Thermal gelation of myofibrillar proteins from aquatic organisms

ABSTRACT

Gelling ability is attributed to myosin, which is the main myofibrillar protein. Therefore, its integrity is very important. However, a gel with good textural characteristics and stability depends on the inherent characteristics of its proteins, as well as on external factors (primarily temperature, pH, protein concentration and added salt). The best gels from aquatic organism proteins are obtained at a pH value of approximately 7.0. However, the concentration of salt is often variable. In contrast, when proteins are recovered using acid/alkaline dissolution, gels with good textural characteristics are obtained without salt. Hydrophobic interactions, disulfide bonds, hydrogen bonds, and electrostatic interactions are the main interactions that stabilize the protein gel. Thus, this review focuses on the study of the main factors involved in protein gelation, as well as on the extraction method effect on the gelling capacity of proteins from aquatic organisms.

RÉSUMÉ

Miosina es la principal proteína miofibrilar a la cual se le atribuye la habilidad gelificante, por lo tanto su integridad es muy importante. Sin embargo, las propiedades texturales y estabilidad de los geles depende de las características inherentes de sus proteínas, así como de factores externos (principalmente temperatura, pH, concentración de proteína y sal añadida). En general, los mejores geles proteicos obtenidos de organismos acuáticos se obtienen a pH cercano a 7.0, mientras que la concentración de sal suele ser variable; sin embargo, cuando la recuperación de proteínas se realiza a través de disoluciones ácidas/alcalinas, es posible obtenerse geles con buenas características texturales en ausencia de sal. Las principales interacciones involucradas en la estabilización de geles proteicos son: interacciones hidrofóbicas, puentes disulfuro, puentes de indrógeno e interacciones electrostáticas. Por esta razón, la presente revisión estudia los principales factores involucrados en la gelificación proteica, así como el efecto del método de extracción, sobre la capacidad gelificante de proteínas de organismos acuáticos.

KEYWORDS:

• Protein gelation
• myofibrillar proteins

PALABRAS CLAVES:

• gelificación proteica
• proteínas miofibrilares

Introduction

A gel is a three-dimensional structure that engages and immobilizes the liquid phase inside itself (Pilosof, 2000). Gels are obtained when proteins are denatured. Subsequently, the functional groups interact to form a three-dimensional network. Denaturation can be caused using chemical (such as salts) and physical (such as heat and pressure) agents. Heat treatment is the most common method used to make such products (Hwang, Lai, & Hsu, 2007; Martínez et al., 2014).

Protein gelation is important for the food industry because the gelling property is used in food systems such as dairy (cheese), sausages, surimi, and vegetable proteins among others. Protein gelation is an important feature in food because it determines the organoleptic properties of food, especially its texture, which, in turn, determines whether a product is accepted by the consumer (Mulvihill & Kinsella, 1987). Gelation provides texture to the food, improves water absorption, has thickening effects, helps to stabilize the system, and determines the appearance of food (Damodaran, 1994; Kinsella, Damodaran, & German, 1985). The above-mentioned factors determine the acceptance of food by a consumer. Gelling is important for the surimi industry because surimi is the intermediate product for making many gelled products from aquatic organisms based on a concentrate of myofibrillar proteins (Guenneugues & Morrissey, 2005). Specifically, surimi manufacturing is an important industry for producing gelled products from aquatic species because surimi is an intermediate in their production. Alaska Pollock (Theragra chalcogramma) and Pacific whiting (Merluccius productos) are the main species used for this purpose. However, currently, surimi is made using a large number of species, especially the lean and white species (Suzuki, 1981) because of their superior gelling characteristics, such as increased gel strength and water-holding capacity (WHC).

Functional properties, such as gelation, are attributed to myofibrillar proteins, especially myosin (Kristinsson & Hultin, 2003). However, this functionality depends on inherent protein characteristics (amount of sulfhydryl groups, amino acid types, hydrophobicity, charge, and others), as well as external factors (gelling temperature, protein concentration, concentration of added salt, pH, and others) (Meng, Ching, & Ma, 2002; Molina-Ortiz & Wagner, 2002). Therefore, the present study aims to review different characteristics of protein gels, as well as the effects of temperature, pH, ionic strength, hydrophobic interactions, and sulfhydryl groups on the gelling property of proteins from aquatic organisms.

