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To improve the naringinase production of Aspergillus tubingensis UA13, shorten the fermentation period, and verify its industrial application value, naringinase production conditions were optimized, and 5 L scale-up study in stirred tank bioreactor was carried out. Parameters, including carbon, nitrogen sources and inducer, optimal seed age, inoculum amount, temperature and pH, were adjusted and optimized in shaking flask. Keeping pH at the optimal value 6 in bioreactor, dissolved oxygen was monitored during the fermentation and the optimal stirring rate was investigated. In 5 L scale-up study, the highest naringinase activity was 72.62 U/mL, which was 1.75 times higher than that (41.52 U/mL) in shaking flask and the fermentation period was shortened by 24 h.

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.

Four strains of Lactobacillus and Bifidobacterium including L. plantarum 01, L. fermentum D13, L. rhamnosus B01725, and B. bifidum B7.5 exhibiting naringinase production were applied in grapefruit juice fermentation. All investigated strains grew well in grapefruit juice without nutrition supplementation. In all cases, cell counts were 108–109 CFU ml−1 after 24 hours of fermentation. The highest lactic acid and acetic acid productions were observed in the case of strain L. plantarum 01. The L. plantarum 01 and L. fermentum D13 strains prefer glucose over fructose and sucrose, whereas fructose was the most favoured sugar for L. rhamnosus B01725 and B. bifidum B7.5. At the end of the fermentation process, antioxidant activity and total polyphenol content of grapefruit juice decreased in all cases, but the changes were not significant. Significant decrease of naringin was observed in the case of L. plantarum 01, 28% naringin in grapefruit juice was removed after fermentation. This result is promising for development of technology for production of probiotic grapefruit juice.

Resumen: El objetivo de esta investigación fue optimizar la inmovilización de naringinasa de Penicillium decumbens mediante atrapamiento en criogeles de alcohol polivinílico y polietilenglicol. En primer lugar, se determinó el efecto de las características de los componentes de la matriz sobre la eficacia de inmovilización y la estabilidad mecánica de los biocatalizadores obtenidos. Se empleó la herramienta de diseño de experimentos y superficie de respuesta para analizar el efecto del pH (3,0-8,0), de las concentraciones de alcohol polivinílico (5,3-8,7%) y polietilenglicol (7,0-12,0%) sobre la eficacia de inmovilización, y para optimizar estos parámetros. Adicionalmente, se estudió la capacidad de reutilización de los inmovilizados en zumos sintéticos. Se consiguieron biocatalizadores inmovilizados que conservaban un 45-47% de la enzima activa y que, tras tres ciclos de reutilización en zumo sintético, mantenían un 54,3% de la actividad inicial. Los resultados obtenidos mostraron que las condiciones de inmovilización afectaban a la eficacia de inmovilización y a la estabilidad operativa de la naringinasa atrapada.

In commercial citrus juice, bitterness causes serious challenges in fruit juice manufacturing industries. The prime cause of bitterness is due to the presence of naringin compound. It is noted that microbial enzyme retains some specific catalytic reactions of physicochemical and biological properties possessing high degree of industrial and medical applications. The microbial enzyme naringinase can be immobilized for industrial use to reduce the cost of debittering of juice. Since environment friendly industrial biocatalysts are economically more viable, so the focus on the present study is debittering of juice at low cost without using chemicals which alter the nutrient composition. In the present study, four strains of naringin degrading bacteria were isolated from the soil of Psidium guajava L. and Terminalia chebula Retz in Dibrugarh University Botanical Garden and were investigated for naringinase activity of soil microbes and their growth conditions at different parameters. The turbidity of cell culture and concentration of proteins in the culture media have been utilized for the optimization of various growth conditions, like temperature and pH for microbial growth. The optimal growth of the four isolates was observed in a media of pH 6 and selected for further study. All the four isolates showed good extracellular naringinase activity. Among the four isolates, oval and rod shaped gram positive bacteria showed the highest specific activity (405.31 U/mg) and lowest activity was shown by rod shaped gram negative bacteria (231.77 U/mg). Furthermore, rod shaped isolate exhibited maximum growth and highest protein content among the four isolates. These results suggested that in addition to the naringinase enzyme, some other proteins were also produced by the isolates. These proteins might have some significance in supporting the growth of the microorganisms.

