Effect of physicochemical factors on glycerol production by simultaneous cultures of wine micro-organisms using the response surface method.

AIM: To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). METHODS AND RESULTS: Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (10(9) CFU ml(-1)), while O. oeni reached 10(7) CFU ml(-1). At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l(-1) SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l(-1) SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. CONCLUSIONS: We propose a predictive condition to improve the performance of mixed cultures for must fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics.

Malolactic enzyme in Lactobacillus murinus.

The malolactic enzyme of Lactobacillus murinus is inducible. The induction is produced by L-malic acid only in the presence of glucose and amino acids and occurs at the transcription level. The enzyme, purified to homogeneity, has a Mr of 220,000 and consists of 2 apparently identical subunits (Mr = 110,000) that were observed after treatment with sodium dodecyl sulphate. NAD+ protected the enzyme against inactivation and its addition, after dissociation, restored the malolactic activity. Maximum enzyme activity was observed at 37 degrees C and pH 5.5. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 8,000 and 16,200 cal mol-1 for temperatures above and below 30 degrees C, respectively. Malolactic enzyme catalyzes the NAD+ and manganese-dependent reaction; L-malate----L-lactate + CO2. The stoichiometry of the reaction was confirmed. The malolactic transformation occurs by a compulsory-order mechanism. NAD+ bound first to the protein, independently of malate concentration. Mn2+ acts as an allosteric activator. Malate bound to the complex enzyme-NAD-Mn2+. Oxamate, fructose 1,6-diphosphate and malonate acted as non-competitive inhibitors, whereas citrate and L-tartrate produced a competitive inhibition. This enzyme can be distinguished from the malic enzyme of pigeon liver (E.C.1.1.1.40) and from the true malic enzymes (E.C.1.1.1.38 and E.C.1.1.1.39).

Polysaccharide production by Pediococcus pentosaceus from wine.

Two polymer-forming strains of Pediococcus pentosaceus from Argentinian wines were grown under different conditions. Polysaccharides were produced independently of carbon source at the early logarithmic phase of growth. With the presence of ethanol and SO2, the specific polymers production was greater. The polysaccharides comprised of glucose, fructose and galactose, with predominant linkages of alpha-1,4- and alpha-1,6-glucosidic with a ratio 1:1. They contained approximately 5500 hexose molecules.

Short-chain fatty acids modulate growth of lactobacilli in mixed culture fermentations with propionibacteria.

Three strains of Lactobacillus (L. helveticus ATCC 15009 and CRL 581, and L casei LC 3) were paired with three strains of Propionibacterium (P. freudenreichii AP8, P. freudenreichii GP6 and P. acidipropionici CRL 756) and grown in individual and mixed cultures in a complex medium. Bacterial growth, carbohydrate consumption, and production of acids was determined and compared after mono and binary culture. Propionibacterium strains were inhibited in mixed cultures that rapidly reached low pH values, and stimulated in those with slow pH reduction. In several pairs Lactobacillus strains were stimulated by mixed cultivation, while in others they were unaffected or inhibited. Lactic acid reduction by Propionibacterium consumption was not always enough to produce a stimulatory effect on growth of lactobacilli. The behaviour of Lactobacillus strains in individual cultures in media with propionic acid was similar to that of mixed cultures with Propionibacterium. As propionic acid concentration increased in the medium and glucose was consumed, both individual and mixed cultures of lactobacilli showed inhibition of the growth and increase of the fermentation activity.

Pediocin N5p from Pediococcus pentosaceus: adsorption on bacterial strains.

Pediocin N5p is a bacteriocin produced by Pediococcus pentosaceus isolated from wine. It is absorbed on both sensitive and resistant Gram-positive and Gram-negative bacteria in non specific (non lethal) sites and higher values, up to 30% are observed in sensitive strains suggesting the presence of particular (lethal) receptors. Cell death without lysis is produced by the action of pediocin on sensitive strains. The cations Mg2+ and Mn2+ increase the pediocin binding by 80 and 100%, respectively. Treatment of sensitive cells with proteolytic enzymes and 1% SDS increases the subsequent binding of bacteriocin by 100 and 25%, respectively. As a lipid moiety of pediocin N5p is critical to its activity, probably a hydrophobic interaction with the peptidic receptor, stabilized by Mn2+ or Mg2+ might be established. Pediocin N5p adsorption is not affected by ethanol and SO2, two factors involved in vinification.

