RESUMO
The fruit-origin strain Fructobacillus tropaeoli CRL 2034 can biotransform selenium into seleno-nanoparticles and selenocysteine. The proteomic analysis of F. tropaeoli CRL 2034 exposed to 5 and 100 ppm of Se showed a dose-dependent response since 19 and 77 proteins were deregulated, respectively. In the presence of 5 ppm of Se, the deregulated proteins mainly belonged to the categories of energy production and conversion or had unknown functions, while when cells were grown with 100 ppm of Se, most of the proteins were grouped into amino acid transport and metabolism, nucleotide transport and metabolism, or into unknown functions. However, under both Se conditions, glutathione reductases were overexpressed (1.8-3.1-fold), while mannitol 2-dehydrogenase was downregulated (0.54-0.19-fold), both enzymes related to oxidative stress functions. Mannitol 2-dehydrogenase was the only enzyme found that contained SeCys, and its activity was 1.27-fold increased after 5 ppm of Se exposure. Our results suggest that F. tropaeoli CRL 2034 counteracts Se stress by overexpressing proteins related to oxidative stress resistance and changing the membrane hydrophobicity, which may improve its survival under (food) storage and positively influence its adhesion to intestinal cells. Selenized cells of F. tropaeoli CRL 2034 could be used for producing Se-enriched fermented foods. KEY POINTS: ⢠Selenized cells of F. tropaeoli showed enhanced resistance to oxidative stress. ⢠SeCys was found in the Fructobacillus mannitol 2-dehydrogenase polypeptide chain. ⢠F. tropaeoli mannitol 2-dehydrogenase activity was highest when exposed to selenium.
Assuntos
Selênio , Selênio/química , Frutas/metabolismo , Manitol Desidrogenases/metabolismo , Proteômica , Estresse OxidativoRESUMO
The enzymes D- and L-lactate dehydrogenase are involved in the reduction of pyruvate to D(+)- and L(-)-lactate, respectively. The fig-origin strain Fructobacillus tropaeoli CRL 2034 produces D- and L-lactic acids in a 9:1 ratio. In this work, two D-ldh (ldh1 and ldh2) and one L-ldh (ldh3) genes were found in the CRL 2034 genome. ldh1 and ldh2 are homologous (79% identity) and organized as contiguous operons, each gene containing 996 base pair (bp) and encoding for a 331-amino acid (aa) protein (74% identity). In contrast, ldh3 is a 927-bp gene coding for a 308-aa protein. The identity between ldh1/ldh2 and ldh3 was lower than 48%. To elucidate the role of these genes in the synthesis of lactic acid by the Fructobacillus strain, plasmid insertion mutants in each gene were generated and characterized. The growth kinetic parameters were affected only in CRL2034 ldh1::pRV300 cells, this mutant showing the lowest total lactic acid production (4.50 ± 0.15 versus 6.36 ± 0.67 g/L of wild-type strain), with a D/L ratio of 7.1:2.9. These results showed that the ldh1 gene is primarily responsible for lactic acid production by the studied strain. A comparative analysis among strains of the five Fructobacillus species revealed that the identity of D-LDH proteins was higher than 70%, while the identity of L-LDH was over 60%. Finally, phylogenetic analysis of D- and L-LDHs revealed that only D-LDH phylogeny was consistent to the phylogenetic evolution among Fructobacillus and evolutionarily related genera. Key Points â¢F. tropaeoli CRL 2034 harbors three ldh genes in its genome. â¢ldh1 and ldh2 encode D-lactate dehydrogenase; ldh3 encodes L-lactate dehydrogenase. â¢Gene ldh1 plays the major role in lactic acid production by strain CRL 2034. â¢Fructobacillus D-LDH phylogeny was consistent to phylogenetic evolution.
Assuntos
L-Lactato Desidrogenase , Ácido Láctico , Isoenzimas , L-Lactato Desidrogenase/genética , Leuconostocaceae , FilogeniaRESUMO
Lactic acid bacteria (LAB) are capable of converting carbohydrate substrates into organic acids (mainly lactic acid) and producing a wide range of metabolites. Due to their interesting beneficial properties, LAB are widely used as starter cultures, as probiotics, and as microbial cell factories. Exploring LAB present in unknown niches may lead to the isolation of unique species or strains with relevant technological properties. Autochthonous rather than allochthonous starter cultures are preferred in the current industry of fermented food products, due to better adaptation and performance of autochthonous strains to the matrix they originate from. In this work, the lactic microbiota of eight different wild tropical types of fruits and four types of flowers were studied. The ability of the isolated strains to produce metabolites of interest to the food industry was evaluated. The presence of 21 species belonging to the genera Enterococcus, Fructobacillus, Lactobacillus, Lactococcus, Leuconostoc, and Weissella was evidenced by using culture-dependent techniques. The isolated LAB corresponded to 95 genotypically differentiated strains by applying rep-PCR and sequencing of the 16S rRNA gene; subsequently, representative strains of the different isolated species were studied for technological properties, such as fast growth rate and acidifying capacity; pectinolytic and cinnamoyl esterase activities, and absence of biogenic amine biosynthesis. Additionally, the strains' capacity to produce ethyl esters as well as mannitol was evaluated. The isolated fruit- and flower-origin LAB displayed functional properties that validate their potential use in the manufacture of fermented fruit-based products setting the background for the design of novel functional foods.
RESUMO
Mannitol is a natural low-calorie sugar alcohol produced by certain (micro)organisms applicable in foods for diabetics due to its zero glycemic index. In this work, we evaluated mannitol production and yield by the fruit origin strain Fructobacillus tropaeoli CRL 2034 using response surface methodology with central composite design (CCD) as optimization strategy. The effect of the total saccharide (glucose + fructose, 1:2) content (TSC) in the medium (75, 100, 150, 200, and 225 g/l) and stirring (S; 50, 100, 200, 300 and 350 rpm) on mannitol production and yield by this strain was evaluated by using a 22 full-factorial CCD with 4 axial points (α = 1.5) and four replications of the center point, leading to 12 random experimental runs. Fermentations were carried out at 30 °C and pH 5.0 for 24 h. Minitab-15 software was used for experimental design and data analyses. The multiple response prediction analysis established 165 g/l of TSC and 200 rpm of S as optimal culture conditions to reach 85.03 g/l [95% CI (78.68, 91.39)] of mannitol and a yield of 82.02% [95% CI (71.98, 92.06)]. Finally, a validation experiment was conducted at the predicted optimum levels. The results obtained were 81.91 g/l of mannitol with a yield of 77.47% in outstanding agreement with the expected values. The mannitol 2-dehydrogenase enzyme activity was determined with 4.6-4.9 U/mg as the highest value found. To conclude, F. tropaeoli CRL 2034 produced high amounts of high-quality mannitol from fructose, being an excellent candidate for this polyol production.