Postbiotics combined with Caesalpinia sappan L. extract inhibit bovine mastitis bacterial pathogens, promote anti-inflammation and in vitro wound healing property

Biological activities on inhibition of bovine mastitis-associated bacterial pathogens and bacterial biofilm, anti-inflammation, and wound healing effects of lactic acid bacteria (LAB); Lactiplantibacillus plantarum TISTR 2070 (LP) and Lacticaseibacillus casei TISTR 1340 (LC) supernatants were investigated in this study. Moreover, Caesalpinia sappan extract, and their combinations with L. plantarum (LPC) and L. casei (LCC) were determined for their biological properties. Antibacterial assays revealed that LPC and LCC exhibited the lowest MIC (0.024–3.125 mg/mL) and MBC (0.098–3.125 mg/mL) values, that showed significant antibacterial activity stronger than LP and LC. In biofilm assays, the LPC and LCC combinations (0.8–1.6 mg/mL) inhibited preformed biofilm of Streptococcus agalactiae O4 (61.97–73.49%), Staphylococcus haemolyticus (38.74–52.65%), and Streptococcus uberis (24.78–58.05%) whereas C. sappan extract alone strongly inhibited both preformed and mature biofilms of all pathogens. Cytotoxicity evaluation showed that LPC and LCC were non-toxic to bovine endothelial (CPAE) and macrophage (RAW 264.7) cells at concentration ≤ 12.5 µg/mL. Furthermore, novel co-culture of CPAE and RAW 264.7 cells was applied in this study, LPS (5 ng/mL) significantly induced nitric oxide (NO) production compared with monoculture. In this model, LPC and LCC markedly suppressed LPS-induced NO production by 60.13–77.38% and 74.64–95.30%, respectively, while C. sappan extract achieved NO inhibition by 80.33–92.79%. Furthermore, LPC and LCC significantly downregulated IL-6 and TNF-α inflammatory genes expression, with LCC showing the strongest effect. Wound healing assay demonstrated that LP, LC, C. sappan extract and the combinations promoted complete closure of wound within 12 h. Therefore, these findings indicate that the combinations of LAB postbiotics with C. sappan extract exhibit strong antibacterial, anti-biofilm, anti-inflammatory, and wound healing activities, supporting their potential as alternative therapeutics against bovine mastitis.