Novel Approach to Recover Natural Antioxidants from Oil Seed Meal

ARTICLE pubs.acs.org/IECR

Novel Approach to Recover Natural Antioxidants from Oil Seed Meal in Ultrafiltration-Nanofiltration-Based Technique Ranjana Das,† Chiranjib Bhattacharjee,*,† and Santinath Ghosh‡ † ‡

Department of Chemical Engineering, Jadavpur University, Kolkata-700032 Department of Chemical Technology, Calcutta University, Kolkata-700009 ABSTRACT: This study describes an innovative approach for recovery and concentration of sesame glucosides from defatted sesame cake using membrane separation route. Simple aqueous extraction of the seed meal at suitable pH value followed by two steps membrane fractionation (ultrafiltration-nanofiltration) was done to obtain sesame glucoside concentrate. In this process, about 0.7% ( 0.29 recovery of total meal glucoside was achieved along with coproduct sesame protein isolate at yield value of 36.50%. Several in vitro analyses were carried out to assay the antioxidative potential of recovered glucosides in different reaction models. Recovered glucosides have shown comparable α,α0 -diphenyl-1-picryl-hydrazyl radical scavenging activity and Cu2+ ion chelation capacity with solvent extracted glucosides. Sesame glucosides have shown unique antioxidant activity for 29 h in an induced oxidation system of linoleic acid emulsion. This study includes a detail analysis on membrane performance parameters and fouling characteristics of ultrafiltration membrane, which are crucial factors to consider for industrial relevance. All findings point toward the potentiality of ultrafiltration-nanofiltration-based technique for recovery of polar antioxidants from various raw materials.

1. INTRODUCTION Antioxidants are considered beneficial because of their protective role against oxidative stress, which is involved in pathogenesis of multiple diseases such as cancer and coronary heart disease. At present, enormous researches are involved in investigating the components those are active in conferring protection against major diseases, for improving quality of life in a nonpharmacological way. Food industries are now searching for safe, natural and effective antioxidants to meet recent consumer appeal as, the use of synthetic antioxidants are restricted by legislative rules.13 In the field of current nutraceuticals research exploration, characterization and utilization of natural antioxidants from comparatively cheap sources has become an innovative branch of study. Sesame glucosides are important class of polar natural antioxidants that constitute one of the largest and most ubiquitous groups of phytochemicals. These non-nutritive plant chemicals protect body cells against oxidative damage and are associated with several salutary effects in higher animal species, such as prevention of coronary artery diseases.4,5 Epidemiological studies have shown an inverse correlation between antioxidant enriched diet and reduced risks of cardiovascular diseases and inhibition to carcinogenesis.6,7 Among various cheap sources, oil seed meals (byproduct of vegetable oil industry) are reported as a potential source of natural phytochemicals having unique antioxidant activity.8 Sesame seed is well-known to the researchers for more than 20 years being a rich source of micronutrients labeled as sesame glucosides (Figure 1), which are recognized as key components for exhibiting antioxidant activity.911 Presence of biologically active components in sesame has already been reported by scientists1214 with an effort to increase the yield of glucosides from the meal by fermentation using Bacillus circulans strain.15 This study aims at the recovery of sesame glucosides from the seed meal by aqueous extraction and their concentration by r 2011 American Chemical Society

ultrafiltration. Studies have shown that isolation process play a crucial role on the applicability of final product and its bioactivity. Nature of the extractant, nature of chemicals and methodology are major deciding factors of efficient isolation processes. In most of the studies reported previously, different organic solvents with varying proportion of water were utilized as an efficient extraction medium for sesame glucosides, but use of organic solvents, involve health concerns regarding human consumption and also on quality of processed product.16 This health concern has driven the researchers to develop a new method of extraction that would reduce the use of organic solvents in the extraction procedure. Aqueous extraction of “polar” micronutrients definitely eliminates the safety concerns, but makes the extraction and concentration process complicated because of the interference of watersoluble proteins, tannins and sugars. Membrane processing is one of the methods that eliminates the use of organic solvents and concentrates the final product.17 Several authors18,19 have explored membrane processing as an emerging technology in extraction and concentration of natural antioxidants from a variety of sources. During the aqueous extraction of the sesame glucosides, seed meal proteins impede a lot in membrane processing, as these are the “major foulants” of membrane and because of its interactions with glucosidic components.20 Sesame protein is rich in sulfur containing amino acid methionine; exhibits well-defined nutraceuticals characteristics, including antioxidative, hypo-cholesterolemic, hepato-protective activity and prevention of hypertension.21 So, attempt was taken to recover sesame protein as ‘process coproduct’ along with isolation of sesame glucosides. Though immense studies are available on Received: March 11, 2011 Accepted: August 31, 2011 Revised: August 10, 2011 Published: August 31, 2011 12124

dx.doi.org/10.1021/ie200485a | Ind. Eng. Chem. Res. 2011, 50, 12124–12133

Industrial & Engineering Chemistry Research

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Figure 1. Chemical structures of sesame glucosides.

Table 1. Properties of Membranes Used in This Study membrane

MWCO

GR95PP UF

2000 Da

specifications operational pH range: 113 operational Pr. Range: 110 maximum temperature: 75 °C

TFC-SR2

400 Da

active surface area: 0.0041 m2 98% NaCl rejection operational pH range: 49 maximum operating pressure: 3.45 MPa maximum process temperature: 45 °C active surface area: 0.0041 m2

the isolation and evaluation of sesame antioxidant (sesame lignans), considering importance of sesame glucosides this study involves a novel approach to recover sesame glucosides. This study also includes biological evaluation of sesame glucosides by several in vitro methods in comparison to the organic solvent extracted sesame glucosides and assessment of fouling characteristics for membranes in use.

