阅读说明：本技术 从甲壳类捕获物中生产蛋白质磷脂复合物的方法 (Method for producing protein phospholipid complex from crustacean capture ) 是由 英奇·布鲁海姆 斯蒂格·瑞莫伊 于 2018-11-30 设计创作，主要内容包括：本发明请求保护一种从含蛋白质的甲壳类捕获物中生产蛋白质磷脂复合物的方法,其中水解反应发生,而甲壳类捕获物中的蛋白质未发生实质性变性。还要求保护一种从甲壳类捕获物中生产稳定蛋白质磷脂乳液的方法。还要求保护一种生产甲壳动物油的方法,其中该油分离自经水解的甲壳类捕获物。请求保护其它生产蛋白质磷脂复合物的方法,其涉及不去壳、去壳、以及去壳后再加回壳以形成该蛋白质磷脂复合物。(The present invention claims a method for producing protein phospholipid complexes from protein-containing crustacean traps, wherein hydrolysis occurs without substantial denaturation of the proteins in the crustacean traps. A method of producing a stable protein phospholipid emulsion from a crustacean trap is also claimed. Also claimed is a process for producing a crustacean oil, wherein said oil is separated from a hydrolysed crustacean trap. Other methods of producing protein phospholipid complexes are claimed which involve no shelling, and adding the shell back after shelling to form the protein phospholipid complex.)

1. A method of producing a protein phospholipid complex comprising the steps of:

a) comminuting the crustacean trap to provide a comminuted crustacean trap comprising protein;

b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolysed crustacean trap without substantial denaturation of the protein; and

c) separating the hydrolyzed crustacean trap to provide a protein phospholipid complex.

2. The method of any one of the preceding claims, wherein the contacting further comprises a second enzyme.

3. The method according to any of the preceding claims, wherein the second enzyme comprises chitinase, collagenase or another proteolytic enzyme.

4. The method according to any one of the preceding claims, wherein the proteolytic enzyme is a mixture of acidic, neutral and alkaline proteases.

5. The method of any one of the preceding claims, wherein the proteolytic enzyme comprises a first protease mixture and a second protease mixture.

6. The method according to any one of the preceding claims, wherein the first protease mixture comprises at least one alkaline protease; and the second protease mixture comprises an acid protease, a neutral protease and an alkaline protease.

7. A process according to any one of the preceding claims, wherein the first protease mixture comprises 0.3-0.5% by weight of the total crushed crustacean-trap; and the second protease mixture accounts for 0.03-0.05% of the total weight of the crushed crustacean-trap.

8. The method of any one of the preceding claims, wherein the protease is from bacillus licheniformis.

9. The method of any one of the preceding claims, wherein the contacting step (b) comprises contacting with a cell wall degrading enzyme.

10. A process according to any one of the preceding claims, wherein the comminuted crustacean trap is contacted with a proteolytic enzyme for more than 100 minutes to provide a hydrolysed crustacean trap.

11. The process according to any one of the preceding claims, wherein the crustacean catch comprises krill.

12. The method of any of the preceding claims, wherein the comminuting comprises one or more of pulping, milling, grinding, or fragmenting.

13. The method of any one of the preceding claims, wherein the contacting comprises an organic solvent.

14. A method according to any one of the preceding claims, wherein said contacting comprises incubating the comminuted crustacean trap at a temperature in the range of 45-75 ℃.

15. The method according to any one of the preceding claims, wherein the water content of the protein phospholipid complex is less than 15% w/w.

16. A method according to any one of the preceding claims, wherein the crustacean trap proteins include digestive enzymes and other proteins.

17. A method of producing a stable protein phospholipid emulsion comprising the steps of:

a) comminuting the crustacean trap to provide a comminuted crustacean trap comprising protein;

b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolyzed crustacean trap; and

c) separating the hydrolyzed crustacean trap to provide a stable protein phospholipid emulsion.

18. The method of claim 17, wherein the contacting further comprises a second enzyme.

19. The method of claim 17 or 18, wherein the second enzyme comprises chitinase, collagenase, or another proteolytic enzyme.

20. The method of claims 17-19, wherein the proteolytic enzyme is a mixture of acidic, neutral and alkaline proteases.

21. The method of claims 17-20, wherein the crustacean catch comprises krill.

22. The method of claims 17-21, wherein the comminuting comprises one or more of slurrying, milling, grinding, or fragmenting.

23. The method of claims 17-22, wherein the contacting comprises an organic solvent.

24. A method according to claims 17-23, wherein said contacting comprises incubating the comminuted crustacean trap at a temperature in the range of 45-75 ℃.

25. The method of claims 17-24, wherein the stable protein phospholipid emulsion comprises a water content of 45-55 v/v%.

26. A stable protein phospholipid emulsion comprising: water, protein and lipid.

27. A process for producing a crustacean oil comprising the steps of:

a) comminuting the crustacean trap to provide a comminuted crustacean trap comprising protein;

b) contacting the comminuted crustacean trap with one or more proteolytic enzymes to provide a hydrolysed crustacean trap without substantial denaturation of the proteins; and

c) separating the hydrolyzed crustacean trap to provide a crustacean oil.

28. The process according to claim 27, wherein the crustacean oil is krill oil and the crushed crustacean catch is crushed krill.

29. The method of claims 27 and 28, wherein the proteolytic enzyme comprises a mixture of acidic, neutral and alkaline proteases.

30. The method of any one of claims 27-29, wherein the method is performed without extraction.

31. A nutritional supplement comprising a crustacean oil and another oil selected from the group consisting of linseed oil, pumpkin seed oil, rapeseed oil, soybean oil, walnut oil, fish oil, seal oil, microalgal oil, mussel oil and shrimp oil.

32. The nutritional supplement of claim 31, wherein the another oil is perna canaliculus oil.

33. The nutritional supplement according to claim 31 or 32, wherein the crustacean oil is krill oil.

34. A method of producing a protein phospholipid complex comprising the steps of:

a) comminuting the crustacean trap to provide a comminuted crustacean trap comprising protein;

b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolysed crustacean trap without denaturation of the protein; and

c) separating the hydrolyzed crustacean trap to provide a protein phospholipid complex, wherein the shell is not removed from the hydrolyzed crustacean trap in the separating step and the shell is part of said protein phospholipid complex.

35. The process of claim 34, wherein the crustacean catch is krill.

36. A method of producing a protein phospholipid complex comprising the steps of:

a) comminuting the crustacean trap to provide a comminuted crustacean trap comprising protein;

b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolysed crustacean trap without denaturation of the protein; and

c) separating the hydrolyzed crustacean trap to provide a protein phospholipid complex, comprising the steps of: (1) separating the shell from the hydrolyzed crustacean trap; (2) pasteurizing the hydrolyzed crustacean trap to inactivate enzymes; (3) removing excess water by separation; and (4) adding the shell back to the hydrolyzed crustacean trap to provide a protein phospholipid complex.

37. A process as claimed in claim 36, wherein, in step (1), the shell is separated from the hydrolysed crustacean catch using a filter or sieve.

38. A method according to claim 36 or 37, wherein in step (3) the excess water is removed using a horizontal centrifuge.

39. The method of claims 36-38, wherein in step (4), the shell is added back with insoluble particles for practical purposes.

Technical Field

This application claims the benefit of U.S. provisional application serial No. 62/594,105 filed on 12/4/2017, which is incorporated herein by reference.

Background

In recent years, scientific research has shown that diets rich in omega-3 (omega-3) fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are beneficial for health. These fatty acids have been shown to be essential for maintaining brain health and studies have shown that they are also capable of promoting cardiovascular health, reducing inflammation, preventing arthritis, improving stomach health, while also having antidepressant effects.

Omega-3 fatty acids are naturally found in a variety of foods, including certain fish, crustaceans, nuts and seeds, and the like. Furthermore, the concentrated form of omega-3 fatty acids constitutes an important part of the vitamin and supplement industry. Among animal derived sources of omega-3 fatty acids, fish oil is gaining importance as a popular vitamin source. However, fish oil capsules have a disadvantage in that "fishy smell" is often left.

Scientists are studying alternative sources of omega-3 fatty acids to determine if they are more bioavailable. For example, some studies have preliminarily shown that krill oil derived from antarctic krill may be a superior source of omega-3 fatty acids because it is more bioavailable than fish oil. See rampraash et al, published in lipid in health and disease (2013, 12:178), "a robust increase in the omega-3 index relative to fish oil after 4 weeks of n-3 fatty acid supplement obtained from krill oil for healthy people, and Schuchardt et al, published in lipid in health and disease (2011, 10:145)," binding of EPA and DHA in plasma phospholipids in different omega-3 fatty acid formulations-comparative study of bioavailability of fish oil to krill oil ". It is speculated that these omega-3 fatty acids are attached to phospholipids, thereby providing a higher bioavailability than fish-derived omega-3 fatty acids attached to triglycerides. See Schuchardt et al. Moreover, krill is the largest biomass in the world, while only less than 1% of the marine krill is harvested annually, and is therefore a sustainable resource; krill is also considered to have no aftertaste in market promotion, and its mercury content is not measurable; and the krill contains the antioxidant astaxanthin.

While krill oil appears to be a good source of omega-3 fatty acids, there remains a need for a better method of processing krill to harvest the omega-3 fatty acids and produce a product that is bioavailable, low in water content and other contaminants, and allows further processing downstream to produce good quality krill oil and other products from krill protein. More specifically, there is a need for a new process for producing protein phospholipid complexes in which digestive enzymes are inactivated by hydrolysis without substantial denaturation of the proteins in krill during the hydrolysis step, and for a new process for producing stable protein phospholipid emulsions in which the emulsions do not separate and the moisture content is sufficiently low to prevent microbial growth.

Disclosure of Invention

One aspect of the present invention relates to a method for producing a Protein Phospholipid Complex (PPC), comprising the steps of: a) pulverizing the crustacean-trap to provide a pulverized crustacean-trap comprising protein; b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolyzed crustacean trap without substantial denaturation of the proteins; and c) separating the hydrolyzed crustacean trap to provide PPC.

In one embodiment, the contacting step comprises a second enzyme. Preferably, the second enzyme may comprise chitinase, collagenase or another proteolytic enzyme. The proteolytic enzyme is preferably a mixture of an acid protease, a neutral protease and an alkaline protease.

In one embodiment, the proteolytic enzyme in the contacting step comprises at least one metalloendopeptidase. In another embodiment, the proteolytic enzyme does not comprise an exopeptidase.

In one embodiment, the contacting step comprises contacting with a first protease mixture and a second protease mixture. The first protease mixture contains at least one alkaline protease, and the second protease mixture contains an acidic protease, a neutral protease, and an alkaline protease.

Preferably, the first protease mixture comprises 0.3 to 0.5% by weight of the total weight of the crushed crustacean-trap; and said second protease mixture comprises 0.03-0.05% by weight of the total weight of the pulverized crustacean-trap.

In a preferred embodiment, the protease is from Bacillus licheniformis (Bacillus L icheniformis).

In one embodiment, the contacting step comprises contacting with a cell wall degrading enzyme.

Preferably, the comminuted crustacean trap is contacted with a proteolytic enzyme for more than 100 minutes to provide a hydrolysed crustacean trap. The contacting step may include an organic solvent. The contacting step may comprise incubating the comminuted crustacean trap at a temperature in the range of 45-75 ℃.

