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Tenderness of Freeze-Dried Chicken Treated with Proteolytic Enzymes
Tenderness of Freeze-Dried Chicken Treated with Proteolytic Enzymes L. E. DAWSON AND G. H. WELLS 1 Department of Food Science, Michigan State University, East Lansing, Michigan 48823 (Received for publication May 22, 1968)
INTRODUCTION
D
1 Now located at the Pillsbury Company Research Labs^ Minneapolis, Minnesota. Michigan Agricultural Experiment Station Journal Article No. 4839.
PROCEDURE
Source and preparation of meat samples: White Leghorn females, 17 months of age, were slaughtered, scalded in a Rotomatic (basket-type) scalder at 49°C. for 70 seconds and feathers removed mechanically. After evisceration, carcasses were chilled in ice slush for 24 hours. The ready-to-cook birds, in lots of 15, were simmered in a steam-jacketed kettle 64
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URING the freeze drying process, cooked poultry meat becomes less tender, and this difference in tenderness is greatest in muscle from young birds (Wells and Dawson, 1966). The use of proteolytic enzymes in the rehydration solution can result in tender meat (Penny, 1960). Attempts to introduce the enzymes into carcass meat prior to cooking and freeze drying have resulted in nonuniform penetration and enzyme action and in some flavor changes. Auerbach (1960) reported that enzyme-treated meat may show over-tenderization and a mushy appearance on the exterior, but little or no effect on the interior of the sample. Lower enzyme concentrations were reported necessary in chicken meat than in beef to produce acceptable tenderness (Sosebee et al., 1963) and the desirable amounts of enzyme depended on initial meat tenderness (Wang el al., 1957). A concentration of 0.0002% ficin, bromelin or papain, and a concentration of 0.02% Rhozyme P-ll was needed for optimum tenderness in beef (Wang et al., 1957). Sosebee et al. (1963) reported optimum tenderness in chicken muscle using 0.03% papain and 0.02% Rhozyme P-ll. Temperature of rehydration solution affects enzyme activity and subsequent product tenderness. Weiner et al. (1957) found that the optimum temperature for enzymatic reaction was related to the
time of the reaction. In general, short digestion times required high optimum temperatures for the reaction. Optimum temperatures for ficin, bromelin, papain and Rhozyme P-ll were reported as 30-50°C, 30-60°C, and 43-60°C, 6085°C, respectively (Anonymous, 1963). The pH of the rehydration solution is another factor which affects enzyme activity. Maximum solubilization of all beef protein fractions occurs at pH 7.0 (80°C.) when using ficin and bromelin (El-Gharbawi and Whitaker, 1963). Addition of buffer to change pH of fresh raw beef was not practical; however, use of a buffer in rehydration of freeze-dried meat was suggested. Optimum pH's for meat digestion were also reported by Kimmel and Smith (1957), Wang and Birkner (1957), and Yatco-Manzo and Whitaker (1962). This research was conducted to evaluate the effects of enzyme concentration (ficin, bromelin, papain and Rhozyme P-ll) and temperature and pH of reconstitution solution on tenderness of meat from old hens, and to discover one optimum combination of factors for these three variables.
TENDERNESS OF FREEZE-DRIED CHICKEN
Buffer solutions: The diced, freeze-dried samples were rehydrated in various buffer solutions as follows: pH 4.0 Combination of 0.2 M acetic acid and 0.2 M sodium acetate 5.0 Combination of 0.2 M acetic acid and 0.2 M sodium acetate 6.0 Combination of 0.2 M monobasic sodium phosphate and 0.2 M dibasic sodium phosphate 7.0 Combination of 0.2 M monobasic sodium phosphate and 0.2 M dibasic sodium phosphate 8.0 Combination of 0.2 M tris (hydroxymethyl) amino methane and 2 MHC1 9.0 Combination of 0.2 M tris (hydroxymethyl) amino methane and 2 M HC1
The above solutions were made up according to the specifications of Gomori (1955). Two hundred ml. of each buffer were placed in a 500 ml. wide-mouthed Erlenmeyer flask, which was attached to an air condenser to prevent water evaporation. Thus, it prevented changes in pH due to evaporation, or changes in the water-to-sample ratio which would affect rehydration. Enzyme activation and temperature: The enzymes used in this study were papain, ficin, bromelin and Rhozyme P-ll. The first three enzymes were obtained from the Nutritional Biochemical Corporation and the latter one from the Rohm and Haas Company. All enzymes were stored at 2°C. at low relative humidity. Enzymes were added to buffer solutions at 40, 50, 60, 70 and 80°C, to which sufficient cysteine was added to give a concentration of 0.0125 M. Cysteine was added to activate the enzymes. Immediately after the addition of cysteine to the buffer, a predetermined amount of enzyme was added to a preweighed freeze-dried meat sample. The reaction mixture was allowed to incubate at the desired temperature for five minutes. After incubation, the reaction flask was removed from the bath, a water cooled condenser was inserted into the neck, and contents heated to boiling over a hot flame and boiled gently for 3 1/4 minutes to stop the reaction. The meat sample was weighed prior to and following rehydration to determine the amount of water absorbed. Protein nitrogen: The commercial proteolytic enzymes used were sold as crude mixtures which were diluted with a filler to a specific activity. Previous workers, when dealing with meat tenderization through the use of proteolytic enzymes, expressed the enzyme used in terms of
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to an internal temperature of 82°C. in the breast. Thermocouples, connected by a series circuit, were inserted in six of the 15 birds in each lot, and an average temperature from the six birds was obtained (Brown 16 point recording potentiometer). After cooking, the birds were cooled in water for five minutes. The two pectoralis major muscles from each bird were removed, packaged in polyvinylidene-chloride bags, frozen at — 35°C. for four hours, and transferred to — 18°C. for storage. After 24 hours of storage at — 18°C, each breast was diced into 1 cm. cubes by a power meat saw. The samples were freeze-dried for 18 to 20 hours in a Stokes freeze-dryer, Laboratory Model 2003 F-2, in which pressure was reduced to 100 to 150 microns of mercury and plate temperature was 30°C. (final average percent moisture was 0.78). The freezedried meat was packaged under partial vacuum in polyvinylidene-choride bags and stored at — 18°C. until used. Freeze-dried control samples were prepared by the same methods and were subjected to the same rehydration procedure as the enzyme treated freezedried samples.
