Malolactic Bacteria Research Articles

Role of malolactic fermentation in lactic acid bacteria

Although decarboxylation of malate to lactate by malolactic enzyme does not liberate biologically available energy ( e.g., ATP, NADH), the growth rate of many malolactic bacteria is greatly enhanced by malolactic fermentation. The deacidification of the medium due to malate dissipation cannot fully account for this situation. The chemiosmotic theory postulates that another form of energy could generated by translocation of protons through the membrane coupled to end-product efflux. Konings et al. showed that this theory is indeed applicable to lactate efflux in Streptococcus cremoris at pH 7.0 A similar mechanism could account for the observed increased activity in malolactic bacteria. The study in wild type and mutant strains of Streptococcus lactis unable to carry out malolactic fermentation led us to the following conclusions: (1) under glucose non-limiting conditions, malolactic fermentation helps to maintain pH of the medium at a certain level; (2) during glucose limited growth, malolactic fermentation could be coupled with an energetic process independent from that mentioned above.

The Interaction of Sulfur Dioxide, pH, and Dimethyl Dicarbonate on the Growth ofSaccharomyces cerevisiaeMontrachet andLeuconostoc oenosMCW

Wine yeast <i>Saccharomyces cerevisiae</i> Montrachet and malolactic bacteria <i>Leuconostoc oenos</i> MCW were grown in and acclimatized to wine with significant amounts of sulfur dioxide. The cultures were transferred to wine at different pH levels with varying amounts of SO₂ and dimethyl dicarbonate (DMDC). Both compounds were inhibitors to yeast and bacteria growth, and there was a synergistic activity between them. With a free SO₂ level of 25 mg/L and the addition of 50 mg/L DMDC, control of the yeast and the bacteria was excellent at pH 3.6 or lower in a sweet white table wine and a dry red table wine.

A numerical taxonomic study of Leuconostoc oenos strains from wine

Phenotypic data of 108 tests conducted on 70 malolactic bacteria, including 54 presumptive Leuconostoc oenos strains, five presumptive pediococci isolated from wine and 11 reference strains, were analysed by numerical taxonomic techniques. Using the simple matching coefficient, 58 strains were grouped in six clusters at the 87% S level. Cell wall analysis of the interpeptide bridges and morphology was also used to differentiate between the strains. No L. oenos reference strains were found to group in any cluster. The hypothetical median organism (HMO) of cluster A was related at only the 63% S level to the L. oenos type strain and it is proposed that these strains be regarded as ‘L. gracile’. The majority of the L. oenos strains (35) grouped together at above the 91% S level in cluster B, with the HMO of this cluster related at the 75% S level to the L. oenos type strain. Results indicate that the majority of L. oenos strains included in this study are closely related, but more research is needed to justify the separation of these strains into more than one species.

SENSORY PANEL TRAINING AND SCREENING FOR DESCRIPTIVE ANALYSIS OF THE AROMA OF PINOT NOIR WINE FERMENTED BY SEVERAL STRAINS OF MALOLACTIC BACTERIA

ABSTRACT.A sensory panel was selected and trained to describe the aroma of Pinot noir wines whose only difference was that they were fermented by different strains of malolactic bacteria. The panel developed a ballot consisting of 33 aroma descriptors and rated the wines using a balanced incomplete block statistical design. A final screening of the individual panelist's data was accomplished by use of correlation analysis. Significant differences were found in twenty of the aroma descriptors, showing that the strain of malolactic bacteria selected for use can effect aroma perception.

Inhibition of the Malolactic Bacterium,Leuconostoc oenos(PSU-1), by Decanoic Acid and Subsequent Removal of the Inhibition by Yeast Ghosts

The influence of decanoic acid, a metabolite produced by wine yeasts, on the growth and malolactic activity of <i>Leuconostoc oenos</i> (PSU-1) in grape juice was investigated. Decanoic acid was dissolved in ethanol and added to diluted Aurore grape juice before inoculation with <i>Leuconostoc oenos</i> (PSU-1). Although low concentrations of decanoic acid (1 mg/L) did not significantly influence cell viability or the rate of malolactic fermentation (MLF), bacterial growth and MLF were inhibited at acid concentrations of 5.0 to 10.0 mg/L. Rapid bacterial death and complete inhibition of MLF was observed at 30.0 mg/L decanoic acid. Addition of yeast ghosts (1000 mg/L) to juice containing lethal concentrations of decanoic acid was found to remove the bacterial inhibition, presumably by adsorption of the acid.

