AU2021218018A1

AU2021218018A1 - Feed Compositions and Uses thereof

Info

Publication number: AU2021218018A1
Application number: AU2021218018A
Authority: AU
Inventors: Christopher Guy Carter; Mohamed Basseer CODABACCUS; Quinn FITZGIBBON; Gregory George Smith
Original assignee: Ornatas Pty Ltd; University of Tasmania
Current assignee: Ornatas Pty Ltd; University of Tasmania
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2023-03-02
Also published as: AU2022331633A1; WO2023019302A1

Abstract

The present invention relates to feed compositions for aquatic species and uses thereof.

Description

FEED COMPOSITIONS AND USES THEREOF

Field

The present invention relates generally to feed compositions for the culturing and development of invertebrates and vertebrate aquatic species, such as crustacean species.

Background

A practical understanding of the key nutritional requirements for complete development of invertebrates and vertebrate aquatic species (such as aquatic crustacean and lobster species) presents a major challenge for the development of a commercially successful aquaculture venture. An example of this is the tropical rock lobster (TRL), Panulirus ornatus, which in recent years has commanded high market prices mainly due to increased demand from consumers and shortages of wild stocks (due in part to over fishing). These crustacean species possess many favourable attributes for the commercial aquaculture industry yet also presents many challenges due in large part to the nutritional demand placed on the species undergoing successive moulting cycles and morphological changes. To date, there have been no successful commercial feeds available for growing juvenile lobsters. Thus, the growout (culture) of the lobster (TRL) as practised in South-East Asia, particularly in Vietnam, relies heavily on fresh seafood such as fish, and other crustaceans and molluscs as the primary source of nutrition. This is not sustainable and commercially viable in an aquaculture setting.

Prior to attaining juvenile stages Palinurid decapod crustaceans have a complex life cycle which is characterised by a series of moults and morphological transformations that occur throughout the pelagic larval stage. Up to 24 morphological increments have been documented for P. ornatus and these are typically divided into 11 distinct stages via the identification of specific morphological milestones. Nutrition has a major influence on the timing and success of these intricate processes, largely dictating the survival of the larvae, postlarvae (puerulus) and their competency as a juvenile.

The moult cycle of crustaceans is divided into 4 recurrent stages: proecdysis (premoult), which begins the process of withdrawing the epidermis from the cuticle; ecdysis, whereby the old cuticle is removed; metecdysis (post-moult), which incorporates the absorption of water and the accumulation of organic reserves; and anecdysis (intermoult), which is the main period of tissue growth. The provision of a suitable feed is required to sustain this process, conversely, an unsuitable feed that results in poor feed intake and/or an inappropriate nutritional profile will be evident with malnourished juveniles displaying sporadic and incomplete moulting processes, leading to poor growth, susceptibility to disease, failure to metamorphose and ultimately death.

It is widely recognised that the use of fresh blue mussel (BM - Mytilus galloprovincialis) represents a highly palatable and nutritious standard to grow TRL. The edible portion of blue mussels is the mantle, viscera, and gonad material, when fed to juvenile lobsters it is often split open and fed as a half shell (HS). The use of BM-HS as a benchmark feed has been widespread across aquaculture facilities, this has been useful in the development and assessment of formulated feeds as it has permitted the comparison of data geographically and temporally, but as a nutritional feed BM-HS is far from complete and many other minerals and nutrients are required.

What is required for invertebrates and vertebrate aquatic species for instance lobsters and particularly, juvenile lobsters, is a complete nutritional feed composition which overcomes one or more of the shortcomings of the prior art formulations, and achieves high feed intake, growth and animal health.

Summary of Invention

Some of the previously proposed feed compositions have been predominantly manufactured using extrusion technology which heat and cook ingredient mixes during production, with a focus on carbohydrate-based binders, rather than as outlined in this composition the use of a protein-based binder. There are also some feeds manufactured using older pressed-pellet mills to bind feeds. Although lobsters are omnivores it has been recognised that they have a high ability to utilise protein therefore, the present invention is predicated, in part, to the use of a protein-based binder, rather than carbohydrates binder, as it may be nutritionally more functional. The present inventors have recognised that the texture of the presented feed compositions is critical in terms of increasing attractiveness and palatability which in turn aids in growth and development. To achieve the desired feed texture transglutaminase (TG), has been used in the present invention. TG is an enzyme widely utilised in the processing of food for human consumption.

