Maite Dueñas

Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking.

Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 degrees C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents.

Exopolysaccharide production by Pediococcus damnosus 2.6 in a semidefined medium under different growth conditions

Ropy Pediococcus damnosus (strain 2.6) was used for production of exopolysaccharide (EPS) in a semidefined medium. From a kinetic point of view, an experiment conducted in SMD medium containing 30 g l(-1) glucose and 5 g l(-1) Bacto casamino acids (Difco Laboratories, Detroit, MI), without pH control, showed that EPS production took place mainly during the growth phase. The viscosity of the cultures developed in parallel to the EPS synthesis until 94 h of incubation; after 200 h of fermentation, viscosity decreased. The effect of glucose, Bacto casamino acid concentrations and temperature on growth and EPS production was determined by using a full factorial design. Within the domain of experimental conditions considered, the concentration of glucose and Bacto casamino acids has a significant effect on the production of exopolysaccharide. The incubation at 12 degrees C produced a prolonged lag phase and due to the lower growth yield, higher specific EPS production was found at this temperature. At 25 degrees C the EPS production was mainly enhanced by the increase in glucose concentration. The increase in nitrogen concentration from 5 to 15 g l(-1) did not yield greater EPS production. However, at 12 degrees C optimal EPS production was obtained when both higher glucose and nitrogen concentrations were used.

Characterization and DNA plasmid analysis of ropy Pediococcus spp. strains isolated from Basque Country ciders

Ten strains of Pediococcus spp. causing ropiness were isolated from Basque Country spoiled ciders and characterized with regards to growth at different pHs, temperatures, and ethanol and SO2 concentrations. They grew well at pHs above 3.7 and failed to grow at temperatures over 37°C. In addition, they were tolerant to 10% ethanol and to 15, 25, and 50 mg/l total SO2 (pH 3.8). A selected ropy strain was used to carry out plasmid DNA analysis; the agarose gel pattern showed the presence of 6 bands of plasmid DNA. Plasmid-curing experiments with ethidium bromide and novobiocin suggested that the ropy phenotype was encoded by plasmid DNA. In addition, this nonropy strain lost its resistance to oleandomycin after curing trials.

Supramolecular Structure and Conformation of a (1→3)(1→2)-β-D-Glucan from Lactobacillus suebicus CUPV221 as Observed by Tapping Mode Atomic Force Microscopy

Tapping mode atomic force microscopy (TM-AFM) has been used to analyze the supramolecular structure and conformation of the (1-->3)(1-->2)-beta-D-glucan produced by Lactobacillus suebicus CUPV221 isolated from cider. Solutions for TM-AFM observation were prepared by dispersing the solid glucan in distilled water and in alkaline aqueous solutions. It was found that from the distilled water at 10 mg/L or higher concentrations, the (1-->3)(1-->2)-beta-D-glucan forms networks. The heat resistance of the networks depends on the concentration. From the alkaline aqueous solutions, different supramolecular structures were observed depending on the pH. From the weakest alkaline solution, a fairly rough morphology with a high density of spikelike growth features was revealed. As the ionic force of the medium increased, the sizes of the spikelike growth features diminished, and even many disaggregated fibers could be found. At 0.4 M NaOH (pH 13.16), the aggregates had disappeared almost totally. NaOH aqueous solutions (0.1 and 0.4 M) were used to carry out the study of conformation. At 0.1 M NaOH, the aggregates were partially detached, and many free microfibers were found to which a helical conformation could be assigned due to their stiffness and rodlike character. At 0.4 M NaOH, the beginning of the dissociation of the helical structures was seen.

Bicarbonate gradients modulate growth and colony morphology in Aspergillus nidulans

Fungal colonies expand through apically extending peripheral hyphae. These long and relatively unbranched leaders leave behind cells that show a higher degree of branching and lower growth rates. Towards the centre of the colony, quiescent, but viable cells sustain processes such as transport of water, solutes and organelles, and support the concerted genesis of specialized reproductive structures. A survey of chemical signals controlling changes in hyphal growth and branching of Aspergillus nidulans in solid minimal medium revealed that progressive accumulation of bicarbonate from the periphery to the centre of the colony effected a dual modulation of both parameters. At areas immediately behind the periphery, bicarbonate induced progressive branching without a significant change in the specific growth rate. At distal and central regions, increased branching was accompanied by progressive inhibition of the specific growth rate. The results provide new evidence that chemical cues participate in the control of colony morphology and function, which are likely to vary with changing environmental conditions.

Note. Histamine production by some lactic acid bacteria isolated from ciders / Nota. Producción de histamina por algunas bacterias lácticas aisladas a partir de sidras

Histidine decarboxylase activity has been investigated in 23 strains of lactic acid bacteria, belonging to Oenococcus oeni, Lactobacillus brevis and Lactobacillus spp., isolated during the cidermaking process at different stages of fermentation. After 14 days at 25 °C on a semisynthetic medium supplemented with L-histidine, 13 strains were histamine producers and six of these strains were selected to study the kinetics of growth and histamine production at different temperatures. The results showed that histamine accumulation was maximal at 25 °C after 10-15 days of growth. Comparison of growth and histamine level curves demonstrated a delay time of eight days between maximal growth and the highest histamine content of the cultures.