Fish hydrolysate composition and preparation method thereof

Abstract

The invention relates to a method for preparing a fish hydrolysate composition and a fish hydrolysate composition obtained by the method. The invention also relates to an animal feed, a fertilizer or a soil enhancer comprising the fish hydrolysate composition of the invention.

Description

Technical Field

[0001] The present invention relates to a method for preparing a fish hydrolysate composition and a fish hydrolysate composition obtained by the method. The present invention also relates to an animal feed, a fertilizer or a soil enhancer comprising the fish hydrolysate composition of the present invention. Background Art

[0002] Recent studies predict that the world's population is expected to increase to 8.5 billion by 2030 and further to 9.7 billion by 2050. Consequently, meat consumption is expected to continue to grow due to this population growth and the accompanying global economic expansion. Studies indicate that as personal wealth and household income grow in developing economies, per capita meat consumption in these countries will also increase. Meanwhile, meat consumption in developed countries will also remain high. This will have a downstream impact on global demand for animal protein, with estimates suggesting that global agricultural greenhouse gas (GHG) emissions could increase by 58% by 2050.

[0003] Concentrations of one greenhouse gas, methane, continue to rise in the atmosphere. The mole fraction of methane reached its highest level in four decades in 2020, with a global average increase of 14.7 parts per billion (ppb). Relatively speaking, methane has been rising twice as fast as carbon dioxide since 1750 and is currently 2.5 times higher than pre-industrial levels. Agriculture plays a significant role in rising methane levels. In 2017, agriculture-related sources (such as farming ruminants) accounted for about two-thirds of global anthropogenic methane emissions, with the remainder coming from fossil fuels. Agricultural activities are also a major source of anthropogenic nitrous oxide emissions.

[0004] Ruminants contribute 16% to 25% of global greenhouse gas emissions, and methane concentrations on dairy farms are known to be up to 1,000 times higher than average. Similarly, nitrous oxide is 300 times more potent than CO2 over a 100-year timescale.

[0005] Recent reports indicate that dietary supplements (e.g., animal feed additives) such as essential oils and red algae can reduce methane emissions from ruminants, but this can sometimes come at the expense of feed digestion and fermentation efficiency. Furthermore, because these dietary supplements often increase variable costs for farmers, there are economic barriers to adopting methane-reducing animal feed technologies. This is particularly true given that national laws do not require the agricultural sector to reduce methane output. Therefore, reducing GHG emissions from ruminants (and the broader agricultural sector) in the form of reduced methane and nitrous oxide remains a challenge.

[0006] While methane-reducing compositions for ruminant feeds are known, such as certain species of red marine macroalgae, such as Asparagopsis A Taxiformis, many of these have processing problems and are therefore not suitable for large or frequent use in animal feeds.

[0007] It is another object of the present invention to obviate or mitigate one or more disadvantages of the prior art, whether described herein or elsewhere. Summary of the Invention

[0008] The present invention relates to a method for preparing a fish hydrolysate composition using fish input. The inventors have determined that the fish hydrolysate composition of the present invention has unexpected and surprising methane-reducing properties when used in animal feed products. Without being bound by theory, the reduction of methane production in animals (e.g., ruminants) occurs via different means. These include reducing methanogenic processes in the animal's digestive cycle, limiting / preventing enzymes involved in methane production, or reducing / destroying the presence of methanogenic microorganisms in the animal's digestive tract. The inventors have also observed that the fish hydrolysate composition of the present invention, in addition to its beneficial use in animal feed applications, also has excellent effects in the agricultural and horticultural fields when incorporated into fertilizers and soil enhancers.

[0009] A first aspect of the present invention provides a method for preparing a fish hydrolysate composition, comprising:

[0010] providing fish inputs;

[0011] conducting an intrinsic enzymatic degradation step in the absence of additive enzymes to provide a hydrolysate intermediate; and

[0012] The hydrolysate intermediate is fermented in the presence of a lactic acid producing bacterial culture to produce a fish hydrolysate composition.

[0013] A second aspect of the present invention provides a fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof.

[0014] A third aspect of the present invention provides an animal feed comprising a fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof, or a fish hydrolysate composition according to the second aspect of the present invention or any embodiment thereof.

[0015] A fourth aspect of the present invention provides a fertilizer or soil enhancer comprising the fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof, or the fish hydrolysate composition according to the second aspect of the present invention or any embodiment thereof.

[0016] A fifth aspect of the present invention provides a method for reducing methane and / or improving metabolic efficiency in an animal, comprising administering to an animal a fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof, or a fish hydrolysate composition according to the second aspect of the present invention or any embodiment thereof, or an animal feed according to the second aspect of the present invention or any embodiment thereof; optionally, wherein the animal is a ruminant.

[0017] A sixth aspect of the present invention provides a composition for use in a method for reducing methane and / or improving metabolic efficiency in an animal, the method comprising administering to an animal a fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof, or a fish hydrolysate composition according to the second aspect of the present invention or any embodiment thereof, or an animal feed according to the second aspect of the present invention or any embodiment thereof; optionally, wherein the animal is a ruminant.

[0018] A seventh aspect of the present invention provides a use of a fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof, a fish hydrolysate composition according to the second aspect of the present invention or any embodiment thereof, or a fertilizer or soil enhancer according to the fourth aspect of the present invention or any embodiment thereof for use in horticulture or agriculture.

[0019] As used herein, "fish input" refers to any fish-based starting material. It should be understood that the terms "fish input" and "fish input material" are used interchangeably herein. Fish input can include whole fish or by-products thereof. In some embodiments, fish input includes whole fish, fish meat, fish trimmings, fish viscera, fish bones, fish cartilage, fish scales, or any combination thereof. In some embodiments, the fish input is whole fish. It should be understood that fish input can include input material obtained from any suitable fish species. In some embodiments, the fish input comprises input material obtained from blue whiting, sprat, herring, mackerel, horse mackerel, cod, bass, plaice, turbot, brill, pollack, bream, sole, halibut, floater, dab, coalfish, garfish, pelagic, demersal, or any combination thereof. In other embodiments, the fish input is whole fish and is of the species blue whiting. The fish input may also be a freshwater fish species.

