Tenderization of meat by natural enzyme control

Method for accelerating the tenderization of meat by natural enzymes by antemortem injection of selected activators of specific natural enzymes and/or binding agents for enzyme inhibitors naturally present in meat, promptly slaughtering the animal or fowl after injection and holding the meat within the optimum temperature range of the natural enzyme for about 2 days.

This invention relates to a method for tenderizing meat by controlling the 
natural enzymes indigenous to meat and, more particularly, relates to a 
method for accelerating the proteolytic activity of enzymes naturally 
present in meat by treating the enzymes with specific activators and 
inhibitors to the enzyme inhibitors or binding agents of enzyme inhibitors 
and thereafter holding the meat within the optimum temperature range of 
the natural enzyme for about two days. 
It has long been known that meat contains natural enzymes which act to 
tenderize it whenever held for sufficient time under the proper 
conditions. For instance U.S. Pat. No. 2,169,081 to James teaches a 
process for utilizing naturally occurring enzymes to tenderize meat. In an 
effort to reduce the time required to obtain the desired degree of 
tenderness, we have identified, in meat, the proteases and other factors 
naturally present which may contribute to increasing the tenderness of 
meat and devised means and methods for enhancing and accelerating their 
meat tenderizing activity.

Therefore, a principal object of the present invention is to provide a 
method for reducing the time required to tenderize meat from animals and 
fowl by accelerating the proteolytic activity of specific enzymes 
naturally present in meat. 
Another object of the present invention is to provide a method for the 
intravenous treatment of meat bearing animals and fowl to effect 
accelerated post-mortem tenderization of the meat. 
Yet another object of the present invention is to provide a method for the 
intravenous injection of specific enzyme activators and deinhibitors 
suitable for accelerating the proteolytic activity of specific enzymes 
naturally present in meat and thereafter holding the meat within the 
optimum temperature range of the natural enzymes for sufficient time to 
permit their proteolytic action to tenderize the meat. 
Other objects and advantages of the invention will become apparent as the 
description proceeds. 
Among a number of enzymes naturally present in meat, one enzyme in 
particular has been found to be a potential major contributor in 
increasing the tenderness of meat. This protease was identified as a 
sulfhydryl enzyme which has an optimum pH of 7.0-7.5 and was found to 
require the presence of free calcium ions to make available its 
tenderizing activity. This protease is hereafter referred to as the 
neutral enzyme. In the absence of an activator, such as calcium, the 
neutral enzyme exhibits no detectable activity. For optimum activity, this 
protease requires 1 mM or more of free calcium ion concentration in the 
reaction mixture. Although normal muscle generally contains calcium at a 
relatively constant level of approximately 40 ppm, practically all of this 
calcium is bound up with subcellular components present in the muscle 
fiber and is, therefore, not available as free calcium ions. 
It was discovered that there are also naturally occurring inhibitory 
elements present in meat tissue; and agents may be selected to suppress 
that inhibitory effect. For instance, it was found that free zinc ions 
normally present in muscle fiber act as a potent inhibitor to the 
proteolytic activity of the neutral enzyme but may be controlled with a 
chelating agent. The levels of zinc in different animals and fowl vary 
from less than 0.1 mM up to about 1 mM. Interestingly, animals and fowl 
which naturally exhibit a high degree of tenderness contain very low 
levels of zinc, and those which are generally considered tough possess 
relatively high levels of zinc. At a concentration of 0.01 mM zinc (0.65 
ppm), the proteolytic activity of the neutral enzyme is approximately 50%, 
while at a concentration of 0.1 mM zinc (6.5 ppm) approximately 90% of the 
proteolytic activity of the neutral enzyme is inhibited. 