65
66
L. E. DAWSON AND G. H. WELLS
TABLE 1.—Effects of proteolytic enzyme concentration on tenderness and acceptability of freeze-dried chicken breast as determined by a panel
Enzyme
Concentration Average tender(weight/ ness volume) Score8
Acceptable?
3.2 2.9 2.6 2.2 1.7
Yes Yes Yes ? No
Ficin Ficin Ficin Ficin
0.0001 0.0005 0.0010 0.0020
4.2 3.3 2.4
Yes Yes No No
Bromelin Bromelin Bromelin Bromelin
0.0005 0.0010 0.0020 0.0030
4.1 3.2 2.7 1.8
Yes Yes Yes No
Papain Papain Papain Papain
0.001 0.002 0.003 0.005
3.3 2.6 2.3 1.3
Yes Yes No No
P-ll P-ll P-ll P-ll P-ll
b
° 7 point Hedonic Score card used. 1—Extremely tender; 7—Very tough. b Too "mushy" to give to the panel and was given an automatic unacceptable rating.
percentage concentration (weight/volume). As a result, it was necessary to conduct protein-nitrogen determinations on the commercial preparations to specify the concentrations of enzyme used. For each enzyme, six protein-nitrogen determinations were made. All determinations were made using the micro-Kjeldahl procedure (Ogg, 1960). Boric acid was used as the receiving agent. Non-protein nitrogen determinations were used as a method of determining enzyme inactivation. A procedure was developed in which the reaction mixture was heated to 100°C. for three minutes and then blended for 60 seconds. Two 10-ml. aliquots were deposited in two large test tubes. Twenty ml. of 20% trichloracetic acid was added to each of the test tubes, which were then shaken vigorously to precipitate the protein. The samples were held for five minutes and
Tenderness measurements: An Allo-Kramer shear press with a Model SP-12 recording attachment was used to measure the tenderness of rehydrated chicken breast muscles. Wells et al. (1962) found that the peak of the pressure-time curve was as accurate as the area under the curve for measuring tenderness of rehydrated freeze-dried chicken. All of the samples were placed in a single layer in the shearing cell with the grain of the meat perpendicular to the shearing blades. Measurements were based on the pounds of force required to shear the sample. Samples were further evaluated using a five member panel, selected and trained, using a seven point hedonic score card. RESULTS AND DISCUSSION
The effects of enzyme concentration on tenderness and acceptability (panel
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0.010 0.015 0.020 0.025 0.030
Rhozyme Rhozyme Rhozyme Rhozyme Rhozyme
filtered twice through Whatman No. 3 filter paper. Ten ml. of the filtrate was pipetted directly into a micro-Kjeldahl flask for nitrogen determination. During the five-minute time interval mentioned, the remaining blended mixture was transferred to a clean 500 ml. wide-mouthed Erlenmeyer flask and returned to the constant temperature water bath (40, 50, 60, 70 or 80°C). Additional duplicate aliquots of this mixture were taken at intervals of 30 and 60 minutes. pH measurements: The pH of each rehydration solution was measured after meat samples were reconstituted, using a Beckman-Zeromatic pH meter. The pH of each freeze-dried chicken breast was measured after rehydrating for five minutes in distilled water at a water-tosample ratio of 6:1 and then blending in a Waring blendor for 60 seconds. A 25-ml. aliquot of the blended mixture was used to determine pH.
67
T E N D E R N E S S OF F R E E Z E - D R I E D C H I C K E N
7.2
V-C0NTR0L
8.1
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X
r
o u.
o u.
PAPAIN
6.3 5.4
^-FICIN
CONTROL
-
^*».
BROMELIN
4.5 3.6
-
-^-
7.2
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FICIN
-•^
2.7
a
i
5.0
I
7.0
i
1
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40
8.0
50
60
i
i
70
TEMPERATURE C O
PH
FIG. 3. Shear values of freeze-dried chicken rehydrated in ficin (0.008%) and bromelin (0.002%) solutions at different temperatures.
