A comparative study between the electro-activation

Food Bioscience 11 (2015) 56 –71

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/fbio

A comparative study between the electro-activation technique and conventional extraction method on the extractability, composition and physicochemical properties of canola protein concentrates and isolates Alina Gerzhovaa,b, Martin Mondorb,c, Marzouk Benalid, Mohammed Aiderb,e,n a

Department of Food Sciences and Nutrition, Université Laval, Quebec, QC, Canada G1V 0A6 Institute of Nutrition and Functional Foods (INAF), Université Laval, Quebec, QC, Canada G1V 0A6 c Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant Boulevard West, Saint-Hyacinthe, Quebec, Canada J2S 8E3 d Natural Resources Canada/CanmetENERGY, 1615 Lionel-Boulet Blvd., PO Box 4800, Varennes, Quebec, Canada J3X 1S6 e Department of Soil Sciences and Agri-Food Engineering, Université Laval, Quebec, QC, Canada G1V 0A6 b

ar t ic l e in f o

abs tra ct

Article history:

A novel technology of electro-activation was used for protein extraction from canola meal.

Received 18 September 2014

An alkaline solution was generated in the cathodic compartment under the influence of

Received in revised form

electric field. It has been reported to have improved extractive properties when compared

23 March 2015

to chemically alkalized solutions. The study aims to verify the efficiency of electro-

Accepted 8 April 2015

activated solutions for protein extraction from canola oil cake by analyzing the effect of

Available online 30 April 2015

extraction method on the extractability rates, composition, and secondary structure of

Keywords:

extracted proteins.

Canola proteins

The tested parameters included NaCl concentration (0.01–1 M), duration of electro-

Electro-activation

activation (10–60 min), and current intensity (0.2, 0.3 A). The electro-activation was

Extraction

performed in a three-compartment cell separated by ion exchange membranes, after

SDS-PAGE

which the obtained solutions were used for 1-h extraction. Maximal protein extractability

FTIR

was 34.3271.21% obtained with the electro-activated solution generated under 0.3 A irrespective of the activation time. The conventional extraction under the same conditions (pH 7–10) yielded 31.1871.89% of proteins. Electrophoretic profiles of electro-activated protein concentrates and isolates analyzed by SDS-PAGE were clearly more distinguishable compared to those obtained by conventional method. FTIR study revealed considerable difference in proteins’ secondary structures between different treatment conditions (pH and salt concentration) as well as between conventional and electro-activated samples, showing less denatured spectra for the latter. & 2015 Elsevier Ltd. All rights reserved.

n

Corresponding author at: Institute of Nutrition and Functional Foods (INAF), Université Laval, Quebec, QC, Canada G1V 0A6. E-mail address: [email protected] (M. Aider).

http://dx.doi.org/10.1016/j.fbio.2015.04.005 2212-4292/& 2015 Elsevier Ltd. All rights reserved.


1.

Introduction

The interest towards new protein sources has grown dramatically in the past few years. Increasing malnutrition in developing countries, high cost of proteins from animal sources, health concerns such as intolerance to animal proteins and a conscious choice of many to refrain consuming animal proteins has led to a substantial search for alternative sources of proteins which could replace conventional ones. Alternative sources have been thoroughly studied in recent years with proteins derived from plants, bacteria and yeasts being the most promising ones. Among them oilseeds are interesting options as the protein rich oil cake left after oil extraction is a byproduct which can be valorized. Canola has become an important agricultural crop in Canada and around the world. It was developed in Canada primarily as a source of edible oil. Canola is also used for the production of biodiesel and its byproduct, the protein rich canola oilcake also finds use as forage for livestock. Other possible applications of canola meal proteins include adhesives, plastics, and biocomposites (Gillberg & Tornell, 1976). The protein content of an oilcake left after oil extraction accounts for 20–50% on a dry weight basis, similar to soybean which is extensively used in food industry (Tan, Mailer, Blanchard, & Agboola, 2011a). However, at present its utilization is limited to the production of animal feed. Numerous studies done on canola proteins’ physicochemical, functional and bioactive properties indicate potential for it to be used in the food industry (Aider & Barbana, 2011; Fleddermann et al., 2012; Ghodsvali, Khodaparast, Vosoughi, & Diosady, 2005; Khattab & Arntfield, 2009; Moure, Sineiro, Domínguez, & Parajó, 2006; Rodrigues, Coelho, & Carvalho, 2012; Wanasundara, 2011; Yoshie-Stark, Wada, Schott, & Wäsche, 2006; Yoshie-Stark, Wada, & Wäsche, 2008). Currently the most common extraction technique is a direct alkaline extraction method which comprises protein solubilization at a highly alkaline pH Z10 with subsequent precipitation either at its isoelectric point or by the use of membrane technologies. Processing in a highly alkaline medium allows to extract up to 60% (Ghodsvali et al., 2005) or even up to 80% of total proteins (Gillberg & Tornell, 1976; Pedroche et al., 2004) depending on the canola variety but at the same time causes undesirable modifications such as protein denaturation, reduction of digestibility and loss of essential amino acids (Rodrigues et al., 2012; Sari, Bruins, & Sanders, 2013). Furthermore, the presence of antinutritive factors is another limiting factor. However, the amount of antinutritive factors can be significantly reduced by the use of ultrafiltration and diafiltration (Ali, Mondor, Ippersiel, & Lamarche, 2011; Xu & Diosady, 2002). In order to increase the protein yield and improve the qualitative characteristics of the protein extracted from different vegetable sources, the effect of various conditions and reagents such as salt (Eromosele, Arogundade, Eromosele, & Ademuyiwa, 2008; Karaca, Low, & Nickerson, 2011), temperature (Gillberg & Tornell, 1976), time of extraction, and meal to solvent ratio (Nioi, Kapel, Rondags, & Marc, 2012) has been studied. Alternative methods to direct alkaline extraction has also also investigated such as protein micellar mass (Ismond & Welsh, 1992), the use of enzymes (Sari et al., 2013), and Osborne

