Marine protein hydrolysate with low fluoride and trimethylamine content

Abstract

Provided are marine protein hydrolysates for use in pharmaceuticals, dietary supplements, functional foods, foods, beverages, and animal feed, as well as methods for making the marine protein hydrolysates. [Solution] A krill protein hydrolysate is provided, which has, on a dry weight basis, a protein content of greater than 85%, a fat content of less than 5%, a fluoride content of 0.1-200 mg / kg hydrolysate, a TMAO (trimethylamine N-oxide) content of 0.1-200 mg N / 100 g hydrolysate, a TMA (trimethylamine) content of 0.1-200 mg N / 100 g hydrolysate, a TVN (total volatile nitrogen) content of 0.1-200 mg N / 100 g hydrolysate, a sodium content of 0.01-4.0 g / 100 g hydrolysate, and a calcium content of 100-25,000 mg / kg hydrolysate.

Description

[Technical Field]

[0001] The present invention provides methods for making protein hydrolysates and marine hydrolysates for use in pharmaceuticals, dietary supplements, functional foods, foods, beverages, and animal feeds. [Background technology]

[0002] Bioactive peptides from marine secondary products have attracted growing interest as they offer great potential for incorporation into functional foods and for medicinal purposes. Bioactive peptides from marine sources have been shown to exhibit a wide range of physiological functions, including antioxidant, antihypertensive, antibacterial, immunomodulatory, anticancer, and antidiabetic2 activities, among others. Most of the research has focused on the preparation and characterization of hydrolysates for commercial use in health and functional foods.

[0003] Krill hydrolysates for use in marine feed and as human supplements have been described (see, for example, WO2010030193; WO2013102792; Kolkovski et al., J. World Aqua. Soc., Volume 31, Issue 1 (2000) pp. 81-88). These krill hydrolysates are generally produced from sources that contain large amounts of lipids. Summary of the Invention

[0004] The present invention provides protein hydrolysates for use in pharmaceuticals, dietary supplements, functional foods, foods, beverages, and animal feeds, as well as methods for making marine protein hydrolysates.

[0005] In some preferred embodiments, the present invention provides protein hydrolysates characterized by having a protein content of greater than 85% on a dry weight basis, less than 5% fat on a dry weight basis, and one or more of the following properties: a. A fluoride content of the hydrolysate of 0.1 to 200 mg / kg on a dry weight basis; b. TMAO (trimethylamine N-oxide) content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis; c. TMA (trimethylamine) content of 0.1 to 200 mg N / 100 g of hydrolyzate on a dry weight basis; d. TVN (total volatile nitrogen) content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis; e. Sodium content of 0.01 to 4.0 g / 100 g of hydrolysate on a dry weight basis; f. A calcium content of the hydrolysate of 100 to 25,000 mg / kg on a dry weight basis; g. 6.30-10.30 g lysine / lysine content of 100 g total amino acids, h. Threonine content of 3.04 to 7.04 g / 100 g of total amino acids; i. Isoleucine content of 3.39 to 7.39 g / 100 g of total amino acids; j.6.27~10.27g of leucine / 100g of total amino acids, k. Histidine content of 0.94 to 3.94 g / 100 g of total amino acids l. 2.76-6.76g phenylalanine / 100g total amino acids phenylalanine content, m. 2.39-6.39g tyrosine / 100g total amino acids tyrosine content, n. 3.69-7.69g valine / 100g total amino acids valine content, o.3.76~7.76g alanine / 100g total amino acids alanine content p. 3.90-7.90g arginine / 100g total amino acids arginine content, q. 9.68~13.68g of aspartic acid and asparagine / 100g of total amino acids, aspartic acid / asparagine content, r. 11.54~17.54g glutamic acid and glutamine / glutamic acid / glutamine content of 100g total amino acids, s. 2.50-6.50g glycine / 100g total amino acids glycine content, t. 1.84~5.84g of proline / 100g of total amino acids, u.2.35~6.35g of serine / 100g of total amino acids, v. 1.22-5.22g methionine / 100g methionine content of total amino acids, w. 0.24 to 1.04 g of cysteine ​​and cystine / cysteine ​​and cystine content of 100 g of total amino acids, x.0.76~1.76g tryptophan / tryptophan content per 100g of total amino acids.

[0006] In some preferred embodiments, the protein hydrolysate has a fluoride content of 0.1 to 30 mg / kg on a dry weight basis. In some preferred embodiments, the protein hydrolysate has a TMAO content of 0.1 to 10 mg N / 100 g hydrolysate on a dry weight basis. In some preferred embodiments, the protein hydrolysate has a TMA content of 0.1 to 30 mg N / 100 g hydrolysate on a dry weight basis. In some preferred embodiments, the protein hydrolysate has a TVN content of 0.1 to 60 mg N / 100 g hydrolysate on a dry weight basis.

[0007] In some preferred embodiments, the protein hydrolysate has two of the properties a, b, c, d, e, and f. In some preferred embodiments, the protein hydrolysate has three of the properties a, b, c, d, e, and f. In some preferred embodiments, the protein hydrolysate has four of the properties a, b, c, d, e, and f. In some preferred embodiments, the protein hydrolysate has five of the properties a, b, c, d, e, and f. In some preferred embodiments, the protein hydrolysate has properties a, b, c, d, e, and f. In some preferred embodiments, the protein hydrolysate further has all of the properties g, h, i, j, k, l, m, n, o, p, q, r, s, t, u, v, w, and x.

[0008] In some preferred embodiments, the hydrolysate has an essential amino acid content of 39.36 to 49.36 g essential amino acids / 100 g total amino acids. In some preferred embodiments, the hydrolysate has a branched chain amino acid content of 16.35 to 22.35 g branched chain amino acids / 100 g total amino acids. In some preferred embodiments, the hydrolysate has a sulfur-containing amino acid content of 2.86 to 4.86 g sulfur-containing amino acids / 100 g total amino acids.

[0009] In some preferred embodiments, the protein hydrolysate has one or more of properties a, b, c, and d, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysate has property a, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysate has properties a and b, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysate has properties a, b, and c, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysate has properties a, b, c, and d, and properties g, h, i, j, k, l, n, v, and x.

[0010] In some preferred embodiments, the protein hydrolysate has one or more of properties a, b, c, and d, and properties i, j, and n. In some preferred embodiments, the protein hydrolysate has property a, and properties i, j, and n. In some preferred embodiments, the protein hydrolysate has properties a and b, and properties i, j, and n. In some preferred embodiments, the protein hydrolysate has properties a, b, and c, and properties i, j, and n. In some preferred embodiments, the protein hydrolysate has properties a, b, c, and d, and properties i, j, and n.

[0011] In some preferred embodiments, the protein hydrolysate has one or more of properties a, b, c, and d, and properties v and w. In some preferred embodiments, the protein hydrolysate has property a, and properties v and w. In some preferred embodiments, the protein hydrolysate has properties a and b, and properties v and w. In some preferred embodiments, the protein hydrolysate has properties a, b, and c, and properties v and w. In some preferred embodiments, the protein hydrolysate has properties a, b, c, and d, and properties v and w.

[0012] In some preferred embodiments, the protein hydrolysate is further characterized by having a protein content of greater than 89%, 90%, 91%, 92%, 93%, 94%, or 95% on a dry weight basis and a fat content of 2% on a dry weight basis. In some preferred embodiments, the protein hydrolysate has a moisture content of less than 3%. In some preferred embodiments, the protein hydrolysate is further characterized by a neutral, non-fishy taste.

