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Title:
A METHOD AND CONTROL SYSTEM FOR OPTIMISING THE QUALITY OF MEAT
Document Type and Number:
WIPO Patent Application WO/2015/020537
Kind Code:
A1
Abstract:
Described herein is a meat quality control process and meat product. More specifically, a method of optimising meat quality including a combination of steps of fast freezing, thawing and aging is described that produces a meat with comparable attributes to chilled (never frozen) meat. The meat product itself and control system for optimising production of the meat product are also described.

Inventors:
KIM YUAN H BRAD (NZ)
LEATH SHANE (NZ)
SALERNO MONICA SENNA (NZ)
BALAN PRABHU (NZ)
KEMP ROBERT (NZ)
Application Number:
PCT/NZ2014/000160
Publication Date:
February 12, 2015
Filing Date:
August 05, 2014
Export Citation:
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Assignee:
AGRES LTD (NZ)
International Classes:
A23B4/07; A23L3/365; F25D11/02; F25D23/12; F25D29/00
Domestic Patent References:
WO2005060449A22005-07-07
Foreign References:
JP2002000247A2002-01-08
US8474279B22013-07-02
JP2005201533A2005-07-28
US20110302942A12011-12-15
Other References:
MUELA, E. ET AL.: "Effect of freezing method and frozen storage duration on instrumental quality of lamb throughout display", MEAT SCIENCE, vol. 84, 2010, pages 662 - 669
HILDRUM, K. I. ET AL.: "Combined effects of chilling rate, low voltage electrical stimulation and freezing on sensory properties of bovine M. longissimus dorsi", MEAT SCIENCE, vol. 52, 1999, pages 1 - 7
DUCKETT, S. K. ET AL.: "Effect of freezing on calpastatin activity and tenderness of callipyge lamb", JOURNAL OF ANIMAL SCIENCE, vol. 76, 1998, pages 1869 - 1874
PERLO, F. ET AL.: "Meat Quality Evaluation of Broiler Breast Fillets Affected by Aging Time and Marination", INTERNATIONAL JOURNAL OF POULTRY SCIENCE, vol. 9, no. 11, 2010, pages 1063 - 1068
MARTINO, M. N. ET AL.: "Size and Location of Ice Crystals in Pork Frozen by High- Pressure-Assisted Freezing as Compared to Classical Methods", MEAT SCIENCE, vol. 50, no. 3, 1998, pages 303 - 313
Attorney, Agent or Firm:
SNOEP, Robert et al. (PO Box 21-445Christchurch, 8143, NZ)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1. A method of extending the shelf life of meat and optimising meat quality by the steps of:

a) obtaining a piece or pieces of meat;

b) fast freezing the meat;

c) storing the meat in a frozen form for a period of time;

d) thawing the frozen meat; and

e) aging the thawed meat.

2. The method as claimed in claim 1 wherein the meat piece or pieces are finished cuts.

3. The method as claimed in claim 1 or claim 2 wherein the meat is packaged prior to step (b) or step (c).

4. The method as claimed in any one of the above claims wherein the meat piece or pieces are fast frozen in step (b) within 6-72 hours post slaughter, the rate of freezing in step (b) being sufficiently fast to prevent and/or minimise the formation of extracellular ice crystals.

5. The method as claimed in claim 4 wherein no single extracellular ice crystal is larger than 50% of the size of a cell within the meat.

6. The method as claimed in claim 4 wherein no combination of extracellular ice crystals are larger than 50% of the size of a cell within the meat.

7. The method as claimed in claim 4 wherein no single extracellular ice crystal is larger than 0.1 mm.

8. The method as claimed in claim 4 wherein no combination of extracellular ice crystals are larger than 0.1 mm.

9. The method as claimed in any one of the above claims wherein the internal meat temperature in step (b) is less than -5°C within at least approximately 12 hours.

10. The method as claimed in claim 9 wherein the time period is less than approximately 3 hours.

11. The method as claimed in any one of the above claims wherein the meat temperature is approximately -18°C within approximately 12 hours.

12. The method as claimed in any one of the above claims wherein storing in step (c) is completed at a temperature to minimise ice crystal growth during the storage time period and for up to 48 months. 3. The method as claimed in any one of the above claims wherein thawing in step (d) is completed until any ice crystals in the meat have substantially melted.

14. The method as claimed in any one of the above claims wherein the rate of thawing in step (d) is sufficient to avoid crystal growth during thawing and avoid agglomeration of ice crystals in order to minimise any extracellular damage to the meat.

15. The method as claimed in any one of the above claims wherein aging in step (e) is completed under controlled conditions for a time period sufficient to cause the desired level of degradation of cytoskeletal myofibrillar proteins, minimise microbial growth and reach a desired meat colour.

16. The method as claimed in claim 15 wherein aging occurs at a temperature of -1 ,5°C to 7°C under ambient pressure.

17. The method as claimed in claim 16 wherein the time period of aging ranges from

approximately 1 to 90 days.

18. The method as claimed in any one of the above claims wherein the meat is derived from livestock of genus selected from: Bovine, Ovine, Porcine, Cervine, Caprine, Equine, Dromadine.

19. The method as claimed in any one of claims 1 to 17 wherein the meat is derived from poultry including chickens, duck, emu, ostrich, turkey.

20. The method as claimed in any one of the above claims wherein one or more steps in the above method is or are completed during transportation of the meat piece or pieces.

21. The method as claimed in any one of the above claims wherein one or more steps in the above method is/are initiated by a remote control system that sends a signal initiating, adjusting and/or terminating one or more of the above steps.

22. A meat piece that has been frozen, thawed and aged and which exhibits a histology showing intracellular cryo-damage and irregularities in muscle fibre distribution compared to chilled meat.

23. The meat piece as claimed in claim 22 wherein the meat has less than or equal to 30% extracellular cell damage.

24. A meat piece that has been frozen, thawed and aged and which exhibits one or more varied metabolite concentrations compared to meat that has (a) only been chilled and aged and (b) meat that has been slow frozen and thawed.

25. The meat piece as claimed in claim 24 wherein the metabolite is selected from: malate, inosine monophosphate, inosine, alanine, carnitine, carnosine, acetate, lactate, pyruvate, and combinations thereof.

26. The meat piece as claimed in any one of claims 22 to 25 wherein the purge loss from the meat is 0.2 to 2.5 times that of chilled and aged meat.

27. The meat piece as claimed in any one of claims 22 to 26 wherein the hue angle of the meat is the same or less than the hue angle of chilled and aged meat.

28. A control system for optimising the quality of a meat piece or pieces including:

a controlled environment into which a meat piece or pieces are placed;

an adjustable unit operatively connected to the controlled environment;

a sensor or sensors in the controlled environment; and

a remotely situated control apparatus that receives a signal or signals from the sensor or sensors and which on command sends a signal to the adjustable unit to adjust the conditions in the controlled environment;

wherein the remotely situated control apparatus is used to initiate, adjust and/or terminate one or more of the steps of:

(a) fast freezing of the meat in the environment;

(b) storing of the meat in a frozen form for a period of time in the environment;

(c) thawing the frozen meat in the environment; and

(d) aging the thawed meat in the environment.

29. The control system as claimed in claim 28 wherein the environment is temperature controlled.

30. The control system as claimed in claim 28 or claim 29 wherein the adjustable unit adjusts the temperature in the controlled environment.

31. The control system as claimed in any one of claims 28 to 30 wherein the sensor or sensors monitor parameters selected from: temperature, pressure, fluid flow, weight, meat crystal size, and combinations thereof.

32. The control system as claimed in any one of claims 28 to 31 wherein the controlled environment is different for one or more steps (a) to (d).

33. The control system as claimed in claim 32 wherein the controlled environment or environments are transportable.

Description:
A METHOD AND CONTROL SYSTEM FOR OPTIMISING THE QUALITY OF

MEAT

RELATED APPLICATIONS

This application derives priority from New Zealand patent application number 613975 incorporated herein by reference.

TECHNICAL FIELD

Described herein is a meat quality control process and meat product. More specifically, a method of optimising meat quality including a combination of steps of fast freezing, thawing and aging is described that produces a meat with comparable attributes to chilled (never frozen) meat. The meat product itself and control system for optimising production of the meat product are also described.

