Thermal gelling properties and mechanism of porcine myofibrillar protein containing flaxseed gum at various pH values

ABSTRACT Flaxseed gum (FG) was reported to enhance the water holding capacity and thermal stability of myofibrillar protein (MP). However, the role of pH in the interaction of MP and FG is still unclear. The aim of this study was to evaluate gelling properties and physicochemical properties of MP-FG gel at various pH values, to explore the mechanism. The results reveal that higher pH value could increase the water holding capacity of MP-FG gels and the highest gel strength is achieved at pH 6.5. Raman spectroscopic analysis reveals that decreasing the pH from 7.5 to 5.5 induces the partial transformation of α-helices into β-sheets. At the same pH value, addition of FG also leads to the transformation of α-helices into β-sheets, affecting the formation of gel. The storage modulus (G’) values and gelation rates during gelation process gradually declines with the increasing pH, which might be conducted to form a fine dense network.


Introduction
Recently, hydrocolloids, such as κ-carrageenan, chitosan and locust bean gum, derived from a variety of plants and microorganisms, have been applied as one of the most effective fat substitutes for developing low-fat meat products and to impart desirable binding characteristics, textures and appearance in ground meat products ). An understanding of the gelation properties of porcine MPs containing hydrocolloids additives is beneficial for the development of comminuted meat products as well as maintaining quality in meat products.
Flaxseed gum (FG) is one of the hydrocolloids, which has been used extensively in the food industry. FG is a heteropolysaccharide containing anionic polysaccharide and neutral polysaccharide (Qian, Cui, Wu, & Goff, 2012). It has shown considerable potential in the meat product due to its good hydrophilicity and emulsibility. It was found in previous studies that, FG significantly enhanced the water holding capacity (WHC) of MPs (Sun, Li, Xu, & Zhou, 2011) and salt-soluble meat protein (SSMP) gels (Chen, Xu, & Wang, 2007), as well as increased the emulsification properties of soybean protein isolate (SPI) (Wang, Li, Wang, & Adhikari, 2011). Addition of FG increased thermal stability of SSMP, suggesting that an interaction between FG and SSMP could have occurred (Chen et al., 2007). The results obtained from the study of adding destabilizer to SSMP gels indicated that electrostatic interaction seemed to be the main force involved in the formation and stability of protein-polysaccharide gel. Sun and other researchers (2011) found that the improvement effect of FG on WHC of heatinduced MP gel was concentration dependent, which was achieved by the formation of a finer gel network, lower relaxation time, and stronger electrostatic attraction.
However, most of studies were conducted in simple characterization of the appearance of MPs system containing FG, and few researches focused on the mechanism under the phenomenon. In the comminuted meat systems, the heatinduced gelation of MP results in the formation of a 3dimensional network structure that holds water and gives rise to a unique texture of meat products. During thermal gelation, the interactive effects of MP, the predominant factor in controlling the extent of formation and behavior of the matrix, were supposed to depend upon pH (Hong, Min, & Chin, 2012;Jiang & Xiong, 2013). Therefore, it is suggested that the gelation properties of MPs are influenced by pH (Jang & Chin, 2011;Lesiów & Xiong, 2003).
At the isoelectric point (pI), proteins have a net charge of zero and retain the least amount of water. Most proteins aggregate and are least soluble at their pI, which results in poor gels or even prevents gel formation (Smith, 2001). Aggregation of proteins prior to heating inhibits gel formation of MPs, and its extent depends basically upon the electrostatic charge on the molecules (Boyer, Joandel, Roussilhes, Culioli, & Ouali, 1996), which in turn is affected by pH and ionic strength. The optimum pH for gelation of MPs is about 6.0, and might vary slightly with species (Sun & Holley, 2011). There is limited report available on the mechanism of interaction between meat proteins and flaxseed gum under the different pH treatments.
Therefore, the objective of the present study is to evaluate: (i) the effects of pH levels on the properties of heatinduced MP-FG gel; (ii) the changes responsible for these effects at molecular level and to propose a mechanism of thermal gelation for MP-FG as affected by pH.

