JP7352671B2 - Water-soluble mussel extract - Google Patents

Description

This application claims priority from New Zealand Provisional Application No. 706298, filed on 24 March 2015, which is incorporated herein by reference.
1. FIELD OF THE INVENTION The present invention relates to water-soluble mussel extracts, compositions containing them and their use in nutraceutical and pharmaceutical applications.

2. BACKGROUND OF THE INVENTION Shellfish, particularly mussels, are known to be rich in compounds that maintain health. Shellfish extracts are an established category of nutraceuticals on the global market.

Some extracts of shellfish have anti-inflammatory and other medicinal effects, effects often claimed to arise from the lipid fraction. Inflammation is associated with a wide variety of conditions including arthritis, atherosclerosis and cancer. Current medical treatment of inflammation relies heavily on non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, many of which have undesirable side effects. Therefore, nutraceutical shellfish preparations with fewer side effects are highly desirable.

A number of nutraceutical preparations of shellfish have been described. The actual chemical composition of such preparations is
(a) the raw materials, including the species of shellfish, its age, and the environmental conditions under which it was grown; and (b) the process used to obtain the preparation - for example, selectively excluding or retaining certain types of compounds. The disruption or alteration step will depend on many factors, including which will affect the chemical makeup of the final preparation.

To date, shellfish nutraceutical preparations have focused on whole animal extracts or the lipid fraction of the material. Supercritical _CO2 extraction of freeze-dried materials is widely used to produce mussel lipid extracts, particularly green-shelled mussel lipid extracts, such as those sold under the trade name Lyprinol™. has been done.

However, whole mussel powder is not suitable for inclusion in many foods because it is insoluble and generally has an unpleasant "fishy" flavor. Mussel lipid extracts are also insoluble and the "fishy" flavor/odor can be exacerbated by oxidation, which is likely to occur during many standard food processing steps. They generally must be administered as lipid-filled capsules to mask unpleasant odors and flavors.

It would therefore be advantageous to provide a water-soluble mussel extract with medicinal properties that does not suffer from the disadvantages associated with corresponding whole body mussel powders and/or lipid extracts.

It is an object of the present invention to provide such an extract, or at least to provide the public with a useful selection.

3. SUMMARY OF THE INVENTION In one aspect, the invention provides a water-soluble mussel extract.
In one embodiment, the water-soluble mussel extract comprises at least about 45% by weight, on a solids basis, peptides, preferably low molecular weight peptides.

In one embodiment, the water-soluble mussel extract comprises from about 45% to about 65% peptides by weight on solids, preferably from about 50% to about 60% peptides by weight on solids.

In one embodiment, more than 90%, preferably more than 95% by weight of the peptide is less than 5 kDa.
In one embodiment, more than 75%, preferably more than 80% by weight of the peptides are less than 2 kDa.

In one embodiment, more than 35%, preferably more than 40% by weight of the peptides are less than 1 kDa.
In one embodiment, the water-soluble mussel extract contains about 5% or less lipid by weight on a solids basis.

In one embodiment, the water-soluble mussel extract has anti-inflammatory activity.
In another aspect, the invention provides a method of producing a water-soluble mussel extract, comprising:
(a) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes;
(b) denaturing the enzyme in the hydrolysis mixture;
(c) removing insoluble materials from the hydrolysis mixture to provide a water-soluble mussel extract.

In one embodiment, the hydrolysis mixture is adjusted to pH 4 after denaturation of the enzyme.
In one embodiment, the water-soluble mussel extract is purified and/or concentrated.
In one embodiment, the mussel is greenshell mussel (or green-lipped mussel) (GSM) - Perna canaliculus, blue mussel - Mytilus edulis, and ribbed mussel (Maori name kopakopa) - selected from the group including, but not limited to, Aulacomya atra maoriana. Preferably the mussel is GSM.

In one embodiment, the mussel material is fresh whole mussel. In another embodiment, the mussel material is mussel powder produced by drying fresh mussel whole bodies. In another embodiment, the mussel material is mussel supercritical marc produced by supercritical fluid extraction of mussel powder or other mussel material.