BACKGROUND ART

Freezing has been practiced for decades in the meat industry and is considered to be one of the most effective methods of food preservation/prolonging shelf-life of meat products. The entire freezing/thawing process consists of four steps: 1 ) aging; 2) freezing of the product i.e. the reduction of the product temperature to the temperature at which it will be stored; 3) frozen storage; and 4) thawing of the product prior to subsequent cooking or further processing. The four steps can each affect meat quality attributes and thus must be controlled to maintain optimal product quality.

Although freezing has merit in terms of preservation, it can lead to a deterioration of meat quality primarily due to cell disruption and destruction of muscle fibre through cryo-damage. In particular, meat quality deterioration associated with prolonged frozen storage and/or repeated freezing-thawing of meat can occur due to the osmotic removal of water, protein denaturation and subsequent oxidation (both lipid and protein) and/or mechanical damage by ice crystal formation. As the water is restricted within and between the muscle fibres of the meat, compartments are formed in the tissue, which complicates the process.

The rate of freezing can play a fundamental role in determining size, location and shape of ice crystals within the muscle fibre. Slow freezing causes the formation of extracellular crystals that can result in damage to the muscle, thereby causing a decrease in myofibrillar protein solubility and water-holding capacity and an increase in weight loss on thawing.

A consumer perception survey conducted by Cryovac (Sealed Air Corporation) also showed that consumers heavily favour chilled meat products rating chilled meats two to three times better in terms of quality, tenderness, juiciness and flavour. Due to both the perceived and real quality differences, chilled meat attains higher prices than frozen meat.

One problem with chilled (never frozen) meat is that it has a limited shelf-life. This shorter shelf life is problematic when meat products are transported and/or limits time on a retail shelf prior to sale and time stored by a consumer prior to use. A further problem with chilled only meat is that the market may be left unsatisfied when meat is out of season.

It should therefore be appreciated that it would be an advantage to have a method of producing a product that utilised freezing steps of meat to attain the long term storage advantages, yet, where the meat post freezing also attained "chilled meat quality" attributes.

Aging meat for a certain period of time also can substantially improve meat quality attributes such as tenderness and water-holding capacity through myofibrillar protein degradation by endogenous proteases. Proteolytic enzymes such as calpains and cathepsins weaken the overall structure of the myofibril by degrading cytoskeletal myofibrillar proteins, subsequently increasing post mortem meat tenderisation during extended aging periods. Further, increased myofibrillar protein degradation during aging can result in improved water-holding capacity by minimising the rigor-induced lateral shrinkage of myofibrils, associated with the formation of drip and also with the re-uptake of previously expelled water. Aging is therefore a common value add step made to meat prior to freezing (or chilling). Aging is not however completed post freezing, most likely because the active proteins may be deactivated or their activity lessened post freezing. It is also possible that damage which occurs during traditional freezing also negates the advantages gained from aging. In any case, the inventors are unaware of art describing use of aging post freezing.

Further aspects and advantages of the process and product will become apparent from the ensuing description that is given by way of example only.

SUMMARY

Described herein is a meat quality control process and meat product. More specifically, a method of optimising meat quality including a combination of steps of fast freezing, thawing and aging is described that produces a meat with comparable attributes to chilled (never frozen) meat. The meat product itself and control system for optimising production of the meat product are also described.

The inventors have unexpectedly found that the combination and order of steps of fast freezing, then thawing, and then aging, may result in final meat characteristics comparable to or at least closer in nature to chilled meat. This combination is not predictable from the art and teaches away from current practice for example, aging does not occur post freezing.

In a first aspect there is provided a method of extending the shelf life of meat and optimising meat quality by the steps of: (a) obtaining a piece or pieces of meat;

(b) fast freezing the meat;

(c) storing the meat in a frozen form for a period of time;

(d) thawing the frozen meat; and

(e) aging the thawed meat.

In a second aspect, there is provided a meat piece that has been frozen, thawed and aged and which exhibits a histology showing intracellular cryo-damage and irregularities in muscle fibre distribution as compared to meat that has (a) only been chilled and aged or (b) meat that has been slow frozen and thawed.

In a third aspect, there is provided a meat piece that has been frozen, thawed and aged and which exhibits one or more varied metabolite concentrations compared to meat that has (a) only been chilled and aged and (b) meat that has been slow frozen and thawed.

In a fourth aspect there is provided a control system for optimising the quality of a meat piece or pieces including:

a controlled environment into which a meat piece or pieces are placed;

an adjustable unit operatively connected to the controlled environment;

a sensor or sensors in the controlled environment; and

a remotely situated control apparatus that receives a signal or signals from the sensor or sensors and which on command sends a signal to the adjustable unit to adjust the conditions in the controlled environment;

wherein the remotely situated control apparatus is used to initiate, adjust and/or terminate one or more of the steps of:

(a) fast freezing of the meat in the environment;

(b) storing of the meat in a frozen form for a period of time in the environment;

(c) thawing the frozen meat in the environment; and

(d) aging the thawed meat in the environment.

Briefly, advantages of the above method utilising a combination of fast freezing, then thawing and then aging include the production of chilled meat quality meat with the advantage of long term storage possible via freezing. The meat produced may be similar to chilled and aged meat that has never been frozen. The meat produced may have similar characteristics including look or colour (no or minimal oxidation or browning changes); flavour (e.g. avoid rancid lipid oxidation flavours); and mouth feel (for example as measured via moisture retention in the meat). Despite the similarities, as noted above there are selected markers that may be used to detect whether the method has been used or not including cryo-damage patterns and the presence of selected metabolites being different in the meat produced via the above methods.

A further advantage of the above method is that it is versatile as it can be utilised on a wide range of meat species and sized piece or cut. In this way, the meat product may be readily packaged and on sold in a convenient form to a consumer.

Further, the entire method or part steps thereof can occur during transportation or storage such that the meat is ready for distribution and/or consumption on delivery, thus reducing processing time and costs to the manufacturer and hence saving costs to the consumer. Finally, the one or more steps in the above method may be initiated by a remote control system that may send a signal initiating, adjusting and/or terminating one or more of the above steps. For example, a remote signal may be sent to a transportation source (containing the meat in a controlled environment) such as within a container of ship's cargo hold such that all or part of the method may be initiated prior to the meats point of destination such as an overseas processing plant. Again, an advantage being the meat may be ready for distribution and/or consumption on its point of delivery, thus reducing manual processing time, and hence decreased costs to the manufacturer which may be passed on to the consumer.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the process and product will become apparent from the following description that is given by way of example only and with reference to the accompanying figures in which:

Figure 1 illustrates a transverse section histology image of a piece of meat that had been frozen via traditional slow freezing methods. The image was obtained from

'Histological Measurements of Ice in Frozen Beef (J. Fd Technol. (1979) 14, 237 - 251 ; A. Bevilacqua, N. E. Zaritzky, and A. Calvelo). The piece was cut in a direction parallel to the fibres showing extracellular damage from conventional slow freezing;

Figure 2 illustrates five transverse section histology images (marked (a) to (e)) of muscles post different freezing and/or aging conditions being: (a) fast-frozen only ('FF'), (b) slow-frozen only ('SF'), (c) fast-frozen, thaw-aged for 2 weeks ('FFA2'), (d) slow- frozen then thaw-aged for 2 weeks ('SFA2'), (e) aged for 2 weeks (never frozen and chilled at -1.5°C) (Ά2'). The stars in Figure 2(b) and 2(d) point to compromised muscle fibre structures due to the formation of extracellular ice crystals between the fibres, whereas the black arrows in Figures 2(a) and 2(c) indicate damage due to intra-cellular ice crystal formation within muscle fibre;

Figure 3 is a graph illustrating three different methods of freezing meat and the speed of temperature reduction;

Figure 4 is a graph showing lipid oxidation levels for aged samples at -1.5°C comparing fresh unfrozen meat to levels observed for varying freezing rates;

Figure 5 is a graph showing lipid oxidation levels for aged samples at 1.0°C comparing fresh unfrozen meat to levels observed for varying freezing rates; Figure 6 is a graph showing hue angle readings (colour) for aged samples at -1.5°C comparing fresh unfrozen meat to hue angles (colour) observed for varying freezing rates;

Figure 7 is a graph showing hue angle readings (colour) for aged samples at 1.0°C

comparing fresh unfrozen meat to hue angles (colour) observed for varying freezing rates;

Figure 8 is a table comparing histology images of meat processed in different ways

illustrating changes in meat structure through processing;

Figure 9 is a graph showing the variation in purge loss between different methods of handling with all samples aged at 1.0°C;

Figure 10 is a graph showing the variation in purge loss between different methods of handling with all samples aged at -1.5°C;

Figure 1 1 illustrates two graphs showing the relative increase in metabolite malate in very fast frozen (VFF) and aged meat compared to control (un-frozen and aged) meat; and

Figure 12 illustrates two graphs showing the relative increase in metabolite malate in fast frozen (FF) and aged meat compared to control (un-frozen and aged) meat.

