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. 2020 Sep 7;58(7):2528–2537. doi: 10.1007/s13197-020-04759-x

Effect of three different proteases on horsemeat tenderness during postmortem aging

Yimei Cheng 1, Xiaofeng Jiang 2, Yufei Xue 1, Fengmin Qi 1, Zhiwei Dai 1, Dong Guan 3, Lingming Kong 1,✉
PMCID: PMC8196178  PMID: 34194089

Abstract

In this study, the semitendinosus of horse meat was used as the raw material. The study assessed the variation of the tenderness of horse meat during postmortem aging through the injection of papain, bromelain and fungal protease. The cooking loss of the horse meat became worse during postmortem aging. Low concentration of protease improved water retention properties of horse meat. Papain, bromelain and fungal protease had significant influence on MFI and shear force. MFI increased obviously but the shear force decreased significantly with the addition of more protease (p <0.05). During postmortem aging, many small molecules popeptide appeared in treatment group. Myosin light chain 2, 20 KDa, 32 KDa and 75 KDa bands appeared at first, however later they disappeared in the group with high concentration of protease treatment in addition to the disappearance of Desmin and Troponin I. The muscle fiber, perimysium and endomysium were degraded because of papain, bromelain and fungal protease treatment. More muscle fiber fragments appeared during postmortem aging.Thus, the tenderness and eating quality of horse meat were improved by adding three kinds of protease.

Keywords: Horse meat, Papain, Bromelain, Fungal protease, Tenderness

Introduction

With the improvement of people's living standards, the requirements for meat quality are becoming higher and higher. Generally, the edible quality of meat refers to the comprehensive reflection of the physical and chemical properties such as the appearance and texture, taste and nutritional value of meat products after the processing of raw meat (Pieszka et al. 2016). Among the various indicators to evaluate the edible quality of meat, the tenderness of meat is the most concerned and valued by consumers. Therefore it is important that a commercially applicable method can be developed to ensure a consistently tender product in order to improve the consumer acceptance for meat products (Doneva et al. 2015). There is considerable interest in the development of methods to produce meat with consistent tenderness and to improve the tenderness of tougher cuts of red meat while maintaining meat quality (Koohmaraie 1996). Eating quality has been regarded as the most critical characteristic in this aspect, because it can influence the repeated purchase behaviours of consumers. Many surveys have shown that tenderness was the most important palatability meat trait for consumers to repeat their purchasing behaviours (Shackelford et al. 2001). The animal husbandry is one of major industries in Xinjiang province, China. In addition, it is also the main area for the horse breeding centres. Horse meat production including smoked horse intestine and smoked horse meat is one of the most popular animal products in Xinjiang. However, the tenderness of such horse meat products has yet to be improved. Currently, the use of exogenous proteases to improve meat tenderness has attracted much interest. Plant enzymes (such as papain, bromelain, and ficin) have been extensively used as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to the controlled meat tenderization and other advantages. These enzymes can digest muscle proteins when they are mixed with meat. They also can hydrolyze collagen and elastin, which help plant thiol proteases to tenderize meat.Therefore, the purpose of this study was to compare the effects of papain, bromelain and fungal protease on tenderization and eating quality of horse meat.

Materials and methods

Materials and reagents

Horse meat (semi-tendinosus) (male llihorses, 36–60 months old, body weight (300 ± 50) kg), purchased from Xinjiang Zhaosu County Green Source Food Co., Ltd. These were the meat from 1 h after slaughter. The meat were rapidly transported to the laboratory wrapped in polyethylene bags where they were stored at 4 °C. Papain (800,000 U/g) and bromelain (500,000 U/g) were bought from Nanning Pangbo Biological Engineering Co. Ltd., China. Fungal protease (conc400) (100,000 U/g) was obtained from Enzyme Solutions Pty. Ltd., Australia. All experimental reagents used were analytical pure unless otherwise stated.

