Nutritional formulations and uses thereof

Abstract

The present invention relates generally to nutritional compositions for aquatic crustacean and fish species development. More specifically, a nutritional formulation for tropical eye spot lobsters comprising n-3 and n-6 fatty acids and a live artemia (Artemia).

Description

[0001] field

[0002] The present invention generally relates to nutritional compositions for the development of aquatic crustacean and fish species.

[0003] background

[0004] Elucidating and providing the key nutritional requirements for complete larval development of aquatic crustaceans and fishes presents a major challenge to the development of robust commercial aquaculture. An example of this is the tropical eyed lobster, Panulirus ornatus, which has earned high market prices in recent years, primarily due to increased consumer demand and wildlife shortages (partly due to overfishing). These crustacean species have many favorable attributes for commercial aquaculture, but also present numerous challenges, largely due to the nutritional requirements of species that undergo continuous molting cycles and morphological changes.

[0005] Likewise, most decapod crustaceans have a complex life cycle characterized by a series of molting and morphological transformations that occur throughout the planktonic larval stage. Nutrition has a major influence on the timing and success of these complex processes, largely determining the prosperity of larvae and their likelihood of reaching the larval stage. As the hatchery stage presents a significant bottleneck to the establishment of a commercial aquaculture sector for lobsters, understanding the requirements for molting and complete larval development in hatchery crustaceans is a key prerequisite for commercial viability.

[0006] For P. ornatus, up to 24 morphological increments have been recorded, and these are usually divided into 11 different stages by identifying specific morphological milestones. The complete molting cycle is divided into 4 cyclical stages: premolting (premolting), the process of removing the epidermis from the cuticle begins; molting, whereby the old epidermis is removed; postmolting (postmolting), which combines the absorption of water and the accumulation of organic reserves; and the nonmolting period (endomolting), which is the main period of tissue growth.

[0007] Healthy phyllodes will repeat molting many times throughout the larval life cycle, allowing growth and regeneration. Conversely, malnourished phyllodes will show sporadic and incomplete molting processes, resulting in poor growth, susceptibility to disease, failure to metamorphose and ultimately death. Although there is some understanding of the nutritional requirements of benthic juvenile Splendid lobsters (P. ornatus), this information is scarce for the planktonic larval stage, which is key to developing the closed life cycle production of this species. Successful phyllodes development and metamorphosis are highly dependent on the efficient utilization of energy reserves and the reconstruction of nutrient reserves required for viable larvae. Although larval lobsters may have requirements for specific nutrients, their quantitative requirements and available forms are largely unknown. As important nutrients, total lipid content, the proportion and composition of individual lipids and their constituent fatty acids may affect the health, development and physiology of phyllodes. Lipids are also believed to have a high impact on the molting process, and in many studies, it has been observed that the total lipid content in larval crustaceans follows a cyclic pattern during each molting cycle. This constitutes the accumulation of lipids throughout the intermolt and premolt phases, followed by a decrease in the postmolt phase.Although the expense of molting is largely minor compared to growth and metabolism, its successful completion is critical for larval development.

[0008] Proper lipid nutrition with regard to the distribution of specific lipid species has been shown to accelerate the larval stage and increase the likelihood of successful metamorphosis. Phospholipids are thought to be required for cell membrane integrity and cholesterol transport during the molting process, improving 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 result in poor survival of crustacean larvae, longer intermolt periods, and narrower crustacean widths in crustacean larvae. Elucidation of the nutritional requirements during the larval stage is necessary to establish an optimal diet for phyllodes of Panulirus ornatus. Researchers have focused on the chemical composition of phyllodes in the pre-molting and post-molting (i.e., before and after molting) stages to provide insight into the crude nutritional trends during the larval molting cycle. However, late-stage phyllodes of Panulirus ornatus are also thought to undergo dietary changes.

[0009] Temperate spiny rock lobster (Jasus edwardsii) often experiences periods of inconsistent larval quality while maintaining catchability.

[0010] What is needed in the industry is a complete nutritional formulation (or feed) which overcomes one or more of the disadvantages of prior art formulations, particularly in achieving high metamorphosis success in crustacean species, particularly crabs and spiny lobsters produced in hatcheries. SUMMARY OF THE INVENTION

[0012] The present invention is based on the discovery that a high success rate of metamorphosis can be achieved by a formulation having a specific combination of ingredients. In particular, the present invention increases the viability of the larvae and the progression of development beyond the larval molt. The compositions disclosed herein include and allow for the inclusion of ingredients that alter the form and balance of the nutrients provided.

[0013] Thus, in one aspect, the present invention provides a nutritional formulation comprising:

[0014] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source;

[0015] (ii) live Artemia (non-enriched);

[0016] (iii) at least one sterol;

[0017] (iv) at least one binder;

[0018] (v) at least one emulsifier;

[0019] (vi) at least one mineral;

[0020] (vii) at least one vitamin;

[0021] (viii) at least one protozoan source; and

[0022] (ix) at least one antioxidant.

