Characterization of a lactic acid bacterium-derived ß-glucosidase for the production of rubusoside from stevioside.

Rubusoside, which is used as a natural sweetener or a solubilizing agent for water-insoluble functional materials, is currently expensive to produce owing to the high cost of the membrane-based technologies needed for its extraction and purification from the sweet tea plant (Rubus suavissimus S. Lee). Therefore, this study was carried out to screen for lactic acid bacteria that possess enzymes capable of bio-transforming stevioside into rubusoside. Subsequently, one such rubusoside-producing enzyme was isolated from Lactobacillus plantarum GS100. Located on the bacterial cell surface, this enzyme was stable at pH 4.5-6.5 and 30-40 °C, and it produced rubusoside as a major product through its stevioside-hydrolyzing activity. Importantly, the enzyme showed higher ß-glucosidase activity toward the ß-linked glucosidic bond of stevioside than toward other ß-linked glucobioses. Under optimal conditions, 70 U/L of the rubusoside-producing enzyme could produce 69.03 mM rubusoside from 190 mM stevioside. The ß-glucosidase activity on the cell surface was high at 35 h of culture. This is the first report detailing the production of rubusoside from stevioside by an enzyme derived from a food-grade lactic acid bacterium. The application of this ß-glucosidase could greatly reduce the cost of rubusoside production, hence benefiting all industries that use this natural product.

Nutritional Composition and Bioactive Compounds in Three Different Parts of Mango Fruit.

Mango (Mangifera indica L.), known as the king of fruits, has an attractive taste and fragrance and high nutritional value. Mango is commercially important in India, where ~55% of the global crop is produced. The fruit has three main parts: pulp, peel, and kernel. The pulp is the most-consumed part, while the peel and kernel are usually discarded. Mango pulp is a source of a variety of reducing sugars, amino acids, aromatic compounds, and functional compounds, such as pectin, vitamins, anthocyanins, and polyphenols. Mango processing generates peels and kernels as bio-wastes, though they also have nutraceutical significance. Functional compounds in the peel, including protocatechuic acids, mangiferin and ß-carotene are known for their antimicrobial, anti-diabetic, anti-inflammatory, and anti-carcinogenic properties. The mango kernel has higher antioxidant and polyphenolic contents than the pulp and peel and is used for oil extraction; it's possible usage in combination with corn and wheat flour in preparing nutraceuticals is being increasingly emphasized. This review aims to provide nutraceutical and pharmacological information on all three parts of mango to help understand the defense mechanisms of its functional constituents, and the appropriate use of mangoes to enhance our nutrition and health.

Poly-(γ-glutamic acid) Production and Optimization from Agro-Industrial Bioresources as Renewable Substrates by Bacillus sp. FBL-2 through Response Surface Methodology.

We optimized culture conditions using Bacillus sp. FBL-2 as a poly-(γ-glutamic acid) (PGA) producing strain isolated from cheonggukjang. All experiments were performed under aerobic conditions using a laboratory scale 2.5 L fermentor. We investigated the effects of fermentation parameters (temperature, pH, agitation, and aeration) and medium components (glutamic acid, citric acid, and yeast extract) on poly-(γ-glutamic acid) production, viscosity, and dry cell mass. A non-optimized fermentation method (1.5 vvm, 350 rpm, and 37 °C) yielded PGA, viscosity, and dry cell mass at levels of 100.7 g/L, 483.2 cP, and 3.4 g/L, respectively. L-glutamic acid, citric acid, and yeast extract supplementation enhanced poly-(γ-glutamic acid) production to 175.9 g/L. Additionally, the production of poly-(γ-glutamic acid) from rice bran and wheat bran was assessed using response surface methodology (central composite rotatable design). Agricultural byproducts (rice bran and wheat bran) and H2SO4 were selected as factors, and experiments were performed by combining various component concentrations to determine optimal component concentrations. Our experimentally-derived optimal parameters included 38.6 g/L of rice bran, 0.42% of H2SO4, 28.0 g/L of wheat bran, and 0.32% of H2SO4. Under optimum conditions, rice bran medium facilitated poly-(γ-glutamic acid) production of up to 22.64 g/L, and the use of wheat bran medium yielded up to 14.6 g/L. Based on a validity test using the optimized culture conditions, poly-(γ-glutamic acid) was produced at 47.6 g/L and 36.4 g/L from these respective mediums, and both results were higher than statistically predicted. This study suggests that rice bran can be used as a potential alternative substrate for poly-(γ-glutamic acid) production.

Enzymatic Synthesis of Glucosyl Rebaudioside A and its Characterization as a Sweetener.

Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001 in Escherichia coli BL21 (DE3), forming single O-α-D-glucosyl-(1″→6') rebaudioside A with yield of 86%. O-α-D-glucosyl-(1″→6') rebaudioside A was purified using HPLC and Diaion HP-20 and its properties were characterized for possible use as a food ingredient. Almost 98% of O-α-D-glucosyl-(1″→6') rebaudioside A was dissolved after 15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O-α-D-glucosyl-(1″→6') rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus, O-α-D-glucosyl-(1″→6') rebaudioside A could be used as a highly pure and improved sweetener with high stability in commercial drinks. PRACTICAL APPLICATION: The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation. Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks. This method can be applied to obtain high value-added bioactive compounds by enzymatic modification.

Optimized Production of Poly(γ-Glutamic acid) By Bacillus sp. FBL-2 through Response Surface Methodology Using Central Composite Design.

In the present study, the optimization of poly(γ-glutamic acid) (γ-PGA) production by Bacillus sp. FBL-2 was studied using a statistical approach. One-factor-at-a-time method was used to investigate the effect of carbon sources and nitrogen sources on γ-PGA production and was utilized to select the most significant nutrients affecting the yield of γ-PGA. After identifying effective nutrients, response surface methodology with central composite design (CCD) was used to obtain a mathematical model to identify the optimum concentrations of the key nutrients (sucrose, L-glutamic acid, yeast extract, and citric acid) for improvement of γ-PGA production. The optimum amount of significant medium components appeared to be sucrose 51.73 g/l, L-glutamic acid 105.30 g/l, yeast extract 13.25 g/l, and citric acid 10.04 g/l. The optimized medium was validated experimentally, and γ-PGA production increased significantly from 3.59 g/l (0.33 g/l/h) to 44.04 g/l (3.67 g/l/h) when strain FBL-2 was cultivated under the optimal medium developed by the statistical approach, as compared to non-optimized medium.

Gamma-Aminobutyric Acid Production from a Novel Enterococcus avium JS-N6B4 Strain Isolated from Edible Insects.

Gamma-aminobutyric acid (GABA)-producing strains were isolated from four edible insects and subjected to 16S rRNA sequence analysis. Among the four GABA-producing bacteria, Enterococcus avium JS-N6B4 exhibited the highest GABA-production, while cultivation temperature, initial pH, aerobic condition, and mono-sodium glutamate (MSG) feeding were found to be the key factors affecting GABA production rate. The culture condition was optimized in terms of glucose, yeast extract, and MSG concentrations using response surface methodology (RSM). GABA production up to 16.64 g/l was obtained under the conditions of 7 g/l glucose, 45 g/l yeast extract, and 62 g/l MSG through the optimization of medium composition by RSM. Experimental GABA production was 13.68 g/l, which was close to the predicted value (16.64 g/l) calculated from the analysis of variance, and 2.79-fold higher than the production achieved with basic medium. Therefore, GABA-producing strains may help improve the GABA production in edible insects, and provide a new approach to the use of edible insects as effective food biomaterials.

Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties.

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization-time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141mM, as determined by response surface methodology. CHG possessed a 65% increased water solubility and 2-fold browning resistance while it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation.

Synthesis and Functional Characterization of Caffeic Acid Glucoside Using Leuconostoc mesenteroides Dextransucrase.

Caffeic acid was modified via transglucosylation using sucrose and dextransucrase from Leuconostoc mesenteroides B-512FMCM. Following enzymatic modification, a caffeic acid glucoside was isolated by butanol separation, silica gel chromatography, and preparative HPLC. The synthesized caffeic acid glucoside had a molecular mass-to-charge ratio of 365 m/z, and its structure was identified as caffeic acid-3-O-α-d-glucopyranoside. The production of this caffeic acid-3-O-α-d-glucopyranoside at a concentration of 153 mM was optimized using 325 mM caffeic acid, 355 mM sucrose, and 650 mU mL-1 dextransucrase in the synthesis reaction. In comparison with the caffeic acid, the caffeic acid-3-O-α-d-glucopyranoside displayed 3-fold higher water solubility, 1.66-fold higher antilipid peroxidation effect, 15% stronger inhibition of colon cancer cell growth, and 11.5-fold higher browning resistance. These results indicate that this caffeic acid-3-O-α-d-glucopyranoside may be a suitable functional component of food and pharmaceutical products.

Cerium oxide-catalyzed multicomponent condensation approach to spirooxindoles in water.

An efficient and facile green synthesis of spirooxindole derivatives bearing pyrano[2,3-c]pyrazole moiety has been achieved via a [Formula: see text]-NPs catalyzed four-component reaction in water. The protocol offers an environmentally benign and effective approach to highly functionalized and biologically interesting spiro[indoline-3,4[Formula: see text]-pyrano[2,3-c]pyrazole] derivatives. The synthesized compounds exhibit potent antioxidant and antibacterial activities.

