Recyclable enzymatic recovery of pectin and punicalagin rich phenolics from waste pomegranate peels using magnetic nanobiocatalyst

Recyclable enzymatic recovery of pectin and punicalagin rich phenolics from waste pomegranate peels using magnetic nanobiocatalyst

Food Hydrocolloids 89 (2019) 468–480 Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd ...

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Food Hydrocolloids 89 (2019) 468–480

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Recyclable enzymatic recovery of pectin and punicalagin rich phenolics from waste pomegranate peels using magnetic nanobiocatalyst

T

Sachin Talekara,b, Antonio F. Pattic, R. Vijayraghavanc, Amit Aroraa,b,∗ a

IITB-Monash Research Academy, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India Bioprocessing Laboratory, Centre for Technology Alternatives for Rural Areas (CTARA), Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India c School of Chemistry, Monash University, Wellington Road, Clayton, Victoria, 3800, Australia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Immobilized enzyme Magnetic nanoparticles Pomegranate peels Hydrocolloid Phenolics

We demonstrate a novel recyclable enzymatic approach of extraction of pectin and punicalagin rich phenolics from the waste pomegranate peels (WPP) using the magnetic nanobiocatalyst technology. First, the cellulase was cross-linked with the iron oxide magnetic nanoparticles using glutaraldehyde to prepare the magnetic nanobiocatalyst which was characterized using FT-IR, FE-SEM, and TEM. Then, ultrasonically pre-treated WPP were hydrolyzed with the magnetic nanobiocatalyst at cellulase loading of 75 U/g of peel powder, pH 6 and 50 °C for 5 h to obtain maximum recovery of pectin (19.4%) and total phenolics (8.8%) comparable to non-green conventional methods. The magnetic nanobiocatalyst was easily recovered by the application of a magnetic field and recycled for five cycles with no change in pectin and total phenolics yields and cellulase activity. In addition, IR, 1H NMR, and TGA analysis showed that the chemical nature of pectin was the same in each cycle. The uronic acid content (72–74%), degree of esterification (62–64%), molecular weight (140–143 kDa) and polydispersity (1.5–1.6) of pectin from each cycle were also in the similar range. The total phenolics obtained in each cycle were rich in punicalagin (75–78% of total phenolics) with similar content of 6.42–6.65 g/100 g WPP. These results indicated the excellent stability and reusability of the cellulase magnetic nanobiocatalyst for the recovery of WPP pectin and phenolics of consistent yield and chemical nature/composition.

1. Introduction Over the last 10 years, the pomegranate processing industry has significantly expanded due to the popularity and demand of pomegranate juice which imparts numerous health benefits (Hasnaoui, Wathelet, & Jiménez-Araujo, 2014; Talekar, Patti, Singh, Vijayraghavan, & Arora, 2018a). Production of each ton of concentrated pomegranate juice generates up to 5–5.5 tons of waste pomegranate peels (WPP) which is a major environmental concern for the pomegranate processing industry (Hasnaoui et al., 2014). The WPP constitute two products of commercial value: pectin (20–25%) and phenolics (10–20%) (Talekar, Patti, Vijayraghavan, & Arora, 2018b). Pectin is a valued commercial hydrocolloid used for gellation, thickening, stabilization, and encapsulation of the cosmetic, food, medical and personal care products (Ciriminna, Chavarría-Hernández, Inés Rodríguez Hernández, & Pagliaro, 2015). WPP phenolics, especially punicalagin, possesses a variety of human health beneficial effects due to their antioxidant potential, thus are used as active component in

neutraceutical, cosmetic and pharmaceutical products (Viuda-Martos, Fernández-López, & Pérez-Álvarez, 2010). The WPP, therefore, represents a resource for the recovery of phenolics and pectin as potentially marketable products using a biorefinery concept. However, most of the industrial practices and research carried out on pomegranate peel utilization nearly target only the extraction of the phenolics. In addition, these extracted phenolics are standardized to ellagic acid content and devoid of punicalagin, a key phenolic compound responsible for most of the pomegranate health benefits (López, Streitenberger, Peñalver, & Martínez, 2010). Few reports present the extraction of punicalagin rich phenolics but still employ non-food grade toxic organic solvents (Lu, Ding, & Yuan, 2010; Lu, Wei, & Yuan, 2007), which create environmental problems due to their emission, require high infrastructure cost for safety and multiple refining steps to eliminate them from the final product. The pectin has been recovered from the WPP using mostly the conventional mineral acid environment (Abid et al., 2017; Moorthy, Maran, Muneeswari, Naganyashree, & Shivamathi, 2015; Pereira et al., 2016) and recently the hydrothermal

∗ Corresponding author. Bioprocessing Laboratory, Centre for Technology Alternatives for Rural Areas (CTARA), Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India. E-mail address: [email protected] (A. Arora).

https://doi.org/10.1016/j.foodhyd.2018.11.009 Received 3 August 2018; Received in revised form 22 October 2018; Accepted 5 November 2018 Available online 10 November 2018 0268-005X/ © 2018 Elsevier Ltd. All rights reserved.

