Physicochemical, microstructure and bioactive characterization of gels made from crayfish protein

1 Physicochemical, microstructure and bioactive characterization 1 of gels made from crayfish protein 2 M. Felixa, A. Romeroa,, T. Rustadb, A. Guerreroa,* 3 a Departamento de Ingeniería Química, Universidad de Sevilla,, 41012 Sevilla, Spain. 4 b Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway. 5 Abstract 6 Crayfish proteins are valuable active ingredients for food products, mainly due to 7 its protein quality and antioxidant activity. A highly soluble crayfish protein 8 concentrate (CF2L) obtained from crayfish surpluses was used to evaluate gelling 9 properties at three different pH values (2.0, 6.5 and 8.0). Thermal gelation processes 10 were monitored by Small Amplitude Oscillatory Shear (SAOS) measurements. 11 Subsequently, gels were characterized by viscoelastic properties, water-holding 12 capacity (WHC) and a selective solubility. All the systems exhibited a gel-like 13 behavior, showing a strong dependence on pH. 14 Antioxidant measurements were performed by using three different reagents 15 (DPPH, ABTS and Folin-Ciocalteu) and revealed an interesting potential for human 16 food. The pH effect was evaluated, showing a remarkable dependence of ABTS on its 17 value. 18 The results show that gels made from crayfish surpluses have potential for use in 19 human nutrition, not only based on the physical properties, but also on its protein 20 quality and antioxidant ability. 21 Keywords: Bioactive properties, Crayfish proteins, Thermal gelation, 22 Viscoelastic properties. 23 24 2 _______________________ 25 *A. GUERRERO 26 Departamento de Ingeniería Química 27 Universidad de Sevilla, Facultad de Química 28 41012 Sevilla (Spain) 29 E-mail: [email protected] 30 Phone: +34 954557179; fax: +34 954556447. 31 1. Introduction 32 In the middle of last century, the freshwater red-swamp crayfish (Procambarus 33 clarkii) was introduced in the Guadalquivir marshlands. Due to weather conditions, 34 abundant food and the lack of predators, the crayfish population underwent a fast 35 and widespread growth and was soon considered as an invasive species. The use of 36 crayfish-meat surpluses to produce a non-denatured protein concentrate may be a 37 useful method to preserve and utilize the crayfish for later use, from which is 38 possible to produce different food products, e.g. in the form of emulsions or gels 39 (Bengoechea et al. 2008; Romero et al. 2009a). 40 In the last few years, there have been an interest in the nutritional value of food 41 industry by-products since it is possible to develop food products that are 42 interesting in the health and snack food markets (Glew et al. 2006; Sah et al. 2015). 43 These added-value may be considered on the basis of both their amino-acid 44 composition and bioactive properties such as antioxidant or hypertensive activity 45 (Dey and Dora 2011). 46 Organic compounds in humans are made up to around 85 wt. % of proteins. Thus, 47 they are an essential part of human diet, not only in infants for growth and 48 development, but also for adults. Protein quality can be determined by the amino 49 3 acid composition and especially the content of the 9 essential amino acids (Reeds 50 2000). 51 Additionally, in a healthy diet, a sufficient intake of antioxidant compounds is also 52 important, since free radicals are continuously produced as part of the human 53 metabolism. These may induce damages to biomolecules that may promote changes 54 in DNA and, as a consequence, serious health problems (Gey 1993). Antioxidants, 55 present naturally in food, have also been postulated as antiaging agent (Brown 56 2005). In addition, antioxidant peptides have been found in numerous food products 57 including milk, wheat, potato and fungi (Suetsuna et al. 2000; Sun et al. 2004). More 58 recently, some studies have been focused on antioxidant peptides from fish 59 (Sakanaka et al. 2005; Elias et al. 2008). 60 Proteins may therefore be used as potential antioxidants (Sa-ard et al. 2014). The 61 antioxidant activity of proteins is attributed to complex interactions amongst their 62 ability to inactivate reactive oxygen species, scavenge free radicals, chelate 63 prooxidative transition metals, reduce hydroperoxides, enzymatically elimination 64 specific oxidants, and alter the physical properties of food systems. As a 65 consequence, proteins are considered unique compared to other food antioxidants, 66 because they can inhibit different steps in lipid oxidation (Elias et al. 2008; Irshad et 67 al. 2013). 