57

scheme (Manamperi, Chang, Wiesenborn, & Pryor, 2012; Tan et al., 2011a; Tan, Mailer, Blanchard, and Agboola, 2011b). One of the most promising methods for the protein extraction is the use of direct electric current. Water subjected to an electric field known as electro activated solution (EAS) was claimed to possess improved extracting, cleaning and disinfecting properties (Aider, Gnatko, Benali, Plutakhin, & Kastyuchik, 2012b). The energy supplied by electricity transforms water to a metastable state, characterized by abnormal physicochemical properties (Leonov, Prilytskiy, & Bakhir, 1999; Plutakhin, Aider, Koshchaev, & Gnatko, 2013). Electroactivation (EA) may be explained by the phenomenon of water electrolysis and oxido-reduction reactions which take place on the electrodes under the influence of electric field. This leads to drastic changes in physicochemical properties in the near electrode layer, resulting in the formation of an acid solution in the anodic side and an alkaline solution in the cathodic side. The products of oxido-reduction reactions responsible for physicochemical activity of the solutions have been identified: (1) stable products of electro-chemical reactions responsible for pH changes; (2) non-stable over reactive substances such as ОН , Н3 О2 , Н2, НО2 , НО2 , О2 ; (3) other products formed near the electrode surface in the form of free structural complexes or hydrated shells of ions, molecules, radicals and atoms (Sprinchan, Bologa, Stepurina, & Polikarpov, 2011). The simultaneous formation of aqua complexes which can also increase the reactivity of the medium has also been documented (Leonov et al., 1999). The abnormal properties of EAS were studied by analyzing the Raman spectra and fluorescence and comparing them with that of chemically alkalized water (Belovolova, Glushkov, Vinogradova, 2006; Pastukhov and Morozov, 2000). The authors concluded that the metastable state of the near-electrode solutions was the cause of EAS activity (Aider, Gnatko, Benali, Plutakhin, & Kastyuchik, 2012a). It was also reported that the physicochemical properties of EAS could be manipulated by using various configurations of the reactor, membranes, and the time of treatment (Liato, Labrie, Benali, & Aïder, 2015). The EAS as mentioned before in addition to their acid and alkaline properties contain other substances which increase their reactivity (Prilutskii & Bakhir, 1997; Tomilov, 2002). These substances are very unstable, yet they might contribute to the extraction processes. In the food industry electro-activation EA has been used for protein extraction from sunflower seed; protein solution was pumped through the cathodic chamber to extract and subsequently through the anodic chamber to precipitate the extracted proteins (Plutakhin, 2005). Comparing the yield, the authors found that chemical extraction at (10.2%), was not significantly higher than electro-chemical extraction (8.9% ) showing the sufficiently high efficiency of extraction of sunflower proteins by means of EA. The aim of the current study was to use the catholyte from cathodic compartment for protein extraction from canola oilcake and to compare them with conventional extraction in terms of protein and total dry matter extractability, subunit composition and secondary structure of extracted proteins, amount of total phosphorus (as an estimate of phytic acid content), and the amount of free amino-acids. In order to find the optimal conditions the effect of the type of configuration, salt concentration, time of treatment, and current intensity was studied.

58


Conventional extraction was put through similar conditions in order to compare the efficiency of new technique.

2.

Materials and methods

2.1.

Chemicals

Defatted canola meal was kindly provided by Bunge ETGO, Becancour, Québec, Canada. Sodium hydroxide (NaOH) was purchased from VWR International LLC (West Chester, PA, USA). Concentrated hydrochloric acid (HCl) and sodium sulphate (Na2SO4) were purchased from Fisher Scientific (Mississauga, ON, Canada) and sodium chloride (NaCl) was purchased from Caledon Laboratories LTD (Georgetown, ON, Canada).

2.2.

for the cathodic one. The values of the applied voltage and the intensity of the electric current were measured using a current power supply (CSI12001X, Circuit Specialists, Inc. Mesa, AZ, USA).

2.4.

Experimental

2.4.1.

Protocol of EA

The EA was carried out at two constant electric current intensities of 0.2 and 0.3 A with total time of treatment of 10, 30, and 60 min. To ensure the current flow salt solutions were circulated in all three compartments. The cathodic compartment was filled with NaCl solution of the following concentrations 0.01, 0.1, and 1 M. Anodic and central compartments were always filled with Na2SO4 solution of 0.25 M concentration.

Ion-exchange membranes 2.4.2.

Anion exchange membrane (AEM) AM-40 and cation exchange membrane (CEM) CM-40 were purchased from the Publicly Traded Company Schekina-Azot (Shchekina, Russian Federation). Prior to utilisation both membranes were washed with 96%-ethanol to remove the wax and prepared according to manufacturer’s instructions by soaking them in the salt solutions before their use in the electro-activation reactor.

2.3.

Configurations of the electro-activation reactor

Electro-activation reactor was made of transparent plexiglass columns with the dimensions of 50 50 120 mm (L W H). Two types of configurations of the three-compartmental reactor were used (Fig. 1). For the first configuration referred as C1 further in the text, the AEM separated the anode and central parts whereas CEM was placed on the cathodic side separating it from central section. The second configuration (C2) had the same three compartments but this time the membranes were swapped places. The RuO2–IrO2–TiO2 electrodes 120 34 1 mm with working active area of 40 cm2 were placed at the extremities of the cells and were connected to the positive side of a direct electric current power supply for the anodic compartment and to the negative side

Extraction by EA

Industrially defatted canola meal was weighed and dispersed in EAS, 10% (w/w). Extractions were carried out at room temperature for 1 h, under constant agitation using a magnetic stirrer. After that the slurry was centrifuged at 10,000 g during 30 min at room temperature so as to separate insoluble material using Eppendorf centrifuge 5804R (Eppendorf AG, Hamburg, Germany). The supernatant was collected as electro activated protein concentrate, further referred as EAPC. All the extractions were performed in triplicate and the mean values were determined. To obtain electro activated protein isolate, further referred as EAPI the pH of the supernatant was adjusted to pH 4.3. The precipitate was collected by centrifugation (10,000 g, 10 min with the help of Eppendorf centrifuge 5804R) and washed three times with distilled water. The pellets were freeze-dried.

2.4.3.

Conventional extraction

Industrially defatted canola meal was dispersed in aqueous NaCl solutions of the following concentrations 0.01 M, 0.1 M, and 1 M. The pH of the slurries was adjusted by adding 2 M NaOH solution dropwise so as to reach pH 7–10 and the extraction was held 1 h with agitation. After centrifugation the supernatant was collected as conventional protein

Fig. 1 – Configurations of the reactor used for EA corresponding to: (a) Configuration 1 (C1); (b) Configuration 2 (C2).

59


concentrate (CPC). The conventional protein isolates (CPI) were obtained by isoelectric precipitation as aforedescribed.

2.5.

Chemical analysis

Proximate analysis of the industrial canola meal was performed according to AOAC International (2012). Moisture and total dry matter were determined by drying the weighed sample in a Fisher Isotemp Vacuum Oven (Fisher Scientific, Montreal, QC, Canada) according to method 925.09. Ash content was determined according to method 923.03 by combusting a weighed portion of 5 g in the muffle oven until constant weight and the ash content was expressed as the ratio between the weight sample after combustion and its dry weight before combustion. Crude protein was determined following the Dumas method with LECO Truspec FP-428 (Leco Corp., St. Joseph, MI, USA) according to the method 992.23 using a conversion factor of 6.25. Residual oil in the defatted meal was determined by Soxhlet extraction according to method 925.85 using petroleum ether as extraction solvent. Carbohydrates were determined by difference (Karaca et al., 2011). Phytic acid being the principal storage form of phosphorus in canola was estimated from the ash residues (Stack, 1996) as the total phosphorus content and was analyzed according to Plaami and Kumpulainen (1991). The amount of free amino-acids was analyzed by EZ-Faast (Phenomenex, Torrance, USA) using GC-FID. All analyses were conducted in triplicate and mean values were calculated.

