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Primary Amino Nitrogen Assay Kit (PANOPA)

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0:05 Introduction
0:50 Principle
1:24  Reagent Preparation
2:34 Procedure
4:52 Calculation

Primary Amino Nitrogen Assay Kit (PANOPA)
Primary Amino Nitrogen Assay Kit PANOPA K-PANOPA
   
Product code: K-PANOPA
€127.00

100 assays (manual) / 1000 assays (microplate) / 1100 assays (auto-analyser)

Prices exclude VAT

Available for shipping

Content: 100 assays (manual) / 1000 assays (microplate) / 1100 assays (auto-analyser)
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: Nitrogen, Primary Amino Nitrogen, YAN
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.2 to 10 µg of amino nitrogen per assay
Limit of Detection: 2.59 mg N/L
Reaction Time (min): ~ 15 min
Application examples: Grape juice, must, wine and other materials.
Method recognition: Novel Method (Under Patent: U.S. Pat. No. 9,738,920)

The Primary Amino Nitrogen (PANOPA) Assay Kit is suitable for the measurement and analysis of primary amino nitrogen 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).

Primary Amino Nitrogen Assay Kit PANOPA K-PANOPA Scheme

Advantages
  • Simple format (absorbances read at 340 nm) 
  • 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 
  • Suitable for manual, microplate and auto-analyser formats
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

Augmentation of chemical and organoleptic properties in Syzygium cumini wine by incorporation of grape seeds during vinification.

VenuGopal, K. S., Cherita, C. & Anu-Appaiah, K. A. (2018). Food Chemistry, 242, 98-105.

The role of grape seed tannins on improving organoleptic properties and its involvement in color stabilization in red wine are well established. The addition of grape seeds as the source of condensed tannins in fruit wine may provide a solution for its color instability and improvement of sensory attributes. Syzgium cumini is traditionally known for its therapeutic properties. In the current study, the influence of yeasts and grape seed addition during fermentation on the chromatic, phenolic and sensory attributes of the wine was accessed. Grape seed addition improved the color characteristics of wine and increased overall phenolic composition. Analysis by HPLC revealed 6 major anthocyanins, among which 3, 5-diglucoside form of delphidin and petunidin was found to be the major components. Cluster and PLSR analysis explained the impact of seed addition on the yeasts, as well as on the perception of panelists, with bitterness and astringency as the dominating attributes.

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Publication
Fermentation of grapes throughout development identifies stages critical to the development of wine volatile composition.

Boss, P. K., Kalua, C. M., Nicholson, E. L., Maffei, S. M., Böttcher, C. & Davies, C. (2017). Australian Journal of Grape and Wine Research, 24(1), 24-37.

Background and Aims: Many variables affect the volatile profile of wine during production, and grape composition is an important source of varietal characters and metabolites for yeast during fermentation. In order to manage wine style through changes in grape composition, the knowledge of when important changes occur in the berries is essential. This study sought to identify stages of berry development that are most critical in defining wine volatile composition. Methods and Results: Cabernet Sauvignon and Riesling grapes at several stages throughout development were fermented, after normalising sugar content, and the volatile compounds in the resulting wines were profiled. For both cultivars, the compounds were grouped into six clusters, which best described the changes in concentration of the volatile compounds in the wines. Some varietal compounds showed significant differences in wines made from berries at the various developmental stages; however, many fermentation-derived volatile compounds, especially esters, were also significantly affected by berry developmental stage. Conclusions: Dynamic changes occur in the volatile profile of wines made from grapes at several stages of development, but a few common patterns of change are seen for several volatile compounds. Significance of the Study: Several distinct phases of berry development associated with either an increasing or decreasing concentration of wine volatile compounds were identified, which will inform the timing of future strategies designed to alter wine composition through interventions in the vineyard.

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Hydrogen sulphide production during cider fermentation is moderated by pre‐fermentation methionine addition.

Boudreau, T. F., Peck, G. M., Ma, S., Patrick, N., Duncan, S., O'Keefe, S. F. & Stewart, A. C. Journal of the Institute of Brewing, 123(4), 553-561.