2. MATERIALS AND METHODS 2.1. Materials. Authentic sesame (Sesamum indicum L.) seed (brown variety) was purchased from super market. Folin Ciocalteau’s phenol reagent (AR grade, 2 N) and pure linoleic acid (99%) were supplied by (SRL) Sisco Research Laboratory, India. DPPH was purchased from Sigma Aldrich Chemical Co., U.S.A. Amino acid standard (AA-S-18) was purchased from Sigma Chemical Co., Louis, MO, USA. Diethyl ethoxymethylenemalonate (98%) was purchased from Lancaster, Weastgate, White Lund, Morecabe, England. All other chemicals, except otherwise stated, were purchased from SRL. Ultrafiltration (UF)

(GR95PP) and nanofiltration (NF) membranes were supplied by Koch Membrane Systems (San Diego, CA). According to the manufacturers, UF membrane is composed of polyethersulfone (PES), and NF membrane is polyamide thin-film composite with a microporous polysulfone supporting layer. Table 1 shows specifications of the membranes used. All experiments were performed using deionized (DI) water from Arium 611, of Sartorius AG, Germany. Absorbance was measured in a Shimadzu (1601A) UVvis Spectrophotometer. 2.1.1. Statistical Analysis. All results are expressed as mean ( Standard error of mean (S.E.M) for three determinations. Statistical analysis was done by Analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant. MATLAB 7.0 software was used for statistical analysis. 2.2. Experimental Processes. 2.2.1. Recovery of Glucosides from Meal. In the present context, a novel approach of recovering sesame glucosides from sesame meal has been described. Recovery of antioxidative glucosides was done by both conventional solvent extraction method and by UF-NF route to study the effect of extraction procedure on bioactivity of extracted active components. Solvent extraction of sesame glucosides (SGs) was done following the method described by Moazzami et al.22 The ground dehulled sesame seeds were defatted by solvent extraction using food grade hexane (boiling point = 6366 °C) in laboratory scale “Soxhlet” apparatus and sesame glucosides were extracted from seed meal using 80% aqueous methanol. Briefly, the seed meal (1 g) was mixed with 10 mL of aqueous 80% methanol and mixture was stirred for 30 min. The seed mealsolvent mixture was filtered into a 50 mL volumetric flask through Watman-42 filter paper. The process was repeated for four times and final volume was made up with the respective solvent. The experimental scheme for aqueous extraction and recovery of sesame glucosides (SGm) by UF-NF is illustrated in Figure 2. 12125

dx.doi.org/10.1021/ie200485a |Ind. Eng. Chem. Res. 2011, 50, 12124–12133

Industrial & Engineering Chemistry Research Seed meal was subjected to aqueous extraction at pH 7.4, with the water-meal ratio of 18:1(v/w) for 1 h at 45 ( 2 °C. After extraction, meal residue was separated by centrifugation (6000 g, 10 min) followed by washing with 6 volumes of distilled water and finally wash solution was mixed with the

Figure 2. Scheme for recovery of sesame glucosides from seed meal using ultrafiltration-nanofiltration based technique.

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supernatant of alkaline extraction, which mostly contains sesame glucosides along with soluble proteins. Seed meal extract after pretreatment with 0.05(N) NaCl, was ultrafiltered (using 2 kDa PES membrane) up to volume reduction factor (VRF) of 4 to separate soluble proteins from sesame glucosides. Salt pretreatment helps to neutralize the interaction between phenolic glucosides and soluble protein; enhancing phenolic removal in accordance to the observation of Xu et al.23 during preparation of high quality canola protein isolate. UF retentate was subjected to discontinuous diafiltration in same module, up to dia-volume (DV) of 3 to achieve better separations of sesame glucosides from soluble protein. The diafiltered retentate was freeze-dried (in Freeze-dryer, EYELA FDU-110, Japan) to get the soluble sesame protein isolate (SPIMPI). Permeate from first UF-DF step, was subjected to NF in similar membrane module with VRF-10. NF retentate was collected and dried in rotary evaporator (Cyber Lab, Singapore) to obtain sesame glucosides rich fraction (SGm), almost free of any soluble protein. 2.2.2. Membrane Module and Methodology Used during Recovery of Glucosides. Two stage membrane separations were performed to recover sesame glucosides from the sesame meal. First stage involves separation of soluble protein from aqueous extract of sesame glucosides followed by three step “discontinuous diafiltration” in same setup to get better yield of glucosides and second stage involves concentration of glucosides by nanofiltration (NF). In this study, UF and NF was carried out in rotating disk membrane module (Figure 3), manufactured by Gurpreet Engineering works, Kanpur, UP, INDIA as per specified design. Details of the membrane module and methodology have already been reported in our previous study.24,25 Choice of the membranes was done according to the molar masses of the components to be separated. According to the membrane supplier’s recommendation, new membranes were conditioned before use. Membranes were kept immersing in DI water overnight and washed thoroughly in running DI water. Washed membranes were subjected to compaction

Figure 3. Schematic diagram of rotating disk membrane module (in-house fabricated). 12126



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Table 2. Performance Parameters of Two Membrane Separation Stagesa Rm (m1)

VRF

CF(Pr)

Rf(Pr)