scores) are reported in Table 1. A sample was considered acceptable when four of the five panel members scored it acceptable. When more t h a n two members rated the sample too tender, it was not acceptable. Optimum enzyme concentrations for later use were determined as follows:
based on preliminary findings.) Ficin, bromelin and Rhozyme P - l l were most effective at p H 5.0, and papain at p H 7.0. Figures 3 and 4 show the pounds of force to shear each gram of sample, when samples of freeze-dried breast muscle were rehydrated at temperatures from 40° to 80°C. Optimum rehydration temperatures of 50°, 50°, 60° and 70°C. were selected for papain, Rhozyme P - l l , bromelin and ficin, respectively. Unlike the pH-shear force curves, temperature-shear force curves showed no sharp peaks. Variations in the temperature of the rehydrating solution did not appear to be as critical as variations in p H in the effect on tenderness. Ficin and bromelin were quite active at all tem-
Rhozyme P - l l Ficin Bromelin Papain
0.02% 0.0008% 0.002% 0.002%
To determine optimum p H for the rehydration solution, the above enzyme concentrations were used. Tenderness of breast meat, as influenced by p H , is shown in Figs. 1 and 2. (Temperatures of rehydration were 50°C. for Rhozyme P - l l and 70°C. for all others. These were
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\
'<*RHOZYME
P-ll
3.6
3.6
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2.7
RHOZYME P-ll
i
9.0
pH FIG. 2. Shear values of freeze-dried chicken rehydrated in bromelin (0.002%) and Rhozyme P - l l (0.02%) solutions at different pH values.
i
i
i
50
60
70
i
TEMPERATURE (°C) FIG. 4. Shear values of freeze-dried chicken rehydrated in papain (0.002%) and Rhozyme P - l l (0.02%) solutions at different temperatures.
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FIG. 1. Shear values of freeze-dried chicken rehydrated in papain (0.002%) and ficin (0.0008%) solutions at different pH values.
68
L. E. DAWSON AND G. H. WELLS TABLE 2. Change in pH of rehydration solutions ; reconstitution Initial pH
Average pH after reconstitution
4.0 5.0 6.0 7.0 8.0 9.0
3.9 5.0 5.6 6.7 7.5 8.4
Difference -0.1
0.0 -0.4 -0.3 -0.5 -0.6
free acidic groups from proteolysis. This was checked by noting no change in pH of solution without added meat. The control samples were most tender when reconstituted in solutions at pH 7.0. This is probably related to an increase in water absorption at this pH (Fig. 5). Auerbach et al. (1954) found the highest level of rehydration of freeze-dried beef in solutions near pH 7.0. The shear values of meat reconstituted in enyzme solutions near pH 7.0 may also be partly due to the increase in water absorption. The percentage of absorbed water 178
y 7.2
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184
WATER UPTAKE
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185 186
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187
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188 l
8.0
i
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PH
FIG. 5. Influence of pH on shear values and percentage water uptake for freeze-dried chicken rehydrated in solutions without added enzymes.
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peratures used. Papain and Rhozyme P-ll possessed little or no activity at 80° C. In general, papain was less active than the other enzymes. The tenderness of control samples was not affected by rehydration temperature. An optimum temperature of 50°C. was found for papain. Tappel et al. (1956a) and Weiner et al. (1957) reported that the optimum temperature for papain digestion of beef was 60° to 80°C. Tappel et al. (1956a) and Weiner et al. (1957) also stated that the optimum temperature for any enzymatic reaction was closely related to the length of time which that reaction covered. Weiner et al. (1957) used a three-minute digestion period. Since a five-minute digestion period was used in the present study, it was expected that the optimum temperature would be comparatively lower. Ficin showed a wide range of pH activity (pH 5.0 to 9.0) with greater activity at pH 5.0 and pH 7.0 at 70°C. Yatco-Manzo and Whitaker (1962) found that ficin-catalyzed hydrolysis of elastin was optimum at a pH 5.0 to 5.5 and at a temperature of 55°C. Rhozyme P-ll was most active at 50°C. and pH 5.0 or 7.0, within the range reported previously (Anonymous, 1963). An optimum temperature between 50° and 60°C was found for bromelin, similar to earlier studies (Anonymous, 1963). The determination of optimum pH for enzymatic action on freeze-dried meat included consideration of both pH and degree of rehydration. This optimum pH for activity is not the same as that found by gelatin or hemoglobin assay by Wang etal. (1958). The pH values for rehydration solutions were determined before and after reconstitution (Table 2). A change in pH (lower) was attributed to the lower pH of meat (pH 5.8) and to an increase in
TENDERNESS OF FREEZE-DRIED CHICKEN
increases in non-protein nitrogen with time after heat treatment. Differences were attributed to experimental error. A direct linear increase in non-protein nitrogen occurred in samples held up to 60 minutes of incubation for ficin, bromelin, papain, and Rhozyme P - l l . This was probably due to the reaction rates of the individual enzymes used. When the five-minute rehydration period was extended to 30 and 60 minutes, the reaction rate of ficin was greater than the reaction rate of Rhozyme P - l l . SUMMARY
The optimum enzyme concentration, temperature and pH of rehydration solution were determined for production of satisfactory tenderness in freeze-dried white meat from old hens. Papain, ficin, bromelin and Rhozyme P - l l were incorporated directly into the rehydration solutions and freeze-dried samples were rehydrated in the enzyme solutions for five minutes. A three-minute heating time at 100°C. was used to in200 I activate the enzymes. Inactivation was determined by the micro-Kjeldahl 2 method; non-protein nitrogen did not ^ \ increase after three minutes of heating. (t 190 - \ Panel scores and Allo-Kramer shear press values were obtained to detect conditions of enzyme concentration, pH «> 180 and temperature, which would produce 5 the most tender yet acceptable chicken. g Panel results indicated that enzyme O ft concentrations (weight/volume) of UJ 0. 0.02%, 0.0008%, 0.002% and 0.002% 170 were suitable for Rhozyme P - l l , ficin, bromelin and papain, respectively. Various buffers were used to control the pH 1 1 1 U [60U of rehydration solutions. Shear press 40 50 60 70 80 values showed that Rhozyme P - l l , ficin, TEMPERATURE C O and bromelin were most active at pH FIG. 6. Influence of temperature of rehydration 5.0. Papain was most active at pH 7.0. solutions (without enzymes) on percentage water Optimum reaction temperatures were uptake by freeze-dried chicken.
