L-Malic Acid Assay Kit (Manual Format)

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L-Malic Acid Assay Kit Manual Format K-LMAL Scheme
Reference code: K-LMAL-116A
SKU: 700004311


116 assays (manual) / 1160 assays (microplate)

Content: (K-LMAL-58A)
58 assays (manual) / 580 assays (microplate)
116 assays (manual) / 1160 assays (microplate)
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: L-Malic Acid
Assay Format: Spectrophotometer, Microplate
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.5 to 30 µg of L-malic acid per assay
Limit of Detection: 0.25 mg/L
Reaction Time (min): ~ 3 min
Application examples: Wine, beer, fruit juices, soft drinks, candies, fruit and vegetables, bread, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by AOAC, EEC, EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN, NBN, ISO and MEBAK

The K-LMAL-58A pack size has been discontinued (read more)

L-Malic Acid (Regular) Assay Kit, for the specific assay of L-malic acid (L-malate) in beverages and food products.

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).

Need other assay kits? View our full list of organic acid assay kits.

Scheme-K-LMAL-116A LMAL Megazyme

  • PVP incorporated to prevent tannin inhibition 
  • Both enzymes supplied as stable suspensions 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation 
  • Very rapid reaction (~ 3 min) 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included 
  • Extended cofactors stability 
  • Suitable for manual and microplate format
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator Product Performance 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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Physicochemical changes during controlled laboratory fermentation of cocoa (CCN-51) with the inclusion of fruits and on-farm inoculation.

Peña González, M. A., Ortiz Urgiles, J. P., Santander Pérez, F. A., Lazo Vélez, M. A. & Caroca Cáceres, R. S. (2023). Brazilian Journal of Food Technology, 26, e2023013.

Fermentation is key to developing the organoleptic characteristics of cocoa beans, as dynamic changes in metabolites have a significant impact on flavors and aromas, hence modifications of this process have been investigated. In this research, the mucilage of CCN-51 cocoa beans was replaced by a mixture of passion fruit (Passiflora edulis) and plantain (Musa paradisiaca L.) pulp, and a controlled fermentation of this mixture was carried out after its spontaneous on-farm inoculation. The physicochemical changes and correlations during the five days of fermentation were evaluated. At the end of the process, the temperature reached 47 ºC in the fermentation mass and pH 5.64 was recorded in the cotyledon. In the first 48 hours, citric acid and fructose were high but at the end of fermentation were 71% and 41.17% lower than at the start of fermentation, respectively. As glucose and fructose were consumed during fermentation, acetic acid and lactic acid levels increased from day two onward, reaching values at the end of the process of 22.48 mg/g and 16.01 mg/g, respectively. In contrast, the bromatological parameters did not show greater variability when comparing each day of fermentation. The data generated and results presented in this study will contribute to the knowledge of possible sensory improvements achieved with the inclusion of pulp fruits in the fermentation stage.

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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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Transient acetaldehyde production by SO2-producing Saccharomyces cerevisiae promotes the survival of Oenococcus oeni during co-fermentation.

Costello, P. J., Kolouchova, R., McCarthy, J., Nandorfy, D. E., Likos, D. & Schmidt, S. A. (2023). OENO One, 57(2), 399-415.

Stuck or sluggish malolactic fermentation (MLF) can be problematic in stressful wine conditions, particularly white and sparkling base musts/wines. In these cases, knowledge of yeast-bacteria strain compatibility and the amount of sulfur dioxide (SO2) a yeast strain produces are important considerations for successful MLF. Here, laboratory- and pilot-scale co-fermentations in Chardonnay were used to investigate the effect of yeast-derived SO2 on Oenococcus oeni survival. Although yeast-derived SO2 is generally inhibitory, we show that SO2 production (to approximately 65 mg/L) can be uncoupled from O. oeni survival in the early stages of co-fermentation. Bacterial survival in the presence of specific SO2-producing yeast strains was correlated with the early, transient formation of high acetaldehyde concentrations. Oenococcus oeni survival coincided with molecular SO2 concentrations remaining below an extremely low threshold of inhibition, which exponentially increased from approximately 3–6 µg/L in the first three days of co-fermentation. Strain-dependent sensitivity of O. oeni to bound SO2 remains a possibility, although the extent and mechanism of such inhibition by the SO2 adduct during co-fermentation remain unclear. The choice of co-inoculation yeast strain also influenced wine diacetyl concentration, which was only detected in wines co-inoculated with high SO2-producing S. cerevisiae strains. The wines with high diacetyl concentrations were found to be distinct by a sensory panel, with comparatively high citation frequency for a buttery sensory attribute. Both the SO2- and acetaldehyde production capacity of yeasts are, therefore, seen as meaningful co-inoculation selection criteria. The range of yeast strains suitable for MLF induction by co-inoculation could be widened to include SO2-producing strains that transiently produce an early, high concentration of acetaldehyde. The effects of low, equilibrium concentrations of molecular SO2 should also be considered in conjunction with total SO2 as a measure of SO2 toxicity towards O. oeni following co-inoculation.

