Primary Amino Nitrogen Assay Kit (PANOPA)

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00:05 Introduction
00:50 Principle
01:24  Reagent Preparation
02:34 Procedure
04:54 Calculations

Primary Amino Nitrogen Assay Kit K-PANOPA Scheme
Reference code: K-PANOPA
SKU: 700004323

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

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

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).

Display our complete list of nitrogen test kits.

View White paper - Free Amino Nitrogen.

Scheme-K-PANOPA PANOPA Megazyme

  • 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
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator Validation Report
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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Agaricus bisporus chitosan influences the concentrations of caftaric acid and furan-derived compounds in Pinot noir juice and base wine.

Mederios, J., Xu, S., Pickering, G. & Kemp, B. (2023). Oeno One, 57(3), 255-268.

Chitosan is a fining agent used in winemaking, although its use in juice and wine beyond fining has been limited until now. Therefore, this study's first aim was to determine if chitosan derived from Agaricus bisporus (button mushrooms) could reduce caffeic and caftaric acid concentrations in Pinot noir grape juice (Study A). The second aim was to determine if chitosan, when added to base wine, could influence the synthesis of furan-derived compounds during storage (Study B). In Study A, Pinot noir grape juice was stored at 10°C for 18 hours after the following treatments: control (no addition), bentonite/activated charcoal (BAC), low molecular weight (< 3 kDa; LMW) chitosan, med. MW (250 kDa; MMW) chitosan, and high MW (422 kDa; HMW) chitosan (all 1 g/L additions). Caftaric acid was decreased, and total amino acid concentration was increased in the LMW chitosan-treated juice, while the estimated total hydroxycinnamic acid content, turbidity, and browning were decreased in the MMW chitosan-treated juice compared to the control. In Study B, Pinot noir base wine destined for sparkling wine was stored at 15 and 30°C for 90 days with the following treatments: control (no addition), LMW chitosan, MMW chitosan, and HMW chitosan (all 1 g/L additions). The three chitosan treatments stored at 30°C had increased furfural, homofuraneol, and 5-methylfurfural formation in the base wine compared to the control. At 15°C, furfural and homofuraneol had greater concentrations in all chitosan-treated wines after 90 days of storage. Our results demonstrate the potential of mushroom-derived chitosan to remove caftaric acid from grape juice and suggest that chitosan can influence the synthesis of furan-derived compounds in wine after short-term storage.

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Sequential inoculation of flocculent Torulaspora delbrueckii with Saccharomyces cerevisiae increases color density of Pinot Noir wines.

McCullough, K. S., Yang, Y., Lindsay, M. A., Culley, N. & Deed, R. C. (2023). Yeast, 40(10), 493-505.

Pinot noir grapes require careful management in the winery to prevent loss of color density and promote aging stability. Winemaking with flocculent yeast has been shown to increase color density, which is desirable to consumers. This research explored interspecies sequential inoculation and co-flocculation of commercial yeast on Pinot noir wine color. Sedimentation rates of six non-Saccharomyces species and two Saccharomyces cerevisiae strains were assayed individually and in combination. The most flocculent pairings, Torulaspora delbrueckii BIODIVA with S. cerevisiae RC212 or VL3, were used to ferment 20 L Pinot noir must. Sequential fermentations produced wines with greater color density at 420 + 520 nm, confirmed by sensory panel. Total and monomeric anthocyanin concentrations were decreased in sequentially fermented wines, despite being the main source of red wine color. BIODIVA adsorbed more anthocyanins than S. cerevisiae, indicating a greater number of cell wall mannoproteins in flocculent yeast, that could then result in a later release of anthocyanins and enhance copigment formation in red wines.

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Fruitlet thinning improves juice quality in seven high-tannin cider cultivars.

Zakalik, D. L., Brown, M. G. & Peck, G. M. (2023). HortScience, 58(10), 1119-1128.

