Glycerol GK Assay Kit

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00:05  Introduction
00:58  Principle
01:49   Reagent Preparation
02:28   Procedure
05:42   Calculations

Glycerol GK Assay Kit K-GCROLGK Scheme
Reference code: K-GCROLGK
SKU: 700004293

70 assays (manual) / 700 assays (microplate) / 600 assays (auto-analyser)

Content: 70 assays (manual) / 700 assays (microplate) / 600 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: Glycerol
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 1.0 to 35 µg of glycerol per assay
Limit of Detection: 0.37 mg/L
Reaction Time (min): ~ 7 min
Application examples: Wine (and grape juice), beer, spirits, vinegar, marzipan, fruit juices, soft drinks, toothpaste, honey, tobacco, paper (and cardboard), cosmetics, pharmaceuticals, soap and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Novel method

The Glycerol GK test kit is a simple, reliable and accurate method for the measurement and analysis of glycerol in beverages, foodstuffs and other material. Based on use of ADP-glucokinase and increase in absorbance on conversion of NAD+ to NADH.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

Explore our complete range of alcohol assay kit products.

Scheme-K-GCROLGK GCROLGK megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Novel tablet format for increased stability 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years as supplied 
  • Very rapid reaction 
  • Positive reaction (assay proceeds with an increase in absorbance) 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included 
  • Suitable for manual, microplate and auto-analyser formats
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator

Towards green chemicals and edible coatings from barks and peels with near critical extraction of suberin.

Lecart, B., Baumsteiger, C., Monie, F., Di Maria, A., Detrembleur, C., Richel, A. & Vanderschuren, H. (2023). Green Chemistry, 25(22), 9282-9291.

Due to their complex structures, industrial suberoligneous by-products such as barks and peels remain poorly exploited. In this study, we demonstrated the efficient extraction of suberin from barks (11.3%) and peels (20.85%) via supercritical and near-supercritical transesterification without the need for catalysts. When applied in non-isocyanate polyurethane (NIPU) foams, suberinic extracts allowed the increase of the biomass content by up to 20% and facilitated the foaming process by improving pre-curing viscosity. When sprayed on fruits, the suberinic extracts effectively reduced the water loss rate by 25%, extending the shelf-life of the produce. A life cycle analysis for the latter application confirmed the competitive potential of our process against plastic sealing. This work uncovers the potential of suberoligneous biomass (SBM) to provide sustainable solutions to emerging societal challenges.

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Co-fermentation of non-Saccharomyces yeasts with Lactiplantibacillus plantarum FST 1.7 for the production of non-alcoholic beer.

Nyhan, L., Sahin, A. W. & Arendt, E. K. (2022). European Food Research and Technology, 289, 1-15.

The non-alcoholic beer (NAB) sector has experienced steady growth in recent years, with breweries continuously seeking new ways to fulfil consumer demands. NAB can be produced by limited fermentation of non-Saccharomyces yeasts; however, beer produced in this manner is often critiqued for its sweet taste and wort-like off-flavours due to high levels of residual sugars and lack of flavour metabolites. The use of Lactobacillus in limited co-fermentation with non-Saccharomyces yeasts is a novel approach to produce NABs with varying flavour and aroma characteristics. In this study, lab-scale fermentations of Lachancea fermentati KBI 12.1 and Cyberlindnera subsufficiens C6.1 with Lactiplantibacillus plantarum FST 1.7 were performed and compared to a brewer’s yeast, Saccharomyces cerevisiae WLP001. Fermentations were monitored for pH, TTA, extract reduction, alcohol production, and microbial cell count. The final beers were analysed for sugar and organic acid concentration, free amino nitrogen content (FAN), glycerol, and levels of volatile metabolites. The inability of the non-Saccharomyces yeasts to utilise maltotriose as an energy source resulted in extended fermentation times compared to S. cerevisiae WLP001. Co-fermentation of yeasts with lactic acid bacteria (LAB) resulted in a decreased pH, higher TTA and increased levels of lactic acid in the final beers. The overall acceptability of the NABs produced by co-fermentation was higher than or similar to that of the beers fermented with the yeasts alone, indicating that LAB fermentation did not negatively impact the sensory attributes of the beer. C. subsufficiens C6.1 and L. plantarum FST 1.7 NAB was characterised as fruity tasting with the significantly higher ester concentrations masking the wort-like flavours resulting from limited fermentation. NAB produced with L. fermentati KBI12.1 and L. plantarum FST1.7 had decreased levels of the undesirable volatile compound diacetyl and was described as ‘fruity’ and ‘acidic’, with the increased sourness masking the sweet, wort-like characteristics of the NAB. Moreover, this NAB was ranked as the most highly acceptable in the sensory evaluation. In conclusion, the limited co-fermentation of non-Saccharomyces yeasts with LAB is a promising strategy for the production of NAB.

