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In the present work, enzymes pectinase and naringinase were simultaneously co-immobilized on an eco-friendly chitosan coated magnetic nanoparticles (chitosanMNPs) by cross-linking using chitosan as a macro-molecular cross-linker. The maximum activity recovery of both enzymes in the co-immobilized form was obtained at chitosanMNPs to enzymes ratio of 1:3, 3% cross-linker concentration and 150 min cross-linking time. The synthesized MNPs before and after co-immobilization were characterized using different techniques. The prepared biocatalyst was found spherical with an average size below 200 nm and showed supermagnetic property with saturation magnetization of 38.28 emu/g. The optimum pH and temperature of both enzymes in co-immobilized form was found at 5.5 and 65 °C. The prepared biocatalyst exhibited an improved thermal stability with 1.8-fold increase in the half-life. The secondary structural analysis revealed that, prepared co-immobilized biocatalyst undergone changes in the conformational and structural rigidity due to macro-molecular cross-linker. The co-immobilized biocatalysts were evaluated for one pot clarification and debittering of grapefruit juice and found ~52% reduction in turbidity and ~85% reduction in the naringin content. The co-immobilized enzymes were recycled up to 7th cycle and can be easily stored at room temperature for 30 days retaining up to 64% and 86% residual activities respectively.

Naringenin, a natural plant flavonoid found in citrus fruits, has been reported to exhibit a wide range of pharmacological functions, including anticancer, antioxidant, antiatherogenic, antithrombotic, and vasodilator activities. Naringenin can be produced from the naringinase (NGase)-catalyzed enzymatic hydrolysis of naringin. However, the poor solubility of naringin in aqueous systems considerably limits the efficiency of naringenin biocatalysis. In this work, a novel substrate adsorption system was proposed for naringin adsorption to increase the efficiency of naringin hydrolysis and naringenin production. Three Amberlite macroporous resins, namely, XAD-4, XAD-7HP and XAD-16, were investigated for their naringin adsorption capacities and effects on NGase hydrolysis. Results indicated that the physical properties of the resins played a critical role in naringin adsorption and naringenin enzymatic synthesis. Naringin hydrolysis was carried out using free and adsorbed substrates. The substrate adsorption strategy could increase the catalytic efficiency at a high naringin concentration. In addition, the reaction conditions for enzymatic naringenin synthesis were optimized, and naringenin was prepared at a liter scale with a high substrate concentration. These results suggested that substrate adsorption is a promising strategy to increase the enzymatic hydrolysis efficiency of naringenin in aqueous systems.

Compound K is a type of protopanaxadiol-type ginsenosides (PPDs) that has strong bioactivities due to fewer glycosyls. However, compound K is not present in raw and unprocessed ginseng. Some PPDs have the same structure with gypenosides, and could be obtained from Gynostemma pentaphyllum. The enzymolysis of PPD-type gypenosides of G. pentaphyllum by naringinase has been reported for the first time in this research. In addition, isolation and identification of enzymolysis end product, and the optimization of enzymolysis parameters were investigated. The results showed that compound K was produced from the enzymolysis of PPD-type gypenosides by naringinase, and could be isolated and purificated by HP-20 macroporous resin and C-18 column chromatography. The optimum enzymolysis conditions determined by the response surface methodology (RSM) are pH 4.1, 50 °C, and 71 h, with a yield of 65.44 ± 4.52% for compound K. These results demonstrated that enzymolysis could be a promising method for producing compound K from the biotransformation of PPD-type gypenosides of G. pentaphyllum.

In this research, the preparation and characterization of a novel biocatalyst comprising nano-magnetic cross-linked enzyme aggregates of naringinase (NM-NGase-CLEAs), which was covalently bounded to lysine-assisted magnetic nanoparticles, were studied. The Schiff base formed between ɛ-amino groups of the lysine residues and aldehyde groups of glutaraldehyde was reduced by ascorbic acid. Among the six different precipitants, tert-butanol performed the best for naringin hydrolysis. The optimal conditions for the immobilization process required 10 mM glutaraldehyde, 1:10 ratio of lysine/enzyme, and 3 h crosslinking at 3–4 °C. The morphology of the NM-NGase-CLEAs implied a non-uniform, semi-pyramid and semi-cubic rods. The dynamic light scattering (DLS) results showed that the nanomagnetite particle size was around 81.9–96.5 nm, with a polydispersity index (PDI) of 0.238. After NM-NGase-CLEAS formation, the particle size was reduced to around 13.2–15.3 nm, with PDI of 0.177, respectively. Moreover, the Ȥ-potential of −28 mV also confirms the improvement of CLEAs stability. The NM-NGase-CLEAs kept 73% of its original activity after 10 cycles, which proposes strong operational stability. In conclusion, the NM-NGase-CLEAs are thermo-stable, reusable, and efficient nanobiocatalyst for debittering of citrus juices.