Metabolic Interactions BetweenSaccharomyces cerevisiaeandLeuconostoc oenosin a Model Grape Juice/Wine System

Metabolic interactions between wine yeast (<i>Saccharomyces cerevisiae</i> Montrachet No. 522) and a malolactic bacterium (<i>Leuconostoc oenos</i> PSU-1) were investigated by comparing their growth in both pure and mixed culture in a model grape juice/wine system. Yeast inhibited early bacterial growth, and growing bacteria accelerated the death phase of the yeast growth cycle. Ornithine was not produced by the growing bacteria; thus, this compound was not responsible for the bacterial antagonism of yeast. Dilution of the grape juice from 20° to 10°Brix with water reduced antagonistic effects, making it possible to obtain both organisms in stationary phase simultaneously. Ethanol and acetaldehyde-bound sulfur dioxide were investigated as possible yeast produced bacterial inhibitors. They were added to the grape juice/wine system sequentially in levels previously determined to be produced by normal yeast growth in the juice. Ethanol stimulated early bacterial growth when concentrations were less than two percent, but inhibition of growth occurred later, when levels exceeded six percent. The low concentration of acetaldehyde-bound sulfur dioxide added (7.8 mg/L) had little effect on bacterial growth, but it did negate the early stimulation of growth by ethanol when the two compounds were added together. The most severe bacterial inhibition resulted from actual yeast growth, which indicated that the early inhibition of bacteria in the juice/wine system was more complex than the factors studied.

Characterization of Leuconostoc oenos Isolated from Oregon Wines

This study was designed to characterize isolates of Leuconostoc species from Oregon wines. Gram-positive cocci were isolated, and their biochemical properties and abilities to decompose malic acid were determined. All of the isolates were heterofermentative, catalase negative, and facultatively anaerobic and occurred in pairs and chains. They produced acid from glucose, fructose, mannose, ribose, cellobiose, trehalose, and salicin but not from sucrose or lactose. They did not produce ammonia from arginine or dextran from sucrose. They grew at pH values of less than 4 and in 10% ethanol. Most but not all strains produced lactic acid and carbon dioxide from malic acid, as determined by paper chromatography and respirometry, respectively. These malolactic bacteria were considered to be strains of Leuconostoc oenos. We compared these isolates with reference strains for relative growth at pH values of 4.0, 3.5, 3.0, and 2.8 at 22 degrees C. The isolates were similar in their growth responses at the two highest pH levels. At pH 3.0 and 2.8, however, the strains failed to grow but revealed variable abilities to dissimilate malic acid.

Transformation of Citric Acid to Acetic Acid, Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn2+, Mg2+, Co2+ and Zn2+ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg2...

Transformation of citric acid to acetic acid, acetoin and diacetyl by wine making lactic acid bacteria.

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307, a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid. Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cellfree extract were optimal at pH 6.0. Divalent cations such as Mn2+, Mg2+, Co2+ and Zn2+ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg2+ and Ag2+. The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl2, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malolactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.

Effect of pH and Sulfur Dioxide on the Rate of Malolactic Fermentation in Red Table Wines

The pH of three must samples from Chancellor, a red French hybrid, was adjusted to 3.3, 3.5, and 3.7, respectively. Musts at each pH were treated with 25, 50, and 75 ppm sulfur dioxide, respecitvley, and then inoculated with a wine yeast, <i>Saccharomyces cerevisiae</i> Montrachet #522. When the fermenting musts reached 5°Brix, each was inoculated with a malolactic bacteria, <i>Leuconostoc oenos</i> PSU-1. For each level of SO₂, rates of malolactic fermentation were nearly the same at pH 3.5 and 3.7, but fermentation was much slower at pH 3.3. At a given pH, malolactic fermentation was faster at the lower SO₂ levels. Following inoculation, the bacterial population decreased, and the lowest bacterial count was observed in the presence of low pH and high SO₂.

Effect of Insoluble Solids on the Sulfur Dioxide Content and Rate of Malolactic Fermentation in White Table Wines

The insoluble solids of several white grape juices were adjusted from 0% to 15%. After treating with 30 mg/L of sulfur dioxide, each juice was inoculated with a wine yeast culture, Montrachet #522. When the wines reached dryness, they were also inoculated with a malolactic bacteria, <i>Leuconostoc oenos</i> PSU-1. The results indicated that the rate of malolactic fermentation was influenced by the level of insoluble solids. Wines with the highest insoluble solids experienced the fastest rate of malolactic fermentation. In addition, the insoluble solids appeared to have an effect upon the sulfur dioxide content. Wines with the highest insoluble solids tended to be lowest in sulfur dioxide.

