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.     

Degradation of malic acid by Issatchenkia orientalis KMBL 5774, an acidophilic yeast strain isolated from Korean grape wine pomace

Several yeast strains degrading malic acid as a sole carbon and energy source were isolated from Korean wine pomace after enrichment culture in the presence of malic acid. Among them, the strain designated as KMBL 5774 showed the highest malic acid degrading ability. It was identified as Issatchenkia orientalis based on its morphological and physiological characteristics as well as the nucleotide sequences of the internal transcribed spacer (ITS) I-5.8S rDNA-ITS II region. Phylogenetic analysis of the ITS I-5.8S rDNAITS II sequences showed that the KMBL 5774 is the closest to I. orientalis zhuan 192. Identity of the sequences of the KMBL 5774 was 99.5% with those of I. orientalis zhuan 192. The optimal pH of the media for the growth and malic acid degradation by the yeast was between 2.0 and 3.0, suggesting that the strain is an acidophile. Under the optimized conditions, the yeast could degrade 95.5% of the malic acid after 24 h of incubation at 30 degrees in YNB media containing 2% malic acid as a sole carbon and energy source.     

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum

A milk–soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk–soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk–soymilk. The fermented beverage was stored at 4°C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2×10⁹ CFU/ml and 6.3×10⁸ CFU/ml, respectively, after 14 days of storage.     

Optimization of medium for lipase production by Acinetobacter haemolyticus from healthy human skin

A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the "one variable at a time" and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone--1, yeast extract--0.5, sodium chloride-1, olive oil-1, Tween-80 1, manganese sulphate--5 mM, sucrose--1, pH-7. It was found that maximum production occurred in late log phase, i.e., after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3% (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.     

Availability of substratum enhances ethanol production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Novel additives that act as substratum for attachment of the yeast cells, increased ethanol production in Saccharomyces cerevisiae. The addition of 2 g rice husk, straw, wood shavings, plastic pieces or silica gel to 100 ml medium enhanced ethanol production by 30–40 (v/v). Six distillery strains showed an average enhancement of 34 from 4.1 (v/v) in control to 5.5 (v/v) on addition of rice husk. The cell wall bound glycogen increased by 40–50 mg g ^–1 dry yeast while intracellular glycogen decreased by 10–12 mg g^–1 dry yeast in cells grown in presence of substratum     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Production and recovery of an alkaline exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation using statistical approach

The empirical models developed through two independent RSM (RSM-I, 2(3); RSM-II, 2(5)) in terms of effective operational factors of inoculum age, inoculum volume, wheat bran-to-moisture ratio (RSM-I) and contact time, extraction temperature, agitation, fermented bran-to-solvent ratio and SDS (RSM-II) were found adequate to describe the optimization of exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation (SSF) conditions. Through the analysis of RSM-I, wheat bran-to-moisture ratio and inoculum volume were found to be the most significant factors and an increment in both had a positive effect in enhancing enzyme yield, while in RSM-II all the factors significantly affected enzyme recovery except fermented bran-to-solvent ratio, which had the least impact within the ranges investigated in enhancing enzyme recovery. Based on contour plots and variance analysis, optimum operational conditions for maximum exo-polygalacturonase yield were achieved when 1.5% (v/w) of 24h old (OD(600 nm) approximately 2.7+/-0.2) B. subtilis RCK cells were inoculated on moistened wheat bran (1:7 solid substrate-to-moisture ratio) and enzyme was harvested by addition of solvent (1:6 fermented bran-to-solvent ratio) under shaking conditions (200 rpm) in presence of SDS (0.25% w/v) for 15 min at 35 degrees C. An over all 3.4 fold (1.7-fold RSM-I; 2.0 fold RSM-II) increase in enzyme production was attained because of optimization by RSM.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

The degradation of malic acid by high density cell suspensions of Leuconostoc oenos

Washed cell suspensions of Leuconostoc oenos catalysed the degradation of L-malic acid to L-lactic acid. Cell suspensions of 10₁₀ cfu ml_-1 degraded 90–95% of the malic acid in a buffer assay system and in wine within 30 min. A reaction time of 6 h was needed to obtain the same extent of degradation with suspensions of 10₉ cfu ml_-1. With a reaction period of 6 h and an initial malic acid concentration of 3 g 1_-1, reaction variables of pH 2.5-4.0, temperature 10–30°C, ethanol up to 15%, and L-lactic acid up to 4 g 1_-1 did not decrease the degradation of malic acid to below 90–95%. Total SO₂ at 100 mg 1_-1 decreased the degradation of malic acid to 80%. The degradation (%) of malic acid was decreased when the concentration of malic acid was decreased below 2 g 1_-1. The results indicate the prospect of using high densities of Leuc. oenos cells in membrane bioreactor systems for the rapid, continuous, deacidification of wine.     

