Gelatin-based aquaculture feeds and methods of preparation

Abstract

Aquaculture feeds tend to break down upon delivery in water, which risks fouling the water before the feed can be consumed by the intended animal, such as by shrimp or prawns. A method of preparing an aquaculture feed is described herein comprising the steps of: mixing gelatin, a fatty acid source, and water to form a mixture, and irradiating the mixture with plasma, such as with microwaves, to improve the structural integrity of the feed. The feed formed by extruded pellets of 0-5% moisture or by cake from 20-80% moisture. Advantageously the described feeds can maintain structural integrity in water for 24 hours or longer, while still being digestible to the intended animals.

Description

GELATIN-BASED AQUACULTURE FEEDS AND METHODS OF PREPARATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 582,737 filed on September 14, 2023 entitled PLASMA IRRADIATED GELATIN FOR AQUACULTURE FEEDS, the entire content of each being hereby incorporated by reference.FIELD

[0002] The present disclosure relates generally to aquaculture feeds. More particularly, the present disclosure relates to aquaculture feeds containing gelatin and methods of preparation.BACKGROUND

[0003] Manufactured aquaculture feeds are an important part of modern commercial aquaculture, providing the balanced nutrition needed by farmed fish. The feeds provide the animals with nutrition in a stable and concentrated form, and is delivered directly into the water, enabling the fish to feed efficiently.

[0004] Fish feeds may be made by grinding and mixing ingredients such as fishmeal, vegetable proteins and binding agents such as wheat. Fish feed extruders play a key role in production lines.Although most of the process of fish feed production occurs in the extruder, grinding and mixing can substantially affect the quality of the final product. Typically, water is added and the resulting paste is extruded through holes in a metal plate. Cost and consistency can both be major factors in the production and quality of aquaculture feeds and their resulting usage. However, feed formulations can lose stability and dissolve when delivered in water if the feed is not consumed swiftly, and the water may foul as a result.

[0005] Gelatin is a known protein and has been used as a feed component, such as in US Patent No. 8,343,753 B2 which describes a feed product made from bovine gelatin and soy; US Patent Publication No. US 2022 / 0025334 A1 which describes a synthetic food composition containing gelatin; and US Patent Publication No. US 2020 / 0405807 A1 which describes formulations for nutritive polypeptides for animal feed. However, there is a need to stabilize such feeds to prevent dissolution when used in water for aquaculture applications.

[0006] It is desirable to provide an aquaculture feed with stability in water.SUMMARY

[0007] It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous aquaculture feeds and methods for the preparation thereof.

[0008] Described herein is a method of preparing an aquaculture feed comprising the steps of: mixing gelatin, a fatty acid source, and water to form a mixture, and irradiating the mixture with plasma to form the aquaculture feed; wherein: the aquaculture feed is shaped in a form to be consumed by animals in water; and optionally the feed is dried to a selected water content.

[0009] The mixture formed in the mixing step may be extruded into a pellet form prior to the step of irradiating.

[0010] The plasma used in the irradiating step may comprise exposure to a gaseous plasma, exposure to microwaves, exposure to radiant heat, or a combination thereof. An exemplary form of plasma irradiation is exposure to microwaves. The plasma may be applied at a level of from 5kJ to 1000 GJ, 5 kJ to 100 kJ, or from 10 kJ to 50 kJ.

[0011] The fatty acid source may comprise soy, canola, or fish.

[0012] The aquaculture feed may be dried to a selected water content of: from 10 to 90%, 20% to 80%, 30% to 70%, or from 33% to 67% for wet pellet or cake feed; or from 0-10% for dry feed.

[0013] The aquaculture feed may be dried to the selected water content using a dehydrator, an oven, a fan, light, air drying, air circulation, convection, application of vacuum, heating, freeze drying, or a combination thereof. An exemplary type of drying the feed is by air drying.

[0014] An exemplary method of forming an aquaculture feed as described herein, comprises the steps of: mixing gelatin; a fatty acid source selected from the group consisting of soy, canola and fish; and water to form a mixture; extruding the mixture to form pellets; and exposing the pellets to plasma irradiation, wherein the plasma irradiation comprises microwaves, to form the aquaculture feed; and optionally including air drying the aquaculture feed to reduce water content.

[0015] An aquaculture feed is described herein comprising gelatin, a fatty acid source, and water, to which plasma irradiation has been applied at a level of from 5 kJ to 1000 GJ, and having a water content of from 10% to 80% by weight for cake or wet pellet feed or 0% to 10% for dry feed; wherein the aquaculture feed maintains its structure in water for at least 2 hours.

[0016] The plasma irradiation may comprises exposure to a gaseous plasma, exposure to microwaves, exposure to radiant heat, or a combination thereof. An exemplary form of plasma irradiation comprises exposure to microwaves. The plasma may be applied at a level of from 5 kJ to 100 kJ or 10 kJ to 50 kJ.

[0017] The fatty acid source used in the pelleted feed comprises soy, canola, or fish.

[0018]

[0019] 17. The pelleted aquaculture feed according to claim 12, wherein:

[0020] the fatty acid source is soy or canola and the ratio of gelatin to soy or canola is about2:1 ;

[0021] the water content is from 20 to 80%; and

[0022] the plasma irradiation comprises exposure to microwaves;

[0023] wherein for aquaculture feed is formulated for consumption by shrimp or prawns.

[0024]

[0025] 18. The pelleted aquaculture feed according to an one of claims 12 to 19 wherein the pelleted aquaculture feed maintains its structure in water for at least 4 hours.

[0026] As described herein, aquaculture feeds comprising bovine gelatin are strengthened by plasma irradiation, which advantageously renders the feed particles more stable in water so as to deter deterioration and dissolution of the feed particles in water when provided to animals. Plasma irradiation advantageously increases the melting point of the gelatin. Other aspects and features of the presentdisclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments.

[0027] Advantageously, the aquaculture feeds formed as described herein advantageously deter the fouling of the water with dissolved feed, leading to cleaner water and less bacterial growth for animals therein.BRIEF DESCRIPTION OF THE FIGURES

[0028] All illustrations of the drawings are for the purpose of describing selected variations or examples, and are not intended as limiting. Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0029] Figure 1 is a a photographic image of prepared samples in Example 1 based on a formulation of bovine gelatin with soy meal.

[0030] Figure 2 shows an image of the plasma machine used in Example 1 .

[0031] Figure 3 depicts the apparatus used for the disintegration test in Example 1 .

[0032] Figure 4 provides a depiction of a sample untreated with plasma (0 kJ) at 200x magnification and observed at 10 kV.

[0033] Figure 5 depicts a sample untreated with plasma (0 kJ) at 450x magnification and observed at 10 kV.

[0034]

[0035] Figure 6 shows the result of sample treatment with 12 kJ of plasma.

[0036] Figure 7 depicts a sample treated with 12 kJ plasma at 450x magnification and observed at 10 kV.

[0037] Figure 8 shows a sample irradiated at 24 kJ and viewed at 200x magnification at 10 kV.

[0038] Figure 9 depicts a sample irradiated with 24 kJ and viewed at 450x magnification at 10 kV.

