Properties and oxidative stability of emulsions prepared with myofibrillar protein and lard diacylglycerols

Meat Science 115 (2016) 16–23

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Properties and oxidative stability of emulsions prepared with myofibrillar protein and lard diacylglycerols Xiaoqin Diao a,b, Haining Guan a,b, Xinxin Zhao a, Qian Chen a, Baohua Kong a,c,⁎ 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 Synergetic Innovation Center of Food Safety and Nutrition, Harbin, Heilongjiang 15000, China

a r t i c l e

i n f o

Article history: Received 17 September 2015 Received in revised form 21 December 2015 Accepted 4 January 2016 Available online 8 January 2016 Keywords: Lard diacylglycerols Myofibrillar proteins Emulsifying property Oxidation stability

a b s t r a c t The objective of this study was to investigate the emulsifying properties and oxidative stability of emulsions prepared with porcine myofibrillar proteins (MPs) and different lipids, including lard, glycerolized lard (GL) and purified glycerolized lard (PGL). The GL and PGL emulsions had significantly higher emulsifying activity indices and emulsion stability indices than the lard emulsion (P b 0.05). The PGL emulsion presented smaller droplet sizes, thus decreasing particle aggregation and improving emulsion stability. The static and dynamic rheological observations of the emulsions showed that the emulsions had pseudo-plastic behavior, and the PGL emulsion presented a larger viscosity and a higher storage modulus (G′) and loss modulus (G′′) compared with the other two emulsions (P b 0.05). The formation of thiobarbituric acid-reactive substances, carbonyl contents and total sulfhydryl contents was not significantly different between the emulsions with PGL, GL and lard (P b 0.05). In general, lard diacylglycerols enhanced emulsifying abilities and had no adverse effects on the oxidation stability of the emulsions prepared with MPs. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction In the meat industry, emulsion-type meat products are very popular and are produced by comminuting raw meat in the presence of water and lipids and forming an aqueous protein phase in which fat droplets are dispersed. In meat products, myofibrillar proteins (MPs) have better emulsion properties for keeping water and oil together (Sarma, Vidya Sagar Reddy, & Srikar, 2000), while fat droplets that are immobilized in the protein matrix play an important role in decreasing purge and cooking loss, improving water holding capacity and providing good mouthfeel and juiciness (Mendoza, García, Casas, & Selgas, 2001). However, diseases caused by consuming too much fat have aroused significant attention worldwide. Decreasing animal fat intake helps in preventing obesity, hypertension, hyperglycemia and cardiovascular heart diseases (Nejat, Polotsky, & Pal, 2010). Fat substitutes have been commonly used to develop low fat meat products, which have helped to improve human health. Diacylglycerols (DAGs) are esters of glycerol in which two of the hydroxyl groups are esterified with fatty acids (FAs). DAGs, particularly 1,3-DAG, have been reported to significantly suppress abdominal and visceral fat accumulation (Maki et al., 2002; Meng, Zou, Shi, Duan, & Mao, 2004), decrease postprandial serum triacylglycerol (TAG) levels ⁎ Corresponding author at: College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 15000, China. E-mail address: [email protected] (B. Kong).

http://dx.doi.org/10.1016/j.meatsci.2016.01.001 0309-1740/© 2016 Elsevier Ltd. All rights reserved.

