AU725573B2 - Improvements in and relating to controlling the tenderness of meat - Google Patents

Description

I P/00/01i1 Regulation 3.2 AUSTRALI1A Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT

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Improvements in and Relating to Controlling the Tenderness of Meat The following statement is a full description of this invention, including the best method of performing it known to me/us: 0 0 1 00 0* 0 *0 0 0..0 4*006 S C 0 S FHPMEL069820501 1k IMPROVEMENTS IN AND RELATING TO CONTROLLING THE TENDERNESS OF MEAT TECHNICAL FIELD The present invention is directed to methods and products affecting the tenderness of meat in slaughtered animals. Preferred embodiments of the invention affect the cellular messenger nitric oxide (NO) to influence tenderness in post-mortem meat.

BACKGROUND ART Toughness associated with meat from slaughtered animals has been of concern to the meat and related industries for some time. Virtually every consumer has at some stage experienced cuts of meat which have ranged from unbelievable tenderness, to a degree of "2 15 toughness which has made them wonder if it had been fit for human consumption.

Accordingly, an aim of the meat industry has long been to identify and control factors which determine the toughness or tenderness of meat for the consumer.

The causes of toughness in meat are not well understood. There is a complex interrelationship of genetic factors, pre-slaughter handling regimes, and processing decisions such as timing and temperature for electrical stimulation or chilling of carcasses. The precise mechanisms are only now beginning to be elucidated.

It has however been known for some time that cold shortening is one cause of toughness S 25 in meat. As research has progressed, it has been identified that below a particular pH cold shortening does not occur.

Consequently, to lower pH, electrical stimulation is often applied to carcasses. Providing the correct type of stimulation is applied, muscle glycogen is converted to lactic acid. The S 30 effect of this is to lower the overall pH.

Research by the applicant has identified other mechanisms which will have an influence on tenderness. One mechanism is the regulation of peroxidation pathways in tissue, particularly through regulation of the cellular messenger nitric oxide, and membrane maintenance/disruption. Generally, peroxidation results in a loss of membrane integrity.

Nitric oxide has previously been used in association with meat products for curative purposes. For example, in US Patent No. 3,873,740 the use of nitrogen oxide producing salts as curing agents is disclosed. The historically recognised role of nitric oxide in the curing process is as an antibacterial agent, or to provide pink colouration. Nitric oxide can be a generator of free radicals with associated deleterious effects when added to meats such as promoting rancidity. Recent concerns over nitrosamines (potential carcinogens) means that the use of nitric oxide producing salts is now contraindicated other than as a flavouring agent.

Therapeutic applications for nitric oxide have been recognised. US 5,508,045 discusses the control of the preterm labour in pregnant women and US 5,536,241 suggests that nitric oxide can be used for localised decrease of smooth muscle contraction in non-respiratory tract organs such as the uterus or bladder in an animal. This patent also teaches that haemoglolin rapidly combines with nitric oxide rendering it unavailable to relax smooth muscle and hence the need to administer the NO to a biological fluid other than blood.

NO is generally administered in gas form and at low concentrations (0.01 /M to 1000ptM o 15 or 1 to 100,000 ppm). There is no recognition in the therapeutic art area of direct S: 0 applications of nitric oxide to relax red muscle tissue, nor any suggestion to do so post- *Joe 0 •mortem.

4, we* S* US Patent No. 5,543,430 discusses the use of compositions comprising L-arginine and venous dilators in the production of NO to effect vasodilation or vasorelaxation to prevent or treat diseases such as hypertension, heart disease, cardiovascular disease, 0° cerebrovascular disease and renovascular ischemia. Again, this is a different art area with 00 no recognition of a general role of nitric oxide in meat tenderness, and certainly not in 0 post-mortem applications.

Physical and enzymic processes of meat tenderisation are known in the art. The "Hydrodyne process" is one physical process that has been suggested. In this process meat is vacuum packed and placed in water where a small explosive charge is detonated.

S The shock wave from the explosion damages the meat structure making it more tender.

0.o00 30 This involves significant additional processing and practical use will depend on developing packaging that will withstand the force of the detonation.

There is still a need for practical methods of tenderising meat which produces a consistent result and are easy to carry out.

It is an object of the present invention to provide an alternative method for influencing meat tenderness or at least to provide the public with a useful choice.

-3- Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

SUMMARY OF INVENTION According to one aspect of the invention there is provided a method for influencing the quality of meat comprising the administration of stimuli sufficient to affect peroxidation pathways in the meat tissue.

According to another aspect of the present invention, there is provided a method substantially as described above, in which the quality influenced is meat tenderness.

In one embodiment the meat is in the form of a carcass.

