Relationship between interfacial and foaming properties of a Porphyra dioica seaweed protein concentrate

1 Relationship between interfacial and foaming properties of 1 a Porphyra dioica seaweed protein concentrate 2 Manuel Felix1,*, Eva Puerta1, Carlos Bengoechea1, Cecilio Carrera-Sánchez2 3 1Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 41011 Sevilla, 4 Spain. 5 2Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41011 Sevilla, Spain. 6 ------------------- 7 *Manuel FELIX 8 Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 9 41011 Sevilla, Spain. 10 E-mail: [email protected] 11 Phone: +34 954557179; fax: +34 954556447. 2 Abstract 12 Porphyra dioica macroalgae can be regarded as a food-grade protein source that can be used 13 for the stability of dispersed food systems. This work is focused on the foaming properties of a 14 seaweed protein concentrate (SWE) from Porphyra dioica. The characterisation of air-water 15 (A/W) interfacial layers formed after the SWE adsorption was carried out by means of 16 interfacial tension, interfacial viscoelastic measurements and step deformation tests. These 17 results were related to those obtained for SWE stabilized foams. Interfacial shear measurements 18 indicate that not extensive protein-protein interactions at the A/W interface were developed (G’s 19 = 1.0 ± 0.1·10-2 mPa·m), whereas the dilatational response (E’s = 23.5 ± 0.5 mN/m) match with 20 the formation of a rigid interface as a result of a densely packed structure. Even if the foaming 21 properties obtained for SWE (overall foam capacity, OFC, = 0.74 ± 0.01 mL/s) are similar to 22 those reported for other commercial protein systems, its lower stability (t1/2 = 204 ± 4 s) would 23 indicate the need of adding stabilizers for an adequate shelf life of the final food product. The 24 suitability of combining dilatational and interfacial shear rheology to predict the dynamics of 25 dispersed systems was proven to be relevant to acquire insights of complex interfaces. 26 27 28 29 Keywords: Interfacial shear rheology; Protein adsorption; Foam capacity; Surface properties. 30 3 1. Introduction 31 Edible seaweeds or macroalgae have been traditionally consumed in the Asian diet, being more 32 restricted in western countries, where they are mainly used as a source of techno-functional 33 polysaccharides (Venugopal, 2008). Macroalgae species, such as Nori or Zicai (Porphyra, a red 34 alga), Kombu, Kunbu or Haidai (Laminaria or Saccharina, a brown alga) and Wakame 35 (Undaria, also a brown alga), are mostly grown by aquaculture (96 % of the global seaweed 36 production per year) in Japan, China and Korea, and in the Indian Ocean (Cherry et al., 2019). 37 Different studies have pointed out the relevant protein content of macroalgae, and particularly 38 red algae (Harnedy and Fitzgerald, 2011), being sometimes higher than that of conventional 39 protein sources, like soybean, cereals, eggs or fish (Jelen, 2006; Kaliaperumal, 2003). Porphyra 40 dioica is a commercial seaweed globally consumed as nori sheets used during the preparation 41 of "sushi" (da Costa et al., 2018). Moreover, benefits associated to its consumption (e.g. anti-42 obesity effect, antitumoral, source of bioactive compounds, antioxidant activity) have 43 facilitated their greater presence in diets (Soler-Vila et al., 2009). These health benefits of 44 seaweed have been supported by both in vitro and animal studies, although still some potential 45 risks should not be neglected such as the presence of metal concentrations which are generally 46 below toxic levels (Cherry et al., 2019). Porphyra C. agardh is a red algal genus, commonly 47 known as nori or laver, that contains a high protein content (⁓ 45 % in dry matter basis), being 48 also rich in iron, zinc, sodium, potassium and calcium (Dawczynski et al., 2007; Rao et al., 49 2007; Sánchez-Machado et al., 2004). Porphyra is one of the most valuable crops produced by 50 aquaculture globally, with a total