Naringinase Activity Research Articles

Impact of submerged culture different parameters on growth and naringinase activity of Bacillus amyloliquefaciens D1 isolated from citrus gardens of North-East India

The study aims at screening, isolation, and characterization of naringinase enzyme producing bacterial strains from soil samples. In this study, 45 bacterial isolates were screened for naringinase activity, and among them, 20 bacterial isolates are found to be capable of naringinase production in liquid mineral medium. Bacterial isolate D1 exhibited the highest naringinase activity in liquid mineral medium. Based on the morphological, biochemical, fatty acid profile, and molecular characterization, bacterial isolate D1 was identified as Bacillus amyloliquefaciens. Impact of different submerged culture parameters were evaluated in different experimental conditions. The study revealed that 48 hrs of incubation periods, pH 6.0 and temperature 350 C is optimal for naringinase activity when the culture medium is supplemented with starch and yeast extract as carbon and nitrogen sources. Thus, it can be concluded that due to the potential naringinase activity of the strain Bacillus amyloliquefaciens D1, further the identifying strain may be used in citrus processing and bioprocess industries upon large scale field applications.

Isolation and Molecular Characterization of the Naringinase Producing Micro-organisms for the Bio-transformation of Flavonoid

Naringin is a flavanone found in citrus fruits and vegetables which are responsible for the asperity, due to which citrus fruit are unable to consume and from which we are unable to get the valuable nutrition in our body. To overcome this asperity in citrus fruits, present investigation is carried out, which provides a detailed report of isolation and screening of naringinase producing micro-organisms from different samples collected from Kalyan -Karnataka region, India. A sum of around 50 strains were isolated from 20 different samples which showed the positive result for naringinase activity, among 50 strains obtained 42 strains are fungi and remaining 8 were bacteria which showed good to moderate results in preliminary screening by 1% FeCl3 method, further 5 fungi isolates which showed a good naringinase activity in submerged fermentation were assayed by Spectrophotometric method. As a result, KLA-80 showed a maximum result with an activity of 559 U/ml. Further from the identification of the isolate KLA-80 by BLAST analysis found to be having similarity with Aspergillus flavus and the accession number of OQ152018 is obtained.

Production of Prunin and Naringenin by Using Naringinase from Aspergillus oryzae NYO-2 and Their Neuroprotective Properties and Debitterization.

Naringin is a flavanone glycoside in citrus fruits that has various biological functions. However, its bitterness affects the quality, economic value, and consumer acceptability of citrus products. Deglycosylation of naringin using naringinase decreases its bitterness and enhances its functional properties. In this study, eight microbial strains with naringinase activity were isolated from 33 yuzu-based fermented foods. Among them, naringinase from Aspergillus oryzae NYO-2, having the highest activity, was used to produce prunin and naringenin. Under optimal conditions, 19 mM naringin was converted to 14.06 mM prunin and 1.97 mM naringenin. The bitterness of prunin and naringenin was significantly decreased compared to naringin using the human bitter taste receptor TAS2R39. The neuroprotective effects of prunin and naringenin on human neuroblastoma SH-SY5Y cells treated with scopolamine were greater than that of naringin. These findings can widen the potential applications of deglycosylation of naringin to improve sensory and functional properties.

Simultaneous production and sustainable eutectic mixture based purification of narringinase with Bacillus amyloliquefaciens by valorization of tofu wastewater

The current investigation is being executed for sustainable one-pot production and purification of naringinase using natural deep eutectic solvent-based extractive fermentation. Five natural deep eutectic solvents were prepared and their physicochemical properties were determined as a function of temperature. Tofu wastewater was used as a low-cost substrate for naringinase production and simultaneous in-situ purification of the enzyme was accomplished by employing NADES. Optimal conditions of influential factors like concentrations of NADES (74.5% w/w), Na2SO4 (15% w/v) and tofu wastewater (1.5% w/w) resulted in an effective yield of naringinase (249.6 U/ml). Scale-up of naringinase production with a 3 l custom made desktop bioreactor was accomplished and effective regeneration of NADES was established. NADES exhibits selectivity during extraction even after the fifth cycle proving it to be tailor-made. The resulting active enzyme was quantified by size exclusion chromatography (736.85 U/mg). Ultrapure enzyme fraction was obtained with anion exchange chromatography yielding maximum purity of (63.2 U/ml) and specific naringinase activity of (3516 U/mg). The in-vitro debittering activity of the resulting ultrapure enzyme fraction was determined with grape juice resulting in naringin and limonin removal of [23.4% (w/w)] and [64.3% (w/w)] respectively.

Immobilization of naringinase on asymmetric organic membranes: Application for debittering of grapefruit juice

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 relevanceThe 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.

Optimization of process parameters for naringinase production by Aspergillus tubingensis UA13 and pilot scale-up study

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.