[0020] It will be appreciated that the fish input can be provided in any suitable form capable of undergoing the intrinsic enzymatic degradation described herein. In some embodiments, the fish input is in the form of a liquid, a solid, a slurry, or any mixture thereof. The fish input can be in a dry form, suspended in an aqueous medium, or formed into a frozen block. In some embodiments, the fish input can comprise material that is optionally unfrozen at room temperature. It will be appreciated that the fish input can optionally be subjected to the maceration described herein prior to undergoing the intrinsic enzymatic degradation.

[0021] In some embodiments, the fish input is in the form of frozen blocks, wherein the frozen blocks have a weight per block of about 1 kg to about 100 kg, per block of about 1 kg to about 50 kg, per block of about 1 kg to about 25 kg, per block of about 50 kg to about 100 kg, per block of about 75 kg to about 100 kg, or per block of about 10 kg to about 30 kg. In some embodiments, the fish input is in the form of frozen blocks, wherein the frozen blocks have a weight per block of about 15 kg to about 20 kg. In some embodiments, the fish input comprises frozen blocks having a weight per block of about 15 kg to about 20 kg, and wherein the fish input material is from blue whiting (e.g., whole fish of the species Blue whiting).

[0022] The method for preparing a fish hydrolysate according to the present invention includes conducting an intrinsic enzymatic degradation step in the absence of additive enzymes to provide a hydrolysate intermediate. It should be understood that the intrinsic enzymatic degradation step refers to the enzymatic cleavage of the fish input material (i.e., the cleavage of molecular bonds within the fish input material) using enzymes inherent to the initial fish input material. In other words, the intrinsic enzymatic degradation step involves the use of enzymes naturally present in the fish input material without the addition of additive enzymes. This includes at least the natural enzymes originally present in the digestive tract of the specific fish species used, on which the fish input material is based. In this intrinsic enzymatic degradation step, no additive enzymes are present. In other words, no enzymes that are not naturally present in the initial fish input material are present or added in the intrinsic enzymatic degradation step. The inventors have discovered that by subjecting the fish input material to a moderate intrinsic enzymatic degradation using only intrinsic enzymes, either prior to or as an alternative to additive enzymatic degradation, superior final fish hydrolysate compositions with improved methane reduction properties are produced. This is supported by the methane reduction testing described herein.

[0023] In some embodiments, the intrinsic enzymatic degradation step is performed in an aqueous environment. In such embodiments, the presence of water is required to form the aqueous environment. During the intrinsic enzymatic degradation step, the water in the aqueous environment facilitates enzymatic digestion of the fish input material by hydrolytically cleaving molecular bonds present in the fish input material. It will be appreciated that the water can be brought into contact with the fish input material (added to the fish input material or vice versa) during the intrinsic enzymatic degradation step, or the water can already be a part of the fish input material prior to the intrinsic enzymatic degradation step (e.g., the fish input material already contains a substantial amount of water). When the intrinsic enzymatic degradation step is performed in an aqueous environment, the weight ratio of the fish input material to the water (w / w ratio) is from about 1:1 to about 1:5. Preferably, the weight ratio of the fish input material to the water (w / w ratio) is from about 1:2 to about 1:4, or the weight ratio of the fish input material to the water (w / w ratio) is 1:2.

[0024] In some embodiments, the intrinsic enzymatic degradation step comprises adding frozen fish input material (e.g., a frozen block of fish input that has optionally undergone maceration) to a preheated water bath maintained at a temperature of about 60° C. to about 70° C. As the frozen block thaws, this creates an aqueous environment in which intrinsic enzymatic degradation occurs. The preheated water allows the target intrinsic enzymatic degradation temperature (e.g., about 30° C. to about 50° C.) to be reached more quickly due to the enhanced thawing process of the fish input.

[0025] In some embodiments, the intrinsic enzymatic degradation step is performed at a temperature of about 30°C to about 50°C, optionally wherein the intrinsic enzymatic degradation step is performed for a duration of about 0.5 hours to about 5 hours.

[0026] The intrinsic enzymatic degradation step may be performed at a temperature of about 30° C. to about 50° C., about 35° C. to about 50° C., about 40° C. to about 50° C., about 45° C. to about 50° C., about 30° C. to about 45° C., about 30° C. to about 40° C., or about 30° C. to about 35° C. Preferably, the intrinsic enzymatic degradation step may be performed at a temperature of about 35° C., about 37° C., or about 40° C.

[0027] In other embodiments, the intrinsic enzymatic degradation step is performed for a duration of about 0.5 hours to about 5 hours, about 1 hour to about 5 hours, about 2 hours to about 5 hours, about 3 hours to about 5 hours, about 4 hours to about 5 hours, about 0.5 hours to about 4 hours, about 0.5 hours to about 3 hours, about 0.5 hours to about 2 hours, about 0.5 hours to about 1 hour, about 1 hour to about 4 hours, or about 2 hours to about 3 hours. In some embodiments, the intrinsic enzymatic degradation step is performed for a duration of about 2 hours, about 2.5 hours, or about 3 hours.

[0028] In further embodiments, the intrinsic enzymatic degradation step is performed at a temperature of about 30° C. to about 50° C. for a duration of about 0.5 hours to about 5 hours. In other embodiments, the intrinsic enzymatic degradation step is performed at a temperature of about 30° C. to about 40° C. for a duration of about 2 hours to about 3 hours.