2.6.

Total dry matter and protein extractability

In order to determine the total extractability the aliquot of the supernatant in each case was weighed and dried in the oven until constant weight. The extractability was calculated as a ratio of dry matter in supernatant excluding the salt added during the extraction over total dry matter content of the canola meal. Nitrogen content of the supernatant was measured using Dumas method with LECO Truspec FP-428. The protein extractability was expressed as a function of nitrogen content in the supernatant in relation to nitrogen content in canola meal on a dry weight basis.

2.7.

SDS PAGE

To assess the impact of tested conditions on the polypeptide profiles of extracted proteins the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) was performed following the method of Aluko and McIntosh (2001). Initially protein concentrates were dialyzed at 4 1C using Spectra/Por 3 dialysis tubing with 3.5 kDa cutoff against water refreshing several times. After that, concentrates were freeze-dried and 1% protein solutions (w/w) were prepared and left overnight, followed by dilution with deionized water so that the amount of

proteins loaded onto the gel was approximately 20 mg. Similarly 1% solutions were prepared from protein isolates. Subsequently, diluted samples were dissolved in Tris–HCl buffer solution containing SDS for non-reduced conditions. For reduced conditions samples were dissolved in Tris–HCl buffer with addition of 5% (v/v) β-mercaptoethanol. Afterwards, the samples were boiled for 5 min, vortexed and 10 ml of each sample were loaded onto 4– 20% gradient gels (#456-1093, Bio-Rad, Canada). Precision plus Protein Kaleidoscope Standards of 10–250 kDa (#161-0375, BioRad, Canada) were used as molecular weight standards and the electrophoresis was run at 15 mA until the tracking dye reached the bottom of the gel. After staining in 2.5% Coomassie Brilliant Blue R250 in water–ethanol–acetic acid (4:5:1, v/v/v) and destaining with water–methanol–acetic acid (10:4:1, v/v/v) the gels were scanned, using Chemi Doc XRS 170-807 (Bio-Rad Laboratories, Inc, Hercules, CA, United States of America).

2.8.

FTIR

FTIR was performed as described by Ellepola, Choi, and Ma (2005). The infrared spectra were recorded at room temperature (22 1C) using a Nicolet Magna IR 560 spectrometer (Thermo Scientific, Madison, WI, USA) with continuous nitrogen supply. Canola protein dispersions (5% w/w) were dissolved in 0.01 mol/l deuterated phosphate buffer (pD 7.4). All protein dispersions were prepared in D2O instead of H2O since D2O has greater transparency in the infrared region, 1600–1700 cm 1. To ensure complete H/D exchange, samples were prepared the day before and left overnight at 4 1C prior to infrared measurements. Samples were placed between two CaF2 windows separated by 25 mm polyethylene terephthalate film spacer for FTIR measurement. A total of 256 scans were averaged at 4 cm 1 resolution. To study the amide I’ region of the protein, Fourier self-deconvolutions were performed using the software OMNIC 9.2.98. Band narrowing was achieved with a full width at half maximum of 22 cm 1 and with a resolution enhancement factor of 2.2 cm 1.

2.9.

Statistical analysis

All tests were performed in triplicate, and the means7standard deviations were used. Data collected were subjected to analysis of variance at 5% confidence level. When significant difference between treatments were found by ANOVA analysis, Tukey’s multiple range test was carried out for multiple comparison of the means using Minitab Software.

3.

Results and discussion

Proximate chemical composition of canola oil cake is shown in Table 1. On the whole these values are well correlated with those reported in the literature before. It had 43.36% of proteins which is in accordance with those reported by

Table 1 – Proximate chemical composition of canola oil cake on a dry weight basis. Moisture (%)

Protein (Nn6.25, %)

Ash (%)

Fat (%)

Carbohydrate (%, by difference)

Total phosphorus (%)

10.5170.01

43.3670.71

7.1970.01

3.5870.07

35.36

2.2170.48

60


Pedroche et al. (2004) and Tan et al. (2011b). When harvested, canola seeds contain 19.0% of protein and 54.2% of fat (Yoshie-Stark et al., 2008). After defatting procedure (made industrially) in our case the oil cake still had fat traces in the amount of 3.58%. Ash and moisture were also in the range of values reported for canola oil cake. Phytic acid is the major storage form of phosphorus in canola. Depending on the variety it accounts for 3.0–6.0 g/100 g (Bell, 1993; Gilani, Wu Xiao, & Cockell, 2012; Tan et al., 2011a). In spite of its known adverse effect such as binding minerals and proteins leading to mineral depletion and deficiency it was also reported to have a beneficial effect. Phytic acid is an effective chelator of iron and it prevents the formation of free radicals, thus working as an antioxidant. In addition it seems to bind heavy metals such as cadmium and lead helping to prevent their accumulation in the body (Lott, Ockenden, Raboy, & Batten, 2000).

3.1.

Changes in pH

3.1.1.

pH increase during EA

(Fig. 2) due to the oxido-reduction reactions on the electrodes. After 10 min further increase is also observed, however it is not that abrupt. In previous study the effect of current intensity, time of treatment, salt concentration and type of configuration on the pH and the strength of the catholyte (alkalinity) was studied. It was shown that time of treatment and current intensity had the most important impact on the alkalinity of the catholyte for C1. For C2, in addition to the aforementioned factors, salt concentration had a significant impact. The pH increase for the C1 was mostly dependent on the time of treatment and to a lesser extent on the current intensity. The effect of salt concentration was also present for the C2. Properties of chosen solutions for I¼ 0.3 A are shown in Table 2. Properties of the solutions obtained under lower current intensities were shown previously. The most important differences between two different configurations are observed at minimal NaCl concentration of 0.01 M (Gerzhova, Mondor, Benali, & Aider, 2015).

3.1.2. During the EA pH in the cathodic chamber increases drastically within first 10 min regardless of the reactor’s configuration

13

13

12

12

11

11

10 pH

10

pH

pH decrease during extraction

Solutions chosen for the extraction all had alkaline pH. During the extraction a pH decrease was observed which can be explained by the buffer capacity of the canola oilcake.

