THE REACTION OF PROTEINASES ON FRESH AND PROCESSED ANIMAL AND VEGETABLE PROTEIN SUBSTRATES TO PRODUCE HYDROLYSATES THAT ELICIT A STRONG SUSTAINED FEEDING RESPONSE IN MARINE FISHES, AND TO USE THESE HYDROLYSATES TO INCREASE THE METABOLIC RATE OF AQUATIC ORGANISMS.

TECHNICAL FIELD This invention relates to a process for manufacturing a berley composition, a berley composition, a process of preparing a berley/bait for marine vertebrates and/or marine invertebrates, a method of attracting marine vertebrates and/or marine invertebrates, a method of catching marine vertebrates and/or marine invertebrates, a method of increasing the weight of marine vertebrates and/or marine invertebrates, a method of increasing the metabolic rate of marine vertebrates and/or marine invertebrates, a method of increasing the growth rate of marine vertebrates and/or marine invertebrates, a method of increasing the feeding response of marine vertebrates and/or marine invertebrates, a method of increasing the sexual behaviour of marine vertebrates and/or marine invertebrates, a method of increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates, a method of increasing the stress response behaviour of marine vertebrates and/or marine invertebrates and a method of increasing the motor activity of marine vertebrates and/or marine invertebrates.

BACKGROUND ART Traditionally, berley consists of compressed blocks of finely minced fish solids which are usually preserved by freezing. This berley often attracts fish but there are times when fish are not attracted in great numbers due to tidal factors, time of day, water temperature and seasonal factors. Thus, it is desirable to have a choice of different types of berleys which can be used as an alternative to the traditional berley or if they have greater fish attracting power, in preference to traditional berley.

OBJECTS OF INVENTION It is an object of this invention to provide a process for manufacturing a berley composition and a process of preparing a berley/bait for marine vertebrates and/or marine invertebrates.

Another object is to provide a berley composition. Further objects are to provide a method of attracting marine vertebrates and/or marine invertebrates, a method of catching marine vertebrates and/or marine invertebrates, a method of increasing the weight

of marine vertebrates and/or marine invertebrates, a method of increasing the metabolic rate of marine vertebrates and/or marine invertebrates, a method of increasing the growth rate of marine vertebrates and/or marine invertebrates, a method of increasing the feeding response of marine vertebrates and/or marine invertebrates, a method of increasing the sexual behaviour of marine vertebrates and/or marine invertebrates, a method of increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates, a method of increasing the stress response behaviour of marine vertebrates and/or marine invertebrates and a method of increasing the motor activity of marine vertebrates and/or marine invertebrates.

DISCLOSURE OF INVENTION

As used herein the expression "modifying agent" refers to a chemical agent such as a hydrolytic agent or physical means that is capable of hydrolyzing a protein, peptide, polypeptide or polypeptide bond or modifying a protein, peptide, polypeptide or polypeptide bond by the hydrolysis of a protein, peptide, polypeptide or polypeptide component or breaking the protein, peptide, polypeptide or polypeptide component into smaller polypeptides, smaller peptides and/or protein subunits.

As used herein the expression "fish oil hydrolysing agent" refers to a chemical agent such as a hydrolytic agent or physical means that is capable of hydrolyzing the lipids in a fish oil into smaller chain fatty acids and derivatives thereof.

As used herein the expression "fish oil" refers to an oil derived from a marine vertebrate including a marine mammal, or a marine invertebrate.

As used herein the expression "fish oil modified mixture" refers to a mixture derived from a fish oil/fat which has been modified by a lipase or 1ipolytic enzyme as follows: fish fat/oil + lipase + H ₂ 0 => mono/and diglycerides + lipase + H D

=> glycerol + _2Q _ ₂₄ fatty acids fish fatty acid esters + lipase + H„0 => alcohol + C _{20 24} fatty acids

As used herein the expression "buffering agent" refers to any agent which can resist changes in pH.

As used herein the term "endopeptidase" refers to an enzyme which hydrolyzes internal peptide bonds of proteins.

Proteins + Endopeptidases => Polypeptides + water + Endopeptidase water

As used herein the term "exopeptidase" refers to an enzyme which hydrolyzes the peptide bonds adjacent to free carboxyl or amino groups.

Polypeptides + Exopeptidases => Amino acids + water + Exopeptidase water

The reactions do not continue to completion. Thus the product of enzyme digestion is a protein modified mixture. As used herein the expression "protein modified mixture" refers to mixtures of: proteins and polypeptides; or proteins, polypeptides and amino acids; or polypeptides; or polypeptides and amino acids which are prepared by modifying proteins with a modifying agent.

Fungal proteases (proteinases) exhibit both exo and endopeptidase activity. Asperαillus orvzae Protease is a mixture of exo and endopeptidases.

As used herein the expression "protein modified surface" refers to the surface of a protein-based marine vertebrates and/or marine invertebrates bait which has been modified by a modifying agent whereby the modified surface has mixtures of: proteins and polypeptides; or proteins, polypeptides and amino acids; or polypeptides; or polypeptides and amino acids which are prepared by modifying proteins with a modifying agent.

According to a first embodiment of this invention there is provided a process for manufacturing a berley composition, comprising: modifying proteins and/or polypeptides by reacting a modifying agent with the proteins and/or polypeptides to form said berley composition comprising a protein modified mixture, as herein defined.

Generally the protein modified mixture comprises polypeptides and/or proteins having molecular weights in the range 200 to 8,000 daltons. Typically the process of the first embodiment further includes shaping/molding at least a portion of the mixture into a shape such as a sausage shape for use as a berley.

The process of the first embodiment can include: placing the composition in a container capable of use as a berley container.

The process of the first embodiment can include: combining the composition with a carrier, diluent or binder. For example, the process can include: combining 99% to 5% by weight of the composition with from 1% to 95% of a binding composition comprising from about 10% to about 35% by weight

flour, from about 30% to about 70% by weight bran, from about 5% to about 40% by weight water, from a trace to about 14% by weight of one or more water soluble alkali metal or alkaline earth metal salts or combinations thereof and from a trace to about 20% by weight fish oil. Generally this process is performed by: mixing the bran with the fish oil to form a bran/fish oil mixture; mixing the flour, the water and the alkali metal or alkaline earth metal salt or combinations thereof with the bran/fish oil mixture to form said binding composition; and mixing said binding composition with the protein modified mixture.

The process of the first embodiment can also include the step of: separating proteins and polypeptides from the protein modified mixture according to molecular weight in the range 200 to 8,000 daltons wherein said separated proteins and polypeptides comprise said berley composition.

The process of the first embodiment can also include separating the protein modified mixture according to ionic properties, solubility, hydrophobicity and/or molecular charge.

The process of the first embodiment can further include the step of heating the berley composition. Typically the composition is heated at a temperature in the range of about 30°C to about 75°C for between 30 minutes to 14 hours. Desirably a temperature of 50°C is employed.

According to a second embodiment of this invention there is provided a process for manufacturing a berley composition, comprising: modifying proteins and/or polypeptides by reacting a modifying agent with the proteins and/or polypeptides to form a protein modified mixture, as herein defined; modifying fish oil with a fish oil hydrolyzing agent to form a fish oil modified mixture; and combining the protein modified mixture with the fish oil modified mixture to form the berley composition or wherein the protein modified mixture is in combination with the fish oil modified mixture to from the berley composition.

Typically fish oils such as tuna, mackeral, sand eel, menhaden, anchovy, sardine, horse mackeral, salmon, herring, cod, capelin, pilchard, sprat, whale, Pacific oyster, Norway pout, seal oil, sperm whale oil or any

mixture thereof are particularly suitable. The fish oil will generally be present in an amount ranging from a trace to about 20% by weight more preferably from 3% to 5% by weight of the composition.

Reacting native protein, I.e. protein which has not been denatured, with a modifying agent is generally the process used for preparing the berley composition.

Typically the modifying agent employed is a protease (proteinase).

According to a third embodiment of this invention there is provided a berley composition whenever prepared by the process of the first or second embodiments.

According to a fourth embodiment of this invention there 1s provided a berley composition comprising a protein modified mixture as herein defined.

Also provided according to this invention are the following compositions:

(a) A composition for attracting marine vertebrates and/or marine invertebrates comprising: a marine vertebrates and/or marine invertebrates attracting amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(b) A composition for inducing a feeding response in marine vertebrates and/or marine invertebrates comprising: a marine vertebrates and/or marine invertebrates feeding response inducing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(c) A composition for increasing the metabolic rate of marine vertebrates and/or marine invertebrates comprising: a marine vertebrates and/or marine invertebrates metabolic rate increasing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(d) A composition for increasing the growth rate of marine vertebrates and/or marine invertebrates comprising: a marine vertebrates and/or marine invertebrates growth rate increasing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(e) A composition for increasing the sexual behaviour of marine vertebrates and/or marine invertebrates comprising:

a marine vertebrates and/or marine invertebrates sexual behaviour inducing amount of the berley composition of the third or fourth embodiments together with÷-a carrier, diluent and/or binding agent.

(f) A composition for increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates comprising: a marine vertebrates and/or marine invertebrates aggressive behaviour inducing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(g) A composition for increasing the stress response behaviour of marine vertebrates and/or marine invertebrates comprising: a marine vertebrates and/or marine invertebrates stress response behaviour inducing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent, (h) A composition for increasing the motor activity of marine vertebrates and/or marine invertebrates comprising: a marine vertebrates and/or marine invertebrates motor activity increasing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

Also provided according to this invention are the following processes for making compositions:

(a) A process for making a composition for attracting marine vertebrates and/or marine invertebrates comprising: mixing a marine vertebrates and/or marine invertebrates attracting amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(b) A process for making a composition for inducing a feeding response in marine vertebrates and/or marine invertebrates comprising: mixing a marine vertebrates and/or marine invertebrates feeding response inducing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(c) A process for making a composition for increasing the metabolic rate of marine vertebrates and/or marine invertebrates comprising: mixing a marine vertebrates and/or marine invertebrates metabolic rate increasing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(d) A process for making a composition for increasing the growth rate of marine vertebrates and/or marine invertebrates comprising:

- i - mixing a marine vertebrates and/or marine invertebrates growth rate increasing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(e) A process for making a composition for increasing the sexual behaviour of marine vertebrates and/or marine invertebrates comprising: mixing a marine vertebrates and/or marine invertebrates sexual behaviour inducing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(f) A process for making a composition for increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates comprising: mixing a marine vertebrates and/or marine invertebrates aggressive behaviour inducing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

(g) A process for making a composition for increasing the stress response behaviour of marine vertebrates and/or marine invertebrates comprising: mixing a marine vertebrates and/or marine invertebrates stress response behaviour inducing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent, (h) A process for making a composition for increasing the motor activity of marine vertebrates and/or marine invertebrates comprising: mixing a marine vertebrates and/or marine invertebrates motor activity increasing amount of the berley composition of the third or fourth embodiments together with a carrier, diluent and/or binding agent.

Generally the protein modified mixture comprises polypeptides and/or proteins having molecular weights in the range 200 to 8,000 daltons.

Generally the berley composition further comprises a carrier, diluent or binder.

Generally carriers include flour, bran, ground rice, or mixtures thereof, and other like carriers.

Typically the selection of an appropriate carrier will depend on the desired formulation. Suitable solid carriers e.g. for dusts and dispersible powders are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. The addition of highly dispersed silicic acid or absorbent polymers can be added to improve the physical properties. Suitable carriers for granule formulations are normally absorptive carriers which are porous for example pumice, broken brick, sepiolite or bentonite. Many other pregranulated inorganic or

organi c materi al s can al so be used e . g. dolomi te or pul veri sed pl ant resi dues .

Other suitable carriers include: adsorbents such as diatomaceous earths or charcoal; magnesium silicates, for example talcs; magnesium aluminum silicates, for example attapulgites and vermiculites; aluminium silicates, for example kaolinites, montmorillonites and micas; calcium carbonate; calcium sulphate; synthetic hydrated silicon oxides and synthetic calcium or aluminium silicates; natural and synthetic resins, for example coumarone resins, polyvinyl chloride, and styrene polymers and copolymers; solid polychlorophenols; bitumen; waxes, for example beeswax, paraffin wax, and chlorinated mineral waxes.

Typically diluents include calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, kaolin, distilled water, a salt solution, a sugar solution, a glycol such as polyethylene glycol or propylene glycol, or an oil such as sesame oil, olive oil, or soybean oil, and other like diluents.

Generally binders can include such substances as starch, corn starch, gluten, alginate, carboxymethylcellulose, agarose, polyvinyl alcohol, hydroxypropylcellulose, gelatin, carrageenan, locust bean gum, pectin, guar gum, gum acacia, gum tragacanth, tamarind, hypnean, furcellaran, and other like binders.

Slow release formulations can be prepared using suitable sustained released adducts including agarose, agar, flour, cellulose, hemicellulose including xylan, carboxymethylcellulose, dextran, dextrin-starches including a ylose, glucans like lichenin, nigeran and glycogens, fructans including inulin, galactans, mannans, polyuronides including gums and mucilages, fucoidin, and derivatives thereof.

A slow release berley of the invention can comprise a mixture of from 99% to 5% by weight of a composition of the invention together with from 1% to 95% of a composition comprising from about 10% to about 35% by weight flour, from about 30% to about 70% by weight bran, from about 5% to about 40% by weight water, from a trace to about 14% by weight of one or more water soluble alkali metal or alkaline earth metal salts or combinations thereof and from a trace to about 20% by weight fish oil. Generally the berley is prepared by a first step of forming a bran/fish oil mixture by mixing the bran with the fish oil and a second step of mixing the flour, the water and the alkali metal or alkaline earth metal salt or combinations

thereof with the bran/fish oil mixture to form a mixture which is then mixed with the composition of the second embodiment to form the berley.

The composition of the invention optionally includes from a trace to about 20% by weight proteins and/or polypeptides with at least about 80% by weight if the reactants are in the same state. (Fluids are the best states of matter for reaction).