[0039] Figure 10 provides a view of a sample treated with 48 kJ plasma at 200x magnification and observed at 5 kV.

[0040] Figure 11 depicts a first view of the smooth surface of a sample to which 48 kJ of plasma was applied.

[0041] Figure 12 provides a second view of the smooth surface of sample to which 48 kJ of plasma was applied.

[0042] Figure 13 depicts dried pellets formed as a result of the described method.

[0043] Figure 14 shows the beginning of the disintegration test for gelatin-containing samples with slight deterioration.

[0044] Figure 15 shows bovine gelatin-containing samples in a state of disintegration.

[0045] Figure 16 depicts total disintegration time for plasma treated bovine samples with a moisture content of 60.6%, with and without HCI (1% HCI by volume).

[0046] Figure 17 shows total disintegration time results for bovine gelatin samples with a moisture content of 30.3% (1% HCI by volume).DETAILED DESCRIPTION

[0047] Generally, the present disclosure provides an aquaculture feed comprising soy meal and bovine gelatin, the combination of which is irradiated with plasma. Plasma may be applied by a variety of acceptable means. The plasma irradiation applied in the present invention changes the gelatin so that its disintegration in water is slowed, reduced or prevented.

[0048] The aquaculture feed described herein, comprises irradiated soy meal and bovine gelatin, which can be placed in water (freshwater) or saltwater.

[0049] The plasma irradiation applied to the formed pellet (or shaped feed formulation) increases the thermal and structural integrity of the feed when placed in water.

[0050] Advantageously, the aquaculture feed described herein utilizes an economical source of protein while not fouling the water involved in aquaculture in which the animals are raised or farmed. For example, a gelatin-containing aquaculture feed that lasts for 4-6 hours in the 35°C saltwater to accommodate the time and conditions of shrimp digestion can be prepared.

[0051] A variety of formulations of bovine gelatin samples can be treated with a wide variety of levels of plasma and moisture content to achieve desired disintegration profiles depending on such parameters as the type of animals to be fed, specialized formulation of the feed, size and shape of the feed pellets or feed format, the optimal temperature, feeding frequency, overall volume in which the animals are housed and the density of animals therein, among others. Adjustment of these parameters can be utilize to determine optimal plasma irradiation for the desired strength of the pellets or feed form. Each distinct aquaculture situation can achieve the desired parameters in a bespoke manner by adjusting the gelatin in the feed and the extent to which the feed is exposed to plasma irradiation.

[0052] Feed Components. The aquaculture feed formed as described herein comprises gelatin, and preferable bovine gelatin, mixed together with ingredients appropriate for nutrition standards of the intended animal, which may be fish, crustacean, or any animal for which delivery of feed in water is desired. While gelatin provides a protein component to the feed, a wide variety of other components may be included. Gelatin may be utilized as dry gelatin powder ad 1% by weight or more of the initial mixture, including water. To supply desired polyunsaturated fatty acids, such as omega-3 or omega-6 fatty acids, soy meal may be added, or an animal source such as fishmeal. Feed ingredients may include fishmeal, starch, fatty acids, and fatty acid-containing ingredients such as soy and canola. Generous amounts of filler components can be included in the feed as appropriate for the intended application and the subject animals. Appropriate vitamin and minerals components can be included within the composition in amounts appropriate for the animal to be fed. When soy or canola is used with gelatin, it may be present in a gelatin:[soy or canola] ratio of from 1 :2 to 10:1 , such as from 2:1 to 5:1 , such as from 2:1 to 3:1 . In preparation of the feed mixture, hot water (such as boiling water) may initially be added to assist in dissolving and activating the properties of the gelatin, while cold water may be added afterward.

[0053] Plasma. Plasma is applied to the feed mixture or solution containing the feed components. Plasma may be applied either before or after pelleting / shaping or cutting the feed to an appropriate edible size. Plasma application effects the cross linkages in the gelatin, rendering a morestructurally strong form that is resistant to dissolving in water, upon delivery of the feed within the water of the animals to be fed. Plasma may be applied by a variety of methods such as by electrified gas (plasma torch or plasma machine), by microwaves applied in a bench-top scale (such as a kitchen microwave) or on an industrial scale, by application of high temperature radiant heat, such as in an oven ranging from bench-top to industrial scale size, or by any combination of these. This process of applying plasma renders resistance to dissolution, but permits the feed to remaining digestible to animals once consumed, as confirmed by pH testing conducted in the Examples described herein. Application of plasma may be interchangeably referred to herein as “application” (of the plasma form), “plasma irradiation”, “microwaving”, or “radiant heat”, all of which fall within the broader term of “plasma” as used herein.

[0054] The mixture can be irradiated with, for example from 5 kJ to 1000 GJ, 5 kJ to 100 kJ, such as 10 kJ - 50 kJ, for example 12 kJ, 24 kJ or 48 kJ. The plasma may be imparted for example, using microwaves or using hydrogen plasma, or a combination thereof. Plasma irradiation may be applied in a vacuum, under an inert gas, or in air, depending on the selected type of plasma utilized.

[0055] Water Content and Drying. The water content of the feed components during mixing and preparation is adequate to permit the gelatin to form a thickened liquid, gel, paste, or semi-sold, and for such a thickened liquid, gel, paste, or semi-sold, to be shaped or extruded into a shaped form. Initial use of warm or hot water, such as water at about the boiling point, advantageously activates the gelforming properties of the gelatin, and may then be cooled by adding supplemental water at a cooler temperature to promote solidification of the feed.

[0056] Once formed (and either before or after being exposed to plasma), the shaped feed or pellets can be dried, either through dehydrators, ovens, fans, light, air circulation or convection, application of vacuum, heating, freeze drying, a combination of these, or any other appropriate means to achieve a lowered water content consistent with the intended usage of the feed, the desired shelf life and storage conditions. Appropriate sized chambers within ovens, dehydrators or other drying apparatus may be utilized as would be apparent to a skilled person. If a slightly or fully hydrated feed is desired, such as a “cake format”, the feed so formed can be dried to a level of 10% to 90%, for example feed may have a water content of from 20% to 80% formed as a result of the process, such as with a water content of 30% to 70%, or from 33% to 67%. Exemplary final water content in the feed may be 30%, 40%, 50% or 60%, depending on desired applications of the feed. Alternatively, if a more dried feed is desired, such as “dry pellet” form, which may be desirable to extend shelf life prior to use or to accommodate lighter weights for shipping purposes, the feeds may be dried to a very low moisture content having a dry structure, having from 0-10%, such as for example 0 - 5% water content. Both forms can be prepared according to the described method, as suited to the desired application.

[0057] Notably, any type of accepted aquaculture feed ingredient can be used with gelatin, which serves as a binder agent. Once exposed to plasma irradiation, the feed is more stable in water, and can serve as a slow release pellet technology as, advantageously, the pellet does not immediately dissolve in the aquaculture environment. This technology is envisioned as useful for all aquaculture species, for example shrimp and prawns. Optionally, the moisture content may be dried as low as 0-5%final moisture content if dry pellets are desired, or may remain higher, such as 20-80% water (by weight), for example if the feed is to be rendered in cake format. A wide range of moisture content is acceptable during shaping or pelletization, for example from 10-90% water (by weight).