9

9 8

8 7

0.01M NaCl 0.1M NaCl

6

7

0.01M NaCl 0.1M NaCl

6

1M NaCl

1M NaCl

5

5 0

10 20 30 40 50 60 70

0

10 20 30 40 50 60 70

time of EA, min

time of EA, min

Fig. 2 – Changes in pH during EA for I¼ 0.3 A: (a) C1; (b) C2. Table 2 – Comparison of pH and alkalinity between two configurations for I¼ 0.3 A and within different NaCl concentrations. C (NaCl), M

Configuration 1

0.01

τ (min) 10 30 60 τ (min) 10 30 60 τ (min) 10 30 60

0.1

1

* Sd: Standard deviation.

C configuration 2 pH7Sd 11.3270.01 11.7870.06 12.0970.01 pH7Sd 11.3870.01 11.8170.02 12.0370.02 pH7Sd 11.4470.02 11.9470.04 12.2570.01

Alkalinity7Sd 8.570.99 21.0470.08 44.570.99 Alkalinity7Sd 7.0770.04 21.6470.03 43.3070.57 Alkalinity7Sd 7.0370.01 21.0570.07 43.2170.28

pH7Sd 11.1970.01 11.4570.04 11.6570.02 pH7Sd 11.5770.01 11.9570.01 12.1370.01 pH7Sd 11.6270.04 12.0970.01 12.3370.01

Alkalinity7Sd 3.9570.35 9.5470.03 14.2770.04 Alkalinity7Sd 6.2970.21 17.9070.28 32.3570.35 Alkalinity7Sd 6.9070.06 20.4070.28 43.0470.06


61

Fig. 3 – Changes in pH during extraction by EAS: (a) C1, 0.01 M NaCl; (b) C1, 0.1 M NaCl; (c) C1, 1 M NaCl; (d) C2, 0.01 M NaCl; (e) C2, 0.1 M NaCl; (f) C2, 1 M NaCl.

The pH decrease is shown in Fig. 3. Solutions obtained under I ¼0.2 A and I ¼0.3 A had the same tendency but were significantly weaker, so not to overload the paper only the results obtained for I ¼0.3 A were shown. No statistically significant difference was observed for the C1 between different salt concentrations, whereas for the C2 a considerable difference was noted between 0.01 M NaCl and the other two concentrations. Regarding 0.1 and 1 M NaCl concentrations for the C2 only the solution treated 60 min was significantly different from the other ones. Time of treatment had the major impact on the changes in pH during extraction. A 10-min solution was the weakest, which was in agreement with alkalinity measurements (Table 2). Its pH decreased dramatically down to pH 6 once it was mixed with canola oilcake and was kept within this value during the extraction. Such behaviour was observed for all treated solutions regardless their NaCl concentration or the type of configuration. For the 30-min solution there was a difference between configurations. This solution was twice as strong as the 10-min according to its alkalinity. The pH of the 30-min solution dropped down not that abruptly and was maintained around pH 8 for C1. For C2 there was a significant difference between all three concentrations. Finally, the solutions treated 60 min were the strongest for the C1 which was also well correlated with their alkalinity. In C2 the strength of the solutions increased with an increase in salt concentration. In this case it seems that the salt concentration was the most important parameter as even after 60 min of treatment the pH of the solution made with 0.01 M NaCl decreased faster and to a lesser value in comparison with

solutions of higher salt content (Fig. 3d–f). Thus, the strength of 60-min treated solution in C2 was lower than the strength of solutions treated 30 min in the same configuration but with higher salt content. Comparing two configurations it is possible to conclude that for the C1 the higher the time of treatment and the current intensity the lesser the pH decrease is, which means the stronger the solution is. For C2 only the solution with 1 M NaCl solution could maintain the pH within the scope of C1. The ability to maintain the pH is related to the strength of the solution, to its alkalinity or buffering capacity in other words. Although the concentration of OH is sufficient for producing the same value of pH as in chemical analogues it is insufficient to maintain it within the constant value, its buffering capacity is lower in comparison with strong alkali such as NaOH. In order to increase the pH of extraction so as to simulate the conditions similar to the conventional extraction the higher current intensity and the longer time of treatment are required. The high pH can possibly be maintained when there is a constant generation of OH ions. This can be reached when performing the extraction directly in the cell. However, there is a risk of membrane fouling or precipitating on the electrodes.

3.2.

Extractability

According to the previous study the pH has the major effect on the extractability which is in accordance with other publications (Ghodsvali et al., 2005; Klockeman, Toledo, and Sims, 1997; Nioi et al., 2012). Canola proteins are characterized as very complicated due to the diversity of their

62


molecular weights and isoelectric points. Thus, Quinn and Jones (1976) reported over 30 protein species with two major proteins—cruciferin being a neutral protein of a high molecular weight (300–310 kDa) and an isoelectric point around pH 7 and napin, a small molecular weight protein (12.5–14.5 kDa) characterized by strong alkalinity with an isoelectric point around pH 10–11 (Aider & Barbana, 2011; Karaca et al., 2011). Minimal solubility was reported to be around pH 4.3 and the majority of plant proteins have their isoelectric points within slightly acid pH region (pH 4–5). With an increase in pH of extracting medium the amount of extracted proteins increases dramatically especially in the pH range of 10–12 due to the increase in proteins ionization.

3.2.1.

Total dry matter extractability

Total dry matter extractability is shown in Fig. 4. C1 and C2 (Fig. 4b and c) did not show an important difference in comparison with conventional extraction (Fig. 4a). Extractability increases with an increase in NaCl concentration, however the increase is not considerable. Between different times of EA the difference was also not important which means that even with the solution obtained after 10 min of EA good results can be obtained. The effect of current intensity was also present. Minimal amounts of extracted dry solids under I¼ 0.2 A was 19.6771.83% obtained in C1 with 10-min solution of 0.01 M NaCl which increased to 29.5271.30% for 60-min solution of 1 M NaCl. Second configuration showed slightly lower results, however the difference was not significant (not shown). Higher current intensity (I¼ 0.3 A) showed the same tendency and the results were 24.1570.51% to 32.0374.97% for C1 and 23.6970.18% to 30.5471.35% for C2 (Fig. 4b and c).

3.2.2.

