Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry     

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 degrees Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 degrees C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 degrees 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.     

Degradation of malic acid in wine by immobilized Issatchenkia orientalis cells with oriental oak charcoal and alginate

Aims: 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. Methods and Results: 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. Conclusions: 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. Significance and Impact of the study: 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.     

The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni

Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5-5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine. The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 x 10(9) cfu sponge(-)(1) in at least four subsequent batch fermentations.     

Expression of the Malolactic Enzyme Gene (<Emphasis Type="Italic">mle</Emphasis>) from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> 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.     

CONTINUOUS ALCOHOL/MALOLACTIC FERMENTATION OF GRAPE MUST IN A BIOREACTOR SYSTEM USING IMMOBILIZED CELLS

Three cultures immobilized by entrapping within alginate gel beads and packed in near-horizontal acrylic columns (15.0° angle) were used for alcohol/malolactic fermentation of grape must. Immobilized cells of Saccharomyces cerevisiae spp. chablis were placed in the 1st column, S. cerevisiae cells (an alcohol-sucrose-tolerant yeast) in the 2nd and the Lactobacillus delbrueckii cells in the 3rd column. Grape must with different levels of sugar(s), were each fed to the bioreactor columns at dilution rate of 0.74 h⁻¹ and recycled at 37.0C. The percent fermentation efficiency and yield using the 1st and 2nd columns for grape must containing 33.3% sugar(s) were 92.9 and 91.5%, respectively, and the wine had 15.5% alcohol after 23 cycles (∼ 50 h fermentation). The viability of the immobilized yeast cells in the alginate gel-bead was 84%± 4.0. Immobilized Lactobacillus delbrueckii cells were then added to the 3rd column (in series 37.0C) and the three cultures resulted in alcohol/malolactic fermentation of the grape must, evidenced by the high level of alcohol formed and simultaneous transformation of malic to lactic acid. Sensory evaluation of the wine scored high (7.8 ± 2.0 based on a value of 10.0) and indicated the potential of using multiple immobilized cells of two specific yeast cultures and a malolactic Lactobacillus for wine production.     

Synthesis of antibacterial PVA/CM-chitosan blend hydrogels with electron beam irradiation

A series of excellent hydrogels were prepared from poly(vinyl alcohol) (PVA) and carboxymethylated chitosan (CM-chitosan) with electron beam irradiation (EB) at room temperature. Electron spectroscopy analysis of the blend hydrogels revealed that good miscibility was sustained between CM-chitosan and PVA. The properties of the prepared hydrogels, such as the mechanical properties, gel fraction and swelling behavior were investigated. The mechanical properties and equilibrium degree of swelling improved obviously after adding CM-chitosan into PVA hydrogels. The gel fraction determined gravimetrically showed that a part of CM-chitosan was immobilized onto PVA hydrogel. The further analyses of FTIR and DSC spectra of the prepared gels after extracting sol manifested that there was a grafting interaction between PVA and CM-chitosan molecules under irradiation. The antibacterial activity of the hydrogels against Escherichia coli was also measured via optical density method. The blend hydrogels exhibited satisfying antibacterial activity against E. coli, even when the CM-chitosan concentration was only 3 wt%.     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine

Grape must was fermented by a mixed culture of Saccharomyces cerevisiae W-3 (a wine yeast) and Issatchenkia orientalis KMBL 5774 (a malic acid-degrading yeast). Co-fermentation with 1:1 (v/v) inoculum ratio of W-3 and KMBL 5774 decreased malic acid to 0.33 mg/ml from 1.1 mg ml with W-3 alone. Ethanol production was the same in both cases (7.8%, v/v). Acetaldehyde, 1-propanol, 2-butanol and isoamyl alcohol all decreased, with an increase in methanol, in the co-fermented wine. Sensory evaluation showed a higher score in the wine fermented with 1:1 (v/v) inoculum ratio than those obtained by 4:1 (v/v) inoculum ratio or W-3 alone.     

