Therapeutic alternatives for mastitis prevention have been gaining prominence to reduce antibiotic dependence and bacterial resistance, making milk production more sustainable. Mimosa tenuiflora (jurema- preta) has antimicrobial properties (Santos et al., 2022). Copaiba oil (Copaifera martii), extracted from trees of the genus Copaifera, is known for its anti-inflammatory activities in the treatment of skin diseases (Símaro et al., 2020) and is also effective in the treatment of mastitis, with an antibiotic effect and tissue repair (Oliveira et al., 2020).

The microalgae Chlorella vulgaris has antioxidant, antimicrobial, and anti-inflammatory properties, inhibit- ing bacteria that cause mastitis (Silva-Junior et al., 2020). Some pigments in microalgae, such as chloro- phyll, may have photosensitizing properties, generating reactive species or singlet oxygen, which cause cell dam- age, in a treatment known as photodynamic therapy (Campanholi et al., 2022).

Considering the problems caused by bovine mas- titis, antimicrobial photodynamic therapy (aPDT) uses light-activated photosensitizing compounds to generate reactive oxygen species, inhibiting microorganisms and biofilms, contributing to milk quality (Teichert et al., 2002). In situ studies confirm the aPDT efficacy in post-dipping (Silva Junior et al., 2019); (Rodrigues et al., 2022). The present study evaluated a new and innova- tive formulation combined with photodynamic inacti- vation of microorganisms, proposing a photosensitive disinfectant for use as post-dipping, containing natural extracts incorporated into a polymeric matrix, aiming to reduce Staphylococcus aureus contamination in the milk and teats of animals.

The formulations were considered stable in the pre- liminary and accelerated stability studies, without phase separation and with adequate incorporation of copaiba oil. The emulgel EJO maintained structural stability, coloration, and absence of phase separation for up to eight months. The formulations demonstrated desirable viscosity, consistent appearance, physical stability, and satisfactory adhesion. For texture analysis, a significant difference was observed in the variables (p<0.0001) except for elasticity. The required strength for hardness (N), compressibility (N.mm), and adhesiveness (N.mm) was higher for the EJO emulgel. In the accelerated stabil- ity test, the EJOC showed instability, possibly due to the photodegradation of C. vulgaris.

There was a significant difference for the variables analyzed regarding bioadhesion in the ex vivo skin (p<0.0001). The bioadhesiveness of the EJOC was higher, demonstrating greater compatibility with cutaneous adhesiveness Table 1.The mean values for total polyphenols were 16.976 mg EAG/L for jurema-preta and 16.1571 mg EAG/L for

C. vulgaris. The flavonoid values were 983.941 µg/mL equivalent quercetin and 134.2084 µg/mL equivalent quercetin, respectively.

The emulgels showed pseudoplastic behavior and nonlinear viscosity (non-Newtonian, n<1) of the thixotropic type, presenting a small hysteresis area of 714.60 Pa.s (EJO) and 4414.33 Pa.s (EJOC), characterized by the ability to increase the material viscosity after application of tension. EJO presented the yield stress. There was a significant difference for the K and n variables (p<0.05) Figure 1.

The emulgels showed G’>G’’ behavior, presenting an elastic nature, conferring viscoelastic properties, and presenting themselves as a structured system, with interactions between their constituents Figure 2. A dose-dependent effect was observed in citotoxicity analysis Figure 3.

Regarding the photoinactivation of these isolates in milk, the results showed significant differences between the strains (p<0.0001) and for the exposure times (p<0.0001) Figure 4, with the light exposure time of 10 minutes being more efficient for isolates 1, 4, 9, and 10.

For the in vitro evaluation against S. aureus, the mix of extracts reduced 100% microbial count at a concentration of 25 mg using light. The standard strain (S. aureus ATCC 25923) submitted and not submitted to illumination presented values of 7.90 and 8.00 Log CFU/ mL, respectively. When testing different concentrations of C. vulgaris and jurema-preta extract, the one that most inhibited growth was 12.5 mg with light, and the decimal reduction value of the control strain was 5.33 Log CFU/mL and 5.34 Log CFU/mL for both compounds. For copaiba oil, the concentration of 100 mg with light had the greatest reduction, with 4.92 Log CFU/mL. C. vulgaris has great antimicrobial potential, varying with the type of solvent and the bacterial species tested.

The extract of jurema-preta and C. vulgaris promoted bactericidal activity against the isolates in preformed biofilm Table 2. These compounds inhibit the synthesis of polysaccharides, altering the integrity of the bacterial membrane and causing the rupture of the preformed biofilms. This strategy may be promising for the development of natural treatments against bacterial infections in biofilms, especially in veterinary settings. Highlight for the lowest concentrations of tested extracts (0.78125 µg), which were efficient in breaking 70.36% and 66.62% of preformed biofilm. It was observed that the milk parameters evaluated did not present a statistically significant difference, except for the Staphylococcus count in the teats Table 3.

In the accelerated stability test, the EJOC formulation showed instability due to photodegradation. Light radiation can alter the color and odor of the emulgel components, and evaporation by heating results in a more fluid texture, which is reversed with cooling.

For texture analysis, adhesiveness favors the emulgel permanence in the teats. The emulgel cohesiveness affects the ability to maintain its integrity; however, adhesion to the skin depends more on the adhesiveness of the emulgel than on its cohesiveness. Therefore, the EJOC lack of cohesiveness does not necessarily preclude its adhesion. Cohesiveness indicates greater viscosity and structure (Oliveira et al., 2021). EJOC has higher bioadhesive properties compared to EJO, which are related to its mechanical characteristics and ability to interact with the skin surface. This ensures that the active ingredients are released at the application site, increasing contact time and, consequently, efficacy. Studies about bioadhesive gels with herbal principles, using polymers such as Carbopol and natural bioactive substances, such as oils and chlorophyll, show great therapeutic potential (Campanholi et al., 2022).

