Journal of Integrative Agriculture 2017, 16(0): 60345-7 Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
The effect of dehydrogenase enzymes activity in glycolysis on the colour stability of mutton during postmortem XIN Jian-zeng1, 2, LI Zheng2, LI Xin2, LI Meng2, WANG Ying2, YANG Fu-min1*, ZHANG De-quan2* 1 2
College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, P.R.China Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
Abstract This study investigated the influence of activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase-B (LDH-B) on the colour stability of mutton. From 60 sheep, 15 M. longissimus dorsi (LD) muscles were selected on the basis of colour stability (R630/580 and a* value) during the storage and classified into three groups (5 for one group) as high colour stability (HCS), intermediate colour stability (ICS) and low colour stability (LCS). The activities of GAPDH and LDH-B, instrumental colour attributes, nicotinamide adenine dinucleuotide (NADH) content and lactate content were measured. The samples in HCS had higher activities of GAPDH and LDH-B than the sample in the LCS, and the samples in HCS also possessed higher NADH content and lower lactate content. The higher activity of dehydrogenase enzyme can result in more NADH and colour stability. The results suggested that the activity of GAPDH and LDH-B may also play a role in maintaining the colour stability. Keywords: mutton, meat colour, GAPDH, LDH-B, NADH
year (Joseph et al. 2012; Suman and Joseph 2013). The
1. Introduction Meat colour is one of the most important quality attributes which influence consumer purchasing decisions, because consumers often consider the cherry-red colour of red meat as an indicator of wholesomeness at the point of sale (Joseph et al. 2012). Discolouration of fresh meat will lead to the rejection by consumer and it was estimated that the United States meat industry incurs more than one billion dollars in lost opportunity due to the discolouration every
metmyoglobin reducing system was considered as the most important factor that maintains the stability of meat colour during postmortem (Ledward et al. 1985; Kim et al. 2009a). Nicotinamide adenine dinucleuotide (NADH) is the ultimate reducing equivalent for the metmyoglobin reduction of enzyme and non-enzyme, which have been shown to be vital for delaying the discolouration in the postmortem muscle (Yuan et al. 2006; Ramanathan et al. 2011). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase B (LDH-B) are important enzymes in muscle glycolysis which plays a key role in forming NADH and maintaining the equilibrium of NADH
Received 27 December, 2016 Accepted 9 March, 2017 Correspondence YANG Fu-min, E-mail:
[email protected]; ZHANG De-quan, E-mail:
[email protected] © 2017, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(16)61622-2
pool in cytoplasm (Barron et al. 1998; Li et al. 2015). Kim et al (2006, 2009ab) reported that addition of lactate could enhance the colour stability through the replenishment of NADH catalyzed by LDH-B activity. In addition, the colour stability tends to be higher in bovine Longissimus lumborum
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muscles compared with Semimembranosus, and Psoas major, due to higher LDH-B (Kim et al. 2006, 2009a, b). Based on the above report, for colour stability of fresh muscle, LDH-B activity and its substrates are important factors because of the regeneration of NADH. In addition, GAPDH is involved in another formation pathway of NADH. In the previously study, glyceraldehyde-3-phosphate, the substrates of GAPDH, was proved reduce the metmyoglobin in beef ground (Saleh and Watts 1968). However, the studies about the effects of GAPDH activity on meat colour are limited. And whether the activity of GAPDH in muscles with different color stability is the same was unclear. We hypothesized that the activity of GAPDH in meat with different colour stability is inconsistent, so the objective of the current study was to determine the GAPDH activity and other biochemical characteristics of different colour stability of M. Longissimus dorsis (LDs). And also to evaluate the potential probability of GAPDH regulating the meat colour stability.
2. Materials and methods 2.1. Raw materials and preparation Sixty male sheep (Bayannur mutton sheep, 8 months of age) with an average carcass weight of (24.74±2.05) kg from a commercial plant (Inner Mongolia Grassland Hongbao Food Co. Ltd., China) were slaughtered following the industrial practice. At 30 min postmortem, both loins ((LDs) were collected (yielding 120 loins in total) from 60 lamb carcasses. Each muscle was divided into 4 steaks and placed on the styrofoam trays, and wrapped with oxygen-permeable polyvinylchloride (PVC, 35×300 type, Nantong South Asia Plastic Film Co., Ltd, Nantong, Jiangsu, China; oxygen transmission rate=25 mL m–2 24 h–1 0.1 Mpa–1) film. Then the sample trays were stored in an industrial refrigerator at (4±2)°C and were taken out for analysis after 2 h, 6 h, 1, 2, 4, 6 and 8 d storage, respectively. One steak was used for determination of instrumental colour and pH during the storage. Other seven steaks were frozen in liquid nitrogen at specific time, and then stored at –80°C until used for biochemical analysis.
