Hydrogen peroxide

Glucose (Duksan Pure Chemicals Co., Seoul, South Korea) was used as carbon source. A mixture of yeast extract (Becton, Dickinson and Company, Sparks, Unitesd States) and peptone (Becton, Dickinson, Sparks, United States) were used as nitrogen sources. The following chemicals were used without further purification: acetic acid (Duksan Pure Chemicals Co., Seoul, South Korea), sodium acetate (Sigma, Saint Louis, MO, United States), hydrogen peroxide (Duksan Pure Chemicals Co., South Korea), aniline (Junsei Chemical Co. Ltd., Tokyo, Japan), KHCF (Fisher Scientific, Loughborough, United Kingdom), AOT (Tokyo Chemical Industry Co., Tokyo, Japan). Laccase (EC 1.10.3.2.) from Myceliophthora thermophila was obtained from Novozymes (Bagsvaerd, Denmark). Citric acid monohydrate (Sigma, Saint Louis, MO, United States), sodium phosphate dibasic dehydrate (Riedel-de Haën, Seelze, Germany), and CMC (Sigma, Saint Louis, MO, United States).

The Gram staining, catalase activity, and acidification ability of each LAB isolate were assessed for phenotypic identification. Gram staining was conducted by following the method described in [18 ]. For the catalase activity of purified single isolates, 3% hydrogen peroxide (Duksan Pure Chemicals Co., Ltd., Ansan, Republic of Korea) was mixed separately on a clean microscope slide with the pure isolates. Positive reactions were evidenced by immediate effervescence (bubble formation) due to the catalase hydrolyzing the hydrogen peroxide [19 ]. The acidification ability of each purified isolate was assessed by inoculation (6 log CFU/mL) in MRS broth and incubation at 30 °C for 24 h, then measuring its pH [20 (link)].

V. vulnificus strains (MO6-24/O, Δfur, Δfur complemented with pRK415-fur, ΔkatG) were cultured overnight in LB medium, washed, and sub-cultured in fresh LB medium. All cells were harvested at exponential phase (A₆₀₀ value of approximately 1.0) and sonicated using Ultrasonic Homogenizer (KUS-650, KBT, Seongnam, Korea) in 10 cycles with 1 s of sonication followed by 2 s of rest for each cycle. After centrifugation, the supernatant was concentrated using Amicon Ultra-0.5 mL Centrifugal Filter Units (10,000 NMWL, UFC501024, Merck-Millipore, Germany). Each of 50 μL sample was mixed with 10 mM hydrogen peroxide (Duksan, Ansan, Korea). The reaction tubes were vortexed, incubated for 2 min at 37°C, and 600 μL of working solution was added. The working solution consisted of 100 mL cobalt (II) solution (20.3 g / 1 L DIW), 100 mL sodium hexametaphosphate solution (10 g / 1 L DIW), 800 mL sodium bicarbonate solution (180 g/ 2 L DIW) (Hadwan, 2018 (link)). The tubes were vortexed for 5 s and then kept at room temperature for 10 min in the dark. After 10 min, the catalase activities were measured at 440 nm using a Multimode Plate Reader (PerkinElmer, Waltham, MA, United States).

Tissues were fixed overnight in 10% formalin and processed in the tissue core facility at the Mayo Clinic. To compare with the histopathologic features of tumor tissue, both human and mouse tumor tissues were divided into 10 um sections and stained with hematoxylin and eosin (H & E). For immunohistochemistry, deparaffinized and rehydrated 10 um sections were retrieved in pH 6.0, or 9.0 citrate buffer (Agilent Technologies, Inc., Santa Clara, CA, USA). Then, the sections were inactivated of endogenous peroxidase with 3% hydrogen peroxide (Duksan, Seoul, Korea) and incubated with 5% BSA. Next, the cells were incubated with the primary antibody for 1 h at room temperature. The secondary antibody was applied for 30 min at room temperature, followed by detection using 3,3′-diaminobenzidine chromogen solution (Agilent Technologies, Inc.). The sections were mounted.

The fixed specimens were washed for 1 h in running water, and then stored in 3% potassium hydroxide (JUNSEI, Tokyo, Japan) solution. 3% aqueous potassium hydroxide solution containing 0.2 mL of 3% hydrogen peroxide (DUKSAN, Ansan, Korea) per 100 mL. The fixed specimens became partially transparent after 3 to 4 weeks of storage, during which time the potassium hydroxide solution was replaced daily.