15 According to another aspect of the present invention there is provided a method for affecting the tenderness of meat derived from slaughtered animals comprising the administration of stimuli sufficient to affect peroxidation pathways in the meat tissue.

According to another aspect of the present invention there is provided a method, substantially, as described above, in which the affected peroxidation pathways are those acting on the lipid structure of tissue membrane and its contained cellular organelles.

According to another aspect of the present invention there is provided a method, substantially as described above, in which the targeted meat tissue is muscle tissue.

According to another aspect of the present invention there is provided a method, substantially as described above, in which the effects on peroxidation pathways is triggered by influencing the levels of the gaseous cellular messenger Nitric Oxide (NO) S in the tissue.

According to another aspect of the present invention there is provided a method, substantially as described above, in which influencing levels of NO is accomplished by affecting the NO synthesising enzyme Nitric Oxide Synthase (NOS).

According to another aspect of the present invention there is provided a method, substantially as described above, in which NO levels are influenced by effecting at least one of the following stimuli: -4adjusting ante-mortem stress of an animal to the extent that NO levels are altered to a level optimising the required degree of tenderness in meat derived from that animal; adjusting post-mortem metabolism in an animal to the extent that NO levels are altered to a level optimising the required degree of tenderness in meat derived from that animal; causing an animal to experience specific ante-mortem behaviours to the extent that NO levels are altered to a level optimising the required degree of tenderness in meat derived from that animal; administering additives to an animal to the extent that NO levels are altered to a level optimising the required degree of tenderness in meat derived from that animal; the post-mortem introduction into meat tissue of additives such that NO levels are "altered to a level optimising the required degree of tenderness in meat derived from *15 that animal.

According to another aspect of the present invention there is provided a method, substantially as described above, in which the level of antemortem NO levels are determined or monitored prior to slaughter of an animal from which meat is to be derived.

According to another aspect of the present invention there is provided a method, substantially as described above, in which tags attached to an animal are used to monitor *03 NO or other chemical levels within the animal.

25 According to another aspect of the present invention there is provided a method of S predicting meat quality, the method comprising measuring levels of NO as an indicator of meat quality.

S According to another aspect of the present invention there is provided a method, 30 substantially as described above, in which in response to determined or monitored NO levels prior to slaughter, at least one of the following steps is implemented: the animal is additionally subjected to stimuli, or the level of stimuli altered, in order to achieve the desired level of NO corresponding to the degree of tenderness desired; the animal is additionally subjected to additives, or the level of administered additives altered, in order to achieve the desired level of NO corresponding to the degree of tenderness desired; timing the steps of additive administration, and/or stimulation to optimise desired levels of NO.

According to another aspect of the present invention there is provided a method, substantially as described above, in which NO levels are influenced by the administration or introduction of at least one NO influencing substance.

Desirably, the NO influencing substances are selected from NOS inhibitors and NO enhancers.

Preferably, the NO influencing substance is at least one substance selected from the group consisting ofN-nitro-L-arginine, N-nitro-L-arginine methyl ester, N-arginine methyl ester, or salts of all of these, superoxide dismutase, and antioxidants or combinations thereof.

em e' o 15 Desirably, the antioxidants are selected from vitamin E, vitamin C and erythorbate.

According to another aspect of the present invention there is provided a method, ee substantially as described above, in which there is administered each of N-arginine methyl see. ester or salt thereof, superoxide dismutase, and vitamin E, either separately or in combination.

According to another aspect of the present invention there is provided a method, m "substantially as described above, in which additives affecting NO levels are administered on a slow release basis prior to slaughter.

So According to another aspect of the present invention there is provided a method, substantially as described above, in which slow release additives are in the form of a bolus b. administered to the animal.

30 According to another aspect of the present invention there is provided a method, substantially as described above, in which stimulation comprises electrical stimulation at a level sufficient to affect peroxidation anid/or NO pathways in the animal.

According to another aspect of the present invention there is provided a method, substantially as described above, in which electrical stimulation comprises post-mortem high voltage or low voltage electrical stimulation.

According to another aspect of the present invention there is provided a method, -6substantially as described above, in which NO levels are influenced by the steps of either or both post-mortem anoxia, and nutrient deprivation.

According to a further aspect of the present invention there is provided a method of affecting the tenderness of meat in a slaughtered animal by the method comprising altering either or both the animal's NO levels, and free radical levels.

According to a further aspect of the present invention there is provided a slow release bolus comprising one or more components able to affect NO levels within the animal, and in a quantity sufficient to affect those NO levels.

Preferred components able to affect NO levels are NOS inhibitors and NO enhancers.