2.2. Instrumental colour The colour of the samples over wrapped with PVC was measured through the PVC film after storage at 4°C for 2 h, 6 h, 1, 2, 4, 6, and 8 d. Colour was measured using a Minolta CM-600d spectrophotometer (Konica Minolta SensingInc., Osaka, Japan) with specular reflectance excluded, 8 mm diameter measuring aperture, illuminant D65, 10° standard observer and Commission Internationale de L’Eclairage
3
(CIE ) L*, a*, b* colour scale. The average value of four measurements on the meat surface was used. The Minolta instrument recorded reflectance values in the range of 360 to 740 nm at 10 nm intervals. Reflectance values that were not directly measured by the colour instrument at specific wave lengths (474, 525 and 572 nm) were calculated by integrations. The percentage of myoglobins was determined as described by Hunt and King (2012) using the formulas: A572–A700 Metmyoglobin%=(1.395– )×100 A525–A700 A473–A700 )×100 A525–A700 Oxymyoglobin%=100%–(MMb%+DMb%) Where, reflex attenuance (A)=log(1/R), A473, A525, A572 and A700 is the reflex attenuance at 473, 525 and 700 nm, respectively. R is reflectance. In addition, the ratio of reflectance at 630 and 580 nm (R630/580) was calculated as an indirect estimate of surface colour stability, a greater ratio indicates a lesser amount of metmyoglobin brown discolouration and thus higher colour stability. Instrumental color data on steaks from the sixty carcasses were ranked based on the R630/580 and a* value from day 4 to 8. From this ranking, the five (n=5) high colour-stable (HCS, R630/580, (2.74±0.13)), five (n=5) intermediate colour-stable (ICS, R630/580, (2.21±0.22)) and five (n=5) low color-stable (LCS, R630/580, (1.98±0.18)) steaks were identified to examine the molecular basis of animal-to-animal variation in color stability. Deoxymyoglobin%=(2.375–
2.3. pH value The pH values were measured by inserting the glass calomel probe and a temperature sensor of pH meter (Testo205 pH meter, Lenzkirch, Germany) directly into the raw sample. The device was calibrated with three buffers (pH 4, 7 and 10). All measurements were analyzed in triplicates from which an average was calculated.
2.4. GAPDH activity The enzyme was extracted from samples as described by the method (Baibai et al. 2007) with slight modifications. All steps were carried out on ice during the extraction. The muscle sample (approximately 2 g, fresh weight) was ground and homogenized using an IKA Ultra-Turrax homogenizer T10 basic S25 (IKA, Germany) in 25 mmol Tris-HCl buffer (pH 7.5), containing 5 mmol EDTA, 10 mmol 2-mercaptoethanol at a ratio of 4 mL g–1 fresh tissue. The supernatant (soluble protein fraction) obtained after centrifugation at 15 000×g for 45 min was considered as the extracted enzyme. The GAPDH activity of samples was measured with Sci-
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enCell colourimetric GAPDH assay kit (Catalog # 8148). It was determined spectrophotometrically by monitoring NADH generation at 340 nm.
2.5. LDH-B activity The activity of LDH in the sample was determined based on the method described by Kim (2009a, b). The muscle sample (2 g) was homogenized in 8 mL of sodium buffer (0.01 mol L–1, pH7.5) for 30 s and held on ice. The homogenate was filtered through Whatman # 42 filter paper after centrifuged at 13 823×g for 30 min at 4°C. The filtered liquid was used as enzyme extract to be measured. The reaction system for the determination of LDH activity contained filtered supernatant (0.1 mL), 172 mmol L–1 NAD (0.1 mL) and 2.4 mL of Tris (112 mmol L–1), KCl (170 mmol L–1), L-lactate (56 mmol L–1) (pH 9.3). The increased absorbance at 339 nm was measured (UV-2401 PC Spectrophotometer, Shimadzu, Kyoto, Japan) from 30 to 120 s and used for calculating LDH activity. The NADH content in fresh muscle (nmol g–1) was calculated. Units for the determination of enzyme activity were expressed as 1 mol min–1 g–1 sample.
