# Effects of Isochoric Freezing on the Quality Characteristics of Raw Bovine Milk
## 3 Results and Discussion  
### Microbiological Evaluation  
Given that raw milk is not inherently sterile, our initial steps included conducting microbiological assessments to evaluate the impact of different treatments and storage conditions on indigenous microorganisms. Psychotropic bacteria are known to be key indicators of milk spoilage. These critical spoilage species include gram-negative bacteria, such as Pseudomonas fluorescens and Pseudomonas fragi [25; 26; 27; 28]. As key indicators of milk quality, these spoilage species can be effectively assessed through total bacteria count (TPC) and _Pseudomonas_ spp. (PS) microbiological quality indexes. Table 1 shows microbial populations found in RF, HTST, and IF-treated milk samples. The initial TPC and PS of raw milk were 6.98 \(\pm\) 0.05 log CFU mL\({}^{-1}\) and 7.22 \(\pm\) 0.13 log CFU mL\({}^{-1}\), respectively. After 2 weeks, the RF sample presented higher counts for both TPC and PS, 8.26 \(\pm\) 0.19 and 8.07 \(\pm\) 0.09 log CFU mL\({}^{-1}\), respectively. Both initial and storage values were consistent with a previous study [25]. The RF samples in this study were well past their microbial shelf-life after 2 weeks (\(\geq\)5.5 log CFU mL\({}^{-1}\)), and no further analyses were performed for these samples. Immediately after HTST treatment, the milk sample reached counts below 1 log CFU mL\({}^{-1}\) (detection limit) for both TPC and PS, demonstrating HTST's effectiveness in deactivating microorganisms. Interestingly, after 2 weeks of refrigerated storage at 4 \({}^{\circ}\)C, both TPC and PS for the HTST sample had increased by more than 5 log CFU mL\({}^{-1}\). As expected, the HTST sample stored for 5 weeks at 4 \({}^{\circ}\)C contained higher microbial load levels, well above the acceptable threshold (\(\geq\)5.5 log CFU mL\({}^{-1}\)). A previous study reported similar results for refrigerated HTST treated milk [6].  
Isochoric freezing affects microbial viability by potentially disrupting non-covalent bonds and cell membranes at high pressures. Prolonged exposure to these pressures can ultimately result in cell destruction and microbial deactivation [26]. After 2 weeks, the IF (\(-5\)\({}^{\circ}\)C) treatment reduced the TPC and PS counts to 3.60 \(\pm\) 0.12 log CFU mL\({}^{-1}\) and to 4.85 \(\pm\) 0.26 log CFU mL\({}^{-1}\), respectively. The IF (\(-10\)\({}^{\circ}\)C) treatment resulted in even greater reductions in TPC and PS counts after 2 weeks, 1.26 \(\pm\) 0.24 and 3.45 \(\pm\) 0.04 log CFU mL\({}^{-1}\), respectively. After prolonged exposure to pressure, microbial counts decreased even further. After 5 weeks, IF (\(-5\)\({}^{\circ}\)C) treatment resulted in more than 5 log CFU mL\({}^{-1}\) reduction for both TPC and PS counts. For the IF (\(-10\)\({}^{\circ}\)C) treatment after 5 weeks, both TPC and PS values were below the detection limit. The positive effect of isochoric freezing on the reducing native microbial populations has been reported in a previous study [18]. A significantly higher reduction was achieved at higher pressures of IF (\(-10\)\({}^{\circ}\)C) treatment compared to IF (\(-5\)\({}^{\circ}\)C) treatment. [PERSON] and [PERSON] [27] also found that HPP at 400 MPa and 600 MPa for 10 min reduced TPC in raw milk by 2 and 5 log CFU/mL, respectively. Another study showed that HPP treated homogenized milk stored at 4\({}^{\circ}\)C had a microbiological shelf-life similar to that of pasteurized (90 \({}^{\circ}\)C for 15 s) milk [28]. This study demonstrated that within just two weeks, IF treatment significantly reduced microbial load levels below those of both RF (refrigeration) and HTST-treated milk. These findings suggest that IF treatment could extend the microbiological shelf-life beyond that of untreated and pasteurized milk.