Gypenoside XLVI (gyp XLVI) is one of the major dammarane-type triterpenoid saponins from Gynostamma pentaphallum with glucosyls at C-3 and C-20 positions, which may constrain its bioactivities. The enzymatic conversion of gyp XLVI by naringinase, and the cytotoxicity of enzymolysis product on SMMC7721 and Bel7402 hepatoma cells were investigated. The results showed that gynosaponin TN-1 (gyp TN-1) was produced from the enzymatic conversion of gyp XLVI by naringinase. The optimum enzymolysis conditions were pH 4.2, 47.3 °C, and 16 h, with a yield of 73.44 ± 6.52% for gyp TN-1. In addition, gyp TN-1 exhibited higher inhibitory activities on SMMC7721 and Bel7402 hepatoma cells than gyp XLVI. Results from methyl thiazolyl tetrazolium (MTT) assay and acridine orange (AO)/ethidium bromide (EB) double staining were highly consistent. These results demonstrated that enzymatic conversion could be a promising method for producing gyp TN-1 from the biotransformation of gyp XLVI and the preparation of gyp TN-1 might provide a reference for the acquisition of novel anticancer drugs.

Naringinase is a kind of glycosidase that catalyzes the biotransformation of naringin into naringenin. It is a multi-compound enzyme that provides the activity of α-L-rhamnosidase and β-glucosidase. In this study, an artificial naringinase system was constructed by co-immobilizing α-L-rhamnosidase (Aspergillus oryzae FJ0123, Ao-rha) and β-glucosidase (Thermotoga maritima MSB8, Tm-glu) on magnetic silica-based chitosan microspheres (MSC). The molar ratio of enzymes and the preparation conditions were optimized for improving synergistic effect and immobilization efficiency. Under optimized conditions (the molar ratio of Ao-rha and Tm-glu, 3:1; temperature, 25 °C; glutaraldehyde concentration, 2.0%; pH, 3.0; time, 9 h), the immobilization yield and activity recovery were 61.4% and 37.3% for Ao-rha and 90.1% and 56.3% for Tm-glu, respectively. Biochemical characterization indicated that MSC-naringinase had better adaptability and stability than free enzymes. The activity of MSC-naringinase was maintained at 58.7% after 10 times of recycling. The catalytic conditions were also investigated. The cascade reactions of naringin to prunin and prunin to naringenin by using MSC-naringinase resulted in a yield of 0.62 mg/mL and a conversion rate of 96.9%, respectively, without prunin accumulation. These results indicated the great potential of MSC-naringinase as an efficient artificial naringinase system for cooperative enzymatic synthesis of naringenin.

An enzymatic membrane reactor (EMR) is assembled for the immobilization of naringinase on a polyethersulfone ultrafiltration membrane, based on fouling-induced method. The effects of molecular weight cut-off, membrane configuration, applied pressure, enzyme concentration and pH are studied in terms of permeate rate, immobilization efficiency, and biocatalytic conversion. The 10 kDa membrane operating in reverse mode, 0.2 MPa, 0.3 gL−1 of enzyme in acetate buffer at pH 5 and cross-linking with 0.25% glutaraldehyde showed the highest naringin conversion (73%). It was determined that the intermediate pore blocking model was the predominant fouling mechanism for the enzymatic immobilization. The EMR was applied for debittering of grapefruit juice, achieving a conversion of naringin below bitterness threshold and maintaining the antioxidant capacity of the juice. Furthermore, the biocatalytic activity of immobilized enzyme was retained at a high level at least during three consecutive reaction runs, with overnight storage at 4 °C after each run. Industrial relevance The potential of membrane technologies in the juice industries is widely recognized today. The development of EMR with naringinase activity is an attractive option for reducing bitterness that could replace current techniques, due to its high specificity and effectiveness, possibility of repeated and continuous use, and in order to retain the properties of juice as much as possible. The research therefore represents an advance in the application of biocatalytic membranes as technological alternative for juice debittering.

Naringinase plays a rather important role in reducing the bitterness of juice by hydrolyzing naringin. A novel extracellular naringinase was purified from Aspergillus oryzae 11250 cultured in the presence of orange peel. A 26.78-fold purification rate was achieved by salt-induced precipitation, followed by anion-exchange and gel filtration chromatography with 32% recovery and specific activity of 2194.62 units per mg protein (U/mg). The optimum pH and temperature for naringinase activity were 5.0 and 45 °C, respectively. This enzyme was stable at 30 °C for 5 h. The K m and V max of naringinase toward naringin determined by Lineweaver-Burk method were 1.60 ± 0.13 mM and 126.21 ± 5.52 μmol/(min mg), respectively. The enzyme activity was inhibited completely by Ag + at 10 mM. Naringinase is capable of hydrolyzing naringin, neohesperidin, and some other glycosides. A supplement of 6 U/mL of this naringinase in citrus juice sufficiently removed naringin to relieve the bitterness of citrus juice. These properties make the enzyme an ideal candidate for commercial application in the debitterization of orange juice.

