There remains a very wide variation in the degree of tenderness/toughness in meat produced for human consumption. This inconsistency in meat tenderness remains a major problem in the industry, and is particularly relevant to beef, pork and lamb (mutton).

Efforts have been made to eliminate this problem through standardisation of animal breeding, and through nutrition and husbandry regimes, but this approach has not been totally successful and, for example, 17% of all loin chops sold in the United Kingdom are unacceptable to consumers due to toughness and dryness of the meat (Warkup et al, "Consumer perceptions of texture; the most important quality attribute", in "Expression of tissue proteinases and regulation of protein degradation as related to meat quality" ed Ouali et al, pp 225-237 ECCEAMST, Urecht, The Netherlands). In recent years much research has focused on the problem of variable meat tenderness in an attempt to determine the physiological factors

involved.

It has been postulated (see Koohmaraie, 1994, Meat Sci.

36:93; and Dransfield, 1994, Meat Sci. 37:391) that the calpain/calpastatin proteolytic system may (at least in part) contribute to myofibrillar degradation post mortem, thus affecting the rate and degree of meat tenderness. Calpains are naturally occurring calcium- dependent thiol proteases which occur in two major isoforms -calpain and m-calpain. Calpains are specifically inhibited by their naturally occurring inhibitor, calpastatin. Skeletal muscle fibres are known to contain both y- and m-calpain (which are able to cause myofibril disassembly and hydrolysis of myofibrillar proteins - although actin and myosin are not affected) and calpastatin. Initial studies indicate that, post mortem, m-calpain is rather stable whilst the activity of jt-calpain and calpastatin decline gradually. The activity of calpastatin has been shown to be highly variable and may be a heritable trait (see Koohmaraie et al, "Calpastatin-based Methods for Predicting Meat Tenderness", 1995, "Expression of tissue proteinases and regulation of protein degradation as related to meat quality" ed Ouali et al, pp 395-412 ECCEAMST, Urecht, The Netherlands). An increase in calpastatin has been associated with reduced meat tenderness and most research in this area has thus concentrated on the role of calpastatin (see Koohmaraie, 1992, Biochimie 74: 239-245; Sensky et al, 1996, Journal of Animal Science 74: 380-387; Sensky et al, 1996, Animal Science 62: 663-664). However, to date it has not been possible to predict the degree of tenderness/toughness in the meat from any particular animal.

Our research looked at the activity of each component of the calpain-calpastatin system in the pig very shortly after death and again prior to the onset of rigor mortis. The most striking observation was that the activity of m-calpain consistently increased at approximately 2 hours post mortem and thereafter declined for those animals which provided tender meat, whilst in animals providing tough meat no such increase in m-calpain activity was found. Our observations (see Figs 1 and 2) enable the tenderness of meat to be accurately predicted for the first time.

The ubiquitous and highly conserved nature of the calpain-calpastatin system across species means that our observations in pigs will also be relevant to other species. Of particular importance to the meat industry are cattle and sheep, but other meat-producing animals (eg horse, deer, chicken, rabbit, pheasant etc) are not excluded.

Thus the present invention provides a predictive assay for meat tenderness, the said assay comprising analysing the activity or amount of m-calpain post mortem. Where the activity of m-calpain is analysed, the assay will include determining whether the said activity increases post mortem and, optionally, comparing the activity found to a known standard.

Where the amount of m-calpain is analysed this amount will be compared to a known standard.

Generally, it will be necessary to analyse m-calpain only once at a specific time point post mortem and then to compare the level of activity to a known standard.

However, under particular circumstances it may be preferable to analyse the activity or amount of m- calpain at death, as well as analysing m-calpain

activity or amount over a relevant time period. For accuracy, it may be preferable to determine the ratio of m-calpain activity at the relevant time point:m- calpain activity at death. Alternatively the ratio of m-calpain amount at the relevant time point:m-calpain amount at death may be obtained. The value obtained may then be compared to a known standard to determine whether the meat is of a suitably tender quality.

References to the m-calpain activity or amount "at death" means that the activity or amount is determined as soon as is practicably possible after death, generally within 30 minutes from the time of death. In practical terms statutory testing of meat intended for human consumption means that assays for m-calpain cannot normally be determined immediately after death.