[0029] During the intrinsic enzymatic degradation step, the fish input material (when suspended in or forming the aqueous environment) may be agitated or circulated to ensure that any solids / particulate matter / sediment is mobilized. Agitation or circulation may also prevent stratification of the fish input material, which may be detrimental to enzymatic degradation.

[0030] It is understood that the intrinsic enzymatic degradation step includes at least one intrinsic enzyme that is inherent to and naturally present in the initial fish input material. For example, when the fish input is a blue cod-based material, the intrinsic enzymatic degradation step includes at least one intrinsic enzyme that is inherent to the blue cod species (e.g., at least one intrinsic enzyme present in the digestive tract of blue cod).

[0031] In some embodiments, the intrinsic enzymatic degradation step comprises degradation by at least one intrinsic enzyme comprising a proteolytic enzyme, a carbohydrase, a lipolytic enzyme, a phosphatase, a transaminase, an amino acid decarboxylase, a glutamate dehydrogenase, or any combination thereof. In some embodiments, the protease comprises at least one cathepsin. The proteolytic enzyme may comprise at least one peptidase.

[0032] The proteolytic enzyme may be selected from the group consisting of trypsin, carboxypeptidase A and carboxypeptidase B, or any combination thereof. The carbohydrase may be selected from the group consisting of maltase, amylase, or any combination thereof. The lipolytic enzyme may be lipase. The phosphatase may be alkaline phosphatase.

[0033] In some embodiments, the intrinsic enzymatic degradation step is followed by the addition of an additive proteolytic enzyme, and proteolytic degradation is then carried out until a pH of 6 or less is reached to provide a hydrolysate intermediate.

[0034] It should be understood that the addition of the additive proteolytic enzyme after the intrinsic enzymatic degradation step is performed to ensure that the fish input material undergoes a milder intrinsic enzymatic degradation before undergoing proteolytic enzymatic degradation in the presence of the additive proteolytic enzyme. In embodiments, there may be an optional pasteurization step between the intrinsic enzymatic degradation step and the addition of the additive proteolytic enzyme. Typically, the optional pasteurization step is performed at 65° C. to 75° C. for approximately 3 hours to destroy or denature the intrinsic enzymes.

[0035] Proteolytic degradation is performed until a pH of 6 or less is reached to provide a hydrolysate intermediate. In embodiments, proteolytic degradation is performed until a pH of 5.5 or less, 5 or less, 4.5 or less, 4 or less, 3.5 or less, or 3 or less is reached. Typically, proteolytic degradation is performed until a pH of about 4 to about 6 or about 5.5 is reached to provide a hydrolysate intermediate.

[0036] In some embodiments, proteolytic degradation is carried out in the presence of an additive proteolytic enzyme for any suitable period of time until the desired pH is reached. Proteolytic degradation can be carried out in the presence of an additive proteolytic enzyme for a period of about 1 hour to about 12 hours, about 2 hours to about 12 hours, about 3 hours to about 12 hours, about 4 hours to about 12 hours, about 5 hours to about 12 hours, about 6 hours to about 12 hours, about 1 hour to about 11 hours, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, or about 1 hour to about 6 hours.

[0037] In some embodiments, the proteolytic degradation step is performed at a temperature of about 30°C to about 50°C in the presence of an additive proteolytic enzyme, optionally wherein the proteolytic degradation step is performed for a duration of about 1 hour to about 12 hours in the presence of an additive proteolytic enzyme.

[0038] The proteolytic degradation step in the presence of an additive proteolytic enzyme may be performed at a temperature of about 30° C. to about 50° C., about 35° C. to about 50° C., about 40° C. to about 50° C., about 45° C. to about 50° C., about 30° C. to about 45° C., about 30° C. to about 40° C., or about 30° C. to about 35° C. Preferably, the proteolytic degradation step in the presence of an additive proteolytic enzyme may be performed at a temperature of about 35° C., about 37° C., or about 40° C.

[0039] In some embodiments, the proteolytic degradation step is performed in the presence of an additive proteolytic enzyme at a temperature of about 30° C. to about 50° C. for a duration of about 1 hour to about 12 hours. In other embodiments, the proteolytic degradation step is performed in the presence of an additive proteolytic enzyme at a temperature of about 30° C. to about 40° C. for a duration of about 2 hours to about 10 hours.

[0040] During the proteolytic degradation step in the presence of the additive proteolytic enzyme, the fish input material (when suspended in or forming an aqueous environment) can be agitated or circulated to ensure that solid / particulate matter and sediment are mobile. Agitation or circulation can also prevent stratification of the fish input material, which can be detrimental to enzymatic degradation, and ensure that the additive proteolytic enzyme is thoroughly mixed through the fish input material.

[0041] During the proteolytic degradation step in the presence of an additive proteolytic enzyme, the additive proteolytic enzyme is added in an amount of about 0.01 wt % (based on the weight of the fish input) to about 1 wt % (based on the weight of the fish input). For example, if 100 kg of fish input material (e.g., 100 kg of frozen blocks of blue cod) is used (e.g., Figure 1 In another embodiment, the additive proteolytic enzyme is added in an amount of about 0.01% by weight of the fish input to about 0.5% by weight of the fish input, about 0.01% by weight of the fish input to about 0.25% by weight of the fish input, about 0.01% by weight of the fish input to about 0.1% by weight of the fish input, about 0.01% by weight of the fish input to about 0.05% by weight of the fish input, or about 0.02% by weight of the fish input to about 0.04% by weight of the fish input. Preferably, the additive proteolytic enzyme is added in an amount of about 0.03% by weight of the fish input.