(Tada, Watanabe, Matsuo, Tokimitsu, & Okazaki, 2001; Taguchi et al., 2000), and reduce body weight. The safety of DAGs has been proved previously in many animal and human studies in which DAGs were used as edible oils for human consumption (Morita & Soni, 2009). In addition, DAGs can be emulsified more easily than TAGs because DAGs have hydrophilic polar groups in their molecular structure and exhibit higher surface activity as well as interfacial properties (Shimada & Ohashi, 2003). Therefore, DAGs have advantageous effects in emulsions and are suitable for use as emulsifiers (Long et al., 2015). In addition, DAGs can improve food texture due to their higher melting points compared with TAGs (Miklos, Xu, & Lametsch, 2011). Lard is cheap and is very important for producing meat products, and DAGs can be prepared by lard glycerolysis. Some reports have shown that DAGs from plant oil and pork fat can be used as fat replacers in fermented sausages and meat emulsions (Miklos et al., 2011; Mora-Gallego et al., 2013). However, to the best of our knowledge, no information about the interaction between DAGs and myofibrillar proteins in emulsions has been reported. Miklos, Zhang, Lametsch, and Xu (2013) investigated the melting and crystallization properties of lardbased diacylglycerols in blends with lard. The results showed that with high concentrations of DAGs, the β-crystal form was more enhanced in the blends, which proved that mixing DAGs and triacylglycerols (TAGs) can impart specific functional properties in products containing solid fats. Proteins can be adsorbed onto the oil–water droplet interface by hydrophobic interactions with stabilizing emulsions (Hu, McClements, & Decker, 2003; Lee, Lefèvre, Subirade, & Paquin,

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2009). Therefore, it is important to understand the compatibility of binary blends of fat globules and proteins in emulsions. DAGs can be produced by direct esterification, partial hydrolysis, or chemical/enzymatic glycerolysis of fats/oils with glycerol. Our previous experiment revealed that enzymatic glycerolysis and molecular distillation can be used to prepare highly purified DAGs from lard. Under the optimum reaction conditions, the conversion rate of TAGs and the content of DAGs in the reaction mixture reached 76.26% and 61.76%, respectively, and the DAGs content in PGL reached 82.03% by molecular distillation (unpublished results). The objective of this study was to evaluate the emulsifying properties (emulsifying activity, emulsion stability, ζ-potential, particle size distribution, viscosity, and microstructure) and oxidation stability [thiobarbituric acid reactive substances (TBARS), carbonyl and total sulfhydryl contents] of the emulsion formed by myofibrillar proteins and lard DAGs.

2.4. Emulsion preparation

2. Materials and methods

2.5. Emulsion characteristics

2.1. Materials

2.5.1. Emulsifying activity indices (EAI) and emulsion stability indices (ESI) Aliquots (50 μL each) of freshly prepared emulsions were pipetted 0.5 cm from the bottom of the container at 0 and 10 min and were dispersed in 5 mL of 0.1% sodium dodecyl sulfate (SDS) solution. Absorbances of different emulsions were measured at 500 nm by a UT-1800 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China), and a 0.1% SDS solution served as the blank. The EAI (m2/g) and ESI (%) were calculated as follows, respectively:

Fresh pork backfat and pork loin muscle were purchased from Beidahuang Meat Corporation (Harbin, Heilongjiang, China). Glycerol with a purity of greater than 99.0% was obtained from Tianjin Chemical Reagent Factory (Tianjin, China). Commercial immobilized lipases Lipozyme RMIM from Rhizomucor miehei [lipase activity 275 Inter Esterase Units Novo (IUN)/g] were purchased from Novozymes A/S (Bagsvaerd, Denmark). 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB), 2,4-Dinitrophenylhydrazine (DNPH), and fluorescent dyes, including nile red and fluorescamine were purchased from SigmaChemical Co. (St. Louis, MO, USA). All reagents were of analytical grade.

2.2. Preparation of lard diacylglycerols Lard DAGs were produced according to our previous method (unpublished paper). Lard was extracted by heating the backfat at 120 °C and stirring constantly. The liquid lard was filtered through four layers of cotton cloth to remove oil residues and was subsequently solidified and stored at 4 °C. The reaction mixture, including melted lard and glycerol was utilized in the glycerolysis reaction that employed Lipozyme RMIM under the following conditions: 14:100 (W/W) of enzyme to lard substrate ratio, 1:1 M ratio of lard to glycerol, and 500 rpm magnetic stirring speed. The reaction mixture was first incubated at 65 °C for 2 h and then transferred to 45 °C for 8 h. After the reaction, the enzyme was filtered to yield the glycerolized lard (GL), in which the content of DAGs was 61.76%. The GL was purified by two-step wiped film molecular distillation (SPE10, manufactured in Haiyuan Biochemical Equipment Co. Ltd., Wuxi, China). The components of glycerol, free fatty acids, and monoacylglycerols in the light phase were separated from GL in the first step. The purified glycerolized lard (PGL), which had a higher DAG content (82.03%), was obtained in the second purification step. The conditions for the distillation process were as follows: evaporation temperatures, 185 and 255 °C, and evaporator vacuum, 60 and 25 Pa for the first and second steps, respectively. The other distillation conditions were the same for both steps: feeding flow rate, 2 kg/h; scraper speed, 350 rpm.