2.6. NADH concentration NADH was extracted based on the methodology described by Klingenberg (1974) with slight modifications. Two grams of ovine muscle sample was mixed with 16 mL of 0.5 mol L–1 cooled alcoholic KOH solution. The mixture was vortexed for 30 s, then agitated in a 90°C water bath for 5 min, and cooled rapidly to 0°C in an ice bath for 5 min. The pH value of the muscle mixture was adjusted to 7.8 by adding 12 mL triethanolamine-HCl-phosphate mixture (0.5 mol L–1 triethanolamine hydrochloride, 0.4 mol L–1 KH2PO4, 0.1 mol L–1 K2HPO4). After holding at room temperature for 10 min to flocculate the denatured protein, the muscle mixture was centrifuged at 25 000×g for 10 min at 4°C (J2-21, Beckman Instruments, Inc., Palo Alto, CA) to get the clear supernatant. NADH concentration was measured based on the methodology described by McCormick an Wright (1971). Muscle extract supernatant (0.1 mL) was mixed with 2.5 mL of 17.5 μg mL–1 dichlorophenolindophenol (DCPIP), 0.5 mL of 0.1 mol L–1 pH 7.4 sodium phosphate buffer, 0.05 mL of 1 mg mL–1 phenazine methosulfate (PMS), 0.1 mL ethanol, and 0.05 mL alcohol dehydrogenase (E1.1.1.1, Sigma, USA, containing 0.3 mg protein). The change of the absorbance at 600 nm was measured (UV-2401 PC Spectrophotometer, Shimadzu, Kyoto, Japan) for 20 min to determine the NADH concentration. The NADH content in fresh muscle tissue (nmol g–1) was calculated with respect to the standard curve.
2.7. Lactate and pyruvate concentration The lactate and pyruvate assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). For lactate assays, the muscle sample was weighed precisely and added physiological saline with the ratio 1 g to 9 mL. The mixture was homogenized in an ice water bath and then centrifuged at 1 000×g for 10 min, and the supernatant was collected for further measurement. Then the lactate concentration was measured with lactate assay kit (A020) at the absorbance of 530 nm and calculated with respect to the lactate standard sample. The extract of pyruvate in the sample was carried out by lactate method. The pyruvate concentration was measured with pyruvate assay kit (A081) at the absorbance of 505 nm and calculated using the pyruvate standard sample.
2.8. Statistical analysis Instrument colour data, pH values, lactate data, GAPDH activity data, LDH-B activity data were analyzed using the SPSS 22.0 software. The results were expressed as means±standard error. Means were compared with ANOVA (one-way analysis of variance) to determine the levels of statistical significance at 0.05 level.
3. Results 3.1. a* values and R630/580 The a* values (redness) and R630/580 of LD steaks in different colour stability groups were shown in Fig. 1. The a* value of sample in different groups increased and then decreased. Steaks in the HCS group had higher a* value than the samples in the other two groups from day 2 to 8 during storage, and the difference was significant from day 4 to 8. The variation of R630/580 value was consistent with a* value, which also increased at first day and then decreased. The R630/580 value of samples in HCS group was significantly higher than the samples in the other two groups from day 2 to 8.
3.2. Proportions of myoglobin redox forms Myoglobin redox forms (%) were presented in Table 1. The relative metmyoglobin percentage of meat sample in three different colour stability groups decreased within 1 d and then increased during the remaining storage time. The accumulation of metmyoglobin in the sample of HCS group was lower than that in the LCS group. The relative deomyoglobin percentage of steaks in three groups decreased
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continuously, and there was no significant difference among the samples in the three groups. Oxymyoglobin (%) of steaks in different groups increased during the original 1 or 2 d after postmortem and then decreased. The sample in HCS had higher percentage of oxymyoglobin than the sample in LCS and ICS on the late storage time. The variation of oxymyoglobin was consistent with the a*-value
HCS
A 16
ICS
LCS a
a* value
14
a
b
12 10
a
b
a
c
8
b
c
6 4
2h
6h
1d 2d 4d Storage time
b
ab
2.5 b
2.0
6d
a
a b
c
b c
1.5
2h
6h
1d
8d
a
a
3.0 R630/580
and R630/580.
3.3. L* value, b* value, chroma and hue angle The L* value (lightness), b* value (yellowness), chroma and hue angle were presented in Table 2. L* value was increased at the first storage time and then decreased, however, no significant difference between the three groups in the same storage time. The b* value of sample in the HCS was higher than samples in the LCS group during the whole storage time except day 1, and the difference was significant from day 4 to day 8. The chroma value of steak in HCS was significant higher than the sample in the other two groups, except day 1. For the hue angle, the sample in the HCS group was higher than the other two groups.
