Gelation of Myofibrillar Protein - ACS Symposium Series (ACS

Chapter 18

Gelation of Myofibrillar Protein 1

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E. Allen Foegeding , Clark J. Brekke , and Youling L. Xiong

Downloaded by STANFORD UNIV GREEN LIBR on July 1, 2012 | http://pubs.acs.org Publication Date: February 19, 1991 | doi: 10.1021/bk-1991-0454.ch018

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Department of Food Science, North Carolina State University, Raleigh, NC 27695-7624 Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376 Department of Animal Sciences, University of Kentucky, Lexington, KY 40546-0215

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Myofibrillar protein fractions from poultry white (breast) and red (thigh or leg) meat form gels with different rheological properties. Firm, deformable gels are produced with breast myofibrillar protein, while the gels formed with red muscle myofibrillar protein are lower in textural properties. The differences in gelation of myofibrillar protein at equal pH does not appear to be due to temperature of protein unfolding or protein extractability. Rather, the association process, bonding within the gel matrix and the geometry of the gel matrix are involved in the superior gelling ability of breast myofibrillar protein. The "art" of making processed meats, which has been with us since antiquity, has produced a cornucopia of products. The quality of these products, like any food item, is judged by the desirability of color, flavor and texture. Variation in product quality is endemic to "art" based processes. Therefore, it has been a goal of food scientists to understand the physical/chemical mechanisms which produce both desirable and undesirable characteristics in meats. Protein gelation has been used as a model system to understand the mechanisms responsible for texture and water holding, with most direct application to finely comminuted products (1, 2). In the investigation of muscle protein gelation, one has to make a choice on complexity of the system to study. Early research on the functional properties of muscle proteins established that myosin was the main protein responsible for binding together sausage structure (3, 4). Therefore, myosin gelation has been the subject of many investigations; for recent reviews see Asghar et al. (2) and Ziegler and Acton (5). A myosin suspension would represent the simplest system for investigating functionality of meat proteins. At the other extreme, a meat product such as a frankfurter is the most complex in that it contains numerous different muscle proteins and other ingredients that would affect gelation (6). In order to develop a fundamental understanding of the 0097-6156/91/0454-0257$06.00A) © 1991 American Chemical Society In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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INTERACTIONS OF FOOD PROTEINS

chemical/physical events that occur during the manufacturing of processed meat products, investigations on myosin and frankfurters, along with intermediate systems, are required. Salt soluble protein suspensions, composed of extracted (ca. 0.5-0.6 ionic strength) myofibrillar proteins, and myofibril suspensions, composed of isolated myofibrils (ca. 0.1-0.3 ionic strength), are preparations which are intermediate between myosin and meat that have been used to investigate the functionality of meat proteins. The main difference between these preparations and meat is that the connective tissue, lipids and cell membranes have been removed. The myofibril contains the contractile system of skeletal muscle. Proteins which participate in contraction (actin and myosin), regulators of contraction (troponins and tropomyosin) and structural proteins (alpha-actinin, C-protein, M-protein, nebulin, titin) are found within the sarcomere organized in ordered biological structures: the thick filaments, thinfilaments,Z-line and M-line (7). The function of salt and chopping in processed meat manufacturing is to extract the myofibrillar proteins from their biological structures. Myofibrils are similar to muscle in that protein extraction is related to functionality of both. Salt soluble protein (SSP) is used to describe proteins extracted from muscle by saline solutions. Unlike myofibrils, there is no standard for this preparation so the proteins comprising SSP will depend on the isolation method. In comparison to myofibrils, SSP represents the proteins extracted and soluble under defined conditions. Factors which are associated with texture and water holding of muscle protein gels are: pH (8, 9, 10, 11), ionic strength (8, 9, 10, 12), protein extractability (13), protein solubility (8, 9), protein concentration (10), myosinractomyosin (actin) ratio (14, 15, 16, 17) and protein isoforms (18, 19, 20). This work will discuss the role of these factors in the gelation of myofibrils and salt soluble protein. Gelation of Myofibrils and Salt Soluble Protein Protein Concentration. That protein concentration would have an effect on properties of thermally-induced protein gels is obvious and well-documented. However, proteins from different muscle systems may differ in their reaction to equal changes in protein concentration. For example, in Figure 1, the effect of concentration on gel penetration stress for chicken breast and leg myofibril suspensions is shown. It is apparent from the data that there is a definite concentration effect for both breast (white) and leg (red) proteins. In addition, it is also obvious that there are differences in the slopes of the two curves between red and white myofibril gels. Similar protein concentration effects on gel shear stress at failure (strength) are seen with turkey SSP (8) and myosin/actomyosin (14) (Table I). Shear strain at failure (deformability) also increases with protein concentration, although to a much smaller extent (Table I). These results indicate that there are definite differences between gelation properties of red and white myofibrils and SSP. By looking further at some of the factors which contribute to this difference, it should be possible to postulate and define factors important to the basis of SSP gelation.

