From waste to wealth: High recovery of nutraceuticals from pomegranate seed waste using a green extraction process

Industrial Crops & Products 112 (2018) 790–802

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From waste to wealth: High recovery of nutraceuticals from pomegranate seed waste using a green extraction process Sachin Talekara,c, Antonio F. Pattib, Ramkrishna Singha,c, R. Vijayraghavanb, Amit Aroraa,c,

T

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a

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

A R T I C L E I N F O

A B S T R A C T

Keywords: Green extraction Protease Pomegranate seed Oil Protein Insoluble fibres Waste valorization

Waste pomegranate seed (WPS) from the pomegranate juice industry is an interesting substrate for the food processing industry as it contains high quality oil which is rich in conjugated fatty acids, high quality proteins and dietary fibres. However, the majority of the work reported extraction of only oil and employed organic solvents which decrease the nutritional quality of pomegranate seed protein and fibres. Therefore, in this study, a one pot enzymatic green process was investigated for recovery of high quality oil, food-grade proteins and fibres from WPS. The WPS were treated with protease followed by centrifugation to recover the oil, protein and insoluble fibres in different phases. The mechanism of extraction was confirmed by FTIR-imaging and scanning electron microscopy analysis of WPS. The highest oil recovery of 22.9% (out of which 97.4% in free form) and protein recovery of 13.2% (out of which 90.2% in free form) were obtained, when WPS was incubated with protease at a concentration of 50 U/g for 14 h, at 45 °C and pH 7.2. The remaining WPS residue was rich in insoluble fibres (97.6 g per 100 g WPS residue). The protease-derived oil had 2.3% higher content of conjugated fatty acids and 1.4 times higher total phenolic content than the hexane-extracted oil. Moreover, the proteasederived oil displayed 4% higher antioxidant activity than the hexane-extracted oil. The extracted free proteins were in protein hydrolysate form and had high values of the essential amino acid index (91.6%), protein efficiency ratio (5) and biological value (88.5) confirming their high quality. The insoluble fibre rich WPS residue possessed improved water and oil holding capacity, glucose absorption capacity and glucose dialysis retardation index compared to raw WPS.

1. Introduction India is the world’s largest pomegranate producer with 2.3 million tons produced in 2016 (NHB, 2016). In recent years, the production and consumption of processed pomegranate products, especially juice, have greatly increased throughout the world due to the health-promoting effect of different components of pomegranates. Pomegranate (Punica granatum L.) seed is a waste produced from the pomegranate juice industry (Kalaycıoğlu and Erim, 2017). After juice extraction, the waste pomegranate seeds (WPS) accounts for 10 wt% of the fruit most of which are not utilized (Abbasi et al., 2008). The WPS can be considered as a source of nutraceuticals such as high quality oil (12–24%) (Liu et al., 2012; Tian et al., 2013), protein (10–20%) (Elfalleh et al., 2012) and insoluble fibres (30–50% is cellulose and hemicellulose) (Uçar et al., 2009; Taher-Maddah et al., 2012). Pomegranate seed oil is a rich source of polyunsaturated fatty acids (PUFAs), especially the conjugated fatty acid punicic acid (18:3 n-5) with IUPAC name

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9Z,11E,13Z-octadeca-9,11,13-trienoic acid due to which it possesses many health promoting effects (Aruna et al., 2016). Several studies have demonstrated that pomegranate seed oil displays variety of functional and medicinal effects, such as antioxidant and radical scavenging activity; anticancer activity against prostate cancer, breast cancer, colon cancer, skin cancer; neuroprotective activity against neurodegenerative diseases; antidiabetic and antiobesity property; nephroprotective effects against nephrotoxicity; protective effect against atherosclerosis; anti-inflammatory activity preventing intestinal damage and bone loss; modulating immune function, etc. (Aruna et al., 2016). Pomegranate seed proteins are rich in essential amino acids and meet WHO requirements of essential amino acids for adult humans (Elfalleh et al., 2012). Therefore, WPS proteins could be used as protein resource for human nutrition. As insoluble fibres are dietary fibres responsible for increasing fecal bulk, improving peristalsis, providing laxative effect and anti-hyperglycemic activity in diabetes (Maphosa and Jideani, 2016), WPS being rich in insoluble fibres could also be

Corresponding author at: IITB Monash Research Academy, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India. E-mail address: [email protected] (A. Arora).

https://doi.org/10.1016/j.indcrop.2017.12.023 Received 28 September 2017; Received in revised form 27 November 2017; Accepted 10 December 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.