Materials
FG (powder, purity 99.8%) was provided by Sinkiang Luqi Biotechnology Ltd. (Sinkiang Province, China). The viscosity of 10 g/L FG solution was 17,000 mPa.s at 25°C. The fresh pork longissimus dorsi was purchased from SuShi Meat Corporation through a local supermarket.

Extraction of MPs
MPs were extracted from pork longissimus dorsi at 4°C using a modified procedure reported by Doerscher, Briggs, and Lonergan (2004). At first, 200 g pork longissimus dorsi was ground by a Waring grinder (Modelnr 8010ES, Waring Commercial, New Hartford, Conn., U.S.A.) for 7 sec at 2,000 rpm twice. The paste was mixed with 800 mL cold extracting solution (100 mmol/L KCl, 20 mmol/L potassium phosphate, 2 mmol/L MgCl 2 , 1 mmol/L EGTA, pH 7.0) homogenized for 1 min at 10,000 rpm by a homogenizer (T25 digital Ultra-Turrax, IKA, Germany) and centrifuged at 1500 × g for 15 min. The procedure was repeated twice. After that, the sediment was treated with extracting solution and homogenized. Ten g/Kg Triton X-100 was then added and stirred for 10 min with an agitator. The extraction was centrifuged at 2000 × g for 10 min. Then, the sediment was treated with 100 mmol/L KCl solution and centrifuged at 2,000 × g for 10 min, which was repeated twice. The final step was to mix the sediment with 4 volumes of 100 mmol/L NaCl solution, which was centrifuged at 2,000 × g for 10 min. The resulting sediment was regarded as MPs. MPs purity was checked using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 1) and the protein concentration was measured using the Biuret method (Gornall, Bardawill, & David, 1949). MPs were stored at 4°C and tested within 3 days.

Preparation of MP-FG sols
FG was added to MP to make MP-FG sols with an isolation buffer as the protein extraction (MP concentration of 4%, and FG concentration of 0 or 0.4%). After standardizing all sols in 0.6 mol/L NaCl, they were stirred and homogenized. The pH of MP-FG sols was adjusted to 5.5, 6.0, 6.5, 7.0 and 7.5 respectively using 0.5 mol/L HCl or NaOH solution. The samples were stored at 4°C prior to measurements.

Preparation of MP-FG gels
MP-FG sols prepared as described above were heated in a water bath from 20°C to 80°C and kept at 80°C for 20 min. The obtained gels were stored at 4°C overnight (12 h) prior to determinations of WHC and scanning electron microscopy (SEM) images.

Dynamic rheological measurements
The dynamic oscillatory measurements were performed using a method described by Westphalen, Briggs, and Lonergan (2005) with slight modifications on a rheometer (Anton Paar, Physica MCR 301, Austria) in oscillatory mode. A 25 mm-parallel steel plate geometry with a 500 μm gap was used. A oscillation constant frequency of 0.1 Hz and a strain of 0.3% were applied to monitor the storage modulus (G'). The temperature was increased from 20°C to 80°C at a 2°C/ min rate and decreased from 80°C to 20°C at a 4°C/min rate for sample treating. All data were collected from triplicate treatments.

Raman spectroscopic analysis
The Raman spectrum of each sample was measured on an FRA 106/S FT-Raman spectrometer equipped using a modified procedure from Li, Kang, Zhao, Xu, and Zhou (2014) and Wang et al. (2015). The spectra were obtained in the range of 400 to 3,600 cm −1 . Each spectrum of the samples was obtained under the following conditions: three scans, 30 sec exposure time, 2 cm −1 resolution, sampling speed 120 cm −1 min −1 , and data collection every 1 cm −1 . The spectra were smoothed, baseline-corrected, and normalized against the phenylalanine band at 1003 cm −1 . The secondary structures of each sample were determined as percentages of α-helix, β-sheet, β-turn, and unordered conformations (Alix, Pedanou, & Berjot, 1988).

WHC
The WHC was measured using the protocol described by Shao, Zou, Xu, Zhou, and Sun (2015). Five g gel in centrifuge tube was centrifuged at 10,000 × g at 4°C for 10 min. The WHC was the percentage of the gel's weight retained after centrifugation relative to its initial weight. All data were collected in triplicate.