According to another aspect of the present invention there is provided a bolus, 15 substantially as described above, which includes at least one of N-nitro-L-arginine, Nnitro-L-arginine methyl ester, N-arginine methyl ester, or salts of all of these, superoxide dismutase, and antioxidants or combinations thereof.

Preferred antioxidants are selected from vitamin E, vitamin C and erythorbate.

According to a further aspect of the present invention there is provided the use of chemicals affecting NO pathways in an animal, other than a human, administered in quantities, and timed, such that the tenderness of meat after slaughter of said animal can be altered according to a user's wishes or within a predetermined range of tenderness.

According to a further aspect of the present invention there is provided the use of external stimuli and exercise on an animal, other than a human, the extent and timing of such being that the tenderness of the meat after slaughter of said animal can be altered according to S a user's wishes or within a predetermined range of tenderness.

Meat produced in accordance with the above methods of the invention forms a further aspect of the present invention.

Although g-calpain appears to be the main protease responsible for meat tenderisation (Utterhaegen et al, 1994) there are some unexplained aspects such as tenderness variability, the quadratic relationship of tenderness and ultimate pH which appear to affect the rate of tenderisation (Watanabe et al, 1996), partial inactivation of calpains at temperatures above or below 15 C (Simmons et al, 1996) and non enzymic mechanisms -7- (Takahashi et al 1987; Takahashi, 1992). Nitric oxide (NO) a gaseous intercellular messenger has recently been proposed in ubiquitous roles, including those within normal functioning skeletal muscle. NO has not, however, been considered as a component of changes in post-mortem muscle. To the knowledge of the inventors it has not been manipulated or measured in meat.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

DETAILED DESCRIPTION *9 According to one method of the present invention there is provided means for influencing 15 the quality, especially the tenderness of meat by affecting the peroxidation pathways so 0 :of. which occur within the meat tissue. Preferably, this is achieved by influencing the levels of the gaseous cellular messenger nitric oxide (NO).

The term "influencing" as used herein encompasses adjusting peroxidation pathways, particularly NO levels, either up or down to achieve a pathway or NO level corresponding to a desired level of tenderness in meat.

Current thinking suggests that NO levels relative to meat tenderness exhibits a U- or nshaped curve such that low or high levels of NO may be associated with unacceptable 25 meat tenderness, with moderate levels of NO representing an optimised tenderness range.

Hence, the requirement to adjust NO levels either up or down.

In New Zealand, tenderness standards are prescribed by the MIRINZ AC A (accelerated conditioning and ageing) standard. According to this standard, 95% of meat must have 30 a shear force below 10 kgF. A mean acceptable shear force is 8 kgF, preferably the shear force is in the range of 4-7 kgF for optimised tenderness. Shear force may be measured using a MIRINZ tenderometer as described in New Zealand Patent No. 190945 incorporated herein by reference. NO levels can be correlated with shear force values to provide a NO standard against which to compare subsequent readings of NO levels. In this way, NO levels can be used as a predictor of meat quality and tenderness in particular.

According to the present invention there are a number of ways by which peroxidation pathways or NO levels may be adjusted. In some methods of the present invention these -8techniques will be performed ante-mortem, while in other methods they may be performed post-mortem. In addition, there may also be a combination of ante-mortem and postmortem techniques.

At a generic level, NO levels may be optimised by genetic selection of animals exhibiting favourable NO levels, or by manipulation to alter genomic expression and control of levels of NO according to methods known in the art, for example by genetic modification of NO products or breakdown products thereof.

In the ante-mortem techniques, NO levels may be influenced in a number of manners. For example, animals transported to a processing plant and fed there for a week are more tender than animals similarly transported and fasted according to normal practice. In the absence of obvious methods of stress (the ultimate pH level was the same), it is the applicant's current belief that this reflects a change in metabolism related to NO that S 15 affects the pathways of tenderisation. By placing an animal under certain types of stress, NO levels may be adjusted to a degree which favourably affects the tenderness in meat derived from a slaughtered animal. However, because the body is a constantly changing system, such ante-mortem techniques are generally best performed within a relatively short period before slaughter, preferably within the 48 hours, more preferably at least within 24 hours, and more preferably within 6 hours preceding slaughter.

Altering NO levels by external influences such as stress or exercise is one method of the present invention. This may involve exercising the animals, such as walking them, by an S amount sufficient to bring NO levels within the range corresponding to the desired level 25 of postmortem tenderness. Alternatively, periods of stress may be introduced, which may include known methods which would place an animal under stress. This may even include electrical stimulation, though not at a level sufficient to kill or seriously injure the animal.

Consideration will also need to be given to what is considered humane, though in most S case the stress levels involved will not correspond to extreme levels of stress.

