Ammonia Assay Kit (Rapid)

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00:04  Introduction
00:55   Principle
01:33    Reagent Preparation
02:10    Procedure
04:52    Calculations

Ammonia Assay Kit Rapid K-AMIAR Scheme
Reference code: K-AMIAR
SKU: 700004261

96 assays (manual) / 960 assays (microplate) / 960 assays (auto-analyser)

Content: 96 assays (manual) / 960 assays (microplate) / 960 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: Ammonia, Nitrogen, YAN
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Decrease
Linear Range: 0.2 to 7 µg of ammonia per assay
Limit of Detection: 0.07 mg/L
Reaction Time (min): ~ 5 min
Application examples: Grape juice, wine, fruit juices, soft drinks, dairy products (e.g. milk), dietetic food, soy sauce, eggs and egg products, cheese, meat, processed meat, seafood, bakery products (and baking agents), fertilisers, pharmaceuticals, tobacco, cosmetics, water, Kjeldahl analysis, paper (and cardboard), water and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by MEBAK

Ammonia Assay Kit, for the rapid measurement and analysis of ammonia in all samples, including grape juice and wine (and other foods/beverages).

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).

Browse all nitrogen assay kits.

Scheme-K-AMIAR K-AMIAR megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Very rapid reaction due to use of uninhibited glutamate dehydrogenase 
  • Enzyme supplied as stabilised suspension 
  • Very competitive price (cost per test)  
  • All reagents stable for > 2 years as supplied  
  • 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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Effect of Yogurt Acid Whey on the Quality of Maize Silage.

Palamidi, I., Paraskeuas, V. V., Kotsampasi, B., Hadjigeorgiou, I., Politis, I. & Mountzouris, K. C. (2023). Fermentation, 9(12), 994.

The increasing popularity of Greek yogurt generates large amounts of acid whey worldwide. The use of yogurt acid whey in animal nutrition is limited. The aim of this study was to determine the effect of a yogurt acid whey powder (YAWP) addition to maize forage prior to ensiling on the nutritional, microbial and fermentation quality of maize silage. Depending on the addition level of the YAWP to maize forage, there were the following four experimental treatments: YAWP 0, 2.5, 5 and 10% w/w. An increasing YAWP inclusion level linearly increased the maize silage dry matter, crude protein and ash concentrations, whereas it reduced the crude fiber, neutral-detergent fiber and acid-detergent fiber concentrations. The silage pH decreased quadratically with the increasing YAWP level, with the lower plateau noted for the YAWP 5% addition. Concentrations of total bacteria in the silage and Lactobacillus spp. decreased linearly with the YAWP increase. The silage acetic acid content decreased linearly, whereas propionic acid, lactic acid and the ratio of lactic to acetic acid increased linearly with the increasing YAWP level. The ammonia-N content decreased linearly with the increasing YAWP level. In conclusion, the incorporation of the 5 and 10% YAWP addition in silage preparation improved the nutritional and fermentative quality of the produced silage.

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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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Survey on Yeast Assimilable Nitrogen Status of Musts from Native and International Grape Varieties: Effect of Variety and Climate.

Bouloumpasi, E., Skendi, A. & Soufleros, E. H. (2023). Fermentation, 9(8), 773.

Yeast assimilable nitrogen (YAN), besides the oenological parameters (sugar content, titratable acidity, and pH) in grape musts of sixteen native and international varieties of Vitis vinifera cultivated in six regions of Northern Greece, was assessed in the frame of the present study. Low levels of YAN are frequently thought to be the cause of problematic fermentations and originate significant changes in the organoleptic aspects of the finished product. The objective of this multi-variety study was to assess factors affecting the YAN amount and composition in technologically mature grapes and, therefore, to evaluate the necessity of YAN supplementation with ammonium salts in musts across different native and international grape varieties. Free amino nitrogen was measured colorimetrically, ammoniacal nitrogen was measured enzymatically, and their values for each must sample were summed to obtain the total amount of YAN. Statistical analysis was carried out including principal component analysis (PCA) to discover relationships among must samples and the parameters studied. PCA analysis classified samples depending on grape varieties and region of origin, bringing knowledge about native and international cultivars of great commercial interest. Moreover, these findings could help to understand how commercial varieties can behave in different climates in the climate change context.

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Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids.

Pöschel, L., Guevara-Martínez, M., Hörnström, D., van Maris, A. J. & Buchhaupt, M. (2023). Applied Microbiology and Biotechnology, 107(20), 6219-6236.

Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches.

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The Effect of Dietary Fermented Grape Pomace Supplementation on In Vitro Total Gas and Methane Production, Digestibility, and Rumen Fermentation.

Kara, K. & Öztaş, M. A. (2023). Fermentation, 9(8), 741.