Electrospinning is a facile method to fabricate nanofibers, in terms of their high specific surface area and porous structure. Electrospun nanofibrous mats are excellent candidates for immobilization of enzymes. In this study, a simple route based on electrospinning and layer-by-layer (LBL) self-assembly processes has been developed to prepared naringinase/alginate multilayer coated electrospun cellulose acetate nanofibers. The content of immobilized naringinase could be tuned by adjusting the number of multilayers. XPS results indicated that naringinase was successfully immobilized on cellulose acetate nanofibers. SEM images showed the nanofibers maintain their sharp but became rougher after multilayer coating. Besides, the surface area of electrospun cellulose acetate nanofibers decreased and mesopores reduced. The major bitter components of grapefruit juice are naringin and limonin, naringin could be slightly removed by hydrolysis with naringinase and limonin might be removed by adsorption with cellulose acetate nanofibers.

An intracellular naringinase from Bacillus amyloliquefaciens 11568 isolated from soil was purified, identified, and characterized. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified enzyme gave a single protein band corresponding to a molecular mass of 32kDa. The optimum pH and temperature for naringinase and its α-l-rhamnosidase and β-d-glucosidase activities were pH 7.5 and 45°C, respectively. The enzymes were stable below 45°C between pH 3.5 and 8.5. The Km and the Vmax of the isolated naringinase were 0.95mmol/L and 3847.3mmol/(L·min), respectively. The isolated naringinase was capable of hydrolyzing naringin, neohesperidin, and other glycosides. Additionally, a concentration of 4U/mL of the enzyme in citrus juice was sufficient to remove the naringin and alleviate the bitterness of the juice. These results provide an in-depth insight into the structure of the naringinase and the hydrolysis of naringin and other flavonoids.

Isoquercitrin, a rare flavonol glycoside with wide biological activities and key synthetic intermediate for the production of enzymatically modified isoquercitrin (EMIQ), was conducted by naringinase-catalyzed conversion of rutin under ultrasound irradiation. The maximum yields were obtained to 98.35 ± 3.13% and 95.20 ± 2.52% under conventional heating and ultrasound irradiation, respectively. The optimal results under ultrasound irradiation were obtained under the following conditions: rutin concentration 0.8 g/L, naringinase concentration 3000 U/L, reaction temperature 40 °C for 20 min, which was more economical than that with conventional heating. The reaction time was reduced from 60 min to 20 min, and the apparent kinetic parameter ( V m / K m ) was increased 3.72-fold. The lower activity energy E a under ultrasonic irradiation was 0.7-fold of that in an incubator reactor, which could easily initiate the enzymatic reaction. The association saturation constant K a was 1.98-fold higher than that with conventional heating, showed a better affinity between rutin and naringinase detected by surface plasmon resonance (SPR) analysis. These results suggest that ultrasound irradiation can accelerate the enzymatic synthesis of isoquercitrin from rutin.

Herein is reported an innovative and reproducible systemic approach for the fabrication of customized 3D hollow microspheres of polyvinyl alcohol (PVA) based on the use of aromatic boronic acids (ABAs) as crosslinkers. A dedicated experimental set-up was developed to enable microsphere formulation. Most of the boronic acids tested were able to produce hollow microspheres. Depending on the ABA used and the production conditions, hollow PVA/ABA microspheres with different morphologies, mass transfer characteristics and unprecedented thermal (up to 121 °C) stability were obtained. The crosslinking temperature proved to be an operational parameter to control membrane thickness. The hollow microspheres were evaluated in biocatalysis, with naringin hydrolysis as model system, using encapsulated naringinase. The results highlighted a naringinase high operational stability, namely in PVA microspheres crosslinked with 1,4-phenylenediboronic acid and with phenylboronic acid, with no evident loss of activity throughout seven consecutive runs, theoretically resulting in an infinite half-life.

Polyvinyl alcohol (PVA) electrospun nanofibers were produced using an electrospinning technique. Key parameters (e.g. collectors, distance from needle tip to collector, among others) that influence the structure and morphology of fibers were optimized. The naringinase entrapped in PVA nanofibers retained over 100% of its initial activity after 212h of operation, at 25°C. Chemical crosslinking with several boronic acids further increased the hydrolysis temperature (up to 85°C) and yielded nanofibers with thermal stability up to 121°C. A mini packed bed reactor (PBR) developed to establish the feasibility for continuous enzymatic operation, ran for 16days at 45°C. Highest naringenin biosynthesis was attained at a flow rate of 10mLh(-1). Highest volumetric (78molL(-1)h(-1)) and specific (26molh(-1)genzyme(-1)) productivities were attained at 30mLh(-1). The activity of NGase in electrospun nanofibers remained constant for almost 16days of operation at 10mLh(-1).

The immobilization of naringinase in PVA lens-shaped particles, a cheap and biocompatible hydrogel was shown to provide an effective biocatalyst for naringin hydrolysis, an appealing reaction in the food and pharmaceutical industries. The present work addresses the operational stability and scale-up of the bioconversion system, in various types of reactors, namely shaken microtiter plates (volume ⩽ 2 mL), batch stirred tank reactors (volume400 mL) and a packed-bed reactor (PBR, 6.8 mL). Consecutive batch runs were performed with the shaken/stirred vessels, with reproducible and encouraging results, related to operational stability. The PBR was used to establish the feasibility for continuous operation, running continuously for 54 days at 45°C. The biocatalyst activity remained constant for 40 days of continuous operation. The averaged specific productivity was 9.07 mmol h(-1) g enzyme(-1) and the half-life of 48 days.

