Meat Science 121 (2016) 333–341

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Meat Science

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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
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 15000, China
b
College of Food and Pharmaceutical Engineering, Suihua University, Suihua, Heilongjiang 152061, China
c
College of Animal Science, Northeast Agricultural University, Harbin, Heilongjiang 15000, China

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to investigate the physicochemical and structural properties of composite gels
Received 28 April 2016 prepared with porcine myofibrillar protein (MP) and lard, glycerolized lard (GL) or purified glycerolized lard
Received in revised form 2 July 2016 (PGL). The gels prepared with MP and GL or PGL had significantly higher penetration force and water-holding ca-
Accepted 7 July 2016
pacity (WHC) than the gel with lard (P b 0.05) and formed a more compact and orderly microstructure. Com-
Available online 08 July 2016
pared with the distributions of T2 relaxation times of the pure MP gel, T21 and T22 of the gels that were
Keywords:
prepared with GL or PGL moved in the direction of slower relaxation time, which suggests that the water mobility
Lard diacylglycerols in the gel system was restricted. The presence of lard, GL and PGL did not affect the participating proteins in com-
Myofibrillar protein posite gels. The presence of GL and PGL altered the secondary and tertiary structures of MP in composite gels,
Gel properties which changed the gel properties. In general, the composite gels that were prepared with MP and GL or PGL
Structural changes showed improved gel quality.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Myofibrillar protein (MP) has notably important biological func-
tions, 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 three-
dimensional 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 en-
trapment 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 cardiovas-
cular 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. Previ-
ous 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: kongbh63@hotmail.com (B. Kong).
(2011) reported that DAGs could improve the food texture and water
retention. In addition, DAGs have been reported to significantly sup-
press 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 emulsi-
fying 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 ox-
idation 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 determi-
nant 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) re-
ported 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 de-
scribe the effect of lard DAGs on the gel strength, water-holding capac-
ity, gel-forming proteins and microstructures of composite gels that

http://dx.doi.org/10.1016/j.meatsci.2016.07.002
0309-1740/© 2016 Elsevier Ltd. All rights reserved.
334 X. Diao et al. / Meat Science 121 (2016) 333–341
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.
2. Materials and methods
2.1. Materials
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 com-
mercial 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 de-
scribed procedure (Xia, Kong, Liu, & Liu, 2009). The final protein concen-
tration 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 compos-
ites 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
investigate the secondary and tertiary structural changes of MP in com-
posite gels using Fourier transform infrared spectra and intrinsic fluo-
rescence measurement.
2.5. Gel strength
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).
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 sur-
face of the gels was soaked using filter paper. WHC (%) was expressed as
the ratio between the gel weights after centrifugation and before centri-
fugation, 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 reso-
nance 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 electropho-
resis (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% ac-
rylamide 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). Dif-
ferent dissolving solutions were used: 8 M urea + 50 mM sodium phos-
phate (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 ho-
mogenized 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 gel-
forming protein, chilled to room temperature and subsequently
 X. Diao et al. / Meat Science 121 (2016) 333–341 335

centrifuged at 10,000 ×g for 15 min. The protein content in the superna-
tant 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 calcu-
lated using the relative protein content in the supernatants in compar-
ison 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).
3. Results and discussion

2.10. Microstructure of composite gels 3.1. Gel strength and water-holding capacity

The microstructure of the composite gels was examined using a The gel strength is an important quality property of muscle protein,
scanning electron microscope (SEM). The gel samples which is notably related to the texture and sensory quality of muscle
(5 × 5 × 5 mm3) were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M food. To elucidate the effect of lard DAGs on MP gel properties, gels
phosphate buffer (pH 7.2) at 4 °C overnight and subsequently washed that were prepared with different fat contents were assessed using a
three times with the above phosphate to remove the glutaraldehyde penetration test. As displayed in Fig. 1A, the addition of lard, GL and
fluid. The composite gels were sequentially dehydrated in ethanol PGL significantly increased the gel strength (P b 0.05), and the extent
with a series of concentrations of 50, 70, 80, 90 and 100% (v/v) for of increase was proportional to the fat addition. MP gels with GL and
10 min each. To remove the ethanol, the dehydrated samples were suc- PGL tended to be more rigid than those with lard, and the difference
cessively immersed in tertiary butanol-ethanol (100%) (1:1) and tertia- was significant (P b 0.05). The gel strength was improved because the
ry butanol for 15 min and freeze-dried. The dried samples were fat globules might behave as “fillers” in the voids of the gels to make
mounted on a bronze stub and sputter-coated with gold (Sputter coater the gels have a more compact structure. As proven in our previous find-
SPI-Module, West Chester, PA, USA). Then, the specimens were ob- ing, PGL and GL have smaller fat globules (Diao et al., 2016), which can
served and photographed with an SEM (S-3400N, Hitachi, Tokyo, adequately fill the gel network structure. Increased the gel strength by
Japan) at an accelerating voltage of 5 kV. addition of lard DAGs can improve the texture and sensory quality of
cooked meat products, so lard DAGs have a good application prospect
2.11. Fourier transforms infrared spectra in the meat products.
A well-structured protein gel can entrap and immobilize a large
The Fourier transform infrared spectra (FT-IR) of the freeze-dried amount of water, fat and other food components through the capillary
samples were recorded using a Perkin-Elmer Spectrum 100 (Perkin- effects of its matrices. WHC is one of the most important functional
Elmer 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 compo-
nents 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). Fi-
nally, the GCF was adjusted to give the best least-squares fit of the indi-
vidual bands to each deconvoluted spectrum. The relative amounts of
α-helix, β-sheet, β-turn and random coil structures of MP in the com-
posite 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.