3.4. pH and lactate
B 3.5
1.0
5
2d 4d Storage time
6d
c
8d
Fig. 1 Surface redness (a* value) (A) and ratio of reflectance at 630 to 580 nm (R630/580) (B) of ovine muscle during storage for 8 days at 4°C. HCS, high colour stability group. ICS, intermediate colour stability group. LCS, low colour stability group. Means between groups within the same storage day with different letters (a–c) are different (P<0.05). Error bars represent the standard error of the mean.
There was no significant difference of pH45 among the three groups as showed in Table 3. However, the pH24 of LD steaks in HCS was significantly higher (P<0.05) than the other two groups on the first day after postmortem. The lactate content of steaks in different groups was presented in Fig. 2. The lactate content in all samples increased within 1 day and then were stable during the storage time. The lactate concentration in the sample of HCS was lower than the sample in ICS and LCS during storage time except day 8.
3.5. NADH, LDH-B, and GAPDH The concentration of NADH in steaks of different groups are shown in Fig. 3. The NADH concentration in steaks of HCS was higher than sample in LCS on the time 2 h, 6 h, 1, 2, and 6 d. The difference of NADH in steaks between ICS and LCS was not completely significant. The activity of LDH-B in samples of different groups was
Table 1 Mb forms (%) of ovine muscle at 2 h, 6 h, 1, 2, 4, 6 and 8 d postmortem (4°C) Pigment Category1) %MetMb
%DeoMb
%OxyMb
1)
HCS ICS LCS HCS ICS LCS HCS ICS LCS
2h 25.71±0.35 by 27.09±0.45 aby 28.84±1.11 axy 53.72±4.79 ax 53.57±2.54 ax 49.19±2.06 ax 20.57±4.87 az 19.35±2.51 az 21.97±1.85 ay
6h 24.36±0.23 by 25.97±1.18 abxy 29.38±1.60 axy 51.22±3.89 ax 53.91±1.73 ax 56.03±1.83 aw 24.42±4.04 az 20.12±1.36 az 14.59±2.51 az
Time of storage 1d 2d 4d 6d 8d 18.30±0.78 az 23.27±0.58 by 25.59±1.33 by 29.03±1.20 bx 32.05±1.17 bw 18.93±0.24 az 23.64±0.57 by 27.33±0.32 ax 31.40±0.71 aw 37.21±1.82 au 20.19±0.54 az 27.06±0.81 ay 27.13±0.12 ay 31.30±0.61 ax 37.43±1.93 aw 19.14±1.95 ay 12.17±0.47 ay 11.21±0.59 az 11.76±0.56 az 11.37±0.39 az 18.96±1.18 ay 13.81±0.81 ay 12.97±0.52 az 12.04±0.63 az 11.28±0.90 az 19.31±0.99 ay 13.11±0.74 az 12.98±0.62 az 12.03±0.43 az 11.28±0.89 az 62.57±1.26 axy 64.56±0.61 ax 63.20±1.04 axy 59.21±0.86 axy 56.58±0.91 ay 62.11±1.29 aw 62.55±0.96 abw 59.70±0.82 bwx 56.56±1.06 ax 51.52±1.65 by 60.51±1.19 aw 59.84±1.05 bw 59.69±0.83 bw 57.58±1.08 aw 51.30±1.72 bx
HCS, high colour stability group; ICS, intermediate colour stability group; LCS, low colour stability group. Results are expressed as the mean±standard error. Means within the same group across storage time with different letters (u–z) are different (P<0.05). Means between groups within the same storage time with different letters (a–c) are different (P<0.05).
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illustrated in Fig. 4. The activity of LDH-B in sample of HCS decreased during the storage time. The sample in the HCS had higher activity than the sample in LCS, and significant difference were on the 6 h, 1 d, 4 d and 6 d. The activity of LDH-B between the HCS and LCS was not significant during the most storage time. The activity of GAPDH in steaks of different groups was shown in Fig. 5. The activity of GAPDH in the sample of HCS and ICS was higher than the sample in LCS, and the difference was found significant on the 2 h, 6 h, 1 d, 2 d, 4 d after the postmortem.
4. Discussion
In partial agreement with our result, King et al. (2011) reported that steaks with stable colour possessed higher a* value. During storage, steaks in three groups demonstrated a decrease on a* value. In agreement with our result, previous study Joseph et al. (2012) also documented a decrease in redness of beef muscle. The result of R630/580 suggested that the steaks in HCS group had higher colour stability. Corroborating our result on a* value, the R630/580 value of sample in the three groups decreased during storage. Joseph et al. (2012) also reported that 630/580 value of two muscles (Longissimus lumborum (LL) and PM) decreased throughout the retail display.