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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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Picrotoxin Delineates Different Transport Configurations for Malate and γ Aminobutyric Acid through TaALMT1.

Ramesh, S. A., Long, Y., Dashtbani-Roozbehani, A., Gilliham, M., Brown, M. H. & Tyerman, S. D. (2022). Biology, 11(8), 1162.

Gamma aminobutyric acid (GABA) a non-protein amino acid is an archaic signalling molecule for cellular communication across all kingdoms of life with distinct signalling roles in animals. Emerging research establishes GABA as a signalling molecule in planta. GABA modulates numerous developmental processes and negatively regulates root growth, stomatal aperture, and anion flux under abiotic stress. A putative GABA binding site with homology to the mammalian GABA binding site has been identified in a family of proteins (Aluminium Activated Malate Transporters -ALMT) involved in mediating anion flux and conferring plant aluminium tolerance. Studies have shown that ALMTs are regulated by GABA and transport both anions and GABA. To gain further insights into the mechanism of GABA regulation, it is essential to differentiate between the two transport modes. Pharmacological agents used to characterise mammalian GABA receptors have proved useful in gaining insights into GABA regulation of plant processes. In this study, picrotoxin an inhibitor of anion flux in mammalian GABA receptors was used to investigate the pathways for anion and GABA transport in ALMTs. Results suggest that picrotoxin inhibits anion flux but not GABA flux and can be used to gain further insights into mechanism of GABA regulation of plant proteins.

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Effect of inoculation strategy with autochthonous Oenococcus oeni strains on aroma development in Rioja Alavesa Tempranillo wines.

Diez-Ozaeta, I., Lavilla, M. & Amárita, F. (2022). LWT, 162, 113399.

The potential use as malolactic starters of four indigenous strains of Oenococcus oeni was evaluated under different inoculation regimes. Among others, the fermentative capacity of strains, their degree of implantation, the main oenological parameters as well as their ability to modulate the aromatic profile of wines, were analysed. Main results elucidated that co-inoculation led to the prompt consecution of malolactic fermentation (MLF), highlighting the performance of indigenous Oenococcus oeni P2A strain and commercial O. oeni Viniflora OENOS strain which finished the process 20-30 days earlier compared to batches that undergo sequential inoculation. Moreover, inoculation strategy did also have an important influence on the volatile profile of wines. Co-inoculated wines significantly showed less concentration of volatile compounds. Main reduction was detected in higher alcohols and acids. Lower concentration of acids and higher alcohols may prevent the masking of desired aroma attributes. Indeed, in co-inoculated wines, the perception of ripe fruit aroma was highlighted over the others, and was extensively perceived by panellists in comparison with their respective sequentially inoculated wines. Above all, it was elucidated the suitability of the strain P2A, resulting an advantageous alternative to significantly reduce the overall winemaking time as well as to better control the fermentative process.

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Molecular and Physiological Properties of Indigenous Strains of Oenococcus oeni Selected from Nero di Troia Wine (Apulia, Italy).

Cappello, M. S., Falco, V., Curcio, R., Mita, G. & Zapparoli, G. (2022). Microorganisms, 10(4), 795.

The characterization of Oenococcus oeni strains isolated from Nero di Troia wine (Apulia, Italy) sampled in two distinct production areas was carried out. The two indigenous populations, consisting of 95 and 97 isolates, displayed high genetic diversity when analyzed by amplified fragments length polymorphisms (AFLP). Based on the UPGMA dendrogram obtained by AFLP analysis, the two populations displayed similar genotypes that grouped in the same clusters with a high level of similarity (>95%). One genotype was found in only one of the two areas. Representative strains of each cluster were analyzed for their enzymatic activities (esterase, β-glucosidase, and protease), assayed in whole cells, and tested for their metabolic properties (consumption of L-malic acid, citric acid, acetaldehyde, and arginine) and growth parameters. Significant differences among strains, including the reference strain ATCC BAA-1163, were observed for all of these properties. Principal component analysis evidenced phenotypic differences among strains, and well separated some of them belonging to different genotypes. Strains exhibiting the best performances in most of these traits could be further investigated in order to select possible candidates as malolactic starters for Nero di Troia wine. This study provided insights on the population structure of O. oeni of a local winemaking area useful to the understanding of the regional diversity of this bacterium, an issue not yet completely resolved

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Impact of microoxygenation on Pinot noir wines with different initial phenolic content.

