L-Arginine/Urea/Ammonia Assay Kit

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00:09  Introduction
01:05   Principle
02:04   Reagent Preparation
03:24   Procedure
08:21   Calculations

L-Arginine Urea Ammonia Assay Kit K-LARGE Scheme
Reference code: K-LARGE
SKU: 700004309

50 Assays per kit

Content: 50 assays per kit
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 1 year under recommended storage conditions
Analyte: Ammonia, L-Arginine, Nitrogen, Urea, YAN
Assay Format: Spectrophotometer
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Decrease
Linear Range: 1.0 to 35 mg of L-arginine, 0.2 to 7.0 μg of ammonia and 0.3 to 14 µg of urea per assay
Limit of Detection: 0.07 mg/L (ammonia),
0.13 mg/L (urea),
0.37 mg/L (L-arginine) 
Reaction Time (min): ~ 20 min [ammonia (2 min), urea (6 min), L-arginine (7 min)]
Application examples: Grape juice, wine must, wine and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Improved method

The L-Arginine/Urea/Ammonia test kit is specific and a rapid measurement and analysis of L-arginine, urea and ammonia in grape juice/must and wine.

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).

Display all of our nitrogen assay kit products.

Scheme-K-LARGE LARGE Megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Improved assay format 
  • Very rapid reactions due to use of uninhibited glutamate dehydrogenase 
  • All enzymes supplied as stabilised suspensions 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator
Megazyme publication

Megazyme “advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

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Megazyme publication

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

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Exploring the influence of grape tissues on the concentration of wine volatile compounds.

Blackford, C. L., Trengove, R. D. & Boss, P. K. (2021). Australian Journal of Grape and Wine Research, In Press.

Background and Aims: Knowledge of varietal wine flavour and aroma compounds has improved, but gaps exist concerning how grape composition impacts wine style. This work aimed to explore the influence that different grape tissues can have on the volatile profiles of wines. Methods and Results: Riesling and Cabernet Sauvignon berries were separated into skin, flesh and seeds. Two sets of fermentations were performed using separated tissues: one using an equal mass of each tissue and another where the amount of each tissue in 25 g of berries was fermented. When an equal mass of tissue was used, the seed-derived wines had a higher concentration of esters than that produced from other grape tissues. Those produced using skins had the highest concentration of lipoxygenase pathway-derived compounds, and, for Riesling, a higher concentration of monoterpenes. When the proportional amounts of each tissue found per berry were used, the flesh-derived wines generally had a higher concentration of many wine volatiles compared to the other tissues. This reflects the greater proportion of flesh tissue in the berry compared to skin and seeds. Conclusions: Seed-derived compounds can enhance ester biosynthesis during fermentation and skins appear to have high lipoxygenase pathway activity. Nevertheless, the flesh makes up such a large proportion of the whole berry that it has the major influence on volatile profiles of whole berry fermentations. Significance of the Study: Different berry tissues can alter wine composition in unique ways, and this can inform strategies to alter wine composition through vineyard management or the selection of new germplasm.

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Arginase activity characterization during alcoholic fermentation by sequential inoculation with non-Saccharomyces and Saccharomyces Yeast.

Benucci, I. & Esti, M. (2021). Food and Bioprocess Technology, 1-8.

Arginine uptake and yeast arginase activity were studied throughout the alcoholic fermentation of a white grape must (Vitis vinifera L. cv Fiano), carried out by sequential inoculation (Torulaspora delbrueckii and S. cerevisiae, TD-SC) and compared with a S. cerevisiae single fermentation (SC). In both samples, yeast assimilable nitrogen (YAN) consumption was mainly during the early phase of alcoholic fermentation (before S. cerevisiae addition in the sequential fermentation). T. delbrueckii alone and S. cerevisiae similarly metabolized YAN, which was further consumed in TD-SC sample following the S. cerevisiae inoculation. The only relevant arginine uptake was found about 3 days after the first inoculum (simultaneously with the greatest YAN consumption) and it appeared to be higher in SC (0.24 g/L) than in TD-SC (0.12 g/L). The kinetic parameters, estimated by means of the Hill equation, and in particular Vmax values (56.0 ± 1.6 U/mgBSAeq at 48 h for SC and 73.3 ± 2.7 U/mgBSAeq at 66 h for TD-SC) indicated the maximum arginase activity at the same point of time corresponding to the lowest YAN amount (48 h for S. cerevisiae and 66 h for T. delbrueckii).