[0013] In some preferred embodiments, the protein hydrolysate is further characterized by dispersing into a clear solution when added to water or other aqueous medium.In some preferred embodiments, the OD590 of a solution of 4.5g of dry weight of the protein hydrolysate in 100g of water is less than 0.4.In some preferred embodiments, the protein hydrolysate is more than 80% water-soluble when assayed by dissolving 4.5g of dry weight of the protein hydrolysate in 100g of water.In some preferred embodiments, the protein hydrolysate is more than 60% water-soluble when assayed by dissolving 4.5g of dry weight of the protein hydrolysate in 100g of water and heating to 85°C for 5 minutes.

[0014] In some preferred embodiments, the protein hydrolysate is a marine protein hydrolysate. In some preferred embodiments, the marine protein hydrolysate is a krill protein hydrolysate. In some preferred embodiments, the krill protein hydrolysate has an astaxanthin content of less than 50 ppm.

[0015] In some preferred embodiments, the present invention provides a process for producing a protein hydrolysate. In particularly preferred embodiments, the process comprises obtaining a marine meal; grinding the marine meal; washing the ground meal with water and / or aqueous citric acid to obtain a washed meal; treating the washed meal with a protease to obtain a protein hydrolysate; and filtering the hydrolysate by microfiltration using a filter having a pore size of 0.1 to 10 μm and / or nanofiltration using a filter having a pore size of 1 to 10 nm to obtain a protein hydrolysate and a permeate. In some preferred embodiments, the meal is a solvent-extracted meal. In some preferred embodiments, the process further comprises a step of inactivating the protease before filtration. In some preferred embodiments, the process further comprises a step of drying the protein hydrolysate to obtain the protein hydrolysate. In some preferred embodiments, the marine meal is krill meal. The krill meal may preferably be whole-fat krill meal or defatted krill meal. In some preferred embodiments, the protease is non-native relative to the starting marine meal (ie, the protease does not naturally occur in the marine organism used to make the meal).

[0016] In another preferred embodiment, the present invention provides a composition comprising a krill protein hydrolysate concentrate characterized by having a protein content of greater than 85% on a dry weight basis, less than 2% fat on a dry weight basis, and less than 4% moisture.

[0017] In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by having a protein content of greater than 89% by dry weight, less than 1% fat by dry weight, and less than 3% moisture. In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by having an arginine content of 5-7% (g AA / 100g protein). In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by having a leucine content of 8-10% (g AA / 100g protein). In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by having a combined branched chain amino acid (i.e., leucine, isoleucine, and valine) content of 17-19.5% (g AA / 100g protein). In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by having a combined methionine and cysteine ​​content of 2-4% (g AA / 100g protein). In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by comprising 50% to 80% peptides of 2 to 20 amino acids on a w / w basis (peptides of free amino acids and peptides of 2 to 20 amino acids in length / total weight of polypeptides).

[0018] In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by containing less than 60 ppm trimethylamine oxide. In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by containing less than 50 ppm fluoride. In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by containing less than 1% salt. In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by having a neutral taste and no fishy taste. In some preferred embodiments, the krill protein hydrolysate concentrate is further characterized by dispersing into a clear or transparent solution when added to water or other aqueous media.

[0019] In some preferred embodiments, the present invention provides a process for producing a krill protein hydrolysate concentrate, the process comprising obtaining a solvent extracted krill meal, washing the solvent extracted krill meal with water and / or citric acid to obtain a washed solvent extracted krill meal, treating the washed solvent extracted krill meal with a protease to obtain a krill protein hydrolysate, and filtering the hydrolysate by microfiltration and / or nanofiltration to obtain a krill protein hydrolysate concentrate and permeate characterized by comprising 50% to 80% by w / w of peptides of 2 to 10 amino acids (peptides of 2 to 10 amino acids by length / total weight of peptides and polypeptides of free amino acids).

[0020] In some preferred embodiments, the process further comprises milling the solvent extracted krill meal before washing. In some preferred embodiments, the process further comprises inactivating proteases before filtering. In some preferred embodiments, the process further comprises drying the krill protein hydrolysate concentrate to obtain a granular krill protein hydrolysate concentrate.

[0021] In some preferred embodiments, the granular krill protein hydrolysate is characterized by having a protein content of greater than 85% by dry weight, less than 2% fat by dry weight, and less than 4% moisture. In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by having a protein content of greater than 89% by dry weight, less than 1% fat by dry weight, and less than 3% moisture. In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by having an arginine content of 5-7% (g AA / 100g protein). In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by having a leucine content of 8-10% (g AA / 100g protein). In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by having a combined branched chain amino acid content of 17-19.5% (g AA / 100g protein). In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by having a combined methionine and cysteine ​​content of 2-4% (g AA / 100g protein).

[0022] In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by comprising less than 60 ppm trimethylamine oxide. In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by comprising less than 50 ppm fluoride. In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by comprising less than 1% salt. In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by having a neutral taste and no fishy taste. In some preferred embodiments, the granular krill protein hydrolysate concentrate is further characterized by dispersing into a clear solution when added to water or other aqueous medium.

[0023] In some preferred embodiments, the present invention provides a krill protein hydrolysate concentrate made by the above process. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a bar graph showing the amino acid profile of a krill protein hydrolysate according to the present invention. [Figure 2] 1 is a bar graph showing the peptide size distribution of krill protein hydrolysates according to the present invention. [Figure 3] 1 is a flow chart of a process according to the present invention.

[0025] definition As used herein, the term "hydrolysate" refers to a mixture of amino acids and peptides of different chain lengths that results from the enzymatic hydrolysis of a source protein with additional enzymes, such as exogenous proteases or proteases that are non-native to the source protein.

[0026] As used herein, the term "oral delivery vehicle" refers to any means of orally delivering a pharmaceutical agent, including, but not limited to, capsules, pills, tablets, and syrups.

[0027] As used herein, the term "food" refers to any food or feed suitable for consumption by humans, non-ruminant animals, or ruminant animals. A "food" can be a prepared and packaged food (e.g., mayonnaise, salad dressing, bread, or cheese food) or an animal feed (e.g., extruded and pelleted animal feed or coarse mixed feed). A "prepared food" means any packaged food approved for human consumption.

[0028] As used herein, the term "foodstuff" refers to any substance suitable for human or animal consumption.

[0029] As used herein, the term "functional food" refers to a food to which a biologically active supplement has been added.

[0030] As used herein, the term "dietary supplement" refers to a food formulated as a dietary or nutritional supplement to be used as part of the diet.

[0031] As used herein, the term w / w (weight / weight), unless otherwise specified, refers to the amount of a given substance in a composition on a weight basis, expressed as a percentage of the total composition weight.

[0032] As used herein, the term "krill meal" refers to a powder made from krill. Examples of krill meal include, but are not limited to, dried krill meal (e.g., krill meal having a moisture content of 3-15%), defatted krill meal (e.g., krill meal from which the fat has been removed by solvent extraction), full-fat krill meal (krill meal that has not been solvent extracted), and sub-fractions of krill meal.

[0033] As used herein, the term "protein," when used in reference to the protein content of a hydrolysate or composition, refers to the content of polypeptides, peptides, and amino acids in the hydrolysate or composition. Unless otherwise specified, protein content is determined, for example, by measuring the total nitrogen content of the hydrolysate and multiplying by a conversion factor of 6.25. Any suitable method for determining total nitrogen content may be utilized. For example, in some preferred embodiments, the Kjeldahl method is utilized. The content of amino acids in a hydrolysate may be expressed as grams of amino acids (e.g., determined by high performance liquid chromatography (HPLC)) per 100 grams of total protein (e.g., determined by the Kjeldahl method) or as grams of amino acids per 100 g of total amino acids (e.g., measured by HPLC of the 20 common amino acids found in proteins).

[0034] As used herein, the term "essential amino acids" refers to the nine amino acids that cannot be synthesized by humans: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

[0035] As used herein, the term "branched chain amino acids" refers to leucine, isoleucine, and valine.

