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

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5. Link to Article Read Abstract It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as \"stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

Megazyme \"advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5. Link to Article Read Abstract 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.

Are Enterococcus populations present during malolactic fermentation of red wine safe? (K-LMAL-58A)

Pérez-Martín, F., Seseña, S., Izquierdo, P. M. & Palop, M. L. (2014). Food Microbiology, 42, 95-101. Link to Article Read Abstract The aim of this study was the genetic characterisation and safety evaluation of 129 Enterococcus isolates obtained from wine undergoing malolactic fermentation. Genetic characterisation by randomly amplified polymorphic DNA-PCR displayed 23 genotypes. 25 isolates representative of all genotypes were identified as Enterococcus faecium by species-specific PCR and assayed for antibiotic resistance, presence of virulence genes and aminobiogenic capacity, both in decarboxylase medium and wine. The aminobiogenic capacity in wine was analysed in presence (assay 1) and absence (assay 2) of Oenococcus oeni CECT 7621. Resistance to tetracycline, cotrimoxazol, vancomycin and teicoplanin was exhibited by 96% of the strains, but none of them harboured the assayed virulence genes. All of the strains harboured the tyrosine decarboxylase (tdc) gene, while 44% were positive for tyramine in decarboxylase medium. Only five out of 25 strains survived in wine after seven days of incubation, and when concentrations of biogenic amines in wines were determined by HPLC, only those wines in which the five surviving strains occurred contained biogenic amines. Histamine, putrescine and cadaverine were detected in wines from both assays, although concentrations were higher in assay 2. Tyramine and phenylethylamine were detected only in absence of O.oeni. This research contributes for the knowledge of safety aspects of enterococci related to winemaking.

Genetic diversity of Oenoccoccus oeni isolated from wines treated with phenolic extracts as antimicrobial agents. (K-LMAL-58A)

García-Ruiz, A., Tabasco, R., Requena, T., Claisse, O., Lonvaud-Funel, A., Bartolomé, B. & Moreno-Arribas, M. (2013). Food Microbiology, 36(2), 267-274. Link to Article Read Abstract Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n= 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds.

Anaerobic organic acid metabolism of Candida zemplinina in comparison with Saccharomyces wine yeasts. (K-LMAL-58A)

Magyar, I., Nyitrai-Sárdy, D., Leskó, A., Pomázi, A. & Kállay, M. (2014). International Journal of Food Microbiology, 178, 1-6. Link to Article Read Abstract Organic acid production under oxygen-limited conditions has been thoroughly studied in the Saccharomyces species, but practically never investigated in Candida zemplinina, which seems to be an acidogenic species under oxidative laboratory conditions. In this study, several strains of C. zemplinina were tested for organic acid metabolism, in comparison with Saccharomyces cerevisiae, Saccharomyces uvarum and Candidastellata, under fermentative conditions. Only C. stellata produced significantly higher acidity in simple minimal media (SM) with low sugar content and two different nitrogen sources (ammonia or glutamic acid) at low level. However, the acid profile differed largely between the Saccharomyces and Candida species and showed inverse types of N-dependence in some cases. Succinic acid production was strongly enhanced on glutamic acid in Saccharomyces species, but not in Candida species. 2-oxoglutarate production was strongly supported on ammonium nitrogen in Candida species, but remained low in Saccharomyces. Candida species, C. stellata in particular, produced more pyruvic acid regardless of N-sources. From the results, we concluded that the anaerobic organic acid metabolisms of C. zemplinina and C. stellata are different from each other and also from that of the Saccharomyces species. In the formation of succinic acid, the oxidative pathway from glutamic acid seems to play little or no role in C. zemplinina. The reductive branch of the TCA cycle, however, produces acidic intermediates (malic, fumaric, and succinic acid) in a level comparable with the production of the Saccharomyces species. An unidentified organic acid, which was produced on glutamic acid only by the Candida species, needs further investigation.

