Isolation, identification, and fermentation condition optimization for antimicrobial activity of an endophytic bacterial strain Z10 from lemon

Abstract:

[Background] In recent years, mining antimicrobial components from the fermentation products of endophytes has become an important way to discover new bioactive components. [Objective] To find new endophytes with high antimicrobial activity, explore the optimum fermentation conditions for the production of bioactive components, and decipher their antimicrobial mechanisms. [Methods] The endophytes were isolated from lemon peels and seeds by tissue culture and streaking method. The strains with strong inhibitory effects on pathogenic microorganisms were screened by filter paper and Oxford cup methods and then identified by morphological observation and 16S rRNA gene sequencing. The antimicrobial effect of the fermentation liquid extract (B02c) of the active strain was examined by the microplate method. Single factor tests and response surface experiment were carried out to optimize the fermentation conditions. Propidium iodide/SYTO-9 probe marking combined with flow cytometry were employed to measure the effect of B02c on the cell membrane of the tested strains. [Results] A strain Z10 with significant inhibitory effect on Staphylococcus aureus was isolated from lemon seeds and identified as Bacillus sp. The optimal fermentation conditions of strain Z10 were as follows:addition of orange peel residue at 1.6 g/100 mL, pH 6.8, inoculum amount of 10.0%, rotation speed of 172 r/min, and culture for 36 h. After optimization, the obtained B02c showed the inhibition rate reaching 84.36%, increased by 16.36% compared with that of the fermentation liquid obtained at the original conditions. B02c showed the MIC of 3.125 mg/mL against S. aureus and destroyed the cell membrane integrity of S. aureus at this dose. [Conclusion] B02c of the lemon endophyte Z10 under optimized fermentation conditions demonstrates a strong inhibitory effect on S. aureus, serving the screening and industrial production of new and efficient natural bioactive substances.