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.

This study aimed to develop a method of naringinase biosynthesis by Aspergillus niger KMS on an optimized culture medium. The concentration of the six medium components in shake flasks was optimized by the Box and Wilson factor gradient method. Naringinase’s substrate, naringin, powdered albedo, flavedo, and red grapefruit segment membranes were used to stimulate naringinase biosynthesis. Rhamnose was chosen as the carbon source, while the nitrogen source was yeast extract and sodium nitrate. Naringinase biosynthesis was most favorable in the culture medium with the following composition (g 100 mL): 3.332—NaNO3; 3.427—yeast extract; 0.184—KH2PO4; 0.855—red grapefruit albedo; 0.168—naringin; 2.789—rhamnose. The obtained Aspergillus niger KMS culture fluid was concentrated, thereby precipitating the protein. As a result, a naringinase preparation with high activity, equal to 816 µmol × min−1 × g−1, was obtained.

Made available in DSpace on 2022-04-29T08:32:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Naringin, considered as the main bitter component of grapefruit, requires the use of enzymes to reduce the level of this substance during juice processing. For this reason, it has been the focus of many studies. In this study, to increase the production of naringinase by Aspergillus niger cultivated in solid-state fermentation (SSF), a three-component simplex-centric mixing design along with a response surface methodology (RSM) was applied to generate statistical models and analyze the dataset. First, grapefruit peel, rice bran, and wheat bran were used for substrate selection for naringinase production and, finally, selected the best of the three inducers or their mixtures to remove the bitterness of grapefruit juice. Cultivation with 2.3 g of grapefruit peel, 2.5 g of rice bran, and 5.2 g of wheat bran and medium supplementation with a mixture of naringin, rutin, and hesperidin in the concentration of 2, 5, 4.5, and 3.0 g/L, respectively, resulted in a maximum activity of 28 U/mL. The results indicate that the sequencing procedure, which allowed the definition of an optimal mixture of components, is a new way for microorganisms to have a high naringinase yield, in particular by SSF, since our data showed a 96% increase in the production of naringinase. This dataset can help other researchers apply a mixing design to increase enzyme production. Department of Biochemistry and Biotechnology State University of Londrina Departamento de Tecnologia Faculdade de Ciências Agrárias E Veterinárias UNESP - Univ Estadual Paulista, Rod. Prof. Paulo Donato Castellane km 5,SP Departamento de Tecnologia Faculdade de Ciências Agrárias E Veterinárias UNESP - Univ Estadual Paulista, Rod. Prof. Paulo Donato Castellane km 5,SP CAPES: #2015

The naringinase enzyme was isolated from Citrus macroptera peel and pomace using Aspergillus niger for immobilization and debittering of juice. A comparative study of the effect of debittering using immobilized enzyme and resin on the physicochemical and phytochemical properties of the juice were carried out. The optimum hydrolytic conditions and Michaelis constant for an immobilized enzyme were observed at pH 4, 70℃, and 16.75 μg/ml, respectively. Adsorption of naringin onto resin followed pseudo‐first‐order with a regression coefficient of 0.92. Naringin content was reduced up to 73.28% in 4 min by resin and 79.76% in 120 min by the enzyme. We reutilized resin and immobilized enzyme effectively to debitter the juice up to seven cycles. Treatment for less than 2 min and 90–120 min using resin and enzyme, respectively, could retain the sizable amount of bioactive components in juice and immobilization retained 62% enzymatic activity during 2 months of storage at 4°C. PRACTICAL APPLICATIONS: Pomelo being a rich source of bioactive nutrients faces slow commercialization due to immediate bitterness (naringin), thus in the present study, Citrus macroptera waste including peel and pomace was efficiently utilized to produce naringinase enzyme for the debittering of pomelo juice. Also, adsorption kinetics of naringin using macroporous resin is presented, which will further help in the selection of optimum time and dose for the removal of naringin from the juice. More importantly, their effect on juice composition helped in the selection of the potential candidate for the industrial purpose with negligible or minimum loss of nutrients.