L-Lactic acid production by combined utilization of agricultural bioresources as renewable and economical substrates through batch and repeated-batch fermentation of Enterococcus faecalis RKY1.

Enterococcus faecalis RKY1 was used to produce l-lactic acid from hydrol, soybean curd residues (SCR), and malt. Hydrol was efficiently metabolized to l-lactic acid with optical purity of >97.5%, though hydrol contained mixed sugars such as glucose, maltose, maltotriose, and maltodextrin. Combined utilization of hydrol, SCR, and malt was enough to sustain lactic acid fermentation by E. faecalis RKY1. In order to reduce the amount of nitrogen sources and product inhibition, cell-recycle repeated-batch fermentation was employed, where a high cell mass (26.3g/L) was obtained. Lactic acid productivity was improved by removal of lactic acid from fermentation broth by membrane filtration and by linearly increased cell density. When the total of 10 repeated-batch fermentations were carried out using 100g/L hydrol, 150g/L SCR hydrolyzate, and 20g/L malt hydrolyzate as the main nutrients, lactic acid productivity was increased significantly from 3.20g/L/h to 6.37g/L/h.

Anti-tyrosinase, antioxidant, and antibacterial activities of novel 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans.

Novel 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans (7a-k) were synthesized using ceric ammonium nitrate (CAN)-catalyzed formal [3 + 2] cycloaddition. Synthesized compounds were evaluated for their tyrosinase inhibitory, antioxidant, and antibacterial activities. A modified spectrophotometric method using l-DOPA as substrate was used to determine tyrosinase inhibitory activities, and a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was used to evaluate antioxidant properties. Antibacterial activities against gram-negative Escherichia coli (KCTC-1924) and gram-positive Staphylococcus aureus (KCTC-1916) were evaluated using the disc diffusion technique. Of the synthesized compounds, 7b with a 4-acetyl and an electron-enriched dihydronaphthofuran ring showed the highest tyrosinase-inhibition activity (IC50 = 8.91 µg/mL), which was comparable with that of standard kojic acid (IC50 = 10.16 µg/mL), potent antioxidant activity (IC50 = 3.33 µg/mL), which was comparable with that of BHT (IC50 = 34.67 µg/mL), and excellent antibacterial activities (MICs: 0.50 µg/mL against E. coli and S. aureus strains). A mechanistic analysis of 7b demonstrated that its tyrosinase inhibitory activity was reversible and competitive. Compounds 7c and 7d showed potent antioxidant activities (IC50: 6.30 and 5.01 µg/mL), and compound 7d also exhibited potent inhibitory activity against E. coli with a MIC of 0.5 µg/mL. Furthermore, compounds 7a, 7e, 7f, and 7i showed potent antibacterial activities against S. aureus with MICs of 0.5 µg/mL, which was comparable to that of ampicillin (MIC = 0.5 µg/mL).

Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid.

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.

Microwave-assisted synthesis of diverse pyrrolo[3,4-c]quinoline-1,3-diones and their antibacterial activities.

With the aim of developing a general and practical method for library production, a novel and efficient two-phase microwave-assisted cascade reaction between isatins and ß-ketoamides in [Bmim]BF4/toluene was developed for the synthesis of pyrrolo[3,4-c]quinoline-1,3-diones. The features of this methodology are, the use of microwave-assisted rapid synthesis, mild reaction conditions, high yields, operational simplicity, facile product separation, and recyclability. Furthermore, the antibacterial activities of the pyrrolo[3,4-c]quinoline-1,3-dione derivatives produced were evaluated against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Enterobacter aerogenes) and Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus). These derivatives showed antibacterial activities against Gram-positive strains that were at least equivalent to that against Gram-negative strains. Compound 7{3,5} displayed the most potent antibacterial activity against P. aeruginosa (MIC = 0.5 µg/mL) and greater activity than standard ampicillin (MIC = 1 µg/mL). Compound 7{4,7} exhibited the best inhibitory activity against E. coli and E. aerogenes (MIC = 1 and 0.5 µg/mL), compared with the standard ampicillin (both MICs = 1 µg/mL). The synthesized pyrrolo[3,4-c]quinoline-1,3-diones are expected to be widely used as lead compounds for the development of new antibacterial agents.

Cell-recycle continuous fermentation of Enterococcus faecalis RKY1 for economical production of lactic acid by reduction of yeast extract supplementation.

Both lactic acid productivity and cell growth were linearly correlated with yeast extract supplementation in batch fermentation. During conventional continuous operation, although fresh feed was introduced into the bioreactor with a significantly low dilution rate (0.04 h(-1)), the amount of yeast extract employed was not enough to maintain the growth of microorganism. However, when the fresh feed contained 100 g/l glucose and 2 g/l yeast extract during cell-recycle continuous operation at a dilution rate of 0.04 h(-1), more than 90 g/l lactic acid was continuously produced, with the average productivity of 3.72 g/l·h. In this experiment, 82 g of yeast extract (77% of reduction yield) could be reduced for the production of 1 kg of lactic acid compared with batch fermentation of a similar volumetric productivity.