In another aspect, the invention provides a nutraceutical composition comprising a water-soluble mussel extract of the invention and one or more ingestible excipients.
In one embodiment, the nutraceutical composition includes maltodextrin and/or an antioxidant.

In one embodiment, the nutraceutical composition comprises a food composition, a food additive composition, a dietary supplement, or a medical food.
In another aspect, the invention provides a pharmaceutical composition comprising a water-soluble mussel extract of the invention and one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises an oral dosage form, preferably a powder.
In another aspect, the invention provides a method of reducing inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract of the invention.

In another aspect, the invention provides a method for preventing, treating, or managing a condition associated with inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract of the invention. Provide a method including.

In one embodiment, the condition associated with inflammation is a chronic condition.
In one embodiment, the condition associated with inflammation is asthma, encephalitis, inflammatory bowel disease including Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergic disease, fibrosis, juvenile arthritis, Arthritis including psoriatic arthritis, rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendinitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis. selected from the group including sclerosis, heart disease, obesity, diabetes, cancer and Alzheimer's disease.

In one embodiment, the condition associated with inflammation increases the levels of inflammatory TNFα and/or IL-1β.
In one embodiment, the water-soluble mussel extract of the invention is administered orally.

Embodiments of the invention will now be described with reference to the drawings.

4. Brief description of the drawing
Figure 2 is a graph showing positive (dexamethasone) and negative (dimethyl sulfoxide, DMSO) assay controls for inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of a water-soluble extract from GSM residue powder on inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of two water-soluble extracts from GSM whole body powder on inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. A combination of two enzymes was tested for protein hydrolysis. Figure 2 is a graph showing the effect of a water-soluble extract from fresh whole body mussel on the inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of aqueous extracts from fresh whole body GSM (with a defatting step) on the inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of a water-soluble extract from fresh whole body Copacopa on the inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 3 is a graph showing positive (dexamethasone) and negative (DMSO) assay controls for inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of a water-soluble extract from GSM residue powder on inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of two water-soluble extracts from GSM whole body powder on inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of a water-soluble extract from fresh whole body mussels on the inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of aqueous extracts from fresh whole body GSM (with a defatting step) on the inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of a water-soluble extract from fresh whole body Copacopa on the inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of a water-soluble extract (pilot scale) from GSM residue powder on inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of a water-soluble extract (pilot scale) from GSM residue powder on the inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of unhydrolyzed GSM residue powder on inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of non-hydrolyzed GSM systemic powder on inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of unhydrolyzed GSM residue powder on inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a graph showing the effect of non-hydrolyzed GSM systemic powder on inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells. Figure 2 is a positive product ion based chromatogram generated by LC-MS analysis of aqueous extract PAR58. Figure 3 is a positive product ion-based chromatogram generated by LC-MS analysis of unhydrolyzed GSM residue powder. Positive product ion-based chromatogram generated by LC-MS analysis of unhydrolyzed GSM whole body powder. Figure 2 is a negative product ion based chromatogram resulting from LC-MS analysis of aqueous extract PAR58. Figure 3 is a negative product ion-based chromatogram produced by LC-MS analysis of unhydrolyzed GSM residue powder. Negative product ion-based chromatogram produced by LC-MS analysis of unhydrolyzed GSM whole body powder.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Definitions As used herein, "a" or "an" means at least one, unless explicitly stated otherwise.

As used herein, the term "about" refers to a value within 10% above or below the value modified by the term.
The term "comprising," as used herein and in the claims, means "consisting at least in part of." When interpreting statements in this specification and claims that include the term "comprising," other features may also be present beyond the features that follow this term in each statement. Related terms such as "comprising" and "including" should be construed in the same manner.

As used herein, the term "therapeutically effective amount" means, in the context of administering therapy to a subject, an amount that reduces or ameliorates the severity, duration, or one or more symptoms of a condition. , to prevent the progression of the condition, to cause regression of the condition, to prevent the recurrence, development or onset of the condition, or to enhance or improve the prophylactic or therapeutic effects of another therapy. means sufficient quantity.

As used herein, the terms "manage," "administering," and "administration" refer to administration of therapy to a subject that does not result in a cure for the condition, but that the subject obtains from the therapy. Refers to beneficial effects. For example, managing the condition includes preventing the condition from worsening.

