Malic Acid Degradation Research Articles

Prevalent lactic acid bacteria in cider cellars and efficiency of Oenococcus oeni strains

Malolactic fermentation (MLF) is an important step in cider production in order to allowing for improvement of microbiological stability and organoleptic characteristics of cider. Induction of this fermentation by using starter cultures enables a better control over this bioprocess, but although it is a common practice in winemaking, starters specifically focussed for cider MLF are not yet commercially available. Proper starter cultures need to present the ability to degrade l-malic acid conferring pleasing sensory characteristics while avoiding toxicological risks. In this work, lactic acid bacteria (LAB) were first isolated from MLF industrial cider samples, obtained in a cellar in the main cider-producing region of Spain, Asturias. Isolates, identified by molecular tools, belonged to the Lactobacillus brevis and Oenococcus oeni species. After a phylogenetic analysis, representative strains of both identified species were evaluated in order to determine their fermentation capacity, showing O. oeni the best behaviour in this cider fermentation, as previously demonstrated for wine in the literature. Consequently, and with the aim to test the influence at strain level, selection of O. oeni isolates as starters for cider fermentation has been undergone. In order to check the influence of geography over biodiversity, O. oeni strains from six different industrial cellars representing the distinct producing areas in the region (located in a ratio of 30 km) were analyzed by using a specific RAPD method. In this way, isolates were typed in five distinct groups, mainly corresponding to each producing area. All strains isolated from the same cellar showed the same RAPD profile revealing the significance of geographical origin in the indigenous cider LAB. Molecular tools were applied to reject those isolates exhibiting presence of genes related to organoleptic spoilage (exopolysaccharides and acrolein production) or food safety (biogenic amine production), as key selection criteria. Representative strains of each of the five O. oeni RAPD groups were tested as pure cultures to evaluate their technological utility for cider production. Experimental data of malic acid degradation and cell concentration obtained were fitted to previously selected kinetic models aimed to optimization and prediction of bioprocess performance. Four strains revealed as suitable potential starter cultures for conducting MLF in cider production.

Simultaneous and Sequential Fermentations with <i>Saccharomyces cerevisiae</i> DJ02 and <i>Lactobacliius acidophilus</i> WS in Pear Wine

The two controlled inoculation procedures of simultaneous and sequential fermentations with Saccharomyces Cerevisiae DJ02 and Lactobacliius acidophilus WS were studied in pear wine. The sugar consumption and ethanol product were delayed in the course of simultaneous fermentation contrary to the single fermentation. However in the sequential fermentation the effect was less than the simultaneous fermentation. In this work the function of malic acid degradation in the sequential fermentation was better than the simultaneous fermentation. The pH value of the pear wine came to 3.9 after the sequential fermentation. The change tendency of the yeast population after adding Lactobacliius acidophilus WS was similar to the yeast single fermentation in the simultaneous and sequential fermentation. But the cell population was went down in the course of the fermentation. The population of Lactobacliius acidophilus WS in the course of the two kinds of fermentation methods showed no significantly differences.

Expression of the Malolactic Enzyme Gene (mle) from Lactobacillus plantarum Under Winemaking Conditions

Malolactic fermentation (MLF) plays an important role in the production of wine, especially red wines, resulting in microbial stability, deacidification, as well as contributing to the aroma profile. MLF can be influenced by a number of factors. In this study, the influence of pH and ethanol on expression of the structural malolactic enzyme gene (mle) from Lactobacillus plantarum was investigated in a synthetic wine media, as well as in wine using quantitative PCR. Expression of mle was shown to be inducible by the presence of malic acid, with increased expression in the middle of MLF. Expression of mle was also shown to be increased at low pH values and decreased in the presence of ethanol. This indicates the role of MLF in acid tolerance and the negative impact of ethanol on the completion of MLF. The results therefore provide further evidence that L. plantarum should be applied as co-inoculation for MLF where alcohol will initially not have a negative impact on the malic acid degradation.