DETAILED DESCRIPTION

As noted above, a meat quality control process and meat product including a combination of freezing, then thawing and then aging steps is described. The meat product itself and control system for optimising production of the meat product are also described.

For the purposes of this specification, the term 'about' or 'approximately' and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % to a reference quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length.

For the purpose of this specification the term 'comprise' and grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements.

The term 'extracellular damage' or grammatical variations thereof refers to damage between cells caused by formation of ice crystals. The crystals associated with extracellular damage are typically greater than 0.1 mm wide or alternatively, greater than about 50% of a cell size. These crystals cause damage (termed 'cryo-damage') as they grow by causing tissue material to deform, causing cell walls to break or lyse, and/or causing cells to deform or loose their integrity. When such damage is observed under a microscope, noticeable changes may include one or more of the following: voids between cells, lysed or broken cell walls, crumpled cell shapes and reduced water content within the cells. At a macro level, extracellular damage is associated with a tougher texture, a darker colour and potential flavour changes associated with oxidation. Extracellular damage is common in traditional frozen and then later thawed meat products leading to the customer perceptions and studies that chilled meat is preferable over frozen meat. A further characteristic of extracellular damage is a greatly increased purge and drip loss from the meat - this is observed by a dry feeling when the meat is eaten versus a juicy texture meat product with no extracellular damage. The combined purge and drip loss from a frozen meat may be double or even triple that of chilled meat never frozen. In one embodiment, the histology of a piece of meat with extracellular damage may look as shown in Figure 1 obtained from 'Histological Measurements of Ice in Frozen Beef (J. Fd Technol. (1979) 14, 237 - 251 ; A. Bevilacqua, N. E. Zaritzky, and A. Calvelo).

The terms 'minimising' and 'reducing' or grammatical variations thereof may be used interchangeably and, in the context of extracellular damage, refer to reducing the presence of extracellular damage by a substantial level relative to a piece of meat that has been frozen at a temperature of -18°C over a period of 48 hours and then later thawed.

The term 'substantial' or grammatical variations thereof refers to at least about 50%, for example 60%, or 70%, or 80%, or 90%.

The term 'meat' may refer to the carcass or animal flesh that is eaten as food mainly composed of water and protein, muscle, associated fat, bone and other tissues. This term should not be seen as limiting as amongst mammalian species (beef, lamb, venison, pigs etc.) raised and prepared for human consumption also includes poultry, and other animals or produce.

The term 'meat piece(s)' refers to a cut of meat such as a primal cut, sub-primal cut, finished cut or finished portion.

By way of clarification, the primal cut may be pieces of meat initially separated from a carcass. When the primal cut is divided into smaller sections usually by separating out the major portions, a sub-primal cut results, examples of sub-primal cuts of meat are the top round, whole tenderloin and rib eye. 'Finish cuts' or finished portions of meat refers to the meat a consumer may receive after excess fat and bone are removed.

In relation to poultry and the like, 'meat piece(s)' for example, may refer to halves (the bird is split from front to back through the backbone and keel to produce two halves of approximately equal weight), breast quarters (halves may be further cut into which include the wing), split breast (breast quarter with the wing removed), split breast without back (a breasts with wing and back portion removed), boneless skinless breast (split breast that has been skinned and deboned), 8- piece cut (whole bird is cut into two breast halves with ribs and back portion, two wings, two thighs with back portion and two drumsticks), whole chicken wing (all white meat portion composed of three sections; the drumette, mid-section and tip), wing drummettes (the first section between the shoulder and the elbow), wing mid section with tip (the flat centre section and the flipper (wing tip)), wing mid section (the section between the elbow and the tip), whole chicken leg (the drumstick-thigh combination), boneless skinless leg (whole chicken leg with skin and bone removed), thigh (the portion of the leg above the knee joint), boneless skinless thigh (thigh with skin and bone removed), drumsticks (the lower portion of the leg quarter), and giblets (includes heart, liver and neck).

For the purposes of the specification, the terms 'fast freezing' or 'very fast freezing' or grammatical variations thereof refers to the process whereby the meat is frozen sufficiently fast to minimise or avoid the formation of substantial amounts of extra cellular ice crystals.

The term 'slow freezing' or grammatical variations therefore refers to the process whereby meat is frozen at a rate sufficiently slow that substantial amounts of extra-cellular ice crystals form. The term 'thaw' or grammatical variations thereof refer to a process in which ice crystals within a piece of meat are allowed to melt. Thawing may be complete when ice crystals in the meat have substantially melted.

The term 'aging' or grammatical variations thereof refers to the process of preparing meat for the improved meat quality such as tenderness, juiciness and/or flavour for consumption, mainly by naturally occurring endogenous enzymes breaking down the cytoskeletal myofibrillar proteins and/or connective tissue.

The term 'meat quality attribute' or grammatical variations thereof refers to one or more distinguishing characteristics of the meat such as tenderness, water-holding capacity and colour stability.

The term 'transportation' or grammatical variations thereof refers to any means or system of transporting the meat at any stage throughout the meat process from one place to another. For example, the transportation may include but not be seen as limited to freight within a container transported by air, land or sea during some steps in the process, during parts of some steps in the process or during all steps in the process.

In a first aspect there is provided a method of extending the shelf life of meat and optimising meat quality by the steps of:

(a) obtaining a piece or pieces of meat;

(b) fast freezing the meat;

(c) storing the meat in a frozen form for a period of time;

(d) thawing the frozen meat; and

(e) aging the thawed meat.

The inventors have unexpectedly found that the combination and order of steps of fast freezing then thawing and then aging may result in final meat characteristics comparable to or at least closer in nature to chilled meat. Characteristics include similar visual look e.g. colour; and similar taste and mouth feel e.g. tenderness by avoidance of oxidation compounds and avoidance in loss of moisture. This combination is not predictable from the art and teaches away from current practice for example, aging does not occur post freezing. A further unexpected effect identified by the inventors is the potentially superior shear force or tenderness resulting from the above method. Fast freezing, then thawing and then aging may result in lower shear force values compared to aged only meat (never frozen). Without being bound by theory, this observation may be linked with the histology analysis as discussed further below where fast freezing may allow the formation of intracellular crystals between degraded myofibrillar proteins, which may induce some additional muscle fibre fragmentation during thawing resulting in increase in meat tenderness.

Also, muscle proteases, such as, μ-calpain, m-calpain and calpastatin activities may be found to be stable in frozen meat and these proteases may make the meat tender during the thaw-aging period. Moreover, the formation of small intracellular ice crystals as shown in Figure 2c may have increased the rate of aging probably by the release of protease enzymes.

The inventors have also unexpectedly found that, post fast freeze/thaw, the endogenous proteolytic enzymes remain active allowing aging post freezing by degradation of cytoskeletal myofibrillar proteins through continuous proteolytic enzyme activity.

The meat obtained for the method may be post rigor. The onset of rigor mortis and its resolution may partially determine the tenderness of the meat as muscle continues to contract and relax after death. Without oxygen, lactic acid may build up and the muscle filaments may permanently lock together. Also, if the meat post mortem is immediately chilled a phenomenon known as cold shortening may occur where the muscle sarcomeres may shrink to a third of their original length. Cold shortening is caused by the release of stored calcium ions from the sarcoplasmic reticulum of muscle fibres in response to the cold stimulus. The calcium ions trigger powerful muscle contraction aided by ATP molecules. To prevent cold shortening, a process known as electrical stimulation may be carried out, especially in beef carcasses, immediately after slaughter and skinning. In this process, the carcass may be stimulated with alternating current, causing it to contract and relax, which may deplete the ATP reserve from the carcass and prevent cold shortening.

The meat piece or pieces may be finished cuts. In this way, the meat product may be readily packaged and on sold in a convenient form to a consumer.

The meat may be packaged prior to step (b) or step (c). The packaging may act to limit exposure of the meat to oxygen and the environment. The packaging may be selected from: vacuum packaging, heat shrink packaging, plastic wrap, modified atmosphere packaging, anti-microbial packaging, and combinations thereof.

