METHOD OF CONDITIONING FISH FIELD OF THE INVENTION

The present invention relates to a method of conditioning fish. It is also relates to the further processing of said conditioned fish in non aquatic or in aquatic conditions according to need. The invention also relates to apparatus for processing fish in non aquatic conditions and fish which have been treated by the methods of the invention. DESCRIPTION OF THE PRIOR ART

The market for fish for human consumption is very lucrative with live fish gaining a premium price due to freshness, intrinsic flesh characteristics, quality and delicate flavour.

There are a number of factors which may define the flesh quality of fish, including fat content, the distribution of fat within the body, flesh colour, and flesh texture. These characteristics are determined in part by the genetic makeup of the fish, and in part by aspects related to the feeding, handling and processing of the fish.

The quality of fish fiesh appears to be related to a number of factors associated with the biochemical pathways of ths metabolism of the creature including muscle glycogen levels, lactic acid levels and muscle pH. In particular, if muscle glycogen levels are reduced excessively prior to death, then the level of muscle metabolism post mortem is reduced resulting in the production of less lactic acid and a high muscle pH. This can affect flesh quality, especially the appearance and water retention capacity of muscle. If post mortem muscle metabolism is too high, or adenosine triphosphate ("ATP") levels are depleted excessively before death, then a reduction of muscle pH occurs and the rate of rigor mortis (muscle stiffening) is increased. The rate of rigor mortis is an important factor in maximising fillet yield because the yield is reduced if fish are processed while in rigor.

The processes associated with slaughter of fish from capture through transport, storage, handling, and slaughter, are stressful events. The effect of triggering a stress response in fish, particularly in terms of metabolism can be described as a change from an anabolic state (tissue creation) to a catabolic state

(tissue breakdown). It is general considered that stress and exertion associated

with disturbances prior to and during slaughter will lead to reduced flesh quality in fish. In unstressed fish, the reduction of muscle ATP levels and consequently the onset of rigor mortis will be delayed relative to fish stressed prior to slaughter. Thus, it is suggested that practices prior to slaughter can have measurable effects upon the biochemistry of muscle tissue in fish and that these changes may translate into adverse effects on flesh quality.

In order to limit the negative side effects on flesh quality induced by stress, non-aquatic processing, more particularly the storage and/or transportation of fresh fish and shellfish has been previously attempted by packing the creature in a container with crushed ice or salt ice without freezing. It is however impossible to resuscitate the fish and shellfish thus stored or transported. For this reason, transportation of shellfish, and in particular, fish has recently been conducted by placing them in water to keep them alive.

However, under aquatic transport conditions, decline in water quality is a major problem (ie lowering of oxygen content, carbon-dioxide build-up, detrimental changes in pH and build-up of fish waste). Under aquatic transport conditions, live fish swim about in a small container or are shaken during the transportation, and so tend to be damaged. In addition, it is necessary to transport a large quantity of water together with the fish. This method is therefore also very expensive due to the additional weight of the water. To reduce transportation costs, an increased number of live fish have been placed in one container. However, after the fish are transported in this manner, they are found to be considerably more damaged.

The processing, more particularly the transporting of large and spiny fish generally requires the use of anaesthetics to prolong a state of low metabolism or activity. This minimizes oxygen uptake and prevents jerking or sudden motion which accounts for damage to the fish and the holding containers. When plastic bags are used for example, punctures resulting from sudden motion of the fish result in loss of oxygen during transport, and subsequent decline in the quality of the fish.

The two most common commercially used anaesthetics in the market are quinaldine (2-methyl chinolin) and MS222 (Tricaine Methanesulfonate). These require great care in handling for health and safety reasons. Both anaesthetics

require treated fish to be subject to a holding period prior to human consumption. MS222 generally requires a 30 day holding period before the fish are suitable for human consumption. OBJECT OF THE INVENTION It is an object of the present invention to provide a method of conditioning fish so as to improve flesh quality, and so as to enable live processing that overcomes, or at least substantially ameliorates, at least one of the aforementioned disadvantages and shortcomings of the prior art. Another object of the invention is to provide a method of processing live fish in non-aquatic conditions, even when the processing period is many hours. Other objects and advantages of the present invention will become apparent from the following description, taken in connection with the accompanying drawings, wherein, by way of illustration and example, embodiments of the present invention are disclosed. SUMMARY OF THE INVENTION

According to one embodiment, there is provided a method of conditioning fish so as to improve flesh quality comprising the step of:

(a) inducing a hibernation state in the fish by exposing the fish to a solution of carbonic acid. According to another aspect, there is provided a method of processing live fish comprising the steps of:

(a) inducing a hibernation state in the fish by exposing the fish to a solution of carbonic acid prior to transport;

(b) transferring the live fish in the induced hibernation state into a container, wherein the container is adapted to maintain the live fish in the induced hibernation state under non-aquatic conditions; and

(c) transporting the live fish under the induced hibernation state within the container.

