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Physicochemical and structural properties of composite gels prepared with myofibrillar protein and lard diacylglycerols
Meat Science 121 (2016) 333–341
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Physicochemical and structural properties of composite gels prepared with myofibrillar protein and lard diacylglycerols Xiaoqin Diao a,b, Haining Guan a,b, Xinxin Zhao a, Xinping Diao c, Baohua Kong a,⁎ a b c
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 15000, China College of Food and Pharmaceutical Engineering, Suihua University, Suihua, Heilongjiang 152061, China College of Animal Science, Northeast Agricultural University, Harbin, Heilongjiang 15000, China
a r t i c l e
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Article history: Received 28 April 2016 Received in revised form 2 July 2016 Accepted 7 July 2016 Available online 08 July 2016 Keywords: Lard diacylglycerols Myofibrillar protein Gel properties Structural changes
a b s t r a c t The objective of this study was to investigate the physicochemical and structural properties of composite gels prepared with porcine myofibrillar protein (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL). The gels prepared with MP and GL or PGL had significantly higher penetration force and water-holding capacity (WHC) than the gel with lard (P b 0.05) and formed a more compact and orderly microstructure. Compared with the distributions of T2 relaxation times of the pure MP gel, T21 and T22 of the gels that were prepared with GL or PGL moved in the direction of slower relaxation time, which suggests that the water mobility in the gel system was restricted. The presence of lard, GL and PGL did not affect the participating proteins in composite gels. The presence of GL and PGL altered the secondary and tertiary structures of MP in composite gels, which changed the gel properties. In general, the composite gels that were prepared with MP and GL or PGL showed improved gel quality. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Myofibrillar protein (MP) has notably important biological functions, and its gel-forming ability plays a key role in processed meat products and significantly affects the texture and sensory characteristics of the final products (Sun & Holley, 2011). Ziegler and Acton (1984) noted the MP gel formation is responsible for the formation of a threedimensional gel matrix because of the association of protein during heat processing. The formed protein gels are notably important for their contribution to meat binding, fat immobilization and water entrapment in meat products (Wu, Xiong, Chen, Tang, & Zhou, 2009). In addition, Mendoza, García, Casas, and Selgas (2001) have reported that animal fats play an important role in providing good mouthfeel and juiciness in meat products and are stabilized by a proteinaceous membrane (Gordon & Barbut, 1992). However, overconsumption of fat causes health problems such as obesity, hypertension and cardiovascular heart diseases (Nejat, Polotsky, & Pal, 2010). To improve human health, low-fat meat foods have been developed in the meat industry without compromising on the texture and mouthfeel. Triacylglycerols (TAGs) are the main component of pork fat. Previous studies have found that TAGs can be converted into diacylglycerols (DAGs) through the enzymatic glycerolysis of fat with glycerol (Cheong, Zhang, Xu, & Xu, 2009; Miklos, Xu, & Lametsch, 2011). Miklos et al. ⁎ Corresponding author. E-mail address: [email protected] (B. Kong).
http://dx.doi.org/10.1016/j.meatsci.2016.07.002 0309-1740/© 2016 Elsevier Ltd. All rights reserved.
(2011) reported that DAGs could improve the food texture and water retention. In addition, DAGs have been reported to significantly suppress abdominal and visceral fat accumulation and reduce body weight (Maki et al., 2002; Meng, Zou, Shi, Duan, & Mao, 2004). Meanwhile, the safety of DAGs has been confirmed through several animal and human studies (Morita & Soni, 2009). Therefore, DAGs may totally or partially replace animal fat in the processing of meat products. Some reports show that lard-based diacylglycerols can be applied as a fat replacer in meat emulsions and fermented sausages (Miklos et al., 2011, 2014; Mora-Gallego et al., 2013). Our previous study investigated the emulsifying properties and oxidative stability of emulsions of MP and different lipids (lard, GL and PGL). The results revealed that lard diacylglycerols enhanced the emulsifying abilities and had no adverse effects on the oxidation stability of the emulsions that were prepared with MP (Diao, Guan, Zhao, Chen, & Kong, 2016). The interaction between fat and the MP gel matrix plays a determinant role in the stability of cooked meat products. Xiong and Kinsella (1991) revealed that fat globules of various sizes and concentrations could reinforce the milk protein-based gel matrix. Wu et al. (2009) reported that lipid types and concentrations could alter the rheological and microstructural properties of MP-lipid composite gels. However, to our knowledge, no literature has been reported about the interaction between myofibrillar proteins and DAGs in composite-gel formation during the heating process. Therefore, in this study, we attempt to describe the effect of lard DAGs on the gel strength, water-holding capacity, gel-forming proteins and microstructures of composite gels that
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were prepared with MP and lard DAGs. The secondary and tertiary structures of MP, mobility of water molecules and molecular forces in composite gels were elucidated.
investigate the secondary and tertiary structural changes of MP in composite gels using Fourier transform infrared spectra and intrinsic fluorescence measurement.
2. Materials and methods
2.5. Gel strength
2.1. Materials
The MP and fat composite gels were penetrated with a flat-surface cylindrical probe (P/0.5, 12 mm in diameter), which was attached to a Model TA-XT2 texture analyser (Stable Micro Systems Ltd., England, U.K.) at a test speed of 1 mm/s over a 10 mm displacement. The required penetration force (N) to rupture the gels was expressed as the gel strength (Xiong & Brekke, 1991).
Fresh pork back fat and pork loin muscle were obtained from the Beidahuang Meat Corporation (Harbin, Heilongjiang, China). Sodium dodecyl sulfate (SDS), piperazine-1 and 4 bisethanesulfonic acid (PIPES) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents in this work were obtained from commercial sources and were of analytical grade. 2.2. Preparation of lard diacylglycerols (DAGs) Lard DAGs were prepared according to Diao et al. (2016). Lard was extracted by heating the backfat at 120 °C. The reaction mixture, which included glycerol, melted lard and Lipozyme RMIM, was used in the glycerolysis reaction under the following conditions: 1:1 M ratio of lard to glycerol, 14:100 (W/W) of enzyme-to-lard substrate ratio and 500 rpm magnetic stirring speed. First, the reaction mixture was incubated at 65 °C for 2 h and transferred to 45 °C for 8 h. After the reaction, the enzyme was filtered to yield the glycerolized lard (GL), whose DAG content was 61.8%. The GL was purified using the two-step wiped film molecular distillation (SPE10, manufactured in Haiyuan biochemical equipment Co. Ltd., Wuxi, China). The purified glycerolized lard (PGL), which had a higher DAG content (82.0%), was obtained in the second purification step. 2.3. Preparation of myofibrillar protein (MP) MP was extracted from the pork loin muscle according to the described procedure (Xia, Kong, Liu, & Liu, 2009). The final protein concentration was measured using the Biuret method with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as a standard. The MP was maintained at 2–4 °C and used within 48 h. 2.4. Preparation of MP and fat composite gel Predetermined amounts of melted fats (lard, GL and PGL at 45 °C) were separately mixed with a myofibril solution (1% protein in 0.6 M NaCl, 50 mM PIPES, pH 6.0). These mixtures were placed in a 35 °C water bath for 5 min to guarantee that the fats maintained liquid and were subsequently homogenized at 10,000 rpm for 1 min with an IKA T18 Ultra-Turrax (IKA-Werke GmbH & Co., Staufen, Germany). The pre-emulsified fats were immediately used after preparation. A predetermined amount of MP was dissolved in 50 mM PIPES (pH 6.0), which contained 0.6 M NaCl, to form a protein solution. Then, each specific amount of pre-emulsified fat was added into the protein solution by gently stirring with a glass rod to produce composites with fat contents of 4%, 8% and 12% (w/w). Simultaneously, the total amount of MP was maintained constant (4%). Then, aliquots of 15 g of MP and lard, GL and PGL composites were separately poured into 25 mm (inner diameter) × 40 mm (length) glass vials and covered with aluminum foil. These composites were stored at 2–4 °C for one night to reach maximum protein solubility (Ramírez-Suárez, Xiong, & Wang, 2001), subsequently equilibrated at room temperature (25 ± 1 °C) for 30 min and heated in a water bath at 72 °C for 10 min. After heating, the formed gels were cooled and stored in crushed ice for 2 h prior to further analysis. Some prepared gel samples were allowed to equilibrate at ambient temperature (approximately 23 °C) for 30 min to measure these gel properties: gel strength, water-holding capacity, molecular forces, gel-forming proteins, low-field NMR analysis and gel microstructure. Other aliquots of gel samples were lyophilized to