2.3. Myofibrillar protein preparation MPs were prepared according to the procedure of Xia, Kong, Liu, & Liu, (2009). The final prepared MPs were kept at 2–4 °C and utilized within 48 h. Protein concentrations were measured by the biuret method with bovine serum albumin as the standard.

Emulsions were prepared with melted pork fat (lard, GL or PGL) and 1% MP solution (0.6 M NaCl, 50 mM sodium phosphate, pH 6.2). The diluted MP solution was first prepared using a vortex mixer until complete dissolution. Then, 2 mL each of lard, GL or PGL was added to 8 mL of an MP solution and homogenized at 16,000 rpm for 60 s using an IKA T18 Ultra-Turrax (IKA-Werke GmbH & Co., Staufen, Germany). Aliquots of the prepared emulsions were maintained at room temperature (approximately 23 °C) to measure the emulsifying properties, including emulsifying activity and emulsion stability, ζ-potential, particle size distribution, viscosity and microstructural characterization. Other aliquots of the emulsions were placed in tightly sealed vials and stored in the dark at room temperature (22–24 °C) for 0, 2, 4, 6, and 8 days to investigate the oxidative stabilities of the emulsions by determining TBARS, carbonyl contents and total sulfhydryl contents.


 2  2:303  A500  dilution factor EAI m2 =g ¼ C  ð1−φÞ104 A10 ESIð%Þ ¼  100 A0 where C (g/mL) is the protein concentration before emulsification, φ (v/v) is the oil volume fraction of the emulsion, and A0 and A10 represent the absorbances at 500 nm after emulsification for 0 and 10 min, respectively. 2.5.2. ζ- potential measurements The ζ-potentials of different fresh emulsions were measured at room temperature using a ZetaSizer Nano-ZS instrument (Malvern Instruments Co. Ltd., Worcestershire, UK). Each measurement was repeated three times. 2.5.3. Measurements of droplet mean diameters and particle size distributions The droplet mean diameters and particle size distributions of different emulsions were analyzed by the laser light scattering method using a mastersizer 2000 instrument (Malvern Instruments Ltd., Worcestershire, UK). The emulsion was dispersed in distilled water until the shading degree reached 15% to avoid multiple scattering. The refractive indexes of the continuous and dispersed phases were set at 1.330 and 1.475, respectively. The droplet average diameters were calculated using the following equations: d3;2 ¼ d4;3 ¼

X X

3 X 2 ni di = nd X i i3 4 ni di = ni di

where di is the droplet diameter, and ni is the number of droplets with diameter di. The measurement results can be expressed as Dv10, Dv50, Dv90, d3,2 and d4,3, where Dv10, Dv50 and Dv90 are the droplet volumes with diameters smaller or equal to these values, which accounted for 10, 50 and 90% of all of the particles in the entire emulsion, respectively. d3,2 is the surface-weighted mean particle diameter, and d4,3 is the volumeweighted mean particle diameter.