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Aroma and Sensory Profiles of Sauvignon Blanc Wines from Commercially Produced Free Run and Pressed Juices.

Parish-Virtue, K., Herbst-Johnstone, M., Bouda, F., Fedrizzi, B., Deed, R. C. & Kilmartin, P. A. (2021). Beverages, 7(2), 29.

Sauvignon blanc is the most important grape cultivar within the New Zealand wine industry, and wines from the Marlborough region are renowned for their intense aromas including tropical, passionfruit, and green capsicum. Quality Sauvignon blanc wines are usually made from free run juice, although press fractions can be included. The chemical aroma composition and sensory profiles of two wine sets made from three press fractions (free run, light press and heavy press) were compared. The compounds 3-mercaptohexan-1-ol and 3-mercaptohexyl acetate were found to decrease between free run and heavily pressed wines while hexyl acetate, hexanol, and benzyl alcohol increased. The accompanying sensory analysis showed that free run wines were marked by aromas of Passionfruit/sweaty, Boxwood and Fresh green capsicum, while the heavy pressed wines were described by French vanilla/bourbon, Floral and Banana lolly attributes, consistent with the aroma chemical composition.

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Nanocurcumin and arginine entrapped injectable chitosan hydrogel for restoration of hypoxia induced endothelial dysfunction.

Mohandas, A. & Rangasamy, J. (2020). International Journal of Biological Macromolecules, 166, 471-482.

Hypoxia is a condition that gradually leads to ischemic damages in organs which is marked by poor tissue perfusion. Depending on the severity of the condition, revascularisation therapies are needed for reducing the risk of organ dysfunction. This study was aimed at developing an injectable nanocurcumin and arginine incorporated chitosan hydrogel (nC/R) that can prevent hypoxia induced endothelial damage. The prepared hydrogel has shear thinning, stable and injectable nature. The (nC and nC/R) hydrogels showed significant antioxidant activity and biodegradation in vitro. The release of curucmin and arginine from the nC/R was found to be higher at acidic pH, which predominates in an ischemic site. To mimic low oxygen environment, an in vitro hypoxic endothelial dysfunction model was developed which showed decreased expressions of phosphorylated eNOS (serine 1177) when compared to the cells cultured in normoxic condition. In vitro tube formation assay demonstrated the protective effect of nC/R towards hypoxia induced reduction of tube width. The nC/R hydrogel was found to enhance phosphorylation of eNOS at serine 1177 site in cultured endothelial cells subjected to hypoxia. Therefore, nC/R hydrogel could effectively deliver both curcumin and arginine and therapeutically reduce the effect of hypoxia induced endothelial dysfunction.

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Functional Applications of Polyarginine-Hyaluronic Acid-Based Electrostatic Complexes.

Kale, N. R., Dutta, D., Carstens, W., Mallik, S. & Quadir, M. (2020). Bioelectricity, 2(2), 158-166.

Background: Electrostatic complexes of poly (l-Arginine) (pArg) and hyaluronic acid (HA) have been investigated for their functional applications to supply free or polymeric form of l-Arginine (Arg) to target cells. As a vital amino acid, Arg plays significant role in multitude of pathophysiological processes ranging from wound healing to cancer. However, serum arginase expression and toxicity of Arg at cellular level renders exogenous delivery of this amino acid a challenging task. We showed that polyarginine-hyaluronic acid ionic nanocomplexes (pArg-HA iNCs) could be an effective way to deliver Arg to target cell populations. Materials and Methods: These electrostatic complexes were prepared by mixing HA (average m.w. of 200 kDa) with pArg (m.w. 5-15 kDa; Sigma) in aqueous solutions and purifying over glycerol. Nanocomplexes were characterized for their particle size, surface charge, capacity to release l-Arg, and intracellular uptake of complexes. Results: Synthesized nanocomplexes showed hydrodynamic diameter ranging from 140-306 nm depending on the content of pArg or HA within the formulation. With surface charge (ζ-potential) of -29 mV, the nanocomplexes showed pH-dependent release of Arg. At pH 7.4, pArg-HA iNCs released 30% of the total Arg-content, while at pH 5.0, 60% of Arg was released after 24 h. These electrostatically stabilized complexes were found to promote growth of human dermal fibroblasts (HDF) in wound-healing assay and increased nitric oxide (NO) activity in these cells in a time-dependent manner. Nanocomplexes also showed cellular uptake and enhanced dose-dependent toxicity against two pancreatic cancer cell lines, i.e. MIA PaCa-2 and Panc-1. Interestingly, the cytotoxic effect was synergized upon pre-treatment of the cells with a frontline chemotherapeutic agent, gemcitabine (GEM), and was not observed when the cells were treated with Arg alone. Conclusion: As such, this communication shows the prospect of pArg-HA iNC electrostatic nanocomplexes to interact and interfere with intracellular Arg metabolic machinery conducive to rescuing different pathological conditions.