2.6. Gel water-holding capacity The water-holding capacity (WHC) of the gels was determined using a centrifugal method. Briefly, the gel samples (5 g) were placed into a centrifuge tube and centrifuged at 10,000 ×g for 15 min at 4 °C. The surface of the gels was soaked using filter paper. WHC (%) was expressed as the ratio between the gel weights after centrifugation and before centrifugation, which was multiplied by 100. The supernatant was collected and used to analyse the gel-forming proteins in section 2.8. 2.7. Low-field nuclear magnetic resonance analysis The nuclear magnetic resonance relaxation of the gel samples was measured using an LF-NMR analyser minispec mq 20 (Bruker Optik GmbH, Germany) to determine the mobility and proportion of different fractions of water molecules in the gel system without destroying the gel structure. Approximately 2 g of composite gel was placed in an NMR glass tube (1.8 cm in diameter and 18 cm in height). The analyser was operated at a magnetic field strength of 0.47 T and a proton resonance frequency of 20 MHz. The transverse relaxation time (T2) was measured using the Carr-Purcell-Meiboom-Gill pulse sequence (CPMG). For each sample, 16 scans were obtained at a 2 s interval with 3000 echoes in total. The relaxation data were treated using the CONTIN software that was provided with the equipment which resulted in the corresponding distributions of relaxation times from the decay curve. The mean apparent relaxation time (T2i) and amplitude (A2i) for each detected population in CONTIN were recorded. 2.8. Identification of gel-forming proteins The solutions that gathered from the WHC measurement were used to perform the sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the composition of uncoagulated proteins in the complex gels according to the method of Laemmli (1970). For SDS-PAGE, a resolving gel of 12% acrylamide and a stacking gel of 5% acrylamide were used. The amount of loaded samples per lane was 12 μL. The following proteins were used as the molecular weight standards: myosin (200.0 kDa), β-galactosidase (116.0 kDa), phosphorylase b (97.2 kDa), serum albumin (66.4 kDa), ovalbumin (44.3 kDa), carbonic anhydrase (29.0 kDa) and trypsin inhibitor (20.1 kDa). 2.9. Molecular forces in composite gels The major molecular forces that were involved in the composite gels were evaluated according to the method of Jiang and Xiong (2013). Different dissolving solutions were used: 8 M urea + 50 mM sodium phosphate (pH 7.0) to analyse the hydrogen bond; 0.5% (w/v) SDS + 50 mM sodium phosphate (pH 7.0) to analyse the total non-covalent forces; 0.25% (v/v) β-mercaptoethanol +50 mM sodium phosphate (pH 7.0) to analyse the disulfide bands. Samples (1 g) of composite gels were homogenized in 9 mL of various solvents at a speed of 13,500 rpm for 20 s. The homogenates were heated at 80 °C for 1 h to dissolve the gelforming protein, chilled to room temperature and subsequently
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centrifuged at 10,000 ×g for 15 min. The protein content in the supernatant was determined using the biuret method. The relevant molecular forces in the protein gels can be expressed based on the gel solubility after different dissolving buffer treatments. The gel solubility was calculated using the relative protein content in the supernatants in comparison to that in the original suspension, which was taken to prepare the protein gel.
in gels were analysed using a mixed model. In this model, each triplicate was included as a random term, and the fat contents (0%, 4%, 8% and 12%) and fat types (lard, GL or PGL) were included as fixed terms. The data were analysed using the General Linear Models procedure of the Statistix 8.1 software package (Analytical Software, St. Paul, MN, USA).
2.10. Microstructure of composite gels
3.1. Gel strength and water-holding capacity
The microstructure of the composite gels was examined using a scanning electron microscope (SEM). The gel samples (5 × 5 × 5 mm3) were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C overnight and subsequently washed three times with the above phosphate to remove the glutaraldehyde fluid. The composite gels were sequentially dehydrated in ethanol with a series of concentrations of 50, 70, 80, 90 and 100% (v/v) for 10 min each. To remove the ethanol, the dehydrated samples were successively immersed in tertiary butanol-ethanol (100%) (1:1) and tertiary butanol for 15 min and freeze-dried. The dried samples were mounted on a bronze stub and sputter-coated with gold (Sputter coater SPI-Module, West Chester, PA, USA). Then, the specimens were observed and photographed with an SEM (S-3400N, Hitachi, Tokyo, Japan) at an accelerating voltage of 5 kV.
The gel strength is an important quality property of muscle protein, which is notably related to the texture and sensory quality of muscle food. To elucidate the effect of lard DAGs on MP gel properties, gels that were prepared with different fat contents were assessed using a penetration test. As displayed in Fig. 1A, the addition of lard, GL and PGL significantly increased the gel strength (P b 0.05), and the extent of increase was proportional to the fat addition. MP gels with GL and PGL tended to be more rigid than those with lard, and the difference was significant (P b 0.05). The gel strength was improved because the fat globules might behave as “fillers” in the voids of the gels to make the gels have a more compact structure. As proven in our previous finding, PGL and GL have smaller fat globules (Diao et al., 2016), which can adequately fill the gel network structure. Increased the gel strength by addition of lard DAGs can improve the texture and sensory quality of cooked meat products, so lard DAGs have a good application prospect in the meat products. A well-structured protein gel can entrap and immobilize a large amount of water, fat and other food components through the capillary effects of its matrices. WHC is one of the most important functional
2.11. Fourier transforms infrared spectra The Fourier transform infrared spectra (FT-IR) of the freeze-dried samples were recorded using a Perkin-Elmer Spectrum 100 (PerkinElmer Corp., Norwalk, CT) at 4 cm−1 resolution in the wave number range of 4000 to 400 cm−1 with KBr pellets. The peak fitting procedure was used to quantitatively analyse the secondary structural components of MP. The characteristic peak of the amide I band was analysed in the region of 1700–1600 cm− 1 using the Peak Fit v 4.12 software. In the study, a baseline was first corrected to accurately measure the band areas of the second-derivative spectra in amide I. Then, the Fourier self-deconvolution, which affects the number, position and intensity of the bands, was further performed using a Gaussian curve fit (GCF). Finally, the GCF was adjusted to give the best least-squares fit of the individual bands to each deconvoluted spectrum. The relative amounts of α-helix, β-sheet, β-turn and random coil structures of MP in the composite gels were determined from the second derivative of amide I by manually computing the areas under the bands that were assigned to a particular substructure.
3. Results and discussion
2.12. Intrinsic fluorescence measurement The intrinsic fluorescence emission spectra of different samples were determined using an F-4500 fluorescence spectrofluorometer (Tokyo, Japan). The concentration of MP in the composite gels was fixed at 0.2 mg/mL in 10 mM phosphate buffer (pH 7.0). The emission spectra of tryptophan were recorded from 300 to 400 nm with an excitation wavelength of 295 nm. The excitation and emission slit widths were set at 5 nm. A solution of 10 mM phosphate buffer (pH 7.0) was used as negative control. 2.13. Statistical analysis Three batches of composite gels were independently prepared to study the effects of MP and different pork fats (lard, GL or PGL) on the physicochemical and structural properties of the gels. For each batch of gel samples, all specific experiments were conducted in triplicate (triplicate observations). The data are presented as the mean ± standard errors. The analysis of variance (ANOVA) with Tukey's multiple comparison was used to measure the significance of the main effects (P b 0.05). Data regarding physicochemical and structural properties
Fig. 1. Penetration force (A) and water-holding capacity (B) of composite gels that were prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL). The lowercase letters (a–c) indicate significant differences (P b 0.05) among different types of fat at identical fat contents.
