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10 protocols using malic acid

1

Anaerobic Bacterial Growth Optimization

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The bacterial species Desulfovibrio desulfuricans was purchased from ATCC (#27774). The bacterial species Desulfovibrio piger was purchased from ATCC (#29098). The vial was handled and opened per ATCC instructions for anaerobic bacteria and cells were grown in Desulfovibrio media described previously (21 (link)). Media was composed of NH4Cl (1 g/L) (Fisher Chemical), Na2SO4 (2 g/L) (Fisher Chemical), Na2S2O3•5H2O (1 g/L) (Sigma), MgSO4•7H2O (1 g/L) (Fisher Chemical), CaCl2•2H2O (0.1 g/L) (Fisher Chemical), KH2PO4 (0.5 g/L) (Fisher Bioreagents), Yeast Extract (1 g/L) (Amresco), Resazurin (0.5 mL/L) (Sigma), cysteine (0.6 g/L) (Sigma), DTT (0.6 g/L) (Sigma), NaHCO3 (1 g/L) (Fisher Chemical), pyruvic acid (3 g/L) (Acros Organics), malic acid (3 g/L) (Acros Organics), ATCC Trace Mineral Mix (10 mL/L), ATCC Vitamin Mix (10 mL/L) and adjusted to pH of 7.2. Bacteria were grown for 48 hrs in an anaerobic chamber (Coy Labs) and stored in growth media containing 25% glycerol at 70°C. 2.5×108 bacterial CFUs were added to 250 μL of drinking water of mice for 1 week.
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2

Evaluating Matrix and Time Effects on Analyte Recovery

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Other parameters were studied to evaluate the susceptibility of the method to changes that might occur during routine analysis (use a different matrix or an extended extraction time). The matrix effect was evaluated using two different matrices mimicking a wine and a vinegar, respectively, by adding the same volatile compounds under evaluation at an intermediate concentration. Apart from volatile compounds, the synthetic wine comprises ethanol (12% by volume, Fisher, 99.8%), tartaric acid (5 g/L, Sigma, 99.5%), glycerol (7.5 g/L, Himedia, 99.5%), and malic acid (2 g/L, Acros Organics, 99%). Synthetic vinegar was prepared using 10 g/L of citric acid (Panreac, 99.5%) and 50 g/L of acetic acid (Sigma). Three replicates were carried out for each matrix. The effect of the change of the duration of the extraction time was also evaluated. Accordingly, three replicates of the extraction procedure were done by stirring the sample for 30 min instead of 15 min of the proposed method. Recovery (Rec) of target compounds, expressed as a percentage, was evaluated by calculating the measured concentration ( Cmeasur ) vs. the expected concentration ( Cexpect ), as stated in Equation (4).
Rec%=CmeasurCexpect×100
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3

Metabolomic Analysis of Diverse Compounds

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Sucrose, glucose, fructose, mannose, galactose, malic acid, inositol, citric acid, quinic acid, benzoic acid, fumaric acid, glycine, alanine, valine, leucine, isoleucine, theronine, proline, glutamine, methionine, cystein, histidine, tyrosine, arginine, lysine, asparagine, aspartic acid, phenylalanine, glutamic acid, serine, threonine, tyrosine, γ-aminobutyric acid, methoxyamine hydrochloride solution (MOX) in pyridine (2%), N-methyl-(N-trimethylsilyl) trifluoracetamide (MSTFA), methylchloroformate (MCF), sodium hydroxide, pyridine, methanol, N,N-dimethylformamide, chloroform, sodium bicarbonate, and sodium ethylenediaminetetraacetic acid (EDTA) were purchased from Fisher Scientific (Pittsburg, PA, USA). Hydrindantin, ninhydrin, lithium hydroxide, and N-methyl-N- [tert-butyl dimethylsilyl]-trifluroacetamide (MTBSTFA), and amino acid standard mix were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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4

Silicate Glass Characterization

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The three silicate glasses (Glass A, B, and C) in this paper were from Corning® Gorilla® Glass families with silica content of 63.76, 67.37, and 70.9 mol%, respectively. Glass A and B are denoted as Glass 4 and Glass 1 in US Patent 20180282201A121 . Glass C is denoted as Ex. H in US Patent US10259746B2 listed in Supplementary Table S129 . ACS reagent grade chemicals were purchased from Fisher Scientific Inc. including citric acid anhydrous, malic acid, lactic acid, tartaric acid, 37% hydrochloric acid, and Iron(III) chloride hexahydrate, aluminum chloride, as well as other alkali metal chlorides, and alkaline earth metal chlorides evaluated in this work. Sodium hydroxide (NaOH) of 50 wt% solution was acquired from Fisher Scientific Inc. and further diluted to 10 wt% for leaching layer removal. DI water of 15 MΩ cm−1 was employed for solution preparation, glass rinsing and cleaning.
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5

Quantitative Analysis of Sugars and Organic Acids

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Submicron-filtered HPLC-grade water was used. D-glucose, D-fructose, maltose, maltotriose, and sulfuric acid purchased from Sigma-Aldrich (St Louis, MO, USA) were used. HPLC-grade lactic acid, acetic acid, succinic acid, citric acid, malic acid, tartaric acid, propionic acid, ethanol, and acetonitrile purchased from Fisher (Pittsburgh, PA, USA) were used.
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6