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used as a source of dietary fibres. When the agro-industrial processing waste is rich in multiple bioactive compounds and nutraceuticals, it can be utlized as a feedstock for the extraction of these products which reduces the waste generation and pollutant loadings (Arshadi et al., 2016; Banerjee et al., 2017). This also provides an additional revenue to agro-processing industries contributing to their sustainable development and achievement of a circular economy based on zero waste (Clark et al., 2016; Mohan et al., 2016). However, the research carried out on WPS utilization has always focused on extraction of a single component, such as the oil and the remaining major WPS organic fraction, rich in proteins and insoluble fibres, would be still disposed of into the environment. In addition, the methods used for WPS oil extraction either yield less oil, employ toxic organic solvent or are not economical. For instance, cold pressing requires significant energy input and the WPS oil extraction yield is only 40–50% (Khoddami et al., 2014). Therefore, the yield loss and energy requirement in cold pressing can be a major factor in the overall cost consideration (Dominguez et al., 1994). Soxhlet extraction (Abbasi et al., 2008), ultrasound (Goula, 2013; Kalamara et al., 2015; Barizão et al., 2015) and microwave (Çavdar et al., 2017) assisted extraction employ organic solvent (mainly hexane) and obtained higher yields (95–99%) of WPS oil. However, because of the safety, environmental contamination and human health hazards associated with the use of hexane, the cost of construction and operating costs of hexane extraction facilities are high (Rosenthal et al., 1996). Supercritical CO2 extraction of WPS oil avoids the use of hexane and a 60–70% oil yield could be obtained (Liu et al., 2009; Liu et al., 2012; Đurđević et al., 2017). However, supercritical CO2 extraction technology requires a high capital investment and, if the protein and insoluble fibre co-products are desired, would still need a separate protein and insoluble fibre extraction facility (Wilken et al., 2016). Drastic mechanical processing, thermal treatment and organic solvents used during the above WPS oil extraction methods not only affect the oil quality but also denature the proteins and decrease their economic and nutritional value (Latif et al., 2011). Therefore, in the scope of industrial application, a mild one pot green process acheiving the extraction of oil, proteins and dietary fibres from WPS in the same facility would be an attractive proposition. An emerging technology involving aqueous enzymatic processing has attracted significant attention for one pot green extraction of both oil and proteins from oilseeds (Yusoff et al., 2015). The aqueous enzymatic process involves treatment of oilseeds with single enzyme or mixture of enzymes in an aqueous medium, followed by centrifugation to separate the slurry into free oil, emulsion (oil rich), aqueous (protein rich) and residual solid phases (Liu et al., 2016a). Recently, Yusoff et al. (2015) and Liu et al. (2016a) reviewed application of protease, carbohydrases and their mixtures for the extraction of oil and protein from oilseeds by the aqueous enzymatic process. The challenge in using this process is to improve the recovery of free oil and protein with no or little emulsion formation (Yusoff et al., 2015). When protein is the dominant barrier for removal of oil, proteolytic enzymes seem to be effective as they hydrolyze all proteins, including the lipophilic protein surrounding the oil bodies, thereby decreasing the emulsion formation and enabling removal of free oil and proteins (De Moura et al., 2008; Jiang et al., 2010; Latif and Anwar, 2011; Latif et al., 2011; Zhang et al., 2011). In addition, the recovery of oil and proteins would concentrate insoluble fibres in the seed residue. Due to the functional properties such as water and oil holding capacity, glucose adsorption and dialysis retardation, the insoluble fibres can be used as dietary fibres (Maphosa and Jideani, 2016). Therefore, proteolytic enzymes not only separate oil and proteins but also allow the utilization of the remaining seed residue as a potential source of dietary fibre, leading to a zero waste process. Recently, Goula et al. (2018) applied cell wall degrading enzymes cellulase and pectinase to recover only oil from pomegranate seeds despite the availability of other natural product such as protein and dietary fibres of high commercial value therewith. Due to the use of cell wall degrading enzymes dietary fibres are degraded, hence loosing

this product. In addition, oil recovery of 70% of hexane-extracted oil (which is less than that obtained in present work) and use of two enzymes may increase the cost of overall process. Our preliminary histochemical analysis of pomegranate seeds showed that the oil bodies are enmeshed in a cytoplasmic network rich in proteins. We hypothesized that the protein is a major component holding oil and acting as a barrier for its extraction. Therefore, we employed a protease to break the cytoplasmic network of pomegranate seed cells to release the oil and proteins and obtain pomegranate seed residue rich in dietary fibres. So far as we know, no previous studies have been conducted on the recovery and concentration of nutraceuticals such as oils, proteins, and dietary fibres from WPS in a single pot. Therefore, the objective of this work was to develop a one pot green process using protease for simultaneous recovery of oil, proteins and insoluble fibres. The effects of protease concentration and incubation time were evaluated for oil yields, fatty acid composition and antioxidant activity of the obtained oil, and these product parameters were compared with hexane-extracted oil. The proteins were characterized in terms of nutritional value based on their amino acid composition, antioxidant activity and molecular weight distribution. The seed residue remaining after oil and protein extraction was characterized in terms of insoluble fibre content and dietary fibre properties, such as water and oil holding capacity, glucose adsorption capacity and glucose dailysis retardation index. Furthermore, light microscope and FTIR-images, and SEM images of seed samples before and after protease treatment were taken in order to understand the extraction mechanism. 2. Materials and methods 2.1. Materials Protease from Aspergillus oryzae (500 U/mL), Folin-Ciocalteu reagent and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, St. Louis (USA). Other reagents used were of analytical grade and obtained either from Sigma-Aldrich or Merck. Fresh pomegranates (5 kg) of Bhagava cultivar were procured from a local market in Mumbai, India. The pomegranate arils were manually separated from the fruit and crushed to separate the seeds from the juice. The seeds were carefully washed with 1 L tap water three times for the removal of pomace residues and dried in hot air oven (model RDHO-50, Remi, India) at 50 °C for 48 h. The dried seeds were ground into a powder (mean particle size 500 mm) in a mixer grinder (model Bajaj GX8 500, India) and kept in a sealed bag at 4 °C until use. 2.2. Conventional soxhlet extraction The pomegranate seed powder (10 g on dry weight basis) was packed into a cellulose thimble, and this thimble was placed in a Soxhlet extractor fixed with a 250 mL round-bottom flask and a water condenser. The round-bottom flask was filled with 150 mL of hexane. The extraction process was executed at 60 °C for 4 h (Tian et al., 2013). After extraction, hexane was removed by evaporation at 40 °C under reduced pressure using a rotary evaporator. The obtained oil was weighed and the extraction yield was calculated by dividing the mass of oil obtained per 100 g of seed powder on dry weight basis. The recovered oil was considered as total oil content of pomegranate seed powder and stored at 4 °C, until use for further experiments. 2.3. One pot extraction of oil, protein and insoluble fibres from WPS The extraction process presented in this study comparises protease treatment of WPS followed by centrifugation for separation of the oil, protein and insoluble fibres in separate phases as depicted in Fig. 1. The details of extraction are as follows: Ten grams (dry basis) of pomegranate seed powder was added to 79 mL of 50 mM sodium phosphate buffer at pH 7.2 and incubated for 791

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Fig. 1. Schematic presentation of protease-assisted aqueous extraction of oil, protein and insoluble fibres from WPS.

as described above. Raw pomegranate seed powder, the aqueous phase and the solid residue phase obtained after protease treatment were analyzed for protein contents by the Kjeldahl method (Horwitz et al., 1970). Raw pomegranate seed powder and solid residue obtained after protease treatment were also analyzed for neutral detergent fibres (NDF), acid detergent fibres (ADF) using the Van Soest method (Goering and Van Soest, 1970) and lignin using NREL method (Sluiter et al., 2010). ADF denotes cellulose and lignin, while NDF corresponds to ADF fraction and hemicellulose. Oil, protein, cellulose, hemicellulose and lignin contents of raw pomegranate seed powder are given in Table 1.