As used herein, the terms "prevent," "preventing," and "prophylaxis" refer to the recurrence, onset, or development of a condition resulting from administration of therapy in the context of administration of therapy to a subject. refers to the prevention or inhibition of

As used herein, the terms "treat," "treatment," and "treating" refer to the progression, severity, or severity of a condition resulting from administration of the therapy, in the context of the administration of therapy to a subject. refers to a reduction or improvement in sex and/or duration, or improvement in one or more of its symptoms.

As used herein, the term "water soluble" when applied to a material means that the material has a solubility of at least 45% (w/v).
Where references are made herein to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, references to such external documents shall be construed as an admission that such documents or sources of information are prior art or part of the common knowledge in the art on any authority. It shouldn't be done.

The description herein may refer to subject matter that is not within the scope of the claims of this application. The subject matter should be readily identifiable by those skilled in the art and may assist in practicing the invention as defined in the claims of this application.
5.2 Methods of Producing Water-Soluble Mussel Extracts of the Invention Water-soluble mussel extracts of the invention are readily prepared using conventional processing techniques, as described below.

The present invention is a method for producing a water-soluble mussel extract, comprising:
(a) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes;
(b) denaturing the enzyme in the hydrolysis mixture;
(c) removing insoluble materials from the hydrolysis mixture to provide a water-soluble mussel extract.

The term "mussel" as used herein refers to an edible bivalve of the marine family Mytilidae. In one embodiment, the mussel used in the method of the invention is a New Zealand mussel.

In one embodiment, the mussel is selected from the group including, but not limited to, GSM, mussel, and Copacopa. Preferably the mussel is GSM.
In one embodiment, the mussel material is "fresh" mussel "whole body." This can be obtained by collecting the material that remains after the removal of the mussel shell, legs and whiskers. In another embodiment, the mussel material is dried mussel powder. This is generally obtained by drying fresh whole mussels, usually by freeze-drying, and grinding them into a powder.

In another embodiment, the mussel material is mussel supercritical residue. The term "residue" generally refers to organic material that remains after extraction of biological material, such as plant or animal material. As used herein, the term "supercritical residue" means the organic material that remains after supercritical fluid extraction.

As used herein, the term "mussel supercritical residue" means the organic material that remains after supercritical fluid extraction of mussel meat or mussel powder.
In one embodiment, the mussel material is mussel supercritical residue, preferably GSM supercritical residue.

In the first step of the method, an aqueous suspension of mussel material is hydrolyzed using one or more proteolytic enzymes.
Hydrolysis is carried out by incubating an aqueous suspension of mussel material with proteolytic enzyme(s) under conditions optimized for the enzyme or enzyme system used. Typically, the pH should be adjusted to about 5.5 to about 8. Optionally, an antioxidant, such as Oxyless U, can be added to the aqueous suspension.

The aqueous suspension is typically heated to about 30 to about 65° C. for 1 to 24 hours, depending on the enzyme system used. Generally, the mussel material is suspended in cold water and the mixture is heated to the required temperature. However, the mussel material can alternatively be suspended in already heated water.

During hydrolysis, proteins present in the mussel material are hydrolyzed into low molecular weight peptides. Hydrolysis must be performed enzymatically to deliver peptides of the appropriate molecular weight range. Chemical hydrolysis, for example with acids, is non-selective. Without being bound by theory, it is believed that the anti-inflammatory properties of the water-soluble mussel extract of the present invention reside in the specific low molecular weight peptides present. Processing steps that destroy these peptides or prevent their formation will alter the anti-inflammatory activity of the extract.

Proteolytic enzymes can be of any type, such as endo-, exo-proteases/peptidases, aminopeptidases, serine proteases, metalloproteases, cysteine proteases, and the like. In one embodiment, the proteolytic enzymes each have a slightly acidic to about neutral pH optimum. In one embodiment, the proteolytic enzyme is a non-animal enzyme.