S The potential difficulty of using external stress to alter NO levels is that it may be difficult to gauge, for different herds and cattle species, what level of stress or exercise is sufficient. The condition of the animals at the time, as well as events leading up until this period, will all potentially have an influence on the NO levels. Accordingly it is desirable, when such methods of external influence are used, that there is also a regime in which at least one or a representative sample of the group has their NO levels monitored.

Preferably this will be through the use of a device able to give virtually instantaneous test -9results a test which only provides results 6 hours later would, for instance, be of little use. Accordingly, a preferred method of monitoring is the use of a tag or other transponder attached appropriately to the monitored animals. The inventors have, in an alternative patent application (International Patent Appln. No. PCT/NZ98/00065 incorporated herein by reference), described a tag able to monitor biochemical levels or changes within a system which would be suitable for use as a monitoring device in the present invention.

However this does not preclude the monitoring of NO levels according to other techniques and methods such as rapid kit formats known in the art. For example, a useful electrode sensor probe for NO is disclosed in US Patent No. 5,603,820 incorporated herein by reference.

Another alternative to exercise or stress, which may also be used in conjunction with these methods, is the administration of a composition able to affect NO levels. In some respects this may be more predictable and consistent in the manner by which it works. Similarly, 15 there may be less need for monitoring animals within the group though it is still considered to be good practice to provide some monitoring in the event that some outside condition causes an unexpected alteration in NO levels.

SA further consideration is the method of administration of these compositions. While these may be administered in a number of ways, including intravenous, orally or otherwise, in most cases meat processing plant, abattoirs, and slaughter yards wish to minimise handling of animals. Accordingly, unless automated, the intravenous administration of suitable compositions at the meat processing plant, abattoirs, and slaughter yards is unlikely to be a popular option. An alternative is for the farmer to intravenously administer chemicals while cattle are being loaded for transport though a potential difficulty here is that unless the transport is relatively prompt and the animals are processed upon arrival, any effects from an intravenous administration may have dissipated before slaughter occurs.

Accordingly, other embodiments of the present invention include compositions which can be more effectively (in terms of handling times) administered to the cattle prior to slaughter. In one option there is provided a slow release bolus which is administered to the cattle. It is envisaged that this is likely to be administered by the farmer prior to transport to the freezing works/slaughter yard. Due to the slow release characteristics of such a bolus, the effects can be tailored to last for several days which should cover conceivable delays in transport and processing.

As can be envisaged, bolus construction is relatively well known and it would be within the expertise of a skilled addressee of the art to prepare a suitable bolus having release characteristics ranging from over a period of hours to even months. This latter option would probably not be that commonly used except for live cattle shipments overseas.

Even in such cases, it would perhaps be preferable to use graduated bolus construction so that during the initial period little or no active components were released, and it was only within a particular period towards the anticipated life of a bolus that effective amounts of the active components were released.

In another option available for administration is to incorporate active components into feedstuffs which are fed to the animals. Again this may be readily performed with little handling time, merely requiring food to be made available for consumption by the cattle.

It should also be appreciated that while the majority of this description is directed to cattle, its applications can be applied to virtually any mammalian animal bred and slaughtered 15 for their meat. It is also envisaged that the present invention will likely also be suitable for use with non-mammalian animals such as poultry.

More specifically, it is contemplated that the present invention can be applied to animals including: cattle, sheep, pigs, goats, rabbits, moose, deer, buffalo and horses; as well as to poultry including chickens, turkeys, ducks and geese.

Active components according to the present invention comprise any substance able to affect NO levels within the animal. Compounds useful in the present invention for influencing NO levels include NOS inhibitors, for example, N-nitro-L-arginine, N-nitro- 25 L-arginine methyl ester, N-arginine methyl ester, salts of all of these, superoxide 0 dismutase and antioxidants. Preferred antioxidants include vitamin E, vitamin C and erythorbate, although any antioxidants known in the art which influence NO levels may •be used.

30 NO enhancers useful in the present invention include S-nitroso-N-acetylpenicillamine, diethylamine/nitric oxide adduct, 5,5-dinitrosodithiol, spermine nanoate and dephostatin, but are not limited thereto. Other useful compounds for influencing NO levels are disclosed in US 5,536,241 and US 5,543,430).

Preferred substances comprise N-nitro-L-arginine, N-arginine methyl ester, N-nitro-Larginine methyl ester, or salts of each of these, superoxide dismutase, and vitamin E or combinations of these. This list is not exhaustive but comprises preferred components for use with the present invention.

-11 It has also been previously mentioned that there are post-mortem techniques associated with the present invention. In such cases, this will involve techniques involving external stimulation of the carcass. While once again components affecting peroxidation pathways or NO related reactions continuing in the post-mortem carcass may be relied upon, the difficulty lies with the effective, administration of active components into the carcass.

