Sergio E. Pasteris

Glycerol Metabolism of Lactobacillus rhamnosus ATCC 7469: Cloning and Expression of Two Glycerol Kinase Genes

Lactobacillus rhamnosus ATCC 7469 was able to grow in glycerol as the sole source of energy in aerobic conditions, producing lactate, acetate, and diacetyl. A biphasic growth was observed in the presence of glucose. In this condition, glycerol consumption began after glucose was exhausted from the culture medium. Glycerol kinase activity was detected in L. rhamnosus ATCC 7469, a characteristic of microorganisms which catabolize glycerol in aerobic conditions. Genetic analysis revealed that this strain possesses two glycerol kinase genes: gykA and glpK, that encode for two different glycerol kinases GykA and GlpK, respectively. The glpK geneis associated in an operon with α-glycerophosphate oxidase (glpO) and glycerol facilitator (glpF) genes. Transcriptional analysis revealed that only glpK is expressed when L. rhamnosus was grown on glycerol.

Sugar―Glycerol Cofermentations by Lactobacillus hilgardii Isolated from Wine

Glycerol catabolism was studied in Lactobacillus hilgardii X(1)B from wine, growing on glycerol and limiting glucose or fructose concentrations in anaerobiosis and microaerophilia. Glycerol consumption occurred simultaneously with sugar use, and it was higher with fructose as a cofermenting sugar in microaerophilia. Enzymatic activities of the glycerol kinase and glycerol dehydratase pathways were detected in both incubation conditions. In anaerobiosis, the main products were lactate, acetate, ethanol, and the intermediary product of the glycerol dehydratase pathway, 3-hydroxypropionaldehyde. However, in microaerophilia, 1,3-propanediol was also detected. In anaerobic glucose + glycerol and fructose + glycerol cultures as in microaerophilic glucose + glycerol cultures, glycerol was degraded mainly through the reductive pathway. However, when L. hilgardii X(1)B was grown on fructose + glycerol cultures in microaerophilia, glycerol dissimilation occurred mainly via the glycerol kinase way. According to these results, L. hilgardii X(1)B can degrade glycerol by producing 3-hydroxypropionaldehyde and acetic acid, both undesirable products for wine sensorial quality.

In vitro inhibition of Citrobacter freundii, a red-leg syndrome associated pathogen in raniculture, by indigenous Lactococcus lactis CRL 1584

Red-leg syndrome (RLS) is one of the main infectious diseases that cause economic losses in Lithobates catesbeianus hatcheries, Citrobacter freundii being an etiological agent. Treatment or prevention with therapeutics or chemicals results in modifications of the indigenous microbiota, development of antibiotic resistance, presence of their residues in food and enhancement of production costs. Thus, probiotics could be used as an alternative therapy. Lactic acid bacteria are part of the indigenous microbiota of healthy frogs and can prevent pathogen colonization by different mechanisms, including the production of antagonistic substances. In this work, the evaluation and characterization of the inhibition of C. freundii CFb by Lactococcus lactis subsp. lactis CRL 1584, a potentially probiotic candidate, were carried out. This strain produced lactic acid, H(2)O(2) and bacteriocin in static and shaken conditions and inhibited pathogen growth in associative cultures, with an earlier inhibition under agitated conditions. The elimination of each of the antimicrobial metabolites partially abolished the inhibition of the pathogen, suggesting that the inhibitory effect could be attributed to a combined action of the three antagonistic molecules. Electron microphotographs revealed the damage caused by L. lactis CRL 1584 supernatants to C. freundii cells. The addition of pure lactic acid, H(2)O(2) and bacteriocin to the culture media showed that each metabolite caused different morphological modifications in C. freundii, in agreement with the effect on viable cell counts. The results support the possibility that L. lactis CRL 1584 might be considered as a probiotic to be used in the prevention of RLS in raniculture.

Cultivable microbiota of Lithobates catesbeianus and advances in the selection of lactic acid bacteria as biological control agents in raniculture

The cultivable microbiota of skin and cloaca of captive Lithobates catesbeianus includes microorganisms generally accepted as beneficial and potentially pathogenic bacteria. In order to select a group of potentially probiotic bacteria, 136 isolates were evaluated for their surface properties and production of antagonistic metabolites. Then, 11 lactic acid bacteria (LAB) strains were selected and identified as Lactobacillus plantarum, Lb. brevis, Pediococcus pentosaceus, Lactococcus lactis, L. garvieae and Enterococcus gallinarum. Studies of compatibility indicate that all the strains could be included in a multi-strain probiotic, with the exception of Ent. gallinarum CRL 1826 which inhibited LAB species through a bacteriocin-like metabolite. These results contribute to the design of a probiotic product to improve the sanitary status of bullfrogs in intensive culture systems, to avoid the use of antibiotics and thus to reduce production costs. It could also be an alternative to prevent infectious diseases during the ex situ breeding of amphibian species under threat of extinction.

