Hydroxycinnamic acid

All used reagents and solvents were analytical or higher grade and purchased from Sigma (Sigma-Aldrich GmbH, Steinheim, Germany), Alkaloid AD (Skopje, North Macedonia), Merck (Darmstadt, Germany), Fluka (Buch, Switzerland), and Kemika (Zagreb, Croatia). The solutions of hydroxybenzoic acids (protocatechuic, gentisic, gallic, vanillic, and syringic) and hydroxycinnamic acids (p-coumaric, caffeic, ferulic, sinapic, and rosmarinic) (Sigma-Aldrich GmbH, Steinheim, Germany) shown in Table 1 were dissolved in an ethanol/water mixture (80:20, by volume) to the final concentration of 1000 µM. The experiment was divided in two parts. Firstly, all phenolic acids were individually tested for antioxidant activity at the concentrations of 2.5 and 5 µM in the ORAC assay, and 100, 500, and 1000 µM in the FRAP assay. Thereafter, the phenolic acids were mixed in binary, ternary, quaternary, and quinary equimolar combinations to reach the concentrations of 5 µM for ORAC and 100, 500, and 1000 µM for the FRAP assay.

The following hydroxycinnamic acids and their derivatives were purchased from Sigma-Aldrich (Steinheim, Germany) and were used as received: 3,4-dihydroxycinnamic acid (caffeic acid, CAF), 3-methoxy-4-hydroxycinnamic acid (ferulic acid, FA), 4-hydroxycinnamic acid (p-coumaric acid, PCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid, SA), 3,4-dihydroxyhydrocinnamic acid (hydrocaffeic acid, HCA), and 3-methoxy-4-hydroxyhydrocinnamic acid (hydroferulic acid, HFA). Cyclodextrin (CD) hosts employed in the study included heptakis(2,6-di-O-methyl)-β-CD (DIMEB or DMB), heptakis(2,3,6-tri-O-methyl)-β-CD (TRIMEB or TMB), and hexakis(2,3,6-tri-O-methyl)-α-CD (TRIMEA or TMA).

An overview of the food ingredients and additives (n = 30) obtained from the food industry and used for the initial screening of substances modulating cereulide synthesis is given in Table S1. Based on the results from the initial screening, a panel of phytochemicals (n = 40) including benzene derivatives, monoterpenes, hydroxycinnamic acids and vitamins, was generated and purchased from Sigma Aldrich, (St. Louis, MO, USA) (see Table 1). The substances were dissolved in 99.9% ethanol, except sodium benzoate (dissolved in dH₂O) and menaquinone (dissolved in dimethyl sulfoxide; DMSO), according to the manufacturer’s instruction, to prepare stock solutions. The following substances were only available in liquid formulation (>95%): 4-anisaldehyde, anisole, benzaldehyde, (S)-(+)-carvone, (R)-(-)-carvone, citral, cuminaldehyde, eucalyptol, geraniol, menthol, nerol, α-phellandrene, sabinene, α-tocopherole. Working solutions were obtained by serial dilutions of stock solutions or by evaporation using a “SpeedVac miVac Duo Plus” vacuum centrifuge (Genevac Ltd., Ipswich, UK).

The analytic phenolic components as followed: resveratrol, hydroxybenzoic acids (i.e., salicylic acid, p-hydroxybenzoic, syringic acid, gallic acid, vanillic acid, 3,4-dimethoxybenzoic acid, protocatechuic acid, and gentisic acid), hydroxycinnamic acids (i.e., p-coumaric acid, o-coumaric acid, m-coumaric acid, caffeic acid, ferulic acid, sinapinic acid, and chlorogenic acid), and flavonoids (i.e., naringenin, hesperidin, naringin, kaempferol, and rutin) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. Reagent chemicals used in the present study were of analytical grade.

Luria–Bertani (LB) medium was used for cell cultivation of E. coli. Hydroxycinnamic acids and the corresponding 4-vinylphenol derivatives, including FA, p-coumaric acid, caffeic acid, 4-vinylguaiacol, and 4-vinylphenol, were purchased from Sigma–Aldrich (St. Louis, MO, United States). Isopropyl-β-D thiogalactopyranoside (IPTG) was also obtained from Sigma–Aldrich. Yeast extract and tryptone were purchased from Oxoid (Basingstoke, England). Kanamycin was provided by Amresco (Solon, OH, United States). All the organic solvents used for whole-cell catalysis and high-performance liquid chromatography (HPLC) were of HPLC grade. The other chemicals used in this study were of analytical grade and were commercially available.