The pulverized crustacean is preferably krill. In the above-mentioned pulverizing step, the crustacean-captured material may be treated by pulping, milling, grinding and/or fragmenting (shredding).

The PPC produced by this process has a water content of less than 15% w/w.

The proteins in krill may include digestive enzymes and other proteins (except digestive enzymes).

Another aspect of the present invention relates to a method of preparing a stable protein phospholipid emulsion comprising the steps of: a) pulverizing the crustacean catch to provide a pulverized crustacean catch; b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolyzed crustacean trap; and c) separating the hydrolyzed crustacean trap to provide a stable protein phospholipid emulsion.

In one embodiment, the contacting step comprises a second enzyme. Preferably, the second enzyme may comprise chitinase, collagenase or another proteolytic enzyme. The proteolytic enzyme is preferably a mixture of an acid protease, a neutral protease and an alkaline protease.

The pulverized crustacean is preferably krill. In the above-mentioned pulverizing step, the crustacean-captured material may be treated by pulping, milling, grinding and/or fragmenting.

In one embodiment, the proteolytic enzyme in the contacting step comprises at least one metalloendopeptidase. In another embodiment, the proteolytic enzyme does not comprise an exopeptidase. The contacting step may further comprise an organic solvent.

The contacting step may comprise incubating the comminuted crustacean trap at a temperature in the range of 45-75 ℃.

The protein phospholipid emulsion produced according to the method may comprise a water content of from v/v45 to 55% v/v.

Another aspect of the invention relates to a stable protein phospholipid emulsion comprising water, protein, and lipid.

Another aspect of the present invention relates to a method for producing a crustacean oil, comprising the steps of: a) comminuting the crustacean trap to provide a comminuted crustacean trap comprising protein; b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolyzed crustacean trap without substantial denaturation of the proteins; and c) separating the hydrolyzed crustacean catch to provide a crustacean oil.

Preferably, the crustacean oil is krill oil and the crushed crustacean catch is crushed krill. Preferably, the proteolytic enzyme comprises a mixture of an acid protease, a neutral protease and an alkaline protease. In one embodiment, the method is performed without an extraction (extraction) step.

Another aspect of the invention relates to a nutritional supplement comprising a crustacean oil and another oil selected from the group consisting of: linseed oil, pumpkin seed oil, rapeseed (canola oil), soybean oil, walnut oil, fish oil, seal oil, microalgal oil, mussel oil, and shrimp oil. Preferably, the oil is perna canaliculus oil. In addition, the preferred crustacean oil is krill oil.

Another aspect of the present invention relates to a method of producing a protein phospholipid complex comprising the steps of a) comminuting a crustacean trap to provide a comminuted crustacean trap comprising protein; b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolysed crustacean trap without denaturing the proteins; and c) separating the hydrolyzed crustacean trap to provide a protein phospholipid complex, wherein in the above separation step the shell is not removed from the hydrolyzed crustacean trap and is part of the protein phospholipid complex. Preferably, the crustacean catch is krill.

Another aspect of the present invention relates to a method of producing a protein phospholipid complex comprising the steps of a) comminuting a crustacean trap to provide a comminuted crustacean trap comprising protein; b) contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolysed crustacean trap without denaturing the proteins; and c) separating the hydrolyzed crustacean trap to provide a protein phospholipid complex, comprising the steps of (1) separating the shell from the hydrolyzed crustacean trap; (2) pasteurizing the hydrolyzed crustacean trap to inactivate enzymes; (3) removing excess water by separation; and (4) adding a shell to the hydrolyzed crustacean trap to provide a protein phospholipid complex.

Preferably, in step (1), the shell may be separated from the hydrolyzed crustacean catch with a filter or a sieve. In another preferred embodiment, excess water may be removed in step (3) by a horizontal centrifuge. In step (4), for practical purposes, it is preferred that the shell be added back alone or with the insoluble particles.

An advantage of the present invention is that the hydrolysis parameters (including enzymes) can be selected to inactivate (i.e., hydrolyze) the digestive enzymes while minimizing damage to all proteins, i.e., without substantial denaturation and for future use, thereby maximizing the value per capture. Furthermore, partially hydrolysed proteins are able to retain more water in a form that is detrimental to biological growth than denatured proteins, which means that PPC may contain higher levels of water, which is beneficial for overall processability and storability, without increasing biological activity. The hydrolyzed protein also has high digestibility and nutritional value for aquatic species, so that krill oil production by-products can be integrated into aquatic feed.

Another advantage is the preparation of a stable protein phospholipid emulsion that does not separate at high temperatures, and contains low water content to prevent microbial activity, and may be advantageous for transportation. Both the PPC and the stabilized protein phospholipid emulsion can be further processed to extract high quality crustacean oil and the remaining hydrolyzed protein can be further utilized.

Drawings

FIG. 1 is a flow diagram of one embodiment of a method for producing a protein phospholipid complex. In this flowchart, the dashed lines represent optional steps. For example, the exoskeleton components may optionally be removed after the hydrolysis step, and optionally added back after the hydrolyzed krill is pasteurized and excess water removed.

FIG. 2 is a flow diagram of one embodiment of a method of producing an emulsion or oil.

Detailed Description

One aspect of the present invention relates to a method of producing a Protein Phospholipid Complex (PPC). In particular, the process parameters of the process ensure that the naturally occurring digestive enzymes in krill are inactivated during hydrolysis while maintaining high quality proteins that are not substantially denatured by the hydrolysis-related processing conditions.

Crustacean capture

The first step involves providing a crustacean trap. Crustaceans refer to any marine organism having an exoskeleton and classified as part of the crustacean subdivision. Crustacean traps include, but are not limited to, krill, shrimp, lobster, crab, water fleas, and/or barnacle. Preferably, the crustacean catch is krill, such as antarctic krill (Euphausia Superba). The crustacean-trap may be fresh or previously frozen. However, fresh krill captured within 60 minutes is the preferred crustacean catch to be processed, and fresh krill captured within 30 minutes is the more preferred crustacean catch to be processed.

Pulverizing crustacean-captured material

The crustacean catch is crushed (for the just caught krill, immediately) to form a crushed crustacean catch. The comminuting includes mechanically breaking the crustacean catch into smaller pieces or smaller particle sizes to facilitate subsequent processing steps. The following values may be combined in any manner to produce a minimum value, a maximum value, or a range thereof for the particle size of the comminuted crustacean catch: 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, 18mm, 19mm, 20mm, 21mm, 22mm, 23mm, 24mm, and 25 mm. For example, the particle size of the comminuted crustacean trap may be about 1-25mm, preferably about 3-15mm, more preferably about 3-6 mm.

The crustacean trap may be comminuted using any conventional method to achieve a particular particle size range. For example, the comminuting device can grind, pulp, mill, and/or fragment the crustacean catch. Examples of comminuting devices include, but are not limited to, knife shredders, blenders, and homogenizers.

The temperature at which the crushing process takes place is approximately the ambient temperature of the water at which the crustacean catch is fresh (e.g. krill caught within 60 minutes). Thus, the temperature may be between about-2 ℃ and about + l ℃, preferably, about 0 ℃ to about +6 ℃.

Hydrolysis

The second step involves contacting the comminuted crustacean trap with one or more proteolytic enzymes to provide a hydrolyzed crustacean trap. The hydrolyzed crustacean-derived material is formed when the crushed crustacean-derived material undergoes a hydrolysis reaction. Hydrolysis is a chemical reaction or process that can be caused or mediated by biological agents, such as proteolytic enzymes, to shorten the native protein sequence (i.e., e.g., by breaking peptide bonds of the primary structure of the amino acid sequence) to form smaller peptides and free amino acids.

The crushed crustacean trap needs to be hydrolyzed in order to inactivate digestive enzymes (e.g., lipase and phospholipase) released by the crustacean trap upon death. If these digestive enzymes are not inactivated after release, they will destroy phospholipids and fatty acids in the crustacean trap. In order to inactivate digestive enzymes, the comminuted crustacean catch is contacted with a proteolytic enzyme under conditions to form a hydrolyzed crustacean catch. Proteolytic enzymes are selected specifically to target digestive enzymes with minimal damage to other proteins in the krill which can be utilized downstream while separating some phospholipids and peptides from the exoskeleton of the comminuted crustacean catch.

The hydrolysis conditions, such as choice of enzyme, temperature, pH and time, are selected to obtain a partially hydrolyzed crustacean trap with a particular degree of hydrolysis. The partially hydrolyzed capture is preferred, so that digestive enzymes can be inactivated, while other proteins can be broken down into smaller peptides and free amino acids. In contrast, a fully hydrolyzed crustacean trap is not preferred, as this would mean that all the protein in the trap is broken down into free amino acids.

The degree of hydrolysis may be determined by methods known in the art, such as pH-stat, trinitrobenzene sulfonic acid (TNBS), ortho-phthalaldehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formaldehyde titration. The following percentages may be combined into a range or used alone as a minimum or maximum to designate the degree of hydrolysis: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%. For example, more than about 30% of the protein in the crustacean trap is hydrolyzed. More preferably, more than about 40% of the protein in the crustacean trap is hydrolyzed. In another embodiment, the degree of hydrolysis may be as high as 90% to indicate that the hydrolysis is nearly complete.

In addition, partially hydrolyzed proteins can increase the digestibility of proteins. For example, the digestibility (peptidic digestility) of a partially hydrolysed protein is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. Preferably, the partially hydrolyzed protein has a digestibility of about 91%. Increased digestibility means that the partially hydrolysed proteins are nutritional for animals that are unable to synthesise essential amino acids. There is therefore much interest in using partially hydrolysed protein by-products as feed supplements for aquatic species and/or pets.

Digestibility can be measured by standard in vivo procedures or by more recent in vitro procedures. Previously, researchers have used rats, roosters, and/or chickens (chicken) to determine the amount of protein that an animal digests when fed a protein-containing compound, thereby determining digestibility in vivo. Analysis of the nitrogen content of animal faeces can indicate the amount of digestible protein in the product. Newer methods determine protein digestibility in vitro by adding spectroscopic reagents to the hydrolyzed protein solution reacted with amine or carboxylic acid functionality, so that the amount of amino acid released during enzymatic digestion can be assessed optically.

In addition, the proteolytic enzymes, hydrolysis conditions and duration of hydrolysis may be specifically selected to inactivate the digestive enzymes without substantially denaturing any proteins in the krill (including digestive enzymes and other proteins, i.e., proteins other than digestive enzymes). Denaturation occurs when a protein loses its original quaternary, tertiary and/or secondary structure.

In another embodiment, the digestive enzymes are not denatured or other proteins are not denatured. Partially hydrolyzed proteins that are not denatured are preferred because they have been found to retain a large amount of water in a form unsuitable for biological growth, 2,3, 4,5, 6, 7,8, 9, or 10 times more time than denatured proteins that retain less water in a form unsuitable for biological growth. The water retention capacity can improve the utilization rate of PPC by improving the fluidity and storage property of the product. It would therefore be advantageous to be able to allow PPC or any other downstream product containing partially hydrolysed proteins to retain large amounts of water over a longer period of time without promoting biological growth.