[0058] Structural Integrity in Water. The exposure of the feed mixture or solution to plasma strengthens the feed form to reduce or prevent dissolution in water. Pellets formed without plasma may be observed to last 30 mins in water before breaking down or dissolving to a small particulate that may foul the water of the aquaculture environment. However, the result of plasma exposure / irradiation, such as by microwave, can result in feed pellets maintaining structural integrity and form for up to at least 48 hours, without dissolving in water. Strengthened integrity for lengthy time periods, and is able to maintain structural for at least 1 hour, at least 2 hours, at least 4 hours, at lest 5 hours, at least 10 hours, at least 20 hours, at least 24 hours (1 day) at least 30 hours, at least 40 hours, for at least 48 hours (2 days) or more can be achieved, depending on the desired strength for the intended application, which can vary depending on the amount of plasma applied. Nevertheless, when exposed to the acidic pH of animal digestion, the structure is able to be dissolved for digestion within the animal. The difference in structural integrity of a feed pellet may represent a 100% - 500% or greater disintegration time, relative to conventional feed disintegration time. Notably, disintegration in an acidic environment, such as with 1% HCI by volume, still occurs such that the feed remains digestible to the animal.

[0059] Example 1

[0060] Increasing Structural Integrity of Gelatin by Plasma Irradiation of Aquaculture Feeds

[0061] Overview. In this Example, bovine gelatin is strengthened by plasma irradiation so it can last longer in 35°C waters before disintegration. A major problem with aquaculture is that the feed is usually in a dry pellet format and fouls the water to which it is added, if not eaten before it disintegrates. Fouled water leads to disease propagation and high filtration costs. The goal of this Example was to confirm efficacy of a feed for aquaculture that is nutritionally comprehensive, does not foul the waters but also is digestible by the species fed. Trends were found that showed increased plasma irradiation of bovine gelatin containing soy meal resulted in longer disintegration of the sample in 35°C seawater. This Example also verifies the disintegration period of the sample in 1% hydrochloric acid (HCI) and seawater solution to simulate digestion by an aquatic species. Samples were found to disintegrate sooner in the 1% HCI seawater than seawater without HCI, confirming that the increased structural integrity of a feed in an animal’s aqueous environment should not deter digestion of the feed by the animal, once consumed.

[0062] INTRODUCTION. Shrimp farming is seen all over the world. Pacific Whiteleg Shrimp (Litopenaeus Vannamei) and Giant Tiger Prawns (Penaeus Monodori) comprises the majority of all the shrimp farmed in the world. Typically, shrimp are farmed in open ponds in tropical climates where it takes them 3-6 months to reach market size. Indoor recirculating ponds are becoming popular in North America as there is a large demand for locally produced foods since the COVID-19 pandemic (Nemes et al., 2021). For example, Sask Shrimp Ltd. (Saskatoon, Saskatchewan, Canada) is developingrecirculating shrimp ponds that are not proximal to maritime or a coastal environment. The omnivorous shrimp species studied in this Example is the Pacific Whiteleg Shrimp.

[0063] It is understood that aquatic creatures have a better growth rate if they receive animal protein. Kumar, Barman et al., (2012) found that the inclusion of high-protein grains in feeds for omnivorous fish can greatly reduce the need for fishmeal. High levels of plant proteins usually result in reduced growth caused by reduced feed consumption, essential amino acid deficiency, and the presence of anti-nutritional factors in the vegetable-based feeds. Previous studies showed that omnivorous animals cannot grow well on vegetable-based proteins alone. It is understood that animal proteins are needed for healthy and fast growth in these animals. Most carnivorous and omnivorous aquatic species are fed fishmeal as the animal-protein source. However, the price of fishmeal has become very expensive as ocean stocks dwindle (Luo, 2017). The Wilson Center containing an Integrated Regional Information Networks (IRIN) report estimates that 70 % of fish populations are overused, or in crisis because of overfishing or warmer waters, projecting crisis levels by 2050 if current fishing and global warming trends continue (Luo, 2017).

[0064] This Example establishes the use of bovine gelatin as a main source of animal protein in a feed for Pacific Whiteleg Shrimp. Pacific Whiteleg Shrimp grow in 30-35 °C seawater. Shrimp and prawns may be grown in either open ponds or in recirculating systems. One of the biggest issues with farming shrimp is mitigating disease. Shrimp available from a supermarket typically comprise antibioticresistant bacteria. Disease usually occurs in farmed shrimp from fouled waters that are not filtered properly. Water is fouled from uneaten feed, excrement or animal mortality (Sharma et al., 2021).

[0065] Advantageously, using bovine gelatin as an ingredient in aquaculture feed is that it can be strengthened by plasma irradiation so that it does not break down easily in the water when the feed is uneaten. Using bovine gelatin in the aquaculture feed described reduces filtration costs and aids in disease prevention.

[0066] Previously, weight gain and growth rate were shown to be higher for shrimp fed a diet containing pollock oil (Zhou et al., 2007). It was hypothesised that this success was due to the polyunsaturated fatty acid (PUFA) profile of pollock oil. AlaskOmega® refined pollock oil is estimated to have 18 g / 100g of omega-3 PUFA. Although shrimp grow faster eating a feed containing a higher amount of Omega-3 fatty acids, bovine gelatin does not contain omega-3 fatty acids but it has a high protein content and is more economically viable than pollock oil. Advantageously, bovine gelatin is a viable option for shrimp feeds, and aquaculture feeds generally, in terms of replacing the protein component. PUFA content can be introduced by adding soy meal to a feed. For example, a PUFA content of 1 .045 g / 100g Omega-3 may be estimated, and the bovine gelatin provides the animal protein needed for this aquatic species. Such a feed results in a strong growth performance and healthy weight gain in omnivorous aquaculture species.

[0067] Objective. Objectives of this example are outlined herein. Traditional dried feed pellets used for aquaculture result in fouled water when such pellets are not eaten quickly, because of pellet disintegration. The inclusion of bovine gelatin improves the ability of a pellet to maintain its integrity longer in the water, when treated / irradiated with plasma. Bovine gelatin is a protein-based gel. It is produced by hydrolysis of collagen, a protein powder extracted from cow tissue such as skin, bones andhooves. Bovine gelatin does not have a fatty acid content, and is primarily protein. Bovine gelatin has a melting point of 31-35 °C (Osorio et al., 2007) and is soluble in both fresh water and sea water. Melting point is used in this example, in terms of when the bovine gelatin starts to denature to a liquid. When bovine gelatin reaches its melting point, it denatures from an amorphous solid to a liquid. This suggests it would no longer be consumed by the aquaculture species fed. Water temperature needed for shrimp to survive is approximately 29-31 °C. Therefore, the natural state of bovine gelatin would quickly denature in the shrimp water. To increase the melting point of a gelatin, increased polymer bonding by cross-linking, may be conducted (Osorio et al., 2007; Suzuki et al., 2022).