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(based on the dry weight) decreased as the temperature of the rehydration solution increased (Fig. 6). Steinberg (1960 a, b) and Norman and Auerbach (1963) reported similar results with freeze-dried beef. Although water uptake by control samples was higher at the lower temperatures used, tenderness remained fairly constant (Figs. 3 and 4). Thus shear force (tenderness) is probably not directly related to water uptake. However, Deatherage (1959) reported that tenderness of meat was related to the ability of meat proteins to hold water. He also found that water-binding capacity decreased with increasing temperatures of the rehydrating solution. When determining enzyme inactivation, the control samples were not heattreated, and proteolysis was allowed to continue to provide a basis for comparing the heat-treated replicates. In all heattreated samples, there were only slight
69
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L. E. DAWSON AND G. H. WELLS
REFERENCES Anonymous, 1963. Enzyme topics. Special Products Department of Rohm and Haas Company, Philadelphia. Auerbach, E., 1960, Meat preservation: dehydration. The Science of Meat and Meat Products. The American Meat Institute Foundation, ed. W. H. Freeman and Company, San Francisco, p. 295-296. Auerbach, E., H. Wang, N. Maynard, D. M. Doty and H. R. Kraybill, 19S4. A histological and histochemical study of beef dehydration. V. Some factors influencing the rehydration level of frozendried muscle tissue. Food Res. 19: 557-563. Deatherage, F. E., 1959. Ion-protein interrelationships affecting the quality of dehydrated meat. Quartermaster Food and Container Institute of the Armed Forces. Report A-316 No. 17. El-Gharbawi, M., and J. R. Whitaker, 1963. Factors affecting enzymatic solubilization of beef proteins. J. Food Sci. 28: 168-172. Gomori, G., 1955. Preparation of buffers for use in enzyme studies. Methods in Enzymology 1. S. P. Colowick and N. O. Kaplan, ed. Academic Press, Inc., New York, p. 138. Kimmel, J. R., and E. L. Smith, 1957. The properties of papain. Advances in Enzymology and Related Subjects of Biochemistry 19. F. E. Nord, ed. Interscience Publishers, Inc., New York, p. 267-334.
Norman, W., and E. Auerbach, 1963. Enhancement of rehydration of precooked freeze-dried meat. Proceedings 15th Research Conference, American Meat Institute Foundation, p. 18. Ogg, C. L., 1960. Determination of nitrogen by the micro-Kjeldahl method. J. Assoc. Offrc. Agr. Chemists, 43: 689-693. Penny, I. F., 1960. Up-grading of low-grade meat. Chem. Ind. 11:288-289. Sosebee, M. E., K. N. May and S. C. Schmittle, 1963. The histological effects of proteolytic enzyme addition on freeze-dehydrated chicken meat. Poultry Sci. 43: 553-559. Steinberg, M. P., 1960 a. Development of techniques for the objective description of freeze dehydrated, cooked beef. Quartermaster Food and Container Institute for the Armed Forces. Res. and Eng. Command, Report A-333 No. 7. Steinberg, M. P., 1960 b. Development of techniques for the objective description of freeze dehydrated, cooked beef. Quartermaster Food and Container Institute for the Armed Forces. Res. and Eng. Command. Report A-333 No. 8. Tappel, A. L., D. S. Miyada, C. Sterling, and V. P. Maier, 1956. Application of meat tenderizer. California Agr. 10: 10. Wang, H., and M. Birkner, 1957. Action of proteolytic enzyme preparations on elastic fibers in beef muscles. Anat. Rec. 127: 384. Wang, H., C. E. Weir, M. L. Birkner and B. Ginger, 1957. The influence of enzyme tenderizers on the structure and tenderness of beef. Proceedings of 9th Research Conference. Am. Meat Inst. Foundation, p. 69-82. Weiner, S., M. Mangel, L. Maharg and G. C. Kelley, 1957. Effectiveness of commercial papain in meat tenderization. Food Technol. 12: 248-252. Wells, G. H., and L. E. Dawson, 1966. Tenderness and juiciness of freeze-dried chicken meat as related to maturity of birds. Poultry Sci. 45: 1004-1008. WeUs, G. H., K. N. May and J. J. Powers, 1962. Taste panel and shear press evaluation of tenderness of freeze- dried chicken as affected by age and pre-slaughter feeding of ions. Food Technol. 16: 137-139. Yatco-Manzo, E., and J. R. Whitaker, 1962. Ficincatalyzed hydrolysis of elastin. Arch. Biochem. Biophys. 97: 122-127.
MAY 26-30. AUSTRALASIAN POULTRY SCIENCE CONVENTION, QUEENSLAND, AUSTRALIA MAY 27-28. AMERICAN FEED MANUFACTURERS ASSOCIATION CONVENTION, MUNICIPAL AUDITORIUM, KANSAS CITY, MISSOURI
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50°, 50°, 60° and 70°C. for Rhozyme P - l l , papain, bromelin and ficin, respectively. The optimum conditions for tenderization by the enzymes used were found to be affected by a combination of water uptake and pH or temperature. Control samples were significantly more tender when rehydrated at pH 7.0 that at higher or lower pH values. This may have been due to a simultaneous increase in water uptake at pH 7.0 during rehydration. In control samples, significant increases in water uptake were found with decreasing rehydration temperatures.