[0023] In an embodiment, the live Artemia (unenriched) is a species in the nauplius developmental stage.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a photograph showing various parts of an extrusion gun for producing a noodle-shaped preparation according to an embodiment of the present invention.

[0026] Figure 2 is a photograph showing an extrusion machine gun for producing a noodle-shaped preparation according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless otherwise defined, 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 purposes of the present invention, the following terms are defined below.

[0029] As used herein, the articles "a" and "an" refer to one or to more than one (ie, 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.

[0030] As the first component of the nutritional formulation of the present invention, there is an effective amount of n-3 and n-6 ​​fatty acids esterified in a high-content phospholipid source. As used herein, the term "n-3 fatty acids" is also referred to as ω-3 or ω-3 fatty acids, and is characterized by a polyunsaturated fatty acid (PUFA) with a double bond at the third carbon atom from the end of the carbon chain. As used herein, the term "n-6 fatty acids" is also referred to as ω-6 or ω-6 fatty acids, and is characterized by a polyunsaturated fatty acid (PUFA) 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:

[0031]

[0032]

[0033] With regard to the present invention, the most common n-3 fatty acids are EPA and DHA, wherein a smaller amount of n-6 fatty acids are all present in aquatic oils. However, for the present invention, the inventors have found that this fatty acid esterified in a high-content phospholipid source provides benefits in obtaining high viability and sustainability of crustaceans during metamorphosis. In particular, it has been found that this advantage is achieved by utilizing the high phospholipid and low triacylglycerol (TAG) ratio source of aquatic fatty acids. For example, fish oils from aquatic sources that are convenient for EPA, DHA and ARA have these fatty acids incorporated into TAG or ethyl esters. Therefore, fish oils generally have a higher TAG content. On the contrary, krill oil (an alternative source of n-3 / n-6 fatty acids) has EPA, DHA and ARA esterified in phospholipids, particularly phosphatidylcholine. Therefore, krill oil is a convenient source of n-3 and n-6 ​​fatty acids esterified with a high phospholipid and low TAG ratio. Another potential source is squid skin oil because it has a higher phospholipid and TAG ratio.

[0034] Thus, the term "effective" as used herein refers to an amount that is capable of increasing the viability (survival) of developing crustacean larvae during metamorphosis relative to a nutritional formulation utilizing fish oil as a fatty acid source. This may also include additional saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA), such as n-9 fatty acids, highly unsaturated fatty acids (HUFA), other PUFAs (e.g., long chain PUFAs). These fatty acids can serve as an important additional energy source and can be additionally provided by known animal and plant sources.

[0035] In embodiments, the nutritional formulations of the invention comprise about 0.5 to 5% wt / wt 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 embodiments, the formulation is prepared by initially forming an emulsion into which krill oil (or other source of desired fatty acids) is incorporated. The krill oil component may comprise about 15 to 25% of the emulsion. The emulsion may comprise about 6 to 15% of the final formulation (ie, a range of about 0.97 to 3.3 wt / wt % to 0.97 to 3.8 wt / wt %).

[0036] The formulation of the present invention also comprises live Artemia (non-enriched) as an ingredient. Artemia is a type of aquatic salt shrimp. In an embodiment, the nutritional formulation of the present invention utilizes Artemia species in the nauplii development stage. The present invention should be distinguished from other feed compositions comprising Artemia adults, which have been enriched with beneficial nutritional products including phospholipids, ascorbic acid (AA) and antibiotics before formulation. Artemia species in the nauplii development stage are an example of non-enriched live Artemia species. In an embodiment, the nutritional formulation of the present invention comprises about 3 to 60% wt / wt live Artemia (non-enriched), for example, 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.

[0037] As a third component, the formulation comprises at least one sterol. In an embodiment, the at least one sterol is cholesterol. "Sterol" as used herein includes known sterols. In an embodiment, the sterol is an animal-based sterol. In an embodiment, the nutritional formulation of the present invention comprises about 0.05 to 0.5% wt / wt of sterol. In an embodiment, a sterol such as cholesterol is mixed in the above-mentioned emulsion. Cholesterol can be added at about 1.5% of the emulsion.

[0038] As another component, the preparation comprises at least one adhesive.In embodiments, adhesive is alginate (alginic acid), which is an anionic polysaccharide derived from brown algae cell wall.Another benefit of using alginate as adhesive is that it is also used as immunostimulant.In embodiments, nutraceutical of the present invention comprises about 0.2 to 3%wt / wt, for example, 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 at least one adhesive of 2.9%wt / wt.In embodiments where adhesive is alginate, it exists as liquid (aqueous) extract with about 1 to 4% concentration. In such embodiments, the % amount of such extracts in the formulation may range from 15 to 25% wt / wt of the total composition.