No substantial denaturation of other proteins occurs and may be defined as a proportion of denaturation of other proteins in krill of 0%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 20%, less than 25%, or less than 30%.

Preferably, the hydrolysis reaction occurs when the comminuted crustacean-captured material is contacted with a proteolytic enzyme and incubated. The optimal temperature for incubation is the temperature at which the particular enzyme is activated, which facilitates hydrolysis by the digestive enzymes without denaturing other proteins in the crustacean trap. The temperature may be obtained during the treatment by any method known in the art. Preferably, the proteolytic enzyme is added to hot water and then mixed with the crushed crustacean trap under agitation. Alternatively, the proteolytic enzyme may be added to water and the mixture heated, or the enzyme, water and the crushed crustacean trap may be mixed together and heated.

The following percentage values may be combined to determine the minimum, maximum, or range of amounts of water added in the contacting step based on the weight of the comminuted capture material: 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% and 55%. For example, water may be added in an amount up to about 50% by weight of the comminuted harvest. In another example, about 45% to about 50% water is added based on the weight of the comminuted prey.

The following values may be combined to determine the minimum, maximum, or range of optimal hydrolysis (i.e., incubation) temperatures for the comminuted crustacean trap: 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃,5 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃,6 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃,7 ℃, 72 ℃, 73 ℃, 74 ℃ and 75 ℃. For example, the contacting step comprises incubating the comminuted crustacean trap at a temperature in the range of about 45 ℃ to about 75 ℃. These temperatures may be applied to the temperature of water or to the temperature of water, enzymes and/or comminuted crustacean mixture. For example, the water may be heated to a temperature of about 60 ℃ prior to mixing the water with the proteolytic enzyme. In another example, the enzyme may function optimally at about 45 ℃ to about 65 ℃.

The amount of enzyme used can be determined by one of ordinary skill in the art to inactivate sufficient digestive enzymes without substantially denaturing other proteins in the krill and to separate some phospholipids and peptides from the exoskeleton of the comminuted crustacean catch.

For example, the proteolytic enzyme may be used in an amount of less than about 0.1% by weight based on the total weight of the comminuted crustacean trap. The following values may be combined in any manner to determine the range of the amount of proteolytic enzyme as a percentage of the total weight of the comminuted crustacean including the minimum and maximum values: 0.3%, 0.29%, 0.28%, 0.27%, 0.26%, 0.25%, 0.24%, 0.23%, 0.22%, 0.21%, 0.2%, 0.19%, 0.18%, 0.17%, 0.16%, 0.15%, 0.14%, 0.13%, 0.12%, 0.11%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, and 0.01%. In one embodiment, proteolytic enzymes may be used in step b) in an amount of about 0.01% to 0.1% based on the total weight of the comminuted crustacean trap.

The pH of the solution may be adjusted to ensure that hydrolysis proceeds in an optimal manner based on the particular proteolytic enzyme used.

The hydrolysis step may require any reasonable time to generate the hydrolyzed crustacean trap. Factors that influence the time required for hydrolysis include the temperature and pH of the mixture, and whether the reaction is under stirring conditions, and how strong the stirring is.

For example, hydrolysis may take less than 100 minutes. The following values in minutes may be combined in any manner to determine the minimum and maximum range of time required for hydrolysis: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40. For example, hydrolysis may take about 15-18 minutes, or hydrolysis may take less than about 45 minutes.

In another embodiment, hydrolysis may take more than 100 minutes. The following values in minutes may be combined in any manner to determine the minimum and maximum range of time required for hydrolysis: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235 and 240. For example, hydrolysis may require about 100-.

Proteolytic enzymes

Proteolytic enzymes useful in the present invention are food grade enzymes that cleave large protein molecules into small molecules by hydrolyzing peptide bonds along the protein backbone. As used herein, the terms "proteolytic enzyme", "protease" and "peptidase" are used interchangeably. As used herein, the term "exopeptidase" refers to a hydrolase that removes the terminal amino acids of a peptide or protein by cleaving peptide bonds. The terminal amino acid is an amino acid within about 10 amino acids of the N-terminus or C-terminus of a protein or peptide. As used herein, the term "endopeptidase" refers to an enzyme that catalyzes the cleavage of peptide bonds within a polypeptide or protein. Peptidase means that it acts on peptide bonds, while endopeptidases means that these are internal bonds.

Proteolytic enzymes include, but are not limited to, esterases such as carboxylic ester hydrolase, thioester hydrolase, phosphomonoesterase hydrolase, phosphatase, phosphodiester hydrolase, triphosphoric monoester hydrolase, sulfatase, diphosphonoester hydrolase, and phosphotriester hydrolase; glycosylases, such as glycosidases, i.e.enzymes which hydrolyze O-and S-glycosyl compounds and enzymes which hydrolyze N-glycosyl compounds; enzymes acting on ether bonds (e.g. hydrolysis of thioethers and trialkylsulphonic acids); peptidases, including exopeptidases, such as aminopeptidases, dipeptidyl peptidases and tripeptidyl peptidases, carboxypeptidases (serine-type carboxypeptidases, metallocarboxypeptidases, cysteine-type carboxypeptidases), dipeptidases, omega peptidases and peptidyl dipeptidases, and endopeptidases, such as serine endopeptidases, cysteine endopeptidases, aspartic endopeptidasesMetalloendopeptidase (e.g., Ab Enzymes Co., Ltd.)7089, CAS 9001-92-7), and threonine endopeptidase; enzymes that hydrolyze halocarbon compounds in a single subclass; an enzyme acting on a phosphorus-nitrogen bond; an enzyme acting on a sulfur-nitrogen bond; an enzyme that hydrolyzes the C-phosphate group; an enzyme acting on a sulfur-sulfur bond; and enzymes acting on carbon-sulfur bonds.

Carboxylic ester hydrolase includes carboxylesterase, arylesterase, triacylglycerol lipase, phospholipase A2, lysophospholipid lipase, acetyl esterase, acetylcholinesterase, cholinesterase, toffee esterase, pectinesterase, sterol esterase, chlorophyllase, L-arabinolactonase, glucuronosylase, uroaldehyde lactonase, tannase, hydroxybutyrate dimer hydrolase, acylglycerol lipase, 3-oxomalate enollactase, l, 4-lactase, galactolipase, 4-pyridoxine lactonase, acylinositol hydrolase, aminoacyl tRNA hydrolase, D-arabinolactonase, 6-phosphogluconolactonase, Al, 6-acetylglucose deacetylase, lipoprotein lipase, dihydrocoumarin hydrolase, citric acid-D-cyclo-lactonase, steroid lactonase, triacetyl lactonase, actinomycin lactonase, Orsselinyl-deppsylase, C-acetylesterase, phosphoesterase-D-lactonohydrolase, phosphoesterase, esterase, phosphoesterase, esterase, phosphoesterase, esterase, phosphoesterase, esterase-D.

Thioesterase enzymes include acetyl-CoA hydrolase, palmitoyl-CoA hydrolase, succinyl-CoA hydrolase, 3-hydroxyisobutyryl-CoA hydrolase, hydroxymethylglutaryl-CoA hydrolase, hydroxyacylglutamine hydrolase, glutathione thioesterase, formyl-CoA hydrolase, acetoacetyl-CoA hydrolase, S-formylglutathione hydrolase, S-succinyl-glutathione hydrolase, oleoyl- [ acyl carrier protein ] hydrolase, citrate lyase deacetylase, (S) -methylmalonyl-CoA hydrolase, ADP-dependent short-chain acyl-CoA hydrolase, ADP-dependent medium-chain acyl-CoA hydrolase, dodecyl- [ acyl carrier protein ] hydrolase, palmitoyl [ protein ] hydrolase, 4-hydroxybenzoyl-CoA thioesterase, phenylacetyl-CoA hydrolase, bilirubin-CoA hydrolase, 1, 4-dihydroxy-2-naphthoyl-CoA hydrolase, fluoroacetylCoA thioesterase, (3S) -maloyl-CoA thioesterase, dihydromozurine-ketorolysin- [ L ] thioesterase, and non-aminobenzoyl-A-esterase.

Phosphomonoesterases include alkaline phosphatase, acid phosphatase, phosphoserine phosphatase, phosphatidic acid phosphatase, 5 '-nucleotidase, 3' (2'),5' -bisphosphate nucleotidase, 3-phytase, glucose-6-phosphatase, glucose-1-phosphatase, fructose bisphosphatase, trehalose phosphatase, phosphoglycerol phosphatase, histidine phosphatase, protein-serine/threonine phosphatase, [ phosphorylase ] phosphatase, phosphoglycolate phosphatase, glycerol-2-phosphatase, phosphoglycerate phosphatase, glycerol-1-phosphatase, mannitol-1-phosphatase, sugar phosphatase, sucrose phosphate phosphatase, inositol phosphate phosphatase, 4-phytase, phosphatidylglycerol phosphatase, ADP-phosphoglycerate phosphatase, N-acylneuraminic acid-9-phosphatase, nucleotidase, polynucleotide 3 '-phosphatase, polynucleotide 5' -phosphatase, deoxynucleotide 3 '-phosphatase, thymidylate 5' -phosphatase, phosphoinose 5-phosphatase, heptulobisphosphatase, 3-phosphoglycerate phosphatase, streptomycin-6-phosphatase, guanidine phosphatase, phosphoserine phosphatase, phosphophosphophosphophosphoinositidine phosphatase, phosphophosphophosphophosphophosphophosphophosphophosphoinositidine phosphatase, phosphoinositide phosphatase, phospho.

Phosphodiesterase hydrolases include phosphodiesterase I, glycerophosphocholine phosphodiesterase, phospholipase C, phospholipase D, phosphoinositide phosphodiesterase C, sphingosine phosphodiesterase, serine ethanolamine phosphodiesterase, [ acyl carrier protein ] phosphodiesterase, 2',3' -cyclic nucleotide 2 '-phosphodiesterase, 3',5 '-cyclic nucleotide phosphodiesterase, 3',5 '-cyclic GMP phosphodiesterase, 2',3 '-cyclic nucleotide 3' phosphodiesterase, glycerophosphocholine phosphocholine phosphodiesterase, alkylglycerophosphoethanolamine phosphodiesterase, CMP-N-acylneuraminic acid phosphodiesterase, sphingomyelin phosphodiesterase D, glycerol-1, 2-cyclic phosphodiesterase, glycerophosphoinositide phosphodiesterase, N-acetylglucosamine-l-phosphodiesterase α -N-acetylglucosaminidase, glycerophosphodiester phosphodiesterase, long-p-pyelose phosphophosphodiesterase, long-p-mannose phosphodiesterase, glycosylphosphatidylinositol D, glucose-l-phospho-D-yl phosphodiesterase, cyclic nucleotide phosphodiesterase, phosphodiesterase-3 ',5' -cyclic nucleotide phosphodiesterase, phosphodiesterase-3 ',3' -dihydrophosphodiesterase, phosphoinositide phosphodiesterase, phosphoinositide phosphodiesterase, N-l-3 '-phosphodiesterase, phosphodiesterase-3', 3 '-adenyl-3' -phosphodiesterase, phosphodiesterase.

The monophosphorous monoesterase enzyme comprises dGTP enzyme.