INTRODUCTION
D
1 Now located at the Pillsbury Company Research Labs^ Minneapolis, Minnesota. Michigan Agricultural Experiment Station Journal Article No. 4839.
PROCEDURE
Source and preparation of meat samples: White Leghorn females, 17 months of age, were slaughtered, scalded in a Rotomatic (basket-type) scalder at 49°C. for 70 seconds and feathers removed mechanically. After evisceration, carcasses were chilled in ice slush for 24 hours. The ready-to-cook birds, in lots of 15, were simmered in a steam-jacketed kettle 64
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney on March 13, 2015
URING the freeze drying process, cooked poultry meat becomes less tender, and this difference in tenderness is greatest in muscle from young birds (Wells and Dawson, 1966). The use of proteolytic enzymes in the rehydration solution can result in tender meat (Penny, 1960). Attempts to introduce the enzymes into carcass meat prior to cooking and freeze drying have resulted in nonuniform penetration and enzyme action and in some flavor changes. Auerbach (1960) reported that enzyme-treated meat may show over-tenderization and a mushy appearance on the exterior, but little or no effect on the interior of the sample. Lower enzyme concentrations were reported necessary in chicken meat than in beef to produce acceptable tenderness (Sosebee et al., 1963) and the desirable amounts of enzyme depended on initial meat tenderness (Wang el al., 1957). A concentration of 0.0002% ficin, bromelin or papain, and a concentration of 0.02% Rhozyme P-ll was needed for optimum tenderness in beef (Wang et al., 1957). Sosebee et al. (1963) reported optimum tenderness in chicken muscle using 0.03% papain and 0.02% Rhozyme P-ll. Temperature of rehydration solution affects enzyme activity and subsequent product tenderness. Weiner et al. (1957) found that the optimum temperature for enzymatic reaction was related to the
time of the reaction. In general, short digestion times required high optimum temperatures for the reaction. Optimum temperatures for ficin, bromelin, papain and Rhozyme P-ll were reported as 30-50°C, 30-60°C, and 43-60°C, 6085°C, respectively (Anonymous, 1963). The pH of the rehydration solution is another factor which affects enzyme activity. Maximum solubilization of all beef protein fractions occurs at pH 7.0 (80°C.) when using ficin and bromelin (El-Gharbawi and Whitaker, 1963). Addition of buffer to change pH of fresh raw beef was not practical; however, use of a buffer in rehydration of freeze-dried meat was suggested. Optimum pH's for meat digestion were also reported by Kimmel and Smith (1957), Wang and Birkner (1957), and Yatco-Manzo and Whitaker (1962). This research was conducted to evaluate the effects of enzyme concentration (ficin, bromelin, papain and Rhozyme P-ll) and temperature and pH of reconstitution solution on tenderness of meat from old hens, and to discover one optimum combination of factors for these three variables.
TENDERNESS OF FREEZE-DRIED CHICKEN
Buffer solutions: The diced, freeze-dried samples were rehydrated in various buffer solutions as follows: pH 4.0 Combination of 0.2 M acetic acid and 0.2 M sodium acetate 5.0 Combination of 0.2 M acetic acid and 0.2 M sodium acetate 6.0 Combination of 0.2 M monobasic sodium phosphate and 0.2 M dibasic sodium phosphate 7.0 Combination of 0.2 M monobasic sodium phosphate and 0.2 M dibasic sodium phosphate 8.0 Combination of 0.2 M tris (hydroxymethyl) amino methane and 2 MHC1 9.0 Combination of 0.2 M tris (hydroxymethyl) amino methane and 2 M HC1
The above solutions were made up according to the specifications of Gomori (1955). Two hundred ml. of each buffer were placed in a 500 ml. wide-mouthed Erlenmeyer flask, which was attached to an air condenser to prevent water evaporation. Thus, it prevented changes in pH due to evaporation, or changes in the water-to-sample ratio which would affect rehydration. Enzyme activation and temperature: The enzymes used in this study were papain, ficin, bromelin and Rhozyme P-ll. The first three enzymes were obtained from the Nutritional Biochemical Corporation and the latter one from the Rohm and Haas Company. All enzymes were stored at 2°C. at low relative humidity. Enzymes were added to buffer solutions at 40, 50, 60, 70 and 80°C, to which sufficient cysteine was added to give a concentration of 0.0125 M. Cysteine was added to activate the enzymes. Immediately after the addition of cysteine to the buffer, a predetermined amount of enzyme was added to a preweighed freeze-dried meat sample. The reaction mixture was allowed to incubate at the desired temperature for five minutes. After incubation, the reaction flask was removed from the bath, a water cooled condenser was inserted into the neck, and contents heated to boiling over a hot flame and boiled gently for 3 1/4 minutes to stop the reaction. The meat sample was weighed prior to and following rehydration to determine the amount of water absorbed. Protein nitrogen: The commercial proteolytic enzymes used were sold as crude mixtures which were diluted with a filler to a specific activity. Previous workers, when dealing with meat tenderization through the use of proteolytic enzymes, expressed the enzyme used in terms of
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney on March 13, 2015
to an internal temperature of 82°C. in the breast. Thermocouples, connected by a series circuit, were inserted in six of the 15 birds in each lot, and an average temperature from the six birds was obtained (Brown 16 point recording potentiometer). After cooking, the birds were cooled in water for five minutes. The two pectoralis major muscles from each bird were removed, packaged in polyvinylidene-chloride bags, frozen at — 35°C. for four hours, and transferred to — 18°C. for storage. After 24 hours of storage at — 18°C, each breast was diced into 1 cm. cubes by a power meat saw. The samples were freeze-dried for 18 to 20 hours in a Stokes freeze-dryer, Laboratory Model 2003 F-2, in which pressure was reduced to 100 to 150 microns of mercury and plate temperature was 30°C. (final average percent moisture was 0.78). The freezedried meat was packaged under partial vacuum in polyvinylidene-choride bags and stored at — 18°C. until used. Freeze-dried control samples were prepared by the same methods and were subjected to the same rehydration procedure as the enzyme treated freezedried samples.
