Preparation of Mustard (Brassica juncea L.) Protein Isolate and

2807) was supplied by Viral Rasayan, West Bengal, India. .... and dried in rotary evaporator (Cyber Laboratory, Singapore) to get the phenolic-acid-ri...
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Ind. Eng. Chem. Res. 2009, 48, 4939–4947

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Preparation of Mustard (Brassica juncea L.) Protein Isolate and Recovery of Phenolic Compounds by Ultrafiltration Ranjana Das,† Chiranjib Bhattacherjee,*,‡ and Santinath Ghosh† Department of Chemical Technology, UniVersity of Calcutta, 92, A.P.C. Road, Kolkata-700009, India, and Department of Chemical Engineering, JadaVpur UniVersity, Kolkata-700032, India

Mustard (Brassica juncea L.) seed meal is good source of protein (28-36%) and phenolic antioxidants like sinapic acid and sinapine. In this work an ultrafiltration-diafiltration-based technique is developed for simultaneous preparation of protein isolate and recovery of phenolic rich fraction from seed meal using a high shear rotating disk membrane module with emphasis on reduced membrane fouling effect. The yield of membrane process found less (31.4% on dry basis) compared to precipitation process (42% on dry basis), but a high purity of protein isolate (96%) with improved functional properties was obtained. The phenolic content in the protein was found to be reduced from 7.23 mg/g of protein to as low as 0.6 mg/g of protein in this process. The phenolic-acid-rich fraction recovered shows about 87.24% radical scavenging and 34.3% chelation capacity per 100 ppm concentration. So, this process can be commercially exploited for protein isolate and phenolic antioxidant-rich product preparation after proper scale up. 1.Introduction Mustard meal has a high protein content of about 40% and a nutritional study shows that mustard protein has a reasonably well-balanced amino acid composition.1 Newkirk et al.2 have worked on the nutritional evaluation of the mustard meal for a broiler diet. Bhattacharya et al.3 also reported about the nutritional aspect of mustard seed protein. As mustard protein has excellent water and fat binding functionality, the meal should be considered as potential source of food grade protein as often used in processed meats.4 Aluko et al.5 have reported the nature and functional properties of the polypeptides from brown mustard meal and protein concentrate. Mustard meal is also a good source of phenolic compounds. These compounds were previously considered undesirable because the presence of phenolic compounds can cause bitterness and astringency and dark colors in protein products, but these are now emerging as value added products, as they exhibit antioxidant property.6 Sinapic acid (SA) (MW-224.2 Da), the main phenolic compound in mustard meal, constitutes over 73% of free phenolic acids and about 80-99% of the total phenolic acids mainly occurring as an ester of sinapic acid, sinapine (MW-275 Da), and glucosides. Sinapic acid and sinapine are major water-soluble antioxidant components in the mustard meal. Saleemi et al.7 have compared the antioxidant properties of low pungency ground mustard seed with synthetic antioxidants like butylated hydroxyl tolulene (BHT) and tertiary butyl hydroquinone (TBHQ) on the stability of comminuted pork. The addition of 1.5% and 2% low pungency ground mustard seed was as effective as 200 ppm of the BHT or 30 ppm of TBHQ in controlling oxidative rancidity as measured by thiobarbituric acid (TBA). Further work by these researchers showed that 85% of the methanolic extract from low pungency ground mustard seed exhibited the strongest antioxidant activity due to the presence of much higher levels of phenolics compared to either water or 10% methanolic extracts. Amarowicz et al.8 have observed that the antioxidant activity of ethanolic extract of * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: +91 92305 62975. Fax: +91 33 2414 6203. † University of Calcutta. ‡ Jadavpur University.

mustard correlated well with the total content of the phenolic compounds in its isolated fractions. Conventionally, protein isolates from seed protein is produced by precipitation process, which involve some quality deterioration, like partial hydrolysis and denaturizing due to the use of strong chemicals.9 In bioprocess industries, dealing with protein purification, concentration, improvement of functional properties and fractionation of protein, membrane technology is now gaining importance because of the usage of less chemical reagent. In the field of seed protein, membrane technology is widely used for improvement of the functional properties of the respective protein isolate. Ghodsvali et al.10 have reported a membrane-based process of protein isolate preparation. The soluble protein isolate was prepared by ultrafiltration (UF) followed by diafiltration (DF) and drying. Protein isolate preparation from yellow mustard meal, almost free of glucosinolate and low level of phytate has been reported by Xu et al.11 Production of the mustard protein isolate from oriental mustard seed (Brassica juncea L.) by membrane-based process and evaluation of its performance in comparison to the commercial soy protein isolate in terms of texture, flavor, and color have been reported by Diosady et al.12 Ethanol (70%) was reported as extracting solvent for sinapic acid and sinapine from seed meals.13-15 The use of organic solvents points at the issue of health aspects regarding the human consumption and also on the quality of the processed product. Use of water definitely eliminates the safety concerns but makes the extraction and concentration process complicated. Considering the importance of the mustard protein and meal antioxidants our objective in this work is to prepare mustard protein isolate using a simple technology, hardly using any chemicals, and to simultaneously recover sinapic acid and the sinapine-rich fraction from mustard meal. 2. Materials Mustard seed (brown variety) was purchased from district Hooghly, West Bengal, India. Folin-Ciocalteau’s Phenol reagent (AR grade, 2 N, batch no. X/729505) was supplied by SISCO Research Laboratory Private Limited, Mumbai, India. Papain (activity 6000NF unit batch no. 2807) was supplied by Viral

10.1021/ie801474q CCC: $40.75  2009 American Chemical Society Published on Web 04/09/2009

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Figure 1. Schematic diagram of rotating disk membrane module (in-house fabricated).

