Nutritional formulations and uses thereof

Abstract

The present invention relates generally to a nutritional composition for the development of aquatic crustaceans and fish species. More specifically, a nutritional formulation comprising n-3 and n-6 fatty acids and live Artemia for the tropical spiny rock lobster.

Description

NUTRITIONAL FORMULATIONS AND USES THEREOFFIELD

[0001] The present invention relates generally to a nutritional composition for the development of aquatic crustaceans and fish species.BACKGROUND

[0002] Elucidation and provision of the key nutritional requirements for complete larval development of aquatic crustaceans and fish species presents a major challenge for the development of robust commercial aquaculture. An example of this is the tropical spiny rock lobster, 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.

[0003] Likewise the majority of decapod crustaceans have a complex life cycle that is characterised by a series of moults and morphological transformations that occur throughout the pelagic larval stage. Nutrition has a major influence on the timing and success of these intricate processes, largely dictating the prosperity of the larvae and their likelihood of reaching the juvenile stage. Since the hatchery phase presents a significant bottleneck for the establishment of a commercial aquaculture sector for palinurid lobsters, understanding the requirements for moulting and complete larval development of cultured crustaceans are critical prerequisites for commercial viability.

[0004] 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. The complete moult cycle 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.

[0005] Healthy phyllosoma will repeat moulting many times throughout the larval life cycle, enabling growth and regeneration. Conversely, malnourished phyllosoma will display sporadic and incomplete moulting processes, leading to poor growth, susceptibility to disease, failure to metamorphose and ultimately death. Although there is some understanding of the nutritional requirements for benthic, juvenile P. ornatus, this information is scarce for the pelagic larval stages, which is key to the development of closed-life cycle production of this species. Successful phyllosoma development and metamorphosis are highly dependent on both the efficient use of energy reserves and on having the necessary nutrient reserves for re-building a viable juvenile. Whilst larval lobsters are likely to have requirements for specific nutrients, their quantitative requirement and available forms are largely unknown. As critical nutritional components, total lipid content, the proportion and composition of individual lipid classes, and their constituent fatty acids, may affect the health, development and physiology of phyllosoma. Lipids are also thought to be highly influential on the moulting process and in many investigations total lipid content in larval crustaceans has been observed to follow a cyclical pattern during each moult cycle. This constitutes an accumulation of lipid throughout the intermoult and premoult stages, followed by a decrease at the post-moult stage. Although the expense of moulting is energetically minor in comparison to that of growth and metabolism, its successful completion is critical for larval development.

[0006] Adequate lipid nutrition with respect to specific lipid class profiles has been shown to accelerate the larval phase and increase the likelihood of successful metamorphosis. Phospholipids are thought essential for cell membrane integrity and cholesterol transport during moulting, improved growth and successful metamorphosis. Deficiencies in the fatty acids, eicosapentaenoic acid (20:5n-3, EPA), docosahexaenoic acid (22:6n-3, DHA) and arachidonic acid (20:4n-6, ARA), have been reported to cause poor survival, longer intermoults and a narrower carapace width in crustacean larvae. Elucidation of the nutritional requirements during the larval phase is essential to the establishment of optimal diets for P. ornatus phyllosoma. Researchers have focussed on the chemical composition of phyllosoma immediately prior and post-ecdysis (i.e. pre- and post-moult) in order to provide insight into the crude nutritional trends during the larval moult cycle. However, late stage spiny rock lobster phyllosoma are also thought to undergo dietary shifts.

[0007] The temperate spiny lobster Jasus edwardsii often experiences periods of inconsistent larval quality when maintained in captivity.