ηr (Pr)

CF(SGm)

Rf(SGm)

ηp (SGm)

first stage UF

4.462 1013

4

1.80 ( 0.21

0.85b

0.88b

0.41 ( 0.03

0.61

0.57 ( 0.02

second Stage NF

1.12 10

stages

a

14

9.7 ( 0.12

10

1

0.98b

Values are expressed as mean ( SEM, n = 3. SEM < 0.0001. b

at transmembrane pressure (TMP) higher than operating TMP to avoid permeate flux decline under high pressure differential during processing. A feed volume of 500 mL with protein concentration 400 mg/L was subjected to ultrafiltration at TMP of 4.9 105Pa, Nm of 1.67 ( 0.01 rps and temperature of 24 ( 1 °C without any stirrer rotation. Diafiltration (DF) was also performed under the similar process condition with similar membrane arrangements. Permeate collected from the first stage UF-DF was used as feed for second stage processing in NF under high TMP to enhance permeate flux and better concentration of desired components, as evident from the Rm values of the two respective membranes (Table 2). High-temperature (5060 °C) condition sometimes helps to maximize permeation flux, but considering product quality, experiments were performed at a low temperature (24 ( 1 °C) to avoid heat degradation of protein and natural antioxidants during processing. Same membranes were used in repetitive runs (for three times), as the membranes were almost completely (99 ( 0.7%) regenerated after performing the prescribed cleaning cycle as mentioned in our previous study.24 2.2.3. Estimation of Glucoside Content As Total Polyphenol and Identification by Mass Spectroscopy. Amount of glucosides (as total polyphenols) was measured according to the standard method of Singleton et al.26 using gallic acid as standard at 760 nm. Preliminary identification of components was done by FT-IR (JASCO FT-IR 670 plus spectrophotometer); further compositional mass analysis of the seed meal extract was done to confirm the presence of those “micronutrients” in final processed extract. Mass spectroscopic analysis of SGm was done in LC (Waters alliance, 2695 separation module) coupled with a photodiode array detector (PDA, 2996, Waters) and MS (MSMICROMASS, Quattro micro API, Micromass. Reverse-phase, symmetric C18 column (4.6 150 mm, 5 μm, Varian) with flow rate 1 mL and column temperature 30 °C was used. The eluents used were (A) acetonitrile with 0.05% HCOOH and (B) water with 0.05% HCOOH. Elution condition maintained as, 05.8 min, A/B (5:95 to 95:5); 5.87.8 min, A/B (95:5 to 5:95); 7.89 min isocratic, A/B (5:95). The molecular weight distribution of protein isolate was determined by SDS-polyacrylamide gel electrophoresis according to the method of Laemmli27 using 12% polyacrylamide gel. 2.2.4. DPPH• Radical-Scavenging Property and Cu(II) Chelation Capacity of the Seed Meal Extract. The free radical scavenging activities of seed meal extracts were assayed in DPPH• system using the method of Oktay et al.28 Meal extracts (10100 mg/L) were added to 3 mL DPPH• solution (0.1 m mol/L) in methanol and after 30 min of reaction, absorbance was measured at 517 nm. Radical scavenging activity was expressed as ‘inhibition percentage’ and was calculated as, percent radical scavenging = (Absorbance of the control Absorbance of sample)/ Absorbance of control 100. To determine the chelating capacity of SGm, 1 mL of 100 mg/L test solution was added to 20 mL of standard 1.0 mM CuSO4 solution and allowed to stand for 30 min to complete the chelation.

Concentrations of free Cu (II) ions in the standard and test solution were determined spectrophotometrically. For spectroscopic analysis, 0.2 mL of 1% (w/v) sodium diethyl dithiocarbamate solution, and 20 mL of 1.5 (N) NH3 solutions were added to the test sample (1 mL) and diluted to 25 mL with double-distilled water. Absorbance of yellow colored solution was read at 460 nm and percent chelation capacity was determined with respect to the control. 2.2.5. Lipid Oxidation Inhibitory Effect of Seed Meal Extract. Linoleic acid emulsion was prepared by homogenizing 2.5 mL linoleic acid, 22.5 mL water, and 130 μL Tween 20. The emulsion was incubated at 38 °C and 1 mL aliquots were removed at several time intervals, followed by phase separation using CHCl3/CH3OH (2:1 v/v). For aqueous phase, thiobarbituric acid reactive substance (TBARS) absorbance was measured at 532 nm to assay the antioxidant activity following method of Coupland et al.29 For organic-phase “diene” content was measured spectrophotometrically at 233 nm,30 after dissolving the sample in isooctane. Percent diene content was calculated using standard AOCS method [Th 1a-64 (09)]. 2.2.6. Estimation of Protein Content and Nutritional Evaluation of Sesame Protein Isolates. Soluble protein content of all fractions was determined according to standard method of Lowry et al.31 using bovine serum albumin (BSA) as standard at 750 nm. Nutritional evaluation of sesame protein isolate (SPIMPI) was done in comparison to standard soy protein isolate (SoPI) and precipitated sesame protein isolate (SPIPPI), by analyzing their respective amino acid compositions. Amino acid composition was analyzed following the method of Alaiz et al.32 with some modifications. Separation was carried out in a 4.6 150 mm Symmetry C18 reversed phase column (Waters) using a binary gradient system. The solvent used were (A) 25 mM sodium acetate containing 0.02% sodium azide (pH-6) and (B) acetonitrile. The solvent was delivered at a flow rate of 1 mL/ minute as follows: time 02 min, elution with A/B (95:5); from 2 to 40 min with linear gradient A/B (95:5) to A/B (70:30); from 40 to 45 min A/B (70:30) to A/B (60:40); from 45 to 50 min A/B (60:40) to A/B (90:10). 2.2.7. Estimation of Performance Indicators of Two Separation Stages. For each stage, volume reduction factor (VRF), concentration factor for glucosides and protein (CFSG and CFPr), retention factor at end of run (RFf), and relative recoveries of the desired components in permeate (ηP) and in retentate (ηR) were calculated using standard methods and defined as VRF ¼ CF ¼

