Influence of lard-based diacylglycerol on rheological and physicochemical properties of thermally induced gels of porcine myofibrillar protein at different NaCl concentrations

Food Research International 127 (2020) 108723

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Influence of lard-based diacylglycerol on rheological and physicochemical properties of thermally induced gels of porcine myofibrillar protein at different NaCl concentrations Xinxin Zhao, Ge Han, Rongxin Wen, Xiufang Xia, Qian Chen, Baohua Kong

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College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Lard diacylglycerol Myofibrillar protein Rheological property Physicochemical property Structural change NaCl concentration

This study investigated the role of lard-based diacylglycerol (DG) on the rheological and physicochemical properties of pork myofibrillar protein (MP) gels at different NaCl concentrations. Incorporated unpurified diacylglycerol (UDG) or purified diacylglycerol (PDG) and increased NaCl concentration significantly enhanced gel compression force, water-holding capacity, storage modulus values (G′), and loss modulus values (G″) (P < 0.05). Scanning electron microscopy results showed that the incorporation of DG promoted a more compact and uniform three-dimensional gel network filled with protein-coated fat globules. The increase in NaCl concentration and addition of UDG or PDG significantly increased the immobilized water contents and decreased the free water contents (P < 0.05) and also caused the increases in the amount of α-helix and β-sheet concomitant with decreases in β-turn and random coil contents. Overall, the increase in NaCl concentration and the incorporation of DG exhibit an enhancing role on the rheological and physicochemical of porcine MP.

1. Introduction Myofibrillar protein (MP) is an important functional protein in muscle which is responsible for the thermally induced gelation properties of meat products. MP plays a critical role in producing the threedimensional viscoelastic gel network via interactions among the proteins by hydrogen bonds, disulfide bonds, and hydrophobic interactions during thermal processing. For comminuted meat products, MP can provide a cohesive force to stabilize fat droplets and water in the threedimensional network structure and bring meat particles together during thermal processing (Guo et al., 2019). The formed protein gels are notably important for their ability to bind water, stabilize fat, and form cohesive membranes on the surface of fat globules in emulsion systems (Wu, Xiong, Chen, Tang, & Zhou, 2009). The gel formation of MP is influenced by many factors, and one of the most important factors is salt concentration. Myosin accounts for nearly 43% of MP in muscle tissue, and the molecule is highly sensitive to the ionic strength (Yates, Greaser, & Huxley, 1983). At low ionic strength (< 0.3 M), myosin molecules aggregate and produce a filament, whereas at high ionic strength (> 0.3 M), myosin molecules disperse individually and occur as monomers (Sano, Noguchi, Matsumoto, & Tsuchiya, 1990). Moreover, high salt concentration is the pre-condition for the solubilization which is followed by the gelation of MP. Hegg (1982), revealing that

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the proper concentration of salt plays a positive role in protein-protein interactions and enhanced quality of the heat-induced gels. NaCl as the most important brine ingredient plays an important role in providing desirable flavour and texture characteristics in comminuted meat products (Gordon & Barbut, 1992). Addition of NaCl to meat products not only influences MP gel behaviour and gel formation but also influences flavour release (Feng, Cao et al., 2018). The perceived saltiness was owing to the Na+ and Cl-, and the flavor intensity depended on salt concentration in meat products (Feng, Cao et al., 2018). However, overconsumption of salt has been shown to lead to degenerative diseases such as hypertension, stroke, cerebrovascular, and cardiovascular diseases (He, Burnier, & Macgregor, 2011; Kloss, Meyer, Graeve, & Vetter, 2015). Fat is an important ingredient in meat products. Addition of animal fat to meat products can provide good textural characteristics, attractive flavour, and high sensory acceptability (Yoo, Kook, Park, Shim, & Chin, 2007). However, excessive animal fat intake could bring about lifestyle-related diseases, particularly obesity, hypertension, and coronary heart disease (Yoo et al., 2007). Triacylglycerol (TG) is a major component, while diacylglycerol (DG) is a minor component in various plant oils and animal fats. Cheong, Zhang, Xu, and Xu (2009) showed that lard is comprised of 97.90% TG and 2.10% DG. In our previous study, the acylglycerol profile of lard, unpurified DG (UDG), and

Corresponding author. E-mail address: [email protected] (B. Kong).

https://doi.org/10.1016/j.foodres.2019.108723 Received 25 April 2019; Received in revised form 24 September 2019; Accepted 28 September 2019 Available online 08 October 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved.

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ultrasonic pretreatment conditions were: molar ratio of lard to glycerol 1:1, ultrasonic pretreatment time 5 min, ultrasonic pretreatment temperature 45 °C, ultrasonic power 250 W, and Lipozyme RMIM-to-lard substrate ratio, 4:100 (w/w). After ultrasonic pretreatment, the mixtures were transferred to a shaking water-bath at 50 °C for 4 h with a constant speed of 180 r/min. After the reaction, lipase was removed by filtration using six layers of cheesecloth, and samples (UDG) were collected. Acylglycerol composition were analysed by high-performance liquid chromatography according to the method of Zhao et al. (2018). The DG content (%) in samples was expressed as a ratio of the amounts of DG to the total acylglycerols, multiplied by 100. The UDG was purified by two-step molecular distillation (SPE10, manufactured in Haiyuan Biochemical Equipment Co. Ltd., Wuxi, China). The UDG was heated and completely melted before being pumped to the feed reservoir. In the first separation step, the condenser temperature was set at 60 °C after the degasifying, evaporation temperature was set at 205 °C. The completely melted UDG (1 L) was fed into the system at a feeding rate of 2.67 L/h. The evaporator vacuum degree and scraper speed were 60 Pa and 350 r/min, respectively. The feed was divided into two fractions after molecular distillation: the distillate stream and residue stream. The light components such as glycerol, free fatty acids and MG were collected in the distillate stream while DG and TG were collected in the residue stream, due to their present higher boiling temperatures (Fregolente et al., 2007). In the two separation step, the residue from the first step distillation was again fractionated by feeding it to the distillation column at a feeding rate of 2.67 L/h. The other operation variables were fixed as follows: 22 Pa as evaporator vacuum, 350 r/min as scraper speed, 280 °C as evaporation temperature, and 60 °C as condenser temperature. The DG contents in UDG and PDG samples were 46.91% (w/w) and 83.10% (w/w), respectively. In this study, three states of pork lard, including untreated lard, unpurified lard-based diacylglycerol (UDG), and purified diacylglycerol (PDG), were used to evaluate their effect on rheological and physicochemical properties of thermally induced gels of porcine myofibrillar protein at different NaCl concentrations.