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properties of protein gel. The WHC of the gels that were prepared with lard, GL or PGL increased with the increase in fat concentration (Fig. 1B). The gel that was prepared with lard had lower WHC than those prepared with GL or PGL (P b 0.05). The gels that were prepared with GL and PGL retained more water possibly because the hydrophilic polar group in the molecular structure of GL and PGL enhanced the interaction between water and protein (Nakajima, 2004). Xu, Han, Fei, and Zhou (2011) and Sun, Wu, Xu, and Li (2012) revealed that the interactions between proteins and water could bind more water molecules in the capillaries of the insoluble protein matrix. Wu et al. (2009) also reported that those gels prepared with fat and oil could have improved WHC due to their compact structure because of the space-filling effect of the lipid droplets. WHC is generally used to objectively evaluate the quality of meat products (Rosenvold & Andersen, 2003). A higher amount of water in cooked meat products can improve their juiciness. 3.2. Low-field NMR analysis The low-field pulsed NMR T2 relaxation times are sensitive to the mobility of water molecules in the gel system (Zhang, Yang, Tang, Chen, & You, 2015). In the gel system, T2b, T21 and T22 are suggested as the relaxation components. The T2b component represents bound water that is closely associated with macromolecules, the T21 component reflects trapped water in the gel structure, i.e., the immobilized water, and the T22 component corresponds to free water outside the gel structure (Shaarani, Nott, & Hall, 2006). Fig. 2A, B and C show the measured distributions of T2 relaxation times of the composite gels
that were prepared with MP and lard, GL or PGL. Three relaxation components were detected in all gels: a minor component at 1–5 ms (T2b), a main component at 6–100 ms (T21), and a component at 100–600 ms (T22). Compared with the distributions of T2 relaxation times of the pure MP gel, T21 and T22 of the gels that were prepared with MP and lard, GL or PGL moved in the direction of slower relaxation time, and T21 (major component) became wider with a decrease in intensity, which suggests that the water mobility in the gel system was restricted. Noronha, Duggan, Ziegler, O'Riordan, and O'Sullivan (2008) showed that shorter relaxation time corresponded to less mobile water fraction. T21 of the gels that were prepared with MP and GL or PGL had broader distribution probably because of a greater variation in the chemical and physical properties of the gels (Salomonsen, Sejersen, Viereck, Ipsen, & Engelsen, 2007). The change in T21 relaxation times of the gels that were prepared with different fat contents is shown in Fig. 2D, and the corresponding area of these T21 distributions is shown in Fig. 2E. The relaxation time T21 of the gels that were prepared with different fats decreased with increased fat contents (P b 0.05). The observation is consistent with the result of Andersen, Frøst, and Viereck (2010), who noted that higher fat content in cream cheeses decreased the relaxation time. There is no significant difference for composite gels at 8% and 12% fat levels (P N 0.05). The gel prepared with PGL had significantly lower relaxation time T21 (P b 0.05) than those prepared with GL and lard, which indicates that PGL significantly increased the ability of protein-binding hydrogen protons. The result can be attributed to the development of a three-dimensional network structure, which holds water in a less
Fig. 2. Distributions of LF NMR T2 relaxation times of composite gels that were prepared with myofibrillar proteins (MP) and lard (A), glycerolized lard (GL) (B) or purified glycerolized lard (PGL) (C) and relaxation time T21 (D) and the corresponding peak area A21 (E). The lowercase letters (a-c) indicate significant differences (P b 0.05) among different types of fat at identical fat contents.
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mobilized state (Ishioroshi, Samejima, & Yasui, 1979). The corresponding areas A21 of composite gels increased with increasing fat content (P b 0.05), and composite gels with PGL had significantly higher A21 than that with lard at 4%, 8% and 12% fat levels and that with GL at 8% and 12% fat levels (P b 0.05). This result indicates an increase of water content in the intramyofibrillar space and suggests that the higher PGL can combine more water in the gel matrix. The T2b components were almost invisible in the MP gel without fat and appeared in the gel prepared with different types of fats (Fig. 2A, B and C), particularly that with higher fat concentrations, which suggests that more water molecules were constrained in the gel structure as immobilized water because of the fat addition. The T22 component of the gels that were prepared with different types of fat decreased with increased fat contents, particularly in PGL samples (P b 0.05). The gels without fat had obviously higher T22 intensity than the other groups (P b 0.05), which shows that there was more free water in the gels without fat. The gel with fats, particularly PGL, obviously reduced the intensity of T22 (P b 0.05), which shows that the PGL addition can trap more free water in the gel matrix and transform it into combined or immobilized water.
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presence of disulfide interactions, hydrogen bonds and hydrophobic contacts in MP gel. Jiang and Xiong (2013) suggested that with the addition of certain chemical reagents into the protein gel, the constituent protein that contributed to gel formation would dissolve. When MP and fat are mixed, the balance of these forces in the gels is destroyed. Therefore, the protein solubility of composite gels that were prepared with MP and lard, GL or PGL after the treatments with different forcedisruption chemicals was measured to clarify possible physicochemical bonds and interactions that stabilized the composite gels. The changes of three molecular forces in MP gels with lard, GL or PGL are described in Fig. 4. The gels that were prepared with different fats were solubilized in 8 M urea, which destroys intermolecular hydrogen bonds, and the protein solubility significantly increased compared to the gel without fat (P b 0.05). Moreover, Fig. 4A shows that the protein solubility increased when the fat additive amount increased from 4% to 12% (P b 0.05), and the gel with GL or PGL had significantly higher protein solubility than the gel with lard (P b 0.05) possibly because GL and PGL have more hydrophilic polar groups in their molecular structure. However, no significant difference (P N 0.05) was observed between the gels with identical GL and PGL contents. The result is consistent
3.3. Identification of gel-forming proteins Weakly bound and noncontributing proteins in the gel matrix can be easily removed by centrifugation (Jiang & Xiong, 2013). SDS-PAGE of the supernatants of centrifuged composite gels was performed to observe the proteins that remained extractable (Fig. 3). The myofibrillar protein band mainly contained myosin heavy chains (MHC), actin, troponin-T and tropomyosin. Notably, MHC and actin were undetectable in the supernatants of all gels, which indicated that they were a key component of the heat-induced protein gel network, and the presence of lard, GL and PGL did not affect the participating proteins in composite gels. In addition, compared with the gel without fat, the electrophoretic patterns of all gels with fat were consistent, which shows that troponin T and tropomyosin were present in the supernatant after centrifugation, and they were not the active gel-building substance. This result is similar to that observed by Wu et al. (2009). Xiong and Brekke (1991) also reported that troponin T and tropomyosin could not coagulate and did not contribute to the muscle protein gel network formation. 3.4. Molecular forces in composite gels Lefevre, Fauconneau, Ouali, and Culioli (2002) attributed the formation of a three-dimensional network of protein gel to the equilibrium of intermolecular forces. Wu, Xiong, and Chen (2011) reported the
Fig. 3. SDS-PAGE of the supernatants of centrifuged (10,000 ×g) composite gels that were prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL). MW: protein standard with the indicated marker protein molecular weights. Lane 1: gel with 0% fat; lanes 2, 3 and 4: gels with lard at 4%, 8% and 12% fat levels; lanes 5, 6 and 7: gels with GL at 4%, 8% and 12% fat levels; lanes 8, 9 and 10: gels with PGL at 4%, 8% and 12% fat levels.
Fig. 4. Solubility of composite gels that were prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL) in 50 mM phosphate buffer (pH 7.0), which contained different chemicals: A, 8 M urea to analyse the intermolecular hydrogen bonds; B, 0.5% sodium dodecyl sulfate (SDS) to analyse the total non-covalent forces; and C, 0.25% β-mercaptoethanol (βME) to analyse the disulfide bands. The means of the samples with different lowercase letters (a-f) significantly differed from one another (P b 0.05).