[0068] In the present Example, it is shown that feed pellets containing bovine gelatin irradiated with plasma maintain a longer-lasting cohesive structure, when such feed pellets are exposed to water, such as seawater. Without wishing to be limited by theory, the improvement correlates to an increase in the gelatin melting point and an improved structural integrity of the pellets, exhibiting additional crosslinking.

[0069] Numerous factors can increase the structural integrity of (bio)polymers (Osorio et al., 2012). In this Example, structural integrity changes occurring in bovine gelatin samples containing 33.3 vol% soy meal are examined as a result of plasma irradiation. Plasma irradiation is the act of distributing a scattering of electrons. When a polymer is subjected to this plasma irradiation / radiation treatment, molecular cross linking or more molecular bonds may be formed, such as covalent bonds in polymer chains attributable to van der Waal forces. A biopolymer may be physically cross-linked by an excited hydrogen plasma gas, or other forms of plasma irradiation. Polymer chains are “linked” by intermolecular forces such as hydrogen, ionic or covalent. This Example illustrates an aquaculture feed that disintegrates less readily in water so as to cause less fouling of the water of the animal stock, but which also is able to be digested once consumed by shrimp. Advantageously, the aquaculture feed described herein is inexpensive, animal-protein rich, and is compliant with feed standards (such as PUFA requirements).

[0070] In this this Example bovine gelatin samples containing soy meal are treated with hydrogen plasma; samples are viewed under a scanning electron microscope (SEM); the cross linking density of samples are viewed with a nuclear magnetic resonance (NMR) spectroscope; disintegration trials of the plasma irradiated gelatin are conducted and the data is reported herein; and assessment is conducted for feeds of 2 different moisture contents.

[0071] Background. Gelatin, a protein ingredient obtained by the thermal denaturation of collagen, is an important industrial gelling biopolymer because of its ability to form gels with water and can also revert back to a liquid state (Osorio et al., 2007). Increasing the structural integrity of gelatin polymers by plasma irradiation has been previously studied. For example, one study reported that the tensile strength of fish gelatin films can be increased by 30% when submitted to 60 kGy (kilograys) of plasma irradiation (BenbettaTeb et al., 2016).

[0072] Plasma Irradiation. Plasma irradiation treatment is also known to increase cross-linking in (bio)polymers by adding a scattering of electrons to the material (Osorio et al., 2007). Plasma irradiation can be used to achieve a better structural integrity in gelatin than standard methodologies.Cross-linking of gelatin has been explored for applications in the biomedical industry (Osorio et al., 2007).

[0073] When a high energy electron beam (plasma) is used to cross-link gelatin hydrogels, polymer chain linking occurs by formation of free radicals that eventually form bonds between the bovine gelatin polymer chains. In fact, when plasma is applied on gelatin solutions, hydroxyls are formed after the reaction of oxygen in the water to eventually form cross-linking bonds in the (bio)polymer of hydrogen bonds (Chen et al. 2020; Higaki et al., 2006). In this Example, it is found that the cross-linking in the bovine gelatin and soymeal biopolymer occurs on the protein polymer chains by covalent bonding, upon adding electrons via plasma irradiation.

[0074] This Example uses hydrogen plasma irradiation as the type of plasma for bovine gelatin cross-linking. Plasma is defined as any excited electron field or high temperature gas such as may be induced by an energy source, for example lightning or fire, or microwave energy or radiant heat energy such as from an oven.

[0075] Plasma irradiation by a gas is used in this study to increase the structural integrity of bovine gelatin. This prevents the gelatin from denaturing in water as well as increases its melting point. From physics accelerated electrons are sources of electromagnetic radiation (Boyd and Sanderson 2003). Electrons accelerate, then create plasma. External gas fields (hydrogen in this case) interacting with the plasma and acceleration of electrons occur. This phenomenon is known as a “scattering of electrons”. The acceleration of electrons creates a plasma mass or plasma fields.

[0076] Gelatin Material. The molecules in gelatin are insoluble fibrous proteins. Collagen molecules are right-handed helices, formed by three parallel left-handed alpha-chains. The chains are continuous amino acid arrangements of Gly-Xaa-Yaa. Xaa and Yaa are used in the chain as a variable of any amino acid. Herein, Gly is glycine, Xaa is proline and Yaa is hydroxyproline.

[0077] To create bovine gelatin, collagen must be resourced. Collagen can be resourced by rendering down bovine skins, hooves or bones either chemically or with solution (Matinong et al., 2022). Collagen may be derived by the use of a pretreatment of enzymatic, acid or alkaline treatment of skins, bones or hooves. Then extraction to a powder form is conducted by hydrolysis, where it can be rehydrated into a gelatin. The source animal (bovine), and its condition during extraction can affect the quality of the gelatin. After the collagen is extracted in solution, filtration, evaporation, drying, grinding, and sifting is performed to finally produce a powder or sheets. Bovine gelatin solidifies at approximately 20°C and melts at approximately 30°C, depending on the concentration of collagen in the batch. Bovine gelatin is a substance that is biologically compatible, economically viable and readily available on the market.

[0078] Shrimp Digestion. The digestive system of shrimp in general can be divided into three parts: foregut, midgut and hindgut. The anatomy of shrimp includes an intestine extending from the stomach, located proximal to the hepatopancreas and heart (Duarte-Restrepo et al., 2020).

[0079] Prawns do not secrete stomach acid however they are found to have an acidic stomach environment of about 4.0 - 6.0 pH. Imaizumi et al. (2021) conducted an experiment where the shrimp stomach acid after feeding was measured. To measure stomach pH, shrimp were fed commercial feed and were dissected 30 min after the start of feeding. A pH test paper was placed on the stomachepithelium and indicated the pH was 5.0. Shrimp digestion is completed by the secretion of enzymes. Similarly, salmon stomach acid was found to be between pH values of 2 and 6. These pH levels are similar for most fish species. Vertebrates secrete hydrochloric acid (HCI) in the digestive system which aids in the breaking down of food. It is understood that the prawns are invertebrates, however the stomach pH of prawns is found to be similar to that of fish.

[0080] For all practical purposes of this study 1% HCI in the seawater is used to simulate fish and shrimp digestion of the plasma irradiated bovine gelatin. It is desirable to have an aquaculture feed product that does not foul the water but which maintains the ability to dissolve and digest adequately in the shrimp or fish gut.

[0081] EXPERIMENTAL. The tasks to be completed in this Example are as follows: to find an amount of the plasma treatment on bovine gelatin versus ultimate disintegration in seawater at 35°C with and without 1% HCI; to explore the surface appearance of the plasma irradiated bovine gelatin containing soy meal at 200x and 450x magnification under scanning electron microscope (SEM); and to examine the density of cross-linking by nuclear magnetic resonance (NMR) spectroscopy based on a spectral intensity reading (data not shown).

[0082] Materials. The materials used in this study are the following:

[0083] 1) Bovine gelatin, dry powder to be hydrolyzed. The bovine gelatin used is Dr. Oetker™ Gelatin Powder (28g), purchased from Walmart™ (Saskatoon, Canada).

[0084] 2) Soy meal was provided by Federated Coop Feeds as a product line with a title of “Soy” (Saskatoon, Canada).