Suitable proteolytic enzymes include papain, Alcalase (subtilisin), Enzidase FP (endo/exopeptidase derived from selected strains of Aspergillus oryzae var.) and Enzidase Neutral (Bacillus amyloliquefaciens). metalloneutral endopeptidases derived from controlled fermentation of non-genetically modified strains of

In one embodiment, the proteolytic enzyme is selected from the group including papain, Enzidase FP, Enzidase Neutral, Alcalase, and the like.
In one embodiment, the proteolytic enzyme includes papain and Enzidase FP.

In another embodiment, the proteolytic enzymes include Alcalase, Enzidase Neutral and Enzidase FP.
The temperature of the aqueous suspension of mussel material during hydrolysis should not be so hot as to degrade the enzyme or inhibit its activity. The duration of hydrolysis depends on the activity of the enzyme(s) used and the mussel material.

The hydrolysis step is performed with agitation to optimize contact between the enzyme(s) and the mussel material.
After hydrolysis is complete, the enzyme is denatured. In one embodiment, the hydrolysis mixture is heated under conditions sufficient to denature the proteolytic enzyme(s). In one embodiment, the hydrolysis mixture is heated at about 80° C. to about 95° C. for about 30 to about 40 minutes. In another embodiment, the hydrolysis mixture is heated at about 80° C. to about 95° C. for about 20 to about 40 minutes.

In one embodiment, the aqueous suspension of hydrolyzed mussel material can optionally be treated to remove lipids prior to denaturing the enzymes. Lipid removal may be accomplished using any method known in the art, for example using physical techniques such as centrifugation or chemical extraction.

Chemical extraction involves removing water from an aqueous suspension of hydrolyzed mussel material and then contacting it with a solvent in which the lipid fraction is soluble. The defatted product (mussel residue) is then resuspended in water to denature the hydrolytic enzymes.

In another embodiment, an aqueous suspension of hydrolyzed mussel material is lyophilized to form a powder, which is rinsed with an organic solvent to remove lipids. Examples of solvents that can be used include, but are not limited to, acetone, ethanol, isopropyl alcohol, hexane, ethyl acetate and dimethylformamide.

In another embodiment, an aqueous suspension of hydrolyzed mussel material is dried and extracted with a supercritical solvent such as _CO2 or _CO2 /ethanol to remove the lipid fraction. The defatted product (mussel residue) is then resuspended in water to denature the hydrolytic enzymes.

An optional defatting step may be used if the mussel material is fairly high in lipids. The defatting step can be omitted if the mussel material has a low lipid content (e.g. fresh whole body or powder of mussel) or if mussel residue is used.

After denaturation of the hydrolytic enzymes, the pH of the mixture may be adjusted to about 3.5 to about 4.2, preferably about 4, to prevent microbial spoilage. Agents such as phosphorinic acid, citric acid or hydrochloric acid can be used to adjust the pH.

Insoluble materials present in the hydrolysis mixture are then removed, resulting in the water-soluble mussel extract of the present invention.
Insoluble material can be removed by any method known in the art. In one embodiment, insoluble material is removed by centrifuging the hydrolysis mixture and collecting the supernatant.

Generally, the pH is adjusted before removing the insoluble material, but if the latter contains shell fragments, this insoluble material should be removed first to prevent solubilization of the shell by the acid.
The water-soluble mussel extract of the invention may be treated to remove impurities that contribute to undesirable color and/or odor, for example, by contacting with activated carbon. Charcoal can be contained in the container and the product passed through it. Alternatively, carbon, remaining fines and lipid residues can be removed using a diatomaceous earth coated filter press or equivalent.

Water-soluble mussel extracts may also be concentrated by removing some or all of the water present.
In one embodiment, the water-soluble mussel extract is dried to provide a powdered extract. Drying can be performed using any technique known in the art, including lyophilization.

Agents such as maltodextrins and antioxidants may be added to the water-soluble mussel extract of the present invention.
Examples 1-8 describe methods for producing water-soluble mussel extracts of the present invention.
5.3 Water-soluble mussel extract of the invention The method of the invention described above produces a water-soluble mussel extract that is rich in peptides, especially low molecular weight peptides. As used herein, the term "low molecular weight peptide" refers to a peptide that weighs 5 kDa or less.