68 Functional properties of crayfish protein isolates such as gel and emulsifying 69 properties have been recently evaluated (Romero et al. 2009a; Romero et al. 2009b; 70 Romero et al. 2011). However, its nutritional value and bioactivity potential have 71 not previously been investigated. The protein quality together with bioactive 72 properties such as antioxidant activity are important for these ingredients to 73 contribute to a healthy diet (FAO 1985). 74 4 The overall objective of the present work was to evaluate the gelling properties 75 and bioactive potentials of gels made from non-denatured crayfish protein 76 concentrate (CF2L) at three different pH values (2.0, 6.5 and 8.0). To achieve these 77 objectives, a physical and chemical characterization of CF2L was carried out. 78 Subsequently, heat-induced gelation of CF2L was monitored by Small Amplitude 79 Oscillatory Shear (SAOS) as a function of pH. Further characterization of the final gel 80 was performed by means of WHC and selective solubility in order to evaluate the 81 involved interactions. Finally, antioxidant activity of the different gels was evaluated 82 by three different methods. 83 2. Material and methods 84 2.1 Materials 85 86 Figure 1 shows the process to obtain different protein fractions from crayfish 87 (CF) meat. Initially, CF meat was separated from the shell by grinding and sieving 88 and, subsequently, CF pulp was kept frozen until use. This first stage was carried out 89 by ALFOCAN (Isla Mayor, Sevilla, Spain). After thawing at 4 ºC, CF pulp was 90 homogenized and subjected to centrifugation at 15,000 xg for 15 min, obtaining 91 three different phases. A heavy phase (CF1P), which mainly consists of solid 92 materials (some parts of the exoskeleton) and muscle tissue. This phase represents 93 about 10 wt. % of the CF pulp, while its protein content is c.a. 14 wt.%, which 94 constitutes the 8.6 wt.% of the total CF-pulp protein amount. An intermediate phase 95 (CF2P), which mainly consists of water (c.a. 70 wt. % of the CF-pulp) was also 96 obtained. The protein content of this phase is c.a. 80 wt.% and represents the 89.2 97 wt.% of the total protein amount in the CF-pulp. Eventually, a light phase (CF3P), 98 which mainly consists of lipids (ca. 20 wt. % of the CF-pulp) was obtained. The 99 5 protein content of this lipophilic phase is c.a. 27 wt.%, which represents the rest of 100 the protein contributed by the CF-pulp (2.2 wt.%). Moreover, some hydrophobic 101 compounds such as astaxanthin and E vitamin are present in this phase. The CF2P 102 was the selected phase because it is the water soluble protein fraction and shows 103 the highest protein content. In addition, both myofibrillar proteins and sarcoplasmic 104 proteins are present in this phase. Finally, the intermediate phase (CF2P) was 105 freeze-dried in order to obtain a flour-fraction rich in proteins, the protein powder 106 was named CF2L. 107 The protein content of the CF2L was determined in quadruplicate as % N x 6.25 108 using a LECO CHNS-932 nitrogen micro analyzer (Leco Corporation, St. Joseph, MI, 109 USA). In the same way, lipid, moisture and ash contents were determined according 110 to A.O.A.C. (1945). 111 DPPH, ABTS, Folin-ciocalteu, HCl, NaOH reagents were of analytical grade, 112 purchased from Sigma–Aldrich Chemical Company (St. Louis, USA). Milli-Q 113 ultrapure water was used for the preparation of all solutions. 114 2.2 Protein powder characterisation 115 116 2.2.1 Amino acid characterization 117 CF2L samples were dissolved in 6.0 M hydrochloric acid and were incubated in 118 an oven at 110°C for 24 h. After hydrolysis, pH was adjusted to 7 using 6M NaOH 119 and samples were filtered through a Whatman glass microfibre filter (GF/C). Finally, 120 samples were diluted (1:500) by adding doubly distilled water. 121 Reverse phase HPLC by precolumn fluorescence derivatization with o-122 phtaldialdehyde (SIL-9A Auto Injector, LC-9A Liquid Chromatograph, RF-530 123 Fluorescence HPLC Monitor, all parts from Shimadzu Corporation, Japan) was 124 performed using a NovaPak C18 cartridge (Waters, Milford, MA, USA). 125 6 Glycine/arginine and methionine/tryptophan were determined together, as their 126 peaks merged. By this procedure, it is only possible to detect: Alanine (Ala), Aspartic 127 acid (Asp), Glutamic acid (Glu), Histidine (His), Serine (Ser), Glycine (Gly), 128 Arginine (Arg), Threonine (Thr), Tyrosine (Tyr), Methionine (Met), Valine (Val), 129 Phenylalanine (Phe), Isoleucine (Ile), Leucine (Leu) and Lysine (Lys). The other 130 amino acids were not included in the results of this study because they are 131 completely destroyed by acid hydrolysis or cannot be directly determined from acid 132 hydrolyzed samples. 