Preferably these will be introduced into an animal's system prior to slaughter, so that effective levels remain in the post-mortem tissue.

Accordingly, commercially implemented post-mortem techniques may comprise the use of electrical stimulation in a manner to effectuate a change in free radical levels, NO levels, or peroxidation processes in a manner yielding a product of the desired degree of quality. This, as in other aspects of the invention, may comprise either an increase or decrease from existing levels to produce the desired levels. In many instances this requires high voltage stimulation, voltages typically being in the range of 200 to 4000V. Preferably 15 200 to 1100V. Particularly preferred voltages for the AC A process in New Zealand is 1100V and in Australia 700V. Low voltage stimulation with a voltage peak in the range eQ°• o of 30 to 100V, typically 40V or 80V, is also contemplated. These voltage ranges are typical of what may be employed but are not restricted to these values.

While electrical stimulation has been applied in the prior art, in most cases at relatively low voltages of around 200 volts or less, there has been no feedback or monitoring of the system and generally the parameters of stimulation are chosen to correspond to what is thought to result in a reduction in cold shortening, not optimisation of NO levels.

S 25 Desirably, when post-mortem electrical stimulation is applied according to the present O invention, there is monitoring of NO levels within the tissue to provide feedback about the desired parameters (such as voltage, pulsing, duration, timing of application (early or later o in processing) previous electrical inputs (from stunning), appropriate pulse shapes, etc.) of the stimulation so that the desired levels of NO are achievable. This monitoring may be prior, during, and/or after stimulation. From the results of such monitoring, the feedback will then be used to either alter or decide the desired parameters for stimulation and/or to repeat or continue the stimulation process. It is considered that by the monitoring of a measurable component such as NO, a meat product which is more consistently optimised to user requirements is generally attainable.

Accordingly, meat products produced in accordance with the above described methods also forms a further aspect of this invention.

12- BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: Figure la represents the time course of tenderness changes in meat that has been exposed to saline containing L-arginine (filled circles; control), saline containing N-nitro-L-arginine and N-nitro-L-arginine methyl ester hydrochloride (filled squares; NOS inhibitors) and saline containing diethylenetriamine/nitric oxide adduct and S-nitroso-N-acetylpenicillamine (filled triangles; NO enhancers). The error bars show the standard deviation, and 15 Figure lb represents the concentration of NO (RM) at different times during the ageing period in another set of samples of meat treated as in Figure la.

Symbols as above.

w BEST MODES FOR CARRYING OUT THE INVENTION The enzyme responsible for the synthesis of NO, NO synthase (NOS), has been localised in skeletal muscle, concentrated mostly in fast twitch fibres (Kobzic et al, 1994) so would e' be available for synthesis of NO in muscle. The inhibition of NOS, or the provision of NO enhancers or donors suggests that physiologically, NO may be involved in muscle 25 relation, a role mediated by cyclic GMP. However, under conditions of muscular exertion, NO also may be responsible for muscle damage via modulation of free radical activity.

At low levels of NO, and in the presence of superoxides, NO can bind to and inactivate them, and recent work suggest that in uncontracted skeletal muscles this occurs.

30 However, at higher concentrations of either superoxide or NO, peryoxynitrite is formed which decomposes to extremely reactive free radicals capable of breaking down fibre structure or its integrity leading to undesirably pulpy meat.

NO can be modulated by stress and by p-adrenergic activation (Garthwaite, 1995) and shows a strong relationship to calcium levels and its regulation, calmodulin and a number of other enzymes. Given these roles NO could have an important influence on postmortem muscle biochemistry. In the present study, the effect of the addition of NO enhancers and inhibitors on post-mortem muscle tenderisation is shown as is free radical 13activity measured by a microprobe.

EXAMPLE 1 The effects of addition of NO enhancers and inhibitors on meat tenderisation Five striploins longissimus lumborum), selected with an ultimate pH 5.5-5.6, were obtained within two hours of slaughter, from 24 month old bulls. These striploins were cut into three em-long strips, pricked with 20 G needles (to facilitate penetration of solutions) and randomly divided equally into one of three test solutions.

These solutions were: saline containing L-arginine (control); saline containing N-nitro-L-arginine and N-nitro-L-arginine methyl ester 15 hydrochloride (NOS inhibitors), or saline containing diethylenetriamine/nitric oxide adduct and S-nitroso-Nacetylpenicillamine (NO enhancers).

e **All chemicals were obtained from Research Biochemicals International (Natick, MA 01176000-2447. USA) and all solutions were made up to 0.1 mol 1-1 concentrations. The meat samples placed in each solution were completely covered and were separated from each other. Samples in test solutions were left at room temperature (18°C) for 15 hours to allow rigor, before being placed at 2 C for storage for the remainder of the experiment.