10 min at 45 °C. Then protease was added to obtain the enzyme concentrations (5–100 U/g pomegranate seed powder dry basis). As previously reported by Latif and Anwar, 2011, the mixture was kept at 45 °C for 4–16 h with constant shaking at 110 rpm in orbital shaker (model CIS-24 Plus, Remi, India), followed by centrifugation at 7000 rpm for 20 min. After centrifugation four distinct phases were obtained: (i) free oil phase (ii) creamy phase (emulsion) (iii) aqueous phase and (iv) residual solid phase. Most of the top oil phase was first withdrawn as free oil using a micro-pipette, followed by the removal of the creamy and aqueous phases, leaving the solid residue at the bottom. The remaining free oil was collected of the hexane wash by following previously described method (De Moura et al., 2008). The creamy phase was rinsed with 2 mL of hexane and the hexane wash was collected. Then the free oil was separated from collected hexane by evaporation at 40 °C under reduced pressure using a rotary evaporator. Hexane was used only to ensure that the free oil was removed completely in order to get its accurate quantification. However, we do not envision using hexane on an industrial scale. The remaining creamy phase was used to determine the quantity of proteins trapped in the emulsion. The aqueous phase contains free proteins in the form of a protein hydrolysate. No residual oil was found in the aqueous phase. Finally, the wet solid residue obtained was mixed thoroughly, dried at 50 °C for 48 h and used for determination of insoluble fibre, residual oil and protein content. The oil trapped in the emulsion was estimated by subtracting the residual oil in the solid residue and the free oil from the total oil content of pomegranate seed powder. A control without protease addition was run under the same conditions and treated identically to obtain similar four phases, with free proteins in the unhydrolysed form present in the aqueous phase. The yields of pomegranate seed oil and protein were calculated using the following Eqs. (1) and (2):

Total oil yield (%) mass of free oil + mass ofoil in emulsion = × 100 mass ofpomegranate seed powder taken for extraction

2.5. Surface morphology of pomegranate seed powder In order to examine the morphological alterations of pomegranate seed powder after protease treatment, and also to propose the extraction mechanism, the Quanta-200 environmental scanning electron microscope system (FEI Company, USA) operated at 20 kV has been utilized. The pomegranate seed powder samples before and after protease treatment were fixed on a specimen holder with a carbon tape and sputtered with a thin layer of platinum prior to examination under low vacuum condition. 2.6. Fatty acid analysis of oil The fatty acid composition of the protease-derived oil and hexaneextracted oil was analyzed as fatty acid methyl esters (FAMEs). The FAMEs were prepared according to the AOAC official method 969.33. AOAC (1995) Methanol (19 mL) and sulfuric acid (1 mL) were added to 0.2 g of each of the oil samples separately and stirred for 5 min. The mixtures were incubated at 80 °C under reflux for 2 h. After cooling to room temperature, FAMEs were extracted with hexane (20 mL). Finally, FAMEs were collected by evaporation of the hexane under nitrogen to avoid oxidation of unsaturated fatty acids.

(1)

Total protein yield (%) mass of free protein + mass of protein in emulsion

Table 1 Oil, protein, cellulose, hemicellulose and lignin content of pomegranate seeds.

− mass of enzyme protein = × 100 mass of pomegranate seed powder taken for extraction (2) 2.4. Oil, protein and insoluble fibre content analysis The oil content of raw pomegranate seed powder and solid residue obtained after protease treatment was determined by Soxhlet extraction 792

Component

g/100 g dry basis

Oil Protein Cellulose Hemicellulose Lignin Total insoluble fibre (cellulose + hemicellulose)

23.5 13.6 13.7 19.8 22.0 33.5

± ± ± ± ± ±

0.9 0.4 0.3 0.5 0.2 0.4

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The diluted FAMEs were analyzed by using an Agilent Technologies 7890A GC system equipped with a Mass Spectrometer Jeol AccuTOF GCV JMS-T100GCV, the GC column was Hewlett-Packard HP-5MS (30 m × 90.25 mm × 90.25 mm). Injector temperature (250 °C) and ion source temperature (200 °C) were set and 2 mL of the FAME sample was injected into the column using the split mode (split ratio 5:1). Helium was used as a carrier gas with flow rate 1 mL/min. Mass fragments were monitored over mass/charge range 50–600. FAME separation was achieved following an oven temperature program: 70 °C initially held for 5 min followed by an increase of 10 °C/min until 200 °C. The temperature program was then increased again by 5 °C/min to a final temperature 275 °C, so the total run time was 32 min. Each sample was injected twice. Fatty acid composition (%) was calculated using peak normalization method, assuming equal detector response. The unknown fatty acids were identified by comparing their retention times with standard fatty acid methyl esters and also on the basis of computer matching of their mass spectrum with National Institute of Standards and Technology (NIST, 3.0) libraries installed in the computer controlling the GC–MS system. From the database, the name, molecular weight and structure of the fatty acids were ascertained. The relative proportions of the fatty acids were expressed as percentages obtained by peak area normalization.

[Lys × Thr × Val × (Met + Cys ) × Ile × Leu × (Phe + Tyr ) EAAI =

× His × Trp]ph 9

[Lys × Thr × Val × (Met + Cys ) × Ile × Leu × (Phe + Tyr ) × His × Trp]sc (3)

Where ph* represents the content (%) of amino acids in protein hydrolysates and sc* represents the content (%) of the same amino acids in standard protein (Casein), respectively. 1 Protein efficiency ratio (PER) was calculated using the Eqs. (4) and (5) developed by Alsmeyer et al. (1974):

PER1 = −0.468 + 0.454(Leu) − 0.105(Tyr )

(4)

PER2 = −1.816 + 0.435(Met ) + 0.78(Leu) + 0.211(His ) − 0.944(Tyr ) (5) 1 Biological value (BV) was calculated using the equation of Oser (1959).

BV = 1.09 × EAAI − 11.7

(6)

2.7. Physico-chemical properties of oil

2.9. Estimation of total phenolics (TP)

Free acidity, peroxide and iodine values of protease-derived oil and hexane-extracted oil were determined by American Oil Chemists’ Society (AOCS) (1993) standard methods Ca 5a-40, Ja 8-87 and Cd 1c85, respectively.