Gel strength
The strength of the gels was analyzed using a TA.XT Plus Texture Analyzer (TA.XT Plus, Stable Micro Systems, UK) at ambient temperatures (approximately 20°C). The gel was subjected to a compression test using a cylindrical probe (P/0.5 in., aluminum) at a trigger type button with a 1.5 mm s −1 pretest speed, a 1.0 mm s −1 test speed, a 1.0 mm s −1 posttest speed, a 4.0 mm distance and a −5 g trigger force.
Peak load after compressing were recorded. The maximum sustained compression force was described as the gel strength . The experiments were conducted in eight replicates.

SEM measurements
The samples were fixed in a 0.1 M phosphate buffer (pH 7.0) containing 25 ml/L glutaraldehyde at 4°C for 2 days as described by Han, Zhang, Fei, Xu, and Zhou (2009). The SEM observations were performed on a SEM (S-3000, Hitachi Science System Ltd., Hitachinaka, Japan) with an accelerating voltage of 7 kV. Two fields from each treatment were examined, and one of the two was presented.

Statistical analysis
Statistical analysis was conducted using Statistical Analysis System for windows (SAS 8.2, SAS Inst. Inc., Cary, N.C., U.S. A., 2000). All data are expressed as mean ± standard error. And the experiments were conducted at least for triplicates.

Effects of pH on the WHC of thermal MP-FG gel
It can be seen from Table 1 that, addition of FG to the MP gel formulation significantly (P < 0.05) improves the WHC of MP gel, even in that prepared at a low pH. Similar results were also observed in our previous study, and the maximum WHC improvement was achieved with addition of 0.4% FG , which is also the additive amount in present study. With the addition of FG, the WHC of MP gel around the pI point (pH 5.5) of the protein is significantly improved to be similar to that under the pH value between 7.0 and 7.5. The WHC indicates a protein's capability to bind water and is generally used to evaluate the quality and yield of meat products. When pH is adjusted to the pI point, the net charge on the protein is zero and most proteins will aggregate (Huff-Lonergan & Lonergan, 2005), which would make negative influence on the network formation, resulting in a reduction in the amount of water that could be attracted and held by the MP gel matrix. The results obtained in present study means that the addition of FG can effectively overcome the loss of the WHC of protein gel caused by the approaching of pH value to its pI point.
In addition, the WHC of the MP-FG gel is increased significantly (P < 0.05) from 86.80 % to 98.73% when the pH value increases from 5.5 to 7.5 (Table 1). This phenomenon is similar with the effect of pH on the WHC of thermal MP gel, a lower pH value is associated with a lower WHC (Bertram, Kristensen, & Andersen, 2004). An increase of pH away from the pI of MP leads to more charged groups on the surface of the protein, rendering MPs better solubility and mobility, thus more surrounding water would be exposed to protein sites via hydrogen bonding. In addition, the electrostatic interaction between MP and FG may be enhanced due to Table 1. Water holding capacity of myofibrillar protein (MP) containing 0 or 0.4% flaxseed gum (FG) as affected by different pHs (5.5, 6.0, 6.5, 7.0, and 7.5).
the exposure of more net charged groups. Consequently, higher pH could facilitate improving WHC of MP-FG gel.

