Research Article Received: 19 December 2014
Revised: 21 March 2015
Accepted article published: 7 May 2015
Published online in Wiley Online Library: 8 June 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7243
The effect of adenosine 5′-monophosphate (AMP) on tenderness, microstructure and chemical–physical index of duck breast meat Daoying Wang,a* Shaoying Deng,a,b Muhan Zhang,a Zhiming Geng,a Chong Sun,a Huan Bian,a Weimin Xu,a* Yongzhi Zhu,a Fang Liua and Haihong Wua Abstract BACKGROUND: Adenosine 5′ -monophosphate (AMP) is often used in meat and poultry soups as a flavor enhancer (flavor modifier), or as food additives for specific nutritional purposes. Our previous research as well as evidence from others showed that actomyosin could be dissociated into myosin and actin by AMP in extracted muscle solution. However, there is no report available on the application of AMP to dissociate actomyosin and to improve meat tenderness. The objectives of this study were to evaluate the effect of AMP on duck meat tenderness and other quality traits and to explore the mechanism of the action of AMP on meat tenderness. RESULTS: Duck breast muscle was treated with 0, 10, 20, 30, 40 mmol L−1 AMP at 5 ∘ C for 10 h and examined for shear force, microstructure, actomyosin dissociation, myofibril fragmentation index (MFI), pH, water content, cooking loss, CIE* color (L*, a*, b*), inosine monophosphate (IMP) and free amino acid (FAA) contents. Results showed that shear force, cooking loss, L* and b* of the muscles significantly decreased after AMP treatment (P < 0.05); actomyosin dissociation, MFI, pH, water content, fiber diameter, sarcomere length, IMP and ammonia significantly increased (P < 0.05); no significant change in a* or other FAA content was observed (P > 0.05), and muscle shrinkage in transverse and longitudinal directions were restrained after AMP treatment. CONCLUSION: The results suggest that AMP could notably improve meat tenderness, and this effect was probably mainly through increasing muscle pH, promoting actomyosin dissociation and disrupting the Z-line; meanwhile, the conversion of AMP to IMP may contribute to the flavor of meat. © 2015 Society of Chemical Industry Keywords: adenosine 5′ -monophosphate; tenderness; microstructure; physicochemical index; duck
INTRODUCTION
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has been proposed that weakening of the bonds between actin and myosin may contribute to the increase in tenderness during the immediate post-rigor period.7 Okitani et al. 8 found that actin liberated from myofibrils may give rise to a greater tenderness in beef, pork and chicken meat when heated at low temperature for a long time. Our previous research also suggested that actomyosin dissociation improves the tenderness of duck meat during the cooking process.9 These findings indicate that actin and myosin dissociation may have a significant effect on meat structure and tenderness.
∗
Correspondence to: Daoying Wang or Weimin Xu, Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, People’s Republic of China. E-mail: [email protected] (Wang); [email protected] (Xu)
a Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, People’s Republic of China b Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
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Adenosine 5′ -monophosphate (AMP), also known as 5′ -adenylic acid, is an ester of phosphoric acid and the nucleoside adenosine (Fig. 1). AMP is an endogenous purine nucleotide, which is a structural component of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and can be found in all living organisms involved in energy metabolism. In food processing, AMP is often used in meat and poultry soups as a flavor enhancer (flavor modifier) or as food additives for specific nutritional purposes.1 Meat tenderness is an important indicator to assess meat quality2,3 and also the major contributor to consumer acceptability of meat.4 Myosin and actin, which are assembled into thin and thick filaments respectively, are the two most abundant proteins in the skeletal muscle myofibril. The combination of myosin and actin results in the formation of actomyosin, which provides the force for muscle contraction and causes stiffness of muscle. In post-rigor muscle, the depletion of adenosine triphosphate (ATP) leads to more permanent actomyosin bonds.5 Although some researchers6 found that actin, myosin heavy chain and myosin light chain I undergo degradations post mortem, actomyosin remains almost unchanged in the post-mortem aging process.5 It
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pH value Two grams of duck meat were homogenized at 5000 rpm with an Ultra Turrax homogenizer (T25, IKA, Labortechnik, Staufen, Germany) in 18 mL distilled water, and the pH of the homogenate was measured using a pH meter equipped with an electrode.14 Myofibril fragmentation index (MFI) MFI was determined by the method of Hopkins et al.,15 with slight modifications. Figure 1. Structural formula of AMP.
It was found that actomyosin can be dissociated into myosin and actin by AMP and inosine monophosphate (IMP) in muscle solution, whereas, such dissociations cannot take place in the presence of adenosine diphosphate (ADP), inosin or adenosine.10,11 Nakamura et al.12 proposed that some nucleoside monophosphates may enhance the extraction of myosin and actin from porcine meat owing to its ability to dissociate actomyosin. However, there is no report available on application of AMP to improve meat tenderness, and whether and how AMP improves meat tenderness remains largely unknown. Our previous research found that actomyosin dissociation was improved in the presence of AMP and IMP at a concentration of 0–40 mmol L−1 , and AMP demonstrated a more remarkable effect than IMP.11 The objectives of this study were: (a) to evaluate the effect of AMP on duck meat tenderness; (b) to investigate the variations of duck meat quality indexes (e.g. microstructure, actomyosin dissociation) and physicochemical indexes (e.g. CIE* color, free amino acids) caused by AMP; and (c) to elucidate the mechanism for the effect of AMP on meat tenderness.
MATERIALS AND METHODS Sample preparation Fifteen lean-type Cherry Valley ducks from a commercial feedlot were slaughtered humanely in a commercial meat-processing plant (Jiangsu Furun Food Ltd, Xuzhou, China), each of which weighed about 2.0 kg. Skinless, de-boned pectoralis major muscles from both sides of breasts were removed from carcasses and subjected to 12 h aging in a refrigerator (4 ± 1 ∘ C). Duck fillets were trimmed of all visible fat and connective tissues, and cut to 7 × 5 × 1.5 cm rectangular-shaped samples with an average weight of 50 ± 1 g. Thirty samples were placed into plastic bags individually and randomly divided into five groups, and immersed in 50 mL 0, 10, 20, 30, 40 mmol L−1 AMP (adenosine 5′ -monophosphate disodium salt, Sigma-Aldrich Chemical Co.) dissolved in 0.9% (w/w) saline solution respectively. After 10 h treatment at 5 ∘ C, samples were removed from the solution and wiped with a paper towel. A portion of samples was taken for the measurement of water content, pH, MFI, actomyosin dissociation and free amino acid content (2 g for each test), and 4 g were taken for the determination of IMP and AMP; all samples were stored at −40 ∘ C. Remaining portions were subjected to color, shear force, cooking loss and microstructure analysis immediately after the AMP treatment. All tests were performed in triplicate.