The aim of this study comprises the effect of fermented grape pomace (FGP) in experimental total mixed rations (TMR) at different rates (0, 7.5%, 15%, and 22.5%) on the in vitro cumulative gas production (6th, 12th, 18th and 24th hours), methane production, ruminal fermentation values, pH and ammonia-nitrogen and straight and branched short-chain fatty acids (SCFA and BCFA) concentration. The method of in vitro total gas production was carried out in glass syringes. Ruminal in vitro methane production linearly decreased by adding up to 22.5% FGP in experimental TMR (p < 0.05). The molarities of acetic, propionic, butyric, and valeric acids in the in vitro fermentation fluid linearly decreased with the addition of FGP to TMR (p < 0.05). FGP up to 22.5% in experimental TMRs decreased the molarity of iso-valeric acid and iso-butyric acid from BSCFAs (p < 0.05). As a result, it was concluded that the use of FGP, containing a low level of total condensed tannins (TCTs), up to 22.5% in the experimental TMR based on dry matter (DM) did not adversely affect the in vitro ruminal fermentation value and had an anti-methanogenic effect. In addition, some SCFA (acetic, propionic, butyric, and valeric acids) molarities and iso-acid BSCFA (iso-butyric and iso-valeric acid) did not change up to 15% rate of FGP in the ration. Still, these values decreased by using a 22% rate of FGP. The dose-dependent effect of FGP on ruminal iso-acids has been associated with the ability of TCTs to inhibit ruminal protein degradation partially.

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Assessment of Maize Silage Quality under Different Pre-Ensiling Conditions.

Serva, L., Andrighetto, I., Segato, S., Marchesini, G., Chinello, M. & Magrin, L. (2023). Data, 8(7), 117.

Maize silage suffers from several factors that affect the final quality and, to some extent, pre-ensiled conditions that can be potentially tuned during harvesting. After assessing new indices for silage quality under lab-scale conditions, several trials have been conducted to find associations between fresh maize characteristics and silage features. Among the first, we included field input levels, FAO class, maturity stage, use of bacterial inoculants, sealing delay and chemical traits, whereas, among the latter, we assessed density and porosity, pH, fermentative profile, dry matter loss and aerobic stability. The trials were conducted using vacuum bags or mini silo buckets. More than 1500 maize samples harvested in Northeast Italy were analysed during the 2016-2022 period. Moreover, to evaluate silage aerobic stability, the fermentative profile and temperature were measured 14 days after the opening of the silo. The association between silage quality and aerobic stability was assessed, and a prognostic risk score was used to calculate the probability of aerobic instability. The dataset could provide baseline information to promote the continuous improvement of maize silage management from different botanical and crop fields, thus improving agronomic and animal farm resource allocation from a precision agriculture perspective.

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Automatic control of chemolithotrophic cultivation of Cupriavidus necator: Optimization of oxygen supply for enhanced bioplastic production.

Lambauer, V., Permann, A., Petrášek, Z., Subotić, V., Hochenauer, C., Kratzer, R. & Reichhartinger, M. (2023). Fermentation, 9(7), 619.

Gas fermentation is an upcoming technology to convert gaseous substrates into value-added products using autotrophic microorganisms. The hydrogen-oxidizing bacteria Cupriavidus necator efficiently uses CO2 as its sole carbon source, H2 as electron donor and O2 as electron acceptor. Surplus CO2 is stored in microbial storage material poly-(R)-3-hydroxybutyrate. O2 supply is the most critical parameter for growth and poly-(R)-3-hydroxybutyrate formation. A narrow O2 optimum between ~0.2 and ~4 mg/L was previously reported. Here, a standard benchtop bioreactor was redesigned for autotrophic growth of C. necator on explosive mixtures of CO2, H2 and O2. The bioreactor was equipped with mass flow control units and O2 and CO2 sensors. A controller for automated gas dosage based on a mathematical model including gas mass transfer, gas consumption and sensor response time was developed. Dissolved O2 concentrations were adjusted with high precision to 1, 2 and 4% O2 saturation (0.4, 0.8 and 1.5 mg/L dissolved O2, respectively). In total, up to 15 g/L cell dry weight were produced. Residual biomass formation was 3.6 ± 0.2 g/L under all three O2 concentrations. However, poly-(R)-3-hydroxybutyrate content was 71, 77 and 58% of the cell dry weight with 1, 2 and 4% dissolved O2, respectively.

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Undigested glycated lentil proteins modulate the gut microbiota profile but not the metabolites in vitro.

Boachie, R. T., Capuano, E., Oliviero, T., Udenigwe, C. C. & Fogliano, V. (2023). Journal of Functional Foods, 107, 105667.