[0036] As used herein, the term "sulfur-containing amino acids" refers to methionine and cysteine ​​(which may be expressed as cysteine ​​and cystine in the assessments reported herein). DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention provides protein hydrolysates for use in pharmaceuticals, dietary supplements, functional foods, foods, beverages, and animal feed, as well as methods for making the protein hydrolysates. In particularly preferred embodiments, the protein hydrolysate is a marine protein hydrolysate. The marine protein hydrolysate may be prepared from any suitable marine starting material containing protein. In some preferred embodiments, the marine protein hydrolysate is prepared from whole krill (e.g., fresh or frozen krill), deshelled krill, or krill meal. In some particularly preferred embodiments, the marine protein hydrolysate is prepared from krill meal, including but not limited to, dried krill meal, defatted krill meal, full-fat krill meal, and fractions of krill meal. The present invention is not limited to the use of any particular krill species. For example, the krill species may be Euphausia In other preferred embodiments, the marine protein hydrolysate is prepared from suitable marine biomass such as fish (e.g., herring, salmon, cod, wild fish, farmed fish), squid, fish roe, algae, shrimp, Calanus, crab, lobster, and mollusks, by-products resulting from the processing of these marine biomass, and meals prepared from these marine biomass, including full-fat and defatted meals resulting from solvent extraction of Calanus, squid, herring, herring roe, or algae, dried and / or solvent extracted or defatted meals.

[0038] During the development of the present invention, it was discovered that it was difficult to process meals such as krill meal to obtain hydrolysates with acceptable taste and odor, along with properties such as low fluoride content and a unique amino acid profile. Previous krill hydrolysates have been described, for example, in WO 2010 / 030193. That application describes a method for reducing fluoride, but the resulting product (believed to be sold as Rimfrost krill powder) contains large amounts of fat and only about 55-60% protein. References such as Zhang et al., Fisheries Sci. (2002) 68:672-679, describe krill hydrolysates, but without evidence of fluoride or TMAO reduction. The quality of the protein produced in Zhang et al. also appears to be low, as evidenced by the reported amino acid composition of the hydrolysate.

[0039] The present invention addresses issues associated with taste, smell, and fluoride levels, providing a product with a unique amino acid composition by utilizing a multi-step process involving washing and / or grinding the meal prior to hydrolysis and subsequent filtration steps, including nanofiltration. These compositions and processes are exemplified with krill meal but are equally applicable to other meals. As shown in the examples, the resulting hydrolyzed product is characterized by a unique amino acid profile, while also possessing improved taste and smell (and associated low levels of TMAO (trimethylamine N-oxide), TMA (trimethylamine), and TVN (total volatile nitrogen)) and low fluoride levels. The amino acid profile reveals that the protein hydrolysate of the present invention is a complete protein source, with sufficient levels of the nine essential amino acids: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. As a non-limiting example, the amino acid score of the krill protein hydrolysate of the present invention has been calculated to be in the range of 129-159, compared to a score of 121 for egg protein and a score of 115 for whey hydrolysate. Intact proteins have a score of 100 or greater on this scale. Amino acid scores are described in detail in WHO (2007): Protein and amino acid requirements in human nutrition, which is incorporated herein by reference in its entirety. Thus, the protein hydrolysate of the present invention is an excellent source of essential amino acids and may be used alone as a complete source of essential amino acids or to supplement other protein sources that may be lower in essential amino acids.

[0040] 1. Starting Materials As discussed above, the present invention is not limited to the use of any particular biological starting material. In a particularly preferred embodiment, the marine protein hydrolysate is prepared from a starting meal, such as a krill meal, such as a full-fat krill meal or a solvent-extracted meal (defatted krill meal) resulting from the extraction of oil from Euphausia superba or Euphausia pacifica krill meal. The krill meal is preferably made by cooking and drying fresh, most preferably live, krill on a fishing vessel to obtain the krill meal. This process yields an exemplary krill meal that may have an approximate protein content of 70%, an approximate fat content of 20%, and an approximate moisture content of less than 8%. In other preferred embodiments, the marine protein hydrolysate is prepared from suitable marine biomass (e.g., biomass of fish (e.g., herring, salmon, cod, wild fish, farmed fish), squid, fish roe, algae, shrimp, Calanus, crab, lobster, or mollusk), marine by-products derived from these biomass, or meal prepared from biomass.

[0041] In some preferred embodiments, the biological starting material is krill meal. Krill meal can preferably be made by any standard marine meal process. Generally, krill meal is produced by cooking freshly caught krill at low temperature (about 80-85°C), drying to reduce the moisture content to about 5-8%, and then grinding. In embodiments where the product is intended for human consumption, it is preferred to package and store the meal under nitrogen without the addition of antioxidants. In some particularly preferred embodiments, the krill meal is solvent extracted or defatted krill meal. Solvent extracted krill meal can be the residual meal from any type of oil extraction process, for example, extraction using supercritical solvents such as ethanol, methanol, heptane, or carbon dioxide with or without an entrainer. In some preferred embodiments, the solvent extracted krill meal is the residual defatted krill meal resulting from the extraction process described in PCT / GB2008 / 001080 or PCT / IB2016 / 000208, both of which are incorporated herein by reference in their entirety.

[0042] In some preferred embodiments, the krill meal is mixed with a suitable solvent to extract lipids from the meal. In contrast to prior art methods, the present invention utilizes conditions that favor the extraction of the maximum amount of lipids from the krill meal, at the expense of increased amounts of contaminants in the initial solvent extract. In preferred embodiments, the solvent is an organic protic solvent, although other solvents known for use in the extraction of food-grade lipids, such as acetone, hexane, etc., may also be used. Suitable organic protic solvents include, but are not limited to, n-butanol, n-propanol, isopropanol, nitromethane, ethanol, and methanol. In a particularly preferred embodiment, the protic solvent is ethanol.

[0043] In a preferred embodiment, the concentration of the protic solvent used in the initial solvent extraction step is at least 90%, or preferably about 94%-98%, more preferably about 95%-97%, and most preferably about 96% (e.g., 96% ethanol or methanol).

[0044] In some embodiments, the protic solvent is mixed with the biological starting material in a ratio of protic solvent:biological starting material of about 1:1 to 10:1, preferably about 3:1 to 6:1, more preferably about 4:1 to 5:1, and most preferably about 4.4:1.

[0045] In preferred embodiments, the biological starting material is extracted with a protic solvent at a temperature of about 5° C. to about 65° C., about 20° C. to about 60° C., preferably about 30° C. to 50° C., more preferably about 30° C. to 50° C., and most preferably about 40° C. In some embodiments, the extraction time (i.e., the length of time the biological starting material is in contact with the solvent) is about 10 minutes to about 2 hours, preferably about 15 minutes to 60 minutes, more preferably about 20 minutes to about 45 minutes, and most preferably about 30 minutes.

[0046] After the extraction step, the crude krill lipid solution containing soluble lipids from the krill meal is separated from the solvent / krill meal mixture, for example by decantation and / or filtration. The insoluble material, including proteins and other useful materials, is then dried to recover the ethanol. The remaining defatted krill meal may then be used to make a hydrolysate according to the present invention. In some preferred embodiments, the defatted krill meal contains less than 15% or 12% wt / wt fat, more than 65% or 70% wt / wt crude protein, and / or less than 5% or 3% w / w moisture.

[0047] Thus, the process of the present invention may be used with a wide variety of starting materials. The remainder of the process discussion will generally refer to the use of marine meals such as solvent extracted krill meal as the starting material. However, it will be understood that any of the starting materials contemplated herein can be substituted for krill meal in the described process.