[0008] What is required in the industry is a complete nutritional formulation (or feed) which overcomes one or more of the shortcomings of the prior art formulations, and especially achieves high attainment in metamorphosis success in crustacean species, and in particular crabs and clawed lobsters which are produced in hatcheries.SUMMARY OF THE INVENTION

[0009] The present invention is predicated on the discovery that attaining metamorphosis at a high success rate can be achieved by a formulation with a specific combination of ingredients. In particular the invention increases juvenile viability and the progression of development beyond juvenile moult. The compositions disclosed herein include and allow for the inclusion of ingredients to change the form and balance of nutrients supplied.

[0010] Accordingly, in an aspect the invention provides a nutritional formulation comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source;(ii) live Artemia (non-enriched);(iii) at least one sterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.In an embodiment the live Artemia (non-enriched) is species at the nauplii stage of development.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a photograph showing the individual components of an extruder gun used to produce noodle shaped formulations according to one embodiment of the invention.

[0012] Figure 2 is a photograph showing an extruder gun used to produce noodle shaped formulations according to one embodiment of the invention.DETAILED DESCRIPTION OF THE INVENTION

[0013] 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.

[0014] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0015] As a first component of the nutritional formulation accordingly to the present invention there is an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source. As used herein the term "n-3 fatty acids" are also referred to as omega-3 or co-3 fatty acids and are characterised as polyunsaturated fatty acids (PUFA’s) with a double bond at the third carbon atom from the end of carbon chain. As used herein the term “n-6 fatty acids” are also referred to as omega-6 or co-6 fatty acids and are characterised as polyunsaturated fatty acids (PUFA’s) with a double bond at the sixth carbon atom from the end of the carbon chain. Common n-3 and n-6 fatty acids include:

[0016] In relation to the present invention the most common n-3 fatty acids are EPA and DHA, with lesser amounts of n-6 fatty acids which are all present in marine oil. For the present invention however the inventors have found that such fatty acids esterfied in a high content phospholipid source provide for benefits in attaining high viability and sustainability of crustacean survival during metamorphosis. In particular it has been found that this advantage is realised with high phospholipid: low triacylglycerol (TAG) ratio sources of marine fatty acids. For instance, fish oils which are a convenient marine source of EPA, DHA and ARA have these fatty acids incorporated in TAG or ethylesters. As such, fish oils generally have a higher TAG content. In contrast, krill oil (an alternative source of n-3 / n-6 fatty acids) has EPA, DHA and ARA esterified in phospholipids, in particular phosphatidylcholine. As such, krill oil is a convenient source of n-3 and n-6 fatty acids esterified with high phospholipid: low TAG ratio. Another potential source is squid skin oil as this has a higher ratio of phospholipid to TAG.

[0017] Accordingly, the term “effective” used herein refers to an amount which is able to increase the viability (survival) of crustacean larval development during metamorphosis relative to a nutritional formulation which utilises fish oil as a source of fatty acids. This may also include additional saturated fatty acids (SFA) and mono unsaturated fatty acids (MUFA), such as n-9 fatty acids, highly unsaturated fatty acids (HUF A), other PUFA’s (such as long chain PUFA’s). These fatty acids may contribute as an important additional energy source and may be additionally provided from known animal and plant sources.

[0018] In an embodiment, the nutritional formulations of the present invention comprise from about 0.5-5% wt / wt of krill oil, such as about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8 or about 4.9% wt / wt. In an embodiment the formulation is prepared by initially forming an emulsion which incorporates the krill oil (or other source of the desired fatty acids). The krill oil component may comprise about 15-25% of the emulsion. The emulsion may comprise about 6-15% of the final formulation (i.e., range of about 0.97-3.3 to 0.97-3.8 wt / wt%).

[0019] The present formulation also incorporates live Artemia (non-enriched) as an ingredient. Artemia is a genus of aquatic brine shrimp. In an embodiment the nutritional formulation of the present invention utilises Artemia species at the nauplii stage of development. The present invention should be distinguished from other feed compositions which comprises Artemia adults which have been enriched with beneficial nutritional products including phospholipids, ascorbic acid (AA) and antibiotics prior to formulation. Artemia species at the nauplii stage of development. are an example of non-enriched live Artemia species. In an embodiment, the nutritional formulations of the present invention comprise from about 3-60% wt / wt of live Artemia, (non-enriched) such as about 4, 5 ,6 ,7 ,8 ,9 ,10 ,11 ,12 ,13 ,14 ,15 ,16 ,17 ,18 ,19 ,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 to about 59% wt / wt.