Implications of new research and technologies for malolactic fermentation in wine

The initial conversion of grape must to wine is an alcoholic fermentation (AF) largely carried out by one or more strains of yeast, typically Saccharomyces cerevisiae. After the AF, a secondary or malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) is often undertaken. The MLF involves the bioconversion of malic acid to lactic acid and carbon dioxide. The ability to metabolise l-malic acid is strain specific, and both individual Oenococcus oeni strains and other LAB strains vary in their ability to efficiently carry out MLF. Aside from impacts on acidity, LAB can also metabolise other precursors present in wine during fermentation and, therefore, alter the chemical composition of the wine resulting in an increased complexity of wine aroma and flavour. Recent research has focused on three main areas: enzymatic changes during MLF, safety of the final product and mechanisms of stress resistance. This review summarises the latest research and technological advances in the rapidly evolving study of MLF and investigates the directions that future research may take.     

Growth and Arginine Metabolism of the Wine Lactic Acid Bacteria Lactobacillus buchneri and Oenococcus oeni at Different pH Values and Arginine Concentrations

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.     

Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.     

Growth and arginine metabolism of the wine lactic acid bacteria Lactobacillus buchneri and Oenococcus oeni at different pH values and arginine concentrations

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.     

Comparative traits of Lactobacillus brevis, Lact. fructivorans and Leuconostoc oenos immobilized cells for the control of malo-lactic fermentation in wine

Decarboxylation of L-malic to L-lactic acid by heterolactic bacteria and formation of secondary products in table wine were studied using immobilized cells of Lactobacillus brevis, Lact. fructivorans and two strains of Leuconostoc oenos in a continuous flow bioreactor. The conversion ratios were 51.2–53.9%, while the decreases in malic and titratable acidity were equivalent to 62.1–74.7% and 16.4–27.3%, respectively. Upon completion of malo-lactic fermentation, pH increased from 3.15 to 3.28–3.35. The conversion ratio and bioreactor efficiency differed according to the strain tested. Gel beads, prepared with cells immobilized in 2% K-carrageenan in the presence of 5% bentonite silica, contained up to 7–8 × 10¹⁰ cfu/g; the concentration of viable cells was relatively stable over 24–48 h bioreactor operation when Lact. brevis was used and decreased in all other cases. The formation of secondary products affecting wine sensory properties, particularly volatile acids and aromatic compounds, was strain-dependent.     

Biodegradation of dichloromethane by the polyvinyl alcohol-immobilized methylotrophic bacterium Ralstonia metallidurans PD11

A dichloromethane (DCM)-degrading bacterium, Ralstonia metallidurans PD11 NBRC 101272, was immobilized in a polyvinyl alcohol (PVA) gel to use in a bioreactor for DCM treatment. After 4-month incubation of PVA gel beads with R. metallidurans PD11 and DCM in a mineral salt medium, the cells were tightly packed in the mesh of the gel. Forty beads of the gel in 10 ml of a batch system model showed effective activity degrading 500 and 1,000 mg l⁻¹ DCM within 2 and 3 h, respectively. Although reduction of pH due to accumulation of chloride ion liberated from DCM decreased the activity, it was recovered by adjustment to neutral pH. The activity of the immobilized cells was not affected by addition of nutrients which were preferentially utilized by R. metallidurans PD11, unlike the activity of the free-living cells. A continuous flow system with a column was more effective for rapid degradation of DCM. Thus, the PVA gel–immobilized cell of R. metallidurans PD11 is thought to be a prospective candidate to develop the bioreactor.     

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

AIMS: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). METHODS AND RESULTS: 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. Conclusions: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.     

Continuous malolactic fermentation in red wine using free Oenococcus oeni

Malolactic fermentation (MLF) of wine in continuous culture was obtained by using Oenococcus oeni (formerly Leuconostoc oenos). The maximum malic acid degradation in our bioreactor system was reached at a dilution rate of 0.016h^–1, and 92–95% of the malic acid (3.9–4.0g/l) was converted to lactic acid and CO₂.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.