[0048] 2. Hydrolysis Process and Composition In some preferred embodiments, the present invention provides a process for producing a protein hydrolysate, comprising obtaining a meal, washing the meal with water and / or citric acid to obtain a washed meal, treating the meal with a protease to obtain a hydrolysate, and filtering the hydrolysate by microfiltration and / or ultrafiltration and / or nanofiltration or diafiltration. In some preferred embodiments, the meal is krill meal. In some particularly preferred embodiments, the krill meal is defatted krill meal or whole krill meal. In other preferred embodiments, the meal is whole or defatted fish (e.g., herring or salmon), fish roe (e.g., herring roe), squid, Calanus, or algae meal. In some preferred embodiments, where the meal is solvent-extracted krill meal, the hydrolysate obtained after filtration is characterized by containing 50% to 80% peptides of 2 to 10 amino acids (free amino acid peptides and peptides of 2 to 10 amino acids in length / total weight) and permeate on a w / w basis. Further characteristics of the preferred krill protein hydrolysate produced by this process are described in detail below.

[0049] In some preferred embodiments, the process includes one or more additional steps, as shown in Figure 3. In some preferred embodiments, the meal is milled, preferably wet-milled, before washing to reduce particle size. In some preferred embodiments, the milling step reduces the particle size of the meal to less than 100 μm, preferably about 50 μm. The meal is then washed with water or another suitable aqueous medium (e.g., an acidic solution such as a citric acid solution, a salt solution, or a basic solution). In some embodiments, the washing step includes a combination of water and acidic washing steps. For example, in some embodiments, the solvent-extracted meal is first washed with several volumes of water, then washed with citric acid, and then rinsed one or more times with water.

[0050] The present invention is not limited to the use of a particular protease or peptidase. In some preferred embodiments, the protease is a serine protease. Suitable serine proteases include, for example, subtilisin A and other subtilisins obtained from Bacillus subtilis. Suitable commercially available proteases are sold by Novozymes and include, but are not limited to, ALCALASE™ 2.4L FG, ALCALASE™ 2.5L, SAVINASE™ 12T, SAVINASE™ 16L, and ESPERASE™ 8.0L. In some preferred embodiments, the process comprises treating the washed and / or milled solvent extracted krill meal with ALCALASE™ at a temperature of 30-70°C, more preferably 50-60°C, and most preferably about 60°C, at a pH of 5-9, preferably 7-8, and most preferably 7.5, for up to 24 hours (e.g., 30 minutes to 24 hours), such as 1-3 hours, about 2 hours, or about 1 hour. Other suitable proteases include, but are not limited to, cysteine ​​proteases, threonine proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and asparagine peptide lyases known in the art.

[0051] In some preferred embodiments, the process includes a step of inactivating proteases before filtration. In further preferred embodiments, the process includes a step of drying the protein hydrolysate to obtain a granular protein hydrolysate. The drying step may be performed by methods known in the art, including spray drying. As depicted in FIG. 3, the process may also preferably include additional steps of vacuum concentration, pasteurization, and filtration. The present invention is not limited to the use of any particular filtration step. In preferred embodiments, a combination of ultrafiltration and nanofiltration is utilized. Ultrafiltration typically utilizes filters, such as membrane filters, with a molecular weight cutoff of about 100 kDa. Nanofiltration typically utilizes filters, such as membrane filters, with a pore size of about 1-10 nanometers, e.g., a molecular weight cutoff of about 300 Da. In some embodiments, a diafiltration step may be utilized in addition to or instead of the nanofiltration step.

[0052] The process of the present invention produces marine protein hydrolysates with improved organoleptic properties, including improved taste and olfaction (as evidenced by lower TMA, TMAO, and TVN levels) and lower fluoride levels compared to the starting material. In some preferred embodiments, the marine protein hydrolysates are characterized by having a protein content of greater than 85% on a dry weight basis, less than 2% fat on a dry weight basis, and less than 4% moisture. In some preferred embodiments, the marine protein hydrolysates are further characterized by having a protein content of greater than 89% on a dry weight basis, less than 1% fat on a dry weight basis, and less than 3% moisture.