[0020] As a third component the formulation comprises at least one sterol. In an embodiment the at least one sterol is cholesterol. As used herein “sterol” includes known steroid alcohols. In an embodiment the sterol is an animal based sterol. In an embodiment the nutritional formulation of the present invention comprises about 0.05-0.5% wt / wt of sterol. In an embodiment the sterol, such as cholesterol, is mixed in the aforementioned emulsion. Cholesterol may be added at about 1.5% of the emulsion.

[0021] As a further component the formulation comprises at least one binder. The binder in one embodiment is alginate (alginic acid) which is an anionic polysaccharide derived from the cell walls of brown algae. One of the additional benefits of using alginate as a binder is that it also serves as an immune stimulant. In an embodiment the nutritional formulation of the present invention comprises from about 0.2-3% wt / wt, such as about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or about 2.9% wt / wt of the at least one binder. In an embodiment where the binder is alginate, it is presented as a liquid (aqueous) extract at a concentration of from about 1-4%. In such an embodiment the % amount of such an extract in the formulation may be in the range of from 15-25% wt / wt of the total composition.

[0022] As a further component the formulation comprises at least one emulsifier. The emulsifier in one embodiment is lecithin. In another embodiment the emulsifier is Acacia gum. In an embodiment the at least one emulsifier in the formulation is present in a range of about 0.2-0.8% wt / wt, such as or about 0.3, 0.4, 0.5, 0.6 or 0.7% wt / wt of the total formulation.

[0023] As a further component the formulation comprises at least one mineral component. Suitable mineral components can be 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 formulation comprises CaCh.

[0024] The mineral component is typically added as a salt component(s) to the formulation during manufacture. In an embodiment the at least one mineral component is added to the aforementioned emulsion at around 0.5% of the emulsion; that is about 0.064% of the total formulation. In this regard the at least one mineral component in the formulation is present in the range of about 0.04-0.1% wt / wt of the total formulation such as about 0.05, 0.06, 0.07, 0.08 to about 0.09% wt / wt.

[0025] As a further component the formulation comprises at least one vitamin. Added vitamins include ascorbic acid (vitamin C), vitamin A, vitamin B -complex, vitamin D and / or vitamin K. In an embodiment the at least one vitamin is also added to the aforementioned emulsion at around 1.2% of the emulsion. In this regards the at least one vitamin component is preferred in the formulation in a range of about 0.05 %-0.5% wt / wt of the total formulation such as 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48 or about 0.49% wt / wt.

[0026] As a further component the formulation comprises at least one anti-oxidant. A preferred antioxidant includes astaxanthin which is a beta-carotenoid. It is sold under the trade name Carophyll®Pink (Roche). In an embodiment Carophyll®Pink is added to the aforementioned emulsion at around 0.4%. In this regard the at least one anti-oxidant is preferred in the formulation in a range of about 0.01-0.1% wt / wt of the total formulation such as 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08 or about 0.09% wt / wt. In a further embodiment the invention also comprises a colour pigment but the pigment may also be the antioxidant such as Carophyll®Pink.

[0027] As a further component the formulation comprises at least one probiont source. A preferred probiont source includes Sanolife®. In an embodiment the probiont source is in the form of an activated solution in the formulation and is in the range of about 1-2% wt / wt of the total formulation such as 1.1, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or about 1.9% wt / wt. The present inventors have found that the inclusion of a probiont source helps prevent white gut. This is a disease of bacterial origin with the primary site of infection being the mid-gut of early to mid-stage phyllosoma. This disease can rapidly be passed between individuals and may infect an entire tank. Established antibiotic treatment protocols are not effective in modulating this disease once an outbreak has occurred.