Effect of cultivation conditions on proteinase production by Lactobacillus murinus.

Proteinase production by Lactobacillus murinus was influenced by temperature, glucose concentration, initial pH and nitrogen sources. Maximum proteinase production occurred at 45 degrees C, pH 6.6 and with 0.5% (W/V) glucose. Tryptone, peptone and gelatin inhibited it.

beta-D-galactosidase of Lactobacillus species.

The activity of beta-D-galactosidase was studied in 13 strains of lactobacilli (groups Streptobacterium, Thermobacterium and Betabacterium). Using 2-nitrophenyl galactopyranoside as substrate, the enzyme activity varied with the strain. The values found in the Thermobacterium group were superior to those in the Streptobacterium group. The optimum pH for the species belonging to the Thermobacterium group was uniform, in contrast to the pH for those from the Streptobacterium which varied according to the species. The optimum temperature was quite uniform within each group and higher in the Streptobacterium. Lactose acted as a competitive inhibitor. MgCl2 protected the enzyme from thermal denaturation. The calcium ions inhibited the activity in all cases. The behaviour of the protectors of the SH groups varied according to the strain. 6-Phospho-beta-D-galactosidase activity was also determined, levels lower than beta-D-galactosidase were found, except in Lactobacillus plantarum ATCC 8014 and 14917.

Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems.

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production.

Characterization of bacteriocin produced by Pediococcus pentosaceus from wine.

Twenty strains of Pediococcus pentosaceus isolated from wine were examined for production of bacteriocins. Only two of them showed inhibitory activity, Ped. pentosaceus N4p against the indicator strains of the same species and N5p against 19 strains of the three genera of lactic acid bacteria from wine. The antimicrobial substance from N5p strains was removed by membrane (0.2 micron) filtration, destroyed by organic solvents and proteolytic enzymes. It was stable for 60 min at 100 degrees C. The bacteriocin was produced early in the growth cycle and its production was maximum after 48 h of culture in tomato juice medium at an initial pH of 6.5. The bactericidal effect was observed.

D-(+)-lactate dehydrogenase from Lactobacillus murinus.

D-(+)-Lactate dehydrogenase from Lactobacillus murinus was purified 670-fold. The Mr was 140,000 as determined by gel filtration. Maximum enzymatic activity was observed at 25 degrees C and pH 6.0 in 200 mM Na2KPO4 buffer. When the temperature was increased from 60 to 65 degrees C, the enzyme was completely inactive in 5 min. The apparent Km for pyruvate and NADH were 4.7 x 10(-4) and 1 x 10(-5) M, respectively. Pyruvate analogs such as oxalate, oxamate, 2-oxobutyrate, and malonate acted as a competitive inhibitors. L-Lactate and L-malate were noncompetitive inhibitors.

Production and stability of pediocin N5p in grape juice medium.

The production and stability of pediocin N5p from Pediococcus pentosaceus, isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml-1) were observed at a initial pH of 7.5 and 30 degrees C. The activity of the inhibitory substance was stable between pH values from 2.0 to 5.0 at 4 degrees and 30 degrees C. At pH 10.0 it was completely inactivated. When submitted to 30 min at 80 degrees, 100 degrees and 115 degrees C, maximal stability was observed at pH 2.0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40-80 mg 1-1 SO2, independently or combined with different ethanol concentrations, affect inhibitory activity.

Sugar and malic acid utilization and acetic acid formation byLeuconostoc oenos.

Leuconostoc oenos ST8, isolated from an Argentinian red wine, utilized glucose, fructose and malic acid and produced acetic acid. Fructose was preferred to glucose as a source of carbohydrate. Malic acid was almost completely degraded in 3 days at 25°C. Acetic acid formation correlated with fructose utilization.