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 exci-
tation 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 ± stan-
Fig. 1. Penetration force (A) and water-holding capacity (B) of composite gels that were
dard errors. The analysis of variance (ANOVA) with Tukey's multiple prepared with myofibrillar proteins (MP) and lard, glycerolized lard (GL) or purified
comparison was used to measure the significance of the main effects glycerolized lard (PGL). The lowercase letters (a–c) indicate significant differences
(P b 0.05). Data regarding physicochemical and structural properties (P b 0.05) among different types of fat at identical fat contents.
336 X. Diao et al. / Meat Science 121 (2016) 333–341
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 pre-
pared 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 be-
tween proteins and water could bind more water molecules in the cap-
illaries 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 compo-
nent 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
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.
that were prepared with MP and lard, GL or PGL. Three relaxation com-
ponents 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 in-
creased 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 indi-
cates that PGL significantly increased the ability of protein-binding hy-
drogen protons. The result can be attributed to the development of a
three-dimensional network structure, which holds water in a less
 X. Diao et al. / Meat Science 121 (2016) 333–341 337

mobilized state (Ishioroshi, Samejima, & Yasui, 1979). The correspond-
ing 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 with-
out fat. The gel with fats, particularly PGL, obviously reduced the inten-
sity 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.
presence of disulfide interactions, hydrogen bonds and hydrophobic
contacts in MP gel. Jiang and Xiong (2013) suggested that with the ad-
dition 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 force-
disruption 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 in-
creased 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 ob-
serve the proteins that remained extractable (Fig. 3). The myofibrillar
protein band mainly contained myosin heavy chains (MHC), actin, tro-
ponin-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 simi-
lar 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 forma-

tion 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).
338 X. Diao et al. / Meat Science 121 (2016) 333–341
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 hydropho-
bic interactions also play an active role in the composite gels. During the
gel formation process, when the fat concentration increases, the hydro-
phobic 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 penetra-
tion force. Wu et al. (2011) reported that an interactive protein film sur-
rounding 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 disul-
fide bonds in the fat globule membrane and between the membrane
and the continuous protein gel matrix are also significant for the stabil-
ity 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
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).
(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
 X. Diao et al. / Meat Science 121 (2016) 333–341
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 α-
helix, 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 β-
sheet favoured the gelation of porcine myosin. Sano et al. (1994) re-
vealed that the α-helix structure was an important conformation to
maintain the secondary structure of native protein and was mainly sta-
bilized 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 com-
posite gels. With the addition of lard, GL and PGL, the contents of α-
helix, β-sheet and β-turn of MP in the composite gels gradually
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).
339
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 em-
bedment of protein-coated GL and PGL in a continuous, three-dimen-
sional 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 pen-
etration force of composite gels. In conclusion, lard, GL and PGL in com-
posite 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 interac-
tion 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 fluores-
cence energy (Burstein, Vedenkina, & Ivkova, 1973). At 280 nm, the
tryptophan residues of MP can be excited; therefore, the changes of ter-
tiary 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 un-
folded, the tryptophan residues become exposed to the hydrophilic en-
vironment, which decreases the fluorescence intensity (Pallarès,
Vendrell, Avilés, & Ventura, 2004). In the folded state, tryptophan resi-
dues are generally located in the hydrophobic environment of the pro-
tein with high fluorescence intensity. Puscasu and Birlouez-Aragon
(2002) also reported that tryptophan fluorescence had good correla-
tions with the tryptophan concentration. As shown in Fig. 7, the trypto-
phan 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 de-
crease 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 com-
posite gels.
4. Conclusions
The types and concentrations of fat significantly affect the physico-
chemical and structural properties of MP composite gels. The gels that
were prepared with MP and GL or PGL had significantly higher penetra-
tion 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 ob-
vious 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 interac-
tions between the protein and fat are responsible for the juiciness, ten-
derness 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.
340 X. Diao et al. / Meat Science 121 (2016) 333–341
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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