Pure flavonoid glycosides and those present in orange ( Citrus sinensis ) and lime ( Citrus latifolia ) juices were structurally modified by enzymatic de-glycosylation to obtain functional derivatives with higher antioxidant activity. The enzymatic reactions were carried out using commercial rhamnosidases (hesperidinase and naringinase) and β-D-glucosidase. An experimental design and statistical tools for the data analysis were employed for each enzyme to evaluate the independent variables (pH and temperature) that significantly affected the enzyme activity (dependent variable), and the antioxidant activity was evaluated before and after bioconversion using DPPH and FRAP assays. Aliquots of controls and enzyme-treated samples were taken at different times and analyzed by UPLC–MS. The antioxidant activity of both treated juices was higher than that of the untreated juices, confirming that the bioconversion reaction conditions used were efficient and produced an increment in the antioxidant activity. After the 4 h-hesperidinase reaction 60% of the hesperidin was converted into hesperetin in the orange juice. The antioxidant capacities of the glycosylated standards were also increased by the enzyme treatment. The results presented herein are a step forward in the use of α-L-rhamnosidases to produce functional beverages by way of de-glycosylation reactions of their flavonoid glycosides.

a b s t r a c t New rhamnolipids were obtained via the development of a synthesis procedure consisting of two bio- catalyzed steps. In the first step, naringinase was used to introduce a primary alcohol function onto rhamnose by glycosylation of 1,3-propanediol. In the second step, immobilized lipase B from Candida antarctica catalyzed the esterification of the primary hydroxyl group with mono- and di-carboxylic fatty acids of increasing chain length (from C8 to C14). For the monoic acids, the initial rate and 24 h yield decreased with increasing chain length. For the dioic acid, the number of carbon atoms of the acid did not influence these parameters. The new rhamnolipid obtained with tetradecanoic acid showed very good surface properties. At pH 5, it had a very low critical aggregation concentration of 1.70 M and it dimin- ished water's surface tension to 27.6 mN/m. It was also able to form stable insoluble monolayers. On the other hand, the rhamnolipid formed with tetradecanedioic acid showed far less interesting surface properties.

Novel ionic liquid (IL) sol–gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α - l -rhamnosidase and β - d -glucosidase activities expressed by naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol–gel matrices and used on naringinase immobilization. The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α -rhamnosidase expressed by naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM] > [BMIM] > [EMIM] > [C 2 OHMIM] > [BIM] and [OMIM] ≈ [E 2 -MPy] ≫ [E 3 -MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α -rhamnosidase efficiencies: [BMIM]BF 4 ≫ [C 2 OHMIM]BF 4 ≫ [BIM]BF 4 . Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E 3 -MPy] and [E 2 -MPy] allowed an impressive difference in the α -rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α -rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF 6 − , BF 4 − and Tf 2 N − ) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL. The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf 2 N] and TMOS/Glycerol@[C 2 OHMIM][PF 6 ] and on TMOS/Glycerol. An improved stability and efficiency of α - l -rhamnosidase and β -glucosidase expressed by encapsulated naringinase on TMOS/Glycerol@[OMIM][Tf 2 N] and TMOS/Glycerol@[C 2 OHMIM][PF 6 ] were achieved. In addition to these advantageous, with ILs as sol–gel templates, environmental friendly processes can be implemented.

Antioxidant capacity, radical scavenging activity, as well as protective effect on lipoperoxidation, glutathione oxidation and DNA damage, were evaluated in grapefruit juice subjected to bitterness removal by naringinase or by physical adsorption with Amberlite®IRA-400. The results showed a reduction in naringin content for the naringinase-treated juice (N-PJ) and those processed with the exchange resin (R-PJ), which made both juices acceptable to consumers. Total antioxidant capacity, measured by ABTS and FRAP assays, was lower in R-PJ samples. The highest superoxide and hydroxyl radical scavenger activity was observed in N-PJ. With regard to inhibitory effect of juice samples on lipoperoxidation, N-PJ also provided the greatest effectiveness. In addition, R-PJ showed the lowest levels of GSH. The results showed a protective effect on DNA oxidative damage for all juice samples tested. In summary, enzymatic technology was more effective than physical adsorption in preserving the antioxidant and biomolecule protection capacity of fresh grapefruit juice.

Naringinase was bound to mesoporous silica MCM-41 via adsorption with glutaraldehyde and used to debitter white grapefruit. K m value of the immobilized naringinase was lower than that of free naringinase. The immobilized catalysts showed excellent thermal stability and storage stability and could be recycled 6 times retained about 44.57% activities. The unaltered structural order of the prepared catalyst was characterized with reference to bulky and surface properties by infrared spectroscopy (FT-IR), elemental analysis and nitrogen adsorption–desorption isotherms analysis.