Protein gelation

Gelation is a phenomenon that occurs via crosslinking of polymers using chemical interactions. Therefore, the formed three-dimensional structures, which are able to trap water and low-molecular-weight substances, are called gels (Fligner & Mangino, 1991; Mulvihill & Kinsella, 1987; Pilosof, 2000).

The mechanism by which proteins form a gel is not well understood. However, two theories are proposed. First, the Flory–Stockmayer theory suggests that gelation is a sudden event that is achieved via some degree of crosslinking between polymers and reaches a critical value called the gel point, in which the viscosity diverges to infinity. Second, the Percolation theory assumes that monomers form small aggregates and, at some point, reach the gel point (Pilosof, 2000).

Gels vary in their microstructure and mechanical properties and are difficult to define. Nevertheless, gels are microstructurally classified into aggregate gels and gels with fine filament networks. The former are transparent due to the thinness of filaments and because of the orderly arrangement of proteins. This allows gels to have a good WHC and to be thermoreversible. Gelatin is an aggregate gel example. In addition, globular proteins exhibit a fine filament structure by forming filaments with small particles. Moreover, aggregate gels are formed when proteins are close to their isoelectric point, are more opaque, and have a lower WHC (Hermansson & Langton, 1994; Kim, Park, & Choi, 2003) due to protein aggregation, which favors a weaker protein–water interaction.

Protein gels can be improved with chemical agents, such as salt, organic acids, transglutaminase, and others, and with physical agents, such as high pressure and heat. The latter is the most common approach (Hwang et al., 2007; Martínez et al., 2014; Totosaus, Montejano, Salazar, & Guerrero-Legarreta, 2002). Therefore, the present review addresses the heat treatment gelling process.

Thermal gelation of the myofibrillar proteins of aquatic organisms

Muscle proteins are classified into three groups based on their solubility in saline solutions: stromal, sarcoplasmic, and myofibrillar. The latter is the most abundant (50% of muscle proteins) (Kijowski, 2001) and is primarily composed of myosin, actin, tropomyosin, and troponin (Hashimoto, Watabe, & Kono, 1979). Out of all myofibrillar proteins, myosin represents the highest percentage (55–60%), and it is mainly composed of two heavy and four light chains. Using scission, the light chain can be split into light meromyosin (LMM), heavy meromyosin (HMM), and subfragments 1 and 2 (S1 and S2) (Mathews, Van Holde, & Ahern, 2002).

Myosin is the myofibrillar protein to which the functional properties, such as gelling, are attributed because it contributes to water retention capacity, gel hardness, cohesiveness, and elasticity, among other properties (Kristinsson & Hultin, 2003). Thus, it is important to maintain the integrity of myosin because the molecular size decreases its gelling ability. Consequently, the technologies used to obtain protein concentrates from aquatic organisms are focused on concentrating myofibrillar proteins and removing sarcoplasmic proteins. The sarcoplasmic fraction contains enzymes, such as proteases, which act on myosin, producing HMM and LMM, while other enzymes cleave myosin subfragments S1 and S2. It is believed that this degradation leads to deterioration of the thermal gel (Konno, Young-Je, Yoshioka, Shinho, & Seki, 2003; Nagashima, Ebina, Nagai, Tanaka, & Taguchi, 1992). Furthermore, other myofibrillar proteins, such as actin, regulatory and cytoskeletal proteins, do not form gels. Nonetheless, they affect viscoelastic properties of myosin gels (Ramírez, Martian-Polo, & Bandman, 2000).