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Water content Water content was determined following the International Organization for Standardization (ISO) recommended method 1442 (ISO, 1997).13
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Actomyosin dissociation The method for actin extraction was performed according to Okitani et al.,8 with slight modifications. Two grams of minced duck meat were mixed with 20 mL Weber–Edsall solution (0.6 mol L−1 KCl; 0.04 mol L−1 NaHCO3 ; 0.01 mol L−1 Na2 CO3 ; pH 7.2) and homogenized at 12000 rpm for 30 s twice at intervals of 10 s. The obtained homogenate was transferred to a beaker and shaken for 24 h at 4 ∘ C. The stirred homogenate was mixed with distilled water to lower the KCl concentration to 0.2 mol L−1 and centrifuged at 12 000 × g for 20 min at 4 ∘ C. The protein concentration was measured with a Bradford protein assay kit (Jiancheng Bioengineering Co., Nanjing, China). Samples were mixed with the sample buffer at a ratio of 2:1 (v/v) and boiled for 5 min. The samples with equal content of proteins (around 20 μL) loaded on each well were subjected to electrophoresis on 12% sodium dodecyl sulfate–polyacrylamide gels at a constant voltage of 200 V using a Mini PROTEAN Tetra cell (Bio-Rad Laboratories, Hercules, CA, USA). Gels were transferred to a 0.45 μm PVDF membrane (Millipore) using a semi-dry electrophoretic transfer cell (Bio-Rad Laboratories). Proteins were detected with polyclonal antibody to actin (Sigma Aldrich) at 1:1000 in 5% skim milk powder solution. After overnight incubation at 4 ∘ C, the membrane was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit and anti-mouse secondary antibodies at 1:5000 dilutions. Images of DAB detection were captured by a gel imager and the intensities of bands in each lane were quantified using Quantity One software (Bio-Rad Laboratories).9 IMP and AMP Four grams of duck meat sample were homogenized with 20 mL of 5% perchloric acid solution at 10 000 rpm using an Ultra Turrax (T25, IKA), followed by dilution to a volume of 25 mL with 5% perchloric acid. 20 mL of the homogenate were centrifuged for 10 min at 15 000 × g at 4 ∘ C (Allegra 64R, Beckman, USA), and the supernatant was collected and 4.5 mL of 1 mol L−1 KOH added. After centrifuging again for 10 min at 15 000 × g at 4 ∘ C, the supernatant was collected for analysis by high-performance liquid chromatography (HPLC). The sample was analyzed in an E2695 HPLC system (equipped with an autoinjector, HPLC workstation; Waters, USA) using a C18 column (5 μm, 250 mm × 4.6 mm i.d., Xbridge, Ireland) operating at 30 ∘ C. Invariable elution with 85% solution A (0.05 mol L−1 KH2 PO4 , 1.5 mmol L−1 tetrabutylammonium hydrogen sulfate, pH 7.0) and 15% B (methanol) was used at a flow rate of 1.0 mL min−1 . Chromatographic peaks were detected with a Waters 2998 PDA detector at 254 nm. The peaks were identified by comparing their retention times with those of the standards; the content of IMP and AMP was determined by the peak areas.16 Free amino acid (FAA) The extraction of FAA was performed as described by Wang et al.,17 with slight modifications. 0.50 g minced muscle was mixed with
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Effect of AMP on duck meat quality
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Table 1. Effect of AMP treatment on shear force, fiber diameter and sarcomere length of cooked duck breast meat AMP treatment Shear force (kg) Fiber diameter (μm) Sarcomere length (μm)
Control 0.94 ± 0.02a 13.74 ± 0.39e 1.40 ± 0.03d
10 mmol L−1
20 mmol L−1
30 mmol L−1
40 mmol L−1
0.85 ± 0.01b 14.58 ± 0.46d 1.48 ± 0.04c
0.70 ± 0.01c 15.54 ± 0.53c 1.56 ± 0.05b
0.63 ± 0.01d 16.68 ± 0.48b 1.69 ± 0.07a
0.53 ± 0.01e 17.45 ± 0.61a 1.72 ± 0.05a
Means in the same row with different letters differ significantly (P < 0.05). Data are expressed as mean ± SD (n = 6).
with 1.0 mL petroleum ether, and the mixture was allowed to stand for 1 h. Then 1.0 mL of the lower phase was collected for analysis of FAA. Identification and quantification of amino acids were carried out using an automatic amino acid analyzer (Hitachi L-8900, Japan) coupled with a BioBasic SCX cation exchange resin analytical column (4.6 mm × 60 mm, 5 μm). Meat color Duck breast meat color (L*, a*, b*) was measured using a colorimeter (CR 400, Minolta, Japan). Cooking loss To measure the cooking loss of samples, each breast fillet was weighed accurately prior to cooking. Samples were cooked in plastic bags individually in a water bath kettle set at 80 ∘ C until the internal meat temperature reached 75 ∘ C. After cooking, the breast fillets were cooled in tap water to an internal temperature of room temperature, then wiped with paper to remove excess water and weighed immediately. Cooking loss was calculated as: cooking loss (%) = [(raw weight − cooked weight) / raw weight] × 100.13 Shear force Shear force was determined through the application of the Meullenet–Owens razor shear (MORS) test,18 using a texture analyzer (TVT-300XP; TexVol Instruments, Viken, Sweden) equipped with a razor blade. The crosshead speed was set at 2 mm s−1 , and the test was triggered by a 10 g contact force. The shear was perpendicular to the axis of the muscle fibers.14 In each treatment, the MORS test value was determined in triplicate at predetermined locations on each of the fillets.
Figure 2. Effect of AMP treatment on duck breast muscle after cooking, observed by SEM and TEM.
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5.0 mL of 10 % sulfosalicylic acid, followed by grinding in a mortar to precipitate the proteins in the sample. The mixture was washed into a 50 mL volumetric flask. The pH of the solution was adjusted to 2 with 2.0 mol L−1 sodium hydroxide solution, and distilled water added to a total volume of 50 mL. 3.0 mL of the solution were taken from the flask and centrifuged at 12 000 rpm for 20 min at 10 ∘ C. 2.0 mL of the supernatant were collected to mix
Microstructure A scanning electron microscope (S-3000N; Hitachi HighTechnologies Corporation, Tokyo, Japan) and transmission electron microscope (H-7650; Hitachi) were employed for microstructural study of the duck meat. After shear force measurement, pieces (5 mm × 5 mm × 2 mm) for analysis by scanning electron microscopy (SEM) were excised from samples and fixed in 2.5% glutaraldehyde in 0.1 mol L−1 phosphate buffer (pH 7.3) at room temperature. The specimens were then rinsed with 0.1 mol L−1 phosphate buffer (pH 7.3) and dehydrated in 50%, 70%, 80% and 90% ethanol, respectively, for 15 min in each solution and 30 min in absolute ethanol three times. The specimens were freeze-dried, mounted on aluminum stubs and coated with gold for examination and photography using SEM. Six micrographs at a magnification of 600× were taken from each sample to determine the fiber diameter. For analysis by transmission electron microscopy (TEM), pieces (1 cm × 1 cm × 2 mm) were fixed in 2.5% glutaraldehyde in 0.1 mol L−1 phosphate buffer (pH 7.3) followed by a secondary fixation with 2% osmium tetroxide at room
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300 b 250
c d
MFI
200 e 150 100 50 0 control
10 mmol L–1 20 mmol L–1 30 mmol L–1 40 mmol L–1
AMP treatment Figure 4. Effect of AMP treatment on MFI of duck breast meat. Figure 3. Effect of AMP treatment on actomyosin dissociation of duck breast meat as shown by western blot, along with its relative values.
temperature. The specimens were then dehydrated in 50%, 70%, 80% and 90% ethanol, respectively, for 15 min in each solution and 30 min in absolute ethanol three times. The samples were embedded in epoxy resin (Durcupan) and the resin was allowed to cure at 70 ∘ C for 24–48 h. The cured resin blocks were cut in an ultra-cut ultramicrotome. The ultra-cuts were stained using a solution of 4% uranyl acetate in ethanol for 10 min, followed by an aqueous solution of Reynolds’ lead (7 min). Six micrographs at a magnification of 2500× were taken for each sample to determine the fiber diameter.19 Statistical analysis Statistical analysis of the differences between each group was evaluated by one-way analysis of variance (ANOVA) using SPSS 18.0. Differences were regarded as significant at P < 0.05. All data are expressed as mean ± standard error.
RESULTS AND DISCUSSION
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Effect of AMP on shear force of duck breast meat after cooking Meat tenderness is affected mainly by two components, i.e. myofibril and connective tissue. The former forms the actomyosin toughness, while the latter forms the background toughness. Compared with myofibrils, connective tissue structure seems to be unchanged during the post-mortem period,20 Many studies have been done on the impacts of heating and aging processes on meat tenderness, and relatively less attention has been paid to the effect of curing agent on tenderness development. As for AMP, there are almost no reports available on its application for improvement of meat tenderness. Therefore this study was carried out to evaluate the effect of AMP on improvement of tenderness. Tenderness can be determined by a trained panel or by physical methods, in which determination of shear force is the most widely used method.21 In the present study, shear force values of duck breast meats treated with different AMP concentrations were given by a texture analyzer, and the results are listed in Table 1. With increasing AMP concentration from 0 to 40 mmol L−1 , the shear force value decreased from 0.94 kg to 0.53kg, and every change of 10 mmol L−1 in AMP concentration resulted in a significant variation of shear force (P < 0.05). This result indicated that AMP could remarkably improve duck meat tenderness.