Yang, Y., Deed, R. C., Araujo, L. D. & Kilmartin, P. A. (2021). OENO One, 55(4), 83-100.

Microoxygenation (MOX) is used to improve wine colour and sensory quality; however, limited information is available for Pinot noir wines and wines with different initial phenolic content. In this study, MOX was applied to two Pinot noir wines, with either a low or a high phenolic content, at two doses (0.50 and 2.11 mg/L/day) for 14 days. With the sterile filtration applied, acetaldehyde formation during MOX was very low, supporting the influence of yeast on acetaldehyde production during MOX. The MOX dosage rate did not significantly affect colour development, while the Pinot noir wine with higher phenolics benefited more from MOX, significantly increasing colour intensity and SO2 resistant (polymeric) pigments. However, these changes did not guarantee colour stability, as a final SO2 addition (100 mg/L) largely erased the improvement to colour in all wines. This could be due to the lower acetaldehyde formation, thus less ethyl-bridged stable pigments resistant to SO2 bleaching. MOX also decreased the flavan-3-ols and anthocyanin monomers, which differed between the two Pinot noir wines, reflecting the initial phenolic content. Lastly, MOX generally increased the measured tannin concentration and affected the proportion of tannin subunits, with a decrease in tannin mass conversion and proportion of (-)-epigallocatechin extension units. Some of these changes in phenolic compounds could potentially increase astringency, suggesting that MOX should be applied to Pinot noir and other low phenolic wines with caution.

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Assessment of β-D-glucosidase activity and bgl gene expression of Oenococcus oeni SD-2a.

Li, Y., Wang, Y., Fan, L., Wang, F., Liu, X., Zhang, H. & Zhou, J. (2020). Plos One, 15(10), e0240484.

Glycosidases enhance flavor during wine-making by mediating the enzymatic release of aroma molecules. In order to better understand the aroma enhancement potential of Oenococcus oeni SD-2a, β-D-glucosidase (βG) activities in the culture supernatant, whole cells, and disrupted cell lysate were assessed at mid log, late log and stationary growth phase. The enzymatic activity was also compared further from cell cultures with 5 different carbon sources (glucose, cellobiose, arbutin, glucose and cellobiose, glucose and arbutin) at late log phase. Correspondingly, expression levels of 3 bgl genes, OEOE-0224, OEOE-1210, and OEOE-1569 were investigated from cell cultures of the 3 growth phases, and the 5 cell cultures with different carbon sources. Finally, the volatile aroma compounds released by O. oeni SD-2a in synthetic wines with natural glycosides were evaluated by GC-MS. Results showed βG of O. oeni SD-2a was not extracellular enzyme, and the location of it didn’t change with the change of growth phase and carbon source studied. βG activities in the whole cells and disrupted cell lysate were similar and constant at the 3 growth phases. As for the carbon sources, βG activities of whole cells and disrupted lysate were positively affected by cellobiose. While arbutin displayed positive and negative effect on βG activity of whole cells and disrupted lysate, respectively. It is probably that bgl genes OEOE-0224 and OEOE-1210 were related to βG activity of SD-2a whole cells, while OEOE-1569 was responsible for βG activity of disrupted lysate. More kinds of volatile compounds and higher total concentration were released by SD-2a in synthetic wine compared with control. Thus, SD-2a showed a great potential for flavor enhancement under wine-like conditions. This study provides more information for further study of βG activity from O. oeni SD-2a.

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Nutrients, bioactive compounds, and minerals in the juices of 16 varieties of apple (Malus domestica) harvested in Austria: A four-year study investigating putative correlations with weather conditions during ripening.

Tschida, A., Stadlbauer, V., Schwarzinger, B., Maier, M., Pitsch, J., Stübl, F., Müller, U.,Lanzerstorfer, P., Himmelsbach. M., WrusS. J., Klaner, G., Schurr, J., Wurm, L., Rosner, F., Höglinger, O., Winkler, S. & Weghuber, J. (2020). Food Chemistry, 338, 128065.

This study was conducted to examine putative correlations between weather parameters during April-September and the amounts of nutrients, minerals and bioactive compounds in the juices of 16 apple varieties from four harvest years in Lower Austria. For most sugar-parameters, negative correlations were found with the total precipitation (r between −0.42 and −0.64). Conversely, positive correlations were observed with the mean air temperature (r between 0.32 and 0.66), the global radiation (r between 0.32 and 0.61) and the number of tropical days (r between 0.39 and 0.51). The sum of 14 polyphenols (HPLC quantitation) was positively correlated with the mean air temperature and global radiation (rs 0.44 and 0.42). Negative correlations were observed between the global radiation and potassium, magnesium and calcium contents (correlation coefficients −0.49, −0.68 and −0.69). We conclude that increased temperatures and global radiation can be correlated with enhanced sugar synthesis and polyphenol formation.