133 2.2.2 Free and total sulfhydryls. 134 Free and total sulfhydryl groups of CF2L samples were determined using the 135 method developed by Beveridge et al. (1974). Samples were suspended (10 mg/mL) 136 in 0.086 mol/L Tris-HCl – 0.09 mol/L glycine – 4 mmol/L EDTA – 8 mol/L urea – pH 137 8.0 buffer. Dispersions were stirred at 25ºC for 10 min at 500 rpm in a thermomixer 138 and centrifuged at 15,000 xg (10 min, 10ºC). The supernatant was incubated with 139 Ellman’s reagent (DTNB) (4mg DTNB/mL methanol) and 1 mL of 2-nitro-5-140 thiosulfobenzoate (NTSB) was used in the case of the total sulfhydryls. Absorbance 141 at 412 nm was measured in a Genesis-20 spectrophotometer (Thermo Scientific, 142 USA). The molar extinction coefficient of NTB (13,600 L·mol-1·cm-1) was used. 143 Protein concentration of extracts was determined by the modified Lowry method 144 (Markwell et al. 1978). 145 2.2.3 Protein solubility 146 Protein solubility was determined as a function of pH in the range 2 to 10. 147 Aqueous dispersions at 1mg/mL were prepared and pH of different aliquots was 148 adjusted with 6 N NaOH and 6 N HCl at alkaline and acid pH values, respectively. 149 Samples were homogenized and centrifuged for 15 min at 15,000 × g. The total 150 7 protein content (protein + peptides) was determined in quadruplicate by a modified 151 Lowry method (Markwell et al. 1978). Percentages of soluble protein (calculated as 152 protein content of supernatant x 100/weight of CF2L) were plotted. 153 2.2.4 SDS-PAGE Electrophoresis 154 CF2L composition was analyzed by sodium dodecyl sulfate-polyacrylamide gel 155 electrophoresis (SDS-PAGE). Continuous and stacking gels of 10 and 3.5% of 156 acrylamide, respectively, were prepared. A buffer containing 2 M Tris-base, 157 containing 0.15% SDS pH 8.8 was used for the separating gel. A running buffer 158 consisted of 0.027 M Tris-base, 0.38 M glycine pH 8.3 with the addition of 0.15% 159 SDS, was utilized. Coomassie Brilliant Blue was used as colorant agent, and β-160 MercaptoEthanol was used in the sample buffer. Precision Plus Protein standards 161 (Bio-Rad-Calibration kit, Richmond, CA, USA) containing ten protein bands were 162 used: 10 kDa, 15 kDa, 20 kDa, 25 kDa, 37 kDa, 50 kDa, 75 kDa,100 kDa,150 kDa, 163 and250 kDa. 164 2.3. Gelation process 165 Aqueous dispersion of 12 wt. % protein concentration were subjected to thermal 166 gelation in three different stages: (i) The first step was carried out at a constant 167 heating rate (5 °C/min) from 20 °C to 90 °C; (ii) After the first step, an isothermal 168 step was performed at 90 °C for 30 min; (iii) Subsequently, a cooling stage was 169 carried out at a constant cooling rate (5 °C/min) from 90 °C to 20 °C. Gels were 170 performed at three different pH values: 2.0, 6.5 and 8.0. 171 2.4 Gel characterisation 172 2.4.1Viscoelastic measurements of gels 173 Small Amplitude Oscillatory Shear (SAOS) measurements were performed in a 174 controlled-stress rheometer (Kinexus Ultra +) from Malvern Instruments (Malvern, 175 8 Worcestershire, United Kingdom). In a preliminary experiment, stress sweep tests 176 were performed in order to establish the linear viscoelasticity range. The gelation 177 process was simulated through in situ heating in the rheometer. All stages were 178 performed at constant frequency and gap (6.28 rad/s-1). Finally, frequency sweep 179 tests (0.06 - 64 rad/s-1) at 20ºC were carried out in order to obtain the mechanical 180 spectra of the final gels. All rheological measurements were carried out at constant 181 gap (1mm). 182 2.4.2 Protein interactions 183 Solubility of CF2L gels in a number of selected solutions was used to determine 184 ionic bond, hydrogen bond, hydrophobic interaction and disulfide bond according 185 to the method of Gomez-Guillen et al. (1997). The selected solutions were: 0.05 186 mol/L NaCl (SA), 0.6 mol/L NaCl (SB), 0.6 mol/L NaCl + 1.5 mol/L urea (SC), 0.6 187 mol/L NaCl + 8 mol/L urea (SD) and 0.6 mol/L NaCl + 8 mol/L urea + 0.5 mol/L β-188 mercaptoethanol (SE) solutions. Quantification of ionic bonds was obtained from 189 the difference between protein solubilized in SB and protein solubilized in SA; 190 hydrogen bonds were quantified by the difference between protein solubilized in SC 191 and protein solubilized in SB; hydrophobic interactions were obtained from the 192 difference between protein solubilized in SD and protein solubilized in SC and, 193 finally, disulfide bonds were expressed as the difference between protein 194 solubilized in SE and protein solubilized in SD. The protein concentration was 195 determined with a modified Lowry method (Markwell et al. 1978). 