[0028] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) at least one sterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0029] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0030] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0031] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0032] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0033] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant which comprises astaxanthin.

[0034] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0035] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0036] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0037] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0038] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0039] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

[0040] In an embodiment the nutritional formulation comprises:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant which comprises astaxanthin.

[0041] The invention also contemplates nutritional formulation (or feed) which comprises one or more of the following additional ingredients:(i) Antibiotic(s) - this may assist in the control of disease states such as white leg. Suitable antibiotics include enrofloxacin and cyprofloxacin (at for instance 0.5%);(ii) Free amino acids - as an added energy source;(iii) Other protein and peptides - such as gelatine, bovine serum albumin, and caseinate;(iv) Carbohydrates and derivatives thereof - such as cellulose, maltose, chitosan, and gellan;(v) Chemo attractants - such as glycine (also a free amino acid);(vi) Further colouring pigments / dyes - which may assist in having an attractant ability; and(vii) pH buffering agents- such as citric acid and sodium bicarbonate.

[0042] The nutritional formulations of the present invention are preferably produced in the form of noodles which are extruded to a diameter size in the range of about 0.3- 1.0mm, such as about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and about 1.0mm. When not in use as a feed the formulation is preferably kept at a temperature of about 4°C or less. The invention contemplates the preparation of the feed (nutritional composition) by initially forming an emulsion with many of the components as discussed above. The emulsion is then added to the remainder of the ingredients to form the feed which is then extruded to form the ultimate nutritional formulation. The inventors have found that when the emulsion constitutes greater than about 12% of the total formulation and the formulation is in the form of a noodle, the nutritional formulation remains neutrally buoyant within the feeding tank which is advantageous to the survival of the rearing larva.

[0043] In a further embodiment the feed formulation is in the form of neutrally buoyant noodles, this means that it has a slow sinking rate, i.e., in the range of about 0.02-2cm s’1, such as about 0.02, 0.05, 0.07, 0.09, 0.12, 0.15, 0.17, 0.19, 0.20, 0.22, 0.25, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0,40, 0.42, 0.46, 0.48, 0.50, 0.55, 0.58, 0.60, 0.64, 0.66, 0.70, 0.75, 0.78, 0.80, 0.85, 0.87, 0.90, 0.92, 0.94, 0.98, 1.0, 1.1, 1.15, 1.2, 1.3, 1.35, 1.4, 1.5, 1.55,1.6, 1.7, 1.75, 1.8, 1.85, 1.9, or about 2 cm s’1(or a range produced by any two of the above figures) The hydrodynamics of the water flow in the culture vessel is such that the feed and the phyllosoma are evenly distributed throughout the water column to allow the phyllosoma to feed by ‘bumping’ into the feed - as they do not actively hunt their foods source / prey. Accordingly, another advantage of the present feed formulation is that it behaves like the feed in the natural environment increasing the contact incidence time which in turn allows for increased feed uptake and increased development.

[0044] In order to maintain palatability and increase the nutrient value of the formulation it is an important feature to maintain the pH of the nutrient formulation to between about 5 to about 10, such as about 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8 ,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or about 10

[0045] Accordingly the present invention also provides an extruder gun device to extrude noodle shaped nutritional formulations comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source;(ii) live Artemia (non-enriched);(iii) at least one sterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant, wherein the extruder gun device is operationally managed by regulated compressed air.

[0046] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following nonlimiting examples.