Transglutaminase acts by inducing the process of cross-linking between polypeptide chains containing lysine and glutamic acid residues. The TG catalytic reaction result in changes in the physical and chemical properties of proteins. The use of different proteins and/or the mix of proteins in combination with specific percentage inclusion of TG impacts on lobster feed intake and subsequent growth. In the present invention TG, in combination with other specific substrates, is demonstrated to induce modifications in the final texture of composition (for instance, viscosity, thermal stability, elasticity and resilience) to produce a pelletised feed with a specific texture and nutritional content that works in synergy to promote, for instance, lobster development and growth.

Thus, the present invention involves the manufacture of novel feed compositions which utilise a specific combination of protein-based binder in the form of an enzyme and specific amino acid substrates to produce formulated feeds with unique properties suited to lobster feeding ability and nutritional requirements. More specifically, the inventors have developed two classes of formulated feeds based on the same binder combination for a nursery and a juvenile feed. The nursery feed (INF), designed for early TRL juveniles has been successfully manufactured on a laboratory scale and tested. Growth was found to be either comparable or superior to BM-HS (see Table 3, Figure 1). The testing of growth performance with the juvenile feed (IJF), designed for growing TRL juvenile is also demonstrated.

The unique properties of the pelletised feed of the present invention is characterised by synergies between its physical, biochemical, raw ingredient and manufacturing features which function in combination to achieve sustained growth performance of TRL juveniles. In particular the feed composition of the present invention improves in particular lobster growth and viability promoting survival during the juvenile phases of growth.

Accordingly, in a first aspect, the invention provides a feed composition for invertebrates and vertebrate aquatic species, such as crustaceans, in the form of pellets comprising:

1) sodium caseinate at about 5-45% wt/wt of the total composition; 2) transglutaminase at about 0.5-5% wt/wt of the total composition; and 3) one or more nutritional ingredients;

wherein the pellets are characterised with a peak tension (gF) of about 20-50 and firmness (Kg) of about 0.1-0.4 when measured after 30 minutes immersion in seawater at about 260 C.

In a second aspect, the invention provides a feed composition in the form of pellets comprising:

1) sodium caseinate at about 5-45% wt/wt of the total composition; 2) soy protein concentrate and/or isolate; 3) transglutaminase at about 0.5-5% wt/wt of the total composition; and 4) one or more nutritional ingredients

In an embodiment and with specific reference to the second aspect the composition comprises both soy protein concentrate and soy protein isolate.

In a third aspect, the invention provides a feed composition in the form of pellets comprising:

1) soy protein concentrate and/or isolate; 2) transglutaminase; and 3) one or more nutritional ingredients;

In an embodiment and with specific reference to the third aspect the composition comprises both soy protein concentrate and soy protein isolate.

In a fourth aspect, the invention provides a feed composition in the form of pellets comprising:

1) soy protein concentrate and/or isolate; 2) squid meal and/or krill meal; 3) transglutaminase at about 0.5-5% wt/wt of the total composition; and 4) one or more nutritional ingredients;

In an embodiment and with specific reference to the fourth aspect the composition comprises both soy protein concentrate and soy protein isolate.

In an embodiment and with specific reference to the fourth aspect the composition comprises both squid and krill meal.

In an embodiment and with specific reference to the fourth aspect the composition comprises soy protein concentrate, soy protein isolate, squid meal and krill meal.

In a fifth aspect, the invention provides a feed composition in the form of pellets comprising:

1) sodium caseinate at about 5-45% wt/wt of the total composition; 2) soy protein concentrate and/or isolate; 3) squid meal and/or krill meal; 4) transglutaminase at about 0.5-5% wt/wt of the total composition; and 5) one or more nutritional ingredients;

In an embodiment and with specific reference to the fifth aspect the composition comprises both soy protein concentrate and soy protein isolate.