Protein extractability

The tested parameters impacted the protein extractability comparatively more than the total yield (dry solids) with the current intensity being the most significant factor (po0.0001). Maximum extractability under I ¼ 0.2 A was 19.9371.39% obtained in C1, 1 M NaCl concentration and with the solution treated 60 min which was significantly lower in comparison with conventional extraction. Other parameters with 0.2 A current intensity gave even lower values (not shown). With I ¼ 0.3 A results were comparable to those obtained with conventional extraction (Fig. 5). Overall, the tendency of the changes in extractability as a function of other parameters (time of EA, salt concentration, type of configuration) was the same for both tested current intensities, that is why only the higher one (I ¼ 0.3 A) will be discussed. The effect of time of EA was lower than expected. According to the alkalinity studies the strength of the 10 min and 30 min solutions increased by 3 times and for the 30 min and 60 min solutionsby 2 times. However, the difference in the amount of proteins extracted by each of these solutions was not significant. Considering the pH of the extraction medium as a key factor these results become logic. Apparently, the protein extractability has low sensitivity to pH changes within the region of pH 7–10 and the increase of the extractability is observed at pH values higher than 10. Thus, the pH of the solution, electro-activated during 10 min was maintained around 7 during the extraction and the values obtained in the first configuration are perfectly correlated with extraction held at pH 7 adjusted by the addition of aqueous NaOH. For C1 the extractability was 26.2970.25% for 0.01 M NaCl, 31.1871.20% for 0.1 M NaCl and 32.7572.59% for 1 M NaCl. Conventional

Fig. 4 – Total dry matter extractability for I¼ 0.3 A: (a) conventional extraction; (b) extraction with EAS in C1; (c) extraction with EAS in C2.


63

Fig. 5 – Protein extractability for I¼ 0.3 A: (a) conventional extraction; (b) extraction with EAS in C1; (c) extraction with EAS in C2. extraction held at pH 7 gave 24.8470.93%, 28.4171.88% and 31.1871.89% for the same NaCl concentrations. A 30 min solution maintained pH around 8 and allowed to extract 27.7270.92%, 30.8270.61% and 32.0772.42% whereas the results for the conventional extraction held at pH 8 were 23.2870.90%, 25.3070.52% and 27.7870.27% which is slightly lower. Finally, the 60-min solution with pH around 9–10 gave 33.8270.59%, 36.1071.24% and 38.0670.13% which was substantially higher in comparison with conventional extraction at pH 10 (23.6770.19%, 27.9870.47% and 29.9771.69%) which can be ascribed to unusual properties of EAS. An increase in protein extractability with an increase in salt concentration is due to the ”salting-in” effect for conventional extraction and for the solutions obtained in the C1. As for C2, more pronounced increase is observed (Fig. 5c). The positioning of the AEM and CEM also significantly affected the protein extractability by the EAS. In C2, alkalinity of the solution increased with an increase in salt concentration as described in previous work (Gerzhova et al., 2015). Briefly, an anion exchange membrane which was placed in the cathodic chamber, separating it from the middle section allowed the migration of hydroxyl ions, responsible for the alkalinity into the neighboring compartment. Cathodic chamber was the zone of depletion (desalting) that led to water dissociation on the membrane in order to supply the current carriers. A generation of Hþ ions in the cathodic compartment limited the increase in alkalinity by neutralizing OH ions. This was especially pronounced when 0.01 M NaCl concentrations was used. With an increase in salt concentration the effect of water dissociation decreased which is supported by an increased alkalinity and the higher amount of extracted proteins (Fig. 5).

When 1 M NaCl concentration was used the effect of ion migration through the membrane was minor for such high salt concentration. This explains the difference in the amount of extracted proteins between the two configurations.

3.3.

Composition

3.3.1.

Composition of protein concentrates

The composition of protein concentrated is shown in Table 3. The proteins extracted through conventional method (conventional protein concentrates) were designated with the code CPC followed by the pH of extraction (e.g. CPC_10 corresponds to conventional protein concentrate extracted at pH 10). The protein extracted with EAS (electro-activated protein concentrates) were designated EAPC, followed by the type of configuration, and the time of treatment (e.g. EAPC_C1_10 means conventional protein concentrate, obtained by extraction with EAS, treated in the configuration 1 for 10 min). The highest protein concentration was obtained by conventional extraction at pH 12. First configuration after 60 min treatment yielded the amount of proteins statistically similar to conventional one at pH 10. All the other treatments were not statistically different. All protein concentrates extracted with 1 M NaCl by either conventional method or by electro-activation had significantly lower amounts of proteins on a dry weight basis. This can be explained by the presence of the considerable amount of salt and thus an increased mineral content. Only concentrates extracted by conventional extraction at pH 12 had statistically higher values, whereas no difference was observed for EA concentrates and conventional ones at pH 10. Ash content of the extracts treated with the addition of 1 M NaCl ranged

64


Table 3 – Comparative characteristics of protein concentrates. C (NaCl), M

CPC_10 CPC_12 EAPC_1_10 EAPC_1_60 EAPC_2_10 EAPC_2_60

Protein content (%)

Ash (%)

0.01

1

0.01

35.1171.29b 49.5271.17a 29.8870.17c 34.0472.53b 27.9571.60c 29.1670.76c

14.2270.62b 17.1270.81a 12.7570.77b 13.7471.41b 12.7870.78b 14.3770.40b

11.6871.68b 16.1570.52a 12.4071.37b 11.3171.75b 11.3670.27b 9.8770.42b


Free amino acids (total) (%)

1

0.01

1

0.01

1

65.9871.10a 63.9270.84a 69.0170.57a 66.5873.45a 63.6974.90a 66.9771.05a

0.4170.02b 0.3570.05b 0.7470.26b 0.6770.33b 1.3370.24a 0.8070.19b

0.0370.02d 0.0670.01c 0.1570.02a 0.0470.02c,d 0.0970.00b 0.0370.00d

3.5770.32a 2.4670.42a 4.5170.97a 3.5470.4a 4.9071.03a 4.2070.55a

0.5970.23a 0.4970.16a 0.7670.06a 0.6270.05a 0.7770.1a 0.6770.16a

* Results represent the average of three determinations7SD, values in the same column with different letters are significantly different (po0.05).

Table 4 – Comparative characteristics of protein isolates. C (NaCl), M

CPI_10 CPI_12 EAPI_1_10 EAPI_1_60 EAPI_2_10 EAPI_2_60

Protein content (%)

Ash (%)


0.01

1

0.01

1

0.01

1

89.6770.90a 86.1670.54b 90.8771.00a 82.4370.47c 91.6270.32a 85.2770.04b

97.9571.60a 89.9971.88c 94.5170.49a,b 94.1670.46a,b 90.0870.43b,c 91.6770.37b,c

1.4370.06b 1.9670.04a 0.8470.08c 1.1370.4b,c 0.3070.05d 2.2870.17a

1.5470.09b 2.3270.47a 1.1970.15b 1.2870.02c 1.1170.25b 1.1870.01b

1.1770.38a 1.6870.41a 1.5870.1a 1.1370.23a 1.2670.04a 1.4270.29a

0.8570.11b 0.9770.22b 1.7270.01a 0.8270.1b 1.0470.07a,b 0.6270.03b

* Results represent the average of three determinations7SD, values in the same column with different letters are significantly different (po0.05).