196 2.4.3 Water-holding capacity of gels 197 Each gel (0.3–1.3 g) was equilibrated at room temperature and placed on a nylon 198 membrane (5.0 mm pores, Micronsep, New York, N.Y., U.S.A.) maintained in the 199 middle position of a centrifuge tube. Water loss was determined by weighing before 200 9 and after centrifugation at 120 × g for 5 min at 5°C. Water-holding capacity (WHC) 201 was expressed as the percentage of the initial water remaining in the gel after 202 centrifugation. 203 2.5 Bio-active properties 204 2.5.1 DPPH Assay 205 The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was performed as described 206 by Brand-Williams et al. (1995) with some modifications. Briefly, the day before 207 analysis, 0.1 mM methanolic DPPH* working solution was prepared and kept on a 208 magnetic stirrer overnight at 4oC. A series of 0-750 μM methanolic working 209 solutions of Propyl Gallate and gel solutions at 10 wt. % in methanol were prepared. 210 An aliquot of DPPH* solution (2.9 mL) was mixed well with 0.1 mL of a sample or 211 methanol (blank). After 20 min of incubation at room temperature, the absorbance 212 at 515 nm was recorded. Results were expressed in propyl gallate (PG) equivalents. 213 2.5.2 ABTS Assay. 214 The 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt 215 (ABTS) assay was performed as described by Nenadis et al. (2004) with a few 216 modifications: ethanol was replaced with methanol and the amount of sample added 217 to the ABTS∙+ solution was 200 μL. For the analysis, a series of 0-55 μM working 218 solutions of Propyl Gallate and gels at 1 wt. % were prepared from stock gels. To 219 compare the antioxidant activities, the absolute values for each antioxidant and each 220 assay were recalculated into Propyl Gallate equivalents. 221 2.5.3 Folin-Ciocalteu (FC) Assay 222 The FC assay was performed as described by Singleton et al. (1999) with some 223 modifications. Briefly, a series of 0-4 mM working solutions of Propyl Gallate and 224 suitable gel solutions were prepared. Deionized water (10 mL), antioxidant solution 225 16 consequence of acid hydrolysis. Finally, although the gel at pH 8.0 also exhibits 373 strong gel behavior, tan δ at high frequency rise up to 0.36, exhibiting an 374 intermediate gel-like structure between those found at pH 6.5 and 2.0. 375 The gel structure has been evaluated by quantifying the interactions among 376 different protein chains present in gels (Figure 5A) and the water holding capacity 377 (WHC) for each gel for three different pH values (Figure 5B). 378 Interactions depend strongly on pH values (Fig. 5A). Ionic bonds, hydrogen 379 bonds, hydrophobic interactions and disulphide bonds are highly involved in the 380 formation of the gel network (Cofrades et al. 1996; Gomez-Guillen et al. 1997). Thus, 381 at acid pH, interactions are low and, as a result, the gel is weak. These results are 382 consistent with the above mentioned viscoelastic properties (low G’ and G’’ values). 383 At pH close to the IEP (pH 6.5), ionic interactions are the most important in 384 absolute terms, which suggests that weak interactive forces are active. This effect is 385 probably related to the absence of repulsive electrostatic interactions as a 386 consequence of the proximity to the IEP. However, it does not suffice to explain the 387 gel strength, taking place at this pH. The remarkable gel strength found in the 388 rheological study is probably attributed to disulphide bonds which are the main 389 responsible for the gel strength (Lanier et al. 2004). 390 Finally, at alkaline pH (pH 8), hydrophobic interactions are the most important 391 interactions, in spite of the large number of disulphide bonds, compared to pH 2.0. 392 These high values for hydrophobic interactions were usually attributed as the most 393 important bond in surimi gels (Gomez-Guillen et al. 1997). The heating process 394 might induce unfolding of native proteins and exposure of protected polar groups 395 and, as a result, hydrophobic protein-protein interaction (Lanier et al. 2004). 396 17 All these results indicate that disulphide bonds show the greatest effect on gel 397 strength. However, formation of disulphide bonds seems to be counterbalanced by 398 repulsive interactions taking place as a result of charged protein surfaces (far from 399 the IEP). Ionic interactions, being particularly relevant at the IEP, and low repulsive 400 electrostatic interactions, thus contribute to facilitate formation of S-S bonds. 