Therefore, it was determined that the amount of free calcium ions present 
in meat needed to be increased in order to activate the natural 
proteolytic activity and, at the same time, zinc chelating compounds were 
needed to bind the free zinc ions naturally present in animal muscle 
fiber. To diminish the requisite time it was decided to employ antemortem 
injection of aqueous solutions of calcium salts to promote the proteolytic 
activity of the neutral protease. Unfortunately, intravenous injection of 
calcium salts in concentrations sufficient to promote the desired 
proteolytic activity results in death to the animals and fowl, caused by 
calcium tetany. However, it was discovered that calcium tetany of the 
animals and fowl can be avoided by injection of an aqueous solution 
containing a relatively unionized, soluble calcium salt, such as calcium 
acetate, with disodium calcium EDTA. In such a mixture most of the 
injected calcium is bound. Thus, the level of free calcium ions in the 
blood stream is not elevated enough to cause calcium tetany. The mixture 
is more effective in preventing tetany than either salt injected alone. 
Moreover, the disodium calcium EDTA is a chelating agent which selectively 
binds the free zinc ions naturally present in the muscle fiber, which zinc 
ions would normally inhibit the accelerated proteolytic activity of the 
neutral enzyme. 
The temperature profile of the neutral protease acting on three different 
substrates showed the neutral enzyme as having the most consistent 
proteolytic activity over a wide temperature range for all substrates of 
any of the other natural proteases tested. The neutral enzyme digested 
casein in the range of 5.degree. to 70.degree. C. with the optimum at 
15.degree. to 20.degree. C., myofibril protein in the range of 5.degree. 
to 45.degree. C., with the optimum at 30.degree. to 45.degree. C., and 
native collagen in the range of 5.degree. to 45.degree. C., with the 
optimum at 25.degree. to 35.degree. C., respectively. 
The tenderizing effect of the neutral enzyme made available by antemortem 
injection of calcium acetate and Na.sub.2 CaEDTA solutions in cattle and 
the effect of aging the meat at the optimum temperature of the neutral 
enzyme was tested. Sixteen uniform animals of choice grade were selected 
and divided into two groups of eight. The eight animals of one group were 
each given a vascular injection of a solution of 0.4 M calcium acetate and 
1.0 M Na.sub.2 CaEDTA at a rate of 200 ml per 100 pounds live weight. The 
other eight animals were used as a non-injected control. All cattle were 
slaughtered within thirty minutes after injection. Following slaughter, 
one carcass half from each of the animals in both groups was chilled and 
held at 35.degree. F., under normal packing house conditions, while the 
other half carcasses were held at 72.degree. F. for two days. Thereafter 
all carcasses were processed identically. All carcasses were broken into 
sub-primal cuts, "Cry-O-Vac" (trademark) film packaged, and the chucks 
shipped to a laboratory for tenderness evaluations. After allowing about 
one week for normal shipping time, the meat in the form of 5-6 pound 
roasts was cooked to an internal temperature of 155.degree. F. Subsequent 
laboratory analysis, standardized mechanical testing for tenderness and 
testing by a panel of experts clearly showed that the carcasses receiving 
antemortem injection of calcium acetate and Na.sub.2 CaEDTA and held at 
72.degree. F. for two days were substantially more tender. Other 
comparative results also showed that holding the meat at 72.degree. F. 
without injection was not as effective in producing tenderness as the 
combination of both treatments. Similar results were obtained from meat 
injected but not held at the elevated temperature. A portion of meat from 
each animal was analyzed for total and ionic calcium content. The results 
showed that antemortem vascular injection of calcium acetate and Na.sub.2 
CaEDTA is effective in increasing the total and ionic calcium level in the 
meat. 
A second test on cattle using the same procedure as in the first test was 
made except that half of the carcasses of both groups were subjected to 
electric stimulation for 10 minutes (768 volts, 15 cycles/sec.) and 
thereafter all carcasses were chilled under normal packing house 
conditions. Top round muscles were filleted out for roasts. After cooking 
the roasts were analyzed in a laboratory and tested mechanically and by a 
taste panel of experts for tenderness. Unlike the results from the first 
cattle test, there were no differences in the tenderness scores among the 
different treatments. There was a similar increase in the muscle calcium 
level. 