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with the WHC measurement. As a consequence, the hydrogen bond plays an important role in the composite-gel formation. When the gels were dissolved in the 0.5% SDS solution (Fig. 4B), a significantly higher amount (P b 0.05) of protein was extracted by SDS from the composite gels with different concentrations of lard, GL or PGL than that from the gel without fat, which indicates that hydrophobic interactions also play an active role in the composite gels. During the gel formation process, when the fat concentration increases, the hydrophobic groups become more exposed, which contributes to the increase in hydrophobicity, and the unfolded protein molecules aggregate to form a gel. The observation is consistent with the results of the penetration force. Wu et al. (2011) reported that an interactive protein film surrounding a fat droplet was conductive to form a composite gel. To clarify the role of disulfide bond cross-linking, which results from sulfhydryl group explosion (Sano, Ohno, Otsuka-Fuchino, Matsumoto, & Tsuchiya, 1994), the gels were treated with βME (Fig. 4C). There were some differences in protein solubility between the MP gel without fat and those with GL or PGL (P b 0.05). The results suggest that the disulfide bonds in the fat globule membrane and between the membrane and the continuous protein gel matrix are also significant for the stability of the composite gels. However, the gels in the βME solution had lower protein solubility than those treated with urea and SDS (P b 0.05), which shows that disulfide bonds are not a significant force in composite gels. O'Kane, Happe, Vereijken, Gruppen, and van Boekel
(2004) reported that hydrophobic and hydrogen bonds were the main forces in the network formation of legumin proteins, and disulfide bonds had minimum involvements. 3.5. Microstructures of composite gels The microstructures of the composite gels that were prepared with MP and lard, GL or PGL are shown using SEM in Fig. 5. Obvious variations were observed in the microstructure of composite gels with different types and concentrations of fat. Compared with the MP gel without fat, the gels with GL and PGL formed more compact and homogeneous three-dimensional network structures. Moreover, when the fat additive amount increased from 4% to 12%, the composite gels showed a more compacted gel network, whereas the pure MP gel exhibited a large amount of void spaces or pores. Youssef and Barbut (2010) showed that oil could function as fillers in the protein network in composite gels and reduce the empty spaces. It was noted that the gels with GL or PGL had a more compacted and smoother gel network than the gels with lard, which indicates that the addition of GL or PGL was more beneficial to the gel network formation. Our previous experiment reveals that GL and PGL are smaller than lard in emulsion. Hence, PGL can evenly disperse in the MP network and result in a compacted and homogeneous microstructure. These structural features explain why MP gels with GL and PGL had stronger penetration forces and water
Fig. 5. Scanning electron micrographs (magnification: 2000×) of the composite gel that was prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL).
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binding. Chen and Dickinson (1998) attributed the reinforcement of composite milk protein gels to the interaction of the protein membrane of active filler particles with protein in the gel matrix. 3.6. Changes in secondary structures To gain insight into the gelation mechanisms of composite gels, the molecular conformation of MP was measured using FTIR to discuss the changes in secondary structures of protein. The percentages of αhelix, β-sheet, β-turn and random coil structures of MP in composite gels with lard, GL or PGL are shown in Fig. 6. Liu, Zhao, Xiong, Xie, and Qin (2008) indicated that the unfolding of α-helix and formation of βsheet favoured the gelation of porcine myosin. Sano et al. (1994) revealed that the α-helix structure was an important conformation to maintain the secondary structure of native protein and was mainly stabilized by hydrogen bonds between carbonyl oxygen (\\CO\\) and amino hydrogen (\\NH\\) of a polypeptide chain. Meng, Ma, and Phillips (2003) reported that β-sheet was an important conformational component in the aggregated globular protein. Fig. 6 shows that α-helix and β-sheet was the predominant secondary structure of MP in composite gels. With the addition of lard, GL and PGL, the contents of αhelix, β-sheet and β-turn of MP in the composite gels gradually
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increased, whereas the random-coil content reduced (P b 0.05), which indicates that the types and concentrations of fat have an essential effect on the secondary structure of MP. In addition, the composite gels with GL and PGL had slightly higher α-helix and β-sheet fractions than those with lard (P b 0.05). This difference may be attributed to the embedment of protein-coated GL and PGL in a continuous, three-dimensional myofibrillar protein gel matrix, which transformed the unordered structure into an ordered structure during the gel formation (Gordon & Barbut, 1990). Several reports have shown that the WHC of myosin gel is positively correlated with the α-helix fraction, a large amount of α-helices prior to heating is beneficial for the WHC of myosin gel, and a large amount of β-sheet can improve the gel strength (Choi & Ma, 2007; W. Liu, Z. Q. Zhang et al., 2010; R. Liu, S. M. Zhao et al., 2010). These observations are consistent with our results of the WHC and penetration force of composite gels. In conclusion, lard, GL and PGL in composite gels can lead to more α-helix and β-sheet at the expense of random coil structures. Furthermore, the higher additive fat content can induce the more obvious secondary structural changes of MP in composite gels. 3.7. Intrinsic tryptophan fluorescence The fluorescence spectra were measured to investigate the interaction between MP and fats in composite gels. Tryptophan is an essential amino acid with relevant biological functions in muscle protein (Lu, Li, Yin, Zhang, & Wang, 2008), and its location in protein affects the fluorescence energy (Burstein, Vedenkina, & Ivkova, 1973). At 280 nm, the tryptophan residues of MP can be excited; therefore, the changes of tertiary structure of the protein can be determined from the fluorescence fluctuations (W. Liu, Z. Q. Zhang et al., 2010; R. Liu, S. M. Zhao et al., 2010; Shen & Tang, 2012). When a protein is partially or completely unfolded, the tryptophan residues become exposed to the hydrophilic environment, which decreases the fluorescence intensity (Pallarès, Vendrell, Avilés, & Ventura, 2004). In the folded state, tryptophan residues are generally located in the hydrophobic environment of the protein with high fluorescence intensity. Puscasu and Birlouez-Aragon (2002) also reported that tryptophan fluorescence had good correlations with the tryptophan concentration. As shown in Fig. 7, the tryptophan fluorescence intensity of MP in the composite gels with lard, GL or PGL remarkably decreased compared with that of the pure MP, which indicates that adding lard, GL and PGL can make the protein unfold. In addition, MP in the composite gel with PGL had significantly (P b 0.05) lower fluorescence intensity values than that with lard at identical fat concentrations. The higher fat content causes a further decrease in fluorescence intensity, which indicates further unfolding and possible interactions between fat and tryptophan residues. The results show that lard, GL and PGL can alter the tertiary structure of MP in composite gels. 4. Conclusions
Fig. 6. Secondary structure fractions of composite gels that were prepared with myofibrillar proteins (MP) and lard (A), glycerolized lard (GL) (B) or purified glycerolized lard (PGL) (C).
The types and concentrations of fat significantly affect the physicochemical and structural properties of MP composite gels. The gels that were prepared with MP and GL or PGL had significantly higher penetration force and WHC as well as more compact and orderly microstructure than other treatments. This effect was largely attributed to the stronger interaction of MP and PGL through hydrogen bonds and hydrophobic association. Furthermore, the additive content of GL and PGL had no obvious effect on the participating proteins in composite gels but changed the secondary and tertiary structures of MP in the gelation of composite gels, which changed the gel properties. Such physicochemical interactions between the protein and fat are responsible for the juiciness, tenderness and mouthfeel of low-fat meat products. In general, the composite gels that were prepared with MP and GL or PGL improve the gel quality, and lard DAGs have the potential for meat product applications.
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References
Fig. 7. Tryptophan fluorescence of MP in composite gels that were prepared with myofibrillar proteins (MP) and lard (A), glycerolized lard (GL) (B) or purified glycerolized lard (PGL) (C).
Acknowledgements This study was funded by the Science and Technology Major Projects in Heilongjiang (grant no. GA15B302) and National Natural Science Foundation of China (grant no. 31471599).