annual production value of more than US$2 billion in Japan 51 alone, where around one-fifth of meals contain seaweed (Cherry et al., 2019) 52 Food foams are aerated products which generally possess a low caloric content and are usually 53 stabilised by high molecular weight surfactants, particles, or even animal and plant proteins 54 (Foegeding et al., 2006; Pizones Ruiz-Henestrosa et al., 2008). However, there are scarce 55 4 studies about the use of algae as foaming agents (Schwenzfeier et al., 2013), in spite of the 56 recent interest for using macro- and micro-algae as a possible protein source (Caporgno and 57 Mathys, 2018; Fradinho et al., 2020). In order to succeed in the foam stabilisation by proteins 58 present in algae, solubility should be maximized to provide adequate functional properties 59 (Doğan et al., 2020; Jambrak et al., 2008), as they often display low solubility when processed 60 in order to remove their characteristic green colour and bitter taste (Mouritsen et al., 2012). 61 Protein solubility is highly dependent on pH, which should be carefully selected when 62 processing food foams to be within the range permitted by both food regulations and consumer 63 acceptance (Doğan et al., 2020; Pelegrine and Gasparetto, 2005). 64 Being dispersed systems, foams are thermodynamically unstable but can reach kinetic stability 65 when the interfacial tension is conveniently reduced. Thus, the development of foams would 66 benefit from the understanding of the air-water interfacial properties after the adsorption of 67 surface active molecules (i.e., proteins). The morphology and mechanical properties of these 68 complex interfaces stabilizing foams play a key role in the formation and stabilization of these 69 systems (Carrera and Rodríguez Patino, 2005; Shrestha et al., 2006). In this sense, the response 70 detected when a mechanical load or deformation is applied to an interface has been reported to 71 be highly sensitive to the interactions among the different molecules adsorbed at that interfacial 72 layer (Wilde et al., 2004). Thus, interfacial dilatational and shear rheology have proven to be 73 complementary and essentials tools for understanding the thermodynamic phenomena that take 74 place in dispersed systems like foams (Dickinson, 1999; Morris et al., 2001; Sagis, 2013; 75 Verwijlen et al., 2013). 76 The main objective of this work was the assessment of interfacial properties of a seaweed 77 protein concentrate (SWE) obtained from Porphyra dioica and how they are related to its 78 foaming properties. For this purpose, surface properties were measured during protein 79 adsorption (10,800 s) and after protein adsorption by two complementary techniques: a droplet 80 5 tensiometer (Tracker) and an interfacial shear rheometer (DHR-3). Thus, time sweep tests were 81 performed during protein adsorption, while frequency step deformation tests after protein 82 adsorption. Finally, these results were related to those obtained during foam formation and 83 subsequent destabilization carried out by a FOAM SCAM device. 84 2. Material and methods 85 2.1. Materials 86 Suralgas (Cadiz, Spain) supplied Porphyra dioica. Fresh samples were rinsed with sea water 87 (removing sand and other solid components present in samples). Subsequently, sample water 88 was removed by freeze-drying and macroalgae was ground with a Cyclotec Mill (1 mm screen, 89 FOSS Tecator AB, Sweden). The freeze-dried and milled seaweed specimens (SW) were stored 90 in darkness at room temperature. 91 Chemical reagents (i.e., HCl, NaOH) were purchased from Sigma-Aldrich Inc. (St. Louis, 92 Missouri, USA). Milli-Q grade water was used for all measurements. 