The extract has an acceptable flavor without bitterness. The extract can be frozen without cryoprecipitation. The powder solubility characteristics of mussel extract do not change during dry storage.

The water-soluble mussel extract of the present invention mainly contains peptides, especially low molecular weight peptides. As can be seen from the molecular weight profiles of the extracts produced in Examples 1 to 8, more than 90% of the peptides present in the extracts are smaller than 5 kDa.

The water-soluble mussel extract of the present invention is as can be seen from Figures 19 to 24, which show the results of LC-MS analyzes performed on the water-soluble GSM mussel extract of the present invention, as well as the starting materials of GSM residue and whole body powder. , containing more compounds than the unhydrolyzed starting material.

In one embodiment, the water-soluble mussel extract comprises at least about 45% by weight, on a solids basis, peptides, preferably low molecular weight peptides.
In another embodiment, the water-soluble mussel extract comprises at least about 45% to about 65% peptides, preferably about 50% to about 60% peptides by weight on a solids basis.

In one embodiment, more than 90%, preferably more than 95% by weight of the peptide is less than 5 kDa.
In one embodiment, more than 75%, preferably more than 80% by weight of the peptides are less than 2 kDa.

In one embodiment, more than 35%, preferably more than 40% by weight of the peptides are less than 1 kDa.
Some carbohydrate materials, lipids and other materials and minerals may also be present.

The peptide content of the extract is calculated based on the "solids" present so that it remains unaffected by its concentration. The "solids" present in the extract constitute the material that remains when any solvent present, including water, is removed. The "solids" present include peptides, carbohydrates and lipids and any other non-solvent materials.

The solubility of the extract of the invention is at least 45% (w/v). Solubility can be determined by saturating water with extract (1:1 ratio), centrifuging, subsequently drying the resulting supernatant and determining the weight dissolved.

In one embodiment, the water-soluble mussel extract of the invention is a dry powder.
The weight percent of peptides present in the extract is calculated excluding solid excipients added to the extract such as maltodextrins and antioxidants.

In one embodiment, the invention provides a water-soluble mussel extract comprising at least about 45% by weight peptides on a solids basis, wherein more than 90% by weight of the peptides are less than 5 kDa. provide.

In another embodiment, the invention provides a water-soluble mussel extract comprising from about 50% to about 60% by weight peptides on a solids basis, wherein more than 95% by weight of the peptides are less than 5 kDa. Provides sex mussel extract.
5.4 Use of Water-Soluble Peptide Extracts of the Invention Inflammation is a process involving a complex biological cascade of molecular and cellular signals that alter the physiology of an organism. While acute inflammation protects and heals the body after physical injury or infection, chronic inflammation produces changes that play a key role in many degenerative diseases. Chronic inflammation is primarily mediated by monocytes and long-lived macrophages. Macrophages release chemical mediators including IL-1, the IL-6 family, TNFα and prostaglandins. These mediators induce upregulation of other proinflammatory cytokines. IL-1, IL-6 and TNFα are major inflammatory biomarkers found at higher levels in inflammatory cells than in quiescent cells.

Phagocytosis of bacteria or foreign particles is associated with increased uptake of oxygen by neutrophils, called the respiratory burst. During this period, reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions, singlet oxygen and hydrogen peroxide are generated. These species kill invading microorganisms and parasites.

Nutraceuticals may inhibit the expression of pro-inflammatory cytokines such as IL-1 and TNFα through several mechanisms, including inhibiting ROS-generating enzyme activity, or increasing ROS scavenging capacity. Inflammatory processes can be inhibited or reduced.

As presented in Example 9, the water-soluble mussel extract of the present invention was found to be active against known markers of inflammation and is therefore believed to reduce or prevent inflammation. As shown in Tables 10 and 11, the activity shown is several times higher than the anti-inflammatory activity of the non-hydrolyzed starting mussel material, such as whole body powder or residue.

In one aspect, the invention provides a method of reducing inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract of the invention.
In another aspect, the invention provides a method for preventing, treating, or managing a condition associated with inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract of the invention. Provide a method including.