Thermal gelation of fish muscle proteins occurs in three stages: suwari setting (40°C), modori (60°C), and gelling (80–90°C) (Gill, Conway, & Evrovski, 1992). During the setting stage, hydrophobic protein–protein interactions occur primarily via unfolding of the myosin heavy chain α-helix (MHC) (Pérez-Mateos, Amato, & Lanier, 2004). Softening occurs by incubating the protein paste with salt (sol) at approximately 60°C. This results in a gel that is brittle, nonelastic, and irreversible. The modori phenomenon is attributed to three causes: coagulation of myofibrillar proteins during heating, myosin degradation via heat-activated proteolytic enzymes, and the participation of nonenzymatic globular proteins, such as transport proteins and some structural proteins (Niwa, 1992). Finally, gelation occurs in two successive steps. First, denaturation occurs, which results in reactive groups being exposed. This allows them to interact and leads to crosslinking or protein aggregation, thus, forming a three-dimensional network that absorbs water (Mulvihill & Kinsella, 1987). Benjakul, Visessanguan, and Chantarasuwan (2004) evaluated different setting times at 40°C in tropical fish species and observed that the increasing setting time resulted in a higher breaking strength of gels. However, the best setting time differed between species because the maximum gel strength in 1 h was obtained for Nemipterus bleekeri, whereas in 2 h it was obtained for Priacanthus tayenus. For barracuda Sphyraena jello, the best setting time was one and a half hours, and for Pennahia macrophthalmus it was 3 h. This breaking force increase was attributed to the formation of covalent bonds. Thus, allowing proteins to remain for an appropriate time at 40°C was a good alternative for improving the gelling property of tropical species.

Temperature is the most important factor that influences gelling properties of myofibrillar proteins because heat is required to denature and unfold the proteins. Additionally, the heating rate plays an important role in gel properties because, if protein aggregation occurs slower than denaturation, more elastic gels are obtained. This suggests that slow heating favors more protein–protein interactions, which results in stronger gels (Camou, Sebranek, & Olson, 1989; Dong & Holley, 2011; Hermansson, 1979).

Differential scanning calorimetry (DSC) studies are useful for evaluating the folding–unfolding transitions of proteins. You, Pan, Shen, and Luo (2012) evaluated the heat treatment effect on the physicochemical properties of actomyosin from Carp (Aristichthys mobilis). The researchers reported two endothermic transitions at 41 and 63.4°C. The first transition is related to myosin denaturation, and the second is related to actin denaturation. Liu, Gao, Ren, and Zhao (2014) reported optimal unfolding of proteins at 40°C in gels made from silver carp surimi. Likewise, other studies performed with other fish and cephalopods reported similar denaturation temperatures at approximately 40 and 60°C, which were attributed to myosin and actin denaturation, respectively, and which could be related to the setting and modori phenomena. Interestingly, myofibrillar proteins from Atlantic salmon are more thermostable. Thus, their denaturation and aggregation occur at higher temperatures, which could be the reason why salmon proteins have a low gelling capacity (Lefevre, Fauconneau, Thompson, & Gill, 2007).

Dynamic oscillatory measurements show a decrease in storage modulus (G´) at approximately 40°C. This behavior has been attributed to the actomyosin complex dissociation and LMM denaturation and is directly related to the thermal properties obtained in the DSC analysis. Yin and Park (2014) reported the same behavior for the Alaska pollock (T. chalcogramma). After G´ has decreased, an exponential increase up to 80 and 90°C is observed. This increase is attributed to the formation of chemical interactions to form gel. Studies in other aquatic organisms indicate the same behavior in the storage modulus (Gómez-Guillén, Martínez-Alvarez, & Montero, 2003; Tolano-Villaverde et al., 2013).

As shown above, proteins from aquatic organisms undergo changes in their structure during thermal gelation, which is similar to proteins from terrestrial organisms. Nevertheless, terrestrial proteins are more stable. Therefore, the surimi industry incorporates cryoprotectants to reduce protein denaturation. These additives increase the surface tension of water and its binding energy, which prevents the removal of water from surface proteins, thus, stabilizing them during freezing (Park, 1994; Thawornchinsombut & Park, 2006; Yoon & Lee, 1990). However, temperature is not the only factor that effects the formation of protein gels. This is discussed in the next section.

The effect of pH and ionic strength on the gelling property of myofibrillar proteins

The gelation of proteins consists of the solid-state protein transformation into the gel state. The forces involved in network formation between polypeptide chains are fundamentally attractive (hydrophobic, electrostatic, hydrogen bonding and/or disulfide bonds) and repulsive (electrostatic and water–protein interactions) (Fennema, 2000). The pH and the type and concentration of salt used affect interactions between proteins because they change their tertiary structures and charge distributions, which alters the nature and structure of the gel (Dihort-Garcia et al., 2015).