Glycerol catabolism by Pediococcus pentosaceus isolated from beer

Among the lactic acid bacteria isolated from beer at different stages of elaboration, Pediococcus pentosaceus was the predominant species and the only that used glycerol as sole carbon source. Its utilization was studied in CAg strain growing on glycerol or on glycerol and limited concentration of glucose. Glycerol kinase and glycerol dehydratase pathways were responsible for glycerol degradation. On glycerol alone, the enzymatic activities of both pathways were expressed simultaneously and after glycerol consumption, the main products were acetate, 2,3-butanediol (2,3-BD) and 1,3-propanediol (1,3-PD). When the carbon sources were glycerol and glucose, glycerol was firstly degraded by the reductive pathway and after glucose consumption the activities of the glycerol kinase pathway were expressed. In this condition, glycerol was transformed into lactate, acetate, 2,3-BD and 1,3-PD. According to the enzymatic activities and fermentation balances, the production of acetate, providing ATP, can be correlated to lactate degradation, and the NAD required by these transformations could be obtained by the formation of 1,3-PD and 2,3-BD. Glycerol degradation should be correlated with the sensitivity of a beer to bacterial spoilage, and P. pentosaceus CAg considered as spoilage strain because it produces high volatile acidity and aroma compounds from glycerol conferring unacceptable flavour to beer.

Characterization of the beneficial properties of lactobacilli isolated from bullfrog (Rana catesbeiana) hatchery.

The present work addresses the isolation and partial identification of the microbial population of a R. catesbeiana hatchery in spring and summer as well as some beneficial properties of Lactobacillus strains isolated in different seasons and hatchery areas. The bacterial population was grouped into the following taxa: Lactobacillus spp., Pediococcus spp., Enterococcus faecalis and Ent. faecium, and Enterobacteriaceae (Enterobacter spp., Escherichia coli) while Pseudomonas aeruginosa and Staphylococcusepidermidis were isolated from frogs displaying red-leg syndrome. The Lactobacillusplantarum and L. curvatus strains isolated showed to inhibit the growth of red-leg syndrome associated pathogens and food-borne bacteria by organic acids. While L. plantarum CRL 1606 also inhibited red-leg syndrome related pathogens by hydrogen peroxide, meat spoilage bacteria were only inhibited by acidity. However, by using a MRS medium added with tetramethyl-benzidine and peroxidase, a high percentage of H2O2-producing lactobacilli were detected. The surface properties of Lactobacillus strains showed that a few strains were able to agglutinate ABO human erythrocytes, while the highest number of strains had a low to medium degree of hydrophobicity. This paper constitute the first study related to the beneficial properties of Lactobacillus isolated from a bullfrog hatchery, as well as the selection criteria applied to a group of strains, which could help to control or prevent bacterial infectious diseases in raniculture.

Characterization of glycerol kinase and NAD‐independent glycerol‐3‐phosphate dehydrogenase from Pediococcus pentosaceus N5p

The pathway involved in glycerol dissimilation in Pediococcus pentosaceus N5p, a strain isolated from wine, includes glycerol kinase and NAD‐independent glycerol‐3P dehydrogenase. The properties of these enzymes were studied. Glycerol kinase activity was maximal at 28 °C and pH 7·5 in 50 mmol l−1 Tris‐HCl buffer. The end‐products of the reaction acted as competitive inhibitors while fructose‐1,6‐diphosphate was a non‐competitive inhibitor. Mg2+ was required for optimal enzyme activity. The Km values for both substrates were 0·11 and 0·37 mmol l−1 for glycerol and ATP, respectively. NAD‐independent glycerol‐3P dehydrogenase activity was maximal at 37 °C and pH 7·5 in 100 mmol l−1 Tris‐HCl buffer. The enzymatic activity was activated by KCN and bivalent cations as Mg2+ and Ca2+, but it was strongly inhibited by others. Dihydroxyacetone phosphate acted as competitive inhibitor while ATP and phosphenolpyruvate were non‐competitive inhibitors.

Survival and beneficial properties of lactic acid bacteria from raniculture subjected to freeze-drying and storage

To evaluate the effect of freeze‐drying and storage conditions on the viability and beneficial properties of lactic acid bacteria (LAB) for raniculture.

Freeze-Drying of Wine Yeasts and Oenococcus oeni and Selection of the Inoculation Conditions after Storage

Fil: Ale, Cesar Emmanuel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Tucuman. Instituto Superior de Investigaciones Biologicas. Universidad Nacional de Tucuman. Instituto Superior de Investigaciones Biologicas; Argentina

Glycerol production by Oenococcus oeni during sequential and simultaneous cultures with wine yeast strains.

Growth and fermentation patterns of Saccharomyces cerevisiae, Kloeckera apiculata, and Oenococcus oeni strains cultured in grape juice medium were studied. In pure, sequential and simultaneous cultures, the strains reached the stationary growth phase between 2 and 3 days. Pure and mixed K. apiculata and S. cerevisiae cultures used mainly glucose, producing ethanol, organic acids, and 4.0 and 0.1 mM glycerol, respectively. In sequential cultures, O. oeni achieved about 1 log unit at 3 days using mainly fructose and L‐malic acid. Highest sugars consumption was detected in K. apiculata supernatants, lactic acid being the major end‐product. 8.0 mM glycerol was found in 6‐day culture supernatants. In simultaneous cultures, total sugars and L‐malic acid were used at 3 days and 98% of ethanol and glycerol were detected. This study represents the first report of the population dynamics and metabolic behavior of yeasts and O. oeni in sequential and simultaneous cultures and contributes to the selection of indigenous strains to design starter cultures for winemaking, also considering the inclusion of K. apiculata. The sequential inoculation of yeasts and O. oeni would enhance glycerol production, which confers desirable organoleptic characteristics to wines, while organic acids levels would not affect their sensory profile.