[0047] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0048] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0049] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.EXAMPLES

[0048] Formulation Examples (in the shape of noodles)

[0049] Preparation exampleAlginate solution1. To make an alginate solution, add Alginate to deionised water.2. Stir the alginate solution into the water until well mixed.3. Warm up the alginate solution in a water bath.Wet whole ingredientsLive Artemia nauplii (non-enriched)1. Decant the necessary volume of live Artemia nauplii from the bucket onto a clean and disinfected 50 micron mesh screen.2. Rinse the Artemia nauplii with deionised water, to rinse the salt water away from the nauplii. . Prepare the trace mineral mix4. Prepare the mineral mix - TRL M2Wet premixThe wet premix can be used as one of the main components of the diets. For a single feed, follow the standard recipe procedures.1. Weigh out the alginate into the mixing bowl.2. Weigh out the choline chloride solution into the mixing bowl.3. Weigh out the deionised water into the mixing bowl.4. Weigh out the autoclaved algal paste into the mixing bowl.5. Weigh out the probiotic solution into the mixing bowl.6. Mix slowly with a spatula until all of the ingredients are mixed through consistently.Preparation of Lecithin - Cholesterol paste1. Weigh out the required amount of liquid lecithin into a beaker.2. Weigh out the required amount of cholesterol powder into the same beaker.3. Leave the solution on a hot plate.4. Stir the solution regularly to incorporate the cholesterol into the lecithin.Preparation of the nutrient solution:5. Measure out 10% of the required amount of deionised water into a clean beaker.6. Add the required amount of carophyll pink into the beaker.7. Mix the carophyll pink.8. Add the vitamin mix, mineral mix and Vitamin C to the beaker.9. Mix the nutrient solution until all components are completely dissolved.10. Add the krill oil to the mix and gently stir.11. Add the lecithin-cholesterol paste to the beaker.Dry premixesVitamin premix (TRL V2)Prepare the trace mineral mixPrepare the mineral mix - TRL M2EmulsionsKrill oil: Emul.KOv3Ingredients:Deionised waterCarophyll® pinkVitamin mix (TRL-V2) *zero choline chlorideMineral mix (TRL-M2)Rovimix 35 (“VitaminC”)Krill oilCholesterol-Lecithin paste (1:2 ratio)Preparation of cholesterol-lecithin paste1. Weigh out the required amount of liquid lecithin into a beaker.2. Weigh out the required amount of cholesterol powder into the same beaker.3. Leave the solution on a hot plate.4. Stir the solution regularly to incorporate the cholesterol into the lecithin as the lecithin softens.Preparation of the Emulsion KQV3:1. Weigh out the deionised water into the beaker.2. Add the required amount of carophyll pink, vitamin mix, mineral mix and Rovimix 35 to the deionised water and mix.3. Weigh the krill oil into the mixing bowl.4. Add part of the nutrient water mixture and mix.,5. Add another part of the nutrient water mixture and mix further.6. Add the cholesterol: lecithin paste and mix.7. When the cholesterol: lecithin mix is well incorporated into the oil water solution, gradually continue to add the remainder of the water while the emulsion is mixing.Setting bathPreparing the setting bath:1. Add Calcium Chloride (CaCh) to carboy (0.2%).2. Fill each carboy with 1 pm filtered seawater3. Autoclave the carboys using the large autoclave.Krill oil: Emul.KOv2Ingredients:Multiply the total emulsion volume required by the percentage inclusion of each ingredient to determine inclusion amounts.Deionised waterCarophyll® pinkVitamin mix (TRL-V2) *zero choline chlorideMineral mix (TRL-M1)Rovimix 35Krill oilCholesterol / Lecithin paste (1:2 ratio)Preparation of Lecithin - Cholesterol paste1. Weigh out the required amount of liquid lecithin into a beaker.2. Weigh out the required amount of cholesterol powder into the same beaker.3. Leave the solution on a hot plate.4. Stir the solution regularly to incorporate the cholesterol into the lecithin as the lecithin softens.Preparation of the nutrient solution:1. Measure out the required amount of deionised water into the clean beaker.2. Add the required amount of carophyll® pink to the beaker.3. Mix the carophyll pink until it is completely dissolved.4. Add the vitamin mix, mineral mix and Rovimix 35 to the beaker.5. Mix the nutrient solution.6. Add the lecithin-cholesterol paste to the beaker.7. Add the krill oil to the nutrient mix and stir.Preparation of the Emulsion KO:1. Cover the pre-emulsion with plastic wrap and allow to cool down2. Place the beaker in an ice water bath.3. Sonicate the emulsion three times Stir the emulsion vigorously between each burst of sonication.4. Place the finished emulsion in a clean and sterilised container.LNN8V3-PB (Formulation)Preparatory Work:1. Hydrate the probiotic powder and place it in the 30°C water bath.2. Place all extruder parts (dies, chamber, sleeves, plunger, O-rings) in a 10% chlorine bath for disinfection.3. Retrieve the Emulsion KO V3.4. Make the premix in an adequately sized container for the total premix volume. . Add the alginate, the choline chloride