In the present study, highly pure rhamnolipids (RLs) was produced using biocatalysts immobilized on amino-functionalized chitosan coated magnetic nanoparticles. Upon immobilizing naringinase and Candida antarctica lipase B (CaLB) under the optimized conditions, an enhanced operational stability with biocatalytic loads of 935 ± 2.4 U/g (naringinase) and 825 ± 4.1 U/g (CaLB) were achieved. Subsequently, the immobilized biocatalysts were utilized sequentially in a two-step RLs synthesis process. The key parameters involved in RLs production were optimized using artificial neural network (ANN) coupled genetic algorithm (GA) and were compared with composite central design (CCD). On validating the efficiency of both models, mean square errors of 1.58% (CCD) and 1.04% (ANN) were obtained. Optimization of parameters by ANN-GA resulted in 1.2-fold increase in experimental RLs yield (80.53%), which was 1.05-fold higher when compared to CCD model. Further, to establish the efficiency of RLs as a bioremediation agent, it was utilized as washing agent. It was observed that at a soil to RLs volume of 1:05, RLs concentration of 0.4 mg/mL, a 95.35 ± 1.33% removal of Total Petroleum Hydrocarbons (TPHs) was obtained at 35 ℃ and 160 rpm in 75 min. Thus, this strategy provides an efficient biocatalytic toolbox for RLs synthesis, which can be effectively used as a bioremediation agent.

Naringinase was mainly obtained by microbial fermentation, and mutagenesis was a major way for obtaining excellent mutants. The aim of this study was to screen out a high naringinase yielding mutant to enhance the potential application value of its industrialization and compare the effects of different mutagenic methods on the enzyme activity of the strain. A novel producing naringinase strain, Aspergillus tubingensis MN589840, was isolated from mildewed pomelo peel, later subjected to mutagenesis including UV, ARTP and UV-ARTP. After five rounds iterative mutagenesis, the mutants U1, A6 and UA13 were screened out with 1448·49, 1848·71, 2475·16 U mg-1 enzyme activity, the naringinase productivity raised by 79·08, 123·56 and 206%, respectively. In addition, the naringinase activity of three mutants rose after each round of iterative mutagenesis. These results indicated that the mutagenesis efficiency of UV-ARTP was higher than that of single ARTP, and both are better than UV. In summary, the iterative UV-ARTP mutagenesis is an effective strategy for screening high naringinase-producing strains.

Silica material has high specific surface area and excellent chemical stability, which make it useful for enzyme immobilization. In this work, naringinase was immobilized from fermentation broth of Aspergillus niger FFCC uv-11 by silica materials with different pore diameters of 2 nm (MCM-41), 7.7 nm (SBA-15) and 80 nm (silica gel). It was shown that SBA-15 had the highest naringinase activity, and this was chosen as a suitable carrier material for naringinase immobilization. First, SBA-15 was modified by glutaraldehyde at a concentration of 7% at 25 °C for 2 h, and it was then used for the immobilization of naringinase. At pH 3.5, the immobilized naringinase activity reached 467.62 U g−1 at 40 °C for 4 h when the initial naringinase activity was 89.04 U mL−1. Furthermore, at the optimal reaction temperature of 45 °C and pH of 4.5, the binding efficiency, activity recovery rate and specific activity of the immobilized naringinase were 63.66%, 87.64% and 517.43 U g−1, respectively. Compared with free naringinase, in naringin hydrolysis, the immobilized naringinase performed over a wide pH application range and had good thermal stability. Even more important, the immobilized naringinase retained 61.81% of the residual naringinase activity after eight consecutive cycles, and kept 80.95% of the residual naringinase activity after one month of storage. This study provides an ideal carrier material and some basic data for naringinase immobilization technology, which will greatly promote the application of naringinase in industrial fruit juice processing.

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.