Formulation and Stability of Rebaudioside A Beverage

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A mixture of 300 g deuterium oxide (99% D) (Sigma Aldrich, Saint Louis, MO, USA), 540 mg rebaudioside A, 135 mg citric acid, 27 mg malic acid, 54 mg potassium sorbate, and 27 mg sodium benzoate (Fisher Scientific, Waltham, MA, USA) was prepared in a 250 mL bottle (pH = 3.5). Dry nitrogen was purged to remove oxygen, and the sample was stored at 35 °C for 6 weeks. Samples were prepared aseptically in a climate-controlled hood and monitored for microbial growth during storage.
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7

Determination of Phytochemicals in Fruits

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Only chemicals of analytical and HPLC grade were used. Standards (ascorbic acid, citric acid, dehydroascorbic acid, malic acid, tartaric acid, gallic acid, (+)-catechin, (-)-epicatechin, procyanidin B 2 , (-)-epigallocatechin-3-Ogallate,, 5-hydroxymethylfurfural), formic acid, sodium carbonate, iron (III) chloride anhydrous, hydrochloric acid and sodium acetate trihydrate were purchased from Fisher Scientific (UK); Folin-Ciocalteu phenol reagent, potassium dihydrogen phosphate (KH 2 PO 4 ), potassium persulphate, ferric chloride hexahydrate, 2,4,6-tris-2pyridyl-s-triazine (TPTZ), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1picrylhydrazyl (DPPH) and metaphosphoric acid (HPO 3 ) were from Sigma (USA); 6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid (Trolox) was from Calbiochem (Germany); acetonitrile, ethanol and methanol were from BDH chemicals (UK).
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8

Natural Deep Eutectic Solvents Extraction

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BSG or MD biomass were obtained from Alken-Maes brewery (Alken, Belgium) and immediately dried at 65 °C until the residual water content was approximately at or below 5 wt%. Malic acid, glycerol, and choline chloride, used to prepare NADES formulations, were obtained from Thermo Fischer Scientific (Waltham, MA, USA). For the HPLC analysis, ferulic acid (FER; Thermo Scientific, Waltham, MA, USA), caffeic acid (CAFF; Thermo Scientific, Waltham, MA, USA), p-coumaric acid (COUM; Thermo Scientific, Waltham, MA, USA), and syringaldehyde (SYR; TCI chemicals, Tokyo, Japan) were used as standards for prevalent phenolic compounds. LC-grade formic acid (LiChropur; Merck, Darmstadt, Germany) was used to acidify the samples for HPLC analysis. Analytical reagent grade acetone (AnalaR NORMAPUR) and analytical reagent grade methanol (HiPerSolv CHROMANORM for LC-MS) were obtained from VWR (Radnor, PA, USA). Ultrapure (UP) water (18 MΩ·cm) was obtained using an Arium Pro System (Sartorius, Göttingen, Germany). Fresh UP water was prepared every day to prevent impurities.
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9

Curcuminoid Extraction and Characterization

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The synthetic phytochemical curcumin (purity > 97%) and bisdemethoxycurcumin (purity > 98%) were bought from TCI (Eschborn, Germany), demethoxycurcumin (purity > 98%) from Sigma-Aldrich (Darmstadt, Germany). The dried rhizome powder of C. longa was purchased from Kwizda (Linz, Austria).
The hydrogen bond acceptors acetylcholine chloride (AcCh, purity > 99%), choline chloride (ChCl, purity > 99%) were bought from Sigma-Aldrich (Darmstadt, Germany) and proline (Pro, purity > 99%) was bought from TCI (Eschborn, Germany). The hydrogen bond donors citric acid (Cit, purity > 99.5%), lactic acid (Lac, purity > 85%, FCC), levulinic acid (Lev, purity > 98%) were obtained from Sigma-Aldrich (Darmstadt, Germany), malic acid (Mal, purity > 99%) from Alfa Aesar (Thermo Fisher GmbH, Kandel, Germany), and oxalic acid dihydrate (Ox, purity > 99%) from Merck (Darmstadt, Germany). To see the corresponding structures and abbreviations of the hydrogen bond donors and acceptors, regard Table A1 of the Appendix A.
The solvents ethanol (EtOH, purity > 99%), acetonitrile (purity > 99%, HPLC grade), acetic acid (purity > 99%), and triacetin (TriA, purity > 99%, food-grade) were purchased from Merck (Darmstadt, Germany). Water was deionized using a Millipore Milli-Q purification system (Merck Millipore, Billerica, MA, USA).
All chemicals were used without further purification.
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10

Comprehensive Wine Quality Analysis

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Conventional enological parameters were determined for the bottled wines after one month.
Alcohol content was measured using an Alcoquick L200 infralyser (Unisensor, Karlsruhe, Germany) and pH with a Titromatic pHmeter (Hachlange, Düsseldorf, Germany). Titratable (?) acidity was measured according to the OIV method (2009) . A Konelab Arena 20 sequential analyzer (Thermo Electron Corporation, Waltham, USA) using enzyme kits provided by several suppliers was used to determine volatile acidity (Megazyme, Wicklow, Ireland) and malic acid (Thermo Fisher Scientific, Waltham, USA). Potassium determination was done by flame photometry (Bio Arrow, France) according to the OIV method ( 2009) and tartaric acid determination by colorimetric titration (Hill and Caputi 2009) . Anthocyanins and the Total Phenolic Index (TPI) were quantified according to the techniques described by Ribéreau-Gayon and Stonestreet (1965) and Ribéreau-Gayon (1970) , respectively, using an Evolution 100 spectrophotometer (Thermo Electron Corporation, Waltham, USA). Absorbance was measured at 420, 520 and 620 nm and Color Hue and Color Intensity were calculated by the A420/A520 ratio and by summing the three color components (A420-yellow, A520-red, and A620-blue), respectively. All determinations were carried out in duplicate.
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