L-Malic Acid Assay Kit (MegaQuant™ Format)

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.

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.

Aims: The aim of this work was to evaluate the effect of acclimation on the viability, membrane integrity and the ability to consume malic acid of three oenological strains of Lactobacillus plantarum. Methods and Results: Cultures in the stationary phase were inoculated in an acclimation medium (Accl.) containing 0, 6 or 10% v/v ethanol and incubated 48 h at 28°C. After incubation, cells were harvested by centrifugation and inoculated in a synthetic wine, containing 14% v/v ethanol and pH 3•5 at 28°C. Viability and membrane integrity were determined by flow cytometry (FC) using carboxyfluorescein diacetate (cFDA) and propidium iodide. Bacterial growth and malic acid consumption were monitored in a synthetic wine during 15 days. In nonacclimated strains, the damage of bacterial membranes produced a dramatic decrease in microbial viability in synthetic wine. In contrast, survival of strains previously acclimated in Accl. with 6 and 10% v/v ethanol was noticeable higher. Therefore, acclimation with ethanol increased the cultivability in synthetic wine and consequently, the consumption of L-malic acid after 15 days of growth. Conclusion: Acclimation of oenological strains in media containing ethanol prior to wine inoculation significantly decreases the membrane damage and improves viability in the harsh wine conditions. The role of membrane integrity is crucial to warrant the degradation of L-malic acid. Significance and Impact of the Study: The efficiency of multiparametric FC in monitoring viability and membrane damage along with the malic acid consumption has a strong impact on winemaking because it represents a useful tool for a quick and highly reliable evaluation of oenological parameters.