The sulfate hydrolase includes arylsulfatase, steroid sulfatase, saccharide sulfatase, N-acetylgalactosamine-6-sulfatase, cholinesterase, cellulose polysulfate, cerebroside sulfatase, chondroitin-4-sulfatase, and chondroitin-6-sulfatase, dithioglucamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, iduronate-2-sulfatase, N-acetylglucosamine-6-sulfatase, N-sulfoglucamine-3-sulfatase, monomethyl sulfatase, D-lactate-2-sulfatase, glucuronate-2-sulfatase, and (R) -specific secondary alkylsulfatase.

The monophosphoryl diphosphates hydrolases include prenyl diphosphatase, guanosine-3 ',5' -bis (diphosphate) 3' -diphosphatase, monoterpene-based diphosphatase, geranylgeranyl diphosphatase, farnesyl diphosphatase, tuberculol synthase, isonicotinin synthase, (13E) -labda-7, 13-dien-15-ol synthase, geranyl diphosphatase, and (+) -kolavivolol synthase.

The phosphotriester hydrolases include aryl dialkyl phosphatases and diisopropyl fluorophosphases.

Exodeoxyribonucleases that produce 5' -phosphomonoesters include exodeoxyribonuclease I, exodeoxyribonuclease III, exodeoxyribonuclease (lambda induced), exodeoxyribonuclease (phage SP3 induced), exodeoxyribonuclease V, exodeoxyribonuclease VII, adenosine-5 ' -diphosphate-5 ' - [ DNA ] diphosphatase, and guanosine-5 ' -diphosphate-5 ' - [ DNA ] diphosphatase.

Exodeoxyribonucleases that produce 3 '-phosphate monoesters include 5' -3 'exodeoxyribonucleases (nucleoside 3' -phosphate formation), as well as DNA-3 '-diphosphate-5' -guanosine diphosphate.

Exoribonucleases that produce 5' -phosphomonoesters include exoribonuclease II, exoribonuclease H, oligonucleotidase, poly (A) -specific ribonuclease, and ribonuclease D.

Exoribonucleases that produce 3' -phosphomonoesters include yeast ribonucleases.

Exonucleases having ribose or deoxyribose nucleic acid activity and producing 5' -phosphate monoesters include venom exonucleases.

Exonucleases having ribonucleic acid or deoxyribonucleic acid activity and producing 3' -phosphate monoesters, such as splenic exonuclease;

the endoglucanase producing the 5' -phosphomonoester includes DNase I, DNase IV, type I site-specific DNase, type II site-specific DNase, type III site-specific DNase, CC-preferred endoglucanase, DNase V, T₄Deoxyribonucleases II and T₄Deoxyribonuclease IV.

The 3' -phosphate monoester-producing endodeoxyribonuclease includes deoxyribonuclease II.

Aspergillus deoxyribonuclease K1 included a cross-linked endoglucanase and deoxyribonuclease X.

Site-specific endodeoxyribonucleases specific for the altered base include deoxyribonucleases (pyrimidine dimers).

Endoribonucleases that produce 5' -phosphomonoesters are also included.

The velvet fungus multiheaded ribonuclease comprises ribonuclease α, ribonuclease III, ribonuclease H, ribonuclease P, ribonuclease IV, ribonuclease P4, ribonuclease M5, ribonuclease [ poly (U) -specific]Ribonuclease IX, tRN enzyme Z, ribonuclease E, and retroviral ribonuclease H;endoribonucleases producing 3' -phosphomonoesters, e.g. ribonuclease T₂。

Bacillus subtilis ribonucleases including ribonuclease T₁Ribonuclease U₂And pancreatic ribonucleases.

Enterobacter ribonucleases include ribonuclease F, ribonuclease V, and rRNA endoribonuclease.

Also included are endoribonucleases active with ribose or deoxyribose nucleic acid and producing 5' -phosphate monoesters, aspergillus nucleases Sl and serratia marcescens nucleases.

Endoribonucleases active with ribose or deoxyribose nucleic acid and producing 3' -phosphate monoesters include micrococcal nucleases.

Also included are glycosylases.

A glycosidase, i.e., an enzyme that hydrolyzes O-and S-glycosyl compounds, including amylase, glucanase, D-4-0-glucosidase, cellulose, endo-l, 3- (4) -00-glucanase, inulase, endo-l, 4-01-xylanase, oligo-l, 6-glucosidase, dextranase, chitinase, polygalacturonase, lysozyme, exo-04-sialidase, 05-glucosidase, 02-glucosidase, 08-galactosidase, 03-galactosidase, 12-mannosidase, 06-mannosidase, 07-fructosidase, 18, 29-trehalase, 09-glucuronidase, endo-1, 3-xylanase, starch-3-1, 6-glucosidase, hyaluronidase, 1, 4-10-xylosidase, 11-D-glucosidase, 1, 3-1-xylosidase, 2-mannosidase, 6-mannosidase, 2-D-mannosidase, 6-D-glucosidase, 2-D-glucosidase, 6-D-glucosidase, 2-D-glucosidase, 6-D-glucosidase, 4-0-glucosidase, 6-glucosidase, 4-0-glucosidase, 6-glucosidase, 4-0-glucosidase, 6-glucosidase, 4-glucosidase, 6-D-glucosidase, 6-glucosidase, 2-glucosidase, 6-endoglucanase, endo-2-D-4, 6-glucosidase, 2-D-glucosidase, 6-glucosidase, 2-glucosidase, 6-D-glucosidase, 2-D-glucosidase, 6-glucosidase, 2-glucosidase, 6-D-glucosidase, 2-endoglucanase, 12-D-glucosidase, 4-glucosidase, 6-D-glucosidase, 12-glucosidase, 4-D-glucosidase, 2-glucosidase, 4-D-glucosidase, 4-glucosidase, 2-D-glucosidase, 4-D-glucosidase, 4-glucosidase, 6-xylosidase, 4-D-glucosidase, 4-glucosidase, 6-D-glucosidase, 2-glucosidase, 6-xyloglucanase, 6-glucosidase, 2-glucosidase, 6-xyloglucanase, 2-D-glucosidase, 6-glucosidase, 12-glucosidase, 2-glucosidase, 6-glucosidase, 2-glucosidase, 12-glucosidase, 6-fructofuranosidase, 6-glucosidase, 12-glucosidase, 6-fructofuranosidase, 12-fructosidase, 6-mannosidase, 6-fructofuranosidase, 12-mannosidase, 6-mannosidase, 12-mannosidase, 4-mannosidase, 2-mannosidase, 4-mannosidase, 6-mannosidase, 12-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 4-mannosidase, 6-mannosidase, 2-mannosidase, 12-mannosidase, 2-mannosidase, 4-mannosidase, 6-mannosidase, 4-mannosidase, 2-mannosidase, 6-mannosidase, 4-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 4-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 4-mannosidase, 2-mannosidase, 4-mannosidase, 6-mannosidase, 2-mannosidase, 8-mannosidase, 2-mannosidase, 4-mannosidase, 2-mannosidase, 8-mannosidase, 2-mannosidase, 8-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 8-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 4-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 6-mannosidase, 2-mannosidase, 4-mannosidase, 2-mannosidase, 4-mannosidase, 6-mannosidase, 2-mannosidase, 4-mannosidase, 2-D-mannosidase, 2-mannosidase, 8-mannosidase, 2-D-mannosidase, 8-mannosidase, N-mannosidase, 2-mannosidase, 8-mannosidase, 2-D-mannosidase, 2-D-mannosidase, N-D-mannosidase, 2-D-mannosidase, 2-D-mannosidase, 2-D-mannosidase, 2-mannosidase, 4-mannosidase, 2-mannosidase.

Hydrolyzing N-glycosyl compounds include purine ribosidase, inosinase, uridine ribosidase, adenylate ribosidase, NAD + glycohydrolase, ADP-ribocyclase/cyclic ADP-ribohydrolase, adenosine ribosidase, ribopyrimidine ribosidase, adenosylcysteine ribosidase, pyrimidine-5' -nucleosidase, β -aspartate-N-acetylglucosaminidase, inosinase, 1-methyladenosine ribosidase, NMN ribosidase, DNA deoxyribonuclease, methylthioadenylase, deoxyribopyrimidine endoribosidase, [ protein ADP riboarginine ] hydrolase, DNA-3-methyladenine glycosylase I, DNA-3-methyladenine glycosylase II, rRNAn-glycosylase, DNA-formamidopyrimidine glycosylase, ADP-ribo- [ diazepoxide reductase ] hydrolase, N-methylnucleosidase, futalosine hydrolase, uracil DNA glycosylase, double stranded uracil DNA glycosylase, thymine DNA glycosylase, and lotidylase.

Thioether and trialkyl sulfonic acid hydrolases include adenosylhomocysteinase and adenosylmethionine hydrolase.

Ether hydrolases include isocohismatase, lysosome-producing enzymes, trans-epoxysuccinate hydrolase, leukotriene-A4 hydrolase, hepatic oxigenin-epoxyhydrolase, limonene-l, 2-epoxyhydrolase, microsomal epoxyhydrolase, soluble epoxyhydrolase, cholesterol-5, 6-oxide hydrolase, oxepitrien-CoA hydrolase, chorismatase, 2, 4-dinitroanisole O-demethylase, and trans-2, 3-dihydro-3-hydroxyanthranilate synthase.

Aminopeptidases include leucyl aminopeptidase, membrane alanyl aminopeptidase, cystyl aminopeptidase, tripeptide aminopeptidase, prolyl aminopeptidase, aminopeptidase B, glutamyl aminopeptidase, Xaa-Pro aminopeptidase, bacterial leucyl aminopeptidase, clostridial aminopeptidase, cytosolic alanyl aminopeptidase, aminopeptidase Y, Xaa-Trp aminopeptidase, tryptophanyl aminopeptidase, methionyl aminopeptidase, D-stereospecific aminopeptidase, aminopeptidase Ey, aspartyl aminopeptidase, aminopeptidase I, PepB aminopeptidase, aminopeptidase S, Xaa-His dipeptidase, Xaa-Arg dipeptidase, Xaa-methyl-His dipeptidase, Glu-Glu dipeptidase, Xaa-Pro dipeptidase, Met-Xaa dipeptidase, non-stereospecific dipeptidase, cytoplasmic non-specific dipeptidase, membrane dipeptidase, b-Ala-His dipeptidase, dipeptidase E and D-Ala-D-Ala dipeptidase.

Dipeptidyl peptidase and tripeptidyl peptidase include dipeptidyl peptidase I, dipeptidyl peptidase II, dipeptidyl peptidase III, dipeptidyl peptidase IV, dipeptidyl dipeptidase, tripeptidyl peptidase I, tripeptidyl peptidase II, Xaa-Pro dipeptidyl peptidase and Xaa-Xaa-Pro tripeptidyl peptidase, peptidyl dipeptidases, such as peptidyl dipeptidase A, peptidyl dipeptidase B, peptidyl dipeptidase Dcp and phycocyanin, serine type carboxypeptidase, such as lysosomal Pro-Xaa carboxypeptidase, serine type D-Ala-D-Ala carboxypeptidase, carboxypeptidase C and carboxypeptidase D, metallocarboxypeptidase, such as carboxypeptidase A, carboxypeptidase B, lysine carboxypeptidase, Gly-Xaa carboxypeptidase, alanine carboxypeptidase, muramyl pentapeptide carboxypeptidase, carboxypeptidase E, glutamate carboxypeptidase, carboxypeptidase M, muramyl tetrapeptide carboxypeptidase, zinc D-Ala-D-Ala carboxypeptidase, carboxypeptidase, Carboxypeptidase A2, membrane Pro-Xaa carboxypeptidase, tubulinyl-Tyr carboxypeptidase, carboxypeptidase T, carboxypeptidase Taq, carboxypeptidase U, glutamate carboxypeptidase II, metallocarboxypeptidase D and angiotensin converting enzyme 2.