[0039] As another component, the formulation comprises at least one emulsifier. In an embodiment, the emulsifier is lecithin. In another embodiment, the emulsifier is gum arabic. In an embodiment, at least one emulsifier in the formulation is present in a range of about 0.2 to 0.8% wt / wt of the total formulation, for example or about 0.3% wt / wt, 0.4% wt / wt, 0.5% wt / wt, 0.6% wt / wt or 0.7% wt / wt.

[0040] As another component, the preparation comprises at least one mineral component. Suitable mineral components can be selected from calcium, chloride, phosphorus, potassium, iodine, sodium, zinc, magnesium, sulfur, iron, cobalt, selenium, chromium, manganese and copper. Mineral components exist usually in the form of mineral salts or inorganic compounds. For example, in one embodiment, the preparation comprises CaCl .

[0041] The mineral component is typically added to the formulation as a salt component during the manufacturing process. In an embodiment, at least one mineral component is added to the above emulsion at about 0.5% of the emulsion; this 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 to 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.

[0042] As another 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, at least one vitamin is also added to the above emulsion at about 1.2% emulsion. In this regard, the at least one vitamin component in the formulation is preferably about 0.05% to 0.5% wt / wt of the total formulation, for example 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.14, 0.15, 0.16, 0.17, 0.17, 0.18, 0.19, 0.20, 0.21, 0.2 44, 0.45, 0.46, 0.47, 0.48 or about 0.49 % wt / wt.

[0043] As another component, the formulation includes at least one antioxidant. Preferred antioxidants include astaxanthin, which is a beta-carotenoid. It is marketed under the trade name Pink (Roche). In one embodiment, about 0.4% of Pink is added to the above emulsion. In this regard, the at least one antioxidant in the formulation is preferably about 0.01 to 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 another embodiment, the present invention also includes a color pigment, but the pigment can also be an antioxidant, such as Pink.

[0044] As a further component, the formulation comprises at least one protozoan source. Preferred protozoan sources include In embodiments, the protist source is in the form of an activated solution in the formulation and is about 1 to 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 inventors have found that the inclusion of a protist source helps prevent white bowel. This is a disease of bacterial origin, and the primary site of infection is the midgut of early to mid-stage phyllodes. This disease can spread rapidly between individuals and can infect entire tanks. Once an outbreak has occurred, established antibiotic treatment regimens are ineffective in regulating this disease.

[0045] In an embodiment, the nutritional formulation comprises:

[0046] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source;

[0047] (ii) live Artemia (non-enriched);

[0048] (iii) at least one sterol;

[0049] (iv) at least one binder;

[0050] (v) at least one emulsifier;

[0051] (vi) at least one mineral;

[0052] (vii) at least one vitamin;

[0053] (viii) at least one protozoan source; and

[0054] (ix) at least one antioxidant.

[0055] In an embodiment, the nutritional formulation comprises:

[0056] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil;

[0057] (ii) live Artemia (non-enriched);

[0058] (iii) cholesterol;

[0059] (iv) at least one adhesive comprising alginate;

[0060] (v) at least one emulsifier comprising lecithin;

[0061] (vi) at least one mineral;

[0062] (vii) at least one vitamin;

[0063] (viii) at least one protozoan source; and

[0064] (ix) at least one antioxidant.

[0065] In an embodiment, the nutritional formulation comprises:

[0066] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil;

[0067] (ii) live Artemia (non-enriched);

[0068] (iii) cholesterol;

[0069] (iv) at least one adhesive comprising alginate;

[0070] (v) at least one emulsifier comprising lecithin;

[0071] (vi) at least one mineral;

[0072] (vii) at least one vitamin;

[0073] (viii) at least one protozoan source; and

[0074] (ix) at least one antioxidant.

[0075] In an embodiment, the nutritional formulation comprises:

[0076] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil;

[0077] (ii) live Artemia (non-enriched);

[0078] (iii) cholesterol;

[0079] (iv) at least one adhesive comprising alginate;

[0080] (v) at least one emulsifier comprising lecithin;

[0081] (vi) at least one mineral comprising calcium chloride;

[0082] (vii) at least one vitamin;

[0083] (viii) at least one protozoan source; and

[0084] (ix) at least one antioxidant.

[0085] In an embodiment, the nutritional formulation comprises:

[0086] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil;

[0087] (ii) live Artemia (non-enriched);

[0088] (iii) cholesterol;

[0089] (iv) at least one adhesive comprising alginate;

[0090] (v) at least one emulsifier comprising lecithin;

[0091] (vi) at least one mineral comprising calcium chloride;

[0092] (vii) at least one vitamin including vitamin C;

[0093] (viii) at least one protozoan source; and

[0094] (ix) at least one antioxidant.