93 2.2. Methods 94 2.2.1. Protein extraction 95 Soluble protein from SW system was extracted according to the method followed by Stack et 96 al. (2017). Briefly, SW system was resuspended in water (1:20), being the dispersion 97 magnetically stirred at 500 rpm over night at 4 ºC. Non-soluble components (IF-1) were 98 separated from soluble fraction (SF) by centrifugation at 4000 x g over 15 min in a Selecta BL-99 S centrifuge (Selecta, Barcelona, Spain). IF-1 fraction was resuspended in 0.12 M NaOH and 100 again magnetically stirred at 500 rpm for 1 h at room temperature. Alkali soluble (AS) and non-101 soluble components (IF-2) were separated by centrifugation at 4000 x g over 15 min in a Selecta 102 BL-S centrifuge (Selecta, Barcelona, Spain). Eventually, IF-2 was also resuspended in water 103 (1:20) and pH was adjusted under continuous magnetic stirring (500 rpm) at 4.0 by the addition 104 of HCl (isoelectric point of SW), obtaining an insoluble protein fraction (IPF) and a soluble 105 6 fraction (SPF). Both fractions were separated by centrifugation in a Selecta BL-S centrifuge 106 (Selecta, Barcelona, Spain). SF, AS and IPF fractions were mixed and freeze-dried, obtaining 107 the seaweed extract (SWE) protein system. 108 2.2.2. Composition of SWE 109 The Dumas method was followed to determined SWE protein content as % 𝑁𝑁× 6.25 in 110 quadruplicate by using the CHNS-932 elemental analyser (Leco Corporation, St. Joseph, 111 Michigan, USA). Moreover, A.O.A.C. approved methods (A.O.A.C., 2000) were used to 112 determine lipid, moisture and ash contents. 113 2.2.3. Protein solubility and Zeta-potential 114 The protein solubility of the SWE system was determined following the Lowry et al., (1951) 115 method for protein solubility, with some minor modifications (Markwell et al., 1978). Briefly, 116 protein solutions (1 mg/mL) were prepared as a function of pH (between 2.0 and 10.0). Samples 117 were centrifuged for 15 min at 15,000 × g using a BL-S centrifuge (Selecta, Barcelona, Spain). 118 SWE supernatants (0.1 mL at 1 mg/mL) were mixed with 3 mL of a solution containing sodium 119 carbonate (2%), sodium dodecyl sulphate (1%), potassium sodium tartrate (1.6%) and Copper 120 (II) sulphate (0.04 %). Subsequently, Folin-ciocalteu reagent (1N) was added and samples were 121 incubated for 40 min. Absorbance was measured at 660 nm, bovine serum albumin (BSA) 122 protein was used as standard. 123 The same aqueous protein solutions were used to determine Zeta-potential of proteins using a 124 Zetasizer Nano Z from Malvern Instruments (Malvern, UK). The Smoluchowski equation (1.5 125 for Fka value) was used for these measurements. 126 2.2.4. Surface characterisation 127 Determination of saturation concentration 128 Air-water (A/W) surface tension values were obtained as a function of SWE concentration 129 (0.00025, 0.0025, 0.05, 0.5, 1.0, 2.0 and 4.0 wt.%) using a pendant droplet tensiometer 130 7 (TRACKER, IT Concept, France). The pH value was fixed at 7.0 by using Trizma-base buffer 131 (50 mM). Measurements were performed at 20 ºC. 132 Dilatational measurements 133 Transient A/W surface tension and dilatational moduli (E’s and E’’s) were determined for 1 134 wt.% SWE protein adsorption during 10,800 s, using the TRACKER pendant droplet 135 tensiometer (IT Concept, France). Dynamic amplitude (10% strain amplitude) tests at 0.1 Hz 136 were carried out to obtain the E’s and E’’s viscoelastic moduli. After 10,800 s protein adsorption, 137 E’s and E’’s moduli were obtained as a function of frequency (from 0.0075 to 0.1 Hz) at 10% 138 strain amplitude. A 8 μL droplet was formed into an optical glass cuvette (8 ml). Experimental 139 setup was thermostated at 20.0 ± 0.1 °C. 140 Dilatational step/deformation tests were also performed after protein adsorption. A droplet 141 expansion of 10 or 20 % was imposed, monitoring the changes in interfacial tension over 1,500 142 s, obtaining a constant tension value (± 0.2 mN/m in 5 min). Subsequently, the area was 143 compressed 10 or 20 % (compression step). The evolution of the surface tension was again 144 monitored over 1,500 s. Results were fitted to the following exponential equation which 145 combines a Kohlrausch-Williams-Watts stretched exponential term with a regular exponential 146 term (Yang et al., 2020): 147 𝛾𝛾(𝑡𝑡)=𝑎𝑎𝑎𝑎−(𝑡𝑡/𝜏𝜏1)𝛽𝛽+𝑏𝑏𝑎𝑎−𝑡𝑡/𝜏𝜏2+𝑐𝑐 (𝐸𝐸𝐸𝐸. 1) 148 where γ is the interfacial tension over adsorption time (t), 𝜏𝜏1 is the initial relaxation time, β is 149 the stretch exponent and 𝜏𝜏2 is the characteristic time of protein rearrangements, whereas a, b, 150 and c are constants required for the fitting. 