Effect of pH on gelation

Proteins are macromolecules with a large number of ionizable groups. Proteins have a net charge that depends on pH. Acidic pH results in a positive charge, and alkaline pH results in a negative charge. In both cases, there is a strong charge repulsion. However, at their isoelectric point (pI), lower repulsion exists, due to the balance between charges. Electrostatic interactions are the major interactions that stabilize and form a gel, and pH is significant for the formation of this type of colloid. It has been reported that protein gels from chicken meat show good textural characteristics when gels are produced at a pH value of 6.0. However, protein gels from aquatic organisms are often superior when they are produced at a pH value of 7.0 (Lesiów & Xiong, 2003; Xiong & Brekke, 1991). This could be because the isoelectric point of myofibrillar protein is approximately 5.0–5.5, and at a pH similar to pI, proteins are attracted, and the protein network is more compact and produces an elastic gel. In contrast, when the pH is not similar to pI, a weak gel is formed because a large net charge causes the molecules to repel, and the formed gel is disordered (Hermansson & Langton, 1994). Dong et al. (2014) has reported that protein gels obtained using actomyosin from Patinopecten yessoensis exhibited the best results at a pH value of 7.0 because they showed a higher WHC and were stronger than gels produced at other pH values (6.0 and 8.0). Therefore, most studies on protein gelling from fish proteins have been performed at approximately neutral pH values (Sun & Holley, 2011).

However, it is possible to obtain gels with lower or higher pH values. Specifically, Venugopal et al. (2002) have produced protein gels from shark meat at a pH value of 3.5. However, protein gelation depended on the protein concentration and temperature. Riebroy, Benjakul, Visessanguan, Eriksonc, and Rustadd (2009) have reported that actomyosin acidification from Gadus morhua and Lota lota using d-gluconic acid-δ-lactone after 48 h acquired a pH value of 4.6. This led to aggregation because the pH value of 4.6 was similar to the pI value of those proteins. The latter causes an increase in particle size, turbidity, hydrophobicity and G′. These gels are formed using acid induction at room temperature and are stabilized via electrostatic and hydrophobic interactions as well as disulfide bonds. In another study, performed by Brenner, Johannsson, and Nicolai (2009), the same behavior of protein aggregation from G. morhua has been reported. When proteins are solubilized at a pH value of 11 and, then, the pH is adjusted to lower than 8.0, this offers an alternative for protein gelation without using high temperatures.

Effect of pH extraction

The manner in which myofibrillar protein concentrate is obtained from aquatic organisms is significant. A traditional method based on washing cycles with water has drawbacks. Thus, Kelleher and Hultin developed and patented a method for producing protein concentrates based on the acid or alkaline solubilization and subsequent isoelectric precipitation, followed by neutralization (Hultin & Kelleher, 1999). This has led many studies to use this methodology. However, the pH shifts can cause protein changes in structure and functionality.

Goto, Takahashi, and Fink (1990) have reported that the induced unfolding using acidic solution depends on the pH of the medium, while refolding depends on the type of ions in the acidic medium. Liu et al. (2014) have reported that the gelation rate of myosin silver carp (Hypophthalmichthys molitrix) decreases with an increase in pH. In other studies, Jafarpour, Shabnpour, Filabadi, and Shabani (2014), when applying an acidic solution to silver carp proteins, have observed that MHC was degraded, while the alkaline solution caused minimal changes in MHC. Furthermore, Yongsawatdigul and Park (2004) reported the same behavior when they applied an acidic treatment to myosin rockfish (Sebastes flavidus). This could be attributed to cathepsin L activity, which produced low-quality gels. The same behavior has been reported for Euphausia superba (Chen & Jaczynski, 2007). Alkaline and acidic solutions cause denaturation. Furthermore, under alkaline conditions, the formation of disulfide bonds was promoted (Yongsawatdigul & Park, 2004).

Most studies that used acidic or alkaline solubilization reported gels with better textural characteristics compared with gels produced using alkaline treatment (Cortés-Ruiz, Pacheco-Aguilar, Lugo-Sánchez, Carvallo-Ruiz, & García-Sánchez, 2008; Dihort-Garcia et al., 2011; Ingadottir & Kristinsson, 2010; Vareltzis & Undeland, 2012). This was rationalized as follows. At acidic pH values, proteolysis is promoted as cathepsin L, which acts on the actomyosin complex. Therefore, acid dissolution is an inconvenience for species with a high cathepsin L activity. However, alkaline pH values promote disulfide bond formation (Yongsawatdigul & Park, 2004).