[0085] 3) Sea water was provided from a reservoir from “Bayside Corals”, a saltwater aquarium store (Saskatoon, Canada).

[0086] 4) A fish tank heater was obtained, “Top Fin®” brand submersible aquarium heater Model H150 with 150 watts (W) power at 120 Volts (V).

[0087] 5) A water tank which was a “Coleman™” insulated 2 gallon beverage cooler.

[0088] 6) The HCI used in this study is from Kencro Chemicals “Muriatic acid” purchased at Construction Fasteners in Saskatoon, Canada.

[0089] A 1% HCI acid was added in seawater to simulate shrimp digestion. This is to understand whether or not the bovine gelatin containing soymeal was still digestible to the animal being fed after it is plasma treated. The below calculations show the pH derived from the 1% HCI solution as well as the chemical equation used for introducing HCI in seawater. It is assumed the composition of seawater for this analysis is sodium chloride and water.

[0090] A 1% HCI and seawater solution is found to have a pH of 5.56 according to the below computation of Equation 1 through Equation 6:

[0091] Molarity of 1% HCI

[0092] 1g HCI in 100 g NaCI H2O; H Atomic mass: 1 .0078 u; Cl Atomic mass: 35.453 u

[0093] 1+35.453 = 36.453 g / mol [Equation 1]

[0094] Mole = 1 g + 36.45 g / mol = 0.0274 mol HCI in solution [Equation 2]

[0095] Molarity = 0.0274 mol HCI + 0.1 kg NaCI H2O= 0.274 mol / kg [Equation 3]

[0096] where: u = g / mol; g = gram; mol = moles.

[0097] Mass Percent

[0098] Hydrogen mass percent = (1 .0078 g / mol + 1 g / mol HCI)100 =

[0099] 1 .0078 g / mol - 36.453 g / mol = (0.0277)100 = 2.77% [Equation 4]

[0100] Hydrogen mol = 0.0000277 kg + 0.1 kg seawater = 0.00000277 kg [Equation 5]

[0101] pH Calculation

[0102] pH = -log(0.00000277) = 5.56 [Equation 6]

[0103] Equation 7 is the chemical equation for addition of HCI in sea water (NaCI +H2O).

[0104] [Equation 7]

[0105] Preparation of Samples. Samples were prepared by mixing 1 part soy meal to2 parts bovine gelatin. The mixture was first dried and then 50 ml of boiling (~100°C) water and 50 ml of cold water (~5°C) was added per 15 ml of bovine gelatin powder. Then the sample solution was manually mixed. The mixture was then poured onto a flat tray and allowed to solidify in a refrigerator for 2 hours. Samples were cut into rectangles of 250 mm x 40 mm x 10 mm (I x w x h) before plasma treatment.

[0106] Figure 1 depicts a photographic image of the fully prepared samples based on a formulation of bovine gelatin with soy meal.

[0107] After the samples were prepared they were treated with hydrogen plasma. After plasma treatment half the samples were dried in an oven at 177°C for 30 minutes to reduce the moisture from 60.6% to 30.3%.

[0108] Plasma Irradiation of Samples. Hydrogen gas was excited with electricity to produce a hydrogen plasma scattering of electrons. This field or mass of plasma is then vacuumed to the surface of the bovine gelatin sample where cross-linking can occur. The plasma machine used in this experiment was a Microwave Plasma-Enhanced CVD reactor from Plasmionique, Inc. (Sainte-Julie, Quebec, Canada).

[0109] Figure 2 shows an image of the plasma machine used, with the sample stage indicated in the upper left, and the controller depicted in the lower right.

[0110] The plasma machine comprises an energy source, a chamber, a reactor, a gas and a vacuum pump. As the machine runs, the energy or electrons ignite the hydrogen gas and are vacuumed onto the sample surface. The samples are irradiated with 12 kJ, 24 kJ and 48 kJ of hydrogen plasma.

[0111] The plasma machine operates at 2.45 gigahertz (GHz), 800 W power max. The vacuum system base pressure is no less than 10A-5 Torr, and the operating pressure is about 30 Torr. The process gas used (hydrogen gas) is deemed as a food safe substance. This machine provision was provided by the physics department at the University of Saskatchewan in Saskatoon, Saskatchewan, Canada.

[0112] Scanning Electron Microscopy (SEM). After samples were treated with plasma, they were gold coated to 10 nm to improve their electrical conductivity and were viewed under the SEM. The bovine gelatin samples were viewed at 200x and 450x magnification at 5 kV and 10 kV.The 5 kV and 10 kV rating is how much voltage is used to shine light on the image. The higher the voltage the brighter the light shines, but too high of a voltage can burn the samples. At times, however, a dimmer light reveals more of the sample surface texture. The SEM is used to see very small particles at the micro level. The SEM microscope projects a deeply magnified image by using an electron beam instead of a lightbulb (as in an optical microscope) to see the specimen. As this beam travels, it is manipulated by filters that focus the beam. This causes the beam to shine across a sample. As the beam shines on the sample, electrons are refracted. Detectors in the chamber harness these refracted electrons to produce a microscopic image of the sample surface.

[0113] Nuclear Magnetic Resonance (NMR). NMR spectroscopy uses the magnetic properties of a carbon nuclei to align with a generated magnetic field then absorb and release the energy in its immediate environment. The NMR reads the energy refracted from a reference chemical and then repeats the exercise to the sample being tested. The difference in the two readings can be used in analysis of the sample. Many different aspects can be analyzed with NMR. This Example utilized NMR to test the spectral intensity reflected off the bovine gelatin samples irradiated with different amounts of energy.

[0114] Access to NMR spectroscopy was provided by the University of Saskatchewan,Canada. The NMR spectrometer used is a13C Solid State NMR.13C solid state NMR spectra was obtained with a 4 mm DOTY CP-MAS probe with a Bruker AVANCE III HD™ spectrometer operating at 125.77 MHz (1H frequency at 500.13 MHz). The13C CP / TOSS (Cross Polarization with Total Suppression of Spinning Sidebands) spectra was obtained at a sample spinning speed of 7.5 kHz, a1H 90° pulse of 5 ps, and a contact time of 2.0 ms, with a ramp pulse on the1H channel. Spectral acquisition employed ca. 2500 scans with a recycle delay of 1 s, along with a 50 kHz SPINAL-64 decoupling sequence.13C NMR chemical shifts were referenced externally to adamantane at 38.48 ppm via the low field signal.

[0115] In this Example, the spectral intensity reading from the NMR was assessed. The spectral intensity is the amount of energy read by the NMR. The NMR creates an energetic frequency and pulsates this energy. What is refracted off the sample is read a reference against the reference chemical adamant in this case. In data not shown, it was established that same spectral intensity was observed for each sample.