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2. Materials and methods

processing (Talekar, Patti, Vijayraghavan, & Arora, 2018b). However, these pectin extraction processes may hydrolyze punicalagin along with other phenolics due to high temperature and acidic conditions (López et al., 2010) and thus are not favourable for the recovery of punicalagin. In addition, the conventional mineral acid extraction of pectin generates acidic effluent that requires neutralization before disposal and causes corrosion, rapid wearing out of the equipment and undesirable breakdown of the pectin chain (Adetunji, Adekunle, Orsat, & Raghavan, 2017). Thus, there exists a need to provide a holistic approach to derive both pectin and punicalagin rich phenolics from WPP, wherein the use of high temperature, acidic conditions, and the toxic organic solvent is eliminated. An attractive strategy is the enzymatic extraction of pectin and phenolics from WPP since the enzymes work in an aqueous medium at ambient temperature and physiological pH (Sheldon & Woodley, 2017). The application of enzymes that selectively degrade the cell wall components has been proposed for the pectin extraction from citrus and apple waste in an acid-free aqueous medium at low temperature (Adetunji et al., 2017). If applied on WPP, it would also release punicalagin in intact form as a co-product as a result of cell wall degradation at mild conditions. Thus, the treatment of WPP with selective cell wall component degrading enzyme would allow co-recovery of pectin and punicalagin rich phenolics, without using hazardous chemical reagents/organic solvents, generation of minimal chemical waste, saving of time, and simplification of the practical aspects in compliance with the green chemistry basic principles (Sheldon, 2017). Nevertheless, enzymatic processes are still costly due to poor recovery, reusability, stability, and the high production cost of enzymes, thus pegging back their industrial application (Sheldon, 2017). To overcome these limitations in industrial applications, enzyme immobilization onto the solid support can be a sustainable approach as it improves the operational lifetime, stability, recovery, and reusability of enzymes thus reduces enzyme costs per kilogram of product (Sheldon, 2017). However, the major challenges for the application of immobilized enzymes are tedious separation of solid immobilized enzymes from solid biomass (such as WPP in the present study) after the reaction and low mass transfer efficiency of immobilized enzymes in the highly viscous reaction mixture of solid biomass substrate (S. H. Hosseini, Hosseini, Zohreh, Yaghoubi, & Pourjavadi, 2018; Khoshnevisan et al., 2017). To overcome these challenges, immobilized enzyme onto magnetic nanoparticles (MNPs) could be an elegant approach owing to the easy and selective enzyme separation after the reaction with a magnetic field and high dispersion with improved mass transfer of immobilized enzyme on MNPs (Cipolatti et al., 2016; Khoshnevisan et al., 2017). Complementary to that, MNPs have tunable surface functionalities, outstanding stability and high surface enzyme loading capacity (Cipolatti et al., 2016; Rossi, Costa, Silva, & Wojcieszak, 2014; Safarik, Pospiskova, Baldikova, & Safarikova, 2016; Vaghari et al., 2016). Thus, to explore this potential of MNPs, for the first time, we demonstrate a novel enzymatic extraction of pectin and punicalagin rich phenolics from WPP using the recyclable magnetic nanobiocatalyst (Fig. 1). In this process, the cell wall degrading enzyme cellulase was immobilized on MNPs to prepare the cellulase magnetic nanobiocatalyst and then, WPP was treated with the cellulase magnetic nanobiocatalyst in an aqueous medium to release pectin and punicalagin rich phenolics. Before the cellulase magnetic nanobiocatalyst treatment, the WPP was pretreated by ultrasound to enhance the access of nanobiocatalyst to cell wall cellulose which improves the cell disintegration and thus increases the overall extraction rate. In order to check the durability and industrial feasibility, the reusability of the magnetic nanobiocatalyst was studied over five cycles of extraction of pectin and punicalagin rich phenolics.

2.1. Materials The cellulase (EC 3.2.1.4) from Trichoderma reesei, DNS (3,5Dinitrosalycyclic acid), ferric chloride hexahydrate (FeCl3.6H2O), Folin-Ciocalteu reagent, ferrous sulphate heptahydrate (FeSO4.7H2O), commercial citrus pectin, glutaraldehyde (25%, v/v), carboxymethyl cellulose, 3-aminopropyl triethoxysilane (APTES), punicalagin, gallic acid, ellagic acid, D2O, etc., were obtained from Sigma Aldrich. Fresh WPP were provided by a pomegranate processing company in Australia. The peels were oven dried for 24 h at 50 °C, ground into powder (particle size < 150 μm) using a laboratory grinding mill and stored in plastic bags for further use.

2.2. Preparation of cellulase magnetic nanobiocatalyst The alkaline coprecipitation of FeCl3 and FeSO4 was employed to prepare the magnetic nanoparticles (MNPs) as described previously (Talekar et al., 2017). Typically, the FeSO4.7H2O and FeCl3.6H2O solutions (100 mL each) were mixed together in Fe2+:Fe3+ molar ratio of 1:2, heated to 90 °C and slowly added with 25 mL of sodium hydroxide (5 M) under rapid stirring. The dark solution obtained was further incubated for 30 min and decanted to discard the supernatant. The black precipitate of MNPs was washed with DI water to pH 7 and vacuum dried. Next, the dry MNPs were coated with amino groups via the APTES silanization previously described (Liu, Li, Li, & He, 2013): MNPs (0.5 g) were added to the 100 mL solution containing 50 mL DI water and 50 mL ethanol and 2 mL APTES, shaken for 4 h at 60 °C, decanted by magnetic field, washed several times with ethanol-DI water mixtures followed by drying at 50 °C to obtain amino coated MNPs (MNP-NH2). Finally, the cellulase was immobilized onto MNP-NH2 by glutaraldehyde (GA) cross-linking. The MNP-NH2 (18 mg) was added into 50 mM sodium citrate buffer of pH 4.8 containing cellulase (50 U with 72 mg protein) and glutaraldehyde (70 mM) and the mixture was shaken at 180 rpm and 25 °C for 6 h. The cellulase bound MNPs (magnetic nanobiocatalyst) were separated using a magnetic field, three times washed with sodium citrate buffer (50 mM, pH 4.8), freeze-dried and stored at 4 °C. The washings and the leftover reaction mixture were assayed for the cellulase activity as described below. The cellulase activity recovery in the magnetic nanobiocatalyst was determined as the percent ratio of cellulase activity of magnetic nanobiocatalyst to the free enzyme's cellulase activity of taken for preparation of the magnetic nanobiocatalyst.

2.3. Cellulase activity assay The 1% (w/v) sodium carboxymethyl cellulose in 50 mM sodium citrate buffer of pH 4.8 was used as a substrate for the determination of the cellulase activity (Ghose, 1987). The DNS methodology was used to measure the released reducing sugars equivalent to glucose (Miller, 1959). The cellulase activity (1U) was calculated as the quantity of cellulase that produces 1 μmol of reducing sugar per minute at 50 °C.

2.4. Characterization of the magnetic nanobiocatalyst The ATR-IR (FT-IR Agilent Technologies Spectrophotometer model Cary 640) analysis of neat MNPs and magnetic nanobiocatalyst was performed for the confirmation of the cellulase binding onto MNPs. The field emission scanning electron microscopy (FESEM, ZEISS Gemini SEM 500) and transmission electron microscopy (TEM, PHILIPS CM200) were used to study the morphology and size of the neat MNPs and magnetic nanobiocatalyst. 469

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Fig. 1. Schematic presentation of extraction of pectin and phenolics from WPP using the recyclable magnetic nanobiocatalyst.

765 nm (R2 = 0.99). The yield of pectin and phenolics was determined using eqns. (1) and (2), respectively.