V0 V0 Vp Vp0

CR C0

ð2Þ

RFf ¼ 1 12127

ð1Þ

CPf CR

ð3Þ



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Table 3. Fouling Characteristics of PES Membrane during Operation in Terms of Resistance Parameters resistance parametersa 1

in first stage UF

Rm (10 m )

4.462 ( 0.19

Rf (1013 m1) Rp (1013 m1)

0.527 ( 0.02 0.073b

Rwr (1013 m1)

0.454 ( 0.02

13

1

Rcr (10 m )

0.927 ( 0.04

Rc (1013 m1)

1.381 ( 0.05

Rt (1013 m1)

6.370 ( 0.33

13

3. RESULTS AND DISCUSSION 3.1. Yield of Sesame Glucosides and Sesame Protein Isolate. Recovery of glucosides was done from the seed meal

Values are expressed as average of three determination ( standard error of mean (SEM). b SEM less than 0.01.

a

ηP ¼

mP mF

ð4Þ

ηR ¼

mR mF

ð5Þ 0

where V0, VP, and VP symbolizes initial feed volume, permeate volume, and permeate volume loss during processing; C0, CR, CPf are the initial feed concentration, retentate concentration, and final permeate concentration for proteins and glucosides; ηP and ηR are the relative recoveries of glucosides in permeate and protein in retentate; and mR, mP, and mF are the masses of the desired components in retentate, permeate, and feed. All these performance indicators bear lots of importance in understanding the overall methodology and hence were estimated and illustrated in Table 2 for better understanding of the process. 2.2.8. Fouling Characteristics of PES Membrane during Operation. The extent of membrane fouling was quantitatively estimated using the resistance in series model eq 6 J ¼

ΔP η 3 Rt

ð6Þ

where J is average permeate flux in (L/m2h); ΔP is the transmembrane pressure; η stands for the viscosity of respective streams at 25 °C; Rt is total resistance Rt ¼ Rm þ Rf þ Rc

recording the SEM images. Experimental conditions were 510 kV, 500 magnification.

ð7Þ

Intrinsic membrane resistance (Rm), resistance due to the solute adsorption into the membrane pores/walls and chemically reversible cake (Rf), resistance due to pore plugging (Rp), resistance due to water recoverable cake (Rwr), chemically recoverable cake (Rcr), total cake resistance formed by cake layer deposited over the membrane surface (Rc),were estimated to know the membrane fouling characteristics (Table 3). Estimations of resistance parameters were done using equations as mentioned in Appendix 1 and average permeate flux values were used in each case as permeate flux declines with time during operation . Scanning electron microscopic (SEM) analysis was also done on Hitachi S-3400 N (Japan) scanning electron microscope to visualize fouling behavior of UF membrane. Membrane samples were initially washed with water to remove the storage chemicals from membrane surface. All fresh and used membrane samples were dried overnight at 30 °C before preparing 3 mm 3 mm strips for gold sputter coating (using Hitachi E-1010 ion sputter). The gold-coated strips were used for

extract by UF-NF based technique. First UF (2 kDa) was done for complete separation of soluble protein from glucosides. Since, SDS-PAGE analysis of meal extract revealed the presence of protein molecules with molecular weight above 4 kDa, accordingly 2 kDa membrane was chosen for effective separation of protein. Similar observation was also reported by Bandopadhya et al.33 Soluble protein was concentrated about 1.8 times in the retentate with recovery yield of 0.88 (Table 2) in the retentate. UF permeates gave a negative response to total protein test indicating complete separation of proteins from glucosides. Results indicate efficient separation of soluble protein, which improves the “bioavailability” of glucosides. Unrecovered mass (12%) in this scheme may be due to adsorption of protein molecules on the membrane surface causing the phenomenon of concentration polarization. Since feasibility of any technique depends on product yield, so a comparative study was done between the conventional approach and newly proposed technique of sesame glucosides extraction Seed meals on solvent extraction (aqueous 80% methanol), yields 2.1 mg of sesame glucosides (SGs) per g meal; whereas, that for aqueous extract (SGm) was 1.24 mg per g of the meal. Comparatively lesser yield of SGm than SGs is probably due to protein-glucoside interactions, arising between the phenolic functional groups with peptide bonds of insoluble protein molecules. To improve SGs yield, breaking of the protein-glucosides interaction necessitates the use of electrolyte NaCl23 which ultimately generates freer glucosidic components that appear in 2 kDa permeate. During membrane processing, pretreatment of feed with 0.05 (M) NaCl shows about 2.88 times more recovery of bonded glucosides and higher (62.44%) glucosides content in permeate (0.719 mg/g) as compared to the retentate (0.449 mg/g) with recovery efficiency (ηSGm) of 0.57 (Table 2). Without NaCl treatment, glucosides distributions were 0.85 mg/g and 0.25 mg/g for retentate and permeate respectively. Similar recovery yield was also reported by Xu et al.23 In the present context, diafiltration was done to recover accompanying glucosides from the 2 kDa retentate with the overall increase of 36.3% yield. It was conducted under similar process conditions like ultrafiltration with average flux values to some extent higher than corresponding UF flux value. In three stages of diafiltration, average permeates flux values were 27.87 ( 0.84, 26.9 ( 1.07, and 25.75 ( 0.77 L m2 h1, respectively, under transmembrane pressure of 4.9 105 Pa at 25 °C. In three successive stages yield increased as 0.84, 0.92, and 0.98 mg/g, respectively. With further steps of diafiltration, no significant changes in yield values were observed and hence processing was ended for three stage diafiltration. To concentrate the diafiltered permeate, second stage processing was done with 400 Da NF membrane up to VRF 10. About 49.59% of the total aqueous extracted glucosides and 29.25% of the total solvent extracted glucosides appeared in NF retentate. Considering commercial importance of “co-product” protein isolates (98.9% pure) and minimal usage of organic solvents, proposed scheme can be utilized as an alternative to solvent based extraction of sesame glucosides. 12128