Rasayan, West Bengal, India. Amino acid standard (AA-S-18) was purchased from Sigma Chemical Co., Louis, MO. Diethyl ethoxymethylenemalonate (98%) was purchased from Lancaster, Weastgate, White Lund, Morecabe, England. Ethanol (batch no. K32295683) was purchased from Merck, Germany, and sodium hypochlorite (approx. 4% w/v available chlorine, batch no. HG3H530443) was purchased from Merck, Mumbai. 2,2Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma Aldrich Chemical Co. All other chemicals, except otherwise stated, were purchased from SRL (Sisco Research Laboratory, India). The absorbance was measured in a Shimadzu (1601A) UV-Vis spectrophotometer. The ultrapure deionized water used was obtained from Arium 611DI ultrapure water system (Sartorius AG., Go¨ttingen, Germany). Cellulose triacetate CTA membrane of 5 kDa molecular weight cut of (MWCO) (cat no. 14529-76-D; diameter, 76 mm) was imported from Millipore Corporation, Bedford, MA, through its Indian counterpart (Millipore India Limited). All results are expressed as average of three determinations. 3. Experimental Processes 3.1. Membrane Module and Methodology. Figure 1 shows the schematic diagram of the experimental setup for the rotating disk module (capable of being used as fixed disk module also) used during the ultrafiltration operations. The module made of SS316 was manufactured by Gurpreet Engineering works, Kanpur, UP, India, as per specified design. The module was equipped with two motors with speed controllers to provide rotation of the stirrer (AC motor, HP ) 1/20, maximum rpm4000) and membrane housing (direct current motor, HP ) 0.25, maximum rpm ) 1500). The module has the facility to rotate the membrane and the stirrer in the opposite direction to provide maximum shear in the vicinity of the membrane. Adequate mechanical sealing (capable of withstanding maximum pressure of 10 kg/cm2) mechanism was provided to prevent leakage from the rotating membrane assembly. The magnetic drive stirrer mechanism prevents any leakage possibility from the top of the stirrer. Air compressor was used to provide compressed air for pressurization of the cell. An intermediate air reservoir coupled with an on-off controller based on a pressure sensor was provided which maintains the pressure within the reservoir between 10-12 kg/cm2. A differential pressure regulator was used to set the pressure at the desired

level within the module. Detail information regarding the membrane module set up is given elsewhere.16 Before the experiment started, the membrane was subjected to compaction for about 1 h with ultrapure water at a pressure of 6 kg/cm2, higher than the highest operating pressure, (4 kg/ cm2), to prevent any possibility of change of the membrane hydraulic resistance (Rm) during ultrafiltration. Once the water flux becomes steady, it is concluded that the full compaction of the membrane has taken place. After the experiment, the membrane was thoroughly washed for 20 min under running water, soaked in deionized water for 6 h, and thoroughly washed again for 20 min. 3.2. Experimental Conditions. Hydrophilic cellulose triacetate (CTA) (5 kDa) were used during this experiment. Membrane hydraulic resistance (Rm) of 5 kDa CTA was 4.38 × 1013 m-1. Ultrafiltration operation was carried out at 4 kg/ cm2 pressure with steady state permeate flux (Js) of 0.98 × 10-5 m sec-1 for process feed protein concentration of 12.78 mg/ mL. Membrane rotation (Nm) was maintained at 100 rpm during the run with no stirrer rotation. Hydrophilic CTA membrane was used to get minimum fouling and better permeate flux, which was the primary objective of this step. 3.3. Preparation of Protein Isolate and Recovery of Phenolic Components from Meal. The experimental scheme for the preparation of mustard protein isolate and simultaneous recovery of phenolic components by a membrane-based process is presented in Figure 2. Dehulling was done in low speed laboratory grinder followed by air classification. The defatted meal is prepared by solvent extraction in laboratory Soxhlet apparatus for 24 h using food grade hexane, and the solvent was removed in a vacuum oven at 50 °C at 4 mmHg for 5 h.17 Protein was extracted from the defatted mustard meal using aqueous NaOH at pH 11 with a water-meal ratio of 18 for 1 h at 50 °C. After the extraction the meal residue was separated by centrifugation (6000g, 10 min). The residual solid was washed with 6 volume of distilled water thrice, and the wash solution was mixed with the supernatant of alkaline extraction. The resulting alkaline extract was ultrafiltered (using 5 kDa CTA membrane) up to volume concentration factor (VCF) of 4 after pretreatment with 0.05 N NaCl. The retentate of ultrafiltration was subjected to diafiltration in same module, up to dia-volume (DV) of 3. The diafiltered retentate was freezedried (in Freeze-dryer, EYELA FDU-110, Japan) to get the