Aspergillus sojae isolated from a traditional Korean fermented soybean product exhibited strong naringinase activity. The naringinase enzyme was purified and had a molecular weight of 70 kDa. The α- l -rhamnosidase activity of this enzyme was optimal at pH 6.0 and stable in the pH range of 5.5–8.0. The purified enzyme also had β- d -glucosidase activity, but the activity was relatively weak compared to the activity of the naringinase from Penicillium decumbens . The enzymatic bioconversion by A. sojae naringinase of naringin to prunin was efficiently performed with a 91% yield and a negligible amount of naringenin. The bioconversion was achieved by repetitive batch reactions with enzyme recycling. Prunin exhibited a markedly enhanced solubility compared to naringenin and naringin while maintaining the in vitro inhibition of HMG-CoA reductase. The results reported in this paper show that the naringinase produced by A. sojae will be useful in enhancing the potential bioavailability of naringin by efficiently converting it to prunin as a food component in citrus.

The hydrolysis of naringin, a bitter flavonone glycoside, with naringinase leads to reducing sugars (rhamnose and glucose), to prunin and to the aglycone, naringenin. To overcome the low solubility of naringin in the enzymatic reaction media, the effect of different solvents was studied, in order to improve the productivity and yield of the system. The effect of increasing concentration of co-solvents on the stability of both soluble and immobilized naringinase expressing α- l -rhamnosidase and β- d -glucosidase activities was evaluated. The enzyme was immobilized onto sol–gel matrices of tetramethoxysilane and glycerol. Combining the higher naringin solubility, and the higher residual activity of both α- l -rhamnosidase and β- d -glucosidase expressed by naringinase, eight solvents were chosen for stability and activity studies: dimethyl sulfoxide, N,N-dimethylmethanamide, methanol, ethanol, acetone, tetrahydrofurane, 1,2-dimethoxyethane and 1,4-dioxane. Deactivation of soluble naringinase was analyzed according to a first-order kinetic model. For the sol–gel immobilized enzyme the two-step deactivation model, of Henley and Sadana, was adjusted. Sol–gel immobilization stables naringinase in all tested co-solvents systems. This effect was specially pronounced at higher co-solvent concentration (10%). The half-life of α- l -rhamnosidase and β- d -glucosidase expressed by naringinase even increased 21- and 59-fold, respectively, in aqueous co-solvation with tetrahydrofurane. These are high innovative and sounding results showing the protective effect of immobilization onto sol–gel (tetramethoxysilane + glycerol) matrices with naringinase in co-solvent systems, which is a great advantage for non-conventional biocatalysis.

Naringinase is an enzyme complex, expressing α- l -rhamnosidase and β- d -glucosidase activities. The impact of high pressure and temperature on naringinase activity and stability were studied, in order to assess the potential of enzyme thermostability on glycosides hydrolyses. To a better understanding of these effects on naringinase enzyme complex, they were also evaluated over α- l -rhamnosidase and β- d -glucosidase activities, using specific substrates, p-nitrophenyl α- l -rhamnopyranoside (4-NRham) and p-nitrophenyl β- d -glucopyranoside (4-NGluc), respectively. Hydrolysis rate of 4-NRham and naringin increased with pressure from 0.1 to 150 MPa. The equilibrium constants for α- l -rhamnosidase and β- d -glucosidase reactions, at pressures of 0.1–200 MPa and 40 °C, were determined and best fitted with the model of Baliga and Whalley equation. Accordingly, reaction volumes of 93 and 64 mL mol −1 were obtained for α- l -rhamnosidase and β- d -glucosidase reactions, respectively. Reaction rate constants were also determined at the same experimental conditions and well fitted to the models of Golinkin, Laidlaw, Hyne and of Burris and Laidler. A negative Δ V ≠ of −7.7 ± 1.5 and −20.0 ± 5.2 mL mol −1 were obtained for α- l -rhamnosidase and naringinase reactions, correspondingly, which reflect the accelerating effect of pressure on the biocatalysis. Moreover, the K M , k cat and k cat / K M values, on naringin hydrolysis under atmospheric (0.1 MPa) and high pressure (150 MPa) conditions at different temperatures (25–80 °C) were determined. A 3-fold and 4-fold increase on naringinase thermostability was observed under 150 MPa at 70 and 80 °C, respectively, compared to 0.1 MPa experiments. In addition, a 15-fold increase of k cat / k M values from experimental conditions of 0.1 MPa and 30 °C to 150 MPa and 70 °C was observed. In fact, high pressure showed to be a powerful tool to increase stability of naringinase against thermal denaturation. In conclusion, the effect of amplification of pressure effects on reaction rates by temperature could have a pragmatic use for accelerating enzymatic reactions.

