**3. Results**

### *3.1. Central Composite Design and Response Surface*

*L. monocytogenes* log reduction (Y1) varied from no reduction to 1.74 log CFU/g, and LAB log reduction (Y2) varied from none to 2.44 log CFU/g. The Chroma value (Y3) ranged from 14.51 to 26.90 (Table 1). The analysis of variance (ANOVA) indicates that the best-fitted model (*p* < 0.0001) for *L. monocytogenes* log reduction was the quadratic model, and for LAB log reduction and meat color the best fitted model was the linear (Table 2).

**Table 2.** Summary of the regression analysis of the three responses.


For *L. monocytogenes* log reduction (Y1), the quadratic model had a significance of *p* < 0.0001 and an *R*<sup>2</sup> value of 0.9038, explaining 90.38% of the variability in the response. The similarity between the *R*<sup>2</sup> and adjusted *R*<sup>2</sup> values showed the adequacy of the model to predict the corresponding response. The resulting signal-to-noise ratio, measured by the term "adequate precision" (above 4), indicated that the model could be used to navigate the design space (Table 2). The lack of fit of the quadratic model was not significant (F = 0.26, *p* = 0.8522).

For LAB log reduction (Y2), the linear model had a significance of *p* < 0.0001, an *R*<sup>2</sup> value of 0.5774 similar to the adjusted *R*2, and a non-significant lack of fit (F = 2.43, *p* = 0.0903). However, the low *R*<sup>2</sup> value (Table 2) indicates that the model has low precision in the predictions.

The adjusted linear model for meat color (Y3) had a significance of *p* < 0.0001 with an *R*<sup>2</sup> value of 0.8002, similar to the adjusted *R*<sup>2</sup> (Table 2). The lack of fit of this model was significant (F = 6.74, *p* = 0.0026), suggesting that besides LA and UV-C, there are other factors affecting meat color that were not considered in the experimental design.

For *L. monocytogenes* and LAB log reduction, both LA and UV-C were significant factors. For color, UV-C was not a significant factor in our system. The generated equations for each response, including only the terms with statistical significance (*p* < 0.05), were as follows (Equations (2)–(4)):

$$\mathbf{Y}\_1 = -0.03381\mathbf{9} + 0.35890\,\mathbf{X}\_1 + 6.054 \times 10^{-3} \,\mathbf{X}\_2 - 0.043525 \,\mathbf{X}\_1^2 - 9.17 \times 10^{-6} \,\mathbf{X}\_2^2 \tag{2}$$

$$\text{Y}\_2 = 0.24423 + 0.24127 \,\text{X}\_1 + 2.04052 \times 10^{-3} \,\text{X}\_2 \tag{3}$$

$$\chi\_3 = 23.63579 - 1.41044 \,\text{\AA}\_1 \tag{4}$$

The 3D response surface plots allow one to visualize the response in the design space (Figure 1). Both LA and UV-C in the ranges studied have a positive effect on LM 100A1 and LAB reduction (Figure 1a,b). For *L. monocytogenes* reduction the 3D response surface plot reflects a curvature according to the quadratic terms in the equation model (Figure 1a). The LAB 3D response surface plot does not present curvature (Figure 1b).

**Figure 1.** 3D response surface plots generated from the Central Composite design showing: (**a**) effect of lactic acid concentration and UV-C dose on *L. monocytogenes* reduction (log CFU/g); (**b**) effect of lactic acid concentration and UV-C dose on LAB reduction (log CFU/g); (**c**) effect of lactic acid concentration and UV-C dose on meat color (expressed as Chroma value).

For Chroma value according to Equation (4), the 3D response surface plot showed no changes in Chroma value due to UV-C and a negative effect by LA (Figure 1c).

### *3.2. Optimization and Model Validation*

Based on the model generated using the Design Expert software with a desirability factor close to 1, the optimal conditions that satisfy the constraints applied (maximize *L. monocytogenes* reduction; maximize LAB reduction; Chroma value > 20) were: 2.6% lactic acid solution and UV-C dose of 330 mJ/cm2. Using these conditions, the model predicted a *L. monocytogenes* reduction of 1.55 ± 0.41 log CFU/g and a LAB reduction of 1.55 ± 1.15 log CFU/g.

