FORM 2
THE PATENTS ACT, 1970 (39 OF 1970)
PATENTS RULES, 7006
PROVISIONAL SPECIFICATION (SECTION 10; RULE 13)
TITLE: IMMOBILIZED ENZYME MEDIATED PROCESS EOR RECOVERY OF PECTIN AND PUNICALAGIN RICH POLYPHENOLS FROM WASTE POMEGRANATE PEELS
Applicant : IITB MONASH RESEARCH ACADEMY Nationality: INDIAN Address : IIT Bombay
Powai, Mumbai 400 076, India
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

TECHNICAL FIELD:
This invention relates to a novel bio-refinery process for the recovery of pectin and punicalagin rich polyphenols from waste pomegranate peels.
BACKGROUND:
The global production of the pomegranate reached 4 million tons in 2015-16 harvest, with India alone produced 2.2 million tons (Board, 2016). In addition, pomegranates are available throughout a year in India. The pomegranate is considered as a super fruit with numerous medical properties and health benefits due to which the global demand for processed pomegranate products (ready to eat pomegranate arils and fresh juice) has spiked in recent years. Thus, the pomegranate processing industry is significantly expanding in the world's main pomegranate producers such as India, Iran, China, Turkey, United States, Spain, South Africa, Peru, Chile, and Argentina (Hasnaoui et al., 2014). Processing of 1 ton of fresh pomegranates generates 500-550 kg waste peels as a major by-product which has a low pH (3-4), high water content (around 70-/5%) and high organic matter content (around 96% of total solids) (Clu et al., 2009). Under European regulations (Directive 2008/98/EC, 2008), these characteristics mean that waste pomegranate peels should not be disposed of on land or in landfills as it presents a significant risk to local water courses and leads to uncontrolled greenhouse gas production. Consequently, the disposal of waste pomegranate peels is becoming a major problem for many pomegranate processing factories. The waste pomegranate peels can be dried and sold as a cattle feed. But with a protein content of only 3-5%, it is a poor cattle feed supplement which is marginally profitable as drying of waste pomegranate peels is very energy-intensive and costly. Currently, there is a growing recognition that the problems of fruit processing waste management can be solved together through a more efficient utilization of waste as a resource for the production of added-value products using a fruit processing waste (FPW) biorefinery concept (Banerjee et al,, 2017). In addition, production of added-value products also provides an additional revenue to fruit processing industries (Lin et al,, 2013). Waste pomegranate peels are a rich source of commercially important products such as polyphenols (10-20%) (Akhtar et al., 2015) and pectin (20-25%) (Moorthy et al., 2015). Pomegranate polyphenols punicalagin and ellagic acid exert beneficial effects such as antioxidant, anticancer, anti-atherosclerosis, anti-inflammatory, anti-influenza and prebiotic activities on human health (Viuda-Martos et al., 2010), Among the polyphenols, punicalagin are mainly responsible for most of the