2.5. Recovery of pectin and punicalagin rich phenolics from WPP Prior to the magnetic nanobiocatalyst treatment, the WPP powder was pretreated with ultrasound to improve the porosity of peels which would further improve the access of magnetic nanobiocatalyst to peel's cellulosic structure. Typically, WPP powder (2 g) was suspended in a 50 mM K-phosphate buffer of pH 6 at 15 mL/g liquid-solid ratio and subjected to ultrasound treatment at a fixed frequency of 37 kHz and 50 °C for 20 min. The resulting suspension was added with the magnetic nanobiocatalyst (cellulase dosage: 75 U/g WPP peel powder) and stirred for 5 h at 180 rpm and 50 °C. Then, the magnetic nanobiocatalyst was recovered using an external magnet followed by the centrifugation (20 min at 2840 g) of the reaction mixture to separate the aqueous solution containing pectin and phenolics from solids. The pectin precipitation was done by mixing the aqueous solution with an equal volume of ethanol under rapid stirring for 5 min and incubating for 12 h at 4 °C. Then, the precipitated pectin was separated using centrifugation in a previous way, ethanol washed and oven-dried at 60 °C to constant weight. Then, the total phenolic content (TPC, equivalent to gallic acid milligrams) of the ethanolic aqueous extract obtained after pectin removal was measured using the Folin-Ciocalteu method (Ainsworth & Gillespie, 2007). For this, a gallic acid calibration curve in the range of 0.2–10 mg/mL was developed by spectrophotometric measurements at

Pectin yield (%) =

Weight of pectin extracted (g db) × 100 Weight of WPP used (g db)

Phenolics yield (%) =

Weight of phenolics extracted (g db) × 100 Weight of WPP used (g db)

(1)

(2)

Finally, the ethanolic aqueous extract was distilled to remove ethanol which was recycled for pectin precipitation. The remaining aqueous phenolic extract was kept at 4 °C under dark until the LC-UV/ MS analysis. The pectin and phenolics from WPP were obtained by the free cellulase for comparison. The WPP powder (2 g) was suspended in 50 mM sodium citrate buffer of pH 5 at 15 mL/g liquid-solid ratio and subjected to ultrasound treatment at a fixed frequency of 37 kHz and 50 °C for 20 min. The free cellulase was added to this suspension at a dosage of 65 U/g of peel powder and shaken at180 rpm and 50 °C for 4 h followed by the centrifugation (20 min at 2840 g) of the reaction mixture to separate the aqueous solution containing pectin and phenolics from solids which was treated similarly as given in the case of the magnetic nanobiocatalyst. 470

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centrifugation to collect the supernatant. The 40 μL of 4 M sulfamic acid/potassium sulfamate was mixed with 400 μL of supernatant or galacturonic acid standard followed by the addition of 2.4 mL of 75 mM sodium tetraborate/sulfuric acid and heating for 20 min at 100 °C. After cooling, 80 μL of 0.5% NaOH containing 8.8 mM m-hydroxydiphenyl was added and mixed for 10 min to develop the uronic acid colouring. To eliminate the sugar colouring the mixture added with 80 μL of plane 0.5% NaOH was used as the sample control. The absorbance of all the samples was measured at 525 nm.

2.6. Recycling and reusing of magnetic nanobiocatalyst The recycling capacity of the magnetic nanobiocatalyst was evaluated by subjecting it to the extraction of pectin and punicalagin rich phenolics from WPP in batch mode as described above. After 5 h batch extraction, the magnetic nanobiocatalyst was recovered using a magnet, washed twice using 50 mM K-phosphate buffer of pH 6 and then applied for a new extraction reaction. At the end of each batch reaction cycle, the yields of pectin and total phenolics were determined by isolating the pectin with ethanol precipitation of reaction mixture and measuring the TPC in remaining ethanolic aqueous extract as described above. The pectin analysis was done in terms of FTIR, NMR, TGA, degree of esterification, molecular weight analysis, and total phenolics were characterized in terms of their phenolic composition using LC-UV/MS analysis. The reusability of magnetic nanobiocatalyst was assessed based on yields and characteristics of pectin and total phenolics obtained from each batch reaction cycle. After each cycle, the residual cellulase activity of the magnetic nanobiocatalyst was also measured by considering the cellulase activity of the fresh magnetic nanobiocatalyst as 100%.

2.8.5. DE by 1H NMR For the determination of the DE by the 1H NMR method of MüllerMaatsch et al. (2014), the pectin was de-esterified with 0.4 M NaOH in D2O for 2 h at 30 °C in presence of maleic acid (0.1 mg/mL) as an internal standard. As shown in Fig. S1 the 1H NMR spectra of the clear reaction mixture (obtained by centrifugation followed by filtration with 0.2 μm nylon filter) were run on a Bruker Advance II 400 NMR spectrometer under 7196.8 Hz spectral width, temperature at 25 °C, 5 s relaxation delay and number of scans 64. The methanol was quantified by the comparision of integrated methanol peak (3.358 ppm) with the peak area of the maleic acid and conversion of these integrals to mass (mg) as described previously (Caligiani, Acquotti, Palla, & Bocchi, 2007). Based on the amount of uronic acid previously determined and methanol, the DE was calculated as the methanol moles: uronic acid moles percentage ratio.

2.7. Conventional extraction of pectin and phenolics Conventional acid treatment was employed for pectin extraction by stirring WPP powder at 85 °C with 0.02 M aqueous HCl of pH 1.7 (liquid-solid ratio 15 mL/g) for 2 h as reported previously (Talekar, Patti, Vijayraghavan, & Arora, 2018b). After that, the aqueous solution containing pectin was separated by centrifugation (20 min at 2840 g). Pectin was then isolated with ethanol precipitation of aqueous solution as described for magnetic nanobiocatalyst. As described previously, WPP phenolics were conventionally extracted with a Soxhlet extractor using methanol for 4 h (Negi, Jayaprakasha, & Jena, 2003) and the TPC was determined after the filtration of extract through Whatman No. 1. The yield of pectin and phenolics was determined as given in eqns. (1) and (2). The remaining phenolic extract was kept at 4 °C under the dark until the LC-UV/MS analysis.

2.8.6. Gel permeation chromatography (GPC) The pectin was analyzed as described previously on a Tosoh HighPerformance EcoSEC HLC-8320 gel permeation chromatography system consisting of serially connected three TSKgel columns (pore size 100 nm, > 100 nm, and 10–100 nm) for the determination of their molecular weights. The calibration curve of log molecular weight of standards versus elution time was set up using PEG/PEO standards (Agilent) of molecular weights from 106 to 1,000,000 g/mol. The pectin samples (1 mg/mL in eluent) were filtered, injected into the column and eluted at 40 °C and 1.0 mL/min flow rate using aqueous solution (eluent) of 0.1 M NaNO3 and 0.1 M NaHCO3. The samples were analyzed in duplicate and the molecular weight (MW) and polydispersity (PD) were determined by analyzing the data using EcoSec Analysis software.