solution and the algal paste and stir thoroughly. When the probiotic is fully activated, add the probiotic solution to the rest of the premix and again stir thoroughly.Preparing the Artemia nauplii:5. Carefully pour the live Artemia nauplii from the bucket onto a clean and disinfected screen.6. Swirl the screen to remove most of the seawater.7. Rinse the Artemia nauplii with deionised water.Preparing the LNN8V3 premix:8. Weigh out the probiotic premix solution.9. Weigh out the deionised water.10. Weigh out the Emulsion K0V3.11. Add the necessary number of drops of acetic acid to the mixing bowl.12. Mix.Adding the Artemia nauplii to the LNN premix:13. Carefully weigh out the dried Artemia nauplii straight into the stainless steel mixing bowl.14. Mix.15. Check the pH of the final diet mix. Adjust if required.Extruding the Formulation:16. Assemble the plunger by attaching the two O-rings.17. Attach the plunger to the end of the chamber and, invert the chamber onto the bench and push the plunger using a tube to the other end.18. Invert the chamber and assemble the throttle with an O-ring, secured with a sleeve.19. Attach the chamber to the arm.20. Attach the compressed air line to the throttle assembly.21. Adjust the regulator.22. Load the extruder with the required amount of diet mixture..23. Assemble the die, spray it with ethanol and secure it to the sleeve. Place the O-ring and the sleeve on the extruder and clamp on until rubber seal has pressure on it.24. Pour calcium chloride bath water into a large plastic tub .25. Submerge the head of the large extruder in the calcium chloride bath and press the throttle.26. Press the pressure release lever while removing the noodle gun from the bath.Cutting and rinsing the diet:27. Dietary lengths are cut for maximizing feeding and efficient flushing.Extruder gunThere are two extruder guns (see figures 1 and 2).Compressed air is used to drive the extrusion plunger within the extruder gun facilitating the extrusion of large batches of noodles. The air pressure is controlled with regulators, to ensure a constant air pressure on the internal plunger, and therefore a constant rate of noodle extrusion. This is important in obtaining consistent and well-formed noodles, without putting too much pressure on the live Artemia.Follow these instructions for operation:1. Assemble the plunger by attaching the two O-rings.2. Attach the plunger to a lubricated end of the chamber and, invert the chamber onto the bench and push the plunger to the other end using a plastic stick. Push the plunger back and forth until the plunger slides freely within the chamber. Leave the plunger at the bottom end of the chamber.3. Invert the chamber and assemble the throttle with an O-ring and fasten it on with the metal sleeve.4. Attach the extruder to the arm bracket mounted on the bench top. Mount the extruder in a position where when inverted it will sit in the top of the calcium seawater.5. Attach the compressed air line to the throttle assembly.6. Adjust on the air pressure regulator within the laboratory to about 6-10psi.7. Load the extruder with the diet mixture until full. If the chamber is not full, carefully and slowly press the throttle to push the mixture up to near the top of the chamber. Be careful to stop before the mixture overflows the extruder.8. Assemble the die into the die housing and spray it with ethanol. Place the rubber O- ring on the die housing and place it on the top of the extruder and clamping it on until rubber seal has pressure on it.9. Using a large scoop create a strong circular current in the bath.10. In one smooth motion, position the noodle gun in the top left hand side of the container, submerge the noodle gun in the bath and press the throttle, while continuing to stir vigorously. Hold the lever down until all of the noodle mixture has been extruded, stirring throughout the extrusion.11. Press the pressure release lever when removing the noodle gun from the bath.12. Repeat the process of loading the gun and extruding until all of the noodle mixture has been extruded.13. Cut the diet to the appropriate length using single or multiple blade scissors.14. Drain the diet onto a screen and rinse with tap water followed by deionised water.15. Dry the diet by placing the screen on paper towel, and then place in containers and then into the fridge.Comparison with fish oil formulation productsWhen fish oil was the lipid source tropical rock lobsters (TRL) phyllosoma grew through all of the larval stages taking between 150 - 180+ days to attempt metamorphosis.Metamorphosis success from the final stage phyllosoma larval stage to puerulus was 0% on the fish oil diets.When Krill oil was utilised as the lipid source (as in the formulations of the present invention) larval development time was reduced to between 90-120+ days. Metamorphosis success from the final phyllosoma larval stage to puerulus was 30- 70%.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A nutritional formulation comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source;(ii) live Artemia (non-enriched);(iii) at least one sterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.