between 63.6974.90% and 69.0170.57% in comparison with 9.8770.42% and 16.1570.52% for 0.01 M NaCl. Although the amount of total phosphorus in the sample treated with 0.01 M NaCl was statistically equal apart from the EAPC_C2_10, a slight decrease with an increase in pH can be noticed. According to Gillberg and Tornell (1976) and Ghodsvali et al. (2005) the extraction of phytic acid and nitrogen takes place concomitantly and nitrogen extractability is influenced by the phytic acid. Ghodsvali et al. (2005) stated that maximum interactions were at pH lower than pI, when anionic groups of phytic acid bind to cationic groups of proteins as proteins are charged positively and the phytic acid is charged negatively. In neutral region it reacts with metal ions and proteins to form a soluble ternary phytic acid–cation–protein complex. At alkaline pH, both the protein and the phytic acid are negatively charged and the protein–phytate interaction takes place only in the presence of multivalent cations and through a salt linkage or an alkaline-earth ion bridge (Champagne et al., 1985). At high pH (411) the interaction between protein and phytic acid decreases and in the presence of sufficient amount of Ca or Mg ternary the complex will precipitate (Champagne et al., 1985). In addition, it has been reported that at high pH, the insoluble phytate exists in a fine colloidal suspension which can be removed by centrifugation (Tzeng, Diosady, & Rubin, 1990). Lower amounts of total phosphorus (phytic acid) in extracts treated with 1 M NaCl solution can be explained by the presence of considerable amounts of salt and decreased protein content. Working in high salt concentration also weakens the electrostatic interactions of phytates with the proteins (Schwenke, 1994) which explains the lower amounts

of phytic acid in concentrates extracted with 1 M NaCl solution in comparison with 0.01 M. The amount of free amino acids can be useful and practical indices for evaluating the protein quality. No statistically significant difference was noticed within the same salt concentration. However, smaller values were obtained in the solutions with 1 M NaCl compared to 0.01 M which can be related to the total amounts of proteins as the latter had considerably higher protein contents.

3.3.2.

Composition of protein isolates

The protein, mineral and total phosphorus contents of protein isolates are shown in Table 4. The conventional isolates were named similarly to concentrates: CPI followed by the pH of extraction. Electro activated protein isolates were designated EAPI, followed by the type of configuration and the time of treatment. The protein content of all isolates was rather high and the ash content was reduced significantly in comparison with concentrates which means a higher purity of final product. Interestingly, the higher protein content was observed in samples with milder treatment conditions. Thus CPI_10, EAPI_C1_10 and EAPI_C2_10 had higher protein content compared to other isolates. Regarding samples treated with 1 M NaCl CPI_10, EAPI_C1_10 and EAPI_C1_60 had the highest protein content. In addition samples of 1 M NaCl concentration had significantly higher protein amount in comparison with those of 0.01 M NaCl. High amounts of total phosphorus (phytic acid) can be explained by the higher purity of the sample compared to protein extracts which contained other interfering substances such as salts and carbohydrates. In addition higher salt concentrations help to reduce the electrostatic interactions of phytates with proteins


resulting in lesser amounts of phytic acid found in the isolate extracted with 1 M NaCl (Schwenke, 1994).

3.4.

SDS PAGE

3.4.1.

SDS PAGE of protein concentrates

Fig. 6 shows polypeptide compositions of canola protein concentrates with their major components analyzed by SDS-PAGE in the absence (non-reducing) and presence (reducing) of β-mercaptoethanol. Major bands in non-reducing conditions were identified between 37 and 50 kDa (as 45 kDa), two between 26 and 37 kDa (27 kDa and 32 kDa), and two between 15 and 20 kDa (as 16 kDa and 18 kDa). This was consistent with Tan et al. (2011b) who observed 5 main fraction with the following molecular weights: 16, 18, 26, 30, 45, and 53 kDa. However, the last subunit of 53 kDa was not present in our proteins. After comparing all three graphs it is noteworthy that in spite of different extraction techniques the proteins have more or less similar composition, apart from the CPC_12 that is distinguished by weaker marked

65

bands or their absence. However, it had a band of higher molecular weight around 250 kDa which was not present in other fractions. Similar phenomenon was noticed by Tan et al. (2011b) who used different solvents in order to extract separately albumin, globulin, glutelin and prolamin fractions. The authors managed to extract higher molecular weight subunits in glutelin fraction which were not observed in albumin and globulin fractions. Therefore, it is possible that under higher pH, subunits of higher molecular weight could be extracted (Quinn and Jones, 1976). The smeared pattern of CPC_12 is an indication of reduced protein solubility as a result of protein denaturation due to harsh alkaline conditions. The similarity in obtained profiles is due to the complexity of canola protein composition and the simultaneous co-extraction of different protein profiles. It is known that canola proteins have complicated composition. Its two major components 12S globulin (cruciferin, 300 kDa) and 2S albumin (napin, 14 kDa) considerably differ by their molecular weight, isoelectric point and functionalities (Manamperi et al., 2012; Wu & Muir, 2008). Cruciferin being salt-soluble

Fig. 6 – Molecular profiles as analyzed by SDS PAGE: (a) EAPC extracted with 0.01 M electro-activated NaCl solution; (b) EAPC extracted with 1 M electro-activated NaCl solution; (c) CPC extracted with 0.01 M and 1 M NaCl solutions.

66


however tend to be co-extracted together with albumin during the conventional extraction as well as napin was found in extracts obtained with 1 M NaCl concentration. That is why minor difference was noticed between profiles extracted with 0.01 M NaCl and 1 M NaCl. Thereby extracted fractions, in fact, are a mixture of two main proteins found in canola such as cruciferin and napin. There was a noticeable difference in SDS-PAGE profiles between the non-reduced and reduced state. Under the reducing conditions two of the most distinctive profiles of 16 and 45 kDa disappeared and smaller fractions of 10, 12, and 22 appeared indicating that the former fractions were linked by disulfide bonds and the latter are the result of other than S–S interactions. Other bands of 18, 27 and 32 kDa were not affected by the presence of β-mercaptoethanol and became more pronounced under reducing conditions which means that other than S–S bonds are predominant. In reducing conditions new bands are more marked in 0.01 M NaCl concentrations. This was consistent with the work of Tan et al. (2011b) who stated that globulin bands had less intensity compared to the polypeptide profiles of albumins but on the whole polypeptide profiles of globulin and albumin fractions were very similar. A comparison with conventional extracting technique revealed the difference in terms of 27, 32 and 44 kDa fractions which are similar in all EAPC and conventional ones extracted at pH 10 (CPC_0.01_10 and CPC_1_10) but disappears at pH 12. Napin was reported to appear on the gel in 14 kDa and 27.5 kDa (a dimer of napin) fractions according to Wu and Muir (2008) who studied the purified samples of both major proteins from canola. In our case it explains why the bands at 27 and 32 kDa are more pronounced with a lesser amount of salt. The 14 kD fraction was not observed in our study however the band observed at 16 kDa is presumably napin that upon the contact with βmercaptoethanol give rise to two new bands of 10 kDa and 6 kDa. It is also present in conventional concentrate extracted at pH 12 which is explained by napin’s rigid structure low prone to structural changes at different pH (Krzyzaniak, Burova, Haertlé, & Barciszewski, 1998). The band at 44 kDa is present in all EAPC and in CPC_10; however it disappears in the extract obtained under higher pH (pH 12). This phenomenon is linked to the protein structure as 12 S globulin is an oligomeric protein which dissociates into smaller subunits under the influence of pH or ionic strength (Schwenke, 1994).