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Nonlinear Behavior of Protein and Tannin in Wine Produced by Cofermentation of an Interspecific Hybrid (Vitis spp.) and vinifera Cultivar.

Norton, E. L., Sacks, G. L. & Talbert, J. N. (2020). American Journal of Enology and Viticulture, 71(1), 26-32.

Wines produced from red interspecific hybrid grape cultivars (Vitis spp.) typically have lower tannin concentrations than wines produced from vinifera cultivars, which can be attributed to the lower concentration of tannins and higher concentration of tannin-binding proteins of interspecific cultivars. Tannin in wines produced from hybrid cultivars may be increased by blending hybrids with vinifera. We hypothesized that blending of grapes prior to fermentation (cofermentation) would result in final wine tannin concentrations lower than those predicted from the individual components due to protein-tannin binding, but that this effect would be absent from monovarietal wines blended postfermentation. To evaluate this hypothesis, a high tannin V. vinifera cultivar (Cabernet Sauvignon) was blended with an interspecific hybrid (Marquette) at different ratios either before (cofermentation) or after fermentation over a two-year period. The tannin and protein concentrations of the wines were measured by methyl cellulose precipitation assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Tannin and protein concentrations in blended wines were compared to values predicted from the linear combination of the two monovarietal wines. Cofermented blends with a high proportion of Marquette had up to 25% lower tannin than predicted, but observed and predicted tannin concentrations did not differ for most cofermentations and postfermentation blends. However, protein concentrations for many of the blends-especially from cofermentation-were lower than predicted values (>50% in some cases). Loss of protein due to adsorption to tannin was well modeled by a Freundlich adsorption isotherm.

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The Transcriptome and Flux Profiling of Crabtree‐Negative Hydroxy Acid‐Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism.

Jessop‐Fabre, M. M., Dahlin, J., Biron, M. B., Stovicek, V., Ebert, B. E., Blank, L. M., Budin, I., Keasling, J. D. & Borodina, I. (2019). Biotechnology Journal, 14(9), 1900013.

Saccharomyces cerevisiae (S. cerevisiae) is the yeast cell factory of choice for the production of many biobased chemicals. However, it is a Crabtree‐positive yeast and so shuttles a large portion of carbon into ethanol. Ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. It is not yet well understood how PDC‐negative yeasts are affected when engineered to produce other products than ethanol. In this study, pathways are introduced for the production of three hydroxy acids (lactic, malic, or 3‐hydroxypropionic acid [3HP]) into an evolved PDC‐negative strain. These strains are characterized via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. Expression of lactic and malic acid biosynthesis pathways improved the maximum specific growth rate (μmax) of the strain by 64% and 20%, respectively, presumably due to nicotinamide adenine dinucleotide regeneration. All strains show a very high flux (> 90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The study, for the first time, directly compares the flux and transcriptome profiles of several hydroxy acid‐producing strains of an evolved PDC‐negative S. cerevisiae and suggests directions for future metabolic engineering.

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Assessment of yeasts for apple juice fermentation and production of cider volatile compounds.

Lorenzini, M., Simonato, B., Slaghenaufi, D., Ugliano, M. & Zapparoli, G. (2019). LWT, 99, 224-230.

At present, yeasts suitable for apple juice fermentation to produce cider have received scarce attention with respect to wine yeasts. In this study, Saccharomyces and non-Saccharomyces strains were investigated for their capacity to ferment apple juice and to influence the volatile compound production in cider. In a first fermentation trial, seven out of 18 yeasts, belonging to Saccharomyces cerevisiae, S. uvarum, Torulaspora delbrueckii, Hanseniaspora osmophila, H. uvarum, Starmerella bacillaris and Zygosaccharomyces bailii, were selected according to their fermentative performance. The effects of these strains on the volatile composition of cider, produced in a second apple fermentation trial, were then evaluated. Significant differences on the production of alcohols, esters and fatty acids were observed. Large amounts of 2-phenylethanol were found in S. uvarum cider. Hanseniaspora uvarum was the greatest producer of hexyl and isoamyl acetate among non-Saccharomyces yeasts. Ciders were well discriminated by principal component analysis. This study provides insights into the actual capacity to produce volatile compounds that the different yeast species that could be used in single or mixed apple juice fermentation for cider production.

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Safety Information
Symbol : GHS07
Signal Word : Warning
Hazard Statements : H319
Precautionary Statements : P264, P280, P305+P351+P338, P337+P313
Safety Data Sheet
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