In this work, a biocatalytic system was chosen to enable the deglycosylation of natural glycosides in order to improve their biological activity. Naringinase, which has both alpha- l -rhamnosidase and beta- d -glucosidase activities, was the enzyme used. Naringin, the glycoside substrate used in this bioconversion, and its aglycone product naringenin are compounds with interesting pharmacological activities such as anti-oxidant and anti-inflammatory; naringenin also acts as an anti-carcinogenic and neuroprotective agent, demonstrating a high potential for use by the pharmaceutical industry. Sol–gel, an innovative technique performed in aqueous media, was developed for naringinase immobilization in lens-shaped particles. Different sol–gel precursors, including tetramethoxysilane (TMOS), methyltrimethoxysilane (MTS), 3-aminopropyltrimethoxysilane (APS) and diglycerylsilane (DGS) with different aging times were tested in five consecutive trials. The best results were obtained with TMOS and TMOS/DGS after 4 h aging time and TMOS/glycerol and DGS at 14 h aging time. The characterization of these matrices was performed with respect to their diameter, volume, naringin and naringenin partition coefficients, and the optimum temperature and pH for naringinase activity. The operational stability of the bioencapsulated naringinase in the selected sol–gel matrices was studied through successive reutilizations. The naringinase sol–gel matrix (TMOS, DGS and TMOS/DGS) deactivation studies followed the Sadana model. The matrix TMOS/glycerol showed a constant 100% residual activity after 50 consecutive runs.

Naringin is the dominant flavonoid bitter principle in grapefruit juice. The aim of this work was the modeling of the enzymatic hydrolysis of naringin, with naringinase immobilised in Ca-alginate beads, under high pressure, in order to optimize this technique for removal of the bitter taste from juices, using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed involving two variables (pressure and temperature) at five levels (−√2, −1, 0, +1, +√2). The second-order polynomial equations with R 2 values above 0.9 showed good agreement between experimental and predicted, respectively, naringinase activity, naringin conversion and naringenin formation. The higher naringin conversion of 81% was obtained using 205 MPa and 60 °C, during a process time of 30 min. It was found that pressure and temperature, as well as their interactions, had significant effects ( p These are promising results to optimize this technique for the removal of the bitter taste and, after future microbial studies to be addressed, as a sterilisation/pasteurization process of citrus juices.

Dangyuja is a citrus fruit of Cheju Island in Korea, which is known to have a high content of flavanone glycosides, such as naringin and neohesperidin. Flavanone glycosides of Dangyuja (Citrus grandis Osbeck) extract were converted into their aglycones by naringinase and hesperidinase, and into their hydroxylated forms by Aspergillus saitoi. Dangyuja extract treated with A. saitoi had significantly higher antioxidant and antidiabetic activity than both its glycosides and aglycones in oxygen radical absorption capacity (ORAC) and supercoiled DNA strand scission assay, rat intestinal α-glucosidase and porcine pancreatic α-amylase inhibition, suggesting that the fermentation of flavanone aglycones of Dangyuja extract with A. saitoi can increase antioxidant and antidiabetic activity, compared to their glycosides and aglycones. This result suggests that Dangyuja extract prepared with enzymatic and microbial treatments could be used for the development of pharmaceutical foods to control the blood glucose level of diabetic patients by inhibiting α-amylase and α-glucosidase in the intestinal tract.

The aim of this work was the hydrolysis of naringin towards naringenin with immobilized naringinase and the evaluation of their anti-inflammatory activity. An acute local inflammation model (rat paw edema induced by X-carrageenan) was chosen to evaluate the contribution of antioxidant properties to a possible anti-inflammatory effect. Grapefruit juice was processed with naringinase immobilized in k-carrageenan (2%) beads (approximate to 3 mm). A 95% naringin conversion in grapefruit juice was obtained with immobilized naringinase (1000 mg L-1), with an activity of 19.5 mg mL(-1) min(-1) and the formation of 215 mg L-1 of naringenin. Ascorbic acid and indometacine were used as positive anti-inflammatory controls. All results were analysed using ANOVA with Dunnett's Post-test. The results show that rats (n = 9) pre-treated with a solution of naringin, rats (n = 9) pre-treated with a solution of naringenin (in concentrations equal as in grapefruit juice) and rats (n = 9) treated with a solution of naringenin and naringin, revealed a significant reduction on edema formation, 6h after X-carrageenan injection. Naringenin demonstrated a high in vivo anti-inflammatory activity, only 8% of paw edema (p0.001) was observed in rats pre-treated with a solution of naringenin. Comparability studies, in rats administered orally with grapefruit juice (before and after processing), showed that enzymatic processing did not affect the anti-inflammatory properties of the juice. (C) 2007 Elsevier B.V. All rights reserved.

Efficient syntheses are reported of the α- l -rhamnosidase inhibitors l -swainsonine [(1 R ,2 S ,8 S ,8a S )-octahydroindolizine-1,2,8-triol], (6 R )- C -methyl- l -swainsonine (1 R ,2 S ,6 R ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol, (6 S )- C -methyl- l -swainsonine (1 R ,2 S ,6 S ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol and related 6- C -methyl hydroxycastanospermines [(1 R ,2 S ,6 R ,7 R ,8 R ,8a R )-6-methyl-octahydroindolizine-1,2,6,7,8-pentaol and (1 R ,2 S ,6 S ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol]. (6 R )- C -Methyl- l -swainsonine [ K i = 0.032 μM] is a significantly more potent naringinase inhibitor than l -swainsonine [ K i = 0.45 μM].