Yeast assimilable nitrogen (YAN) concentration and composition impact hydrogen sulphide (H2S) production and fermentation kinetics during wine fermentation, but this phenomenon has not been extensively studied in cider fermentation. Our hypothesis was that H2S production during cider fermentation could be decreased through pre-fermentation modification of concentrations of individual amino acids. Apple juice (53 mg L-1 YAN) was supplemented with asparagine, arginine, methionine or ammonium and fermented with EC1118 and UCD522 yeast strains. No difference in H2S production among fermentations was observed with addition of asparagine, arginine or ammonium. Methionine addition of 5 mg L-1 decreased H2S production by yeast strain EC1118 at 53 mg L-1 YAN. With 153 mg L-1 initial YAN, only methionine addition of 50 mg L-1 decreased H2S production, and no tested methionine rates decreased H2S production with 253 mg L-1 initial YAN. Supplementation to 153 mg L-1 YAN resulted in increased H2S production at all methionine concentrations tested. Sensory differences in aroma were detected in samples supplemented with ammonium and methionine, and these differences were correlated with observed differences in H2S production. Our results indicate that supplementing cider fermentations with methionine leads to lower H2S formation, especially in apple juice containing low YAN.

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The interactive effect of fungicide residues and yeast assimilable nitrogen on fermentation kinetics and hydrogen sulfide production during cider fermentation.

Boudreau, T. F., Peck, G. M., O'Keefe, S. F. & Stewart, A. C. (2017). Journal of the Science of Food and Agriculture, 97(2), 693-704.

BACKGROUND: Fungicide residues on fruit may adversely affect yeast during cider fermentation, leading to sluggish or stuck fermentation or the production of hydrogen sulfide (H2S), which is an undesirable aroma compound. This phenomenon has been studied in grape fermentation but not in apple fermentation. Low nitrogen availability, which is characteristic of apples, may further exacerbate the effects of fungicides on yeast during fermentation. The present study explored the effects of three fungicides: elemental sulfur (S0) (known to result in increased H2S in wine); fenbuconazole (used in orchards but not vineyards); and fludioxonil (used in post-harvest storage of apples). RESULTS: Only S0 led to increased H2S production. Fenbuconazole (≥0.2 mg L-1) resulted in a decreased fermentation rate and increased residual sugar. An interactive effect of yeast assimilable nitrogen (YAN) concentration and fenbuconazole was observed such that increasing the YAN concentration alleviated the negative effects of fenbuconazole on fermentation kinetics. CONCLUSION: Cidermakers should be aware that residual fenbuconazole (as low as 0.2 mg L-1) in apple juice may lead to stuck fermentation, especially when the YAN concentration is below 250 mg L-1. These results indicate that fermentation problems attributed to low YAN may be caused or exacerbated by additional factors such as fungicide residues, which have a greater impact on fermentation performance under low YAN conditions.

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A survey of yeast from the Yarrowia clade for lipid production in dilute acid pretreated lignocellulosic biomass hydrolysate.

Quarterman, J., Slininger, P. J., Kurtzman, C. P., Thompson, S. R. & Dien, B. S. (2016). Applied Microbiology and Biotechnology, 101(8), 3319-3334.

Yarrowia lipolytica is an oleaginous yeast species that has attracted attention as a model organism for synthesis of single cell oil. Among over 50 isolates of Y. lipolytica identified, only a few of the strains have been studied extensively. Furthermore, 12 other yeast species were recently assigned to the Yarrowia clade, and most are not well characterized in terms of cell growth and lipid accumulation, especially in industrially relevant conditions. In the present study, we investigated biomass and lipid production by 57 yeast isolates, representing all 13 species in the Yarrowia clade, on a non-detoxified dilute acid-pretreated switchgrass hydrolysate under highly aerobic conditions. The objective was to compare yeast physiology during growth in an abundant, low-cost biomass feedstock and to expand diversity of genetically tractable, oleaginous yeasts available for lipid research. Screening of 45 Y. lipolytica isolates demonstrated considerable variation within the species in terms of lipid accumulation (min = 0.1 g/L; max = 5.1 g/L; mean = 2.3 g/L); three strains (NRRL YB-420, YB-419, and YB-392) were especially promising for cellulosic biomass conversion with average improvements of 43, 57, and 64%, respectively, in final lipid titer as compared to control strain W29. Subsequently, evaluation of strains from 13 distinct species in the Yarrowia clade identified Candida phangngensis PT1-17 as the top lipid producer with a maximum titer of 9.8 g/L lipid, which was over twofold higher than the second-best species in the clade (Candida hollandica NRRL Y-48254). A small set of the most promising strains from the screenings was further characterized to evaluate inhibitor tolerance, lipid production kinetics, and fatty acid distribution. We expect that the results of this study will pave the way for new biotechnological applications involving previously overlooked and under-characterized strains within the Yarrowia clade.