Over 3 years (2016-18), tree productivity, biennial bearing, return bloom, and fruit quality were evaluated for seven high-tannin cider apple (Malus ×domestica Borkh.) cultivars. Five treatments were evaluated on each of the seven cultivars: hand-thinned of all fruit (a zero crop load treatment); hand-thinned to crop densities of three, six, or nine fruit/cm2 trunk cross-sectional area (TCSA); or left unthinned. In this paper, we report on the fruit maturity and juice quality properties that were analyzed for the three nonzero crop load treatments and the unthinned control. The effects of crop load on fruit maturity, as measured by starch pattern index and preharvest drop, were cultivar dependent. Crop density (fruit/cm2 TCSA) had a significant effect on all fruit maturity and juice quality variables, although effects were weakest in the “off” year (2017) for the whole planting when initial fruit set was low. As crop density increased, total poly phenols, titratable acidity, soluble solids, and primary amino nitrogen decreased in the juice of all seven cultivars. A partial budget analysis indicated that the reduced costs of nitrogen supplements due to increased primary amino nitrogen concentration alone would not justify cost of chemical or hand-thinning. By extrapolating the spring flowering density in the fourth year to potential fruit yields at harvest, we found that reducing crop load was projected to increase cumulative total polyphenol yields per tree over the long term. For the cultivars in this experiment, a target crop density of nine fruit/cm2 was found to adequately decrease biennial bearing while also not diminishing juice quality for hard cider production. High-tannin cider apple growers should consider juice quality, particularly tannin production, when making crop load management decisions.

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The Effect of Yeast Inoculation Methods on the Metabolite Composition of Sauvignon Blanc Wines.

Pinu, F. R., Stuart, L., Topal, T., Albright, A., Martin, D. & Grose, C. (2023). Fermentation, 9(8), 759.

Evidence from the literature suggests that different inoculation strategies using either active dry yeast (ADY) or freshly prepared yeast cultures affect wine yeast performance, thus altering biomass and many primary and secondary metabolites produced during fermentation. Here, we investigated how different inoculation methods changed the fermentation behaviour and metabolism of a commercial wine yeast. Using a commercial Sauvignon blanc (SB) grape juice, fermentation was carried out with two different inoculum preparation protocols using Saccharomyces cerevisiae X5: rehydration of commercial ADY and preparation of pre-inoculum in a rich laboratory medium. We also determined the effect of different numbers of yeast cells inoculation (varying from 1 × 106 to 1 × 1012) and successive inoculation on fermentation and end-product formation. The yeast inoculation method and number of cells significantly affected the fermentation time. Principal component analysis (PCA) using 60 wine metabolites showed a separation pattern between wines produced from the two inoculation methods. Inoculation methods influenced the production of amino acids and different aroma compounds, including ethyl and acetate esters. Varietal thiols, 3-mercaptohexanol (3MH), and 4-methyl-4-mercaptopentan-2-one (4MMP) in the wines were affected by the inoculation methods and numbers of inoculated cells, while little impact was observed on 3-mercaptohexyl acetate (3MHA) production. Pathway analysis using these quantified metabolites allowed us to identify the most significant pathways, most of which were related to central carbon metabolism, particularly metabolic pathways involving nitrogen and sulphur metabolism. Altogether, these results suggest that inoculation method and number of inoculated cells should be considered in the production of different wine styles.

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Unravelling copper effect on the production of varietal thiols during Colombard and Gros Manseng grape juices fermentation by Saccharomyces cerevisiae

Dournes, G., Dufourcq, T., Suc, L., Roland, A. & Mouret, J. R. (2023). Frontiers in Microbiology, 14, 1101110.

Nowadays the rapidly increasing organic vineyard management with the utilization of copper as sole fungal control pesticide against downy mildew raises once again the question of copper impact on varietal thiols in wine. For this purpose, Colombard and Gros Manseng grape juices were fermented under different copper levels (from 0.2 to 3.88  mg/l) to mimic the consequences in must of organic practices. The consumption of thiol precursors and the release of varietal thiols (both free and oxidized forms of 3-sulfanylhexanol and 3-sulfanylhexyl acetate) were monitored by LC–MS/MS. It was found that the highest copper level (3.6 and 3.88  mg/l for Colombard and Gros Manseng respectively) significantly increased yeast consumption of precursors (by 9.0 and 7.6% for Colombard and Gros Manseng respectively). For both grape varieties, free thiol content in wine significantly decreased (by 84 and 47% for Colombard and Gros Manseng respectively) with the increase of copper in the starting must as already described in the literature. However, the total thiol content produced throughout fermentation was constant regardless of copper conditions for the Colombard must, meaning that the effect of copper was only oxidative for this variety. Meanwhile, in Gros Manseng fermentation, the total thiol content increased along with copper content, resulting in an increase up to 90%; this suggests that copper may modify the regulation of the production pathways of varietal thiols, also underlining the key role of oxidation. These results complement our knowledge on copper effect during thiol-oriented fermentation and the importance of considering the total thiol production (reduced+oxidized) to better understand the effect of studied parameters and differenciate chemical from biological effects.

