and the dilution factor were used to determine the concentration (log (CFU/g)) of C. sporogenes during challenge testing with cheeses and the slope (α) of this relation was used to determine the amplification efficiency (E = 10 − 1/α -1) of the assay.…”