[0116] Moisture Content. The moisture content of the sample was calculated fromEquation 8:

[0117] H2O vol (total volume)100 = % moisture [Equation 8]

[0118] 100 ml H2O - 165 ml total = (0.606)100 = 60.6% moisture

[0119] Samples were tempered in the oven for 30 minutes at 350 F (177 °C) to decrease the moisture content from about 60% to about 30%. The samples tempered were about 80 mm x 120 mm x 20 mm in size. After oven tempering (e.g. drying) the percent moisture was determined by Equations 9 to 11 :

[0120] ((Initial mass - final mass) + (initial mass))100 = change in %moisture [Equation 9]

[0121] ((24.2 g-12.1 g)-24 g)100% = 50% [Equation 10]

[0122] Therefore: (60.6%) 0.5= 30.3% moisture after tempering [Equation 11]

[0123] Disintegration Test. Samples were placed in a 35°C insulated 2-gallon container containing seawater. Disintegration trials were run with and without the presence of 1% HCI. The fish tank heater was placed in this insulated container to maintain the seawater temperature at 35°C. This temperature was verified by the internal thermometer of the heater. The samples were then timed until ultimate disintegration (determined by visual observation). Figure 3 photographically depicts the apparatus used for the disintegration trial. Disintegration testing was conducted in the depicted insulated containment tank, supplied with a heater.

[0124] Samples at 60.6% humidity and 30.3% humidity were tested for structural integrity. A disintegration test was performed where samples of 20 mm x 20 mm x 20 mm squares were immersed into 35°C salt water with and without 1% HCI and timed until ultimate disintegration. This process was used to determine if the plasma treatment increased structural integrity by visually observing the thermal denaturation of the biopolymer.

[0125] RESULTS & DISCUSSION. It was evaluated whether a trend exists between plasma irradiation amount and increase in structural integrity. This is assumed by the period of time the samples take to ultimately disintegrate in the seawater with and without 1% HCI after treatment of the plasma irradiation.

[0126] Below is the discussion surrounding the findings of this Example. These observations establish that the plasma irradiation increased cross-linking and structural integrity of the bovine gelatin containing soy meal. Also, data was found for how much plasma irradiation contributed to the structural integrity improvements of the bovine gelatin containing soy meal as described below.

[0127] S EM Images. The SEM images of the gelatin samples irradiated with 0 kJ, 12 kJ, 24 kJ and 48 kJ are described herein. Digital photos showing the surface appearance at 200x and 450x magnifications were evaluated for each sample type. It was visually observed that the more the gelatin is treated with plasma (time and strength), the smoother the surface appearance looks. This indicates that more bonding is occurring at a molecular level as per the increased treatment with plasma energy (kJ).

[0128] Sample Treated with 0 kJ Plasma at 200x Magnification and observed at 10 kV.

[0129] Figure 4 shows a bovine gelatin sample containing soy meal that is not irradiated (0 kJ plasma) at 200x magnification and viewed at 10 kV. The SEM image looks as though the gelatin has formed multiple independent globular modules and each one is bonded to its neighbour. This suggests that there is no conformity of cross-linking apparent. Thus independent modules or bubbles form without the addition of scattered electrons. This microstructure present is easily breakable, filled with micro boundaries which makes the substance easy to fissure or crack. This visual observation correlates to the weakness of the material and low melting point in this format of the gelatin sample.

[0130] Sample Treated with 0 kJ Plasma at 450x Magnification and Observed at 10 kV.

[0131] Figure 5 shows a gelatin sample not irradiated (0 kJ) at 450x magnification with 10 kV. It is even more pronounced in this image that the gelatin naturally forms its own bodies based on the limited number of electrons available for bonding. This has created a random assortment of globular bodies. Again, this structure is also weak in strength because it is a containment of self-forming bodies instead of a congruent matrix.

[0132] Sample Treated with 12 kJ Plasma at 200x Magnification and Observed at 10 kV.

[0133] Figure 6 shows the result of treatment with 12 kJ of plasma. The surface appearance of the gelatin is becoming smoother with the application of plasma irradiation. More cross-linked bonds have been made between independent globular modules giving the gelatin a smoother surface from the previous sample. There are still voids and fractional surfaces seen as the gelatin was not irradiated at a high energy level.

[0134] Sample Treated with 12 kJ Plasma at 450x Magnification and Observed at 10 kV.

[0135] Figure 7, shows the result of treatment with 12 kJ plasma, in which the surface is slightly smoother than as seen in Figure 6, with less independent large-sized globular modules, although there are still some present. Here, the sporadic nature of the oil-filled, small-sized, independent modules are more distinguishable. However, fissures or cracks formed from surface fragmentation are clearly observed (indicated with an arrow), showing that fragmentation of the sample occurred on more of a linear plane.

[0136] Sample Treated with 24 kJ Plasma at 200x Magnification and Observed at 10 kV.

[0137] Figure 8 shows the sample irradiated at 24 kJ and viewed at 200x magnification at 10 kV. It can be observed that more of the globular bodies (indicated with an arrow) are starting to bond with their neighbour and form smooth cohesive congruences. The long strands observed are from the soybean meal. There are lower amounts of defined fractured lines which suggest that the sample is conjoined in a more ductile fashion. There are numerous random shapes as shown in the sample which indicates the level of plasma introduction has not found a level of uniform cross-linking in the sample. However, uniformity is more prevalent from previous samples that were irradiated at lower energies.

[0138] Sample Treated with 24 kJ Plasma at 450x Magnification and Observed at 10 kV.

[0139] Figure 9 shows a sample that was irradiated with 24 kJ and viewed at 450x magnification at 10 kV. At a higher level of magnification, the surface has larger regions of smoothness. This suggests that the cross-linking has taken effect, however there are still independent globular bodies throughout the surface. This indicates that the material is stronger compared to samples that were not irradiated but still have the potential to be made stronger. Next the level of energy in sample treatment was doubled to 48 kJ.

[0140] Sample Treated with 48 kJ Plasma at 200x Magnification and Observed at 5 kV.

[0141] Figure 10 shows the 200x magnification at 5 kV energy beam of the gelatin sample irradiated with 48 kJ of plasma. At this irradiation level, the plasma has made the surface very smooth. This suggests the bonding is becoming uniform in the gelatin. This is where the structural integrity of the gelatin increases. In the molecular bonding, there are more bonds made in the molecule lattices of the polymer chains. It can be compared to a bridge made with more cross-supports which strengthens the overall structure of the bridge. The highest amount of plasma treatment produced the structurally strongest gelatin. Based on how smooth the sample has become, it is expected that not much more plasma can be introduced without either burning the sample or producing any further improvements. If all the free bonds are now cross-linked at this level of plasma, additional scattering of electrons would not have a purpose or effect.

[0142] Sample Treated with 48 kJ Plasma at 450x Magnification and Observed at 5kV.

[0143] Figure 11 and Figure 12 show the smooth surface from the applied 48 kJ of plasma onto the sample at a higher magnification. This shows at the micro level the surface is smooth and has formed a congruent matrix. The SEM results show that a stronger bovine gelatin can be achieved with a higher melting point.

[0144] Disintegration Test. The disintegration test consisted of the insulated tank, seawater, fish tank heater, HCI, a stopwatch and a 40 mm x 40 mm x 10 mm sized sample. The samples were placed in the seawater and timed until ultimate disintegration.

[0145] Figure 13 depicts dried sample pellets formed as a result of the described method.