Sample preparation

After removing tendons from horse meat, the meat was cut into 3 cm×3 cm×6 cm meat 648 pieces. Papain, bromelain and fungal protease solutions were prepared using phosphoric acid buffer of pH 7.0 and then they were placed in 100 mL beaker, respectively. Three protease solutions were injected into horse meat using a disposable syringe (10 mL/100 g according to the weight of meat). The meat pieces were then divided into 13 samples with different treatments as follows: Sample C was the horse meat without any treatment; sample P1, P2, P3, and P4 samples of horse meat were injected with 10, 20, 30, and 40 U/g papain, respectively; sample B1, B2, B3, and B4 were the samples of horse meat injected with 10, 20, 30, and 40 U/g bromelain, respectively; sample F1, F2, F3, and F4 were the samples of horse meat injected with 10, 20, 30, and 40 U/g fungal protease, respectively.

After that, the samples were sealed with a self-sealing bag (12 cm×8 cm) and placed in the vacuum rolling machine(GR-20, Zhucheng Meibang Machinery Co., Ltd, C, China) for 30 min. Finally, they were placed in the refrigerator at 4 °C All indexes were measured respectively after 1, 3, 7 and 14 d storage.

Cooking loss

Before cooking, its weight was m1 The samples were cooked individually in plastic bags at 90 °C using a water bath (HH-420, Shanghai lichen Bangxi Instrument Technology Co., Ltd., China) until the core temperature reached 70 °C. During heating, the digital thermometer (DM6801A, Shenzhen Victor Hi-Tech Co. Ltd., China) was inserted into the samples individually to track the core temperature. After cooking, the samples were cooled at room temperature for 1 h. Next, the filter paper (12.5 cm diameter) was used to absorb the water on the surface of sample, and at this time its weight is m2 Cooking loss of sample was measured by the following equation:

Cooking loss(%)=m1-m2/m2∗100

Shear Force Measurements

Warner–Bratzler shear force (WBS) was determined using a texture analyzer (Stable Micro System co. Ltd., Surrey, UK) equipped with a Warner–Bratzler shear device. Briefly, after cooking losses were measured, the cooked samples were cut into 5 strips (5 cm × 1 cm × 1 cm) parallel to the longitudinal orientation of muscle fiber. After that, they were sheared perpendicular to the long axis of the cores. Shearing procedure was performed with a pre-test speed of 2 mm/s, test speed of 2 mm/s and post-test speed of 10 mm/s, respectively.

Determination of myofibril fragmentation index (MFI)

Myofibril fragmentation index was determined by the modified method of Culler et al. (1978). Meat samples (4 g) without visible fat and connective tissue were homogenized for 30 s at 4 °C in 40 mL buffer containing 100 mM KCl, 11.2 mM K2HPO4, 8.8 mM KH2PO4, 1 mM EGTA, 1 mM MgCl2 and 1 mM NaN3 at 4 °C. The homogenate was centrifuged (H1850, Hunan Xiang Yi, China) at 10,000 r/min for 2 min and the supernatant was discarded. The sediment was re-suspended in 40 mL buffer before centrifuging and the supernatant was discarded again as the procedures above. The sediment was re-suspended in 10 mL buffer and filtered through a copper screen (40 mesh) to remove fat and connective tissues. The myofibrillar protein concentration of suspension was determined according to the Biuret method (Gornall et al. 1949). Then, the suspension was diluted to a protein concentration of 0.5 mg/mL with buffer. And this was also used as a sample for SDS-PAGE analysis. The absorbance of the myofibrillar suspension was measured at 540 nm using a UV spectrophotometer (UV1780, Shimadzu) using a standard curve constructed from bovine serum protein and multiplied by a factor of 200 in order to obtain MFI value.

Electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 4% stacking gel and 12% separating gel. The diluted myofibrillar protein samples (prepared during the determination of MFI was mixed with the sample buffer (1:5 = v/v) containing 10% of β-mercaptoethanol (β-ME). The mixture was then boiled for 5 min. Each well of gel was loaded with 10 μ L of samples. After electrophoresis, gels containing protein bands were stained with 0.1% coomassie brilliant blue. After clearing the free dye, the gels were photographed with a Gel Doc XRTM System (Bio-Rad Laboratories, Hercules, CA).