Methanol utilization by a strain of Aspergillus niger: influence on the synthesis and activity of pectinases.

During the production of pectinases by a strain of Aspergillus niger isolated from rotten lemons, methanol was liberated into the medium due to the cleavage of the pectin molecule used as the carbon source. The methanol was subsequently consumed by the microorganism but neither the synthesis nor the activity of pectinesterase and polygalacturonase was affected. Although not studied in detail, the mechanism involved in the utilization of methanol is similar to that described for methylotrophic yeasts.

Effect of actinomycin D and chloramphenicol on the induction of the malate decarboxylase system in Lactobacillus thermobacterium 5 (CNRZ) 313.

This paper presents data on the induction of malate decarboxylase in Lactobacillus thermobacterium and the effect of chloramphenicol and actinomycin D on the induction.

Purification and properties of malolactic enzyme from Lactobacillus murinus CNRZ 313.

The malolactic enzyme of Lactobacillus murinus was purified 79 fold. Mr = 220,000 as determined by gel filtration and gradient gel electrophoresis. The enzyme consists of two apparently identical subunits (Mr = 110,000) that were observed after treatment with sodium dodecyl sulfate. NAD protected the enzyme against inactivation and its addition, after dissociation, restored the malolactic activity. The apparent Km's for malate, NAD, and Mn2+ were 2.31 X 10(-2), 4.5 X 10(-4), and 1.4 X 10(-4) mM, respectively. Maximum enzymatic activity was observed at 37 degrees C and pH 5.5 in 0.2 M phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 8000 and 16,200 cal mol-1 for the temperature values more than and less than 30 degrees C, respectively. Malolactic enzyme catalyzes the NAD and manganese-dependent reaction L-malate----L-lactate + CO2. Therefore, this enzyme can be distinguished from the well-known malic enzymes [L-malate: NAD+ oxidoreductase, oxaloacetate decarboxylating (EC 1.1.1.38) or decarboxylating (EC 1.1.1.39)].

Competitive Inhibition of Propionibacterium acidipropionici by Mixed Culturing with Lactobacillus helveticus.

Lactobacillus helveticus and Propionibacterium acidipropionici were grown in pure and mixed cultures in a complex medium to assess the associative interaction. The specific growth rates, substrate consumption coefficient, substrate utilization and product formation rates were determined in each case. Propionibacterium acidipropionici utilized glucose preferably when it grew in a medium containing a mixture of glucose and lactate. Its growth rate was higher on glucose than on lactate in pure culture. However, lactic acid was the substrate utilized by propionibacteria in the associative growth. The fast pH reduction produced by the growth of lactobacilli and the slow lactate utilization by propionibacteria in mixed culture determined the inhibition of propionic acid bacteria in associative growth.

Isolation and properties of beta-galactosidase of a strain of Lactobacillus helveticus isolated from natural whey starter.

beta-Galactosidase has been isolated from Lactobacillus helveticus of a strain isolated from natural starters for the manufacture of Argentine hard cheeses and its properties have been studied. The enzyme was purified 14-fold (by chromatography on DEAE-cellulose and Sepharose 6B-DEAE-cellulose columns and by affinity chromatography in agarose-p-aminophenyl-beta-D-thiogalactoside). The purified extract exhibited a single band following polyacrylamide gel electrophoresis. Maximum enzymatic activity was observed at 42 degrees C and pH 6.5 in 50 mM phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The Km's for o-nitrophenylgalactoside (ONPG) and ONPG + 10 mM of lactose were 4.46 X 10(-5) and 8.9 X 10(-5) M, respectively. Glucose, galactose, galactose 6-phosphate, and lactate acted as noncompetitive inhibitors; MgCl2 protected the enzyme from thermal denaturation. The activation energy of enzymatic hydrolysis of ONPG was 11,400 cal/mol. The Mr was estimated to be 250,000. It is an oligomeric enzyme made of 4 subunits of Mr 65,000.