[0042] The method for preparing a fish hydrolysate according to the present invention comprises fermenting a hydrolysate intermediate in the presence of a lactic acid-producing bacterial culture to produce a fish hydrolysate composition. It will be appreciated that the hydrolysate intermediate may optionally be subjected to a first pasteurization step (as described herein) to destroy or denature enzymes present within the fish input material during intrinsic enzymatic and proteolytic degradation in the presence of additive proteolytic enzymes.

[0043] It should be understood that the step of fermenting the hydrolyzate intermediate in the presence of a lactic acid-producing bacterial culture medium involves further degradation of the hydrolyzate intermediate material by enzymes produced by bacteria in the lactic acid-producing bacterial culture medium. This fermentation step must be carried out under conditions that allow the bacteria to remain alive and active. The lactic acid-producing bacterial culture medium comprises at least one lactic acid-producing bacterial species (i.e., at least one lactic acid bacteria) and may optionally comprise a sugar component. In an embodiment, the lactic acid-producing bacterial culture medium comprises at least one lactic acid-producing bacterial species (i.e., at least one lactic acid bacteria), at least one acetic acid-producing bacterial species (i.e., at least one acetic acid bacteria), and may optionally comprise a sugar component.

[0044] The lactic acid bacteria mentioned herein may be selected from the genus group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, Weissella, or any combination thereof. In an embodiment, the lactic acid bacteria may include at least one bacterial species selected from the genus Lactobacillus and / or Pediococcus.

[0045] In a further embodiment, the bacterial culture medium for producing lactic acid comprises at least one species from the genus Lactobacillus, and optionally a sugar component. In other embodiments, the bacterial culture medium for producing lactic acid comprises at least one species from the genus Lactobacillus and / or the genus Pediococcus, and optionally a sugar component. In an embodiment, the bacterial culture medium may comprise Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis. In such embodiments, the bacterial culture medium optionally comprises a sugar component.

[0046] In some embodiments, the lactic acid producing bacterial culture comprises at least one homofermentative bacterial strain and / or at least one heterofermentative bacterial strain.

[0047] "Sugar component" as referred to herein includes any suitable sugar-containing material or sugar source which is consumed by the at least one bacterial species present in the lactic acid producing bacterial culture during the fermentation step of the hydrolysate intermediate. In a preferred embodiment, the sugar component comprises molasses.

[0048] In embodiments, the sugar component is present in an amount of about 5% by weight (based on the weight of the fish input) to about 50% by weight (based on the weight of the fish input), about 10% by weight (based on the weight of the fish input) to about 50% by weight (based on the weight of the fish input), about 20% by weight (based on the weight of the fish input) to about 50% by weight (based on the weight of the fish input), about 30% by weight (based on the weight of the fish input) to about 50% by weight (based on the weight of the fish input), about 40% by weight (based on the weight of the fish input) to about 50% by weight (based on the weight of the fish input), about 5% by weight (based on the weight of the fish input), or about 10% by weight (based on the weight of the fish input) to about 15% by weight (based on the weight of the fish input). In some embodiments, the sugar component is used in an amount of about 25% by weight of the fish input to about 50% by weight of the fish input. Typically, the sugar component is used in an amount of about 30% by weight of the fish input. In such embodiments, the sugar component comprises molasses.

[0049] In some embodiments, there is a step of fermenting the hydrolysate intermediate in the presence of a lactic acid producing bacterial culture to produce a fish hydrolysate composition, wherein the lactic acid producing bacterial culture comprises Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis and molasses in an amount of about 30% by weight (based on the weight of the fish input).

[0050] The step of fermenting the hydrolysate intermediate in the presence of a lactic acid-producing bacterial culture medium to produce a fish hydrolysate composition can be carried out at a temperature of about 30°C to about 50°C; optionally, wherein fermenting the hydrolysate intermediate in the presence of a lactic acid-producing bacterial culture medium to produce a fish hydrolysate composition can be carried out for a duration of about 120 hours to about 240 hours.

[0051] In embodiments, fermenting the hydrolysate intermediate in the presence of a lactic acid-producing bacterial culture to produce a fish hydrolysate composition is carried out at a temperature of about 30° C. to about 50° C., about 35° C. to about 50° C., about 40° C. to about 50° C., about 45° C. to about 50° C., about 30° C. to about 45° C., about 30° C. to about 40° C., or about 30° C. to about 35° C. Preferably, fermenting the hydrolysate intermediate in the presence of a lactic acid-producing bacterial culture to produce a fish hydrolysate composition is carried out at a temperature of about 35° C., about 37° C., or about 40° C.

[0052] In some embodiments, fermentation of the hydrolysate intermediate in the presence of a lactic acid producing bacterial culture to produce a fish hydrolysate composition can be carried out for about 120 hours to about 240 hours, about 24 hours to about 240 hours, about 48 hours to about 240 hours, about 60 hours to about 240 hours, about 72 hours to about 240 hours, about 84 hours to about 240 hours, about 96 hours to about 240 hours, about 108 hours to about 240 hours, about 132 hours to about 240 hours, about 144 hours to about 240 hours, about 156 hours to about 240 hours, about 168 hours to about 240 hours, about 180 hours to about 240 hours, or about 192 hours to about 240 hours. A duration of about 204 hours to about 240 hours, about 216 hours to about 240 hours, about 228 hours to about 240 hours, about 120 hours to about 228 hours, about 120 hours to about 216 hours, about 120 hours to about 204 hours, about 120 hours to about 192 hours, about 120 hours to about 180 hours, about 120 hours to about 168 hours, about 120 hours to about 156 hours, about 120 hours to about 144 hours, or about 120 hours to about 132 hours.

