Potassium hydroxide

Standard reagents, namely, fatty acid methyl standards, Vitamin E reference standards (d-α-, β-, γ-, and δ-tocopherol), 2,2,5,7,8-pentamethyl-6-hydroxychroman, and (E)-2-decenal, were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). A custom alkane blend standard of C6–C16 was obtained from Restek Corporation (Bellefonte, PA, USA). Hydrochloric acid was purchased from Kanto Chemical Company Limited (Tokyo, Japan). Hexane, methanol, acetic acid, isopropanol, butylated hydroxytoluene, potassium hydroxide, 2,4-dinitrophenylhydrazine (2,4-DNPH), and n-butanol were purchased from FUJIFILM Wako Pure Chemical Corporation. Among them, Hexane and isopropanol were chromatography grade and all other chemicals were analytical grade.
The camellia oil and frozen par-fried French fries were purchased in the local market (Akita, Japan). The frying process involved frying French fries at 180 °C in a restaurant-style stainless steel electric fryer (TF-20A, Taiji & Company Limited, Kanagawa, Japan) using three liters of oil. The temperature of the frying oil was kept constant at 180 °C during frying, and 75 g of French fries were fried for 3 min at 27 min intervals. This operation was repeated for 5 h per day for 5 consecutive days, with 150 mL of frying oil taken every 2.5 h, without adding additional oil to the fryer. Oil samples were stored at −18 °C before analysis.

Pyrogallol (C₆H₃(OH)₃, > 99%), sodium citrate hydrate (C₆H₅Na₃O₇·2H₂O, > 99%), polyvinpyrrolidone (PVP, > 99%), methanol (CH₃OH, > 99.8%), sodium hydroxide (NaOH, > 97%), potassium hydroxide (KOH, > 85%), lead (II) nitrate (Pb(NO₃)₂, > 99.9%), and nitric acid (HNO₃, 69%) were purchased from Wako Pure Chemical Industries, Ltd. All chemical were used as received without further purification. Deionized water with a specific resistance of 18.2 MΩ cm was obtained by reverse osmosis followed by ion exchange and filtration.

Bacterial cell components were prepared from heat-killed b240. Intact cell walls (ICW) of b240 were prepared according to the method described by Shida et al. [20] (link) with minor modifications. Briefly, heat-killed b240 was suspended in a 0.3% solution of sodium dodecyl sulfate (Wako, Osaka, Japan) and boiled for 15 min. After centrifugation, the precipitate was washed with a 2∶1 mixture of methanol (Wako) and distilled water (Otsuka, Tokyo, Japan), methanol, and acetone (Wako). The b240 was treated with actinase E (Kaken, Tokyo, Japan) and delipidated with methanol, and a 1∶1 mixture of methanol and chloroform (Wako). The delipidated material was treated with DNase I (Sigma) and RNase A (Sigma), followed by treatment with actinase E. The insoluble material was washed with distilled water (Otsuka), lyophilized, and then used as ICW. To remove sugar from the ICW, the ICW was suspended in 2% potassium hydroxide (Wako) and boiled for 1 h. potassium hydroxide-treated ICW was washed with distilled water and lyophilized, and then used as KOH-treated ICW. Neutral sugars in ICW and KOH-treated ICW were determined using the phenol-sulfuric acid method. Approximately 98% of the neutral sugars in the ICW were removed by KOH-treatment.

Hexane, methanol, acetic acid, isopropanol, butylated hydroxytoluene, potassium hydroxide, 2,4-dinitrophenylhydrazine (2,4-DNPH), and butanol were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Hydrochloric acid was purchased from Kanto Chemical Company Limited (Tokyo, Japan). Standard reagents include fatty acid methyl standards, Vitamin E reference standards (d-α-, β-, γ-, and δ-tocopherol), 2,2,5,7,8-pentamethyl-6-hydroxychroman, and (E)-2-decenal were purchased from FUJIFILM Wako Pure Chemical Corporation. Custom alkanes blend standard of C6–C16 was provided by Restek Corporation (Bellefonte, PA, USA). Hexane and isopropanol were of high-performance liquid chromatography grade and all other chemicals were of analytical grade.