Naringin is a flavonoid naturally present in citrus fruits. It is present in orange, grapefruit, lemon and other fruits of citrus family. Naringin is the major cause of bitterness in citrus fruit juices. This flavonoid may cause interference during the citrus fruit juice processing. Naringinase (E.C.3.2.1.40) is an enzyme which catalyzes the hydrolysis of naringin into prunin and then into naringenin, which is non-bitter and tasteless. Naringinase is produced by many microorganisms but the commercial production of this microbial enzyme is still the area of interest. Naringinase is chiefly produced by various fungal strains and it has been reported for elimination of bitter flavour (due to naringin) in citrus fruit juice. Few bacterial sources have also been reported for naringinase. Besides removal of bitterness, naringainase has potent application in enhancement of aroma in wine making. This review focuses over microbial sources, production and applications of naringinase in food industries.

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.

Naringinase which was extracted from the fermented broth of Cryptococcus albidus was purified about 42-folds with yield 0.7% by sulfate fractionation and chromatography on Toyopearl HW-60, Fractogel DEAE-650-s, and Sepharose 6B columns. Molecular weight of protein determined by gel filtration and SDS-PAGE was 50 kDa. Naringinase of C. albidus includes high content of the dicarbonic and hydrophobic amino acids. Enzyme contains also carbohydrate component, represented by mannose, galactose, rhamnose, ribose, arabinose, xylose, and glucose. The enzyme was optimally active at pH 5.0 and 60 °C. Naringinase was found to exhibit specificity towards p-nitrophenyl-α-L-rhamnose, p-nitrophenyl-β-D-glucose, naringin, and neohesperidin. Its K m towards naringin was 0.77 mM and the V max was 36 U/mg. Naringinase was inhibited by high concentrations of reaction product—L-rhamnose. Enzyme revealed stability to 20% ethanol and 500 mM glucose in the reaction mixture that makes it possible to forecast its practical use in the food industry in the production of juices and wines.

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.

The crude naringinase from Penicillium decumbens and a purified naringinase with high α-L-rhamnosidase activity could be covalently immobilized on two-dimensional zeolite ITQ-2 after surface modification with glutaraldehyde. The influence of pH and temperature on the enzyme activity (in free and immobilized forms) as well as the thermal stability were determined using the specific substrate: p-nitrophenyl-alpha-L-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on ITQ-2 were applied in the hydrolysis of naringin, giving the flavonoids naringenin and prunin respectively with a conversion '90 % and excellent selectivity. The supported enzymes showed long term stability, being possible to perform up to 25 consecutive cycles without loss of activity, showing its high potential to produce the valuable citrus flavonoids prunin and naringenin. We have also succeeded in the application of the immobilized crude naringinase on ITQ-2 for debittering grapefruit juices in a continuous process that was maintained operating for 300 h, with excellent results., The authors acknowledge financial support from PGC2018‐097277‐B‐100 (MCIU/AEI/FEDER,UE) project and Program Severo Ochoa (SEV‐2016‐0683). Jilin agreement 111 Project (Grant No. B17020). JMC thanks to Universitat Politècnica de València for predoctoral fellowships.

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.

A pure yeast Clavispora lusitaniae, isolated from whey beverage, phenotypically characterized and molecularly characterized by sequencing of D1/D2 domain of 26S rRNA and Internal Transcribed Spacer (ITS) region was used to produce low alcoholic naturally carbonated fermented debittered beverage from Grapefruit. C. lusitaniae produces enzyme naringinase. This enzyme is a mixture of α-L-rhamnosidase and β-D-glucosidase. The bitter component in citrus fruit, naringin can be hydrolyzed by α-L-rhamnosidase to rhamnose and prunin then by β-glucosidase to glucose and naringenin. The freshly prepared fermented Grapefruit beverage had TSS 14 °B, pH 4.7, acidity 0.26%, brix acid ratio 53.85, total sugars 11.6%, reducing sugars 3.34%, ascorbic acid 21.9 mg/100 ml, naringin 643.2 ppm, alcohol 0.00% (v/v), CO2 0.00 bar and total yeast count 5.83 (Log no.of cells/ml). Physico-chemical changes recorded after three months of storage at refrigerated temperature revealed TSS 12.0 °B, pH 4.2, acidity 0.54%, brix acid ratio 22.22, total sugars 8.97%, reducing sugars 1.94%, ascorbic acid 18.45 mg/100 ml, naringin 365.2 ppm, alcohol 0.76 % (v/v), CO2 1.35 bar and total yeast count 8.54 (Log no.of cells/ml). Naturally produced CO2 by C. lusitaniae during fermentation adds effervescence, sparkle, tangy taste to the beverage in addition to its antimicrobial properties. Thus bio-enzymatic debittering by C. lusitaniae may become the new direction of citrus juice processing in the future, due to its economical viability with strong ability to remove the bitter taste from citrus juice beverage.

