# Effects of Isochoric Freezing on the Quality Characteristics of Raw Bovine Milk
## 3 Results and Discussion
### Protein Content and SDS-PAGE  
The total protein content of all samples was determined using the Lowry protein assay. The initial total protein content of raw milk was 32.16 \(\pm\) 3.33 mg/mL, similar to values reported in reference [44]. The total protein content in HTST treated milk showed an overall significant decrease in value to 23.53 \(\pm\) 2.33 mg/mL, which was attributed to an increase  
Figure 3: Effects of refrigeration (RF), HTST, and isochoric freezing (IF) on lipid oxidation (\(\mathrm{\SIUnitSymbolMicro g}\) MDA/mL) of raw bovine milk during storage. Values are the means of data sets, and the standard deviations are indicated by the vertical error bars. a, b: Different letters for the same parameter indicate significantly different means (ANOVA; \(p<0.05\)). Figure 2: Effects of refrigeration (RF), HTST, and isochoric freezing (IF) on the viscosity values of raw bovine milk during storage. Values are the means of data sets, and the standard deviations are indicated by the vertical error bars. a, b, c, d: Different letters for the same parameter indicate significantly different means (ANOVA; \(p<0.05\)). in proteolytic activity produced by the microflora of milk. In comparison, the protein contents after IF treatments showed no significant changes when compared with the initial values and remained stable throughout the storage period. The total protein contents of the IF (\(-5\)\({}^{\circ}\)C) sample were 30.57 \(\pm\) 0.20 mg/mL after 2 weeks and 30.77 \(\pm\) 0.42 mg/mL after 5 weeks. Furthermore, the total protein contents of the IF (\(-10\)\({}^{\circ}\)C) sample were 30.50 \(\pm\) 0.66 mg/mL after 2 weeks and 32.17 \(\pm\) 0.46 mg/mL after 5 weeks (Figure 4). A previous study also found that 60 days of hyperbaric storage at a pressures of 75-100 MPA did not affect the protein contents in milk [12]. Figure 5 shows the SDS-PAGE results for the milk samples under native (A) and reducing (B) conditions. The protein bands for RF milk at week 2 and HTST-treated milk at week 5 appeared with low intensity, indicating a decrease in soluble protein content attributed to an increase in the microbial load levels (Table 1). Surprisingly, under native conditions, all samples showed large molecular weight proteins near the top that could not diffuse into the gel as well as in the absence of whey proteins. The \(\alpha\)-casein and \(\beta\)-casein seemed to be unaffected by HTST treatment. Under reducing conditions, all the large molecular weight proteins disappeared, and the \(\kappa\)-casein and whey proteins appeared in the gels. This pattern was also previously observed in heat-treated bovine milk [45]. The large aggregates that could not diffuse into the gels were predominantly composed of disulfide-bonded \(\alpha\)-la, \(\beta\)-lg, and \(\kappa\)-casein [46; 47]. The IF (\(-5\)\({}^{\circ}\)C) and IF (\(-10\)\({}^{\circ}\)C) samples also displayed a lower intensity for the casein bands under native conditions when compared to fresh milk. However, these bands became visible in the gels after the application of the reducing reagent. The generation of aggregates following high pressure treatments was previously reported by [PERSON] et al. [48]. \(\beta\)-lg is the most sensitive whey protein to pressure and can undergo denaturation under certain pressure [48; 49]. Previous studies have found that pressure treatments between 50 and 100 MPa led to a predenatured state of \(\beta\)-lg, which can be reversible [50]. Higher pressure treatments, such as 400 MPa, can induce intermolecular-disulfide bonds between \(\beta\)-lg and \(k\)-casein. HPP treatment with longer durations or higher pressures can also induce interactions between \(\beta\)-lg and casein micelles that lead to sedimentation [40]. Under reducing conditions, the casein proteins appeared as sharp bands, suggesting that the denatured proteins did not sediment out of the solution. [PERSON] et al. [40] reported that the level of sedimentable denatured \(\beta\)-lg increases with pressures greater than 100 MPa.