[0095] In an embodiment, the nutritional formulation comprises:

[0096] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil;

[0097] (ii) live Artemia (non-enriched);

[0098] (iii) cholesterol;

[0099] (iv) at least one adhesive comprising alginate;

[0100] (v) at least one emulsifier comprising lecithin;

[0101] (vi) at least one mineral comprising calcium chloride;

[0102] (vii) at least one vitamin including vitamin C;

[0103] (viii) at least one protozoan source; and

[0104] (ix) at least one antioxidant comprising astaxanthin.

[0105] In an embodiment, the nutritional formulation comprises:

[0106] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation;

[0107] (ii) live Artemia (non-enriched);

[0108] (iii) cholesterol;

[0109] (iv) at least one binder;

[0110] (v) at least one emulsifier;

[0111] (vi) at least one mineral;

[0112] (vii) at least one vitamin;

[0113] (viii) at least one protozoan source; and

[0114] (ix) at least one antioxidant.

[0115] In an embodiment, the nutritional formulation comprises:

[0116] (i) an effective amount of n-3 fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation;

[0117] (ii) live Artemia present in the formulation at about 30 to 60% wt / wt of the total formulation;

[0118] (iii) cholesterol;

[0119] (iv) at least one binder;

[0120] (v) at least one emulsifier;

[0121] (vi) at least one mineral;

[0122] (vii) at least one vitamin;

[0123] (viii) at least one protozoan source; and

[0124] (ix) at least one antioxidant.

[0125] In an embodiment, the nutritional formulation comprises:

[0126] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation;

[0127] (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation;

[0128] (iii) cholesterol;

[0129] (iv) at least one adhesive comprising alginate;

[0130] (v) at least one emulsifier;

[0131] (vi) at least one mineral;

[0132] (vii) at least one vitamin;

[0133] (viii) at least one protozoan source; and

[0134] (ix) at least one antioxidant.

[0135] In an embodiment, the nutritional formulation comprises:

[0136] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation;

[0137] (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation;

[0138] (iii) cholesterol;

[0139] (iv) at least one adhesive comprising alginate;

[0140] (v) at least one emulsifier comprising lecithin;

[0141] (vi) at least one mineral;

[0142] (vii) at least one vitamin;

[0143] (viii) at least one protozoan source; and

[0144] (ix) at least one antioxidant.

[0145] In an embodiment, the nutritional formulation comprises:

[0146] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation;

[0147] (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation;

[0148] (iii) cholesterol;

[0149] (iv) at least one adhesive comprising alginate;

[0150] (v) at least one emulsifier comprising lecithin;

[0151] (vi) at least one mineral comprising calcium chloride;

[0152] (vii) at least one vitamin;

[0153] (viii) at least one protozoan source; and

[0154] (ix) at least one antioxidant.

[0155] In an embodiment, the nutritional formulation comprises:

[0156] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation;

[0157] (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation;

[0158] (iii) cholesterol;

[0159] (iv) at least one adhesive comprising alginate;

[0160] (v) at least one emulsifier comprising lecithin;

[0161] (vi) at least one mineral comprising calcium chloride;

[0162] (vii) at least one vitamin including vitamin C;

[0163] (viii) at least one protozoan source; and

[0164] (ix) at least one antioxidant.

[0165] In an embodiment, the nutritional formulation comprises:

[0166] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation;

[0167] (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation;

[0168] (iii) cholesterol;

[0169] (iv) at least one adhesive comprising alginate;

[0170] (v) at least one emulsifier comprising lecithin;

[0171] (vi) at least one mineral comprising calcium chloride;

[0172] (vii) at least one vitamin including vitamin C;

[0173] (viii) at least one protozoan source; and

[0174] (ix) at least one antioxidant comprising astaxanthin.

[0175] The present invention also relates to a nutritional formulation (or feed) comprising one or more of the following additional ingredients:

[0176] (i) Antibiotics - this can help control disease states such as white leg. Suitable antibiotics include enrofloxacin and ciprofloxacin (e.g. 0.5%);

[0177] (ii) Free amino acids - as an added energy source;

[0178] (iii) other proteins and peptides - such as gelatin, bovine serum albumin, and caseinate;

[0179] (iv) carbohydrates and their derivatives, such as cellulose, maltose, chitosan and gellan gum;

[0180] (v) Chemical attractants - such as glycine (also a free amino acid);

[0181] (vi) other color pigments / dyes - may contribute to attractant properties; and

[0182] (vii) pH buffers - such as citric acid and sodium bicarbonate.

[0183] The nutritional formulation of the present invention is preferably produced in the form of noodles extruded into a diameter of about 0.3 to 1.0 mm, such as about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and about 1.0 mm. When not used as a feed, the formulation is preferably kept at a temperature of about 4°C or lower. The present invention contemplates preparing a feed (nutritional composition) by initially forming an emulsion having many of the above-mentioned components. The emulsion is then added to the remaining ingredients to form a feed, which is then extruded to form the final nutritional formulation. The inventors have found that when the emulsion accounts for greater than about 12% of the total formulation and the formulation is in the form of noodles, the nutritional formulation remains neutrally buoyant in the feeding tank, which is beneficial to the survival of the reared larvae.