Statistical analysis

The entire experiment was carried out in at least three times. The results were expressed as mean ± standard deviation. The obtained data were analyzed by using one-way analysis of variance (ANOVA) (SPSS 20.0, Chicago, IL, USA). The data were then compared using Duncan’s multiple range tests at 5% or 1% significance level.

Results

Variation of cooking loss of horse meat with different protease during postmortem aging

The water holding capacity of meat is usually measured by cooking loss. It has a direct effect on the quality of meat, such as the flavor, taste, juiciness, tenderness and color (Ngapo et al. 1999).

When compared with the blank group, the cooking loss of the P1 and P2 groups was reduced, but the cooking loss of P3 and P4 was significantly increased (Table 1). Among all papain treatment groups, the cooking loss of P4 group was the highest. In the bromelain treatment group, the cooking loss of the B1 treatment group was decreased compared with the blank group, but the cooking loss of the other treatment groups was significantly increased. The cooking loss of F1 was the smallest among all the group of fungal protease treatments with an increasing concentration of fungal protease, the cooking loss significantly increased (p<0.05).

Table 1.

Variation of cooking loss of horse meat with different protease during postmortem aging

Enzymes Enzymes concentration/postmortem time Cooking loss (%)
1 d 3 d 7 d 14 d
Papain Control 34.4 ± 1.0efghi 35.9 ± 0.5def 35.5 ± 1.3defgh 36.7 ± 0.4cdef
10 U/g 31.8 ± 1.4i 32.6 ± 1.0hi 33.6 ± 1.2fghi 34.3 ± 0.7efghi
20 U/g 32.6 ± 1.1ghi 34.6 ± 1.1efghi 35.7 ± 0.9defg 36.7 ± 1.2cde
30 U/g 36.0 ± 0.6def 36.1 ± 0.9def 37.1 ± 1.2cde 37.8 ± 0.5cd
40 U/g 38.3 ± 1.1cd 39.5 ± 1.1bc 41.9 ± 1.0ab 42.7 ± 1.2a
Bromelain Control 33.0 ± 0.2hijk 35.9 ± 0.2defg 35.5 ± 1.3efgh 39.0 ± 0.5ab
10 U/g 30.7 ± 0.9k 31.6 ± 0.7jk 32.6 ± 1.2ijk 33.8 ± 0.9ghij
20 U/g 33.9 ± 1.7ghik 34.7 ± 0.7fghi 34.2 ± 0.5fghij 36.0 ± 0.3defg
30 U/g 36.0 ± 1.3cdefg 36.5 ± 0.9bcdefg 36.5 ± 1.1bcdefg 36.6 ± 0.9bcdef
40 U/g 39.4 ± 0.8a 38.1 ± 0.5abcde 38.7 ± 0.8abc 38.3 ± 0.7abcd
Fungal Control 33.0 ± 1.1hijk 35.9 ± 0.4defg 35.5 ± 1.3efgh 39.0 ± 0.9ab
10 U/g 30.7 ± 3.02k 31.6 ± 1.6jk 32.6 ± 1.1ijk 33.8 ± 1.2ghij
20 U/g 33.9 ± 1.0ghij 34.7 ± 2.2fghi 34.2 ± 0.9fghij 36.0 ± 1.0defg
30 U/g 36.0 ± 0.5cdefg 36.5 ± 0.9bcdefg 36.5 ± 1.3bcdefg 36.6 ± 0.8bcdef
40 U/g 39.4 ± 1.3a 38.1 ± 0.8abcde 38.7 ± 0.9abc 38.3 ± 1.1abcd
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From Table 1, we can see that papain, bromelain and fungal protease had a significant effects on the cooking loss of horse meat (p < 0.01). The effect of maturity time on cooking loss was also significant (p < 0.05). With the extension of the maturity time, the cooking loss of the control group and treatment group showed a gradually increasing trend. Due to the meat maturation process, the skeleton protein tertiary structures degrade and water is released (Labeit and Kolmerer 1995), which cause the meat become less juicy. It can also be seen from Table 1 that as the protease concentration increased, the cooking loss first decreased and then increased.