401 Hydrophobic interactions, typically enhanced over thermal heating, as well as 402 hydrogen bonds, play a role at the cooling stage, both contributing to reinforce the 403 gel network structures. Thus, G’ and G’’ profiles undergo a noticeable increase upon 404 cooling at pH 8, where hydrophobic interactions are relevant. 405 In addition, the results from WHC tests are in agreement with those obtained 406 from linear viscoelasticity and interactions measurements, all of them reflecting 407 different aspects of gel strength. In this sense, the gel with the highest value of WHC 408 is the gel made at pH 6.5. This corresponds to the gel showing higher viscoelastic 409 moduli (higher G’ and G’’ and lower tan δ) and higher amount of disulphide bonds. 410 3.2.2. Antioxidant Activity 411 Figure 6 shows the antioxidant activity of the gels formed at different pH values 412 compared to the reference (propyl gallate, PG) measured according to three 413 different methods: DPPH, Folin–Ciocalteu and ABTS. For the DPPH method, all the 414 gels show a similar radical scavenging activity at around 310 mM of PG equivalents 415 and it is not pH-dependent. This antioxidant properties will be not influenced on the 416 pH of the gel performed, what is very interesting. Most of the studies on DPPH 417 scavenging activity have been focused on different vegetable oils, grapes and wines 418 (Arranz et al. 2008; Espinoza et al. 2009; Spatafora et al. 2013), even some 419 researchers have found this property for proteins such as egg yolk and fish proteins, 420 which can scavenge free radicals (Sakanaka et al. 2005; Elias et al. 2008; Kristinova 421 18 et al. 2009). Recently some stuydes have been based on fish proteins and 422 hydrolysates (Kristinova et al. 2009), however the pH influcence was not evaluate 423 with proteins. 424 Figure 6 shows that these gels have a high ABTS antioxidant activity. In contrast 425 to DPPH, the pH has a large effect, the activity varied between 500 and 5500 mM PG 426 equivalents. This strong dependence on pH cannot be justified on the basis of 427 protein interactions, since the trend followed by the ABTS assay is not in accordance 428 to the gel interactions and rheology measurements. Thus, whereas gels exhibit a 429 higher ABTS activity when an increase in pH values takes place, structural 430 characterization showed that stronger interactions (and as a consequence higher 431 gel strength) occurred at pH 6.5. This pH effect is also contradictory for other 432 authors, Floch et al. (2007) found the highest activity in soil at low pH values. 433 However, Lemanska et al. (2001), found the same pH effect for hydroxyflavones, this 434 means that the ABTS assay is strongly dependent on the nature of the compound 435 studied. 436 As may be observed in Fig 6 all gels show a FC antioxidant activity with a slight 437 increase if the pH value rises from 2.0 to 8.0. This antioxidant activity is quite 438 different from the ABTS radical cation assay, which also was sensitive to phenol 439 compounds. This difference may be explained because the FC reagent is not capable 440 to measure lipophilic antioxidants due to the high affinity of the FC chromophore 441 towards water (Huang et al. 2005). 442 Besides the different behavior of protein gels, many studies have been reported 443 showing this effect that depends on the active agent and the type of assay (DDPH, 444 ABTS, FC) (Wootton-Beard et al. 2011). 445 4. CONCLUSIONS 446 19 The CF2L protein concentrate corresponds to a highly soluble fraction from 447 crayfish surpluses. CF2L exhibits a significant amount of the essential amino acids 448 with excellent nutritional importance that makes this product as a potential food 449 ingredient (i.e. gelation). 450 SAOS measurement shows a high dependence of the gel strength on pH. Thus, 451 near the IEP, the absence of net charges leads to a gel with strong viscoelastic 452 properties (showing the highest values for G’ and G’’), with higher amount of 453 disulphide bonds and enhanced WHC. However at pH 8.0 and 2.0, hydrophobic 454 interactions are more relevant, leading to a gel with lower viscoelastic properties 455 and WHC. This fact is related to the presence of repulsive interactions among 456 charged protein surfaces, which inhibit the development of S-S bonds. Gels at pH 2.0 457 need special attention since a weak gel is obtained, probably due to the protein 458 hydrolysis with a large increase in the relative amount of the peptide fraction. 459 Finally, antioxidant activity was found using three different compounds. 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