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Figure 2. Scheme for the preparation of mustard protein isolate and recovery of phenolic-acid-rich fraction. Table 1. Characteristics of Mustard Protein Isolate (MPI)a characteristics

MPIPPI

MPIMPI

protein (%) phytate (%) phenolics g/100 g protein (as gallic acid)

84.1 ( 1.4% 0.2 ( 0.02 1.4 ( 0.07

96.0 ( 0.3% N/Db 0.2 ( 0.05

a Values are expressed as mean ( SEM, where n ) 3. detected.

b

N/D ) Not

Table 2. Polyphenol Content in Mustard Meal and Different Fractions Produced during Preparation of Mustard Protein Isolatea

Figure 3. SDS-PAGE patterns of mustard meal: (lane1) alkaline extract, (lanes 2 and 5) protein isolate, (lane 3, 4, and 6) UF permeate, (lane 7) standard marker.

soluble protein isolate. To get the precipitated protein isolate the supernatant is adjusted to pH 3.5 (isoelectric point) using 1 N HCl under stirred conditions for 30 min at 37 °C and then centrifuged at 8000g for 10 min. The solid mass was collected as precipitated protein isolates (MPIPPI) and freeze-dried. Permeate from ultrafiltration operation was collected and dried in rotary evaporator (Cyber Laboratory, Singapore) to get the phenolic-acid-rich fraction.

product name

salt concentration (M)

polyphenol content (mg/g of meal)

seed meal (70% ethanol) alkaline extract MPI (precipitate) MPI (supernatant) MPI (5 kDa retentate) 5 kDa Permeate

0 0 0 0 0.05 0.05

17.11 ( 0.02 7.23 ( 0.01 4.94 ( 0.01 2.18 ( 0.03 0.60 ( 0.05 6.53 ( 0.02

a

Values are expressed as mean ( SEM, n ) 3.

3.4. Determination of Protein Content. Protein contents of the feed, retentate, and permeate were determined according to Folin-Lowry method of protein assay18 at 750 nm against an appropriate blank. Briefly, 5 mL of alkaline solution (50 parts of 2% Na2CO3 in 0.1 M NaOH + 1 part of 0.5% CuSO4 in 1% sodium potassium tartarate solution) was added to 1 mL of the test solution, mixed thoroughly, and allowed to stand for 10 min at room temperature. Diluted Folin-Ciocalteau reagent (0.5 mL) was rapidly added to the above solution with immediate

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Table 3. Emulsifying Properties of Mustard Protein Isolate (MPI)a product name

EAI (m2/g)

ESI (min)

MPI PPI MPI MPI

111.60 ( 0.63 129.23 ( 0.91

37.76 ( 0.60 48.70 ( 0.72

a Values are expressed as mean ( SEM, n ) 3; EAI ) emulsion activity index; ESI ) emulsion stability index.

mixing, and the mixture was allowed to stand for 30 min. Color was then measured at 750 nm. 3.5. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis of Protein. The molecular weight distributions of protein isolate were determined according to the method of Laemmli.19 Polyacrylamide gel (12%) was found most suitable for this purpose. The standard molecular weight (MW) in the range of 2-205 kDa was used to determine the molecular weight distribution of mustard protein. The gel was soaked in an excess of staining solution (0.1% Coomassie brilliant blue R-250, 40% methanol, and 10% acetic acid) for 30 min. The gel was then destained with excess of destaining solution (10% acetic acid, 30% methanol, and 60% water). The destaining solution was changed several times until the background had been satisfactorily removed. 3.6. Determination of Emulsifying Property. Emulsification properties were measured by the method of Pearce and Kinsella.20 Pure soybean oil (2 mL) and 6 mL of 0.1% protein solution (pH-8) were homogenized in a mechanical homogenizer at the highest setting for 1 min. The emulsion (5 mL) was pipetted from the bottom of the container at 0 and 10 min after homogenization. Each portion was diluted with 5 mL of 0.1% SDS (sodium dodecyl sulfate) solution. Absorbance of these diluted solutions was measured at 500 nm with the aid of UV-vis spectrophotometer against the appropriate blank. The absorbance was measured immediately (A0) and 10 min (A10) after emulsion formation. The following equations were used to calculate emulsifying activity index (EAI) and the emulsion stability index (ESI). EAI (m2/g) ) 2T[(A0 × dilution factor)/(c × φ × 10000)] (1) where c is the weight of the protein per unit volume of the aqueous phase before the emulsion formed. T ) 2.303, and φ ) oil volume fraction of the emulsion. ESI (min) ) A0 × ∆t/∆A