Our results were consistent with the findings reported by other studies which studied the effects of papain on the tenderness of beef and camel meat, respectively (Ming et al. 2009; Ma et al. 2012). The authors reported that the papain at low concentration reduced the cooking loss of meat. When the concentration of papain was too high, the cooking loss increased significantly.

Variation of MFI value of horse meat with different protease treatment during postmortem aging

As shown in Table 2, with increasing maturity time, the myofibril fragmentation index (MFI) gradually increased. This indicated that myofibrillar integrity became worse with the extension of maturity time, and the tenderness of the meat became better. Table 2 shows that the addition of three different proteases made MFI value increase, indicating that the three proteases improved the maturity speed and tenderness of meat.

Table 2.

Variation of MFI value of horse meat with different protease treatment during postmortem aging

Enzymes Enzymes concentration/postmortem time MFI
1 d 3 d 7 d 14 d
Papain Control 26.3 ± 1.9k 35.4 ± 1.2j 42.1 ± 0.6i 49.8 ± 1.4gh
10 U/g 32.7 ± 1.4j 42.3 ± 1.7i 51.6 ± 1.2g 64.7 ± 1.0de
20 U/g 35.6 ± 1.1j 46.5 ± 1.2h 55.6 ± 1.0f 72.7 ± 1.6c
30 U/g 35.4 ± 0.6j 50.9 ± 1.3g 61.6 ± 1.2e 77.2 ± 1.4b
40 U/g 36.3 ± 0.5j 56.9 ± 1.6f 68.0 ± 0.6d 83.4 ± 2.1a
Bromelain Control 43.5 ± 1.2m 56.8 ± 1.5jk 58.4 ± 0.8ij 60.7 ± 1.0hi
10 U/g 48.9 ± 0.6l 54.5 ± 1.1k 62.0 ± 1.7gh 64.2 ± 0.6fg
20 U/g 54.4 ± 0.7k 60.7 ± 1.0hi 66.2 ± 1.2ef 66.8 ± 1.0def
30 U/g 58.5 ± 1.1ij 64.5 ± 0.9fg 68.7 ± 1.0de 72.6 ± 0.9bc
40 U/g 63.6 ± 1.0fgh 69.6 ± 0.8cd 74.6 ± 0.8ab 76.9 ± 1.0a
Fungal Control 43.5 ± 1.2m 56.8 ± 1.5kl 58.4 ± 0.8jk 60.7 ± 1.0ij
10 U/g 46.6 ± 0.8m 63.4 ± 1.6hi 65.6 ± 0.7gh 66.5 ± 0.8gh
20 U/g 54.5 ± 0.8l 68.6 ± 0.9fg 71.5 ± 0.9ef 73.0 ± 0.4cde
30 U/g 59.8 ± 1.5jk 72.4 ± 0.6de 75.2 ± 1.2bcd 76.4 ± 2.2abc
40 U/g 64.3 ± 0.7h 75.8 ± 1.1bcd 78.3 ± 1.3ab 79.4 ± 0.8a
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In the papain treatment group, there was no significant difference between the treatment groups on 1st day of maturity (p > 0.05) except on 3rd, 7th and 14th days of maturity (p < 0.05). However, the effect of maturity time on each treatment was significant (p < 0.05). In the bromelain treatment group, the concentration and the maturity time of bromelain had a significant effect on the MFI value (p < 0.01). In the fungal protease treatment group, there was no significant difference in MFI value between F3 and F4 during maturity period (p > 0.05), and other treatment groups were significant (p < 0.05). With the extension of maturity time, MFI value increased, but the change was not significant. During muscle maturity, myofibrils break off from Z-line, which leads to myofibril fragmentation. MFI value is a very important indicator for evaluating tenderness (Davey and Gilbert 1969; Taylor et al. 1995; Whipple et al. 1990). It is an indicator of the integrity of myofibrils and their skeletal proteins in muscle cells. The greater the MFI value, the greater the degree of damage to the integrity of myofibrils.