Effects of pH on the gel strength of thermal MP-FG gel
Different gel strength caused by addition of FG under different pH conditions are shown in Table 2. The effect of pH seemed to be different for each system (with or without flaxseed gum). It can be seen from Table 2 that, among MP samples, the highest gel strength appeared at pH 6.0, and the maximum gel strength of MP-FG occurred at pH 6.5. The gel strength declines significantly with movement of the pH away from 6.0 or 6.5 (P < 0.05). When pH is lower than 6.5, the MP-FG gels show lower gel strength than MP gels. However, the gel strength of MP-FG is higher than that of MP as pH raised to 7.0 and 7.5. The change of gel strength of MP-FG gel is insignificant (P > 0.05) when pH value increased from 5.5 to 6.5, which is in consistence with the effects of pH on the WHC of MP-FG gel. Variations in gel strength as affected by pH cannot be fully explained by differences in protein solubility, which might also be ascribed to the change of myosin isoforms (Morita, Choe, Yamamoto, Samejima, & Yasui, 1987) and the different interactions between protein-protein and protein-FG (Chen et al., 2007;Lesiów & Xiong, 2003). It is expected that increasing pH away from the pI point can enhance the protein solubility via electrostatic repulsion force. The high solubility of protein is thought to only alter the quantity of the effective gelling components, rather than affecting the specific protein bonds in the gel matrix. This hypothesis is supported by the observation that the proteins form substantially weaker gels as the pH is raised from 6.0 to 7.0 (Xiong, 1994). Thus, we propose that the different gel strength of MP-FG induced by pH is ascribed to the protein-protein and protein-FG interactions that form the gel structures with various texture properties.
On the basis of the results of the effects of pH on the WHC and gel strength of MP-FG gel, the following research will focus on the gel samples prepared under pH 5.5, 6.5 and 7.5 conditions to explore the possible gelling mechanism of the MP-FG gel influenced by pH.

Effects of pH on the rheological properties of MP-FG gel
Dynamic rheological tests have been used extensively to study heat-induced gelation of MPs. The viscoelastic measurement of the storage modulus (G') is used to characterize the MP gel. The G' value estimates the stored energy of the elastic portion (Cao, Xia, Zhou, & Xu, 2012). G 0 in Table 3 is defined as the initial G' before heating. And the rate of gelation from 60 to 80°C is extracted from the slope of the storage modulus versus time (Westphalen et al., 2005), which is associated with the degree of protein unfolding and aggregation. In consequence, it would affect gel properties.
The effect of pH on rheological properties of MP-FG is shown in Figure 2 and Table 3. As pH increases from 5.5 to 7.5, the G' decreases. At each pH level, addition of 0.4% FG Table 2. Gel strength of myofibrillar protein (MP) containing 0 or 0.4% flaxseed gum (FG) as affected by different pHs (5.5, 6.0, 6.5, 7.0, and 7.5).
3. a-e Different letters in the same row of the sample with adding 0% or 0.4% FG indicate statistically significant differences at P < 0.05; one-way ANOVA and Duncan's multiple range test. 1. Los datos están expresados como promedio ± SD, n = 8.
2.* in the column indicate MP and MP-FG are statistically significant differences at P < 0.05; T-test.
3. a-c Different letters in the same row of the sample with adding 0% or 0.4% FG indicate statistically significant differences at P < 0.05; one-way ANOVA and Duncan's multiple range test.
generates a higher G 0 value as we could found in Table 3, indicating that an electrostatic interaction between MP and FG occurs before heating (Chen et al., 2007). At pH 5.5, the G 0 of MPs and MP-FG exhibits insignificant differences (P > 0.05) in Table 3. The negative and positive charges inside protein molecules are approximately equal under this pH condition close to pI point and the electrostatic interactions between them are weak. As the pH (6.5, 7.5) deviates from the pI point, the interactions are enhanced, thus significantly higher G 0 is detected in MP-FG (Table 3). The gelation rate from 60 to 80°C of MP-FG are significantly different compared with MP, indicating FG has a noteworthy impact on the mixture. On the other hand, as the pH value increases from pH 5.5 to pH 7.5, the gelation rate of MP-FG decreases from 19.62 Pa/°C to 9.87 Pa/°C, respectively. The same phenomenon was also observed in porcine myosin gels (Liu, Zhao, Xiong, Xie, & Qin, 2008) and fish myosin gels (Liu et al., 2010). It seems that MPs at lower pH always possesses a higher gelation rate during the heating stage (Liu et al., 2008;Westphalen et al., 2005). The results probably arise from the closer association of proteins at lower pH or the increased strength of hydrophobic interactions and disulphide bonds (Riebroy, Benjakul, Visessanguan, Erikson, & Rustad, 2008;Westphalen et al., 2005). Slow gelation rate might give rise to full unfolding and aggregation which is conducive to the formation of the orderly gel network with good gel properties.