bioactivities of pomegranate polyphenols. Due to their high water solubility punicalagin have high bioavailability inside the body where they are broken down into two powerful antioxidants: ellagic acids and urolithins (Bialonska et al., 2009; Espin et al., 2007), When free ellagic acid is administrated orally, it is poorly absorbed due to its low water solubility at physiological pH which significantly decreases its bioavailability (Lei et al., 2003; Seeram et al., 2004). The only way to absorb ellagic acid and urolithins in bloodstream is through punicalagin (Larrosa et .al., 2010). Thus, for harnessing the real power of pomegranate peel polyphenols there is a need of extraction of polyphenols rich in punicalagin from pomegranate waste peels.
Pectin is a valued commercial hydrocolloid widely used as a gelling, thickening and stabilizing agent in the food, cosmetic and pharmaceutical industries (Ciriminna et al., 2015). In addition, pectin used in functional foods binds to cholesterol, glucose, lead and mercury in the gastrointestinal tract and reduces their absorption in blood. It is used as a drug encapsulating agent in medicine and skin anti-aging agent in cosmetic and personal care products (Noreen et al., 2017). At present, limited pectin manufacturing units are available in India, the known one being Krishna Pectins which produces pectin from sunflower heads. The majority of pectin is imported in India. Pectin rich fruit processing waste such as citrus, apple and mango as a feedstock for pectin recovery has attracted a lot of research and industries in the last few decades (Ciriminna et al., 2015). Thus, in view of the pomegranate availability in India and huge quantity of pomegranate peels generated from production of pomegranate juice and arils, the peels can be used as a novel source for the recovery of pectins. However, the research on pectin extraction from waste pomegranate peels is very limited and requires further investigation to develop an industrial process for pectin extraction.
Thus, waste pomegranate peels represent a potential resource of added-value products: pectin and polyphenols within the context of a FPW bio-refinery.
OBJECTS OF THE INVENTION:
The waste pomegranate peels generated by pomegranate processing industries contain two major products of high commercial value: pectin and polyphenols. Worldwide, the pomegranate polyphenol market is seen to be advancing significantly forward and segmented into cosmetics, dietary supplements, food & beverages, nutraceuticals and others. The present global pectin market size is greater than USD 1 billion whereby industrial pectin is obtained mainly from apple, sunflower or citrus pomace. Considering

the potential of pomegranate peels for pectin extraction, they could very well be used as a novel source for pectin production. Most of the waste pomegranate peel extraction technologies invented in patents and published in the literature report the recovery of only one product polyphenols therefrom. Among polyphenols, punicalagin is a key phenolic compounds responsible for most of the bioactivity of pomegranate polyphenols. Although few patents report the method for extraction of punicalagin from pomegranate peels they require the use of organic solvents, even toxic ones such as methanol, acetone and ethyl acetate. In addition, they extract only punicalagin rich polyphenols and not pectin from pomegranate peels. Recently, some researchers attempted to extract pectin from waste pomegranate peels but using hot (80-90) and aqueous acidic conditions (pH 1-2) ultimately generating large volumes of acidic waste detrimental to large-scale industrial operation. The hot and highly acidic conditions also reguire special equipment which may increase capital cost. These attempts only extract pectin from waste pomegranate peels and no attention has been given to polyphenol extraction- Moreover, punicalagin being sensitive to high temperature and acidic conditions, hot and acidic conditions used during pectin extraction could hydrolyze punicalagin.
Therefore, the objective of the present invention is to provide a holistic approach to recover both pectin and punicalagin rich polyphenols from waste pomegranate peels, wherein the process ensures no use of high temperature, acidic conditions, and toxic solvents. Thus, in India, being world's largest pomegranate producer, the present invention could be useful for pomegranate processing industries to obtain high market value pectin and polyphenol extracts rich in punicalagin from waste pomegranate peels. The process is also applicable internationally for any pomegranate processing industry,
PRIOR ART:
Prior art on extraction of pectin from pomegrante peel
More recently, some researchers embarked on attempts to extract pectin from waste pomegranate peels but using hot (60-90°C) and aqueous acidic conditions (pH 1.2-2.5) (ill INVALID CITATION !!!). Use of acidic conditions generates large volumes of acidic waste detrimental to large-scale industrial FPW biorefinery operation. In addition, it requires special equipment to withstand acidic environment that would result in increase in capital cost. Instead of acidic conditions, Chinese patent CN106008744A discloses the method tor preparing pectin from pomegranate peel residues using hot (70°C) alkaline conditions which further requires neutralization with mineral acid before the purification of pectin. This process also employed the