Cysteine-type carboxypeptidases include cathepsin X.

Omega peptidases include acylaminoacyl peptidase, peptidyl glycinamidase, pyroglutamyl peptidase I, b-aspartyl peptidase, pyroglutamyl peptidase II, N-formylmethionyl peptidase, g-glutamyl hydrolase, g-D-glutamyl-m-diaminopimelate peptidase and ubiquitylhydrolase 1.

Serine endopeptidases include chymotrypsin, chymotrypsin C, metridin, trypsin, thrombin, factor Xa, plasmin, enteropeptidase, acrosin, a-lyases endopeptidase, glutamineendopeptidase, cathepsin G, factor VIIa, factor IXa, cucumber protease, prolyl oligopeptidase, factor Xia, brachyuran, plasma kallikrein, tissue kallikrein, pancreatic elastase, leukocyte elastase, factor XIIa, chymotrypsin, complement subfraction Clr, complement subfraction Cls, classical complement pathway C3/C5 convertase, complement factor I, complement factor D, complement pathway C3/C5 convertase, cerevisin, hypodermin C, lysyl endopeptidase, endopeptidase L a, G-renin, venomombin endopeptidase, leucyl endopeptidase, trypsin-like protease, kexilysin, Orxin, Oryzin, subtilisin, lysozymin, phosphopeptidase K, heat-peptidase, trypsin-endopeptidase, trypsin-peptidase, trypsin-related endopeptidase A-peptidase, trypsin-related endopeptidase A endopeptidase, trypsin peptidase, trypsin-related endopeptidase, trypsin-peptidase IV peptidase, trypsin-related endopeptidase-peptidase, trypsin-related endopeptidase-A endopeptidase, trypsin-peptidase, trypsin-related endopeptidase-peptidase IV peptidase, trypsin-I, trypsin-I, trypsin-I, trypsin-II, trypsin-I, trypsin-I, trypsin-II, trypsin-II, trypsin-I, trypsin-II, trypsin.

Cysteine endopeptidases include cathepsin B, papain, ficin, chymopapain, asclesin, clostripain, streptomycin, kiwi fruit proteinase, cathepsin L, cathepsin H, cathepsin T, glycyl endopeptidase, cancer coagulants, cathepsin S, picornain3C, picornanin 2A, caricin, bromelain, stem bromelain, fruit bromelain, bean protease, cathepsin, caspase-1, porphyromonas gingivalis protease R, cathepsin K, adenain, bleomycin hydrolase, cathepsin F, cathepsin O, cathepsin V, nuclear inclusion body a endopeptidase, accessory component protease, L peptidase, porphyromonas gingivalis protease K, staphopain, separatase, V-cathepsin, cruzipain, calpain-L, calpain-2, calpain-3, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-1, caspase-7, caspase-protease, caspase-10, caspase-1, caspase-protease (caspase-1), caspase-9, caspase-7, caspase-1, caspase-9, caspase-1, caspase-1, and caspase.

Aspartic endopeptidases include pepsin A, pepsin B, pepsin, chymosin, cathepsin D, nepenthesin, renin, HIV-l retropeptin, pro-opiomelanocortin transferase, Aspergillus pepsin I, Aspergillus pepsin II, Penicillium pepsin, Rhizopus pepsin, endo-capsid pepsin, Mucor pepsin, Candida pepsin, saccharopepsin, Rhodotorula pepsin, acyclindropeptin, polypopepsin, Microporus pepsin, scytalidopisin A, scytalidopeptosin B, cathepsin E, barrierpepsin, Signal peptidase II, plaspepsin I, plasminsin II, phytepsin, yapsin 1, thermomoprin, prepromasin peptidase, nodavirus endopeptidase, Membrane aspartic protease 1, Membrane aspartic protease 2, HIV-2 retropeptin, plasminogen activator, plasmin, Hydropotexzyme endopeptidase I, human retrovirus endopeptidase I.

Metalloendopeptidases include macrocephalospira metalloendopeptidase A, microbial collagenase, leukolysin, interstitial collagenase, neprilysin, envlaysin, IgA-specific metalloendopeptidase, procollagen N-endopeptidase, thimet oligopeptidase, lysin, stromelysin 1, transmembrane peptidase A, procollagen C-endopeptidase, peptidyl L ys metalloendopeptidase, astaxanthin, stromelysin 2, stromelysin, gelatinase A, Vibriolysin, pseudohemolysin, thermosol, bacteriocin, aurolysin, aureolysin, cocolyysin, mycolysin, B lysobactopeptidase, Asp metalloendopeptidase, neutrophil collagenase, gelatinase B, lissima, saccharolysin, gasololysin, deuterolysin, serralysin, creosolysin endopeptidase B, macrocephalosporanopeptidase B, transmembrane endopeptidase 2, macrocephalosporanopeptidase, cathepsin, endopeptidase F endopeptidase, endopeptidase I-endopeptidase, endopeptidase I-I, endopeptidase I-P-S-I, endopeptidase I-I, endopeptidase B-I, endopeptidase B-I, endopeptidase B-I, endopeptidase, endop.

Threonine endopeptidases include the protease endopeptidase complex and HslU-HslV peptidase.

Endopeptidases of unknown catalytic mechanism are also included.

The linear amides acting on carbon-nitrogen bonds other than peptide bonds include asparaginase, glutaminase, ω amidase, urease, β -urealyllaminase, urealyosuccinase, formylaspartic acid deformylase, arylformamidase, formyltetrahydrofolate deformylase, penicillinamidase, biotinases, arylamidase, N-acyl-aliphatic-L-amino acid amidohydrolase, asparaginase, acetylornithine deformylase, acyllysine deformylase, succinyl-diaminopimelate dessuccinylase, nicotinamidase, sphingomyelinase, N-acetyl- β -alanine deformylase, pantothenate, ceramidase, cholylase, cholylglycan, N-acetylglycidyl-hydrolase, N-acetylglucosaminyl-6-phosphate deformylase, N4- (β -N-acetylglucosaminyl-glucosyl) -L-monophosphoryl amidase, N-acetylmuramyl-3-alanine amidase, 2- (acetylaminomethyl) amidase, N-aminoacylase, N-aminoacylamidase, N-aminoacylamidase, N-succinyl-2-aminoacylamidase, N-aminoacylamidase, N-2-N-aminoacylamidase, N-acyl-serine-aminoacylamidase, N-serine-hydrolase, N-serine-hydrolase, N-serine-hydrolase, N-serine-6-serine-aminoacylase, N-serine-6-aminoacylase, N-serine-aminoacylase, serine-hydrolase, serine-hydrolase, serine-hydrolase, serine-hydrolase, serine-hydrolase, serine-hydrolase, serine-hydrolase, serine-hydrolase, serine-hydrolase, serine-.

Among cyclic amides, barbiturates, dihydropyrimidinases, dihydroorotases, carboxymethylhydantoinases, allantoases, β -lactamases, imidazolinylacrylases, 5-oxoprolinases (ATP hydrolysis), creatinases, L-lysine-lactamases, 6-aminocaproic acid-cyclodimer hydrolases, 2, 5-dioxopiperazine hydrolases, N-methylhydantoinases (ATP hydrolysis), cyanuric amide hydrolases, maleimide hydrolases, hydroxyisouricases, enamidases, streptomycin hydrolases, isatin hydrolases are included.

Linear amidines include arginase, guanidinoacetic acid enzyme, creatinase, allantoase, carbamoyllactic acid deaminase, arginine deaminase, guanidinobutylase, carbamoylglutamic acid deaminase, allantoic acid deaminase, D-arginase, agmatinase, agmatine deaminase, carbamoylglutamic acid deaminase, aspartase, protein-arginine deaminase, methylguanidine, guanylpropanase, dimethylanilinohydrolase, diguanidinobutanase, methylenediamine deaminase, proclorazinate aminodihydrolase, N-succinylarginine dihydrolase, N1-aminopropylguanylguanidine urea hydrolase, N omega-hydroxy-L-arginine aminohydrolase and (S) -uridine glycinohydrolase.

The cyclic amidines include cytosine deaminase, adenine deaminase, guanine deaminase, adenosine deaminase, cytidine deaminase, AMP deaminase, ADP deaminase, aminoimidazole, methylenetetrahydrofolate cyclohydrolase, IMP cyclohydrolase, pterin deaminase, dCMP deaminase, dCTP deaminase, guanosine deaminase, GTP cyclohydrolase I, adenosine deaminase, ATP deaminase, ribose phosphate AMP cyclohydrolase, cysteamine deaminase, creatinine deaminase, l-pyrroline-4-hydroxy-2-carboxylic acid deaminase, blastinomycin-S deaminase, sepiapterin deaminase, GTP cyclohydrolase II, diaminophenylribosyl aminopyrimidine deaminase, methylenetetrahydromethylpterin cyclohydrolase, S-adenosine homocysteine deaminase, GTP cyclohydrolase IIa, dCUMTP deaminase (forming dTP P), S-methyl-5' -thioadenosine, 8-oxoguanine deaminase, tRNA (adenine 34) deaminase, tRNAAla (adenine 37) deaminase, tRNA (cytosine 8) deaminase, mRNA (cytosine 6666) deaminase, double stranded RNA adenine deaminase, single stranded DNA cytosine deaminase, GTP cyclohydrolase IV, aminodeoxyribofuranosine deaminase, 5' -deoxyadenosine deaminase, N-isopropylacrylamide isopropylamine deaminase, hydroxydeoxychloroatrazine hydrochloramine hydrosylate, tetrahydropyrimidine hydrolase, melamine deaminase, cAMP deaminase.

Included among the nitriles are nitrilase, ricinine nitrilase, cyanoalanine nitrilase, arylacetonitrilase, bromoxynil nitrilase, aliphatic nitrilase and thiocyanic acid hydrolase.

Other compounds include riboxanthinase, aminopyrimidine aminohydrolase, 2-amino-muconic acid deaminase, glucosamine-6-phosphate deaminase, l-aminocyclopropane-l-carboxylic acid deaminase, 5-nitro-anthranilate aminohydrolase, 2-nitroimidazole nitrohydrolase, 2-iminobutyrate/2-iminopropionic acid deaminase, and 2-amino-muconic acid deaminase (2-hydroxy muconate formation).

