Extracellular glucose and ethanol from experiments conducted in defined minimal medium (ZMM) were quantified via HPLC using an Agilent 1100 series system (Agilent Technologies, Germany) equipped with a Rezex-ROA column (Phenomenex, USA). 2 µl of the samples were injected onto the column by an autosampler and analyzed using isocratic elution with 4 mM H2SO4 at a flow rate of 0.5 mL/min at 60 °C and was detected with an RID detector. Extracellular lactate and alanine were quantified with an Inertsil ODS-3 column (GL Sciences, Germany). 10 µl of the samples were injected onto the column by an autosampler and analyzed using isocratic elution with 0.1 M ammonium phosphate monobasic (pH 2.6) at a flow rate of 0.1 mL/min at 40 °C and was detected with a DAD detector. Concentrations were calculated based on the peak area using standard solutions. For experiments conducted in undefined complex medium (ZCM), extracellular glucose, ethanol and lactate were quantified using the respective enzymatic assay kits from Megazyme (Ireland).
Enzymatic assay kit
Manufactured by Megazyme
Sourced in Ireland
The Enzymatic Assay Kit is a laboratory instrument designed to perform quantitative analysis of specific chemical compounds or enzymes in a sample. It provides a standardized and automated method for measuring the concentration or activity of target analytes using enzymatic reactions.
Automatically generated - may contain errors
Lab products found in correlation
Rezex roa column, by Phenomenex (1 mentions)
Inertsil ods 3 column, by GL Sciences (1 mentions)
Agilent 1100 series system, by Agilent Technologies (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Biochrom 30 amino acid analyser, by Harvard Bioscience (1 mentions)
Phosphate assay kit, by Merck Group (1 mentions)
Mp220, by Mettler Toledo (1 mentions)
Gago20, by Merck Group (1 mentions)
Hpx 87h column, by Bio-Rad (1 mentions)
Agilent 1200 high, by Agilent Technologies (1 mentions)
Sevencompact s220, by Mettler Toledo (1 mentions)
Fp 528, by Leco (1 mentions)
15 protocols using enzymatic assay kit
1
Quantification of extracellular metabolites
2
Comprehensive Nutritional Analysis of Foods
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The nutrients were analysed for the percentage of moisture, proteins, fats, dietary fibre, and ash. The nutritional value was determined using the procedures described by the Association of Official Analytical Chemists [25 ]. Dietary fibre was determined using a gravimetric method [26 (link)]. Total carbohydrates were calculated as the residual difference after subtracting protein, ash, moisture, total fibre, and crude fat content.
Total energy was determined by the calculation of energy values of carbohydrate, fat, protein, and fibre: Energy, KJ = 37 (crude fat content, g) + 17 (protein content, g + carbohydrate content, g) + 8 (fibre content, g). Cellulose determination was performed according to SRPS ISO 6541:1997 [27 ]. Resistant starch, beta-glucan, arabinoxylan, fructan, and sugars were quantified by a spectrophotometric method using enzymatic assay kits, Megazyme, Bray, Ireland. Analyses were performed according to an instruction manual of a kit producer [28 (link),29 (link)].
Fibre fractions intake were calculated using 3-day food records, applying data on the fructan, arabinoxylan, cellulose, resistant starch, and beta-glucan content in the foods fromTable 2 which were previously analysed for fibre and RS content [22 (link),23 (link),30 ].
Total energy was determined by the calculation of energy values of carbohydrate, fat, protein, and fibre: Energy, KJ = 37 (crude fat content, g) + 17 (protein content, g + carbohydrate content, g) + 8 (fibre content, g). Cellulose determination was performed according to SRPS ISO 6541:1997 [27 ]. Resistant starch, beta-glucan, arabinoxylan, fructan, and sugars were quantified by a spectrophotometric method using enzymatic assay kits, Megazyme, Bray, Ireland. Analyses were performed according to an instruction manual of a kit producer [28 (link),29 (link)].
Fibre fractions intake were calculated using 3-day food records, applying data on the fructan, arabinoxylan, cellulose, resistant starch, and beta-glucan content in the foods from
3
Metabolite Analysis of Cell Culture
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We measured changes in glucose and lactate media concentration taking 100 µL of spent culture medium at the respective time points using a blood gas analyser (ABL90 FLEX, Radiometer MedicalApS, Brønshøj, Denmark). The concentration of pyruvate and a panel of amino acids were measured with their respective enzymatic assay kits (Megazyme, Wicklow, Ireland) following the microplate assay procedures supplied by the manufacturer. Absorbance was measured using a microplate reader (Spectramax M3, Molecular Devices, Sunnyvale, CA, USA).