Relationship between mineralization kinetics and mechanistic pathway during malic acid photodegradation

The photocatalytic degradation of malic acid was studied under ambient conditions in an aqueous TiO 2 suspension. The results demonstrated the presence of four distinct mineralization rate variations over the course of its degradation. Differences in the mineralization rate were governed by the dominant intermediate present in the solution at that time. Initial mineralization was rapid (∼175 μg C/min) resulting from the swift extraction of a carbon from strongly adsorbed malic acid via the photo-Kolbe mechanism. The mineralization rate then slowed down (∼50–70 μg C/min) with the continued formation of an intermediate product (believed to be malonaldehydic acid), malonic acid and acetic acid in the solution. The findings provide greater insight into the photocatalytic degradation mechanism, illustrating the importance of intermediates and their attributes in relation to the overall rate of organic removal.

Evidence of Distinct Populations and Specific Subpopulations within the SpeciesOenococcus oeni

Among the lactic acid bacteria (LAB) present in the oenological microbial ecosystem, Oenococcus oeni, an acidophilic lactic acid bacterium, is essential during winemaking. It outclasses all other bacterial species during malolactic fermentation (MLF). Oenological performances, such as malic acid degradation rate and sensorial impact, vary significantly according to the strain. The genetic diversity of the O. oeni species was evaluated using a multilocus sequence typing (MLST) scheme. Seven housekeeping genes were sequenced for a collection of 258 strains that had been isolated all over the world (particularly Burgundy, Champagne, and Aquitaine, France, Chile, South Africa, and Italy) and in several wine types (red wines, white wines, and champagne) and cider. The allelic diversity was high, with an average of 20.7 alleles per locus, many of them being rare alleles. The collection comprised 127 sequence types, suggesting an important genotypic diversity. The neighbor-joining phylogenetic tree constructed from the concatenated sequence of the seven housekeeping genes showed two major phylogenetic groups, named A and B. One unique strain isolated from cider composed a third group, rooting the phylogenetic tree. However, all other strains isolated from cider were in group B. Eight phylogenetic subgroups were statistically differentiated and could be delineated by the analysis of only 32 mutations instead of the 600 mutations observed in the concatenated sequence of the seven housekeeping genes. Interestingly, in group A, several phylogenetic subgroups were composed mostly of strains coming from a precise geographic origin. Three subgroups were identified, composed of strains from Chile, South Africa, and eastern France.

Development of a reliable and easy method for screening <em>Oenococcus</em> carbohydrate consumption profile

<p style="text-align: justify;"><strong>Aims</strong>: The aim of this study was to develop a colorimetric test to determine <em>Oenococcus</em> carbohydrate consumption profile.</p><p style="text-align: justify;"><strong>Methods and results</strong>: A semi-defined growth medium which enabled efficient bacterial growth and medium acidification in the presence of glucose was developed. The inoculum size and the presence of citrate in the medium were optimized. Acidification of the medium was revealed by adding bromocresol green and was shown to be perfectly correlated with D-glucose, D-fructose, L-arabinose, D-xylose, L-rhamnose, D-cellobiose or D-trehalose consumption by 23 distinct <em>O. oeni</em> strains and one <em>O. kitaharae</em> strain (confirmed with HPLC analysis).</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Efficient bacterial growth is essential before malic acid degradation occurs in wine. The method developed here will facilitate the comparison of numerous <em>O. oeni</em> strains and their ability to grow by using the various carbohydrates present in wine prior to malolactic fermentation.</p>

Degradation of malic acid in wine by immobilizedIssatchenkia orientaliscells with oriental oak charcoal and alginate

To test degradation of malic acid content in wine by immobilized Issatchenkia orientalis KMBL 5774 cells recently isolated from Korean wine pomace as a malic acid-degrading yeast. I. orientalis KMBL 5774 cells were immobilized using a mixture of oriental oak (Quercus variabilis) charcoal with sodium alginate. When the immobilized yeast cells were observed on a scanning electron microscope, cells were efficiently immobilized on the surface area of the charcoal. A Korean wine containing a high level of malic acid was treated with the immobilized yeast cells. The HPLC analysis of the malic acid content in the treated wine showed the malic acid content was reduced to 0.75 mg ml(-1) after treatment from the original content of 8.96 mg ml(-1), representing 91.6% of the malic acid was degraded during the treatment. The immobilization of the malic acid-degrading yeasts with oriental oak charcoal and sodium alginate is useful for degradation of malic acid in wines containing a high level of malic acid with no significant increase in other acids. Malic acid is sometimes detrimental to the quality of wines when present at high concentrations in some varieties. The immobilized I. orientalis KMBL5774 cells appear to be a promising candidate in view of developing biotechnological methods for reduction of malic acid contents in wine.