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2.5.4. Viscosity measurements of emulsions Static and dynamic rheological measurements of different emulsions were recorded using a Bohlin Gemini HR nano rheometer (Malvern Instruments Ltd., Worcestershire, UK) with a stainless steel parallel plate system at 25 °C. Emulsions (1 mL) were loaded onto the lower plate, and the upper plate (40 mm in diameter) was slowly lowered to narrow the distance between the two parallel plates to 1 mm. Excess sample leaked out of the plates was trimmed off. Steady shear rheological properties were determined by the control shear rate, which was increased from 0.01 to 100 s−1 within 5 min. The relationship between the shear rate of the emulsion and the apparent viscosity was recorded. The dynamic viscoelastic properties of all emulsions were evaluated using a small amplitude oscillatory frequency sweep mode. The frequency was oscillated from 0.1 to 10 rad/s with a strain of 0.4% within the linear viscoelastic region. The storage modulus (G′) and loss modulus (G′′) were recorded. All measurements were obtained in three replicate experiments for each sample. 2.5.5. Microstructural characterization of emulsions Microscopic images of droplets in different emulsions were captured at room temperature with a light microscope (Olympus BX50, Optical Co. Ltd., Tokyo, Japan). A drop of sample was placed on a clean microscope slide and gently covered with a cover slip before microscopic observation at a magnification of 100 ×. Images were recorded using a camera connected to a computer. Confocal imaging observations of emulsions were performed using a confocal laser scanning microscope (CLSM) (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany) at room temperature with an HCX PL APO 40 × objective. Emulsions for micro structural analyses were stained with 0.01% nile red in ethanol and 0.1% fluorescamine in acetone to label the lipids and MPs, respectively. The stained emulsions were smeared onto microscope slides, covered with cedar oil-coated cover slips and examined with a 40 magnification lens. The fluorescent dyes were excited by a Helium Neon Red laser (HeNe-R) at 543 nm for lipid analysis and a Helium Neon Green laser at 488 nm for protein analysis, and the emission wavelengths were collected at 598 and 540 nm, respectively. Multiple fields were viewed, and representative images were acquired.

modifications. Diluted emulsions (1 mL) of 2 mg/mL MP concentrations were reacted with 1 mL of 10 mM DNPH in 2 M HCl with agitation for 1 h at room temperature; another emulsion (1 mL) mixed with 2 M HCl (1 mL) was used as a control. After the reaction, 20% TCA (1 mL) was added, and then the mixtures were centrifuged for 10 min at 8500 ×g. The sediments were washed three times with 1 mL of ethyl acetate: ethanol (1:1 v/v) and subsequently dissolved with 6 M guanidine hydrochloride (3 mL) at 37 °C for 15 min. After centrifugation (8500 ×g, 3 min), the absorbances of the supernatants were measured at 370 nm. The carbonyl formation was expressed as nmol carbonyl/mg protein using an absorption coefficient of 22,000 M− 1 cm− 1 for protein hydrazones.

2.7.2. Total sulfhydryl contents Sulfhydryl (SH) contents were assessed as described by Ellman (1959). Diluted emulsions (1 mL) of 2 mg/mL myofibril protein concentrations were vortexed with 8 mL of buffer (0.086 M Tris, 0.09 M glycine, 4 mM EDTA at pH 8.0). The mixtures (4.5 mL) and 0.5 mL of Ellman's reagent (10 mM DTNB) were incubated in the dark for 30 min at room temperature. After centrifugation (l0,000 ×g, 15 min), the absorbances of the supernatants were measured at 412 nm. The SH contents were expressed as nmol SH/mg of protein using a molar extinction coefficient of 13,600 M−1 cm−1.

2.8. Statistical analyses Three batches of emulsions were prepared independently to study the effects of myofibrillar proteins and different pork fats (lard, GL or PGL) on the emulsifying properties and oxidation stabilities of emulsions. For each batch of emulsion samples, all of the specific experiments were conducted in triplicate (triplicate observations). The results are expressed as the mean values ± standard deviations. The data were analyzed using the General Linear Model procedure of the Statistix 8.1 software package (Analytical Software, St. Paul, MN, USA). The significance of the main effects was determined by analysis of variance (ANOVA), and significant differences (P b 0.05) among the means were identified by the Tukey procedure.