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Effect of AMP on microstructure of duck breast meat after cooking In order to study the impacts of AMP on microstructure of duck meat, SEM and TEM analyses were employed in this paper. Both muscle fiber diameter and sarcomere length increased with the concentration of AMP from 0 to 40 mmol L−1 , and every increase of 10 mmol L−1 in AMP led to significant changes in fiber diameter and sarcomere length (P < 0.05) except for sarcomere length, when AMP concentration increased from 30 to 40 mmol L−1 (see Table 1). Myofibrillar structures of AMP-treated duck breast meats are shown in Fig. 2. On transverse sections, gaps between muscle fibers were plainly visible in the control (Fig. 2A1), which was similar to those reported by Wattanachant et al.,22 who examined the microstructure of chicken meat. With increasing AMP concentration, muscle fibers became increasingly larger (Fig. 2B1–E1). Contrarily, the gaps between muscle fibers became smaller and almost disappeared when treated with 40 mmol L−1 ATP. On longitudinal sections, AMP exhibited the same effect on the microstructure of duck meat as on the transverse sections (Fig. 2A2–E2). In the control sample, myofibrils were closely packed, with the I band and A band being clearly distinguished, and Z-line and M-line being plainly visible. However, remarkable structural changes were observed in AMP-treated samples, and weakening of myofibrils and loss of Z-disks occurred when the sample was treated with 40 mmol L−1 AMP. These results indicated that AMP treatment could restrain the shrinkage of muscle fiber transversally and longitudinally, and could make the meat tender. Such a finding, from another perspective, supported the study by Li et al.,19 who reported transverse and longitudinal shrinkage of muscle fiber, resulting in tougher meat. Effect of AMP on actomyosin dissociation and MFI of duck breast meat Actomyosin dissociation occurs during the immediate post-rigor period and heating process, which improves meat tenderness.8,9,15,23 In the present paper, actomyosin dissociations in duck breast meats treated with different AMP concentrations were investigated and the results are shown in Fig. 3. In the control sample, only a low-density band of actin was observed, while the densities of actin bands were increased significantly in samples treated with AMP, and the density increased with the increasing of AMP level, which indicated that actomyosin dissociation was very weak in duck breast meat and AMP favored actomyosin dissociation. The result was consistent with that by Okitani et al.,8 who found that more actin was liberated from actomyosin of chicken, beef and pork skeleton muscles treated with AMP and IMP. Previous studies suggest that the binding of myosin motor domain to
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Effect of AMP on duck meat quality
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Table 2. Effect of AMP treatment on pH, water, cooking loss, AMP and IMP content of duck breast meat AMP treatment
Control
10 mmol L−1
20 mmol L−1
30 mmol L−1
40 mmol L−1
pH Water (%) Cooking loss (%) AMP (mg/g) IMP (mg/g)
5.89 ± 0.08e 80.17 ± 0.09e 33.24 ± 0.62a 0.03 ± 0.01a 1.55 ± 0.02e
6.31 ± 0.03d 81.18 ± 0.06d 29.84 ± 0.56b 0.04 ± 0.01a 2.41 ± 0.07d
6.59 ± 0.10c 81.56 ± 0.03c 20.24 ± 0.48c 0.03 ± 0.01a 3.88 ± 0.19c
6.73 ± 0.05b 81.78 ± 0.08b 15.22 ± 0.42d 0.04 ± 0.02a 5.90 ± 0.17b
6.87 ± 0.06a 82.45 ± 0.11a 10.61 ± 0.47e 0.03 ± 0.01a 7.52 ± 0.14a
Means in the same row with different letters differ significantly (P < 0.05). Data are expressed as mean ± SD (n = 6).
Effect of AMP on pH, water content and cooking loss of duck breast meat pH, water content and cooking loss of duck breast muscle treated with different AMP concentrations are listed in Table 2. Both pH and water content increased with increasing AMP, while cooking loss varied in the opposite direction. For all of these factors, every change of 10 mmol L−1 in AMP resulted in significant variations (P < 0.05). Meat pH is an important determinant of meat quality.29 – 31 An abnormally low pH may cause denaturation of sarcoplasmic and myofibrillar proteins and affect the integrity of skeletal muscle membrane, resulting in lower water content and less tenderness.29 Water content and cooking loss are related closely to pH; the pH of early post-mortem meat can explain 72% of the variation in water content.32 The pH is responsible for a large part of muscle shortening, which could lead to expulsion of water from the myofibrillar structure into the extracellular space and ultimately out of the muscle.33 As AMP could increase the muscle pH and restrain muscle shrinkage, it increases the space available for water within the myofibril. The improved water retention of meat could also partially be attributed to the increased ionic strength by AMP, which alters the conformation and solubility of myofibrillar proteins that bind and entrap water.
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L*
a
a*
b
50
c
b* d
d
40 30 20 10 0
a a
control
a
b
a
a c
a d
e
10 mmol L–1 20 mmol L–1 30 mmol L–1 40 mmol L–1
AMP treatment
Figure 5. Effect of AMP treatment on CIE* color of duck breast meat.
in remarkable variations of the values (P < 0.05), except for L* value when treated with 30 and 40 mmol L−1 AMP. As for a* value, no significant variation was observed. The low L* value has been reported to be related to higher water-holding capacity.35 Our results show that AMP treatment caused a significant decrease in brightness and yellowness of duck meat (P < 0.05), while redness showed no significant change (P > 0.05). This is in agreement with several studies demonstrating that breast meat lightness and yellowness were significantly negatively correlation with meat pH.35,36 According to Qiao et al.,37 the breast fillet of broiler is grouped to normal when L* is in the range of 48–53. We obtained similar results, which implied that AMP may have no negative impact on meat color. Effect of AMP on IMP and FAA content of duck breast meat AMP is an endogenous purine nucleotide, and can be converted to IMP and ammonia by adenosine deaminase (AMPD), which is abundant in all kinds of animal meats.38 In this paper, in order to understand the fate of the AMP employed to treat duck meat, contents of AMP, IMP and FAA in duck meats before and after AMP treatment were quantitatively analyzed, and the results are listed in Table 3. AMP level in meat sample was found unchanged when treated with 0–40 mmol L−1 AMP, while IMP level increased with increasing AMP concentration, and each increase of 10 mmol L−1 in AMP resulted in a significant variation of IMP in meat (P < 0.05). In meats treated with AMP, FAA content remained almost unchanged, except for ammonia, which increased significantly with each increase of 10 mmol L−1 in AMP (P < 0.05). These results indicate that the exogenous AMP permeating through duck meat, no matter how high the concentration, was totally converted to IMP and ammonia by AMPD in duck meat. IMP has an umami flavor and is widely distributed in animals. There is about 2 mg g−1 IMP in poultry and livestock muscles,39,40 whereas in fish IMP can reach 3–8 mg g−1 ; thus fish is of more
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Effect of AMP on CIE* color of duck breast meat Meat color is an important quality trait of meat products which affect the consumer’s decision in purchasing meat product; it is highly correlated with the metmyoglobin formation, muscle fiber type and water distribution.34 In the present paper, the effect of AMP on CIE* color of duck meat was investigated, and the results are shown in Fig. 5. AMP yielded significant impacts on L* and b*values, and every change of 10 mmol L−1 in AMP levels resulted
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Color of meat
actin depends on the nucleotide or nucleotide analogue bound to myosin subfragment.24,25 With bound nucleotide, the interaction of myosin with actin becomes weak. MFI is a useful indicator of both rupture of the I-band and breakage of intermyofibril linkages.26,27 MFI reflects the integrity of muscle fibers and its skeleton proteins. The larger MFI value indicates the increased damage that the internal structure of the myofibrils is exposed to.28 In this paper, MFI of duck breast muscles treated with different AMP concentrations were quantitatively analyzed and the results are presented in Fig. 4. MFI was significantly increased with increasing AMP concentration from 0 to 40 mmol L−1 (P < 0.05). The variation of MFI indicated that AMP promoted I-band rupture and breakage of intermyofibril linkages. Just as observed in microstructure, Z-line structure was lost and the internal structure of myofibrils was destroyed when duck meat samples were treated with AMP, which might result in the increase in MFI.