Antimicrobial phenolic extracts able to inhibit lactic acid bacteria growth and wine malolactic fermentation. (K-LMAL-58A)

García-Ruiz, A., Cueva, C., González-Rompinelli, E. M., Yuste, M., Torres, M., Martín-Álvarez, P. J., Bartolome, B. & Moreno-Arribas, M. (2012). Food Control, 28(2), 212-219. Link to Article Read Abstract The purpose of this study was to determine whether phenolic extracts with antimicrobial activity may be considered as an alternative to the use of sulfur dioxide (SO₂) for controlling malolactic fermentation (MLF) in winemaking. Inhibition of the growth of six enological strains (Lactobacillus hilgardii CIAL-49, Lactobacillus casei CIAL-52, Lactobacillus plantarum CIAL-92, Pediococcus pentosaceus CIAL-85, Oenococcus oeni CIAL-91 and O. oeni CIAL-96) by phenolic extracts (n= 54) from different origins (spices, flowers, leaves, fruits, legumes, seeds, skins, agricultural by-products and others) was evaluated, being the survival parameter IC₅₀ calculated. A total of 24 extracts were found to significantly inhibit the growth of at least two of the LAB strains studied. Some of these extracts were also active against two acetic acid bacteria (Acetobacter aceti CIAL-106 and Gluconobacter oxydans CIAL-107). Transmission electron microscopy of the bacteria cells after incubation with the phenolic extract confirmed damage of the integrity of the cell membrane. Finally, to test the technological applicability of the extracts, the eucalyptus extract was added (2 g/L) to an industrially elaborated red wine, and the progress of the MLF was evaluated by means of residual content of malic acid. Addition of the extract significantly delayed the progress of both inoculated and spontaneous MLF, in comparison to the control wine (no antimicrobial agent added), although not as effective as K₂S₂O₅ (30 mg/L). These results demonstrated the potential applicability of phenolic extracts as antimicrobial agents in winemaking.

Impact of γ-irradiation on antioxidant capacity of mango (Mangifera indica L.) wine from eight Indian cultivars and the protection of mango wine against DNA damage caused by irradiation.

Kondapalli, N., Sadineni, V., Variyar, P. S., Sharma, A. & Obulam, V. S. R. (2014). Process Biochemistry, 49(11), 1819-1830. Link to Article Read Abstract The present study aims to evaluate the effect of gamma-irradiation on the total phenolic content (TPC), total flavonoid content (TFC), antioxidant and radioprotective properties of the mango wine. γ-Irradiation resulted in an increase in TPC and TFC in a dose dependent manner and their concentrations were in the range of 226.8–555.3 mg/L and 68.6–165.1 mg/L, respectively, in 3 kGy irradiated wine samples. There was a significant increase in the concentration of certain polyphenolic compounds with the exception of ellagic acid, which was unaltered and a significant decrease in the ferulic and synapic acids as measured by HPLC. Treatment with γ-irradiation resulted in overall reduction in microbial loads; further, no microbe was detected with a dose of 3 kGy in all wine samples, indicating improvement in the quality of mango wine. The DPPH radical scavenging activity of mango wine varied from 97.14 (Sindhura) to 83.64% (Mulgoa) and the DMPD scavenging capacity varied from 95.27 (Banginapalli) to 77.8% (Mulgoa) at 100 µL and 3 kGy dose. However, the FRAP activity of mango wine varied from 33.96 (Sindhura) to 27.38 mM/L (Mulgoa), and the NO scavenging capacity from 88.2 (Banginapalli) to 74.44% (Mulgoa) at 500 µL and 3 kGy dose. These scavenging activities were significantly increased with the irradiation dose and also with concentration. Mango wine was also demonstrated to protect DNA against UV + H₂O₂ and γ-irradiation (500 Gy) induced DNA damage, confirming its protective actions in vitro and thus could be a valuable source of antioxidants.

Water stress and abscisic acid treatments induce the CAM pathway in the epiphytic fern Vittaria lineata (L.).

Minardi, B. D., Voytena, A. P. L., Santos, M. & Randi, Á. M. (2014). Photosynthetica, 52(3), 404-412. Link to Article Read Abstract Among various epiphytic ferns found in the Brazilian Atlantic Forest, we studied Vittaria lineata (L.) Smith (Polypodiopsida, Pteridaceae). Anatomical characterization of the leaf was carried out by light microscopy, fluorescence microscopy, and scanning electron microscopy. V. lineata possesses succulent leaves with two longitudinal furrows on the abaxial surface. We observed abundant stomata inside the furrows, glandular trichomes, paraphises, and sporangia. We examined malate concentrations in leaves, relative water content (RWC), photosynthetic pigments, and chlorophyll (Chl) _a fluorescence in control, water-deficient, and abscisic acid (ABA)-treated plants. Plants subjected to drought stress (DS) and treated by exogenous ABA showed significant increase in the malate concentration, demonstrating nocturnal acidification. These findings suggest that V. lineata could change its mode of carbon fixation from C₃ to the CAM pathway in response to drought. No significant changes in RWC were observed among treatments. Moreover, although plants subjected to stress treatments showed a significant decline in the contents of Chl a and b, the concentrations of carotenoids were stable. Photosynthetic parameters obtained from rapid light curves showed a significant decrease after DS and ABA treatments.

Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine. (K-LMAL-116A)

Mazzoli, R., Lamberti, C., Coisson, J. D., Purrotti, M., Arlorio, M., Giuffrida, M. G., Giunta, C. & Pessione, E. (2009). Amino acids, 36(1), 81-89. Link to Article Read Abstract Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine–histamine conversion.

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines. (K-LMAL-116A)

Rodríguez‐Nogales, J. M., Vila‐Crespo, J. & Fernández‐Fernández, E. (2013). Biotechnology Progress, 29(1), 60-65. Link to Article Read Abstract Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell-loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell-loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels.

Optimization of air-blast drying process for manufacturing Saccharomyces cerevisiae and non-Saccharomyces yeast as industrial wine starters.

Lee, S. B., Choi, W. S., Jo, H. J., Yeo, S. H. & Park, H. D. (2016). AMB Express, 6(1), 105. Link to Article Read Abstract Wine yeast (Saccharomyces cerevisiae D8) and non-Saccharomyces wine yeasts (Hanseniaspora uvarum S6 and Issatchenkia orientalis KMBL5774) were studied using air-blast drying instead of the conventional drying methods (such as freeze and spray drying). Skim milk—a widely used protective agent—was used and in all strains, the highest viabilities following air-blast drying were obtained using 10% skim milk. Four excipients (wheat flour, nuruk, artichoke powder, and lactomil) were evaluated as protective agents for yeast strains during air-blast drying. Our results showed that 7 g lactomil was the best excipient in terms of drying time, powder form, and the survival rate of the yeast in the final product. Finally, 7 types of sugars were investigated to improve the survival rate of air-blast dried yeast cells: 10% trehalose, 10% sucrose, and 10% glucose had the highest survival rate of 97.54, 92.59, and 79.49% for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. After 3 months of storage, S. cerevisiae D8 and H. uvarum S6 demonstrated good survival rates (making them suitable for use as starters), whereas the survival rate of I. orientalis KMBL5774 decreased considerably compared to the other strains. Air-blast dried S. cerevisiae D8 and H. uvarum S6 showed metabolic activities similar to those of non-dried yeast cells, regardless of the storage period. Air-blast dried I. orientalis KMBL5774 showed a noticeable decrease in its ability to decompose malic acid after 3 months of storage at 4°C.

Metabolomic Measurements at Three Time Points of a Chardonnay Wine Fermentation with Saccharomyces cerevisiae.

Richter, C. L., Kennedy, A. D., Guo, L. & Dokoozlian, N. (2015). American Journal of Enology and Viticulture, 66(3), 294-301. Link to Article Read Abstract The transformation of grape juice to wine is a complex metabolic relationship between two species, the grape plant Vitis vinifera and yeast, primarily Saccharomyces cerevisiae. The final molecular composition resulting from the grape–yeast relationship contributes to the flavor, aroma, and mouthfeel of the wine. In this study, we examined this complex relationship by determining the exo- and endo-metabolome (the collection of metabolites present extra- and intracellularly, respectively) of yeast at three time points (days 4, 9, and 15) of a Chardonnay wine fermentation. We identified and tracked 227 metabolites in the exo-metabolome and 404 metabolites in the endo-metabolome, and each metabolite was grouped into metabolic pathways or into metabolite families. Considerable metabolic variation was present at each stage of the fermentation, illuminating metabolic patterns suggesting that regulation of the yeast metabolic pathways is coupled to the fermentation progress. Analysis of the differential utilization and production of primary and secondary metabolites during a wine fermentation in this work provides a key understanding of cell-communication mechanisms relevant to metabolic engineering and industrial biotechnological processes.