Comparison of Procedures for Isolation of Malolactic Bacteria from Wine

Malolactic bacteria were isolated from wine by direct streaking on modified Rogosa agar plates followed by aerobic incubation or incubation in a high CO₂ atmosphere, or enrichment in modified Rogosa broth before streaking and aerobic incubation (designated aerobic, high CO₂ and enrichment incubations, respectively). The incubation temperature was 30°C throughout. More cocci were isolated with the aerobic and high CO₂ incubations than with the enrichment incubation. Preliminary identification tests indicated that 41 lactobacilli, 34 leuconostocs and 15 pediococci were isolated by the aerobic and high CO₂ incubations while 45 lactobacilli and only two leuconostocs and one pediococcus were isolated with the enrichment incubation. The high C0₂ incubation seemed the most suitable for the isolation of leuconostocs.

Microbial Inhibition Caused by p-Hydroxybenzoate Esters in Wine

A method is given for analysis of n-hexyl, n-heptyl, and n-octyl alcohol p-hydroxybenzoates in wine. The effectiveness of these compounds and others is discussed as inhibitors of yeasts and bacteria in wine. Solubility generally limits the effectiveness of the individual esters, but combinations of the 6-, 7-, and 8-chain alcohol esters have additive effects and prevent yeast growth effectively. The heptyl ester also appears to be a potent antimicrobial agent against malo-lactic bacteria. Sensory tests indicate a slight bitterness associated with these compounds.

The Effect of Fumaric Acid on Malo-Lactic Fermentation in Wines from Warm Areas

Wines made from grapes from warm areas such as the San Joaquin Valley would seldom seem to benefit from malo-lactic fermentation. Not only is acidity lost but off-odors and off-flavors can develop which are sometimes difficult to remove. Most wines from these areas are sold young, without aging, and part of their value is their fresh fruitiness. Fumaric acid has been shown (3, 8) to be effective in delaying or inhibiting the growth of malo-lactic bacteria in wine. Earlier work (5) had indicated that quality difference in wines might depend on the species of malo-lactic bacteria present. This paper demonstrates that routine addition of fumaric acid at the time of the first racking, along with a normal addition of sulfur dioxide, will in general improve wine quality significantly by inhibiting malo-lactic fermentation as well as adding needed acid to the wines. In addition, the usual recommended malo-lactic strain (<i>Leuconostoc oenos</i> ML-34) was tested to see whether it did improve wine quality over that obtained by allowing a natural flora to develop.

Characterization and Energetics of Leuconostoc Oenos ml 34

Characteristics of the malo-lactic bacterium <i>Leuconostoc oenos</i> ML 34 are reported indicating that this organism, previously designated <i>L. citrovorum</i> ML 34, should be placed in the new species <i>L. oenos</i> Garvie 1967. Of 26 compounds tested, energy sources for the organism were D-glucose, D-fructose, D-ribose, trehalose, and cellobiose. L-malic and citric acids, not energy sources themselves, provided a slight increase in cell yield in the presence of limited carbohydrate. Production of one of the end products, D-lactic acid, greatly exceeded that expected on the basis of the fermentation of glucose and L-malic acid substrates. It was concluded not only that sugar utilization is required for malo-lactic fermentation, but that malo-lactic fermentation also has a profound influence on the metabolism of the organism and implies a stimulation in utilization of carbon sources. Also discussed are the nomenclature and taxonomy of the wine leuconostocs.

The Effect of Grape Skin Treatments on Induced Malo-Lactic Fermentation in Ohio Wines

Induced malo-lactic fermentation was attempted in grape musts prepared by cold pressing, hot pressing, and fermentation "on the skins" for 1, 3, and 5 days before pressing. The musts were inoculated with the malo-lactic bacterium <i>Leuconostoc citrovorum</i> (ML 34) and otherwise treated by a standard vinification procedure. Results revealed that fermentation "on the skins" had a profound effect on the occurrence and rate of malo-lactic fermentation. The grape musts prepared by cold pressing, hot pressing, and fermentation "on the skins" for one day supported only limited growth of the inoculated bacteria, and no malo-lactic fermentation was defected after one year. The wines that were fermented "on the skins" for 3 days underwent an incomplete malo-lactic fermentation. Wines that were fermented 5 days "on the skins" completed malo-lactic fermentation by 11.3 weeks after the bacterial inoculation. In addilion, model grape musts were prepared which simulated the grape musts prepared by hot and cold pressing as well as by fermentation "on the skins." Isolates prepared from these model musts were demonstrated to contain substance(s) stimulatory to the growth of <i>L. citrovorum</i> (ML 34). Microbiological assays indicated that the isolates from the skins caused a greater growth stimuation than the isolates from the model musts stimulating hot- and cold-pressed musts.

