RESUMEN
Naturally fermented pickles harbour many lactic acid bacteria (LAB). Forty-three LAB strains with conjugated linoleic acid (CLA)-producing ability were isolated from three naturally fermented pickle brines. Of these isolates, lp15 identified as Lactobacillus plantarum by API 50 CHL system and full-length 16S rDNA sequence analysis exhibited the highest CLA-producing ability (26.1% conversion) at 48 h in de Man Rogosa Sharpe (MRS) broth in the presence of 100 µg/ml of linoleic acid (LA). Compared to other strains, L. plantarum strain lp15 showed the highest tolerance upon increased levels of LA in the medium, i.e., up to 600 µg/ml. This strain converted about 25% of LA into CLA isomers [predominantly cis-9, trans-11 CLA (9-CLA) and trans-10, cis-12 CLA (10-CLA)], of which 75% was 9-CLA. Interestingly, though the conversion rate of LA into CLA by lp15 remained stable between 100 to 600 µg/ml LA levels in the medium, it dropped sharply at 1000 µg/ml. Taken together, the lp15 strain displayed relatively high LA tolerance with higher conversion rate, which implies that this strain is a valuable candidate for enhancing the CLA content in food-sources like pickles.
Asunto(s)
Cucumis sativus/microbiología , Lactobacillus plantarum/aislamiento & purificación , Lactobacillus plantarum/metabolismo , Ácidos Linoleicos Conjugados/biosíntesis , ADN Bacteriano/química , ADN Bacteriano/genética , Fermentación , Microbiología de Alimentos , Lactobacillus plantarum/genética , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genéticaRESUMEN
The aim of this work was to construct a novel food-grade industrial arming yeast displaying beta-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The beta-1,3-1,4-glucanase gene from Bacillus subtilis was fused to alpha-agglutinin and expressed under the control of the GAL1 promoter. alpha-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the alpha-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of beta-1,3-1,4-glucanase. The analysis of beta-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that beta-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed beta-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of beta-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.
Asunto(s)
Biotecnología/métodos , Endo-1,3(4)-beta-Glucanasa/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Membrana Celular/metabolismo , ADN/metabolismo , Galactoquinasa/biosíntesis , Vectores Genéticos , Microbiología Industrial/métodos , Modelos Genéticos , Fosfoglicerato Quinasa/biosíntesis , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Temperatura , alfa-Galactosidasa/biosíntesis , alfa-Galactosidasa/metabolismoRESUMEN
We investigated the higher structure of konjac glucomannan (KGM) in the amorphous state and solution using a laser particle size analyzer and a water activity meter. The results show that the thermodynamic structures of native KGM were primarily composed of the lamella structure units, which involve both granular crystalline and amorphous regions, and that the connection zones of such units contained both loose and tight aggregation regions. The value of surface tension (sigma) of native KGM, resting with the density of its hydroxyl groups' self-association, was an important parameter to analyze the higher structures of native KGM in the thermodynamic swelling model of native KGM.