Experimental responses using the optimal conditions to treat meat samples were compared to the predicted results from the fitted models to evaluate the precision of the polynomial equations. The experimental values for *L. monocytogenes* and LAB reduction were 1.24 ± 0.18 log CFU/g and 1.20 ± 0.20 log CFU/g respectively. Both *L. monocytogenes* and LAB reduction experimental values were within the 95% CI of the predicted outcome by the models.

#### *3.3. Evolution of L. monocytogenes, LAB, pH and Meat Color Treated with 2.6% of LA and 330 mJ/cm<sup>2</sup> of UV-C Dose Vacuum Packed and Stored at 4* ◦*C for 8 Weeks* 3.3.1. *L. monocytogenes* and LAB Counts

Application of 2.6% of LA and 330 mJ/cm<sup>2</sup> of UV-C reduced *L. monocytogenes* and LAB initial log counts by 1.2 and 1.3 log compared to control. Treated meat samples had *L. monocytogenes* and LAB log counts significantly lower (*p* < 0.05) than the control samples throughout the 8 weeks (Figure 2a,b). *L. monocytogenes* counts in LA/UV-C treated meat decreased from 3.6 log to 3.0, while in control samples an increase from 4.86 to 7.38 log CFU/g was observed (Figure 2a). LAB counts in treated samples remained constant until week 4 (*p* > 0.05), then an increase was observed at week 6, reaching 6.89 log CFU/g in week 8. In control samples, LAB counts remained unchanged during the first two weeks, and then increased over time up to 7.85 log CFU/g (Figure 2b).

**Figure 2.** Bacterial count evolution in vacuum packed meats stored at 4 ◦C (**a**) *L. monocytogenes* (LM) (**b**) Lactic Acid Bacteria (LAB). Light grey and dark grey represent samples treated with 2.6% of LA/330 mJ/cm<sup>2</sup> of UV-C and no treatment control, respectively. Mean ± SD (*n* = 3) of the values are presented. Different capital letters indicate significant differences at *p* ≤ 0.05 among the means over time for each treatment, and different small letters indicate significant differences at *p* ≤ 0.05 between control and treated samples for each time point.

### 3.3.2. pH Values

LA/UV-C treatment decreased (*p* < 0.05) superficial meat pH from 5.78 to 3.70. Then, the treated sample's pH increased, reaching 5.29 at week 2. After week 2, the superficial pH

of treated and untreated samples decreased over time. The pH value of LA/UV-C treated meat was always lower (*p* < 0.05) than the control. The final pH for control samples was 5.55 and for treated samples was 5.07 (Figure 3).

**Figure 3.** Superficial pH evolution in vacuum packed meats stored at 4 ◦C. Light grey and dark grey represent samples treated (2.6% of LA and 330 mJ/cm<sup>2</sup> of UV-C) and control, respectively. Mean ± SD (*n* = 3) of the values are presented. Different letters indicate a significant difference at *p* ≤ 0.05 across time for each treatment.

### 3.3.3. Color Measurements

Changes in CIE L\*, a\*, b\* and Chroma values (C\*) at weeks 0 and 8 are shown in Table 3. At the initial time, L\*, b\* and C\* values of LA/UV-C treated and untreated meat did not show variations from each other (*p* > 0.05); the a\* value of control meat was higher (*p* < 0.05) than the value of LA/UV-C treated meat. At week 8, treated and untreated meat had non-significant differences in b\* and C\* values. However, the L\* and a\* values of LA/UV-C treated meat were lower (*p* < 0.05) than the control.

**Table 3.** Instrumental color parameters (L\*, a\*, b\*) measured and Chroma value (C\*) treated LA/UV-C and control meat samples at initial time and at 8 weeks.


Different superscripts within a row show significant results (*p* ≤ 0.05). Data recorded as Mean ± Standard Deviation.

For both control and LA/UV-C treated meat, CIE L\*, a\*, b\* and C\* values were lower (*p* < 0.05) at 8 weeks compared to values at week 0, except the L\* value of control samples that showed no significant change (*p* > 0.05).