The meat piece or pieces may be fast frozen in step (b) within 6-72 hours post slaughter. The rate of freezing in step (b) may be sufficiently fast to prevent and/or minimise the formation of extracellular ice crystals.

The rate of freezing appears to play a fundamental role in determining size, location and shape of ice crystals within the muscle fibre. In fact, the above process may cause intracellular damage which appears to produce an unexpected effect and contrary to the norm where any damage to the cellular structure of the meat would be considered undesirable. Fast freezing may allow the formation of intracellular fine crystals between degraded myofibrillar proteins, which may induce some additional muscle fibre fragmentation during thawing resulting in an increase in meat tenderness. Therefore, an advantage of fast freezing compared to slow freezing is that slow freezing causes the formation of extracellular large crystals resulting in significant damage to tissue, thus causing a decrease in myofibrillar protein solubility and water-holding capacity and an increase in weight loss on thawing.

As above, different freezing methods substantially influence the freezing rate and hence the quality attributes of the meat. Examples of fast freezing methods may include any one of the selected methods and/or combinations thereof: a cryogenic gas; a cooling fluid/powder; a low freezing point fluid (non-limiting examples including glycols, alcohols, esters, oils); water with a freezing point depression agent (e.g. calcium chloride, sodium chloride, sulphates or other ionic salt, a glycol, a sugar, organic acids; slurry (e.g. water-ice, fluidised beds-beads); aerated fluid or deluge; and/or sublimating powder.

The rate of freezing may be sufficiently fast that no or substantially no single extracellular ice crystal and/or combination of extracellular ice crystals may be larger than 30, or 35, or 40, or 45, or 50, or 55, or 60% of the size of a cell within the meat. In one embodiment, extracellular ice crystals may be larger than approximately 50% of the size of a cell within the meat. Alternatively, no single and/or combination of extracellular ice crystal(s) may be larger than approximately 0.05, or 0.06, or 0.07, or 0.08, or 0.09, or O.10, or 0.1 1 , or 0.12, or 0.13, or O.14, or 0.15 mm. In one embodiment, no single and/or combination of extracellular ice crystal(s) may be larger than approximately 0.1 mm. As noted above, an advantage of reducing the size and formation of extracellular large crystals is to minimise damage to tissue, and hence improve meat quality attributes.

It has been found that a histology study of pieces of meat frozen under controlled conditions show that the different ice morphology i.e. sizes of the crystals formed are one of the factors responsible for the changes in meat quality attributes. The histology cross-section of the finished meat product post step (b) may have intracellular ice crystals that may be of a fine and small size in the outer 1 -15mm (excluding fat cover) of depth of the meat cut graduating to a relatively coarser and larger size deeper into the meat cut.

In one embodiment, fast freezing may be defined by the internal meat temperature in step (b) being less than or equal to approximately -5°C in less than approximately 3, or 2.5, or 2 hours of the cooling process commencing. In one embodiment, very fast freezing may be completed where the temperature is less than or equal to approximately -5°C in less than approximately 1.5, or 1.25, or 1 hour. In one embodiment, very fast freezing may reduce the temperature to less than or equal to -5°C within approximately 30 minutes. Note that reference to temperature above in respect of fast freezing refers to a core meat temperature,

Alternatively fast freezing may be defined by the meat temperature being approximately - 8°C within at least approximately 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12 hours of cooling commencing from a starting temperature of approximately 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10°C. Very fast freezing may in an alternative be defined by the meat temperature being approximately -18°C within approximately 1.5, or 2, or 2.5 hours of cooling commencing from a starting temperature of approximately 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10°C. Note that reference to temperature in respect of fast freezing or very fast freezing generally refers to a core meat temperature. This temperature rate of reduction compares with slow freezing which, from a similar starting temperature, may take more than 35 hours to reach a temperature of - 15°C. It should be appreciated that similar effects may also be achieved where the temperature noted above relates to a temperature away from the core such as the scenario of a fast or very fast frozen outer layer. It is understood that this rate is associated with the formation of extracellular crystals resulting in cryo-damage to the tissue. Further, fast or very fast freezing reduces the total meat processing/handling time for a processing plant, thus reducing time and energy costs compared to conventional slow freezing. Note that for prolixity, the term 'fast freezing' in the description herein also encompasses very fast freezing unless otherwise noted. Storage time may be an important aspect of the method as well, an objective being to store the meat in a frozen form as long as possible in order to supply high quality meat products to markets worldwide throughout the full year without compromising important meat quality attributes. In one embodiment, storing in step (c) may be completed at a temperature sufficient to minimise ice crystal growth during the storage time period. The inventors have found that storing in step (c) may be completed at a temperature of less than or equal to approximately - 10, or -11 , or -12, or -13, or -14, or -15, or -16, or -17, or -18, or -19, or -20, or -21 , or -22, or - 23, or -24, or -25°C. Storage may be for up to approximately 6, or 12, or 18, or 24, or 30, or 36, or 42, or 48 months.

Thawing rates in the method above may also affect the meat quality attributes. Thawing of the fast frozen meat in step (d) may be completed until any ice crystals in the meat have substantially melted. Also, the rate of thawing in step (d) may be sufficient to avoid crystal growth during thawing and avoid agglomeration of ice crystals in order to minimise any extracellular damage to the meat. Furthermore, the thaw rate may be sufficient to avoid and/or minimise microbial growth. The inventors have found that temperatures over approximately 7°C may affect the microbiological shelf-life of the meat. In one embodiment, the meat may be allowed to thaw by holding the meat piece or pieces at a temperature of between approximately -2 and 7°C until all ice crystals have melted. In selected embodiments, the temperature for thawing may be approximately -1.5, or -1 , or -0.5, or 0, or 0.5, or 1°C, these temperatures being approximately equivalent to current industry best practice to optimise aging and food safety. Upon thawing, the meat may be aged for a certain period of time (depending upon type of muscle cuts/species (i.e. beef, lamb, chicken or venison) and at a chilling temperature to enhance meat quality attributes. For example, tenderness and water-holding capacity may be substantially improved via aging through naturally occurring endogenous proteolytic enzymes.

The aging in step (e) may be completed under controlled conditions for a time period sufficient to cause the desired level of degradation of cytoskeletal myofibrillar proteins, minimise microbial growth and reach a desired meat colour.

As noted above, aging may occur at a temperature of approximately -2°C to 7°C. It should be appreciated that the temperature conditions and/or other conditions such as water activity may be altered to minimise microbial growth and achieve desired aging.

The time period of aging may range from approximately 1 , or 5, or 10, or 15, or 20, or 30, or 40, or 50, or 60, or 70, or 80, or 90 days. In one embodiment, aging may occur at a temperature of approximately -1.5, or -1 , or -0.5, or 0, or 0.5, or 1 °C for at least 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21 days under ambient pressure conditions. Aging may alternatively occur at a temperature of approximately 2, or 2.5, or 3, or 3.5, or 4°C for at least 5, or 6, or 7, or 8, or 9, or 10 days under ambient pressure. The aim of the temperature and time relationship may be to optimise activation of the endogenous proteolytic enzymes yet maintain acceptable food safety standards.

Upon aging, the meat temperature may be reduced to a further chilled temperature to enhance the meat's retail storage and display case shelf life. For example, microbial growth and colour degradation may be substantially minimised by completing this temperature reduction.

In alternative embodiments, partial aging may occur after step (a) and before step (b). The partial aging may be completed at a temperature of between approximately 3°C to 7°C for a time period sufficient to cause the desired level of degeneration of cytoskeletal myofibrillar proteins and to minimise microbial growth. The inventors have found that accelerated aging/chilling conditions such as 3°C to 7°C for 8 days of the meat prior to freezing may result in equivalent meat attributes such as meat tenderness and water-holding capacity compared to typical aged/frozen meat without risking microbiological shelf-life of the frozen/thawed meat.

It is known that meat quality attributes, (mainly tenderness and water-holding capacity) may be substantially improved during aging by altering meat ultrastructure and degradation of the protein cytoskeletal proteins. Accelerated chilling however, during aging, may induce more degradation of cytoskeletal myofibrillar proteins through the increased proteolytic enzyme activity and hence may be advantageous.

The meat produced may be similar to chilled and aged meat that has never been frozen. The meat produced may have similar characteristics including look or colour (no or minimal oxidation or browning changes); flavour (e.g. avoid rancid lipid oxidation flavours); and mouth feel (for example as measured via moisture retention in the meat).