Although the control of pH is important in the Hultin and Kelleher (1999) process, ionic strength also affects the solubility and functionality of the muscle protein. Thus, Lin and Park (1998) have found that myosin from salmon (Oncorhynchus tshawytscha) was slightly soluble at pH > 7 or < 4 in the absence of salt. The solubility was higher at the pH values of 2 and 8–10 due to the increased hydrophobic surface and sulfhydryl content, as well as the decrease of α-helix.

The effect of ionic strength on gelation

Ionic bonds attract positively and negatively charged areas on the protein surface. Ionic interactions are considered the most important forces in the formation of thick filaments, and the addition of salt interferes with electrostatic attraction and leads to the breakdown of thick filaments and a greater dispersion of myosin or actomyosin (Lanier, Carvajal, & Yongsawatdigul, 2005).

For good surimi gel, salt should be added to break ionic bonds and assist in protein dispersion, which is necessary for developing an elastic structure during heating. Salt ions (Na+, Cl–) selectively bind to exposed charged groups on the protein surface. Calcium ions have a divalent charge (Ca2+) and can form ionic bonds between two adjacent negatively charged areas in proteins. Hence, the addition of calcium ions may contribute to the ionic strength of intermolecular interactions (Lanier et al., 2005; Pilosof, 2000).

Arfat and Benjakul (2012) have reported a higher aggregation of actomyosin from Selaroides leptolepis when zinc sulfate (ZnSO₄) was added because zinc ion (Zn2+) is a divalent ion and forms electrostatic interactions between two negatively charged residues (Chawla, Venugopal, & Nair, 1996; Xiong, 1997). The formation of large aggregates frequently favors the preparation of elastic gels (Chan, Gill, & Paulson, 1992). Thus, ZnSO₄ is often added to improve the gelling capacity of surimi paste. Techaratanakrai, Okazaki, and Osako (2012) have reported a higher breaking strength in protein gels obtained from Toradores pacificus, when sodium gluconate, sodium citrate, sodium succinate, and sodium acetate were added. However, gels containing sodium acetate showed a lower breaking strength because this salt has no chelating ability to inhibit metalloproteinase (Kuwahara, Osako, Okamoto, & Konno, 2006).

Electrostatic interactions play an important role in gel formation where salt concentration has a significant influence because the higher the salt concentration is, the more pronounced the electrostatic interactions are. Nevertheless, the addition of salt is limited due to sensory issues (Ramírez, Del Angel, Uresti, Velásquez, & Vázquez, 2007). Sodium chloride (NaCl) is the most commonly used salt, and it is tolerated up to 2% or 3% (Xiong & Brekke, 1991). The effect of ionic strength on gel hardness is observed with an increase of G´ over a temperature sweep (Wu, Hamann, & Foegeding, 1991).

To improve the gelling properties of proteins from jumbo squid (Dosidicus gigas), Gómez-Guillén, Borderias, and Montero (1997) have studied the effect of salt on gelling capacity. They have found that salt content of 1.5% was better than 2.5%. However, Tahergorabi, Beamer, Matak, and Jaczynski (2012), while working with Alaska pollock surimi, found that 3% added salt was better than 1.5%, based on the shear force and rheology experiments (Tahergorabi & Jaczynski, 2012).

To decrease sodium content in gelling products, Tahergorabi et al. (2012) have studied replacement of NaCl with KCl in frozen Alaska pollock surimi. The research showed that NaCl could be replaced with KCl, but a slight decrease in hardness was detected when KCl was used. Covalent cations shifted the onset of myosin transition to higher temperature and resulted in larger myosin peaks. This suggests that in the presence of salt or salt substitute (KCl) myosin requires more heat (i.e., higher temperature) to initiate unfolding. Moreover, even though the same molar concentration of NaCl and KCl was used, thermal behavior differences (using DSC and rheology) have been observed (Tahergorabi & Jaczynski, 2012).

Protein isolates produced using the traditional surimi method have good textural characteristics when accompanied by a greater salt concentration. However, Kim and Park (2008) have reported that protein isolates from Alaska pollock (T. chalcogramma), obtained using acidic or alkaline solutions, showed the opposite effect because better textural characteristics were obtained when no salt was added. The researchers attributed this behavior to aggregate formation caused by the pH shift effect, which is not disturbed by the addition of 3% salt.