[0053] In some particularly preferred embodiments, the krill hydrolysate is characterized by having a protein content of greater than 85% on a dry weight basis, less than 5% fat on a dry weight basis, and one or more of the following properties: a. a fluoride content of F of 0.1 to 200 mg / kg on a dry weight basis, preferably 0.1 to 30 mg / kg on a dry weight basis, more preferably 0.1 to 10 mg / kg, even more preferably 0.1 to 5 mg / kg on a dry weight basis, and most preferably 0.1 to 3 mg / kg on a dry weight basis, or less than 200, 30, 10, 3, or 1 mg / kg on a dry weight basis; b. a TMAO content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis, preferably 0.1 to 100 mg N / 100 g hydrolysate on a dry weight basis, more preferably 0.1 to 30 mg N / 100 g hydrolysate on a dry weight basis, even more preferably 0.1 to 25 mg N / 100 g hydrolysate on a dry weight basis, and most preferably 0.1 to 10 mg N / 100 g hydrolysate on a dry weight basis; c. a TMA content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis, preferably 0.1 to 100 mg N / 100 g hydrolysate on a dry weight basis, more preferably 0.1 to 50 mg N / 100 g hydrolysate on a dry weight basis, even more preferably 0.1 to 30 mg N / 100 g hydrolysate on a dry weight basis, and most preferably 0.1 to 10 mg N / 100 g hydrolysate on a dry weight basis; d. a TVN content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis, preferably 0.1 to 100 mg N / 100 g hydrolysate on a dry weight basis, more preferably 0.1 to 60 mg N / 100 g hydrolysate on a dry weight basis, and most preferably 0.1 to 20 mg N / 100 g hydrolysate on a dry weight basis; e. a sodium content of 0.01 to 4.0 g / 100 g hydrolysate on a dry weight basis, more preferably 0.1 to 1.0 g / 100 g hydrolysate on a dry weight basis, and most preferably 0.01 to 0.2 g / 100 g hydrolysate on a dry weight basis; f. a calcium content of 100 to 25,000 mg / kg of hydrolysate on a dry weight basis, more preferably 10,000 to 25,000 mg / kg of hydrolysate on a dry weight basis, and most preferably 12,000 to 16,000 mg / kg of hydrolysate on a dry weight basis; g. A lysine content of 6.30 to 10.30 g lysine / 100 g total amino acids, preferably 7.30 to 9.30 g lysine / 100 g total amino acids, more preferably 7.80 to 8.80 g lysine / 100 g total amino acids, or 6.29 to 10.29 g lysine / 100 g protein, preferably 7.29 to 9.29 g lysine / 100 g protein, more preferably 7.79 to 8.79 g lysine / 100 g protein; h. a threonine content of 3.04 to 7.04 grams threonine / 100 g total amino acids, preferably 4.04 to 6.04 g threonine / 100 g total amino acids, or more preferably 4.54 to 5.54 g threonine / 100 g total amino acids, or 3.36 to 7.36 g threonine / 100 g protein, preferably 4.36 to 6.36 g threonine / 100 g protein, more preferably 4.86 to 5.86 g threonine / 100 g protein; i. an isoleucine content of 3.39 to 7.39 g isoleucine / 100 g total amino acids, preferably 4.39 to 6.39 g isoleucine / 100 g total amino acids, more preferably 4.89 to 5.89 g isoleucine / 100 g total amino acids, or 3.38 to 7.38 g isoleucine / 100 g protein, preferably 4.38 to 6.38 g isoleucine / 100 g protein, more preferably 4.88 to 5.88 g isoleucine / 100 g protein; j. a leucine content of 6.27 to 10.27 g leucine / 100 g total amino acids, preferably 7.27 to 9.27 g leucine / 100 g total amino acids, or more preferably 7.77 to 8.77 g leucine / 100 g total amino acids, or 6.37 to 10.37 g leucine / 100 g protein, preferably 7.37 to 9.37 g leucine / 100 g protein, or more preferably 7.87 to 8.87 g leucine / 100 g protein; k. A histidine content of 0.94 to 3.94 g of histidine / 100 g of total amino acids, preferably 1.44 to 3.44 g of histidine / 100 g of total amino acids, more preferably 1.94 to 2.94 g of histidine / 100 g of total amino acids, or 1.09 to 4.09 g of histidine / 100 g of protein, preferably 1.59 to 3.69 g of histidine / 100 g of protein, more preferably 2.09 to 3.09 g of histidine / 100 g of protein; l. A phenylalanine content of 2.76 to 6.76 g phenylalanine / 100 g total amino acids, preferably 3.76 to 5.76 g phenylalanine / 100 g total amino acids, more preferably 4.26 to 5.26 g phenylalanine / 100 g total amino acids, or 3.05 to 7.05 g phenylalanine / 100 g protein, preferably 4.05 to 6.05 g phenylalanine / 100 g protein, more preferably 4.55 to 5.55 g phenylalanine / 100 g protein; m. A tyrosine content of 2.39 to 6.39 g of tyrosine / 100 g of total amino acids, preferably 3.39 to 5.39 g of tyrosine / 100 g of total amino acids, more preferably 3.89 to 4.89 g of tyrosine / 100 g of total amino acids, or 2.46 to 6.46 g of tyrosine / 100 g of protein, preferably 3.46 to 5.46 g of tyrosine / 100 g of protein, more preferably 3.96 to 4.96 g of tyrosine / 100 g of protein; n. A valine content of 3.69 to 7.69 g valine / 100 g total amino acids, preferably 4.69 to 6.69 g valine / 100 g total amino acids, more preferably 5.19 to 6.19 g valine / 100 g total amino acids, or 3.88 to 7.88 g valine / 100 g protein, preferably 4.88 to 6.88 g valine / 100 g protein, more preferably 5.38 to 6.38 g valine / 100 g protein; o. An alanine content of 3.76 to 7.76 g of alanine / 100 g of total amino acids, preferably 4.76 to 6.76 g of alanine / 100 g of total amino acids, or more preferably 5.26 to 6.26 g of alanine / 100 g of total amino acids, or 3.80 to 7.80 g of alanine / 100 g of protein, preferably 4.80 to 6.80 g of alanine / 100 g of protein, or more preferably 5.30 to 6.30 g of alanine / 100 g of protein; p. An arginine content of 3.90 to 7.90 g arginine / 100 g total amino acids, preferably 4.90 to 6.90 g arginine / 100 g total amino acids, or more preferably 5.40 to 6.40 g arginine / 100 g total amino acids, or 3.98 to 7.98 g arginine / 100 g protein, preferably 4.98 to 6.98 g arginine / 100 g protein, more preferably 5.48 to 6.48 g arginine / 100 g protein; q. an aspartic acid / asparagine content of 9.68 to 13.68 g of aspartic acid and asparagine / 100 g of total amino acids, preferably 10.68 to 12.68 g of aspartic acid and asparagine / 100 g of total amino acids, or more preferably 11.18 to 12.18 g of aspartic acid and asparagine / 100 g of total amino acids, or 10.37 to 14.37 g of aspartic acid and asparagine / 100 g of protein, preferably 11.37 to 13.37 g of aspartic acid and asparagine / 100 g of protein, more preferably 11.87 to 12.87 g of aspartic acid and asparagine / 100 g of protein; r. A glutamic acid / glutamine content of 11.54 to 17.54 g of glutamic acid and glutamine / 100 g of total amino acids, preferably 12.54 to 16.54 g of glutamic acid and glutamine / 100 g of total amino acids, or more preferably 13.54 to 15.54 g of glutamic acid and glutamine / 100 g of total amino acids, or 11.66 to 17.66 g of glutamic acid and glutamine / 100 g of protein, preferably 12.66 to 16.66 g of glutamic acid and glutamine / 100 g of protein, more preferably 13.66 to 15.66 g of glutamic acid and glutamine / 100 g of protein; s. A glycine content of 2.50 to 6.50 g of glycine / 100 g of total amino acids, preferably 3.50 to 5.50 g of glycine / 100 g of total amino acids, more preferably 4.00 to 5.00 g of glycine / 100 g of total amino acids, or 2.70 to 6.70 g of glycine / 100 g of protein, preferably 3.70 to 5.70 g of glycine / 100 g of protein, more preferably 4.20 to 5.20 g of glycine / 100 g of protein; a proline content of 1.84 to 5.84 g proline / 100 g total amino acids, preferably 2.84 to 4.84 g proline / 100 g total amino acids, or more preferably 3.34 to 4.34 g proline / 100 g total amino acids, or 2.27 to 6.27 g proline / 100 g protein, preferably 3.27 to 5.27 g proline / 100 g protein, or more preferably 3.77 to 4.77 g proline / 100 g protein; a serine content of 2.35 to 6.35 g serine / 100 g total amino acids, preferably 3.35 to 5.35 g serine / 100 g total amino acids, more preferably 3.85 to 4.85 g serine / 100 g total amino acids, or 2.64 to 6.64 g serine / 100 g protein, preferably 3.64 to 5.64 g serine / 100 g protein, more preferably 4.14 to 5.14 g serine / 100 g protein; v. A methionine content of 1.22 to 5.22 g methionine / 100 g total amino acids, preferably 2.22 to 4.22 g methionine / 100 g total amino acids, or more preferably 2.72 to 3.72 g methionine / 100 g total amino acids, or 1.69 to 5.69 g methionine / 100 g protein, preferably 2.69 to 4.69 g methionine / 100 g protein, more preferably 3.19 to 4.19 g methionine / 100 g protein; wt. cysteine ​​and cystine / 100g of total amino acids, preferably 0.44 to 0.84 g of cysteine ​​and cystine / 100g of total amino acids, or more preferably 0.54 to 0.74 g of cysteine ​​and cystine / 100g of total amino acids, or 0.249 to 1.049 g of cysteine ​​and cystine / 100g of protein, preferably 0.449 to 0.849 g of cysteine ​​and cystine / 100g of protein, more preferably 0.549 to 0.749 g of cysteine ​​and cystine / 100g of protein; x. A tryptophan content of 0.76 to 1.76 g tryptophan / 100 g total amino acids, preferably 0.96 to 1.56 g tryptophan / 100 g total amino acids, or more preferably 1.06 to 1.46 g tryptophan / 100 g total amino acids, or 0.8 to 1.8 g tryptophan / 100 g protein, preferably 1.0 to 1.6 g tryptophan / 100 g protein, or more preferably 1.1 to 1.5 g tryptophan / 100 g protein;

[0054] As such, ranges of total amino acid content are provided as grams of a particular amino acid per 100 grams of total amino acids (of the 20 amino acids found in protein) as measured by HPLC, or as grams of amino acid (measured by HPLC) per 100 grams of total protein (e.g., as measured by the Kjeldahl method).

[0055] In some further preferred embodiments, the hydrolysates have a protein content of greater than 90%, 91%, 92%, 93%, 94%, or 95% on a dry weight basis and / or a fat content of less than 3%, 2%, or 1% on a dry weight basis.

[0056] In some preferred embodiments, the hydrolysate further has a fluoride content of 0.1 to 30 ppm, and / or a TMAO content of 0.1 to 10 ppm; and / or a TMA content of 0.1 to 30 ppm; and / or a TVN content of 0.1 to 60 ppm.