Results Concerning the Evolution of Bacterial Population during the Malolactic Fermentation

During this experiment, the evolution of inner lactic bacteria microflora was monitored, during a spontaneous and conducted malolactic fermentation developed in the fall of the year 2006 at red wines obtained in Minis – Maderat wine yard. Thereby was monitored the bacterial population evolution, immediately after finishing the alcoholic fermentation (before developing the malolactic fermentation), through standard cultural method and through direct counting methods (counting with Thoma board and counting through Breed method).&#x0D; Results show that wines, at the end of alcoholic fermentation present bacterial loads between 102 and 104 cells/ml, after which in the exponential growing phase of the lactic bacteria registered at 5 days after sowing the selected malolactic bacteria, the bacterial density of wines to grow to 106 – 107 cells/ml, and at the end of malolactic fermentation, which matches the decline phase of lactic bacteria, the bacterial density of wines to get back to 105 cells/ml.

Chemical Characterization of Wines Fermented with Various Malo-lactic Bacteria

Six malo-lactic strains of lactic acid bacteria were isolated from California wines and identified as Lactobacillus delbrueckii, L. buchneri, L. brevis, Leuconostoc citrovorum , and two strains of Pediococcus cerevisiae . Malo-lactic fermentation was induced in separate lots of wine by inoculation of each lot with one of the strains of bacteria. Malo-lactic fermentation had occurred in each inoculated wine within 2 months. The resultant wines were subjected to chemical analysis, including gas chromatographic examination of concentrated extracts of the wines. Only a few differences in composition were found when the malo-lactic wines were compared one with another. The differences that were found were in volatile acidity and in concentrations of acetoin (plus diacetyl) and probably diethyl succinate.

Proper Use of Words

The effects of wine yeasts and malolactic bacteria on the anthocyanin profile of Sangiovese wines produced by both commercial- and laboratory-scale winemaking processes were investigated. The mean anthocyanin profile of the wines obtained from commercial winemaking processes, carried out in several wineries in Tuscany, showed a distinctive anthocyanin pattern characterized by high percentages of malvidin-3-glucoside, without appreciable levels of acylated anthocyanins. This anthocyanin pattern, also shared by all wines produced in the laboratory, reproduces the peculiar profile of Sangiovese grapes and distinguishes Sangiovese wines from Merlot and Cabernet Sauvignon wines. Principal component analysis of all anthocyanin data showed that the Sangiovese wines did not group on the basis of wine-producing area, harvest year, or winery but were distributed along a longitudinal axis, owning to the variability of cyanidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside percentages. This variability was independent of the yeast ecology during fermentation, at least if the yeast ecology was described at the level of yeast species. In both commercial and experimental wines, higher percentages of vitisin A, a malvidin-3-glucoside derivative, were dependent on the growth and dominance of <i>Candida zemplinina</i> in the early stages of vinification. The anthocyanin profile of the Sangiovese wines was also maintained after malolactic fermentation.

SIR ROBERT MORANT

The effects of wine yeasts and malolactic bacteria on the anthocyanin profile of Sangiovese wines produced by both commercial- and laboratory-scale winemaking processes were investigated. The mean anthocyanin profile of the wines obtained from commercial winemaking processes, carried out in several wineries in Tuscany, showed a distinctive anthocyanin pattern characterized by high percentages of malvidin-3-glucoside, without appreciable levels of acylated anthocyanins. This anthocyanin pattern, also shared by all wines produced in the laboratory, reproduces the peculiar profile of Sangiovese grapes and distinguishes Sangiovese wines from Merlot and Cabernet Sauvignon wines. Principal component analysis of all anthocyanin data showed that the Sangiovese wines did not group on the basis of wine-producing area, harvest year, or winery but were distributed along a longitudinal axis, owning to the variability of cyanidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside percentages. This variability was independent of the yeast ecology during fermentation, at least if the yeast ecology was described at the level of yeast species. In both commercial and experimental wines, higher percentages of vitisin A, a malvidin-3-glucoside derivative, were dependent on the growth and dominance of Candida zemplinina in the early stages of vinification. The anthocyanin profile of the Sangiovese wines was also maintained after malolactic fermentation.