2.8. Pectin characterization 2.8.1. Degree of esterification (DE) by titrimetry The DE of pectin was measured titrimetrically by the saponification of pectin described by the Food Chemical Codex. The DE was determined in terms of the methyl ester content as the percentage ratio of esterified carboxylic group titer (V2) to the sum of the free carboxylic group titer (V1) and V2.

2.8.7. Thermo-gravimetric analysis (TGA) The thermal behavior of pectin was studied by TGA with the following settings: 50 mL/min nitrogen gas flow, 25 °C–550 °C temperature range, 10 °C/min heating rate on Mettler Toledo TGA analyzer, sample: 10 mg pectin in an aluminium pan, reference: an empty aluminium pan.

2.8.2. Attenuated total reflection infrared spectroscopy (ATR-IR) ATR-IR analysis was performed using the following conditions: 4000 cm−1 to 600 cm−1 wavelength range, 2 cm−1 resolution, 64 scans, and attenuated total reflectance mode on Agilent Cary 640 spectrophotometer.

2.9. Characterization of phenolic extract The phenolics extract derived from the magnetic nanobiocatalyst, free cellulase, and conventional method was analyzed using an Agilent 1260 Infinity liquid chromatography system consisting of a Phenomenex Kinetex® 5 μm C18 100 Å column (250 mm × 4.6 mm) connected to a 6120 series quadrupole electrospray mass spectrometer as presented in previous work (Talekar, Patti, Vijayraghavan, & Arora, 2018b). The 10 μL of sample was injected (triplicate injections per sample) into the column and the phenolic compounds were separated under gradient elution (0% acetonitrile −0 min, 20% acetonitrile −30 min, 60% acetonitrile −50 min, 100% acetonitrile −55 min, 100% acetonitrile −70 min, 0% acetonitrile −73 min, 0% acetonitrile −76 min) at 30 °C with two solvents (acetonitrile and water each containing 0.5% acetic acid) at 1.0 mL/min and monitored at 210 nm, 254 nm, 280 nm, 320 nm, 378 nm with UV detector. Electrospray ionization mass spectrometer was used under 100–1200 Da mass range, 12 l/min drying gas at 350 °C, 35 psi nebulizer pressure, 3000 V

2.8.3. 1H NMR analysis The 1H NMR spectra of pectin (20 mg) dissolved in D2O (2 mL) was acquired with Bruker Advance II 400 NMR spectrometer under 800 Hz spectral width, temperature at 25 °C, 2 s relaxation delay and number of scans 80 according to the previously published method (Talekar, Patti, Vijayraghavan, & Arora, 2018b). The chemical shifts were calibrated by referring to the D2O signal at 4.80 ppm. 2.8.4. Uronic acid content of pectin The amount of uronic acid in pectin was spectrophotometrically measured as galacturonic acid equivalent using m-hydroxydiphenyl method (Melton & Smith, 2001). Pectin was hydrolyzed to release uronic acid by the concentrated H2SO4 (1 mL × 2) under stirring, cooled, added with 0.5 mL and 5 mL DI water followed by the 471

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magnetic nanobiocatalyst (p > 0.05). Therefore, 20 min was chosen for further experiments. It can also be seen that the combination of ultrasonic and free cellulase/magnetic nanobiocatalyst treatment greatly increased the extraction yields compared to ultrasonic, free cellulase or magnetic nanobiocatalyst treatment alone. The reaction conditions, such as pH and temperature, strongly determine the enzyme's catalytic activity which is crucial to the disintegration of the cell wall to release pectin and phenolics from WPP. Therefore, we examined the impact of pH and temperature on cellulase activities and yields of pectin and total phenolics obtained by the magnetic nanobiocatalyst and free cellulase treatment of WPP. It was found that pH notably influences the yields of pectin and total phenolics (Fig. 3a–b). The pectin and total phenolics yields improved significantly with the increment of pH. The free cellulase showed enhanced performance in acidic pH and attained the best yields of pectin (20.1%) and total phenolics (9.4%) at pH 5 (Fig. 3b). In contrast, the magnetic nanobiocatalyst performed better at near neutral pH, for which the best yields of pectin (19.9%) and total phenolics (8.6%) were obtained at pH 6 (Fig. 3a). However, the best yields of pectin and total phenolics obtained by both biocatalysts did not differ significantly (p > 0.05). This result appears to be of special industrial interest, where the risk of corrosion and rapid wearing out of the equipment and generation of acidic waste impose the use of less acidic conditions for pectin extraction (Adetunji et al., 2017). Moreover, the total phenolics obtained at pH 6 by the magnetic nanobiocatalyst are expected to contain intact punicalagin which is prone to hydrolysis at acidic conditions. The yields of pectin and total phenolics decreased markedly above the pH 5 for the free cellulase and pH 6 for the magnetic nanobiocatalyst. It was discovered that the cellulase activity of both biocatalysts increased with pH and at pH 5 the free cellulase showed the maximum activity which was shifted to pH 6 for the magnetic nanobiocatalyst (Fig. 3a–b). This shift in pH maxima for cellulase activity to higher values could be due to the generation of negative charge on the cellulase surface as a result of glutaraldehyde cross-linking of cellulase to MNPs via its surface amino groups (Khoshnevisan et al., 2011). Thus, the reduced yields of pectin and total phenolics below and above the pH 5 for the free cellulase and pH 6 for the magnetic nanobiocatalyst could be attributed to their lower cellulase activity. Additionally, alkaline degradation of pectin may also contribute to the decreased yields of pectin at alkaline pH (Kravtchenko, Arnould, Voragen, & Pilnik, 1992). Fig. 3c–d shows the influence of the reaction temperature (30°C70 °C) on the extraction of WPP pectin and total phenolics by the magnetic nanobiocatalyst and free cellulase. The highest yields of pectin (19.7%) and total phenolics (8.8%) were obtained in the range of 50–60 °C for the magnetic nanobiocatalyst, whereas the free cellulase achieved the best yields of pectin (19.9) and total phenolics (9.2%) at 50 °C. This could be correlated to the maximum cellulase activity of the magnetic nanobiocatalyst and free cellulase. These highest pectin and total phenolics yields obtained by the magnetic nanobiocatalyst and free cellulase did not differ significantly (p > 0.05). However, the yields decreased to pectin (16%) and total phenolics (5.8%) for the magnetic nanobiocatalyst and pectin (7.4%) and total phenolics (3.7%) for the free cellulase at 70 °C, possibly due to the decrease in their cellulase activities at this temperature (Fig. 3c–d). The higher yields of the magnetic nanobiocatalyst than free cellulase in the region of high temperature can be attributed to the retention of higher cellulase activity in the magnetic nanobiocatalyst, which could be further explained by the protection of the cellulase in the magnetic nanobiocatalyst due to covalent binding between cellulase and MNPs which restricts its conformational mobility (Talekar et al., 2017). Although the magnetic nanobiocatalyst showed similar cellulase activity within 50–60 °C the thermostability of the magnetic nanobiocatalyst was higher at 50 °C in contrast to 60 °C. Over 28 h, the magnetic nanobiocatalyst retained greater than 96% of its original cellulase activity at 50 °C, whereas, at 60 °C, it was continuously diminished up to 58% (Fig. S3). Considering the necessity of long-term stability of enzyme for