3.4.2.

SDS PAGE of protein isolates

The non-reduced (without β-mercaptoethanol) and reduced (with β-mercaptoethanol) SDS-PAGE patterns for protein isolates and canola meal displayed as CM are presented in Fig. 7. Similar to protein concentrates the protein isolates profiles look comparable, however new bands of higher molecular weight revealed on the gel. These bands of 75 kDa, 100 and 110 kDa were not present in the concentrates and could result from protein aggregation during the precipitation with HCl. These aggregates are linked together by disulfide bonds which can be confirmed by their absence in reduced condition. In addition a partial hydrolysis of high molecular weight proteins may take place with an increase in extracting pH. Also the 12S subunits were reported to dissociate in the presence of 2–8 M urea solutions or at pH levels below 3.5

(Goding, Bhatty, & Finlayson, 1970). As it can be noted in Fig. 7c and d the band corresponding to 100 kDa disappeared at pH 12 and is lighter at pH 11 in comparison with conventional extraction held at pH 10 and to those performed with EAS. This was also observed by Jarpa-Parra et al. (2014) when lentil protein was subjected to different pH during extraction. In their case an alkaline pH created a progressive dissociation of the 11S protein to lower molecular weight subunits (7S and 2S). An evident difference between protein profiles of CPI and EAPI can be noticed which increases with an increase in pH suggesting proteins denaturation (Fig. 7a–d). In the work of Mwasaru, Muhammad, Bakar, and Che Man (1999) subunit composition of proteins isolates from cowpea and pigeon pea was not affected by the extraction technique. In the study the authors used micellar isolation and isoelectric protein methods and concluded that regardless of the extraction technique and pH conditions extracted proteins exhibited similar electrical mobility. In the other work where the influence of pH of extraction on the molecular profiles was tested samples extracted at pH 8, 9, and 10 showed similar SDS-PAGE patterns. The authors suggested that the protein compositions of the three extracts were similar and mainly composed of globulin proteins (Jarpa-Parra et al., 2014). Consequently, it can be concluded that protein composition is not influenced by the extraction technique in the range of pH 7–10, however further pH increase provokes proteins denaturation which can be observed by fading away of the bands in Fig. 7c and d.

3.5.

FTIR

Although total protein concentration is an important parameter the understanding of protein secondary structure is crucial for the understanding of its nutritive quality, functionalities, availability and digestive behavior which determines its further utilization. Even if the protein concentration is the same, the quality may differ due to the difference in the secondary structures. For the proteins to maintain its biological and functional values it should be folded in the same way as in the native state. The changes in environment influence protein conformation and changes in pH and ionic strength are strong factors affecting it.

3.5.1.

Secondary structure of protein concentrates

Fig. 8 shows the infrared spectra of canola protein concentrates in the amide I’ region (1700–1600 cm 1). It depends on the secondary structure of the backbone and is hardly affected by the nature of the side chain, thereby being the most commonly used region in monitoring the changes in secondary structure. It is composed mainly of C¼ O stretching vibrations and is extremely sensitive to changes in hydrogen bonding so that even subtle changes in the secondary structure can be observed (Barth, 2007; Kong & Yu, 2007). Bands of βpleated sheets arise in easily distinctive peaks between approximately 1620 and 1640 cm 1 and in some cases even below 1620 cm 1, showing more than one component and one weaker band between 1670 and 1680 cm 1. Amide I’ bands disposed between approximately 1650 and 1658 cm 1 are generally considered to be a characteristic of α-helical structures. The peaks around 161673 and 168974 cm 1 are


67

Fig. 7 – Molecular profiles as analyzed by SDS PAGE: (a) EAPI extracted with 0.01 M electro-activated NaCl solution; (b) EAPI extracted with 1 M electro-activated NaCl solution; (c) CPI extracted with 0.01 M NaCl solution; (d) CPI extracted with 1 M NaCl solution.

Fig. 8 – Deconvulved FTIR spectra: (a) Protein concentrates extracted with 0.01 M NaCl; (b) Protein concentrates extracted with 1 M NaCl.

68


normally due to the formation of an intermolecular hydrogenbonded antiparallel β-sheet structure. Finally, the peak around 1645 cm 1 points at the presence of a disordered structure known as random coil (Dong, Prestrelski, Allison, & Carpenter, 1995; Surewicz, Mantsch, & Chapman, 1993; Susi & Byler, 1988; Tang & Ma, 2009). Five main bands were observed in the spectra after deconvultion, which, according to the literature are β-structures predominantly (Fig. 8). The main protein fractions in canola proteins are cruciferin and napin as shown previously in the SDS PAGE profiles. The secondary structure of cruciferin is performed mainly by β-sheet (50%) and low content of α-helix (10%) conformations and was found similar to other l1S globulins (Schwenke, Raab, Plietz, & Damaschun, 1983). Napin, on the other hand is characterized by a high content of α-helix structure (40–46%) and a low content of β-sheet conformation (12%) (Schwenke, 1994). The quality of protein can be judged by their peak intensities, a protein in the native state would have clear peaks revealing α- and β-forms. A decrease in peak intensity, band shifting, an appearance of new bands associated with aggregation or an increase in random coil structure would indicate protein unfolding. When the protein is completely unfolded it loses its secondary structure and together with it its value and functionalities. An evident decrease in peak intensities can be observed in Fig. 8a from the top to the bottom as reflected by the protein unfolding. The most pronounced secondary structure is carried by two top proteins EAPC_C1_0.01_10 and EAPC_C2_0.01_10, their bands are identical, therefore secondary structures are comparable. The most prominent peak is the one at 1634 cm 1 attributed to β-sheet structure, followed by 1651 cm 1 associated with α-helix structure. The presence of two peaks at 1618 cm 1 and 1688 cm 1 was claimed to be due to the formation of an intermolecular hydrogen-bonded antiparallel β-sheet structure (Chehín et al., 1999; Dong et al., 1995). Thus, protein aggregation judged by an increase in non-native intermolecular β-sheet structures is the beginning of the loss of native protein which is a common reaction to the application of thermal, physical or chemical stresses (Chehín et al., 1999; Chi, Krishnan, Randolph, & Carpenter, 2003). The growth of aggregation bands is accompanied by the rise of another broad band at 1645 cm l, which can be assigned to unordered structure or random coil (Dong et al., 1995). This was noticed for CPC_0.01_10 (Fig. 8a), which still has some secondary structure; however the peaks are significantly diminished and the appearance of a broad platform at 1645 cm 1 is observed. Finally, at pH 12 (CPC_0,01_12) more loss in the secondary structure was observed, the protein was denatured with a broad spectrum within Amide I’ distributed between 1600 and 1700 cm 1. Structural differences in Fig. 8a are induced by the different characteristics of the solvent which in one case is EAS and in the other case saline solutions adjusted to desired pH. Clearly, the properties of a solvent play an important role. The pH of a solution determines the charge on the protein molecule. Proteins are normally stable against aggregation over narrow pH ranges and may aggregate rapidly in solutions with pH outside these ranges. Thus, in the work of Jarpa-Parra et al. (2014) the pH change in the range of 7–9 did not have an impact on the secondary structure of lentil proteins. However, at extreme values protein unfolded exposing its buried sites and changing