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Contents lists available at ScienceDirect
Meat Science journal homepage: www.elsevier.com/locate/meatsci
Physicochemical and structural properties of composite gels prepared with myofibrillar protein and lard diacylglycerols Xiaoqin Diao a,b, Haining Guan a,b, Xinxin Zhao a, Xinping Diao c, Baohua Kong a,⁎ a b c
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 15000, China College of Food and Pharmaceutical Engineering, Suihua University, Suihua, Heilongjiang 152061, China College of Animal Science, Northeast Agricultural University, Harbin, Heilongjiang 15000, China
a r t i c l e
i n f o
Article history: Received 28 April 2016 Received in revised form 2 July 2016 Accepted 7 July 2016 Available online 08 July 2016 Keywords: Lard diacylglycerols Myofibrillar protein Gel properties Structural changes
a b s t r a c t The objective of this study was to investigate the physicochemical and structural properties of composite gels prepared with porcine myofibrillar protein (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL). The gels prepared with MP and GL or PGL had significantly higher penetration force and water-holding capacity (WHC) than the gel with lard (P b 0.05) and formed a more compact and orderly microstructure. Compared with the distributions of T2 relaxation times of the pure MP gel, T21 and T22 of the gels that were prepared with GL or PGL moved in the direction of slower relaxation time, which suggests that the water mobility in the gel system was restricted. The presence of lard, GL and PGL did not affect the participating proteins in composite gels. The presence of GL and PGL altered the secondary and tertiary structures of MP in composite gels, which changed the gel properties. In general, the composite gels that were prepared with MP and GL or PGL showed improved gel quality. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Myofibrillar protein (MP) has notably important biological functions, and its gel-forming ability plays a key role in processed meat products and significantly affects the texture and sensory characteristics of the final products (Sun & Holley, 2011). Ziegler and Acton (1984) noted the MP gel formation is responsible for the formation of a threedimensional gel matrix because of the association of protein during heat processing. The formed protein gels are notably important for their contribution to meat binding, fat immobilization and water entrapment in meat products (Wu, Xiong, Chen, Tang, & Zhou, 2009). In addition, Mendoza, García, Casas, and Selgas (2001) have reported that animal fats play an important role in providing good mouthfeel and juiciness in meat products and are stabilized by a proteinaceous membrane (Gordon & Barbut, 1992). However, overconsumption of fat causes health problems such as obesity, hypertension and cardiovascular heart diseases (Nejat, Polotsky, & Pal, 2010). To improve human health, low-fat meat foods have been developed in the meat industry without compromising on the texture and mouthfeel. Triacylglycerols (TAGs) are the main component of pork fat. Previous studies have found that TAGs can be converted into diacylglycerols (DAGs) through the enzymatic glycerolysis of fat with glycerol (Cheong, Zhang, Xu, & Xu, 2009; Miklos, Xu, & Lametsch, 2011). Miklos et al. ⁎ Corresponding author. E-mail address: [email protected] (B. Kong).
http://dx.doi.org/10.1016/j.meatsci.2016.07.002 0309-1740/© 2016 Elsevier Ltd. All rights reserved.
(2011) reported that DAGs could improve the food texture and water retention. In addition, DAGs have been reported to significantly suppress abdominal and visceral fat accumulation and reduce body weight (Maki et al., 2002; Meng, Zou, Shi, Duan, & Mao, 2004). Meanwhile, the safety of DAGs has been confirmed through several animal and human studies (Morita & Soni, 2009). Therefore, DAGs may totally or partially replace animal fat in the processing of meat products. Some reports show that lard-based diacylglycerols can be applied as a fat replacer in meat emulsions and fermented sausages (Miklos et al., 2011, 2014; Mora-Gallego et al., 2013). Our previous study investigated the emulsifying properties and oxidative stability of emulsions of MP and different lipids (lard, GL and PGL). The results revealed that lard diacylglycerols enhanced the emulsifying abilities and had no adverse effects on the oxidation stability of the emulsions that were prepared with MP (Diao, Guan, Zhao, Chen, & Kong, 2016). The interaction between fat and the MP gel matrix plays a determinant role in the stability of cooked meat products. Xiong and Kinsella (1991) revealed that fat globules of various sizes and concentrations could reinforce the milk protein-based gel matrix. Wu et al. (2009) reported that lipid types and concentrations could alter the rheological and microstructural properties of MP-lipid composite gels. However, to our knowledge, no literature has been reported about the interaction between myofibrillar proteins and DAGs in composite-gel formation during the heating process. Therefore, in this study, we attempt to describe the effect of lard DAGs on the gel strength, water-holding capacity, gel-forming proteins and microstructures of composite gels that
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were prepared with MP and lard DAGs. The secondary and tertiary structures of MP, mobility of water molecules and molecular forces in composite gels were elucidated.
investigate the secondary and tertiary structural changes of MP in composite gels using Fourier transform infrared spectra and intrinsic fluorescence measurement.
2. Materials and methods
2.5. Gel strength
2.1. Materials
The MP and fat composite gels were penetrated with a flat-surface cylindrical probe (P/0.5, 12 mm in diameter), which was attached to a Model TA-XT2 texture analyser (Stable Micro Systems Ltd., England, U.K.) at a test speed of 1 mm/s over a 10 mm displacement. The required penetration force (N) to rupture the gels was expressed as the gel strength (Xiong & Brekke, 1991).
Fresh pork back fat and pork loin muscle were obtained from the Beidahuang Meat Corporation (Harbin, Heilongjiang, China). Sodium dodecyl sulfate (SDS), piperazine-1 and 4 bisethanesulfonic acid (PIPES) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents in this work were obtained from commercial sources and were of analytical grade. 2.2. Preparation of lard diacylglycerols (DAGs) Lard DAGs were prepared according to Diao et al. (2016). Lard was extracted by heating the backfat at 120 °C. The reaction mixture, which included glycerol, melted lard and Lipozyme RMIM, was used in the glycerolysis reaction under the following conditions: 1:1 M ratio of lard to glycerol, 14:100 (W/W) of enzyme-to-lard substrate ratio and 500 rpm magnetic stirring speed. First, the reaction mixture was incubated at 65 °C for 2 h and transferred to 45 °C for 8 h. After the reaction, the enzyme was filtered to yield the glycerolized lard (GL), whose DAG content was 61.8%. The GL was purified using the two-step wiped film molecular distillation (SPE10, manufactured in Haiyuan biochemical equipment Co. Ltd., Wuxi, China). The purified glycerolized lard (PGL), which had a higher DAG content (82.0%), was obtained in the second purification step. 2.3. Preparation of myofibrillar protein (MP) MP was extracted from the pork loin muscle according to the described procedure (Xia, Kong, Liu, & Liu, 2009). The final protein concentration was measured using the Biuret method with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as a standard. The MP was maintained at 2–4 °C and used within 48 h. 2.4. Preparation of MP and fat composite gel Predetermined amounts of melted fats (lard, GL and PGL at 45 °C) were separately mixed with a myofibril solution (1% protein in 0.6 M NaCl, 50 mM PIPES, pH 6.0). These mixtures were placed in a 35 °C water bath for 5 min to guarantee that the fats maintained liquid and were subsequently homogenized at 10,000 rpm for 1 min with an IKA T18 Ultra-Turrax (IKA-Werke GmbH & Co., Staufen, Germany). The pre-emulsified fats were immediately used after preparation. A predetermined amount of MP was dissolved in 50 mM PIPES (pH 6.0), which contained 0.6 M NaCl, to form a protein solution. Then, each specific amount of pre-emulsified fat was added into the protein solution by gently stirring with a glass rod to produce composites with fat contents of 4%, 8% and 12% (w/w). Simultaneously, the total amount of MP was maintained constant (4%). Then, aliquots of 15 g of MP and lard, GL and PGL composites were separately poured into 25 mm (inner diameter) × 40 mm (length) glass vials and covered with aluminum foil. These composites were stored at 2–4 °C for one night to reach maximum protein solubility (Ramírez-Suárez, Xiong, & Wang, 2001), subsequently equilibrated at room temperature (25 ± 1 °C) for 30 min and heated in a water bath at 72 °C for 10 min. After heating, the formed gels were cooled and stored in crushed ice for 2 h prior to further analysis. Some prepared gel samples were allowed to equilibrate at ambient temperature (approximately 23 °C) for 30 min to measure these gel properties: gel strength, water-holding capacity, molecular forces, gel-forming proteins, low-field NMR analysis and gel microstructure. Other aliquots of gel samples were lyophilized to