purified DG (PDG) was analysed by HPLC (Diao, Guan, Kong, & Zhao, 2017). The results revealed that lard was composed of 100% TG; UDG was comprised of 14.50% monoacylglycerol (MG), 61.76% DG, and 23.74% TG; and PDG was comprised of 8.85% MG, 82.03% DG, and 9.12% TG. Compared with TG, DG can reduce postprandial serum TG elevation and suppress fat accumulation in the body (Yasukawa & Katsuragi, 2004). The differences in metabolic characteristics between DG and TG result from differences in metabolic pathways in the small intestinal cells (Yasukawa & Katsuragi, 2004). Moreover, numerous studies in animal and human clinical trials have revealed that DG exerted no adverse effects on health (Morita & Soni, 2009). In addition, compared with TG, DG exerts unique physicochemical properties related to hydrogen bonding due to the presence of a hydrophilic group (hydroxyl group) in DG (Zhao, Sun, Qin, Liu, & Kong, 2018). DG can also improve product texture due to its higher melting point (Yasukawa & Katsuragi, 2004). Meanwhile, the taste, flavour, and texture of DG are similar to those of TG (Nishide, Shimizu, Tiffany, & Ogawa, 2004). Therefore, total or partial substitution of porcine fatback with lardbased DG in meat products may represent an alternative method to improve functional and nutritional properties. Miklos et al. (2014) reported that partial substitution of pork back fat with lard-based DG in fermented sausages could improve the quality of meat products. Our previous experiments studied the preparation of DG from lard by lipase-catalysed glycerolysis (Diao et al., 2017). Furthermore, we evaluated the influence of ultrasonic pretreatment on DG preparation by enzymatic glycerolysis of lard (Zhao, Sun, Liu, & Kong, 2018). The results revealed that the lard samples, after 4 h of glycerolysis reactions with ultrasonic pretreatment, had similar DG contents to 11 h of glycerolysis reactions without ultrasonic pretreatment, which showed that ultrasonic pretreatment promotes DG preparation from lard. The UDG was prepared by a ultrasonic pretreatment method. The UDG was purified by molecular distillation and labelled as PDG. The melting point of UDP was between 29 °C and 42 °C, and melting point of PDG was between 30 °C and 44 °C (Diao, Guan, Kong, Han, & Zhao, 2016). In addition, lard, UDG, and PDG had very similar fatty acid compositions with C14:0 (myristoleic, 1.10–1.20%), C16:1 (palmitoleic, 1.79–1.92%), C16:0 (palmitic, 23.97–24.60%), C18:2 (linoleic, 7.75–8.03%), C18:1 (oleic, 47.64–48.09%), and C18:0 (stearic, 12.95–13.05%) (Diao et al., 2017). However, there were limited reports about the influence of DG on gelation and processing properties of MP. The aim of this work was to investigate rheological and physicochemical properties of porcine MP as influenced by the addition of lard-based DG at different NaCl concentrations, and the changes in gel microstructure, water distribution, and protein secondary structure were also analysed.

2.3. Extraction of myofibrillar protein Myofibrillar protein (MP) was extracted according to the method of Xia, Kong, Liu, and Liu (2009) with some modifications. Fresh pork loin muscle were minced, minced muscle was suspended in 4 vol (w/v, based on muscle weight) of isolation buffer (0.1 M NaCl, 10 mM sodium phosphate, 2 mM MgCl2, 1 mM glycol-bis-(2-aminoethylether)N,N,N′,N′-tetraacetic acid, pH 7.0) and homogenized with a JJ-2 Waring blender (Ronghua Instrumental Manufacturing Co., Ltd., Changzhou, China) at maximum speed for 60 s. The muscle homogenate was centrifuged at 2200 g for 15 min, and the supernatant was discarded. The pellet was washed two more times with 4 vol of the same isolation buffer using the same blending and centrifugation condition as indicated above. The myofibril pellet was then washed three more times with 4 vol of 0.1 M NaCl under the same condition as above except that in the last wash. The myofibril suspension was filtered through four layers of cheese cloth to remove connective tissue and its pH was adjusted to 6.0 with 0.1 M HCl prior to centrifugation. The MP was kept at 4 °C and utilized within three days. Protein concentration was measured using the Biuret method with bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as the standard.

2. Materials and methods 2.1. Materials Fresh pork backfat and fresh pork loin muscle were purchased from the Beidahuang Meat Corporation (Harbin, Heilongjiang, China). Piperazine-1, 4 bisethanesulfonic acid (PIPES) was obtained from the Shanghai Yuanye Chemical Technology Co. Ltd. (Shanghai, China). All reagents in this work were of analytical grades. 2.2. Preparation of lard-based diacylglycerol

2.4. Preparation of myofibrillar protein composite gels

Lard was obtained by heating fatback at 120 °C with constant agitation. Lard-based diacylglycerol (DG) was synthesized according to the procedure of Zhao et al. (2018). Before the enzymatic glycerolysis reactions, the ultrasonic pretreatment was used first. The completely melted lard (10 g) and glycerol in a molar ratio of 1:1 were mixed in a centrifuge tube, and the lipase was added. The tube was first incubated in an ultrasonic bath and the microtip probe was immersed into the mixture to a depth of over 5 mm for ultrasound pretreatment. The

To prepare the emulsion gels, 10 mg/mL MP solution and 60 mg/mL MP solution were prepared in 50 mM PIPES (pH 6.0) at various NaCl concentrations (0, 0.1, 0.3, and 0.6 M). Then, 2.1 g of completely melted pork fats (lard, UDG and PDG) were incorporated into 8 g of 10 mg/mL MP solution containing various concentrations of NaCl. The mixtures were placed in a 32 °C water bath for 2 min, ensuring that fats 2

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The samples were freeze-dried, mounted on a bronze stub and sputtercoated with gold (Sputter coater SPI-Module, West Chester, PA, USA).

(lard, UDG and PDG) remained liquid. The mixtures were then homogenized at 17,000 r/min for 1 min using an IKA T18 Ultra-Turrax homogenizer (IKA-Werke GmbH & Co., Staufen, Germany) to obtain pre-emulsions. Each pre-emulsion was immediately mixed into 60 mg/ mL MP solutions containing corresponding NaCl concentrations to prepare composites with a final concentration of 40 mg/mL protein and 8% (w/w) fat. MP and fat composites were stored at 4 °C overnight to ensure maximum protein solubility. Gel samples of MP and fat composites were formed with 30 mm (inner diameter) × 50 mm (length) glass vials by heating in a water bath at 75 °C for 20 min and cooled immediately in crushed ice. Prior to measurement, gels were equilibrated at room temperature for 30 min.