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Table 3. Effect of AMP treatment on free amino acid content of duck breast meat (expressed as mg g−1 ) AMP treatment Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Ammonia Histidine Arginine Proline Total
Control 0.07 ± 0.01a 0.30 ± 0.02a 0.17 ± 0.03a 0.16 ± 0.01a 0.11 ± 0.02a 0.21 ± 0.02b 0.01 ± 0.02a 0.06 ± 0.01a 0.04 ± 0.01a 0.05 ± 0.01a 0.09 ± 0.02a 0.07 ± 0.01a 0.07 ± 0.01a 0.06 ± 0.02b 0.08 ± 0.01e 0.03 ± 0.01a 0.09 ± 0.03a 0.07 ± 0.01b 1.71 ± 0.06b
10 mmol L−1 0.10 ± 0.01a 0.25 ± 0.01a 0.15 ± 0.01a 0.15 ± 0.02ab 0.10 ± 0.01a 0.24 ± 0.03ab 0.01 ± 0.01a 0.08 ± 0.02a 0.05 ± 0.01a 0.06 ± 0.01a 0.10 ± 0.01a 0.06 ± 0.01a 0.08 ± 0.02a 0.08 ± 0.01ab 0.16 ± 0.02d 0.04 ± 0.02a 0.11 ± 0.02a 0.09 ± 0.02ab 1.90 ± 0.22ab
20 mmol L−1 0.11 ± 0.02a 0.29 ± 0.03a 0.18 ± 0.02a 0.14 ± 0.02b 0.14 ± 0.03a 0.27 ± 0.02a 0.01 ± 0.03a 0.08 ± 0.01a 0.04 ± 0.02a 0.06 ± 0.02a 0.09 ± 0.01a 0.08 ± 0.02a 0.07 ± 0.01a 0.11 ± 0.02a 0.23 ± 0.02c 0.03 ± 0.01a 0.11 ± 0.01a 0.12 ± 0.01a 2.12 ± 0.23a
30 mmol L−1 0.10 ± 0.01a 0.27 ± 0.03a 0.16 ± 0.01a 0.11 ± 0.01b 0.13 ± 0.02a 0.28 ± 0.01a 0.01 ± 0.02a 0.07 ± 0.01a 0.05 ± 0.01a 0.04 ± 0.02a 0.08 ± 0.03a 0.07 ± 0.02a 0.06 ± 0.03a 0.10 ± 0.02a 0.31 ± 0.03b 0.03 ± 0.03a 0.12 ± 0.02a 0.10 ± 0.03a 2.13 ± 0.12a
40 mmol L−1 0.09 ± 0.01a 0.23 ± 0.01a 0.15 ± 0.02a 0.11 ± 0.01b 0.12 ± 0.02a 0.28 ± 0.03a 0.01 ± 0.02a 0.07 ± 0.02a 0.05 ± 0.01a 0.06 ± 0.01a 0.09 ± 0.02a 0.06 ± 0.01a 0.07 ± 0.02a 0.09 ± 0.01a 0.38 ± 0.02a 0.04 ± 0.02a 0.12 ± 0.03a 0.11 ± 0.02a 2.14 ± 0.10a
Means in the same row with different letters differ significantly (P < 0.05). Data are expressed as mean ± SD (n = 6).le
umami taste.41 In our previous studies, IMP was found to be able to promote dissociation of actomyosin, but its effect was much weaker than that of AMP.11 As many studies reported,38,40 ammonia is another product of AMP hydrolysis by AMPD; however, what role it plays in the dissociation of actomyosin originated by AMP remains unknown. We suppose that the generation of ammonia could increase the muscle pH, which might result in the increase of water retention and also the potential disruption of the Z-line of muscle fibers.
CONCLUSIONS The treatment of duck breast muscle with AMP may result in multiple biological and chemical reactions: dissociation of actomyosin directly by AMP, conversion of AMP to IMP and ammonia by AMPD and restraint of muscle fiber shrinkage. The series of reactions contributes to the quality improvement of duck breast meat. However, the mechanism regarding the impact of AMP on actomyosin dissociation and meat quality is still poorly understood, and further studies are needed to detail the mechanism and to enhance the function of AMP in meat products.
ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (31101312, 31271891), Natural Science Foundation Program of Jiangsu Province (BK2012785) and Innovation of Agricultural Science and Technology of Jiangsu Province (CX(12)5030).
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1 Koguchi T, Nakajima H, Koguchi H, Wada M, Yamamoto Y, Innami S et al., Suppressive effect of viscous dietary fiber on elevations of uric acid in serum and urine induced by dietary RNA in rats is associated with strength of viscosity. Int J Vitam Nutr Res 73:369–376 (2003).
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2 Lee YS, Saha A, Xiong R, Owens CM and Meullenet JF, Changes in broiler breast fillet tenderness, water-holding capacity, and color attributes during long-term frozen storage. J Food Sci 73:E162–E168 (2008). 3 Moeller SJ, Miller RK, Edwards KK, Zerby HN, Logan KE, Aldredge TL et al., Consumer perceptions of pork eating quality as affected by pork quality attributes and end-point cooked temperature. Meat Sci 84:14–22 (2010). 4 Myers AJ, Scramlin SM, Dilger AC, Souza CM, McKeith FK and Killefer J, Contribution of lean, fat, muscle color and degree of doneness to pork and beef species flavor. Meat Sci 82:59–63 (2009). 5 Huff-Lonergan E, Zhang W and Lonergan SM, Biochemistry of postmortem muscle-Lessons on mechanisms of meat tenderization. Meat Sci 86:184–195 (2010). 6 Lametsch R, Roepstorff P, Møller HS and Bendixen E, Identification of myofibrillar substrates for μ-calpain. Meat Sci 68:515–521 (2004). 7 Taylor RG, Geesink GH, Thompson VF, Koohmaraie M and Goll DE, Is Z-disk degradation responsible for post- mortem tenderisation. J Anim Sci 73:1351–1367 (1995). 8 Okitani A, Ichinose N, Itoh J, Tsuji Y, Oneda Y, Hatae K et al., Liberation of actin from actomyosin in meats heated to 65∘ C. Meat Sci 81:446–450 (2009). 9 Wang DY, Dong H, Zhang MH, Liu F, Bian H, Zhu YZ et al., Changes in actomyosin dissociation and endogenous enzyme activities during heating and their relationship with duck meat tenderness. Food Chem 141:675–679 (2013). 10 Okitani A, Ichinose N, Koza M, Yamanaka K, Migita K and Matsuishi M, AMP and IMP dissociate actomyosin into actin and myosin. Biosci Biotechnol Biochem 72:2005–2011 (2008). 11 Deng SY, Wang DY, Zhang MH, Bian H, Wu HH, Zhu YZ et al., Actomyosin dissociation as influenced by Ca2+ , ATP and its degradation products. Food Sci 24:325–329 (2014). [In Chinese] 12 Nakamura Y, Migita K, Okitani A and Matsuishi M, Enhancing effect of IMP on myosin and actin extraction from porcine meat. Biosci Biotechnol Biochem 76:1611–1615 (2012). 13 Wang DY, Zhang MH, Xu WM, Bian H, Liu F, Geng ZM et al., Changes in chemical–physical index and microstructure during dry-cured duck processing. J Poult Sci 51:221–227 (2014). 14 Fernandez X, Neyraud E, Astruc T and Sante V, Effects of halothane genotype and pre-slaughter treatment on pig meat quality. Part 1. Post mortem metabolism, meat quality indicators and sensory traits of M. Longissimus lumborum. Meat Sci 62:429–437 (2002).