2.6. Lipid oxidation Lipid oxidation was evaluated by measuring TBARS, which is a secondary oxidation product, according to the method of Qiu, Zhao, Decker, and McClements (2015) with slight modifications. Emulsions (1.0 mL) were mixed with thiobarbituric acid solution (2.0 mL), vortexed, and subsequently heated for 15 min in a boiling water bath. The reaction products were cooled to room temperature and centrifuged at 1800 g for 10 min. The absorbances of the supernatants were measured at 532 nm. The TBARS values were calculated using the following equation and expressed as milligrams of malonaldehyde (MDA) per kilogram of oil sample: TBARS ðmg MDA=L oilÞ ¼

A532  M  V  1000 ε1v

where A532 is the absorbance at 532 nm; M is the molecular weight of MDA (72 g); V is the emulsion volume (1 mL); ε is the molar extinction coefficient (156,000 M−1 cm−1) of the red TBA reaction product; 1 is the optical path (1 cm) and v is the oil volume (mL). 2.7. Protein oxidation 2.7.1. Carbonyl content Carbonyl formations were evaluated by reacting emulsions with DNPH to form protein hydrazones using the method described by Oliver, Ahn, Moerman, Goldstein, and Stadtman (1987) with slight

3. Results and discussion 3.1. Emulsifying properties Generally, EAI is dictated by protein–lipid and protein–protein interactions, and ESI is associated with continuous and dispersed phases (Wu, Xiong, Chen, Tang, & Zhou, 2009). The possibility of forming a stable emulsion depends on protein molecules remaining at the interface after adsorption to stabilize oil droplets (Sakuno, Matsumoto, Kawai, Taihei, & Matsumura, 2008). EAI and ESI of emulsions with lard, GL and PGL are shown in Fig. 1. EAI of the emulsion with lard was 4.76 (m 2/g), which was significantly lower than those with GL (12.0 m2/g) and PGL (14.9 m2/g) (P b 0.05). The EAI value of the emulsion with PGL was higher than that with GL, but there was not a significant difference between them (P N 0.05). The ESI values of different emulsions had a similar behavior as the EAI values. The maximum ESI value was obtained from the emulsion with PGL. The emulsion with lard had a significantly lower ESI compared with those of GL and PGL. The results indicated that GL and PGL could more efficiently disperse in emulsions than lard, which may be attributed to the hydrophilic groups in DAGs that can more strongly associate with water droplets dispersed throughout emulsions (Shimada & Ohashi, 2003). The acylglycerol structures of GL and PGL are more flexible than lard and could interact more intensely with proteins at the water–oil interface.

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emulsions are expected to affect the chemical and physical stabilities of the emulsions. The solution with “0” electrical charges (ζ-potential) have the lowest emulsifying stability, and increase the absolute value of ζ-potential will increase the emulsifying stability. Low electrical charges (ζ-potential) of emulsion droplets decreased the electrostatic repulsion between droplets, which was no longer strong enough to overcome various attractive interactions, thus causing droplet aggregation (Guzey, Kim, & McClements, 2004), and an unstable emulsion was eventually formed. 3.3. Droplet mean diameters and droplet size distributions of emulsions

Fig. 1. Emulsifying activity index (EAI) and emulsion stability index (ESI) of emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL). Error bars represent standard deviations obtained from triplicate sample analyses. Means in the same indexes with different letters (a–b) differ significantly (P b 0.05).

3.2. ζ- potential measurements ζ-potential can reflect the potential stability of emulsion systems according to its magnitude. The ζ-potential values (negative) of all emulsions with different types of fats are shown in Fig. 2. The negative ζpotentials in the molecular structures of different emulsions can be mainly attributed to the negatively charged MPs. The absolute values of the ζ-potentials of the GL and PGL emulsions were 31.2 and 35.2 mV, which were significantly higher than that of the lard emulsions (18.3 mV), and the ζ-potential of the PGL emulsion was significantly higher than that of the GL emulsion (P b 0.05). Sakuno et al. (2008) reported that differences in ζ-potential do not result from the components of different fats in the emulsions but can be attributed to the layer of different proteins adsorbed on droplet surfaces. Dickinson and Iveson (1993) showed that lower oil polarities can result in higher protein surface coverage in emulsions. Therefore, the adsorbed protein amount was higher in the lard emulsions than in the GL and PGL emulsions, causing the plane of shear to be pushed farther away from the oil– water interface, leading to lower absolute values of ζ-potential in the lard emulsions. The magnitude and sign of the droplet charges in

Fig. 2. ζ- potentials of emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL). Error bars represent standard deviations obtained from triplicate sample analyses. Means with different letters (a–c) differ significantly (P b 0.05).

