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L-Arginine/Urea/Ammonia Assay Kit

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L-Arginine Urea Ammonia Assay Kit K-LARGE Scheme
   
Product code: K-LARGE
€173.00

50 Assays per kit

Prices exclude VAT

Available for shipping

Content: 50 assays per kit
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: 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.

Advantages
  • 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
Publications
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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Publication
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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Publication
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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Publication
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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Publication
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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Publication
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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Publication
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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Publication
Biguanide related compounds in traditional antidiabetic functional foods.

Perla, V. & Jayanty, S. S. (2013). Food Chemistry, 138(2-3), 1574-1580.

Biguanides such as metformin are widely used worldwide for the treatment of type-2 diabetes. The identification of guanidine and related compounds in French lilac plant (Galega officinalis L.) led to the development of biguanides. Despite of their plant origin, biguanides have not been reported in plants. The objective of this study was to quantify biguanide related compounds (BRCs) in experimentally or clinically substantiated antidiabetic functional plant foods and potatoes. The corrected results of the Voges–Proskauer (V–P) assay suggest that the highest amounts of BRCs are present in green curry leaves (Murraya koenigii (L.) Sprengel) followed by fenugreek seeds (Trigonella foenum-graecum L.), green bitter gourd (Momordica charantia Descourt.), and potato (Solanum tuberosum L.). Whereas, garlic (Allium sativum L.), and sweet potato (Ipomea batatas (L.) Lam.) contain negligible amounts of BRCs. In addition, the possible biosynthetic routes of biguanide in these plant foods are discussed.

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Publication
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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Publication
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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Publication
Stabilized and Immobilized Bacillus subtilis Arginase for the Biobased Production of Nitrogen‐Containing Chemicals.

Könst, P. M., Turras, P. M. C. C. D., Franssen, M. C. R., Scott, E. L. & Sanders, J. P. M. (2010). Advanced Synthesis & Catalysis, 352(9), 1493-1502.

L-Ornithine could serve as an intermediate in the biobased production of 1,4-diaminobutane from L-arginine. Using the concept of biorefinery, L-arginine could become widely available from biomass waste streams via the nitrogen storage polypeptide cyanophycin. Selective hydrolysis of L-arginine to L-ornithine is difficult to perform chemically, therefore the stabilization and immobilization of Bacillus subtilis arginase (EC 3.5.3.1) was studied in a continuously stirred membrane reactor system. Initial pH of the substrate solution, addition of L-aspartic acid and reducing agents all appeared to have an effect on the operational stability of B. subtilis arginase. A remarkably good operational stability (total turnover number, TTN=1.13.108) at the pH of arginine free base (pH 11.0) was observed, which was further improved with the addition of sodium dithionite to the substrate solution (TTN>1.109). B. subtilis arginase was successfully immobilized on three commercially available epoxy-activated supports. Immobilization on Sepabeads EC-EP was most promising, resulting in a recovered activity of 75% and enhanced thermostability. In conclusion, the stabilization and immobilization of B. subtilis arginase has opened up possibilities for its application in the biobased production of nitrogen-containing chemicals as an alternative to the petrochemical production.

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Publication
Regulatory role of nitric oxide in the reduced survival of erythrocytes in visceral leishmaniasis.

Chowdhury, K. D., Sen, G. & Biswas, T. (2010). Biochimica et Biophysica Acta (BBA)-General Subjects, 1800(9), 964-976.

Background: Nitric oxide (NO) plays a vital role in maintaining the survivability of circulating erythrocytes. Here we have investigated whether NO depletion associated with visceral leishmaniasis (VL) is responsible for the reduced survival of erythrocytes observed during the disease. Methods: Infected hamsters were treated with standard anti-leishmanial sodium stibogluconate (SAG) and NO donor isosorbide dinitrate (ISD). Erythrophagocytosis by macrophages was determined by labelling the cells with FITC followed by flow cytometry. Aggregation of band3 was estimated from band3 associated EMA fluorescence. Caspase 3 activity was measured using immunosorbent assay kit. Phosphatidylserine (PS) externalization and cell shrinkage were determined using annexin V. Aminophspholipid translocase and scramblase activities were measured following NBD-PS and NBD-PC internalization, respectively. Results: Impairment of both synthesis and uptake of NO resulted in decreased bioavailability of this signaling molecule in erythrocytes in VL. NO level was replenished after simultaneous treatment with ISD and SAG. Combination treatment decreased red cell apoptosis in infected animals by deactivating caspase 3 through s-nitrosylation. Drug treatment prevented infection-mediated ATP depletion and altered calcium homeostasis in erythrocytes. Improved metabolic environment effectively amended dysregulation of aminophospholipid translocase and scramblase, which in turn reduced cell shrinkage, and exposure of phosphatidylserine on the cell surface under the diseased condition. Conclusion and general significance: In this study, we have identified NO depletion to be an important factor in promoting premature hemolysis with the progress of leishmanial infection. The study implicates NO to be a possible target for future drug development towards the promotion of erythrocyte survival in VL.

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Publication
Changes in the volatile compound production of fermentations made from musts with increasing grape content.

Keyzers, R. A. & Boss, P. K. (2010). Journal of Agricultural and Food Chemistry, 58(2), 1153-1164.

Wine is a complex consumer product produced predominately by the action of yeast upon grape juice. Model must systems have proven to be ideal for studies into the effects of fermentation conditions on the production of certain wine volatiles. To clarify the contribution of grape juice to the production of wine volatiles, we have employed a model must system spiked with increasing amounts of grape juice (Riesling or Cabernet Sauvignon). The resulting fermented wines were analyzed by SPME-GC-MS and the data obtained grouped using ANOVA and cluster analyses to reveal those compounds that varied in concentration with reproducible trends relative to juice concentration. Such grouping highlights those compounds that are grape-dependent or for which production is modulated by grape composition. In some cases, increasing the proportion of grape juice in the fermentations stimulated the production of certain esters to levels between 2- and 140-fold higher than those seen in fermentations made with model grape juice media alone. The identification of the grape components responsible for the increased production of these wine volatiles will have implications for the impact of grape production and enology on wine flavor and aroma.

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Publication
The determination of urea in wine – a review.

Francis, P. S. (2006). Australian Journal of Grape and Wine Research, 12(2), 97-106.

The concentration of urea in wine is not routinely measured in Australian laboratories, but has been examined in studies of yeast metabolism and the formation of ethyl carbamate, a known carcinogen. For alcoholic beverages that may contain high levels of urea, steps have been taken to reduce the concentration of urea and therefore prevent ethyl carbamate production. Methods for the determination of urea in wine can be grouped into three categories that indicate how selectivity for urea is achieved; those based on colour-forming reactions, enzymatic hydrolysis and chromatographic separation. The two dominant methods used by research groups over the past fifteen years for the determination of urea in wine are based on the urea/ammonia test kit available from Boeringer Mannheim/R-Biopharm and the reaction of urea with 1-phenyl-1,2-propanedione-2-oxime; both are time-consuming and labour-intensive, but involve relatively straightforward and well-established procedures. However, other options are available that may be better suited to the desired application and the instrumentation available in any particular laboratory.

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Safety Information
Symbol : GHS07, GHS08
Signal Word : Warning
Hazard Statements : H302, H315, H319, H360, H361, H362, H412
Precautionary Statements : P201, P202, P260, P263, P264, P270, P273, P280, P301+P312, P302+P352, P305+P351+P338, P308+P313, P330, P405, P501
Safety Data Sheet
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