2.6. Gel water-holding capacity The water-holding capacity (WHC) of the gels was determined using a centrifugal method. Briefly, the gel samples (5 g) were placed into a centrifuge tube and centrifuged at 10,000 ×g for 15 min at 4 °C. The surface of the gels was soaked using filter paper. WHC (%) was expressed as the ratio between the gel weights after centrifugation and before centrifugation, which was multiplied by 100. The supernatant was collected and used to analyse the gel-forming proteins in section 2.8. 2.7. Low-field nuclear magnetic resonance analysis The nuclear magnetic resonance relaxation of the gel samples was measured using an LF-NMR analyser minispec mq 20 (Bruker Optik GmbH, Germany) to determine the mobility and proportion of different fractions of water molecules in the gel system without destroying the gel structure. Approximately 2 g of composite gel was placed in an NMR glass tube (1.8 cm in diameter and 18 cm in height). The analyser was operated at a magnetic field strength of 0.47 T and a proton resonance frequency of 20 MHz. The transverse relaxation time (T2) was measured using the Carr-Purcell-Meiboom-Gill pulse sequence (CPMG). For each sample, 16 scans were obtained at a 2 s interval with 3000 echoes in total. The relaxation data were treated using the CONTIN software that was provided with the equipment which resulted in the corresponding distributions of relaxation times from the decay curve. The mean apparent relaxation time (T2i) and amplitude (A2i) for each detected population in CONTIN were recorded. 2.8. Identification of gel-forming proteins The solutions that gathered from the WHC measurement were used to perform the sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the composition of uncoagulated proteins in the complex gels according to the method of Laemmli (1970). For SDS-PAGE, a resolving gel of 12% acrylamide and a stacking gel of 5% acrylamide were used. The amount of loaded samples per lane was 12 μL. The following proteins were used as the molecular weight standards: myosin (200.0 kDa), β-galactosidase (116.0 kDa), phosphorylase b (97.2 kDa), serum albumin (66.4 kDa), ovalbumin (44.3 kDa), carbonic anhydrase (29.0 kDa) and trypsin inhibitor (20.1 kDa). 2.9. Molecular forces in composite gels The major molecular forces that were involved in the composite gels were evaluated according to the method of Jiang and Xiong (2013). Different dissolving solutions were used: 8 M urea + 50 mM sodium phosphate (pH 7.0) to analyse the hydrogen bond; 0.5% (w/v) SDS + 50 mM sodium phosphate (pH 7.0) to analyse the total non-covalent forces; 0.25% (v/v) β-mercaptoethanol +50 mM sodium phosphate (pH 7.0) to analyse the disulfide bands. Samples (1 g) of composite gels were homogenized in 9 mL of various solvents at a speed of 13,500 rpm for 20 s. The homogenates were heated at 80 °C for 1 h to dissolve the gelforming protein, chilled to room temperature and subsequently
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centrifuged at 10,000 ×g for 15 min. The protein content in the supernatant was determined using the biuret method. The relevant molecular forces in the protein gels can be expressed based on the gel solubility after different dissolving buffer treatments. The gel solubility was calculated using the relative protein content in the supernatants in comparison to that in the original suspension, which was taken to prepare the protein gel.
in gels were analysed using a mixed model. In this model, each triplicate was included as a random term, and the fat contents (0%, 4%, 8% and 12%) and fat types (lard, GL or PGL) were included as fixed terms. The data were analysed using the General Linear Models procedure of the Statistix 8.1 software package (Analytical Software, St. Paul, MN, USA).
2.10. Microstructure of composite gels
3.1. Gel strength and water-holding capacity
The microstructure of the composite gels was examined using a scanning electron microscope (SEM). The gel samples (5 × 5 × 5 mm3) were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C overnight and subsequently washed three times with the above phosphate to remove the glutaraldehyde fluid. The composite gels were sequentially dehydrated in ethanol with a series of concentrations of 50, 70, 80, 90 and 100% (v/v) for 10 min each. To remove the ethanol, the dehydrated samples were successively immersed in tertiary butanol-ethanol (100%) (1:1) and tertiary butanol for 15 min and freeze-dried. The dried samples were mounted on a bronze stub and sputter-coated with gold (Sputter coater SPI-Module, West Chester, PA, USA). Then, the specimens were observed and photographed with an SEM (S-3400N, Hitachi, Tokyo, Japan) at an accelerating voltage of 5 kV.
The gel strength is an important quality property of muscle protein, which is notably related to the texture and sensory quality of muscle food. To elucidate the effect of lard DAGs on MP gel properties, gels that were prepared with different fat contents were assessed using a penetration test. As displayed in Fig. 1A, the addition of lard, GL and PGL significantly increased the gel strength (P b 0.05), and the extent of increase was proportional to the fat addition. MP gels with GL and PGL tended to be more rigid than those with lard, and the difference was significant (P b 0.05). The gel strength was improved because the fat globules might behave as “fillers” in the voids of the gels to make the gels have a more compact structure. As proven in our previous finding, PGL and GL have smaller fat globules (Diao et al., 2016), which can adequately fill the gel network structure. Increased the gel strength by addition of lard DAGs can improve the texture and sensory quality of cooked meat products, so lard DAGs have a good application prospect in the meat products. A well-structured protein gel can entrap and immobilize a large amount of water, fat and other food components through the capillary effects of its matrices. WHC is one of the most important functional
2.11. Fourier transforms infrared spectra The Fourier transform infrared spectra (FT-IR) of the freeze-dried samples were recorded using a Perkin-Elmer Spectrum 100 (PerkinElmer Corp., Norwalk, CT) at 4 cm−1 resolution in the wave number range of 4000 to 400 cm−1 with KBr pellets. The peak fitting procedure was used to quantitatively analyse the secondary structural components of MP. The characteristic peak of the amide I band was analysed in the region of 1700–1600 cm− 1 using the Peak Fit v 4.12 software. In the study, a baseline was first corrected to accurately measure the band areas of the second-derivative spectra in amide I. Then, the Fourier self-deconvolution, which affects the number, position and intensity of the bands, was further performed using a Gaussian curve fit (GCF). Finally, the GCF was adjusted to give the best least-squares fit of the individual bands to each deconvoluted spectrum. The relative amounts of α-helix, β-sheet, β-turn and random coil structures of MP in the composite gels were determined from the second derivative of amide I by manually computing the areas under the bands that were assigned to a particular substructure.
3. Results and discussion
2.12. Intrinsic fluorescence measurement The intrinsic fluorescence emission spectra of different samples were determined using an F-4500 fluorescence spectrofluorometer (Tokyo, Japan). The concentration of MP in the composite gels was fixed at 0.2 mg/mL in 10 mM phosphate buffer (pH 7.0). The emission spectra of tryptophan were recorded from 300 to 400 nm with an excitation wavelength of 295 nm. The excitation and emission slit widths were set at 5 nm. A solution of 10 mM phosphate buffer (pH 7.0) was used as negative control. 2.13. Statistical analysis Three batches of composite gels were independently prepared to study the effects of MP and different pork fats (lard, GL or PGL) on the physicochemical and structural properties of the gels. For each batch of gel samples, all specific experiments were conducted in triplicate (triplicate observations). The data are presented as the mean ± standard errors. The analysis of variance (ANOVA) with Tukey's multiple comparison was used to measure the significance of the main effects (P b 0.05). Data regarding physicochemical and structural properties
Fig. 1. Penetration force (A) and water-holding capacity (B) of composite gels that were prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL). The lowercase letters (a–c) indicate significant differences (P b 0.05) among different types of fat at identical fat contents.
