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.
Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers.
Slininger, P. J., Dien, B. S., Kurtzman, C. P., Moser, B. R., Bakota, E. L., Thompson, S. R., , O'Bryan, P. J., Cotta, M. A., Balan, V., Jin, M., Sousa, L. D. C. & Dale, B. E. & Sousa, L. D. C. (2016). Biotechnology and Bioengineering, 113(8), 1676-1690.
Oleaginous yeasts can convert sugars to lipids with fatty acid profiles similar to those of vegetable oils, making them attractive for production of biodiesel. Lignocellulosic biomass is an attractive source of sugars for yeast lipid production because it is abundant, potentially low cost, and renewable. However, lignocellulosic hydrolyzates are laden with byproducts which inhibit microbial growth and metabolism. With the goal of identifying oleaginous yeast strains able to convert plant biomass to lipids, we screened 32 strains from the ARS Culture Collection, Peoria, IL to identify four robust strains able to produce high lipid concentrations from both acid and base-pretreated biomass. The screening was arranged in two tiers using undetoxified enzyme hydrolyzates of ammonia fiber expansion (AFEX)-pretreated cornstover as the primary screening medium and acid-pretreated switch grass as the secondary screening medium applied to strains passing the primary screen. Hydrolyzates were prepared at ~18–20% solids loading to provide ~110 g/L sugars at ~56:39:5 mass ratio glucose:xylose:arabinose. A two stage process boosting the molar C:N ratio from 60 to well above 400 in undetoxified switchgrass hydrolyzate was optimized with respect to nitrogen source, C:N, and carbon loading. Using this process three strains were able to consume acetic acid and nearly all available sugars to accumulate 50–65% of cell biomass as lipid (w/w), to produce 25–30 g/L lipid at 0.12–0.22 g/L/h and 0.13–0.15 g/g or 39–45% of the theoretical yield at pH 6 and 7, a performance unprecedented in lignocellulosic hydrolyzates. Three of the top strains have not previously been reported for the bioconversion of lignocellulose to lipids. The successful identification and development of top-performing lipid-producing yeast in lignocellulose hydrolyzates is expected to advance the economic feasibility of high quality biodiesel and jet fuels from renewable biomass, expanding the market potential for lignocellulose-derived fuels beyond ethanol for automobiles to the entire U.S. transportation market.
Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.
Milne, N., Luttik, M. A. H., Rojas, H. C., Wahl, A., Van Maris, A. J. A., Pronk, J. T. & Daran, J. M. (2015). Metabolic Engineering, 30, 130-140.
In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe vgene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni2+-dependent enzyme and Saccharomyces cerevisiae does not express native Ni2+-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2 Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min-1 mg protein-1 and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni2+ concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni2+-dependent enzymes can be functionally expressed in S. cerevisiae.
Urea and lipid extraction treatment effects on δ15N and δ13C values in pelagic sharks.
Li, Y., Zhang, Y., Hussey, N. E. & Dai, X. (2016). Rapid Communications in Mass Spectrometry, 30(1), 1-8.
Rationale: Stable isotope analysis (SIA) provides a powerful tool to investigate diverse ecological questions for marine species, but standardized values are required for comparative assessments. For elasmobranchs, their unique osmoregulatory strategy involves retention of 15N -depleted urea in body tissues and this may bias δ15N values. This may be a particular problem for large predatory species, where δ15N discrimination between predator and consumed prey can be small. Methods: We evaluated three treatments (deionized water rinsing [DW], chloroform/methanol [LE] and combined chloroform/methanol and deionized water rinsing [LE+DW]) applied to white muscle tissue of 125 individuals from seven pelagic shark species to (i) assess urea and lipid effects on stable isotope values determined by IRMS and (ii) investigate mathematical normalization of these values. Results: For all species examined, the δ15N values and C:N ratios increased significantly following all three treatments, identifying that urea removal is required prior to SIA of pelagic sharks. The more marked change in δ15N values following DW (1.3 ± 0.4‰) and LE+DW (1.2 ± 0.6‰) than following LE alone (0.7 ± 0.4‰) indicated that water rinsing was more effective at removing urea. The DW and LE+DW treatments lowered the %N values, resulting in an increase in C:N ratios from the unexpected low values of <2.6 in bulk samples to ~3.1 ± 0.1, the expected value of protein. The δ13N values of all species also increased significantly following LE and LE+DW treatments. Conclusions: Given the mean change in δ15N (1.2 ± 0.6‰) and δ13N values (0.7 ± 0.4‰) across pelagic shark species, it is recommended that muscle tissue samples be treated with LE+DW to efficiently extract both urea and lipids to standardize isotopic values. Mathematical normalization of urea and lipid-extracted δ15NLE+DW and δ13CLE+DW values using the lipid-extracted δ15NLE and δ13CLE data were established for all pelagic shark species.