4.2. L* value and b* value
4.1. a value and R630/580 *
The a* value is one of the most important indicators of the colour stability of fresh muscle, and the redness of fresh muscle is mainly influenced by the proportion of oxymyoglobin (Mancini and Hunt 2005). The a* value and proportion of oxymyoglobin in steaks of HCS were higher compared with that in the other two groups from day 2 to 8 in the present study.
L* value is another key indicator of the colour stability of fresh muscle, in the current research steaks in three groups exhibited no difference on L* value during storage except day 8. In support, previous study (King et al. 2011; Canto et al. 2015) demonstrated that beef muscle which has different colour stability shows no difference on lightness (L* value) during storage. Muscles from different source that
Table 2 Instrumental colour of ovine muscle at 2 h, 6 h, 1-, 2-, 4-, 6-, and 8-day postmortem (4°C). Parameters1) Category2) L*
b*
Chroma
Hue
HCS ICS LCS HCS ICS LCS HCS ICS LCS HCS ICS LCS
2h
6h
1d
34.08±0.35 az 33.96±0.26 az 33.28±0.86 ay 6.27±0.23 az 5.40±0.36 az 5.23±0.31 ay 10.73±0.43 az 9.53±0.44 ay 9.53±0.41 az 35.77±0.60 ay 34.44±1.07 az 33.31±1.33 az
34.43±0.82 ay 36.87±1.60 ay 33.21±0.41 ay 6.10±0.26 az 5.31±0.47 az 5.47±0.22 ay 10.32±0.39 az 9.07±0.73 ay 9.67±0.24 az 36.28±1.03 ay 36.37±2.31 az 34.39±1.23 az
40.22±1.38 ax 41.29±0.40 ax 38.75±0.77 ax 10.56±0.60 ay 10.63±0.32 ay 10.57±0.49 ax 15.83±0.96 ay 15.59±0.53 ax 15.38±0.57 ax 41.91±0.62 ax 43.05±0.69 ay 43.41±1.09 ay
Time of storage 2d
4d
42.27±1.01 ax 40.89±0.61 ax 42.60±0.84 ax 42.07±0.80 ax 40.88±0.52 ax 39.73±0.71 ax 12.47±0.65 ax 13.12±0.60 ax 11.77±0.49 axy 12.05±0.51 abx 10.46±0.62 ax 10.67±0.24 bx 18.43±0.66 awx 19.34±0.59 aw 17.33±0.68 abx 17.49±0.72 ax 15.07±0.75 bx 15.11±0.37 bx 42.53±1.31 ax 42.68±1.03 ax 42.84±1.00 ay 43.54±0.54 axy 43.93±1.28 axy 44.97±0.71 axy
6d
8d
39.60±0.33 ax 41.45±0.48 ax 39.50±0.82 ax 12.29±0.33 ax 11.86±0.44 axy 9.59±0.39 bx 18.03±0.43 awx 16.83±0.62 ax 13.11±0.40 by 42.96±0.55 bx 44.83±0.51 abxy 46.98±0.82 awx
39.30±0.57 bx 41.96±0.71 ax 39.41±0.77 abx 12.01±0.22 ax 11.74±0.37 axy 10.36±0.33 bx 17.46±0.25 axy 16.11±0.54 ax 13.56±0.34 by 43.43±0.44 cx 46.88±0.98 bx 49.81±0.80 aw
1)
Chroma=(a*2+b*2)0.5, Hue angle=ATAN (b*/a*)×(180/π). HCS, high colour stability group; ICS, intermediate colour stability group; LCS, low colour stability group. Results are expressed as the mean±standard error. Means within the same group across storage time with different letters (w–z) are different (P<0.05). Means between groups within the same storage time with different letters (a–c) are different (P<0.05).
2)
Table 3 pH of muscle samples in three groups at 45 min and 24 h postmortem.
have inconsistent colour stability also had no significant
Category1) HCS ICS LCS
pH24 5.94±0.02 ay 5.87±0.02 by 5.83±0.01 by
2015). On contrary, Neethling et al. (2016) reported that
HCS, high colour stability group; ICS, intermediate colour stability group; LCS, low colour stability group. Results are expressed as the mean±standard error. Means within the same group across storage time with different letters (x and y) are different (P<0.05). Means between groups within the same storage time with different letters (a and b) are different (P<0.05).