[0146] Figure 14 shows the beginning of the disintegration test where the samples have slightly deteriorated. The samples were also tested in 1% HCI / seawater solution using the same apparatus.

[0147] Once the samples are no longer solid it is considered that ultimate disintegration has occurred.

[0148] Figure 15 shows the samples in a state of ultimate disintegration of bovine gelatin capsules, in which the stopwatch for the trial was stopped and length of time was recorded.

[0149] From the disintegration test it was found that samples at 60.6% moisture, from least to most plasma treatment, had an increased disintegration time of 614% in seawater (without HCI). The samples that were not irradiated lasted 7.02 mins + / - 5s and samples irradiated with 48kJ plasma lasted 43.1 mins + / - 4s. Samples at 60.6% moisture in 1% HCI / seawater lasted 5.2 mins + / - 4s with no plasma irradiation and samples irradiated with 48 kJ lasted 23.1 mins + / - 18s in the HCI / seawater solution which constituted an increase of 460% disintegration time from least to most plasma irradiated.

[0150] Figure 16 shows the results for the total disintegration time as a function of plasma treated bovine samples with moisture contents of 60.6%, with and without 1% (volume / volume) HCI.

[0151] From the second disintegration test it was found that samples at 30.3% moisture had an increased disintegration time of 292% in seawater (with HCI). Samples not plasma irradiated lasted 13.2 mins + / - 11s and samples irradiated with 48 kJ of plasma lasted 38.1 mins + / - 14s. Samples at 30.3% moisture in 1% HCI / seawater solution lasted 7.01 mins + / - 4s with no plasma irradiation and samples irradiated with 48 kJ lasted 21 .94 mins + / - 16s which constitutes an increase of 314% disintegration time.

[0152] Figure 17 shows the total disintegration time results for bovine gelatin samples prepared with moisture contents of 30.3%, with and without HCI at 1% (by volume).

[0153] From the disintegration tests, it is established that the structural integrity of the bovine gelatin samples containing soy meal is increased when level of plasma irradiation increases. Thus, the plasma irradiation increased the melting point and structural integrity of the bovine gelatin-containing feed samples. Plasma irradiation increased the structural integrity in the gelatin-containing material.

[0154] CONCLUSION. The findings establish that the plasma treatment increased the structural integrity of bovine gelatin materials, generally in a linear fashion, such that the more the samples were exposed to plasma, the higher the structural integrity. The plasma conditioning did not change the density of the samples. However, the moisture concentration changed the structural integrity even after the plasma treatment. Interestingly, the samples at a lower moisture content (e.g. 30.3%) had a stronger increase from least to most plasma treated when exposed to the seawater with 1 % HCI thanthe samples in the seawater without HCI. Thus, the samples at the lower moisture content took longer to digest when exposed to more plasma. For the samples to be optimally digested by the shrimp, a higher moisture content may be advantageous. This has impacts on manufacturing and storage as, typically, a higher moisture content product may have a shorter shelf-life.

[0155] These data illustrate that samples moisture content and plasma treatment can be optimized to produce an aquaculture feed for a desired shelf life and disintegration regime.

[0156] Example 2

[0157] Formulation for Plasma Irradiation of Bovine Gelatin Pellets by Microwave Extends Structural Integrity in Water

[0158] In this Example, pellets are made by extrusion. Pellet sizes, ranging 0.5 mm to 5 cm are formed. Formed pellets are plasma irradiated by microwave at a level of from 60 kJ to 120 MJ. Desired strengthening properties can be observed in the gelatin, depending on the application.

[0159] Pellets formed without irradiation are observed to last 30 mins in water before dissolving, while the result of plasma irradiation by microwave was observed to result in pellets maintaining structural integrity up to at least 48 hours, without dissolving in water.

[0160] Plasma irradiation may alternatively be applied to feed pellets by a variety of methods such as by electrified gas (plasma torch or plasma machine), by microwaves applied in a bench-top scale (such as a kitchen microwave) or on an industrial scale, or by application of high temperature radiant heat, such as in an oven ranging from bench-top to industrial scale size. This process of applying plasma irradiation to the formed gelatin-containing pellets strengthens the pellets, rendering resistance to dissolution while remaining digestible to animals once consumed.

[0161] Pellets irradiated by microwaves were then found to last for approximately 48 hours instead of approximately 30 mins observed in control pellets prepared without irradiation.

[0162] The derivation of gelatin through hydrolysis of collagen results from an annealing of collagen (through a process of heat in the presence of moisture), thereby forming gelatin. Amalgamating constituents of the gelatin via plasma irradiation, such as with microwaves, creates stronger bonds and increases the integrity of pellet structure by strengthening of the integrity of the gelatin cross-linking which is present throughout the pellet.

[0163] The pellets can be formulated to include bovine gelatin plus any desirable ingredient to be consumed by the intended aquaculture animal stock, such as soy which provides appropriate fatty acids.

[0164] Aquaculture pellets formed as described herein contain, in addition to gelatin, at least one of: fishmeal, starch, fatty acids, and fatty acid-containing ingredients such as soy and canola. Such pellets may be formulated with ingredients and extruded to sizes as are normally utilized in the aquaculture industry, based on recipes that vary from species to species. The formulation is extruded into pellet form, and subsequently strengthened by plasma irradiation, for example as may be delivered by microwave.

[0165] A formulation intended for a large fish, such as salmon, would different in composition, size, and shape than a formulation intended as a shrimp or prawn diet, although both diets can containgelatin together with any protein source such as soy or fishmeal. The formulation is prepared with ingredients that contains an appropriate level of omega fatty acids, such as those present in soy or fishmeal.

[0166] Advantageously, the use of the pellets formed in this example, which remain stable in water at 24 hours, and at 48 hours, gives more water stability to the aquaculture farmer, as compared with using a traditional feed which breaks down and dissolves in the water quickly, thereby increasing the likelihood that the feed would dissolve and foul the water, breeding bacteria before the feed can be consumed by the animals for which it is intended.

[0167] Example s

[0168] Use of Microwave For Preparation of Dry Pellets for Shrimp

[0169] Extruded pellets are prepared for feeding shrimp in aquaculture, using ingredients described in Example 1 .

[0170] Pellets are were prepared by mixing soy meal and bovine gelatin powder to form a powdered feed mixture. Boiling water is added to the ingredients and mixed thoroughly into the powdered mixture, following which cold water (~5°C), is added and mixed to form a feed solution.

[0171] The addition of bovine gelatin powder is followed by extrusion at a high moisture content. Pellets are air dried, for about one day and then microwaved by a standard 1000 watt (W) microwave suitable for home / kitchen use. Pellets are observed to last at least 48 hours in 35 °C water and 4 months on the shelf. Final moisture content of pellets stored is 0% - 5%.

[0172] Appropriate feed-sized pellets are formed by extrusion through a metal plate onto a microwaveable tray, prior to application of microwaves.

[0173] Pellets are stored in an air-tight container prior to use as an aquaculture feed.

[0174] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required. References cited herein are incorporated by reference.

[0175] Although explained herein in relation to select or preferred embodiments, it is to be understood that many other possible modifications and variations can be made without departing from the intended spirit and scope. The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[0176] References.