In an embodiment and with specific reference to the fifth aspect the composition comprises both squid and krill meal.

In an embodiment and with specific reference to the fifth aspect the composition comprises soy protein concentrate, soy protein isolate, squid meal and krill meal.

In relation to any one of the above aspects certain embodiments of the feed composition comprise (when present):

-sodium caseinate at about 5-45% wt/wt of the total composition;

-soy protein concentrate at about 5-25% wt/wt of the total composition;

-soy protein isolate at about 5-30% wt/wt of the total composition;

-squid meal at about 5-30% wt/wt of the total composition;

-krill meal at about 5-30% wt/wt of the total composition; and

-transglutaminase at about 0.5-5% wt/wt of the total composition.

-sodium caseinate at about 10-40% wt/wt of the total composition;

-soy protein concentrate at about 10-20% wt/wt of the total composition;

-soy protein isolate at about 10-25% wt/wt of the total composition;

-squid meal at about 10-25% wt/wt of the total composition;

-krill meal at about 10-25% wt/wt of the total composition; and

-transglutaminase at about 1-4% wt/wt of the total composition.

It will be appreciated that present compositions may comprise one or more "nutritional ingredients" which include ingredients selected from vitamins, minerals, oils, supplements, attractants, preservatives, and colorants/pigments.

In certain embodiments the feed compositions of the present invention includes one or more nutritional ingredients selected from:

Green shell mussel meal Fish meal Poultry meal Meat and bone meal Blood meal Clam meal Scallop meal Insect meal Lipid Hydrolysates Betaine Cholesterol powder Pigment Choline chloride Vitamins Minerals Glycogen or derivatives Chitin Prebiotics Probiotics Organic acids

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes two or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes three or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes four or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes five or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes six or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes seven or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes eight or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes nine or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention further includes ten or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes eleven or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes twelve or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes thirteen or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes fourteen or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes fifteen or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes sixteen or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes seventeen or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes eighteen or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes nineteen or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes twenty or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes twenty-one or more of the above listed ingredients.

With respect to all aspects, in certain embodiments the feed compositions of the present invention includes twenty-two or more of the above listed ingredients.

With respect to aspects one to five, in certain embodiments the feed compositions of the present invention comprises all of the following nutritional ingredients:

Green shell mussel meal Fish meal Poultry meal Meat and bone meal Blood meal Clam meal Scallop meal Insect meal Transglutaminase Lipid Hydrolysates Betaine Cholesterol powder Pigment Choline chloride Vitamins Minerals Glycogen or derivatives Chitin Prebiotics Probiotics Organic acids

The feed compostions of the present invention are produced in the form of pellets, which are extruded to a diameter size in the range of about 0.3-6.0 mm, such as 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.8, 2.0, 2.4, 3.0, 3.5, 4.0, 5.0, or 6.0 mm (or any range between two of these figures).

In another aspect the invention also provides a process for manufacturing a feed composition (as defined in any one of the above aspects) in the form of pellets, said process comprising:

1) milling all dry nutritional ingredients with, when present, sodium caseinate, soy protein concentrate, soy protein isolate, squid meal, and krill meal, to achieve a combined milled product with an average particle size of less than 200pm; 2) mixing the combined milled product with transglutaminase; 3) adding and mixing lipid to the product of step 2); 4) adding water and continue mixing at a temperature of about 40-500 C to form a substantially homogeneous dough; 5) extruding said dough into pellets through a cold press extruder at a temperature of less than about 50°C; 6) allowing the pellets to set overnight airtight in fridge; and 7) allowing said pellets to dry.

In an embodiment the pellets are allowed to dry using air drying techniques.

In an embodiment the pellets are allowed to dry in an oven at less than 50°C for about 4 h.

In an embodiment the pellets are allowed to dry in an oven at less than 50°C for about 4 h followed by drying at an elevated temperature for a shorter period, eg around 80°C for about 2h.