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15 Hopkins DL, Martin L and Gilmour AR, The impact of homogenizer type and speed on the determination of myofibrillar fragmentation. Meat Sci 67:705–710 (2004). 16 Hernández-Cázares AS, Aristoy MC and Toldrá F, Nucleotides and their degradation products during processing of dry-cured ham, measured by HPLC and an enzyme sensor. Meat Sci 87:125–129 (2011). 17 Wang DY, Zhang MH, Bian H, Dong H, Xu WM, Xu XL et al., Proteolysis and cathepsin activities in the processing of dry-cured duck. Poult Sci 93:687–694 (2014). 18 Meullenet J, Jonville E, Grezes D and Owens CM, Prediction of the texture of cooked poultry pectoralis major muscles by near-infrared reflectance analysis of raw meat. J Texture Stud 35:573–585 (2004). 19 Li C, Wang DY, Xu WM, Gao F and Zhou GH, Effect of final cooked temperature on tenderness, protein solubility and microstructure of duck breast muscle. LWT – Food Sci Technol 51:266–274 (2013). 20 Nishimura T, The role of intramuscular connective tissue in meat texture. Anim Sci J 81:21–27 (2010). 21 Combes S, Lepetit J, Darche B and Lebas F, Effect of cooking temperature and cooking time on Warner–Bratzler tenderness measurement and collagen content in rabbit meat. Meat Sci 66:91–96 (2004). 22 Wattanachant S, Benjakul S and Ledward DA. Effect of heat treatment on changes in texture, structure and properties of Thai indigenous chicken muscle. Food Chem 93:337–348 (2005). 23 Tornberg E, Effects of heat on meat proteins: implications on structure and quality of meat products. Meat Sci 70:493–508 (2005). 24 Spudich JA, The myosin swinging cross-bridge model. Nat Rev Mol Cell Biol 2:387–392 (2001). 25 Gordon AM, Homsher E and Regnier M, Regulation of contraction in striated muscle. Physiol Rev 80:853–924 (2000). 26 Volpelli LA, Failla S and Sepulcri A, Calpain system in vitro activity and myofibril fragmentation index in fallow deer (Dama dama): effects of age and supplementary feeding. Meat Sci 69:579–582 (2005). 27 Volpelli LA, Valusso R and Morgante M. Meat quality in male fallow deer (Dama dama): effects of age and supplementary feeding. Meat Sci 65:555–562 (2003). 28 Veiseth E, Shacklford SD and Wheeler TL. Indicators of tenderization are detectable by 12 h postmortem in ovine longissimus. J Anim Sci 82:1428–1436 (2004).
29 Huff-Lonergan E and Lonergan SM, Mechanisms of water holding capacity of meat: the role of postmortem biochemical and structural changes. Meat Sci 71:194–204 (2005). 30 Zamora F, Aubry L, Sayd T, Lepetit J, Lebert A, Sentandreu MA et al., Serine peptidase inhibitors, the best predictor of beef ageing amongst a large set of quantitative variables. Meat Sci 71:730–742 (2005). 31 Juncher D, Ronn B, Mortensen ET, Henckel P, Karlsson A and Skibsted LH, Effect of pre-slaughter physiological conditions on the oxidative stability of color and lipid oxidation during chill storage of pork. Meat Sci 58:347–357 (2001). 32 Schafer A, Rosenvold K, Purslow PP, Andersen HJ and Henckel P. Critical post mortem pH and temperature values in relation to drip loss in pork, in Proceedings of the International Congress on Meat Science and Technology, Vol. 47, Krakow Poland, pp. 206–207 (2001). 33 Honikel KO, Kim CJ, Hamm R and Roncales P, Sarcomere shortening of prerigor muscles and its influence on drip loss. Meat Sci 16:267–282 (1986). 34 Froning GW, Color of poultry meat. Poult Avian Biol Rev 6:83–93 (1995). 35 Barbut S, Colour measurement for evaluating the pale soft exudative (PSE) occurrence in turkey meat. Food Res Int 26:39–43 (1993). 36 Allen CD, Russell SM and Fletcher DL, The relationship of broiler breast meat color and pH to shelf life and odor development. Poult Sci 76:1042–1046 (1997). 37 Qiao M, Fletcher DL, Smith DP and Northcutt JK, The effect of broiler breast meat color on pH, moisture, water holding capacity, and emulsification capacity. Poult Sci 80:676–680 (2001). 38 Hiroshi S, Hirofumi O, Yoshihiro M and Hachiro N, Interaction of rat muscle AMP deaminase with myosin I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle. Biochim Biophys Acta 566:335–344 (1979). 39 Li JS and Wang YM, Determination of nucleotide’s metabolites in chicken by HPLC. Bull Sci Technol 18:323–326 (2002). [in Chinese] 40 Michel A, Linda JF, James TK, Bruce WM and Alan G. Post-slaughter changes in ATP metabolites, reducing and phosphorylated sugars in chicken meat. Meat Sci 94:55–62 (2013). 41 Liu X, Wang J, Zhang W, Du JY and Su YQ, Analysis of inosine monophosphate in six fish musculatures. Marine Sci 32:22–25 (2008). [in Chinese]
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Revised: 21 March 2015
Accepted article published: 7 May 2015
Published online in Wiley Online Library: 8 June 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7243
The effect of adenosine 5′-monophosphate (AMP) on tenderness, microstructure and chemical–physical index of duck breast meat Daoying Wang,a* Shaoying Deng,a,b Muhan Zhang,a Zhiming Geng,a Chong Sun,a Huan Bian,a Weimin Xu,a* Yongzhi Zhu,a Fang Liua and Haihong Wua Abstract BACKGROUND: Adenosine 5′ -monophosphate (AMP) is often used in meat and poultry soups as a flavor enhancer (flavor modifier), or as food additives for specific nutritional purposes. Our previous research as well as evidence from others showed that actomyosin could be dissociated into myosin and actin by AMP in extracted muscle solution. However, there is no report available on the application of AMP to dissociate actomyosin and to improve meat tenderness. The objectives of this study were to evaluate the effect of AMP on duck meat tenderness and other quality traits and to explore the mechanism of the action of AMP on meat tenderness. RESULTS: Duck breast muscle was treated with 0, 10, 20, 30, 40 mmol L−1 AMP at 5 ∘ C for 10 h and examined for shear force, microstructure, actomyosin dissociation, myofibril fragmentation index (MFI), pH, water content, cooking loss, CIE* color (L*, a*, b*), inosine monophosphate (IMP) and free amino acid (FAA) contents. Results showed that shear force, cooking loss, L* and b* of the muscles significantly decreased after AMP treatment (P < 0.05); actomyosin dissociation, MFI, pH, water content, fiber diameter, sarcomere length, IMP and ammonia significantly increased (P < 0.05); no significant change in a* or other FAA content was observed (P > 0.05), and muscle shrinkage in transverse and longitudinal directions were restrained after AMP treatment. CONCLUSION: The results suggest that AMP could notably improve meat tenderness, and this effect was probably mainly through increasing muscle pH, promoting actomyosin dissociation and disrupting the Z-line; meanwhile, the conversion of AMP to IMP may contribute to the flavor of meat. © 2015 Society of Chemical Industry Keywords: adenosine 5′ -monophosphate; tenderness; microstructure; physicochemical index; duck
INTRODUCTION
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has been proposed that weakening of the bonds between actin and myosin may contribute to the increase in tenderness during the immediate post-rigor period.7 Okitani et al. 8 found that actin liberated from myofibrils may give rise to a greater tenderness in beef, pork and chicken meat when heated at low temperature for a long time. Our previous research also suggested that actomyosin dissociation improves the tenderness of duck meat during the cooking process.9 These findings indicate that actin and myosin dissociation may have a significant effect on meat structure and tenderness.
∗
Correspondence to: Daoying Wang or Weimin Xu, Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, People’s Republic of China. E-mail: [email protected] (Wang); [email protected] (Xu)
a Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, People’s Republic of China b Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
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Adenosine 5′ -monophosphate (AMP), also known as 5′ -adenylic acid, is an ester of phosphoric acid and the nucleoside adenosine (Fig. 1). AMP is an endogenous purine nucleotide, which is a structural component of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and can be found in all living organisms involved in energy metabolism. In food processing, AMP is often used in meat and poultry soups as a flavor enhancer (flavor modifier) or as food additives for specific nutritional purposes.1 Meat tenderness is an important indicator to assess meat quality2,3 and also the major contributor to consumer acceptability of meat.4 Myosin and actin, which are assembled into thin and thick filaments respectively, are the two most abundant proteins in the skeletal muscle myofibril. The combination of myosin and actin results in the formation of actomyosin, which provides the force for muscle contraction and causes stiffness of muscle. In post-rigor muscle, the depletion of adenosine triphosphate (ATP) leads to more permanent actomyosin bonds.5 Although some researchers6 found that actin, myosin heavy chain and myosin light chain I undergo degradations post mortem, actomyosin remains almost unchanged in the post-mortem aging process.5 It
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pH value Two grams of duck meat were homogenized at 5000 rpm with an Ultra Turrax homogenizer (T25, IKA, Labortechnik, Staufen, Germany) in 18 mL distilled water, and the pH of the homogenate was measured using a pH meter equipped with an electrode.14 Myofibril fragmentation index (MFI) MFI was determined by the method of Hopkins et al.,15 with slight modifications. Figure 1. Structural formula of AMP.
