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.
Fermentation of stalk juices from different Nigerian sorghum cultivars to ethanol.
Nasidi, M., Agu, R., Yusuf Deeni, Y. & Walker, G. (2013). Bioethanol, 1(1), 20-27.
For improved production of ethanol from sorghum stalk juice fermentation, cultivation location and cultivar type are important factors to consider. In the present study, SSV2 and KSV8 sorghum cultivars were cultivated in Kano and Kaduna states in Nigeria that exhibit notably different rain precipitation and diurnal temperatures. The crude stalk juices (without pre-treatment or nutrient supplementation) were extracted from these sorghum samples and fermented with a distiller’s strain of the yeast, Saccharomyces cerevisiae. Sugar consumption and alcohol production were determined by HPLC and GC-MS, respectively. When it was grown in the Kaduna site, SSV2 was identified as the highest yielding sorghum cultivar from which we extracted the maximum levels of extractable sugars (161.50 g l-1) that yielded favourable ethanol levels of 80.56 g l-1 following fermentation. Our findings show that relatively colder and wetter cultivation sites are preferred for sorghum stalk juice destined for bioethanol production.
Sensor combination and chemometric variable selection for online monitoring of Streptomyces coelicolor fed-batch cultivations.
Ödman, P., Johansen, C. L., Olsson, L., Gernaey, K. V. & Lantz, A. E. (2010). Applied Microbiology and Biotechnology, 86(6), 1745-1759.
Fed-batch cultivations of Streptomyces coelicolor, producing the antibiotic actinorhodin, were monitored online by multiwavelength fluorescence spectroscopy and off-gas analysis. Partial least squares (PLS), locally weighted regression, and multilinear PLS (N-PLS) models were built for prediction of biomass and substrate (casamino acids) concentrations, respectively. The effect of combination of fluorescence and gas analyzer data as well as of different variable selection methods was investigated. Improved prediction models were obtained by combination of data from the two sensors and by variable selection using a genetic algorithm, interval PLS, and the principal variables method, respectively. A stepwise variable elimination method was applied to the three-way fluorescence data, resulting in simpler and more accurate N-PLS models. The prediction models were validated using leave-one-batch-out cross-validation, and the best models had root mean square error of cross-validation values of 1.02 g l-1 biomass and 0.8 g l-1 total amino acids, respectively. The fluorescence data were also explored by parallel factor analysis. The analysis revealed four spectral profiles present in the fluorescence data, three of which were identified as pyridoxine, NAD(P)H, and flavin nucleotides, respectively.
Analysis of protein and total usable nitrogen in beer and wine using a microwell ninhydrin assay.
Abernathy, D. G., Spedding, G. & Starcher, B. (2009). Journal of the Institute of Brewing, 115(2), 122-127.
In this study we present a ninhydrin based microwell assay that can be utilized in place of the traditional Kjeldahl method for the determination of the protein content of beer or wine. In addition, the assay is ideal for the determination of free amino acids in beer (FAN), a term understood and used by brewers, and yeast assimilable nitrogen (YAN) used by enologists. The assay only measures alpha amino acids and ammonia so other nitrogen sources are not detected, resulting in a 30% reduction in total protein of a variety of beers compared to the Kjeldahl method, which measures nitrogen from all sources. The results also showed that only 25% of the total “protein” in beer is actually derived from peptides larger than 3,500 Kd. Analysis of beer or wine with the microwell assay for total usable nitrogen was compared to the standard FAN and YAN methods and conditions were determined for maximal efficiency and precision. Superior results were obtained with low reaction volumes and a stable sodium acetate buffered ninhydrin reagent at pH 5.5. As an alternative, for use with cuvettes, a reduced volume FAN assay using the same pH 5.5 sodium acetate buffered ninhydrin reagent gave comparable results. The assay is economical, rapid, accurate and applicable to large numbers of samples.
Gene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectors.
Shan, Y., Luo, T., Peng, C., Sheng, R., Cao, A., Cao, X., Shen, M., Guo, R., Tomas, H. & Shi, X. (2012). Biomaterials, 33(10), 3025-3035.