Detailed Description of the Invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

As used herein "sodium caseinate" refers to the most common form of casein which is a family of related phosphoproteins. In the present invention not only does it serve as a feed source supply of amino acids, carbohydrates and essential elements of phosphorus and sodium, it also serves as a texture and stabilising agent for the formed pellets of the present invention. Commercially available "sodium caseinate" products include Excellion "I (FrieslandCampina), or spray-dried sodium caseinate from Armor Proteines S.A.S.

As used herein "soy protein concentrate" refers soy protein which is about 40-70% of defatted soybean protein, (the remainder being carbohydrates and fats). In the present invention not only does it serve as a feed source it also serves as a texture and glue for the formed pellets of the present invention.

As used herein "soy protein isolate" refers to protein isolated from soybean with about 90% protein. Like the concentrate it is made from soybean meal that has been dehulled and defatted but is further processed to increase the protein content and reduce the amount of carbohydrate and fat. In the present invention not only does it serve as a feed source it also serves as a texture and glue for the formed pellets of the present invention.

As used herein "transglutaminase" (TG) refers to an enzyme that catalyses the formation of isopeptide bonds between proteins. It serves as a cross-linking property is widely used in various processes to the manufacture of cheese and other dairy products. The present invention utilises TG with enzyme activity at a range of between about 60-100 units/gram, such as about 60, 65, 70, 75, 80, 85, 90, 95, or about 100 units/gram. Such commercially available TG can be purchased from BDF Australia as BDF PROBIND

In certain embodiments and relevant to all the above aspects, the present invention is characterised with transglutaminase at an activity range of between about 60-100 units/gram, and in an amount of about 0.5-5% wt/wt of the total composition.

In certain embodiments and relevant to all the above aspects, the present invention is characterised with transglutaminase at an activity range of between about 70-90 units/gram, and in an amount of about 0.5-5% wt/wt of the total composition.

In certain embodiments and relevant to all the above aspects, the present invention is characterised with transglutaminase at an activity range of between about 60-100 units/gram, and in an amount of about 1-4% wt/wt of the total composition.

In certain embodiments and relevant to all the above aspects, the present invention is characterised with transglutaminase at an activity range of between about 60-100 units/gram, and in an amount of about 1-3% wt/wt of the total composition.

In certain embodiments and relevant to all the above aspects, the present invention is characterised with transglutaminase at an activity range of between about 70-90 units/gram, and in an amount of about 1-4% wt/wt of the total composition.

In certain embodiments and relevant to all the above aspects, the present invention is characterised with transglutaminase at an activity range of between about 70-90 units/gram, and in an amount of about 1-3% wt/wt of the total composition.

In certain embodiments and relevant to all the above aspects, the present invention is characterised with transglutaminase at an activity range of between about 80 units/gram, and in an amount of about 1-3% wt/wt of the total composition.

The present invention may include one or more of fish, meat, crustacean, insect or blood meal which are all presented as "dry ingredients". The meal may be selected from the group consisting of squid meal, krill meal, green shell mussel meal, fish meal, poultry meal, meat and bone meal, blood meal, clam meal, scallop meal, and insect meal. When added, the meal is preferably added within the composition in an amount of about 2-30% wt/wt of the total composition based on each of the meal ingredients, such as about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or about 28% wt/wt of the total composition, or any range between any of these two of these %units. For instance, the composition may contain squid meal in an amount between about 10-30% wt/wt of the total weight of the composition.

The present invention may also include an amount of lipid, which may be sourced from either one or combinations of commercially available oils such as krill oil, fish oil, squid oil, chicken fat, vegetable oil and soy lecithin.

The feed composition may also comprise an amount of hydrolysates. Preferably the hydrolysate is a protein hydrolysate. In certain embodiments feed compositions comprise an amount of hydrolysate at about 2-20% wt/wt of the total composition based on each of the lipid ingredients, such as about 4, 6, 8, 10, 12, 14, 16, 18, or about 28 % wt/wt of the total composition, or any range between any of these two %units. For instance, the composition may contain krill oil in an amount between about 10-20% wt/wt of the total weight of the composition.