defatting pomegranate peels with toxic ethyl acetate prior to pectin extraction. In addition, the hot acidic and alkaline conditions utilized in these reports hydrolyze the key active pomegranate peel polyphenolic compound i.e. punicalagin.
Prior art on extraction of punicalagin rich polyphenols from pomegranate peel
WO2005/097106 (which is also published as EP1734949A1, EP.1734949A4, EP1734949B1, US7638640, US7897791, US7919636, US20060211635, US20080318877, US20100173860) and the corresponding paper of (Seeram et al., 2005) disclose the method of purification of punicalagin from pomegranate peels using toxic solvent methanol. CN101974043A and CN101974043B disclose a multi-step lengthy method (more than 24h) for preparing punicalagin and ellagic acid from pomegranate rind which involves extraction with acidic water to obtain extract; re-extraction of obtained extract with toxic organic solvent like ethyl acetate then hot: (100-105°C) acid hydrolysis and purification. Similarly, CN106977559A describes a multi-step lengthy method (more than 13 h) for separating punicalagin and gallic acid from pomegranate peels which comprises soaking in hot toxic methanol or ethanol, ultrasound treatment and bleaching to obtain extract, then re-extraction of obtained extract with toxic ethyl acetate and purification. (Lu et al., 2010) employed non-food grade toxic organic solvents such as ethyl acetate and acetone, respectively for the separation of punicalagin from pomegranate peels. CN101747388A and CN101747388B disclose a method for extracting punicalagin and ellagic acid from pomegranate peels using organic solvent extraction with subsequent acid/alkaline treatments of obtained extract, However, the acidic treatment needs special equipment and alkaline treatment generates large amount of salts which are tedious to remove from final product. Similar drawback applies to (Amyrgialaki et al., 20,14) who applied acidified ethanol for obtaining polyphenols rich in punicalagin and ellagic acid from pomegranate peels.
The patents CN106349301A and CN102180916A employed non-toxic organic solvent; ethanol for the extraction and purification of punicalagin. (Kazemi et al., 2016) applied pulsed ultrasound treatment using 70% ethanol but obtained polyphenols rich in both punicalagin and ellagic acid. Although in few reports water was used as green solvent for extraction of pomegranate peel polyphenols using methods such as stirring (Qu el al,, 2010), pressurized water (Cm & Hisil, 2010) and ultrasound treatment (Pan et al., 2011) but the extraction was focused on total polyphenols and not on punicalagin. WO2006/127832 (also published as CA2653305A1, CA2653305C, EP1901756A2, EP1901756A4, EP1901756B1, EP259949.1A1, EP2599491B1, US76U738, US86S8220, US9352007, US20060269629, US20100009019, US20150079208, WO2006127832A3) and US20100298250A1 (also published as US8609152 and US20140080776) disclose enzymatic (mixture of cellulose, hemicellulasc and pectinase) treatment to produce an aqueous

pomegranate polyphenol extract from by-products of the pomegranate juice industry. However, this process did not reuse the multiple enzymes and employed pasteurization at 90-115°C to inactivate the enzymes due to which the cost of overall process increases. In addition, due to pasteurization extracted punicalagin may have been destroyed as seen from the presence of broad variety of phenolics including punicalagin, punicalin, ellagic acid, ellagic acid glycosides in their final polyphenol extract, Similarly, (Mushtaq el al., 2015) & 2016) inactivated the enzymes and did not reuse them after extracting pomegranate peel polyphenols by enzymatic treatment with mixture of pectinase, protease and cellulase, Also, they did not find punicalagin in their polyphenol extract,
In addition to the individual downsides as mentioned above, the major drawback of all of the prior art processing technologies developed so far for recovering valuable products from waste pomegranate peels is-that they are aimed at extracting only single product either pectin or polyphenols, Although Schieber et al. (2003) and Berardini et al. (2005) presented the process of combined recovery of pectin and polyphenols from apple pomace and mango peels, respectively but their process involves extraction with hot and highly acidic aqueous medium. Therefore, this process cannot be used for combined extraction of pectin and punicalagin rich polyphenols from pomegranate peels as hot and highly acidic conditions hydrolyze punicalagin. In addition, hot and highly acidic conditions require special equipment which may increase capital cost and generate large amount of acidic waste.
Keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present invention realized that there exists a dire need to provide a holistic approach to derive both pectin and punicalagin rich polyphenols from waste pomegranate peels, wherein the process ensures no use of high temperature, acidic conditions and toxic organic solvent. Such an approach would also improve the overall process economics as more than one product of commercial value is extracted from waste pomegranate peels.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 Schematic presentation of extraction of pectin and phenolics from WPP using the recyclable magnetic
nanobiocatalyst.
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.