65
66
L. E. DAWSON AND G. H. WELLS
TABLE 1.—Effects of proteolytic enzyme concentration on tenderness and acceptability of freeze-dried chicken breast as determined by a panel
Enzyme
Concentration Average tender(weight/ ness volume) Score8
Acceptable?
3.2 2.9 2.6 2.2 1.7
Yes Yes Yes ? No
Ficin Ficin Ficin Ficin
0.0001 0.0005 0.0010 0.0020
4.2 3.3 2.4
Yes Yes No No
Bromelin Bromelin Bromelin Bromelin
0.0005 0.0010 0.0020 0.0030
4.1 3.2 2.7 1.8
Yes Yes Yes No
Papain Papain Papain Papain
0.001 0.002 0.003 0.005
3.3 2.6 2.3 1.3
Yes Yes No No
P-ll P-ll P-ll P-ll P-ll
b
° 7 point Hedonic Score card used. 1—Extremely tender; 7—Very tough. b Too "mushy" to give to the panel and was given an automatic unacceptable rating.
percentage concentration (weight/volume). As a result, it was necessary to conduct protein-nitrogen determinations on the commercial preparations to specify the concentrations of enzyme used. For each enzyme, six protein-nitrogen determinations were made. All determinations were made using the micro-Kjeldahl procedure (Ogg, 1960). Boric acid was used as the receiving agent. Non-protein nitrogen determinations were used as a method of determining enzyme inactivation. A procedure was developed in which the reaction mixture was heated to 100°C. for three minutes and then blended for 60 seconds. Two 10-ml. aliquots were deposited in two large test tubes. Twenty ml. of 20% trichloracetic acid was added to each of the test tubes, which were then shaken vigorously to precipitate the protein. The samples were held for five minutes and
Tenderness measurements: An Allo-Kramer shear press with a Model SP-12 recording attachment was used to measure the tenderness of rehydrated chicken breast muscles. Wells et al. (1962) found that the peak of the pressure-time curve was as accurate as the area under the curve for measuring tenderness of rehydrated freeze-dried chicken. All of the samples were placed in a single layer in the shearing cell with the grain of the meat perpendicular to the shearing blades. Measurements were based on the pounds of force required to shear the sample. Samples were further evaluated using a five member panel, selected and trained, using a seven point hedonic score card. RESULTS AND DISCUSSION
The effects of enzyme concentration on tenderness and acceptability (panel
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0.010 0.015 0.020 0.025 0.030
Rhozyme Rhozyme Rhozyme Rhozyme Rhozyme
filtered twice through Whatman No. 3 filter paper. Ten ml. of the filtrate was pipetted directly into a micro-Kjeldahl flask for nitrogen determination. During the five-minute time interval mentioned, the remaining blended mixture was transferred to a clean 500 ml. wide-mouthed Erlenmeyer flask and returned to the constant temperature water bath (40, 50, 60, 70 or 80°C). Additional duplicate aliquots of this mixture were taken at intervals of 30 and 60 minutes. pH measurements: The pH of each rehydration solution was measured after meat samples were reconstituted, using a Beckman-Zeromatic pH meter. The pH of each freeze-dried chicken breast was measured after rehydrating for five minutes in distilled water at a water-tosample ratio of 6:1 and then blending in a Waring blendor for 60 seconds. A 25-ml. aliquot of the blended mixture was used to determine pH.
67
T E N D E R N E S S OF F R E E Z E - D R I E D C H I C K E N
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BROMELIN
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-
-^-
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i
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7.0
i
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TEMPERATURE C O
PH
FIG. 3. Shear values of freeze-dried chicken rehydrated in ficin (0.008%) and bromelin (0.002%) solutions at different temperatures.