It was found that actomyosin can be dissociated into myosin and actin by AMP and inosine monophosphate (IMP) in muscle solution, whereas, such dissociations cannot take place in the presence of adenosine diphosphate (ADP), inosin or adenosine.10,11 Nakamura et al.12 proposed that some nucleoside monophosphates may enhance the extraction of myosin and actin from porcine meat owing to its ability to dissociate actomyosin. However, there is no report available on application of AMP to improve meat tenderness, and whether and how AMP improves meat tenderness remains largely unknown. Our previous research found that actomyosin dissociation was improved in the presence of AMP and IMP at a concentration of 0–40 mmol L−1 , and AMP demonstrated a more remarkable effect than IMP.11 The objectives of this study were: (a) to evaluate the effect of AMP on duck meat tenderness; (b) to investigate the variations of duck meat quality indexes (e.g. microstructure, actomyosin dissociation) and physicochemical indexes (e.g. CIE* color, free amino acids) caused by AMP; and (c) to elucidate the mechanism for the effect of AMP on meat tenderness.
MATERIALS AND METHODS Sample preparation Fifteen lean-type Cherry Valley ducks from a commercial feedlot were slaughtered humanely in a commercial meat-processing plant (Jiangsu Furun Food Ltd, Xuzhou, China), each of which weighed about 2.0 kg. Skinless, de-boned pectoralis major muscles from both sides of breasts were removed from carcasses and subjected to 12 h aging in a refrigerator (4 ± 1 ∘ C). Duck fillets were trimmed of all visible fat and connective tissues, and cut to 7 × 5 × 1.5 cm rectangular-shaped samples with an average weight of 50 ± 1 g. Thirty samples were placed into plastic bags individually and randomly divided into five groups, and immersed in 50 mL 0, 10, 20, 30, 40 mmol L−1 AMP (adenosine 5′ -monophosphate disodium salt, Sigma-Aldrich Chemical Co.) dissolved in 0.9% (w/w) saline solution respectively. After 10 h treatment at 5 ∘ C, samples were removed from the solution and wiped with a paper towel. A portion of samples was taken for the measurement of water content, pH, MFI, actomyosin dissociation and free amino acid content (2 g for each test), and 4 g were taken for the determination of IMP and AMP; all samples were stored at −40 ∘ C. Remaining portions were subjected to color, shear force, cooking loss and microstructure analysis immediately after the AMP treatment. All tests were performed in triplicate.
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Water content Water content was determined following the International Organization for Standardization (ISO) recommended method 1442 (ISO, 1997).13
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Actomyosin dissociation The method for actin extraction was performed according to Okitani et al.,8 with slight modifications. Two grams of minced duck meat were mixed with 20 mL Weber–Edsall solution (0.6 mol L−1 KCl; 0.04 mol L−1 NaHCO3 ; 0.01 mol L−1 Na2 CO3 ; pH 7.2) and homogenized at 12000 rpm for 30 s twice at intervals of 10 s. The obtained homogenate was transferred to a beaker and shaken for 24 h at 4 ∘ C. The stirred homogenate was mixed with distilled water to lower the KCl concentration to 0.2 mol L−1 and centrifuged at 12 000 × g for 20 min at 4 ∘ C. The protein concentration was measured with a Bradford protein assay kit (Jiancheng Bioengineering Co., Nanjing, China). Samples were mixed with the sample buffer at a ratio of 2:1 (v/v) and boiled for 5 min. The samples with equal content of proteins (around 20 μL) loaded on each well were subjected to electrophoresis on 12% sodium dodecyl sulfate–polyacrylamide gels at a constant voltage of 200 V using a Mini PROTEAN Tetra cell (Bio-Rad Laboratories, Hercules, CA, USA). Gels were transferred to a 0.45 μm PVDF membrane (Millipore) using a semi-dry electrophoretic transfer cell (Bio-Rad Laboratories). Proteins were detected with polyclonal antibody to actin (Sigma Aldrich) at 1:1000 in 5% skim milk powder solution. After overnight incubation at 4 ∘ C, the membrane was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit and anti-mouse secondary antibodies at 1:5000 dilutions. Images of DAB detection were captured by a gel imager and the intensities of bands in each lane were quantified using Quantity One software (Bio-Rad Laboratories).9 IMP and AMP Four grams of duck meat sample were homogenized with 20 mL of 5% perchloric acid solution at 10 000 rpm using an Ultra Turrax (T25, IKA), followed by dilution to a volume of 25 mL with 5% perchloric acid. 20 mL of the homogenate were centrifuged for 10 min at 15 000 × g at 4 ∘ C (Allegra 64R, Beckman, USA), and the supernatant was collected and 4.5 mL of 1 mol L−1 KOH added. After centrifuging again for 10 min at 15 000 × g at 4 ∘ C, the supernatant was collected for analysis by high-performance liquid chromatography (HPLC). The sample was analyzed in an E2695 HPLC system (equipped with an autoinjector, HPLC workstation; Waters, USA) using a C18 column (5 μm, 250 mm × 4.6 mm i.d., Xbridge, Ireland) operating at 30 ∘ C. Invariable elution with 85% solution A (0.05 mol L−1 KH2 PO4 , 1.5 mmol L−1 tetrabutylammonium hydrogen sulfate, pH 7.0) and 15% B (methanol) was used at a flow rate of 1.0 mL min−1 . Chromatographic peaks were detected with a Waters 2998 PDA detector at 254 nm. The peaks were identified by comparing their retention times with those of the standards; the content of IMP and AMP was determined by the peak areas.16 Free amino acid (FAA) The extraction of FAA was performed as described by Wang et al.,17 with slight modifications. 0.50 g minced muscle was mixed with
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Effect of AMP on duck meat quality
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Table 1. Effect of AMP treatment on shear force, fiber diameter and sarcomere length of cooked duck breast meat AMP treatment Shear force (kg) Fiber diameter (μm) Sarcomere length (μm)
Control 0.94 ± 0.02a 13.74 ± 0.39e 1.40 ± 0.03d
10 mmol L−1
20 mmol L−1
30 mmol L−1
40 mmol L−1
0.85 ± 0.01b 14.58 ± 0.46d 1.48 ± 0.04c
0.70 ± 0.01c 15.54 ± 0.53c 1.56 ± 0.05b
0.63 ± 0.01d 16.68 ± 0.48b 1.69 ± 0.07a
0.53 ± 0.01e 17.45 ± 0.61a 1.72 ± 0.05a
Means in the same row with different letters differ significantly (P < 0.05). Data are expressed as mean ± SD (n = 6).
with 1.0 mL petroleum ether, and the mixture was allowed to stand for 1 h. Then 1.0 mL of the lower phase was collected for analysis of FAA. Identification and quantification of amino acids were carried out using an automatic amino acid analyzer (Hitachi L-8900, Japan) coupled with a BioBasic SCX cation exchange resin analytical column (4.6 mm × 60 mm, 5 μm). Meat color Duck breast meat color (L*, a*, b*) was measured using a colorimeter (CR 400, Minolta, Japan). Cooking loss To measure the cooking loss of samples, each breast fillet was weighed accurately prior to cooking. Samples were cooked in plastic bags individually in a water bath kettle set at 80 ∘ C until the internal meat temperature reached 75 ∘ C. After cooking, the breast fillets were cooled in tap water to an internal temperature of room temperature, then wiped with paper to remove excess water and weighed immediately. Cooking loss was calculated as: cooking loss (%) = [(raw weight − cooked weight) / raw weight] × 100.13 Shear force Shear force was determined through the application of the Meullenet–Owens razor shear (MORS) test,18 using a texture analyzer (TVT-300XP; TexVol Instruments, Viken, Sweden) equipped with a razor blade. The crosshead speed was set at 2 mm s−1 , and the test was triggered by a 10 g contact force. The shear was perpendicular to the axis of the muscle fibers.14 In each treatment, the MORS test value was determined in triplicate at predetermined locations on each of the fillets.