Fig. 3 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,
Fig. 4 |R 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.
Fig. 5 H 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.
Fig. 6 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.
DETAILED DESCRIPTION:
The waste pomegranate peels generated by pomegranate processing industries contain two major products of high commercial value: pectin and polyphenols- Most of the waste pomegranate peel extraction technologies invented in patents and published in literature report the recovery of only one product polyphenols therefrom. In many reports, the obtained polyphenol extracts are devoid of the key polyphenol i.e. punicalagin while few employed toxic organic solvents to obtain punicalagin rich polyphenols. Most recently, some reports describe the extraction of pectin from waste pomegranate peels but ignored polyphenols. In addition, these pectin extraction attempts employed highly acidic conditions and high temperature which not only require special equipment but also destroy the key polyphenol i.e. punicalagin. However, in the present invention, both products: pectin and punicalagin rich polyphenols were simultaneously recovered from waste pomegranate peels without employing any toxic organic solvent, acidic conditions and high temperature. This was achieved by aqueous enzymatic treatment of waste pomegranate peel powder using immobilized cellulase onto magnetic nanoparticles. Also, due to magnetic nature cellulase was easily recovered from reaction mixture by magnetic field and reused for

multiple cycles of pectin and punicalagin rich polyphenols recovery without affecting their yield and quality. The products obtained through this process are primary products and can be further processed aiming at specialty applications. The flow chart of the said process developed in the present invention is given in FIg.l
PREPARATION OF MAGNETIC NANOBIOCATALYST
The alkaline co-precipitation of FeCI3 and FeSO4was employed to prepare the magnetic nanoparticles (MNPs) as described previously (Talekar et al., 2017). Typically, the FeSO4.7H2O and FeCI3.6H2O solutions (100 mL each) were mixed together in Fe2+:Fe3+ molar ratio of 1:2, heated to 90QC 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 Dl water to pH 7 and vacuum dried. Next, the dry MNPs were coated with amino groups via the APTFS silanization previously described (Liu et al., 2013): MNPs (0.5 g) were added to the 100 mL solution containing 50 mLDI water and 50 mLethanol and 2 mLAPTES, 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. The 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 (Figs. 2b-e).

PREPARATION OF WASTE POMEGRANATE PEELS (WPP)
Waste pomegranate peels of Ruby variety were procured from the pomegranate processing company, and was frozen until used. Before each extraction process, the frozen pomegranate peels were ground for 30 s using a laboratory blender and dried at 50"C for 24 h. The dried pomegranate peels were then ground to powder (particle size <150 μm).
RECOVERY OF PECTIN AND PUNICALAGIN RICH PHENOLICS FROM WASTE POMEGRANATE PEELS
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. The 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 150 W power and 50°C for 20 m/n. 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 spectrophotometrlc measurements at 765 nm (R2 - 0.99). The yield of pectin and phenolics was determined using eqn. (1) and (2), respectively.