The anhydride-acting phosphorus-containing anhydrides include inorganic bisphosphatase, trimetaphosphase, ATPase, apyrase, nucleoside diphosphatase, acylphosphatase, ATP diphosphatase, nucleotide diphosphatase, endo-polyphosphatase, exopolyphosphatase, dCTP diphosphatase, ADP ribose diphosphatase, adenosine tetraphosphate, nucleoside triphosphatase, CDP glycerodiphosphatase, bis (5' -nucleoside) -tetraphosphate (asymmetric), FAD diphosphatase, 5' -acylphosphoadenosine hydrolase, ADP sugar diphosphatase, NAD + diphosphatase, dUTP diphosphatase, nucleoside phosphorylhydrolase, triphosphatase, CDP diacylglycerol diphosphatase, undecyl diphosphatase, thiamine triphosphatase, bis (5' -adenosine triphosphatase, ribose-ATP diphosphatase, thymidine triphosphatase, guanosine-5 ' -triphosphatase, 3' -diphosphatase, bis (5' -nucleoside) -tetraphosphate (symmetric), guanosine diphosphatase, dolichthyiptase, oligosaccharide-diphosphatase, UDP-diphosphate glucidamole phosphatase, UDP-5 ' -diphosphatase, UDP-D-phosphophosphatase, UDP-D2-phosphophosphatase, UDP-D2-phosphodiphosphatase, phosphoenolphosphatase, UDP-5-D-2-phosphoenolphosphatase, UDP-phosphoenolphosphatase, UDP-5-phosphoenolphosphoenolphosphatase, ATP-D-phosphoenolphosphatase, ATP-D-phosphoenolphosphatase, ATP-phosphoenolphosphoenolphosphatase, ATP-phosphoenolphosphoenolphosphoenolphosphoenolphosphatase, ATP-phosphoenolphosphoenolphosphoenolphosphatase, ATP-phosphoenolphosphoenolphosphoenolphosphoenolphosphatase, ATP-phosphoenolphosphatase, ATP-D-12-phosphoenolphosphatase, ATP-D-phosphoenole, ATP-phosphoenol.

Included among the sulfonyl-containing anhydrides are adenyl sulfatase and phosphoadenyl sulfatase.

Transmembrane motions acting on the anhydride catalysis to catalyze substances include phospholipid translocating atpase, Mg2+ import atpase, Cd2+ export atpase, Cu2+ export atpase, Zn2+ export atpase, H + export atpase, Na + export atpase, Ca2+ transport atpase, Na +/K + exchange atpase, H +/K + exchange atpase, Cl-transport atpase, K + transport atpase, H + transport binodal atpase, Na + transport binodal atpase, arsenite transport atpase, monosaccharide transport atpase, oligosaccharide transport atpase, maltose transport atpase, glycerol-3-phosphate transport atpase, polar amino acid transport atpase, nonpolar amino acid transport atpase, oligopeptides atpase, nickel transport atpase, sulfate transport atpase, nitrate transport atpase, phosphate transport enzyme, molybdate transport enzyme, Fe3+ chloroplast transport enzyme, atpase, ATP transport enzyme, calcium phosphate.

Examples of enzymes that act on anhydrides to promote cellular and subcellular movement include myosin atpase, dynein atpase, microtubule-cleaving atpase, positive end-directed kinesin atpase, negative end-directed kinesin atpase, vesicles that fuse atpases, peroxisome assembly atpase, proteasome atpase, chaperone atpase, non-chaperone atpase, nucleoplasmin atpase, DNA helicase, and RNA helicase.

Examples of GTP-promoting activities include heterotrimeric G-protein GTPases, small monomeric GTPases, protein-synthesizing GTPases, signal-recognizing particulate GTPases, dynamic GTPases, and tubulin GTPases.

Acting on carbon-carbon bonds in ketones include oxaloacetate, fumarylacetoacetylase, kynureninase, phloretin hydrolase, acylpyruvate hydrolase, acetylacetonate hydrolase, b-diketohydrolase, 2, 6-dioxo-6-phenylhexyl-3-enoate hydrolase, 2-hydroxycycloconate-6-semialdehyde hydrolase, cyclohexane-l, 3-diketohydrolase, cyclohexane-1, 2-diketohydrolase hydrolase, cobalt-pregrinine 5A hydrolase, 2-hydroxy-6-oxo-6- (2-aminophenyl) hexyl-2, 4-dienoate hydrolase, 2-hydroxy-6-oxonona-2, 4-dienoate hydrolase, thiolester hydrolase, and the like, 4,5:9, l 0-double cleavage-3-hydroxy-5, 9, 17-trioxandrosta-1 (10), 2-diene-4-acid ester hydrolase, 6-oxocamphorase, 2, 6-dihydroxypseudooxynicotinase, 3-fumaric acid pyruvate hydrolase, 6-oxocyclohex-l-ene-l-carbonyl CoA hydratase, 3D- (3,5/4) -trihydroxycyclohexane-l, 2-diketonoyl hydrogenase (Ring opening) and maleyl pyruvate hydrolase.

Examples of the halogen bond acting on the halogenated carbon compound include alkyl halogenase, (S) -2-halogenoacid dehalogenase, halogenoacetic acid dehalogenase, haloalkane dehalogenase, 4-chlorobenzoate dehalogenase, 4-chlorobenzoyl-CoA dehalogenase, atrazine chlorohydrolase, (R) -2-halogenoacid dehalogenase, 2-halogenoacid dehalogenase (inversion of configuration), and 2-halogenoacid dehalogenase (retention of configuration).

Acting on the phosphorus-nitrogen bond include phosphoamidase, protein arginine phosphatase and phosphohistidine phosphatase.

Acting on the sulfur-nitrogen bond, the sulfohydrolase comprises N-sulfoglucosamine thiohydrolase and sulfohydrolase.

Examples of the enzyme acting on the carbon-phosphorus bond include phosphonoacetaldehyde hydrolase, phosphonoacetate hydrolase and phosphonopyruvate hydrolase. Acting on the thiothio bond is a trisulfate hydrolase.

Acting on carbon-sulfur bond, there are UDP-sulfanylquinolulose synthase, 2' -hydroxybiphenyl-2-sulfite desulfhydrase, 3-sulfite propyl-CoA desulfhydrase, carbon disulfide hydrolase and [ CysO-sulfur carrier protein ] -S-L-cysteine hydrolase.

Preferred commercially available enzyme packagesIncluding thiol proteases (papain from Enzybel International S.A., CAS 9001-73-4), serine endoproteases (Protamex from Novozymes), and neutral proteases comprising endopeptidase activity from Bacillus subtilis cultures, i.e., metalloendopeptidases, subtilisin (neutral protease from Ab enzyme)7089，CAS 9001-92-7)。

In one embodiment, a mixture of proteases may be used. Suitable protease mixtures include one or more acidic, neutral or alkaline proteases.

Acidic proteases are protein digestive enzymes that exhibit maximal activity and stability under acidic conditions (e.g., pH 2.0-5.0, 2.0-3.0, 2.0-4.0, or 3.0-5.0) and are inactivated at pH values above 6.0. Acidic proteases generally have a lower isoelectric point and a lower content of basic amino acids.

Neutral proteases are active over a narrow pH range (pH 5-8) and may have relatively low thermostability. Neutral proteases include cysteine proteases, metalloproteases, and some serine proteases.

The alkaline protease is characterized by high activity at alkaline pH, such as at least pH 9, at least pH 10, at least pH 11. Examples of the alkaline protease include serine protease. They have a wide range of substrate specificities, including ester hydrolysis activity and amidase activity. The isoelectric point of the serine protease is generally between pH 4 and 6. Serine alkaline proteases are active at high alkaline pH and are the largest subgroup of serine proteases.

In one embodiment, the protease mixture comprises a protease from bacillus licheniformis. Acidic, neutral and alkaline proteases from Bacillus licheniformis are known in the art. See, for example, Yilmaz et al, J Enzyme Inhib MedChem, 2016, 31 (6): 1241-1247; rao et al, review in microbiology and molecular biology, 1998, 62 (3): 597-; and Jellouli et al, process biochemistry, 2011, 46 (6): 1248-1256.

In one embodiment, the protease mixture comprises a food grade cell wall degrading enzyme obtained from an organism of the genus aspergillus. Examples of Aspergillus cell wall degrading enzymes include mylase, pectinase, xylanase, and cellulase. Specific examples include b-glucosidase, endoglucanase, filter paper enzyme, polygalacturonase and pectate lyase.

In one embodiment, the method of the present invention comprises adding a first protease mixture having at least one alkaline protease; and a second protease mixture having at least one of an acid protease, a neutral protease and an alkaline protease.

The first protease mixture comprises at least one, at least two, at least three or at least four alkaline proteases. In one embodiment, the alkaline protease is from bacillus licheniformis. In one embodiment, the first protease mixture comprises only endoproteases.

In one embodiment, the first protease mixture comprises an alkaline protease.

In one embodiment, the first protease mixture comprises two alkaline proteases. In one embodiment, the first alkaline protease and the second alkaline protease sum to 100% of the proteases of the first protease mixture. In one embodiment, the first alkaline protease is 1-80% of the protease mixture and the second alkaline protease is 1-80% of the protease mixture. In one embodiment, the first alkaline protease is 1-30% of the protease mixture and the second alkaline protease is 70-99% of the protease mixture. In one embodiment, the first alkaline protease is 1-10% of the protease mixture and the second alkaline protease is 9-99% of the protease mixture. In one embodiment, the first alkaline protease and the second alkaline protease are present in the first protease mixture in the same ratio.

In one embodiment, the first protease mixture comprises three alkaline proteases. In one embodiment, the first alkaline protease, the second alkaline protease and the third alkaline protease total 100% of the proteases of the first protease mixture. In one embodiment, the first alkaline protease is 1-80% of the protease mixture, the second alkaline protease is 1-80% of the protease mixture, and the third alkaline protease is 1-80% of the protease mixture. In one embodiment, the first alkaline protease is 1-30% of the protease mixture, the second alkaline protease is 1-30% of the protease mixture, and the third alkaline protease is 50-98% of the protease mixture. In one embodiment, the first alkaline protease is 1-10% of the protease mixture, the second alkaline protease is 1-30% of the protease mixture, and the third alkaline protease is 10-98% of the protease mixture. In one embodiment, the first alkaline protease, the second alkaline protease and the third alkaline protease are present in the first protease mixture in the same ratio.

The second protease mixture comprises: at least one of an acid protease, a neutral protease and an alkaline protease. In one embodiment, the acid protease, neutral protease or alkaline protease is from bacillus licheniformis. The proteases of the second protease mixture may include exoproteases and endoproteases.

In one embodiment, the second protease mixture includes an acid protease and a neutral protease. In one embodiment, the acidic protease and the neutral protease total 100% of the proteases of the second protease mixture. In one embodiment, the second protease mixture comprises 1-80% acidic protease and 1-80% neutral protease. In one embodiment, the acidic protease is 1-30% of the protease mixture and the neutral protease is 70-99% of the protease mixture. In one embodiment, the acidic protease is 1-10% of the protease mixture and the neutral protease is 9-99% of the protease mixture. In one embodiment, the acidic protease and the neutral protease are present in equal proportions in the second protease mixture.