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Impact of Steam Extraction and Maceration Duration on Wines from Frozen ‘Frontenac’Must.

Svyantek, A., Wang, Z. & Hatterman-Valenti, H. (2023). Fermentation, 9(4), 317.

The enology industry in North Dakota is extremely young, with less than twenty years of existence. At times throughout the development of the North Dakota viticulture and enology industries, commercial wine producers have elected to purchase or store fresh harvested grapes as frozen musts. To investigate the fermentation outcomes related to skin contact for red grapevine musts, a postfreeze fermentation experiment was conducted with fruit from ‘Frontenac’, one of the most widely grown red grapevines in the Upper Midwest U.S. and North Dakota. Four fermentation treatments were applied to frozen ‘Frontenac’ grapevine musts: steam juice extraction, rosé, 1 day after inoculation (DAI) skin contact, and 9 DAI skin contact. Samples were collected daily for ten days and analyzed for fermentation progress and spectrophotometric monitoring of wine color attributes and total phenolics. The final wines were analyzed two years after bottling. Steam-extracted musts were initially darkest; however, they were lighter as final wines than the 9 DAI wines and similar to rosé wines in lightness. Total phenolics were greatest for 9 DAI wines and total red pigments were lowest for steam-extracted wines. While differences between treatments were detected, the wines remained visually similar; this indicates that color extraction within the freeze–thaw processes of musts may obliterate subtly and make it difficult to produce wines of light color when stored under these conditions. Continued work with additional grapevines beyond ‘Frontenac’ may help fine-tune must and fermentation extraction procedures for small-scale wineries growing cold-hardy grapevines.

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Dual-responsive core-shell tecto dendrimers enable efficient gene editing of cancer cells to boost immune checkpoint blockade therapy.

Liu, J., Li, G., Guo, H., Ni, C., Gao, Y., Cao, X., Xia, J., Shi, X. & Guo, R. (2023). ACS Applied Materials & Interfaces, 15(10), 12809-12821.

Immune checkpoint blockade (ICB) therapy has become a promising strategy in treating multiple tumor types, but the therapeutic efficacy is still unsatisfactory due to the temporary and inefficient blocking and the poor immune responsiveness. Herein, we report the development of dual reactive oxygen species (ROS)- and pH-responsive core–shell tecto dendrimers loaded with gold nanoparticles (for short, Au CSTDs) to deliver a plasmid-clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system for the permanent disruption of the programmed death ligand 1 (PD-L1) gene in cancer cells to boost cancer immunotherapy. In our work, Au CSTDs were constructed using lactobionic acid (LA)-modified generation 5 poly(amidoamine) dendrimers entrapped with gold nanoparticles as cores and phenylboronic acid (PBA)-conjugated generation 3 dendrimers as shells via the formation of responsive phenylborate ester bonds between PBA and LA. The plasmid-CRISPR/Cas9 system can be efficiently compacted and specifically taken up by cancer cells overexpressing sialic acids due to the PBA-mediated targeting and be responsively released in cancer cells by the responsive dissociation of the Au CSTDs, leading to the successful endosomal escape and the efficient knockout of the PD-L1 gene. Further in vivo delivery in a mouse melanoma model reveals that the developed Au CSTDs/plasmid-CRISPR/Cas9 complexes can be specifically accumulated at the tumor site for enhanced computed tomography (CT) imaging of tumors, owing to the X-ray attenuation effect of Au, and disrupt the PD-L1 expression in tumor cells, thus promoting the ICB-based antitumor immunity. The designed dual-responsive Au CSTDs may be developed as a versatile tool for genetic engineering of other cell types to achieve different therapeutic effects for expanded space of biomedical applications.

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Scheffersomyces stipitis ability to valorize different residual biomasses for vitamin B9 production.

Mastella, L., Senatore, V., Beltrani, T. & Branduardi, P. (2022). Microbial Biotechnology.