The mean diameters (Dv10, Dv50 and Dv90) of the oil droplets, volume-mean diameters (d4,3) and volume-surface mean diameters (d3,2) of the emulsions with lard, GL and PGL are listed in Table 1, and the particle size distributions are presented in Fig. 3. The Dv10, Dv50 and Dv90 values of the GL and PGL emulsions were significantly lower than those of the lard emulsion, and the PGL emulsion had the lowest Dv10, Dv50 and Dv90 values (P b 0.05). The smaller droplet sizes of the PGL emulsion were possibly derived from the high miscibility of PGL with water because of the presence of free hydroxyl groups in DAGs (Long et al., 2015). Larger droplet diameters result in a sizable interface area between the two phases in emulsions. The higher d3,2 signifies lower specific surface areas, and increases in d4,3 means individual droplets are forming into larger aggregates (Hebishy, Buffa, Guamis, & Trujillo, 2013). The emulsion with lard showed larger oil droplets (d4,3 and d3,2) than those with GL and PGL (P b 0.05), which indicated the instability of the lard emulsion. The results of droplet mean diameters and droplet size were consistent with ζ-potential measurements. The higher electrical charges in GL and PGL emulsions will contribute to the higher repulsive force between the droplets, which will inhibit emulsion droplet aggregation and improve the emulsifying stability. In emulsions, particle size distribution is generally known to be an important parameter for evaluating the quality of an emulsion (HuckIriart, Pizones Ruiz-Henestrosa, Candal, & Herrera, 2013). The droplet sizes of the emulsion with lard show a slight bimodal distribution (Fig. 3), indicating that there may be droplet coalescence (Wang, Li, Wang, & Özkan, 2010). A left shift of a distribution peak of the PGL emulsion toward smaller sizes was detected. The diminishing ratio of larger droplets from the distribution can indicated increased stability of the emulsion. 3.4. Static and dynamic rheological measurements Rheological properties can also be used to evaluate the stability of an emulsion. Flow profiles of emulsions with lard, GL and PGL were plotted in the form of the shear rate dependence of apparent viscosities (Fig. 4A). Obvious shear-thinning behaviors were perceived for the three emulsions with increased shear rate, and the three emulsions displayed non-Newtonian pseudo plastic behaviors. The change in rheological behavior was mainly attributed to the change in the aggregation state of the droplets (Mao & McClements, 2013). The emulsion with lard had a relatively low apparent viscosity and reached a constant value at a shear rate N0.1 s−1. However, the viscosity of the emulsion with PGL was higher than that of the emulsions with lard and GL and reached a constant value when the shear rate was greater than 1 s−1. As protein and fat interact, the protein matrix extends to emulsified fat and limits the aggregation of emulsion droplets, causing the emulsion viscosity to increase (Ömer, 2006; Chen & Dickinson, 1999). High viscosities could hinder emulsion droplets from colliding and prevent their coalescence and aggregation, resulting in better emulsion stability. These observations were consistent with the results of the droplet mean diameters and droplet size distributions of the emulsions. Dynamic oscillatory testing is an important method for studying the viscoelastic properties of emulsions. The storage modulus (G′) and loss modulus (G′′) of emulsions were measured, and plots of G′ and G′′

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Table 1 Droplet mean diameters, d3,2 and d4,3 of emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL). Fat type