Several studies have shown that MFI values were significantly correlated with meat tenderness. A study by Whipple et al. (1990) reported that the fragmentation of myofibrils during meat maturity weakened the Z-line. MFI value was significantly correlated with meat tenderness. The fragmentation index of mature meat was significantly higher than that of immature meat Hu et al. (2010) used papain to treat mutton, and found that papain significantly increased the MFI value of mutton during the maturity process. Another study by Lei et al. (2009) reported that papain had a significant effect on beef tenderness, which increased the MFI value and reduced its shear force.

Variation of shear force of horse meat with different protease treatment during postmortem aging

As shown in Table 3, the effects of papain, bromelain and fungal protease on the shear force were extremely significant (p < 0.01), and the effect of maturity time on shear force was significant (p < 0.05). The shear force decreased with an increase in enzyme concentration. In the papain treatment group, the shear force of the P1 and P2 treatment groups was not significant on 1st day of maturity. The differences were significant (p < 0.05) between 3rd, 7th and 14th days of maturity. In the bromelain treatment group, the shear force of B2 and B3 changed slightly on 1st and 3rd days; no difference (p > 0.05) in the shear force of B4 between 3rd and 11th day. Among all treatment groups, the shear force of the B4 treatment group was always the smallest.

Table 3.

Variation of shear force of horse meat with different protease treatment during postmortem aging

Enzymes Enzymes concentration/postmortem time Shear force (Kg)
1 d 3 d 7 d 14 d
Papain Control 12.12 ± 1.10a 11.21 ± 1.5ab 9.75 ± 0.50bc 9.50 ± 0.84c
10 U/g 6.40 ± 0.54d 5.61 ± 0.46def 4.58 ± 0.39efg 4.08 ± 0.28fgh
20 U/g 6.13 ± 0.26de 4.65 ± 0.40efg 3.78 ± 0.38ghi 3.24 ± 0.33ghij
30 U/g 4.61 ± 0.43efg 3.18 ± 0.32ghij 2.54 ± 0.33hijk 2.20 ± 0.41ijk
40 U/g 3.23 ± 0.36ghij 2.05 ± 0.25jk 1.55 ± 0.27k 1.15 ± 0.22k
Bromelain Control 12.08 ± 1.1a 11.21 ± 1.5ab 9.75 ± 0.50b 9.50 ± 0.84b
10 U/g 6.76 ± 1.13cd 6.90 ± 0.68c 5.49 ± 0.42cde 4.90 ± 0.30defg
20 U/g 5.00 ± 0.53cdefg 5.22 ± 0.40cdef 4.25 ± 0.35efgh 3.97 ± 0.27efghi
30 U/g 3.18 ± 0.44ghijk 3.32 ± 0.40fghij 2.56 ± 0.46hijk 2.53 ± 0.48hijk
40 U/g 1.98 ± 0.39ijk 1.58 ± 0.29jk 1.44 ± 0.37jk 1.22 ± 0.22k
Fungal Control 12.08 ± 1.11a 11.21 ± 1.5ab 9.75 ± 0.51b 9.50 ± 0.85b
10 U/g 6.38 ± 0.61c 3.37 ± 0.43defg 4.20 ± 0.69d 3.58 ± 0.29def
20 U/g 3.99 ± 0.41de 2.07 ± 0.34fghi 2.95 ± 0.42defgh 1.75 ± 0.54ghi
30 U/g 2.25 ± 0.49efghi 1.92 ± 0.41fghi 1.55 ± 0.62hi 1.22 ± 0.28hi
40 U/g 1.19 ± 0.31hi 1.33 ± 0.39hi 1.72 ± 0.34ghi 0.97 ± 0.15i
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In the fungal protease treatment group, there was no significant difference in shear force between F2 and F3 on 3rd day of maturity (p > 0.05). There was no significant difference in shear force in the blank group between 7th and 14th days of maturity (p > 0.05). However, other treatment groups were significantly different during maturity days.