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The impact of postharvest ultra-violet light irradiation on the thiol content of Sauvignon blanc grapes.

Parish-Virtue, K., Herbst-Johnstone, M., Bouda, F. & Fedrizzi, B. (2019). Food Chemistry, 271, 747-752.

Sauvignon blanc grapes were exposed to an ultra-violet (UV) light source post-hand harvest (whole bunches) or post-machine harvest. The thiol precursors S-3-(hexan-1-ol)-L-cysteine (Cys-3MH) and S-3-(hexan-1-ol)-L-glutathione (GSH-3MH) were quantified in the juices before and after UV treatment. Results showed that irradiation of the grapes with UV light had little to no effect on the thiol precursors. Wines were fermented from the corresponding juices and 18 aroma compounds were quantified. Differences were found between UV treatments of the wines for 3-mercaptohexanol, hexan-1-ol, ethyl butanoate, ethyl hexanoate, ethyl octanoate and phenylethyl alcohol. However, these changes were not significant (p  < 0.05) for both grape media trialled. Future studies involving larger sample sizes and replicate numbers should be completed in order to ascertain any changes in aroma chemistry as a result of UV light application to grapes postharvest.

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Volatile profiles and chromatic characteristics of red wines produced with Starmerella bacillaris and Saccharomyces cerevisiae.

Englezos, V., Rantsiou, K., Cravero, F., Torchio, F., Giacosa, S., Ortiz-Julien, A., Gerbi, V., Rolle, L. & Cocolin, L. (2018). Food Research International, 109, 298-309.

The use of mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae is gaining attention in recent years due to their ability to modulate the metabolites production of enological interest. In the present study, four of the most popular planted red grape varieties (Cabernet sauvignon, Merlot, Pinot noir and Shiraz) were fermented using the aforementioned species and two different inoculation protocols (inoculation of S. cerevisiae after 24 and 48 h from the Starm. bacillaris inoculation), in order to evaluate their impact on the volatile composition and chromatic characteristics of wines. Analysis from chemical composition showed that titratable acidity and glycerol content exhibited marked differences among wines after fermentation. For volatile compounds, mixed fermented wines using an inoculation delay of 48 h led to reduction of volatile compounds (mainly esters). A shorter 24 h delay produced wines with higher values of color intensity than pure fermented wines. The differences observed between the inoculation protocols can be explained by the growth dynamics of both species during fermentation. These findings suggest that mixed fermentations posed a great potential in reducing metabolites which are considered negative for wine quality (mainly ethyl acetate and volatile fatty acids) and with an improvement of the chromatic profile of the wines.

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Kinetic characterization of arginase from Saccharomyces cerevisiae during alcoholic fermentation at different temperatures.

Benucci, I., Fiorelli, V., Lombardelli, C., Liburdi, K. & Esti, M. (2017). LWT-Food Science and Technology, 82, 268-273.