The extracts from the protease-derived and hexane-extracted oil were prepared separately by the method of Latif and Anwar (2011) with some modifications. Oil constituents were extracted by adding 3 mL of solvent (methanol: water, 50:50 v/v) to 1 gm of oil in a test tube. The test tube was vortexed for 5 min and then centrifuged at 7000 rpm for 15 min and the supernatant was collected. This procedure was done three times and all three supernatants were combined to give an extract from oil. For the estimation of total phenolics in pomegranate seed protein hydrolysate, a protein hydrolysate sample with a protein concentration of 1 mg/mL was used. For comparison, the free protein sample (protein content 1 mg/mL) from a control experiment without protease was also used. The total phenolic contents of extract from oil, protein hydrolysate and control group free protein sample were determined using the modified Folin-Ciocalteau method (Singleton and Rossi, 1965). Three hundred microliters of sample were mixed with 600 mL of Folin-Ciocalteu reagent (10% v/v freshly prepared) and incubated for 10 min at 30 °C. Then 2400 mL of 700 mM sodium carbonate solution was added and the resulting mixture was mixed vigorously. After 2 h incubation at 30 °C, the absorbance at 765 nm was measured and total phenolics were calculated in gallic acid equivalents (GAE) in milligrams per gram of oil or protein using gallic acid standard curve. Similar method has been used in previous literature for the determintion of total phenolics in the oil (Latif and Anwar, 2011) and the proteins or protein hydrolysate (Siddeeg et al., 2015).

2.8. Amino acid composition and nutritional value of pomegranate seed protein hydrolysate For amino acid analysis, a protein hydrolysate sample containing 4 mg proteins was added to 6N HCl (15 mL) containing 0.1% phenol (to avoid oxidation) and the oxygen was expelled from the sample/acid mixture by passing gaseous nitrogen through the tube for 2 min. The mixture was incubated for 20 h at 110 °C followed by neutralization with 4N NaOH. The neutralized mixture was then filtered using a 0.2 mm membrane (Whatman) and the filtrate was used for amino acid analysis. The amino acid analysis technique by Agilent Technologies was used to analyze the filtrate (Long, 2015). Samples were analyzed using a high performance liquid chromatography system (Agilent 1100 series) equipped with a photodiode array (PDA) detector. The chromatographic separations were performed using an Agilent Poroshell HPH C18 column (4.6 × 100 mm, 2.7 mm dp) supplied by Agilent Technologies. The separation was conducted under binary gradient mode using mobile phase A (10 mM Na2HPO4, 10 mM Na2B4O7, pH 8.2, and 5 mM NaN3) and mobile phase B (acetonitrile:methanol:water; 45:45:10, v:v:v). The gradient used was: 0 min, 2% B; 0.35 min, 2% B; 13.4 min, 57% B, 13.5 min, 100% B; 15.7 min, 100% B; 15.8 min, 2% B; 18 min, 2% B, with a mobile phase flow rate of 1.5 mL/min, and column temperature of 40 °C. PDA detection wavelengths were 262 nm, and 338 nm with peak width settings of > 0.01 min. The injection volume was 20 mL. The unknown amino acids were identified by comparing their retention times with standards available from Agilent and quantified using five concentrations calibration curve of each standard amino acid. The amino acid composition of pomegranate seed protein hydrolysate was used for determination of several nutritional parameters as follows:

2.10. DPPH scavenging activity The antioxidant activities of the protease-derived oil, hexane-extracted oil and protein hydrolysates were measured in terms of their ability to scavenge the commercially available stable organic nitrogen radical DPPH. For comparison with protein hydrolysates, we also studied the ability of free protein sample obtained from a control group without protease treatment to scavenge DPPH radicals. Ascorbic acid was used as a standard reference and prepared with ethanol. The DPPH radical scavenging activity was measured with different concentrations of oil (0.4–22 mg/mL), protein hydrolysate (0.012–0.36 mg/mL) and a control group free protein sample (0.012–0.36 mg/mL) in the reaction mixtures. For oil, 1 mL of the oil sample solution was mixed with 3 mL DPPH solution (4 mg/100 mL ethanol) and for protein hydrolysate and control group free protein samples, 1 mL of the protein hydrolysate or the control group free protein sample solution was mixed with 3 mL

1 Essential amino acid index (EAAI) was calculated according to following equation (Steinke et al., 1980) using the amino acid composition of casein as standard.

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2.12.2. Glucose adsorption capacity The glucose adsorption capacity of pomegranate seed residue remaining after oil and protein extraction was determined according to the method of Ma et al. (2016). Seed residues (1 g) were added into a glucose solution (60 mL) containing 3.34 mmol glucose. After stirring for 5 h at 37 °C, the glucose-adsorbing capacity of seed residues was determined using the glucose estimation kit supplied by Sigma aldrich.

DPPH solution (4 mg/100 mL 95% ethanol). The reaction mixtures were shaken vigorously, incubated in the dark at 30 °C for 30 min, and the absorbance was recorded at 517 nm. DPPH radical-scavenging activities were expressed as inhibition percentage of DPPH calculated according to following equation:

Asample − Ablank ⎞ Inhibition(%) = ⎛1 − × 100 Acontrol ⎠ ⎝

(7) 2.12.3. Glucose dialysis retardation index (GDRI) The glucose dialysis retardation index (GDRI) was determined as described by Liu et al., (2016b) with slight modifications. Seed residues (400 mg) were added to 15 mL of Millipore pure water containing 30 mg glucose, and stirred for 1 h. Then, the mixtures were transferred to previously hydrated dialysis bags (12,000 MWCO, Himedia) and dialyzed against 400 mL of Millipore pure water at 37 °C with constant magnetic stirring for 1 h. At 10, 20, 30, 40, 50 and 60 min, 0.5 mL of the dialysate was collected, and the glucose concentration was determined using the glucose estimation kit supplied by Sigma aldrich. Under the same conditions, dialysis bags containing only a glucose solution were used as the control. GDRI was calculated as follows:

where Ablank is absorbance of oil or protein hydrolysate or control group free protein samples without DPPH while Acontrol is the absorbance of DPPH without any oil or protein hydrolysate or control group free protein samples. The concentration of samples required to scavenge 50% of DPPH radicals (IC50) was also calculated by fitting data to the inbuilt non-linear regression (Dose-response inhibition) equation in GraphPad Prism 7. Experiments were conducted in triplicate for each sample and the data were expressed as means ± standard deviation.

2.11. Size distribution of peptides from protein hydrolysate using mass spectrometry

Total glucose diffused from samples × 100⎞⎟ GDRI = 100 − ⎛⎜ ⎝ Total glucose diffused from control ⎠

For mass spectrometric analysis, protein hydrolysate was desalted and concentrated by dialysis using a C18 reverse phase resin (ZipTip, Millipore, Bedford, MA) according to the manufacturer’s instructions. For comparison, free protein sample obtained from a control group without protease treatment was also desalted and concentrated by ZipTip C18 pipette tips in the same way. Then, protein hydrolysate and control group free protein samples were directly injected into an electrospray ionization-mass spectrometer (ESI–MS) (Bruker Maxis Impact) coupled with tandem mass spectrometry (MS/MS). Spectra were recorded in positive ion mode over the mass/charge (m/z) range of 50–3000. High purity N2 was used as drying gas at a flow rate of 4.0 L/ min and as a nebulizing gas at a pressure of 0.3 bar. The drying heater was 180 °C, the voltage of capillary was 3.8 kV and charging was 2.0 kV.