2.10. Fourier transform infrared spectra Fourier transform infrared spectra (FTIR) of freeze dried gels were studied according to the method of Diao, Guan, Zhao, Diao, and Kong (2016) with some modification. The FTIR measurements from 4000 to 500 cm−1 were performed on an FT-IR Spectrometer 100 (Perkin-Elmer Inc., MA, USA) with 4 scans at a resolution of 4 cm−1. The freeze dried gels were mixed with potassium bromide (KBr) in a mass ratio of approximately 1:60 and milled evenly. Quantitative analysis of secondary structures of MP was carried out by the Peak Fit v 4.12 software. The secondary structural components were calculated from the relative areas of individual assigned bands in the amide I region (1700–1600 cm−1).

2.5. Gel strength Gel strength of composite gels was determined with a Model TA-XT plus texture analyser (Stable Micro Systems Ltd., Godalming, UK) using a flat-surface cylindrical probe (P/0.5, 12 mm diameter) under following parameters: test speed 1 mm/s, trigger force 5 g, distance 8 mm. The penetration force (N), defined as the maximum force required to rupture the gels, was expressed as gel strength.

2.11. Statistical analysis Three batches of MP and fat composites were prepared to investigate the rheological and gelation properties of porcine MP as influenced by the addition of lard-based DG at different NaCl concentrations. Each batch of samples was in triplicate. The data are expressed as the mean ± standard errors. The analysis of variance (ANOVA) was measured to analyse the significance of the main effects (P < 0.05) using Tukey procedures. All data were analysed using the general linear models procedure of the Statistix 8.1 software package (Analytical Software, St. Paul, MN, USA).

2.6. Gel water-holding capacity Gel water-holding capacity (WHC) of composite gels was measured according to the procedure of Wu et al. (2009) with a minor modification. Composite gels were centrifuged at 10,000 g for 15 min at 4 °C. WHC (%) was defined as the gel weight after centrifugation divided by the original gel weight before centrifugation, then multiplying by 100.

3. Results and discussion

2.7. Dynamic rheological analysis

3.1. Gel strength and water-holding capacity

The samples were prepared as described in Section 2.4 without heating treatment. The dynamic rheological properties of MP samples during thermal gelation were tested using a Discovery HR-1 hybrid rheometer (TA Instruments Co., New Castle, DE, USA) equipped with parallel plates (40 mm diameter, 1 mm gap) according to the method of Xia, Ma, Chen, Li, and Zhang (2018). MP and fat composites were heated from 20 °C to 80 °C at a rate of 2 °C/min under a strain of 1% and a fixed frequency of 0.1 Hz. The storage modulus (G′) and loss modulus (G″) were recorded during thermal processing to analyse the rheological characteristics.

Gel strength was examined by gel compression force testing. As shown in Fig. 1A. as the concentration of NaCl increased from 0 M to 0.6 M, the compression forces significantly increased (P < 0.05) and the maximum gel strength appeared at 0.6 M. The compression forces increased from 0.06, 0.11, 0.14 and 0.16 N (0 M NaCl) to 0.38, 0.45, 0.59 and 0.72 N (0.6 M NaCl) for control (MP alone), lard-, UDG- and PDG-composite gels (P < 0.05), respectively. These results likely occurred because protein solubility was enhanced by increasing ionic strength. Desmond (2006) also reported that the addition of NaCl improved the texture of meat products due to the increase in hydration capacity of proteins when NaCl was incorporated, thus leading to an increase in binding properties of proteins. Furthermore, high concentration electrolytes alter the structures of water molecules, further altering the hydrophobic interactions between non-polar groups (McClements, 1999). Xia et al. (2018) revealed that increasing NaCl concentration caused an increase in hydrogen bonding and hydrophobic interactions between proteins during their unfolding. Meanwhile, compared with the pure MP gel, the composite gels with fat resulted in significantly higher (P < 0.05) compression forces at the same NaCl concentrations. These results could be because fats were chopped into small globules during pre-emulsion and then dispersed in the network of the gel matrix (Gordon & Barbut, 1992), making the composite gel structure more compact due to their space-filling effect in the voids of the gel matrix. The UDG- and PDG-composite gels had significantly higher (P < 0.05) compression forces than lard-composite gels, but no significant differences were observed between UDG- and PDG-composite gels at all examined NaCl concentrations (P > 0.05). The size of fat particles and protein-lipid interactions were major factors affecting the stability of the system (Lee, Hampson, & Abdollahi, 1981). The MP emulsions with UDG and PDG exhibited smaller particle sizes (Diao, Guan, Zhao, Chen, & Kong, 2016), thus decreasing droplet aggregation, further sufficiently dispersing and integrating tightly with the MP gel matrix to produce higher gel strength. Throughout the

2.8. Low field nuclear magnetic resonance analysis The water distribution of gels was determined using a low field nuclear magnetic resonance (LF-NMR) analyser (Bruker Corp., Germany). The T2 was measured using the Carr-Purcell-Meiboom-Gill pulse sequence (CPMG) with 8 scans of 0.25 ms pulses between 90° and 180°. Data were analysed by the CONTIN software (Bruker Corp., Germany). 2.9. Scanning electron microscopy Microstructures of the composite gels were determined by a HitachiS-3400N field emission scanning electron microscope (SEM) (Hitachi High Technologies Corp., Tokyo, Japan). Gel samples (2 × 2 × 5 mm3) were first fixed in 2.5% glutaraldehyde (pH 6.8) at 4 °C overnight, then washed three times with 0.1 M phosphate buffer (pH 6.8) for 10 min, followed by post-fix with 1% osmium tetroxide (OsO4) for 1.5 h and three washes with 0.1 M phosphate buffer (pH 6.8) for 10 min each. The fixed samples were then dehydrated by a series of ethanol solutions: 50%, 70%, and 90% for 10 min, and then twice in 100% ethanol for 10 min. The gels were immersed in ethanol-tertiary butanol (1:1) for 15 min, and then were immersed in pure tertiary butanol for 15 min. 3