The feed composition may also comprise an amount of betaine as an attractant. Betaine also, known as trimethylglycine (TMG), is a naturally occurring amino acid derivative most found presented in the hydrochloride salt form. In certain embodiments feed compositions comprise an amount of betaine at about 0.5-5% wt/wt of the total composition, such as about 0.6, 0.8, 1, 2, 3, 4, or about 5 % wt/wt of the total composition, or any range between any of these two %units. For instance, the composition may contain betaine in an amount between about 1-3% wt/wt of the total weight of the composition.

In further embodiments the compositions may further comprise one or more supplements selected from the group consisting of: cholesterol powder, pigment, choline chloride, polysaccharides (eg chitin), vitamin mixtures, mineral mixtures, prebiotics, probiotics, glycogen (or derivatives, such as glucosamines) and organic acids.

The mineral premix may be a premix based on the group selected from calcium, chloride, phosphorus, potassium, iodine, sodium, zinc, magnesium, sulphur, iron, cobalt, selenium, chromium, manganese and copper. The mineral component is typically presented as mineral salts or inorganic compounds. For instance, in one embodiment, the composition comprises CaCl2.

The vitamin premix may be a premix based on the group selected from ascorbic acid (vitamin C), vitamin A, vitamin B-complex, vitamin D, vitamin E and/or vitamin K.

In certain embodiments feed compositions comprise an amount of each supplement at about 0.05-5% wt/wt of the total composition, such as about 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 2, 3, 4 or about 5% wt/wt of the total composition, or any range between any of these two %units. For instance, the composition may contain vitamin premix in an amount between 0.5-2% wt/wt of the total weight of the composition.

The present feed compositions which in the form of pellets, are prepared by initially milling the dry ingredients (besides the TG) to achieve an initial average particle size of less than 200um, such as less than 180, 170, 150, 130, 110 or less than 90um. Once the milled mix is substantially homogeneous the milled product is then mixed with the transglutaminase and further to this an oil (lipid) component may then be added and mixing continued. Water is then added. Preferably the mixing is continued at a temperature of about 40-500 C to achieve a substantially homogeneous consistency of a dough which can be submitted to cold extrusion. As used herein this is done through a cold press extruder at a temperature of less than about 500 C. Preferably diameter size of the extruded pellets are in the range of about 0.3-6.0 mm, such as 0.3, 0.5, 0.7, 1.2, 1.8, 2.4, 3.0, 3.5, 4.0, 5.0, 6.0. Once extruded the moist pellets are allowed to set overnight airtight in a fridge followed by drying, either using air drying techniques or in an oven at less than 50°C for 4-10h and possibly also at an elevated temperature for a shorter period, eg around 80°C for about 2h. The pellets are preferably stored in air tight container in a fridge (4-5 C) and are preferably use within 1 month from manufacture.

As discussed previously the main advantage of the present composition is that the produced pellets possess beneficial texture qualities and in this regard the pellets are characterised with a peak tension (gF) of about 20-50 and firmness (Kg) of about 0.1 0.40 when measured after 30 minutes immersion in seawater at about 260 C. Furthermore, in certain embodiments the pellets are to retain their form (i.e., do not fall apart) within seawater (at room temperature) for at least 24hrs.

In a further aspect the present invention provides a method of feeding juvenile crustacean species in an aquaculture environment including the step of administering an effective quantity of the feed composition according to that defined in any one of the aforementioned aspects or embodiments.

In an embodiment the juvenile crustacean is tropical rock lobster (TRL), Panulirus ornatus.

It will be appreciated that the invention also relates to market ready invertebrates and vertebrate aquatic species (for instance, crustacean species) which have been feed with a feed composition as defined in any one of the aforementioned aspects or embodiments.

In a further aspect the invention provides market ready tropical rock lobster (TRL), Panulirus ornatus, which have been feed with a feed composition as defined in any one of the aforementioned aspects or embodiments.

In a further aspect the invention also provides a method claims of improving/increasing body weight or growth rate of an aquatic species including the step of administering the feed.