Effect of air-blast drying and the presence of protectants on the viability of yeast entrapped in calcium alginate beads with an aim to improve the survival rate

Five yeast strains, Saccharomyces cerevisiae D8, M12, and S13; Hanseniaspora uvarum S6; and Issatchenkia orientalis KMBL5774, isolated from Korean grapes, were entrapped in Ca-alginate beads, which are non-toxic, simple to use, and economical. Ca-alginate beads containing yeast cells were soaked in protective solutions, such as skim milk, saccharides, polyols, and nitrogen compounds, before air-blast drying to improve the yeast survival rate and storage ability. The results showed that both entrapment in Ca-alginate beads and soaking in protective agents favorably affected the survival of all strains. The microenvironment formed by the beads and protective agents can protect the yeast cells from harsh environmental conditions, such as low water (below 10 %). All the yeast strains entrapped in Ca-alginate beads showed greater than 80 % survival and less than 11 % water content after air-blast drying at 37 °C for 5 h. In addition, air-blast dried cells of S. cerevisiae D8, M12, S13; H. uvarum S6; and I. orientalis KMBL5774 entrapped in 2 % Ca-alginate beads and soaked in protective agents (10 % skim milk containing 10 % sucrose, 10 % raffinose, 10 % trehalose, 10 % trehalose, and 10 % glucose, respectively) after air-blast drying at 37 °C for 5 h showed 90, 87, 92, 90, and 87 % viability, respectively. All dried entrapped yeast cells showed survival rates of at least 51 % after storage at 4 °C for 3 months.     

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.     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

High intracellular expression of giant catfish growth hormone under the control of PGK promoter in Saccharomyces cerevisiae

Different yeast plasmid systems containing different promoters such as ADH1, PGK, GAPDH and GAL1, and different selectable markers, such as URA3, TRP1 and leu2-d were compared to obtain the yeast expression system that provides high intracellular expression of giant catfish growth hormone (gcGH). The highest level of gcGH expression was observed in a recombinant yeast under the control of PGK promoter (17.1 mg/l or 1.4 g/0.1 OD). The amount of gcGH was increased six-fold (102.5 mg/l) when cells were grown in a rich medium (YEPD) with the inoculum and medium ratio of 1:1, although the amount of gcGH expression per cell density did not increase (1.0 g/0.1 OD). This indicated that the increased yield of gcGH in rich medium was due to the increased cell density. The aim of the study was to produce high level gcGH in the cells of S. cerevisiae in order to use the yeast cells as potential feed additives to promote growth in giant catfish.     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.     

Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation

A study was taken up to evaluate the role of some fermentation parameters like inoculum concentration, temperature, incubation period and agitation time on ethanol production from kinnow waste and banana peels by simultaneous saccharification and fermentation using cellulase and co-culture of Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC 1077. Steam pretreated kinnow waste and banana peels were used as substrate for ethanol production in the ratio 4:6 (kinnow waste: banana peels). Temperature of 30°C, inoculum size of S. cerevisiae G 6% and (v/v) Pachysolen tannophilus MTCC 1077 4% (v/v), incubation period of 48 h and agitation for the first 24 h were found to be best for ethanol production using the combination of two wastes. The pretreated steam exploded biomass after enzymatic saccharification containing 63 gL⁻¹ reducing sugars was fermented with both hexose and pentose fermenting yeast strains under optimized conditions resulting in ethanol production, yield and fermentation efficiency of 26.84 gL⁻¹, 0.426 gg ⁻¹ and 83.52 % respectively. This study could establish the effective utilization of kinnow waste and banana peels for bioethanol production using optimized fermentation parameters.     

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

AIMS: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. METHODS AND RESULTS: Two yeast expression/secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase/xylanase gene cassette (pEXS) containing the endo-beta-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-beta-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase/xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase/xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase/xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. CONCLUSIONS: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharide-degrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price:quality ratio of wine according to consumer expectations.     

Optimization of <Emphasis Type="Italic">R</Emphasis>-(+)-α-terpineol production by the biotransformation of <Emphasis Type="Italic">R</Emphasis>-(+)-limonene

R-(+)-limonene is an abundant and non-expensive by-product of the citrus industry and is, therefore, a suitable starting material for the production of natural flavor and fragrance compounds. The biotransformation of R-(+)-limonene to R-(+)-alpha-terpineol by Fusarium oxysporum 152b has already been reported, although the influence of the main process parameters on the production has not yet been evaluated. In this paper, a Plackett-Burman screening design was used to define the effects of the medium composition (glucose, peptone, yeast extract, malt extract and pH), the presence of a co-substrate (biosurfactant), the cultivation conditions (temperature, agitation), the substrate concentration and the inoculum/culture medium ratio on the absolute amount of R-(+)-alpha-terpineol resulting from this biotransformation. The process conditions were further optimized applying response surface methodology (RSM). The volatiles were extracted using a SPME device and were subsequently quantified by GC-FID and identified by GC-MS. The best results were obtained using 0.5% (v/m) R-(+)-limonene in pure distilled water as the culture medium with an inoculum/culture medium ratio of 0.25 (m/m) and 72 h cultivation at 26 degrees C/240 rpm. Under these conditions the concentration of R-(+)-alpha-terpineol in the culture medium reached 2.4 g L(-1), a production almost six times greater than in earlier trials. The presence of a biosurfactant (0-500 mg L(-1)) did not significantly increase the yield.     

Improved production of curdlan with concentrated cells ofAgrobacterium sp.

The addition of a limited concentration of yeast extract to a minimal salt medium (MSM) enhanced cell growth and increased the production of curdlan whereas nitrogenlimitation was found to be essential for the higher production of curdlan byAgrobacterium sp. ATCC 31749. As the amount of the inoculum increased, the cell growth as well as the production of curdlan also increased in the MSM without a nitrogen source. The cell growth and production of curdlan increased as the initial pH of the medium decreased as low as 5.0. The conversion rate and concentration of curdlan from 2% (w/v) glucose in the MSM with concentrated cells under nitrogen deletion was 67% and 13.4 g/L, respectively. The highest conversion rate of curdlan under the conditions optimized in this study was 71% when the glucose concentration was 1% (w/v).