The look, as measured via colour, of meat processed according to the above methods may be the same or within approximately 5, or 10, or 15, or 20% of a measured hue angle, as chilled and aged (never frozen) meat.

The flavour may be similar to chilled and aged meat as measured for example by lipid oxidation. Unexpectedly, the inventors found that the lipid oxidation levels may be the same or less than chilled and aged (never frozen) meat. In experiments completed by the inventors, the lipid oxidation levels may be at least 10, or 1 1 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21 , or 22, or 23, or 24, or 25% lower than that measured for chilled and aged meat after 7 days of aging at -1.5°C or 1.0°C.

The purge loss of meat processed via the above methods may be at a level close to chilled and aged meat or at least close enough to still provide a similar taste and mouth feel to chilled and aged meat. In experiments completed by the inventors, the purge loss may be approximately 0.2, or 0.3, or 0.4, or 0.5, or 0.6, or 0.7, or 0.8, or 0.9, or 1 , or 1.1 , or 1.2, or 1.3, or 1.4, or 1.5, or 1.6, or 1.7, or 1.8, or 1.9, or 2, or 2.1 , or 2.2, or 2.3 or 2.4, or 2.5 times that of purge loss from chilled and aged meat. By contrast, the purge loss from slow frozen meat may be 3.5 to 4 times that of chilled and aged meat.

An advantage of the above method is that it is versatile as it can be utilised on a wide range of meat species. The meat may be derived from livestock of genus selected from: Bovine, Ovine, Porcine, Cervine, Caprine, Equine, Dromadine. The meat may be derived from poultry including chickens, duck, emu, ostrich and turkey.

One or more steps in the above method may be completed during transportation of the meat piece or pieces. In this way, the entire method, steps, or part of steps may occur during transportation or storage such that the meat is ready for distribution and/or consumption on delivery or even post purchase by the consumer, thus increasing versatility of the method and end product.

One or more steps in the above method may be initiated by a remote control system that may send a signal initiating, adjusting and/or terminating one or more of the above steps. For example, a remote signal may be sent to a transportation source (containing the meat in a controlled environment) such as within a container of ship's cargo hold such that all or part of the method may be initiated prior to the meats point of destination such as an overseas processing plant. Again, an advantage being the meat may be ready for distribution and/or consumption on its point of delivery, thus reducing manual processing time and inventory costs, and hence decreased costs to the manufacturer which may be passed on to the consumer.

In a second aspect, there is provided a meat piece that has been frozen, thawed and aged and which exhibits a histology showing intracellular cryo-damage and irregularities in muscle fibre distribution as compared to meat that has (a) only been chilled and aged or (b) meat that has been slow frozen and thawed.

As noted above, it has been found that a histology study of pieces of meat frozen under controlled conditions show that the different ice morphology i.e. sizes of the crystals formed, are one of the factors responsible for the changes in meat quality attributes (as shown in the Figures and described later in the specification). A histology cross-section of the finished meat product may have intracellular ice crystals that may be of a fine and small size in the outer 1 -15mm (excluding fat cover) of depth of the meat cut graduating to a relatively coarser and larger size deeper into the meat cut. In relation to extracellular cellular ice crystal formation, the meat piece above may have has less than or equal to approximately 20, or 25, or 30, or 35, or 40% extracellular cell damage. In one example, the meat piece may have has less than or equal to 30% extracellular cell damage

In a third aspect, there is provided a meat piece that has been frozen, thawed and aged and which exhibits one or more varied metabolite concentrations compared to meat that has (a) only been chilled and aged and (b) meat that has been slow frozen and thawed.

As noted, metabolite concentrations may vary between meat that undergoes the above method and meat that is either never frozen or meat that is slow frozen. Some examples of metabolite variations identified by the inventors that vary as measured in purge loss from meat tested include:

• An increase in malate concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

• An increase in inosine monophosphate concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

· An increase in inosine and alanine concentration in very fast frozen meat compared to chilled, slow frozen or fast frozen meat;

• An increase in carnitine concentration in fast or very fast frozen meat compared to

chilled or slow frozen meat;

• A decrease in carnosine concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

• An increase in acetate concentration in very fast frozen meat compared to chilled, slow frozen or fast frozen meat;

• An increase in lactate concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

· An increase in pyruvate concentration in fast or very fast frozen meat compared to

chilled or slow frozen meat.

As may be appreciated the above metabolites may provide a useful finger print of meat processed via the above methods. These metabolites are not, at least at the concentrations present, factors that influence the meat quality attributes in a way that makes the meat look or taste different to chilled (never frozen) meat.

The meat piece quality attributes, such as tenderness, water-holding capacity, and colour stability may be assessed by measuring purge loss, drip loss, shear force, pH, aerobic plate count, surface meat colour. These parameters may be approximately similar to chilled meat or at least closer than is the case for frozen only meat (fast or slow).

It should also be appreciated that other measures such as intracellular fat distribution, selenium quantities along with indole profiling may be used to characterise the meat source and method of processing the of meat.

The meat piece may have a shear force of less than or equal to approximately 3, or 3.5, or 4, or 4.5, or 5 kgF. A reduced shear force may be highly beneficial as a comparatively low value may be an indicator of more tender meat. Surprisingly, fast freezing, then thawing, and then aging may result in lower shear force values compared to aged only meat (never frozen) hence more tender meat. Without being bound by theory, this observation may be linked with the histology analysis as discussed further below that fast freezing may allow the formation of intracellular crystals between degraded myofibrillar proteins, which may induce some additional muscle fibre fragmentation during thawing resulting in increase in meat tenderness. Also, muscle proteases, such as, μ-calpain, m-calpain and calpastatin activities may be found to be stable in meat frozen via fast freezing unlike slow freezing, and these proteases and may be those that make meat tender during the thaw-aging period. Moreover, the formation of small intracellular ice crystals as shown in Figure 2(c) may increase the rate of aging possible by the release of protease enzymes.

The combined purge loss and drip loss from the meat piece may be no more than 40, or 45, or 50, or 55, or 60% different to that observed in chilled meat aged for an equivalent time period. In this way, fast freezing may minimise the water loss of the thawed meat by reducing physical cell damage and chemical changes of proteins through small ice crystal formation. As a comparison, experiments completed by the inventors showed that fast frozen only meat had a combined purge and drip loss twice that of chilled meat while slow frozen meat had almost three times the combined purge/drip loss of chilled meat. Avoiding loss of liquid may be desirable for taste and texture.

As should also be appreciated, a combination of characteristics may be used to characterise a piece of meat as being processed via the above methods or not. For example, techniques including measured purge loss, metabolite concentrations and profiles as well as observing the meat histology may be used to collectively arrive at a conclusion. Reference to any one method of testing noted above should not be seen to limit the use of multiple methods.

The meat piece may be stored up to 12, or 18, or 24, or 30, or 36, or 42, or 48 months old post slaughter. The ability to store the meat in a frozen form as long as possible is a significant advantage as the meat may be supplied throughout the year without compromising important meat quality attributes. Storage may be completed at a temperature to minimise ice crystal growth during the storage time period.

The meat piece may be a finished cut. In this way, the meat product may be readily packaged and on sold in a convenient form to a consumer.

In a fourth aspect there is provided a control system for optimising the quality of a meat piece or pieces including:

a controlled environment into which a meat piece or pieces are placed;

an adjustable unit operatively connected to the controlled environment;

a sensor or sensors in the controlled environment; and a remotely situated control apparatus that receives a signal or signals from the sensor or sensors and which on command sends a signal to the adjustable unit to adjust the conditions in the controlled environment;

wherein the remotely situated control apparatus is used to initiate, adjust and/or terminate one or more of the steps of:

(a) fast freezing of the meat in the environment;

(b) storing of the meat in a frozen form for a period of time in the environment;

(c) thawing the frozen meat in the environment; and

(d) aging the thawed meat in the environment.

The controlled environment may be transportable. For example, the controlled environment may be a shipping container or containers. The controlled environment may alternatively be a controlled insulated and mobile container such as a chilly bin or mobile refrigerator. In this way, the entire meat quality control process or part steps thereof can occur during transportation or storage such that the meat is ready for distribution and/or consumption on delivery, thus reducing processing time and costs to the manufacturer and hence saving costs to the consumer.

The controlled environment may be temperature controlled. For example, the shipping container may be refrigerated and operable by an adjustable unit such as a cooling device that may adjust the temperature in the controlled environment.