The first two tests demonstrate that antemortem injection with calcium 
acetate and Na.sub.2 CaEDTA alone or in combination with electric 
stimulation did not increase the tenderness of meat. The results of these 
two tests show that, even after the calcium level in the muscle tissue has 
been raised to the desired level and the free zinc ions present in meat 
have been effectively bound by chelating agents, temperature should be 
maintained within the optimum range of the neutral enzyme. When the proper 
balance of these factors was maintained immediately after slaughter the 
degree of tenderness obtained was substantially greater in a shorter 
length of time than the tenderness produced when the meat was merely held 
at the elevated temperature without the other factors present. 
Sixteen sheep were selected ranging between 100--120 pounds live weight and 
divided into two groups. The procedure of the first cattle test was used. 
The test group of eight sheep were given an antemortem vascular injection 
of a solution of 0.4 M calcium acetate and 1 M Na.sub.2 CaEDTA. Employing 
the same variety of mechanical tests, laboratory analysis and testing by a 
panel of experts as followed in the first cattle tests, and results were 
substantially identical. 
Separate tests were made on sixteen live chickens each weighing 4 pounds 
and sixteen live turkeys of varying weights. The same procedure as 
employed in the previous tests was followed except that each bird in the 
respective test groups was injected subcutaneously with a solution 
containing 0.2 M calcium acetate and 0.5 M Na.sub.2 CaEDTA at the rate of 
7.5 ml of solution per 4 pounds. The birds were dispatched within 15 
minutes after injection. Both the analysis and test results produced 
findings the same as in the previous tests. 
Antemortem injection is the preferred embodiment of the invention for 
several reasons. Vascular antemortem injection utilizes the vascular 
system of the animals to effect a more even distribution of the solution 
throughout the muscle tissue. It also results in the most efficient use of 
the materials injected. In the case of fowl, subcutaneous injection is the 
most efficient because of the difficulty of injecting directly in the 
birds vascular system and because of the rapid absorption of the solution 
into the vascular system of the bird. In addition, the pH of meat becomes 
somewhat lower the longer the meat is held after slaughter thereby 
resulting in a somewhat lower rate of proteolytic activity of the neutral 
enzyme. However, other means of introducing the activators are also 
effective. 
To demonstrate the effect of postmortem injection of activators into meat, 
a 0.85% NaCl solution containing 33 mM calcium acetate and 23 mM Na.sub.2 
CaEDTA was stitch pumped into one side of beef rounds, and the other side 
of each round was similarly injected with a 0.85% NaCl solution to give a 
4% gain in weight after pumping and a 3% retention upon standing. To 
minimize variability between control and test, 4 paired rounds from the 
left and right legs of 4 animals were tested as a group. A first group of 
eight rounds was taken from rounds which had aged under refrigeration of 
35.degree. F. for 24 hours and a second group was taken from warm rounds 
injected immediately after slaughter. The postmortem pumping of calcium 
acetate and Na.sub.2 CaEDTA resulted in a rise of the total calcium level. 
The warm test rounds of the second group which were injected with calcium 
acetate and Na.sub.2 CaEDTA and held at 72.degree. F. for two days, were 
judged the most tender as determined by a trained test panel, by 
mechanical force measurement and by laboratory analysis of the tissue 
texture. The resulting increase in the tenderness of these injected test 
rounds was somewhat less than that produced by antemortem injection. Also, 
the degree of increased tenderness produced in the warm rounds or in those 
injected immediately after slaughter was greater than those aged under 
refrigeration for 24 hours. The difference in tenderness between these two 
groups clearly shows the importance of holding at the optimum temperature 
of the neutral enzyme and the gradual lowering of the pH in meat after 
slaughter. 
Obviously many modifications and variations of the invention as 
hereinbefore set forth may be made without departing from the spirit and 
scope thereof and, therefore, only such limitations should be imposed as 
are indicated in the appended claims.