Facile preparation of immobilized naringinase on polyethylenimine/dopamine‐coated hydrothermal carbon spheres with high performance

AbstractBACKGROUNDMussel‐inspired surface chemistry is recognized as a simple, efficient, and mild surface modification method, and has become a research hotspot in many fields. Hydrothermal carbon is a bio‐based material that is referred to as environmentally friendly renewable resource. In this study, the polyethylenimine (PEI)/dopamine (DA) was coated on the surface of hydrothermal carbon spheres using the co‐deposition method, making it possible to immobilize naringinase with high activity and operation stability. Subsequently, the optimal modification and immobilization conditions as well as enzyme properties were investigated.RESULTSThe naringinase activity reached up to 461.14 U g–1 carrier, which was much higher than those of the previous works. Besides, the residual naringinase activity still kept 76.44% of the initial enzyme activity after one month of storage, and maintained 62.01% of the initial enzyme activity after eight reuse cycles.CONCLUSIONTherefore, the strategy of the immobilized enzyme on PEI/DA coated hydrothermal carbon could be recognized as a promising and universal enzyme immobilization method with the advantages of high relative enzyme activity, high enzyme loading rate, high enzyme activity recovery rate, good operational stability, and facile and environmental friendly preparation route. © 2021 Society of Chemical Industry (SCI).

Novel Strategy of Mussel-Inspired Immobilization of Naringinase with High Activity Using a Polyethylenimine/Dopamine Co-deposition Method.

Mussel-inspired surface chemistry is recognized as a simple, efficient, and mild surface modification method and has become a research hotspot in many fields. In this study, polyethylenimine/dopamine was coated on the surface of SBA-15 using a co-deposition method, making it possible to immobilize naringinase with high activity and operation stability. The optimal modification and immobilization conditions as well as enzyme properties were investigated. The naringinase activity can reach up to 753.78 U/g carrier, which was much higher than those of the previous works. Besides, the residual naringinase activity still kept 78.91% of the initial activity after one month of storage and maintained 60.79% after 8 cycles. Therefore, the strategy of mussel-inspired enzyme immobilization could be recognized as a promising and universal enzyme immobilization method, with the advantages of high relative enzyme activity, enzyme carrying rate, enzyme activity recovery rate, and good reusability and storage stability.

Controllable biotransformation of naringin to prunin by naringinase immobilized on functionalized silica

AbstractBACKGROUNDMesoporous silica materials, with high specific surface area, tailorable pore size and excellent chemical stability, have wide prospects for application in catalyst carriers. In this work, aldehyde‐modified silica materials were first prepared through silanization reaction of N‐[3‐(trimethoxysilyl)propyl]ethylenediamine and then Schiff base reaction of glutaraldehyde. Subsequently, the resulting materials were used for immobilization of naringinase.RESULTSThe results demonstrated that the activity of immobilized naringinase was optimized as 352.83 U g−1 support. The optimal immobilization conditions were determined as follows: immobilization temperature of 30 °C, immobilization time of 3 h, pH of 3.5 and initial naringinase activity of 200.75 U mL−1. In addition, at the optimal reaction temperature of 50 °C and pH of 8.0, the percentage of immobilization, naringinase activity recovery and naringinase activity of the immobilized naringinase reached 56.41%, 93.42% and 423.22 U g−1 support, respectively. Besides, comparing with free naringinase, in the process of naringin hydrolysis, the immobilized naringinase exhibited wide pH application range and good thermal stability, and still maintained 71.13% residual naringinase activity after 1 month of storage. Even more important, the immobilized naringinase could controllably transform naringin to prunin as the only hydrolysis product, and no naringenin was detected, which represents a new route for controllable preparation of prunin.CONCLUSIONSThis study provides an ideal carrier material and some basic data for naringinase immobilization technology, which will greatly promote the application of naringinase and the development of the pharmaceutical industry. © 2020 Society of Chemical Industry

Identification and iterative combinatorial mutagenesis of a new naringinase-producing strain, Aspergillus tubingensis MN589840.

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, 2475·16Umg-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.

Fermentative Production of Naringinase from Aspergillus niger van Tieghem MTCC 2425 Using Citrus Wastes: Process Optimization, Partial Purification, and Characterization.

Enzymatic hydrolysis of naringin by the action of naringinase is one of the standard practices adopted in the citrus fruit juice industry for debittering. In the present study, a submerged fermentation condition was optimized for producing naringinase from Aspergillus niger van Tieghem MTCC 2425. As per Placket-Burman design, pH (3-5), incubation temperature (26-30°C), and inducer concentration (12-18g·L-1) were the most important factors influencing the naringinase production. Naringin from citrus waste was used as an inducer. A rotatable central composite design was employed on these three variables and the numerical optimization predicted that fermentation at 29.8°C, pH4.7, and inducer concentration of 14.9gL-1 would yield a maximum naringinase activity of 545.2IUg-1. During partial purification, ion exchange chromatography led to a 9.92-fold increase in enzyme activity resulting a specific activity of 5460IUg-1 with an activity recovery of 17%. As reflected by SDS-PAGE profile, the partially purified naringinase showed the molecular weight bands of 10-20, 65, and 80kDa, respectively. The purified form of enzyme showed optimum stability at pH5 and 50°C. The naringinase activity was completely retained up to 150days when stored at 4°C.