capillary voltage, and positive and negative operation mode. The UV and mass analysis data were acquired with Agilent Chemstation and MassHunter Software, respectively. The gallic acid was quantified at 280 nm whereas ellagic acid and punicalagin were quantified at 378 nm using their calibration curves. 3. Results and discussion 3.1. Preparation of cellulase magnetic nanobiocatalyst For obtaining a highly active cellulase magnetic nanobiocatalyst required for the efficient recovery of pectin and punicalagin rich phenolics from WPP, the influence of glutaraldehyde concentration, MNPs:enzyme ratio and immobilization time on cellulase activity recovery in the magnetic nanobiocatalyst was studied. We found an increase in the cellulase activity recovery until the glutaraldehyde concentration reaches up to 70 mM at which the highest activity recovery of 93% was achieved (Fig. S2a). Beyond the 70 mM glutaraldehyde concentration, the decline in the cellulase activity recovery in the magnetic nanobiocatalyst was observed which could be because of the loss of conformational flexibility of the cellulase resulted by the extensive cross-linking (Feng, Yu, Li, Mo, & Li, 2016). The immobilization with the MNPs:enzyme ratio of 1:4 recovered maximum cellulase activity in the magnetic nanobiocatalyst above which it is decreased (Fig. S2b). It could be because of blocking of cellulase active sites by a steric hindrance between cellulase molecules at the surface of MNPs at high cellulase loading of MNPs:enzyme ratio greater than 1:4 (Ladole, Mevada, & Pandit, 2017). It was also observed that the cellulase activity recovery was improved with the immobilization time and reached the maximum of 94% at 6 h and thereafter decreased probably due to the blocking of the free amino groups on the surface of MNP-NH2 required for the cellulase bonding after this time (Fig. S2c). Less cellulase activity recovery at immobilization time lower than 6 h is most likely because of the incomplete immobilization at the shorter cross-linking period (Talekar et al., 2017). Immobilization time exceeding 6 h did not increase activity recovery in the magnetic nanobiocatalyst. Thus, immobilization of cellulase with 70 mM glutaraldehyde concentration and 1:4 MNP:enzyme ratio for 6 h recovered 94% of initial cellulase activity in the magnetic nanobiocatalyst. The as-prepared magnetic nanobiocatalyst had cellulase activity of 2.6 U/mg of magnetic nanobiocatalyst. The immobilization of cellulase onto MNPs was evidenced as the typical protein peaks at 1651 cm−1 (amide I) and 1541 cm−1 (amide II) of free cellulase also appeared in the IR spectrum of the magnetic nanobiocatalyst (Fig. 2a). The scanning and transmission electron micrographs of MNPs and magnetic nanobiocatalyst showed that particle size (10 nm) of MNPs increased to about 18 nm upon cellulase immobilization (Fig. 2b–e). Thus, by virtue of its nano-size, the mass transfer of the magnetic nanobiocatalyst in the viscous reaction medium (such as aqueous medium containing WPP solid particles in the present study) could be improved compared to cellulase immobilized with other macro-carriers (Shylesh, Schünemann, & Thiel, 2010). 3.2. Recovery of pectin and punicalagin rich phenolics from WPP Before the free cellulase and magnetic nanobiocatalyst treatment, WPP was treated by ultrasound to destabilize its cell structure and enhance the diffusion of biocatalysts within cell wall which can increase the cell wall hydrolysis (Arshadi et al., 2016) and thus, the release of pectin and phenolics. Time gradient was set between 0 and 30 min to investigate the influence of ultrasound treatment time on extraction efficiency of pectin and phenolics. For both free cellulase and the magnetic nanobiocatalyst, the yields of pectin and total phenolics increased with increasing ultrasound treatment time up to 20 min, whereas a further increment of ultrasound treatment time to 30 min did not significantly increase the yields (Table 1). Also, no significant differences were seen clearly in yields obtained by the free cellulase and 472

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Fig. 2. (a) FT-IR spectrums of the prepared MNPs, magnetic nanobiocatalyst and free cellulase. FE-SEM image of (b) prepared MNPs and (c) magnetic nanobiocatalyst. TEM image of (d) prepared MNPs and (e) magnetic nanobiocatalyst.

Table 1 Effect of ultrasound treatment of WPP on the recovery of pectin and total phenolics. Ultrasound treatment time (min)

0 10 20 30 Conventional

a

With magnetic Nanobiocatalyst

b

With free cellulase

b

Without magnetic nanobiocatalyst or free cellulase c

Pectin yield (g/ 100 g db)

TPC yield (g/ 100 g db)

Pectin yield (g/ 100 g db)

TPC yield (g/ 100 g db)

Pectin yield (g/ 100 g db)

TPC yield (g/ 100 g db)