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Figure 4. FT-IR analysis of sesame meal extracts (membrane processed).

Figure 5. DPPH radical scavenging activity of sesame meal extract with changing dose.

3.2. Identification of Sesame Glucosides in Seed Meal Extract. To confirm the presence of polyphenolic hydroxyl

groups in extracted components, FT-IR spectrum has been recorded on JASCO FT-IR 670 plus spectrophotometer. Spectrum (Figure 4) showed the presence of monomeric alcohol (strong, broad peak 3400 cm1), aromatic CH bond (2995 cm1), aromatic ring CdC bond (1650 cm1), aliphatic CH2 group (1490 cm1), symmetric stretching for the COC of the ether/CO bond in ethers (1050 cm1), CH phenyl ring substitution band (675 cm1) and confirms the presence of glucosides (Figure 1). Mass spectroscopic analysis also points to the presence of two masses of molecular weights 717.3 and 879.3 Da, corresponding to sesame di glucosides and tri glucosides. 3.3. Radical Scavenging Activity of Seed Meal Extract. Natural antioxidants being highly sensitive to processing conditions (extraction method, nature of solvents and chemicals), in vitro studies on radical scavenging activity (RSA) were carried out to assess the bioactivity of recovered glucosides (SGm) in comparison to solvent extracted glucosides (SGs) and standard antioxidants. Free radicals are considered responsible for propagation of oxidative chain reactions and antioxidants donate hydrogen to the reactive radicals to neutralize radicals and inhibit oxidation.

The antioxidant potential of sesame glucosides (NF retentate) was studied by DPPH• radical scavenging activity. Methanolic DPPH• solution exhibits absorption maxima at 517 nm against an appropriate blank. While DPPH• radical encounters with proton donating substance, absorption at 517 nm reduces indicating the transformation of the active radicals to neutral molecules. The characteristic absorption of DPPH• solution trim down markedly on adding SGm (10 mg/L); strongly suggesting that SGm has the radical scavenging activity even at a very low concentration level like SGs.13,34 Moreover, SGm exhibits significant (p < 0.05) ‘concentration dependent’ radical scavenging activity from 10 to 50 mg/L, followed by a gradual change (Figure 5). Absorption of DPPH• solution changes sharply for SGs, but the change is gradual for SGm, which may be due to the high purity of solvent extracted glucosides, in SGm presence of impurity inhibits scavenging activity that causes a gradual change in absorption value. RSA of SGm was found nearly 3.2% lower than SGs, 5.7% lower than Tertiary Butyl Hydro-Quinone (TBHQ) and 9.8% lower than tocopherol. Almost similar RSA for TBHQ and tocopherol with concentration variation may be due to saturation effect. RSA of aqueous extract (SGm) was about 69% and 82.3% at 50 and 100 mg/L concentration level, respectively, strongly suggesting the effectiveness of the extraction method in recovery of natural phytochemicals with their significant bioactivity. Transition metal ions like iron and copper are major primary catalysts that initiate oxidation in vivo and in vitro. Metal ions play a significant role in accelerating oxidation of important biomolecules and propagating radical chain reaction. Chelating agents, are known to stabilize pro-oxidative transition metal ions either by ion exchange or by complexing them, where unshared pairs of electrons in the molecular structure of chelator promote the complexation.34 At 100 mg/L concentration level for SGs and SGm percent chelation were 84.45 and 82.74, respectively. With dosage of extracts, the chelation capacity increases almost linearly (R2 = 0.98). ANOVA analysis was done to evaluate the effect of doses and extraction procedure on Cu (II) chelation with 95% confidence level. Results indicate that concentrations of extract have significant effect (p < 0.05) on chelation capacity without any significant effects of extraction route. Thus, SGm can serve as an excellent alternative to solvent extracted glucosides and can be used in food formulations devoid of any health concerns. 3.4. Effect of Seed Meal Extract on Induced Lipid Oxidation. The antioxidant activity of a compound can also be illustrated by its ability to delay the onset of auto oxidation by scavenging reactive 12129



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Figure 7. Amino acids profile of SPI isolate.