In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


18. FOEGEDING ET AL.

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Gelation of Myofibrillar Protein

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PROTEIN CONCENTRATION ( m g / m L ) Figure 1

Effect of protein concentration on gel strength of myofibrils isolated from chicken breast and leg muscles. Data points represent means of three replicates. Gelation conditions: 0.6 M NaCl, 50 mM piperazine-N, Ν bis (2-ethane sulfonic acid) (PIPES), 1 mM NaN , pH 6.00; heating rate = l°C/min, from 18 to 70°C (Reprinted with permissionfromref. 23. Copyright 1989.) 3


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Table I. Rheological Properties of Turkey Breast and Thigh Myofibrillar Protein Qgls Protein cone. (mg/ml)

pH

Stress (kPa)

Strain (m/m)

1

25 35

6.0 6.0

3.75" 7.48"

2.11* 2.22"

2

10 15

7.0 7.0

2.60" 5.14*

1.96* 2.06"

40

6.0

8.37"

2.07*

20 25

7.0 7.0

3.54" 5.03

1.75" 2.12"

PROTEIN


Breast Salt Soluble Protein Myosin/Actomyosin Thigh Salt Soluble Protein Myosin/Actomyosin

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*Data from (8). Data from (14). Values for each rheological property within one type of myofibrillar protein with different superscripts are significantly different (P ο

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ω 4 15 Figure 2

25

35 45 55 65 TEMPERATURE (°C)

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ANS-protein fluorescence intensity of salt-soluble proteins (SSP) prepared from postrigor chicken myofibrils as a function of temperature. Data points represent means from three replicates. SSP was suspended (1 mg/mL) in 0.6 M NaCl, 50 mM PIPES, 1 mM NaN , pH 6.00; heating rate = l°C/min (Reprinted with permissionfromref. 23. Copyright 1989.) 3

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Leg

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ZI

0.70

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ω Q

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o

-0.10 15 20 25 30 35 40 45 50 55 60 65 70 TEMPERATURE (°C) Figure 3

Protein-protein interaction (turbidity change) at pH 6.00 for saltsoluble proteins extracted from postrigor chicken breast and leg myofibrils. Assay conditions: 0.3 mg/mL protein, 0.6 M NaCl, 50 mM PIPES, 1 mM NaN , pH 6.00; heating rate = l°C/min (Reprinted with permissionfromref. 23. Copyright 1989.) 3



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initial aggregation and gelation may allow formation of a gel microstructure that is more conducive to gel strength. First derivative plots can also be used to examine in more detail the differences in protein-protein interaction (turbidity change). The plot for both breast and leg at pH 6.0 is shown in Figure 4. There is a dramatic difference in peak heights (5-6 times greater for leg than for breast), indicating a difference in rate of protein-protein interaction. Tml and Tm2 at 43 and 53°C, respectively, are the same for breast and leg. However, breast proteins showed a third peak at about 60°C. This suggests that different protein-protein interaction mechanisms are involved for red vs. white muscle proteins. Protein Extraction From the preceding section, it is apparent that there are some definite differences in the rheological properties of gels made from white (chicken or turkey breast) vs. red (chicken leg or turkey thigh) SSP. However, for SSP to have an effect on gelation and, subsequently, on quality characteristics of processed meat products, it must first be extracted from the myofibril. This section will address how extraction from the myofibrillar structure fits into the overall gelation mechanism. Maximum penetration force for breast and leg SSP is at about 280 and 175 χ 10 dynes, respectively, after 20 mg/ml suspensions are heated to 80°C (Xiong, Y . L . , Brekke, C.J. J. Food Sci.. in press). Under the same conditions, including total protein content, the maximum penetration force for myofibrils from breast and leg is only approximately 160 and 10 χ 10 dynes for breast and leg, respectively (Xiong, Y . L . , Brekke, C.J. J. Food Sci.. in press). The myofibril preparation at 0.6 M NaCl is a mixture of both extracted myofibrillar protein and non-extracted myofibrils, a condition that also would be present in a processed meat system such as a frankfurter. This fact that not all protein has been extracted obviously makes a large difference in gel strength of SSP and myofibril suspensions. Hence, extraction from the myofibril is necessary for maximum gelation of muscle proteins. 3