Effects of pH on the secondary structure of MP and MP-FG in sol condition
The secondary structural contents of proteins in MP-FG samples, as affected by pH, are shown in Table 4. The α-helical content of MP-FG decreases markedly (lower than 40%) as the pH decreases from 6.5 to 5.5, and there is no significant difference on the secondary structures(ɑ-helix, β-sheet, βturn and unordered)between pH 6.5 and 7.5. At different pH values, addition of FG varies the secondary structure of MP (Table 4). At the pI point (pH 5.5), the presence of FG has no significant effects (P > 0.05) on the secondary structures of MP. When the pH value is set at 6.5, addition of FG to MP significantly (P < 0.05) reduces the content of α-helical structure (Table 4). At the same time, the content of βsheet structure increases. The changes in protein conformation occur in the presence of polysaccharides, which might be caused by the promoted interactions between MP and FG (Turgeon, Schmitt, & Sanchez, 2007).
Hydrogen bonds between the carbonyl oxygen (-CO) and amino hydrogen (NH-) of a polypeptide chain mainly account for the stabilization of α-helix structure (Sano, Ohno, Otsuka-Fuchino, Matsumoto, & Tsuchiya, 1994). Electrostatic interactions between amino acids also contribute to the stability of the secondary structures (Satoh, Nakaya, Ochiai, & Watabe, 2006). As the pI point of myosin is about pH 5.5 (Foegeding, Lanier, & Hultin, 1996), reducing the pH value from 7.5 to 5.5 might weaken the electrostatic attraction among protein-protein and protein-FG via charge neutralization, and destabilize the hydrogen bonds. The changes in electrostatic interactions and hydrogen bond stability could in turn lead to the reduction of α-helix content (Liu et al., 2008). The β-sheet content of MP-FG significantly increases as pH decreases from 6.5 to 5.5 (Table 4). Therefore, there might be a α-helical to β-sheet transition with reducing pH from 6.5 to 5.5, probably originating from the conformational changes of myosin (Liu et al., 2010(Liu et al., , 2008. Protein gel properties are closely related to the changes in its conformation. Both the unfolding of αhelices and the formation of β-sheets play an important role in MP gel formation (Liu et al., 2010;Villamonte, Jury, Jung, & de Lamballerie, 2015). It was suggested that α-helices exhibited a negative effect on G' at 90°C whereas β-sheets exhibited positive effects on G'at 90°C (Liu et al., 2010). β-sheets were an important conformational component of the aggregated-state secondary structure of globulin derived from common buckwheat (Choi & Ma, 2007). In the present study, formation of Figure 2. Storage modulus (Gˊ) of myofibrillar protein (MP) containing 0.4% flaxseed gum (FG) as affected by different pHs (5.5, 6.5 and 7.5) during heating from 20°C to 80°C at a rate of 2°C/min and cooling from 80°C to 20°C at a rate of 4°C/min. Figura 2. Módulo de almacenamiento (Gˊ) de proteína miofibrilar (MP) que contiene 0,4% de goma de linaza (FG) afectada por diferentes pH (5,5; 6,5; 7,5) durante calentamiento de 20 a 80°C a un índice de 2°C/min y enfriamiento de 80 a 20°C a un índice de 4°C/min. Table 4. Secondary structure of myofibrillar protein (MP) containing 0 or 0.4% flaxseed gum (FG) as affected by different pHs (5.5, 6.5 and 7.5).
β-sheets occurs simultaneously with the unfolding of αhelical structures as pH value shifts from 6.5 to 5.5 (Table 4). Thus, it can be observed from Figure 2 that a higher G' of MP-FG gel appeared at lower pH of 5.5, which could be attributed to the increased content of β-sheets.
Regarding to the WHC of the myosin gel, it was found in previous studies that plenty of α-helices prior to heating were beneficial for the WHC of fish myosin gel and the WHC of porcine myosin gel was negatively correlated with the β-sheet fraction prior to heating (Liu et al., 2010). However, this correlation couldn't be found when comparing the case of MP gel with that of MP-FG gel. Although MP has higher content of α-helices (Table 4), its WHC is significantly lower than that of MP-FG gel at the same pH condition of pH 6.5 (Table 1). The electrostatic interactions between MP and FG and the strong hydrophilicity of FG might lead to the enhanced WHC.