In another aspect, the invention provides the use of a water-soluble mussel extract of the invention in the manufacture of a medicament for reducing inflammation in a subject in need thereof.
In another aspect, the invention provides the use of a water-soluble mussel extract of the invention in the manufacture of a medicament for preventing, treating or managing a condition associated with inflammation in a subject in need thereof.

The invention also provides a water-soluble mussel extract of the invention for use in reducing inflammation in a patient in need thereof.
The invention also provides a water-soluble mussel extract of the invention for use in preventing, treating or managing conditions associated with inflammation in a patient in need thereof.

In one embodiment, the condition associated with inflammation is a chronic condition.
In another embodiment, the condition associated with inflammation increases the levels of inflammatory markers TNFα and/or IL-1β.

Examples of conditions associated with inflammation that can be prevented, treated or managed by the present invention include asthma, encephalitis, inflammatory bowel disease including Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergies. Sexual diseases, fibrosis, juvenile arthritis, psoriatic arthritis, arthritis including rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendonitis, bursitis, laryngitis, gingivitis, gastritis, otitis , celiac disease, diverticulitis, atherosclerosis, heart disease, obesity, diabetes, cancer, and Alzheimer's disease.

The present invention also provides a method of reducing the levels of inflammatory markers TNFα and/or IL-1β in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract of the present invention. Provide a method including.

A subject in need of prevention, treatment, or management of a condition associated with inflammation is one who has been diagnosed with, is at risk for, or has recovered from such a condition. The subject may be susceptible to and/or at risk for the condition due to genetic and/or environmental factors.

In one embodiment, the subject is a mammal, preferably a human. In another embodiment, the subject is a pet or horse.
Administration of the water-soluble mussel extract of the present invention may be via a pharmaceutical or nutraceutical composition.

In one aspect, the invention provides a dietary supplement composition comprising a water-soluble mussel extract of the invention and one or more ingestible excipients.
In one embodiment, the nutritional supplement composition of the present invention is a food composition, food additive composition, dietary supplement, or medical food composition.

The term "ingestible" as used herein generally means suitable for, or approved by a governmental regulatory agency for, consumption by animals and humans.

The term "food" as used herein means any processed, semi-processed, or unprocessed substance that is intended for consumption by animals, including humans. including, but not limited to, beverages and chewing gum. The food composition of the invention comprises a water-soluble mussel extract of the invention in combination with a food product.

The term "food additive" as used herein refers to any substance that is not normally consumed as a food by itself, but is added to a food for technical purposes. Examples are natural and artificial sweeteners, colorants, setting and pickling agents, flavors, emulsifiers, lipid substitutes, stabilizers, leavening agents, lubricants, humectants, preservatives, stabilizers and thickeners. . The food additive composition of the invention comprises a water-soluble mussel extract of the invention in combination with one or more food additives.

The term "health supplement" as used herein is intended to supplement the diet, containing one or more of the following ingredients: vitamins, minerals, herbs, metabolites, extracts, etc. Refers to the product. Dietary supplements are typically not intended to be used as a stand-alone item in a meal and can be taken independently of any food. The health supplement of the present invention is
A water-soluble mussel extract of the present invention in combination with one or more dietary supplements.

The term "medical food" as used herein refers to a food product that is taken under the supervision of a physician or that is formulated to be administered enterally. Medical foods are intended for the dietary management of conditions with characteristic nutritional requirements. Examples of medical foods include, but are not limited to, single-specified nutritional foods, oral rehydration solutions, and products intended for use in the dietary management of metabolic disorders. The medical food composition of the invention comprises a water-soluble mussel extract of the invention in combination with a medical food.

In one embodiment, the nutraceutical composition of the invention is a food composition. The water-soluble extract of the invention has a pleasant odor and flavor and is therefore particularly suitable for use as a food composition and can therefore be added directly to food products. Unlike other mussel products, it does not need to be encapsulated. Example 11 describes the analysis of volatile organic compounds present in two aqueous extracts of the invention. The results shown in Tables 13 and 14 are consistent with a pleasant flavor composition.

In one embodiment, the food composition is a bakery product including, but not limited to, bread, pizza dough, cake, muffin, donuts, biscuits, tortillas, wraps, naan, noodles, and pasta.