[0184] In another embodiment, the feed formulation is in the form of a neutrally buoyant noodle, which means that it has a slow sinking rate, i.e., between about 0.02 and 2 cms. -1In the range of 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, 0.6 0, 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 2cms -1 (or a range resulting from any two of the above numbers). The hydrodynamics of the water flow in the culture vessel distributes the feed and phyllo larvae evenly throughout the water column to allow the phyllo larvae to be fed by "bumping" into the feed. Because they are not actively looking for their food source / bait. Therefore, another advantage of the feed formulation of the present invention is that it behaves like feed in a natural environment, increasing the time contact occurs, which in turn allows for increased feed intake and increased development.

[0185] In order to maintain the palatability of the formulation and increase the nutritional value of the formulation, it is an important feature to maintain the pH of the nutritional formulation at about 5 to about 10, for example, 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, 8.8, 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.

[0186] Therefore, the present invention also provides an extruder gun device to extrude a noodle-shaped nutritional preparation, the nutritional preparation comprising:

[0187] (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source;

[0188] (ii) live Artemia (non-enriched);

[0189] (iii) at least one sterol;

[0190] (iv) at least one binder;

[0191] (v) at least one emulsifier;

[0192] (vi) at least one mineral;

[0193] (vii) at least one vitamin;

[0194] (viii) at least one protozoan source; and

[0195] (ix) at least one antioxidant,

[0196] Wherein said extruder device is operably controlled by regulated compressed air.

[0197] In order that the invention may be readily understood and put into practical effect, specific preferred embodiments will now be described by way of the following non-limiting examples.

[0198] The disclosures of each patent, patent application, and publication cited herein are incorporated by reference in their entirety.

[0199] Citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" to the present application.

[0200] Throughout the specification, the purpose is to describe the preferred embodiments of the present invention without limiting the present invention to any one embodiment or a specific set of features. Therefore, it will be understood by those skilled in the art that various modifications and changes may be made to the specific embodiments illustrated without departing from the scope of the present invention, based on this disclosure. All of these modifications and changes are included within the scope of the appended claims. Example

[0201] Preparation Example (in the shape of noodles)

[0202] Noodle Name LNN8 Ingredients(W / W) Alginate solution (3.5%) Live Artemia nauplii (AA) Algae pulp: high pressure sterilization (Reed) Deionized water (DIW) Choline chloride solution (5%) Emul.KO3(Krill Oil) Probiotics <![CDATA[CaCl2 (% w / v) in seawater]]>

[0203] Preparation Example

[0204] Alginate solution

[0205] 1. To prepare alginate solution, add alginate to deionized water.

[0206] 2. Stir the alginate solution into the water until well mixed.

[0207] 3. Warm the alginate solution in a water bath.

[0208] Wet monolith

[0209] Live Artemia nauplii (unenriched)

[0210] 1. Transfer the required volume of live Artemia nauplii from the bucket onto a clean and sterilized 50 micron mesh sieve.

[0211] 2. Rinse the Artemia nauplii with deionized water to rinse the salt water away from the nauplii.

[0212] 3. Prepare a trace mineral mixture

[0213] Trace element mixture <![CDATA[CoSO4 7H2O (Cobalt Sulfate)]]> <![CDATA[Sodium selenite]]> KI (Potassium iodide) <![CDATA[CrK(SO4)2·12H2O (Potassium Chromium Sulfate)]]> <![CDATA[FeSO47H2O (Ferrous sulfate)]]> <![CDATA[MnSO4H2O (Manganese Sulfate)]]> <![CDATA[CuSO45H2O (Copper Sulfate)]]>

[0214] 4. Preparation of Mineral Mixture - TRL M2

[0215] TRL-M2 Trace element mixture <![CDATA[ZnSO4 7H2O (Zinc sulfate)]]> <![CDATA[MgSO4 7H2O (Magnesium sulfate heptahydrate)]]> NaCl (Sodium Chloride) <![CDATA[KH2PO4 (Potassium Dihydrogen Phosphate)]]> <![CDATA[CaCO3 (Calcium carbonate)]]>

[0216] Wet premix

[0217] The wet premix can be used as one of the main ingredients of the diet.For single feeds, standard formulation procedures are followed.

[0218] 1. Weigh the alginate into a mixing bowl.

[0219] 2. Weigh the choline chloride solution into a mixing bowl.

[0220] 3. Weigh deionized water into a mixing bowl.

[0221] 4. Weigh the autoclaved algal slurry into a mixing bowl.

[0222] 5. Measure the probiotic solution into a mixing bowl.