In a further aspect therefore the present invention provides an assay for meat tenderness, said assay comprising analysing the post mortem activity or amount of m-calpain at a pre-determined time point.

As explained above, our observations in pigs noted an increase in m-calpain activity at approximately 2 hours post mortem. Naturally, the time at which this increase in m-calpain activity occurs may vary in different types of animals, although determination of the relevant time period can be established by those skilled in the art using known methodologies. We believe that in each animal species the peak in m- calpain activity may be related to the time of death and the onset of rigor mortis.

For the pig, a suitable time period is from one to 4 hours post mortem, especially 90 minutes to 150 minutes post mortem.

For cattle, a suitable time period may be from one to 9 hours post mortem.

As well as analysis of m-calpain activity post mortem, we have established that meat tenderness may be accurately predicted using anti-m-calpain antibodies to measure the amount of m-calpain in the muscle tissue.

Our results are presented in Figure 3 and the experimental work is detailed in Example 2.

In yet a further aspect, the present invention provides an assay for meat tenderness, said assay comprising using anti-m-calpain antibodies to determine the level of m-calpain present post mortem.

Our observations on the behaviour of m-calpain activity and the use of anti-m-calpain antibodies to determine the amount of m-calpain present post mortem has other applications in addition to testing the degree of meat tenderness in any particular animal carcase. For example, the assays of the present invention may be used in a breeding program designed to produce animals yielding tender meat on a consistent basis. Since the assays described are conducted post mortem, the results obtained will provide valuable data on the potential for tender meat production in the close relatives (for example the offspring or siblings) of the animal tested.

Viewed from a further aspect therefore the present invention provides an assay for selecting an animal for use in a conventional breeding program, the assay comprising analysing the activity or amount of m- calpain in a muscle of a close relative of said animal, said assay being conducted post mortem.

Whilst we do not wish to be bound by theoretical considerations, we believe that the observed increase in m-calpain activity or may be due to one or more of the following factors: i. a physical difference in the m-calpain protein or its substrate due to genetic factors; ii. the pre-slaughter handling or stress levels in the animals (adrenaline and fi-adrenergic agonists have been shown to affect meat tenderness - see Koohmaraie et al, 1991, J Anim Sci. 69:4823; Wheeler et al, 1992, J Anim Sci. 70:3035; Parr et al, 1992, Eur J Biochem 208:333; Bardsley et al, 1992, Biochimie 74:267; Sensky et al, J Anim Sci., 1996 74:380-387); iii. pre-slaughter nutrition of the animals, which may affect muscle metabolism and thus levels of calpains or calpastatin.

Further results indicate that there may be a genetic factor underlying the observed increase in m-calpain activity or amount. In particular, a further study (described in Example 3) showed that m-calpain activity and amount was preferentially increased post mortem in cross bred (Duroc x white) pigs compared to purebred pigs.

Thus, the assay of the invention may be of especial utility in determining the meat tenderness of a cross bred animal (in particular a cross bred pig, preferably a Duroc x white pig, in particular a 50:50 Duroc x Large White cross).

The present invention will now be further described

with reference to the following non-limiting Examples and figures in which: Fig. 1: is a graph showing m-calpain activity (x107 fluorescence units/kg) varying over time post mortem for tough, normal and tender meat (pork). The results are pH independent and demonstrate a 0-2 hour post mortem increase in activity for the non-tough samples but a 0-2 hour post mortem decrease in activity for the tough samples.

Fig. 2: shows the relationship between the ratio of m-calpain activity 2 hours after death: immediate post slaughter activity and the final meat toughness as determined by shear force measurements 8 days after slaughter.

Fig. 3: is a graph showing how the amount (yg) of m- calpain alters with time post mortem for tough, normal and tender meat (pork). The results are pH independent.

Fig. 4: shows the ratio of m-calpain activity 2 hours after death:immediate postslaughter activity for purebred Yorkshire, Duroc and Large White pigs and for 50:50 Duroc x Large White cross.

Fig. 5: shows the amount of m-calpain located using anti-m-calpain antibodies for the samples used in Fig. 4. Again the amount of m- calpain is expressed as a ratio of the value obtained 2 hours after death:immediate postslaughter value.