Tuna oil or pilchard oil or mixtures thereof is/are typically used by first mixing with the cereal bran so that in use the area of the oil/water interface is large. This absorption tends to decrease the rate at which the oil is separated from the other components of the berley composition when they are released by the action of water.

The flour can be coarse or fine mesh or a mixture thereof white or wholemeal flour preferably selected from wheat, rye, corn, rice, millet, sorghum, maize, barley or oat flour or any mixture thereof or flour of other like grain or mixture thereof. Preferably high protein wheat flour

(about 10% to 16% by weight protein at about 10% to 14% by weight moisture level) is used.

The bran can be coarse or fine or a mixture thereof of any cereal bran including wheat, rye, corn, rice, millet, sorghum, maize, barley or oat bran or any mixture thereof or bran of other like grain or mixture thereof. A 50:50 mixture of coarse: fine wheat which is present in an amount from about 38% to 65% by weight is especially preferred.

Preferably sodium chloride is used as the alkali metal salt although other salts such as potassium chloride, lithium chloride, cesium chloride, potassium bromide, lithium bromide, cesium bromide, sodium iodide, potassium iodide, lithium iodide, cesium iodide, sodium sulphate, sodium nitrate and other like salts or mixtures thereof can be used. Most preferably sodium chloride is present in an amount of from about 3% to about 14% by weight.

Fine mesh flour and fine mesh cereal bran are also typically used in the composition in addition to coarse flour and bran and to fine mesh dried fish waste solids. As a result fine mesh particles are released when a compressed berley composition is placed in water. These fine particles have a slow settling velocity, typically less than 1 metre per 300 seconds in still water which results in an effective berley gradient from the slow release berley composition.

An advantage of the slow settling rate is that fish are attracted to

the near vicinity of the berley due to the higher concentration of released particles and it has been observed in practice that they remain in the close vicinity until the complete decomposition of the berley which is about four hours. Other forms of berley which are currently used in practice are typically released in pulse form which although stimulate the feeding of fish do not necessarily attract them for a suitably long time at a fixed location.

The flour in the slow release berley composition acts as a binder for the other particles in the berley. Thus the decomposition of the berley has been found in practice to be proportional to the flour/bran ratio. It has also been found that the brine/total solids ratio affects the decomposition time of the berley. The higher the brine/total solids ratio the faster the decomposition rate. It is thought that the sodium chloride modifies the elastic properties of the flour and bran and enables the gluten in the flour to break up in a controlled manner. In addition to sodium chloride, vegetable gelling agents may be used; e.g. modified grain starches.

The sodium chloride also preserves the berley composition. Additionally preservatives such as sodium nitrite and sodium benzoate can be incorporated into the berley composition to inhibit bacterial and fungal action.

In addition the compositions may also include, stabilizers surfactants, salts, buffers, or mixtures thereof. Suitable stabilisers include dehydroacetic acid and salts thereof.

Heating the berley composition at a suitable temperature, also enhances the shelf life of the product. Alternatively the composition may be frozen, typically to -10°C, or freeze dried. Alternatively the composition may be spray dried under vacuum at about 30°C to about 75°C, typically at 60°C.

The composition may be coated onto a suitable substrate e.g. a conventional bait, or an appropriate carrier, and dried. Appropriate carriers may be selected from those described above

Colouring agents such as edible dyes may optionally be added to the berley composition for identification purposes.

Preferably the berley composition is compressed, e.g., by pressure moulding, to form a compressed mass in the form of a selected shape. It is preferred that the compressed berley composition is dried to a total water

- n - content of about 5% to 25% by weight and more preferably about 9% to about 11.5% by weight.

A sausage shape is a particularly preferred selected shape. The selected shape is then preferably dried (e.g., by heating in an oven) to reduce the total water of the compressed mass to about 9% to about 11.5% by weight.

A combination of a selected shape of a berley composition of the invention disposed in a water-permeable container such as a Sarlon netting bag or an extruded plastic or poly-ethylene net bag is preferred.

This combination is an independent berley system which when placed in water breaks down releasing a constant stream of particles for up to four hours. Its use does not require any preparation and there is no mess associated with its application. The berley composition is totally biodegradable. The fine particles released from the berley do not tend to satiate fish since they have to swim harder to gather the fine particles released from the berley and this tends to stimulate their appetite.

The above combination is typically stored in a non porous bag. A thick polyethylene bag having a tropical moisture vapour transfer rate

-2 -1 (M.V.T.R.) rating of less than 6g day moisture transfer under a gradient of 95% R.H. at 25°C is especially suitable.

Preferably an antifungal, antibacterial, fumigant or any mixture thereof is added to the bag just prior to packing the berley and sealing.

Paraformal ehyde has been found to be particularly effective fumigant for preventing mould formation and thereby increasing shelf life. Antifungals and/or antibacterials such as heavy metal salts, carbamates, organo sulphur compounds or halogenated hydrocarbons or other like substances or combinations thereof are also suitable. However, paraformaldehyde is preferred since:-

(a) It breaks down to liberate formaldehyde, an antifungal and fumigant the rate of breakdown increasing as the temperature increases. This is particularly desirable since as temperature increases the amount of moisture liberated from the berley increases leading to more favourable conditions (in the absence of paraformaldehyde) for mould growth in the immediate vicinity of the berley.

(b) Paraformaldehyde is preferable to heavy metal salts, carbamates, organo sulphur compounds or halogenated hydrocarbons because it is less toxic to fish and it is rapidly biodegraded and thus quickly removed from

the environment.

Typically in the berley composition 99% to 5% by weight of the composition is combined with from 1% to 95% of a binding composition comprising from about 10% to about 35% by weight flour, from about 30% to about 70% by weight bran, from about 5% to about 40% by weight water, from a trace to about 14% by weight of one or more water soluble alkali metal or alkaline earth metal salts or combinations thereof and from a trace to about 20% by weight fish oil.

According to a fifth embodiment of this invention there is provided a process of preparing a berley/bait for marine vertebrates and/or marine invertebrates comprising: modifying the surface of the flesh of a protein-based marine vertebrates and/or marine invertebrates berley/bait by reacting a modifying agent with the surface to form a protein modified surface, as herein defined.

Typically the marine vertebrates and/or marine invertebrates berley/bait is selected from the group consisting of prawns, shrimps, yabbies, Balmain bugs, pippies, worms, sea snakes, flesh of turtles, flesh of tortoises, eels, octopus, squid, flesh of lobsters, flesh of crayfish, flesh of crabs, marine mammals and fish.

Generally the process of the fifth embodiment includes: applying a marine vertebrates and/or marine invertebrates attracting substance to the protein modified surface.

According to a sixth embodiment of this invention there is provided a process of preparing a berley/bait for marine vertebrates and/or marine invertebrates comprising: applying a fish oil to the surface of the flesh of a protein-based marine vertebrates and/or marine invertebrates berley/bait; modifying the surface of the flesh of berley/bait by reacting a modifying agent with the surface to form a protein modified surface, as herein defined; and modifying the fish oil with a fish oil hydrolysing agent.

The processes of the fifth and sixth embodiments can also include the step of refrigerating the berley/bait having the modified surface flesh to store the berley/bait prior to use. Typically the refrigeration comprises freezing the berley/bait.

According to a seventh embodiment of this invention there is provided

a berley/bait for marine vertebrates and/or marine invertebrates when produced by the process of the fifth or sixth embodiments.

According to a eighth embodiment of this invention there is provided a method of attracting marine vertebrates and/or marine invertebrates comprising: placing a marine vertebrates and/or marine invertebrates attracting amount of said berley composition of the third or fourth embodiments in a body of water containing marine vertebrates and/or marine invertebrates whereby the berley composition attracts marine vertebrates and/or marine invertebrates in the near vicinity.

According to an ninth embodiment of this invention there is provided a method of attracting marine vertebrates and/or marine invertebrates comprising: placing a marine vertebrates and/or marine invertebrates attracting amount of said berley/bait for marine vertebrates and/or marine invertebrates of the seventh embodiment in a body of water containing marine vertebrates and/or marine invertebrates whereby the berley/bait attracts marine vertebrates and/or marine invertebrates in the near vicinity.

According to a tenth embodiment of this invention there is provided a method of catching marine vertebrates and/or marine invertebrates comprising: placing a marine vertebrates and/or marine invertebrates attracting amount of said berley composition for marine vertebrates and/or marine invertebrates of the third or fourth embodiments in a body of water containing marine vertebrates and/or marine invertebrates whereby the berley composition for marine vertebrates and/or marine invertebrates attracts marine vertebrates and/or marine invertebrates in the near vicinity; and catching by a conventional fishing technique marine vertebrates and/or marine invertebrates that have been attracted to the near vicinity of the berley composition for fish.

According to a eleventh embodiment of this invention there is provided a method of catching marine vertebrates and/or marine invertebrates comprising: placing a marine vertebrates and/or marine invertebrates attracting amount of berley/bait for marine vertebrates and/or marine invertebrates of

the seventh embodiment in a body of water containing marine vertebrates and/or marine invertebrates whereby the berley/bait for marine vertebrates and/or marine invertebrates attracts marine vertebrates and/or marine invertebrates in the near vicinity; and catching by a conventional fishing technique marine vertebrates and/or marine invertebrates that have been attracted to the near vicinity of the berley/bait for ish- According to an twelfth embodiment of this invention there is provided a method of increasing the weight of marine vertebrates and/or marine invertebrates comprising: placing a feeding response inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine vertebrates and/or marine invertebrates whereby a feeding response is induced in the marine verteb ^r ates and/or marine invertebrates; and placing food for the marine vertebrates and/or marine invertebrates into the body of water whereby the marine vertebrates and/or marine invertebrates having the induced feeding response eat the food.

According to a thirteenth embodiment of this invention there is provided a method of increasing the weight of marine vertebrates and/or marine invertebrates comprising: placing a feeding response inducing amount and a feeding amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby a feeding response is induced in the marine vertebrates and/or marine invertebrates and the marine vertebrates and/or marine invertebrates having the induced feeding response eat the berley/bait.

The food for the marine vertebrates and/or marine invertebrates can be placed into the body of water before, at the same time and/or after the feeding response inducing amount of a berley composition of the third or fourth embodiments and/or the berley/bait for marine vertebrates and/or marine invertebrates of the seventh embodiment is placed into the body of water.

According to a fifteenth embodiment of this invention there is provided a method for increasing the metabolic rate of marine vertebrates and/or marine invertebrates comprising: placing a metabolic rate inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine

vertebrates and/or marine invertebrates whereby an increased metabolic rate is induced in the marine vertebrates and/or marine invertebrates.

According to a sixteenth embodiment of this invention there is provided a method of increasing the metabolic rate of marine vertebrates and/or marine invertebrates comprising: placing a metabolic rate inducing amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased metabolic rate is induced in the marine vertebrates and/or marine invertebrates.

According to an seventeenth embodiment of this invention there is provided a method of increasing the growth rate of marine vertebrates and/or marine invertebrates comprising: placing a growth rate inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased growth rate is induced in the marine vertebrates and/or marine invertebrates.

According to a eighteenth embodiment of this invention there is provided a method of increasing the growth rate of marine vertebrates and/or marine invertebrates comprising: placing a growth rate inducing amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased growth rate is induced in the marine vertebrates and/or marine invertebrates.

According to a ninteenth embodiment of this invention there is provided a method of increasing the feeding response of marine vertebrates and/or marine invertebrates comprising: placing a feeding response inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased feeding response is induced in the m?rine vertebrates and/or marine invertebrates.

According to an twentieth embodiment of this invention there is provided a method of increasing the feeding response of marine vertebrates and/or marine invertebrates como-ising: placing a feeding response inducing amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased feeding response is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty first embodiment of this invention there is provided a method of increasing the sexual behaviour of marine vertebrates and/or marine invertebrates comprising: placing a sexual behaviour inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased sexual behaviour is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty second embodiment of this invention there is provided a method of increasing the sexual behaviour of marine vertebrates and/or marine invertebrates comprising: placing a sexual behaviour inducing amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased sexual behaviour is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty third embodiment of this invention there is provided a method of increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates comprising: placing an aggressive behaviour inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased aggressive behaviour is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty fourth embodiment there is provided a method of increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates comprising: placing an aggressive behaviour inducing amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased aggressive behaviour is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty ---fi embodiment there is provided a method of increasing the stress response behaviour of marine vertebrates and/or marine invertebrates comprising: placing a stress response behaviour inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased stress response behaviour is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty sixth embodiment of this invention there is provided a method of Increasing the stress response behaviour of marine vertebrates and/or marine invertebrates comprising: placing a stress response behaviour inducing amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased stress response behaviour is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty eighth embodiment of this invention there is provided a method of increasing the motor activity of marine vertebrates and/or marine invertebrates comprising: placing a motor activity inducing amount of a berley composition of the third or fourth embodiments into a body of water containing marine vertebrates and/or marine invertebrates whereby increased motor activity is induced in the marine vertebrates and/or marine invertebrates.

According to a twenty ninth embodiment of this invention there is provided a method of increasing the motor activity of marine vertebrates and/or marine invertebrates comprising: placing a motor activity inducing amount of a berley/bait of the seventh embodiment into a body of water containing marine vertebrates and/or marine invertebrates whereby increased motor activity is induced in the marine vertebrates and/or marine invertebrates.

According to the invention there is provided the following methods:

(a) A method of attracting marine vertebrates and/or marine invertebrates comprising: placing a marine vertebrates and/or marine invertebrates attracting amount of the composition for attracting marine vertebrates and/or marine invertebrates in a body of water containing marine vertebrates and/or marine invertebrates whereby the composition attracts marine vertebrates and/or marine invertebrates in the near vicinity.

(b) A method of catching marine vertebrates and/or marine invertebrates comprising: placing a marine vertebrates and/or marine invertebrates attracting amount of the composition for attracting marine vertebrates and/or marine invertebrates in a body of water containing marine vertebrates and/or marine invertebrates whereby the berley composition for marine vertebrates and/or marine invertebrates attracts marine vertebrates and/or marine

invertebrates in the near vicinity; and catching by a conventional fishing technique marine vertebrates and/or marine invertebrates that have been attracted to the near vicinity of the berley composition for fish.