Preferably the transport of the fish occurs in noπ aquatic conditions. A number of studies have been performed showing that crushed coral, when mixed with water, is useful for inducing a hibernation state in fish which both reduces stress upon the creature in further processing and also enables the fish to be removed from aquatic conditions for a substantial period of time without

any adverse effect on the fish. Without being bound by theory it is believed that the carbonate (HCO ₃ ) in coral is partially converted in water to carbonic acid (H ₂ CO ₃ ) which in turn is useful for inducing the hibernation state in the fish. It is understood that the distribution of CO ₂ in the blood of fish (and most animals) is as follows:

A. 8% in plasma as gas

B. 20% bound to haemoglobin/amino acids

C. 72% earned by blood as bicarbonate

It is further believed that exposing the fish to a carbonic acid solution will result in increased levels of bicarbonate in the blood of the fish. The increasing bicarbonate levels in the blood affect the thickness of the capillaries, making them expand to expel excess carbon dioxide. In non-aquatic conditions or when water is drawn-out in the vicinity of the gills, these capillaries stay open to accept oxygen freely. In the process of expelling excess COa, the fish develop a defence mechanism to counter the critically low level of CO ₂ such that rhythmic contractions of the capillaries are manifested allowing oxygen to be assimilated by the haemoglobin of the fish. These contractions also enable the fish to produce mucous in the skin which is advantageous when reintroducing the fish to water. Preferably the source of carbonic acid is one or more compounds capable of forming carbonic acid in water. More preferably the one or more compounds capable of forming carbonic acid in water include carbonate. Preferably the source of carbonate is one or more of those which have increased solubility in water such as sodium bicarbonate, calcium carbonate, etc. It has been found that coral forms an excellent source of calcium carbonate having predominately crystalline structures of calcite and aragonite.

The concentration of carbonate suitable for inducing a hibernation state is generally similar for both marine and freshwater fish or tropical and temperate fish and is preferably in the range of 2 and 10 ppt. For carbonate containing corals, between 100 and 300 mg of coral per 10 litres of water can give similar effects. The concentration of carbon dioxide in the solution is preferably 0.24 - 0.45 mg CO ₂ per litre of water.

In inducing the hibernation state the length of time the fish are exposed to carbonic acid solution will generally vary depending on the species of fish (tropica! and temperate) and their native environment (marine or freshwater). Suitable length of time for exposing fish to carbonic acid solution with a view to inducing a hibernation state is as follows: Tropical Fish

(a) Marine 3 - 15 minutes

(b) Freshwater 30 - 60 minutes Temperate Fish (a) Marine 5 - 20 minutes

(b) Freshwater 60 - 120 minutes

Preferably in both the method of conditioning the fish and the method of processing fish there is a further step of evacuating the contents of the fishes stomach, or starving the fish both of which in different ways enable the fish to survive for longer periods of time in non-aquatic conditions. It is believed that the process of evacuating the contents of the fish stomach, or starving the fish assists in lowering the metabolism of the fish thereby enabling the fish to survive for longer periods of time in non-aquatic conditions, and for the quality of the fish flesh to be improved. Preferably the process of evacuating the contents of the fish stomach is achieved by densely packing the fish into a holding tank. More preferably step (d) is assisted by increasing the temperature of the water in the holding tank by 1 - 2 ⁰ C. Where starvation occurs, this may be achieved by starving the fish for up to 48 hours.

Preferably both methods includes a further step of removing ecto-parasites and disease forming bacteria or fungi which further improves the quality of the fish flesh and/or enables the fish to survive for long periods of time in non-aquatic conditions. This may be done by exposing the fish to substantially fresh water for up to 60 minutes. Preferably the fresh water has a salt concentration of 0 ppt. The value of this step is that it provides a "clean fish" particularly in the gill region in preparation for non-aquatic transport and consumption.