(2)

where ∆t ) 10 min and ∆A ) A0 - A10. 3.7. Determination of Foaming Properties. Foaming properties in terms of foam capacity (FC) and foam stability (FS) were determined by the method of Okezie and Bello.21 It was expressed as percent volume increase and calculated using the following formula. (%)

FC ) volume after whipping - volume before whipping 100 volume before whipping

FS was determined by measuring the foam height at 10, 20, 40, and 60 min. Both FC and FS were determined at pH 8 and at room temperature (30 ( 2 °C). 3.8. Determination of Amino Acid Composition by HPLC. Protein hydrolysis was achieved according to the method of Alaiz et al.22 with some modifications. Protein sample was completely hydrolyzed with the 4 mL of 6.0 N HCl. The solution was sealed in tube under nitrogen and incubated in an oven at 110 °C for 24 h and then dried in a desiccator.

Derivatization: To a dried sample of the standard amino acid mixture (amino acid standard solution, AA-S-18) or the protein hydrolysate (200 µg), 1 mL of 1 M sodium borate buffer (pH 6.6) containing 0.02% of sodium azide and 0.8 µL of diethyl ethoxymethylenemalonate was added. The reaction was carried out at 50 °C for 50 min with vigorous shaking. The resulting mixture was cooled to room temperature and 20 µL was injected in to a Waters HPLC system. Separation was done in a 4.6 × 150 mm Symmetry C18 reversed phase column (Waters) using a binary gradient system. The solvents 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/ min as follows: time 0-2 min, elution with A/B (95:5); from 2-40 min linear gradient A/B (95:5) to A/B (70:30); from 40-45 min A/B (70:30) to A/B (60:40); from 45-50 min A/B (60:40) to A/B (90:10). 3.9. Determination of Total Polyphenol Content. The amount of total polyphenol content was measured according to the standard method of Singleton et al.23 Briefly 0.1 mL of the extract was added to 5.9 mL water followed by addition of 0.5 mL Folin-Ciocalteaus’ phenol reagent and 1.5 mL of 20% sodium carbonate solution and final volume was made up to10 mL. The solution was kept for 2 h at room temperature and absorbance of the solution was measured at 760 nm. Gallic acid was used as the standard for calibration curve. Total polyphenol content was expressed as gallic acid equivalent. 3.10. Mass Spectroscopy of the Polyphenolic Extract. Liquid chromatography (LC) (Waters alliance, 2695 separation module) coupled with photodiode array detector (PDA, 2996, Waters) and mass spectroscopy (MS) (MS-MICROMASS, Quattro micro API, Micromass) was used for mass spectroscopic study of the permeate of 5 kDa UF membrane. C18 column (4.6 mm × 50 mm, 5 µm, Varian) with flow rate of 1 mL, maintaining column temperature at 30 °C was used. The eluents used were (A) acetonitrile with 0.05% HCOOH and (B) water with 0.05% HCOOH. The elution conditions were 0-5.8 min A:B (5:95 95:5), 5.8-7.8 min A:B (95:5 - 5:95), 7.8-9 min isocratic A:B (5:95). 4. Results and Discussion 4.1. Characteristics of Membrane Processed Mustard Protein Isolate. Figure 2 shows the steps of the preparation of mustard protein isolate and enhanced recovery of phenolic-acidrich fraction from mustard meal by an ultrafiltration-based process. The salt treatment helps in breaking of some of the protein-phenolic interaction and the phenolics are removed through diafiltration and thereby reducing the phenolic content in protein. Xu et al.24 have reported that phenolic compounds in canola meal bind with proteins through a variety of mechanism in aqueous medium including hydrogen bonding, hydrophobic interactions, covalent bonding, and ionic bonding. According to their studies, sodium chloride treatment (0.05 M) considerably reduced the sinapic acid content in the canola protein. Since the NaCl concentration of 0.05 M was too low to have a significant effect on hydrophobic interactions between phenolic compounds and proteins, the released phenolic acids were ionically bonded to the proteins. Enhanced phenolic removal by salt treatment from canola protein is also reported by the same group Xu et al.25 The diafiltered retentate was collected and freeze-dried to get membrane-processed soluble protein isolate (MPIMPI). The 5 kDa permeate was collected as a polyphenol-rich fraction. In the ultrafiltration step, CTA membrane was used, because of the less fouling effect of the

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a

Table 4. Foaming Properties of Mustard Protein Isolate (MPI)

foam stability (volume after different time at room temperature)

product name

volume before whipping (ml)

volume after whipping (ml) (5 min)

% foam capacity

10 min

20 min

40 min

60 min

MPI PPI MPI MPI

50 50

80.0 ( 0.2 89.7 ( 0.5

60.0 ( 0.2 79.4 ( 0.4

73.3 ( 0.7 75.4 ( 0.5

70.4 ( 0.8 72.8 ( 0.6

67.0 ( 0.5 70.4 ( 0.4

64.0 ( 0.4 68.4 ( 0.7

a

Values are expressed as mean ( SEM, n ) 3.