(8)

2.13. Statistical analysis All the experiments were performed in triplicate and data are expressed as mean values ± standard deviation obtained from triplicate determinations. Statistically significant differences between in the datasets were evaluated (p < 0.05) using a one way analysis of variance (ANOVA) with Tukey test. Analysis was carried out with the help of Minitab 17 (Minitab Inc. USA). 3. Results and discussion

2.12. Measurement of functional properties of pomegranate seed residue

3.1. One pot extraction of oil, protein and insoluble fibres from pomegranate seed by protease treatment

2.12.1. Water and oil holding capacity The water and oil-holding capacity of the pomegranate seed residue remaining after oil and protein extraction were measured, following the procedure described in the literature (Zhang et al., 2017). Seed residues (0.5 g dry basis) were placed into 50 mL centrifuge tubes and 30 mL of either distilled water or soybean oil was added and mixed together. After incubating for 5 h at 37 °C, the samples were centrifuged at 6000 rpm for 20 min, and the supernatant was carefully discarded. The obtained pellet was weighed and its water or oil holding capacity was calculated as grams of water or oil retained per gram of seed residue sample.

The mechanism of one pot extraction of pomegranate seed oil, protein and insoluble fibres is shown in Fig. 2. Results from the light microscopy and FTIR-imaging of pomegranate seeds showed that oil is located within the cytoplasmic matrix which is also composed of proteins (Fig. 3). Therefore, the cytoplasmic matrix proteins can be a major component holding the oil and presenting a barrier of the oil extraction. Protease treatment of pomegranate seeds hydrolyses these proteins which breaks down the cytoplasmic matrix releasing the oil and protein hydrolysates into the surrounding aqueous medium. The protein hydrolysates are solubilized in an aqueous medium, while oil (being insoluble) forms a separate phase above the aqueous medium. The

Fig. 2. A possible mechanism of oil, protein and insoluble fibre extraction from pomegranate seeds by protease treatment.

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Fig. 3. (a) Visible image of the chosen area containing seed coat (SC) and endosperm (ES) of sectioned pomegranate seed (b) Microscopy image of the pomegranate seed section stained with Sudan black, indicating the presence of oil bodies (OB) mostly in the endosperm. (c) Infrared images representing lipid distribution at the absorbance of 1740 cm−1 and (d) protein distribuition the absorbance at 1650 cm−1. A pink area corresponds to a high concentration of component relative to a blue area.

of protease (Zhang et al., 2011). The coincidence of the extraction time required to achieve maximum yields of protein and oil confirmed that the mechanism of the oil release strongly depends on the hydrolysis of proteins. On average, the protease treatment gave an oil yield of 22.9% which was comparable to the oil yield of 23.5% obtained by conventional Soxhlet extraction. As the oil and protein in free form are easy to separate compared to those which are in the emulsion phase, it is important to obtain a maximum fraction of total extracted oil and protein in free form for the economic viability of the process (Yusoff et al., 2015). It was observed that the increase in both protease concentration and extraction time decreased the amount of emulsion formed and increased the free oil and free protein fractions of total extracted oil and protein. When the extraction time at a protease concentration of 50 U/g was increased from 4 to 14 h, the free oil and free protein fractions of total oil and protein extracted at each time were increased (Fig. 5c and d). The maximum free oil (98%) and free protein (93.1%) fractions of total extracted oil and protein were achieved at 14 h and no significant change (p > 0.05) was observed with prolonged extraction (16 h). Hydrolysis products may be inhibiting protease to act further during prolonged extraction. The increase in free oil and free protein fractions with protease concentration and extraction time could be explained by an increase in hydrolysis and solubilization of the protein based membrane that surrounds the oil bodies in emulsion phase, which decreases the integrity of the interfacial film (Wu et al., 2009), and

remaining insoluble residue of pomegranate seeds contains unbroken cell wall insoluble fibres. This is indicative of protease treatment of pomegranate seeds separates oil, protein and insoluble fibres. The proposed mechanism was confirmed by scanning electron microscopic analysis of raw and protease treated pomegranate seed powder. Fig. 4a and 4b clearly show the entrapped oil bodies enclosed within the cell structure. After protease treatment, both the oil bodies and the surrounding matrix disappeared, leaving empty cells with intact cell wall fibre structure (Fig. 4c and d). It indicates that upon protease treatment the matrix was ruptured due to hydrolysis and solubilization of proteins, which resulted in the release of oil. Several combinations of enzyme dosage and time were evaluated to obtain maximum yield of oil. It was observed that enzyme dosages 50 U/g and higher for 14 h yielded the maximum oil recovery. However, increase in enzyme dosage beyond 50 U/g (at 14 h incubation time) did not significantly improve the oil recovery (Fig. 5a). It could be due to substrate saturation (Jiang et al., 2010). Since it was also important to understand how protein recovery varies with enzyme dosagetime combinations, the enzyme dosage was fixed at 50 U/g and it was found that the extraction yields of total oil and protein increased simultaneously up to 14 h, and maximum yields of 22.9 ± 0.8% and 13.2 ± 0.7%, respectively were obtained (Fig. 5b). No further significant increase (p > 0.05) in extraction yields was observed which may be due to the depletion of the substrates and/or product inhibition 795

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Fig. 4. Environmental scanning electron microscopy images of raw pomegranate seed powder at (a) 1000 X and (b) 3000 X and protease treated pomegranate seed powder at (c) 1000 X and (d) 3000 X. OB- oil body, CM- cell matrix, CW- cell wall.