Photocatalytic efficiencies of self-cleaning glasses. Influence of physical factors

Two commercial types of self-cleaning glass (SCG) have been tested to confirm the real photocatalytic nature of their properties. This was done by using four photocatalytic tests: (i) in the gas phase with the total oxidation of acetylene; (ii) in water with the total degradation of malic acid, (iii) in water with the total degradation of methylene blue, and (iv) in the solid phase with the total oxidative degradation of a layer of stearic acid deposited on the self-cleaning glass surface, in contact with the superficial titania coating. The influence of various factors (temperature, humidity, wavelength, radiant flux, presence of inorganic particles stuck at the glass surface) was explained in line with the fundamentals of photocatalysis. The results helped to understand the behaviour of self-cleaning glass.

Compositional Corrections Applied in Obtaining Semi Dry and Semi Sweet White Wines in Iasi Vineyard

Compositional corrections - sugar and acid adjustments, as well as biological degradation of malic acid, wines blending- are used to enhance or diminish the content of certain substances. Feteasca alba, Feteasca regala, Muscat Ottonel and Tamâioasa româneasca grape varieties, harvested in 2008 from Iasi vineyard, center of Copou-Adamachi farm were used. The grapes were processed through the classic technology of obtaining white dry wines. The wines were corrected with rectified concentrated must (RCM) and concentrated must (CM) in order to obtain semi-dry and semi- sweet wines. Tartaric acid was also added. The analyses were done according to international standards and specific literature. The main physical-chemical characteristics of the obtained wines show an increase in reductive sugars, non-reductive extract and density.

Effects of Cluster Light Exposure on 3-Isobutyl-2-methoxypyrazine Accumulation and Degradation Patterns in Red Wine Grapes (Vitis vinifera L. Cv. Cabernet Franc)

The effects of light exposure on 3-isobutyl-2-methoxypyrazine (IBMP) accumulation and degradation in Vitis vinifera L. cv. Cabernet Franc berries were assessed by comparison of shaded and exposed clusters within the same vine throughout a growing season. Twenty-seven vines were shoot-thinned to create regions of high and low cluster-light exposure within each vine. Samples were collected at 10 time points starting from 5 to 130 days postbloom. The experimental design allowed for intravine comparison of IBMP levels between treatments at each time. Vine-to-vine variability of IBMP and the correlation of IBMP to malic acid were also evaluated. Cluster exposure reduced accumulation of IBMP at all preveraison time points by 21-44%, but did not increase postveraison degradation. Significant vine-to-vine variability in IBMP content was observed, with the highest level of IBMP in shaded berries in the most vigorous block of vines. Although IBMP concentration by weight decreased significantly due to dilution just prior to color change (veraison), no significant IBMP degradation per berry occurred until after color change (day 70 postbloom). By contrast, malic acid degradation began prior to color change, and malic acid concentrations were not affected by cluster exposure preveraison, but were affected postveraison. A survey of 13 sites in New York state (Seneca Lake) showed that IBMP concentrations at 2 weeks preveraison were highly correlated (R(2) = 0.936, p < 0.0001) to levels at harvest, whereas classic grape maturity indices at harvest were uncorrelated with IBMP at harvest. In summary, light exposure conditions critically influence IBMP accumulation but not IBMP degradation.

Developmental changes in pulp organic acid concentration and activities of acid-metabolising enzymes during the fruit development of two loquat ( Eriobotrya japonica Lindl.) cultivars differing in fruit acidity

Changes in organic acid concentration and related enzyme activities in loquat ( Eriobotrya japonica Lindl.) pulp were studied, using low-acid ‘Changhong 3’ and high-acid ‘Jiefangzhong’ cultivars. Both titratable acidity (TA) and malic acid concentration increased during the early stages of fruit development and decreased at the later stages. The difference in TA between the two cultivars could be explained by the difference in malic acid concentration, which could result from a difference in NAD-malate dehydrogenase (NAD-MDH) and NADP-malic enzyme (NADP-ME) activities. Although the difference in malic acid concentration between the two cultivars could not result from a difference in phosphoenolpyruvate carboxylase (PEPC) activity, malic acid concentration in both ‘Changhong 3’ throughout fruit development and ‘Jiefangzhong’ at the early stages increased linearly and curvilinearly with increasing PEPC activity, respectively. Therefore, NAD-MDH, NADP-ME and PEPC activities may play significant roles in malic acid biosynthesis and degradation.