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A well-structured gel is characterized by the ability to sufficiently trap and immobilize water within a three-dimensional network structure (Wu et al., 2009). As shown in Fig. 1B, the WHC of the MP gels with or without fat at 0.6 M NaCl were both significantly higher than that of the corresponding control at 0 M NaCl (P < 0.05), which could be due to the formation of more hydrogen bonds in the gel network and increase in the degree of swelling as the ionic strength increased (Offer & Trinick, 1983). High concentrations of NaCl (0.6 M) are able to dissociate myosin filaments, thereby creating a bulky polypeptide matrix for moisture retention (Xiong, 2004). This result is similar to that reported by Xia et al. (2018), who found that WHC of MP gels increased as the concentration of NaCl escalated from 0.3 M to 0.6 M. The disordered aggregations existed in the structure of MP gel at low NaCl concentration may lead to an adverse impact on its WHC and gel strength (Feng, Cao et al., 2018). Meanwhile, the gel WHC values of lard-, UDG- and PDG-composite gels were higher than that of exclusively MP gels (P < 0.05). The UDG- and PDG-composite gels exhibited higher WHC than lard-composite gels (P < 0.05), and no significant differences between UDG- and PDG-composite gels were observed at the same NaCl concentration (P > 0.05). The increased WHC in composite gels may be attributed to the formation of a more compact gel structure caused by the incorporated fats, and the compact composite gels exhibited greater ability to entrap and retain water. Furthermore, the MP with UDG- or PDG-composite gels might immobilize more water due to the presence of a hydrophilic group (hydroxyl group) in the molecular structures of UDG and PDG. During the gel formation process, the UDG or PDG may be bound to water molecules by hydrogen bonds, while the hydrophobic fatty acid chain could be bound to the hydrophobic region of MP by hydrophobic interactions, which may enhance the formation of the MP gel network and increase the gel WHC.

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Concentration of NaCl (M) The effects of NaCl concentration on the rheological properties of MP with or without fat are described by determining the storage modulus (G′, the elastic property of protein gel) and loss modulus (G″, the viscosity property of protein gel) (Fig. 2). The G′ of samples with or without fat at 0.6 M NaCl exhibited typical muscle protein rheological patterns. The G′ of gels with or without fat at 0.6 M NaCl were significantly higher than those at 0, 0.1, and 0.3 M NaCl over the entire temperature range. As shown in Fig. 2A, the G′ of control MP at 0.6 M NaCl showed a typical increase from the beginning until achieving a peak value (337 Pa) at approximately 49 °C, then dropped rapidly until approximately 54 °C, with a subsequent continuous increase to 80 °C. This result was consistent with the results of Chen et al. (2013), and this rheological pattern was typical of MP and reflected the transitions of heavy meromyosin and light meromyosin (Egelandsdal, Fretheim, & Samejima, 1986). The first increase in G′ (peak temperature of approximately 49 °C) was called “gel setting”, which was caused by the denaturation of the myosin head S1 subfragment. The second increase in G′ (peak temperature of approximately 80 °C) was called “gel strengthening”, which was because the majority of the myosin molecule may have unfolded to strengthen the gel matrix and form an irreversible and firm gel network (Li, Kong, Xia, Liu, & Diao, 2013). When the concentrations of NaCl were 0, 0.1 or 0.3 M, the first peak disappeared and MP almost lost the typical G′ profile, indicating the formation of a loose network gel. This result was most likely due to excessive aggregation and low solubility of MP at low NaCl concentration, which could cause poor gel-forming ability. The high ionic strength promoted the solubilization of meat proteins, which might increase G′ value and improve gel network structure (Stanley, Stone, & Hultin, 1994). Similar results were obtained by Stanley et al. (1994), who reported that the gel network structure improved with increasing concentration of NaCl. Feng, Pan et al. (2018) also found that the G′ of porcine MP sols increased as the concentration of NaCl

Fig. 1. Compression force (A) and water-holding capacity (B) of gels prepared with myofibrillar protein (MP) alone or MP combined with lard, unpurified diacylglycerol (UDG) or purified diacylglycerol (PDG) at different NaCl concentrations (0, 0.1, 0.3, and 0.6 M). Uppercase letters (A-C) indicate significant differences (P < 0.05) among the same composite gels at different NaCl concentrations and lowercase letters (a-c) indicate significant differences (P < 0.05) among different composite gels at the same NaCl concentrations.

emulsifying process, fat are chopped into small globules and further stabilized by a salt-soluble MP membrane. These smaller particle sizes of oil globules could be coated by the more salt-soluble MP and interact with the MP through hydrophobic interactions and disulfide bonds, causing improved gel quality (Zhuang et al., 2019). Hu, Xing, and Zhang (2017) studied the effect of regenerated cellulose on thermal gelation and microstructural properties of MP and found that addition of stable regenerated cellulose emulsion increased the gel strength due to its space-filling function. Sikorski (1997) indicated that protein gels with small fat globules had higher gel strength. Youssef and Barbut (2009) also reported that meat products with smaller lipid globules showed greater resistance to compression than products with larger lipid globules. Another explanation could be that DG had higher melting points than TG, thus improving the hardness of meat products (Miklos, Xu, & Lametsch, 2011). Additionally, UDG and PDG have a free hydroxyl group in their structure, and thus UDG and PDG may partially bind MP by hydrogen bonds during the gel formation process, contributing to the improvement of the gel network. These results were in agreement with the results of Wu et al. (2009), who proved that the protein-lipid interactions could immobilize fat and improve the gel strength of MP gels. Diao, Guan, Zhao, Diao et al. (2016) also suggested that gels with DG had higher gel strength than those of pure MP gels and lard-composite gels. 4

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Fig. 2. Storage modulus (G′) (A, B, C and D) and loss modulus (G″) (E, F, G and H) values of gels prepared with myofibrillar protein (MP) alone or MP combined with lard, unpurified diacylglycerol (UDG) or purified diacylglycerol (PDG) at different NaCl concentrations (0, 0.1, 0.3, and 0.6 M).