Figure 6. Lipid oxidation inhibition behavior of sesame meal extracts in linoleic acid emulsion. (a) Variation of TBARS absorbance values as function of time and concentration of glucosides in aqueous phase. (b) Variation of percent diene content as function of time and concentration of glucosides in organic phase.

oxygen species (ROS). The ability of sesame meal extracts in prevention of lipid per-oxidation was studied in linoleic acid emulsion system with time. Progress of oxidation reaction was followed by spectroscopic quantification of “thio-barbituric acid reactive substance” (TBARS) (in aqueous phase) and “conjugated diene” content (in organic phase). In the presence of SGm, formation of TBARS and ‘conjugated diene’ both are inhibited as compared to control (Figure 6). After 29 h of incubation, antioxidant effect of the extracted glucosides was ceased. SGm exhibited lower oxidation inhibition compared to SGs. At concentration level of 1 mg/L, SGs suppress 19% of malonyl dialdehyde (MDA) color intensity (after 28 h of oxidation) compared to 8.13% for SGm. For 2 mg/L dose, these values were 43% and 16%, respectively. The extent of inhibition to conjugated diene formation in organic phase was found relatively higher (2123%) as compared to the MDA formation in aqueous phase (19%) with 1 mg/L dose of the extract. This behavior also gives an idea about the nature of the extracted active molecules and supports the concept of polar paradox (Koleva et al.35), that polar compounds exhibit weaker antioxidant activity in

PPI

of SPI

MPI

and soy protein

aqueous phase than organic phase of the emulsion due to the dilution of these compounds in the aqueous phase. 3.5. Nutritional aspects of sesame protein isolate. To evaluate the nutritional aspect of the sesame protein, amino acid composition of SPIMPI was determined (Figure 7) in comparison to soybean protein isolate (SoPI) and precipitated sesame protein isolate (SPIPPI). Essential amino acid composition of SPIMPI was found significantly different from that of SPIPPI. Amount of proline, alanine and threonine have increased and methionine, valine, tyrosine content have decreased in SPIMPI. Amount of other amino acids were found similar with no significant changes. On basis of amino acid composition, SPIMPI may be of special interest for clinical use particularly in the treatment of patients suffering from leaver disease. This is due to the high ‘Fischer Ratio’ [ratio of (Val + Leu + Ile) to (Tyr + Phe)] value of 2.43 in SPIMPI in comparison to 1.96 in SPIPPI. The amount of aspartic acid and glutamic acid was higher in the precipitated isolate. The Lys/Arg ratio of SPIMPI also found slightly higher (0.43) as compared to precipitated protein isolate (0.39), which is also important parameter to be considered as it also affects the cholesterol metabolism. Total aromatic amino acid percent in SPIPPI was 10.97 compared to 9.07 in SPIMPI. Reduced content of aromatic amino acids like, phenylalanine, methionine, histidine, and tyrosine may make SPIMPI useful in treatment of congenital illness such as phynylketonuria or tyrosinamie, where diets low in aromatic amino acids are recommended. Low level of aromatic amino acid also expands the field of application for SPIMPI and hence makes the proposed technique significantly important. 3.6. Fouling Characteristics of Membrane. From the time of permeate collection until the end of run (up to VRF 4), permeate was collected. The volume was recorded and Javerage evaluated by the relationship in the following equation: Javerage ð%Þ ¼ ½Vp =ðJiw tÞ 100

ð8Þ

Javerage is average permeate flux (%); Vp is total volume of permeate; Jiw is initial pure water flux; and t is total processing time. Jiw and Jfw are two important parameters that are used to 12130



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due to fouling (Rt) is only 1.14%, indicating that, major fouling is due to cake layer and not for pore plugging. Contribution of Rwr and Rcr are 7.13% and 14.55%, respectively, which indicates the cake layer is mostly due to chemically recoverable proteins. Scanning electron microscopic (SEM) analyses were also done to visualize the fouling behavior of UF membrane and are illustrated in Figure 8a and b. Figure 8a shows the nature of the proteins on the membrane surface that are mostly “fibrous” in nature rather than “globular”; presence of fibrous proteins on the membrane surface are typically responsible for surface accumulation rather than pore blocking, which is evident from comparatively low value of Rp (Table 3). Figure 8b shows the regenerated membrane surface with very few deformed foulants, which supports the theoretical observation of 99.7% recovery of pure water flux after proper cleaning cycle. Structural deformation of accumulated proteins helps in easy removal of proteins from membrane surface with water flow. Thus, SEM images clarify the theoretical estimation of fouling parameters very efficiently. Fouling analysis of NF membrane was done on basis of fall in pure water flux, and result shows insignificant fall in water flux and solute accumulation. So, further analyses for fouling parameters were not performed for NF membrane.

Figure 8. (a) Scanning electron microscopic analysis of protein adsorbed membrane to visualize the fouling characteristics of sesame protein isolate. (b) Scanning electron microscopic analysis of chemically washed membrane to visualize the fouling characteristics of sesame protein isolate.

assay the fouling characteristics of the membranes. These parameters were estimated observing the pure water flow rate for fresh compacted membrane and used membrane and values found are 44.3 ( 0.89 and 39.62 ( 0.79 L m2 h1, and the Js value was 25.67 ( 0.51 L m2 h1. Results showed slightly decline in pure water flux, which indicative of the phenomenon of solute accumulation on membrane surface. Large protein molecules being the major foulants in this membrane separation process, analysis of the membrane fouling was done based on the amount of protein molecules lost during UF. In first stage, percent loss of mass (protein) was 27.46 ( 0.51. This protein loss may be considered responsible for the membrane fouling/ concentration polarization phenomenon. These finding strongly reveals the positive corelation between feed concentration and membrane fouling. Several other parameters related to membrane fouling were also estimated to asses the nature of fouling using resistance-in-series model (eqs 6 and 7) and illustrated in Table 3. In first stage UF, contribution of Rp to total resistance