[0223] 6. Mix slowly with a spatula until all ingredients are uniformly mixed.

[0224] Preparation of lecithin-cholesterol paste

[0225] 1. Weigh the required amount of liquid lecithin into a beaker.

[0226] 2. Weigh the required amount of cholesterol powder into the same beaker.

[0227] 3. Leave the solution on the hot plate.

[0228] 4. Stir the solution periodically to incorporate the cholesterol into the lecithin.

[0229] Preparation of nutrient solution:

[0230] 5. Measure out 10% of the required amount of deionized water in a clean beaker.

[0231] 6. Add the required amount of carophyll pink to the beaker.

[0232] 7.Mixed carophyll pink.

[0233] 8. Add vitamin mix, mineral mix and vitamin C to beaker.

[0234] 9. Mix the nutrient solution until all components are completely dissolved.

[0235] 10. Add krill oil to the mixture and stir gently.

[0236] 11. Add the lecithin-cholesterol paste to the beaker.

[0237] Intervention Mixture

[0238] Vitamin Premix (TRL V2)

[0239]

[0240]

[0241] Formulating trace mineral blends

[0242] Trace element mixture <![CDATA[CoSO4 7H2O (Cobalt Sulfate)]]> <![CDATA[Sodium selenite]]> KI (Potassium iodide) <![CDATA[CrK(SO4)2 12H2O (Potassium Chromium Sulfate)]]> <![CDATA[FeSO47H2O (Ferrous sulfate)]]> <![CDATA[MnSO4H2O (Manganese Sulfate)]]> <![CDATA[CuSO45H2O (Copper sulfate)]]>

[0243] Preparation of mineral mixture - TRLM2

[0244] TRL-M2 Trace element mixture <![CDATA[ZnSO4 7H2O (Zinc sulfate)]]> <![CDATA[MgSO4 7H2O (Magnesium sulfate heptahydrate) <!-- 12 -->]]> NaCl (Sodium Chloride) <![CDATA[KH2PO4 (Potassium Dihydrogen Phosphate)]]> <![CDATA[CaCO3 (Calcium carbonate)]]> <![CDATA[Ca(H2PO4)2H2O (Calcium dihydrogen phosphate)]]>

[0245] Emulsion Krill Oil: Emul.KOv3 Element:

[0246] Deionized water

[0247] Pink

[0248] Vitamin Mix (TRL-V2)*Choline Chloride Free

[0249] Mineral Mixture (TRL-M2)

[0250] ROVIMIX 35 ("Vitamin C")

[0251] Krill Oil

[0252] Cholesterol-lecithin paste (1:2 ratio)

[0253] Preparation of cholesterol-lecithin paste

[0254] 1. Weigh the required amount of liquid lecithin into a beaker.

[0255] 2. Weigh the required amount of cholesterol powder into the same beaker.

[0256] 3. Leave the solution on the hot plate.

[0257] 4. As the lecithin softens, stir the solution periodically to incorporate the cholesterol into the lecithin.

[0258] Preparation of emulsion KOV3:

[0259] 1. Weigh deionized water into a beaker.

[0260] 2. Add required amount of carophyllpink, vitamin mix, mineral mix and ROVIMIX 35 to deionized water and mix.

[0261] 3. Weigh the krill oil into a mixing bowl.

[0262] 4. Add part of the nutrient water mixture and mix.

[0263] 5. Add another portion of the nutrient water mixture and mix further.

[0264] 6. Add cholesterol:lecithin paste and mix.

[0265] 7. When the cholesterol:lecithin mixture is well incorporated into the oil-water solution, gradually continue to add the remaining water while mixing the emulsion.

[0266] Sedimentation tank

[0267] Preparation of sedimentation tank:

[0268] 1. Add calcium chloride (CaCl2) to carbon (0.2%).

[0269] 2. Fill each bottle with 1 μm filtered seawater.

[0270] 3. Autoclave the bottle using a large autoclave.

[0271] Krill Oil: Emul.KOv2

[0272] Element:

[0273] Multiply the desired total emulsion volume by the inclusion percentage of each ingredient to determine the inclusion amount.

[0274] Deionized water

[0275] Pink

[0276] Vitamin Mix (TRL-V2)*Choline Chloride Free

[0277] Mineral Mixture (TRL-M1)

[0278] Rovimix 35

[0279] Krill Oil

[0280] Cholesterol / lecithin paste (1:2 ratio)

[0281] Preparation of lecithin-cholesterol paste

[0282] 1. Weigh the required amount of liquid lecithin into a beaker.

[0283] 2. Weigh the required amount of cholesterol powder into the same beaker.

[0284] 3. Leave the solution on the hot plate.

[0285] 4. As the lecithin softens, stir the solution periodically to incorporate the cholesterol into the lecithin.

[0286] Preparation of nutrient solution:

[0287] 1. Measure the required amount of deionized water into a clean beaker.