Lard GL PGL

Droplet mean diameter (μm) Dv10

Dv50

Dv90

21.00 ± 0.35a 12.96 ± 2.55b 9.52 ± 0.61b

214.86 ± 3.35a 51.71 ± 6.10b 32.73 ± 0.63c

448.33 ± 11.29a 140.13 ± 13.12b 70.68 ± 0.38c

d3,2 (μm)

d4,3 (μm)

29.95 ± 0.54a 14.55 ± 2.07b 11.89 ± 0.38b

229.89 ± 4.86a 70.49 ± 6.37b 36.81 ± 0.42c

Different letters in the same column indicate statistically significant differences (P b 0.05). Dv10, Dv50 and Dv90 correspond to cumulative distributions at 10, 50 and 90%; d3,2 indicates the surface-weighted mean particle diameter; d4,3 represents the volume-weighted mean particle diameter.

versus frequency are shown in Fig. 4B. G′ represents the amount of energy stored in the elastic structure or the recoverable energy, and G′′ represents the energy lost by viscous dissipation per cycle of deformation. Both moduli of different emulsions slightly increased as the oscillatory frequency was increased, and the G′ value was greater than the G′′ value in the linear viscoelastic range. These observations were in agreement with the results of Ng, Lai, Abas, Lim, and Tan (2014), who stated that the G′ value was higher than the G′′ value in a palm olein-based diacylglycerol emulsion as the frequency was increased. The increase in G′ was most likely due to the formation of interactions between droplets and proteins (Li, Fu, Luo, & Huang, 2013). Furthermore, Turan, Altay, and Güven (2015) reported that the G′ value was greater than the G′′ value in emulsions, indicating the presence of a weak-gel model, in which weak interactions ensure the stability of emulsions. In this study, the PGL emulsion had the highest G′ and G′′ values among the three emulsions, and the GL emulsion showed higher G′ and G′′ values than the lard emulsion. The higher G′ reflected the presence of some non-covalent physical crosslinks between fats and proteins and suggested that the DAGs played an important role in intensifying the elastic properties of emulsions.

have higher miscibility with water (Long et al., 2015). Moreover, the low affinity of lard for water could induce coalescence of droplets, leading to the generation of large droplets. CLSM observations explained the interaction of fats and proteins in emulsions (Fig. 5B) and showed that all of the particles in emulsions coated with a layer of membrane protein were dispersed throughout the entire emulsion system. Stokes and Telfrod (2004) previously reported that mayonnaise has a complex microstructure consisting primarily of a large volume of oil phase dispersed within a protein-rich aqueous phase. The PGL emulsion had a much smaller oil droplet size (shown in red) than those from lard and GL. The oil droplet size in the GL emulsion was smaller than that in the lard emulsion and larger than that in the PGL emulsion. Careful examination of the microscopic images in the PGL emulsion indicated that the oil droplets were better distributed in the emulsion and little coalescence was present, which

3.5. Microstructures of emulsions To elucidate the micro morphology of the MP emulsions with lard, GL and PGL, optical photomicrographs of all of the emulsions were examined, and typical micrographs of emulsions are presented in Fig. 5A. The light microscopy observations supported the EAI and ESI results. The micrographs revealed that emulsions prepared with different fats varied significantly in particle diameters and volumes. The emulsions prepared from MPs with GL and PGL showed a more effective and even dispersion of oil than the lard emulsion. The emulsion prepared with lard had a relatively larger particle diameter, which was consistent with the droplet size distribution. The current study also suggested that the PGL emulsion has tiny droplets. The differences in the droplet diameters between different emulsions are because DAGs

Fig. 3. Volume-based droplet size distributions of the emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL).

Fig. 4. Plots of viscosity (Pa·s) as a function of shear rate (0.01–100 s−1) (A) and plots of modulus response subjected to frequency sweep at strain amplitudes of 0.4% (B) of different emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL).