8.8 ± 1.2 13.7 ± 1.6 19.1 ± 0.8 19.2 ± 1.1

4.7 6.3 8.6 8.4

9.2 ± 0.7 14.4 ± 1.3 19.9 ± 1.1 19.6 ± 0.8

4.9 6.9 9.4 9.1

3.0 ± 0.4 4.6 ± 0.9 6.2 ± 0.9 7.1 ± 0.7 19.5 ± 1.4

1.9 ± 0.3 2.8 ± 0.5 3.7 ± 0.2 4.9 ± 1.1 10 ± 0.6

± ± ± ±

0.8 0.9 1.0 0.6

± ± ± ±

0.5 0.7 1.2 0.9

Ultrasound treatment was given at a fixed frequency of 37 kHz, liquid-solid ratio of 15 mL/g, 50 °C and pH 5. Both free cellulase and magnetic nanobiocatalyst at a cellulase dosage of 100 U/g of peel powder was added to ultrasound treated WPP and stirred at 180 rpm and 50 °C for 7 h. c Ultrasound treated WPP was directly stirred at 180 rpm and 50 °C for 7 h in absence of free cellulase or magnetic nanobiocatalyst. a

b

473

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Fig. 3. Influence of pH on cellulase activity and yields of pectin and total phenolics obtained by (a) magnetic nanobiocatalyst (b) free cellulase treatment of WPP. Influence of temperature on cellulase activity and yields of pectin and total phenolics obtained by (c) magnetic nanobiocatalyst (d) free cellulase treatment of WPP. The highest cellulase activity was assumed as 100%. In case of influence of pH, the highest cellulase activity was 2.62 U/mg for magnetic nanobiocatalyst and 0.7 U/ mg for free cellulase, and WPP was treated at 50 °C with cellulase loading of 100 U/g peel powder for 7 h. In case of influence of temperature, the highest cellulase activity was 2.56 U/mg for magnetic nanobiocatalyst, and 0.68 U/mg for free cellulase and WPP was treated at pH 5.5 with cellulase loading of 100 U/g peel powder for 7 h. Data points represent the average of triplicates, the error bars represent standard deviations.

alteration of the cellulase flexibility upon covalent bonding to MNPs which was confirmed by slightly increased Km (3.43 mg/mL) and decreased Vmax (2.71 μmol/min) of cellulase in the magnetic nanobiocatalyst compared to the free cellulase Km (3.27 mg/mL) and Vmax (2.90 μmol/min) values (Cui, Ren, Lin, Feng, & Jia, 2018; Ladole et al., 2017). No significant improvements in yields were observed even after a magnetic nanobiocatalyst treatment time of 7 h which could be reasoned either by the loss of cellulase activity of the magnetic nanobiocatalyst or by the fact of the leftover substrate was not easy to access for the magnetic nanobiocatalyst, requiring long time for substrate degradation (Hansen, Kristensen, Felby, & Jørgensen, 2011; Jeong et al., 2014). However, when the cellulase activity of the magnetic nanobiocatalyst was measured after the reaction for each treatment time, it was interestingly stable even after 7 h of treatment (Fig. 4c). This indicates that after 5 h treatment the remaining cellulose in WPP seems to be less accessible for the magnetic nanobiocatalyst. Nonetheless, the maximum yields of pectin and total phenolics obtained by the magnetic nanobiocatalyst are still comparable to the conventional method derived yields of pectin (19.5%) and phenolics (10%).

industrial application, the temperature of 50 °C was employed for the subsequent studies. From the economic consideration, optimizing the cellulase dosage makes the use of the minimum cellulase amount for the achievement of the maximum pectin and total phenolics yields. Therefore, the magnetic nanobiocatalyst and free cellulase at various cellulase dosages, ranging from 25 to 175 U/g WPP powder were used for the treatment of WPP. As shown in Fig. 4a, the highest yields of pectin (19.5%) and total phenolics (8.9%) were observed when the cellulase dosage was 75 U/g WPP powder. Beyond this dosage, the yields of the pectin and total phenolics remained constant, thus reinforcing the economic benefit of using the magnetic nanobiocatalyst at cellulase loading of 75 U/g WPP powder. Although the highest yields of pectin (19.6%) and total phenolics (9.6%) obtained by the free cellulase are not significantly different than those obtained by the magnetic nanobiocatalyst (p > 0.05), they were obtained with lower cellulase dosage of 65 U/g WPP powder (Fig. 4b). But still, the use of the recyclable magnetic nanobiocatalyst can be cost-effective owing to the fact that the enzymes in free form are applied on a single-use, throw-away basis increasing their costs per kilogram of product which can be significantly cut down by recycling enzymes via immobilization (Sheldon, 2017). The catalytic performance of the magnetic nanobiocatalyst and free cellulase was investigated over the different time periods of treatment. The yields of pectin and total phenolics for free cellulase increased with the increasing treatment time from 0.5 to 4 h and the optimum yields of pectin (19.7%) and total phenolics (9.5%) were obtained at 4 h and thereafter remained stable (Fig. 4d). On the other hand, the yields for the magnetic nanobiocatalyst increased slightly slowly as the treatment time increased and the maximum yields of pectin (19.5%) and total phenolics (9.1%) were obtained at 5 h (Fig. 4c), however, not significantly different from yields of free cellulase (p > 0.05). The slower rate of the magnetic nanobiocatalyst can be explained by slightly lowering the substrate accessibility to the cellulase active site due to the

3.3. Recycling of magnetic nanobiocatalyst The reusability of the enzyme for successive reaction cycles is crucial for the economic sustainability of the proposed immobilized enzyme approach for the recovery of pectin and phenolics from WPP (Franssen, Steunenberg, Scott, Zuilhof, & Sanders, 2013). Therefore, to determine the recycling capability of magnetic nanobiocatalyst, the magnetic nanobiocatalyst was separated after the reaction using a magnet (Fig. 5), washed twice with potassium phosphate buffer (50 mM, pH 6) and added to the fresh reaction mixture containing WPP. The magnetic nanobiocatalyst was recycled for five sequential batchwise reaction cycles. The recycling efficiency of the magnetic nanobiocatalyst was assessed based on yields, properties of pectin and 474

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Fig. 4. Influence of cellulase dosage on yields of pectin and total phenolics obtained by (a) magnetic nanobiocatalyst (b) free cellulase treatment of WPP. Conditions of treatment: temperature at 50 °C, time for 7 h, pH 6 for magnetic nanobiocatalyst and pH 5 for free cellulase. Time course of extraction of pectin and total phenolics by (c) magnetic nanobiocatalyst along with variation in its cellulase activity (d) free cellulase treatment of WPP. Conditions of treatment: 50 °C, pH 6, and cellulase loading of 75 U/g of peel powder for magnetic nanobiocatalyst and 50 °C, pH 5, and cellulase loading of 65 U/g of peel powder for free cellulase Data points represent the average of triplicates, the error bars represent standard deviations.