Preferably both methods include a further step of lowering the metabolic activity of the fish which enables the fish to survive for longer periods of time in non-aquatic conditions and/or improves the quality of the fish flesh for human

consumption. The lowering of the metabolic activity of both tropical and temperate fish is preferably performed with the use of water super-saturated with dissolved oxygen. The concentration of dissolved oxygen is preferably between 8 - 9 mg of O ₂ per litre of water. Preferably both methods include a further step of inducing gill apical cell dilation which enables the fish to survive for longer periods of time in non-aquatic conditions and/or improves the quality of the fish flesh. Inducing gill apical cell dilation may be achieved by temperature reduction of preferably 10 - 12 ⁰ C from the optimum environmental temperature of the fish. The difference in temperature between the fish gill and the external environment enables the apical cells of the gills to open, until fish are put back in water, enabling direct assimilation of oxygen by the capillary blood vessels of the apical cells. The process also enables the fish cells to produce "hypothermic peptides" which reduce drastically the physiological process at the cellular level. As an example, tropical fish such as grouper with an optimum environmental temperature of 26 - 28 "C preferably have a lowest hibernation temperature of 14 - 16 ^D C. As another example, temperate fish such as barramundi with an optimum environmental temperature of 14 - 16 ⁰ C preferably have a lowest hibernation temperature range of 2 - 4 °C. As yet another example, temperate fish such as salmon with an optimum environmental temperature of 10 - 12 ⁰ C preferably have a lowest hibernation temperature range of between -2 - 0 ⁿ C. Preferably the temperature is gradually lowered at 2 - 3 ⁰ C per 30 minutes.

The further steps described may be performed individually or in any combination or sequence prior to the step of inducing a hibernation state in the fish by exposing the fish to a solution of carbonic acid.

According to another aspect, there is provided an apparatus for processing live fish in a hibernation state induced by a solution of carbonic acid, the apparatus including a sealable container able to contain the fish in a manner such that: (i) the live fish are in non-aquatic conditions; and (ii) the gills of the live fish are exposed to oxygen.

According to yet another aspect there is provided a live fish in a hibernation state induced by a solution of carbonic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

It will be convenient to further describe the invention by reference to the accompanying drawings which illustrate features of preferred embodiments of the present invention. Other embodiments of the invention are possible, and consequently the particularity of the accompanying drawings is not to be understood as superseding the generality of the preceding description of the invention.

Figure 1 is a side view of a number of groupers commonly cultured in Asia;

Figure 2 is a cross sectional view of Fig 3 showing a fish inside the observation box;

Figure 3 is a perspective view of an observation box used in the method;

Figures 4 and 5 are scatter diagrams for aquatic and non-aquatic transport; and

Figures 6 to 13 are graphs of mean values for opercular actuation and temperatures in aquatic and non-aquatic conditions. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "fish" is intended to refer not only to fish taxonomically classified but also to so-called "fishery products", and includes, for instance, saltwater fish, freshwater fish, crustacean, shellfish and reptilia. As used herein, the term "tropical fish" is intended to include but is not limited to the following fish:

(a) Marine - groupers, wrasse, coral aquarium fish, snapper, sea bass, prawns, molluscs and shells;

(b) Freshwater - tilapia, carp, sea bass, shrimps and shells. As used herein, the term "temperate fish" is intended to include but is not limited to the following fish:

(a) Marine - coral trout, barramundi, molluscs and shells;

(b) Freshwater- rainbow trout, salmon, carp and perch.

As used herein, the term "low density" equates to approximately 3-5 kg or 8 fish, per 20 litres of water, and the term "high density" equates to approximately 6-8 kg or 14 fish, per 20 litres of water.

As used herein, the term "optimum environmental temperature" is intended to denote the temperature of the environment at which the fish normally lived in

immediately prior to harvesting. That is, optimum environmental temperature may refer to the temperature in a wild or cultured environment.

As used herein, the term "operculum" refers to the gill cover of fish; and to a horny or calcareous plate which closes the opening of the shell when the animal is retracted.

As used herein, the term "processing" is intended to include but is not limited to the steps of transporting, storing, harvesting, handling, capturing and slaughtering.

As used herein, the term "oxygen" is intended to include but is not limited to the group of pure oxygen (Oa) and an oxygen mixture such as air. EXAMPLE 1

The specific objective of the example is to show fish survival in non-aquatic and aquatic conditions at various starting temperatures of 6, 12 and 18 "C. The type of fish used in the Example was brown grouper (Epinephalus tauvina). The Example used 6 fish for each starting temperature and condition totalling 36 fish. A total of 36 observation boxes as shown in figure 2 were prepared for holding the fish wherein each box had two double glass slots to serve as windows for observations. The boxes were constructed of Styrofoam in order to maintain desired temperatures to which the fish were exposed. Standard plastic bags (grade 003, 25" x 30") were used to enclose the fish. These plastic bags can hold approximately 0.05 m ³ (2.5g O ₂ ) of medical grade oxygen when inflated. The amount of water utilized in the aquatic transportation conditions was determined by the rule of thumb ratio 1 : 4 (ie 1 kg fish: 4 kg water).