[0057] It will be understood that the above hydrolysates can have one or more of the described characteristics, and it will be readily appreciated that any of the characteristics can be incorporated to identify the claimed compositions. For example, in some preferred embodiments, the hydrolysates have two of characteristics a, b, c, d, e, and f. In some preferred embodiments, the hydrolysates have three of characteristics a, b, c, d, e, and f. In some preferred embodiments, the hydrolysates have four of characteristics a, b, c, d, e, and f. In some preferred embodiments, the hydrolysates have five of characteristics a, b, c, d, e, and f. In some preferred embodiments, the hydrolysates have characteristics a, b, c, d, e, and f. In some preferred embodiments, the hydrolysates have characteristics a, b, c, and d. In some preferred embodiments, the hydrolysates have characteristics a and b. In some preferred embodiments, the hydrolysate further has one or more (e.g., all) of properties a, b, c, d, e, and f, as well as all of properties g, h, i, j, k, l, m, n, o, p, q, r, s, t, u, v, w, and x.

[0058] In some preferred embodiments, the hydrolysate has property a and properties g through x. In some preferred embodiments, the hydrolysate has property b and properties g through x. In some preferred embodiments, the hydrolysate has property c and properties g through x. In some preferred embodiments, the hydrolysate has property d and properties g through x. In some preferred embodiments, the hydrolysate has properties a, b, and g through x. In some preferred embodiments, the hydrolysate has properties a, c, and g through x. In some preferred embodiments, the hydrolysate has properties a, d, and g through x. In some preferred embodiments, the hydrolysate has properties a, b, c, and g through x. In some preferred embodiments, the hydrolysate has properties a, b, d, and g through x. In some preferred embodiments, the hydrolysate has properties b, c, d, and g through x.

[0059] In some preferred embodiments, the protein hydrolysates have one or more of the properties a, b, c, and d, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysates have property a, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysates have property a and b, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysates have property a, b, and c, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysates have property a, b, c, and d, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysates have property a, b, c, and d, and properties g, h, i, j, k, l, n, v, and x. In some preferred embodiments, the protein hydrolysates have one or more of the properties a, b, c, and d, and properties i, j, and n. In some preferred embodiments, the protein hydrolysates have property a, and properties i, j, and n. In some preferred embodiments, the protein hydrolysates have properties a and b, and properties i, j, and n. In some preferred embodiments, the protein hydrolysates have properties a, b, and c, and properties i, j, and n. In some preferred embodiments, the protein hydrolysates have properties a, b, c, and d, and properties i, j, and n. In some preferred embodiments, the protein hydrolysates have one or more of properties a, b, c, and d, and properties v and w. In some preferred embodiments, the protein hydrolysates have property a, and properties v and w. In some preferred embodiments, the protein hydrolysates have properties a and b, and properties v and w. In some preferred embodiments, the protein hydrolysates have properties a, b, and c, and properties v and w. In some preferred embodiments, the protein hydrolysates have properties a, b, c, and d, and properties v and w.

[0060] In some preferred embodiments, the hydrolysate is further characterized by having an essential amino acid content of 39.36 to 49.36 g essential amino acids / 100 g total amino acids, preferably 41.36 to 47.36 g essential amino acids / 100 g total amino acids, more preferably 42.36 to 46.36 g essential amino acids / 100 g total amino acids, and most preferably 43.36 to 45.36 g essential amino acids / 100 g total amino acids.

[0061] In some preferred embodiments, the hydrolysate is further characterized by having a branched chain amino acid content of 16.35 to 22.35 g branched chain amino acids / 100 g total amino acids, 17.35 to 21.35 g branched chain amino acids / 100 g total amino acids, and more preferably 18.35 to 20.35 g branched chain amino acids / 100 g total amino acids.

[0062] In some preferred embodiments, the hydrolysate is further characterized by having a sulfur-containing amino acid content of 2.86 to 4.86 g sulfur-containing amino acids / 100 g total amino acids, preferably 3.16 to 4.56 g sulfur-containing amino acids / 100 g total amino acids, and more preferably 3.46 to 4.26 g sulfur-containing amino acids / 100 g total amino acids.

[0063] In some preferred embodiments, the protein hydrolysate is further characterized by having an arginine content of 5-7% (g amino acids ("AA") / 100g protein). In some preferred embodiments, the protein hydrolysate is further characterized by having a leucine content of 7-10% (g AA / 100g protein). In some preferred embodiments, the protein hydrolysate is further characterized by having a combined branched chain amino acid content of 17-19.5% (g AA / 100g protein). In some preferred embodiments, the protein hydrolysate is further characterized by having a combined methionine and cysteine ​​content of 2-4% (g AA / 100g protein). In some preferred embodiments, the protein hydrolysate is further characterized by having a tryptophan content of 0.1%-3% (g AA / 100g protein), more preferably 0.2%-2% (g AA / 100g protein). In some preferred embodiments, the protein hydrolysate is further characterized by comprising 50% to 80% by w / w of peptides of 2 to 10 amino acids (peptides of 2 to 10 amino acids by free amino acid peptides and polypeptide length / total weight). In some preferred embodiments, the protein hydrolysate is further characterized by comprising 50% to 80% by w / w of peptides of 2 to 20 amino acids (peptides of 2 to 20 amino acids by free amino acid peptides and polypeptide length / total weight). In some preferred embodiments, the protein hydrolysate is further characterized by comprising 50% to 80% by w / w of peptides of 2 to 15 amino acids (peptides of 2 to 15 amino acids by free amino acid peptides and polypeptide length / total weight). In some preferred embodiments, the protein hydrolysate is further characterized by comprising 50% to 80% by w / w of peptides of 2 to 20 amino acids (peptides of 2 to 10 amino acids by free amino acid peptides and polypeptide length / total weight).

[0064] In some preferred embodiments, the marine protein hydrolysate is further characterized by containing less than 60 ppm trimethylamine oxide. In some preferred embodiments, the marine protein hydrolysate is further characterized by containing less than 50 ppm fluoride. In some preferred embodiments, the marine protein hydrolysate is further characterized by containing less than 4%, most preferably less than 1%, sodium. In some preferred embodiments, the marine protein hydrolysate is further characterized by a neutral taste, lacking a fishy taste.

[0065] In some preferred embodiments, the marine protein hydrolysate is further characterized by dispersing into a clear solution when added to water or other aqueous media. For example, in some embodiments, when 4.5 grams of the hydrolysate of the present invention on a dry weight basis is dissolved in 100 g of water at a pH of 3 to 9 (e.g., pH 6), the optical density at 590 nm (OD590) is less than 0.4, preferably less than 0.3. In some embodiments, the hydrolysate preferably has 0.1 to 50 ppm, 0.1 to 10 ppm, or 0.1 to 5 ppm astaxanthin, more preferably less than 50 ppm, less than 10 ppm, or less than 5 ppm astaxanthin.

[0066] In some embodiments, the solubility of a marine protein hydrolysate may be assayed by dissolving 4.5 grams of hydrolysate, on a dry weight basis, in 100 grams of water at 20-25°C (e.g., 23°C), stirring, centrifuging the solution at 1500g for 5 minutes, and measuring the dry weight of the material in the solution after centrifugation. In some preferred embodiments, the solubility of the hydrolysate, as measured by this assay, is 80%, 90%, 91%, 92%, 92%, 94% or greater at pH 3-7 (e.g., pH 3.5), and 75%, 80%, or 90% or greater at pH 9. In some embodiments, the solubility after heat treatment at 85°C for 5 minutes is 60% or greater as measured by the assay at pH 3-9 (e.g., pH 6).

[0067] 3.Hydrolysis products The hydrolysates of the present invention may be incorporated into a variety of products. Thus, in some preferred embodiments, the present invention provides foods, beverages, functional foods, animal feeds, powdered protein supplements, meal replacements, beverage powders, protein shake powders, etc., comprising the hydrolysates of the present invention, in particular krill protein hydrolysates.