4. CONCLUSIONS This work shows that it is possible to recover sesame glucosides from aqueous extract of the seed meal using ultrafiltrationnanofiltration based technique, with the potential to recover seed protein isolate as a process byproduct. Activity profile of the aqueous meal extracts as determined by several in vitro methods specify good antioxidant potency. Extract showed evidence of significant free radical scavenging, with considerable suppression of lipid peroxidation in linoleic acid emulsion. In the present context, though the yield of glucosides found much less than conventional route but activity profiles of the two extracts were found nearly equivalent. Amino acid profile of the membrane processed protein isolate was found comparable to commercial protein isolates with improved Lys/Agr ratio value. This approach also gets rid of the uses of toxic solvents like methanol and eliminates health concern related to the final processed product. All findings point to the probability of the seed meal utilization in getting high value products, which can bring good revenue to seed meal industries after proper modifications and scale up of this novel approach. ’ APPENDIX 1 To estimate the resistance parameters, initially pure water flux (Jiw), pure water flux of used water washed membrane (Jfw), pure water flux of chemically washed membrane (Jcw), and permeate flux of the meal extract was determined experimentally (Js) at particular transmembrane pressure (ΔP). Parameters are calculated using following equations:

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Rm ¼

ΔP ; η ¼ viscosity of pure water at 25 °C η Jiw

Rf ¼

ΔP Rm η Jfw

ð10Þ

Rp ¼

ΔP Rm η Jcw

ð11Þ

ð9Þ


Industrial & Engineering Chemistry Research Rwr ¼ Rf Rp ΔP Rc ¼ ðRm þ Rp þ Rwr Þ ηs Js

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ð12Þ ð13Þ

where ηs = 1.08 103 kg m1 sec1(25 °C) Rcr ¼ Rc Rwr

ð14Þ

Rt ¼ Rm þ Rf þ Rc

ð15Þ

’ AUTHOR INFORMATION Corresponding Author

*Email: [email protected].

’ ACKNOWLEDGMENT We are very much thankful to Council of Scientific and Industrial Research (CSIR) India, for funding the whole work as research associateship. ’ ABBREVIATIONS CFPr = concentration fraction for protein CFSG = concentration factor for sesame glucosides DI = deionized water DF = diafiltration DPPH = α,α0 -diphenyl-1-picryl-hydrazyl Rm = intrinsic membrane resistance (m1) Nm = membrane rotation speed (rps) SGm = membrane processed sesame glucosides NMWCO = nominal molecular weight cutoff (Dalton) PES = polyether sulfone Rf = resistance due to solute adsorption on membrane surface (m1) Rp = resistance due to pore plugging (m1) Rwr = resistance due to water washable cake (m1) Rcr = resistance due to chemically recoverable cake (m1) SPIPPI = sesame protein isolate (precipitated) SPIMPI = sesame protein isolates (membrane processed) SGs = solvent extracted sesame glucosides TMP, ΔP = trans-membrane pressure (Pa) Rt = total resistance (m1) UF = ultrafiltration ’ REFERENCES (1) Branen, A. Toxicology and Biochemistry of Butylated Hydroxyl Anisole and Butylated Hydroxyl Toluene. J. Am. Oil. Chem. Soc. 1975, 52, 59. (2) Linderschrnidt, R.; Trylka, A.; Goad, M.; Witschi, H. The Effect of Dietary Butylated Hydroxytoluene on Liver and Colon Tumor Developed in Mice. Toxycology 1986, 387, 151. (3) Tappel, A. Antioxidant Protection against Peroxidation. Inform 1995, 6, 780. (4) Muldoon, M. F.; Kritchevsky, S. B. Flavonoids and Heart Disease. Brit. Med J. 1996, 312, 458. (5) John Cooke, P. The Cardiovascular Cure; Random House, Inc.: New York, 2002. (6) Curin, Y.; Andriantsitohaina, R. Polyphenol as Potential Therapeutic Agents Against Cardiovascular Diseases. Pharmacol. Rep. 2005, 57, 97. (7) Chung Yang, S.; Lee, M- J.; Chen, L.; Yang, G- Y. Polyphenol as Inhibitor of Carcinogenesis. Environ. Health Perspec. 1997, 105, 971.