(c) A method of increasing the weight of marine vertebrates and/or marine invertebrates comprising: placing a feeding response inducing amount of the composition for inducing a feeding response in marine vertebrates and/or marine invertebrates into a body of water containing marine vertebrates and/or marine invertebrates whereby a feeding response is induced in the marine vertebrates and/or marine invertebrates; and placing food for the marine vertebrates and/or marine invertebrates into the body of water whereby the marine vertebrates and/or marine invertebrates having the induced feeding response eat the food.

(d) A method of increasing the growth rate of marine vertebrates and/or marine invertebrates comprising: placing a growth rate inducing amount of the composition for increasing the growth rate of marine vertebrates and/or marine invertebrates into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased growth rate is induced in the marine vertebrates and/or marine invertebrates.

(e) A method of increasing the feeding response of marine vertebrates and/or marine invertebrates comprising: placing a feeding response inducing amount of the composition for inducing a feeding response in marine vertebrates and/or marine invertebrates into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased feeding response is induced in the marine vertebrates and/or marine invertebrates.

(f) A method of increasing the sexual behaviour of marine vertebrates and/or marine invertebrates comprising: placing a sexual behaviour inducing amount of the composition for increasing the sexual behaviour of marine vertebrates and/or marine invertebrates into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased sexual behaviour is induced in the marine vertebrates and/or marine invertebrates.

(g ⁾ A method of increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates comprising:

placing an aggressive behaviour inducing amount of the composition for increasing the aggressive behaviour of marine vertebrates and/or marine invertebrates into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased aggressive behaviour is induced in the marine vertebrates and/or marine invertebrates.

(h) A method of increasing the stress response behaviour of marine vertebrates and/or marine invertebrates comprising: placing a stress response behaviour inducing amount of the composition for increasing the stress response behaviour of marine vertebrates and/or marine invertebrates into a body of water containing marine vertebrates and/or marine invertebrates whereby an increased stress response behaviour is induced in the marine vertebrates and/or marine invertebrates.

(i) A method of increasing the motor activity of marine vertebrates and/or marine invertebrates comprising: placing a motor activity inducing amount of the composition for increasing the motor activity of marine vertebrates and/or marine invertebrates into a body of water containing marine vertebrates and/or marine invertebrates whereby increased motor activity is induced in the marine vertebrates and/or marine invertebrates.

The food for the marine vertebrates and/or marine invertebrates can be placed into the body of water before, at the same time and/or after the feeding response inducing amount of a berley composition of the third or fourth embodiments and/or the berley/bait for marine vertebrates and/or marine invertebrates of the seventh embodiment is placed into the body of water

Marine vertebrates include fish, marine reptiles such as sea snakes, turtles, eels and marine mammals. Marine invertebrates include prawns, shrimps, lobsters, scallops, crabs, yabbies, Balmain bugs, octopus, squid, crayfish and pippies.

Typically the source of the proteins and/or polypeptides can be from animals, birds, reptiles, fish, plants, insects and/or protists. Animal proteins and/or polypeptides can be obtained from man, camel, cow, sheep, pig, horse, dog, whale, seal and other like animals. Bird proteins and/or polypeptides can be obtained from chicken, sparrow, pigeon, albatross, sea gull, heron and other like birds. Reptile proteins and/or polypeptides can be obtained from snake, crocodile, alligator and other like reptiles. Fish

proteins and/or polypeptides can be obtained from hardiheads, white bait, mullet, sardines, salmon, tuna, trout, bream, black fish, flathead, tailer,

John Dory, schnapper, trevally, sweep, shark, garfish, pike, leatherjacket, wrasse, mulloway, dolphin fish, kingfish, blennies, gobies, toad fish and other like fish. Plant proteins and/or polypeptides can be obtained from rice, barley, oat, rye, corn, wheat, flowering plants such as rose, iris, carnation, daffodil, lily, vegetables such as cabbage, cauliflower, peas, beans, such as soyabeans, lentils, ung beans, lima beans, kidney beans, adzuki beans, broad beans, broccoli, brussel sprouts, peanuts, chickpeas, asparagus, soy and other like plants. Insect proteins and/or polypeptides can be obtained from spiders, cockroaches, ants, beetles, silverfish and other like insects. Protist proteins and/or polypeptides can be obtained from yeast, algae, fungi, bacteria and other like protists.

Generally modifying agents can be proteases, fish oil hydrolysing agents, upases, carbohydrases, esterases, phosphatases, nucleases, acids, alkalis, temperature, organic solvents miscible with water, e.g. acetone, methanol or ethanol , mercaptides, e.g. 2-mercaptoethanol , and mixtures thereof or other like modifying agents. Typically, a protease whether an exopeptidase or an endopeptidase is the modifying agent. Typically proteases include papain, bro elain, lysozyme, trypsin, chymotrypsin, pepsin, renin, subtilisin, carboxypeptidase, aminotripeptidase and other like proteases.

Generally the sources of protease include vegetable extracts, e.g. cereal grain gum, papaya extracts (papain), tropical fig extracts (ficin); fungal extracts, e.g. Asperc Hus oryzae extracts, other fungal sources; microbial and bacterial extracts, e.g. Escherichia coli . other bacteria; animal extracts, e.g. pancreatic enzymes, stomach enzymes, other digestive gland enzymes; or other like sources. Preferably the source of protease is bacterial extracts due to the presence of bacterial enzymes in the gut content of crabs, prawns and sea urchins and fungal extracts. Typically the amount of _ orvzae protease or papain used is from 0.001 wt% to 1.0 wt% more typically is from 0.01 wt% to 0.1 wt%.

Typically fish oil hydrolysing agents include, upases, strong alkalis, or acids or other like hydrolysing agents.

Typical upases include pancreatic lipase, steapsin. General carbohydrases include α-amylase, sucrase, maltase. Typical phosphatases include fructose -1 ,6-diphosphatase, glucose 6-phosphatase. General

nucleases include ribonuclease, deoxyribonuclease. Acids typically include hydrochloric acid, sulphuric acid. Alkalis generally include sodium hydroxide, potassium hydroxide.

Enzymatic activity can be dependent on pH, ionic strength, temperature and metal ion requirements. pH is generally determined and maintained by buffering agents. Examples of buffering agents include phosphate, tris (hydroxymethyl) aminomethane, amino acids, citrate, acetate, succinate, maleate, cacodylate, barbital, carbonate, sulphate or mixtures thereof or other like buffering agents. The pH for enzymatic activity and other modifying agents can range from 0 to 14.

Ionic strength can be maintained by the addition of salts. Examples of salts include sodium chloride, potassium chloride, lithium chloride, cesium chloride, sodium bromide, potassium bromide, lithium bromide, cesium bromide, sodium iodide, potassium iodide, lithium iodide, cesium iodide, sodium sulphate, sodium nitrate and other like salts or mixtures thereof can be used.

The temperature range for enzymatic activity can vary from 0 to 100°C, depending on the type of enzyme and for other modifying agents, the temperature range can vary from -80°C to 110°C.

Enzymatic activity requiring metal ions can be met by addition of calcium, copper, magnesium, manganese, sodium, potassium, zinc and other like metal ions or mixtures thereof.

Many of the proteins in nature with which digestive enzymes react are in solution or at least In a very fine emulsion.

All the convenient protein substrates freely available on the market with the possible exception of albumen and gelatin exist as insoluble particles.

Chemical reaction occurs easiest if the reactants are in the same state. (Fluids are the best states of matter for reaction).

When a solid particle is suspended in water only the surface of the particle is available for reaction with compounds dissolved in the water. The rate of reaction in enzyme systems is governed to a large extent by the concentration of substrate molecules available.

As a particle is ground finer more particles are produced, and the combined surface area of these new particles is much greater than the parent particle. It is possible but not always practical to grind substances so fine that they eventually exhibit properties similar to real

sol ution .

When carrying out enzyme reactions with insoluble protein substrates it is important that these substrates be ground as fine as possible. If there are not enough substrate molecules available for the enzymes they hydrolyze themselves. This slows down the rate of reaction and reduces the efficiency of the digest.

Wheat flour and defatted soy flour are excellent examples of finely ground products which react efficiently with proteases. Typically protein substrates should be ground so that 90% passes a 150 micron sieve. BEST MODE AND OTHER MODE(S) FOR CARRYING OUT THE INVENTION

Freshly caught mullet is washed and scaled. The fish flesh is processed with water and weighed. 0.01 wt% of papain is dissolved in 0.1M sodium (and/or potassium) phosphate buffer, pH7.0. This solution is then allowed to react with the processed mullet flesh by heating to 70°C and maintained for about 2 hours. The reacted mixture is allowed to cool to room temperature over a period of about 1 hour. Once the reacted mixture is at room temperature it is frozen at -10°C until ready for use.

The frozen hydrolyzed mullet flesh is thawed and the following berley composition is prepared:

31.2% Coarse wheat bran (95% + > 2mm)

31.2% Fine wheat bran (-1mm + 11μm)

12.0% High protein wheat flour (13.3% to 14% total protein calculated at 13% moisture level)

16.0% Brine solution (5.0% Total alkali)

6.0% Hydrolyzed mullet

3.6% Tuna oil

(% by weight)

The tuna oil is first mixed with the coarse and fine wheat brans. The other ingredients are then added in the above proportions. After mixing, the sausage-shaped berley (20-150mm in diameter, 30-500mm in length) is manufactured by pressure moulding using teflon or ultra-high density, polyethylene coated moulds.

The sausage is dried to lower the moisture content to equal or slightly less than about 20% by weight. The sausage shaped berley 1s then packed in a thick polyethylene bag having a tropical M.V.T.R. rating of

-2 -1 lleessss than 6g day moisture transfer under a gradient of 95% R.H. at

25 ^β C.

Paraformaldehyde pellets are added to the plastic bag just prior to sealing at 1.5 - 2.0g of paraformaldehyde per kilogram of berley.

The prepared berley is then placed into the water. The berley composition breaks down slowly approximately over 4h. The fish are attracted to the decomposing berley composition and are readily caught.

Embodiments of this invention are described below by way of the following examples. Index 1.0 Aim 2.0 Preamble

2.1 Proteinases

2.2 Protein Substrates

2.2.1 Plant Protein

2.2.2 Animal Protein

2.2.3 Protein Substrates Used 3.0 Results

3.1 Berlies(Chum)

3.2 Baits

3.3 Aquaria 4.0 Experiments

4.1 Preparation of Polypeptide Berlies

4.2 Testing Procedures for Enzyme Modified Berley Mixtures

4.3 Preparation of Enzyme Modified Baits Examples

1.0 AIM

The following examples show that the action of proteolytic enzymes on various protein substrates produce polypeptides that increase the metabolic activity of marine organisms which results in one or more of the following:-

1. General increase in the movements

2. Increased feeding behavior

3. Increased growth rate

4. Increase in aggressive behavior

5. Increased responses to stress.

The polypeptides used in the experiments reported herein are mixtures of polypeptides in the range 200 to 8,000 Daltons. The polypeptides in this molecular size range were produced by carefully controlling the hydrolysis of proteins and larger polypeptides using endopeptidase proteinases.

2.0 PREAMBLE.

2.1 Proteinases

Proteinases are the enzymes responsible for digesting protein. Proteinases are broadly classified into two groups:

1 Endopeptidases. These proteinases break bonds in the interior of protein molecules and polypeptide chains.

2 Exopeptidases. These proteinases break of amino acids sequentially from the ends of proteins. These enzymes are often used to determine the primary structure of protein ie the amino acid sequence.

Fungal proteinases exhibit both exo and endopeptidase activity. Asperqillus orvzae Protease is a mixture of exo and endopeptidases.

2.1.1 Endopeptidases

Papain & Bromelin were used as endopeptidases in the following tests. These enzymes behave in a similar manner to trypsin, and chymotrypsin and therefore can be used as a representative class.

2.1.2 Exopeptidases

Crude preparations of bacterial and fungal proteinases exhibit both endo and exopeptidase activity. Crude fungal proteinase prepared from Asperqillus orvzae was used not because of its exo peptidase activity, but because it was a cheap alternative endopeptidase source to papain. Asperqillus Orizae proteinase is a much more active protease at room temperature. This has definite advantages in minimising production costs.

Proteinases react with protein and peptide substrates to produce low molecular weight peptides and free amino acids. It was theorized that these peptide hydrolysates would give berlies manufactured from marine organisms such as crabs, sea urchins, shrimp, marine worms, marine invertebrates and digestive organs of certain fish and animals that feed on Detritus (biological) such as fish, molluscs, crustacia, echinoderms and cephalopods and other non marine protein sources, superior fish attracting and feeding stimulant properties.

2.2 Protein Substrates

Two classes of protein substrates were chosen as representative of

the protei ns avai l able i n the biosphere namely:

2.2.1 Plant Proteins

(1) Wheat and more particularly wheat gluten as representative of cereal based proteins.

(2) Soy flour and more particularly soy protein isolate as representative of legume protein.

2.2.2 Animal Proteins

(1) Bovine meat meal and more particularly gelatin as representative of mammalian protein.

(2) Fish meal and more particularly mugil cephalus muscle protein as representative of fish protein.

(3) Shrimp protein as representative of invertebrate protein.

2.2.3 Protein Substrates Used

The following protein substrates were chosen as representative of their class.

(a) Sea Mullet, Mugil cephalus: chosen because it is primarily a detritus feeder. This fish was chosen because it grows to a large size, 3.4kg. Because of its size it is possible to fillet this fish carefully without cutting into its digestive tract. Cutting into the digestive tract would release bacteria and proteolytic enzymes which would react with muscle protein producing peptides. With due care it is possible to prepare fillets of mullet free of contamination. The composition of this muscle tissue is near enough to 100% protein.

Sea Mullet represent an ideal detrital feeder organism to use to prepare controls from muscle tissue which are not contaminated with enzymes or bacteria.