[0053] In further embodiments, the hydrolysate intermediate is fermented in the presence of a lactic acid-producing bacterial culture to produce a fish hydrolysate composition until a pH of 4 or less is reached. In embodiments, the hydrolysate intermediate is fermented in the presence of a lactic acid-producing bacterial culture to produce a fish hydrolysate composition until a pH of 2 or less, 3 or less, 5 or less, or 6 or less is reached. It should be understood that in some embodiments, the pH of the fish input material decreases during fermentation due to the production of lactic acid and / or acetic acid during fermentation. This occurs without the need to introduce additives or external acid sources into the fish input material.

[0054] It should be understood that the fish input can be subjected to a soaking step prior to undergoing the intrinsic enzymatic degradation step; optionally, wherein the soaking step can provide a particle size within the fish input of about 2 mm to about 6 mm. One such technique for soaking fish input known to those skilled in the art entails passing the fish input material through a hydraulic soaker, whereby the particle size within the fish input material can be controlled by selecting an appropriately sized screening die plate through which the soaked fish input material is passed. The screening die plate contains a plurality of openings / perforations of a specific size that control the particle size within the fish input during / after soaking. In some embodiments, the soaking step prior to undergoing the intrinsic enzymatic degradation step is industrial grinding, chopping, and / or flaking of the fish input material using techniques known to those skilled in the art.

[0055] It should be understood that the impregnated fish input material can be in the form of a solid, liquid, suspension, or slurry. If the temperature of the fish input material after impregnation is below the target temperature required for the intrinsic enzymatic degradation step (e.g., the impregnated fish input material is produced from a frozen block and has a temperature of less than or equal to 10°C), then, as described herein, the impregnated fish input material is contacted with preheated water (e.g., a preheated water bath) having a temperature of about 60°C to about 70°C (e.g., about 55°C to about 70°C, about 65°C to about 70°C, or about 60°C to about 75°C) prior to the intrinsic enzymatic degradation step. In this way, the target temperature required for intrinsic enzymatic degradation can be reached in a shorter period of time. In such embodiments, once the target temperature has been reached, the intrinsic enzymatic degradation step can be performed in the preheated water. It should be understood that the preheated water is only suitable for warming or thawing the impregnated fish input material to the target temperature without causing damage to the intrinsic enzymes in the fish input material required for intrinsic enzymatic degradation.

[0056] In some embodiments, the soaking step prior to undergoing the intrinsic enzymatic degradation step provides a particle size within the fish input of about 2 mm to about 6 mm, about 3 mm to about 6 mm, about 4 mm to about 6 mm, about 5 mm to about 6 mm, about 2 mm to about 5 mm, about 2 mm to about 4 mm, about 2 mm to about 3 mm, about 1 mm to about 6 mm, or about 1 mm to about 3 mm. In some embodiments, the soaking step prior to undergoing the intrinsic enzymatic degradation step provides a particle size within the fish input of 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, or 1 mm or less.

[0057] In some embodiments, the hydrolysate intermediate is subjected to a first pasteurization, optionally wherein the first pasteurization is carried out under the following conditions: (i) at a temperature of about 60° C. to about 80° C., and / or (ii) for a duration of about 1 hour to about 12 hours. It will be understood that the hydrolysate intermediate will have undergone at least an intrinsic enzymatic degradation step and, optionally, a proteolytic degradation step in the presence of an additive proteolytic enzyme prior to being subjected to the first pasteurization. It will be understood that the hydrolysate intermediate is subjected to the first pasteurization step until the enzymes present in the hydrolysate intermediate (e.g., intrinsic enzymes and or additive proteolytic enzyme materials) are denatured / destroyed and any bacterial strains present in the hydrolysate intermediate are killed. During the first pasteurization step, the hydrolysate intermediate may be agitated (e.g., circulated using a low shear open impeller pump).

[0058] The first pasteurization step may be carried out at a temperature of about 60°C to about 80°C, about 65°C to about 80°C, about 70°C to about 80°C, about 75°C to about 80°C, about 60°C to about 75°C, about 60°C to about 70°C, or about 60°C to about 65°C.

[0059] In embodiments, the first pasteurization step may be performed for a duration of about 1 hour to about 12 hours, about 1 hour to about 10 hours, about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1 hour to about 4 hours, about 2 hours to about 12 hours, about 4 hours to about 12 hours, about 6 hours to about 12 hours, about 8 hours to about 12 hours, or about 10 hours to about 12 hours. The first pasteurization step may be performed for a duration of approximately 3 hours.

[0060] Fermentation may be followed by a second pasteurization to produce a fish hydrolysate composition, optionally wherein the second pasteurization is performed (i) at a temperature of about 55° C. to about 75° C. and / or (ii) for a duration of about 1 hour to about 4 hours. During the second pasteurization step, the fish hydrolysate composition may be agitated (e.g., circulated using a low shear open impeller pump).

[0061] The second pasteurization step may be carried out at a temperature of about 60°C to about 80°C, about 65°C to about 80°C, about 70°C to about 80°C, about 75°C to about 80°C, about 60°C to about 75°C, about 60°C to about 70°C, or about 60°C to about 65°C.

[0062] In some embodiments, the fish hydrolysate composition is filtered after the second pasteurization step. A suitable filtration technique known to those skilled in the art is to filter the fish hydrolysate composition through a mesh screen with a suitable pore size to extract solid fish hydrolysate composition particles of a specific size. Depending on the final application, the mesh screen may comprise an pore size of about 15 microns to about 500 microns. In such embodiments, the fish hydrolysate composition is retained in the filtered aqueous filtrate (liquid). It should be understood that in some embodiments, this optional filtration step provides a final fish hydrolysate composition product with improved water solubility characteristics, which is desirable for certain downstream applications (e.g., where the fish hydrolysate composition forms part of an aqueous fertilizer solution for sprinkler irrigation applications).

[0063] In an embodiment, after filtration, the fish hydrolysate is evaporated to provide a solid. The solid may then be dried using any suitable drying technique, such as oven drying or freeze drying. The fish hydrolysate composition may be pelletized.