scores) are reported in Table 1. A sample was considered acceptable when four of the five panel members scored it acceptable. When more t h a n two members rated the sample too tender, it was not acceptable. Optimum enzyme concentrations for later use were determined as follows:
based on preliminary findings.) Ficin, bromelin and Rhozyme P - l l were most effective at p H 5.0, and papain at p H 7.0. Figures 3 and 4 show the pounds of force to shear each gram of sample, when samples of freeze-dried breast muscle were rehydrated at temperatures from 40° to 80°C. Optimum rehydration temperatures of 50°, 50°, 60° and 70°C. were selected for papain, Rhozyme P - l l , bromelin and ficin, respectively. Unlike the pH-shear force curves, temperature-shear force curves showed no sharp peaks. Variations in the temperature of the rehydrating solution did not appear to be as critical as variations in p H in the effect on tenderness. Ficin and bromelin were quite active at all tem-
Rhozyme P - l l Ficin Bromelin Papain
0.02% 0.0008% 0.002% 0.002%
To determine optimum p H for the rehydration solution, the above enzyme concentrations were used. Tenderness of breast meat, as influenced by p H , is shown in Figs. 1 and 2. (Temperatures of rehydration were 50°C. for Rhozyme P - l l and 70°C. for all others. These were
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pH FIG. 2. Shear values of freeze-dried chicken rehydrated in bromelin (0.002%) and Rhozyme P - l l (0.02%) solutions at different pH values.
i
i
i
50
60
70
i
TEMPERATURE (°C) FIG. 4. Shear values of freeze-dried chicken rehydrated in papain (0.002%) and Rhozyme P - l l (0.02%) solutions at different temperatures.
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FIG. 1. Shear values of freeze-dried chicken rehydrated in papain (0.002%) and ficin (0.0008%) solutions at different pH values.
68
L. E. DAWSON AND G. H. WELLS TABLE 2. Change in pH of rehydration solutions ; reconstitution Initial pH
Average pH after reconstitution
4.0 5.0 6.0 7.0 8.0 9.0
3.9 5.0 5.6 6.7 7.5 8.4
Difference -0.1
0.0 -0.4 -0.3 -0.5 -0.6
free acidic groups from proteolysis. This was checked by noting no change in pH of solution without added meat. The control samples were most tender when reconstituted in solutions at pH 7.0. This is probably related to an increase in water absorption at this pH (Fig. 5). Auerbach et al. (1954) found the highest level of rehydration of freeze-dried beef in solutions near pH 7.0. The shear values of meat reconstituted in enyzme solutions near pH 7.0 may also be partly due to the increase in water absorption. The percentage of absorbed water 178
y 7.2
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FIG. 5. Influence of pH on shear values and percentage water uptake for freeze-dried chicken rehydrated in solutions without added enzymes.
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peratures used. Papain and Rhozyme P-ll possessed little or no activity at 80° C. In general, papain was less active than the other enzymes. The tenderness of control samples was not affected by rehydration temperature. An optimum temperature of 50°C. was found for papain. Tappel et al. (1956a) and Weiner et al. (1957) reported that the optimum temperature for papain digestion of beef was 60° to 80°C. Tappel et al. (1956a) and Weiner et al. (1957) also stated that the optimum temperature for any enzymatic reaction was closely related to the length of time which that reaction covered. Weiner et al. (1957) used a three-minute digestion period. Since a five-minute digestion period was used in the present study, it was expected that the optimum temperature would be comparatively lower. Ficin showed a wide range of pH activity (pH 5.0 to 9.0) with greater activity at pH 5.0 and pH 7.0 at 70°C. Yatco-Manzo and Whitaker (1962) found that ficin-catalyzed hydrolysis of elastin was optimum at a pH 5.0 to 5.5 and at a temperature of 55°C. Rhozyme P-ll was most active at 50°C. and pH 5.0 or 7.0, within the range reported previously (Anonymous, 1963). An optimum temperature between 50° and 60°C was found for bromelin, similar to earlier studies (Anonymous, 1963). The determination of optimum pH for enzymatic action on freeze-dried meat included consideration of both pH and degree of rehydration. This optimum pH for activity is not the same as that found by gelatin or hemoglobin assay by Wang etal. (1958). The pH values for rehydration solutions were determined before and after reconstitution (Table 2). A change in pH (lower) was attributed to the lower pH of meat (pH 5.8) and to an increase in
TENDERNESS OF FREEZE-DRIED CHICKEN
increases in non-protein nitrogen with time after heat treatment. Differences were attributed to experimental error. A direct linear increase in non-protein nitrogen occurred in samples held up to 60 minutes of incubation for ficin, bromelin, papain, and Rhozyme P - l l . This was probably due to the reaction rates of the individual enzymes used. When the five-minute rehydration period was extended to 30 and 60 minutes, the reaction rate of ficin was greater than the reaction rate of Rhozyme P - l l . SUMMARY
The optimum enzyme concentration, temperature and pH of rehydration solution were determined for production of satisfactory tenderness in freeze-dried white meat from old hens. Papain, ficin, bromelin and Rhozyme P - l l were incorporated directly into the rehydration solutions and freeze-dried samples were rehydrated in the enzyme solutions for five minutes. A three-minute heating time at 100°C. was used to in200 I activate the enzymes. Inactivation was determined by the micro-Kjeldahl 2 method; non-protein nitrogen did not ^ \ increase after three minutes of heating. (t 190 - \ Panel scores and Allo-Kramer shear press values were obtained to detect conditions of enzyme concentration, pH «> 180 and temperature, which would produce 5 the most tender yet acceptable chicken. g Panel results indicated that enzyme O ft concentrations (weight/volume) of UJ 0. 0.02%, 0.0008%, 0.002% and 0.002% 170 were suitable for Rhozyme P - l l , ficin, bromelin and papain, respectively. Various buffers were used to control the pH 1 1 1 U [60U of rehydration solutions. Shear press 40 50 60 70 80 values showed that Rhozyme P - l l , ficin, TEMPERATURE C O and bromelin were most active at pH FIG. 6. Influence of temperature of rehydration 5.0. Papain was most active at pH 7.0. solutions (without enzymes) on percentage water Optimum reaction temperatures were uptake by freeze-dried chicken.