Sugar beet pulp (SBP), sugar beet molasses (SBM) and unfermented grape marcs (UGM) represent important waste in the agro-food sector. If suitably pre-treated, hexose and pentose sugars can be released in high quantities and can subsequently be used by appropriate cell factories as growth media and for the production of (complex) biomolecules, accomplishing the growing demand for products obtained from sustainable resources. One example is vitamin B9 or folate, a B-complex vitamin currently produced by chemical synthesis, almost exclusively in the oxidized form of folic acid (FA). It is therefore desirable to develop novel competitive strategies for replacing its current fossil-based production with a sustainable bio-based process. In this study, we assessed the production of natural folate by the yeast Scheffersomyces stipitis, investigating SBM, SBP and UGM as potential growth media. Pre-treatment of SBM and SBP had previously been optimized in our laboratory; thus, here we focused only on UGM pre-treatment and hydrolysis strategies for the release of fermentable sugars. Then, we optimized the growth of S. stipitis on the three media formulated from those biomasses, working on inoculum pre-adaptation, oxygen availability and supplementation of necessary nutrients to support the microorganism. Folate production, measured with a microbiological assay, reached 188.2 ± 24.86 μg/L on SBM, 130.6 ± 1.34 μg/L on SBP and 101.9 ± 6.62 μg/L on UGM. Here, we demonstrate the flexibility of S. stipitis in utilizing different residual biomasses as growth media. Moreover, we assessed the production of folate from waste, and to the best of our knowledge, we obtained the highest production of folate from residual biomasses ever reported, providing the first indications for the future development of this microbial production process.

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Impact of Two Commercial S. cerevisiae Strains on the Aroma Profiles of Different Regional Musts.

Patrignani, F., Siesto, G., Gottardi, D., Vigentini, I., Toffanin, A., Englezos, V., et al. (2022). Beverages, 8(4), 59.

The present research is aimed at investigating the potential of two commercial Saccharomyces cerevisiae strains (EC1118 and AWRI796) to generate wine-specific volatile molecule fingerprinting in relation to the initial must applied. To eliminate the effects of all the process variables and obtain more reliable results, comparative fermentations on interlaboratory scale of five different regional red grape musts were carried out by five different research units (RUs). For this purpose, the two S. cerevisiae strains were inoculated separately at the same level and under the same operating conditions. The wines were analyzed by means of SPME-GC/MS. Quali-quantitative multivariate approaches (two-way joining, MANOVA and PCA) were used to explain the contribution of strain, must, and their interaction to the final wine volatile fingerprinting. Our results showed that the five wines analyzed for volatile compounds, although characterized by a specific aromatic profile, were mainly affected by the grape used, in interaction with the inoculated Saccharomyces strain. In particular, the AWRI796 strain generally exerted a greater influence on the aromatic component resulting in a higher level of alcohols and esters. This study highlighted that the variable strain could have a different weight, with some musts experiencing a different trend depending on the strain (i.e., Negroamaro or Magliocco musts).

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Development and Analysis of an intensified batch-fed wine fermentation process.

Miller, K. V., Arefaine, E., Arikal, A., Cantu, A., Cauduro Girardello, R., Oberholster, A., Heymann, H. & Block, D. E. (2022). Fermentation, 8(6), 268.

White wine fermentations are typically performed in an entirely batchwise manner, with yeast nutrients only added at the beginning of fermentation. This leads to slow (2+ weeks) fermentation cycle times, with large capital expenditures required to increase winery processing capacity. Prior attempts to speed fermentations via increasing temperature have resulted in unpalatable wine, and continuous fermentation processing is uneconomical and impractical in the winery setting. In this work, we measured yeast nutrient consumption as a function of fermentation progression at the 300 mL scale, and from this derived an equation to optimize yeast nutrient concentration as a function of fermentation progression. These findings were applied at the pilot scale in 150 L fermentors, which resulted in a 60% cycle time reduction versus “best practices” control fermentations. The resultant wines were compared via GC-MS as well as by a trained sensory panel. Organoleptic analysis found statistically significant, but overall, small differences in sensory characteristics between the control and process intensified wines. This intensified fermentation process shows great promise for fermented beverage producers wishing to maximize equipment utilization and debottleneck wineries or other beverage fermentation facilities.

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Volatile Profile Survey of Five Apple Varieties Grown in Southwest Colorado from Juice to Finished, Dry-Hopped Cider.