Whilst it is preferable to condition the fish especially wild fish, in enclosed containers intermittently prior to actual transport, fish originating from aquaculture enclosures may be prepared and transported immediately.

A carbonic acid solution was formulated by mixing a carbonate containing crushed coral (200 mg) with freshwater (10 L) which was adequate to produce an appropriate hibernation state in the fish. The salinity of conditioning and transport water was determined using

ATAGO S-1OE refractometer.

The live fish were prepared prior to transportation in non-aquatic conditions using the following steps. The fish were starved for 48 hours inside holding tanks

and were then transferred to re-circulating holding tanks containing 3OL of saline water at 18 ppt. From an initial optimum environmental temperature of 26 ⁰ C, the water temperature was lowered down by 4 °C every hour until the appropriate starting temperature of 6, 12 and 18 ⁰ C was attained. The temperature reductions in this instance were performed by introducing 500 grams of ice per 10L of saline water per 20-30 minutes. The fish were individually exposed to a bath of the carbonic acid solution for 5 minutes prior to transfer to plastic polyethylene bags. Random sampling procedures were utilized In placing fish in pre-numbered Styrofoam boxes. The fish under non-aquatic conditions were transferred to the plastic bags without water whereas the fish under aquatic conditions were transferred to the plastic bags with water. The plastic polyethylene bags were sealed with elastic rubber bands after introducing 0.05 m ³ of medical grade oxygen. Four pieces of ice in plastic bags (8 cm x 30 cm) were placed inside the Styrofoam box in order to maintain a reduced temperature during transportation.

Monitoring of Physical Parameters

Hourly readings of temperature at the bottom portion of the plastic bags and the Styrofoam box ambient temperature were recorded. Holes were made at the top and lower portions of the Styrofoam box to accommodate the insertion of laboratory thermometers.

Fish Observations

Hourly readings of opercular movement were recorded while fish behaviour was determined by digital video camera every two hours. Motion of operculum per minute was counted for each fish. Similarly, fish behaviour was recorded specifically focusing on the position of dorsal, pectoral and caudal fins. Attempts were made to record fish colour, eye movement and body spots. The absence of a consistent benchmark to note changes in colour, eye movement and spots prevented any reliable recordable observation.

The fish survival data and opercular movement data for the above experiment is provided in Table 1 and Figures 4 to 13.

No data means dead for 6°C Aquatic experiment

The data for transportation or storage of fish under non-aquatic conditions shows a trend of decreasing opercular movement over time which is understood to be beneficial for increasing fish survival during transportation or storage. Whereas, increased opercular movement is understood to indicate increased stress levels in the fish.

[EXAMPLE 2 Transporting live fish to markets sometimes several hundred kilometres away is stressful to fish and deserves special attention. In particular, the handling of the fish during and after harvesting should be adapted so as to minimise stress which could weaken the fish.

The fish were conditioned prior to transport to reduce stress by performing the following process steps. The fish were placed into a holding tank having a high density of fish in order to induce the fish to evacuate their stomach contents. In order to hasten the rate the fish evacuate their stomach contents the water temperature of the holding tank was approximately 1-2 ⁰ C above their optimum environmental temperature. The fish were then given a freshwater bath for 2-3 minutes in order to substantially remove ecto-parasites and harmful

bacteria. After the freshwater bath the temperature of the water in the holding tank was gradually reduced by 10 - 12 ⁰ C. The rate of reducing the water temperature was approximately 2 - 3 ⁰ C per 30 minutes. Air was also injected into the water of the holding tank using an air compressor and diffuser in order to raise the dissolved oxygen content. Next, the fish were exposed to the carbonic acid solution having concentration of 2 - 10 ppt for a time sufficient to induce a hibernation state in the fish.

The packaging of the live fish into a transport container for non-aquatic transportation requires a quick and efficient process for performing each of the individual steps. The transport container in this instance comprised an inner container such as a polyethylene plastic bag placed inside a suitable outer container such as a Styrofoam box. Preferably, the hibernating fish are placed into the inner container before placing the fish and inner container into the outer container. The inner container was then filled with a suitable oxygen supply such as pure or medical grade oxygen and the inner container was subsequently sealed with a suitable sealing means such as an elastic band. A suitable amount of a coolant such as ice was then added to the outer container, and then the outer container was covered and suitably sealed.

At the transportation stage an aeroplane pallet would generally accommodate approximately 126 Styrofoam boxes which is equivalent to approximately 450 kg of live fish.

It will be appreciated that the invention is not limited to the examples and embodiments described and that other embodiments may be derived from the invention but which are not illustrated herein but are nevertheless within the scope of this invention.

Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or grou ps thereof.