magnetic nanobiocatalyst after first and fifth cycle of reuse are also similar to that of the fresh magnetic nanobiocatalyst (Fig. S4). This indicates that no significant change in yields of pectin and total phenolics over five cycles of reuse could be due to the retention of activity of magnetic nanobiocatalyst. Additionally, for each cycle, the obtained pectin was characterized in terms of FT-IR, NMR, degree of esterification, molecular weight and polydispersity analysis and total phenolics were characterized in terms

composition of total phenolics obtained in each cycle. From Fig. 6, it can be seen that the magnetic nanobiocatalyst exhibited constant yields of pectin (19.2–19.5%) and total phenolics (8.4–8.6%), even after five cycles of reuse. The magnetic nanobiocatalyst still retained 100% of its original cellulase activity after five cycles of reuse. No free cellulase activity was detected in the reaction mixture after separation of the magnetic nanobiocatalyst in each cycle, demonstrating no leakage of cellulase from magnetic nanobiocatalyst. The FT-IR spectrum of the

Fig. 5. Dispersed magnetic nanobiocatalyst in the reaction mixture (left) and separated magnetic nanobiocatalyst by using an external magnet (right). 475

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carboxylic acid (1630 cm−1) groups, the galacturonic acid hydrogen bonds OeH stretch (3200-3600 cm−1), methyl group CeH absorption and CH2 of pectin (2800-3000 cm−1), methyl ester CH3 bending (1350–1450 cm−1) were observed which suggest the presence of pectin structure (S. S. Hosseini, Khodaiyan, & Yarmand, 2016) and show an excellent correlation with FT-IR spectra of the commercial citrus pectin, free cellulase, and conventional method derived pectin. With reference to the previously reported chemical shifts of pectin, the 1H NMR spectra of the samples from each cycle as shown in Fig. 8 revealed the pectin like structure (Grassino et al., 2016). The characteristic chemical shifts of protons on methyl ester group (3.81 ppm), anomeric carbon C1 (5.1 ppm), C2 (3.74 ppm), C3 (3.99 ppm), C4 (4.44 ppm), and C5 (4.96 ppm) of galacturonic acid were clearly observed in the pectin samples from each cycle and were similar to those of the free cellulase and conventional method derived pectin and commercial citrus pectin. The uronic acid content and the DE of pectin (determined with titrimetry and 1H NMR) obtained in each cycle was in the close range of 72–74% and 62–64%, respectively (Table 2). Since the uronic acid content of pectin for use in food should not be less than 65% (Joint, 2007), the pectin extracted in each cycle can be considered as ideal for food applications. The pectin having a DE < 50% is categorized as low methoxyl pectin (LM), while that having a DE > 50% is categorized as high methoxyl pectin (HM) (Adetunji et al., 2017) and therefore for

Fig. 6. Reusability of the magnetic nanobiocatalyst during the recovery of pectin and phenolics from WPP. Batch reaction cycle conditions: cellulase loading of 75 U/g peel powder, 5 h time at pH 6 and 50 °C. Data points represent the average of triplicates, the error bars represent standard deviations.

of their composition of phenolic compounds. In the FT-IR spectra of pectin from each cycle (Fig. 7), the typical peaks of glycosidic bond CeO stretch (1000–1200 cm−1), methyl ester (1740 cm−1) and

Fig. 7. IR spectra of pectin obtained from each batch reaction cycle during recycling of the magnetic nanobiocatalyst, commercial pectin, pectin derived from free cellulase and conventional acid extraction. Batch reaction cycle conditions: cellulase loading of 75 U/g of peel powder, 5 h time at pH 6 and 50 °C. 476

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Fig. 8. 1H NMR spectra of pectin obtained from each batch reaction cycle during recycling of the magnetic nanobiocatalyst, commercial pectin, pectin derived from free cellulase and conventional acid extraction. Batch reaction cycle conditions: cellulase loading of 75 U/g of peel powder, 5 h time at pH 6 and 50 °C.

Table 2 Uronic acid content and degree of esterification for pectin samples from different batches during recycling of magnetic nanobiocatalyst, free cellulase and conventional acid extraction. Pectin sample

Commercial Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Free cellulase Conventional

Uronic acid (%)

76.0 73.1 71.9 74.1 73.3 72.7 73.7 66.9

± ± ± ± ± ± ± ±

0.7 0.6 0.2 0.8 0.2 0.1 0.4 0.1

Table 3 Molecular weight and polydispersity analysis of pectin samples from different batches during recycling of magnetic nanobiocatalyst, free cellulase and conventional acid extraction.

Degree of esterification

Pectin sample

Mn

Mw

Mw/Mn

Titration

NMR

70.0 62.4 63.8 62.2 62.9 62.5 64.9 59.0

73.3 64.1 62.1 63.8 64.3 64.4 63.0 58.3

Commercial Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Free cellulase Conventional

107225 90323 89317 92882 87484 88828 90203 99479

141538 140905 143801 139324 142600 143902 142522 137281

1.32 1.56 1.61 1.50 1.63 1.62 1.58 1.38

± ± ± ± ± ± ± ±

2.0 2.0 3.8 1.2 4.5 2.4 0.7 2.3

± ± ± ± ± ± ± ±

1.2 0.8 1.3 1.5 2.0 0.7 0.7 2.0

that obtained by the magnetic nanobiocatalyst (Tables 2–3). However, pectin isolated with conventional acid method had lower uronic acid content (66.9%), DE (58.3–60.1%), MW (137.2 kDa), and PD (1.38) (Tables 2–3), which could be because of the de-esterification and breakdown of pectin by an acid (Pereira et al., 2016). The thermal analysis of pectin of each cycle showed similar regions of mass loss: 50–200 °C, 200–400 °C and 400–650 °C and nearly the same rate of mass loss in the TGA curves (Fig. 9). The mass loss in the first region

each cycle, the pectin extracted is classified as high methoxyl pectin (HM). The weight average molecular mass (MW) and polydispersity (PD) of pectin obtained from each cycle were also found to be similar (in the range of 140.3–143.9 kDa and 1.5–1.6, respectively) (Table 3). As expected, the pectin obtained with free cellulase had similar uronic acid content (73.7%), DE (63–65%), MW (142.5 kDa), and PD (1.58) to 477

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Fig. 9. TGA of pectin obtained from each batch reaction cycle during recycling of the magnetic nanobiocatalyst, commercial pectin, pectin derived from free cellulase and conventional acid extraction. Batch reaction cycle conditions: cellulase loading of 75 U/g of peel powder, 5 h time at pH 6 and 50 °C.