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).

To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin.Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-L-rhamnosidase activity while the β-D-glucosidase activity was abrogated. The addition of Mg(2+), Fe(2+) and Co(2+) was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and (1)H-NMR.A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-L-rhamnosidase activity of naringinase and blocked its β-D-glucosidase activity.

[EN] Production of citrus flavonoids prunin and naringenin was performed selectively through the enzyme hydrolysis of naringin, a flavonoid glycoside abundant in grapefruit wastes. To produce the monoglycoside flavonoid, prunin, crude naringinase from Penicillium decumbens was purified by a single purification step resulting in an enzyme with high -rhamnosidase activity. Both crude and purified enzymes were covalently immobilized on graphene oxide. The activity of the immobilized enzymes at different pH levels and temperatures, and the thermal stability were determined and compared with those exhibited by the free naringinases using specific substrates: p-nitrophenyl--d-glucoside (Glc-pNP) and p-nitrophenyl-alpha-l-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on GO were tested in the hydrolysis of naringin, giving naringenin and prunin, respectively, in excellent yields. The supported enzymes can be reused many times without loss of activity. The naringinase stabilized on GO has high potential to produce the valuable citrus flavonoids prunin and naringenin., Authors acknowledge the financial support from MICINN Project CTQ-2015-67592-P and Program Severo Ochoa (SEV-2016-0683). JVC thanks Universitat Politecnica de Valencia for predoctoral fellowships. JY and AC thank the support from the National Natural Science Foundation of China (Grant No. 21320102001) and the 111 Project (Grant No. B17020).

In India, a variety of agricultural products such as bananas, pomegranates, apples, cashew apples, citrus, litchi, mangos, guava, apricots, pineapples, peaches, berries, grapes, watermelons, passion fruit, carrots, amlas, and tomatoes are produced in plenty. As some of the vegetables/fruits are perishable in nature, the improvement of shelf life plays an important role. An alternative strategy adopted for the proper utilization of such fruits/vegetables is juice preparation for appropriate usage of the raw material. In modern fruit juice manufacturing, industrial enzymes are major fundamental components. The functions of enzymes in juice processing are increasing juice yield, clear and visually attractive juice production, debittering of juice, and improvement of the antioxidant and nutritional properties of juice. The use of the enzymes such as pectinase, tannase, carbohydrase, naringinase, and lipase/esterase alone as well as their blends can not only give better juice yield but also help in retaining enhanced nutrition in the juice. In this chapter, the authors will emphasize not only the cost-effective production of industrial enzymes for their selection in appropriate fruit juice production in the pure as well as mixed form, but also accentuate the titer value, shelf life, appropriate enzyme selection, and nutritional upliftment of the final product. A series of enzymes involved in these processes will include laccase, tannase, pectinase, naringinase, carbohydrase, and a few oxido-reductase along with the juice quality and sensory evaluation for maximum wide acceptability of the fruit/vegetable juices.

Naringinase has high industrial importance, and the progress in naringinase research is still quite slow. The unavailability of an effective, simple screening method, which will be applicable to different microorganisms such as bacteria, fungi, and actinomycetes, is one of the main reasons for this gap. Therefore, a simple plate assay was developed for effective screening of microorganisms for naringinase by exposing to iodine vapors. This plate assay will fill the technological void for simple screening method and will lead to screen more potent industrially important naringinase-producing microorganisms.