[0064] Also disclosed herein is a fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof.

[0065] Also provided is an animal feed comprising a fish hydrolysate composition obtained according to the method of the first aspect of the invention or any embodiment thereof, or a fish hydrolysate composition according to the second aspect of the invention or any embodiment thereof.

[0066] In an embodiment, the animal feed comprises the fish hydrolysate composition in an amount of about 5% by weight (based on the total weight of the animal feed) and optionally exhibits a methane reduction (%) of about 20% to about 30% as measured according to a suitable method known to the skilled artisan (e.g., the method described herein under "Methane Reduction Test"). It should be understood that the total weight of the animal feed, taken together, includes the weight of both the animal feed and the fish hydrolysate composition.

[0067] In an embodiment, the animal feed comprises the fish hydrolysate composition in an amount of about 10% by weight (based on the total weight of the animal feed) and optionally exhibits a methane reduction (%) of about 25% to about 35% as measured according to a suitable method known to the skilled artisan (e.g., the method described herein under "Methane Reduction Test").

[0068] In an embodiment, the animal feed comprises the fish hydrolysate composition in an amount of about 20% by weight (based on the total weight of the animal feed) and optionally exhibits a methane reduction (%) of about 70% to about 80% as measured according to a suitable method known to the skilled artisan (e.g., the method described herein under "Methane Reduction Test").

[0069] In an embodiment, the animal feed comprises the fish hydrolysate composition in an amount of about 30% by weight (based on the total weight of the animal feed) and optionally exhibits a methane reduction (%) of at least 95% as measured according to a suitable method known to the skilled artisan (e.g., the method described herein under "Methane Reduction Test").

[0070] Disclosed herein is a fertilizer or soil enhancer comprising a fish hydrolysate composition obtained according to the method of the first aspect of the present invention or any embodiment thereof, or a fish hydrolysate composition according to the second aspect of the present invention or any embodiment thereof.

[0071] Disclosed herein is a method for reducing methane and / or improving metabolic efficiency in an animal, comprising administering to an animal a fish hydrolysate composition obtained according to the method of the first aspect of the invention or any embodiment thereof, or a fish hydrolysate composition according to the second aspect of the invention or any embodiment thereof, or an animal feed according to the second aspect of the invention or any embodiment thereof; optionally, wherein the animal is a ruminant.

[0072] Also disclosed herein is a composition for use in a method of reducing methane and / or improving metabolic efficiency in an animal, comprising administering to an animal a fish hydrolysate composition obtained according to the method of the first aspect of the invention or any embodiment thereof, or a fish hydrolysate composition according to the second aspect of the invention or any embodiment thereof, or an animal feed according to the second aspect of the invention or any embodiment thereof; optionally, wherein the animal is a ruminant.

[0073] It is understood that ruminants include, but are not limited to, the following: cows, bulls, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, and antelopes. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0075] Figure 1 Schematic diagram showing a method for preparing a fish hydrolysate composition according to the present invention.

[0076] Figure 2 : is a bar graph showing the average methane reduction (%) of the fish hydrolysate composition A of the present invention at inclusion rates of 5%, 10%, 20% and 30%.

[0077] Figure 3 It is a bar graph showing the average methane reduction (%) of the fish hydrolysate composition B of the present invention at inclusion rates of 5%, 10%, 20% and 30%. DETAILED DESCRIPTION

[0078] The present invention will now be further described, by way of example only, with reference to the accompanying drawings.

[0079] Figure 1 A method for preparing a fish hydrolysate composition according to the present invention is provided in the schematic diagram shown. Figure 1 The exemplary method shown is described for frozen fish input (blue cod (Micromesistius Poutassou) provided by Ward Fish in 15 kg frozen blocks). Such frozen input may include, but is not necessarily limited to: whole fish, fish trimmings, fish bones, fish scales, or any combination thereof. Figure 1 In the described embodiment, the fish input is provided in the form of frozen blocks weighing approximately 15 kg containing whole blue cod.

[0080] exist Figure 1 During step (2) shown, frozen fish input is fed into a hydraulic impregnation machine at a feed rate of one frozen block of fish input every 30 seconds. The fish input is impregnated to produce a liquid or slurry medium containing water, fish meat, and fish bones. The particle size of any solid matter in the liquid or slurry medium is controlled to a particle size of about 2 mm to about 6 mm. This is achieved by selecting the aperture size of the screening die plate used in the hydraulic impregnation machine. The liquid or slurry medium is squeezed through the screening die plate directly into a hopper, which then transfers the fish medium to a pasteurization tank.

[0081] Typically, the pasteurization tank has a capacity of approximately 5000 liters. The fish medium at this time consists of 1 / 3 fish material and 2 / 3 water. The fish medium from the above hopper can be added to the water located in the tank which is preheated to 60°C to 70°C. The preheating of the water can allow the target dimensions in the tank to be reached in a shorter period of time. The pasteurization tank is equipped with a low shear split impeller pump, which is used to ensure that the fish material in the tank is circulated. The circulation of the material in the tank ensures that the fish-based particulate matter and sediment in the tank is mobile and prevents the fish input material from stratifying in the tank. As Figure 1 As shown in step (3) of the process, the fish material is then kept in circulation at approximately 40°C for 2 hours. Importantly, this allows the intrinsic enzymatic degradation of the fish material to occur. No additive enzymes are added at this stage.

[0082] As shown in step (4), an additive proteolytic enzyme (Protease AP-30L from Enzyme Supplies, liquid formulation (10-12%), CAS No. 9001-92-7, Einecs-CE No. 232-642-4, IUB No. 3.4.23.18) is added to the fish material in the tank at approximately 0.03% by weight based on the weight of the fish input. The material is then circulated at approximately 40°C until a pH of 6 or less is reached within the material. Typically, reaching a pH of 5.5 can take from about 1 hour to about 12 hours, depending on the batch size.