chromatograms and mass spectra are illustrated in Fig. S5. The punicalagin (α + β) and ellagic acid were found in phenolics of each cycle. The punicalagin, an active pomegranate phenolic compound, is highly bioavailable and converted into potent antioxidants ellagic acid and urolithins inside the body (Espín et al., 2007). The consumption of free ellagic acid reduces its capacity to provide antioxidant activity to the body as the free ellagic acid is less bioavailable owing to its less watersolubility at physiological pH (Seeram, Lee, & Heber, 2004). It was interestingly found that the total phenolics of all five batch cycles were rich in punicalagin (Table 4). For all five batch cycles, the amount of punicalagin was also similar in the range of 6.42–6.65 g per 100 gDM of peel powder and represented about 75.3–78.2% of total extracted phenolics. The amount of ellagic acid found in total extracted phenolics

(50–200 °C) corresponds to the loss of volatiles with increasing temperature. The major mass loss of approximately 53% occurred in the second region (200–400 °C) for pectin from all five cycles was consistent with that (50.4–54%) of commercial pectin, free cellulase and conventional method derived pectin occurred in the same region (200–400 °C), which could be correlated to the decarboxylation and thermal decomposition of pectin and formation of solid char. This solid char mostly likely slowly undergoes the thermal degradation in the region of 400–650 °C resulting in a slow mass loss (Zhou, Xu, Wang, & Tian, 2011). Thus, the thermal analysis showed that pectin obtained in each cycle possessed similar thermal stability. The phenolic constituents of total phenolics of each cycle were determined by LC-UV/MS and the respective characteristic HPLC 478

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stability and showed close similarity (based on IR, 1H NMR, and TGA analysis) to commercial pectin. The punicalagin content of phenolics obtained in each cycle was also the same. This excellent enzyme reusability demonstrates the applicability of magnetic nanobiocatalyst approach for the improvement of the cost-effectiveness and sustainability of green enzyme-based value-added product recovery from biomass. This could have a very positive implication on pectin hydrocolloids extraction industry in terms of cost and sustainability. The process optimization for multiple products recovery at scale-up scenarios will be evaluated and presented in future investigations.

Table 4 TPC, punicalagin and ellagic acid content of total phenolics obtained from different batches during recycling of magnetic nanobiocatalyst a, free cellulase b and conventional acid extraction. Values in bracket indicate percentage of TPC. Batch number

TPC (g/100 g db)

Punicalagin (g/ 100 g db)

Ellagic acid (g/ 100 g db)

1 2 3 4 5 Free cellullase Conventional

8.60 ± 0.3 8.53 ± 0.7 8.44 ± 0.4 8.70 ± 0.2 8.57 ± 0.5 9.45 ± 1.3 10.00 ± 1.6

6.65 ± 0.4 (77.3%) 6.42 ± 0.1 (75.3%) 6.60 ± 0.2 (78.2%) 6.65 ± 0.5 (76.4%) 6.59 ± 0.4 (76.9%) 6.92 ± 0.6 (73.3%) 3.88 ± 0.01 (38.8%)

0.48 ± 0.03 0.49 ± 0.02 0.55 ± 0.03 0.50 ± 0.05 0.60 ± 0.01 0.64 ± 0.02 4.93 ± 0.04 (49.3%)

(5.6%) (5.7%) (6.5%) (5.7%) (7.0%) (6.8%)

Acknowledgments AA and ST gratefully acknowledge the IITBMonash Research Academy and Tata Chemicals Innovation Centre to provide financial assistance to ST for his doctoral study (IMURA 0509).

a

Magnetic nanobiocatalyst treatment was given at liquid-solid ratio of 15 mL/g, 50 °C, pH 6 and cellulase dosage of 75 U/g of peel powder for 5 h. b Free cellulase treatment was given at liquid-solid ratio of 15 mL/g, 50 °C, pH 5 and cellulase dosage of 65 U/g of peel powder for 4 h.

Appendix A. Supplementary data

of each batch cycle was very low but still quite similar in the range of 0.48–0.6 g per 100 gDM of peel powder representing about 5.6–7% of total extracted phenolics. This suggests that the treatment of magnetic nanobiocatalyst is able to extract punicalagin rich phenolics-beneficial for human health. Similar to the magnetic nanobiocatalyst, two phenolics: punicalagin and ellagic acid were identified in phenolics extracted with free cellulase. In addition, the amount of punicalagin (6.92 g per 100 gDM of peel powder representing 73.3% of total phenolics) and ellagic acid (0.64 g per 100 gDM of peel powder representing 6.8% of total phenolics) found in phenolics extracted with free cellulase was also similar to that obtained for the magnetic nanobiocatalyst. Nevertheless, phenolics obtained by conventional methanol extraction were identified as a mixture of multiple phenolics such as pedunculagin, punicalagin, gallic acid, ellagic acid, epicatechin, etc. Compared to magnetic nanobiocatalyst, the amount of punicalagin was decreased to 3.88 g per 100 gDM of peel powder representing 38.8% of total phenolics and that of ellagic acid was significantly increased to 4.93 g per 100 gDM of peel powder representing 49.3% of total phenolics for conventional methanol extraction. Thus, the conventional methanol extraction yields phenolics rich in an ellagic acid-the phenolic compound with less bioavailability. These results clearly illustrate that the magnetic nanobiocatalyst exhibited the outstanding consistency in not only the yields but also properties of pectin and composition of total phenolics obtained in each batch cycle of reuse. This excellent reusability could be ascribed to the high stability of cellulase in the magnetic nanobiocatalyst and its specificity and selectivity.

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4. Conclusions For the first time, we have demonstrated the applicability of the recyclable magnetic nanobiocatalyst consisting of cellulase bound to magnetic nanoparticles for the recovery of high-value pectin and punicalagin rich phenolics from pomegranate processing waste. The magnetic nanobiocatalyst was able to recover good quantities of pectin (19.4%) and phenolics (8.8%) in aqueous medium at near neutral (pH 6) and low temperature (50 °C) conditions comparable to those obtained by the free cellulase derived pectin (19.6%) and phenolics (9.2%), conventional hot aqueous acidic extraction of pectin (19.5%) and toxic organic solvent extraction of phenolics (10%). Without further purification, the obtained phenolics were found to be rich in punicalagin (75–78% of total phenolics) which can be of high commercial importance. The magnetic nanobiocatalyst was easily recovered after the reaction using a magnet and recycled for five consecutive batches of simultaneous pectin and phenolics recovery without compromising yields of pectin and phenolics and its cellulase activity. Moreover, the pectin extracted in each cycle possesses a similar degree of esterification, uronic acid content, molecular weight, polydispersity, and thermal 479

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