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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.

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Publication
Partially PEGylated dendrimer-entrapped gold nanoparticles: a promising nanoplatform for highly efficient DNA and siRNA delivery.

Hou, W., Wei, P., Kong, L., Guo, R., Wang, S. & Shi, X. (2016). Journal of Materials Chemistry B, 4(17), 2933-2943.

Exploring a plasmid DNA (pDNA)/small interfering RNA (siRNA) delivery vector with excellent biocompatibility and high gene transfection efficiency still remains a great challenge. In this research, generation 5 (G5) dendrimer-entrapped gold nanoparticles (Au DENPs) partially modified with polyethylene glycol monomethyl ether (mPEG) were designed as non-viral pDNA/siRNA delivery vectors. The pDNA that can encode luciferase (Luc) or enhanced green fluorescent protein (EGFP) and the Bcl-2 siRNA that can knockdown the expression of the Bcl-2 protein were successfully packaged by the partially PEGylated Au DENPs and effectively delivered into HeLa cells. The length of the surface conjugated mPEG chains and the composition of the entrapped Au NPs were systematically altered to explore their influences on the structure, cytotoxicity, and pDNA or siRNA delivery efficiency. We show that the modified mPEG and entrapped Au NPs can significantly improve the encoding of Luc and EGFP or silence the Bcl-2 protein expression, and the {(Au0)50-G5.NH2-mPEG2K} DENPs display the best DNA or siRNA delivery efficiency among all the designed partially PEGylated Au DENPs. The Luc transfection efficiency of the {( Au0)50-G5.NH2-mPEG2K} was about 292 times higher than that of the G5.NH2 dendrimers at an N/P ratio of 5:1, and the Bcl-2 protein was silenced to 15% using the {( Au0)50-G5.NH2-mPEG2K} as a vector relative to the expression level transfected using the G5.NH2 dendrimers (100%). With enhanced pDNA/siRNA transfection efficiency and less cytotoxicity, the PEGylated Au DENPs may hold great promise to be used in pDNA and siRNA delivery applications.

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Publication
RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

Kong, L., Alves, C. S., Hou, W., Qiu, J., Möhwald, H., Tomás, H. & Shi, X. (2015). ACS Applied Materials & Interfaces, 7(8), 4833-4843.

We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

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Publication
Designing and creating Saccharomyces interspecific hybrids for improved, industry relevant, phenotypes.

Bellon, J. R., Yang, F., Day, M. P., Inglis, D. L. & Chambers, P. J. (2015). Applied Microbiology and Biotechnology, 99(20), 8597-8609.

To remain competitive in increasingly overcrowded markets, yeast strain development programmes are crucial for fermentation-based food and beverage industries. In a winemaking context, there are many yeast phenotypes that stand to be improved. For example, winemakers endeavouring to produce sweet dessert wines wrestle with fermentation challenges particular to fermenting high-sugar juices, which can lead to elevated volatile acidity levels and extended fermentation times. In the current study, we used natural yeast breeding techniques to generate Saccharomyces spp. interspecific hybrids as a non-genetically modified (GM) strategy to introduce targeted improvements in important, wine-relevant traits. The hybrids were generated by mating a robust wine strain of Saccharomyces cerevisiae with a wine isolate of Saccharomyces bayanus, a species previously reported to produce wines with low concentrations of acetic acid. Two hybrids generated from the cross showed robust fermentation properties in high-sugar grape juice and produced botrytised Riesling wines with much lower concentrations of acetic acid relative to the industrial wine yeast parent. The hybrids also displayed suitability for icewine production when bench-marked against an industry standard icewine yeast, by delivering icewines with lower levels of acetic acid. Additionally, the hybrid yeast produced wines with novel aroma and flavour profiles and established that choice of yeast strain impacts on wine colour. These new hybrid yeasts display the desired targeted fermentation phenotypes from both parents, robust fermentation in high-sugar juice and the production of wines with low volatile acidity, thus establishing their suitability for wine styles that are traditionally troubled by excessive volatile acidity levels.

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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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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.

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Publication
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.

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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<sub2) 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.

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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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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.

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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.

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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.

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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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