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(based on the dry weight) decreased as the temperature of the rehydration solution increased (Fig. 6). Steinberg (1960 a, b) and Norman and Auerbach (1963) reported similar results with freeze-dried beef. Although water uptake by control samples was higher at the lower temperatures used, tenderness remained fairly constant (Figs. 3 and 4). Thus shear force (tenderness) is probably not directly related to water uptake. However, Deatherage (1959) reported that tenderness of meat was related to the ability of meat proteins to hold water. He also found that water-binding capacity decreased with increasing temperatures of the rehydrating solution. When determining enzyme inactivation, the control samples were not heattreated, and proteolysis was allowed to continue to provide a basis for comparing the heat-treated replicates. In all heattreated samples, there were only slight
69
70
L. E. DAWSON AND G. H. WELLS
REFERENCES Anonymous, 1963. Enzyme topics. Special Products Department of Rohm and Haas Company, Philadelphia. Auerbach, E., 1960, Meat preservation: dehydration. The Science of Meat and Meat Products. The American Meat Institute Foundation, ed. W. H. Freeman and Company, San Francisco, p. 295-296. Auerbach, E., H. Wang, N. Maynard, D. M. Doty and H. R. Kraybill, 19S4. A histological and histochemical study of beef dehydration. V. Some factors influencing the rehydration level of frozendried muscle tissue. Food Res. 19: 557-563. Deatherage, F. E., 1959. Ion-protein interrelationships affecting the quality of dehydrated meat. Quartermaster Food and Container Institute of the Armed Forces. Report A-316 No. 17. El-Gharbawi, M., and J. R. Whitaker, 1963. Factors affecting enzymatic solubilization of beef proteins. J. Food Sci. 28: 168-172. Gomori, G., 1955. Preparation of buffers for use in enzyme studies. Methods in Enzymology 1. S. P. Colowick and N. O. Kaplan, ed. Academic Press, Inc., New York, p. 138. Kimmel, J. R., and E. L. Smith, 1957. The properties of papain. Advances in Enzymology and Related Subjects of Biochemistry 19. F. E. Nord, ed. Interscience Publishers, Inc., New York, p. 267-334.
Norman, W., and E. Auerbach, 1963. Enhancement of rehydration of precooked freeze-dried meat. Proceedings 15th Research Conference, American Meat Institute Foundation, p. 18. Ogg, C. L., 1960. Determination of nitrogen by the micro-Kjeldahl method. J. Assoc. Offrc. Agr. Chemists, 43: 689-693. Penny, I. F., 1960. Up-grading of low-grade meat. Chem. Ind. 11:288-289. Sosebee, M. E., K. N. May and S. C. Schmittle, 1963. The histological effects of proteolytic enzyme addition on freeze-dehydrated chicken meat. Poultry Sci. 43: 553-559. Steinberg, M. P., 1960 a. Development of techniques for the objective description of freeze dehydrated, cooked beef. Quartermaster Food and Container Institute for the Armed Forces. Res. and Eng. Command, Report A-333 No. 7. Steinberg, M. P., 1960 b. Development of techniques for the objective description of freeze dehydrated, cooked beef. Quartermaster Food and Container Institute for the Armed Forces. Res. and Eng. Command. Report A-333 No. 8. Tappel, A. L., D. S. Miyada, C. Sterling, and V. P. Maier, 1956. Application of meat tenderizer. California Agr. 10: 10. Wang, H., and M. Birkner, 1957. Action of proteolytic enzyme preparations on elastic fibers in beef muscles. Anat. Rec. 127: 384. Wang, H., C. E. Weir, M. L. Birkner and B. Ginger, 1957. The influence of enzyme tenderizers on the structure and tenderness of beef. Proceedings of 9th Research Conference. Am. Meat Inst. Foundation, p. 69-82. Weiner, S., M. Mangel, L. Maharg and G. C. Kelley, 1957. Effectiveness of commercial papain in meat tenderization. Food Technol. 12: 248-252. Wells, G. H., and L. E. Dawson, 1966. Tenderness and juiciness of freeze-dried chicken meat as related to maturity of birds. Poultry Sci. 45: 1004-1008. WeUs, G. H., K. N. May and J. J. Powers, 1962. Taste panel and shear press evaluation of tenderness of freeze- dried chicken as affected by age and pre-slaughter feeding of ions. Food Technol. 16: 137-139. Yatco-Manzo, E., and J. R. Whitaker, 1962. Ficincatalyzed hydrolysis of elastin. Arch. Biochem. Biophys. 97: 122-127.
MAY 26-30. AUSTRALASIAN POULTRY SCIENCE CONVENTION, QUEENSLAND, AUSTRALIA MAY 27-28. AMERICAN FEED MANUFACTURERS ASSOCIATION CONVENTION, MUNICIPAL AUDITORIUM, KANSAS CITY, MISSOURI
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50°, 50°, 60° and 70°C. for Rhozyme P - l l , papain, bromelin and ficin, respectively. The optimum conditions for tenderization by the enzymes used were found to be affected by a combination of water uptake and pH or temperature. Control samples were significantly more tender when rehydrated at pH 7.0 that at higher or lower pH values. This may have been due to a simultaneous increase in water uptake at pH 7.0 during rehydration. In control samples, significant increases in water uptake were found with decreasing rehydration temperatures.