The following examples are not intended to limit the invention in anyway:

Examples

Table 1: Growth of TRL early juveniles (J1-J6) fed commercial formulated feeds, and fresh blue mussels.

Source Year Feed Housing Initial Final Initial Final Days SGR stage stag weight weight (% BW e (g) (g) day-') RL J06 2014 KPF Individual J3 J6 0.42 1.9 45 3.0

RL J06 2014 Co-fed Individual J3 J6 0.35 1.9 57 3.0 KPF/BM HS RL JO8 2014 Co-fed Communal J1 J5 0.15 1.78 55 4.5 KPF/BM HS RL J10 2014 BM-HS Individual J1 J3 0.19 0.31 20 2.5

RL J14 2014 BM-HS Individual J1 J5 0.15 1.80 40 6.3

RL J14 2014 Co-fed Individual J1 J5 0.15 1.80 53 4.9 KPF/BM HS RL J15 2014 BM-HS Communal J1 J5/6 0.15 2.1 59 4.3

MS B3 2015 KPF Communal J1 J4 0.16 0.75 37 4.0

RL J15 2016 BM-HS Individual J4 J7/J8 1.08 6.43 48 3.7

RL J15 2016 KPF Individual J4 J6 1.12 2.48 48 1.7

RL J15 2016 RPF Individual J4 J5/J6 1.18 1.87 48 1.0

RL J15 2016 LGLF Individual J4 - 1.00 - 48

BM - blue mussel; BW - body weight; HS - half shell; KPF - kuruma prawn feed; LGLF LobGrow commercial lobster feed; MKPF1 - modified kuruma prawn feed 1; MKPF2 modified kuruma prawn feed 2; RPF - Ridley commercial prawn feed; SGR - standard growth rate.

Table 2: Growth of TRL juveniles fed either formulated feeds (commercial and experimental) only or cofed with fresh seafood.

Source Year Feed Housing Initial Final Initial Final Days SGR (% stage stage weight weight BW day (g) (g) 1) UTAS' 2019 ACIAR ELF Individual - - 1.95 4.37 54 1.5 UTAS' 2019 BM-HS Communal - - 2.05 11.09 54 3.1 UTAS' 2019 ACIAR ELF Individual - - 2.06 5.06 54 1.6 UTAS' 2019 BM-HS Individual - - 1.75 4.91 54 1.9 2 ACIAR 2009 ELF Communal - - 2.0 6.4 60 1.9 ACIAR 2 2009 ELF Individual - - 2.0 4.7 60 1.4 Irwin et 2010 ELF Communal - - 2.1 9.1 49 3.0 3 al.

ACIAR - Australian centre for international agricultural research; BM - Blue mussel; BW - Body weight; ELF - Experimental lobster feed; HS - Half shell; SGR - Specific growth 2 3 rate; '(Marchese et al., 2019); (Irvin and Williams, 2009); (Irvin et al., 2010).

Table 3: Growth of TRL juveniles fed Innovative nursery feed (INF), and BM-HS.

Source Year Feed Housing Initial Final Initial Final Days SGR stage stage weight weight (%BW (g) (g) day-') MS 2a i & 2021 INF1 Communal J3 J6 0.51 3.05 29 6.2 2a iii MS 2a i & 2021 INF2 Communal J3 J6 0.60 3.73 31 6.0 2a iii MS 2a i & 2021 BM-HS Communal J3 J6 0.52 3.41 28 6.7 2a iii MS 2 a iii 2021 INF2 Isolated J3 J6 0.46 2.55 37 4.7 (systems) MS 2 a iii 2021 INF2 Communal - - 0.48 1.77 23 5.7 (systems) (Manhattan) MS 2 a iii 2021 INF2 Communal - - 0.40 2.23 23 7.5 (systems) (blue tub) RL J10 2021 BM-HS Communal J1 J4 0.14 1.41 41 5.6 (Q5) RL J10 2021 INF2 Communal J1 J4 0.14 1.21 39 5.5 (Q5) RL J10 2021 BM-HS Communal J1 J4 0.14 1.29 40 5.5 (Q4) RL J10 2021 INF1 Communal J1 J4 0.14 1.19 40 5.3 (Q4) RL J10 2021 INF2 Communal J1 J4 (Q4)

