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.
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.
Determination of urea using high-performance liquid chromatography with fluorescence detection after automated derivatisation with xanthydrol.
Clark, S., Francis, P. S., Conlan, X. A. & Barnett, N. W. (2007). Journal of Chromatography A, 1161(1-2), 207-213.
A high-performance liquid chromatography (HPLC) method for the determination of urea that incorporates automated derivatisation with xanthydrol (9H-xanthen-9-ol) is described. Unlike the classic xanthydrol approach for the determination of urea, which involves the precipitation of dixanthylurea (N,N′-di-9H-xanthen-9-ylurea), the derivatisation procedure employed in this method produces N-9H-xanthen-9-ylurea, which remains in solution and can be quantified using fluorescence detection (λex = 213 nm; λem = 308 nm) after chromatographic separation from interferences. The limit of detection for urea was 5 × 10-8 M (0.003 mg L-1). This method was applied to the determination of urea in human and animal urine and in wine.
Urea degradation in some white wines by immobilized acid urease in a stirred bioreactor.
Andrich, L., Esti, M. & Moresi, M. (2010). Journal of Agricultural and Food Chemistry, 58(11), 6747-6753.
A purified acid urease preparation was covalently immobilized onto either Eupergit C 250 L or glutaraldehyde-cross-linked chitosan-derivative beads (i.e., Chitopearls BCW-3003 and BCW-3010). The kinetics of urea degradation in two target Italian white (i.e., Grechetto and Sauvignon Blanc) wines, as well as in a model wine solution, by using the above Eupergit C 250 L-, BCW-3003-, or BCW-3010-based biocatalysts, was confirmed to be of the pseudofirst order with respect to the urea concentration in the liquid bulk and not limited by urea mass transfer. In Grechetto and Sauvignon Blanc wines, the corresponding kinetic rate constants were quite similar, being about 7, 18, or 17% of that observed for free enzyme in the model wine solution, respectively. Owing to their minor sensitivity to the phenolic content of the wines tested, the chitosan-based biocatalysts might be potentially employable in the make up of packed-bed cartridges to continuously remove urea from commercial wines.
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.
Energy metabolism of leukemia cells: glycolysis versus oxidative phosphorylation.
Suganuma, K., Miwa, H., Imai, N., Shikami, M., Gotou, M., Goto, M., Mizuno, S., Takahashi, M., Yamamoto, H., Hiramatsu, A., Wakabayashi, M., Watarai, M., Hanamura, I., Imamura, A., Mihara, H. & Nitta, M. (2010). Leukemia & Lymphoma, 51(11), 2112-2119.
For generation of energy, cancer cells utilize glycolysis more vigorously than oxidative phosphorylation in mitochondria (Warburg effect). We examined the energy metabolism of four leukemia cell lines by using glycolysis inhibitor, 2-deoxy-D-glucose (2-DG) and inhibitor of oxidative phosphorylation, oligomycin. NB4 was relatively sensitive to 2-DG (IC50: 5.75 mM), consumed more glucose and produced more lactate (waste product of glycolysis) than the three other cell lines. Consequently, NB4 was considered as a “glycolytic” leukemia cell line. Dependency on glycolysis in NB4 was confirmed by the fact that glucose (+) FCS (−) medium showed more growth and survival than glucose (−) FCS (+) medium. Alternatively, THP-1, most resistant to 2-DG (IC50: 16.14 mM), was most sensitive to oligomycin. Thus, THP-1 was recognized to be dependent on oxidative phosphorylation. In THP-1, glucose (−) FCS (+) medium showed more growth and survival than glucose (+) FCS (−) medium. The dependency of THP-1 on FCS was explained, at least partly, by fatty acid oxidation because inhibitor of fatty acid β-oxidation, etomoxir, augmented the growth suppression of THP-1 by 2-DG. We also examined the mechanisms by which THP-1 was resistant to, and NB4 was sensitive to 2-DG treatment. In THP-1, AMP kinase (AMPK), which is activated when ATP becomes limiting, was rapidly phosphorylated by 2-DG, and expression of Bcl-2 was augmented, which might result in resistance to 2-DG. On the other hand, AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in NB4, which is 2-DG sensitive. These results will facilitate the future leukemia therapy targeting metabolic pathways.