Shrimp were used as the substrate for experiments using proteolytic enzymes to enhance the performance of natural baits. The shrimp (Metapennaus Maclayii) were caught the same day they were treated.

It could be argued that the flesh of detrital feeders is what is attractive to fish. What is claimed is that it is not the flesh itself that does the attracting but the peptide hydrolyzates formed as a result of the action of digestive proteases on the flesh itself.

Experiments using uncontaminated mullet flesh and fresh shrimp as controls verify this hypothesis.

(b) Fish meal prepared from Sardinops neopilchardus was used as a representative ^' dried processed fish meal protein substrate.

(c) Meat meal prepared from beef byproducts was used as a representative animal protein substrate.

(d) Wheat flour was used as a representative cereal protein substrate.

(e) Soy flour as a representative legume protein substrate. Digestive juices contain both endo and exopeptidases. It would be expected that if it were possible to differentiate between the performance of the hydrolyzates then those produced by the action of Asperqillus orvzae would produce a stronger attraction and stimulus in fish. This is because crude preparations of bacterial and fungal peptidases exhibit both endo¬ peptidase and exopeptidase activity. Crude fungal protease prepared from / _. orvzae was used as a typical example of an exopeptidase.

3.0 RESULTS

3.1 Berlies (Chum)

Berley is by definition any material thrown into the water to attract fishsand to stimulate them to feed. The word Berley is synonomous with the word Chum in the US and the word Groundbait in the UK. Three types of experiments were conducted with protein hydrolysed berlies(Chums):

Type 1 Measuring the effect of polypeptides in berley mixtures by direct observation.

Type 2 Measuring the effect of polypeptides in berley mixtures by comparing angling catches.

Type 3 Measuring the effect of polypeptides in berley mixtures by placing them in aquaria.

In each set of experiments polypeptides produced stronger feeding stimuli to marine organisms than the controls.

It is also possible that PAPAIN SOY FLOUR digests elicit sexual behavior in Goldfish. This could be brought about by some polypeptides produced having similar structural components to Protamines which correspond to low molecular wt proteins in fish sperm (Mol Wt C:6000 Daltons) .

3.2 Baits

Fishing baits were also treated with proteinases to observe whether this treatment enhanced their performance.

In every test the treated baits outperformed the control baits in the number of fish caught, the number of species caught, and the total mass of

fish caught. 3.3 Tests 1n Aquaria

Goldfish were exposed to protein hydrolysates. In each case they exhibited a strong feeding response to the presence of protein hydrolysates, and became generally much more active than they were prior to the addition of protein hydrolysates. It was observed that the presence of protein hydrolysates enhanced the feeding response goldfish display when they are fed a proprietary goldfish feed.

Growth rates were measured for two goldfish in an aquarium. They more than doubled in bodyweight in 31 days at a conversion ratio of 2.7. This growth-rate supports the observation that the presence of these hydrolysates Increase the metabolic rate of these fish.

On the basis of the above evidence it would be safe to assume that the general statement; "the action of endopeptidases on suitable protein substrates produce hydrolysates which provide a positive stimulus to feed to marine animals." is correct. There have been no exceptions to this statement, (which would invalidate it), in the experimental results so far.

4.0 EXPERIMENTAL DESIGN

4.1 Preparation of Test Samples

A commercially available extract of papaya latex, crude type 1, 2.5 units per mg, was used to prepare the test samples. The active ingredient in this product is Papain.

The pH of' the reacting media was 7.0 * 0.5 for fresh fish flesh. A sample of fish flesh water mixture was prepared from fresh mullet flesh and water by blending the mixture in a domestic food blender. The hydrolyzate was prepared from this blended stock.

The fresh fish and 0.01 wt% enzyme were mixed together. The mixture was heated for two hours at 70°C. Then cooled to room temperature over about one hour. This was then adsorbed on extruded bran pellets and dessicated.

The fresh fish was caught the same day as it was processed. Extreme care was taken to minimize bacterial contamination. This was an essential step in preparing the negative control. All the mixtures were frozen to -10°C until ready to use.

All digests of papain were done for two hours at 70°C except for the flour and soy protein meals. These were digested at room temperature for three hours.

Digests prepared from 0.01 wt% A. orvzae protease (1 unit will react with casein to produce colour equivalent to 1.0 μ mole, tyrosine per minute at pH7.5 at 37°C) were carried out for two hours at room temperature, 20 - 23°C.

The following digests were carried out with Papain protease: Dried fish meal prepared from sardinops neopilchardus. Fresh fish meal - Mullet flesh. Wheat flour.

The following digests were carried out with / . orvzae protease: Dried fish meal prepared from Sardinops neopilchardus. Dried bovine protein meal. Wheat flour. Soy flour. 4.2 Test Procedure

The negative controls were tested first in the following manner:

(a) A Set & Forget Berley Dispenser was filled with fresh wheat Pollard, (Negative Control BRAN);

(b) The dispenser was placed at the test location and manually manipulated to produce a constant stream of bran particles;

(c) The test continued until a stable population of fish was observed;

(d) The number of fish of a particular species and the number of species was recorded:

⁽ e) The proximity to the berley source and the state of arousal were also recorded for each species;

(f) Steps (a> to (e) were repeated for a fish meal bran mixture as a second negative cor ^o .

This procedure s repeated for the various pollard protein hydrolyzate mixtures.

The recording procedure was carried out as follows. The investigator was underwater wearing a set of aqualungs and negatively weighted with lead weights. The investigator would observe the type and number of fish and their behaviour that were attracted by the berley under test. Once the fish population attracted to the berley was stable the investigator would leave the water together with the berley, write his observations and wait at least 15 minutes before repeating the procedure with the next type of berley. There was very little flushing and repeating control would only

produce a similar response to the last tested berley in this situation.

4.3 Preparation of Enzyme Modified Baits

The surface of peeled shrimp were coated with a mixture of salt, buffer, and Papain. The reaction was allowed to continue for a period of time, then the product was dried by osmosis and refrigerated.

This bait was then offered to fish as described in the examples.

INDEX OF EXAMPLES

1.0 ENZYME MODIFIED BERLEY EXPERIMENTS

1.1 Tests Carried Out At Seal Rocks

1.1.1 Observations in a Tidal Rock Pool

1.1.2 Berley Fishing at Seal Rocks

1.1.3 Tests carried out at one mile beach

1.2 Tests Carried out At Moonie Beach 1.2.1 Berley Fishing at Mooney

1.3 Fishing With Berlies and Polypeptide Berlies

1.4 Tests At South West Rocks

1.4.1 Experiments Conducted On The 25th May 1989

1.5 Comparing Soy and Wheat Flour Digests

1.6 Summary of Experimental Results, Experiments 1 TO 5 1.6.1 Comments on Enzyme modified Berley Experiments

2.0 FISH BERLIED USING SET & FORGET PEPTIDE BERLEYS

3.0 TESTING PROTEINASE BERLIES IN THE US

3.1 Discussion

4.0 Observation at Little Beach Nelson Bay NSW 10 - 1 - 1990 5.0 APPLYING PROTEOLYTIC ENZYME TECHNOLOGY TO BAITS.

5.1 Discussion

5.2 Method

5.3 Natural Bait Enhancement

5.4 Controls

5.5 Feeding Stimulant

6.0 EXPERIMENTAL RESULTS FOR ENZYME MODIFIED BAITS

6.1 Kiama

6.2 Bateman Bay

6.3 Boat Harbour

6.4 B1rub1 Point

6.5 Boat Harbour

6.6 Little Beach Nelson Bay

6.7 Summary of results

7.0 SIX EXPERIMENTS CARRIED OUT IN AN AQUARIUM

7.1 Experiment 1

7.2 Experiment 2

7.3 Experiment 3

7.4 Experiment 4

7.5 Experiment 5

7.6 Experiment 6

8.0 THE EXTRACTION OF GELATIN FROM BOVINE TENDONS AND THE

HYDROLYSIS OF THIS GELATIN WITH PAPAIN BUFFERED AT pH

8.5 USING PHOSPHATE BUFFER

1.0 Enzyme modified berley experiments

Example 1

1.1 Tests carried out at seal rocks

The test location was inside the northern headland at Seal Rocks on the Northern NSW coast. This is an oceanic location which is influenced by wave surge with a visibility of 20 feet. The visibility was limited by a fine red weed which was suspended evenly top to bottom. The water temperature was 19°C. Fish are generally not plentiful when this weed is abundant. It is a dangerous test site because sharks are prevalent in the area. The tests were conducted here because heavy rain and stormy weather had reduced the visibility elsewhere.

The berley compositions were tested in the order listed in Table 1 because the theory was that papain would react with fresh fish flesh faster, and produce a product with a potentially stronger feeding response to that produced by the action of the enzyme on dried fish flesh. It was for this reason that the processed fish product was tested before the fresh fish product.

Table 1 Results of tests.

Mixture Species Number Proxi ity Arousal common names in feet 0 least, 4 most

Negative Toad Fish 20 1 3

Control Wrasse red 2 3+ 1

Wrasse green 1 3+ 1

Papain & Toad Fish >20 0.0 4

Fish Meal Wrasse Red 4 2+ 4

(Dried) Wrasse Green 2 2 2

Blenny 1 5 3

Papain & Toad Fish >20 1 4

Fresh Wrasse Red 10 3+ 4

Mullet Wrasse Green 3 2 4

Flesh Blenny 1 5 3

Eel 1 0.0 4

Positive Toad Fish >20 1-7 4

Control Wrasse Red 10 0.0 4

Ground Wrasse Green 3 0.0 4

Sea Urchin* Pencil Wrasse 1 0.0 4

Eel 1 0.0 4

* Note Ground Sea Urchin is a very effective berley but is very expensive and therefore rarely used in practice. General Comments

The fish became so excited with the enzyme berlies that they began biting the investigator. It was necessary to drop the dispenser and retreat up current a safe distance. The fish were so aroused that they began chasing each other. Identifiable individual fish remained in the berley stream actively feeding for the duration of the tests.

It was surpri si ng that the berley composi tion was not as attracti ve to comb wrasse as expected .

EXAMPLE 2

1.1.1 Observations in a tidal rock pool

Fresh fish meal papain mixtures were sprinkled separately into a large tidal rock pooland the behavior of the marine animals was observed..

These berley compositions were not mixed with bran. An interval of at least 45 min was allowed before the next berley composition was tested.

The hydrolyzed fresh fish flesh had a mild stimulus for the fish and crustaceans but did not wet readily and this may have influenced its effectiveness. In contrast when the hydrolyzed processed fish flesh was used 45 minutes after the hydrolyzed fresh fish flesh, it proved to be very effective as it wet very well. The berley compositions were tested in the order listed in Table 2.

Table 2 Results of tests.

Mixture Species Number Proximity Arousal common names in feet 0 least, 4 most

Before Fish 1 NA 1 Adding Mixtures Fish A NA

Fish 1 10 NA 2

Papain Fish 2 1 NA 2

Fresh Fish A 1 NA 3

Mullet

Papain Fish 1 15+ NA 4

Processed Fish 2 4 NA 4

Fish Fish 3 2 NA 4

Fish A 1 NA 3

Shrimp 2 NA 4

Hermit Crab 1 NA 3

Fishes 1, 2, 3, are either blennies or gobies or the like. Fish A was a free swimming juvenile of a sea perch. Note that the Fresh Fish Papain mixture in this test gave a poorer result than the Processed Fish Papain mixture. This can be explained because the mixture floated on the surface of the water and not in it. Both mixtures had a significant and observable influence on the feeding behaviour of some marine animals in the pool.

Example 3

1.1.2 Berley fishing at Seal Rocks

The hydrolyzed fish flesh were combined into a mixture of loose and pelletised bran. This mixture was then used as a berley. The test location selected was on the headland (Seal Rocks) which is considered the best and safest fishing platform and where it had been observed that only one edible fish of legal size had been caught since Low Tide that day. The prepared berley was placed into position and within 30 minutes 5 bream of average weight of 21bs were caught.

From these results it is clear that proteases produce hydrolyzates which are strong fish feeding stimulants.

Example 4

1.1.3 Tests carried out at one mile beach

Location, Southern head at 1 Mile Beach, Nelson Bay, N.S.W. Test Conditions

Tide falling. Water Temp 21°C. Visibility 8 feet. Wind gusting to 20knots. Overcast. Rain Squalls. Two schools of pelagic fish sighted during the test. Comments

1. Normally garfish are satisfactorily berlied with Set & Forget Fishoil Technology Berlies, but at this location, and on the day, they were not attracted to this product.

2. The Papain, Soy flour product attracted clouds of small fish within 15 minutes.

3. The Set & Forget Fishoil Technology based berley was used in conjunction with the Papain Soy flour product. (It could be argued that the Fishoil product was putting the fish off the bite.) From the results this is clearly not the case.

4. The fish bit the bait freely after the Papain Soy product was placed in the water.

5. The test was terminated after 1 hr using the Papain Soy flour product as an appreciable increase in catch was obvious. Table 3 Results of tests. Bait Enzyme Hydrolysed Shrimp

MIXTURE SPECIES CAUGHT NUMBER TOTAL COMMON NAMES CAUGHT

Negative Crimson Banded

Control Wrasse

Set & Forget Weed Fish

Fishoil Tech Wirrah

Berley

2 Hrs Fishing

Papain Toad Fish Soy Flour Crimson Banded Berley Sausage Wrasse 3 Product , Plus Pencil Wrasse 2 Set & Forget Red Sea Perch 4 Fishoil Tech Mato 1 Berley Wirrah 1 1 Hr Fishing Weed Fish 1

Yellowtail 1

Garfish 7 22

Conclusion

Protein Hydrolysates produced by the action of Papain on Soy protein produced a stronger stimulus to feed than did Set & Forget Fishoil based berley on its own.

Example 5 1.2.1 Berley fishing at Mooney

This test was conducted in a tidal lagoon situated behind rocks at Mooney Beach, just South of Newcastle on the near north coast of New South Wales. It was high tide and the water temperature was 19°C.