Papain, bromelain and fungal protease significantly reduced the shear force, indicating that the three proteases can be used to improve meat tenderness. Among the three proteases, papain had the most obvious tenderization effect, but it had stronger hydrolysis ability on myofibrillar protein. Therefore, the use of papain could easily to cause uneven and excessive tenderness in the tenderized area. The tenderization effect of bromelain was moderate, and its enzyme activity was more stable. The tenderization effect of fungal protease was the most excellent compared with other two proteases, and there was no uneven tenderization phenomenon. Our results were similar to other results (Bagley 2007; Mckeith et al. 1994; Rajagopal et al. 2015). Sullivan and Calkins (2010) used papain, bromelain and fungal protease to treat beef, and reported that the shear force significantly decreased. Another study by Schenkova et al. (2007) used papain and high pressure to treat beef. The authors reported that the shear force of beef significantly decreased, and the tender degree of meat significantly increased.

SDS-PAGE

Papain treatment of horse semitendinosus muscle changes skeleton protein degradation during maturation

As shown in Fig. 1a, on 1st day of maturation, compared with the blank group, the 28 KDa bands of the four treatment groups became more and more blurred; the 32 KDa bands disappeared and smaller molecular bands appeared. Myosin light chain 1 of P2, P3, P4 treatment group disappeared.

Fig. 1.

Fig. 1

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(a–d) represent the effect of papain on the degradation of myofibrils in the semitendinosus of horse meat during the 1st, 3rd, 7th and 14th days of postmortem aging. Lane Maker: protein maker lane C: control, Lane P1 = 10 U/g, P2 = 20 U/g, P3 = 30 U/g, P4 = 40 U/g

As shown in Fig. 1b, on 3rd day of maturity, a number of small molecular weight bands were found in the P1 and P2 treatment groups, and it was most obvious in the P1 treatment group. With increasing papain concentration, the small molecular weight bands increased gradually, and the small molecular weight bands in the blank group were lesser, indicating that these small molecular bands were produced by papain hydrolysis of large molecular proteins. The Troponin I bands of the four treated groups all disappeared and did not appear. As indicated in Fig. 1c, on 7th day of maturity, with increasing papain addition, the Myosin light chain 2 band became more and more blurred l Troponin I bands became more and more blurred; Troponin I bands disappeared in P4 groupl and the 20 KDa bands disappeared in P3 and P4 treatment groups. As shown in Fig. 1d, Troponin I and Troponin C bands disappeared at 20 KDa in P2 and P3 treatment group on 14th day of maturity. With the increasing amount of papain added, the 75 KDa bands of the four treatment groups became clearer and clearer.

Bromelain treatment horse semitendinosus muscle changes of skeleton protein degradation during maturation

As shown in Fig. 2a, on 1st day of maturity, the Troponin I and 32 KDa bands of the four treatment groups all disappeared. Several small molecular bands have been developed accordingly. Myosin light chain 2 and 28 KDa band were significantly weakened, and the 20 KDa band of the B4 treated group was weakened. As shown in Fig. 2b, on 3rd day of maturity, the 32 KDa of the treatment group completely disappeared; 28 KDa and 20 KDa bands of B4 group disappeared; and the Myosin light chain 2 of B4 disappeared completely, but 75 KDa band became clearer.

Fig. 2.

Fig. 2

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(a–d) represent the effect of bromelain on the degradation of myofibrils in the semitendinosus of horse meat during the 1st, 3rd, 7th and 14th days of postmortem aging. Lane Maker: protein maker lane C: control, lane B1 = 10 U/g, B2 = 20 U/g, B3 = 30 U/g, B4 = 40 U/g

As shown in Fig. 2c, on 7th day of maturity, the 20 KDa and Myosin light chain 1 bands completely disappeared, and the α-Actinin band was significantly weakened. Several new bands appeared between 48 and 100 KDa, which might be caused by degradation of α-Actinin. The 28 KDa and 32 KDa bands were very fuzzier than the control group, and the 75 KDa band of the four treatment groups appeared. There were some new bands between 11 and 17 KDa in B3 and B4 treatment groups appeared. As shown in Fig. 2d, the 20 KDa and 28 KDa bands were gone in B3 treatment group and some new bands appeared on 14th days of maturity. Myosin light chain 1 and Myosin light chain 2 of B3 and B4 disappeared.