Synthesis of novel and diverse mollugin analogues and their antibacterial and antioxidant activities.

Novel and diverse mollugin analogues (1-12) were synthesized using PhB(OH)2/AcOH-mediated electrocyclization reaction as a key step. The newly synthesized compounds were screened for antioxidant and antibacterial activities. Compounds 1, 2, 5, 6, 8, and 10-12 showed high antioxidant activities in DPPH inhibition (IC50=0.52-1.11 µM) compared with BHT (IC50=9.67 µM). Compounds 3 exhibited potent antibacterial activity against Staphylococcus aureus (KCTC-1916) bacterial strain at 100 µg/mL. Structures of newly synthesized compounds were confirmed by IR, (1)H NMR, (13)C NMR data and high-resolution mass spectrometry.

Characterization of a novel steviol-producing ß-glucosidase from Penicillium decumbens and optimal production of the steviol.

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing ß-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3-6.0 and at 40-60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and (1)H- and (13)C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other ß-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 µl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel ß-glucosidase, which may be useful for the pharmaceutical and agricultural areas.

Effect of fermentation inhibitors in the presence and absence of activated charcoal on the growth of Saccharomyces cerevisiae.

The acidic hydrolysis of biomass generates numerous inhibitors of fermentation, which adversely affect cell growth and metabolism. The goal of the present study was to determine the effects of fermentation inhibitors on growth and glucose consumption by Saccharomyces cerevisiae. We also conducted in situ adsorption during cell cultivation in synthetic broth containing fermentation inhibitors. In order to evaluate the effect of in situ adsorption on cell growth, five inhibitors, namely 5-hydroxymethylfurfural, levulinic acid, furfural, formic acid, and acetic acid, were introduced into synthetic broth. The existence of fermentation inhibitors during cell culture adversely affects cell growth and sugar consumption. Furfural, formic acid, and acetic acid were the most potent inhibitors in our culture system. The in situ adsorption of inhibitors by the addition of activated charcoal to the synthetic broth increased cell growth and sugar consumption. Our results indicate that detoxification of fermentation media by in situ adsorption may be useful for enhancing biofuel production.

A novel lactic acid bacterium for the production of high purity L-lactic acid, Lactobacillus paracasei subsp. paracasei CHB2121.

Fermentation-derived lactic acid has several potential industrial uses as an intermediate carbon chemical and a raw material for biodegradable polymer. We therefore undertook the identification of a novel bacterial strain that is capable of producing high concentrations of lactic acid and has potential commercial applications. A novel L(+)-lactic acid producing bacterium, Lactobacillus paracasei subsp. paracasei CHB2121 was isolated from soil obtained near an ethanol production factory and identified by 16S rRNA gene sequence analysis and characterization using an API 50 CHL kit. L. paracasei subsp. paracasei CHB2121 efficiently produced 192 g/L lactic acid from medium containing 200 g/L of glucose, with 3.99 g/(L·h) productivity, and 0.96 g/g yield. In addition, the optical purity of the produced lactic acid was estimated to be 96.6% L(+)-lactic acid. The newly identified L. paracasei subsp. paracasei CHB2121 efficiently produces high concentrations of lactic acid, and may be suitable for use in the industrial production of lactic acid.

Mass production of rubusoside using a novel stevioside-specific ß-glucosidase from Aspergillus aculeatus.

Rubusoside (R) is a natural sweetener and a solubilizing agent with antiangiogenic and antiallergic properties. However, currently, its production is quite expensive, and therefore, we have investigated nine commercially available glycosidases to optimize an economically viable R-production method. A stevioside (ST)-specific ß-glucosidase (SSGase) was selected and purified 7-fold from Aspergillus aculeatus Viscozyme L by a two-step column chromatography procedure. The 79 kDa protein was stable from pH 3.0 to pH 7.0 at 50-60 °C. Hydrolysis of ST by SSGase produced R and steviol monoglucosyl ester as determined by (1)H and (13)C nuclear magnetic resonance (NMR). Importantly, SSGase showed higher activity toward ST than other ß-linked glucobioses. The optimal conditions for R production were 280 mM ST and 16.6 µL of SSGase at pH 5.1 and 63 °C. This is the first discussion detailing the production of R by enzymatic hydrolysis of ST and is useful for the food additive and pharmaceutical industries.

Large increase in Leuconostoc citreum KM20 dextransucrase activity achieved by changing the strain/inducer combination in an E. coli expression system.

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction (OD600) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of 17 degrees C. These results may effectively apply to the heterologous expression of other large DexT genes.