2. A nutritional formulation according to claim 1 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) at least one sterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin; and(viii) at least one probiont source; and(ix) at least one anti-oxidant.A nutritional formulation according to claim 1 or 2 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant. A nutritional formulation according to anyone of claim 1 to 3 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.A nutritional formulation according to anyone to anyone of claims 1 to 4 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant. A nutritional formulation according to anyone to anyone of claims 1 to 5 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant.A nutritional formulation according to anyone to anyone of claims 1 to 6 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant which comprises astaxanthin. A nutritional formulation comprising according to anyone to anyone of claims 1 to 7:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched);(iii) cholesterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant.A nutritional formulation comprising according to anyone to anyone of claims 1 to 8:(i) an effective quantity of n-3 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant, A nutritional formulation according to anyone to anyone of claims 1 to 9 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant. A nutritional formulation according to anyone to anyone of claims 1 to 10 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant. A nutritional formulation according to anyone to anyone of claims 1 to 11 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin;(viii) at least one probiont source; and(ix) at least one anti-oxidant. A nutritional formulation according to anyone to anyone of claims 1 to 12 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant. A nutritional formulation according to anyone to anyone of claims 1 to 13 comprising:(i) an effective quantity of n-3 and n-6 fatty acids esterified in a high content phospholipid source which is Krill oil present in the formulation at about 0.5-5.0% wt / wt of the total formulation;(ii) live Artemia (non-enriched) present in the formulation at about 30-60% wt / wt of the total formulation;(iii) cholesterol;(iv) at least one binder which comprises alginate;(v) at least one emulsifier which comprises lecithin;(vi) at least one mineral which comprises calcium chloride;(vii) at least one vitamin which comprises vitamin C;(viii) at least one probiont source; and(ix) at least one anti-oxidant which comprises astaxanthin. A nutritional formulation according to anyone to anyone of claims 1 to 14 in the form of noodles.A nutritional formulation according to anyone to anyone of claims 1 to 14 in the form of noodles with a diameter of 0.3- 1.0mm. Tropical spiny rock lobster which have been fed on the formulation according to anyone of claims 1 to 16. Tropical spiny rock lobster according to claim 17 of species Panulirus ornatus. A method of attaining metamorphosis success in crustacean population in a hatchery environment including the step of feeding said population an effective amount of a formulation according to anyone of claims 1 to 16. A method according to claim 19 wherein the crustacean population is tropical spiny rock lobster, preferably of the species Panulirus ornatus.

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