means protease-derived oil had more unsaturated fatty acid (P < 0.05). The results were confirmed by fatty acid analysis of oils. Six fatty acids were identified by comparison of mass spectra of the compound with the mass spectra available in spectral libraries (Table 3). The percentage of polyunsaturated fatty acid (PUFA) is higher in protease derived-oil in comparison to the conventional hexane-extracted oil (p < 0.05). PUFA consists of two important conjugated fatty acids conjugated linolenic acid (C18:3) and conjugated linoleic acid (C18:2), which are recognized for anti-cancer, antidiabetic, anti-obesity, anti-inflammatory, neuroprotective, nephroprotective activities. The protease derived-oil contained 53.92 ± 0.01% conjugated linolenic acid and 6.74 ± 0.03% conjugated linoleic acid which are higher than those present in hexane-extracted oil (p < 0.05). The punicic acid was a major C18:3 fatty acid in both protease derived-oil and hexane extracted oil followed by a-linolenic acid. The protease derived-oil contained 53.36 ± 0.01% punicic acid and 0.56% a-linolenic acid which are higher than those present in hexane-extracted oil (p < 0.05). Moreover, overall total unsaturated fatty acids were higher in protease derived-oil (86.07 ± 0.07%) than in hexane-extracted oil (85.1 ± 0.17%) (p < 0.05). Usually, unsaturated fatty acids have desirable nutrition properties (Long et al., 2011). Organic solvent extraction is based on the solubility of oil in an organic solvent, and different fatty acids may have different solubilities in hexane (Long et al., 2011). In contrast, in protease treatment, oil extraction is based on the insolubility of the oil in the aqueous medium, rather than on the dissolution of oil. In this case, the soluble

resulted in partitioning of free oil and protein into two phases. The control group extracted for 14 h without any protease treatment had the lowest extraction yields of total oil (7.54 ± 1.3%) and protein (3.23 ± 1.9%). Most of the oil and protein were still in the pomegranate seeds. Protease treatment of pomegranate seeds increased its insoluble fibre proportion. Tables 1 and 2 show that extraction of free oil and protein resulted in concentration of insoluble fibre from 33.5 to 59% (w/w dry basis) in the residue. In contrast, control treatment enriched insoluble fibre content from 33.5 to only 37.7% (w/w dry basis). Total mass balance of oil, protein and insoluble fibres for the proteaseassisted extraction process shows that the protease-assisted one pot extraction process not only maximized the extracts of oil and protein, but also concentrated insoluble fibres in the seed residue. 3.2. Physico-chemical properties and fatty acid analysis of oil The protease-derived oil has lower peroxide value (0.33 ± 0.02 mmol/ kg) in comparison to hexane-extracted oil (1.4 ± 0.3 mmol/kg), which means the protease-derived oil showed less oxidative degradation under the same storage condition (Çavdar et al., 2017). The lower acid value of protease-derived oil (0.42 ± 0.1 mg KOH/g)) compared to hexane-extracted oil (0.87 ± 0.08 mg KOH/g) indicated that protease-derived oil contained less free fatty acids (P < 0.05). The protease-derived oil had higher iodine value (195.8 ± 1.4) than hexane-extracted oil (190.1 ± 1.7), which 796

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Fig. 5. (a) Effect of enzyme concentration and incubation time on the extraction yield of total oils from pomegranate seed (b) Effect of incubation time on extraction yields of oil and protein at optimized enzyme dosage (50 U/g). (c) The free oil and oil in emulsion fractions of total extracted pomegranate seed oil with different incubation times (d) The free protein and protein in emulsion fractions of total extracted pomegranate seed protein at different incubation times. All experiments were conducted at 45 °C and pH 7. The experiments were done in triplicate and the error bars represent the standard deviation. All extraction yields are expressed on dry weight basis.

Table 2 Insoluble fibre recovery in pomegranate seed residue obtained with and without protease treatment. Insoluble fibre

Cellulose Hemicellulose Lignin Total insoluble fibre (cellulose + hemicellulose)

With protease treatment (g/100 g dry basis) 23.6 35.4 38.6 59.0

± ± ± ±

0.3a 0.4a 0.1a 0.3a

Table 3 Fatty acid composition (% of total fatty acids) of protease-derived and hexane- extracted pomegranate seed oil.

Without protease treatment (g/100 g dry basis) 15.4 22.3 25.0 37.7

± ± ± ±

Fatty acid C16:0 C18:2 C18:1 C18:0 C18:3 C18:3 punicic acid C18:3 a-linolenic acid C20:3 SFA UFA PUFA

0.3b 0.5b 0.2b 0.4b

The values not sharing a common superscript small letter in two columns are significantly different (P < 0.05).

components of the oil seeds diffuse into the aqueous medium rather than the oil, thereby releasing the oil which was previously bound in the original structure (Rosenthal et al., 1996). Therefore, the protease treatment resulted in different composition of the oil.

Protease-derived oil a

8.07 ± 0.02 6.74 ± 0.01a 24.89 ± 0.04a 5.84 ± 0.02a 53.92 ± 0.01a 53.36 ± 0.01a 0.56 ± 0.02a 0.52 ± 0.01a 13.91 ± 0.04a 86.07 ± 0.07a 61.18 ± 0.03a

Hexane-extracted oil 8.53 ± 0.03b 5.14 ± 0.07b 26.24 ± 0.02b 5.95 ± 0.05b 53.14 ± 0.03b 52.91 ± 0.03b 0.23 ± 0.05b 0.60 ± 0.01b 14.48 ± 0.08b 85.1 ± 0.17b 58.85 ± 0.07b

The values not sharing a common superscript small letter in two columns are significantly different (P < 0.05). SFA-Saturated fatty acids, UFA-Unsaturated fatty acids, PUFAPolyunsaturated fatty acids.

Table 4. The pomegranate seed protein hydrolysate contained 20 amino acids, including all 9 essential amino acids. The total amount of essential and non-essential amino acids were 41.94 g and 57.37 g per 100 g protein, respectively. Among the essential amino acids leucine, phenylalanine, tyrosine, isoleucine, lysine and valine were predominant. Glutamic acid, aspartic acid, arginine, alanine and serine

3.3. Amino acid composition and nutritional value of pomegranate seed protein hydrolysate The total amino acid profile of the protein hydrolysate obtained from pomegranate seeds by protease treatment is summarized in 797

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of quality plant protein for body builders, over aged adults and Type-2 diabetes patients. The EAAI of pomegranate seed protein hydrolysate was found to be high (91.62%) as compared to that of soy protein (83%) and chicken meat protein (82%). The protein hydrolysate was rated as good quality protein resource based on Oser’s (1959) method when the EAAI is 90%, useful when around 80% and incomplete protein resource when below 70%. Therefore, pomegranate seed protein hydrolysate obtained by protease treatment is a good quality protein source. The predicted PER for pomegranate seed protein hydrolysate was 5 which is higher than that of proteins from soya (1.6), whey (4.7), chicken egg (2.9) and chicken meat (2). As proteins with PER which exceeds 2.7 have been recognized as excellent protein sources (Hoffman and Falvo, 2004), the pomegranate seed protein hydrolysate can be considered as an excellent quality protein. The biological value (BV) of pomegranate seed protein hydrolysate (88.5), which is an estimate of how much of the consumed protein would be incorporated in the body (Hoffman and Falvo, 2004), was quite comparable to that of whey (94) and chicken egg (94.4) proteins and higher than that of soya (78.7) and chicken meat (77.6) proteins.