On day 1, 3, 6 and 8 after rigor mortis, samples were removed for tenderness measurements. The samples were placed in individual Tuflex® bags (Trigon Packaging Systems, Hamilton, New Zealand) and cooked in a water both at 100 0 C until they reached an internal temperature of 75 °C determined by placing a temperature probe (Thermometer Type 3001, Comark Electronics Ltd., England) in the meat. The cooked samples were 30 immediately placed in an ice-water slurry. From the chilled samples, ten 1 cm x 1 cm samples, approximately 3 cm long were cut parallel to the fibre direction. The force required to shear each portion perpendicular to the meat fibre was measured using a tenderometer which corresponds to a Warner Bratzler tenderometer if results are multiplied by 0.7 (Graafhuis et al. (1991)).

The tenderness of each sample was calculated as the mean shear force value of kgF of ten portions. At this point the ultimate pH was obtained to ensure only meat with values lying between 5.5-5.6 were used. Student t test (2 tail) was used either paired on non-paired to follow the day-to-day changes within or between treatment groups respectively.

14- EXAMPLE 2 Measurement of NO activity in meat following addition of enhancers and inhibitors.

A further five striploins were obtained and treated exactly as above with the addition of measuring free radical activity on cut strip samples immediately upon meat obtainment, and on days 1, 3, 6 and 8 after rigor mortis. The measurements were performed using an electrode consisting of a glass insulated carbon fibre coated with porphyrin/naflon (Malinski et al.

(1992) and PCT/NZ98/00065). These probes have a diameter of 50 gm and were inserted in 4 places (at random) along the sample strips. This type of probe allows detection of NO activity through oxidation of NO at the porphyrin surface monitored amperometrically.

The Nafion surface serves to increase the selectivity of the measurement for NO, however contribution from other free radicals cannot be ruled out. Construction of these probes and °15 their use has been described (Malinski and Taha, 1992; Cook, 1997).

$*O6 Ageing rates of meat following addition of inhibitors and enhancers All treatments (Fig. 1) showed a significant decrease in shear force values over time. At days 3 and 6, shear force values were significantly lower in the NO enhanced group than in other groups, whereas with the NO inhibited group on day 6, the shear force values were significantly higher. As the duration of storage increased further to 6 days, there was no significant difference in shear force values between the control group and the NO enhanced group. With the control group and the NO enhanced group, the shear 25 force values were significantly lower than the NO inhibited group. However at the end of the period of storage at 8 days, the tenderness values tended to become similar and all treatment groups were not significantly different.

Changes in NO over time in meat treated with inhibitors and enhancers Probe measurements indicated high free radical activity in all groups immediately after slaughter. Given the probe selectivity it is likely that much of this free radical activity measured is due to NO. In the control and NO enhanced group this activity was still elevated compared to the NO inhibited group on days 3 and 6, although values were less than on day of slaughter. Values of NO were highest in the NO enhanced group. By day 8 NO activity was low in all groups (Fib lb).

The experiments suggest that NO can affect the rate of meat tenderisation as the NO enhanced group was more tender than the NO inhibited group in the early stages of tenderisation, but the differences became less with increasing storage duration (eventually all muscles reached the same tenderness). This pattern was mimicked by the measured activity of NO. This begets the question, however, of whether in normal pre-slaughter or post-mortem conditions, situations arise that ensure NO is produced and able to alter rates of tenderisation.

The mechanism of NO in tenderisation is not known, but NO can mediate its effects by free radicals and calcium changes which in turn affect proteolytic enzymes. The effects of NO inhibitors however, suggest roles additional to those mediated via free radicals. The initial change in tenderness was lower in control samples compared to samples treated with NO inhibitors and speculatively, this possibly could be attributed to effects on endogenous calpains (Utterhaegen et al, 1994). It may be that NO is needed to trigger calpain action, either directly or through secondary actions. Both NO and calpain are 15 influenced by the calcium environment.

6 By day 8, when ageing has almost been completed, the shear force values for all groups were similar, suggesting that there is tenderisation from other causes (NO measurement "differences were also not obvious between the groups at this stage). This could arise from a delayed activity, or a removal of inhibition of calpains, or alternatively it could arise from the emergence of a second enzyme system such as cathepsins which are not involved °o in initial tenderisation (Utterhaegen et al, 1994).

