Background and Aim: The current phytobiotic industry is struggling to determine a proper sterilization method for an herbal formula that comprises multiple plant extracts. Hence, this study aims to investigate the effects of two sterilization methods of herbal formula on phytochemical compounds and antibacterial activity against mastitis-causing bacterial isolates.

Materials and Methods: The herbal formula comprised the extracts of Piper betle leaves, Curcuma domestica, and Curcuma zanthorriza. We applied two sterilization methods – sterilization using 0.45 μM syringe filter and sterilization using an autoclave at 121°C for 15 min. After sterilization, we measured phytochemical contents (phenol and flavonoid) of the herbal formula. Using the disk diffusion method, the antibacterial susceptibility test of the sterilized herbal formula against mastitis-causing bacterial isolates was conducted. Tetracycline, erythromycin, and penicillin – common antibiotics for mastitis therapy in dairy farms – were used as standard antibiotics.

Results: Compared with autoclave sterilization, syringe filter sterilization resulted in less (p<0.05) phenolic and flavonoid contents. Against Escherichia coli and Staphylococcus aureus, the filter sterilized herbal formula (E. coli: 65.9%-73%; S. aureus: 6.2%-18.1%) markedly reduced the antibacterial activity than the autoclave-sterilized herbal formula (E. coli: 2.1%- 3%; S. aureus: 4.5%-10.7%).

Conclusion: This study establishes that autoclave sterilization of the herbal formula is the best sterilization method that exerts minimal adverse effects on the phytochemical compounds and could sustain the antibacterial efficacy against mastitis-causing bacteria. Hence, the herbal formula could be used as an alternative treatment for bovine mastitis.

Keywords: herbal extract, mastitis, phytobiotic, phytochemical compound, sterilization method.