[0068] The hydrolysates of the present invention are preferably administered orally. Thus, in some embodiments, the hydrolysates of the present invention (as described in the previous section) are formulated with an acceptable excipient and / or carrier for oral ingestion. The actual form and composition of the carrier are not critical. The carrier may be a liquid, gel, gelcap, capsule, powder, solid tablet (coated or uncoated), tea, etc. The oral delivery vehicle is preferably in the form of a tablet or capsule. Suitable excipients and / or carriers include vegetable oil, fish oil, krill oil, maltodextrin, calcium carbonate, dicalcium phosphate, tricalcium phosphate, microcrystalline cellulose, glucose, rice flour, magnesium stearate, stearic acid, croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose, vegetable gum, lactose, methylcellulose, povidone, carboxymethylcellulose, corn starch, etc. (including mixtures thereof). Preferred carriers include calcium carbonate, magnesium stearate, maltodextrin, and mixtures thereof. The various ingredients and excipients and / or carriers are mixed and formed into the desired form using conventional techniques. The tablets or capsules of the present invention may be coated with an enteric coating that dissolves at a pH of about 6.0 to 7.0. A suitable enteric coating that dissolves in the small intestine but not in the stomach is cellulose acetate phthalate. Further details of techniques for formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

[0069] In some embodiments, hydrolysate is formulated for oral administration with flavoring or sweetening agent.Examples of useful flavoring agents include, but are not limited to, pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or pure vanilla extract; or volatile oils such as balm oil, bay oil, bergamot oil, cedarwood oil, walnut oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla cookie powder, butterscotch, or toffee.In one embodiment, the dietary supplement comprises cocoa or chocolate.An emulsifier may be added to ensure the stability of the final product.Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., derived from egg or soybean), and / or monoglycerides and diglycerides. Other emulsifiers will be readily apparent to those skilled in the art, and the selection of a suitable emulsifier(s) will depend, in part, on the formulation and final product. In addition to the carbohydrates described above, the dietary supplement may contain natural or artificial (preferably low-calorie) sweeteners, such as sugars, cyclamate, aspartamine, aspartame, acesulfame K, and / or sorbitol.

[0070] Dietary supplements may contain one or more inactive ingredients, especially when it is desirable to limit the number of calories added to a diet by the dietary supplement. For example, dietary supplements of the present invention may also contain optional ingredients, including, for example, herbs, vitamins, minerals, enhancers, colorants, sweeteners, flavorings, inactive ingredients, etc. For example, dietary supplements of the present invention may contain one or more of the following: ascorbates (ascorbic acid, mineral ascorbates, rosehip, acerola, etc.), dehydroepiandosterone (DHEA), green tea (polyphenols), inositol, kelp, dulse, flavonoids, maltodextrin, nettle, niacin, niacinamide, rosemary, selenium, silica (silicon dioxide, silica gel, horsetail, shaved grass, etc.), spirulina, zinc, etc. Such optional ingredients may be in either a naturally occurring or concentrated form.

[0071] In some embodiments, the dietary supplement further comprises vitamins and minerals, including but not limited to calcium phosphate or calcium acetate, tribasic; potassium phosphate, dibasic; magnesium sulfate or magnesium oxide; salt (sodium chloride); potassium chloride or potassium acetate; ascorbic acid; ferric orthophosphate; niacinamide; zinc sulfate or zinc oxide; calcium pantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxine hydrochloride; thiamine mononitrate; folic acid; biotin; chromium chloride or chromium picolinate; potassium iodide; sodium selenate; sodium molybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenate; copper sulfate; vitamin A; vitamin C; inositol; potassium iodide. Suitable dosages of vitamins and minerals may be obtained, for example, by consulting the US RDA guidelines.

[0072] In another embodiment, the present invention provides a dietary supplement (e.g., an energy bar or meal replacement bar or beverage) containing the hydrolysate of the present invention. The dietary supplement serves as a meal or snack replacement and generally provides nutritional calories. Preferably, the dietary supplement provides balanced amounts of carbohydrates, protein, and fat. The dietary supplement may further include carbohydrates, simple, medium-chain length, or polysaccharides, or a combination thereof. The simple sugars can be selected for desirable organoleptic properties. Uncooked cornstarch is an example of a complex carbohydrate. If it is desired to maintain its high molecular weight structure, it should only be included in food formulations or portions thereof that are not cooked or heat-treated, since heating breaks down complex carbohydrates into simple carbohydrates (simple carbohydrates are monosaccharides or disaccharides). In one embodiment, the dietary supplement contains a combination of carbohydrate sources with three levels of chain length (simple, medium, and complex, e.g., sucrose, maltodextrin, and uncooked cornstarch).

[0073] In yet another embodiment, the present invention provides foods, prepared foods, or other foodstuffs (e.g., functional foods) comprising the hydrolysates of the present invention. In preferred embodiments, the foods comprise effective amounts of the components described above. For example, in some embodiments, beverages and solid or semi-solid foods comprising the hydrolysates are provided. These forms can include, but are not limited to, beverages (e.g., soft drinks, milk and other dairy beverages, and diet drinks), baked goods, puddings, dairy products, confectionery, snack foods, or frozen confections or novelties (e.g., ice cream, milkshakes), prepared frozen meals, candy, snack products (e.g., chips), soups, spreads, sauces, salad dressings, prepared meat products, cheese, yogurt, and any other fat- or oil-containing food, and food ingredients (e.g., flour).

[0074] In some preferred embodiments, the hydrolysate is incorporated into a chewable matrix. Preferred chewable matrix jelly candies and gelatin-based gummy candies. Exemplary gummy candies include bear candies, worm candies, frog candies, hamburger candies, cherry candies, soda bottle candies, shark candies, soldier candies, hippo candies, lobster candies, watermelon candies, octopus candies, apple candies, peach candies, and orange candies. The terms "gummy" and "gummy" are used interchangeably herein.

[0075] As will be appreciated, the hydrolysates of the present invention may be delivered as dietary supplements, nutraceuticals, or functional foods, or may be incorporated into foods or beverages. Thus, in some embodiments, the present invention provides a method for preparing a dietary supplement, nutraceutical, functional food, food, or beverage, comprising mixing the protein hydrolysates described above with one or more additional components to form a desired product (i.e., a dietary supplement, nutraceutical, functional food, food, or beverage). The additional components of these products are described in detail above. Furthermore, the present invention provides the use of the protein hydrolysates described herein as an ingredient in the preparation of a dietary supplement, nutraceutical, functional food, food, or beverage.

[0076] In some preferred embodiments, the hydrolysate is incorporated into animal feed and food. Animal feed according to the present invention may be formulated for companion animals such as cats and dogs, as well as domestic and wild animals including poultry, pigs, cattle, sheep, rabbits, and birds, fish such as trout, salmon, catfish, and tilapia, shrimp, and other species produced by aquaculture. Many different feeds may be formulated for animals from many different feed ingredients. Food is generally formulated to provide nutrients in accordance with the standards of the National Research Council. The feed used in the food (in addition to the hydrolysate) can be selected according to market price and availability. Thus, some components of the food may change over time. In the feed of the present invention, the food will contain the hydrolysate according to the present invention as part of the protein component, while other components may vary over time based on the price of the components. For a discussion of feed formulation, actual diets, and NRC guidelines, see Church, Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984) and Feeds and Nutrition Digest, Ensminger, Oldfield and Heineman eds., Ensminger Publishing Corporation, Clovis, Calif. (1990) (incorporated herein by reference).