Figure 2. Effect of AMP treatment on duck breast muscle after cooking, observed by SEM and TEM.
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5.0 mL of 10 % sulfosalicylic acid, followed by grinding in a mortar to precipitate the proteins in the sample. The mixture was washed into a 50 mL volumetric flask. The pH of the solution was adjusted to 2 with 2.0 mol L−1 sodium hydroxide solution, and distilled water added to a total volume of 50 mL. 3.0 mL of the solution were taken from the flask and centrifuged at 12 000 rpm for 20 min at 10 ∘ C. 2.0 mL of the supernatant were collected to mix
Microstructure A scanning electron microscope (S-3000N; Hitachi HighTechnologies Corporation, Tokyo, Japan) and transmission electron microscope (H-7650; Hitachi) were employed for microstructural study of the duck meat. After shear force measurement, pieces (5 mm × 5 mm × 2 mm) for analysis by scanning electron microscopy (SEM) were excised from samples and fixed in 2.5% glutaraldehyde in 0.1 mol L−1 phosphate buffer (pH 7.3) at room temperature. The specimens were then rinsed with 0.1 mol L−1 phosphate buffer (pH 7.3) and dehydrated in 50%, 70%, 80% and 90% ethanol, respectively, for 15 min in each solution and 30 min in absolute ethanol three times. The specimens were freeze-dried, mounted on aluminum stubs and coated with gold for examination and photography using SEM. Six micrographs at a magnification of 600× were taken from each sample to determine the fiber diameter. For analysis by transmission electron microscopy (TEM), pieces (1 cm × 1 cm × 2 mm) were fixed in 2.5% glutaraldehyde in 0.1 mol L−1 phosphate buffer (pH 7.3) followed by a secondary fixation with 2% osmium tetroxide at room
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300 b 250
c d
MFI
200 e 150 100 50 0 control
10 mmol L–1 20 mmol L–1 30 mmol L–1 40 mmol L–1
AMP treatment Figure 4. Effect of AMP treatment on MFI of duck breast meat. Figure 3. Effect of AMP treatment on actomyosin dissociation of duck breast meat as shown by western blot, along with its relative values.
temperature. The specimens were then dehydrated in 50%, 70%, 80% and 90% ethanol, respectively, for 15 min in each solution and 30 min in absolute ethanol three times. The samples were embedded in epoxy resin (Durcupan) and the resin was allowed to cure at 70 ∘ C for 24–48 h. The cured resin blocks were cut in an ultra-cut ultramicrotome. The ultra-cuts were stained using a solution of 4% uranyl acetate in ethanol for 10 min, followed by an aqueous solution of Reynolds’ lead (7 min). Six micrographs at a magnification of 2500× were taken for each sample to determine the fiber diameter.19 Statistical analysis Statistical analysis of the differences between each group was evaluated by one-way analysis of variance (ANOVA) using SPSS 18.0. Differences were regarded as significant at P < 0.05. All data are expressed as mean ± standard error.
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Effect of AMP on shear force of duck breast meat after cooking Meat tenderness is affected mainly by two components, i.e. myofibril and connective tissue. The former forms the actomyosin toughness, while the latter forms the background toughness. Compared with myofibrils, connective tissue structure seems to be unchanged during the post-mortem period,20 Many studies have been done on the impacts of heating and aging processes on meat tenderness, and relatively less attention has been paid to the effect of curing agent on tenderness development. As for AMP, there are almost no reports available on its application for improvement of meat tenderness. Therefore this study was carried out to evaluate the effect of AMP on improvement of tenderness. Tenderness can be determined by a trained panel or by physical methods, in which determination of shear force is the most widely used method.21 In the present study, shear force values of duck breast meats treated with different AMP concentrations were given by a texture analyzer, and the results are listed in Table 1. With increasing AMP concentration from 0 to 40 mmol L−1 , the shear force value decreased from 0.94 kg to 0.53kg, and every change of 10 mmol L−1 in AMP concentration resulted in a significant variation of shear force (P < 0.05). This result indicated that AMP could remarkably improve duck meat tenderness.
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Effect of AMP on microstructure of duck breast meat after cooking In order to study the impacts of AMP on microstructure of duck meat, SEM and TEM analyses were employed in this paper. Both muscle fiber diameter and sarcomere length increased with the concentration of AMP from 0 to 40 mmol L−1 , and every increase of 10 mmol L−1 in AMP led to significant changes in fiber diameter and sarcomere length (P < 0.05) except for sarcomere length, when AMP concentration increased from 30 to 40 mmol L−1 (see Table 1). Myofibrillar structures of AMP-treated duck breast meats are shown in Fig. 2. On transverse sections, gaps between muscle fibers were plainly visible in the control (Fig. 2A1), which was similar to those reported by Wattanachant et al.,22 who examined the microstructure of chicken meat. With increasing AMP concentration, muscle fibers became increasingly larger (Fig. 2B1–E1). Contrarily, the gaps between muscle fibers became smaller and almost disappeared when treated with 40 mmol L−1 ATP. On longitudinal sections, AMP exhibited the same effect on the microstructure of duck meat as on the transverse sections (Fig. 2A2–E2). In the control sample, myofibrils were closely packed, with the I band and A band being clearly distinguished, and Z-line and M-line being plainly visible. However, remarkable structural changes were observed in AMP-treated samples, and weakening of myofibrils and loss of Z-disks occurred when the sample was treated with 40 mmol L−1 AMP. These results indicated that AMP treatment could restrain the shrinkage of muscle fiber transversally and longitudinally, and could make the meat tender. Such a finding, from another perspective, supported the study by Li et al.,19 who reported transverse and longitudinal shrinkage of muscle fiber, resulting in tougher meat. Effect of AMP on actomyosin dissociation and MFI of duck breast meat Actomyosin dissociation occurs during the immediate post-rigor period and heating process, which improves meat tenderness.8,9,15,23 In the present paper, actomyosin dissociations in duck breast meats treated with different AMP concentrations were investigated and the results are shown in Fig. 3. In the control sample, only a low-density band of actin was observed, while the densities of actin bands were increased significantly in samples treated with AMP, and the density increased with the increasing of AMP level, which indicated that actomyosin dissociation was very weak in duck breast meat and AMP favored actomyosin dissociation. The result was consistent with that by Okitani et al.,8 who found that more actin was liberated from actomyosin of chicken, beef and pork skeleton muscles treated with AMP and IMP. Previous studies suggest that the binding of myosin motor domain to
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Table 2. Effect of AMP treatment on pH, water, cooking loss, AMP and IMP content of duck breast meat AMP treatment
Control
10 mmol L−1
20 mmol L−1
30 mmol L−1
40 mmol L−1
pH Water (%) Cooking loss (%) AMP (mg/g) IMP (mg/g)
5.89 ± 0.08e 80.17 ± 0.09e 33.24 ± 0.62a 0.03 ± 0.01a 1.55 ± 0.02e
6.31 ± 0.03d 81.18 ± 0.06d 29.84 ± 0.56b 0.04 ± 0.01a 2.41 ± 0.07d
6.59 ± 0.10c 81.56 ± 0.03c 20.24 ± 0.48c 0.03 ± 0.01a 3.88 ± 0.19c
6.73 ± 0.05b 81.78 ± 0.08b 15.22 ± 0.42d 0.04 ± 0.02a 5.90 ± 0.17b
6.87 ± 0.06a 82.45 ± 0.11a 10.61 ± 0.47e 0.03 ± 0.01a 7.52 ± 0.14a
Means in the same row with different letters differ significantly (P < 0.05). Data are expressed as mean ± SD (n = 6).
Effect of AMP on pH, water content and cooking loss of duck breast meat pH, water content and cooking loss of duck breast muscle treated with different AMP concentrations are listed in Table 2. Both pH and water content increased with increasing AMP, while cooking loss varied in the opposite direction. For all of these factors, every change of 10 mmol L−1 in AMP resulted in significant variations (P < 0.05). Meat pH is an important determinant of meat quality.29 – 31 An abnormally low pH may cause denaturation of sarcoplasmic and myofibrillar proteins and affect the integrity of skeletal muscle membrane, resulting in lower water content and less tenderness.29 Water content and cooking loss are related closely to pH; the pH of early post-mortem meat can explain 72% of the variation in water content.32 The pH is responsible for a large part of muscle shortening, which could lead to expulsion of water from the myofibrillar structure into the extracellular space and ultimately out of the muscle.33 As AMP could increase the muscle pH and restrain muscle shrinkage, it increases the space available for water within the myofibril. The improved water retention of meat could also partially be attributed to the increased ionic strength by AMP, which alters the conformation and solubility of myofibrillar proteins that bind and entrap water.
