Malic acid

T. reesei RUT-C30 (ATCC 56765) was purchased from ATCC (Manassas, VA) and T. atroviride Ta13 (MUT 6701) was isolated from
wheat seeds (Algeria) and deposited at the Mycotheca Universitatis
Taurinensis (MUT, Turin, Italy).
2,6-Dimethoxyphenol, 3,5-dinitrosalicylic
acid, 4-nitrophenyl butyrate, acetonitrile, bovine serum albumin,
carboxymethyl cellulose, citrate solution, citric acid, formic acid,
malic acid, methanol, Na acetate buffer, Na phosphate buffer, Na phosphate–citrate,
potato dextrose agar, trichloroacetic acid, Tris-HCl buffer, and Triton
X-100 were purchased from Merck (Darmstadt, Germany).

A pure standard solution of n‐alkanes (from n‐C7 to n‐C30) for linear retention indices (I^T) calibration and system quality control was from Merck (Milan, Italy) and prepared in toluene at the concentration of 100 mg L⁻¹.
The internal standard (IS) 1,4-dibromobenzene (from Merck, Milan, Italy) solution was prepared in toluene at a concentration of 10 g L⁻¹.
Pure reference standards used for identity confirmation of pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, 2-ketoglutaric acid, 3-hydroxybutyric acid, fumaric acid, 2-keto-3-metilvaleric acid, aspartic acid, hippuric acid, citric acid, uric acid, l-alanine, l-valine, l-leucine, l-proline, glycine, l-threonine, l-tyrosine, l-phenylalanine, l-isoleucine, l-methionine, l-cysteine, l-ornithine, l-tryptophan, xylitol, ribitol, fructose, galactose, glucose, mannitol, myo-inositol, glycerol, palmitic acid, stearic acid, and creatinine were from Merck (Milan, Italy).
Derivatizing agents O-methyl hydroxylamine hydrochloride (MOX), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and LC-grade pyridine, n-hexane, dichloromethane, and toluene used as solvents were all from Merck (Milan, Italy).

Chemical standards of the carbohydrates (fructose, glucose, sucrose, and sorbitol) and organic acids (malic, citric, fumaric, and shikimic acids) were obtained from Fluka (Buchs, Switzerland) except malic acid, which was sourced from Merck (Darmstadt, Germany). The following standards were used for the quantification of individual polyphenolic compounds: chlorogenic acid (5-caffeoylquinic acid) and (−)-epicatechin from Sigma (St. Louis, MO, USA), (+)-catechin from Roth (Karlsruhe, Germany), quercetin 3-O-glucoside, quercetin 3-O-rutinoside, and arbutin from Fluka (Buchs, Switzerland). Methanol, acetonitrile of chromatographic grade quality, and butylhydroxytoluene (BHT) were purchased from Sigma–Aldrich (Steinheim, Germany). Deionized water was obtained using the Milli-Q system (Millipore, Billerica MA, USA).

The metabolite standards were purchased from Sigma-Aldrich or Fluka (Vienna, Austria) except for malic acid which was purchased from Merck (Vienna, Austria). The metabolomics experiment also included an external calibration with a calibration mix of 133 substances and internal standardization with U¹³C-labeled yeast extract. Within every 10 injections, a blank was injected, as well as a pooled QC from extracts (all four groups represented in each: 200 µM KP1339-treated, 20 µM oxaliplatin-treated, control, control with 0.5% dimethylsulfoxid (DMSO)). We used high resolution Orbitrap MS coupled to Vanquish UPLC and with a HILIC separation, as described elsewhere [37 (link)]. MS-data were acquired with positive–negative switching and the extracted ion chromatograms were evaluated with Thermo Trace Finder, with the help of external calibration and the internal standard, absolute amounts were calculated in pmol. The normalization with total protein content resulted in pmol metabolite per µg protein.

Acetone (99.9%), glacial acetic acid (99.8%), acetonitrile (99.9%), absolute ethanol, methanol (LC-MS grade), HCl (37%), chloroform, and sodium hydroxide were obtained from PanReac (Barcelona, Spain). Folin–Ciocalteu reagent, ABTS^•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), DPPH^• (1,1-diphenyl-2-picrylhydrazyl), gallic acid, quercetin, cyanidin-3 glucoside, β-carotene, aluminium chloride, potassium bromide, potassium persulfate, sodium bicarbonate, sodium borate, sodium sulphate, sodium sulphite, cetrimonium bromide, citric acid, malic acid, and PMP (1-phenyl-3-methyl-5-pyrazolone) were acquired from Merck (Madrid, Spain). Pure compounds for the identification of monosaccharides, enzymes and electrolytes for in vitro digestion were also purchased from Merck. Bacterial strains Lactobacillus casei CECT 475 and Lactococcus lactis subsp. lactis CECT 185, as well as their respective culture media, were obtained from the Spanish Type Culture Collection (CECT) and PanReac, respectively.

The activity staining of malate dehydrogenase (MDH, New Delhi, India) was carried out by immersing the gel in 50 mL of 50 mM Tris-Cl (pH 8.5) containing 150 mg of malic acid (Merck Millipore, Burlington, MA, USA), 10 mg of nicotinamide-adenine dinucleotide (NAD) (PanReac AppliChem, Darmstadt, Germany), 10 mg of NBT (VWR Chemicals), and 2 mg of phenazine methosulfate (PMS) (PanReac AppliChem). The gel was incubated at ~22 °C until the indigo bands appeared [38 ].