In one embodiment, the second protease mixture comprises an acid protease and an alkaline protease. In one embodiment, the acidic protease and the alkaline protease sum to 100% of the proteases of the second protease mixture. In one embodiment, the second protease mixture comprises 1-80% acid protease and 1-80% alkaline protease. In one embodiment, the acidic protease is 1-30% of the protease mixture and the alkaline protease is 70-99% of the protease mixture. In one embodiment, the acidic protease is 1-10% of the protease mixture and the alkaline protease is 9-99% of the protease mixture. In one embodiment, the acidic protease and the alkaline protease are present in the second protease mixture in the same ratio.

In one embodiment, the second protease mixture comprises a neutral protease and an alkaline protease. In one embodiment, the first neutral protease and the alkaline protease amount to 100% of the proteases of the second protease mixture. In one embodiment, the second protease mixture comprises 1-80% neutral protease and 1-80% alkaline protease. In one embodiment, the neutral protease is 1-30% of the protease mixture and the alkaline protease is 70-99% of the protease mixture. In one embodiment, the neutral protease is 1-10% of the protease mixture and the alkaline protease is 9-99% of the protease mixture. In one embodiment, the neutral protease and the alkaline protease are present in the second protease mixture in equal proportions.

In one embodiment, the second protease mixture comprises an acidic protease, a neutral protease, and an alkaline protease. In one embodiment, the acidic protease, the neutral protease and the alkaline protease total 100% of the proteases of the second protease mixture. In one embodiment, the second protease mixture comprises 1-80% acid protease, 1-80% neutral protease, and 1-80% alkaline protease. In one embodiment, the second protease mixture comprises 1-10% acid protease, 1-30% neutral protease, and 60-98% alkaline protease. In one embodiment, the acidic protease, the neutral protease and the alkaline protease are present in the second protease mixture in the same ratio.

In one embodiment, the alkaline protease of the first protease mixture is not in the second protease mixture.

In one embodiment, the amount of the first protease cocktail/second protease cocktail is 0.2-0.6% of the first protease cocktail and 0.02-0.06% of the second protease cocktail. In one embodiment, the amount of the first protease cocktail/second protease cocktail is 0.3-0.5% of the first protease cocktail and 0.03-0.05% of the second protease cocktail. In one embodiment, the amount of the first protease enzyme mixture is about 0.4% and the amount of the second protease enzyme mixture is about 0.04%.

A preferred commercially available protease mixture for the first protease mixture comprises Endocut-02L (Tailore food/Tailorezyme.) A preferred commercially available protease mixture for the second protease mixture comprises Exockt-B L (Tailore food/Tailorezyme.) Exockt-B L may comprise a cell wall degrading enzyme from Aspergillus in place of or in addition to one or more proteases.

The following values may be combined in any manner to determine the range of minimum and maximum amounts of the endo protease mixture as a percentage of the total weight of the comminuted crustacean trap: 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, or 1.5%.

The following values may be combined in any manner to determine the range of minimum and maximum amounts of the exout protease mixture as a percentage of the total weight of the comminuted crustacean: 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.25%, or 0.5%.

In one embodiment, the amount of endo/Exosu is 0.2-0.6% of the mixture of endo proteases and 0.02-0.06% of the mixture of Exosu proteases. In another embodiment, the amount of endo/Exosu is 0.3-0.5% of the endo protease mixture and 0.03-0.05% of the Exosu protease mixture. In one embodiment, the amount of endo protease is about 0.4% and the amount of exout protease is about 0.04%.

In one embodiment, the first protease mixture and the second protease mixture are added simultaneously to the comminuted crustacean trap.

In one embodiment, the hydrolyzing step comprises contacting the crustacean trap with the first protease mixture prior to contacting the crustacean trap with the second protease mixture. In this embodiment, the following values in minutes may be combined in any manner to create a range of minimum and maximum values for the time required for hydrolysis of the first protease mixture prior to addition of the second protease mixture: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235 and 240. For example, the crushed crustacean-trap is hydrolyzed using the first protease mixture for 1-10 minutes before the second protease mixture is added.

In one embodiment, the hydrolyzing step comprises contacting the second protease mixture with the comminuted crustacean trap prior to contacting the crustacean trap with the first protease mixture. In this embodiment, the following values in minutes may be combined in any manner to create a range of minimum and maximum values for the time required for hydrolysis of the second protease mixture prior to addition of the first protease mixture: 1. 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235 and 240. For example, the crushed crustacean-trap is hydrolyzed using the second protease mixture for 1-10 minutes before the first protease mixture is added. In another embodiment, a second enzyme comprising chitinase or collagenase is used. Such an enzyme may be used with the first enzyme or a combination of enzymes.

In another embodiment, the enzyme used for hydrolysis does not comprise an exopeptidase.

The contacting step may also include the use of an organic solvent. Examples of useful organic solvents include, but are not limited to, ethanol, acetone, and ethyl acetate.

Terminating the hydrolysis reaction

The hydrolysis reaction may be terminated by inactivating the enzyme. The enzyme may be inactivated by different means, including addition of inhibitors, removal of co-factors (e.g., by dialysis key ions), by heat inactivation, and/or by any other means of inactivation.

The conditions under which each enzyme is inactivated may vary, but generally include raising the pH and temperature of the solution. For example, deactivation7089 it starts at pH>7.5 and a temperature greater than 55 ℃. The conditions are selected such that the enzyme is inactivated at a temperature at which the protein of the crushed crustacean-captured material is not denatured. The following temperatures may be combined in any manner to determine the minimum temperature, maximum temperature or temperature range at which the enzyme is inactivated without denaturing the protein of the crustacean trap: 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 9l ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃, 99 ℃ and 100 ℃. For example, enzyme A may be inactivated at a temperature of about 90 ℃ or preferably between about 92 ℃ to about 98 ℃.

As mentioned above, the hydrolysis is stopped at the correct moment so that the crustacean trap is preferably only partially hydrolyzed to produce a hydrolyzed crustacean trap, wherein the protein has a certain degree of hydrolysis. Furthermore, one of ordinary skill in the art is able to determine the conditions and duration of hydrolysis for a particular enzyme, as well as the conditions under which the hydrolyzed crustacean trap is produced, wherein a percentage of the crustacean trap is hydrolyzed.

Separating to provide a protein phospholipid complex

The third step is to separate the hydrolyzed crustacean trap to provide PPC. In this step, various procedures may be used to ensure that the PPC meets certain criteria. For example, the lipid content of PPC is about 30-55%. In another example, the fluoride level may be reduced to produce PPC with a fluoride content of less than about 5mg/kg PPC.

Methods for reducing the fluorine content are various. For example, the exoskeleton of crustacean traps, i.e., the shell, crustacean and/or shell, typically contains a high amount of fluorine. Removal of exoskeletons (e.g., shells) from hydrolyzed crustacean traps is one method of reducing fluorine content. The shell may be removed by various methods, for example using filters, sieves, decanters or centrifuges. For example, the opening size of the filter or screen may be selected so that all or most of the shell is removed without removing most or any non-shell hydrolysates. Standard woven mesh screens can be used with mesh sizes selected from about 125mm, 25mm, 4mm, 1mm, 250 μm, 45 μm and 20 μm. U.S. patents 8557297 and 9068142, and Bruheim et al, the disclosure of which is incorporated herein by reference in its entirety, disclose methods for reducing fluoride content from crustacean materials.

During this process, the shells may also be removed at different points in time. For example, the shell may be removed with a decanter or sieve immediately after hydrolysis, but before pasteurization and enzyme inactivation.

The fluorine content may also be reduced downstream when krill oil is produced by supercritical extraction and/or subcritical fluid extraction. Thus, the hydrolyzed shell can be removed from the main process line and the enzyme deactivated before introduction into the main process line. In other words, the shell and/or any other insoluble particles may be reintroduced into the main process line prior to drying. Reintroduction of the shell and/or any other insoluble particles into the main process line may facilitate the use of PPC, as the presence of the shell allows the product to be packaged into strong blocks that are more easily broken down and allow greater flowability in downstream processing steps. The insoluble particles are preferably food grade and can be easily removed downstream from the PPC. Examples of insoluble particulates include fibers.

Alternatively, the shell need not be removed at any time during processing, and may be part of the PPC. As noted above, any excess fluorine associated with the shell may be removed downstream by supercritical processing.

In another example, the level may be reduced to prevent microbial activity in the separation step. The water content of PPC refers to the percentage of water in which microorganisms are less likely to grow. The following percentages may be used to create a minimum, maximum, or range value for the PPC moisture content: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% w/w. For example, the PPC has a water content of less than about 15%, based on the total weight of the PPC. Desirably, the water activity (activity) is less than about 0.85, and preferably less than about 0.65.

The water after hydrolysis may be removed by one or more dewatering steps, which may include drying and mechanical removal before, during or after the separation process. Preferred dewatering methods include vacuum low temperature drying and treatment of the soft sediment with a decanter centrifuge or horizontal centrifuge. Any separation method may be used, such as a decanter and a separator. The preferred separator uses a rotational force to rotate the sample and separate the sample into layers.

Horizontal centrifuges refer to any device capable of rotating a mixture in the Z plane (as opposed to the X plane and/or Y plane of a conventional centrifuge). The rotation is caused by the horizontal alignment of the helical conveyor elements within the tubular housing. The induced centrifugal forces then layer the heavier particles to the outer edges of the shell, while the lighter particles form a layer closer to the center of the shell. Some horizontal centrifuges are modified to include an extended separation path and induce high gravitational forces. The preferred horizontal centrifuge is FlottwegThe water can be mechanically separated from the sediment.Is an improved horizontal centrifuge comprising a long horizontal clarification/separation zone, generating high centrifugal forces (5000 to 10000 g).The S3E-3 model filter bowl was 12 "in diameter, rotated at 7750 rpm, and weighed 90" x 28 "x 30" at 2315 pounds.

Protein phospholipid emulsion

Another aspect of the invention relates to a method of producing a stable protein phospholipid emulsion. In this method, specific enzymes and process parameters are selected to ensure that the final product is a stable emulsion. There is a need for a stable emulsion to protect functional ingredients such as phospholipids, fatty acids and hydrolyzed proteins and to improve the handling, stability and efficacy of these functional ingredients.

A stable emulsion is defined as an emulsion that does not separate as determined by visual inspection and/or viscosity measurements at room temperature or slightly elevated temperatures. The following temperatures may represent the lowest or highest temperatures, or may be combined to produce a temperature range for defining an elevated temperature: 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃ and 45 ℃. The following time periods in months may represent minimum or maximum values and may be combined to define a range for the time required for the emulsion to remain stable at the above-mentioned elevated temperatures: 3. 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. For example, a stable emulsion can be defined as an emulsion that does not separate at temperatures up to about 40 ℃ for 4 months, or in another example, does not separate at ambient temperatures (e.g., between 20 ℃ and 25 ℃) for 6 months. In addition, the stable emulsion contains a desirable water content to prevent microbial growth and meet safety guidelines for human consumption.

As described above, the crustacean-captured material is pulverized in the first step. In a second step, the comminuted crustacean trap is contacted with a proteolytic enzyme to provide a hydrolyzed crustacean trap. In this step, the choice of enzyme is critical to ensure that the enzyme does not inhibit the formation of a stable emulsion of phospholipids. The parameters of the contacting step are as described above.

In a third step, the hydrolyzed crustacean trap is separated to provide a stable phospholipid emulsion. The third step may include a fluid extraction process, such as a supercritical process.