151 Interfacial shear measurements 152 Surface viscoelastic moduli (G’s and G’’s) were determined by oscillatory tests (0.1 Hz) during 153 1 wt.% SWE protein adsorption (10,800 s) using the DHR-3 rheometer (TA instruments, 154 8 Illinois, USA). After 10,800 s, dilatational viscoelastic moduli were obtained as a function of 155 frequency (from 0.0075 to 0.1 Hz). All the experiments were carried out within the linear 156 viscoelastic range (LVR), previously determined by amplitude tests. 157 Moreover, creep tests were also performed at constant stress ranging from 6.0·10-6 to 1.4·10-5 158 Pa to evaluate the A/W interfacial behaviour. Creep response was fitted to Burgers model using 159 one single element for the Kelvin-Voigt contribution: 160 𝐽𝐽𝑖𝑖(𝑡𝑡)=𝐽𝐽i,0+𝑡𝑡 𝜂𝜂i,0−𝐽𝐽𝑖𝑖·𝑎𝑎−𝑡𝑡 τ ⁄ (Eq. 2) 161 where 𝐽𝐽i,0 is the instantaneous interfacial creep compliance, 𝐽𝐽𝑖𝑖 the creep compliance that 162 corresponds to a retardation time τ and 𝜂𝜂i,0 is the steady state interfacial viscosity. 163 2.3. Foaming properties 164 Foaming ability and foam stability were assessed using a Foamscan device (Teclis, It Concept, 165 Longessaigne, France). N2 gas (45 mL·min-1) was blowing through a porous glass filter (0.2 166 μm) below the SWE solution (1 wt.%). Once the foam reached a volume of 120 mL and 167 bubbling stopped, foam stability was measured. Conductivity measurements were used to 168 determine foam height. Several parameters were calculated in order to evaluate the foaming 169 capacity and stability: 170 Overall foaming capacity (OFC) is defined as the slope of the foam volume formed until the 171 end of the bubbling process. The foaming capacity (FC) was calculated as follows: 172 𝐹𝐹𝐹𝐹 =𝑉𝑉𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓(𝑓𝑓) 𝑉𝑉𝑔𝑔𝑓𝑓𝑔𝑔 (𝑓𝑓)𝑥𝑥100 (Eq. 3) 173 where 𝑉𝑉𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓(𝑓𝑓) and 𝑉𝑉𝑔𝑔𝑓𝑓𝑔𝑔(𝑓𝑓) were the final foam volume and the final gas volume injected, 174 respectively 175 The relative foam conductivity (𝐹𝐹𝐹𝐹, %) was calculated by Eq. (4): 176 9 𝐹𝐹𝐹𝐹 =𝐹𝐹𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝐹𝐹𝑙𝑙𝑖𝑖𝑙𝑙 𝑥𝑥100 (Eq. 4) 177 where 𝐹𝐹𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 and 𝐹𝐹𝑙𝑙𝑖𝑖𝑙𝑙 were the final foam and liquid conductivity, respectively. 178 The foam maximum density (MD) was determined as Eq. (5) indicates: 179 𝑀𝑀𝑀𝑀 =𝑉𝑉𝑙𝑙𝑖𝑖𝑙𝑙 (𝑖𝑖)−𝑉𝑉𝑙𝑙𝑖𝑖𝑙𝑙 (𝑓𝑓) 𝑉𝑉𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 (𝑓𝑓)𝑥𝑥100 (Eq. 5) 180 Where 𝑉𝑉𝑙𝑙𝑖𝑖𝑙𝑙 (𝑖𝑖) corresponds to the initial liquid volume, 𝑉𝑉𝑙𝑙𝑖𝑖𝑙𝑙 (𝑖𝑖) corresponds to the final liquid 181 volume and 𝑉𝑉𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 (𝑓𝑓) corresponds to the final volume of the foam. 182 The foam half-life (t1/2) indicates the time needed to drain half of the volume of liquid from the 183 foam to the liquid phase (Patino et al., 1997). 184 2.4. Statistical analysis 185 At least three replicates of each measurement were carried out. Standard deviation was used to 186 determined uncertainty. One-way ANOVA tests was used to determine significant differences 187 (p < 0.05). 