Prior to death and during subsequent periods post-mortem, low levels of NO seem 25 unlikely as post-mortem changes would ensure considerable synthesis. This was supported by probe measurements on the day of slaughter. NO elevation also could be a consequence of pre-slaughter stress. Fast twitch fibres which have the highest NOS activity are t* d extremely prone to stress effects. The non-enzymic tenderisation of meat proposed by Takahashi (Takahaski et al, 1987; Takahashi, 1992) and involving calcium ions could, in 30 part, be explained by the actions of NO. While the mechanisms of action of either NO enhancers or inhibitors in meat tenderisation are not yet clear, their effect may elucidate some of the underlying variability in meat tenderness from previously unexplained causes and in particular the role of stressors immediately before slaughter.

16 Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof.

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REFERENCES

1) Cook, C.J. (1997) Real-time measurements of corticosteroids in conscious animals using an antibody-based electrode. Nature Biotechnology 15, 467-472.

2) Garthwaite, J. (1995) Neural nitric oxide signalling. Trends in Neuroscience 18, 51-52.

3) Graafhuis, Honikel, Devine, Chrystall, B.B. (1991) Meat tenderness of different muscles cooked to different temperatures and assessed by different methods. Proceedings 37th International Congress of Meat Science and Technology, Kulmbach, 365-368.

*:15 4) Kobzic, Reid, Bredt, D.S. and Stamler, J.S. (1994) Nitric oxide in skeletal muscle. Nature 372, 546-548.

5) Malinski, T. and Taha, Z. (1992) Nitric oxide release from a single cell measured in situ by a porphyrinic based microsensor. Nature 358, 676-678.

6) Simmons, Singh, Dobbie, P.M. and Devine, C.E. (1996) The effect of prerigor holding temperature on calpain and calpastatin activity and meat tenderness.

Proceedings of the 42nd International Congress of Meat Science and Technology, Lillehammer, pp. 414-415.

7) Takahashi, K. (1992) Non-enzymatic weakening of myofibrillar structures during conditioning of meat. Biochemie 74, 247-250.

8) Takahaski, Kim, O.H. and Yano, K. (1987) Calcium induced weakening of zdiscs in post-mortem skeleta muscle. Journal of Biochemsitry 101, 767-774.

9) Utterhaegen, Claeys, E. and Demeyer, D. (1994) Effects of exogenous protease effectors on beef tenderness development and myofibrillar degradation and solubility. Journal ofAnimal Science 72, 1209-1223.

-18- Watanabe, Daly, C.C. and Devine, C.E. (1996) The effects of the ultimate pH of meat on tenderness changes during aging. Meat Science 42, 67-78.

All references are incorporated herein by reference.

It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

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Claims (36)

1. A method for influencing the tenderness of meat derived from a slaughtered animal, other than a human, comprising the determination of antemortem or postmortem NO levels and the administration of stimuli sufficient to influence (as herein defined) the NO levels in meat tissue.

2. A method according to claim 1 wherein the meat is in the form of a carcass.

3. A method according to claim 1 or 2 in which the NO levels are influenced by stimulation of peroxidation pathways.

4. A method according to claim 3 in which the peroxidation pathways are those acting on the lipid structure of tissue membrane and its contained cellular organelles. 00=

5. A method according to any one of claims 1 to 4 in which the tissue is a muscle tissue. OSS@ *006

6. A method according to any one of claims 1 to 4 in which influencing levels S 15 of NO is accomplished by inhibiting or enhancing the NO synthesising enzyme Nitric Oxide Synthase (NOS). 0 .06

7. A method according to any one of claims 1 to 5 in which NO levels are influenced by effecting at least one of the following stimuli: adjusting ante-mortem stress of an animal to the extent that NO levels are altered to a level producing the required degree of tenderness in meat derived from that animal; adjusting post-mortem metabolism in the animal to the extent that NO levels are altered to a level producing the required degree of tenderness in meat derived from that animal; causing an animal to experience ante-mortem behaviours to the CD/00368683.7 extent that NO levels are altered to a level producing the required degree of tenderness in meat derived from that animal; administering additives to an animal to the extent that NO levels are altered to a level producing the required degree of tenderness in meat derived from that animal; the post-mortem introduction to tissue of additives such that NO levels are altered to a level producing the required degree of tenderness in meat derived from that animal.

8. A method according to any one of claims 1 to 7 in which antemortem NO levels are monitored over a predetermined period of time prior to slaughter of the animal from which the meat is to be derived.

9. A method according to claim 8 in which, in response to determined or monitored NO levels prior to slaughter, at least one of the following steps is implemented: **0 0 o0o 15 the animal is additionally subjected to stimuli, or the level of stimuli altered, in order to achieve a level of NO corresponding to the degree of tenderness required; the animal is additionally subjected to additives, or the level of administered additives altered, in order to produce a level of NO 20 corresponding to the required degree of tenderness; timing the steps of additive administration, and/or stimulation to .00* produce required levels of NO. 0* 0 0 A method according to any one of claims 1 to 9 in which NO levels are influenced by the administration or introduction of at least one NO influencing substance.