Isolation of naringinase producing soil bacteria from <em>Psidium guajava</em> L. and <em>Terminalia chebula</em> Retz and its enzymatic activity

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.

Nano-encapsulation of naringinase produced by Trichoderma longibrachiatum ATCC18648 on thermally stable biopolymers for citrus juice debittering.

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.

The study of the characteristics and hydrolysis properties of naringinase immobilized by porous silica material.

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.

Quality changes in high hydrostatic pressure and thermal pasteurized grapefruit juice during cold storage.

This study evaluated the effects of high hydrostatic pressure (HHP) and thermal pasteurization (TP) on microbial counts, physicochemical properties, antioxidant characteristics, naringin and naringenin contents, and naringinase activity of grapefruit juice during 21 days cold storage period. Results showed that HHP and TP significantly decreased the total microbial, coliform, and yeast counts. No significant differences between HHP-treated grapefruit juice (600MPa/5min) and untreated fruit juice with respect to physicochemical properties such as total titratable acidity, pH, and total soluble solids was observed after 21 days of storage. Although HHP affected the colour and antioxidant characteristics of grapefruit juice, the extent of effect was significantly lower than that for TP-treated fruit juice. This demonstrated that HHP could better maintain the original flavour and quality of grapefruit juice compared to TP. In addition, 92% naringinase activity was maintained in HHP-600 group on Day 21, which increased the degradation of bitter naringin into non-bitter naringenin during the cold storage of grapefruit juice. In summary, HHP can simultaneously maintain the microbiological safety of grapefruit juice along with its original quality characteristics. HHP can effectively extend the storage period and safety during cold chain transport, and hence highly applicable in the grapefruit juice industry.

Characterization of a naringinase from Aspergillus oryzae 11250 and its application in the debitterization of orange juice

Abstract 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.

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

ABSTRACTNaringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04 mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14 mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778 U/L at 34 hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164 U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.

Characterization of best naringinase producing fungus isolated from the citrus fruits

Naringinase enzyme has potential application in food and pharmaceutical industry. Naringin and limonin are principle bitter components in the citrus fruit. The microorganisms that associate with citrus fruit may have the ability to degrade the naringin by extracellular naringinase enzymes that are produced by microorganisms. The objective of the study is to isolate naringinase producing fungus from the citrus fruit to debitter the citrus juice and to characeterize the fungus. Citrus fruits were allowed to spoil under the air and soil and the lesion was used to streak on fresh PDA plates. Out of the eight strains isolated from citrus fruits, five were positive for naringinase enzyme. When all the naringinase producing fungi were subjected to liquid fermentation medium for eight days at room temperature at 200 rpm and the crude enzyme was tested for naringinase enzyme at pH 5 and 50 ºC for 10 minutes, one strain showed the best naringenase activity (1.92 µmol/ml/min). This strain was identified as Aspergillus flavus based on the macroscopic, microscopic and biochemical tests. The culture conditions were optimized to increase the naringinase production via solid state fermentation system using paddy husk as the support. Though naringinase activity of Aspergillus flavus has started on the 2nd day, the highest activity (449.58Ug-1Dry Matter) was obtained on the 8th day. Thereafter the naringinase activity has started to decline. Solid state fermentation using paddy husk as support could be used for large scale naringinase enzyme production.

Optimization of naringinase production by Rhizophus stolonifer in solid state fermentation media using paddy husk as support

The palmyrah (Borassus flabellifer L) tree represents a major socio economic factor in tropics. The economic value of the tree is reduced by fruit pulp, due to the bitter compound present in the fruit pulp but it can be hydrolyzed by the usage of naringinase enzyme. Hence this enzyme plays a key role in the food industry and pharmaceuticals for its debittering nature. Some microbes like bacteria and fungiare involved in the natural production of cost effective naringinase. The aim of this work was to optimize the cultivation conditions and the media to produce naringinase by solid state fermentation of Rhizophus stolonifer, using paddy husk as the support. The highest enzyme activity was obtained on the 7 th day of incubation. Highest naringinase enzyme activity (57.97U/g DM) was obtained on the 7 th day of incubation and after that activity started to decline. The enzyme activity (212.885U/g DM) obtained with 0.75% naringin concentration was significantly higher than that of other naringin concentrations tested. When the regular medium (0.5% glucose) was replaced with (w/v) 1.5% glucose and 1.5% sucrose, the enzyme activity obtained with 1.5% of glucose (484.96U/g DM) was significantly higher than that of 1.5% sucrose (238.88U/g DM). When naringinase activity was tested with different glucose concentrations, activity obtained with 1.5% of glucose concentration was significantly higher than that of other concentrations. Among the nitrogen sources tested, when 0.5 % ammonium nitrate was used significantly higher naringinase was produced by Rhizophus stolonifer than any other sources and their concentrations. The cost to produce 1073.97U/g DM was reduced by 20.15% when conditions and media composition are optimized when compared to the basal medium(57.97U/g DM). Therefore when naringinase was produced by Rhizophus stolonifer under optimized condition, the production cost was marginally reduced.

Highly selective and efficient biotransformation of linarin to produce tilianin by naringinase.

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.