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by Bertram, Kristensen, and Andersen (2004), who found that the T2 relaxation times of major components of myofibrils increased significantly with increasing ionic strength. Meanwhile, as shown in Table 1, the relative area A21 values of control (MP alone) and MP with lard-, UDG- and PDG-composite gels were respectively 13.77%, 16.95%, 18.72% and 20.74% at 0 M NaCl, and they significantly increased to 29.89%, 36.01%, 43.95% and 49.14% at 0.6 M NaCl (P < 0.05). The relative area A22 values of control (MP alone) and MP with lard-, UDG- and PDG-composite gels were 86.17%, 82.92%, 81.20% and 79.17% at 0 M NaCl, and they significantly decreased to 70.09%, 63.81%, 55.79% and 50.72% at 0.6 M NaCl, respectively (P < 0.05). The results indicated that the relative content of entrapped water increased and the relative content of free water decreased. The degrees of myofibril swelling were strongly NaCl concentration-dependent: Cl- bound to the filaments when the NaCl was added, and the electrostatic repulsive force between filaments was increased by enhanced NaCl concentration, widening the distance between myofilaments due to swelling of the filament lattice and causing a decrease in free water content and increase in entrapped water mobility (Offer & Trinick, 1983). Moreover, Bertram, Meyer, Wu, Zhou, and Andersen (2008) also found that the expansion of the filament lattice was increased by enhanced NaCl concentration, and thus the surface area within the myofibril increased, resulting in more exposed macromolecules as sites for water binding. Offer and Trinick (1983) reported that 4.6%-5.8% (w/w) NaCl stimulated swelling of myofibrils and high water uptake. Xiong, Lou, Harmon, Wang, and Moody (1999) also reported that the increase in NaCl concentration from 0.1 M to 0.6 M greatly facilitated muscle fibre (myofibrils comprise approximately 80% of the volume of muscle fibre) swelling. Melander and Horvath (1977) also speculated that the changes in protein conformation at high ionic strength were due to changes in hydrophobic interactions. High ionic strength causes the opening up of the structure of myofibrillar proteins and facilitates the increase of water binding (Gordon & Barbut, 1992). Bertram et al. (2004) also suggested that the increase in WHC could be attributed to the swelling of myofibrils, and thus increased the relaxation time T2. In addition, Han et al. (2009) concluded that the increase in water retention may be due to the fact that more free water could be trapped within the MP gel network and transformed into entrapped water. These results were also consistent with the WHC results. Compared to the pure MP gels, the composite gels with fat resulted in significantly lower (P < 0.05) T21 relaxation times at all of the examined NaCl concentrations. Meanwhile, the relative area in A21 of UDG- and PDG-composite gels were significantly higher than those of control and lard-composite gels (P < 0.05), but no significant differences were observed between UDG- and PDG-composite gels at the same NaCl concentration (P > 0.05). By comparison, the relative area in A22 of UDG- and PDG-composite gels at all four NaCl concentrations studied were significantly lower than those of the corresponding controls and lard-composite gels (P < 0.05). These results indicated that addition of fats resulted in an increase in immobilized water content and reduction in free water content. These results occur probably because a protein coat around fat globules was formed upon incorporation of fat, and protein-coated fat globules were further stabilized by physical entrapment within a gelled protein-water matrix. Structurally, emulsified fats occupy the void spaces in the network of the protein gel matrix and produce a more compact three-dimensional gel network, thereby trapping more water in the gel structure. Zhou, Chen et al. (2019) reported that interfacial protein film can immobilize emulsified fat globules which result in further aggregation of the protein gel network matrix and restrict the flow of fat and water via physical confinement. The similar observation was also reported by Diao et al. (2016), who noted that addition of fat reduced the T21 and T22 relaxation times of MP gels. Xiong et al. (2016) also found that addition of pre-emulsified hot pork fatback resulted in lower T21 and T22 when compared with the chicken liver paste batters.

increased from 0.2 M to 0.6 M, and the highest G′ was obtained at 0.6 M. Meanwhile, the G′ of gels prepared with MP alone or MP combined with lard, UDG or PDG at the same NaCl concentration had a similar rheological trend, although incorporation of lard, UDG or PDG obviously increased G′ over the entire thermal temperature range when the MP sols were transformed into gels, especially for samples at 0.6 M NaCl (Fig. 2B, C, D). At 0.6 M NaCl, when the lard, UDG and PDG were incorporated into the MP sample, the first peak G′ values of control (MP alone) were increased from 337 Pa to 472, 590 and 751 Pa, respectively, and the first peak temperatures were increased by 1–2 °C. These results indicated that fat addition strengthened the cross-linking of protein and increased the G′ values, and these results in agreement with the description of Zhou, Yang, Wang, Wei, and Li (2019). A possible explanation for the higher G′ values of samples combined with fats was that fat globules in the pre-emulsion are dispersed in the protein matrix, the presence of fat globules would generate more friction when sheared during the dynamic rheological measurement, and the fat globules participated in the development of the MP gel networks by acting as fillers. Fats globules interacted with proteins in the continuous phase to produce an amorphous network possessing a viscoelastic characteristic (Wu et al., 2009). Zhou, Yang et al. (2019) studied the effect of low level of fat addition on chicken MP gelation properties, and the results showed that when the fat addition was 0.2%, the first peak G′ values were increased from 173 Pa (no fat addition) to 473 Pa. In addition, UDG- and PDG-composite gels displayed higher G′ values than control and lard gels, which could in part be attributed to the hydrophilic group in the DGs. The rheological changes were also described by the temperaturedependent shifts in G″. The changes in G″ of each sample (0.6 M NaCl) exerted a trend similar to corresponding G′ at approximately 40–49 °C. From approximately 49 to 55 °C, the G″ dropped sharply and levelled off until the temperature reached 80 °C (Fig. 2E, F, G and H). During heat treatment, the G′ of samples with or without fat was always higher than the G″ at the same NaCl concentration, especially when the temperature was greater than 55 °C, indicating that a more elastic or less viscous material during the protein gel matrix formation. Above approximately 55 °C, major structural changes in myosin have already happened. Continuing molecular cross-linking (accounting for a high elasticity) and interactions, which were beneficial to improved elasticity, dominated in the viscoelastic gel system (Li et al., 2013). The higher G′ and G″ values obtained by high NaCl concentration (0.6 M) or the incorporation of fat indicated that high NaCl concentration and added fat could promote the formation of MP gel. 3.3. Low field nuclear magnetic resonance analysis LF-NMR is a useful analytical method due to its rapid, non-invasive, and low cost characteristics for the estimation of water mobility in the meat industry. According to the water molecular mobility, the T2 relaxation times are characterized by three relaxation components (T2b, T21, and T22). The T2b component represents bound water that is tightly associated with macromolecules, the T21 component represents trapped water that is entrapped or immobilized within the myofibrils in the spaces between the thick and thin filaments, and the T22 component represents free water that is located outside the gel structures (Han, Fei, Xu, & Zhou, 2009). Fig. 3 and Table 1 show the distribution of T2 relaxation times and T2 relative peak areas of MP gels with or without fats at different NaCl concentrations. The LF-NMR curves exhibited one or two different forms of trapped water (T21a and T21b). T21a water was more tightly entrapped within the gel network than T21b water (Xiong et al., 2016). No significant changes were found for the T2b values of gels with or without fat at different NaCl concentrations (P > 0.05). The increase in NaCl concentration caused an increase in T21 relaxation times and reduction in T22 relaxation times, indicating that the distance between the thin and thick filaments increased. The similar result was reported 6

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PDG + MP + 0 M NaCl

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T2 relaxation time (ms)

T2 relaxation time (ms)

Fig. 3. Distribution analysis of LF NMR T2 relaxation times of gels prepared with myofibrillar protein (MP) alone or MP combined with lard, unpurified diacylglycerol (UDG) or purified diacylglycerol (PDG) at different NaCl concentrations (0, 0.1, 0.3, and 0.6 M) (A, B, C and D).