CD/00368683.7 21

11. A method according to claim 10 wherein the NO influencing substances are selected from NO enhancers and NO synthase inhibitors.

12. A method according to claim 11 wherein the NO synthase inhibitors are selected from the group consisting of N-nitro-L-arginine, N-nitro-L-arginine methyl ester, N-arginine methyl ester, or salts of all of these, superoxide dismutase, antioxidants or combinations thereof.

13. A method according to claim 12 wherein the antioxidants are selected from vitamin E, vitamin C and erythorbate.

14. A method according to claim 12 in which there is administered each of N- arginine methyl ester, superoxide dismutase, and vitamin E, either separately or in combination.

15. A method according to claim 11 wherein the NO enhancers are selected from the group consisting of S-nitroso-N-acetylpenicillamine, diethylamine/nitric adduct, 5,5-dinitrosodithiol, spermine nanoate, and 15 dephostatin. 0

16. A method according to claim 7 or claim 9 in which additives affecting NO levels are administered on a slow release basis prior to slaughter.

17. A method according to claim 16 in which slow release additives are administered to the animal in the form of a bolus. 0

18. A method according to claim 9 in which stimulation comprises electrical stimulation at a level sufficient to affect peroxidation and/or NO pathways in the animal.

19. A method according to claim 18 in which electrical stimulation comprises post-mortem high or low voltage electrical stimulation.

20. A method according to any one of claims 1 to 9 in which NO levels are Co D/0036683.7 22 influenced by the steps of either or both post-mortem anoxia and nutrient deprivation.

21. A method of affecting the tenderness of meat in a slaughtered animal, the method comprising raising or lowering either or both the animal's NO levels, and NO free radical levels to a level equating with a users wishes or a predetermined range of tenderness.

22. A slow release bolus comprising at least one NO synthase inhibitor able to affect NO levels within the animal, and in a quantity sufficient to affect those NO levels, the NO synthase inhibitor selected from the group consisting of N-nitro-L-arginine, N-nitro-L-arginine methyl ester, N-arginine methyl ester, or salts of all of these, superoxide dismutase, antioxidants (other than vitamin C and vitamin E) or combinations thereof.

23. A bolus according to claim 22 wherein at.least two NO synthase inhibitors are present, wherein the at least one NO synthase inhibitor is other than an 15 antioxidant, and wherein at least one other NO synthase inhibitor is an antioxidant, including but not limited to vitamin C and vitamin E.

24. A bolus according to claim 23 wherein the antioxidant is selected from vitamin E, vitamin C and erythorbate, or a combination of these. o 20

25. A bolus according to claim 23 which comprises two or more of N-arginine 20 methyl ester, superoxide dismutase, and vitamin E.

26. A slow release bolus comprising at least one NO enhancer able to affect NO levels within the animal, and in a quantity sufficient to affect those NO levels, wherein the NO enhancer is selected from the group consisting of S- nitroso-N-acetylpenicillamine, diethyl amine/nitric adduct, dinitrosodithiol, spermine nanoate, and dephostatin.

27. The use of chemicals affecting NO pathways in an animal, other than a human, administered in quantities, and timed, such that the tenderness of SCD/003 68683.7 .23 meat after slaughter of said animal can be altered according to a user's wishes or within a predetermined range of tenderness.

28. The use of external stimuli and exercise on an animal, other than a human, the extent and timing of such being that the tenderness of the meat, as predicted by determination of antemortem or postmortem NO levels, after slaughter of said animal can be altered according to a user's wishes or within a predetermined range of tenderness.

29. A method of predicting meat quality, the method comprising measuring levels of NO as an indicator of meat tenderness.

30. Meat produced according to the method of any one of claims 20 or 29.

31. A method as defined in claim 1 for influencing the quality of meat substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. to

32. A method as defined in claim 4 of affecting the tenderness of meat in a :t 15 slaughtered animal substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

33. A slow release bolus as defined in claim 22 substantially as herein described with reference to the examples thereof. 0 oo.• 20

34. The use as claimed in claim 27 substantially as herein described with reference to the examples with or without reference to the accompanying drawings. The use as claimed in claim 28 substantially as herein described with reference to the examples with or without reference to the accompanying drawings.

CD/00368683.7 24

36. A method as defined in claim 29 of predicting meat quality substantially as herein described with reference to the examples and with or without reference to the accompanying drawings. THE HORTICULTURE AND FOOD RESEARCH INSTITUTE OF NEW ZEALAND LIMITED By its Registered Patent Attorneys Freehills Carter Smith Beadle 17 May 2000 S **s *5 S S *SO SD