The kinetic characterization of arginase activity of a commercial S. cerevisiae strain was carried out for the first time, estimating the kinetic parameters (Vmax, K0.5 and Vmax/K0.5) throughout alcoholic fermentation in order to investigate the catalytic efficiency of the enzyme and its ability in metabolizing arginine to sustain biosynthetic processes. Alcoholic fermentation was carried out at three different temperatures (15, 20, 25°C) in semi-synthetic grape juice added with arginine at usual maximal concentration (1 g L-1) in grape must. Arginine uptake was quite constant throughout fermentation process and it was more effectively assimilated during high temperature fermentation (20 and 25°C) than at 15°C. The sigmoidal behavior of yeast arginase kinetic curves, well fitted to the Hill equation, indicated a mechanism of positive cooperativity for the trimeric enzyme. The highest Vmax (4740.0 U mg-1BSAeq) and the maximal catalytic efficiency (78.87 min-1) were observed when fermentation was at 20°C approximately 3 days after the inoculum. Moreover, the K0.5 value was similar (53–60 mg mL-1) when maximal catalytic efficiency was achieved, thus indicating that the affinity of enzyme for the substrate is not altered by fermentation temperature which only affected product release velocity and therefore Vmax/K0.5 ratio.

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Viable and culturable populations of Saccharomyces cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris (synonym Candida zemplinina) during Barbera must fermentation.

Wang, C., Esteve-Zarzoso, B., Cocolin, L., Mas, A. & Rantsiou, K. (2015). Food Research International, 78, 195-200.

The present study analyzed the viable and/or culturable populations of Saccharomyces cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris (synonym Candida zemplinina) during laboratory grape must fermentation, in order to investigate the interaction between the three species considered. Firstly, population dynamics during wine fermentation were followed by culture-dependent techniques, and non-Saccharomyces yeast became non-culturable at late stages of fermentation when S. cerevisiae dominated. Four different culture-independent techniques were further applied to detect viable yeast cells at the late stage of fermentation. Both quantitative PCR techniques applied, namely ethidium monoazide bromide (EMA)-qPCR and Reverse Transcription (RT)-qPCR, detected H. uvarum and Starm. bacillaris at a concentration of 105 to 106 cells/mL. These non-culturable cells had membranes impermeable to EMA and stable rRNA. The background signals from dead cells did not interfere with the quantification of viable cells in wine samples by EMA-qPCR technique. As a qualitative culture-independent technique, DGGE technique was coupled with EMA treatment (EMA-PCR-DGGE) or with RT (RT-PCR-DGGE). With EMA-PCR-DGGE non-Saccharomyces species during fermentation were detected although it was limited by the predominance of S. cerevisiae.

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In vitro removal of ochratoxin A by two strains of Saccharomyces cerevisiae and their performances under fermentative and stressing conditions.

Petruzzi, L., Bevilacqua, A., Baiano, A., Beneduce, L., Corbo, M. R. & Sinigaglia, M. (2014). Journal of Applied Microbiology, 116(1), 60-70.

Aims: The aim of this research was to study the effect of time, temperature, sugar content and addition of diammonium phosphate (DAP) on ochratoxin A (OTA) removal by two strains of Saccharomyces cerevisiae using a completely randomized design. Methods and Results: The strains were grown in a medium containing OTA (2 µg l-1), two sugar levels (200 and 250 g l-1), with or without DAP (300 mg l-1), and incubated at 25-30°C. The yeasts were able to decrease the toxin amount by c. 70%, with the highest removing effect observed after 3 days at 30°C in the presence of 250 g l-1 of sugars and with DAP; after 10 days, the toxin was partially released into the medium. The strains produced high ethanol and glycerol contents, showed high tolerance to single/combined stress conditions and possessed β-D-glucosidase, pectinase and xylanase activities. Ochratoxin A removal was affected by time, temperature, sugar and addition of DAP. Moreover, the phenomenon was reversible. Conclusions: Ochratoxin A removal was affected by time, temperature, sugar and addition of DAP. Moreover, the phenomenon was reversible.Significance and Impact of the Study: Ochratoxin A removal could be an interesting trait for the selection of promising strains; however, the strains removing efficiently the toxin could release it back; thus, the selection of the starter should take into account both the removal and the binding ability of OTA.

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Sauvignon blanc metabolomics: grape juice metabolites affecting the development of varietal thiols and other aroma compounds in wines.

Pinu, F. R., Edwards, P. J. B., Jouanneau, S., Kilmartin, P. A., Gardner, R. C. & Villas-Boas, S. G. (2014). Metabolomics, 10(4), 556-573.