Effects of pH on the SEM of MP-FG gel
The 3-dimensional network structure of a gel is an important determinant of WHC and gel strength. Figure 3 shows the structure of the MP or MP-FG gels at 3000 × magnification, affected by different pH (5.5, 6.5 and 7.5) treatments.
The addition of FG enhances the density of the MP matrix and eliminates some of the cavities, forming a smooth, continuous and uniform gel matrix (Figure 3). In MP-FG gel samples, there are some membrane structures (marked with arrows) distributing throughout the surface or interacting with the protein matrix (Figure 3 (b1-b3)). We attributed these visible structures to FG matrix in MP-FG gel. Some hydrocolloid (locust bean, guar, xanthan and carboxymethylcellulose) had also been observed to disperse on or inside gel matrix of meat protein by SEM (Montero, Solas, & Pérez-Mateos, 2001;Pérez-Mateos, Solas, & Montero, 2002). It is proposed that the denser network induced by FG may be caused by the electrostatic interaction between MP and FG and the physical entrapment effect.
Irrespective of the FG addition, the matrix of the MP-FG gel demonstrated distinctive appearances under different pH treatment. As displayed in Figure 3 (b1), the MP-FG gel exhibits a network with many large cavities and coarse crosslinked strands at pH 5.5. When pH is increased to 6.5 and 7.5 (Figure 3 (b2,b3)), a slightly denser structure with small cavities and more cross-linkages is revealed. In addition to the influence of pH on the hydration, mobility and solubility of proteins, it is considered that the interaction possibly exists between FG and meat protein (Chen et al., 2007). Higher pH environment might cause enhanced electrostatic interaction between MP and FG, thus forming a denser structure. In addition, the changes in the microstructure MP-FG gels at different pH levels might be explained by the gelation rate (Liu et al., 2010;Wang, Xu, Huang, Huang, & Zhou, 2014). When pH is increased from 5.5 to 7.5, the gelation rate of MP-FG decreases from 19.62 Pa/°C to 9.87 Pa/°C, respectively (Table 3). It could be presumed that low gelation rate is beneficial for formation of a fine dense gel network. Hence, higher pH value leads to a denser gel network for MP-FG. It was well reported that a fine, uniform structure with numerous small pores would benefit for possessing greater WHC. The addition of FG and elevation of pH can both produce high dense network with small pores, and this change is more conducive to water immobilization, hence improving the WHC (Table 1). Although it is complicated to interpret the relationship between gel strength and network structure of MP-FG gel, some investigations might lead us further inside into the mechanism. As described above, there appears to be less interaction between MP and FG at the pH of 5.5. Then a physical entrapping effect is supposed to be the main role in taking part in the MP gel matrix where the FG absorbed water via hydrogen bonding. FG would interfere with the formation of MP gel network through entanglement (Xiong & Blanchard, 1993), inducing gel network with thin cross-linked strands (Figure 3 (b1)), which consequently results in a decreased gel strength of MP-FG at pH 5.5 ( Table 2). As pH increases to 6.5, a strong electrostatic interaction between MP and FG might occur, transforming gel into a highly interactive matrix system that is more resistant to deformation (Feng & Xiong, 2003;. Then the gel strength of MP-FG at pH 6.5 is expected to be significantly higher than that at pH 5.5 (Table 2). However, further pH increment decreases the gel strength of MP-FG (Table 2), which we speculate to be attributed to the thin cross-linked strands ( Figure 3) and high water retention of gel network that weakens its gel strength.

Conclusions
Different responses to pH are found on the thermal gelation process of MP-FG complex. Gel WHC shows a significant increase at high pH values. Maximum gel strength of MP-FG gel is observed at pH 6.5. Roman spectroscopy analysis shows that the addition of FG to MP leads to a α-helical to ß-Sheet transition under the pH conditions away from the pI, which plays an important role in the MP-FG gelation process. Decreasing gelation rate coupled with increasing pH of MP-FG facilitates the formation of a fine dense gel network, hence enhancing WHC and weakening gel strength. These results suggest that pH adjustment is necessary for MP-FG system to meet the textural and WHC requirements for meat processing.

Disclosure statement
No potential conflict of interest was reported by the authors.