Table 5. Amino Acid Composition of Mustard Protein Isolate (g/100 g Protein)a name of amino acid aspartic acidb glutamic acidc serine histidine glycine threonine arginine alanine tyrosine valine proline methionine cystine isoleucine leucine phenylalanine lysine tryptophan

MPI

PPI

7.40 ( 0.10 20.10 ( 0.20 5.00 ( 0.24 3.60 ( 0.30 5.10 ( 0.20 5.31 ( 0.10 7.10 ( 0.13 5.2 ( 0.10 4.47 ( 0.32 4.60 ( 0.20 4.20 ( 0.50 2.50 ( 0.30 4.00 ( 0.10 7.60 ( 0.10 5.24 ( 0.54 6.20 ( 0.31 1.60 ( 0.24

MPIMPI

SPId

7.32 ( 0.12 20.37 ( 0.11 5.18 ( 0.30 3.50 ( 0.13 5.89 ( 0.10 5.13 ( 0.20 7.16 ( 0.10 4.65 ( 0.20 4.52 ( 0.31 4.80 ( 0.40 4.5 ( 0.11 2.30 ( 0.23 4.33 ( 0.11 7.50 ( 0.10 5.12 ( 0.23 5.96 ( 0.20 1.54 ( 0.11

11.19 20.86 4.46 2.47 4.17 3.62 7.68 4.17 3.98 4.84 5.21 1.33 1.23 4.84 7.58 5.02 6.12 1.25

a Values are expressed as mean ( SEM, n ) 3. b >Aspartic acid and asparagine. c Glutamic acid and glutamine. d Soy protein isolate.

process stream (rich in protein) on the hydrophilic CTA membrane.26 SDS-PAGE analysis of the crude alkaline extract of seed meal, retentate, and the 5 kDa permeate was done to know the molecular weight distribution of mustard protein. Figure 3 shows the presence of the eight protein bands in between 20 and 150 kDa. Since the minimum molecular weight of the extracted protein was above 5 kDa, the 5 kDa membrane was used for the separation of protein from phenolic acids. The level of protein in the 5 kDa permeate has been significantly reduced to a concentration below the detection limit for this method, suggesting almost complete separation of protein. Molecular weight distribution for the retentate was similar to that for alkaline extract, which suggested that the membrane-processed mustard protein isolate had the similar molecular weight distribution to that of the precipitated protein isolate. So from a nutritional point of view both the precipitated and membraneprocessed protein isolate were supposed to be equivalent. In membrane-based process the solid residue yield was 58.1% (on dry basis) and that for mustard protein isolate (MPIMPI) was 31.4% (on dry basis). In the precipitation process the yield of solid meal residue and protein isolate (MPIPPI) were 58% and 42% (on dry basis), respectively. Table 1 shows the characteristics of the mustard protein isolates prepared by both precipitation process and membrane process. Protein content of MPIMPI was found higher than MPIPPI. Phytic acid content was also found negligible in MPIMPI than MPIPPI. Phenolic content (as gallic acid equivalent) in MPIMPI was about seven times lower than MPIPPI. Since, the MPIMPI quality was much better than MPIPPI, this membrane-based process could be used as an alternative to the precipitation process of protein isolate preparation. Table 2 shows the distribution of polyphenolic compounds in different fractions. Among the different solvents, 70% ethanol showed the maximum yield of polyphenols (as gallic acid equivalent). The yield of polyphenol is 17.110 mg/g of meal.

In the alkaline extract the yield was 7.23 mg/g. Since phenolics remain bonded with insoluble proteins of seed meal, the yield of phenolics in alkaline extract was comparatively less than in solvent extract (PES). In the MPIPPI and its supernatant the polyphenol content were 4.94 mg/g and 2.18 mg/g, respectively. In the membrane process most of the phenols appear in the permeate part (6.529 mg/g compared to 0.602 mg/g in the MPIMMPI). 4.2. Functional Properties of Protein Isolate. Functional properties of protein products determine their application fields. Table 3 shows the emulsifying properties of the mustard protein isolates. Membrane-processed protein isolate (MPIMPI) showed better emulsifying properties compared to precipitated protein isolate (MPIPPI). Both the EAI and ESI values were found higher for MPIMPI compared to MPIPPI. Improvement of functional properties of membrane processed extract has also been reported by Massoura et al.27 Purity of the protein is of prime importance for improved emulsion properties. Higher EAI of the MPIMPI was due to the higher protein percent in MPIMPI (96%) compared to the MPIPPI (84.1%). Presence of salt (NaCl) also affected the EAI of protein isolate. Inyang et al.28 studied the positive impact of salt concentration on the emulsifying properties of the sesame protein concentrate. The emulsifying activity increased due to the increase in solubilization of the protein with the increasing salt concentration. The presence of salt also helped in the formation of more cohesive protein film which attributed to higher emulsion stability as well as reduced coulumbic interactions between neighboring droplets. A similar observation was reported by Kinsella et al.29 The diffusion rate of protein molecules to the oil-water interface also has a profound effect on emulsion properties. In MPIPPI protein molecules remain agglomerated, which requires more mechanical energy to diffuse at the oil-water interfaces, whereas the MPIMPI being less agglomerated diffused easily at the interfaces and consequently facilitated the emulsion formation. All these factors helped to show the improved emulsion properties of MPIMPI. Table 4 shows the percent foam volume expansion and foam stability of mustard protein isolates. Foams are thermodynamically unstable colloidal systems in which gas is maintained as a distinct dispersed phase in liquid matrix. A kinetic behavior to bubble coalescence and rapture is typically provided by a protein film surrounding the bubble. In foam formation soluble proteins are subjected to an interfacial exposure/adsorption, which alter their structure and allows for subsequent association of other proteins at the interface. Protein conformation is an important parameter contributing to foam stability. Retention of some native protein conformation and structures is important in development of stable foam. The MPIMPI showed higher foaming property than MPIPPI. Foam capacity of the MPIPPI was 60%, whereas for MPIMMPI it was about 79.4%. Foam volume changed sharply with time for MPIPPI than for MPIMPI, clearly indicating that the foam stability of MPIMPI was better than MPIPPI. Thus, over all functional properties of the membrane processed protein isolate (MPIMPI) have been found better than conventional precipitated protein isolate (MPIPPI). These improved foaming properties may be due to higher protein percent