There was moderate current about lO m/second which aided the

dispersal of the berley particles. Superimposed upon this slight current was a considerable surge, this made observations difficult as the fish followed the surge. TEST RESULTS Table 4 Results of Tests

Mixture Species Number Proximity Arousal common names in feet 0 least, 4 most

Negative Bream 15 3-8 1

Control Mullet >20 4-6 1

BRAN Whiting 3 4-8 1

Negative Bream >20 2-8 2

Control Mullet >20 3-6 2

BRAN Whiting 5 3-8 2

MULLET FLESH

Papain Bream >30 2-12 4

Fresh Mullet >30 2-12 4

Mullet Whiting 8 3-8 4

Flesh Tarwhine 1 4 4

Room Temp Black Drummer 2 6-12 3

Papain Bream >30 2-12 4

Bromelain Mullet >30 2-12 4

Fresh Whiting 8 3-8 4

Mullet Tarwhine 0

70°C Black Drummer 1 6 3

Newly hatched >30 4 4

Fry

After this test was completed the unused portions of the berley mixtures were pooled in a large plastic bucket and mixed with an approximately equal quantity of pelletised bran. This mixture was then

taken around the headland to where fishermen were fishing. They had not caught any fish in the three hours they were fishing.

The investigator then berleyed in the gutter where they were fishing. In the next half hour the investigator caught 4 large bream (average weight 950g) and then the investigator caught a black drummer of 475g. Another fisherman caught two sea pike during the same period. This indicates that the product stimulated fish to take a bait. SUMMARY OF RESULTS

It is clear from the table of results that hydrolyzates of protease treated fresh fish flesh provide a greater feeding stimulus to feed than does the untreated fresh fish flesh.

It was hoped to discriminate between the room temperature hydrolyzate and the digest at 70°C. The test procedure was unable to detect a significant difference between these samples.

The freezing process ruptures cell walls and spills their protein contents into the reacting medium during thawing. This may negate the advantages gained by heating. COMMENTS

Low molecular weight hydrolyzates may have to be separated from the larger peptides and tested separately.

Another approach may be to compare the performance of endopeptidases with exopeptidases. CONCLUSION

Although it was not possible to discriminate between the two digests, it was nevertheless possible to discriminate between the fresh fish bran control and the digest.

Both the room temperature digest and the 70°C digest produced a stronger sustained feeding response that the fresh fish flesh, bran mixture. This, together with the increase in fish feeding activity experienced in the half hour after proteinase berley was poured into the gutter, supports the hypothesis that protein hydrolyzates formed as a result of the action of proteolytic enzymes on protein substrates, stimulate fish to feed.

Example 6 1.3 Fishing with berlies and proteinase berlies

AIM: To observe the fish's response to traditional berley mixtures, and to hydrolyzed protein berlies when angling.

BERLEY MIXTURES: A commercially available meat tenderizing product,

Master Foods Meat Tenderizers, was used to prepare the test samples. The active ingredients in this product are papain and bromelain (in a ratio of

1:1) in the presence of salt thickener (wheat starch).

The pH of the reacting media was 7.0 ⁺ ~ 0.5 for fresh fish flesh.

A bulk sample of fish flesh water mixture was prepared from fresh mullet flesh and water by blending the mixture in a domestic food blender. Both hydrolyzates were prepared from this blended stock.

500g processed fresh fish flesh was reacted with 1.5 tablespoons of meat tenderizer at room temperature for 1 hour. The second fish flesh was reacted with the enzyme for two hours at 70°C and then cooled over about one hour to room temperature. Both preparations were then frozen.

Fresh bran, mixed with unreacted protein substrate was used as the negative control in these tests.

The fresh fish was caught the same day as it was processed. Extreme care was taken to minimize bacterial contamination. This was an essential step in preparing the negative control. All the mixtures were frozen to -10 ^β C until ready to use.

LOCATION: Mooney Headland approximately 150m NNW of the point of the headland. Water depth 10m. No discernable current.

TIME OF TEST: The test was carried out 14 May 1989 from 3pm till 5pm.

PROCEDURE: The two berley mixtures were mixed with an equal volume of compressed bran pellets to improve their vertical dispersion in the water column.

Three handfuls of berley were thrown overboard every five minutes for the duration of the tests.

RESULTS Table 5

TABLE OF CAPTURES 1 HOUR FISHING WITH CONTROL AND 1 HOUR FISHING WITH ENZYME HYDROLYZED FRESH FISH PROTEIN BERLEY

BERLEY SPECIES CAUGHT NUMBER TOTAL

Negative WRASSE Control SWEEP FISH + BRAN

BRAN + WRASSE 2

HYDROLYZED SWEEP 4

PROTEIN TARWHINE 1

PIKE 5 12

The control formulated from fresh fish and bran produced four fish during the first hour's angling. 3 wrasse and a sweep. During the second hour's fishing the berley containing the hydrolyzate was used. 2 wrasse, 5 pike,

1 tarwhine, 4 sweep were caught.

CONCLUSION

The berley containing enzyme hydrolyzed protein worked considerably better than the control at producing a feeding response in fish.

Example 7 1.4 Tests at South West Rocks

These experiments were carried out at South West Rocks, a small fishing village east of Kempsy on the northern New South Wales Coast. The aim of these experiments was to observe the results obtained from hydrolyzing various protein substrates with Asperqillus orvzae protease, and to compare these with papain hydrolyzates.

On the first day the tests were carried out on Bait Reef a small reef in 20ft of water just east of the Trial Bay Gaol breakwall. The visibility during this test was 40ft. The water temperature was 22-23°C.

The tests were commenced 30 min after high tide surge was moderate and the current was <0.2 knots.

The following samples were tested:

(1) Meat meal, Fish meal, bran mixture was used as the control.

(2) Meat meal, . orvzae protease, and bran.

(3) Fish meal, A. orvzae protease, and bran.

These hydrolyzates were prepared at room temperature as no suitable heating was available on location.

Table 6 Results of Tests

Mixture Species Number Proximity Arousal common names in feet 0 least, 4 most

Negative Bream >12 5-15 3

Control Sweep >20 3-8 3-4

BRAN Yellowtail 100 2-8 3-4

FISH MEAL Trevaly 1 5- 3-4

MEAT MEAL Maitos >50 3-8 3-4

Pike 2 5-8 3

Slimy Mackeral 20 5-10 3

Asperqillus ; Bream >30 3-10 4 orvzae Sweep >20 2-5 4 protease Yellowtail >100 0-12 4

MEAT Trevaly 1 5- 4

MEAL Maitos >50 3-8 4

Pike 5 5-8 4

Slimy Mackeral >30 1-12 4

Bon1to 8 8-15 4

Tarwhine 2 6 4

Moray Eel 2 5 4

Asperαillus Bream >30 3-10 4 orvzae Sweep >20 2-5 4 protease Yellowtail >100 0-12 4

FISH Trevaly 1 5- 4

MEAL Maitos >50 3-8 4

Pike 5 5-8 4

Slimy Mackeral >30 1-12 4

Bonito 8 8-15 4

Tarwhine 2 6 4

Moray Eel 2 5 4

The fish were keen to feed and this is reflected in the results. There was a slight increase in arousal with the protein hydrolyzates compared with the control. However there was no difference between the responses with the fish meal hydrolyzates and the meat meal hydrolyzates.

Identifiable individual fish remained actively feeding in the berley stream from the time they were recruited until the end of the tests.

It became apparent as the day progressed why this reef was called Bait Reef. Professional fishermen berlied up live bait of this reef. The fish were conditioned to come to the berley stream.

It was interesting to note that the two fishermen jigging for live baits were not catching fish until the investigator began berleying with protein hydrolyzates. The presence of these hydrolyzates stimulated the fish attracted by their berley to their bait jigs.

When the investigator stopped berleying, the other fishermen caught progressively fewer fish until they stopped catching Slimy Mackeral and Bonito altogether.

Because the fish on Bait Reef were conditioned to berley fishing it was decided to test the rest of the berley mixtures at a different location the following day.

Example 8 1.4.1

The aim of these experiments was to observe the effect of vegetable protein extract hydrolyzates on the feeding behaviour of fish.

Two substrates were chosen for these tests Wheat Gluten, and Soy Protein Isolate.

Hydrolysis was carried out at room temperature because there was no suitable heating equipment available.

Test location: On a small reef 450ft out from the caravan park beach at South West Rocks.

Depth: 10ft

Visibility: 20ft

Current: Nil

Surge: Moderate

Temperature: 22°C

Tide: Full at start of test.

The following fish species were visible prior to testing:

Yellowtail

Sweep

Bream

Blackfish

Maitos '

Table 7 Results of Tests

Mixture Species Number Proximity Arousal common names in feet 0 least, 4 most

Negative Bream 20 4+ 2

Control Yellowtail >50 3+ 2

BRAN Blackfish 15 6+ 2

FLOUR Sweep 15 3+ 2

Maitos 10 3+ 2

Papain Bream 20 4+ 2

FLOUR Yellowtail >50 3+ 2

BRAN Blackfish 15 6+ 2

Sweep 15 3+ 2

Maitos 10 3+ 2

Asperqillus Bream >30 2+ 4 orvzae Yellowtail >100 1+ 4 protease Blackfish >20 3+ 4

FLOUR Sweep 30 1+ 4

Maitos 10 2+ 4

Blennies 3 1+ 4

Wrasse 4 3+ 4

Asperqillus Bream >30 2+ 4 orvzae Yellowtail >100 1+ 4 protease Blackfish >20 3+ 4

SOY FLOUR Sweep 30 1+ 4

Maitos 10 2+ 4

Blennies 3 1+ 4

Wrasse 4 3+ 4

Asperqillus Bream >30 2+ 3 orvzae Yellowtail >50 2+ 4 protease Blackfish >20 3+ 3

MEAT Sweep 30 2+ 4

MEAL Maitos 10 2+ 3

Blennies 3 2+ 4

Wrasse 4 3+ 3

There was one bream attracted which had an obvious wound that had healed. This bream was noted actively feeding for the duration of the test.

The papain flour hydrolyzate performed as well as the control. A new brand of papain was used for this test and it would not dissolve. The product was supplied in a course granulated form. It was assumed that these granules would have dissolved but they did not. There would have been negligible reaction between the enzyme and the substrate. Effectively the papain flour mixture could be considered as another control.

The / orvzae protease digests were successful. A physical difference in the property of the suspension was evident at the end of the digest. These digests produced the strongest responses in fish observed to date using dried protein substrates.

A subjective evaluation would rank the / _ orvzae flour and soy digests as effective as the Mullet flesh papain/bromelain digests carried out at 70°C.

This result was predicated from the hypothesis on which these tests were based.

Example 9 1.5 Comparing soy and wheat flour digests

Table 8 Results of Tests

Mixture Species Number Proximity Arousal common names in feet 0 least, 4 most

Negative Bream 15 3-8 2

Control Mullet >20 4-6 3

BRAN FLOUR Whiting 3 4-8 3

SOY FLOUR

AsDerqillus Bream 15 1-12 4 orvzae Mullet >30 1-12 4 protease Whiting 3 2-8 4

SOY FLOUR Tarwhine 5 3 4

Room Temp Black Drummer 2 6-12 3

Sweep 2 3 3

Asperqillus Bream 15 1-12 4 orvzae Mullet >30 1-12 4 protease Whiting 2 3-8 4

WHEAT FLOUR Tarwhine 3 3 4

Room Temp Black Drummer 2 6 3

Sweep 2 4 3

Location Mooney Beach Lagoon

Water Temperature 19°C

Vi si bi l i ty 20ft

Depth 3ft

Tide 30 min after full tide

Current Slight

Surge Slight

There was not a marked increase in feeding response between the control and the digests. The difference is however statistically significant considering that the hydrolyzates consistently produce feeding responses in fish equal to or better than the control in every experiment conducted to date.

There was no difference between the performance of the Soy Flour digests and the Wheat Flour digests.

1.6 SUMMARY OF EXPERIMENTAL RESULTS

(1) In every test conducted to date the berlies containing the various protein hydrolyzates produced feeding responses equal to or greater than the negative controls used.

(2) Papain digests gave similar results to aspergillus digests only when the papain digests were carried out at elevated temperatures for two to three hours and allowed to cool naturally before freezing.

(3) Digests carried out with fresh fish flesh produced superior results with endopeptidases to those carried out with dried fish or meat substrates with endopeptidases.

(4) Vegetable protein digests approximated the performance effectiveness of fresh fish meal digests. This can be explained by the fact that wheat and soy flours are finely divided and have a high surface to weight ratio (high specific surface). It is important that solid protein substrates are crushed extremely fine so that the available surface is maximised;

For Soy Flour 85% should pass 250micron screen.

For Wheat Flour 85% should pass 150micron screen.

For Meat and Fish meals 75% should pass 250micron screen.

Reactions for solid substrates occur on the surface of particles: the greater the surface area, the more complete the reaction.

(5) Asperqillus orvzae protease produced superior results with vegetable protein substrates, dried meat meal substrates, and dried fish meal substrates, when reactions were completed at room temperature.

(6) One of the most important observations recorded was that certain identifiable fish remained in the berley stream actively feeding for the duration of the tests. (The impression given is that most of the fish recruited remain in the berley stream actively feeding. This is impossible to prove however if the fish do not have individual identifying marks).

(7) There were four occasions when the protease berlies were tested by anglers. On each occasion the anglers fished for at least one hour with or without the use of standard - traditional berley mixtures prior to using protein digest based berley products.

After using the protein digests berlies the fishermen in each case reported a dramatic increase in their catches compared with the previous

hour's catches. COMMENTS 1.6.1 COMMENTS

It is clear from the experimental data presented that proteinases react with various protein substrates and peptides to produce hydrolysates which produce a strong feeding response in fish.

Previous art has taught the efficacy of free amino acids for stimulating fish to feed.