Table 4 Total amino acids profile of the protein hydrolysate obtained from pomegranate seeds. Amino acids

Amino acids in protein hydrolysates (g/100 g)

Essential amino acids Histidine Isoleucine Leucine Lysine Methionine + Cysteine Phenylalanine + Tyrosine Threonine Tryptophan Valine

2.14 ± 0.15 4.53 ± 0.35 12.17 ± 0.02 4.7 ± 0.20 2.30 ± 0.10 8.28 ± 0.63 2.61 ± 0.22 0.93 ± 0.04 4.48 ± 0.17

Non- essential amino acids Aspartic acid Glutamic acid Aspargine Serine Glutamine Glycine Arginine Alanine Proline Hydroxyproline

9.21 ± 0.60 20.78 ± 0.80 0.43 ± 0.06 4.38 ± 0.02 1.06 ± 0.01 6.25 ± 0.25 7.37 ± 0.60 7.16 ± 0.28 ND 0.73 ± 0.09

3.4. Antioxidant activity assay The antioxidant activities of the protease-derived oil, hexane-extracted oil and protein hydrolysate were determined as DPPH inhibition rates (%). DPPH inhibition rates (%) at different concentrations of oils and protein hydrolysate are shown in Fig. 6a and b. With increasing oil concentrations, DPPH inhibition rates (%) increased up to a maximum level and thereafter reached a plateau. The maximum level of DPPH inhibition rate for the protease-derived oil was high (98.3 ± 0.5%) compared to the hexane-extracted oil (94.3 ± 0.2%). Next, the concentration of sample needed to scavenge 50% of DPPH present in the test solution (IC50) was also calculated. The IC50 of standard ascorbic acid was 0.02788 ± 0.0080 mg/mL. The IC50 values of protease-derived and hexane-extracted oil were 1.38 ± 0.05 mg/mL and 1.66 ± 0.11 mg/mL, respectively. A lower IC50 value indicates a better DPPH radical scavenging activity. Therefore, the protease derived-oil had a higher DPPH radical-scavenging activity. The total phenolics content of protease derived-oil (64.38 ± 1.91 mg/g oil) was higher than hexane-extracted oil (44.81 ± 0.89 mg/g oil). It could be attributed to the non-polar nature of hexane reducing the extraction of phenolic compounds (Abbasi et al., 2008; Zuorro et al., 2013). In addition, it was also demonstrated that enzyme treatment during aqueous extraction enhances the release of phenolic compounds into the seed oil, thus, contributing to the superior antioxidant activity of such oils (Ballus et al., 2015; Latif and Anwar, 2011).

were abundant in non-essential amino acids. Table 5 presents a comparison of essential amino acids content, EAAI, PER and BV of pomegranate seed protein hydrolysate with those of some other proteins (soy, whey, chicken egg and chicken meat) available in the market. The total essential amino acids content in the hydrolysate was higher than in the soy protein (36.9 g/100 g protein) and the chicken meat protein (35.3 g/100 g protein). The amount of essential amino acids in pomegranate seed protein hydrolysate, whey and chicken egg protein meets the FAO/WHO/UNU requirement (WHO/FAO/UNU, 2007) for all age groups above 3 years whereas soy protein and chicken meat protein do not meet methionine + cysteine (both) and valine (for chicken meat protein) requirement for all age groups above 3 years. In contrast, for the age group below 2 years old, the limiting amino acids are lysine, methionine + cysteine and threonine for pomegranate seed protein hydrolysate, lysine, methionine + cysteine and valine for soy protein, histidine for whey protein, leucine, methionine + cysteine and valine for chicken meat protein. Noticeably, pomegranate seed protein hydrolysate is rich in leucine (12.17 g/100 g protein) compared to soy, whey and chicken egg and meat proteins. As leucine prevents muscle breakdown by activating muscle protein synthesis and improves glycemic control in humans with Type-2 diabetes (Yang et al., 2010), pomegranate seed protein hydrolysate could be a useful dietary source

Table 5 Comparision of the quality of protein hydrolysates obtained from pomegranate seeds with some proteins available in the market. Essential amino acids

Histidine Isoleucine Leucine Lysine Methionine + Cysteine Phenylalanine + Tyrosine Threonine Tryptophan Valine Total EAAI PER BV a

Amount (g/100 g protein)

Age-wise FAO/WHO/UNU amino acid requirement (g/100 g protein)

Pom

Soya

Wheya

Egga

Chicken meata

0.5

1–2

3–10

11–14

15–18

> 18

2.14 4.53 12.17 4.7 2.30 8.28 2.61 0.93 4.48 42.14 91.6 5 88.5

2.30 4.25 6.78 5.32 2.17 7.82 3.13 1.11 4.09 36.97 83 1.67 78.77

1.31 5.60 10.23 9.70 3.76 5.24 7.91 1.88 5.87 51.5 97 4.7 94.03

2.46 5.40 8.56 7.40 5.64 9.24 4.73 1.72 6.60 51.75 97.4 2.9 94.46

2.40 3.40 6.20 7.0 1.50 6.50 3.80 0.90 3.60 35.3 82 2 77.68

2.0 3.2 6.6 5.7 2.7 5.2 3.1 0.85 4.3

1.8 3.1 6.3 5.2 2.5 4.6 2.7 0.7 4.1

1.6 3.0 6.1 4.8 2.3 4.1 2.5 0.66 4.0

1.6 3.0 6.1 4.8 2.3 4.1 2.5 0.66 4.0

1.6 3.0 6.0 4.7 2.3 4.0 2.4 0.63 4.0

1.5 3.0 5.9 4.5 2.2 3.8 2.3 0.6 3.9

USDA National Nutrient Database for Standard Reference.