The pathway for the biogenesis of varietal thiols, such as 3-mercaptohexanol (3MH), 3-mercaptohexyl acetate (3MHA) and 4-mercapto-4-methylpentan-2-one (4MMP) in Sauvignon blanc (SB) wines is still an open question. Varietal thiol development requires yeast activity, but poor correlation has been found between thiols and their putative respective precursors. This research is the first application of metabolomics to unravel metabolites in the grape juice that affect the production of varietal thiols in wines. Comprehensive metabolite profiling of 63 commercially harvested SB juices were performed by combining gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. These juices were fermented under controlled laboratory conditions using a commercial yeast strain (EC1118) at 15°C. Correlation of thiol concentration in the wines with initial metabolite profiles identified 24 metabolites that showed positive correlation (R > 0.3) with both 3MH and 3MHA, while only glutamine had positive correlation with 4MMP. Subsequently, we carried out juice manipulation experiments by adding subsets of these 24 metabolites in a 2011 SB grape juice in order to validate the hypotheses generated by metabolomics. The juice manipulation results confirmed metabolomics hypotheses and revealed grape juice metabolites that significantly impact on the development of three major varietal thiols and other aroma compounds of SB wines.

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Grape contribution to wine aroma: production of hexyl acetate, octyl acetate, and benzyl acetate during yeast fermentation is dependent upon precursors in the must.

Dennis, E. G., Keyzers, R. A., Kalua, C. M., Maffei, S. M., Nicholson, E. L. & Boss, P. K. (2012). Journal of Agricultural and Food Chemistry, 60(10), 2638-2646.

Wine is a complex consumer product produced predominately by the action of yeast upon grape juice musts. Model must systems have proven ideal for studies of the effects of fermentation conditions on the production of certain wine volatiles. To identify grape-derived precursors to acetate esters, model fermentation systems were developed by spiking precursors into model must at different concentrations. Solid-phase microextraction–gas chromatgraphy mass spectrometry analysis of the fermented wines showed that a variety of grape-derived aliphatic alcohols and aldehydes are precursors to acetate esters. The C6 compounds hexan-1-ol, hexenal, (E)-2-hexen-1-ol, and (E)-2-hexenal are all precursors to hexyl acetate, and octanol and benzyl alcohol are precursors to octyl acetate and benzyl acetate, respectively. In these cases, the postfermentation concentration of an acetate ester increased proportionally with the prefermentation concentration of the respective precursor in the model must. Determining viticultural or winemaking methods to alter the prefermentation concentration of precursor compounds or change the precursor-to-acetate ester ratio will have implications upon the final flavor and aroma of wines.

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Genome-wide fitness profiles reveal a requirement for autophagy during yeast fermentation.

Piggott, N., Cook, M. A., Tyers, M. & Measday, V. (2011). G3: Genes, Genomes, Genetics, 1(5), 353-367.

The ability of cells to respond to environmental changes and adapt their metabolism enables cell survival under stressful conditions. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is particularly well adapted to the harsh conditions of anaerobic wine fermentation. However, S. cerevisiae gene function has not been previously systematically interrogated under conditions of industrial fermentation. We performed a genome-wide study of essential and nonessential S. cerevisiae gene requirements during grape juice fermentation to identify deletion strains that are either depleted or enriched within the viable fermentative population. Genes that function in autophagy and ubiquitin-proteasome degradation are required for optimal survival during fermentation, whereas genes that function in ribosome assembly and peroxisome biogenesis impair fitness during fermentation. We also uncover fermentation phenotypes for 139 uncharacterized genes with no previously known cellular function. We demonstrate that autophagy is induced early in wine fermentation in a nitrogen-replete environment, suggesting that autophagy may be triggered by other forms of stress that arise during fermentation. These results provide insights into the complex fermentation process and suggest possible means for improvement of industrial fermentation strains.

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Safety Information
Symbol : GHS07, GHS08
Signal Word : Danger
Hazard Statements : H302, H315, H319, H360
Precautionary Statements : P201, P202, P264, P270, P280, P301+P312, P302+P352, P305+P351+P338, P330, P337+P313, P501
Safety Data Sheet
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