Thailand has vast areas of tropical forests with many indigenous plants, but limited information is available on their phytochemical profile and in vitro inhibitions of enzymatic and nonenzymatic reactions. This study investigated phenolic profiles using liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS), antioxidant activities, and in vitro inhibitory activities of 10 indigenous plants on key enzymes related to obesity (lipase), diabetes (α-amylase and α-glucosidase), and Alzheimer’s disease (cholinesterases and β-secretase). The nonenzymatic anti-glycation reaction was also investigated. The 10 indigenous plants were Albizia lebbeck (L.) Benth, Alpinia malaccensis (Burm.) Roscoe, Careya arborea Roxb., Diplazium esculentum (Retz.) Swartz, Kaempferia roscoeana Wall., Millettia brandisiana Kurz., Momordica charantia, Phyllanthus emblica L., Zingiber cassumunar Roxb, and Zingiber citriodorum J. Mood & T. Theleide. Preparations were made by either freeze-drying or oven-drying processes. Results suggested that the drying processes had a minor impact on in vitro inhibitions of enzymatic and nonenzymatic reactions (<4-fold difference). P. emblica was the most potent antioxidant provider with high anti-glycation activity (>80% inhibition using the extract concentration of ≤6 mg/mL), while D. esculentum effectively inhibited β-secretase activity (>80% inhibition using the extract concentration of 10 mg/mL). C. arborea exhibited the highest inhibitory activities against lipase (47–51% inhibition using the extract concentration of 1 mg/mL) and cholinesterases (>60% inhibition using the extract concentration of 2 mg/mL), while Mi. brandisiana dominantly provided α-amylase and α-glucosidase inhibitors (>80% inhibition using the extract concentration of ≤2 mg/mL). Information obtained from this research may support usage of the oven-drying method due to its lower cost and easier preparation step for these studied plant species and plant parts. Furthermore, the information on in vitro inhibitions of enzymatic and nonenzymatic reactions could be used as fundamental knowledge for further investigations into other biological activities such as cell culture or in vivo experiments of these health-beneficial plants.
β-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant β-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The Km and vmax values for lactose and o-nitrophenyl-β-d-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and oNPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55 °C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of β-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.
In the present study, we isolated and screened thirty strains of GABA (γ-aminobutyric acid)-producing lactic acid bacteria (LAB) from traditional Indonesian fermented foods. Two strains were able to convert monosodium glutamate (MSG) to GABA after 24 h of cultivation at 37 °C based on thin layer chromatography (TLC) screening. Proteomic identification and 16S rDNA sequencing using MALDI-TOF MS identified the strain as Lactobacillus plantarum designated as L. plantarum FNCC 260 and FNCC 343. The highest yield of GABA production obtained from the fermentation of L. plantarum FNCC 260 was 809.2 mg/L of culture medium after 60 h of cultivation. The supplementation of 0.6 mM pyridoxal 5’-phosphate (PLP) and 0.1 mM pyridoxine led to the increase in GABA production to 945.3 mg/L and 969.5 mg/L, respectively. The highest GABA production of 1226.5 mg/L of the culture medium was obtained with 100 mM initial concentration of MSG added in the cultivation medium. The open reading frame (ORF) of 1410 bp of the gadB gene from L. plantarum FNCC 260 encodes 469 amino acids with a calculated molecular mass of 53.57 kDa. The production of GABA via enzymatic conversion of monosodium glutamate (MSG) using purified recombinant glutamate decarboxylase (GAD) from L. plantarum FNCC 260 expressed in Escherichia coli was found to be more efficient (5-fold higher within 6 h) than the production obtained from fermentation. L. plantarum FNCC 260 could be of interest for the synthesis of GABA.
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