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be buried within the protein core where they are inaccessible to DPPH radicals. Protease hydrolysis disrupts the tertiary structure of proteins to form low molecular weight peptides (protein hydrolysate) where solvent accessibility of these amino acids increases, which leads to DPPH radical scavenging activity. Generally, the short peptides with 2–10 amino acids exhibit stronger antioxidant capacity than their parent native proteins or large polypeptides (Elias et al., 2008; Pan et al., 2011). We confirmed the formation of short peptides in protein hydrolysate by mass spectrometry. Low molecular weight peptides in the range from 300 to 1000 Da were formed (Fig. 7a). The highest proportion comprised peaks corresponding to peptides with a molecular weight of 381.06 Da and 543.12 Da. However, mass spectrometry of the control group free protein sample did not show the formation of low molecular weight peptides (Fig. 7b) which could be one more reason for its lower antioxidant activity than protein hydrolysate. 3.5. Functional properties of pomegranate seed residue remaining after oil and protein extraction The important properties of insoluble fibres, which include water holding capacity (WHC), oil holding capacity (OHC), glucose adsorption capacity and GDRI, are suggestive of the possibility of using insoluble fibres, as an ingredient in food products. Table 6 presents the WHC, OHC, glucose adsorption capacity and GDRI of raw pomegranate seed powder and the pomegranate seed residue remaining after protease treatment. As can be observed in Table 6, WHC, OHC, glucose adsorption capacity and GDRI values increased in pomegranate seed residue remaining after protease treatment. The SEM micrographs showed that after protease treatment the surface of pomegranate seeds becomes highly porous (Fig 4c and d). It is known that the appearance of pores increases the area accessible to molecules which enhances their physical entrapment into the porous structure (Ma et al., 2016). Therefore, the increased WHC, OHC, glucose adsorption capacity and GDRI of pomegranate seed residue remaining after protease treatment could be ascribed to the increased entrapment of water, oil and glucose in its porous structure. The increased WHC, OHC, glucose adsorption capacity and GDRI impact both the technological functionalities and physiological effects of insoluble fibre ingredients. Examples of these include: insoluble fibres with high WHC increase the stool weight and slow the digestion process, which is helpful for weight loss (Slavin, 2005); insoluble fibre ingredients with a high OHC allow for the stabilization of high-fat food products and emulsions; and high glucose adsorption capacity and GDRI delay glucose absorption in the gastrointestinal tract which may help to control postprandial blood glucose as a low-calorie ingredient for fibre enrichment and dietetic snacks (Liu et al., 2016b). Therefore, these results demonstrated the potential of the solid pomegranate seed residue remaining after oil and protein extraction as a dietary fibre ingredient for functional foods.

Fig. 6. Antioxidant activities of (a) protease-derived and hexane extracted pomegranate seed oil (b) pomegranate seed protein hydrolysate obtained by protease treatment and free protein sample obtained in control experiment without protease. Antioxidant activities were measured by DPPH radical scavenging assay.

As in the oil samples, the DPPH inhibition rates (%) were also increased with increase in concentrations of protein hydrolysate up to a maximum level and thereafter remained constant (Fig. 6b). In our current study, for comparison with protein hydrolysates, we also studied the ability of free protein samples (unhydrolysed proteins, obtained from a control group without protease treatment) to scavenge DPPH radicals. A similar concentration-dependent behavior of DPPH inhibition rates (%) was also observed in these free protein samples. At equivalent concentrations, the DPPH inhibition rates of protein hydrolysates were significantly higher than those of the control group free protein samples. Protein hydrolysate had 87% maximum level of DPPH inhibition rate, while the control group free protein sample showed only 22% (Fig. 6b). Therefore, it was not possible to determine the IC50 value of the control group free protein sample. The IC50 value of protein hydrolysate was 0.1047 ± 0.0026 mg/mL. The higher DPPH radical scavenging activity of the protein hydrolysate over that of the control group free protein sample could be due to its high total phenolics content (14.39 ± 0.05 mg/g protein) compared to control group free protein sample (9.71 ± 0.02 mg/g protein). Another possible reason for the increased ability of protein hydrolysate to scavenge DPPH radicals could be the availability of amino acids such as cystein, tyrosine, histidine, methionine, phenylalanine and tryptophan, etc. that can scavenge DPPH radicals (Elias et al., 2008). These amino acids can

4. Conclusions This work demonstrated that protease treatment is an effective method for the one pot extraction of high quality oil, food grade-protein and dietary fibres as nutraceuticals from WPS coming from pomegranate juice industries. The effect of protease concentration and time of treatment on free oil and protein recovery was investigated. It was observed that 50 U/g enzyme dosage at 14 h yielded maximum oil recovery (22.9 ± 0.8%, d.b), which was at par with hexane extracted oil (23.5 ± 0.5%, d.b). The conjugated fatty acid content of extracted oil using the protease treatment was higher than that of oil obtained with conventional hexane extraction, which was used as a benchmark. The extracted proteins were in the form of a protein hydrolysate with high nutritional quality. The protease-derived oil had 2.3% higher content of conjugated fatty acids and 1.4 times higher total phenolic content than the hexane-extracted oil. Moreover, the protease-derived oil displayed 4% higher antioxidant activity than the hexane-extracted oil. Both the 799

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Fig. 7. Mass spectrometry spectrum of (a) protein hydrolysate sample (b) control group free protein sample.

in vitro binding capacities (i.e. WHC, OHC, glucose adsorption capacity and GDRI) than WPS powder used for extraction, suggesting its potential for the development of a functional food ingredient. Thereby, in a one pot treatment using protease, multiple products viz. oil, protein and insoluble fibres can be easily and conveniently produced from WPS. The products have an existing potential market demand in the food

oil and protein hydrolysates obtained here showed antioxidant capacity, this is possibly due to the presence of phenolics in them and the presence of low molecular weight peptides in the protein hydrolysate. The protein hydrolysate had high values of the EAAI (91.6%), PER (5) and BV (88.5) confirming their high quality.The insoluble fibre rich WPS residue obtained after oil and protein extraction exhibited higher 800

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Table 6 The in vitro binding capacities of raw pomegranate seed powder and seed residue remaining after protease treatment. Property

Raw pomegranate seed powder

Seed residue remaining after protease treatment

Water holding capacity (g/g) Oil holding capacity (g/ g) Glucose adsorption capacity (mmol/g) GDRI (%)

1.6 ± 0.2a

2.8 ± 0.3b

0.77 ± 0.1a

2.3 ± 0.2b

0.69 ± 0.2a

1.55 ± 0.1b

23 ± 0.4a

45 ± 0.2b

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The values not sharing a common superscript small letter in two columns are significantly different (P < 0.05).

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