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Optimization of Lipid Production by Schizochytrium limacinum Biomass Modified with Ethyl Methane Sulfonate and Grown on Waste Glycerol.

Talbierz, S., Dębowski, M., Kujawska, N., Kazimierowicz, J. & Zieliński, M. (2022). International Journal of Environmental Research and Public Health, 19(5), 3108.

One of the most promising avenues of biofuel research relates to using waste as a starting feedstock to produce liquid or gaseous energy carriers. The global production of waste glycerol by the refinery industry is rising year after year. The aim of the present study was to examine the effect of ethyl methane sulfonate (EMS) on the growth rates and intracellular lipid accumulation in heterotrophically-cultured Schizochytrium limacinum microalgae, grown on waste glycerol as the carbon source. The strain S. limacinum E20, produced by incubating a reference strain in EMS for 20 min, was found to perform the best in terms of producing biomass (0.054 gDW/dm3·h) and accumulating intracellular bio-oil (0.021 g/dm3·h). The selected parameters proved to be optimal for S. limacinum E20 biomass growth at the following values: temperature 27.3 °C, glycerol level 249.0 g/dm3, oxygen in the culture 26%, and yeast extract concentration 45.0 g/dm3. In turn, the optimal values for lipid production in an S. limacinum E20 culture were: temperature 24.2 °C, glycerol level 223.0 g/dm3, oxygen in the culture 10%, and yeast extract concentration 10.0 g/dm3. As the process conditions are different for biomass growth and for intracellular lipid accumulation, it is recommended to use a two-step culture process, which resulted in a lipid synthesis rate of 0.41 g/dm3·h.

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Impact of Lachancea thermotolerans strain and lactic acid concentration on Oenococcus oeni and malolactic fermentation in wine.

Snyder, E. C., Jiranek, V. & Hranilovic, A. (2021). OENO One, 55(2), 365-380.

The yeast Lachancea thermotolerans can produce lactic acid during alcoholic fermentation (AF) and thereby acidify wines with insufficient acidity. However, little is known about the impact of L. thermotolerans on Oenococcus oeni, the primary lactic acid bacterium used in malolactic fermentation (MLF). This study explored the impact of sequential cultures of L. thermotolerans and Saccharomyces cerevisiae on MLF performance in white and red wines. Four L. thermotolerans strains were tested in Sauvignon blanc with sequential S. cerevisiae inoculation, compared to an S. cerevisiae control and the initially un-inoculated treatments. The L. thermotolerans wines showed large differences in acidification, and progression of MLF depended on lactic acid production, even at controlled pH. The highest and lowest lactic acid producing strains were tested further in Merlot fermentations with both co-inoculated and sequentially inoculated O. oeni. The low lactic acid producing strain enabled successful MLF, even when this failed in the S. cerevisiae treatment, with dramatically quicker malic acid depletion in O. oeni co-inoculation than in sequential inoculation. In contrast, a high lactic acid producing strain inhibited MLF irrespective of the O. oeni inoculation strategy. In a follow-up experiment, increasing concentrations of exogenously added lactic acid slowed MLF and reduced O. oeni growth across different matrices, with 6 g/L of lactic acid completely inhibiting MLF. The results confirm the inhibitory effect of lactic acid on O. oeni while highlighting the potential of some L. thermotolerans strains to promote MLF and the others to inhibit it.

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The use of different fermentative approaches on Paracoccus denitrificans: Effect of high pressure and air availability on growth and metabolism.

Mota, M. J., Lopes, R. P., Pinto, C. A., Sousa, S., Gomes, A. M., Delgadillo, I. & Saraiva, J. A. (2020). Biocatalysis and Agricultural Biotechnology, 26, 101646.