In order to regulate the controlled environment, the control system may include a sensor or sensors to monitor parameters selected from: temperature, pressure, fluid flow, weight, meat crystal size, and combinations thereof.

The controlled environment may be different for one or more steps (a) to (d). For example, in one embodiment, execution of the steps of the control system may occur in a country of origin where the meat is obtained prior to shipping. Other embodiments may include execution of the steps or part thereof during transportation such as within a container of a ship's cargo and/or at the meats point of destination such as an overseas processing plant.

The following exemplifies, but should not be seen as limiting on variations where execution of the steps or part steps thereof in the control system may occur:

I. Fast freezing of the meat in the environment, storing of the meat in a frozen form for a period of time in the environment, thawing the frozen meat in the environment (to replicate chilled meat qualities) at country of origin, shipping and aging the thawed meat in the environment enroute to overseas destination; or

II. Fast freezing of the meat in the environment at country of origin, storing of the meat in a frozen form for a period of time in the environment while shipping to overseas destination, thawing the frozen meat in the environment (having several weeks of shelf life remaining), and aging the thawed meat in the environment at overseas destination; or

III. Fast freezing of the meat in the environment at country of origin, storing of the meat in a frozen form for a period of time in the environment while shipping to overseas destination and, signalling the control system, e.g. a week from the overseas destination to execute thawing and aging steps in the environment. An advantage of part of the steps being signalled to occur enroute is that the meat product arrives at port in optimum condition, still has several weeks of shelf life and there is no increase inventory. If for example, sales in a particular market are slow then the thaw/age process can be delayed; or

IV. Fast freezing of the meat in the environment, storing of the meat in a frozen form for an extended period of time in the environment at country of origin, shipping, thawing the frozen meat in the environment (may be controlled by insulated packaging)/aging the thawed meat enroute to overseas destination. An advantage of utilising insulated packaging that may control thaw rate enroute is that the meat industry may be able to supply "as chilled" meat to lucrative markets such as Asia where the cool chain (never frozen, and distinct from the cold (frozen) chain), is limited in supply.

There are a number of advantages associated with this invention that may include:

• Utilising a combination of fast freezing then thaw-aging which may result in an improved water-holding capacity and tenderness of meat product (even relative to aged only

(never frozen) chilled meat) by minimising extracellular ice crystal formation and allowing continued proteolytic tenderisation process even after thawing of the frozen meat;

• The meat may be packaged prior to fast freezing or thawing which may limit exposure of the meat to oxygen and the environment while also providing easier handling of the product throughout the process;

• Fast freezing may allow the formation of intracellular fine crystals between degraded myofibrillar proteins, which may induce some additional muscle fibre fragmentation during thawing resulting in an increase in meat tenderness;

• Fast freezing may reduce the size and formation of extracellular large crystals thereby minimising damage to tissue, and hence improve meat quality attributes;

• Fast freezing may reduce the total meat processing/handling time for a processing plant, thus reducing time and energy costs compared to conventional slow freezing;

• The method may minimise ice crystal growth during the storage time period, thus may allow the storage of meat in a frozen form as long as possible thereby allowing supply of high quality meat products to the premium end of the market throughout the whole year without compromising important meat quality attributes;

• An advantage of aging post fast freeze/thaw suggests the endogenous proteolytic

enzymes may remain active allowing further degradation of cytoskeletal myofibrillar proteins through continuous proteolytic enzyme activity, and hence more tender meat; · The above method is versatile as it may be utilised on a wide range of meat species and the meat piece or pieces may be finished cuts. In this way, the meat product may be readily packaged and on sold in a convenient form to a consumer;

• The entire method or part steps thereof may occur during transportation or storage such that the meat is ready for distribution and/or consumption on delivery, thus reducing processing time and costs to the manufacturer and hence saving costs to the consumer; and,

• The one or more steps in the above method may be initiated by a remote control system that may send a signal initiating, adjusting and/or terminating one or more of the above steps. For example, a remote signal may be sent to a transportation source (containing the meat in a controlled environment) such as within a container of ship's cargo hold such that all or part of the method may be initiated prior to the meats point of destination such as an overseas processing plant. Again, an advantage being the meat may be ready for distribution and/or consumption on its point of delivery, thus reducing manual processing time, and hence decreased costs to the manufacturer which may be passed on to the consumer.

The embodiments described above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the embodiments relates, such known equivalents are deemed to be incorporated herein as of individually set forth,

Where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

EXAMPLE 1

The above described methods of processing meat and meat products are now described by reference to a specific example.

I. MATERIALS AND METHODS

Raw materials and processing

At 24 hours post mortem, both loins (M. longissimus dorsi) from 20 lamb carcasses were obtained, vacuum packaged, placed on ice and transported to a muscle biochemistry laboratory. The loins were weighed and randomly distributed to one of five different freezing/aging conditions:

1 ) fast-frozen only (FF);

2) slow-frozen only (SF);

3) fast-frozen then thaw-aged for 2 weeks (FFA2);

4) slow-frozen then thaw-aged for 2 weeks (SFA2); and 5) aged for 2 weeks (never frozen at -1.5°C; A2).

The loins assigned to fast freezing (FF and FFA2) were placed in an immersion calcium chloride tank at -19°C 1 day post mortem for 24 hours, and then stored for an additional 1 week at -18°C prior to thawing at 3°C overnight. The loins assigned to slow freezing (SF and SFA2) were placed in an -18°C freezer for 1 week then thawed in the same manner. The freezing rate for both fast and slow conditions was monitored at 1 minute intervals. Aged only (A2) and frozen/thawed/aged (FFA2 and SFA2) treatments were accomplished by placing the loins at - 1.5°C for 2 weeks. At the end of the assigned processing periods for each treatment, loins were removed from vacuum packs and then sub-divided to separate cuts for shear force, histology and drip loss.

Histological analysis

The loin samples from each treatment were cut transversally to the muscle fibre at the end of each assigned storage period. The cuts were further sliced on a Cryo-Cut at -20°C (10pm and 20 pm thick). The slices were stained with haematoxylin-eosin and then moulded in Canada balsam. Viewing and photographing were carried out using a Leitz microscope (X200).

Purge and drip loss

Purge loss during the vacuum storage was measured at the end of the assigned

process/storage time by weighing the initial and final weight of the loins under vacuum- packaging. Drip loss was measured after 48 hrs (3±1 °C) according to established methods in the industry. The percentage difference between initial weight and final weight was calculated for the drip loss.

Shear force

The loin cuts were cooked in a water bath set at 99°C to an internal temperature of 75°C (measured by 12 channel Digisense Thermocouple Thermometer). After cooling, 10 mm x 10 mm cross section samples were cut and sheared using a MIRINZ Tenderometer. Ten replicates were measured for each sample. The results were expressed as shear force (kgF).

Data analysis

All statistical analysis was done using the REML directive of GenStat. Least squares means for each attribute were separated (F test, P < 0.05) by using least significant differences. II. RESULTS AND DISCUSSION

Temperature decline and pH

Different freezing methods substantially influenced the freezing rate of the loins (data not shown). Fast freezing resulted in taking less than 6 hours to reach an internal temperature of - 18°C for the meat, whereas the slow freezing took more than 35 hours to get below -15°C. Different freezing methods did not affect pH of the loins (P > 0.05), but the freezing/thaw-aging treatments did influence pH of the loins after the storage (P < 0.05). Histological analysis

Figure 2 shows transverse section of muscles at different freezing (-18°C)/aging (-1.5°C) under conditions A: (a), FF: Fast-frozen only, (b), SF: slow-frozen only, (c), FFA2: fast-frozen, thaw- aged for 2 weeks, (d), SFA2: slow-frozen then thaw-aged for 2 weeks, (e), A2: aged for 2 weeks (never frozen at -1.5°C).

Transverse section of aged only (A2) loins showed more of regular distribution of fibres (Figure 2e), whereas there was an irregular distribution of fibres and a different pattern of cryo-damage in muscle in case of fast and slow frozen loins (Figure 2a, b, c, d). There was clear intracellular and extracellular cryo-damage within the muscle fibres for fast-frozen loins (FF (Figure 2a) and FFA2 (Figure 2c)) whereas typically extracellular cryo-damage was observed between muscle fibres for the slow-frozen loins (SF and SFA2 (Figure 2b and 2d). The observed cryo-damage either within muscle fibres or between muscle fibres is likely due to the different pattern of ice crystal formation. The intracellular cryo-damage formation could be due to the dehydration of the muscle fibres by the growth of extracellular ice crystals which was not enough to compensate for the rapid cooling rate, and consequently, the supercooling in the solution inside the cells causing ice nucleation. This observation confirms that the freezing rate has a fundamental impact on the nature and extent of crystallization within muscle tissue.