Development of highly efficient nonviral gene delivery vectors still remains a great challenge. In this study, we report a new gene delivery vector based on dendrimer-entrapped gold nanoparticles (Au DENPs) with significantly higher gene transfection efficiency than that of dendrimers without AuNPs entrapped. Amine-terminated generation 5 poly(amidoamine) (PAMAM) dendrimers (G5.NH) were utilized as templates to synthesize AuNPs with different Au atom/dendrimer molar ratios (25:1, 50:1, 75:1, and 100:1, respectively). The formed Au DENPs were used to complex two different pDNAs encoding luciferase (Luc) and enhanced green fluorescent protein (EGFP), respectively for gene transfection studies. The Au DENPs/pDNA polyplexes with different N/P ratios and compositions of Au DENPs were characterized by gel retardation assay, light scattering, zeta potential measurements, and atomic force microscopic imaging. We show that the Au DENPs can effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines as demonstrated by both Luc assay and fluorescence microscopic imaging of the EGFP expression. The transfection efficiency of Au DENPs with Au atom/dendrimer molar ratio of 25:1 was at least 100 times higher than that of G5.NH2 dendrimers without AuNPs entrapped at the N/P ratio of 2.5:1. The higher gene transfection efficiency of Au DENPs is primarily due to the fact that the entrapment of AuNPs helps preserve the 3-dimensional spherical morphology of dendrimers, allowing for more efficient interaction between dendrimers and DNA. With the less cytotoxicity than that of G5.NH2 dendrimers demonstrated by thiazoyl blue tetrazolium bromide assay and higher gene transfection efficiency, it is expected that Au DENPs may be used as a new gene delivery vector for highly efficient transfection of different genes for various biomedical applications.
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.
Dendrimer-entrapped gold nanoparticles modified with folic acid for targeted gene delivery applications.
Xiao, T., Hou, W., Cao, X., Wen, S., Shen, M. & Shi, X. (2013). Biomaterials Science, 1(11), 1172-1180.
We report a new use of dendrimer-entrapped gold nanoparticles (Au DENPs) modified with folic acid (FA) as a non-viral vector for targeted gene delivery applications. In this study, amine-terminated generation 5 poly(amidoamine) dendrimers modified with FA via covalent conjugation were used as templates to synthesize gold nanoparticles with an Au salt/dendrimer molar ratio of 25 : 1. The synthesized FA-modified Au DENPs (Au DENPs-FA) were used as a non-viral vector for the delivery of plasmid DNA (pDNA) into a model cancer cell line (HeLa cells) overexpressing high-affinity FA receptors (FAR). The DNA compaction ability of the formed Au DENPs-FA was systematically characterized using a gel retardation assay, zeta potential, and dynamic light scattering. We show that similar to the Au DENPs vector without FA, the Au DENPs-FA vector was able to compact the pDNA encoding enhanced green fluorescent protein (EGFP) at an N/P ratio of 0.5. Transfection results show that the Au DENPs-FA vector enables much higher luciferase and EGFP gene expression in HeLa cells overexpressing FAR than the Au DENPs without FA, demonstrating the role played by FA-mediated targeting for enhanced gene transfection in target cells. With a lower cytotoxicity than that of the Au DENPs without FA proven by a cell viability assay, the developed FA-modified Au DENPs may be used as a promising non-viral vector for safe and targeted gene therapy applications.
Growth and lipid production of Umbelopsis isabellina on a solid substrate—Mechanistic modeling and validation.
Meeuwse, P., Klok, A. J., Haemers, S., Tramper, J. & Rinzema, A. (2012). Process Biochemistry, 47(8), 1228-1242.
Microbial lipids are an interesting feedstock for biodiesel. Their production from agricultural waste streams by fungi cultivated in solid-state fermentation may be attractive, but the yield of this process is still quite low. In this article, a mechanistic model is presented that describes growth, lipid production and lipid turnover in a culture of Umbelopsis isabellina on κ-carrageenan plates containing the monomers glucose and alanine as C-source and N-source, respectively, and improves the understanding of the complex solid-state system. The model includes reaction kinetics and diffusion of glucose, alanine and oxygen. It is validated empirically and describes the different phases of the culture very well: exponential growth, linear growth because of oxygen limitation, accumulation of lipids and carbohydrates after local N-depletion and turnover of lipids after local C-depletion. Extending the model with an unidentified extracellular product improved the fit of the model to the data. The model shows that oxygen limitation is extremely important in solid-state cultures using monomers, and explains the difference in production rate with submerged cultures. However, the results also show that the specific lipid production rate in solid-state cultures is much lower than in submerged cultures, which results in a low lipid yield.
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.
Changes in the volatile compound production of fermentations made from musts with increasing grape content.
Keyzers, R. A. & Boss, P. K. (2009). 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.