If it were solely the presence of amino acids produced in the hydrolysates that were doing the stimulating then one cannot explain why such good results are produced by papain an endopeptidase (product predominantly polypeptides bellow 8,000 Daltons) . Being an endopeptidase papain produces few free amino acids as a result of its action on protein substrates. From the experimental results reported in this example, it is plain that papain markedly improves the fish attracting and stimulating potential of fish protein substrates. It is therefore clear that it is not the presence of amino acids which are doing the stimulating in the case of papain but it is the presence of low molecular weight polypeptides.

Further evidence to support this claim can be inferred from literature published regarding the effect of free amino acids.

1. The feeding stimuli reported for free amino acids and combinations of free amino acids are typically of between a few seconds and a few minutes duration at the most.

2. Mixtures of free amino acids are reported to produce stronger feeding responses than their constituents. These responses are still of short duration.

This must be compared with polypeptide responses reported in this submission. Fish are stimulated to feed for periods in excess of one hour using polypeptides.

It is clear from the experimental data presented that endopeptidases and exopeptidases react with various protein substrates and peptides to produce hydrolyzates which produce a strong feeding response in fish.

Endopeptidase fresh fish meal hydrolyzates produce comparable feeding responses to crude mixtures of Endo and Exopeptidase reactions on wheat and soy protein flour substrates. The protease activity of Asperqillus orvzae protease is much stronger than the papain and papain/bromelain proteases used in these tests.

To compare like with like, it is only fair to compare these hydrolyzates. Because the fresh fish meal/papain protease can be mixed intimately in a domestic food blender, the effectiveness of the enzyme is optimized and its hydrolyzing power optimized. The fresh fish meal/papain digests and the vegetable protein/A. orvzae protease digests produced comparable fish feeding stimuli. This demonstrates that It is the presence of polypeptides themselves which are producing the stimulus.

There is a strong synergism created when berlies based on protein hydrolyzates are mixed with berlies based on fish oil hydrolyzates. The mixed berley outperforms either constituent used separately.

Being an endopeptidase, papain does not produce free amino acids as a result of its action on protein substrates. From experimental results, it is clear that papain markedly improves the fish attracting and stimulating potential of fish protein substrates. It is therefore clear that it is not the presence of amino acids which stimulate in the case of papain but it is the presence of low molecular weight polypeptides.

Asperqillus orvzae protease is both an endo and exopeptidase source.

Exopeptidases produce a complex mixture of free amino acids from polypeptides. Some amino acids produce a short duration strong feeding stimulus in fish. One must contrast the short duration effects of free amino acids with the longer sustained responses produced by the presence of polypeptides. Use of this enzyme is preferred, not because of its exopeptidase activity, but because it is cheaper to purchase and easier to use. It is of fungal origin, it is much more effective on dried protein meals than papain or bromelain or trypsin.

Example 10

2.0 FISH BERLIED USING SET & FORGET PEPTIDE BERLEYS

Following is a list of marine organisms that have displayed feeding responses when exposed to peptide berley stimulants.

Anchovies Crabs

Blackfish Prawns

Blennies

Bream Cuttlefish

Butterfish Octopus

Catfish Squid

Cod

Coral Trout Tubeworms

Drummer

Eels

Filefish

Flathead

Garfish

Groper

Jewfish

Kingfish

Leatherjacket

Longtorn

Maitos

Morwong

Moses Perch

Mullet

Pike

Redfish

Slimey Mackeral

Snapper

Stingrays

Swallowtail

Sweep

Sweet!ip

Tailor

Tarwhine

Trevally

Tuna

Whiting

Wrasse

Yellowtail

Example 11 3.0 TESTING PROTEINASE BERLIES IN THE US

To enhance the performance of Fish Frenzy product 3% soy isolate and PAPAIN an endopepdidase was added. The product therfore had enhanced protease activity. This increased the amount of hydrolysates produced in the berley during manufacture. (Any Standard Text on the subject of protease production and activity will explain why one gets a dramatic increase in protease activity when extra protein is present.)

Papain produces a large proportion of di & tripeptides in its hydrolisates.

Therefore the product used in the following examples had an enhanced proportion of peptide hydrolysates in general, and di & tripeptides in particular.

This being the case it would be anticipated that this product should produce enhanced feeding responses in the fisheries in which the product was tested. Below is a summary of results.

Florida

Panama City, Fl . , August 6, 1989.- Fish Frenzy sausage format was used in to catch Speckled Sea Trout. The existing conditions represented an absolutely ideal forum for testing the effectiveness of Fish Frenzy.

Four fish weighing 13 1/4 lbs, and four fish weighing 5 1/4 lbs were caught.

This result is consistent with results achieved when using ^' the product in Australia.

Pompano Beach Fl . , August 12, 1989 . Fish Frenzy pellets were used while drift fishing in 120 feet depth. The target species were snapper. In the morning fishing was commenced at 07:45 there was only a very slight breeze; less than 1 knot, and no appreciable current for the first hours fishing. There were no fish caught until Fish Frenzy was used.

Then snapper and amberjack were caught until the boat was blown out of our berley trail. The fishing then slowed considerably. The product was again tested in the afternoon when the wind was between 6 & 12 knots. The only fish berlied were surface fish like ballyhoo and bonito. The boat was drifting too fast to successfully berley bottom fish.

Pompano Beach, August 13,. Canal Fishing three species of table fish were caught while using the Fish Frenzy pellets product.

Pompano Beach, Fl . , August 14 1989 - Charter boat fishing. Pellets used. Confirmed August 12 results.

Northeast

Newburyport, Massachusetts, August 19,20 & 23, 1989 - Charter boat bluefishing using predominately sausage format in conjunction with traditional chum (herring pieces) produced excellent results on two of three occasions. It was decided to test the product in the afternoons because it was reported that the fish were reluctant to bite in the afternoon. (It is not good practice to test Fish Frenzy products when fish

are biting freely.)

August 19 - No fish were caught in the first 40 minutes of conventional chumming. Fish Frenzy was then incorporated with the chum. Initially clouds of pollack were chummed to the boat, suddenly they disappeared and within a minute the bluefish began biting. In excess of 100 fish were caught in the next 2 hrs.

The fish went off the bite within 5 minutes of removing Fish Frenzy from the chum stream.

August 20 - No fish were caught in spite of heavy chumming with Fish Frenzy mixed with conventional chum . Water Temp was 4°F below the minimum temp for bluefish. There were no reports of any fish caught in the waters off the Isles of Shoals that day.

August 23 - A strong two to three knot current was flowing at the Isles of Shoals and it was generally difficult to hold fish around the boat under these conditions. Fish Frenzy berley sausages were put in the water the moment the boat was anchored. 15 minutes after anchoring the first fish was caught. The fishing became faster and faster until the boat was in the center of a large school of bluefish in a feeding frenzy. One hundred and ten fish were kept and fish well in excess of 100 were released Within minutes of removing the Fish Frenzy sausages from the water the fish went off the bite.

Newburyport, Mass., August 22, 1989 - Deep sea Bluefin Tuna fishing with Fish Frenzy sausage was extremely successful at berleying bait fish. The only strike that day was on a line to which a Fish Frenzy sausage was attached. No fish were caught.

New Jersey Coast, August 26, 1989 - Deep sea Yellowfin Tuna Fishing at "The Canyons". Fish Frenzy used to berley baitfish. Heavy trolling as a fishing style prevented the opportunity for Fish Frenzy to be realistically tested.

Lake Erie, Ohio, August 31, September 2 & 3, 1989 - charter boat fishing for Smallmouth Bass on Lake Erie. Fish Frenzy Sausage was used.

50 Smallmouth bass & in excess of 100 bugle mouth bass (sheepheads) were caught in three days fishing. This was accomplished during a period when the fishing was generally poor.

The waters of Lake Eire were so Dutrified that they appeared pea green and the fish may have been stressed by periods when DO levels were low.

Summary

All catches reported using Fish Frenzy where a comparison could be made, showed Fish Frenzy producing superior results.

Example 12 4.0 Observation at Little Beach Nelson Bay NSW

Visibility 15 feet

Depth 60 - 80 feet

Light poor (No color on video)

Tide high

Fish Bream & Matos

Water Temp C: 20°C (estimated)

On this occaision enzyme modified sausage was tested against crushed sea urchins.

A school of bream was found at about 80 feet.

The bream were not stimulated to feed when sea urchin was broken open. When the sausage was removed from a sealed plastic bag, the bream began feeding. They began to feed on both the sea urchin and the particles liberated from the enzyme modified sausage.

It was clear that the bream responded to the presence of feed inducing chemicals liberated from the sausage.

The important point to note from this observation is this. Fish Frenzy proteolytic enzyme technology on this occasion outperformed fresh crushed sea urchins as a fish feeding stimulant. This was the first time that the investigator had observed an "artificial" product outperform fresh pulped sea urchin.

Example 13

5.0 APPLYING PROTEOLITIC ENZYME TECHNOLOGY TO BAITS.

5.1 Discussion

Low molecular weight protein hydrolysates produced by the action of proteinases elicit strong sustained feeding stimuli in fish. It was therefore assumed that this technology would improve the performance of natural & artificial baits.

Artificial baits were prepared from gelatin.

Natural baits (Shrimp Metapennaus Maclayii) were coated with proteolitic enzymes and subsequently dried and refrigerated.

Two controls were used in these tests one was the untreated bait. The other was the BROWN BEAR BAIT COMPANY Prawn Flavoured Bait Eggs. Based on

Gel ati n .

The tests were carried out over a two month period. Fishing days were chosen when the fishing was likely to be poor. Weather and local fishing reports were taken into consideration when choosing the day. No target species were chosen before each trip, it was a matter of fishing for what was there.

5.2 Method

The artificial bait was prepared from 5% gelatin solution to which was added aspergillus orizae proteinase. This was incubated at 37°C for 8 hrs. The resultant product did not gel indicating that the enzyme had hydrolysed most of the protein.

It was noted that during the hydrolysis a strong sulphur smell could be detected above the digest. The sulphur smell was largely the result of the manufacturing process for gelatin (there is no cystine in collagen, therefore there should be no sulphur in the protein gelatin. Sulphur adducts on proteins make them unpalatable to fish).

The resultant liquid was boiled to kill the enzyme and 10% gelatin added. This product was then combined with wheat flour and molded into a bait.

5.3 Natural Bait Enhancement

Fresh shrimp were shelled, sprinkled with a mixture of papain and salt.

This was then preserved in a mixture of 50% flossy salt & 50% superfine sugar to which was added a few drops of vanilla and dill extract to improve the odor of the product and fluorescein. The treated shrimp were then placed on sheets of newsprint to dry. The dried product was then refrigerated until used. This product remained "fresh" for 24 hrs when removed from the refrigerator.

5.4 Controls

Fresh shrimp & BROWN BEAR BAIT COMPANY Prawn Flavoured Bait Eggs were used as controls.

5.5 Feeding Stimulant

Set & Forget Berley Pellets were used because they contained no protein digestates, only lipid based feeding stimulants.

Example 14

6.0 EXPERIMENTAL RESULTS FOR ENZYME MODIFIED BAITS

6.1 KIAMA 2 locations 2 controls

TABLE OF CAPTURES 1 HOUR FISHING WITH CONTROL AND 1 HOUR FISHING WITH ENZYME HYDROLYSED FRESH BAIT

BAIT SPECIES CAUGHT NUMBER TOTAL

Control

Shrimp Nil

No Stimulant Nil

Control

Bait Eggs Nil

No Stimulant Nil

Treated Sweep 13

Shrimp Wrasse 3

No Stimulant Black Drummer 1

Trevally 1 18

Control Bream Shrimp with stimulant

Method:

The same gear was used for all the tests. The baits were rotated Bait Eggs first cast, then the treated shrimp second cast, then the untreated shrimp third cast. This was done to remove bias due to time and tide. Comments:

The weather was overcast, wind from the south east, water gray green cold water upwelling from the continental shelf. No sea birds working. Only trash fish biting. No berley was required to stimulate fish to feed with treated shrimp. No bites occurred with or without stimulant using B.B.B.Co Prawn Flavoured bait eggs. Bream and Trevally caught only after heavy

berleying with Set & Forget Berley Pellets. It was observed that fish would bite treated shrimp more quickly than other baits. The bites were definite strikes and the fish were easy to hook.

Conclusion:

Treated Shrimp clearly outperformed the other baits.

6.2 BATEMAN BAY 2 locations 2 controls

TABLE OF CAPTURES 1 HOUR FISHING WITH CONTROL AND 1 HOUR FISHING WITH

ENZYME HYDROLYSED FRESH BAIT

BAIT SPECIES CAUGHT NUMBER TOTAL

Preserved Whiting Beach Worm Nil No Stimulant

Control Bait Eggs Nil No Stimulant Nil

Treated Sweep 17

Shrimp Mullet 3

No Stimulant Trigger Fish 1 Silver Drummer 1 Blackfish 1 Trevally 1 24

Control Sweep 6

Shrimp Wrasse 2

Set & Forget stimulant

Method:

The same gear was used for all the tests. The baits were rotated using Bait Eggs first cast, then the treated shrimp second cast, then the untreated shrimp third cast. This was done to remove bias due to time and tide.

Comments :

The weather was overcast, wind from the south east, water gray green with cold water upwelling from the continental shelf. Oceanic site. No sea birds working. Only trash fish biting. No berley was required to stimulate fish to feed with treated shrimp. No bites occurred with or without stimulant using B.B.B. Co Prawn Flavoured bait eggs.

One Whiting was caught off the beach adjacent to the rock on which the investigator was fishing. This fish was caught on preserved beach worms. It was observed that fish would bite treated shrimp more quickly than other baits. Conclusion

Treated Shrimp clearly outfished controls.