Characteristics of naringinase nano-encapsulated forms on different carrier materials (chitosan and alginate polymers) were investigated in this study. Screening of twelve fungal isolates for naringinase production indicated that Trichoderma longibrachiatum was the most promising. Grapefruit rind was used as a substrate containing naringin for naringinase production. TEM micrographs showed that chitosan nano-capsules were applied for the production of morphologically homogeneous enzymatic nano-particles with high enzyme encapsulation efficiency, small asymmetric sizes (from 15.09 to 27.07 nm with the mean of 21.8 nm) and rough surfaces compared to nano-encapsulated naringinase in alginate which showed nano-particle size (from 33.37 to 51.01 nm with the mean of 43.03 nm). It was revealed that the highest naringinase activity was found in case of chitosan nano-capsule naringinase compared to alginate nano-capsule one. Thermogram analysis (TGA) showed that the free enzyme loses about 92% of its weight at approximately 110°C, while the nano-encapsulated ones show more stability at higher temperatures. Conclusively, the nano-capsulation process improves the kinetics and operational stability so could be useful as a debittering agent for various thermal processing applications in citrus juices industries which makes the fruit juice more acceptable and cost-effective to the consumer.

In this work, the structural thermostabilization of the characterized nanomagnetic cross-linked enzyme aggregates of naringinase have been considered. Comparisons have been made between free and immobilized enzyme by the determination of temperature-dependent half-lives (t1/2), energy barriers of thermal inactivation (Ea(in)) process, and thermodynamic parameters (ΔH*, ΔG*, and ΔS*) in a storage thermostability approach. Samples of NM-NGase-CLEAs were treated at different temperatures in the range of 40–80 °C for 90 min. The Km values of immobilized enzyme was reduced about 10.7 folds compared to the free one. The catalytic efficiency (kcat/Km) was raised about 10.5 folds after immobilization. Enzyme half-life (t1/2) of NM-NGase-CLEAs increased from 18.7 to 52.9 min (about 3 folds) at 80 °C. The thermodynamics study indicated that Ea(in) of the free enzyme increased from 38.51 to 49.14 (KJ·mol−1) and ΔH* increased from 35.57 to 46.20 (KJ·mol−1) after immobilization, which indicates an increase in the thermostability of this multimeric enzyme after nanomagnetic CLEAs fabrication. The NM-CLEAs of naringinase preserved 73% of its original activity after 10 cycles, which implies strong operational stability. Thus, the developed method for nanomagnetic CLEAs preparation has provided an efficient and simple approach for the productive and reusable nanobiocatalyst together with ease in enzyme handling.

Resin adsorption and naringinase treatment represent two major methods for debittering naringin in citrus juices, but their effects on the aroma profiles of citrus juices have not been intensively investigated yet. In this study, the volatiles of the fresh, naringinase-treated and resin-absorbed juices of pummelo were investigated by gas chromatography coupled with a mass spectrometer (GC-MS) and sensory evaluation. The fresh juice was analyzed to have 24 volatiles, among which (E)-3-hexen-1-ol and 1-hexanol had the highest concentrations. After the naringinase treatment, the juice showed 32 volatiles, among which (Z)- and (E)-linalool oxides were the most dominant in concentration; while the resin adsorption captured most of the volatiles. Principal component analysis (PCA) showed that naringinase treatment led to an increase in some compounds, particularly aldehydes, whereas resin adsorption decreased the amounts of a lot of volatile compounds. The sensory evaluation demonstrated the fresh and naringinase-treated juices had similar aroma profiles, whereas the resin-adsorbed juice had a much weaker aroma intensity than its counterparts. These results indicated that the naringinase treatment is more desirable than the resin adsorption for debittering citrus juice because the naringinase treatment could maintain the aroma profile close to the original, fresh juice. Copyright © 2015 John Wiley & Sons, Ltd.

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.