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properties of protein gel. The WHC of the gels that were prepared with lard, GL or PGL increased with the increase in fat concentration (Fig. 1B). The gel that was prepared with lard had lower WHC than those prepared with GL or PGL (P b 0.05). The gels that were prepared with GL and PGL retained more water possibly because the hydrophilic polar group in the molecular structure of GL and PGL enhanced the interaction between water and protein (Nakajima, 2004). Xu, Han, Fei, and Zhou (2011) and Sun, Wu, Xu, and Li (2012) revealed that the interactions between proteins and water could bind more water molecules in the capillaries of the insoluble protein matrix. Wu et al. (2009) also reported that those gels prepared with fat and oil could have improved WHC due to their compact structure because of the space-filling effect of the lipid droplets. WHC is generally used to objectively evaluate the quality of meat products (Rosenvold & Andersen, 2003). A higher amount of water in cooked meat products can improve their juiciness. 3.2. Low-field NMR analysis The low-field pulsed NMR T2 relaxation times are sensitive to the mobility of water molecules in the gel system (Zhang, Yang, Tang, Chen, & You, 2015). In the gel system, T2b, T21 and T22 are suggested as the relaxation components. The T2b component represents bound water that is closely associated with macromolecules, the T21 component reflects trapped water in the gel structure, i.e., the immobilized water, and the T22 component corresponds to free water outside the gel structure (Shaarani, Nott, & Hall, 2006). Fig. 2A, B and C show the measured distributions of T2 relaxation times of the composite gels
that were prepared with MP and lard, GL or PGL. Three relaxation components were detected in all gels: a minor component at 1–5 ms (T2b), a main component at 6–100 ms (T21), and a component at 100–600 ms (T22). Compared with the distributions of T2 relaxation times of the pure MP gel, T21 and T22 of the gels that were prepared with MP and lard, GL or PGL moved in the direction of slower relaxation time, and T21 (major component) became wider with a decrease in intensity, which suggests that the water mobility in the gel system was restricted. Noronha, Duggan, Ziegler, O'Riordan, and O'Sullivan (2008) showed that shorter relaxation time corresponded to less mobile water fraction. T21 of the gels that were prepared with MP and GL or PGL had broader distribution probably because of a greater variation in the chemical and physical properties of the gels (Salomonsen, Sejersen, Viereck, Ipsen, & Engelsen, 2007). The change in T21 relaxation times of the gels that were prepared with different fat contents is shown in Fig. 2D, and the corresponding area of these T21 distributions is shown in Fig. 2E. The relaxation time T21 of the gels that were prepared with different fats decreased with increased fat contents (P b 0.05). The observation is consistent with the result of Andersen, Frøst, and Viereck (2010), who noted that higher fat content in cream cheeses decreased the relaxation time. There is no significant difference for composite gels at 8% and 12% fat levels (P N 0.05). The gel prepared with PGL had significantly lower relaxation time T21 (P b 0.05) than those prepared with GL and lard, which indicates that PGL significantly increased the ability of protein-binding hydrogen protons. The result can be attributed to the development of a three-dimensional network structure, which holds water in a less
Fig. 2. Distributions of LF NMR T2 relaxation times of composite gels that were prepared with myofibrillar proteins (MP) and lard (A), glycerolized lard (GL) (B) or purified glycerolized lard (PGL) (C) and relaxation time T21 (D) and the corresponding peak area A21 (E). The lowercase letters (a-c) indicate significant differences (P b 0.05) among different types of fat at identical fat contents.
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mobilized state (Ishioroshi, Samejima, & Yasui, 1979). The corresponding areas A21 of composite gels increased with increasing fat content (P b 0.05), and composite gels with PGL had significantly higher A21 than that with lard at 4%, 8% and 12% fat levels and that with GL at 8% and 12% fat levels (P b 0.05). This result indicates an increase of water content in the intramyofibrillar space and suggests that the higher PGL can combine more water in the gel matrix. The T2b components were almost invisible in the MP gel without fat and appeared in the gel prepared with different types of fats (Fig. 2A, B and C), particularly that with higher fat concentrations, which suggests that more water molecules were constrained in the gel structure as immobilized water because of the fat addition. The T22 component of the gels that were prepared with different types of fat decreased with increased fat contents, particularly in PGL samples (P b 0.05). The gels without fat had obviously higher T22 intensity than the other groups (P b 0.05), which shows that there was more free water in the gels without fat. The gel with fats, particularly PGL, obviously reduced the intensity of T22 (P b 0.05), which shows that the PGL addition can trap more free water in the gel matrix and transform it into combined or immobilized water.
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presence of disulfide interactions, hydrogen bonds and hydrophobic contacts in MP gel. Jiang and Xiong (2013) suggested that with the addition of certain chemical reagents into the protein gel, the constituent protein that contributed to gel formation would dissolve. When MP and fat are mixed, the balance of these forces in the gels is destroyed. Therefore, the protein solubility of composite gels that were prepared with MP and lard, GL or PGL after the treatments with different forcedisruption chemicals was measured to clarify possible physicochemical bonds and interactions that stabilized the composite gels. The changes of three molecular forces in MP gels with lard, GL or PGL are described in Fig. 4. The gels that were prepared with different fats were solubilized in 8 M urea, which destroys intermolecular hydrogen bonds, and the protein solubility significantly increased compared to the gel without fat (P b 0.05). Moreover, Fig. 4A shows that the protein solubility increased when the fat additive amount increased from 4% to 12% (P b 0.05), and the gel with GL or PGL had significantly higher protein solubility than the gel with lard (P b 0.05) possibly because GL and PGL have more hydrophilic polar groups in their molecular structure. However, no significant difference (P N 0.05) was observed between the gels with identical GL and PGL contents. The result is consistent
3.3. Identification of gel-forming proteins Weakly bound and noncontributing proteins in the gel matrix can be easily removed by centrifugation (Jiang & Xiong, 2013). SDS-PAGE of the supernatants of centrifuged composite gels was performed to observe the proteins that remained extractable (Fig. 3). The myofibrillar protein band mainly contained myosin heavy chains (MHC), actin, troponin-T and tropomyosin. Notably, MHC and actin were undetectable in the supernatants of all gels, which indicated that they were a key component of the heat-induced protein gel network, and the presence of lard, GL and PGL did not affect the participating proteins in composite gels. In addition, compared with the gel without fat, the electrophoretic patterns of all gels with fat were consistent, which shows that troponin T and tropomyosin were present in the supernatant after centrifugation, and they were not the active gel-building substance. This result is similar to that observed by Wu et al. (2009). Xiong and Brekke (1991) also reported that troponin T and tropomyosin could not coagulate and did not contribute to the muscle protein gel network formation. 3.4. Molecular forces in composite gels Lefevre, Fauconneau, Ouali, and Culioli (2002) attributed the formation of a three-dimensional network of protein gel to the equilibrium of intermolecular forces. Wu, Xiong, and Chen (2011) reported the
Fig. 3. SDS-PAGE of the supernatants of centrifuged (10,000 ×g) composite gels that were prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL). MW: protein standard with the indicated marker protein molecular weights. Lane 1: gel with 0% fat; lanes 2, 3 and 4: gels with lard at 4%, 8% and 12% fat levels; lanes 5, 6 and 7: gels with GL at 4%, 8% and 12% fat levels; lanes 8, 9 and 10: gels with PGL at 4%, 8% and 12% fat levels.
Fig. 4. Solubility of composite gels that were prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL) in 50 mM phosphate buffer (pH 7.0), which contained different chemicals: A, 8 M urea to analyse the intermolecular hydrogen bonds; B, 0.5% sodium dodecyl sulfate (SDS) to analyse the total non-covalent forces; and C, 0.25% β-mercaptoethanol (βME) to analyse the disulfide bands. The means of the samples with different lowercase letters (a-f) significantly differed from one another (P b 0.05).