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Figure 4. HPLC analysis of mustard meal extracts (PEM): (peak 1) sinapic acid; (peak 4) sinapine; peaks 2,3,5, and 6 are not identified. Table 6. DPPH• Radical Scavenging Activity in Comparison with Other Dietary Antioxidantsa

a

name of antioxidant

EC50 (mg/mL)

ascorbic acid BHA PEM

0.025 0.035 0.059

EC50 ) concentration required for 50% reduction of DPPH radical.

Table 7. Metal Ion (Cu+2) Chelation Capacity of Mustard Meal Extracta % chelation capacity dose (ppm) 5 25 50 100 a

PEM 8.7 ( 0.8 12.2 ( 1.1 23.5 ( 1.8 34.3 ( 2.3

PES 8.3 ( 0.7 11.4 ( 1.2 22.9 ( 1.5 33.8 ( 2.5

Values are expressed as mean ( SEM, n)3.

in MPIMPI than MPIPPI. The presence of salt (NaCl) has positive impact on foaming properties. A strong repulsive force has developed as a consequence of adsorption of highly charged protein to dilute monolayer coverage, which prevented the approach of additional similarly charged proteins. Furthermore, masking these charges with counterions (from salt) would reduce the electrostatic repulsion and favor protein association beyond a monolayer. This increase contributes to the increased solubilization of protein and hence foaming properties. This observation is in agreement with the observation of Inyang et al.28 4.3. Nutritional Aspects of Mustard Protein Isolate. Mustard proteins were reported as having a well-balanced amino acid composition.1 We have also studied the amino acid composition of mustard protein isolate produced by both isoelectric precipitation process and membrane process. Table

5 presents the essential amino acid profile of mustard protein isolates prepared in different route and commercial soy protein isolates.30 It is clearly noted that mustard protein is comparable to soy protein isolate and also has higher methionine content. Membrane processed mustard protein isolates have similar amino acid compositions to that of precipitated protein isolate, so these are likely to be equivalent from a nutritional point of view. Thus, a good nutritious protein with improved functional properties can be prepared by the membrane process. 4.4. Fouling Behavior of Mustard Protein. Membrane fouling and flux decline are critical factors associated with ultrafiltration operation. Percent fouling of the membrane was determined by measurement of the pure water flux, before and after the run was over: % fouling )

Wi - Wf 100 Wi

where Wi is the pure water flux observed before the run was performed and Wf is the pure water flux observed after washing the membrane. Severe fouling (35%) was observed for 5 kDa CTA membrane during UF of mustard protein with feed concentration of 100 ppm under 2 bar transmembrane pressure. After performing the proposed cleaning protocol, about 98% recovery of initial pure water flux was observed. This fouling is supposed to be due to surface adsorption of protein on membrane surface and not membrane pore plugging. As we have observed from the molecular weight distribution of mustard protein (Figure 3), the minimum molecular weight of protein is about 20 kDa, which cannot enter the pores of 5 kDa membrane. Some small peptides present in the alkaline extract may cause partial pore plugging. Unrecovered pure water flux may be due to this partial pore plugging effect.

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Figure 5. LC-MS analysis of mustard meal extract (PEM) under different conditions: (1) scan ES-, 2.22e5; (2) scan ES+, 1.26e8; (3) scan ES+, 5.44e5; (4) ES+, 8.80 × 6.

Figure 6. DPPH radical cation scavenging activity of mustard meal extracts.