BM - Blue mussel; HS - Half shell; INF - Innovative nursery feed

4.0

3.5

3.0

2.5

1352.0

1.5 0

1.0

0.5

1 2 3 4 5 6 7 Juvenile stage

OINF1&2 OBM-HS *BM-HS&KPF o KPF

Figure 1: Growth summary of TRL juveniles from stage 1 to stage 6 fed UTAS innovative nursery feed (INF), blue mussel half shell (BM-HS), blue mussel half shell and kuruma prawn feed (BM-HS & KPF) and kuruma prawn feed (KPF)

The degree of firmness is defined as the weight in Kg required to press a feed pellet of diameter 2.0 mm by 0.5 mm. The peak tension is defined as the force (g F) required to break a feed strand of diameter 2.0 mm by applying tension. Key texture characteristics of the feed is presented in Table 4.

Table 4: Texture characteristics of feed at 30 and 60 minutes in seawater at 26 °C.

Parameter 30 minutes seawater immersion 60 minutes seawater immersion Min Max Min Max Peak tension force (g 25 45 10 25 F) Firmness (Kg) 0.20 0.30 0.16 0.22

Figure 2. Material Process steps to achieve optimal texture of the pellets:

Steps in forming the diet include the following:

Step 1 Milling of dry ingredients to < 200 um

Step 2 Mechanical mixing of dry ingredients and TG

Step 3 Add oil to dry feed mix and continue mixing until oil is fully incorporated to dry mix

Step 4 ~ Add freshwater and mix at temperature 40-50°C until a homgeneous dough is formed

Step 5 Extrude dough into pellets through a cold pressed extruder, temperature < 50°C (pasta extruder style)

Step 6 Set pellets overnight airtight in the fridge

Step 7 Dry pellets in an oven at 40°C for 8h, then at 80°C for 2h

Step 8 store pellets in air tight container in a fridge (4-5 °C) and use within 1 month from manufacture

Raw ingredients are included in the range of:

Table 5: Raw ingredient list, inclusion rates and function Raw Ingredient Inclusion Function

(%) Texture Nutrition Attractant Supplement Sodium caseinate 10-40 /

/ Soy protein 10-20 /

/ concentrate Soy protein isolate 10-25 /

/ Squid meal 10-30 / /

/ Krill meal 15-25 / /

/ Green shell mussel 15-25 /

/ meal Fish meal 10-25 /

/ Poultry meal 10-20 / Meat and bone meal 5-10 /

Blood meal 5-10 /

Clam meal 5-10 / 5-10 / Scallop meal / /

Insect meal 10-20 /

Transglutaminase 1-3 /

(activity 60-100 u/g) Lipid 5-15 / /

Hydrolysates 1-3 /

Glycogen and 1-3 / /

drivatives Betaine 1-3 /

Cholesterol powder 0.1-0.5 /

Pigment 0.1-1 /

Choline chloride 0.1-0.5 /

Vitamins 0.5-2 /

Minerals 0.5-2 Chitin 1-3 /

Prebiotics 0.1-1 /

Probiotics 0.1-1 /

Organic acids 0.1-1 /

References

IRVIN, S. J. & WILLIAMS, K. C. 2009. Comparison of the growth and survival of Panulirus ornatus seed lobsters held in individual or communal cages. spiny lobster aquaculture in the Asia-Pacific region, 89.

IRVIN, S. J., WILLIAMS, K. C., BARCLAY, M. C. & TABRETT, S. J. 2010. Do formulated feeds for juvenile Panulirus ornatus lobsters require dietary cholesterol supplementation? Aquaculture, 307, 241-246.

MARCHESE, G., FITZGIBBON, Q. P., TROTTER, A. J., CARTER, C. G., JONES, C. M. & SMITH, G. G. 2019. The influence of flesh ingredients format and krill meal on growth and feeding behaviour of juvenile tropical spiny lobster Panulirus ornatus. Aquaculture, 499, 128-139.