188 3. Result and discussion 189 3.1. Proximate composition 190 Results from protein content indicates that this protein system has 42.9 ± 1.1 % proteins, which 191 would confirm the success of the method followed in section 2.2.1. to obtain the seaweed 192 protein extract (SWE), obtaining a protein system which may be used for the stability of 193 dispersed systems. Porphyra dioica protein content has been reported to depend on the 194 harvesting season, being in any case within 9.40 to 21.52 % (Smith et al., 2010; Stack et al., 195 2017). The comparison between the results obtained in this research and previous publications 196 would imply that the protein concentration has increased in a factor of around 4.6 to 2.0 after 197 protein extraction. As for the other components of the protein system, moisture content for SWE 198 was 0.9 ± 0.1 %, lipids reached 2.9 ± 0.2 %, whereas ashes reached 17.2 ± 0.8 %. Eventually, 199 16 According to other authors, interfacial shear measurements can be related to the interactions 349 developed at the interface (Dickinson, 2001). Thus, these results would suggest that the 350 interactions developed at the A/W interface were weaker than in the systems containing the 351 above-mentioned proteins. 352 The mechanical spectra of the interfacial layer formed after SWE adsorption at A/W interface 353 is plotted in Figure 5B. This figure confirms the formation of an interfacial film, exhibiting a 354 weak gel-like response. Similar mechanical spectra and relatively high tan δ0.1 value (0.23 ± 355 0.03) have been previously obtained for a silkworm protein concentrate adsorbed at O/W 356 interface (Felix, Yang, et al., 2019). This suggests that, even if the viscoelastic properties were 357 not at the same level as other above-mentioned proteins (i.e., WPI), SWE system could be used 358 for the stabilization of dispersed systems. 359 Figure 6 shows the linear creep response obtained for the SWE interfacial layer formed at A/W 360 interface, as well as the line-fitting of experimental data to Burgers model (Eq. 4). The 361 interfacial creep response of SWE indicates that the interfacial film formed exhibited a weak 362 elastic response, since it was characterised by high values of compliance (⁓ 4·105 m/N). 363 Moreover, the fact that the experimental response can be reproduced by one single element of 364 the Kelvin-Voigt model is in agreement with a low level of protein-protein interactions. 365 Furthermore, Figure 6 also shows that the recovery response for the interfacial film formed after 366 the creep stage was 56 ± 4 %. This recovery was higher than the obtained after similar creep 367 tests for interfaces stabilized by crayfish protein concentrate (Felix, Romero, Vermant, & 368 Guerrero, 2016), which agree on the high-elastic character of the interfaces developed by SWE 369 system. 370 The steady state interfacial viscosity (𝜂𝜂i,0) obtained for SWE protein after adjusting the creep 371 response to Burger’s model was higher than that the response obtained for crayfish protein 372 concentrate at pH 8.0 (37.2 ± 0.2 vs. 24.2 Pa·m·s) (Felix et al., 2016). Moreover, the creep 373 17 compliance (𝐽𝐽𝑖𝑖) and the a retardation time τ were (3.84 ± 0.3)·105 1/Pa·m and 11.0 ± 0.1 s, 374 respectively. The higher viscosity together with the much higher compliance values obtained 375 for SWE than for crayfish indicate that a greater deformation was suffered by the interfacial 376 layer when applying the same stress level. This result confirms that the interfacial film formed 377 is acting like a viscous liquid, rather than a solid, when they are tested by interfacial shear 378 rheology. 379 Results from dilatational and interfacial shear measurements were complementary, in spite of 380 the different nature of the tests (transient and stationary, respectively) carried out. However, 381 whereas dilatational measurements indicate that the interface was rigid, where adsorbed protein 382 molecules were densely packed, the interfacial shear characterisation indicate that no extended 383 protein-protein interactions occur. Thus, the combination of dilatational and interfacial shear 384 measurements was essential for a complete understanding of complex fluid fluid-interfaces. 