4.5. Identification of Antioxidative Components in the Membrane Processed Extract. HPLC analysis has shown that in PEM, about 16% SA and about 62% SP are present (Figure 4). LC-MS analysis was done with the membrane processed extract (PEM) which confirmed the presence of the two components of molecular weight 224.2 Da and 265 Da which were similar to that of sinapic acid (MW, 224.2 Da) and sinapine (MW, 265 Da) along with some other unknown components. The presence of these components in PEM is shown in Figure 5. Several published literature pieces have reported about the

Figure 7. DPPH radical scavenging activity of mustard meal extract with changing doses from 10-100 ppm.

antioxidant activity of SA- and SP-rich fractions. 14,31,32 Since the PEM also has these two components, several in vitro studies were performed to determine the antioxidant potential of the membrane processed meal extract. 4.6. Antioxidant Activity of Phenolic-Rich Fractions. Mustard meal extract (PEM) has shown equivalent free radical scavenging activity like PES. The blank DPPH solution on scanning between 400-600 nm gives a maximum absorption peak at 517 nm. After adding 50 ppm PEM, the absorption peak

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disappeared, suggesting that the extract had the radical scavenging activity. Some literature have reported good DPPH radical scavenging activity of mustard meal extract (in organic solvent).13 Figure 6 shows that the percent radical scavenging activity of PEM is equivalent to PES. The radical scavenging activity of synthetic antioxidant BHA is about 10-12% higher than PEM. Figure 7 shows how the radical scavenging activity changes with varying concentration of PEM. Initially from 10 to 50 ppm the scavenging activity changed sharply, after 50 ppm the change became gradual; 50 ppm aqueous extract showed about 84.78% scavenging, and 100 ppm extract showed about 87.24% scavenging activity. The EC50 value of PEM in comparison to the other dietary antioxidants is shown in Table 6. Thus, the concentration of PEM required for 50% reduction of DPPH radical was 2 times higher than well-known antioxidant ascorbic acid and about 1.5 times higher than BHA. Metal ions such as iron and copper are major primary catalysts that initiate oxidation in vivo and in vitro.28 Metal ions play an important role in the acceleration of oxidation of important biological molecules; for instance they may catalyze the formation of a first few radicals that may lead to the propagation of the radical chain reaction. Chelating agents are known to stabilize the pro-oxidant metal ions by complexing them. Table 7 shows the dose dependent metal ion chelation capacity of mustard meal extracts. For 100 ppm of PEM and PEM, the percent chelation capacities were 34.3 and 33.8, respectively. With an increase in the dose of PEM and PEM from 5 to 100 ppm on average a 4 times increase in chelation capacity was observed. All these studies indicated that the membrane processed extract (PEM) also had as good a radical scavenging activity and metal ion chelation capacity as the solvent extract (PES). Considering this aspect it can be concluded that the phenolic-acid-rich extract from mustard meal instead of synthetic antioxidants could be utilized for various food and nonfood formulations. 5. Conclusions From all these studies it can be concluded that ultrafiltration can be used to improve the quality of protein isolate as well as recover phenolic antioxidants from mustard meal. In regards to the functional properties and amino acid compositions it can be said that the membrane processed protein isolate is much better than the precipitated protein isolate. The membrane processed protein isolate has better emulsifying and foaming properties with low phenolic content and thus, can be used in food formulations. Membrane processing does not affect the nutritional quality of the protein isolate as evidenced from the amino acid profile. The membrane processed phenolic-acid-rich fraction also has a good radical scavenging and chelation capacity. Finally, it can be said that industrialization of this scheme will open up a potential route of mustard seed meal utilization and would bring an overall contribution to the meal industry. Acknowledgment We are very much thankful to Technical Education and Quality Improvement Program (TEQIP), a World Bank project for funding the work. Some part of this work (UF with rotating disk module, a novel high shear device) was carried out utilizing the infrastructures developed under the Indo-Australian Project, entitled Milk Nutraceuticals: A Biotechnology Opportunity for Australian and Indian Dairy Producers, funded by DBT under Indo-Australian Biotechnology Fund (IABF). The contribution of IABF is gratefully acknowledged.