Pectinase and naringinase were investigated for improving the production of pummelo juice by increasing the juice yield and eliminating juice bitterness. Compared to a control, the enzymatic treatment significantly (p

Naringinase has attracted a great deal of attention in recent years due to its hydrolytic activities which include the production of rhamnose and prunin and debittering of citrus fruit juices. Screening of fifteen marine-derived fungi, locally isolated from Ismalia, Egypt, for naringinase enzyme production, indicated thatAspergillus nigerwas the most promising. In solid state fermentation (SSF) of the agroindustrial waste, orange rind was used as a substrate containing naringin. Sequential optimization strategy, based on statistical experimental designs, was employed to enhance the production of the debittering naringinase enzyme. Effects of 19 variables were examined for their significance on naringinase production using Plackett-Burman factorial design. Significant parameters were further investigated using Taguchi’s (L1645) orthogonal array design. Based on statistical analysis (ANOVA), the optimal combinations of the major constituents of media for maximal naringinase production were evaluated as follows: 15 g orange rind waste, 30 mL moisture content, 1% grape fruit, 1% NaNO3, 0.5% KH2PO4, 5 mM MgSO4, 5 mM FeSO4, and the initial pH 7.5. The activity obtained was more than 3.14-fold the basal production medium.

Five strains of naringin-degrading bacteria were isolated and found to be positive for extracellular naringinase activity. The one that showed highest activity in the selective medium was identified by 16S rRNA analysis as Bacillus methylotrophicus . The best combination of carbon-nitrogen source was determined by employing two-level full factorial analyses, comprising 24 experiments in shake flasks. Sucrose-yeast extract showed significant increase in naringinase activity (7.46 U/L) compared to the basal medium. Naringinase production was found to be inducible and naringin was found to be the best inducer among naringin, naringenin, hesperidin, and L-rhamnose. Inoculum size of 2% (v/v) and age of 48 hr favored naringinase and biomass production. Highest naringinase activity of 8 U/L was observed at the initial medium pH of 6. Response surface modeling was applied based on central composite design to determine the effects of three independent variables (sucrose, yeast extract, and naringin) and their mutual interactions. In total, 20 experiments were conducted and a statistical model was developed, which predicted naringinase production of 10.61 U/L. Subsequently, verification experiments were conducted and validity of the model was verified. Bioreactor studies conducted with the optimized medium showed an enzyme production of 12.05 U/L within 34 hr of fermentation.

An HPLC method that can separate naringin, prunin, and naringenin was used to help accurately measure the activities of naringinase and its subunits (α-L-rhamnosidase and β-D-glucosidase). The activities of the naringinase and β-d-glucosidase were determined through an indirect calculation of the naringenin concentration to avoid interference from its poor solubility. The measured enzymatic activities of the naringinase complex, α-L-rhamnosidase, and β-D-glucosidase were the as same as their theoretical activities when the substrates' (i.e., naringin or prunin) concentrations were 200 μg/mL, and the enzyme concentrations were within the range of 0.06-0.43, 0.067-0.53, and 0.15-1.13 U/mL, respectively. The β-D-glucosidase had a much higher Vmax than either naringinase or α-L-rhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step of the enzyme reaction. The reliability of the method was finally validated through the repeatability test, indicating its feasibility for the determinations of the naringinase complex.

A cost-effective and environmentally friendly approach was developed to improve the extraction of active ingredients from plants, in which a bifunctional enzyme was employed for not only facilitating cell wall degradation but also increasing the bioactivity of target compounds in the extract. In the aqueous extraction of flavonoids from Glycyrrhizae radix, Trichoderma viride cellulase, a commercial cell-wall-degrading enzyme, was found to efficiently deglycosylate liquiritin and isoliquiritin, which are of high content but low bioactivity, into their aglycones that have much higher physiological activities for dietary and medicinal uses. Under optimized conditions, the extraction yield of liquiritigenin and isoliquiritigenin aglycones reached 4.23 and 0.39 mg/g of dry weight (dw) with 6.51- and 3.55-fold increases, respectively. The same approach was expanded to the extraction of flavonoids from Scutellariae radix using Penicillium decumbens naringinase, where enhanced production of more bioactive bacalein and wogonin was achieved via enzymatic deglycosylation of bacalin and wogonoside.