Supernatants obtained from centrifuged cultures were filtered through 0.2 μm polytetrafluoroethylene (PTFE) filters (VWR, Barcelona, Spain). BA were derivatized with diethyl ethoxymethylenemalonate (DEEMM) (Sigma–Aldrich). 100 μl of sample were mixed with 175 μl of 1 M borate buffer (1 M boric acid neutralized with NaOH until pH 9.0), 75 μl of methanol (Merck), 2 μl of L -2-aminoadipic acid as internal standard (2 g/L) (Sigma–Aldrich) and 3 μl of DEEMM. The mixture was incubated at 30°C in an ultrasound bath (Selecta, Barcelona, Spain) for 45 min. Samples were then heated at 70°C for 2 h to allow the complete degradation of excess DEEMM and reagent by-products. Samples were filtered through 0.2 μm PTFE membranes (VWR) before injection into the chromatograph system. Samples were diluted, when necessary, with 0.1 N HCl (Merck). BA were separated and quantified by ultra high performance liquid chromatography (UHPLC) system (Waters, Milford, MA, United States) with an UPLC®BEH C18 1.7 μm column (Waters), following the method previously described (Redruello et al., 2013 (link)). Empower 2 software (Waters) was used to control the system and to analyze the data. Standards were prepared with agmatine, putrescine dihydrochloride (Acros Organics, Geel, Belgium), tyrosine and tyramine in Milli-Q water. The BA concentrations provided are the average of three independent cultures.
Deemm
Manufactured by Merck Group
Sourced in United States
DEEMM is a reagent used in analytical chemistry and biochemistry laboratories. It is a diethyl ester of maleic acid and is primarily used for the derivatization of amino groups in proteins and peptides prior to analysis, such as by mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Empower 2, by Waters Corporation (2 mentions)
L 2 aminoadipic acid, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Ptfe filter, by Avantor (1 mentions)
Uplc beh c18 1.7 μm column, by Waters Corporation (1 mentions)
Uhplc system, by Waters Corporation (1 mentions)
Hplc grade water, by Merck Group (1 mentions)
Bicinchoninic acid (bca), by Merck Group (1 mentions)
Amino acid standard, by Merck Group (1 mentions)
Hplc grade acetonitrile, by Merck Group (1 mentions)
Ptfe membrane, by Avantor (1 mentions)
234 auto injector, by Gilson (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Acquity uplc h class system, by Waters Corporation (1 mentions)
Acquity uplc beh c18 1.7 m column, by Waters Corporation (1 mentions)
Amino acid, by Merck Group (1 mentions)
Hplc grade acetonitrile, by Avantor (1 mentions)
Sodium hydroxide (naoh), by Avantor (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
7 protocols using deemm
1
Quantitative Analysis of Biogenic Amines
2
Quantification of Fecal Glutamate and GABA
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Glutamate and GABA concentrations in feces and colonic contents were determined by high performance liquid chromatography (HPLC) coupled with a photodiode-array (PDA) detector (HPLC-PDA) on fecal waters following diethyl ethoxymethylenemalonate (DEEMM, Sigma-Aldrich) derivatization according to previously described procedures (19 (link)). First, samples were weighed and diluted with 20 volumes of PBS, using Stomacher for homogenization. Then, free amines and amino acids were first extracted using acidification with hydrochloric acid and filtrated using 3-kDa cutoff Amicon filtering units (Millipore-Merck). Finally, extracted samples were subjected to DEEMM derivatization and analyzed by HPLC-PDA as previously described.
Differences between male and female animals were first assessed statistically using two-way ANOVA, and when no differences were detected, treatment groups including both genders were compared using one-way ANOVA.
Differences between male and female animals were first assessed statistically using two-way ANOVA, and when no differences were detected, treatment groups including both genders were compared using one-way ANOVA.
3
Amino Acid Derivatization and Analysis
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DEEMM, BCA, dl- 2-aminobutyric acid (internal standard, I.S.), amino acid standards, water (HPLC grade), and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). γ-Glutamyl-β-cyano-L-alanine was purified from V. sativa as described [18 (link)].