concentration (0.6 M) would facilitate the formation of a fine threedimensional gel network. The results were in accordance with Xia et al. (2018), who suggested that the protein gel microstructure depends on ionic strength. The changes in the microstructure of MP may be due to differences of the MP solubility at different NaCl concentration. The MP gel with low NaCl concentration showed a weak gel structure with large cavities and coarse cross-linked strands, and this loose structure was adverse to WHC; as the increase of NaCl concentration, the MP gel expressed a three-dimensional network caused by aggregation of myosin monomers into a cross-linked structure (Feng, Cao et al., 2018;

3.4. Microstructure of the gels The SEM micrographs of MP with or without fat at different NaCl concentrations are presented in Fig. 4. The pure MP gel at 0 M NaCl showed coarse, porous, disordered structure composed of protein aggregates. The gels appeared as a more fine, compact, homogeneous and smooth gel structure when the NaCl concentration increased from 0 to 0.6 M NaCl. In particular, a three-dimensional network, which consisted of fine strands in a dense arrangement with small void spaces or pores, was observed in MP gels at 0.6 M NaCl, indicating that a higher NaCl

Table 1 Distributions of T2 relative peak areas of gels prepared with myofibrillar protein (MP) alone or MP combined with lard, unpurified diacylglycerol (UDG) or purified diacylglycerol (PDG) at different NaCl concentrations (0, 0.1, 0.3, and 0.6 M). Relative peak area (%)

Sample

Concentration of NaCl (M) 0

0.1

0.3

0.6

A21

MP Lard + MP UDG + MP PDG + MP

13.77 16.95 18.72 20.74

± ± ± ±

0.86cC 0.48bC 0.75abD 0.93aD

21.87 26.53 29.78 32.03

± ± ± ±

1.26bB 1.39aB 1.82aC 1.39aC

24.29 29.20 35.93 39.30

± ± ± ±

1.81cB 0.86bB 1.47aB 1.05aB

29.89 36.01 43.95 49.14

± ± ± ±

1.24cA 2.01bA 0.93aA 2.23aA

A22

MP Lard + MP UDG + MP PDG + MP

86.17 82.92 81.20 79.17

± ± ± ±

0.91aA 0.51bA 0.82bcA 0.25cA

78.06 73.22 69.94 67.77

± ± ± ±

0.72aB 0.48bB 1.46bcB 1.26cB

75.62 70.71 63.84 60.69

± ± ± ±

0.72aBC 1.10aB 1.44bC 2.63bB

70.09 63.81 55.79 50.72

± ± ± ±

2.94aC 1.25abC 1.37bcD 2.67cC

Uppercase letters (A-D) indicate significant differences (P < 0.05) among the same composite gels at different NaCl concentrations, and lowercase letters (a-c) indicate significant differences (P < 0.05) for the same peak among different composite gels at the same varying NaCl concentrations. A21, the relative content of immobilized water; A22, the relative content of free water. 7

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0 M NaCl

0.1 M NaCl

0.3 M NaCl

0.6 M NaCl

MP

MP + Lard

MP + UDG

MP + PDG

Fig. 4. Scanning electron micrographs (magnification: 1000 × ) of gels prepared with myofibrillar protein (MP) alone or MP combined with lard, unpurified diacylglycerol (UDG) or purified diacylglycerol (PDG) at different NaCl concentrations (0, 0.1, 0.3, and 0.6 M).

adequately entrap and immobilize water, fat, non-meat protein and other food components. A similar finding was also found by Diao et al. (2016), who found that gel microstructures of MP with UDG- and PDGcomposite gels were more dense and uniform than those of pure MP gel and lard-composite gels. The SEM micrographs supported the results observed with respect to gel strength and gel WHC.

Zheng, Han, Ge, Zhao, & Sun, 2019). Lanier, Carvajal, and Yongsawatdigul (2004) illustrated that protein gel formation involves partial denaturation of protein, followed by permanent and irreversible protein aggregation, which produces a three dimensional gel matrix. The gel properties are related to the relative rates of denaturation and aggregation. If the rate of aggregation is faster than denaturation, a rougher and more uneven gel structure would be formed (Liu, Zhao, Xiong, Xie, & Qin, 2008), which might be ascribed to the fact that the NaCl concentration played a critical role in MP conformation, and the low solubility of MP could cause excessive aggregation of MP at low NaCl concentrations that did not favour fine gel structure formation. In contrast, more proteins with a more swelled state were formed with increasing NaCl concentration, which resulted from electrostatic repulsion, ultimately leading to a more fine, dense, smooth and uniform gel matrix. Compared with the MP alone, relatively compact and homogeneous protein matrices with fat globules and small pores were observed in the gels with fats. Meanwhile, the presence of protein membranes on the fat globules and embedding of fat particles in a protein matrix suggested that fat globules may serve as interactive fillers that reinforce the gel network. Youssef and Barbut (2010) suggested that oil could reduce void spaces of the composite gels due to its space-filling effect. Gordon and Barbut (1990) found that physical binding of fat globules to the protein matrix took place in frankfurters, and this physical binding may may result from protein-protein interactions between the interfacial protein film and the matrix proteins. They also indicated that protein aggregation during thermal processing increases the immobilization of protein-coated fat globules by binding them to the matrix, thereby further stabilizing these globules and preventing coalescence. Jost, Baechler, and Masson (1986) reported that fat globules covered with adsorbed protein molecules were immobilized by direct reticulation between membrane-forming protein molecules and bulk phase protein. Moreover, smaller fat globules for UDG- and PDG-composite gels were observed compared with that of lard-composite gels, which could contribute to the formation of fine gel structures with the ability to

3.5. Fourier transform infrared spectra FTIR spectroscopy is a powerful method for assessing relative changes in protein secondary structures. The FTIR spectra and relative contents of secondary structure components of MP in gels were influenced by the addition of fats and changing NaCl concentration, as described in Fig. 5. It can be seen that the secondary structures of MP in gels were influenced by the addition of fat. Primary differences in the FTIR spectra were observed at approximately 2850 cm−1 (I) and 724 cm−1 (II), namely, that lard-, UDG- and PDG-composite gels exhibited similar absorption peaks at approximately 2850 cm−1 and 724 cm−1, respectively, whereas MP alone gels showed a minor peak at approximately 2850 cm−1 and had no absorption peaks at approximately 724 cm−1. The peaks at 2850 cm−1 and 724 cm−1 represented –CH2 symmetrical stretching and the –(CH2)n of the swing vibration, respectively (Lerma-García, Ramis-Ramos, Herrero-Martínez, & SimóAlfonso, 2010). The intensity of the C–H stretching band was related to protein denaturation. The increase in the C–H stretching band intensity was an indication of protein denaturation (Ma, Rout, Chan, & Phillips, 2000), which may benefit the formation of gel networks. According to Qi et al. (2017), the bands at 1610–1640 cm−1, 1640–1650 cm−1, 1650–1660 cm−1, and 1660–1690 cm−1 represented the β-sheet, random coil, α-helical, and β-turn structures, respectively. The unfolding of α-helical structure and the formation of β-sheet structure contributed to the aggregation of porcine myosin and the formation of gel networks (Liu et al., 2008). The α-helical structures played a key role in the adsorption and interaction with the oil phase of emulsions and were stabilized by hydrogen bonds between the carbonyl 8