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Extracted Protein Concentration. Extractiontimeis important to the quantity of protein extracted from the myofibrils, with a maximum for both breast and red myofibrils at approximately 11 hr storage (Xiong, Y . L . , Brekke, C.J. J. Food Sci.. in press). The effects of this extraction time on gel strength of the myofibril suspension (including extracted and non-extracted proteins) further supports the correlation between protein extractability and gel strength. The enhanced gel strength with the increased amount of extracted SSP is expected because at a greater concentration of SSP more protein cross-links can be formed. However, whereas extractability plateaus at approximately 11 hr storage, the rapid increase in gel strength plateaus earlier, at about 5 hr storage, indicating that extractability is not the sole explanation for variation in myofibril gel strength due to extraction time (22). pH. Both breast and leg myofibrils exhibit a pH-dependent extraction profile, with extractability being least at pH 5.50 for either breast or leg samples (Figure 5). This



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25

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60

65

70

TEMPERATURE ( ° C ) Figure 4

Derivative curves of the protein-protein interaction at pH 6.00 for saltsoluble proteins of postrigor chicken breast and leg myofibrils. dA /dT = differential change in optical density as a function of temperature (Reprinted with permissionfromref. 23. Copyright 1989.) 320

5.50

5.75

6.00

6.25

6.50

6.75

7.00

PH Figure 5

Effect of pH on protein extractability and gel strength of myofibrils isolated from postrigor chicken breast and leg (mean of 3 replicates). Extractability, ; penetration force, . Conditions: extractability - 5 mg/mL protein, 0.6 M NaCl, 50 mM PIPES, 1 mM NaN ; gelation - 20 mg/mL, 0.6 M NaCl, 50 mM PIPES, 1 mM NaN , heating from 20 to 70°C at l°C/min (Reprinted with permission from ref. 23. Copyright 1989.) 3

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would be expected since myofibrillar proteins collectively have an isoelectric point near pH 5.1 to 5.4. Breast myofibrils show a great dependence of protein extractability on pH between 5.75 and 6.00; at pH greater than 6.00, further change in protein extractability appears to be minor. This suggests an increase in electrostatic repulsion among protein molecules and an increase in protein-solvent interaction as the pH was increased from 5.75 to 6.00. Extraction of proteins from leg myofibrils is less dependent on pH than for breast myofibrils, although from pH 5.50 to 5.75 there is an increased extractability of the leg myofibrillar proteins. Since the pH of chicken leg muscle is generally greater than 6.0, the minimal extractability at reduced pH is not a practical problem in processed chicken products from leg meat. Based on extractability alone, and all other things being equal, it would be expected that the maximum gel strength of breast myofibrils would occur at pH 6.00 or greater with a drastic reduction at pH 5.75. Gel strength for leg myofibrils should not change from pH 5.75 to greater pH values. Some of this seems to be the case, as shown in Figure 5, where pH is seen to have a marked effect on gelation. Obviously, breast myofibril gelation is more pH-dependent than is leg myofibril gelation. As at least partially explained by protein extractability, minimum gel strength for those pH values investigated is obtained with breast myofibrils at pH 5.50, with a maximum at pH 6.00. However, extractability does not explain the additional decrease in breast myofibril strength at pH 6.50. This suggests that other factors, e.g. ionic or electrostatic interactions, are also involved in gel strength. Foegeding (8) also reported that turkey breast SSP (0.5 M NaCl) forms a more rigid gel at pH 6.0 than at 5.0 or 7.0. The gel strength of leg myofibril suspensions does not change from pH 5.50 to 6.50, even though protein extractability increases somewhat between 5.50 and 5.75 (Figure 5). SDS-PAGE shows that the composition of the extracted protein (SSP) is influenced by the pH of the extraction buffer, and this pH effect is dependent upon the muscle type (Xiong, Y . L . ; Brekke, C.J. J. Food Sci.. in press). Actin is the predominant component of the SSP extracted from the breast myofibrils at pH 5.50, with myosin present in a small quantity compared to actin. Proteins extracted at pH 6.00 are similar to those extracted at pH 6.5°. These results are very similar to those reported by Foegeding (8) on postrigor turkey breast SSP, where differences were seen between SSP soluble at pH 5.0 and 6.0, but not between pH 6.0 and 7.0. The pH effect on protein composition is much less for leg myofibril samples, and the relative amounts of the individual proteins appears similar for samples extracted at all three pH values. Thus, both extractability and composition of extracted proteins can be influenced by pH of the environment and impact gelation mechanisms that lead to the differences observed in gel strength. Summary Factors in addition to protein extractability must play a role in the observed differences for gelation properties of red vs. white muscle protein systems. Since chicken SSP gelation follows the sequential changes of protein unfolding to association to gelation, the degree of protein unfolding prior to aggregation and the extent of protein aggregation before gelation (i.e., the amount of aggregated protein