[0177] The following documents are herein incorporated by reference.

[0178] US Patent No.8, 343, 753 B2

[0179] US Patent Publication No. US 2022 / 0025334 A1

[0180] US Patent Publication No. US 2020 / 0405807 A1

[0181] Benbettaieb, Nasreddine, Thomas Karbowiak, et al., "Impact of electron beam irradiation on fish gelatin film properties." Food Chemistry 195 (2016): 11-18.

[0182] Boyd, T. J. M., and J. J. Sanderson. “Plasma Radiation.” Chapter 9. In: The Physics of Plasmas, pages 324-375. Cambridge: Cambridge University Press, 2003. doi: 10.1017 / CBO9780511755750.010.

[0183] Chen, Junfeng, Edzna S. Garcia, and Steven C. Zimmerman. "Intramolecularly crosslinked polymers: from structure to function with applications as artificial antibodies and artificial enzymes." Accounts of Chemical Research 53, no. 6 (2020): 1244-1256.

[0184] Duarte-Restrepo, Edisson, Beatriz E. Jaramillo-Colorado, and Laura Duarte-Jaramillo. "Effects of chlorpyrifos on the crustacean Litopenaeus vannamei." PloS one 15, no. 4 (2020): e0231310.

[0185] Higaki, Yuji, et al,. "A thermodynamic polymer cross-linking system based on radically exchangeable covalent bonds." Macromolecules 39, no. 6 (2006): 2121-2125.

[0186] Imaizumi, Kentaro, et al., "Analysis of microbiota in the stomach and midgut of two penaeid shrimps during probiotic feeding." Scientific Reports 11 , no. 1 (2021): 9936.

[0187] Khalaji, et al., Replacing soybean meal with gelatin extracted from cow skin and corn protein concentrate as a protein source in broiler diets. Poultry Science (2016) 95(2), p287-297.

[0188] Kumar, Barman, et al. “Anti-nutritional Factors in Plant Feedstuffs Used in Aquafeeds.” World Aquaculture (2012) September, pages 64-68.

[0189] Luo, Azua (Zizhan) “Ocean Fish Stocks on “Verge of Collapse,” Says IRIN Report.” New Security Beat, Wilson Centre. (2017) February 28.

[0190] Matinong, Andrea Marie E., Yusuf Chisti, Kim L. Pickering, and Richard G. Haverkamp.2022. "Collagen Extraction from Animal Skin" Biology 11 , no. 6: 905. https: / / doi.Org / 10.3390 / biologyl 1060905

[0191] Nemes, Gusztav, Yuna Chiffoleau, et al. "The impact of COVID-19 on alternative and local food systems and the potential for the sustainability transition: Insights from 13 countries." Sustainable Production and Consumption 28 (2021): 591-599.

[0192] Osorio, Fernando A., Elizabeth Bilbao, Ruben Bustos, and Fresia Alvarez. "Effects of concentration, bloom degree, and pH on gelatin melting and gelling temperatures using small amplitude oscillatory rheology." International Journal of Food Properties 10, no. 4 (2007): 841-851.

[0193] Sharma, Laxmi, Ravinder Nagpal, et al., "Antibiotic-resistant bacteria and gut microbiome communities associated with wild-caught shrimp from the United States versus imported farm-raised retail shrimp." Scientific Reports 11 , no. 1 (2021): 3356.

[0194] Suzuki, Takahisa, Shigeru Tsunoda, et al., "A Simple Preparation Method of Gelatin Hydrogels Incorporating Cisplatin for Sustained Release." Pharmaceutics 14, no. 12 (2022): 2601.

[0195] Zhou, Q-C., et al., "Effects of dietary lipid sources on growth and fatty acid composition of juvenile shrimp, Litopenaeus vannamei." Aquaculture Nutrition 13, no. 3 (2007): 222-229.

Claims

CLAIMS:1 . A method of preparing an aquaculture feed comprising the steps of: mixing gelatin, a fatty acid source, and water to form a mixture, and irradiating the mixture with plasma to form the aquaculture feed; wherein: the aquaculture feed is shaped in a form to be consumed by animals in water; and optionally the feed is dried to a selected water content.

2. The method according to claim 1 , wherein the mixture formed in the mixing step is extruded into a pellet form prior to the step of irradiating.

3. The method according to claim 1 or 2, wherein the plasma used in the irradiating step comprises exposure to a gaseous plasma, exposure to microwaves, exposure to radiant heat, or a combination thereof.

4. The method according to claim 3, wherein the plasma comprises exposure to microwaves.

5. The method according to any one of claims 1 to 4, wherein the plasma is applied at a level of from 5kJ to 1000 GJ, 5 kJ to 100 kJ, or from 10 kJ to 50 kJ.

6. The method according to any one of claims 1 to 4, wherein the fatty acid source comprises soy, canola, or fish.

7. The method of any one of claims 1 to 7, wherein the aquaculture feed is dried to a selected water content of: from 10 to 90%, 20% to 80%, 30% to 70%, or from 33% to 67% for wet pellet or cake feed; or from 0-10% for dry feed.

8. The method of any one of claims 1 to 7, wherein the aquaculture feed is dried to the selected water content using a dehydrator, an oven, a fan, light, air drying, air circulation, convection, application of vacuum, heating, freeze drying, or a combination thereof.

9. The method of claim 8, wherein the feed is dried by air drying.

10. The aquaculture feed prepared according to the method of any one of claims 1 to 9.11 . The method of preparing an aquaculture feed according to claim 1 , comprising the steps of:mixing gelatin; a fatty acid source selected from the group consisting of soy, canola and fish; and water to form a mixture; extruding the mixture to form pellets; and exposing the pellets to plasma irradiation, wherein the plasma irradiation comprises microwaves, to form the aquaculture feed; optionally including air drying the aquaculture feed to reduce water content.

12. An aquaculture feed comprising gelatin, a fatty acid source, and water, to which plasma irradiation has been applied at a level of from 5 kJ to 1000 GJ, and having a water content of from 10% to 80% by weight for cake or wet pellet feed or 0% to 10% for dry feed; wherein the aquaculture feed maintains its structure in water for at least 2 hours.

13. The aquaculture feed according to claim 12, wherein the plasma irradiation comprises exposure to a gaseous plasma, exposure to microwaves, exposure to radiant heat, or a combination thereof.

14. The aquaculture feed according to claim 13, wherein the plasma irradiation comprises exposure to microwaves.

15. The aquaculture feed according to any one of claims 12 to 14, wherein the plasma is applied at a level of from 5 kJ to 100 kJ or 10 kJ to 50 kJ.

16. The aquaculture feed according to any one of claims 12 to 15, wherein the fatty acid source comprises soy, canola, or fish.

17. The aquaculture feed according to claim 12, wherein: the fatty acid source is soy or canola and the ratio of gelatin to soy or canola is about 2:1 ; the water content is from 20 to 80%; and the plasma irradiation comprises exposure to microwaves; wherein for aquaculture feed is formulated for consumption by shrimp or prawns.

18. The aquaculture feed according to an one of claims 12 to 17, wherein the aquaculture feed maintains its structure in water for at least 4 hours.

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