The steaks in the present study significantly increased
1)
pH45 6.96±0.02 ax 6.93±0.04 ax 6.97±0.04 ax
difference on L* value (Joseph et al. 2012; Canto et al. different source muscles from blesbok have inconsistent L* value during storage. during the storage time. In agreement with our result on lightness, Luciano et al. (2009) reported that semimembranosus muscle (SM) from lamb possessed an increase throughout the storage. Likewise, Bao et al. (2016) also
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King et al. (2011) demonstrated that longissimus thoracis steaks with stable colour stability had higher yellowness throughout storage except day 0 and 1. Canto et al. (2016) also reported that LL from Nellore had higher b* than PM. Furthermore, McKenna et al. (2005) demonstrated that muscle that possessed higher colour stability also maintained greater b* value than muscle which had low or very low colour stability. The result of present research exhibited that storage time increased the b* value of steaks in three groups. In support
reported that L* value of patties made from minced beef significantly increased during the storage time. Gao et al. (2013) demonstrated that LD from lamb showed a decrease on L* value during the simulated retail display, and this was not similar with our result. But the L* value of beef steaks in some previous reported studies (McKenna et al. 2005; King et al. 2010; King et al. 2011; Canto et al. 2015) were different with our report and this may due to animal difference. Steaks in HCS group had higher b* value (yellowness) than steaks in LCS group from day 4 to 8. In support,
HCS
120
ICS
LCS ax
Lactate content (μmol g–1)
110 100
ax
90 80
by by
70 60 50
ay
ax
bx
bx
ax
ax
ax
bx
ax
ax
bx
bx
bx
bx
az bz bz
40 30
2h
6h
1d
2d
4d
6d
8d
Storage time
Fig. 2 Lactate concentrations determined in meat sample during storage. HCS, high colour stability group. ICS, intermediate colour stability group. LCS, low colour stability group. Means among groups at the same time points with different letters (a–c) are different (P<0.05). Error bars represent the standard error of the mean. Means within the same group across storage days with different letters (x and y) are different (P<0.05).
100
NADH content (nmol g–1)
90
au bu
80
bu
HCS
au bu bu
av
bv bv
ICS
aw bw
70
LCS
60
cw x
50
x
40
x ay by by
30
z
z
z
20 10 0
2h
6h
1d
2d
4d
6d
8d
Storage time
Fig. 3 The NADH concentration in M. longissimus dorsi steaks in different colour stability groups during the storage time. HCS, high colour stability group. ICS, intermediate colour stability group. LCS, low colour stability group. Means between groups within the same storage time with different letters (a-b) are different (P<0.05). Means within the same group across storage days with different letters (u-z) are different (P<0.05).
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HCS
10
LDH-B Activity (μmol min–1 g–1)
LCS
a
9
a
8 7
a a
6 5
ICS
a a
b
a
4
a
a
a
a
a
b
a
a
a
b
a
3 b
2
a
1 0
2h
6h
1d
2d
4d
6d
8d
Storage time
Fig. 4 LDH-B activity of M. longissimus dorsi steaks in different colour stability groups during the storage time. HCS, high colour stability group. ICS, intermediate colour stability group. LCS, low colour stability group. Means between groups within the same storage time with different letters (a and b) are different (P<0.05).
HCS
ICS
20 GAPDH activity (U g–1)
18 16 14 12
a
a
a
a
b
10
a
a
a
LCS
a b
b
b
8
b
c
6
a
a
a a
a
a
a
4 2 0
2h
6h
1d
2d
4d
6d
8d
Storage time
Fig. 5 GAPDH activity of M. longissimus dorsi steaks in different colour stability groups during the storage time. HCS, high colour stability group. ICS, intermediate colour stability group. LCS, low colour stability group. Means between groups within the same storage time with different letters (a and b) are different (P<0.05).
to our results, previous research (Luciano et al. 2009) had
which can maintain the reduction state of myoglobin and
a similar result. In contrast, Gao et al. (2013) reported that
colour stability (Yin and Faustman 1993; Hunt and King
b value of LD from lamb showed a decrease throughout
2012; Richards 2013).
*
the display.
The accumulation of lactate in muscle was closely related to the change in pH after slaughter (Matarneh et al. 2015).