Claims

The Claims defining the Invention are as follows:

1.A feed composition in the form of pellets comprising:

1) sodium caseinate at about 5-45% wt/wt of the total composition;

2) transglutaminase at about 0.5-5% wt/wt of the total composition; and

3) one or more nutritional ingredients;

wherein the pellets are characterised with a peak tension (gF) of about 20-50 and firmness (Kg) of about 0.1-0.4 when measured after 30 minutes immersion in seawater at about 260 C. 2. A feed composition in the form of pellets comprising:

1) sodium caseinate at about 5-45% wt/wt of the total composition;

2) soy protein concentrate and/or isolate;

3) transglutaminase at about 0.5-5% wt/wt of the total composition; and 4) one or more nutritional ingredients;

3. A composition according to claim 2 which comprises both soy protein concentrate and soy protein isolate.

4. A feed composition in the form of pellets comprising:

1) soy protein concentrate and/or isolate;

2) transglutaminase; and 3) one or more nutritional ingredients;

5. A composition according to claim 4 which comprises both soy protein concentrate and soy protein isolate.

6. A feed composition in the form of pellets comprising:

7. A feed composition according to claim 6 which comprises both soy protein concentrate and soy protein isolate.

8. A feed composition according to claim 6 which comprises both squid and krill meal.

9. A feed composition according to claim 6 which comprises soy protein concentrate, soy protein isolate, squid meal and krill meal.

10. A feed composition in the form of pellets comprising:

11. Afeed composition according to claim 10 which comprises both soy protein concentrate and soy protein isolate.

12. A feed composition according to claim 10 which comprises both squid and krill meal.

13. A feed composition according to claim 10 which comprises soy protein concentrate, soy isolate, squid meal and krill meal.

14. A feed composition according to anyone of claims 1-13 which comprise (when present):

-sodium caseinate at about 5-45% wt/wt of the total composition;

-soy protein concentrate at about 5-25% wt/wt of the total composition;

-soy protein isolate at about 5-30% wt/wt of the total composition;

-squid meal at about 5-30% wt/wt of the total composition;

-krill meal at about 5-30% wt/wt of the total composition; and

-transglutaminase at about 0.5-5% wt/wt of the total composition.

15. A feed composition according to anyone of claims 1-13 which comprises (when present):

-sodium caseinate at about 10-40% wt/wt of the total composition;

-soy protein concentrate at about 10-20% wt/wt of the total composition;

-soy protein isolate at about 10-25% wt/wt of the total composition;

-squid meal at about 10-25% wt/wt of the total composition;

-krill meal at about 10-25% wt/wt of the total composition; and

-transglutaminase at about 1-4% wt/wt of the total composition.

16. A feed composition according to anyone of claims 1-15 wherein the feed compositions of the present invention includes one or more nutritional ingredients selected from:

17. A process for manufacturing a feed composition according to anyone of claims 1-16 for crustaceans in the form of pellets, said process comprising:

1) milling all dry nutritional ingredients with, when present, sodium caseinate, soy protein concentrate, soy protein isolate, squid meal, and krill meal, to achieve a combined milled product with an average particle size of less than 200pm; 2) mixing the combined milled product with transglutaminase; 3) adding and mixing lipid to the product of step 2); 4) adding water and continue mixing at a temperature of about 40-500 C to form a substantially homogeneous dough; 5) extruding said dough into pellets through a cold press extruder at a temperature of less than about 50°C; 6) allowing pellets to set overnight airtight in a fridge; and 7) allowing said pellets to dry.

18. A method of feeding juvenile crustacean species in an aquaculture environment including the step of administering an effective quantity of the feed composition according to anyone of claims 1 to 16.

19. A method according to claim 18 wherein the juvenile crustacean species is tropical spiny rock lobster (TRL), Panulirus ornatus.

20. Market ready tropical rock lobster (TRL), Panulirus ornatus, which have been feed with a feed composition as defined in any one of claims 1 to 16.

21. A method of improving/increasing body weight or growth rate of an aquatic species including the step of administering a feed composition according to anyone of claims 1 to 16.