LW= 100% Large White pure bred D= 100% Duroc pure bred LWxD= 50 Large white x 50% Duroc cross bred Example 1 Post mortem changes in m-calpain activity Post mortem changes in m-calpain activity were measured in samples collected from randomly selected commercially produced pigs, representing diverse husbandry regimes, following slaughter by electrical stunning and severance of the carotid arteries at a commercial slaughterhouse.

Calpain activity was determined for each animal as described in Sensky et al, J Anim Sci, 1996, 74: 380- 387. Samples were measured at 30 minutes, 2 hours, 4 hours, and 24 hours post mortem. Samples were then tested for toughness and graded accordingly. Samples were tested for toughness, using conventional shear force measurements using samples which had been conditioned for 8 days at 20C.

The results are presented in Figure 1. The rapid increase in m-calpain activity at approximately 2 hours is clearly shown for the samples judged to be tender.

Figure 2 shows that determination of the ratio of m- calpain activity 2 hours after death: immediate post slaughter activity and the final meat toughness can be used to predict the final toughness/tenderness of the samples.

Example 2 M-calpain measured using an anti porcine m-calpain antibody An antibody to porcine m-calpain was raised against a fusion protein expressed in E coli.

A cDNA sequence was generated for porcine m-calpain which encoded a region spanning the majority of domain 3. A region equivalent to residues 295-538 of the human m-calpain sequence (Biochemistry 27:8122-8128).

This 244 amino acid peptide was expressed in E coli as a Glutathione S-transferase fusion protein product using the pGEX vector system (Pharmacia). The fusion protein product was used as the antigen. Antibodies (polyclonal) were raised in rabbits and tested against m-calpain purified by anion-exchange chromatography (as in J Anim Sci, 1996, 74:380-387).

The antibody was used to test samples from trial 3 (commercial trial). A crude extract was made of the cell soluble components. These were subjected to SDS- PAGE, Western blotted and then probed with the m- calpain antibody. Detection was done by the Enhanced Chemiluminence System (ECL). Quantification was done by scanning densitometry.

The results are shown in Figure 3. The increased intensity of the m-calpain signal at two hours is an indicator of meat tenderness.

Example 3 The methodologies of Example 1 and 2 were repeated in a further experiment in which the genotype of the animals

undergoing assay were carefully pre-selected and the m- calpain activity and amount were determined only immediately posts laughter and 2 hours post mortem. 11 or 12 animals were present in each group, with the groups being as follows: Y: (100%) Yorkshire pigs LW: (100%) Large White pigs D: (100%) Duroc pigs LWxD:(50:50) Large white x Duroc crosses.

The results are expressed as a ratio of the values obtained 2 hours post mortem: immediately post slaughter and are given in Tables 1 and 2 below and in Figures 4 and 5. n 0 h 2 h 2:0 h ratio Yorkshire 12 2.570 2.362 0.906 Duroc 12 1.980 1.477 0.821 Large White 11 1.858 1.476 0.923 Large White x Duroc 12 1.609 1.872 1.407 SED = 0.398 0.385 0.230 p 0.111 0.082 0.060 Table 1. m-calpain activity Oh and 2h after slaughter and the ratio of activity measured at the two timepoints in 4 different pig genotypes

Table 2. Amounts of m-calpain determined by immunochemistry Large White Duroc Large White x Duroc 0 hours 2 hours 2:0 0 hours 2 hours 2:0 0 hours 2 hours 0:2 hours hours hours ratio ratio ratio 13.4645 7.5288 0.599 7.60862 9.04987 1.189 13.8049 11.2852 0.817 16.3303 16.5399 1.013 11.3734 9.31125 0.819 8.45153 3.89466 0.437 8.73218 4.5717 0.524 24.0365 17.6414 0.734 11.9378 12.0578 1.010 12.4001 11.1111 0.896 14.4884 13.6604 0.943 14.1833 16.9577 1.196 14.5826 11.4039 0.782 15.2552 13.0076 0.853 11.5243 13.3869 1.162 19.5198 14.5866 0.747 14.2764 6.60478 0.463 17.8502 11.5708 0.648 10.1347 7.30908 0.721 12.4752 9.77982 0.784 2.63526 4.45339 1.690 av 0.749 av 0.826 av 0.994 sd 0.173 sd 0.219 sd 0.411 sem 0.065 sem 0.083 sem 0.155