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L*
a
a*
b
50
c
b* d
d
40 30 20 10 0
a a
control
a
b
a
a c
a d
e
10 mmol L–1 20 mmol L–1 30 mmol L–1 40 mmol L–1
AMP treatment
Figure 5. Effect of AMP treatment on CIE* color of duck breast meat.
in remarkable variations of the values (P < 0.05), except for L* value when treated with 30 and 40 mmol L−1 AMP. As for a* value, no significant variation was observed. The low L* value has been reported to be related to higher water-holding capacity.35 Our results show that AMP treatment caused a significant decrease in brightness and yellowness of duck meat (P < 0.05), while redness showed no significant change (P > 0.05). This is in agreement with several studies demonstrating that breast meat lightness and yellowness were significantly negatively correlation with meat pH.35,36 According to Qiao et al.,37 the breast fillet of broiler is grouped to normal when L* is in the range of 48–53. We obtained similar results, which implied that AMP may have no negative impact on meat color. Effect of AMP on IMP and FAA content of duck breast meat AMP is an endogenous purine nucleotide, and can be converted to IMP and ammonia by adenosine deaminase (AMPD), which is abundant in all kinds of animal meats.38 In this paper, in order to understand the fate of the AMP employed to treat duck meat, contents of AMP, IMP and FAA in duck meats before and after AMP treatment were quantitatively analyzed, and the results are listed in Table 3. AMP level in meat sample was found unchanged when treated with 0–40 mmol L−1 AMP, while IMP level increased with increasing AMP concentration, and each increase of 10 mmol L−1 in AMP resulted in a significant variation of IMP in meat (P < 0.05). In meats treated with AMP, FAA content remained almost unchanged, except for ammonia, which increased significantly with each increase of 10 mmol L−1 in AMP (P < 0.05). These results indicate that the exogenous AMP permeating through duck meat, no matter how high the concentration, was totally converted to IMP and ammonia by AMPD in duck meat. IMP has an umami flavor and is widely distributed in animals. There is about 2 mg g−1 IMP in poultry and livestock muscles,39,40 whereas in fish IMP can reach 3–8 mg g−1 ; thus fish is of more
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Effect of AMP on CIE* color of duck breast meat Meat color is an important quality trait of meat products which affect the consumer’s decision in purchasing meat product; it is highly correlated with the metmyoglobin formation, muscle fiber type and water distribution.34 In the present paper, the effect of AMP on CIE* color of duck meat was investigated, and the results are shown in Fig. 5. AMP yielded significant impacts on L* and b*values, and every change of 10 mmol L−1 in AMP levels resulted
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Color of meat
actin depends on the nucleotide or nucleotide analogue bound to myosin subfragment.24,25 With bound nucleotide, the interaction of myosin with actin becomes weak. MFI is a useful indicator of both rupture of the I-band and breakage of intermyofibril linkages.26,27 MFI reflects the integrity of muscle fibers and its skeleton proteins. The larger MFI value indicates the increased damage that the internal structure of the myofibrils is exposed to.28 In this paper, MFI of duck breast muscles treated with different AMP concentrations were quantitatively analyzed and the results are presented in Fig. 4. MFI was significantly increased with increasing AMP concentration from 0 to 40 mmol L−1 (P < 0.05). The variation of MFI indicated that AMP promoted I-band rupture and breakage of intermyofibril linkages. Just as observed in microstructure, Z-line structure was lost and the internal structure of myofibrils was destroyed when duck meat samples were treated with AMP, which might result in the increase in MFI.
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D Wang et al.
Table 3. Effect of AMP treatment on free amino acid content of duck breast meat (expressed as mg g−1 ) AMP treatment Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Ammonia Histidine Arginine Proline Total
Control 0.07 ± 0.01a 0.30 ± 0.02a 0.17 ± 0.03a 0.16 ± 0.01a 0.11 ± 0.02a 0.21 ± 0.02b 0.01 ± 0.02a 0.06 ± 0.01a 0.04 ± 0.01a 0.05 ± 0.01a 0.09 ± 0.02a 0.07 ± 0.01a 0.07 ± 0.01a 0.06 ± 0.02b 0.08 ± 0.01e 0.03 ± 0.01a 0.09 ± 0.03a 0.07 ± 0.01b 1.71 ± 0.06b
10 mmol L−1 0.10 ± 0.01a 0.25 ± 0.01a 0.15 ± 0.01a 0.15 ± 0.02ab 0.10 ± 0.01a 0.24 ± 0.03ab 0.01 ± 0.01a 0.08 ± 0.02a 0.05 ± 0.01a 0.06 ± 0.01a 0.10 ± 0.01a 0.06 ± 0.01a 0.08 ± 0.02a 0.08 ± 0.01ab 0.16 ± 0.02d 0.04 ± 0.02a 0.11 ± 0.02a 0.09 ± 0.02ab 1.90 ± 0.22ab
20 mmol L−1 0.11 ± 0.02a 0.29 ± 0.03a 0.18 ± 0.02a 0.14 ± 0.02b 0.14 ± 0.03a 0.27 ± 0.02a 0.01 ± 0.03a 0.08 ± 0.01a 0.04 ± 0.02a 0.06 ± 0.02a 0.09 ± 0.01a 0.08 ± 0.02a 0.07 ± 0.01a 0.11 ± 0.02a 0.23 ± 0.02c 0.03 ± 0.01a 0.11 ± 0.01a 0.12 ± 0.01a 2.12 ± 0.23a
30 mmol L−1 0.10 ± 0.01a 0.27 ± 0.03a 0.16 ± 0.01a 0.11 ± 0.01b 0.13 ± 0.02a 0.28 ± 0.01a 0.01 ± 0.02a 0.07 ± 0.01a 0.05 ± 0.01a 0.04 ± 0.02a 0.08 ± 0.03a 0.07 ± 0.02a 0.06 ± 0.03a 0.10 ± 0.02a 0.31 ± 0.03b 0.03 ± 0.03a 0.12 ± 0.02a 0.10 ± 0.03a 2.13 ± 0.12a
40 mmol L−1 0.09 ± 0.01a 0.23 ± 0.01a 0.15 ± 0.02a 0.11 ± 0.01b 0.12 ± 0.02a 0.28 ± 0.03a 0.01 ± 0.02a 0.07 ± 0.02a 0.05 ± 0.01a 0.06 ± 0.01a 0.09 ± 0.02a 0.06 ± 0.01a 0.07 ± 0.02a 0.09 ± 0.01a 0.38 ± 0.02a 0.04 ± 0.02a 0.12 ± 0.03a 0.11 ± 0.02a 2.14 ± 0.10a
Means in the same row with different letters differ significantly (P < 0.05). Data are expressed as mean ± SD (n = 6).le
umami taste.41 In our previous studies, IMP was found to be able to promote dissociation of actomyosin, but its effect was much weaker than that of AMP.11 As many studies reported,38,40 ammonia is another product of AMP hydrolysis by AMPD; however, what role it plays in the dissociation of actomyosin originated by AMP remains unknown. We suppose that the generation of ammonia could increase the muscle pH, which might result in the increase of water retention and also the potential disruption of the Z-line of muscle fibers.
CONCLUSIONS The treatment of duck breast muscle with AMP may result in multiple biological and chemical reactions: dissociation of actomyosin directly by AMP, conversion of AMP to IMP and ammonia by AMPD and restraint of muscle fiber shrinkage. The series of reactions contributes to the quality improvement of duck breast meat. However, the mechanism regarding the impact of AMP on actomyosin dissociation and meat quality is still poorly understood, and further studies are needed to detail the mechanism and to enhance the function of AMP in meat products.
ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (31101312, 31271891), Natural Science Foundation Program of Jiangsu Province (BK2012785) and Innovation of Agricultural Science and Technology of Jiangsu Province (CX(12)5030).
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