[0083] The hydrolysate intermediate formed in step (4) is then pasteurized in step (5) at 65 to 75° C. for about 3 hours. This step ensures that the enzymes present in the hydrolysate intermediate are denatured and kills any bacteria in the system.

[0084] The hydrolysate intermediate is then transferred to a biorefinery and cooled to a temperature of approximately 40°C under circulation. Molasses is added to the cooled hydrolysate intermediate. Typically, molasses is added in an amount of approximately 30% by weight based on the weight of the fish input. Figure 1 As shown in step (6), bacterial strains Lactobacillus plantarum (1K20723), Pediococcus pentosaceus (1K2107) and Lactobacillus brevis (1K20723) are added to the intermediate mixture before fermentation. The fermentation process is carried out at a temperature of about 30°C to 40°C for about 5 to 10 days until a pH of 4 or less is reached. This step uses both homofermentative and heterofermentative strains, thereby allowing the strains to work together. Lactobacillus plantarum and Pediococcus pentosaceus quickly reduce the pH level of the fish medium by rapidly producing lactic acid during the front-end main fermentation cycle. The rapid drop in pH helps to control the fermentation step and inhibit anaerobic losses. A secondary fermentation cycle is then carried out to convert the lactic acid into acetic acid, resulting in a stabilized final product with a pH of 4 or less without the need to add additional acid.

[0085] exist Figure 1 In step (7) shown, the fermented fish hydrolysate material is filtered using a sieve of a specific size (eg, about 15 microns to about 500 microns depending on the end application).

[0086] The filtered material is then subjected to a final pasteurization step (8) at 65°C to 75°C for approximately 2 hours. The final fish hydrolysate product may then optionally undergo downstream processing. Evaporation or cryogenic evaporation separation may be used to provide a solid material. The material may then be pelletized and dried.

[0087] Methane reduction testing

[0088] It should be understood that the following methane reduction testing was conducted using an ANKOM RF gas generation system (eg, a GEN 3 RF gas generation system).

[0089] use Figure 1 The methods exemplified and described above prepared exemplary fish hydrolysate compositions A and B. However, exemplary fish hydrolysate composition A was not subjected to a terminal pasteurization step (8), while fish hydrolysate composition B was subjected to a terminal pasteurization step (8).

[0090] Fish hydrolysate compositions A and B were tested for methane reduction according to the following protocol:

[0091] 1. Obtain rumen inoculum fluid from ruminants (cows).

[0092] 2. Prepare test samples in vials or Agilent bottles using the following components:

[0093] i. Feed matrix containing freeze-dried silage (typically 0.7 g).

[0094] ii. Add to pre-warmed vial of Van Soest anaerobic incubation buffer at 39°C - the amount added depends on the amount of fish hydrolysate composition contained.

[0095] iii. Rumen inoculum liquid (100 mL, filtered through two layers of fine cloth and kept in carbon dioxide at 39°C)

[0096] iv. Fish hydrolysate composition - Separate test samples were prepared in the absence of the fish hydrolysate composition (control group) and in the presence of the fish hydrolysate composition at inclusion rates of 5 wt % (based on the total weight of the feed matrix and the fish hydrolysate composition), 10 wt % (based on the total weight of the feed matrix and the fish hydrolysate composition), 20 wt % (based on the total weight of the feed matrix and the fish hydrolysate composition), and 30 wt % (based on the total weight of the feed matrix and the fish hydrolysate composition).

[0097] 3. The vials / bottles were then sealed and incubated in an anaerobic incubator at 100 rpm (stirring) and 39°C.

[0098] 4. Methane was measured in the headspace of each sample using gas chromatography (GC) at time points of 0, 4, 24, and 48 hours.

[0099] 5. Convert the methane measurement to volume units (mL) using the ideal gas law or other conversion techniques known to those skilled in the art.

[0100] 6. Record three replicates for each sample.

[0101] The average methane reduction measurements (eg, averages across all time points) for the fish hydrolysate compositions are shown below in Tables 1 and 2. The methane reduction (%) values ​​are based on the reduction in methane compared to the control sample.

[0102] Inclusion rate % Average methane reduction (%) 5 -4.56％ 10 -29.09％ 20 -64.24％ 30 -88.09％

[0103] Table 1: Average methane reduction (%) for fish hydrolysate composition A at 5%, 10%, 20% and 30% inclusion rates

[0104] Inclusion rate % Average methane reduction (%) 5 -20.7％ 10 -26.2％ 20 -75.3％ 30 -95.7％

[0105] Table 2: Average methane reduction (%) for fish hydrolysate composition B at 5%, 10%, 20% and 30% inclusion rates

[0106] Figure 2 and Figure 3 Included are bar graphs illustrating the data presented in Tables 1 and 2, respectively.

[0107] As shown in Tables 1 and 2, Figure 2 and Figure 3As shown in the data presented in [ 15 ], as the inclusion rate of the fish hydrolysate composition of the present invention in the test sample feed matrix increases, the sample's methane reduction performance readings also increase. For fish hydrolysate compositions A and B, methane reduction (%) significantly increased as the inclusion rate varied from 5%, 10%, 20%, to 30%. While fish hydrolysate composition A exhibited excellent methane reduction (%) curves at different inclusion rates, fish hydrolysate composition B was surprisingly observed to exhibit unexpectedly improved methane reduction results at all inclusion rates. In particular, fish hydrolysate composition B provided a methane reduction value of -20.7% even at a lower inclusion rate of 5%, and a methane reduction value of -95.7% at a 30% inclusion rate.

