Naringinase Production Research Articles

Molecular Characterization of a New Bacterial Source of Naringinase as Paenibacillus stellifer RAMCM-44 and Fermentative Production of Naringinase at Shake-Flask Level

Molecular Characterization of a New Bacterial Source of Naringinase as Paenibacillus stellifer RAMCM-44 and Fermentative Production of Naringinase at Shake-Flask Level

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.

Enzymatic debittering of citrus juices: Optimization, modeling, and characterization of naringinase production from marine Bacillus subtilis strain BSnari

The bitterness of citrus juices is a major obstacle to their industrial acceptance. Bitterness can be reduced using the naringinase enzyme. This study investigated two approaches for producing and purifying naringinase. Bacillus subtilis strain Bsnari was isolated from the Red Sea with accession number OQ576096 and had the highest naringinase productivity (7.09 ± 0.09 U/ml) using sucrose (1.5%) as carbon source, 0.6% w/v of citrus peel powder and 1% w/v of soy bean meal at pH 7.0 and 37 °C. Box-Behnken design has been successfully used for statistical modeling of naringinase production by Bacillus subtilis. Naringinase was partially purified using ammonium sulfate and gel filtration, resulting in a specific activity of 215.106 U/mg, a purification fold of 8.95, and a molecular weight of 73 KDa. Enzymatic hydrolysis reduced bitterness by around 33–36%, with optimal debittering after 4 h at 40–50 °C. A new method for processing citrus juice debittering was proposed using α-l-rhamnosidase and β-d-glucosidase encoded by the genes rhaA and bglA, respectively. The protein structures of β-l-rhamnosidase and β-d-glucosidase were also investigated. The topology of the 3D structure of α-l-rhamnosidase and β-d-glucosidase proteins of Bacillus sp. Predicted and aligned against other bacterial species. These findings provide insights into efficient naringinase production and purification for use in various applications, with implications for commercial production in food and pharmaceutical industries.

Improved naringinase double screen plate assay: progress towards the perfect screening

Naringinase is an important enzyme for commercial purposes due to its dual activity as both α-l-rhamnosidase and β-d-glucosidase. The traditional method for screening microbes that produce naringinase involves growing them on naringin agar, but this method has limitations and result in false positive results. This is because the growth on the naringin agar plate could be due to the presence of other organisms that produce rhamnosidase or other glucosidases, or those that use agar as a carbon source, rather than actual naringinase producers. To address these limitations, a double screen plate assay was developed using synthetic substrates, to separately test for β-d-glucosidase and α-l-rhamnosidase activity. The presence of a yellow zone of p-nitrophenol indicates the action of these enzymes, and the intensity of the yellow colour zone indicates the potential for naringinase production. This new screening method is a significant improvement in identifying real naringinase producers and represents progress towards a more reliable screening assay.

Sequential optimization of aspergillus flavus kla-80 for the production of naringinase using stastical experimental designs

Sequential optimization of aspergillus flavus kla-80 for the production of naringinase using stastical experimental designs

Antioxidant, anti-cancer, and debittering potential of edible fungi (Aspergillus oryzae) for bioactive ingredient in personalized foods

Fungal strain Aspergillus oryzae (MTCC 3107) was screened for antioxidant, anti-cancer, and debittering applications. Three different extraction solvents (water, aqueous ethanol 50%, and methanol 100%) were used to extract bioactive compounds from fungal biomass. Further mycelial mass was recovered, dried, and evaluated for mycochemicals and antioxidant properties. Naringinase production by A. oryzae was observed as 19.28 IU/mL. Treatment of Kinnow juice with crude fungal enzyme results a decrease in naringin and limonin content by 59.6% and 41.2% respectively. Bioactive metabolites (phenolics and condensed tannins) in fungal extracts were ranged from 13.73 to 20.08 mg GAE (gallic acid equivalent)/g and 0.62–1.81 mg CE (catechin equivalent)/g, respectively. Mycochemicals observed in fungal extracts were flavonoids, steroids, tannins, and saponins. Fungal extract showed the presence of gallic acid (1.18–2.43 μg/g), catechol (1.60 μg/g), ascorbic acid (2.9–4.09 μg/g), p-Coumaric acid (0.56–0.83 μg/g), benzoic acid (0.33–0.91 μg/g), and cinnamic acid (0.17 μg/g). An aqueous-alcohol (ethanol 50%) extract prepared from dried fungal biomass (45 °C, 30 min) showed significant antitumor potential against model MCF-7 breast cancer cell line by about 44% inhibition. After toxicological analysis, A. oryzae extracts could be used in the preparation of health benefiting food and pharmaceutical formulations.