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 yield of pectin and phenolics obtained by magnetic nanobiocatalyst were 19.1 ± 0,8 and 8.6 ± 1.0, respectively.
The pectin and phenolics from WPP were also obtained by the free cellulase for comparison. The WPP powder (2 g) was suspended in SO mM sodium citrate buffer of pH 5 at 15 mL/g liquid-solid ratio and subjected to ultrasound treatment at 150 W power 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 at 180 rpm and SOT 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. The yield of pectin and phenolics obtained by free cellulase were 19.9 ± l.land 9.4 ± 1,2, respectively.
Conventional acid treatment was employed for pectin extraction by stirring WPP powder at S5"C with 0.02 M aqueous HCt of pH 1.7 (liquid-solid ratio 15 mL/g) for 2 h as reported previously (Talekar et al,, 2018). After that, the aqueous solution containing pectin was separated by centrifugation (2.0 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 et al., 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 eqn. (1) and (2). The remaining phenolic extract was kept at 4°C under the dark until the LC-UV/MS analysis. The yield of pectin and phenolics obtained by conventional methods were 19.5 ± 1.4 and 10 ± 0.6, respectively.
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 in [026]. 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%.
From Fig. 3, it can be seen that the magnetic nanobiocatalyst exhibited constant yields of pectin (19.?-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.
In the FT-IR spectra of pectin from each cycle (Fig. 4), the typical peaks of glycosidic bond C-O stretch (1000-1200 cm -4), methyl ester (1740 cm-1) and carboxylic acid (1630 cm"1) groups, the galacturonic acid hydrogen bonds O-H stretch (3200*3600 crn-1), methyl group C-H absorption and CH2 of pectin (2800-3000 cm1), methyl ester CH3 bending (1350-1450 cm"1) were observed which suggest the presence of pectin structure (Hosseini et al., 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. 5 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 DF 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 1). 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 (L.M), while that having a DF >50% is categorized as high methoxyl pectin (MM) (Adetunji et al., 2017) and therefore for each cycle, the pectin extracted is classified as high methoxyl pectin (HM).
Table-1 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 Uronic acid (%) Degree of esterification
Titration NMR

Commercial 76.0 ± 0.7 70.0 + 2.0 73.3 + 1.2
Batch 1 73.1 ± 0,6 62.4.+2.0 64,1 + 0.8
Batch 2 71.9 ±0.2 63.8+3.8 62.1 + 1.3
Batch 3 74.1 + 0.8 62.2 + 1.2 63.8 + 1.5
Batch 4 733 + 0.2 62.9+4.5 64.3 + 2.0
Batch 5 72.7 + 0.1 62.5+2.4 64.4 + 0.7
Free cellulase 73.7 + 0.4 64.9 + 0.7 63.0 + 07
Conventional 66.9 + 0.1 59.0 + 2.3 58.3 + 2.0
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) (Fable 1-2), As expected, the pectin obtained with free cellulase had similar uronic acid content (73.7%), DB (63-65%), MW (142.5 kDa), and PD (1.58) to that obtained by the magnetic nanobiocatalyst (Fables 1-2). 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 1-2), which could be because of the de-esterification and breakdown of pectin by an acid (Pereira et al,, 2016).
Table 2 Molecular weight and polydispersity analysis of pectin samples from different batches during recycling of magnetic nanobiocatalyst, free cellulase and conventional acid extraction.

Pectin sample M
n M
w M /M
w n
Commercial 107225 141538 1.32
Batch 1 90323 140905 1.56
Batch 2 89317 143801 1.61
Batch 3 92882
87484 139324 142600 1.50
Batch 4

1.63
Batch 5 88828 143902 142522 1.62
Free cellulase 90203
1.58
Conventional 99479 137281 1.38


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. 6). The mass loss in the first region (50-200°C) corresponds to the loss of volatiles with increasing temperature. The major mass loss of approximately 53% occurred in the second region (200-400T) 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 et al., 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 using external calibration standards. The punicalagin (a + p) and ellagic acid were found in phenolics of each cycle. It was interestingly found that the total phenolics of all five batch cycles were rich in punicalagin (Table 3). 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 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.

Table 3 punicalagin and ellagic acid content of total phenolics obtained from different batches during recycling of magnetic nanobiocatalyst '', free cellulase b and conventional acid extraction. Values in bracket indicate percentage of TPC.