The hydrophobic effect on the gelation of myofibrillar proteins from aquatic organisms

Hydrophobic interactions play a fundamental role in determining the conformation of molecules in solution as well as the association between them. Hydrophobicity is one of the most important variables that affects the protein structure. In addition to electrical and steric parameters, hydrophobicity is the determining factor in functional properties of a specific protein (Alizadeh-Pasdar & Li-Chan, 2000). Hydrophobic regions regulate interactions between proteins based on the number and size of hydrophobic sites on the protein surface. This determines the solubility and susceptibility to aggregation under the physiological conditions of pH, temperature, and ionic strength (Cardamone & Puri, 1992).

Changes in surface hydrophobicity have been used to study the denaturation of fish proteins. In these studies, it has been found that if the protein was treated with acid a higher hydrophobicity was detected compared to an alkaline-treated protein (Kim et al., 2003). Liu et al. (2014) have studied the surimi silver carp and have reported that hydrophobic interactions contribute to gel stability because they significantly increase after denaturation occurs, which indicates that a large interaction between hydrophobic regions occurs after their unfolding. The same behavior has been reported by Yongsawatdigul and Park (2004) and You et al. (2012) for threadfin bream and carp, respectively. Moreover, carp proteins showed a decrease in solubility, which was attributed to the formation of agglomerates. In addition, the formation of agglomerates has already been reported for other aquatic species when a pH shift is used (Kim et al., 2003; Tolano-Villaverde et al., 2013).

The effect of sulfhydryl group on the gelation of myofibrillar protein from aquatic organisms

When heat treatment is applied to a protein, unfolding occurs, which facilitates the SH exposure. Thus, the available SH groups are freed for S-S formation. Sulfhydryl groups (SH) are the most reactive functional groups in proteins (Fennema, 2000; Kim et al., 2003). Some authors have reported that SH and SS play an important role in the formation of rigid structures, such as gels, because they are involved in myosin aggregation mechanisms. Myosin contains 42 SH groups, of which approximately 68% are in the head region and the remaining (32%) are present in the rod (Benjakul, Seymour, Morrissey, 1997; Xiong, 1997). Interestingly, some authors have mentioned that SS formation between polypeptide chains, which are involved in protein gelation, do not act as an initial stabilizer of the three-dimensional network, but only extend the polypeptide chain (Totosaus et al., 2002).

Tolano-Villaverde et al. (2013) have reported a very low number of sulfhydryl groups (2.9 mol/105 g) for giant squid (Dosidicus gigas) compared with other aquatic organisms that have good gelling properties, such as burbot (L. lota), Atlantic pollock (G. morhua), and barracuda (S. jello), which have 5.2, 8.2, and 19.6 mol/105 g of sulfhydryl groups, respectively (Ramachandran, Mohan, & Sankar, 2007; Riebroy et al., 2009). This indicates that the number of sulfhydryl groups can be the cause of low gelling ability of giant squid protein. Several studies have reported a decrease in reactive sulfhydryl groups after exposure to 45°C, which has been attributed to protein unfolding and initiation of the gel network formation, which the possible formation of disulfide bonds could be attributed to (Wen-Ching, Chi-Cheng, & Kuo-Chiang, 2007; Yongsawatdigul & Park, 2004; You et al., 2012). The decrease of SH is consistent with the exponential increase of G´ at 45–50°C.

Conclusions

The muscle from marine sources differs from that from mammalian sources. The marine source muscle has a low content of stromal proteins, and, depending on species, it has proteolytic activity. Moreover, myosin, the main protein to which gelling properties are attributed, appears to have important differences. Therefore, differences in gelation have been attributed to intermolecular interactions between proteins that form the network, such as a low sulfhydryl content. However, the formation and stability of gels, obtained from proteins from aquatic organisms, depend largely on the method used for protein recovery. Although protein gelation from aquatic species is primarily performed using the recovered protein obtained by washing muscle with water, it is important to notice that the acid/alkaline dissolution is widely used at the research level. This process modifies the protein structure and affects the gelation process. Sometimes, this modification is positive and sometimes it is negative. However, the goal of this research is to identify deeper specific differences in the gelation of mammalian proteins. Thus, it is necessary to determine the contribution of overall protein–protein and protein–water interactions to determine the forces that the chemical interactions have.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors thank the CONACyT from México for the scholarship provided to the first author and for the resources to fund this research.