Production of plant-based fermented beverages possessing functional ingredients antioxidant, γ-aminobutyric acid and antimicrobials using a probiotic Lactiplantibacillus plantarum strain L42g as an efficient starter culture.

Plant-based probiotic beverages have gained increasing interest due to demand from health-conscious consumers. In this study, we aimed to isolate and screen lactic acid bacteria possessing functional properties for use as a starter culture of fermented almond and coix beverages. Lactiplantibacillus plantarum L42g isolated from fermented beef was selected. Both intact cells and cell free supernatant of this strain exhibited high antioxidant activity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging at 38.2% and 44.9%, respectively. L. plantarum L42g grown in MRS broth supplemented with 1% (w v-1) monosodium glutamate (MSG) produced a large amount of γ-aminobutyric acid (GABA) at 496.7 µg mL-1. Moreover, strain L42g displayed remarkable antibacterial activity against several potential foodborne bacterial pathogens, including Bacillus cereus, Listeria monocytogenes, Listeria inocua, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, Shigella sp., Vibrio cholerae and Vibrio parahaemolyticus. Strain L42g also possessed additional probiotic properties including abilities to tolerate gastrointestinal conditions, adhere to gut mucosa, co-aggregate with pathogens, be susceptible to antibiotics, and produce protease. Probiotic strain L42g was subsequently employed in fermenting almond and coix juices containing MSG (1%) supplementation. Levels of antioxidant, GABA and antibacterial formation along with cell growth were clearly higher in fermented almond juice than in fermented coix juice. Nonetheless, both fermented almond and coix juices meet the standards required for the consumption of fermented beverages. Therefore, L. plantarum strain L42g represents a promising starter culture for producing functional plant-based probiotic beverages.

Recombinant Expression, Purification and Characterization of Pyridoxal 5'-phosphate Synthase from Geobacillus sp. H6a, Thermophilic Bacterium Producing Extracellular Vitamin B6.

Background: Pyridoxal 5' -phosphate synthase (PLPS) is present in deoxyxylose 5'-phosphate-independent of the de novo vitamin B6 biosynthesis pathway. This enzyme complex consists of PdxS and PdxT, which function as synthase and glutamine amidotranferase respectively to produce PLP. Objectives: This study aimed to clone, express, and purify PLPS of Geobacillus sp. H6a, followed by its characterization. Material and Methods: The PdxS and PdxT genes were amplified from Geobacillus (Gh) sp. H6a. Recombinant vectors pET28a-GhpdxS and pET28a-GhpdxT were constructed and the resulting His-tagged proteins were expressed in E. coli BL21(DE3). The soluble rGhpdxS and rGhpdxT were purified via nickel-affinity chromatography and cation-exchange chromatography. The mixture of rGhpdxS and rGhpdxT was further characterized. Results: The molecular weights of rGhpdxS and rGhpdxT were estimated to be 35 and 23 kDa by SDS-PAGE, respectively. The native form of rGhpdxS showed hexamer and dodecamer, whereas those of rGhpdxT were a monomer upon detection with non-denaturing gel electrophoresis and gel filtration. A molar ratio of 1:1 of rGhpdxS:rGhpdxT showed the highest PLP synthesis activity (4.16 U.mg-1) and was used for analyzing the biochemical properties. The kinetic values were obtained by using glyceraldehyde 3-phosphate, ribose 5-phosphate, and glutamine as the substrates. The rGhPLPS showed pentose phosphate isomerization without triose phosphate isomerase activity. The metal ions affected PLP synthesis activity. The optimum pH and optimum temperature of rGhPLPS were 9 and 70 °C, respectively. The rGhPLPS was active over a broad range of temperatures and pH values. Conclusions: These results support the potential of rGhPLPS as a candidate for industrial application.

Molecular cloning, expression, and properties of an unusual aldo-keto reductase family enzyme, pyridoxal 4-dehydrogenase, that catalyzes irreversible oxidation of pyridoxal.

Microbacterium luteolum YK-1 has pyridoxine degradation pathway I. We have cloned the structural gene for the second step enzyme, pyridoxal 4-dehydrogenase. The gene consists of 1,026-bp nucleotides and encodes 342 amino acids. The enzyme was overexpressed under cold shock conditions with a coexpression system and chaperonin GroEL/ES. The recombinant enzyme showed the same properties as the M. luteolum enzyme. The primary sequence of the enzyme was 54% identical with that of d-threo-aldose 1-dehydrogenase from Agrobacterium tumefaciens, a probable aldo-keto reductase (AKR). Upon multiple alignment with enzymes belonging to the 14 AKR families so far reported, pyridoxal 4-dehydrogenase was found to form a new AKR superfamily (AKR15) together with A. tumefaciens d-threo-aldose 1-dehydrogenase and Pseudomonas sp. l-fucose dehydrogenase. These enzymes belong to a distinct branch from the two main ones found in the phylogenic tree of AKR proteins. The enzymes on the new branch are characterized by their inability to reduce the corresponding lactones, which are produced from pyridoxal or sugars. Furthermore, pyridoxal 4-dehydrogenase prefers NAD(+) to NADP(+) as a cofactor, although AKRs generally show higher affinities for the latter.

An enzymatic fluorometric assay for pyridoxal with high specificity and sensitivity.

An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5'-phosphate by column chromatography.

Purification and characterization of pyridoxal 4-dehydrogenase from Aureobacterium luteolum.

A pyridoxal dehydrogenase was purified to homogeneity from Aureobacterium luteolum, which can use pyridoxine as a carbon and nitrogen source, and characterized. The enzyme was a dimeric protein with a subunit molecular weight of 38,000. It had several properties distinct from those of the partially purified enzyme from Pseudomonas MA-1. The optimum pH (8.0-8.5) was 0.8-1.3 lower than that of the Pseudomonas enzyme. The Aureobacterium enzyme showed much higher and lower affinities for NAD+ (Km, 0.140 +/- 0.008 mM) and pyridoxal (0.473 +/- 0.109 mM), respectively, than those of the Pseudomonas enzyme. The Aureobacterium enzyme could use NADP+ as a substrate: the reactivity was 6.5% of NAD+. The enzyme was much more tolerant to metal-chelating agents. Irreversibility of the enzymatic reaction was shared by the two enzymes. No aldehyde dehydrogenase showed similarity to the amino-terminal amino acid sequence of the enzyme.