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Fig. 5. Optical microscopy images (A) and confocal laser scanning microscopic (CLSM) pictures (B) of emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL). The emulsions were stained with fluorescence dye nile red and fluorescamine green to observe the oil phase and proteins, respectively, in the CLSM pictures.

was in close agreement with the optical microscopy image. As reported by Worrasinchai, Suphantharika, Pinjai, and Jamnong (2006), when the droplets in emulsions are close enough to interact with each other, three-dimensional networks of aggregated droplets can form. The large oil droplets in the lard emulsion tended to aggregate, which may have led to the instability of this emulsion. The small droplets in the GL and PGL emulsions may contribute to the high EAI and ESI values of the emulsions. 3.6. Oxidative stabilities of emulsions 3.6.1. Lipid oxidation Lipid oxidation of the emulsion prepared with MP and pork fat was studied by measuring TBARS (Fig. 6). TBARS production increased significantly (P b 0.05) in the three emulsions during storage, which revealed that longer storage times could obviously increase the formation of malonaldehyde or other secondary products. TBARS productions in the GL and PGL emulsions were higher than that in the lard emulsion over 8 days of storage, but there were no significant differences among the three emulsions (P N 0.05). Some studies have indicated that positively charged interfaces repel prooxidant metals from the droplet surfaces and thereby decrease oxidation, whereas negatively charged interfaces easily attract prooxidant metals and thus enhance oxidation (Waraho, Cardenia, RodriguezEstrada, Julian McClements, & Decker, 2009). Our ζ- potential results indicated that the droplets of all emulsions were negative and that the absolute potential of the emulsion with PGL was higher than those of other emulsions, which also explains why the emulsion with PGL had a higher oxidation activity. However, there were no significant differences (P N 0.05) among the three emulsions during storage. 3.6.2. Protein oxidation Many amino acid side-chains are readily modified by oxidants to form carbonyl derivatives; therefore, carbonyl content is commonly

utilized to assess the extent of protein oxidation (Estévez, 2011). As presented in Fig. 7A, carbonyl formation gradually increased in all emulsions with storage duration (P b 0.05), and the carbonyl contents in the PGL and GL emulsions were higher than that in the lard emulsion during storage, although there were no significant differences (P N 0.05) among the three emulsions. The results revealed that the effect of different types of fat on carbonyl formation was much less significant than the storage time. Carbonylation is a non-enzymatic and irreversible protein modification that results in the formation of carbonyl groups induced by oxidative stress (Estévez, 2011). Emulsions of meat proteins and lipids are very susceptible to oxidative reactions that lead to carbonyl

Fig. 6. The formation of TBARS in emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL) during storage. Error bars represent standard deviations obtained from triplicate sample analyses. Significant differences (P b 0.05) between emulsions within storage days are denoted by different letters.

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4. Conclusions The study indicated that emulsion properties can be significantly improved by DAGs. The emulsion prepared with MP and PGL had a higher emulsifying activity and emulsion stability, low negative electrical charge, small particle mean diameter, narrower droplet size distribution, homogeneous microstructure and low partial fat aggregation compared with the emulsion prepared with lard. The emulsion with PGL also had higher viscosity and higher storage G′ and G′′ moduli than those of the emulsion with lard. The TBARS, carbonyl and sulfhydryl contents in the different emulsions were measured to investigate the oxidation stability of the emulsions and show that the oxidative stability of the PGL emulsion was decreased but was not significantly different compared with the lard emulsion. In general, present results revealed that lard DAGs improved emulsifying abilities of emulsion prepared with MPs and had no obvious adverse effects on the oxidation stability of emulsions. So, lard DAGs have the very good application prospect in emulsion-type meat products, such as frankfurters, bolognas, and wieners. The studies about application of DAGs in emulsion-type sausage, which was a part of the overall goal of the research, will be reported in a separate paper.

Acknowledgments This study was funded by the Science and Technology Major Projects in Heilongjiang (grant no. GA15B302) and the National Natural Science Foundation of China (grant no. 31471599).

References

Fig. 7. Carbonyl contents (A) and total sulfhydryl contents (B) in emulsions prepared with myofibrillar proteins and lard, glycerolized lard (GL), or purified glycerolized lard (PGL) during storage. Significant differences (P b 0.05) between emulsions within storage days are denoted by different letters.

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