The water content of the stabilized protein phospholipid emulsion may be any of the following values, which represent individual minimum and maximum values, and may also be combined into a range: 40% v/v, 41% v/v, 42% v/v, 43% v/v, 44% v/v, 45% v/v, 46% v/v, 47% v/v, 48% v/v, 49% v/v, 50% v/v, 51% v/v, 52% v/v, 53% v/v, 54% v/v, 55% v/v, 56% v/v, 57% v/v, 58% v/v, 59% v/v, and 60% v/v. For example, a preferred water content is about 45-55% v/v.

Another aspect of the invention relates to a stable protein phospholipid emulsion comprising water, protein, and lipid.

Crustacean oil

Another aspect of the present invention relates to a process for producing crustacean oil. The method comprises the following steps: comminuting the crustacean trap to provide a comminuted crustacean trap comprising protein; contacting the comminuted crustacean trap with one or more proteolytic enzymes to provide a hydrolyzed crustacean trap without substantial denaturation of the proteins; and separating the hydrolyzed crustacean trap to provide PPC. These steps are as described above. The process further comprises a fourth step involving extracting crustacean oil from the PPC.

The extraction step preferably involves separating oil from the PPC using fluid extraction. Preferred fluids include supercritical carbon dioxide, ethanol, acetone and/or C₁-C₃A monohydric alcohol. Supercritical carbon dioxide refers to a mixture of carbon dioxide in a fluid state at its critical temperature and critical pressure or higher, which expands in character to fill a container like a gas, but has a density like a liquid. Carbon dioxide becomes supercritical at 31.1 ℃ and above 72.9atm/7.39 mpa.

Some methods of extracting crustacean oil from PPC are described in U.S. Pat. Nos. 9034388 to Bruheim et al, the contents of which are incorporated by reference in their entirety. Supercritical processing is also discussed in U.S. patent nos. 9068142 to Bruheim et al, the contents of which are incorporated by reference in their entirety.

For example, crustacean oil may be extracted from PPC by a two-stage process. In the first stage, neutral lipids are removed by extraction with pure supercritical carbon dioxide or carbon dioxide plus about 1-10% of a co-solvent, such as ethanol. In the second stage, the crustacean oil is extracted with supercritical carbon dioxide and about 10-30% ethanol.

Preferably, the crustacean oil is krill oil and the ground crustacean catch is preferably ground krill.

Preferably, the crustacean oil comprises about 400-500g/Kg phospholipids and about 200-260g/Kg omega-3 fatty acids.

One or more other nutritional oils may be combined with the crustacean oil of the present invention to create a single dosage form with health benefits. The nutritional oil may be extracted from other marine sources, plant sources, or other animal sources. Some examples of nutritional oils include, but are not limited to, vegetable oils (such as linseed oil, pumpkin seed oil, rapeseed oil, soybean oil, or walnut oil), fish oils, seal oil, microalgal oils, mussel oil, and shrimp oil. Another preferred additional marine resource is perna canaliculus oil.

The crustacean oils discussed above are suitable for administration to humans and animals (e.g., dogs) for various health benefits. For example, pets, such as dogs, are given krill oil capsules directly or krill oil is placed directly in their food.

Another aspect of the present invention relates to a process for producing crustacean oil without an extraction step (i.e. without an extraction step). The method comprises the following steps: pulverizing the crustacean-trap to provide a pulverized crustacean-trap comprising protein; contacting the comminuted crustacean trap with a proteolytic enzyme to provide a hydrolyzed crustacean trap without substantially denaturing the proteins; and separating the hydrolyzed crustacean trap to provide free crustacean oil. Preferred proteolytic enzymes are those which provide almost complete hydrolysis to liberate the crustacean oil as free oil. For example, the preferred proteolytic enzyme is a mixture of Bacillus licheniformis acidic, neutral and alkaline proteases.

Reference in the specification to "one embodiment", "an example" or "an example" means that a particular feature, structure or characteristic described in connection with the embodiment or example is included in at least one embodiment of the embodiments of the invention. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," "one example," or "an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable combinations and/or subcombinations in one or more embodiments or examples. Further, it is to be understood that the drawings are for explanation to persons of ordinary skill in the art.

Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not to an exclusive "or". For example, the condition "a or B" is satisfied by any one of: "a is true (or present), B is false (or not present)," a is false (or not present), "B is true (or present)," and "a and B are both true (or present)".

Examples of the invention

Example 1:

within 30 minutes after capture, fresh krill is chopped with a knife into particles of approximately 3-6mm in size. The temperature of the krill is about 1-2 degrees celsius at this point. A mixture of fresh water (up to 50% w/w of krill weight) and enzyme (about 0.2% w/w of krill weight) was added and heated to about 50 ℃. Hydrolysis took approximately 30 minutes. The amount of enzyme, temperature and hydrolysis time are critical to ensure that the krill proteins are not denatured during the hydrolysis process.

After hydrolysis, the shell is separated from the main process line using a sieve or decanter. The hydrolysed krill in the main production line is then pasteurised to ensure hygienic quality and enzyme inactivation. Pasteurization is carried out at 90 ℃ for at least 10 minutes. The hydrolysed krill is then dried using a precipitation separator, excess water is mechanically separated and low temperature vacuum dried. The final product is PCC.

Example 2:

krill was processed as described in example 1, but the decanter step after hydrolysis was removed. The hydrolysed krill bypasses the decanter. Thus, the krill shells remain in the hydrolysed krill, are pasteurized along the main process line, and then are subjected to precipitation separation and low temperature vacuum drying.

Thus, the krill shells remain mixed with the PPC. Mixing the shrimp shell with the product is beneficial because the shell allows the firm product pieces to break easily during processing, increasing flowability. Another benefit is that any small protein fraction that remains associated with the shell after hydrolysis remains stored with the product, rather than being separated from the shell.

Due to the presence of the shell, excess fluoride in the product can be removed downstream during the supercritical extraction.

Example 3:

after krill hydrolysis, the krill shells are separated from the main processing line. However, after bypassing the deactivation process and the precipitation separation process, i.e. before drying, the shell is reintroduced into the main process line.

Thus, while there have been described what are believed to be the preferred embodiments of the invention, those skilled in the art will recognize that other examples may be made without departing from the spirit of the invention, including all further modifications and changes coming within the true scope of the claims set forth herein.

Example 4:

500 kg of krill (chopped to 5 mm) was added to 500 kg of water. The endogut/Exosut protease from Tailorezymes corporation was added at 0.4% and 0.04%, respectively. The reaction was carried out at 55 ℃ for 2 hours. The decanter was operated at 5400 rpm and a flow rate of 800 liters/hour. The separator was run at about 8200 rpm with a flow rate of 1000 liters/hour and released every 300 seconds. The release time refers to the time for the release of the hydrolysate.

Table 1: weight resolution table in example 1.

Product(s)	Weight (kg)	Weight (%)	Dried substance (%)	Yield (%)
					Shell	55	5.7	30	3.44
Hydrolysate	700	73	9.5	13.85
					PPC	160	16.7	25	8.33
Loss of power	45	4.6

Example 5:

500 kg of krill was added to 500 kg of water. The endo cut/Exoct protease was added at doses of 0.4% and 0.04%, respectively. The reaction temperature was 55 ℃ and the reaction time was 2 hours.

The decanter was operated at 5400 rpm with a flow rate of 800 liters/hour. The temperature is raised to 90 ℃, the hydrolysate is treated on a separator, the rotating speed of the separator is 8200 r/min, and the flow rate is 1000 l/h. The release of the hydrolysis products was gradually increased to every 900 seconds.

Table 2: weight resolution table in example 2.

Product(s)	Weight (kg)	Weight (%)	Dried substance (%)	Yield (%)
					Shell	43	7.17	28	4.3
Hydrolysate	409	68.17	9.5	12.95
					PPC	105	17.50	25	8.75
Loss of power	43	7.17

Example 6:

300 kg krill was added to 300 kg water and Corrolase7089 (0.15% dose). The reaction was carried out at a temperature of 55 ℃ for a total reaction time of 1 hour, and then entered a decanter (shelling), followed by continuing the reaction for 2 hours before deactivation.

The decanter was rotated at 2800 rpm and the flow rate was 650 l/h. The separator was rotated at about 8200 rpm and at a flow rate of 1000 liters/hour, releasing every 300 seconds. No free oil was formed.

Table 3: weight resolution table in example 3.

Product(s)	Weight (kg)	Weight (%)	Dried substance (%)	Yield (%)
					Shell	40	4.2	30	2.5
Hydrolysate	750	78	9.5	14.84
					PPC	120	12.5	25	6.25
Loss of power	50	5.3

Example 7:

220 kg krill was mixed with 55 kg water and Corrolase7089 (0.15% dosage). The reaction was carried out at a temperature of 55 ℃ for 1 hour before decantation and then for 2 hours before deactivation.

The decanter was rotated at 2800 rpm and the flow rate was 650 l/h. The separator was rotated at about 8200 rpm and at a flow rate of 1000 liters/hour, releasing every 300 seconds. No free oil was formed.

Table 4: weight resolution table in example 4.

Product(s)	Weight (kg)	Weight (%)	Dried substance (%)	Yield (%)
					Shell	26	9.45	32	3.78
Hydrolysate	178	64.73	14	11.33
					PPC	55	20	25	6.25
Loss of power	16	5.82

Example 8:

500 kg krill was mixed with 125 kg water and Endocut/Exoctu (at 0.4% and 0.04% doses, respectively). The reaction temperature was 55 ℃. The reaction was carried out for 1 hour before decantation and then for 30 minutes before deactivation. A decanter: 2800 rpm and a flow rate of 650 l/h.

Table 5: weight resolution table in example 5.

Product(s)	Weight (kg)	Weight (%)	Dried substance (%)	Yield (%)
					Shell	50	8	32	3.2
Hydrolysate	517	87.2	14	14.48
					PPC	33	5.28	25	1.65
Loss of power	25	4

The separator was rotated at about 8200 rpm and at a flow rate of 1000 liters/hour, and released every 600 seconds.

After some change in the setting (i.e. the counter pressure) the free oil can be separated from the separator. The composition of the oil is as follows (table 6).

Table 6: free oil component formed in example 5.

Example 9:

the hydrolysate from example 1 was further treated by microfiltration and nanofiltration to remove molecules less than 300-400 daltons.

The 50% DM hydrolysate was diluted with water to a hammer value of 8.

The retentate was dried by spray drying.

Mixing the dried product with beverage, and making into odorless and tasteless beverage.

Example 10:

enzymatic hydrolysis was carried out in a Distek Premiere5100 dissolution system at 55 ℃ and 150rpm, enzyme dosages varied from 1kg/ton to 5kg/ton for Endocut-02L and from 0.05kg/ton to 0.4kg/ton for Exocit-B L, both based on the starting materials, the starting materials were processed in a meat grinder using a 4.5mm grind plate and water added as per the following table, after a total reaction time of 60 or 120 minutes, inactivation was carried out by heating (95 ℃) followed by preliminary separation of the shell through a sieve and then filtration of the remaining suspension.

Table 7: experimental setup

Table 8: sensory evaluation of hydrolysates