[0288] 2. Add the required amount of Pink added to beaker.

[0289] 3. Mix carophyll pink until it is completely dissolved.

[0290] 4. Add the vitamin mix, mineral mix and ROVIMIX 35 to the beaker.

[0291] 5. Mix the nutrient solution.

[0292] 6. Add the lecithin-cholesterol paste to the beaker.

[0293] 7. Add krill oil to the nutrient mixture and stir.

[0294] Preparation of Emulsion KO:

[0295] 1. Cover pre-emulsion with plastic wrap and allow to cool.

[0296] 2. Place the beaker in an ice water bath.

[0297] 3. Sonicate the emulsion three times, stirring the emulsion vigorously between each sonication.

[0298] 4. Place the finished emulsion in a clean and sterilized container.

[0299] LN8V3-Pb(Preparation)

[0300] Preparation:

[0301] 1. Hydrate the probiotic powder and place in a 30°C water bath.

[0302] 2. Sterilize all extruder parts (die, chamber, sleeve, plunger, O-rings) in a 10% chlorine bath.

[0303] 3. Take back the lotion KOV3.

[0304] 4. Place the premix in a container of appropriate size to obtain the total premix volume. Add alginate, choline chloride solution and algae slurry and stir thoroughly. When the probiotics are fully activated, add the probiotic solution to the remaining premix and stir thoroughly again.

[0305] Preparation of Artemia nauplii:

[0306] 5. Carefully pour the live Artemia nauplii from the bucket onto the clean and disinfected strainer.

[0307] 6. Rotate the strainer to remove most of the sea water.

[0308] 7. Rinse the Artemia nauplii with deionized water.

[0309] Preparation of LN8V3 premix:

[0310] 8. Weigh out the probiotic premix solution.

[0311] 9. Weigh the deionized water.

[0312] 10. Weigh out the lotion KOV3.

[0313] 11. Add necessary amount of acetic acid dropwise into mixing bowl.

[0314] 12. Mix.

[0315] Add Artemia nauplii to LNN premix:

[0316] 13. Carefully weigh the dried Artemia nauplii directly into the stainless steel mixing bowl.

[0317] 14. Mix.

[0318] 15. Check the pH of the final diet mixture. Adjust if necessary.

[0319] Extrusion Formulations:

[0320] 16. Assemble the plunger by connecting the two O-rings.

[0321] 17. Attach the plunger to the end of the chamber and invert the chamber onto the bench and use the tube to push the plunger to the other end.

[0322] 18. Turn the chamber over and assemble the throttle valve with the O-ring secured with the sleeve.

[0323] 19. Connect the chamber to the arm.

[0324] 20. Connect the compressed air line to the throttle valve assembly.

[0325] 21. Adjust the regulator.

[0326] 22. Charge the extruder with the desired amount of diet mixture.

[0327] 23. Assemble the mold, spray with ethanol, and secure it to the sleeve. Place the O-ring and sleeve on the extruder and clamp until the rubber seal has pressure on it.

[0328] 24. Pour the calcium chloride bath into a large plastic bucket.

[0329] 25. Immerse the head of a large extruder in a calcium chloride bath and squeeze the throttle.

[0330] 26. Press the pressure release lever while removing the noodle gun from the bath.

[0331] Cutting and rinsing food:

[0332] 27. Cut diet length to maximize feeding and effective flushing.

[0333] Extruder gun

[0334] There are two extruder guns (see Figure 1 and 2 ).

[0335] Compressed air is used to drive the extrusion plunger within the extrusion gun, thereby facilitating the extrusion of large batches of noodles. The air pressure is controlled with a regulator to ensure 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.

[0336] Follow the instructions below:

[0337] 1. Assemble the plunger by connecting the two O-rings.

[0338] 2. Attach the plunger to the lubricated end of the chamber and invert the chamber onto the bench and use a plastic stick to push the plunger to the other end. Push the plunger back and forth until the plunger slides freely within the chamber. Leave the plunger at the bottom end of the chamber.

[0339] 3. Turn over the chamber, assemble the throttle valve with the O-ring, and tighten the throttle valve on it with the metal sleeve.

[0340] 4. Attach the extruder to the arm bracket mounted on the bench top. Mount the extruder in a position where it will sit on top of the calcium sea water when it is turned upside down.

[0341] 5. Connect the compressed air line to the throttle valve assembly.

[0342] 6. Adjust the air pressure regulator in the lab to approximately 6 to 10 psi.

[0343] 7. Load the extruder with the diet mixture until full. If the chamber is not full, carefully and slowly press the restrictor to push the mixture to near the top of the chamber. Be careful to stop before the mixture overflows the extruder.

[0344] 8. Assemble the mold into the mold shell and spray it with ethanol. Place the rubber O-ring on the mold shell and place it on top of the extruder and clamp it down until the rubber seal has pressure on it.