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.

Production of Naringinase by using Amla on Solid State Fermentation

Microbial enzymes are widely used in different pharmaceutical, textile and in leather industries, mainly because of vast availability of sources. They could be genetically modified and are considered as more economical in comparison to plant and animal enzymes. Production of microbial enzymes by application of fermentation involves microbial propagation like bacteria, mold and yeast to get useful product. There are two methods of fermentation used to produce microbial enzymes called submerged fermentation and solid-state fermentation. Submerged fermentation involves the production of enzymes by using microorganisms in a liquid state nutrient media. Solid-state fermentation is the cultivation of microorganisms in solid substrate. Nutrients containing carbon compounds are broken down by the microorganisms, which produce the enzymes either intracellular or extracellular. Industries that use enzymes generated by fermentation are the brewing, wine making, baking, cheese making, dairy, beverages, and cereals. In the present study Asperigillus Niger strain was used to produce the extra cellular naringinase enzyme in nutrient medium containing Amla as a solid substrate. Amla is the chief material for the production maximum Naringinase enzyme. The study also involved in the optimization of various physical parameters like temperature, PH, incubation period, mass of inoculum. The study concluded that pH -5.5, temperature of 28oc, incubation period of 96 hrs and 20% of inoculums for maximum naringinase production.

Preparation of Aspergillus niger 426 naringinases for debittering citrus juice utilization of agro-industrial residues.

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 2.3g of grapefruit peel, 2.5g of rice bran, and 5.2g of wheat bran and medium supplementation with a mixture of naringin, rutin, and hesperidin in the concentration of 2, 5, 4.5, and 3.0g/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.

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.

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.

Changes in bitterness, antioxidant activity and total phenolic content of grapefruit juice fermented by Lactobacillus and Bifidobacterium strains

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.

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.

NARINGINASE: MICROBIAL SOURCES, PRODUCTION AND APPLICATIONS IN FOOD PROCESSING INDUSTRY

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.

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.

Optimization of naringinase production from Aspergillus flavus in solid state fermentation media using citrus peel as support

Optimization of naringinase production from Aspergillus flavus in solid state fermentation media using citrus peel as support

Otimização da produção de naringinase de Aspergillus niger por metodologia de superfície de resposta

Citrus juice for export has a slight bitter taste which is characteristic and acceptable by the consumer but high levels of naringin reduce its quality and commercial value. Naringin (a flavonoid linked to a rhamnose and glucose) is the main bitter component of several citrus fruits. The reduction in bitterness may be achieved by naringinase, an enzyme that degrades naringin. This study aimed the production of naringinase of Aspergillus niger by fermentation of low-cost substrates from agroindustry. The experiment was planned according to a 2³ factorial design which included three replications at the center point having as variables (g/L): naringin (0.20; 0.30 and 0.40); soy bean molasses (0.35; 1.05 and 1.75 of total sugars) and sugar cane molasses (0.35; 1.05 and 1.75 of total sugars). Using the results of 144 hours of fermentation the statistical analysis and predictions were carried out. The statistical analysis showed that the significant variables for the production of naringinase were sugar cane molasses and soy molasses, as long as naringin was used at its lowest concentration. The predictive analysis showed a maximum value of 1.35U/mL could be achieved when naringin (0.25g/L), sugar cane molasses (1.75g/L) and soy bean molasses (1.75g/L) were used. When the validity test was carried out a value of 1.344 (99.3% of prevision) was obtained.

Optimization of Process Parameters by Statistical Experimental Designs for the Production of Naringinase Enzyme by Marine Fungi

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.