385 3.4 Foam characterisation 386 Figure 7 shows the liquid foam volume during foam formation and destabilization using 387 seaweed protein concentrate (SWE). This plot indicated that the foam half-life (t1/2) is 204 ± 4 388 s, which was closed to the t1/2 previously obtained for foams stabilized by sucrose/soy globulin 389 under the same conditions (Pizones Ruiz-Henestrosa et al., 2008), but was shorter than the 390 system stabilized by sunflower protein isolate (Patino et al., 2007) This parameter referred to 391 foam stability, however, foam capacity is also important in food products. Table 2 shows 392 parameters obtained during foams formation, which help to have an overview about the 393 suitability of SWE for the stabilization of A/W interfaces. The overall foam capacity (OFC) of 394 SWE system was 0.74 ± 0.01 mL/s, which was slightly lower than the OFC obtained for sucrose 395 (0.8 mL/s) and sunflower (0.8 mL/s) (Patino et al., 2007; Pizones Ruiz-Henestrosa et al., 2008). 396 Moreover, FC, CF and MD parameters obtained in SWE-stabilized foams were also slightly 397 smaller than the corresponding values obtained for sunflower and sucrose/soy globulin systems. 398 18 In this sense, the foamy properties of SWE may be correlated with the interfacial properties 399 previously obtained by means of dilatational and interfacial shear rheology. Dilatational 400 rheology indicated an interfacial film formed with a gel-like response, however interfacial shear 401 rheology suggested that the interactions developed at the interfacial film were weak. 402 Dilatational rheology was previously correlated with short-term stability, whereas interfacial 403 shear rheology was related with long-term stability (Benjamins and Lucassen-Reynders, 1998). 404 Thus, the better dilatational properties predicted that foams can be formed with SWE system, 405 however the poorer interfacial shear response suggested that the interfaces formed would be 406 less stable than the interfaces stabilized by the other protein systems referenced. 407 4 . Concluding remarks 408 Results obtained indicate that SWE system could be revalorised with potential applications in 409 the stabilization dispersed systems. The low protein solubility together net surface charge 410 forced to select pH 7.0 for further analysis. The adsorption of SWE system at A/W interface 411 led to the reduction of surface tension up to 38.5 ± 0.5 mN/m. Dilatational experiments 412 evidenced a rigid interface characterised by an elastic modulus of 23.5 ± 0.5 mN/m, which is 413 higher and exhibited longer relaxation times than similar interfaces stabilized by β-414 lactoglobulin, suggesting that the results obtained for SWE by dilatational measurements are 415 caused by a densely packed structure. On the other hand, interfacial shear rheology indicated 416 that there are not extended protein-protein interactions at the A/W interface, being these results 417 in agreement with interfacial shear creep tests where a lack of interactions is also inferred. The 418 foam capacity and stability estimated is consequence of the previous interfacial 419 characterisation. Therefore, the foam formed has similar levels for foam formation than other 420 foams stabilized by other protein systems, being related to dilatational rheology results. 421 However, the long-term stability of the foam is lower, being associated to the lack of protein-422 protein interactions deduced from interfacial shear rheology tests. 423 19 These results confirm the suitability of SWE protein to be used in dispersed systems, although 424 the use of a stabilizing system would be required for a commercial product requiring a greater 425 stability. The suitability of the combination of both dilatational and interfacial shear rheology 426 to predict the dynamics of dispersed systems is proven to be relevant to acquire a deep 427 knowledge of complex interfaces. 428 Acknowledgments 429 The authors also acknowledge University of Seville for the VPPI-US grant (Ref.-II.5) to 430 Manuel Felix. 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Dynamic heterogeneity in complex interfaces of 581 32 Figure 2 669 670 671 33 Figure 3 672 673 674 34 Figure 5 675 676 35 Figure 4 677 678 679 36 Figure 6 680 681 682 37 Figure 7 683 684