4.3. pH and lactate
The results of our study confirmed this conclusion. The extent of pH decline in steaks of three groups was linearly
The pH value of the muscle 24 hours after slaughter was
associated with the increase of lactate on day 1.
an important factor which influenced the instrumental colour (Calnan et al. 2016). The result of present research
4.4. NADH, LDH-B, and GAPDH
confirmed that steaks in HCS had higher colour stability and pH24. The result may ascribe the reason to higher pH,
The regeneration of NADH play a key role in maintaining the
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colour stability of meat (Ramanathan et al. 2011a, b). The steaks in HCS group had higher NADH than steaks in other two groups. Kim et al. (2008) reported that LL possessed higher colour stability and NADH content than PM, which was partially consistent with our results. GAPDH and LDH-B are important dehydrogenase enzymes which can regenerate the NADH that is necessary for the reduction of metmyoglobin. In support, previous research (Kim et al. 2009a; Ramanathan et al. 2011ab) demonstrated that LDH-B play an important role in maintaining the colour stability of meat. This was corresponding with our study, which also showed that steaks in HCS had higher LDH-B activity. Our present result is in partial agreement with the Kim et al. (2008) reports, which indicated that LL possessed higher colour stability and activity of LDH-B than PM. GAPDH can also catalyze the formation of NADH in the glycolytic pathway. It had been reported that the activity of GAPDH increases the reduction of metmyoglobin in beef ground (Saleh and Watts 1968), and steaks of LL in color-stable group had greater GAPDH content than steaks in color-labile (Canto et al. 2015). The research of Canto speculated that the possible reason is that the muscle in color-stable group which have more content glycolytic enzymes also possess a higher strong glycolytic metabolism and thus can enhance the production of NADH. For the first time, the result of the present study confirmed that the muscle with high color stability also possess higher GAPDH activity. It can be speculated that the muscle possessed higher activity of GAPDH which results in greater content of NADH, then the meat had a lower rate of accumulation of metmyoglobin and consequently higher colour stability.
5. Conclusion The result of present research exhibited that steaks in HCS group had higher activity of GAPDH and LDH-B. These two enzymes possessed higher activity in the more stable colour steaks, and them may play a role in maintaining stable color in coordination, as they can produce NADH. Future studies should focus on the reason what caused the inconsistent activity of these two dehydrogenase enzymes, and the role of them in diverse animal muscle, such as pork and beef, and determine the activity dehydrogenase enzymes in glycolysis of abnormal meat like DFD and PSE with respect to meat color stability.
Acknowledgements We gratefully acknowledge financial support from the National Agricultural Science and Technology Innovation Program, the Special Fund for Agro-scientific Research in the Public Interest (201303083) in China and China Agriculture
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Research System (CARS-39).
References Baibai T, Oukhattar L, Moutaouakkil A, Soukri A. 2007. Purification and characterization of glyceraldehyde-3phosphate dehydrogenase from european pilchard sardina pilchardus. Acta Biochimica et Biophysica Sinica, 39, 947–954. Bao Y, Puolanne E, Ertbjerg P. 2016. Effect of oxygen concentration in modified atmosphere packaging on color and texture of beef patties cooked to different temperatures. Meat Science, 121, 189–195. Barron J T, Gu L, Parrillo J E. 1998. Malate-aspartate shuttle, cytoplasmic nadh redox potential, and energetics in vascular smooth muscle. Journal of Molecular & Cellular Cardiology, 30, 1571–1579. Calnan H, Jacob R H, Pethick D W, Gardner G E. 2016. Production factors influence fresh lamb longissimus colour more than muscle traits such as myoglobin concentration and pH. Meat Science, 119, 41–50. Canto A C, Costa-Lima B R, Suman S P, Monteiro M L, Viana F M, Salim A P, Nair M N, Silva T J, Conte-Junior C A. 2016. Color attributes and oxidative stability of longissimus lumborum and psoas major muscles from Nellore bulls. Meat Science, 121, 19–26. Canto A C, Suman S P, Nair M N, Li S, Rentfrow G, Beach C M, Silva T J, Wheeler T L, Shackelford S D, Grayson A, McKeith R