Fungal protease treatment of semitendinosus in horse skeleton protein degradation during maturation

As shown in Fig. 3a, on 1st day of maturity, the 20 KDa band of the four treatment groups disappear; the Myosin light chain 1 was significantly weakened; and the F3 treated group showed a Desmin band. The 32 KDa and Myosin light chain 2 bands of four treatment groups all disappeared and there was a 75 KDa band. As shown in Fig. 3b, on 3rd day of maturity, Troponin-T appeared in the four treatment groups, but was not very clear. Myosin Light chain 3 disappeared in the control group. Troponin I and Myosin light chain 2 bands of four treatment groups were completely disappeared, and a number of new bands appeared between 63 and 48 KDa.

Fig. 3.

Fig. 3

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(a–d) represent the effect of fungal protease on the degradation of myofibrils in the semitendinosus of horse meat during the 1st, 3rd, 7th and 14th days of postmortem aging. Lane Maker: protein maker lane C: control, lane F1 = 10 U/g, F2 = 20 U/g, F3 = 30 U/g, F4 = 40 U/g

As shown in Fig. 3c, on 7th day of maturity, the 75 KDa band of F2 was very relatively vague, the corresponding 11–17 KDa more than a new band and this band could be generated by the protein of 75 KDa. As shown in Fig. 3d, on 14th days of maturity, Desmin band appeared clearly in the four treatment groups. With the increase in fungal protease concentration, there were more and more new bands appeared between 100 and 48 KDa. In addition, the number of new bands in the two treatment groups of F3 and F4 was more than that of the other groups.

Discussion

Skeletal proteins can maintain the integrity of the structure of the muscle fibers, and many scholars have studied the research on the direction of actin, myosin, desmin and so on (Lonergan et al. 2010). The effect of endogenous protease calpains can cause the degradation of skeletal protein. A study by Lonergan et al. (2010) showed that most of the cytoskeleton proteins including Titin, Nebulin and Desmin were the degradation products of calpains.

The determination of the meat tenderness are the structural integrity of the skeletal muscle, the relationship between regulatory proteins and cytoskeletal proteins and the changes of regulatory proteins (Koohmaraie et al. 1987). In the maturity process, the main structural changes of skeletal muscle cells are the destruction of Titin, Desmin and Troponin T (Koohmaraie et al. 1987; Ho et al. 1996). These proteins have direct relationship with the main structural proteins of the thin filament and thick filamentor which regulate each other and play a key role in the integrity of the muscle cells.

When these proteins are degraded, they can lead to physical, chemical and structural changes in myofibril. Small pieces of myofibril are obvious and the cellular structure integrity are degraded. Subsequently, this improves the tenderness of meat (Huff-Lonergan et al. 1995; Taylor et al. 1995). The appearance of 28 KDa and 32 KDa polypeptide fragments is regarded as a sign of protein hydrolysis and it is closely related to the tenderness of the meat (Macbride and Parrish 1977). It can be concluded that the 28 KDa and 32 KDa peptide fragments were produced by the degradation of Troponin T (Ho et al. 1994; Robson et al. 1991). On 1st day of maturity, in the papain treatment group, the 28 KDa and 32 KDa bands appeared in the blank group, while only the 28 KDa band appeared in the treatment group. The 32 KDa band was very dim. In the subsequent maturation, the 28 and 32KDa bands of treatment group disappeared gradually. The larger the amount of papain, the earlier the two bands disappeared at the 28 KDa and 32 KDa.

On 1st day of maturity, in the bromelain treatment group, with the increase in bromelain concentration, the band of 28 KDa became weaker and 32 KDa band disappeared. In the subsequent maturation, the 28 KDa band gradually disappeared. On 3rd day of maturity, in the fungal protease treatment group, the 28 KDa bands of the four treated groups appeared, and the band did not disappear in the subsequent maturation, which showed that the three kinds of protease had good effects on the tenderness.