Oenococcus oeni cells immobilized on delignified cellulosic material for malolactic fermentation of wine

Oenococcus oeni ATCC 23279 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF). In first, eleven repeated alcoholic fermentation batches of white must of 11–12 °Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 °C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 °C. From the 3rd MLF batch up to 10th, the malic acid degradation was 53.1 up to 67.4% and the cfu of the immobilized cells/g of biocatalyst remained stable. The produced lactic acid was less than the stoichiometric yield and acetic acid content was significantly reduced after MLF not contributing in an important increase of the volatile acidity of wine. Ethanol, higher alcohols acetaldehyde and diacetyl contents in wines after MLF were in acceptable levels.

Malic acid photocatalytic degradation using a TiO 2-coated optical fiber reactor

A photoreactor containing a bundle of TiO 2-coated optical fibers was employed for the photocatalytic degradation of malic acid, a polycarboxylic acid found in fermentation processes and in biomass. TiO 2 film was coated on optical fiber using a dip-coating method. The kinetic behavior of malic acid conversion in this study was found to follow a first-order kinetics with a degradation rate of 6.8 × 10 −2 h −1. Laboratory experiments enabled one to detect six intermediates presented in five degradation pathways. Their evolution during the photocatalytic reaction was also investigated. The major one, malonic acid, is initiated by a decarboxylation (“photo-Kolbe”) reaction in the α-position with respect to the OH group followed by an OH addition. The others intermediates come from the same process or from H-abstraction or oxidation. The total organic carbon removal after 20 h of treatment was 21%. A high photonic efficiency of 6% was achieved for experiments operated in the optical fiber photoreactor due to the optimized light utilization.

Impact of winemaking practices on arginine and citrulline metabolism during and after malolactic fermentation

To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. This study suggests that arginine metabolism is inhibited in oenococci at low pH values (< 3.5) and that in the postalcoholic fermentation phase, citrulline formation from arginine degradation can be avoided if MLF is induced by pure cultures of O. oeni with inhibition of the bacterial biomass after malic acid depletion. Residual yeast lees in the wine have been identified as a significant risk factor for increased citrulline formation. Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.

Modelling malic acid accumulation in fruits: relationships with organic acids, potassium, and temperature

Malic acid production, degradation, and storage during fruit development have been modelled. The model assumes that malic acid content is determined essentially by the conditions of its storage in the mesocarp cells, and provides a simplified representation of the mechanisms involved in the accumulation of malate in the vacuole and their regulation by thermodynamic constraints. Solving the corresponding system of equations made it possible to predict the malic acid content of the fruit as a function of organic acids, potassium concentration, and temperature. The model was applied to peach fruit, and parameters were estimated from the data of fruit development monitored over 2 years. The predictions were in good agreement with experimental data. Simulations were performed to analyse the behaviour of the model in response to variations in composition and temperature.

Volatile Compounds in Cider: Inoculation Time and Fermentation Temperature Effects

A cider fermentation at laboratory scale with controlled inoculation with selected yeasts and malolactic bacteria was performed. The composition of the major volatile compounds with low boiling points (<145°C) was determined by gas chromatography throughout the fermentation process, under the different operating conditions of inoculation time of the microorganisms (simultaneous or sequential) and fermentation temperature (15°C or 22°C). Temperature had a more important effect on the levels of certain volatile compounds when the simultaneous inoculation method was used. It was observed that when fermentation temperature increased to 22°C, using the simultaneous method, the final concentrations of ethyl acetate and some of the higher alcohols decreased, while others maintained similar levels. In the sequential inoculation model, after completion of the alcoholic fermentation at 15°C, an increase in the temperature of the malolactic fermentation (15°C to 22°C) showed no statistically significant differences in the profiles of the volatile compounds tested. Thus, malolactic fermentation could be conducted at 22°C, favouring malic acid degradation, without losses in the major volatile compounds, in relation to the levels measured at the lower temperature. Considering the traditionally recognized preference for low alcoholic fermentation temperatures in cider making, the results allow for the optimisation of the operating conditions.