The high concentration of flavonoids and tannins in jurema-preta confers antimicrobial properties, inhibiting the growth of several bacteria, highlighting its potential as a natural antimicrobial agent (Santos et al., 2022). The values reported in the literature were: jurema-preta extract 5.4989 µg/mL of flavoids, a value lower than the present study, for total phenolics of 50.584 µg/mL (Silva et al., 2021), and for C. vulgaris of 24.95 mg/100 g for total phenolics (Miranda et al., 2001).

The materials analyzed showed pseudoplastic behavior, with adequate viscosity for topical application, facilitating spreadability and adhesion to the skin. They also showed thixotropic behavior. In oscillatory rheology, the emulgels showed an elastic predominance over viscous (G’ > G’’) at low frequencies, indicating adequate mechanical stability. The transition from elastic to viscous behavior was observed at a critical frequency. These results suggest that the formulation has cohesive and stable structures reinforced by the presence of bioactive compounds, such as polysaccharides and phenolic compounds, which contribute to its efficacy and stability (Narvaes et al., 2023).

Regarding toxicity, a dose-dependent effect was observed, i.e., with the increase in the concentration of the extracts, there was a decrease in cell viability. However, this decrease only occurred more prominently at concentrations greater than 1,500 µg/mL Figure 3. Copaiba oil is recognized for its anti-inflammatory and healing properties, and is generally safe in moderate concentrations, but high doses can cause cytotoxicity in some cells. The addition of C. vulgaris to the EJOC emulgel seems to have reduced the toxic effects, increasing cell viability at intermediate concentrations, which is in line with the evidence that C. vulgaris can increase the stability of formulations and offer cellular protection due to the presence of chlorophyll and polysaccharides, which promote tissue repair and reduce oxidative stress (Latif et al., 2021). EJOC emulgel shows advantages in cell viability at higher concentrations, making it suitable for topical applications with low toxicity. These results highlight the importance of combining plant extracts with synergistic activities, such as copaiba oil and C. vulgaris, to optimize the efficacy and safety of formulations.

All 12 isolates amplified the nuc gene, only two isolates for the hla and hlb genes, and three for the sea gene. The presence of the nuc gene is used to characterize the species S. aureus. The presence of the sea gene is essential to produce toxins and contributes to their virulence. The genes hla and hlb encode hemolysins that increase the ability of S. aureus in the host to aggravate infections such as mastitis.

Formation of biofilms by S. aureus is a challenge in the treatment of infections due to the resistance of Gram-positive bacteria. Thus, studies aiming at the bactericidal activity of different compounds against preformed biofilms become necessary. The fractions of the C. urucarana extract eradicated S. aureus in biofilms in a manner equivalent to the antibiotic vancomycin, where the extract (5 mg/mL) was able to inhibit 88.94% of the formation of the bacterial matrix of S. aureus (Nader et al., 2018). These studies demonstrate the importance of several natural extracts in the inhibition and eradication of S. aureus biofilms, indicating the development of new antibacterial agents.

It was observed that the milk parameters evaluated did not present a statistically significant difference. This suggests that the different treatments applied did not influence the composition or physicochemical quality of the milk, since the animals did not present mastitis during the application of the products. In contrast analysis, it was observed that the control treatment showed significantly lower levels of Staphylococcus contamination in the teats compared to the emulgel without illumination (p<0.001). The contrast between emugel treatments proves the efficacy of photodynamic therapy in reducing the microbial load, since the application of light generated singlet oxygen on the surface of the teats, contributing to the reduction of microorganisms (p=0.025). The SCC values obtained were lower than the limits established by IN 77 (Agricultura et al., 2018), which establishes a maximum count of 5.70 log10 (500.000 cells/mL) Table 3. As in the present study, (Silva Junior et al., 2019) demonstrated that the use of an SF hydrogel (photosensitizer) was equivalent to iodine, ensuring microbiological quality and low SCC in milk. The emulgels acted as an antiseptic product, providing adequate teat coverage and as a physical barrier. The addition of carbopol provided better adhesiveness, stability, and consistency, since it is a synthetic hydrophilic polymer derived from polyacrylic acid (Campanholi et al., 2022).

For the blood analysis, there was no significant dif- ference for the parameters evaluated (p>0.05). However, at 28 days, the animals showed a significant reduction in the values of eosinophils (p<0.024) and for the Days x Treatment interaction for the eosinophil variable (%), the lowest count was for the emulgel containing copaiba oil (EJOC) (6.67^b), followed by EJO (12.67^a) and control (9.00^a). In the present study, the values obtained were in accordance with the reference limits for eosinophils; lower values may indicate stress, acute infections, use of corticosteroids, pregnancy, lactation, or severe allergic reactions. In cases of mastitis, the immune response tends to prioritize neutrophil activation, resulting in decreased eosinophil count (Braun et al., 2021).

The emulgels tested showed adequate characteristics of stability, bioadhesiveness, and pseudoplastic behavior, making them a promising option for therapeutic applica- tions. The association of natural extracts combined with photodynamic therapy proved to be effective in reducing Staphylococcus aureus, without presenting cytotoxic effects and with anti-biofilm capacity, reinforcing its potential to fight bacterial infections. In the context of post-dipping application, the tested formulations have efficacy similar to iodine in the SCC maintenance and staphylococci con- trol, evidencing their potential as an antiseptic alternative to prevent bovine mastitis.