4
Quantitative Analysis of Flour and Sourdough
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Carbohydrates and fermentation metabolites were extracted from a homogeneous suspension of flour or sourdough (5 g) and H2SO4 10 mM (45 ml) (Minervini et al., 2012a (link)). Glucose, fructose, maltose, sucrose, lactic acid (only sourdough samples), acetic acid (only sourdough samples) and ethanol (only sourdough samples) were quantified using enzymatic Assay Kits (Megazyme International Ireland Limited, Bray, Co. Wicklow, Ireland), following the manufacturer’s instructions. A second water-soluble extract was prepared for analysis of free amino acids (FAA), using Tris-HCl 50 mM pH 8.8 as homogenizing solution (Minervini et al., 2012a (link)), and analyzing the water-soluble extract through the Biochrom 30 Amino Acid Analyser (Biochrom LTD, Cambridge, United Kingdom) (De Angelis et al., 2007 (link)).
5
Comprehensive Nutrient Analysis of Legume and Wheat Flours
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The Kjeldahl procedure (NF V 03–050, 1970) was used to determine total protein content using a conversion factor of 6.25 for legume flours and 5.70 for wheat semolina. An enzymatic assay kit (Megazyme, Co. Wicklow, Ireland; AACC method 76–13.01) was used to determine total starch content. Amino acid profiles, lipid content and soluble and insoluble fibers were determined by Agrobio (Rennes, France) according to the CEE 152/2009 (2009), decree 08-09-1977, AOAC 991–42 and AOAC 993–19 methods, respectively.
6
Detailed Carbohydrate and Metabolite Analysis
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All liquid samples (anolyte suspension or fresh medium) were filtered through 0.22 μm membranes (Millex, Merck Millipore Ltd., Ireland). Total carbohydrate analysis was performed using a colorimetric phenol/sulphuric acid method (Dubois et al., 1956 (link)). Total phosphate concentration was determined using a phosphate assay kit (Merck, Germany). L -lactate and glycerol were determined using EnzyChrom enzymatic assay kits ECLC-100 and EGLY-100, respectively (BioAssay Systems, USA). The concentrations of glucose and iron were determined using Sigma assay kits GAGO20 and MAK025 (Sigma–Aldrich, USA). The pentosan content in the medium was quantified using a colorimetric method (Finnie et al., 2006 (link)) and xylose concentration was measured using an enzymatic assay kit (Megazyme, Ireland). Acetate, succinate, and propionate in the suspension were determined using 1H NMR spectroscopy as previously described Li et al. (2011) (link). Measurement of pH was performed on 10 ml of anolyte suspension using a pH-meter (Mettler Toledo MP220, Switzerland).
7
Enzymatic Assay for Optical Isomer Analysis
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In order to distinguish between the two optical isomers of LA an enzymatic assay kit (K-DLATE 06/08, Megazyme, Ireland) was used. Starch consumption was measured as the disappearance of blue color intensity given by the starch–iodine complex.20
Cell density was determined in a spectrophotometer by measuring the optical density (OD) at 600 nm. Cell counts were performed in a counting chamber (Thoma) after appropriate dilution. All measurements were performed in duplicate.
Cell density was determined in a spectrophotometer by measuring the optical density (OD) at 600 nm. Cell counts were performed in a counting chamber (Thoma) after appropriate dilution. All measurements were performed in duplicate.
8
Comprehensive Food Composition Analysis
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Moisture and ash contents were analyzed according to ICC Standard Methods No. 110/1 [35 ] and International Standard ISO 2171:2007 [36 ], respectively. Protein content was determined with the Kjeldahl method according to ICC Standard Method No. 105/2 [35 ], using a conversion factor of 6.25. The lipid content was determined by acidic hydrolysis following the AOAC Official Method 922.06 [37 ]. The total dietary fiber was determined using the enzymatic assay kit (Megazyme International Ltd., Bray, Ireland), in accordance with the AOAC Official Method 985.29 [37 ].
9
Chromatographic Analysis of Formate
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Media samples collected during the evolution experiment and batch-growth experiments were first filtered through a 0.22 micron PVDF Millex-GV syringe filter unit (Merck Millipore), and stored at −80°C. After thawing, the media samples they were analyzed with an Agilent 1200 high-performance liquid chromatography system (Agilent technologies, USA) equipped with a refractive index detector and an anion exchange Bio-Rad HPX-87H column (Bio-Rad, USA). The column was eluted with 5 mM sulfuric acid at a flow rate of 0.6 mL/min at 45°C. Samples with a formate concentration below the detection limit of the HPLC were analyzed by an enzymatic assay kit (Megazyme, Ireland). Media samples from the evolution experiments were each measured once. Media samples from the batch growth experiments were measured 3 times, with the mean ± SD is shown in Figure 2 C. The samples analyzed with the enzymatic kit were measured twice; the mean ± SD is reported.
10
Comprehensive Nutritional Analysis of Pea Flour
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Total crude protein content of pea flour was obtained using Kjeldahl procedure with a nitrogen-to-protein conversion factor of 6.25. Total starch content was determined with an enzymatic assay kit (Megazyme, Co., Ireland). Ash content was determined by incineration at 900 °C for 2h according to the French standard (NF 03-720). Total lipids were extracted according to method of Folch et al. (1957) . All measurements were conducted in triplicate. The reported values are averages
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