Professor Li Yalei et al.: Effect of structural proteins on tenderness of Qinchuan beef during post-slaughter maturation

As a low-fat, high-protein food, beef is loved by consumers
.
Among them, tenderness is the main factor affecting consumer satisfaction and acceptance, and it is also one of the key indicators to measure the quality of
beef.
Therefore, the research on the mechanism of meat tenderness change at home and abroad has not stopped
.
Based on the research results of scholars, four main mechanisms affecting the formation of tenderness can be summarized: first, the fragmentation of myofibrillar protein or the change of protein structure leads to the weakening of Z line; The second is the cleavage of proteins through proteases (calpsin, cathepsin) and proteasomes to trigger tenderness changes; Third, glycolytic enzymes regulate tenderness after slaughter; Fourth, apoptosis and heat shock proteins participate in tenderness formation
.

Ma Xuhua, Yang Bo, Li Yalei* from the School of Food and Wine of Ningxia University mainly used 4D-non-labeled quantitative (4D-LFQ) proteomics technology to screen differential proteins, detected the protein components of the longest muscle of Qinchuan cattle dorsal muscle at 0~8 d, analyzed the signaling pathways involved, and explored the effect of structural protein degradation on the tenderization mechanism of Qinchuan cattle after slaughter, so as to study shear force, myofibril small piece index (MFI), total muscle soluble protein, myofibrillar protein, Mechanism of change in sarcoplasmic
protein.
In the experiment, the longitis dorsal muscles of Qinchuan cattle were selected as the research object, and Qinchuan cattle were selected as the dominant characteristic breed in Shaanxi-Gansu-Ningxia region, with delicate meat, high proportion of lean meat, obvious marbling, and its longitus dorsal muscles can be made into high-grade steaks
.
The use of 4D-LFQ proteomics technology to study the tenderness change mechanism is expected to provide corresponding theoretical support
for the control of tenderness in the industrial processing of Qinchuan beef.

1.
Changes in the shear force of the longest muscles of Qinchuan cattle dorsal at different storage times

It can be seen from Figure 1 that the overall shear force during the storage period of 0~8 days shows a trend of first rising and then decreasing, and the amplitude of increase is less than the amplitude
of decline.
The shear force of beef was (120.
25±5.
15)N when stored for 0 d, showed an upward trend when 0~4 d, and was the highest at 4 d, and the shear force was (157.
94±2.
53)N.
After 4 days, it showed a downward trend, and reached the lowest point of the whole storage period at 8 days, and the shear force was (93.
41±5.
08)N
.

2.
Changes in the total soluble protein content of the longest muscle of Qinchuan bovine at different storage times

It can be seen from Figure 2 that the total soluble protein content during the storage period of 0~8 days showed a significant downward trend (P<0.
05).
<b10> The content was (196.
44±2.
10) mg/g when stored for 0 days, and the total soluble protein content reached the lowest point of the whole process ((128.
47±0.
87) mg/g after 8 days of storage, and the overall decrease was 34.
60%.

This result shows that the protein changes during the storage period of Qinchuan beef are obvious, the muscle tissue is metabolized during post-slaughter storage, and the oxidative modification degree of amino acid side chain groups is high, resulting in the aggravation of protein conformation, so that more hydrophobic groups are exposed in the protein molecule and the surface hydrophobicity is increasing
.
At the same time, because free sulfhydryl groups are easily oxidized into disulfide bonds, a large number of proteins are aggregated and precipitated, which degrades
muscle tissue proteins.

It can be seen from Figure 3 that there are many bands of the longest muscle of Qinchuan cow, which can be judged to have many
types of proteins.
With the extension of storage time, the protein band in 150~250 kDa gradually narrowed.
The total soluble protein band of the longest muscle of Qinchuan cow gradually became lighter at 100 kDa of the longest muscle of Qinchuan.
The protein bands in 37~50 kDa gradually became shallow and dispersed.
The 25~37 kDa endomyosin band gradually became shallower, but the total band II became significantly deeper.
The total band III and IV became deeper in 10~25 kDa, and the total band of IV became significantly deeper
.
In general, the high molecular weight protein bands gradually became shallow, and the low molecular weight protein bands gradually deepened, which combined with the total soluble protein analysis results, it can be inferred that the total soluble protein of the longest muscle of Qinchuan has a significant degradation, and the total soluble protein change of its structure may affect the formation
of tenderness.

3.
Changes in the content of sarcoplasmic protein of the longest muscle of Qinchuan cattle at different storage times

It can be seen from Figure 4 that the content of sarcoplasmin increased during the storage period of 0~8 days, from (43.
48±0.
43) mg/g to (52.
97±0.
35) mg/g, an overall increase of 21.
83%.

It can be seen from Fig.
5 that the I sarcoplasm (two protein bands in the range of 150~250 kDa) gradually becomes lighter during the storage period of 0~8 d.
II sarcoplasm (100 kDa protein band) gradually becomes shallow and narrow; III sarcoplasm (37 kDa protein band) gradually changed from 3 protein bands to two, and the middle band disappeared; IV sarcoplasma (25 kDa protein band) gradually deepened significantly in the first 2 days, and did not change significantly during the storage period of 2~8 days.
V sarcoplasm (15 kDa protein band) gradually widens after 2 days, which may be the result of the breakdown of macromolecular proteins; There were no significant changes
in the other bands.
Through the changes of the above 5 bands, it can also be concluded that the high molecular weight protein bands gradually become shallow, and the low molecular weight protein bands gradually become deeper and wider
.
It can be inferred that during the storage process, the high-molecular mass protein in the longest-muscular sarcoplasmic protein of Qinchuan bovine back is decomposed, the content becomes less, and the types of low-molecular mass protein gradually increase, and the concentration continues to increase
.