Desmin is the main structural protein which is connected to the middle line of the Z line (Robson et al. 1991; Koohmaraie and Shackelford 1991). In mature muscle cells, desmin plays an important role in connecting adjacent myofibrils in the Z line position. It plays an important role to maintain the order and integrity of the skeletal muscle cells. Studies have found that destruction of the myofibrils location and integrity is due to the degradation of desmin. When myofibrillar connectivity to the structure of the muscle fiber membrane is destroyed, it improves meat tenderness (O'Halloran et al. 1997; Wheeler and Koohmaraie 1999; White et al. 2006). The bands of P2, P3 and P4 disappeared on 14th day in the papain treatment group. In the bromelain treatment group, the Desmin band on 7th day of maturity was very clear, but disappeared on 14th day. In the fungal protease treatment group, the Desmin band had not obvious change. It showed that the hydrolysis ability of papain and bromelain to the muscle fiber protein was stronger than that of the fungal protease.

Troponin-T is a kind of adjustable muscle protein. In the maturity process of meat, the destruction of actin integrity may be due to the degradation of Troponin T, which then causes the changes of actin and myosin binding strength. Troponin-T band is usually an important sign of improvement in muscle tenderness (Goll et al. 1983). In the treatment group of papain, the Troponin-T band of P4 was weakened significantly. But in the treatment group of bromelain and fungal protease, Troponin-T had not changed obviously in the process of maturation.

The appearance of 20 KDa band was a sign of improving meat tenderness. On 1st day of maturity, the 20 KDa bands all appeared in the four treatments of papain and bromelain, but with the increase in aging time, 20 KDa bands disappeared. The 20 KDa band did not disappear in the four treatment groups of fungal protease, indicating that the hydrolysis capacity of fungal protease was stronger than that of papain and bromelain.

Myosin Light chain 1 also had a great relationship with the tenderness of meat, and its disappearance indicated that meat tenderness was better. On 1st day of maturity, Myosin light chain 1 of P2, P3 and P4 were all disappeared. On 3rd day of maturity, Myosin light chain 1 in the bromelain and fungal protease treatment group disappeared while Myosin light chain 1 did not change significantly in treatment group of fungal protease, which showed that the tenderness of the meat treated with papain and bromelain was improved more significantly.

In the maturity process, myosin light chain 2 band appeared, indicating that it was a sign of meat tenderness change. During initial period of maturity, myosin light chain 2 appeared in each group treated with papain and bromelain. But with the increase in aging time, the band gradually disappeared. The reason might be that proteases hydrolyzed it into smaller polypeptide molecules.

Conclusion

In the meat maturation process, the cooking loss would gradually increase. Compared with the blank group, the low concentration of protease reduced the cooking loss. The excessive amount of the three proteases increased the cooking loss. The maturation time had a significant effect on cooking loss (p < 0.05). Cooking loss decreased gradually with the increase in the maturation time. The effect of papain and bromelain on cooking loss was significant (p < 0.05) but the effect of fungal protease on cooking loss was not significant (p > 0.05). The effects of maturation time and protease addition on MFI were significant (p < 0.05). MFI value gradually increased during maturation process, and the greater the addition of protease, the greater the MFI value. Papain, bromelain and fungus protease were found to influence shear force significantly (p < 0.01). The greater the amount of three kinds of protease, the smaller the shear force value, suggesting that the shear stress in the mature process would reduce gradually.

During the process of maturity, there appeared a lot of small molecular peptides in the treatment group. Myosin light chain 2, 20 KDa, 32 KDa and 75 KDa stripes appeared in the beginning of maturity, but with the extension of time, these bands had different degrees of weakening. Desmin and Troponin I also disappeared in the later stage of maturity. Therefore, adding papain, bromelain, and fungal protease can promote protein degradation.

Funding

The research was financially supported by the National Natural Science Foundation of China (No. 31660440).

Compliance with ethical standards

Conflict of interest

All authors have no conflicts to disclose.

Footnotes

Publisher's Note

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