[0345] 9. Use a large spoon to create a strong circulation in the bath.

[0346] 10. In one smooth motion, position the noodle gun on the upper left side of the container, submerge the noodle gun in the bath and press the throttle while continuing to stir vigorously. Keep the lever down until all the noodle mixture is extruded, stirring throughout the extrusion process.

[0347] 11. When removing the noodle gun from the tub, press the pressure release lever.

[0348] 12. Repeat the process of loading the gun and extruding until all the noodle mixture has been extruded.

[0349] 13. Use single or multiple blade scissors to cut food into appropriate lengths.

[0350] 14. Drain the food into the strainer, rinse it with tap water, and then rinse it with deionized water.

[0351] 15. Dry food by placing a strainer on a paper towel and then place in a container and place in the refrigerator.

[0352] Fish Oil Product Comparison

[0353] When fish oil was the lipid source, tropical rock lobster (TRL) phyllodes grew through all larval stages, taking 150 to 180+ days to attempt metamorphosis. On the fish oil diet, metamorphosis success from the final stage phyllodes stage to the larval stage was 0%.

[0354] When krill oil is used as the lipid source (as in the formulations of the present invention), the larval development time is reduced to between 90 and 120+ days. The success rate of metamorphosis from the final thallus stage to puberty is 30 to 70%.

Claims

1. A nutritional preparation comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high 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 protozoan source; and (ix) at least one antioxidant.

2. The nutritional formulation of claim 1, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being 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; as well as (viii) at least one protozoan source; and (ix) at least one antioxidant.

3. The nutritional formulation according to claim 1 or 2, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil; (ii) live Artemia (non-enriched); (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one protozoan source; and (ix) at least one antioxidant.

4. The nutritional formulation according to any one of claims 1 to 3, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil; (ii) live Artemia (non-enriched); (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one protozoan source; and (ix) at least one antioxidant.

5. The nutritional formulation according to any one of claims 1 to 4, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil; (ii) live Artemia (non-enriched); (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin; (viii) at least one protozoan source; and (ix) at least one antioxidant.

6. The nutritional formulation according to any one of claims 1 to 5, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil; (ii) live Artemia (non-enriched); (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin including vitamin C; (viii) at least one protozoan source; and (ix) at least one antioxidant.

7. The nutritional formulation according to any one of claims 1 to 6, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, the high phospholipid source being krill oil; (ii) live Artemia (non-enriched); (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin including vitamin C; (viii) at least one protozoan source; and (ix) at least one antioxidant comprising astaxanthin.

8. The nutritional formulation according to any one of claims 1 to 7, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 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 protozoan source; and (ix) at least one antioxidant.

9. The nutritional formulation according to any one of claims 1 to 8, comprising (i) an effective amount of n-3 fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation; (ii) live Artemia present in the formulation at about 30 to 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 protozoan source; and (ix) at least one antioxidant.

10. The nutritional formulation according to any one of claims 1 to 9, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation; (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one protozoan source; and (ix) at least one antioxidant.

11. The nutritional formulation according to any one of claims 1 to 10, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation; (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral; (vii) at least one vitamin; (viii) at least one protozoan source; and (ix) at least one antioxidant.

12. A nutritional formulation according to any one of claims 1 to 11, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation; (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin; (viii) at least one protozoan source; and (ix) at least one antioxidant.

13. A nutritional formulation according to any one of claims 1 to 12, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation; (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin including vitamin C; (viii) at least one protozoan source; and (ix) at least one antioxidant.

14. A nutritional formulation according to any one of claims 1 to 13, comprising: (i) an effective amount of n-3 and n-6 ​​fatty acids esterified in a high phospholipid source, wherein the high phospholipid source is krill oil present in the formulation at about 0.5 to 5.0% wt / wt of the total formulation; (ii) live Artemia (non-enriched) present in the formulation at about 30 to 60% wt / wt of the total formulation; (iii) cholesterol; (iv) at least one adhesive comprising alginate; (v) at least one emulsifier comprising lecithin; (vi) at least one mineral comprising calcium chloride; (vii) at least one vitamin including vitamin C; (viii) at least one protozoan source; and (ix) at least one antioxidant comprising astaxanthin.

15. A nutritional formulation as claimed in any one of claims 1 to 14 in the form of noodles.

16. A nutritional formulation as claimed in any one of claims 1 to 14 in the form of noodles having a diameter of 0.3 to 1.0 mm.

17. Tropical lobster fed with the preparation according to any one of claims 1 to 16.

18. The tropical lobster according to claim 17, which is Panulirus ornatus.

19. A method of achieving metamorphosis success in a population of crustaceans in a hatchery environment comprising the step of feeding said population an effective amount of a formulation as claimed in any one of claims 1 to 16.

20. The method of claim 19, wherein the crustacean species is tropical spiny lobsters, preferably Panulirus ornatus.

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