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B

587

4000

3500

3000

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1000

Lard + MP + 0 M NaCl Lard + MP + 0.1 M NaCl Lard + MP + 0.3 M NaCl Lard + MP + 0.6 M NaCl

Transmittance (%)

Transmittance (%)

A

500

4000

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-1

Transmittance (%)

3500

3000

2500

2000

1500

1000

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aA

80

aA

aB aB

bA cA

bB

20

aCaC bC cC

bC bB bC

0

1000

500

aA

80

aA

60

aB aB

α-Helix β-Sheet β-Turn Random coil

bA cA

aCaC

40

bB

20

aCaC bC cC

bC bB bC

0 0

0.1

0.3

0.6

0

Concentration of NaCl (M)

aA

60

aB aB

H

α-Helix β-Sheet β-Turn Random coil

Secondary structure content (%)

aA

80

bA cA

aCaC

40

bB aCaC bC cC

0.1

0.3

0.6

Concentration of NaCl (M)

100

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F 100

α-Helix β-Sheet β-Turn Random coil

aCaC

40

2000

Wave number (cm )

100

60

2500

-1

Secondary structure content (%)

Secondary structure content (%)

1000

Transmittance (%)

D

Wave number (cm )

Secondary structure content (%)

1500

PDG + MP + 0 M NaCl PDG + MP + 0.1 M NaCl PDG + MP + 0.3 M NaCl PDG + MP + 0.6 M NaCl

-1

G

2000

Wave number (cm )

UDG + MP + 0 M NaCl UDG + MP + 0.1 M NaCl UDG + MP + 0.3 M NaCl UDG + MP + 0.6 M NaCl

4000

E

2500

587

-1

Wave number (cm )

C

II (724)

I (2850)

bC bB bC

0

100

aA

80

aA

60

aB aB

α-Helix β-Sheet β-Turn Random coil

bA cA

aCaC

40

bB

20

aCaC bC cC

bC bB bC

0 0

0.1

0.3

0.6

0

Concentration of NaCl (M)

0.1

0.3

0.6

Concentration of NaCl (M)

Fig. 5. Fourier transform infrared spectra (A, B, C and D) of gels prepared with myofibrillar protein (MP) alone or MP combined with lard, unpurified diacylglycerol (DG) or purified diacylglycerol (PDG) at different NaCl concentrations (0, 0.1, 0.3, and 0.6 M) and the corresponding relative contents of secondary structure components (E, F, G and H).

9

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Program during the 13th Five-year Plan in China (2016YFD0401504).

oxygens (–CO) and amino hydrogens (NH–) of a polypeptide chain (Lee, Lefévre, Subirade, and Paquin (2007)). The β-sheet structures were related to the formation of the protein membrane on fat globule surfaces, and fat membranes could boost α-helix formation of polypeptides (Sakuno, Matsumoto, Kawai, Taihei, & Matsumura, 2008). As shown in Fig. 5E, F, G and H, the α-helical and β-sheet were the dominant secondary structures of MP in gels. The content of α-helical structure increased at the expense of fewer β-sheet, β-turn and random coil structures when the NaCl concentration increased from 0 M to 0.6 M. These results were in agreement with the description of Feng, Cao et al. (2018), in which an increase in the content of α-helical structures simultaneously with a decrease in the content of β-sheet structures was observed upon addition of NaCl. Satoh, Nakaya, Ochiai, and Watabe (2006) showed that electrostatic interactions between amino acids favoured the stabilization of the secondary structures. The increase in NaCl concentration facilitated more hydrogen bonding between proteins, along with the enhancement of electrostatic repulsion, which could contribute to the increase in α-helical structure content (Xia et al., 2018). Yang et al. (2017) showed that the increase of α-helix content contributed to increase in WHC. Meanwhile, compared with pure MP gel, the gels with fat increased α-helical and β-sheet structures of MP in gels, whereas β-turn and random coil structures were decreased. Moreover, the amount of α-helical and β-sheet structure percentages of UDG- and PDG-composite gels were higher than that of lardcomposite gel or MP alone. These results might be due to hydrophobic interactions and hydrogen bonding between MP and fat. Howell, Herman, and Li-Chan (2001) analysed the protein-oil interactions and found that the addition of oil can lead to changes in the exposure of hydrophobic amino acid residues and secondary structures of proteins. The exposure of hydrophobic groups facilitated intermolecular combination and gel formation. Another possible explanation for this is the embedding of protein-coated fat in the three dimensional MP matrix, which could contribute to the transformation from the disordered structure to an ordered structure during the gel formation (Gordon & Barbut, 1990). Additionally, the secondary structure differences of MP among lard-, UDG- and PDG-composite gels may be due to the hydroxyl groups in the structures of UDG and PDG.