In another embodiment, the food composition is a liquid food product including milk, fruit juice, smoothies, yogurt, soups and soft drinks. The water-soluble mussel extract of the invention can also be added to dry mixes such as instant soups, beverages, puddings and cake mixes.

In one aspect, the invention provides a pharmaceutical composition comprising a water-soluble mussel extract of the invention and one or more pharmaceutically acceptable excipients.
The term "pharmaceutically acceptable" as used herein means approved by a government regulatory agency for use in animals, particularly humans.

The term "excipient" as used herein refers to a vehicle, carrier, diluent, adjuvant, stabilizer or Contains fillers. Suitable excipients are well known to those skilled in the art of pharmacy and include starch (and its derivatives such as maltodextrin), glucose, sucrose, flour, silica gel, glycerol, sodium chloride, water, ascorbic acid and ethanol. However, it is not limited to these. The suitability of a particular excipient for incorporation into the pharmaceutical compositions of the invention will depend on the manner in which the dosage form is administered.

The pharmaceutical compositions of the invention can be administered by any suitable route including, but not limited to, parenterally, orally, intranasally, topically, transdermally, transmucosally and rectally.
The composition and shape of the dosage form will typically vary depending on the usage. Examples of dosage forms include tablets, capsules, pills, pellets, liquid-containing capsules, troches, lozenges, dispersions, suppositories, nasal sprays or inhalants, gels, suspensions, etc. These include, but are not limited to, clouding agents, emulsions, solutions and elixirs.

In one embodiment, the invention provides a water-soluble mussel extract of the invention in an oral dosage form. Because of their ease of administration, oral dosage forms are preferred, both as nutraceutical compositions and as pharmaceutical compositions. A typical oral dosage form is prepared by combining the dried water-soluble mussel extract of the present invention with at least one excipient suitable for use in a solid oral dosage form. The product is then shaped into the desired dosage form.

Such excipients include, but are not limited to, diluents, granulating agents, wetting agents, suspending agents, fillers, lubricants, binders, and disintegrants.
Examples of binders include corn starch, potato starch or other starches, gelatin, gums such as arabic and guar, sodium alginate, powdered tragacanth, cellulose and carboxymethyl cellulose, pre-gelatinized cellulose, hydroxypropyl cellulose and microcrystalline Examples include, but are not limited to, derivatives thereof, including cellulose. Examples of fillers include, but are not limited to, talc, calcium carbonate granules or powder, microcrystalline cellulose, powdered cellulose, dextrate, kaolin, mannitol, sorbitol, and starch. Disintegrants ensure that the tablet disintegrates when exposed to an aqueous environment. Examples include, but are not limited to, agar, calcium carbonate, alginic acid, croscarmellose sodium, microcrystalline cellulose, pregelatinized starches, clays, and gums. Lubricants include, but are not limited to, calcium stearate, light oil, glycerin, sorbitol, mannitol, polyethylene glycol, sodium lauryl sulfate, hydrogenated vegetable oils, and agar. Wetting agents include, but are not limited to, lecithin and polyoxyethylene stearate. Suspending agents include, but are not limited to, sodium carboxymethylcellulose, methylcellulose, and sodium alginate.

Tablets can be prepared by compression or molding. Oral dosage forms of the invention also include chewable tablets, hard gelatin capsules or soft gelatin capsules. Liquid preparations for oral administration include, but are not limited to, solutions, syrups or suspensions, and may be presented as a dry product for reconstitution with water or another suitable vehicle before use. .

The amount of a water-soluble mussel extract of the invention that is effective in the prevention, treatment or management of conditions associated with inflammation will depend on the nature and severity of the disease or condition and the route by which the extract is to be administered, as well as the subject's Varies depending on subject-specific factors such as age, weight, gender, and medical history.

Generally, dosage forms used in the acute treatment of conditions associated with inflammation will contain larger amounts of water-soluble mussel extract than those used in the treatment of chronic conditions. Similarly, parenteral dosage forms may contain less water-soluble mussel extract than oral dosage forms. The formulation of specific dosage forms is readily understood by those skilled in the pharmaceutical arts. The inventors of Remington's Pharmaceutical Sciences, Allen et al., 22nd edition. Reference is made to ISBN 978-0-85711-062-6.