The performance of fermentation under sub-lethal high pressure (HP) is a strategy for stimulation of microbial growth and/or improvement of fermentation titers, rates and yields. The present work intended to study the possibility of applying HP to Paracoccus denitrificans glycerol fermentation, considering that HP-fermentation usually involves some process constrains, such as limited air volumes. Consequently, the work was divided in two main goals: i) study the effects of air availability on P. denitrificans; ii) assess if the strain is able to grow and maintain metabolic activity under HP (10-35 MPa). Paracoccus denitrificans growth and metabolism were highly affected by air availability. Samples under higher air availability showed considerable cell growth, but no production of ethanol or organic acids. On the other hand, samples without air had lower cell growth, but active metabolic activity (with the production of ethanol and organic acids). Regarding the HP experiments, P. denitrificans was able to grow at 10, 25 and 35 MPa, but to a lower extent compared to atmospheric pressure. Application of HP promoted modifications in the production of ethanol, acetate and succinate, and the fermentative profile varied according to the pressure level. Overall, the present work demonstrated new metabolic features of P. denitrificans at atmospheric pressure and HP conditions. It also opened the way for further studies regarding P. denitrificans fermentation under HP, as well as utilization of this technology for other glycerol fermentations, in particular in the case of high requirements of air availability.

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Characterization of E 472 food emulsifiers-determination of bound and free fruit acids, free glycerol and ash content.

Oellig, C., Link, K. & Schwack, W. (2020). Journal of Chromatography A, 1619, 460946.

Emulsifiers of the type E 472 are esters of fruit acids and mono- and diacylglycerols (MAG and DAG), which are used to adjust techno-functional properties in various food products. The most dominant representatives of E 472 emulsifiers are acetic acid esters (E 472a), lactic acid esters (E 472b), citric acid esters (E 472c), and mono- and diacetyl tartaric acid esters (E 472e). For the determination of fruit acids, a high-performance liquid chromatography method with ultraviolet light (HPLC-UV) detection was developed. Free and total fruit acids were determined by reversed phase HPLC-UV analysis of untreated and saponified emulsifier extracts with 20 mM potassium hydrogen phosphate buffer (pH 2.6) as isocratic eluent. Limits of quantitation of 0.08-0.27 g free fruit acid/kg emulsifier and 4-14 g total fruit acid/kg granted a reliable method with recoveries for free and total fruit acids between 80 and 100% with relative standard deviations (%RSD) below 4%. For the quantitation of free glycerol by spectrophotometry, an enzymatic assay was optimized for the analysis of E 472 providing reliable results with %RSD values below 9%. In addition, the ash content of E 472 emulsifiers was determined.

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Effect of High Pressure on Paracoccus denitrificans Growth and Polyhydroxyalkanoates Production from Glycerol.

Mota, M. J., Lopes, R. P., Simões, M. M., Delgadillo, I. & Saraiva, J. A. (2019). Applied Biochemistry and Biotechnology, 188(3), 810-823.

The performance of fermentation under non-conventional conditions, such as high pressure (HP), is a strategy currently tested for different fermentation processes. In the present work, the purpose was to apply HP (10-50 MPa) to fermentation by Paracoccus denitrificans, a microorganism able to produce polyhydroxyalkanoates (PHA) from glycerol. In general, cell growth and glycerol consumption were both reduced by HP application, more extensively at higher pressure levels, such as 35 or 50 MPa. PHA production and composition was highly dependent on the pressure applied. HP was found to decrease polymer titers, but increase the PHA content in cell dry mass (%), indicating higher ability to accumulate these polymers in the cells. In addition, some levels of HP affected PHA monomeric composition, with the polymer produced at 10 and 35 MPa showing considerable differences relative to the ones obtained at atmospheric pressure. Therefore, it is possible to foresee that the changes in polymer composition may also affect its physical and mechanical properties. Overall, the results of this study demonstrated that HP technology (at specific levels) can be applied to P. denitrificans fermentations without compromising the ability to produce PHA, with potentially interesting effects on polymer composition.

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An accurate description of Aspergillus niger organic acid batch fermentation through dynamic metabolic modelling.

Upton, D. J., McQueen-Mason, S. J. & Wood, A. J. (2017). Biotechnology for Biofuels, 10(1), 258.