Initially exploited in chemistry and physics, high-pressure technology has gained importance in various fields. Flavonoids, namely naringin and naringenin, from citrus, are functional chemicals with important properties in the fields of healthcare, food and agriculture. Naringin, the principal bitter flavonone glycoside and the primary bitter component in grapefruit juice, can be hydrolysed by naringinase into tasteless naringenin. The temperature of 303 K was ideal for maximizing the activity of the naringinase at 160 MPa when compared to atmospheric pressure. The pressure had a positive effect on the reaction rate, with a value of −15.0 ± 1.8 cm 3 mol −1 for the activation volume. Kinetic parameters, k M and V max for naringinase were evaluated, the maximum initial rate at 160 MPa ( V max = 2.7 mM min −1 ) was higher than that at atmospheric pressure ( V max = 0.06 mM min −1 ). This is a promising result for future application of enzymatic hydrolysis of naringin at high pressure, in the citrus juice industry.

The aim of this study was the modelling, under high pressure, of naringin hydrolysis by naringinase. Response surface methodology (RSM) was used to compare the effects of the selected variables on the bioconversion under study. The combined action of temperature (13-61 degrees C) and pressure (80-216 MPa) on the catalytic activity of naringinase was investigated at pH 4.0 using naringin as the substrate. The choice of experimental domains resulted from preliminary studies. Naringinase activity, for naringin hydrolysis at pH 4.0, could be described by a convex surface with a maximum of 0.13 mM min(-1), at 41 degrees C and 158 MPa. After 1 h of reaction time, reducing sugars production could also be described by a convex surface, with a maximum reducing sugars concentration of 8 mM at 38 degrees C and 168 MPa. The interaction temperature-pressure had a significant effect on both naringinase activity and reducing sugar formation after 1 h. Under the optimized conditions, the naringin hydrolysis by naringinase was evaluated. (c) 2007 Elsevier Ltd. All rights reserved.

Naringinase, induced from Aspergillus niger CECT 2088 cultures, was immobilized into a polymeric matrix consisting of poly(vinyl alcohol) (PVA) hydrogel, cryostructured in liquid nitrogen, to obtain biocatalytically active beads. The effects of matrix concentration, enzyme load and pH on immobilization efficiency were studied. Between 95% and 108% of the added naringinase was actively entrapped in PVA cryogel, depending on the conditions of immobilization used. The optimal conditions were: 8% (w/v) PVA at pH 7 and 1.6–3.7 U ml −1 of enzyme load. The pH/activity profiles revealed no change in terms of shape or optimum pH (4.5) upon immobilization of naringinase. However, the optimum temperature was shifted from 60 °C to 70 °C and the activation energy of reaction, E a , was decreased from 8.09 kJ mol −1 to 6.36 kJ mol −1 by immobilization. The entrapped naringinase could be reused through six cycles (runs of 24 h at 20 °C), retaining 36% efficacy for the hydrolysis of naringin in simulated juice.

The fabrication and characterization of a surface plasmon resonance (SPR) based fiber-optic sensor to determine the amount of naringin, which causes bittering effect in citrus fruit juices, have been reported. The sensing region has been fabricated by coating about 1 cm length of the unclad core with silver. To sense naringin, the enzyme, naringinase, has been immobilized over the metallic surface. The spectral interrogation method has been used to characterize the sensor. It has been observed that the SPR wavelength increases with an increase in the concentration of naringin. In addition, the sensitivity, reproducibility and the signal to noise ratio (SNR) of the sensor have been studied. It has been found that the sensitivity increases with an increase in the concentration of naringin, while the reverse is the case for SNR. The fiber-optic SPR sensing probe reported can be repeatedly used. Further, it can be used for remote and in situ monitoring of the naringin concentration.

Aspergillus niger MTCC 1344 was used to produce extracellular naringinase in a complex (molasses, yeast extract and salts) medium. An initial medium pH 4.5 and cultivation temperature 30 °C were optimal for enzyme production. Among various carbon and organic nitrogen sources used, molasses and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when metal ions were added to the medium. Fermentation conditions are described which produced a higher rate of enzyme synthesis. An increase in initial sugar concentration from 6 to 10 g l−1 in the fermentation medium produced decreased naringinase synthesis while cell mass growth increased with the increase of sugar concentration. At a higher sugar level (10 g l−1) the production of cell mass decreased.

Renewable resources, such as rapeseed oil or oleic acid, combined with glucose were used both in a feed-batch and two-stage continuous process for the production of sophorolipids with the yeast Candida bombicola ATCC 22214. High yields >300 g l −1 and increased productivities of 57 g l −1 d −1 (feed-batch) and 76 g l −1 d −1 (continuous mode), respectively, were obtained by using optimized cultivation conditions. The key-factors for enhanced sophorolipid production are presented. The acidic sophorolipid was used for further modification after alkaline hydrolysis and purification. A novel glucoselipid was synthesized by using the enzyme naringinase (EC 3.2.1.40). A (ω-1)-hydroxyfatty acid, commercially not yet available and difficult to prepare by organic synthesis, was released by acidic hydrolysis, purified and is proposed as a precursor for plastics and flavours.

The synthesis and inhibitory properties towards naringinase (L-rhamnosidase) of L-(+)-swainsonine and of a number of more highly oxygenated analogues, and of some monocyclic equivalents, are reported. L-(+)-swainsonine and 1,4,6-trideoxy-1,4-imino-L-mannitol are powerful and specific inhibitors of naringinase.