4.
Changes in myofibrillar protein content of the longest muscle of Qinchuan cattle at different storage times

It can be seen from Figure 6 that the content of myofibrillar protein in the storage period of 0~8 days showed a downward trend, from (136.
72±3.
06) mg/g to (67.
60±0.
84) mg/g
.
The decline rate in the early stage was relatively fast, and the decline rate in the later stage slowed down, with an overall decrease of 50.
56%.

It can be seen from Figure 7 that with the extension of storage time, the protein band with molecular weight in the range of 100~250 kDa gradually becomes deeper, and the protein content gradually increases; The protein band with molecular weight within 37~50 kDa becomes shallow; Within 25~37 kDa, the relatively high molecular weight protein bands became deeper, and the relatively low molecular weight protein bands gradually disappeared or became weaker.
The protein bands with molecular weight within 15~20 kDa gradually deepened; At 10~15 kDa, gradient protein bands
gradually appeared with the extension of storage period.
It can be seen from the above changes in protein bands that the degraded myofibrillar protein appears in the range of 37~75 kDa, while the myofibrillar protein with increased content appears in the range of 10~20 and 100 kDa, and the overall change is more significant, and its change may affect the formation
of protein tenderness.

5.
MFI analysis of the longest muscles of Qinchuan cow with different storage times

It can be seen from Figure 8 that the MFI of the longest muscles of Qinchuan bull showed an upward trend during the whole storage process, and the increase was slower during the storage period of 0~2 d and 6~8 d, and the increase was faster
at 2~6 d.
The MFI was 28.
50±5.
16 when stored for 0 days, and then the MFI increased all the time, reaching the highest point of the whole storage process (99.
98±1.
20) when stored for 8 days, an increase of 250.
81%.

This result is consistent
with the trend of MFI in Li Shengsheng's study on the formation mechanism of smooth muscle tenderness in yaks.

6.
Mechanism analysis of the effect of structural proteins on the tenderization of the longest muscles of Qinchuan cattle after slaughter

It can be seen from Table 1 that MFI, which characterized the degradation degree of muscle fibers during the storage period of 0~8 d, has no significant correlation with shear force (r=-0.
285), and D'Alessandro et al.
also showed the same results
in the study of beef tenderness.
Combined with the tenderness formation proposed by a large number of scholars is closely related to the degradation of myofibrils, the degree of myofibril small pieces in the early storage period can be increased, mainly due to muscle contraction and tissue rigidity caused by changes in energy and pH, which in turn affects the decrease of tenderness and has little
impact on shear force.
During storage, MFI gradually increased, and when the small pieces of myofibrils accumulated to a certain amount in the middle and late stages, it contributed to
the formation of tenderness.

It can be seen from Figure 9 that when stored for 0 days, 69.
60% of myofibrils, 22.
13% of sarcoplasmic protein and 6.
60% of matrix protein were measured in the longest muscle muscle of Qinchuan cattle dorsal muscle, which was in line with the proportion of protein structure composition in
muscle tissue.
In addition, the proportion of myofibrillar protein stored at 0~8 days showed a continuous decreasing trend, gradually decreasing from 69.
60% at 0 d to 52.
61% at 8 days.
The proportion of sarcoplasmic protein continued to increase, gradually increasing from 22.
13% at 0 d to 41.
23% at 8 d, but the content of matrix protein changed only slightly
.
From this, it can be obtained that the protein contained in the muscles is mainly the degradation
of myofibrillar protein.
Myofibrillar protein is mainly composed of actin (45 kDa), myosin heavy chain (200 kDa) and tropomyosin protein (35 kDa), as can be seen from Figure 7, the band to which actin and tropomyosin belong during the storage period of 0~8 days shows a significant trend of shallower, actin and tropomyosin degradation can be obtained, and a new band around 31 kDa may be a decomposition product of actin as a marker
of actin degradation 。 For the degradation of myosin heavy chains, a shallower band around 200 kDa was found in the total soluble protein gel electrophoresis, but no significant shallower was found in myofibrillar protein electrophoresis, so it is impossible to judge whether the myosin heavy chain is degraded
.
Myofibrillar protein degradation may be due to endogenous enzymes (calpain, cathepsin, apoptotic enzyme) during storage, and microorganisms also accelerate actin degradation on muscle contamination and protein decomposition and utilization
.

7.
Differential protein screening

In the experiment, 4D-LFQ was used to detect the changes of the proteome of Qinchuan cattle after slaughter, and the obtained protein secondary structure was searched by mass spectrometry, a total of 1 149 proteins were searched, 1.
2 times was set as the standard difference multiple, a total of 120 differential proteins that met the standards were screened, and the KEGG database was used to perform bioinformatics analysis on these 120 differential proteins, and the obtained data were determined by Pearson correlation analysis in SPSS 25 software The differential proteins are related to the tenderness formation mechanism, as shown in
Table 2.

8.
Bioinformatics analysis

Figure 10 is a network diagram of tenderness-related protein interactions using Cytoscape 3.
6.
1 and String 10.
0 software
.
The clustering coefficient of this network plot is 0.
759, and the protein interaction (PPI) enrichment P value is 1.
0×^10-16
.
ACTN1, MYH9, MYLPF, MYL12B, MYH13, MYL2, MYH6, ACTC1, TNNI1, TNNI2, MYH15 interact strongly
.
The above 11 proteins regulate the physiological state
of cells in myosin binding, calcium ion binding, cytoskeletal protein-bound myofibril assembly, and skeletal muscle tissue development process.

Conclusion