L 8900

For amino acid composition analysis, the amino analysis system L-8900 (Hitachi) was employed as per the manual of operator and the reference of method (Jangra et al., 2018 (link)). In brief, a sufficient amount of active agent was hydrolyzed by 6 N HCl in a sealed ampoule at 110°C for 24 h. Afterward, the solvent was evaporated using a vacuum drying chamber (Yiheng, Shanghai) and resuspended in disodium hydrogen phosphate buffer (pH3.3). Analysis was done by the amino analysis system L-8900 (Hitachi) according to the methods provided by the manufacturer.
Lipopolysaccharide (LPS) is a component of the outer wall of Gram-negative bacterial cells (Huang and Yousef, 2014 (link); Abdelhamid and Yousef, 2019 (link)). LPS derived from E. coli 0111: B4 was purchased from Sigma. A certain amount of LPS were added to the active agent and the supernatant, respectively, then incubated for 1 h at room temperature to determine the antimicrobial activity against E. coli ATCC 25922. The change in antimicrobial activity was observed with or without LPS in the samples. In addition, the antimicrobial spectrum of the supernatant and the active agent was determined using the agar well diffusion method described in section “Antimicrobial activity determination.”

Free amino acid composition in SBPH, SBTH, SBNPH, SBPH-Ca, SBTH-Ca, and SBNPH-Ca was measured using a fully automated amino acid analyzer (L-8900, Hitachi, Japan). The 5 mg/mL samples were prepared and mixed with acetone in a ratio of 1:3 to stand for 60 min. Subsequently, the samples were centrifuged at 10,000 rpm for 5 min, and the resulting reaction solutions were subjected to rotary evaporation, followed by being redissolved with 0.02 M HCl. Finally, the samples were filtered through a 0.45-μm microporous membrane and placed in the Hitachi L-8900 amino acid analyzer for determination by post-column ninhydrin derivatization photometry (Zhu, Zhang, Kang, Zhou, & Xu, 2014 (link)).