Urea degradation kinetics in model wine solutions by acid urease immobilised onto chitosan-derivative beads of different sizes.
Andrich, L., Esti, M. & Moresi, M. (2010). Enzyme and Microbial Technology, 46(5), 397-405.
In this work, a purified acid urease preparation was covalently immobilised onto porous chitosan beads of different size. The covalent binding method was found to be more efficient than the adsorption cross-linkage one whatever the glutaraldehyde-to-chitosan bead ratio (YGA/CHI) used. At the optimal YGA/CHI ratio of 0.625 g g-1 , the specific activity (ABi) of the biocatalysts decreased from circa 300 to 70 IU g-1 wet support, as the bead average diameter (dP) increased from 0.14 to 2.2 mm. Generally, ABi reduced less than 5% after preservation in the wet form at 4°C for 150–170 days. Only the biocatalyst prepared using the Chitopearl BCW-3001 lost about 40% of its initial activity. The kinetics of urea degradation in a model wine solution using these biocatalysts was of the pseudo-first order with respect to the urea concentration in the liquid bulk, the apparent pseudo-first order kinetic rate constant (kIi) ranging from about two thirds to one fifth of that (kIF) pertaining to free acid urease. In the operating conditions tested, the reaction kinetics was estimated as unaffected by the contribution of the external film and intraparticle diffusion mass-transfer resistances. When the model wine solution was enriched with the high-inhibitory tannins extracted from grape seeds, at the maximum level tested (374 ± 2 g GAE m-3) kIi reduced to no more than (58 ± 9)% of kIF), this proving quite a higher protective action against such compounds for the chitosan-based biocatalysts towards free or Eupergit® C 250 L-immobilised acid urease.
Comparative study of colorectal health related compounds in different types of bread: Analysis of bread samples pre and post digestion in a batch fermentation model of the human intestine.
Hiller, B., Schlörmann, W., Glei, M. & Lindhauer, M. G. (2011). Food Chemistry, 125(4), 1202-1212.
Seven different types of wheat and rye bread were analysed for colorectal health related compounds, pre and post digestion, in batch fermentation model of the human intestine. Pre digestion, higher amounts of colorectal health-related dietary fibre compounds (soluble/insoluble/total dietary fibre, arabinoxylans, β-glucans) and phytochemicals (mono-/di-phenolic acids, phytic acid, hydroxymethylfurfural) were detected in wholemeal than in refined flour types of bread, as well as in rye flour types than in wheat flour types of bread. Post digestion, faecal bacterial metabolites of colorectal health promoting (acetate/propionate/butyrate, lactate, free mono-/di-phenolic acids) and impairing (amino metabolites, bile acid metabolites) activities were found in fermentation supernatants of bread samples. All types of bread positively affected faecal bacterial metabolism; among the different types of bread, the highest stimulation of organic acid production (acetate/propionate/butyrate, lactate) and the lowest detrimental bacterial enzyme activities (β-glucuronidase, urease) were detected for wheat flour bread, whereas the strongest retardation of bacterial bile acid degradation and the strongest stimulation of phenolic acid metabolite release (phenylpropionic/phenylpropenoic acid derivatives) were induced by wholemeal rye bread. This study for the first time presents a qualitative and quantitative overview over the broad spectrum of colorectal health related compounds in high- and low-fibre types of bread, pre and post in vitro digestion, and highlights the significance of bread for the preventive nutritional intervention of colorectal cancer.
Assessing heterogeneity of the composition of mare's milk in Flanders.
Naert, L., Vande Vyvere, B., Verhoeven, G., Duchateau, L., De Smet, S. & Coopman, F. (2013). Vlaams Diergeneeskundig Tijdschrift, 82(1), 23-30.
In this study, the effect of farm, time, season and health was evaluated on the composition of mare's milk sold in Flanders. The content of the analyzed components (i.e. fat, fatty acids, protein, lactoferrin, lysozyme, lactose and urea) differed significantly (p < 0.0001) between farms, at a given moment in time. Within each farm, large month-to-month variations for most milk components (p <0.01 to 0.0001) were observed. The variation over time between different farms was smaller. These findings indicate that the composition of the mare's milk consumer portions varies substantially between the different farms and also over time within each farm. Season, nutrition, udder health and worm burden are believed to contribute significantly to this variation.