Overexpression of a C₄-dicarboxylate transporter is the key for rerouting citric acid to C₄-dicarboxylic acid production in Aspergillus carbonarius.

Yang, L., Christakou, E., Vang, J., Lübeck, M. & Lübeck, P. S. (2017). Microbial Cell Factories, 16(1), 43. Link to Article Read Abstract Background: C₄-dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. Previous studies on organic acid production by Aspergillus carbonarius, which is capable of producing high amounts of citric acid from varieties carbon sources, have revealed its potential as a fungal cell factory. Earlier attempts to reroute citric acid production into C₄-dicarboxylic acids have been with limited success. Results: In this study, a glucose oxidase deficient strain of A. carbonarius was used as the parental strain to overexpress a native C₄-dicarboxylate transporter and the gene frd encoding fumarate reductase from Trypanosoma brucei individually and in combination. Impacts of the introduced genetic modifications on organic acid production were investigated in a defined medium and in a hydrolysate of wheat straw containing high concentrations of glucose and xylose. In the defined medium, overexpression of the C₄-dicarboxylate transporter alone and in combination with the frd gene significantly increased the production of C₄-dicarboxylic acids and reduced the accumulation of citric acid, whereas expression of the frd gene alone did not result in any significant change of organic acid production profile. In the wheat straw hydrolysate after 9 days of cultivation, similar results were obtained as in the defined medium. High amounts of malic acid and succinic acid were produced by the same strains. Conclusions: This study demonstrates that the key to change the citric acid production into production of C₄-dicarboxylic acids in A. carbonarius is the C₄-dicarboxylate transporter. Furthermore it shows that the C₄-dicarboxylic acid production by A. carbonarius can be further increased via metabolic engineering and also shows the potential of A. carbonarius to utilize lignocellulosic biomass as substrates for C₄-dicarboxylic acid production.

Non-anthocyanin polyphenolic transformation by native yeast and bacteria co-inoculation strategy during vinification.

Devi, A., Archana, K. M., Bhavya, P. K. & Anu Appaiah, K. A. (2017). Journal of the Science of Food and Agriculture, In Press. Link to Article Read Abstract Background: Co-inoculation has been adapted by many wine-producing countries as it enhances the success of malolactic fermentation and reduces the fermentation cost as well as time. However, wine phenolics have been sparsely highlighted during co-inoculation even though polyphenols are important parameter affecting wine colour, astringency and aroma. Here, we investigate the impact of co-inoculation on non-anthocyanin polyphenol profile for two different grape varieties. Result: Co-inoculation of native yeast strain (AAV2) along with Oenococcus oeni was adapted for Cabernet Sauvignon and Shiraz wine. It was observed that the co-inoculation had minimal yet significant impact on the phenolic composition of wines for both the grape varieties. Color loss as well as fruity aroma development was observed in co-inoculated wines. The wines were at par with the commercial wine as well as wines without MLF in terms of phenolic compounds and overall organoleptic acceptance. PCA and HCA further suggested that the varietal influence on phenolic composition was dominating when compared to inoculation strategies. Among the varieties, the inoculation strategies have significantly influenced the Cabernet wines as compared to Shiraz wines.

Manual format UV-method for the determination of L-Malic Acid in foodstuffs, beverages and other materialsPrinciple: (L-malate dehydrogenase)(1) L-Malic acid + NAD⁺ ↔ oxaloacetate + NADH + H⁺ (glutamate-oxaloacetate transaminase)(2) Oxaloacetate + L-glutamate → L-aspartate + 2-oxoglutarate

Kit size: (K-LMAL-58A) * 58 assays (manual) / 580 (microplate) or (K-LMAL-116A) * 116 assays (manual) / 1160 (microplate)* The number of manual tests per kit can be doubled if all volumes are halved.This can be readily accommodated using the MegaQuant^TM Wave Spectrophotometer (D-MQWAVE).

Method: Spectrophotometric at 340 nmReaction time: ~ 3 minDetection limit: 0.25 mg/LApplication examples: Wine, beer, fruit juices, soft drinks, candies, fruit and vegetables,bread, cosmetics, pharmaceuticals and other materials (e.g. biologicalcultures, samples, etc.)Method recognition: Methods based on this principle have been accepted by AOAC, EEC, EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN and MEBAK

Advantages

• 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