Nutrient broth

All chemical reagents were of analytical grade, used as supplied without further purification unless indicated. Pyrrole, KH₂PO₄, H₃PO4, lysozyme, sodium dodecyl sulfate (SDS), and NaOH were purchased from Wako Pure Chemical Industries (Japan). Nutrient broth was purchased from Eiken Chemical (Japan). Bacterial viability kit L7007 (SYTO9/PI) was purchased from Molecular Probes. Bacteria such as E. coli O157:H7 and E. coli O26:H11 were provided by Prof. Miyake in Osaka Prefecture University. On the other hand, Salmonella enterica, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis were purchased from the Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Japan. Milli-Q grade (>18 MΩ) water with ultraviolet sterilization was used throughout the study.

A commercially pure, medical-grade Ti plate (ISO5832-2) (Ti > 99.5%) was provided by Nilaco Co., Tokyo, Japan. Ti-6Al-4V alloy plate (Ti = balance, Al = 6.18, V = 4.27 mass%) and Ti-15Zr-4Nb-4Ta alloy plate (Ti = balance, Zr = 14.51, Nb = 3.83, Ta = 3.94, Pd = 0.16, and O = 0.25 mass%) were supplied by Kobelco Research Institute, Inc., Hyogo, Japan.
The chemical reagents (NaOH, CaCl₂, ICl₃, ICl, NaI and povidone iodine (PVP-I)) used for surface treatment were reagent-grade and purchased from Kanto Chemical Co., Inc., Tokyo, Japan. Reagent-grade NaCl, NaHCO₃, KCl, K₂HPO₄·3H₂O, MgCl₂·6H₂O, CaCl₂, Na₂SO₄, tris-hydroxymethylaminomethane (CH₂OH)₃CNH₂, and 1 M HCl were purchased from Nacalai Tesque, Inc., Kyoto, Japan, and used for the preparation of simulated body fluid (SBF). Minimum essential medium (MEM) was obtained from Gibco, Thermo Fisher Scientific, Waltham, MA, USA, and used for the cell culture test. Nutrient broth (Eiken Chemical Co., Ltd. Tochigi, Japan) and RPMI 1640 (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) were used for the antibacterial activity test.

V. parahaemolyticus was isolated from the muscle (edible parts), gill and viscera collected from individual Japanese horse mackerel with scissors aseptically. Bacteria were isolated by a qualitative test method recommended by the National Institute of Health Sciences (http://www.nihs.go.jp/fhm/mmef/pdf/protocol/NIHSJ-06_ST4%20201607.pdf). The separation and identification processes are described briefly as follows. Each collected part was diluted 10-fold with alkaline peptone broth (Nissui Pharmaceutical, Co., Ltd., Tokyo Japan) and cultured at 35°C for 18 hours without shaking. A portion of the culture was then spread on CHROMagar (BD Japan, Tokyo, Japan). Vibrio culture medium, and typical light purple colonies were picked up as suspected colonies. Characterization and identification of isolates were performed using 1% NaCl-added TSI culture medium (Nissui Pharmaceutical, Co., Ltd.), 1% NaCl-added LIM culture medium (Nissui Pharmaceutical, Co., Ltd.), Nutrient Broth (BD Japan), 8% NaCl-added Nutrient Broth, 2% NaCl-added VP semi-solid medium (Eiken Chemical, Co., Ltd., Tokyo, Japan) and cytochrome oxidase test filter paper (Nissui Pharmaceutical, Co., Ltd.).