Background: Aspergillus niger fermentation has provided the chief source of industrial citric acid for over 50 years. Traditional strain development of this organism was achieved through random mutagenesis, but advances in genomics have enabled the development of genome-scale metabolic modelling that can be used to make predictive improvements in fermentation performance. The parent citric acid-producing strain of A. niger, ATCC 1015, has been described previously by a genome-scale metabolic model that encapsulates its response to ambient pH. Here, we report the development of a novel double optimisation modelling approach that generates time-dependent citric acid fermentation using dynamic flux balance analysis. Results: The output from this model shows a good match with empirical fermentation data. Our studies suggest that citric acid production commences upon a switch to phosphate-limited growth and this is validated by fitting to empirical data, which confirms the diauxic growth behaviour and the role of phosphate storage as polyphosphate. Conclusions: The calibrated time-course model reflects observed metabolic events and generates reliable in silico data for industrially relevant fermentative time series, and for the behaviour of engineered strains suggesting that our approach can be used as a powerful tool for predictive metabolic engineering.

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Light enhanced calcification in Stylophora pistillata: effects of glucose, glycerol and oxygen.

Holcomb, M., Tambutté, E., Allemand, D. & Tambutté, S. (2014). PeerJ, 2, e375.

Zooxanthellate corals have long been known to calcify faster in the light than in the dark, however the mechanism underlying this process has been uncertain. Here we tested the effects of oxygen under controlled pCO2 conditions and fixed carbon sources on calcification in zooxanthellate and bleached microcolonies of the branching coral Stylophora pistillata. In zooxanthellate microcolonies, oxygen increased dark calcification rates to levels comparable to those measured in the light. However in bleached microcolonies oxygen alone did not enhance calcification, but when combined with a fixed carbon source (glucose or glycerol), calcification increased. Respiration rates increased in response to oxygen with greater increases when oxygen is combined with fixed carbon. ATP content was largely unaffected by treatments, with the exception of glycerol which decreased ATP levels.

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Construction of an efficient xylose-fermenting diploid Saccharomyces cerevisiae strain through mating of two engineered haploid strains capable of xylose assimilation.

Kim, S. R., Lee, K. S., Kong, I. I., Lesmana, A., Lee, W. H., Seo, J. H., Kweon, D. H. & Jin, Y. S. (2013). Journal of Biotechnology, 164(1), 105-111.

Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain. Specifically, we obtained two engineered haploid strains (YSX3 and SX3). The YSX3 strain consumed xylose rapidly and produced a lot of xylitol. On the contrary, the SX3 strain consumed xylose slowly with little xylitol production. After converting the mating type of SX3 from alpha to a, the resulting strain (SX3-2) was mated with YSX3 to construct a heterozygous diploid strain (KSM). The KSM strain assimilated xylose (0.25 g xylose h-1 g cells-1) as fast as YSX3 and accumulated a small amount of xylitol (0.03 g g xylose-1) as low as SX3, resulting in an improved ethanol yield (0.27 g g xylose-1). We found that the improvement in xylose fermentation by the KSM strain was not because of heterozygosity or genome duplication but because of the complementation of the two xylose-metabolic pathways. This result suggested that mating of suboptimal haploid strains is a promising strategy to develop engineered yeast strains with improved xylose fermenting capability. -1

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Dynamic modeling of methylotrophic Pichia pastoris culture with exhaust gas analysis: From cellular metabolism to process simulation.

Niu, H., Daukandt, M., Rodriguez, C., Fickers, P. & Bogaerts, P. (2013). Chemical Engineering Science, 87, 381-392.

A systematic approach to establish a dynamic model of methylotrophic Pichia pastoris culture in bioreactor is presented on the basis of biomass compartmentalization and metabolic stoichiometry simplification. Besides direct state variables (i.e., biomass, glycerol, methanol, and ammonia), CER and OUR calculated from on-line exhaust gas analysis are included in the model. The model is directly and crossly validated with five experimental cultures involving glycerol growth and methanol feeding phases. Model parameters are identified with confidence intervals. Meanwhile, data consistency is straightforward checked between predicted CERs and measured CTRs. In addition, in the light of the model structure, the results of parameter sensitivity analysis verifies the relative “freedom” of biomass initial compartmentalization and the high output sensitivity when the substrate (glycerol or methanol) concentration is close to the affinity constant (KGly or KMeth). With the proposed model, a process control strategy can be accordingly developed, which is based on real-time monitoring and regulating of cellular metabolic state during culture.

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Construction of self-cloning, indigenous wine strains of Saccharomyces cerevisiae with enhanced glycerol and glutathione production.

Hao, R. Y., Liu, Y. L., Wang, Z. Y. & Zhang, B. R. (2012). Biotechnology Letters, 34(9), 1711-1717.

To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19% in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.

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