Glycation enhances plant protein techno-functionality; however, digestibility and the equilibrium between peptides absorbed and those reaching the colon can be altered. This study evaluated how undigested glycated lentil proteins, potentially reaching the colon affect the gut microbiota using batch fermentation and the Simulator of Human Intestinal Microbiome Ecosystem (SHIME®). Lentil protein-fructose mixtures were incubated at 60 °C for 0, 24, or 48 h (conjugates labelled LP+Fr0, LP+Fr1, LP+Fr2). Maillard reaction markers increased by over 10-fold and in vitro protein digestibility decreased by 23.5 % after 48-h incubation. Short- and branched-chain fatty acids produced by 48 h-fermentation of the insoluble hydrolysates of conjugates were comparable. LP+Fr2 hydrolysates caused 42 % relative increase in Bacteroidetes in the proximal colon of Donor 1 whereas 26 % increase was observed with LP+Fr0 hydrolysates. Bacteria population profile in the colon sections was differentially modulated depending on the donor. Our findings show that the extent of glycation does not affect short- and branched-chain fatty acid levels produced in the colon, while the effect on microbiota population is dependent on host and colon section.

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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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Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates.

Mierke, F., Brink, D. P., Norbeck, J., Siewers, V. & Andlid, T. (2023). Fungal Genetics and Biology, 103783.

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source. The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose.

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Effects of plantago species herbage and silage on in vitro ruminal fermentation and microbiome.

Kara, K., Yılmaz, S., Önel, S. E. & Özbilgin, A. (2022). E Italian Journal of Animal Science, 21(1), 1569-1583.

The present study aimed to compare the nutrient composition, in vitro ruminal fermentation values and microbiome in the ruminal fermentation of herbage and silage of the Plantago media, P. major and P. lanceolata species. The lactic acid (LA) content of P. lanceolata silage was higher than those of other plantago silages (p < 0.05). The α-linolenic, w-3, polyunsaturated (PUFA), medium chain (MCFA) and long-chain fatty acids (LCFA) of plantago silages were lower than those of plantago herbages (p < 0.05). The neutral detergent fibre (NDF) and acid detergent fibre (ADF) contents, total gas and methane production, metabolic energy (ME) and organic matter digestion (OMD) values and ammonia-nitrogen concentration in the in vitro fermentation fluid of P. major silage were lower than those of other plantago silages (p < 0.05). The in vitro ruminal methane production and community of archaea Methanobrevibacter in the microbiome of P. major herbage were higher than that of P. media and P. lanceolata herbages. The ensiling process significantly increased the in vitro total gas production, acetic acid concentration and Prevotellaceae bacteria of P. media and P. lanceolate compared their herbages. As a result, P. lanceolata and P. media silages provided high-quality silage fermentation; the nutrient composition was not lost to a great extent in the silage environment and the ruminal fibrolytic bacterial composition was positively affected. Plantago major silage could not provide a good silage quality and the in vitro ruminal fermentation and ruminal fibrolytic bacteria community value were negatively affected.

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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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The film-forming Pichia spp. in a winemaker's toolbox: A simple isolation procedure and their performance in a mixed-culture fermentation of Vitis vinifera L. cv. Gewürztraminer must. 

Scansani, S., van Wyk, N., Nader, K. B., Beisert, B., Brezina, S., Fritsch, S., semmler, H., Pasch, L., Pretorius, I. S., Wallbrunn, C., Schnell, S. & Rauhut, D. (2022). International Journal of Food Microbiology, 365, 109549.

Certain yeast species belonging to the Pichia genus are known to form a distinctive film on grape must and wine. In a mixed-culture type fermentation, Pichia spp. (P. kluyveri in particular) are known to impart beneficial oenological attributes. In this study, we report on an easy isolation method of Pichia spp. from grape must by exploiting their film-forming capacity on media containing 10% ethanol. We isolated and identified two Pichia species, namely Pichia kudriavzevii and Pichia kluyveri, and subsequently co-inoculated them with Saccharomyces cerevisiae to ferment Gewürztraminer musts. Noteworthy differences included a significant increase in the 2-phenethyl acetate levels with the P. kluyveri co-fermentation and a general increase in ethyl esters with the P. kudriavzevii co-fermentation. Both Pichia co-inoculations yielded higher levels of glycerol in the final wines. Based on all the wine parameters we tested, the P. kluyveri strain that was isolated performed similarly to a commercial P. kluyveri strain.

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Safety Information
Symbol : GHS07, GHS08
Signal Word : Danger
Hazard Statements : H302, H315, H319, H360
Precautionary Statements : P201, P202, P264, P270, P280, P301+P312, P302+P352, P305+P351+P338, P330, P337+P313, P501
Safety Data Sheet
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