Screening of matrices for the immobilization of naringinase demonstrated that 2% sodium alginate was an optimal matrix. Upon immobilization, 30 U of naringinase gave 82% naringin hydrolysis in 3 h, broadening of pH optima has attributed desirable flexibility for debittering kinnow juice of varying pHs, and temperature profiles indicated an improved thermostability which could be handy during the reduction in cost of debittering. Alginate permitted attainment of equilibrium readily with no hindrance in the inflow of naringin and the outflow of naringenin/prunin, and adequate mechanical stability, cumulatively indicating the feasibility of its commercial exploitation. The application of kinetic parameters optimized with pure naringin to kinnow juice resulted in 60% debittering. It suggested the desirability of ultrafiltration to minimize product inhibition in order to maximize naringin hydrolysis, and subsequent fortification by the permeate at the end of debittering to formulate sweetened kinnow juice with nourishing and natural characteristics. Efforts to change over from the batch process to a continuous column process are underway.

Five-membered ring azasugars with the L-rhamnose configuration were synthesized as inhibitors of alpha-L-rhamnosidase from Penicillium decumbens. All compounds tested were in the microM or sub-microM range. Substitution at the nitrogen shifted the inhibition mechanism from mixed to competitive.

Synthesis of both enantiomers of deoxyrhamnojirimycin and rhamnonolactam from D- and L-gulonolactones are described. The effects as inhibitors of the enantiomeric deoxyrhamnojirimycins and rhamnonolactams on human liver glycosidases are compared with deoxymannojirimycin and mannonolactam. No significant inhibition of the activity of naringinase (an α-L-rhamnosidase) was caused by any of these compounds.

The use of Rhodococcus fascians for debittering kinnow juice was explored. Its growth and application conditions were optimized. Incubation of kinnow juice with 5% (v/v) R. fascians mass cultivated for 48 h afforded optimal (60%) degradation of limonin at 25°C, pH 4.0, and 150 rev min -1 for 48 h. Coupling of this step with alkaline degradation led to 78% degradation of limonin.

The α- l -rhamnosidase component of naringinase from Penicillium decumbens was purified and characterised as a monomeric enzyme with a mol. wt. of ∼90,000 and with a 50% content of carbohydrate. Digestion of the enzyme with pronase and fractionation of the products on Sephadex G-100 gave two glycopeptides. The larger glycopeptide contained galactofuranose and mannose residues together with lesser proportions of ethanolamine and glucose. Methylation analysis and 13 C-n.m.r. spectroscopy indicated its structure to be similar to that of the cell-wall peptido-galactomannan of P. charlesii . The smaller glycopeptide was of the N -linked type with 10–12 mannose residues per asparagine. The 1 H-n.m.r. spectrum indicated the structure to resemble those reported for Saccharomyces cerevisiae glycopeptides, with the addition of 1–2 galactofuranose residues and 3 ethanolamine substituents to the oligosaccharide unit.

Naringinases from both Penicillium sp. and Aspergillus niger were compared for their enzyme kinetics and the effects of sugars on the enzyme activities. Lineweaver-Burk plots showed that glucose, fructose and rhamnose were all competitive inhibitors for α-rhamnosidase of naringinase from Penicillium sp. and non-competitive inhibitors for the same enzyme from A. niger , When naringinase from Penicillium sp. was immobilized on chitin and used successively for the hydrolysis of p -nitrophenyl- α -rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, it was observed that the enzyme column was very stable. Such results are in contrast to what has bee observed for naringinase fron A. niger. Therefore, it is quite possible that the sugars in the fruit juice which play a role as competitive or non-competitive inhibitors on naringinase may account for the stability of the enzyme column during successive debittering of grapefruits juice.

We now report on the substrate specificity of naringinase, using a number of synthetic mono-and oligo-rhamnoside substrates. The structures of the product of hydrolysis were established by 1 H-n.m.r. spectroscopy at 500 MHz

Robinin and two novel glycosides: isorhamnetin 3-(6-rhamnosylgalactoside)-7-rhamnoside and 3-(2,6-dirhamnosylgalactoside)-7-rhamnoside were isolated from a leaf extract of Rhazya stricta . Naringinase catalysed hydrolysis of both glycosides removed the rhamnose from the 7-hydroxyl. The product isorhamnetin 3-(2,6-dirhamnosylgalactoside) was shown by 1 H NMR to be identical with a glycoside previously isolated from Primula veris .

Naringinase from Penicillium sp. was immobilized on cellulose triacetate by the fiber entrapment method. Although the optimum pH (3.7) and optimum temperature (55°C) of the fiber-entrapped enzyme were similar to those of the native form, the immobilized enzyme had better heat stability. Kinetic studies showed that the immobilized enzyme had higher K m values than its native form. When this immobilized naringinase was successively used in a column reactor for the hydrolysis of ρ-nitrophenyl-α- l -rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, the enzyme column could be operated with satisfactory stability. In addition, when the natural grapefruit juice was recycled through the column reactor, no column blocking or filtering action of the catalyst bed was observed.