# The Fate of _Yersinia pseudotuberculosis_ in Raw Fermented Meat Products
## 2 Materials and Methods
### Physicochemical, Microbiological, and Statistical Analyses  
The water activity and pH were measured daily. The ripening period of the dry fermented sausage was finished when the water activity reached \(\leq\)0.930, in accordance with Czech legislation [11].  
The water activity (a\({}_{\text{w}}\)) value was measured using an AquaLab 4 te Duo machine (METER Group, Pullman, WA, USA) according to ISO 21807, with a resolution of 0.0001. The pH values were measured using a pH meter WTW 3110 with a resolution of 0.001 (Xylem Inc., Rye Brook, NY, USA), equipped with a puncture glass electrode Hamilton Double Pore (Hamilton Comp., Reno, NV, USA) and calibrated using pH standards 4.0 and  
\begin{table}
\begin{tabular}{c c c} \hline
**Ingredient** & **Tewurst** & **Dry Fermented Sausage** \\ \hline Pork shoulder & 1.6 kg & 1.6 kg \\ Beef neck & - & 1.2 kg \\ Pork backfat & 2.4 kg & 1.4 kg \\ Nitrite curing salt & 88 g & 88 g \\ Seasoning mix & 32 g & 32 g \\ Starter culture & 2 g & 2 g \\ \hline Total & 4.122 kg & 4.322 \\ \hline \end{tabular}
\end{table}
Table 1: List of ingredients for the fermented sausages.  
7.0 at 25 \({}^{\circ}\)C. A temperature sensor NTC30 (Mettler Toledo, Greifensee, Switzerland) was used during the measurement of samples for automatic temperature correction.  
Before inoculation, a sample from each batch was taken and analyzed for presence of _Yersinia_ spp. (with a negative result).  
For a microbiological analysis, the sampling of the spreadable fermented sausage took place on Day 0 (raw batter after inoculation), Day 2 (final product), Day 10, Day 20, Day 30, and Day 37. The sampling of the dry fermented sausage took place on Day 0 (raw batter after inoculation), Day 2, Day 4, Day 7 (final product), Day 9, and Day 11. In the preliminary experiment, the sampling period for dry fermented sausage was up to 48 d, but since there was an absence of _Yersinia_ in the final product, we decided to shorten the intervals and monitor the changes during ripening more closely. Three samples per batch were taken on each sampling day. In total, 216 samples were analyzed.  
For the detection of _Y. pseudotuberculosisis_, 25 g of sample in a sterile blender bag with lateral filter (VWR International, Radnor, PA, USA) was homogenized in a Stomacher (SMASHER(r), bioRxivMerieux, Marcy-I'Etoile, France) with 225 mL of Peptone Sorbitol Bile (PSB) broth (M941, Himedia, India) and incubated at 30 \({}^{\circ}\)C for 24 h. The enrichment was followed by streaking on Cefsulodin-Irgasan-Novobiocin (CIN) agar (M843, Himedia, Mumbai, India) and cultivation at 30 \({}^{\circ}\)C for 24 h.  
The enumeration of _Y. pseudotuberculosisis_ was performed by homogenizing 10 g of sample with 90 mL of PSB broth. After resuscitation for 2 h at laboratory temperature, serial dilutions were made when necessary, and 0.1 mL was spread in duplicate on CIN agar and incubated at 30 \({}^{\circ}\)C for 24 h. The number of typical colonies formed was counted and reported as log CFU/g for each sample.  
The results of the analyses are reported as mean values \(\pm\) standard error of the mean (SE). A mixed model ANOVA with Tukey HSD test was applied to analyze the fixed effects and their interaction (initial concentration and days after manufacture), with the batch (A or B) as the random effect. The two types of meat products were compared in a separate model, as the comparison was possible only for Day 0 and Day 2. A level of significance of 0.05 was used. Statistical analyses were performed using the statistical program STATISTICA v. 7.1 (StatSoft, Tulsa, OK, USA). For statistical purposes, values below the limit of detection (LOD) were replaced with a value of 1.0 log CFU/g.