Malolactic fermentation in sea buckthorn (Hippophaë rhamnoides L.) juice processing

Malolactic fermentation (MLF) traditionally used in winemaking was applied to sea buckthorn to reduce the high sourness of the berry juice. Chemical and microbiological aspects, as well as sensory properties of the juice during MLF were studied in order to develop an optimal process. In 1:1 water diluted juice with malic acid content of 15 g/l and pH 2.8, efficient conversion of l-malic acid to l-lactic acid was achieved with direct inoculation of unadapted Oenococcus oeni at a cell density of 109 CFU/ml. The rapid malic acid degradation was performed by non-growing bacterial cells without loss of sugars, vitamin C, or pulp oil. More than 50% of the initial malic acid was metabolized to lactic acid and CO2 already in 12 h of fermentation and a significant decrease in sourness and astringency was noticed. In 24 h fermentation, pH value was increased to 3.1 and only 3 g/l malic acid remained in the juice. Prolonged fermentation had only minor effect on malolactic reaction, but off-flavor of the juice began to increase.

Putrescine Accumulation in Wine: Role of Oenococcus oeni

Putrescine, the most abundant biogenic amine in wine, was proved to be produced by Oenococcus oeni strains in wine not only from ornithine but also from arginine. In this case, putrescine may originate from strains possessing the complete enzyme system to convert arginine to putrescine or by a metabiotic association, with an exchange of ornithine, between strains capable of metabolizing arginine to ornithine but unable to produce putrescine and strains capable of producing putrescine from ornithine but unable to degrade arginine. Putrescine production by this metabiotic association occurred once the malolactic fermentation was completed, whereas conversion of ornithine to putrescine by a single culture of the ornithine decarboxylating strain concurred with the degradation of malic acid. Moreover, in the former case, putrescine formation proceeded more slowly than in the latter. Metabiosis may play an important role in the accumulation of putrescine in wine, arginine being one of the major amino acids found in wine.

Interactions of rare earth elements with bacteria and organic ligands

We investigated the interactions of rare earth elements (REEs) Eu(III) and/or Ce(III, IV) with the common soil bacterium Pseudomonas fluorescens and organic ligands, such as malic acid, citric acid, a siderophore (DFO), cellulose, chitin, and chitosan. Malic acid formed complexes with Eu(III), but degradation of malic acid was observed when the ratio of malic acid to Eu(III) was higher than 100. Citric acid formed a stoichiometric complex with Eu(III) that was not degraded by P. fluorescens. Adsorption of Eu(III) from the DFO complex occurred as a free ion dissociated from DFO and not as the Eu(III)–DFO complex. Cerium(III) was oxidized to Ce(IV) during complexation with DFO to form the Ce(IV)–DFO complex. Time-resolved laser-induced fluorescence spectroscopy (TRLFS) analysis showed that cellulose, chitin, and chitosan, respectively, formed a weak complex, an inner-spherical complex, and an outer-spherical complex with Eu(III). This method also demonstrated that the coordination environment of Eu(III) adsorbed on P. fluorescens possessed similar characteristics to that of chitin, and revealed that adsorption of Eu(III) on P. fluorescens was through a multidentate and predominantly inner-spherical coordination.

Molecular analysis of Oenococcus oeni population dynamics and the effect of aeration and temperature during alcoholic fermentation on malolactic fermentation

SummaryThe effects of aeration and temperature during alcoholic fermentation (AF) on spontaneous and inoculated malolactic fermentation (MLF) of wine have been analysed by following the population dynamics of Oenococcus oeni strains with multiplex random amplified polymorphic DNA‐polymerase chain reaction. It has enabled us to follow precisely the proportion of different autochthonous strains and the starter strain through fermentations. Lack of aeration led to delays in AF, which meant that autochthonous lactic acid bacteria could develop earlier and prevented the starter strain from developing correctly. Temperature was not found to lead to any differences. Two strains were isolated in the same spontaneous MLF, suggesting that, in some cases, multiple strains might be responsible for the degradation of malic acid in wine. It can be concluded that delays in the AF can negatively affect the control of MLF and that this can be studied by following the development of the different strains of O. oeni.