the electrostatic interactions (Chehín et al., 1999). As the pH changes the number of charged groups increases which by turn increases the charge repulsions. This is sufficient to overcome the attractive forces (mostly hydrophobic and dispersive) resulting in protein destabilization and partial unfolding (Chi et al., 2003). First it was thought that aggregation arose from completely unfolded protein, however further research showed that aggregates were formed from partly unfolded proteins (Chi et al., 2003). The protein’s native conformation is flexible and does not exist as a single structure but as an ensemble of conformations and the aggregation may take place even at physiological conditions (Chi et al., 2003). Aggregation band and the presence of random coil both characterize protein unfolding, however, when the presence of aggregates is an intermediate state and protein molecule can refold again as it still has secondary structure, the random coil means the loss of secondary structure and its properties. Therefore the unfolded state is normally devoid of any native structure and exists as a structureless random coil (Salahuddin, 1984; Tanford, 1968). Fig. 8b shows the spectroscopic characteristics of protein concentrates in the presence of 1 M NaCl. The bands of the spectra are similar to those shown in Fig. 8a, however, all bands are shifted to the higher values which indicates the weakening of H-bonding (Zhao, Chen, Xue, & Lee, 2008). This was also observed by Ma, Rout, and Mock (2001) who studied the effect of different salts on the conformation of oat globulins. In addition, an appearance of a new band in EAPC_C1_1_10 which was not present in Fig. 8a was noted. This band centered at 1644 cm 1 as previously discussed was assigned to a random coil conformation and the presence of this band indicates a higher level of disorder in protein’s secondary structure. Similar to Fig. 8a this band grows from the top protein to the bottom one. The decrease in 1672, 1654, and 1635 cm 1 and an increase in 1690, 1644 and 1618 cm 1 peak intensities suggest transition from ordered conformation to random coils and aggregated strands. Salts are known to perturb protein conformation by affecting both electrostatic and hydrophobic interactions via a modification of water structure (Ma et al., 2001). Over 100 years ago Hofmeister discovered that adding salts to egg white protein could alter its solubility (Kunz, Henle, & Ninham, 2004; Schwartz et al., 2010). The degree of the solubility is governed by the type of ions, their concentration and their ability to increase or decrease the hydrophobic interaction, resulting in “salting-in” or “salting-out” effects. This phenomenon became known as “Hofmeister series” or “lyotropic series”. Salts with the ability of the ions to reduce the hydrophobic interaction may enhance the unfolding tendency of proteins were called chaotropes or structure breakers and salts which can increase the hydrophobic interactions were called “kosmotropes”, or “structure makers” (Schwartz et al., 2010). However such classification was argued (Zangi, 2009). NaCl depending on its concentration can cause both “salting-in” and “salting-out effects”, at low concentrations working as a chaotropic salt but with an increase in molar concentration it acts as a kosmotrope and stabilizes protein conformation. Thus, it could precipitate proteins only in the presence of 3.5 N and higher salt concentration (Kunz et al., 2004). In the current work the salt concentration was chosen in order to increase the extractability of proteins which was reflected on the protein spectra.


69

Fig. 9 – Deconvulved FTIR spectra (a) Protein isolates extracted with 0.01 M NaCl; (b) Protein isolates extracted with 1 M NaCl.

3.5.2.

Secondary structure of protein isolates

The spectra of protein isolates were quite different in shape and peak intensities, indicating that proteins were more stable in concentrates. Broader and flatter surface suggests that protein’s native state was more affected in protein isolates in comparison with protein concentrates. As opposed to concentrates all the isolates were subjected to isoelectric precipitation which could explain the difference in their secondary structure. The net charge on the protein due to the titration of all the ionizing groups led to intramolecular repulsion, sufficient to overcome the attractive forces resulting in at least partial unfolding of the protein. This is supported by the presence of the peak close to 1645 cm 1 corresponding to a random coil which was not present in the concentrates (Fig. 9a). Fig. 9b was characterized by the higher level of disordered structure and the band shifting towards the higher frequencies, similar to what was observed in the concentrates and explained by the presence of NaCl. On the whole, the tendency was similar in protein isolates and concentrates. With an increase in the severity of treatment the aggregation bands and those related to random coil increased in intensities, while those related to the native structure decreased. In spite of acidic precipitation step which had certain negative effect on both conventional and EA isolates, the latter managed to retain more of the native structure.

4.

Conclusion

EAS proved to be an effective and a better medium for protein extraction from canola meal. In contrast to conventional techniques which use chemicals, EA is a “green technology”

as it utilizes electric current to generate solutions possessing desired properties. Both quantitative and qualitative analyses were performed comparing the extraction with EAS with the conventional method. Proteins extracted by EAS were of higher quality as seen in the SDS PAGE and FTIR results, also, the protein extractability was higher when compared to conventional extraction for same process parameters (pH and time). It was noted that the protein yield could be increased by increasing the current intensity, time of treatment and salt concentration. Another option is to perform the extraction inside the EA cell, which would ensure a constant generation of OH . However, it would influence the quality of extracted proteins, resulting in the conformational changes and possibly higher denaturation rates. In addition, there is a risk of membrane fouling or precipitation on the electrodes. The structure of proteins dictates its quality and functional properties which determines their further industrial utilization and commercial significance. Therefore, next objective should be to analyze and compare the functional properties of proteins obtained by EAS and conventional extraction.

Acknowledgments This work was financially supported by the innovation in food support program that was funded by contracts through the Growing Forward Program that occurred between the Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (Ministry of Agriculture, Fisheries and Food of Quebec) and Agriculture and Agri-Food Canada.

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