Hinkley, J. L., Bingman, M. T., Lee, J. S., Bradley, C. P. & Cole, C. A. (2021). Journal of the American Society of Brewing Chemists, 1-10.

The volatile organic compounds (VOCs) produced during cider fermentation, maturation, and dry hopping greatly impact enjoyment of the final product. To investigate the effect of apple variety on VOC development in cider, five apple varieties were picked from two orchards in southwest Colorado. Each apple variety was juiced, fermented with Lalvin QA23 Saccharomyces cerevisiae, dry-hopped with Citra hops, and analyzed separately alongside non-dry hopped controls. The aroma attributes of these historical southwest Colorado apples have never been explored, and few studies have chemically profiled the aromas of dry-hopped cider. A total of 73 VOCs were identified using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) from fruit juice to cider. Ethyl esters, acetate esters, higher alcohols, and terpenes were the major aroma contributors observed in dry-hopped cider. The identity and concentrations of VOCs varied greatly between apple varieties, with only 24 common VOCs detected in all samples studied. This indicates the importance of apple variety choice in tuning the aroma profile of the finished cider. This method can be used to more effectively evaluate orchard or cider production treatments on cider quality. This case study also provides practical information for cider makers seeking to improve the quality of their products through consideration of fruit variety and dry hopping.

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Exploring the influence of grape tissues on the concentration of wine volatile compounds.

Blackford, C. L., Trengove, R. D. & Boss, P. K. (2021). Australian Journal of Grape and Wine Research, In Press.

Background and Aims: Knowledge of varietal wine flavour and aroma compounds has improved, but gaps exist concerning how grape composition impacts wine style. This work aimed to explore the influence that different grape tissues can have on the volatile profiles of wines. Methods and Results: Riesling and Cabernet Sauvignon berries were separated into skin, flesh and seeds. Two sets of fermentations were performed using separated tissues: one using an equal mass of each tissue and another where the amount of each tissue in 25 g of berries was fermented. When an equal mass of tissue was used, the seed-derived wines had a higher concentration of esters than that produced from other grape tissues. Those produced using skins had the highest concentration of lipoxygenase pathway-derived compounds, and, for Riesling, a higher concentration of monoterpenes. When the proportional amounts of each tissue found per berry were used, the flesh-derived wines generally had a higher concentration of many wine volatiles compared to the other tissues. This reflects the greater proportion of flesh tissue in the berry compared to skin and seeds. Conclusions: Seed-derived compounds can enhance ester biosynthesis during fermentation and skins appear to have high lipoxygenase pathway activity. Nevertheless, the flesh makes up such a large proportion of the whole berry that it has the major influence on volatile profiles of whole berry fermentations. Significance of the Study: Different berry tissues can alter wine composition in unique ways, and this can inform strategies to alter wine composition through vineyard management or the selection of new germplasm.

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Aroma and Sensory Profiles of Sauvignon Blanc Wines from Commercially Produced Free Run and Pressed Juices.

Parish-Virtue, K., Herbst-Johnstone, M., Bouda, F., Fedrizzi, B., Deed, R. C. & Kilmartin, P. A. (2021). Beverages, 7(2), 29.

Sauvignon blanc is the most important grape cultivar within the New Zealand wine industry, and wines from the Marlborough region are renowned for their intense aromas including tropical, passionfruit, and green capsicum. Quality Sauvignon blanc wines are usually made from free run juice, although press fractions can be included. The chemical aroma composition and sensory profiles of two wine sets made from three press fractions (free run, light press and heavy press) were compared. The compounds 3-mercaptohexan-1-ol and 3-mercaptohexyl acetate were found to decrease between free run and heavily pressed wines while hexyl acetate, hexanol, and benzyl alcohol increased. The accompanying sensory analysis showed that free run wines were marked by aromas of Passionfruit/sweaty, Boxwood and Fresh green capsicum, while the heavy pressed wines were described by French vanilla/bourbon, Floral and Banana lolly attributes, consistent with the aroma chemical composition.

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Safety Information
Symbol : GHS07, GHS08
Signal Word : Danger
Hazard Statements : H315, H317, H319, H360, H412
Precautionary Statements : P201, P202, P261, P264, P272, P273, P280, P305+P351+P338
Safety Data Sheet
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