6.3 BOAT HARBOR 1 location 1 control RESULTS:

TABLE OF CAPTURES 1 HOUR FISHING WITH CONTROL AND 1 HOUR FISHING WITH ENZYME HYDROLYSED FRESH BAIT

BAIT SPECIES CAUGHT NUMBER TOTAL

Prawn Flavour Bait Eggs Nil Nil Nil

Treated Maitos 3 Shrimp Yellowtail 4

Sweep 8

Garfish 23

Wrasse 1 39

Natural Nil

Shrimp Nil

Bait Nil

Natural Yellowtail 1

Shrimp Garfish 11 with Matos 2 14

Set & Forget

Stimulant

Method:

The same gear was used for all the tests. The baits were rotated using Bait Eggs first cast, then the treated shrimp second cast, then the untreated shrimp third cast. This was done to remove bias due to time and tide. Comments:

The weather was stormy. Oceanic site. The water was alive with baitfish. No seagulls or surface fish working. Water clear warm. The investigator concentrated on catching bait fish.

The Brown Bear Bait Co Prawn Flavoured Bait Eggs were chosen as a typical artificial bait. The promotional information recommended them for use in salt water. At the conclusion of this test not one bite had occurred whilst using the bait eggs. Considering the number of fish caught using the treated shrimp it was decided to investigate why this product had failed to produce a bite.

Conclusion

The treated shrimp clearly outperformed the other baits used.

6.4 BIRUBI POINT 1 location 1 control

TABLE OF CAPTURES 1 HOUR FISHING WITH CONTROL AND 1 HOUR FISHING WITH

ENZYME HYDROLYSED FRESH BAIT

BAIT SPECIES CAUGHT NUMBER TOTAL

Control Nil

Prawn

Flavoured Nil

Bait Eggs Nil

Treated Garfish 27

Shrimp Yellowtail 3

Maitos 2

Sweep 5 37

Prepared Nil

Artificial Nil

Bait Nil

Untreated List in transit Shrimp NA NA

Method:

The same gear was used for all the tests. The baits were rotated using Bait Eggs first cast, then the treated shrimp second cast, then the artificial bait which was prepared from gelatin. This was done to remove bias due to time and tide.

Comments

Weather conditions were again stormy. Strong winds constantly changing direction. Oceanic site. No seabirds working. No surface fish chopping. Plenty of baitfish visible. No fish were caught on either the B.B.B. Co Prawn Flavoured Bait Eggs, or the artificial bait. Control prawns lost. Other fishermen in the vicinity using a variety of baits did not catch a fish during the test. Conclusion

Treated shrimp clearly outperformed the other baits used. 6.5 BOAT HARBOR 1 Location 1 control RESULTS:

TABLE OF CAPTURES 1 HOUR FISHING WITH CONTROL AND 1 HOUR FISHING WITH ENZYME HYDROLYSED FRESH BAIT

BAIT SPECIES CAUGHT NUMBER TOTAL

Brown Bear Nil Bait Eggs Nil Control Nil

Prawn Whirras 3 Control Wrasse 2

Treated White Ear 1 Shrimp Wrasse 1 Sweep 10 Eel 1 13

Method:

The same gear was used for all the tests. The baits were rotated using Bait Eggs first cast, then the treated shrimp second cast, then the untreated shrimp third cast. This was done to remove bias due to time and tide. Comments

Clear fine day, moderate northeasterly breeze, water stained with lignum from recent flood. Oceanic site. Small baitfish in close to

rocks. Birds working on current line C:600m off shore. Fish were reluctant to bite until Set & Forget feeding stimulant was used. No fish were caught on bait eggs but 12 bait eggs were bitten off the hook. This was the first time fish attacked this product. They did so only after heavy berleying with Set & Forget pelletised fish feeding stimulant.

Conclusion

Treated Shrimp again was the superior bait.

6.6 LITTLE BEACH 1 Location 2 controls

RESULTS:

(6) TABLE OF CAPTURES 1 HOUR FISHING WITH CONTROL AND 1 HOUR FISHING

WITH ENZYME HYDROLYSED FRESH BAIT

BERLEY SPECIES CAUGHT NUMBER TOTAL

Prawn Control Bream 2 2

Treated Bream 5

Shrimp Puffer 1 6

Method:

The same gear was used for all the tests. The investigator fished exclusively with treated shrimp. Comments

Estuarine site. Night fishing. Commenced low tide. Target Species Bream (MiHo sp.). No fish caught for the first three hours fishing. (Plenty of bites from small fish. ⁾ The big fish began to bite at c:23:00 hrs. This was then chosen as the start of the test. The investigator fished till 12:15. The above table summarises the results. Observation

More bites were felt, more baits were lost, and more fish were caught using treated shrimp. 6.7 Summary of Results for Experiments Designed to Measure the Effect of

Proteolitic Enzyme Activity on Natural & Artificial Baits

(1) The Brown Bear Bait Co Prawn Flavoured Bait Eggs did not appeal to the fish recorded in these experiments.

(2) The artificial bait manufactured, based on gelatin (the same

substance used to manufacture the B.B.B. Co bait eggs), proved unsuccessful as a bait in these experiments.

(3) Fresh Shrimp was effective as a bait in Experiments five & six. Fresh shrimp used in conjunction with Set & Forget Berley Pellets was successful as a bait in Experiments 1, 2, 3, and 4.

(4) Proteinase modified shrimp bait was successful without added stimulant in experiments 1, 2, 3, 4, 5,and 6.

(5) There were 3 edible fish of legal size caught in Experiment 6.1 There were 24 edible fish of legal size caught in Experiment 6.2. There were 23 edible fish of legal size caught in Experiment 6.3. There were 27 edible fish of legal size caught in Experiment 6.4. There were no edible fish of legal size caught in Experiment 6.5. There were 2 edible fish of legal size caught in Experiment 6.6.

In other words there was 79 edible fish caught using enzyme modified shrimp bait over the duration of the test. No extra stimulants were required to achieve this.

Example 15

7.0 Six experiments carried out in aquaria. Discussion

A large production run of proteinase beriey sausages using soy flour as an added protein substrate was manufactured.

The product was tested on two goldfish (Carassius auratus) in a twenty litre aquarium. The water was oxygenated by a pump and a porous stone suspended halfway down the tank. The fish were customarily fed a proprietary goldfish feed at irregular intervals.

The following two experiments were designed to observe the effect of the new enzyme enhanced berley sausage has on these fish.

The first experiment was conducted three hours after they were fed. The second experiment 12 hrs after these fish were fed (3 days after the first test).

7.1 Experiment 1. Carried out at 10:15 Method

The fish were observed from a distance prior to introducing a small piece C: Igrm of enzyme modified protein sausage. The experiment was not started until the fish were observed to be "resting" before starting the experiment. This piece was then introduced into a nearly inaccessible position behind an ornament in the tank. The fish were then observed and

their behavior noted.

Observations

(1) Activity prior to the introduction of enzyme modified material. The fish were quietly hovering about 8 centimeters above the bottom of the tank. Occasionally one or both would circle at the same depth, then return to its previous position and hover motionless.

(2) 27s. After I added the enzyme sausage the smaller fish began to swim into the glass. When it touched the surface, it slowly kept swimming as though it was trying to swim through the glass. This behavior seemed inexplicable.

(3) The larger fish began swimming in circles, smoothly at first and then more erratically. It then stopped in the middle of the tank, erected all its fins, remained in this pose for a few seconds then darted toward the smaller fish and bumped it on the side.

(4) The larger fish kept up this behavior of circulating for a while then aggressively bumping the smaller fish. All this time the smaller fish kept up its behavior of trying to swim through the glass, slowly and deliberately.

(5) 175s. into the test the larger female fish began to exhibit feeding behavior. Simultaneously the smaller fish stopped slowly trying to swim through the side wall of the aquarium and began actively feeding.

(6) This feeding behavior could best be described as vigorous, every 25 to 30s the larger fish would swim to where the smaller fish was feeding and bump it out of the way.

(7) Approximately 350s into the test the fish appeared to be in a regular active feeding mode. At this point I introduced some flaky goldfish food carefully on the surface of the water in the tank.

(8) Goldfish must be preprogrammed to feed off surfaces at or near the bottom. Although there was goldfish food on the surface of the tank the fish appeared unaware of this food.

(9) As the flakes would hydrate they would sink. At first the fish would only recognise this food when it had settled on the bottom.

(10) 400s. into the test the fish began to recognise the flaky food some 80 to 100 mm from the bottom. Still their attention was directed downward.

(11) 450s into the test the fish became aware of the goldfish food, and attacked it ravenously, repeatedly striking the surface like a bass.

(12) 15 minutes after the enzyme modified sausage fragment was added the fish were still actively feeding, even though there was no food apparent; i.e. their behavior was not being rewarded.

Comments

Three distinct behaviors were noted. An initial avoidance response from the smaller fish, an initial aggressive response from the larger fish, and finally an aggressive sustained feeding response from both fish. Conclusion

The combination of Papain & Soy Protein have proved to produce extremely effective feeding stimulus. 7.2 Experiment 2

The second test was conducted in the same manner as the first.

The test was commenced at 21:00 hrs 19 2 90 Observations

(1) 20s after the fragment of sausage was introduced into the tank, the larger fish exhibited aggressive behavior. It began chasing the smaller fish around the tank.

(2) This behavior continued for about 600s. Both fish stopping from time to time to "feed". Except for the accessible particles of Enzyme modified sausage there was no food for them to eat.

(3) 15 minutes after the stimulant was added, the same goldfish food was placed on the surface carefully so that it would float.

(4) The fish within seconds began feeding actively on the goldfish food.

(5) 40 minutes after the introduction of the sausage the fish were still aroused.

⁽ 6) It was impossible for the observer to approach the tank because they recognised his shape and would swim to the glass and look at him in an agitated and excited manner. At a distance of 7 feet they still noticed him and his presence influenced their behavior.

(7) In order to observe them without influencing their behavior, the observer tried lying down near the tank but this did not work.

(8) It was noticed that both fish were defecating.

(9) The smaller fish was observed attacking the larger fish around the gill area. Careful observation revealed some damage to the scales of the larger fish in this area.

⁽ 10) At 0700 hrs on the 20 2 90 the observer tried to approach the

tank to observe the fish. At a distance of C: 7 feet they recognised his shape and altered their behavior pattern, swimming to the front surface of the glass and observing him.

(11) At 14:00 hrs. The observer again tried to approach the fish tank to observe their behavior. They recognised his shape at a distance of 5 feet and swam to the front to observe him. The smaller fish darting from side to side actively anticipating a feed.

(12) At 14:40 the fish were fed again and their response to the goldfish food was an aggressive feeding response. A much stronger response than exhibited at feeding times prior to the commencement of these tests.

At this point it was decided to prolong this test to observe what would happen if repeated doses of stimulant were added.

(13) At 15:00 on the 20 2 90 some more stimulant (enzyme modified product) was introduced . The larger fish again began actively chasing the smaller fish and ignored the presence of the observer who was less than 2 feet from the aquarium. The response may be sexual and not blatantly aggressive. The larger fish was attempting to swim under the smaller fish and brush its ventral regions. These results also coincide with the observations of the arousal of the fish in tests carried out at seal rocks in Example 3.

(14) 5 minutes later i.e. 15:05 the larger fish began passing fecal matter. There was a lot of fresh fecal matter visible on the bottom of the tank.

(15) The fishes movements were much faster and more deliberate than before the stimulant was added.

(16) 10 minutes after the introduction of the stimulant the fish still ignored the approach of the observer to the tank. They were still more interested in feeding and chasing each other around the aquarium.

(17) Twice more stimulant was added 1 gram at a time.

(18) The fish were then left alone for 18 hrs.

(19) When the observer again approached the tank the fishes response was to initially swim to the glass, but then instead of anticipating a feed, they swam slowly to the far end of the tank and tried to swim through the glass in an avoidance response. They were not fed. END OF TEST Conclusion

The fish are extremely aroused by putting a gram of enzyme modified sausage into their tank. They display aggressive feeding responses and

possibly sexual responses to the presence of the protein hydrolysates. This arousal can be induced at least 5 times in a twenty four hour period.

At the end of the test the water was very cloudy possibly sullied by fecal material.

7.3 Experiment 3

Growth Rate Test on Goldfish

Initial Volume (No accurate scales) 27ml for large goldfish. Initial Volume (No accurate scales) 17ml for small goldfish.

During the test the fish were rested for one week. During this time they were fed three times a day.

A total of four cans of fish food were used, (Excelpet Gold Fish Food in 25grm packages).

Three different stimulants were used. All three were endopeptidase protein digests using papain bro elin enzymes.

120 grams of protein modified berley sausages were used during the test. This was available for the fish to eat.

During the test two temperature excursions occurred one to 16 ^β C and one to 27°C. Both were for at least six hours duration. The low temp excursion happened during the resting period.

Final volume of fish was 80ml for larger fish and 45ml for the smaller fish.

Total increase in biomass of fish = 125 - 44 81ml Total amount of added food = lOOg goldfish food + 120 grams of protein modified berley sausage. 220grms average protein 18%. Conversion ratio C.3:l

7.4 Experiment 4 Papain Digests in Gels

The Aim of this test was to ascertain whether it was possible to incorporate papain digests into a vegetable gel. Goldfish were chosen for this experiment because a good database was available for goldfish.

Digestate was prepared from papain/soy milk digest. Arrowroot was used as the vegetable gel. Method

2 Grms. of gel was spread on the inside surface of the aquarium 2" under the surface. The goldfish were then observed. Results:

The goldfish displayed the same behavior as that reported for the digestate alone.

Conclusion

The hydrolysates in the gel diffused into the water raising the level of activity of fish. 7.5 Experiment 5 Digestates in Gel Australian Bass, and Murray Cod in fresh water, Gel stimulant.

A papain, soy flour digest was heated with arrowroot to form a gel. The consistency of the gel was altered by adding digestate until a satisfactory consistency was attained. The product was then heat sterilized. Method

6Grms. of gel were spread over the inside face of an aquarium (3301.) containing a Murray cod fingerling and a juvenile bass. Any behavioral changes in these fish were noted. Results

Prior to the addition of the gel the fish had remained stationary for 10 minutes. 60 seconds after the addition of the gel both fish began aggressively feeding. This response was maintained for at least 14hrs. (Observations concluded) Conclusion

Bass & Murray Cod are stimulated to feed under the influence of the chemicals liberated from the gel. This demonstrates that protein hydrolysates stimulate fresh water predatory fish. Again this supports the findings that the product increased the catch of Smallmouth Bass in the Lake Erie fishery 7.6 Experiment 6 Bromelain/Albumin Hydrolysate Tests in a Saltwater Aquarium.