References Bertram, H. C., Kristensen, M., & Andersen, H. J. (2004). Functionality of myofibrillar proteins as affected by pH, ionic strength and heat treatment-a low-field NMR study. Meat Science, 68, 249–256. Bertram, H. C., Meyer, R. L., Wu, Z. Y., Zhou, X. F., & Andersen, H. J. (2008). Water distribution and microstructure in enhanced pork. Journal of Agricultural and Food Chemistry, 56, 7201–7207. Chen, H. S., Kong, B. H., Guo, Y. Y., Xia, X. F., Diao, X. P., & Li, P. J. (2013). The effectiveness of cryoprotectants in inhibiting multiple freeze-thaw-induced functional and rheological changes in the myofibrillar proteins of common carp (cyprinus carpio) surimi. Food Biophysics, 8, 302–310. Cheong, L. Z., Zhang, H., Xu, Y., & Xu, X. B. (2009). Physical characterization of lard partial acylglycerols and their effects on melting and crystallization properties of blends with rapeseed oil. Journal of Agricultural and Food Chemistry, 57, 5020–5027. Desmond, E. (2006). Reducing salt: A challenge for the meat industry. Meat Science, 74(1), 188–196. Diao, X. Q., Guan, H. N., Kong, B. H., & Zhao, X. X. (2017). Preparation of diacylglycerol from lard by enzymatic glycerolysis and its compositional characteristics. Korean Journal for Food Science of Animal Resources, 37(6), 813–822. Diao, X. Q., Guan, H. N., Kong, B. H., Han, Q., & Zhao, X. X. (2016). Analysis of the thermal properties and structural characteristics of purified glycerolytic lard. Food and Fermentation Industries, 43(4), 98–103. Diao, X. Q., Guan, H. N., Zhao, X. X., Chen, Q., & Kong, B. H. (2016). Properties and oxidative stability of emulsions prepared with myofibrillar protein and lard diacylglycerols. Meat Science, 115, 16–23. Diao, X. Q., Guan, H. N., Zhao, X. X., Diao, X. P., & Kong, B. H. (2016). Physicochemical and structural properties of composite gels prepared with myofibrillar protein and lard diacylglycerols. Meat Science, 121, 333–341. Egelandsdal, B., Fretheim, K., & Samejima, K. (1986). Dynamic rheological measurements on heat-induced myosin gels: Effect of ionic strength, protein concentration and addition of adenosine triphosphate or pyrophosphate. Journal of the Science of Food and Agriculture, 37, 915–926. Feng, J. H., Cao, A. L., Cai, L. Y., Gong, L. X., Wang, J., Liu, Y. G., ... Li, J. R. (2018). Effects of partial substitution of NaCl on gel properties of fish myofibrillar protein during heating treatment mediated by microbial transglutaminase. LWT-Food Science and Technology, 93, 1–8. Feng, M. Q., Pan, L. H., Yang, X., Sun, J., Xu, Y. L., & Zhou, G. H. (2018). Thermal gelling properties and mechanism of porcine myofibrillprotein containing flaxseed gum at different NaCl concentrations. LWT-Food Science and Technology, 87, 361–367. Fregolente, L. V., Fregolente, P. B. L., Chicuta, A. M., Batistella, C. B., Filho, M. R., & WolfMaciel, M. R. (2007). Effect of operating conditions on the concentration of monoglycerides using molecular distillation. Chemical Engineering Research & Design, 85(11), 1524–1528. Gordon, A., & Barbut, S. (1990). Cold stage scanning electron microscopy study of meat batters. Journal of Food Science, 55, 1196–1198. Gordon, A., & Barbut, S. (1992). Mechanisms of meat batter stabilization: A review. Critical Reviews in Food Science and Nutrition, 32, 299–332. Guo, J. J., Zhou, Y. H., Yang, K., Yin, X. L., Li, Z. S., Sun, W. Q., & Han, M. Y. (2019). Effect of low-frequency magnetic field on the gel properties of pork myofibrillar proteins. Food Chemistry, 274, 775–781. Han, M. Y., Fei, Y., Xu, X. L., & Zhou, G. H. (2009). Heat-induced gelation of myofibrillar proteins as affected by pH-a low field NMR study. Agricultural Sciences in China, 42, 2098–2104. He, F. J., Burnier, M., & Macgregor, G. A. (2011). Nutrition in cardiovascular disease: Salt in hypertension and heart failure. European Heart Journal, 32(24), 3073–3080. Hegg, P. O. (1982). Conditions for the formation of heat-induced gels of some globular food proteins. Journal of Food Science, 47, 1241–1244. Howell, N. K., Herman, H., & Li-Chan, E. C. Y. (2001). Elucidation of protein-lipid interaction in a lysozyme-corn oil system by Fourier transform Raman spectroscopy. Journal of Agricultural and Food Chemistry, 49(3), 1529–1533. Hu, Pereira, Xing, Zhou, & Zhang. (2017). Thermal gelation and microstructural properties of myofibrillar protein gel with the incorporation of regenerated cellulose. LWT-Food Science and Technology, 86, 14–19. Jost, R., Baechler, R., & Masson, G. (1986). Heat gelation of oil-in-water emulsions stabilized by whey protein. Journal of Food Science, 51(2), 440–444. Kloss, L., Meyer, J. D., Graeve, L., & Vetter, W. (2015). Sodium intake and its reduction by food reformulation in the European Union-a review. NFS Journal, 1, 9–19. Lanier, T. C., Carvajal, P., & Yongsawatdigul, J. (2004). Surimi gelation chemistry. In J. W. Park (Ed.). Surimi and surimi seafood (pp. 451–470). (second ed.). New York, USA: Marcel Dekker. Lee, C. M., Hampson, J. W., & Abdollahi, A. (1981). Effect of plastic fats on thermal stability and mechanical properties of fat-protein gel products. Journal of the American Oil Chemists' Society, 58(11), 983–987. Lee, S. H., Lefévre, T., Subirade, M., & Paquin, P. (2007). Changes and roles of secondary structures of whey protein for the formation of protein membrane at soy oil/water interface under high-pressure homogenization. Journal of Agricultural and Food Chemistry, 55(26), 10924–10931. Lerma-García, M. J., Ramis-Ramos, G., Herrero-Martínez, J. M., & Simó-Alfonso, E. F. (2010). Authentication of extra virgin olive oils by Fourier-transform infrared spectroscopy. Food Chemistry, 118(1), 78–83. Li, Y. Q., Kong, B. H., Xia, X. F., Liu, Q., & Diao, X. P. (2013). Structural changes of the myofibrillar proteins in common carp (Cyprinus carpio) muscle exposed to a hydroxyl

4. Conclusion Incorporation of lard-based DG and change of NaCl concentrations had a profound effect on rheological and physicochemical gelation properties of porcine MP. The incorporation of UDG or PDG, the gel compression force and gel WHC were enhanced and the rheological properties (G′ and G″) and gel microstructure were improved with the increase in NaCl concentration. LF-NMR analysis revealed that the incorporation of UDG or PDG and the increase in NaCl concentration increased the entrapped water contents and decreased the free water contents. The secondary structure results revealed that the increase in NaCl concentration and the addition of UDG or PDG caused the increases in the amounts of α-helix and β-sheet, concomitant with decreases in β-turn and random coil contents, effects which could contribute to the formation of gel networks. Overall, the rheological and physicochemical properties of porcine MP can be improved by the increase in NaCl concentration and the incorporation of DGs, and lardbased DG displays potential for use in improving the quality of comminuted meat products. Declaration of interest statement The authors declare no competing financial interest. Acknowledgements This study was funded by the National Natural Science Foundation of China (31771990) and National Key Research and Development 10

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