[0108] It will be appreciated that various modifications may be made to the above-described fish hydrolysate compositions and methods for their preparation without departing from the spirit and scope of the present invention, for example, as defined by the appended claims. Furthermore, any one or more of the above-described aspects / embodiments may be combined with one or more features of other aspects / embodiments, and all such combinations are within the scope of this disclosure.

[0109] Optional and / or preferred features may be used in combinations other than those explicitly described herein, and optional and / or preferred features described in relation to one aspect of the invention may also be present in another aspect of the invention where appropriate.

[0110] The described and exemplified embodiments are to be considered illustrative rather than restrictive in nature, and it is to be understood that only preferred embodiments are shown and described, and that all changes and modifications within the scope of the invention as defined by the claims are to be protected. It is to be understood that although the use of words such as "preferred," "preferably," "preferred," or "more preferred" in the specification indicates that the described features may be desirable, they may not be required, and embodiments lacking such features may be understood to be within the scope of the invention as defined by the appended claims. With respect to the claims, the use of words such as "one" or "at least one" before a feature is not intended to limit the claim to only one such feature unless otherwise expressly stated in the claim.

Claims

1. A method for preparing a fish hydrolysate composition, comprising: providing fish inputs; An intrinsic enzymatic degradation step is performed in the absence of additive enzymes to provide a hydrolysate intermediate; and The hydrolysate intermediate is fermented in the presence of a lactic acid producing bacterial culture to produce the fish hydrolysate composition.

2. The method of claim 1, wherein the intrinsic enzymatic degradation step is performed at a temperature of about 30°C to about 50°C; preferably, wherein the intrinsic enzymatic degradation step is performed for a duration of about 0.5 hours to about 5 hours.

3. The method of claim 1 or 2, wherein the intrinsic enzymatic degradation step comprises degradation by at least one intrinsic enzyme comprising a proteolytic enzyme, a carbohydrase, a lipolytic enzyme, a phosphatase, or any combination thereof.

4. The method according to claim 3, wherein: (a) the proteolytic enzyme is selected from the group consisting of trypsin, carboxypeptidase A and carboxypeptidase B or any combination thereof; and / or (b) the carbohydrase is selected from the group consisting of maltase, amylase or any combination thereof; and / or (c) the lipolytic enzyme is a lipase; and / or (d) The phosphatase is alkaline phosphatase.

5. A method according to any preceding claim, wherein the intrinsic enzymatic degradation step is followed by the addition of an additive proteolytic enzyme and then proteolytic degradation is carried out until a pH of 6 or less is reached to provide a hydrolysate intermediate.

6. The method of claim 5, wherein the additive proteolytic enzyme is added in an amount of about 0.01% by weight (based on the weight of the fish input) to about 1% by weight (based on the weight of the fish input).

7. The method according to any of the preceding claims, wherein the lactic acid-producing bacterial culture comprises at least one species from the genus Lactobacillus and optionally a sugar component.

8. The method according to claim 7, wherein: (a) the bacterial culture medium comprises Lactobacillus plantarum, Pediococcus pentosaceus and Lactobacillus brevis; and / or (b) the sugar component comprises molasses; and / or (c) The sugar component is used in an amount of about 25% by weight (based on the weight of the fish input) to about 50% by weight (based on the weight of the fish input).

9. The method of any preceding claim, wherein fermenting the hydrolysate intermediate in the presence of a lactic acid-producing bacterial culture to produce the fish hydrolysate composition is carried out at a temperature of about 30°C to about 50°C; optionally, wherein fermenting the hydrolysate intermediate in the presence of a lactic acid-producing bacterial culture to produce the fish hydrolysate composition is carried out for a duration of about 120 hours to about 240 hours.

10. A process according to any preceding claim, wherein the hydrolysate intermediate is fermented in the presence of a lactic acid producing bacterial culture to produce a fish hydrolysate composition until a pH of 4 or less is reached.

11. The method of any preceding claim, wherein the fish input is subjected to a soaking step prior to being subjected to the intrinsic enzymatic degradation step; optionally, wherein the soaking step provides a particle size within the fish input of about 2 mm to about 6 mm.

12. The process according to any one of the preceding claims, wherein the hydrolysate intermediate is subjected to a first pasteurization, optionally wherein the first pasteurization is carried out under the following conditions: (i) at a temperature of about 60°C to about 80°C; and / or (ii) The duration is from about 1 hour to about 12 hours.

13. The method according to any one of the preceding claims, wherein a second pasteurization is performed after the fermentation step to produce the fish hydrolysate composition, optionally wherein the second pasteurization is performed under the following conditions: (i) at a temperature of about 55°C to about 75°C; and / or (ii) The duration is from about 1 hour to about 4 hours.

14. A fish hydrolysate composition obtained by the method according to any one of claims 1 to 13.

15. An animal feed comprising the fish hydrolysate composition obtained according to the method of any one of claims 1 to 13 or the fish hydrolysate composition according to claim 14.

16. A fertilizer or soil enhancer comprising the fish hydrolysate composition obtained according to the method of any one of claims 1 to 13 or the fish hydrolysate composition according to claim 14.

17. A method of reducing methane and / or improving metabolic efficiency in an animal, comprising administering to an animal a fish hydrolysate composition obtained according to the method of any one of claims 1 to 13, a fish hydrolysate composition according to claim 14, or an animal feed according to claim 15; optionally, wherein the animal is a ruminant.

18. A composition for use in a method of reducing methane and / or increasing metabolic efficiency, comprising administering the fish hydrolysate composition obtained according to the method of any one of claims 1 to 13, the fish hydrolysate composition according to claim 14, or the animal feed according to claim 15 to an animal; optionally, wherein the animal is a ruminant.

19. Use of the fish hydrolysate composition obtained according to the method of any one of claims 1 to 13, the fish hydrolysate composition according to claim 14, or the fertilizer or soil enhancer according to claim 16 for horticulture or agriculture.

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