Table 1 below illustrates the effect of different freezing and aging methods on various meat quality attributes. Table 1 - A FF: Fast-frozen only, SF: slow-frozen only, FFA2: fast-frozen, thaw-aged for 2 weeks, SFA2: slow-frozen then thaw-aged for 2 weeks, A2: aged for 2 weeks (never frozen at -1.5°C).

3 Purge loss (%), °Drip loss (%), D Shear force (kgF), E Standard errors of difference. a bc Treatments with different superscript letters are different (P < 0.05). Trait FF SF FFA2 SFA2 A2 SED E pH 5.67 b 5.65 b 5.6 ab 5.62 a 5.70 c 0.01

PL B 4.81 " 6.91 b 6.69 ab 13.03° 4.28 a 0.89

DL C 6.14° 3.71 b 1.15 a 1.63 a 1.54 a 0.39

SF D 5.83 c 5.63 c 2.71 a 3.99 b 3.75 b 0.21

Water-holding capacity

The different freezing methods significantly affected purge and drip loss of the loin samples (Table 1). Generally, the fast freezing resulted in significantly lower purge and drip loss of the loins compared to the slow freezing regardless of further aging periods after thawing. It is particularly interesting to note that slow freezing then thaw-aged loins (SFA2) had the highest purge loss (almost 2 times greater) than other treatments. This observation could indicate that a combined influence of extracellular cryo-damage by slow freezing and further protein degradation for 2 weeks of aging would result in more increased water loss from the muscle tissue. Taken purge and drip loss together, the SFA2 loins had the highest purge/drip loss followed by FF, SF and FFA2, while A2 had the lowest water loss (P<0.05; Table 1). These results confirm that fast freezing can minimise the water loss of thawed meat possibly by reducing physical cell damage and chemical changes of proteins through small ice crystal formation. Furthermore, the histology results corroborates well as fast frozen loins had small ice crystals within the muscle fibre, whereas slow frozen samples had large ice crystal between the fibres resulting in the highest purge/drip loss for the SFA2 loins (Table 1).

Shear force

The frozen/thawed only loins (FF and SF; without further aging) observed the highest shear force values compared to other aged loins (P < 0.05), but the freezing rate did not affect the shear force values for these two treatments (P > 0.05). This could indicate the freezing rate did not influence meat tenderness of frozen/thawed meat when applied at the early post mortem period. However, and surprisingly fast freezing then thaw-aging (FFA2) resulted in the lowest shear force values among the treatments (Table 1 ; P < 0.05). Unexpectedly, the FFA2 loins had lower shear force values compared to aged only meat (never frozen; A2). Without being bound by theory, this observation could be linked with the histology analysis as discussed above that fast freezing allow the formation of intracellular crystals between degraded myofibrillar proteins, which may induce some additional muscle fibre fragmentation during thawing resulting in increase in meat tenderness. Also, muscle proteases, such as, μ-calpain, m-calpain and calpastatin activities are found to be stable in frozen meat and may be those may have made meat tender during the thaw-aging period. Moreover, the formation of small intracellular ice crystals as shown in Figure 2c may have increased the rate of aging probably by the release of protease enzymes. III. CONCLUSION

The freezing rate has substantial impacts on meat quality attributes particularly water-holding capacity and tenderness by minimizing extracellular cryo-damage of the frozen/thawed meat. More surprisingly, the inventors have found that the combination of fast freezing, then thawing, then aging can result in more improved tenderness and water-holding capacity of the frozen/thawed meat compared to the slow frozen counterpart and even the aged only (never frozen) meat.

EXAMPLE 2

Different freezing regimen tested

Whole lamb racks were chilled in-plant at the AFFCO Rangiuru abattoir to between~ 2-4°C and then transported in chilly bins to AgResearch, Ruakura campus. At 24 h post mortem, both loins ( longissimus dorsi) from each of 45 lamb carcasses were excised (90 loins in total). An extra 18 loins were also collected for temperature data recording. The loins were selected by an ultimate pH24h of 5.8 or below measured by inserting a calibrated Hanna HI99163 with a FC232D combined pH/temperature probe (HANNA instruments, Rl, USA) at two points along each loin. The selected loins were labelled and vacuum packed. The loins were randomly assigned to one of three different freezing conditions:

1. Slow freezing, (SF)

2. Fast Freezing, (FF)

3. Very Fast Freezing, (VFF)

The samples were, also randomly, assigned to one of two different thaw/ageing temperatures after the freezing period:

1. -1.5°C for 2 weeks

2. 1 °C for i week

The frozen samples were kept in a frozen state for a time period of 7 days before commencing thawing and aging steps.

Control (never frozen) and aged meat samples were also kept as a comparison to the above.

The samples above were used in subsequent experiments as described in later Examples below. Freezing Process

The samples assigned to Very Fast Freezing (VFF -40°C) and Fast Freezing (FF -18°C) were placed in a calcium chloride immersion chamber.

For the Fast Freezing (-18°C) regimen, an immersion tank was used. This consisted of a 250 litre insulated tank fitted with a titanium plate heat exchanger and PID controller. Calcium chloride was pumped through the cooling system and also used as the immersion fluid.

For the Very Fast Freezing (-40°C) regimen, the immersion tank was placed in an environmental chamber operating at -40°C at least six days before the trial started, to ensure the immersion fluid temperature had equilibrated to -40°C. Samples assigned to Slow Freezing (SF -18°C) were placed in an air freezer.

The core temperatures of the cuts were monitored to confirm freezing rate and final temperature using a Grant 1000 Series Squirrel Data Logger fitted with T-type thermocouples that had been calibrated in an ice reference. Recorded data was adjusted using the ice point check data for each thermocouple.

The speed of cooling is shown in Figure 3. Fast freezing reduced the temperature to -5°C within approximately 2 to 2.5 hours and -18°C within 5 hours. Very fast freezing reduced the temperature to -5°C within approximately 30 minutes and to -18°C within approximately 1.5 hours. EXAMPLE 3

In this example a lipid oxidation assessment was completed. Lipid oxidation influences a consumers perception of meat quality as lipid oxidation results in fat darkening and, can give a rancid taste to the meat. Lipid oxidation is therefore to be avoided.

Using samples produced from Example 2, the samples were aged for 7 days at -1.5°C and 1.0°C and the extent of lipid oxidation of the loins were determined as follows. Before sampling the finely cut, previously frozen samples, were crushed to a fine powder using Freezer/Mill 6970 EFM USA, 3 minutes run time 9 cycles per second.

Powdered frozen muscle tissues (5.0 g) were homogenised in 15 ml distilled water followed by centrifugation in an Eppendorf 581 OR centrifuge at 2000 rpm for 10 min at 4 °C. An aliquot of the homogenate (2 ml) was transferred to test tubes and 4 ml of trichloroacetic acid and thiobarbituric acid TCA/TBA and 100uL butylated hydroxyl anisole BHA was added.

The samples were incubated for 15 min in a water bath (80°C) and cooled for 10 min in ice cold water. After centrifuging the homogenate one more time at 2000 rpm for 10 min at 25°C, the supernatant was filtered through a Whatman filter paper #4. The absorbance of the supernatant was read at 531 nm using a 96-well plate. (Spectrophotometer Multiskan GO Thermo scientific Finland, skanit 3.2 Research Edition) The lipid oxidation was reported as thiobarbituric acid-reactive substances (TBARS) by expressing mg malonaldehyde per kg of meat.

The findings are shown in Figures 4 (for -1.5°C) and 5 (for 1°C).

These results show that at aging temperatures of -1.5°C and 1°C, lipid oxidation, as measured by TBARS assay, is lower for FF and VFF in comparison to SF regimen. Lipid oxidation influences meat shelf life and taste and therefore shows that the FF and VFF regimen are superior to SF for meat quality attributes relevant to the importer (supermarkets) and the consumer. Further, the lipid oxidation rates for FF and VFF were found to be lower than the control (never frozen meat samples).

EXAMPLE 4

Colour changes

Meat samples produced in Example 2 were maintained in a frozen form for 4 weeks in this trial as this was the minimum time to make the experiment relevant to the industry. Post freezing, aging was completed and hue angle measurements were taken before and after aging to determine whether any negative browning colour change had occurred.