Tests were conducted on tropical saltwater fish in two aquariums one a 200gal tank, and the other a 120 gal tank. In these tanks were a variety of small saltwater tropical fish and shrimp. 3 species of chaetodon, 3 species of wrasse, 2 species Pomacentridae, one trigger fish, a banded coral shrimp, and a pistol shrimp.

These fish were observed prior to the addition of hydrolysate (egg albumin/Bromelain) Their activity was rated from a distance so as not to influence their behavior. On a scale of 1 to 4 the fishes general state of arousal was rated at 2 for the butterfly fish, 2 for the damselfish, 3 for the wrasse and 1 for the shrimp.

After the addition of the hydrolysate the fish were again observed.

The coral shrimp began walking around the tank, threatening the fish with his chella. The fish began actively swimming, attacking the other fish if they swam too close, and searched for food. The pistol shrimp who lives in a network of burrows in the sand on the bottom of the smaller aquarium, began exploding through the surface of the sand, diving into a burrow and then bursting out of the sand again.

The activity of all the animals in the tank would have to be rated 4 plus after the addition of hydrolysate.

Example 16 8.0 THE EXTRACTION OF GELATIN FROM BOVINE TENDONS AND THE HYDROLYSIS OF

THIS GELATIN WITH PAPAIN BUFFERED AT pH 8.5 USING PHOSPHATE BUFFER DISCUSSION

Artificial baits prepared from a gelatine free from contaminants was made as follows. A sufficient quantity of collagen tissues was carefully cut from the shinbones of a cow to manufacture enough gelatine for test.

Once the collagen was converted into gelatin it was hydrolysed at 52 ^β C for 5 hours with a mixture of Papain & Bromel in buffered to a pH 8.5. Gelatine Hydrolysate Experiment

The hydrolysate was then cooled and two drops were poured into an aquarium and its effects on the behavior of two goldfish therein were noted.

The goldfish displayed the same behavior as that described for the enzyme modified sausages. If one scaled the intensity of the stimulus elicited by the enzyme modified sausage at 10; then the behavioral response of the goldfish exposed to the gelatine hydrolysate would be scaled at 8.5-9.0.

This test was repeated in another aquarium with 5 goldfish. These five goldfish exhibited the same behavior as the other two when exposed to the hydrolysate, (the goldfish in this tank had not been exposed to protein hydrolysates before).

Both sets of goldfish continued actively feeding for at least 60 minutes even though their activity was not being rewarded with food. After an hour further observation was considered academic as stimuli reported in the available literature describes feeding activity ceasing after just a few minutes.

Observations of Two Goldfish over Two Week Test Period. Metabolic Rate Observations:

Gill beats increase 20 seconds after administration of Papain digests. Feeding behavior increases; general activity, swimming, chasing

increases.

Production and passing of fecal matter increases. Duration of Increased Activity

This increase in metabolic rate is sustained for a minimum of 1 hr after the stimulant was added. Habituation:

Eight additions of stimulant were added in one day. The only decrease in response appeared to be due to fatigue i.e. the same behavioral responses were exhibited after each addition, peaking at the third addition and then tapering off by the eighth addition. Growth Rate

The fish increased in overall dimension by approximately one third during the two week test period. General Appearance.

The fish look healthy, active, with no visible signs of distress noted as a result of repeated stimuli. Their swimming activity appears swifter more definite characterised by sharp darting movements. Their feeding response is now more aggressive.

Example 17 Table of Results of tests. Swansea Channel, NEWCASTLE under old road bridge.

Mixture Species Number Proximity Arousal common names in feet 0 least, 4 most

Enzyme Whitebait 1000+ 0-15 4

Modified Sweep 100+ 0-10 4

Protein Schnapper 8? 5-15+ 3

Sausage Wrasse 10 3-15+ 4

Wrasse 2 4

Leatherjacket 30+ 4

Oldwife 5 3

Bream 100+ 3

Tarwhine 10? 3

Fortescue 30+ 4

Eels-Moray 2? 8-10 2-3

Conditions:

Weather, clear. Wind to 15 knots. Tide full and just turning, water temperature 23C, current to 1/2 knot. Visibility c:18 feet, depth 20 feet. Comments

No schooling or aggregations of fish at the test site prior to testing. 10 minutes after commencing test it was impossible to take a census of fish attracted as the clouds of fish reduced visibility to less than three feet. Attempts were made to swim through the schools of fish attracted to take a census but this disturbed the feeding fish and no accurate census could be performed. A trial at the same site with a control was not attempted. Again the investigator was nibbled by fish while carrying out this test. Conclusion

This was the first time the effects of an enzyme modified sausage was observed underwater. It attracted clouds of feeding fish, recruiting bream and tarwhine from deeper water. This was the most fish attracted to a test location to date. The enzyme modified sausages appear to be more effective at attracting and stimulating fish to feed than other products.

Example 18 Observations on the Effects of Exposing Recently Caught Fish to the Effects of Low Molecular Weight Polypeptides. Discussion

Following tests using protein hydrolysates to stimulate goldfish, the following rather unexpected reaction was exhibited by goldfish after repeated additions of enzyme hydrolysates. The aim of the experiment was to see if the investigator could repeatedly stimulate the goldfish to feed, when their elicited feeding behavior was not being rewarded with food.

Initially it awas observed that the goldfish were excited by the presence of the investigator moments before the stimulant was introduced. (The moment the polypeptide stimulant was administered the fish would ignore the presence of the investigator and actively feed and chase each other around the tank).

After repeated stimulations a novel behavior was noticed . Instead of the fish recognizing the observer and swimming back and forth excitedly for him to add some more stimulant, as soon as they recognized the observer, they swam to the far end of the tank and tried to swim through the glass, in an obvious avoidance response.

Why should they suddenly exhibit such a different response when the observer approached?

Repeated stimulations that are accompanied with feeding never elicits this fear response; on the contrary the fish get more excited by ones presence.

In order to explain this seemingly aberrant behavior, it was postulated that the presence of the low molecular weight polypeptides may have other effects than just triggering a feeding response. Careful observation of fish that have been exposed to the stimulus for the first time revealed the following.

The general activity of the fish after the addition of polypeptides had increased.

Their breathing rate increased.

They became more physically active, they sometimes exhibited

"aggressive" behavior towards other fish in the tank, and they feed more keenly and for long periods of time.

It was highly probable that the presence of these low molecular weight polypeptides was also increasing the metabolic rate of the fish. A sustained high metabolic rate without nourishment must eventually stress an organism, depleting its energy reserves.

The goldfish now associated the presence of the observer with stress, hence the avoidance behavior. b) Experiment

To test the theory that polypeptides raise the metabolic rate of fish, without using expensive tests and equipment, it was decided to observe the effect of polypeptides on fish that were exhibiting stress responses.

One snapper and three sweep were caught and placed into a large saltwater aquarium. The snapper was obviously stressed.

15 grams of enzyme modified polypeptides were added to the tank and the behavior of the fish noted.

Feed was also added to the tank (peeled Shrimp). One sweep began to feed, the other two sweep began swimming around and around the tank. The snapper however displayed extreme stress response 20 seconds after the polypeptides were added. 3 minutes after addition of the stimulant the snapper was so exhausted by its activity that it lay on its side in the far corner of the tank.

The enzyme product was siphoned out from the tank and replaced by a large volume of the water in the tank. The snapper slowly recovered. c) Conclusion

The addition of the polypeptides significantly enhanced the activity of the fish. The response it elicited from the snapper exhausted the fish.

These responses could best be described as general arousal. They were not feeding responses as three of the four fish ignored the shrimp altogether.

The presence of the polypeptides in this experiment increased the general activity of the organism (not a feeding response).

The evidence is pointing strongly toward the conclusion that the addition of polypeptides also raises the general metabolic rate of fishes.

Observations in Aquaria containing Crustacia & Fish. Discussion

It was observed that hydrolysates prepared from a mixture of proteins; egg albumin and soy isolate, produced a strong stimulus in Crustacea.

Up until this time egg albumin had not been used in testing. On the other hand soy protein isolate had been tested repeatedly in experiments in aquari .

It was decided to prepare hydrolisates from egg albumin and compare these with hydrolysates made from soy protein isolate.

Bromelain was used as the proteinase in these experiments.

Three aquaria were used for these tests, two saltwater aquaria and a fresh water aquarium. The saltwater aquaria contained small tropical fish, coral shrimp, and pistol shrimp. The freshwater aquarium contained a bass, a murray cod fingerling, and 3 large freshwater crayfish. Procedure

No controls were used. Instead the behaviour of the aquarium fishes were observed for twenty minutes before the addition of hydrolysates. The tests were conducted three hours after the fishes morning feed in the saltwater tank. Live food was used in the freshwater tank; the tests in this tank were carried out at 10:00 hours.

The tests were carried out on consecutive days. Normally a couple of days at least were left between tests. However, this was not practical due to limited access to the aquaria.

The fish in the saltwater tank were always fed in the right hand

front corner of the tank. Fish would congregate here when one approached the tank. There was no such bias in the behavious of the freshwater fish.

In the saltwater tank hydrolysates were added to the left hand front corner of the tank. Test Results

Table of Results of tests carried out in two saltwater aquaria containing Fish and Crustacians.

Mixture Species Number Arou! >al Before Arousal+Stimulant common names Test 1 to 4 1 least, 4 most

Bromelain Wrasse 5 3 4

Soy Protein Butterfly Fish 5 2 4 Aggressive

Isolate Damosells 3 3 4

Trigger Fish 1 1 4

Surgeon Fish 1 3 4

Coral Shrimp 2 1 1

Pistol 1 1 1

Bromelain Wrasse 5 3 4

Egg Albumin Butterfly F1sh 5 2 4 Aggressive Damosells 3 4 Trigger Fish 1 4

Surgeon Fish 1 3 4

Coral Shrimp 2 1 4 Aggressive

Pistol Shrimp 1 1 4

The investigator had never before observed the coral shrimp come out his hiding place and march all over the bottom of the tank threatening any fish that came near with his chella.

The pistol shrimp is never visible above ground in the daytime.

After the addition of the egg albumin hydrolysate he continually burst through the sand surface, dived down a burrow and burst through the sand again. This alarming behaviour lasted for 70 seconds. Conclusion

Egg Albumin Hydrolysates had a far greater stimulus on Crustacea than did soy hydrolysates.

Discussion

Following this surprising result, it was decided to test soy isolate hydrolysates and egg albumin hydrolysates on fish and Crustacea in freshwater to see if a similar result could be observed.

Table of Results of tests carried out in a freshwater aquarium containing Fish and Crustacians.

Mixture Species Number Arous ;al Before Arousal+Stimulant common names Test 1 to 4 1 least, 4 most

Bromelain Murray Cod 1 3 4

Soy Protein Australian Bass 1 1 4 Aggressi e

Isolate Freshwater Cray 2 1 1

Bromelain Murray Cod 1 3 4

Egg Albumin Australian Bass 1 1 4

Freshwater Cray 3 1 4

Again it was observed that the egg albumin hydrolysate stimulated crustacia. It appears that different hydrolysates elicit different behaviour in different fish. Below is a list of "unusual" behaviour observed during the testing so far.

(1) Out of season sexual behaviour in goldfish (Papain Gelatin).

(2) Moray Eels leaving their holes/crevices and wrapping themselves around dispensors. As many as four have been observed "cuddling" a dispensor at one time. (Papain Soy Isolate).

(3) Crustacea responding to egg albumin isolates (Egg Albumin, Bromelain).

(4) Increased growth rates and general metabolic activity in goldfish (Papain Soy Isolate).

It is apparent from these observations that a "spectrum" of responses can be elicited from fish and Crustacea by varying reaction conditions. It is simplistic to state that hydrolysates elicit strong feeding responses per se.

The most accurate general statement concerning the effect that enzyme hydrolysed proteins have on fish and crustacia is this. Low molecular weight polypeptides, produced from protein by the action of endopeptidases,

el i ci t a spectrum of responses i n aquati c organi sms .

Example 19

Preservation of Digests

It is important to be able to preserve digests for subsequent use. Three methods of preservation were tried. Refrigeration: Freezing to -10°C Reducing Water Activity: Heat Sterilizing: Freezing:

This has proved a successful means of preservation. The first set of experiments conducted to support this patent application were preserved by freezing. The major drawbacks are:

(1) It requires a freezer for storage and transport.

(2) Any gel products manufactured using this technology cannot be frozen as freezing destroys the gel structure.

Reducing water activity by dessication or incorporating humectants.

Most suitable humectants, be they clays, or carbohydrates compromise water quality in impounded waters and aquaria.

For this reason reducing water activity has limited use, except in drying digestates after adsorption on matrices.

All solid berleys/chu s/fish feeding stimulants used in the experiments reported in this application were biologically stabilized by drying at 50°C.

Adsorption and drying at 50°C has proved very satisfactory. Heat Sterlizing

Autoclaving digests at 110°C destroys their fish stimulating properties. The tests which gave neutral results were those where the digests were heat sterilised. They represent a discreet subset of results, and with those digests carried out with inactive proteases, represent the only tests that produced neutral results.

It was also noted that digests performed with native protein- undenatured protein, gave superior results to those digests carried out with denatured protein. It is theorised that a certain amount of tertiary structure is maintained in polypeptides produced by the enzyme hydrolysis of protein substrates. It is the maintenance of this tertiary structure that accounts for the superior stimulus provided by products based on enzyme hydrolysed proteins.

INDUSTRIAL APPLICABILITY

A berley composition of the invention can be readily utilized to attract fish to any fresh water or sea water location. The technology of enzyme hydrolyzed protein and/or polypeptides to produce hydrolyzates can be incorporated in marine feeds and fish attracting and feeding stimulant products. Such products as aquiculture feeds, baits, berlies, chums, fish lures and scents.