Batch number TPC (g/100 g db) Punicalagin (g/100 gdb) Ellagic acid (g/100gdb)
1 8.60 ± 0.3 6.65 ± 0.4 (77.396) 0.48 + 0.03(5.6%)
2 8.53 ± 6.7 6.42 ± 0.1(75.3%) 0.49 + 0.02 (5.7%)
3 8.44± 0.4 6.60 ± 0.2(78.2%) 0.55± 0.03(6.5%)
4 8.70 ±0.2 8.57 ±0.5 6.65 ± 0.5 (76.4%) 0.50 ± 0.05 (5.7%)
5
6.59 ± 0.4 (76.9%) 0.60 ± 0.01 (7.0%)
free cellullase 9.45 ± 1.3 6.92 ± 0.6(73.3%) 0.64 ±0.02 (6.8%)
Conventional 10.00 ±1.6 3.88 + 0.01(38.8%) 4.93 ± 0.04 (49.3%)
'' Magnetic nanobiocatalyst treatment was given at liquid-solid ratio of 15 ml/g, 50°C, pH 6 and cellulase dosage of 75 U/gof peel powder for 5h.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 4h.
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We Claim:
1. An immobilized enzyme mediated process for the extraction of pectin and punicalagin rich
polyphenols from waste pomegranate peels comprising the steps of:
i) drying the waste pomegranate peels;
ii) grinding the dried waste pomegranate peels into powder;
iii) preparing the slurry of waste pomegranate peel powder in aqeuous buffer;
iv) subjecting the slurry to ultrasound treatment;
v) subjecting the slurry to immobilized enzyme (magnetic naobiocatalyst) treatment;
vi) separation of immobilized enzyme;
vii) isolation of pectin and punicalagin rich polyphenols from the solid residues
2. the process in claim 1 wherein the waste pomegranate peels were dried for 24 h at 50 UC and ground into powder of particle size <150 pm using laboratory mill.
3. the process in claim 1 wherein the slurry was prepared by adding waste pomegranate peels into aqeous buffer (pH 4.5-7) at liquid to solid ratio of 10-25 mL/gdw.
4. the process in claim 1 wherein the slurry was subjected to ultrasound treatment using 100-300 W power at 30-60°C for 0-30 min.
5. the process in claim 1 wherein the immobilized enzymatic treatment of slurry was performed by magnetic nanobiocatalyst at cellulase loading of 25-175 U/g, temperature 30-60°C and pH 5-7 for 0.5-7 h under stirring at 150-200 rpm.
6. the process in claim 3 wherein the magnetic nanobiocatalyst comparised of cellulase from Trichoderma reesei covalently attached to amino propyl tri-ethoxysilan coated iron oxide magnetic nanoparticles at 1:3-1:6 MNP:enzyme ratio using 50-100 mM glutaraldehyde as cross-linker and 3-8 h immobilization time at 20--30°C and pH 4-6.
7. the magnetic nanobiocatalyst in claim 5 was of size around 15-30 nm and consisted of cellulase activity of 1-3 U/mg.
8. the process in claim 1 wherein the magnetic nanobiocatalyst was seprated from reaction mixture by external magnetic field
9. the process in claim 1 wherein the extracted pectin was isolated from the reaction mixture by centrifuging the reaction mixture to collect clear superntant, adding ethyl alcohol (99,3% pure) to

the clear supernatant in 1:1 v/v ratio to form pectin precipitate and removing the pectin precipitate by centrifugation from the alcoholic supernatant
10. the process in claim 8 wherein the collected pectin precipitate was washed with ethyl alcohol (99.9% pure) and dried for 24 h at 50°C
11. the process in claim 1 wherein the aqueous extract punicalagin rich polyphenols was collected by distillation of ethyl alcohol from the alcoholic supernatant obtained in claim 8
12. the process in claim 1 wherein the magnetic nanobiocatalyst was reused for five cycles of extraction of pectin and punicalagin rich phenolics without changing its activity,
13. the process in claim 11 wherein the yield, degree of esterification (determined by titration, FTIR, and NMR), molecular weight (determined by gel permeation chromatography), galacturonic acid content and molecular structure (determined by FTIR and 1H NMR) of pectin obtained in each cycle were same.
14. the process in claim 11 wherein the yield and punicalagin content of polyphenols obtained in each cycle were same.