4
Quantification of Fecal Metabolites
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Quantification of amino acids, derived biogenic amines, ammonium, and GABA was carried out by UHPLC following a derivatization with diethyl ethoxymethylenemalonate (DEEMM, Sigma-Aldrich, St. Louis, MO, USA) in 100 µL of fecal water, by the procedure described in Redruello et al. [33 (link)], adapted and optimized to human fecal cell-free supernatants by Salazar et al. [34 (link)]. Samples were filtered through 0.22 µm pore diameter PTFE membranes (VWR International, Radnor, PA, USA) prior to injection of 1 µL into the UHPLC chromatographic system. Concentrations of the analyzed compounds in fecal supernatants were calculated in millimolar (mM) and referred as concentration in fecal samples.
5
Amino Acid Derivatization and HPLC Analysis
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The amino acid derivatization was performed according to (Alaiz et al. 1992 (link)). Briefly, 400 µl of the amino acid extract obtained from yeast of HepG2 cells was mixed with 1 ml of 150 mM borate buffer pH 9.0 and 0.8 µl DEEMM (Sigma-Aldrich, St. Louis, USA) and subsequently incubated at 50 °C for 1 h by shaking at 1400 rpm. The samples were centrifuged for 15 min at 20,800g. 50 µl of the samples were injected by a Gilson 234 auto injector connected to two Gilson 305 pumps onto a 250 × 4.8 mm Zorbax column connected to a C18 guard column. The UV signal at 280 nm was detected with a photodiode array detector (SPD-20A Shimadzu). Eluent A was 10 mM acetate pH 5.8 in ddH2O and eluent B was 100% acetonitrile. The flow rate was set to 0.9 ml/min. The HPLC program was optimized to obtain an optimal amino acid peak separation by adjusting the eluent B percentage (%) at different time points (Table 1 ).
HPLC program used for amino acids determination
| Time point (min) | 0 | 10 | 20 | 30 | 40 | 45 | 56 | 58 | 60 | 61 | 72 |
| Eluent B (%) | 1 | 5 | 9 | 13 | 18 | 19 | 30 | 50 | 60 | 95 | 1 |
6
Histamine Production Assay for Bacterial Isolates
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To test the capacity of each isolate to produce histamine, all isolates were grown in MRS broth supplemented with 10 mM histamine for 24 h at 37ºC without aeration.
The cells were then removed by centrifugation at 8000 g for 10 min, and 100 µL of the supernatant derivatised with diethyl ethoxymethylenemalonate (DEEMM) (Sigma-Aldrich) to quantify its histamine content by ultra-high performance liquid chromatography (UHPLC) using an H-Class AcquityUPLC system (Waters, Milford, MA, USA) as previously described Redruello et al. (2013) . Separation was performed at 35ºC in a Waters AcquityUPLC BEHC18 1.7 µm column (2.1 x 100 mm). Data were acquired and analysed using Empower 2 software (Waters).
To determine the amount of histamine present in the cheese, 1 g samples were processed to extract the BAs as previously described by Herrero-Fresno et al. (2012) . One hundred microlitres were then derivatised and analysed as described above.
The cells were then removed by centrifugation at 8000 g for 10 min, and 100 µL of the supernatant derivatised with diethyl ethoxymethylenemalonate (DEEMM) (Sigma-Aldrich) to quantify its histamine content by ultra-high performance liquid chromatography (UHPLC) using an H-Class AcquityUPLC system (Waters, Milford, MA, USA) as previously described Redruello et al. (2013) . Separation was performed at 35ºC in a Waters AcquityUPLC BEHC18 1.7 µm column (2.1 x 100 mm). Data were acquired and analysed using Empower 2 software (Waters).
To determine the amount of histamine present in the cheese, 1 g samples were processed to extract the BAs as previously described by Herrero-Fresno et al. (2012) . One hundred microlitres were then derivatised and analysed as described above.
7
HPLC-MS analysis of amino acids and biogenic amines in beer
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HPLC-grade acetonitrile and sodium hydroxide were purchased from VWR (Barcelona, Spain), methanol and hydrochloric acid from Merck (Darmstadt, Germany), boric acid from USB (Cleveland, OH, USA), and ammonium acetate, sodium azide, DEEMM, L-2-aminoadipic acid (internal standard), amino acids, BAs and ammonium chloride from Sigma-Aldrich (Madrid, Spain). All solutions were made with Milli-Q water. Eleven beers (lager or ale) made by different European brewers and produced using different techniques, were chosen for analysis. Four of them (two lager and two ale) were used during the matrix effect analysis (see 2.4.1 section for samples description). All samples were purchased in retail stores in Spain.
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