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with the WHC measurement. As a consequence, the hydrogen bond plays an important role in the composite-gel formation. When the gels were dissolved in the 0.5% SDS solution (Fig. 4B), a significantly higher amount (P b 0.05) of protein was extracted by SDS from the composite gels with different concentrations of lard, GL or PGL than that from the gel without fat, which indicates that hydrophobic interactions also play an active role in the composite gels. During the gel formation process, when the fat concentration increases, the hydrophobic groups become more exposed, which contributes to the increase in hydrophobicity, and the unfolded protein molecules aggregate to form a gel. The observation is consistent with the results of the penetration force. Wu et al. (2011) reported that an interactive protein film surrounding a fat droplet was conductive to form a composite gel. To clarify the role of disulfide bond cross-linking, which results from sulfhydryl group explosion (Sano, Ohno, Otsuka-Fuchino, Matsumoto, & Tsuchiya, 1994), the gels were treated with βME (Fig. 4C). There were some differences in protein solubility between the MP gel without fat and those with GL or PGL (P b 0.05). The results suggest that the disulfide bonds in the fat globule membrane and between the membrane and the continuous protein gel matrix are also significant for the stability of the composite gels. However, the gels in the βME solution had lower protein solubility than those treated with urea and SDS (P b 0.05), which shows that disulfide bonds are not a significant force in composite gels. O'Kane, Happe, Vereijken, Gruppen, and van Boekel
(2004) reported that hydrophobic and hydrogen bonds were the main forces in the network formation of legumin proteins, and disulfide bonds had minimum involvements. 3.5. Microstructures of composite gels The microstructures of the composite gels that were prepared with MP and lard, GL or PGL are shown using SEM in Fig. 5. Obvious variations were observed in the microstructure of composite gels with different types and concentrations of fat. Compared with the MP gel without fat, the gels with GL and PGL formed more compact and homogeneous three-dimensional network structures. Moreover, when the fat additive amount increased from 4% to 12%, the composite gels showed a more compacted gel network, whereas the pure MP gel exhibited a large amount of void spaces or pores. Youssef and Barbut (2010) showed that oil could function as fillers in the protein network in composite gels and reduce the empty spaces. It was noted that the gels with GL or PGL had a more compacted and smoother gel network than the gels with lard, which indicates that the addition of GL or PGL was more beneficial to the gel network formation. Our previous experiment reveals that GL and PGL are smaller than lard in emulsion. Hence, PGL can evenly disperse in the MP network and result in a compacted and homogeneous microstructure. These structural features explain why MP gels with GL and PGL had stronger penetration forces and water
Fig. 5. Scanning electron micrographs (magnification: 2000×) of the composite gel that was prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL).
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binding. Chen and Dickinson (1998) attributed the reinforcement of composite milk protein gels to the interaction of the protein membrane of active filler particles with protein in the gel matrix. 3.6. Changes in secondary structures To gain insight into the gelation mechanisms of composite gels, the molecular conformation of MP was measured using FTIR to discuss the changes in secondary structures of protein. The percentages of αhelix, β-sheet, β-turn and random coil structures of MP in composite gels with lard, GL or PGL are shown in Fig. 6. Liu, Zhao, Xiong, Xie, and Qin (2008) indicated that the unfolding of α-helix and formation of βsheet favoured the gelation of porcine myosin. Sano et al. (1994) revealed that the α-helix structure was an important conformation to maintain the secondary structure of native protein and was mainly stabilized by hydrogen bonds between carbonyl oxygen (\\CO\\) and amino hydrogen (\\NH\\) of a polypeptide chain. Meng, Ma, and Phillips (2003) reported that β-sheet was an important conformational component in the aggregated globular protein. Fig. 6 shows that α-helix and β-sheet was the predominant secondary structure of MP in composite gels. With the addition of lard, GL and PGL, the contents of αhelix, β-sheet and β-turn of MP in the composite gels gradually
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increased, whereas the random-coil content reduced (P b 0.05), which indicates that the types and concentrations of fat have an essential effect on the secondary structure of MP. In addition, the composite gels with GL and PGL had slightly higher α-helix and β-sheet fractions than those with lard (P b 0.05). This difference may be attributed to the embedment of protein-coated GL and PGL in a continuous, three-dimensional myofibrillar protein gel matrix, which transformed the unordered structure into an ordered structure during the gel formation (Gordon & Barbut, 1990). Several reports have shown that the WHC of myosin gel is positively correlated with the α-helix fraction, a large amount of α-helices prior to heating is beneficial for the WHC of myosin gel, and a large amount of β-sheet can improve the gel strength (Choi & Ma, 2007; W. Liu, Z. Q. Zhang et al., 2010; R. Liu, S. M. Zhao et al., 2010). These observations are consistent with our results of the WHC and penetration force of composite gels. In conclusion, lard, GL and PGL in composite gels can lead to more α-helix and β-sheet at the expense of random coil structures. Furthermore, the higher additive fat content can induce the more obvious secondary structural changes of MP in composite gels. 3.7. Intrinsic tryptophan fluorescence The fluorescence spectra were measured to investigate the interaction between MP and fats in composite gels. Tryptophan is an essential amino acid with relevant biological functions in muscle protein (Lu, Li, Yin, Zhang, & Wang, 2008), and its location in protein affects the fluorescence energy (Burstein, Vedenkina, & Ivkova, 1973). At 280 nm, the tryptophan residues of MP can be excited; therefore, the changes of tertiary structure of the protein can be determined from the fluorescence fluctuations (W. Liu, Z. Q. Zhang et al., 2010; R. Liu, S. M. Zhao et al., 2010; Shen & Tang, 2012). When a protein is partially or completely unfolded, the tryptophan residues become exposed to the hydrophilic environment, which decreases the fluorescence intensity (Pallarès, Vendrell, Avilés, & Ventura, 2004). In the folded state, tryptophan residues are generally located in the hydrophobic environment of the protein with high fluorescence intensity. Puscasu and Birlouez-Aragon (2002) also reported that tryptophan fluorescence had good correlations with the tryptophan concentration. As shown in Fig. 7, the tryptophan fluorescence intensity of MP in the composite gels with lard, GL or PGL remarkably decreased compared with that of the pure MP, which indicates that adding lard, GL and PGL can make the protein unfold. In addition, MP in the composite gel with PGL had significantly (P b 0.05) lower fluorescence intensity values than that with lard at identical fat concentrations. The higher fat content causes a further decrease in fluorescence intensity, which indicates further unfolding and possible interactions between fat and tryptophan residues. The results show that lard, GL and PGL can alter the tertiary structure of MP in composite gels. 4. Conclusions
Fig. 6. Secondary structure fractions of composite gels that were prepared with myofibrillar proteins (MP) and lard (A), glycerolized lard (GL) (B) or purified glycerolized lard (PGL) (C).
The types and concentrations of fat significantly affect the physicochemical and structural properties of MP composite gels. The gels that were prepared with MP and GL or PGL had significantly higher penetration force and WHC as well as more compact and orderly microstructure than other treatments. This effect was largely attributed to the stronger interaction of MP and PGL through hydrogen bonds and hydrophobic association. Furthermore, the additive content of GL and PGL had no obvious effect on the participating proteins in composite gels but changed the secondary and tertiary structures of MP in the gelation of composite gels, which changed the gel properties. Such physicochemical interactions between the protein and fat are responsible for the juiciness, tenderness and mouthfeel of low-fat meat products. In general, the composite gels that were prepared with MP and GL or PGL improve the gel quality, and lard DAGs have the potential for meat product applications.
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References
Fig. 7. Tryptophan fluorescence of MP in composite gels that were prepared with myofibrillar proteins (MP) and lard (A), glycerolized lard (GL) (B) or purified glycerolized lard (PGL) (C).
Acknowledgements This study was funded by the Science and Technology Major Projects in Heilongjiang (grant no. GA15B302) and National Natural Science Foundation of China (grant no. 31471599).
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