[0077] The animal feed of the present invention may be characterized according to NRC requirements. NRC requirements may be found in Church, Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984) or other nutritional standards. Animal meals are traditionally balanced using protein and energy requirements, and then adjusted, if necessary, to meet other requirements. The animal meal of the present invention will contain a hydrolysate according to the present invention, e.g., 0.5% to 30% wt / wt, 0.5% to 20% wt / wt, 0.5% to 10% wt / wt, or 0.5% to 5% wt / wt, in addition to other feed ingredients needed to balance the diet to meet NRC requirements for different stages of growth and maintenance. The relative amounts of protein and energy are adjusted to reflect the requirements of the nutritional standards. The amounts of the feed components will vary with the stage of the animal being fed. Growing diets for young animals will have higher protein levels, while finishing diets for market animals will have higher energy values ​​provided by carbohydrates. In some feeding situations, care should be taken to provide adequate amino acid and overall protein content. In most animal diets, energy requirements are met by grain starch. Energy requirements can also be met by adding fat to the diet. In the present invention, CFAP provides a portion of the energy requirements.

[0078] Other ingredients may be added to the feed. These ingredients include, but are not limited to, mineral supplements such as calcium, phosphorus, salt, selenium, and zinc; vitamin supplements such as vitamins A, B, D, E, and K; amino acid supplements such as lysine; anticoccidial agents or growth promoters such as bacitracin or virginamycin; and other active agents such as chlortetracycline, sulfathiozole, and penicillin. For vitamin, mineral, and antibiotic supplement formulations, see Church, Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984).

[0079] In a preferred embodiment, the hydrolysate is incorporated into a pelleted feed for administration to farm animals. The pelleted feed is made by first mixing the feed components and then compressing and extruding the feed components through a die under heat and pressure. The feed is pelleted by methods known in the art, as described in MacBain, *Pelleting Animal Feed*, *American Feed Manufacturers Association*, Arlington, Va. (1974), incorporated herein by reference. When incorporating added fat into pelleted feed, care must be taken to avoid creating powdery pellets. Generally, only about 2% of the fat is added during pelleting, with the remainder added after the pellets have cooled. [Example]

[0080] Example 1 Solvent-extracted krill meal produced according to the process described in PCT / IB2016 / 000208 is milled in a pin mill at 16,000 RPM to an average particle size of 50 μm. The milled protein composition is then washed with citric acid (65°C, 9 minutes, 0.19M, pH 4), followed by three 9-minute washes with hot water at 65°C. The washed protein composition is then treated with ALCALASE at pH 7.5 and 52.5°C for 2.5 hours, followed by a 9-minute wash with hot water at 65°C. The resulting hydrolysate is then microfiltered and nanofiltered (1.25 L / h / m2 / bar, less than 5 bar pressure) to obtain a concentrate. The concentrate is then granulated by spray drying (agglomeration, three-stage drying, inlet temperature 182°C, outlet temperature 82.5°C). The hydrolysate concentrate has the properties described in Tables 1-3 and Figures 1 and 2. [Table 1] [Table 2] [Table 3]

[0081] Example 2 Two processes for producing krill hydrolysate were compared. Except where noted, the process of Figure 3 was utilized. In the first process, there was no wet-grinding or ultrafiltration step. The hydrolysis time was 1 hour. The resulting hydrolysate is referred to as "Batch A." ​​A nanofiltration step was utilized after hydrolysis. The second process utilized wet-grinding before hydrolysis and both ultrafiltration and nanofiltration after hydrolysis. The hydrolysis time was 14.5 hours. The resulting hydrolysate is referred to as "Batch B." A comparison of selected values ​​for Batch A and B is provided in Table 4. Table 5 provides the amino acid composition of Batch B, as well as the average amino acid content across multiple krill hydrolysate batches produced by the process of the present invention. In the third column of Table 5, the amino acid content is expressed as grams amino acids per 100 grams of protein. The grams of amino acids in 100 g of hydrolysate were determined by high-performance liquid chromatography (HPLC). Protein content was determined by the Kjeldahl method. Column 4 of Table 5 lists the amino acid content for mixed batches made from different meals, including defatted krill meal and full-fat krill meal. In column 4, the amino acid content is listed in grams of the indicated amino acid per 100 grams of total amino acids (measured by HPLC). As can be seen, the hydrolysate of Batch B has substantially improved values ​​for fluoride (F) content, TMAO, TMA, and TVN content. A subsequent batch ("Batch C") was found to have an F content of 1.09 mg / kg on a dry weight basis, as well as lower values ​​for TMA, TMAO, and TVN. The hydrolysate has an excellent amino acid composition, particularly with respect to the content and composition of essential and branched-chain amino acids. [Table 4] [Table 5]

Claims

1. A krill protein hydrolysate, a protein content of greater than 85% on a dry weight basis; less than 5% fat on a dry weight basis; Fluoride content of 0.1 to 200 mg / kg of hydrolysate on a dry weight basis; TMAO (trimethylamine N-oxide) content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis; TMA (trimethylamine) content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis; TVN (total volatile nitrogen) content of 0.1 to 200 mg N / 100 g hydrolysate on a dry weight basis; a sodium content of 0.01 to 4.0 g / 100 g of hydrolysate on a dry weight basis; and a calcium content of the hydrolysate of 100 to 25,000 mg / kg on a dry weight basis; A krill protein hydrolysate characterized by having:

2. 2. The krill protein hydrolysate according to claim 1, wherein the krill protein hydrolysate has a fluoride content of 0.1 to 30 mg / kg on a dry weight basis.

3. 3. Krill protein hydrolysate according to claim 1 or 2, wherein the krill protein hydrolysate has a TMAO content of 0.1 to 10 mg N / 100 g hydrolysate on a dry weight basis.

4. 4. The krill protein hydrolysate according to any one of claims 1 to 3, wherein the krill protein hydrolysate has a TMA content of 0.1 to 30 mg N / 100 g hydrolysate on a dry weight basis.

5. 5. The krill protein hydrolysate according to any one of claims 1 to 4, wherein the krill protein hydrolysate has a TVN content of 0.1 to 60 mg N / 100 g hydrolysate on a dry weight basis.

6. 6. Krill protein hydrolysate according to any one of claims 1 to 5, further characterized in that the krill protein hydrolysate has a protein content of more than 89% on a dry weight basis and a fat content of less than 2% on a dry weight basis.

7. 7. The krill protein hydrolysate according to any one of claims 1 to 6, wherein the krill protein hydrolysate has a water content of less than 3%.

8. 8. The krill protein hydrolysate according to any one of claims 1 to 7, wherein the krill protein hydrolysate is more than 80% water soluble when assayed by dissolving 4.5g of dry weight of the krill protein hydrolysate in 100g of water.

9. 9. The krill protein hydrolysate according to any one of claims 1 to 8, wherein the heat-treated protein hydrolysate is more than 60% water soluble when assayed by dissolving 4.5g of dry weight of said protein hydrolysate in 100g of water and heating to 85°C for 5 minutes.

10. 10. Krill protein hydrolysate according to any one of claims 1 to 9, characterized in that the krill protein hydrolysate has a protein content of more than 90%, 91%, 92%, 93%, 94%, or 95% by dry weight and / or a fat content of less than 3%, 2%, or 1% by dry weight.

11. 1. A process for producing filtered krill protein hydrolysate, comprising: - obtaining krill meal, - grinding the krill meal, - washing the ground krill meal with water and / or citric acid to obtain washed krill meal; - treating the washed krill meal with a protease to obtain a krill protein hydrolysate; and - filtering said krill protein hydrolysate by ultrafiltration and nanofiltration using a filter having a pore size of 1 to 10 nm to obtain a filtered krill protein hydrolysate and a permeate; The process comprising:

12. 12. The process of claim 11, wherein the meal is a solvent extracted meal.

13. 13. The process of claim 11 or 12, further comprising the step of inactivating the protease before filtration.

14. 14. The process according to any one of claims 11 to 13, further comprising the step of drying the filtered krill protein hydrolysate to obtain a dried krill protein hydrolysate.

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