Abbreviations UF ) ultrafiltration DF ) diafiltration Rm ) membrane hydraulic resistance MPI PPI ) precipitated mustard protein isolate MPI MPI ) membrane processed protein isolate EAI ) emulsion activity index ESI ) emulsion stability index PEM ) phenolic extract membrane processed PES ) phenolic extract solvent process DPPH ) 2, 2- diphenyl-1-picrylhydrazyl Literature Cited (1) Etten, C. H.; Kwolek, W. F.; Peters, J. E.; Barclay, A. S. Plant Seeds as Protein Source for Food and Feed: Evaluation Based on Amino Acid Composition of 379 Species. J. Agric. Food Chem. 1967, 53, 162. (2) Newkirk, R. W.; Classen, H. L.; Tyler, R. T. Nutritional Evaluation of Low Glucosinolate Mustard Meals (Brassica j uncea) in Boiler Diets. Poultry Science. 1997, 76, 1272. (3) Sen, M.; Bhattacharyya, D. Nutritional Effects of a Mustard Seed Protein on Growing Rats. Int. J. Food Sci. Technol 2003, 38, 225. (4) Prapakornwiriya, N.; Diosady, L. L. Isolation of Yellow Mustard Protein by Microfiltration Based Process. Int. J. Appl. Sci. Eng 2004, 2, 127. (5) Aluko, R. E.; McIntosh, T.; Ketepa-Mupondwa, F. Comparative Study of the Polypeptide Profile and Functional Properties of Sinapis alba and Brassica juncea Seed Meals and Protein Concentrates. J. Sci. Food Agric. 2005, 85, 1931. (6) Sosulski, F. W. Organoleptic and Nutritional Effects of Phenolic Compounds on Oil Seed Protein Products. J. Am. Oil Chem. Soc. 1979, 56, 711. (7) Saleemi, Z. O.; Janitha, P. K.; Wanasundara, P. D.; Shahidi, F. Effects of Low-Pungency Ground Mustard Seed on Oxidative Stability, Cooking Yield and Colour Characteristics of Comminuted Pork. J. Agric. Food Chem. 1993, 41, 641. (8) Amarowicz, R.; Naczk, M.; Shahidi, F. Antioxidant Activity of the Different Fractions of Non-tannin Phenolic of Canola Hulls. J. Agric. Food Chem. 2000, 48, 2755. (9) Ihekoroney, A. I. Nutritional Quality of Acid-precipitated Protein Concentrate from the Nigerian ‘Red Skin’ Groundnut (Arachis hypogaea L.). J. Sci. Food Agric 1987, 38, 49. (10) Ghodsvali, A.; Haddad, M. H.; Khodaparast, M. H.; Vosoughi, M.; Diosady, L. L. Preparation of Canola Protein Materials using Membrane Technology and Evaluation of Meals Functional Properties. Food Res. Int 2005, 38, 223. (11) 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. (12) Marnoch, R.; Diosady, L. L. Production of Mustard Protein Isolates from Oriental Mustard Seed (Brassica juncea L.). J. Am. Oil Chem. Soc 2006, 83, 65. (13) Thiyam, U.; Sto¨ckmann, H.; Felde, T. Z.; Schwarz, K. Antioxidant Effect of the Main Sinapic Acid Derivatives from Rapeseed and Mustard Oil Byproducts. Eur. J. Lipid Sci. Technol. 2006, 108, 239. (14) Amarowicz, R.; Wanasundara, U. N.; Karamac´, M.; Shahidi, F. Antioxidant Activity of Ethanolic Extract of Mustard Seed. Nahrung. 1996, 40, 261. (15) Shahidi, F.; Wanasundara, U. N.; Amarowicz, R. Natural Antioxidants from Low-Pungency Mustard Flour. Food Res. Int. 1994, 27, 489. (16) Das, R.; Ghosh, S.; Bhattacharjee, C. Studies on Membrane Processing of Sesame Protein Isolate and Sesame Protein Hydrolysate using Rotating Disk Module, Sep. Sci. Technol., 2009, 44, 131. (17) Bandopadhyay, K.; Ghosh, S. Preparation and Characterization of Papain Modified Sesame Protein Isolate. J. Agric. Food Chem. 2002, 50, 6854. (18) Lowry, O.; Rosebrough, N. J.; Randal, A. L. Protein Measurement with Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 267. (19) Laemmli, U. K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 1970, 227, 680. (20) Pearce, K. N.; Kinsella, J. E. Emulsifying Properties of Protein: Evaluation of a Turbidimetric Technique. J. Agric. Food Chem. 1979, 26, 716. (21) Okezei, B. O.; Bello, A. B. Physico-chemical and Functional Properties of Winged Bean Flour and Isolate Compared with Soy Isolate. J. Food Sci. 1988, 53, 450.

Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009 (22) 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. (23) Vernon Singleton, L.; 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, 152. (24) Xu, L.; Diosady, L. L. Interactions between Canola Proteins and Phenolic Compounds in Aqueous Media. Food Res. Int. 2000, 33, 725. (25) Xu, L.; Diosady, L. L. Removal of Phenolic Compounds in the Production of High Quality Canola Protein Isolates. Food Res. Int. 2002, 35, 23. (26) Cheryan, M. Ultrafiltration Handbook; Technomic Publishing Company Inc: Lancaster, PA, 1998. (27) Massoura, E.; Vereijken, J. M.; Kolster, P.; Derksen, J. T. P. Isolation and Functional Properties of Protein from Crambe abyssinica Oil Seeds, I. Progress in New Crops; Janick, J., Ed.; ASHS Press: Alexandria, VA, 1996, 322. (28) Inyang, U. E.; Iduh, A. O. Influence of pH and Salt Concentration on Protein Solubility and Foaming Properties of Sesame Protein Concentrate. J. Am. Oil Chem. Soc. 1996, 73, 1663.

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ReceiVed for reView October 1, 2008 ReVised manuscript receiVed December 29, 2008 Accepted March 12, 2009 IE801474Q