Figures 6 and 7 show the colour trial findings. Trials completed showed very significant results. Hue angle is used as indication of discoloration (calculated as [(b*/a*)tan-1]) and a higher value is associated with greater discoloration. As shown in Figures 6 and 7, while FF and VFF regimen generate values similar (or even better) than the control never frozen meat, SF values are significantly higher than control. This higher level for SF samples will impact on shelf life and consumer acceptance of the meat, as darker, browner meat is considered undesirable.

EXAMPLE 5

Histological analysis

The loin samples from each treatment of Example 2 were cut transversally to the muscle fibre at the end of each assigned storage period. Samples were cut at approximately 100 x 100mm, fixed in 10% buffered formalin and embedded in paraffin. Cut sections (7pm) were stained with haematoxylin and eosin for visualisation of muscle structure.

Histological analysis of the samples submitted to the different freezing regimens showed that, when compared to aged only samples (control) FF and VFF samples show obvious cryodamage to the muscle fibres (intracellular damage), observed as holes within the fibres (indicated by right pointing arrow in Figure 8). On the other hand, while no obvious intracellular cryodamage was seen in any of the slow-frozen samples, a bigger gap between the fibres is observed in these samples (indicated by left pointing arrow in Figure 8), and is a consequence of extracellular (between fibres) damage. Extra cellular damage leads to decreased water retention capacity, hence less desirable meat quality, which was confirmed by the larger purge loss measurements from SF meats (see Example 6 below).

EXAMPLE 6

Purge loss

After the thaw-ageing period, loin samples from Example 2 were removed from the vacuum bags, dried on paper towels and weighed (final weight). The purge loss was calculated as weight lost expressed as a percentage of the original sample weight. The purge liquid was collected and frozen at -80°C for metabolomics analysis (see Example 7 below).

The purge loss results are shown in Figures 9 and 10.

Generally, the FF and VFF regimens resulted in lower purge and drip loss than SF samples, regardless of the aging temperature. In particular, in all aging temperatures, SF regimen shows a significant increase in purge loss when compared to control as well as to FF and VFF regimens. Further, the FF and VFF regimens resulted in purge loss levels similar to that of the control (never frozen meat samples). These results agree with the histological finding where cryodamage to the SF samples appear to be occurring between the fibres, leading to decreased water holding capacity of the meat when compared to FF and VFF. Reduced purge loss is desirable to maintain fresh (never frozen) meat quality characteristics and indeed, the trial results show similar findings between FF and VFF regimens versus control meaning the likely meat texture will be comparable to the end consumer.

EXAMPLE 7

Metabolite changes were measured to assess differences between meat processing methods. Sample preparation.

50 lamb loin purge samples collected in Example 5 were used in this study.

Purge samples were randomised and defrosted on ice. 600 pL of purge was transferred to Eppendorf tubes. The samples were centrifuged for 15 min at 13,500g and 4°C. 500 mL of the supernatant was transferred to centrifugal filters and were centrifuged for 2h at 13,500g and 4°C.

200 pL filtered purge was transferred to standard 5 mm NMR tubes. 340 pL 100 mM phosphate buffer (containing 10% D 2 0 and 90% H 2 0, pH 7.0) and 60 pL internal standard solution (containing 5 mM 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (DSS) as a quantitation standard and chemical shift reference and 100 mM imidazole as a pH indicator) was added. When the volume of the filtrate was less than 200 pL the total volume was adjusted to 600 pL by addition of MilliQ water. A quality control (QC) sample was also prepared by pooling a small amount of purge from selected samples and preparing according to the method above. NMR spectroscopy.

1 -dimensional (1D) 1 H-NMR spectra were acquired for each loin extract sample on a Bruker 700 MHz Ultrashield NMR spectrometer using water suppression to supress the large water peak. First, the noesyprl d pulse sequence was used with a recycle delay of 1.5 collecting 256 scans. A 1 D 1H-NMR spectrum was also recorded on the QC sample (consisting of a pool of selected samples) and this was used as a reference spectrum. 2-dimensional NMR spectra (1 H.1 H- TOCSY, 1 H.13C-HSQC) were recorded on the QC sample for metabolite identification.

The 1 D NMR spectra was insufficient to obtain clear results as protein removal was insufficient from the samples causing broad resonances originating from proteins in the NMR spectra of some samples.

Therefore, the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used instead of the noesyprld pulse sequence. The CPMG pulse sequence attenuates signals from

macromolecules such as proteins. This 1 D pulse sequence was used for all the samples to obtain spectra without broad protein resonances.

Spectral processing.

The 1 D 1 H-NMR spectra were processed using the MestreNova 8.1 software: phasing and baseline correction was performed. The spectra were referenced to the alanine doublet at 1.466 ppm. Regions in the spectrum containing peaks from DSS, residual water and imidazole were deleted and the spectra were divided into 0.04 ppm wide 'bins'. Bins containing resonances from pH sensitive compounds (i.e. carnosine) were merged. The peaks in each bin were integrated to find the peak intensities and the sum of all peak intensities in each spectrum was set to 100 (normalisation) to account for the varying amounts of purge in the samples and also potential concentration differences between samples. The resulting intensity matrix (161 bins x 50 samples) was exported to Excel where information about treatment group was added.

Metabolite identification.

Tentative identification of metabolites in the 1 D NMR spectra of the QC sample was carried out using the Chenomx NMR Suite Professional software with an in-built 1 D spectral database. These tentative identifications were then confirmed (or rejected) by comparing the 2D NMR spectra of the QC sample with 2D spectra of individual metabolites from the Human Metabolome Database (http://www.hmdb.ca/). Data analysis.

The data was analysed using the web-based metabolomics software MetaboAnalyst 2.0 (http://www.metaboanalyst.ca/MetaboAnalyst/faces/Home.jsp) using both univariate and multivariate statistical methods. The intensity matrix was uploaded to MetaboAnalyst and the data was filtered, log-transformed and Pareto-scaled before analysis.

Principal Component Analysis (PCA) is an unsupervised multivariate data analysis method which is used to reduce dimensionality and explain variance in large datasets. The outputs from PCA are two plots: the scores plot, showing how similar or different spectra are from each other; and the loadings plot, which shows why the spectra of different treatment groups are different from each other (i.e. which regions of the NMR spectra that make up the difference). PCA is normally used as a first step in the data analysis to get an overview of the dataset.

When a difference between treatment groups could be found using PCA, a supervised multivariate data analysis method called Partial Least Squares-Discriminant Analysis (PLS-DA) was used to further investigate the metabolites responsible for differences between groups. PLS-DA is a supervised method, which means that information about class membership (e.g. freeze thaw and age (FTA) regime) is supplied and a model is then built trying to maximise the differences between the groups. PLS-DA works best for two-group comparisons. Caution needs to be taken when using supervised methods as they are prone to over-fitting, i.e. finding differences between groups when there is in fact no difference. Given this, PLS-DA results were only used in a qualitative way.

T-tests were used to find the regions ('bins') of the NMR spectra that differed significantly between two treatment groups at a time (p=0.05). The large number of variables present in metabolomics data greatly increases the risk of Type I errors (false positives), and therefore False Discovery Rate correction was used with the t-test.

The NMR spectral regions found to significantly differ between two groups using the t-test were examined in more detail and the majority of them could be directly assigned to specific metabolites.

Results

As shown in Figure 11 and Figure 12, levels of the metabolite malate are significantly elevated in comparison to control in the purge liquid from samples submitted to FF and VFF regimen following metabolomics analysis. No differences are seen between SF and control. This can be used as a "marker" to differentiate meat samples submitted to FF or VFF from SF regimen. These results are from samples aged at -1.5°C.

The following further metabolite results were identified:

• An increase in inosine monophosphate concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

• An increase in inosine and alanine concentration in very fast frozen meat compared to chilled, slow frozen or fast frozen meat; • An increase in carnitine concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

• A decrease in carnosine concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

• An increase in acetate concentration in very fast frozen meat compared to chilled, slow frozen or fast frozen meat;

• An increase in lactate concentration in fast or very fast frozen meat compared to chilled or slow frozen meat;

• An increase in pyruvate concentration in fast or very fast frozen meat compared to chilled or slow frozen meat.

The above findings indicate the metabolites may be used as markers of meat processed according to the newly used protocols as ways to distinguish the processed meat from unprocessed or never frozen and aged meat.