O, King D A. 2015. Differential abundance of sarcoplasmic proteome explains animal effect on beef Longissimus lumborum color stability. Meat Science, 102, 90–98. Gao X G, Xie L, Wang Z Y, Li X M, Luo H L, Ma C W, Dai R T. 2013. Effect of postmortem time on the metmyoglobin reductase activity, oxygen consumption, and colour stability of different lamb muscles. European Food Research and Technology, 236, 579–587. Hunt M C, King A. 2012. AMSA Meat Color Measurement Guidelines. American Meat Science Association. Champaign, Illinois, USA 61820. pp. 1–135. Joseph P, Suman S P, Rentfrow G, Li S, Beach C M. 2012. Proteomics of muscle-specific beef color stability. Journal of Agricultural & Food Chemistry, 60, 3196–3203. Kim Y H. 2008. Lactate dehydrogenase regulation of the metmyoglobin reducing system to improve color stability of bovine muscles through lactate enhancement. Dissertation Abstracts International, 69, 1–115. Kim Y H, Hunt M C, Mancini R A, Seyfert M, Loughin T M, Kropf D H, Smith J S. 2006. Mechanism for lactatecolor stabilization in injection-enhanced beef. Journal of Agricultural & Food Chemistry, 54, 7856–7862. Kim Y H, Keeton J T, Smith S B, Berghman L R, Savell J W. 2009a. Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles. Meat Science, 83, 376–382. Kim Y H, Keeton J T, Yang H S, Smith S B, Sawyer J E, Savell
10
*** et al. Journal of Integrative Agriculture 2017, 16(0): 60345-7
J W. 2009b. Color stability and biochemical characteristics of bovine muscles when enhanced with L- or D-potassium lactate in high-oxygen modified atmospheres. Meat Science, 82, 234–240. King D A, Shackelford S D, Kuehn L A, Kemp C M, Rodriguez A B, Thallman R M, Wheeler T L. 2010. Contribution of genetic influences to animal-to-animal variation in myoglobin content and beef lean color stability. Journal of Animal Science, 88, 1160–1167. King D A, Shackelford S D, Rodriguez A B, Wheeler T L. 2011. Effect of time of measurement on the relationship between metmyoglobin reducing activity and oxygen consumption to instrumental measures of beef longissimus color stability. Meat Science, 87, 26–32. Klingenberg M. 1974. Nicotinamide-adenine dinucleotides (NAD, NADP, NADH, NADPH) spectrophotometric and fluorimetric methods. In: Methods of Enzymatic Analysis. 2nd ed. Academic Press, New York and London. pp. 2045–2072. Ledward D A. 1985. Post-slaughter influences on the formation of metyyoglobin in beef muscles. Meat Science, 15, 149–171. Li W, Sauve A A. 2015. NAD(+) content and its role in mitochondria. Methods in Molecular Biology, 1241, 39–48. Luciano G, Monahan F J, Vasta V, Pennisi P, Bella M, Priolo A. 2009. Lipid and colour stability of meat from lambs fed fresh herbage or concentrate. Meat Science, 82, 193–199. Matarneh S K, England E M, Scheffler T L, Oliver E M, Gerrard D E. 2015. Net lactate accumulation and low buffering capacity explain low ultimate pH in the longissimus lumborum of AMPKγ3R200Q mutant pigs. Meat Science, 110, 189–195.
McCormick D B, Wright L D. 1971. Nicotinic acid: Analogs and coenzymes. In: Colowick S P, Kaplan N O (Eds.), Methods in Enzymology, Vol. 18. New York and London: Academic Press. pp. 26–27. McKenna D R, Mies P D, Baird B E, Pfeiffer K D, Ellebracht J W, Savell J W. 2005. Biochemical and physical factors affecting discoloration characteristics of 19 bovine muscles. Meat Science, 70, 665–682. Neethling N E, Suman S P, Sigge G O, Hoffman L C. 2016. Muscle-specific colour stability of blesbok (Damaliscus pygargus phillipsi) meat. Meat Science, 119, 69–79. Ramanathan R, Mancini R A, Dady G A. 2011. Effects of pyruvate, succinate, and lactate enhancement on beef longissimus raw color. Meat Science, 88, 424–428. Ramanathan R, Mancini R A, Joseph P, Yin S, Tatiyaborworntham N, Petersson K H, Sun Q, Konda M R. 2011. Effects of lactate on ground lamb color stability and mitochondriamediated metmyoglobin reduction. Food Chemistry, 126, 166–171. Richards M P. 2013. Redox reactions of myoglobin. Antioxid Redox Signal, 18, 2342–2351. Saleh B, Watts B M. 1968. Substrates and intermediates in the enzymatic reduction of metmyoglobin in ground beef. Journal of Food Science, 33, 353–358. Suman S P, Joseph P. 2013. Myoglobin chemistry and meat color. Annual Review of Food Science and Technology, 4, 79–99. Yin M C, Faustman C. 1993. Influence of temperature, pH, and phospholipid composition upon the stability of myoglobin and phospholipid a liposome model. Journal of Agricultural & Food Chemistry, 41, 853–857. (Managing editor WENG Ling-yun)