(8) Amarowicz, R.; Wanasundara, U. N.; Karamac, M.; Shahidi, F. Antioxidant Activity of Ethanolic Extract of Mustard Seed. Nahrung 1996, 40, 261. (9) Fukuda, Y.; Osawa, T.; Namiki, M.; Ozaki, T. Studies on the Antioxidative Substances in Sesame. Agric. Biol. Chem. 1985, 49, 301. (10) Katsuzaki, H.; Kawasumi, M.; Kawasaki, S.; Osawa, T. Structure of Novel Antioxidative Lignan Glucosides Isolated from Sesame Seed. Biosci. Biotechnol. Biochem. 1992, 56, 2087. (11) Kang, M -H.; Kawai, Y.; Naito, M.; Osawa, T. Dietary Defatted Sesame Flour Decreases Susceptibility to Oxidative Stress in Hypercholesterolemic Rabbits. J. Nutr. 1999, 129, 1885. (12) Shyu, Y -S.; Hwang, L. S. Antioxidative Activity of the Crude Extract of Lignan Glycosides from Unroasted Burma Black Sesame Meal. Food Res. Int. 2002, 35, 357. (13) Suja, K. P.; Jayalekshmy, A.; Arumughan, C. Free Radical Scavenging Behavior of Antioxidant Compounds of Sesame (Sesamum indicum L.) in DPPH 3 System. J. Agric. Food Chem. 2004, 52, 912. (14) Xu, L.; Chen, S.; Hu, Q. Antioxidant Activity of Brown Pigment and Extracts from Black Sesame Seed (Sesamum indicum L.). Food Chem. 2005, 91, 79. (15) Ohtsuki, T.; Akiyama, J.; Shimoyama, T.; Yazaki, S.-I.; Sadaharu, U.; Hirose, Y.; Mimura, A. Increased Production of Antioxidative Sesaminol Glucosides from Sesame Oil Cake Through Fermentation by Bacillus circulans Strain YUS-2. Biosci. Biotechnol. Biochem. 2003, 67, 2304. (16) Nawaz, H.; Shi, J.; Mittal, G. S.; Kakuda, Y. Extraction of Polyphenols from Grape Seed and Concentration by Ultrafiltration. Sep. Purif. Technol. 2006, 48, 176. (17) Moresi, M.; Presti, S. L. Present and Potential Application of Membrane Processing in Food Industry. Ital. J. Food Sci. 2003, 15, 3. (18) Shi, J.; Yu, J.; Pohorly, J.; Bryan, Y. M. Optimization of the Extraction of Polyphenol from Grape Seed Meal by Aqueous Ethanol Solution. J. Food Agric. Environ. 2003, 1, 42. (19) Nawaz, H.; Shi, J.; Mittal, G. S.; Kakuda, Y. Extraction of Polyphenol from Grape Seeds and Concentration by Ultrafiltration. Sep. Purif. Technol. 2006, 48, 176. (20) Xu, L.; Lui, F.; Luo, H.; Diosady, L. L. Production of Protein Isolate from Yellow Mustard Meals by Membrane Process. Food Res. Int. 2003, 36, 849. (21) Nakano, D.; Itoh, C.; Ishii, F.; Kawanishi, H.; Takaoka, M.; Kiso, Y. Effects of Sesamin on Aortic Oxidative Stress and Endothelial Dysfunction in Deoxycorticosterone Acetate-Salt Hypertensive Rats. Biol. Pharm. Bull. 2003, 26, 1701. (22) Moazzami, A. A.; Anderson, R. E.; Eldin, A. K. HPLC Analysis of Sesaminol Glucosides in Sesame Seeds. J. Agric. Food Chem. 2006, 54, 633. (23) Xu, L.; Diosady, L. L. Removal of Phenolic Compounds in the Production of High Quality Canola Protein Isolates. Food Res. Int. 2002, 35, 23. (24) Das, R.; Bhattacharjee, C.; Ghosh, S. Studies on Membrane Processing of Sesame Protein Isolate and Sesame Protein Hydrolysate using Rotating Disk Module. Sep. Sci. Technol. 2009, 44, 131. (25) Das, R.; Sen, D.; Sarkar, A.; Bhattacharyya, S.; Bhattacharjee, C. A Comparative Study on the Production of Galacto-oligosaccharide from Whey Permeate in Recycle Membrane Reactor and in Enzymatic Batch Reactor. Ind. Eng. Chem. Res. 2011, 50, 806. (26) Singleton, L. V.; Orthofer, R.; Lamuela Raventos, R. M. Analysis of Total Polyphenols and other Oxidation Substrates and Antioxidants by Means of Folin-Ciocalteau Reagent. Methods Enzymol. 1999, 299, 15. (27) Laemmli, U. K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 1970, 227, 680. (28) Oktay, M.; Culcin, L.; Kufrevioglu, O. I. Determination of in Vitro Antioxidant Activity of Fennel (Foeniculum Vulgare) Seed Extract. Lebensmittel-Wissenschaft und-Technologie. 2003, 36, 263. (29) Coupland, J. N.; Wan, H.; Clements, Mc D. J.; Nawar, W. W. Droplet Composition Affects the Rate of Oxidation of Emulsified Ethyl Linoleate. J. Am. Oil Chem. Soc. 1996, 73, 795. (30) Recknagel, R. O.; Glender, A. E. Spectrophotometric Detection of Lipid Conjugated Dienes. Methods Enzymol. 1984, 105, 331. 12132



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(31) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randal, R. J. Protein Measurement with Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 265. (32) Alaiz, M.; Navarro, J. L.; Giron, J.; Vioque, E. Amino Acid Analysis by High Performance Liquid Chromatography after Derivatization with Diethyl Ethoxymethylenemalonate. J. Chromatogr. 1992, 591, 181. (33) Bandopadhyay, K.; Ghosh, S. Preparation and Characterization of Papain Modified Sesame Protein Isolate. J. Agric. Food Chem. 2002, 50, 6854. (34) Shahidi, F.; Chandrika, M.; Pathirana, L.; Wall, D. S. Antioxidant Activity of White and Black Sesame Seeds and Their Hull Factions. Food Chem. 2006, 99, 478. (35) Koleva, I.; Van Beek, T. A.; linssen, J. P. H.; Groot, A. de; Evstatieva, L. N. Screening of Plant Extracts for Antioxidant Activity: A Comparative Study on Three Testing Methods. Phytochem. Anal. 2002, 13, 8.

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Novel Approach to Recover Natural Antioxidants from Oil Seed Meal

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