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required for the matrix at gel point) are likely also involved. One must conclude that differences in protein gelation of these two muscle systems is dependent upon differences in mechanisms of protein-protein interaction for each. Literature Cited 1.


2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Acton, J. C.; Ziegler, G. R.; Burge, D. L., Jr. CRC Critical Reviews in Food Sci. and Nutr., 1983,18,99-121. Asghar, Α.; Samejima, K.; Yasui, T. CRC Critical Reviews in Food Sci. and Nutri., 1985, 22, 27-107. Fukazawa, T.; Hashimoto, Y.; Yasui, T. J. Food Sci., 1961, 26, 331-336. Fukazawa, T.; Hashimoto, Y.; Yasui, T. J. Food Sci., 1961, 26, 550-555. Ziegler, G. R.; Acton, J. C. Food Technol., 1984,38(5),77-80-82. Foegeding, Ε. Α.; Lanier, T. C. Cereal Foods World, 1987, 32, 202-205. Bechtel, P.J. In Muscle as Food; Bechtel, P.J., Ed.; Academic: Orlando, FL, 1986; Chapter 1. Foegeding, E. A. J. Food Sci., 1987, 52, 1495-1499. Ishioroshi, M.; Samejima, K.; Yasui, T. J. Food Sci., 1979, 44, 1280-1284. Samejima, K.; Oka, Y.; Yamamoto, K.; Asghar, Α.; Yasui, T. Agric. Biol. Chem., 1986,50,2101-2110. Yasui, T.; Ishioroshi, M.; Nakano, H.; Samejima, K. J. Food Sci., 1979, 44, 1201-1204, 1211. Hermansson, A-M.; Harbitz O.; Langton, M. J. Sci. FoodAgric.,1986, 37, 69-84. Samejima, K.; Egelandsdal, B.; Fretheim, K. J. Food Sci., 1985, 50, 15401543, 1555. Dudziak, J. Α.; Foegeding, Ε. Α.; Knopp, J. A. J. Food Sci., 1988, 53, 1278-1281, 1332. Ishioroshi, M.; Samejima, K.; Arie, Y.; Yasui, T. Agric. Biol. Chem., 1980, 44, 2185-2194. Yasui, T.; Ishioroshi, M.; Samejima, K. J. Food. Biochem., 1980, 4, 6178. Yasui, T.; Ishioroshi, M.; Samejima, K. Agric. Biol. Chem., 1982, 46, 1049-1059. Asghar, Α.; Morita, J.-I.; Samejima, K.; Yasui, T. Agric. Biol. Chem., 1984, 48, 2217-2224. Morita, J.-I.; Choe, I.-S.; Yamamoto, K.; Samejima, K.; Yasui, T. Agric. Biol. Chem., 1987, 51, 2895-2900. Wicker, L.; Lanier, T. C.; Hamann, D. D.; Akahane, T. J. Food Sci., 1986, 51, 1540-1543, 1562. Samejima, K.; Ishioroshi, M.; Yasui, T. Agric. Biol. Chem., 1982, 46, 535-540. Xiong, Y. L.; Brekke, C. J. J. Food Sci., 1989, 54, 1141-1146. Xiong, Y. Ph.D. Thesis, Washington State University, Pullman, WA, 1989.

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Gelation of Myofibrillar Protein - ACS Symposium Series (ACS

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