Those skilled in the art can predict effective doses from dose response curves obtained from in vitro or animal model studies. Generally, an effective amount of the water-soluble mussel extract of the present invention is from about 100 mg to about 3000 mg per day, either as a single daily dose or in divided doses throughout the day.

Various aspects of the invention will now be described in a non-limiting manner with reference to the following examples.

6. Example 6.1 Materials and Methods Oxyless U was obtained from Naturex (Avignon, France). Maltodextrin (Maltrin 100 or 150) is available from New Zealand
It was manufactured by Starch (Auckland, NZ). Celite HyFlo and C
elite 545 is manufactured by Imerys Filtration Minerals Inc. (San Jose, California, USA). Enzidase Papain 6000L, Enzidase FP and Enzidase Neutral were from Zymus (Auckland, NZ). Alcalase was from Novozymes (Bausvaer, Denmark).

All other chemicals and reagents were standard laboratory supplies.
Size exclusion HPLC setup column: Yarra™ 3 μm SEC-2000 (Phenomenex).
Sample preparation:
1. Dissolve the lyophilized extract sample at a concentration of 10 mg/mL in 100 mL of sodium phosphate buffer pH 6.8.

2. Dilute the solution in the ratio of 4 parts sample in phosphate buffer and 1 part 10% SDS to obtain a final concentration of 2% SDS.
3. Heat at 50°C for 5 minutes.

4. Centrifuge at 13000 rpm for 5 minutes.
5. Fill a vial with the supernatant and perform HPLC.
Operating conditions for SE-HPLC of peptides:

6.2 Extraction of Mussels to Produce Water-Soluble Extracts of the Invention Various mussel sources were processed according to the examples provided below. Process and sample codes are summarized in Table 1 below.

Example 1: Extraction of green mussel supercritical residue (GSM residue extract) PAR15
The residue (160 g) resulting from the supercritical _CO2 extraction of freeze-dried green mussel was suspended in 600 ml of water. Oxyless U antioxidant (0.25g) was added. Papain (5ml) was added and the mixture was heated to 55°C in a shaking water bath for 1 hour. Enzidase FP (0.5 g) was then added and hydrolysis continued for an additional hour at 55° C. in a shaking water bath.

The hydrolysis mixture was heated at 90° C. for a further 30 min, cooled to 55° C., and the supernatant (565 ml) was collected after centrifugation at 10000 rpm for 30 min. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through a Seitz 900 filterboard using diatomaceous earth (20 g Precoat (Celite HyFlo) + 20 g Body Feed (Celite 545)). The filtrate was collected (510ml) and mixed with maltodextrin (27.2g) and Oxyless U antioxidant (0.25g) until dissolved. The resulting solution was freeze-dried to obtain 126.2 g of solid product (GSM residue extract).

The GSM residue extract was completely water soluble and had a pleasant flavor. 60% of the solids present in the starting supercritical _CO2 residue were recovered.
The procedure was repeated several times using the same enzyme system (PAR13, 19 and 30), once replacing Enzidase FP with Alcalase (PAR12).

The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 2 below.

Example 2: Fresh Green Mussel Whole Body Extract (Fresh GSM Whole Body Extract) PAR37
Homogenized meat (500 g) from green mussel was suspended in 400 ml of water. Oxyless U antioxidant (0.25g) was added. Alcalase (2.5ml) and Enzidase Neutral (2.5ml) were added and the mixture was heated to 60°C in a shaking water bath for 3 hours. Enzidase FP (0.125 g) was then added and hydrolysis continued for an additional hour at 60° C. in a shaking water bath.

The hydrolysis mixture was heated at 95° C. for a further 40 min, cooled to 55° C., and the supernatant (795 ml) was collected after subsequent centrifugation at 10000 rpm for 30 min. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through a Seitz 900 filterboard using diatomaceous earth (20 g precoat + 20 g bodyfeed). The filtrate was collected (750ml) and mixed with maltodextrin (27.2g) and Oxyless U antioxidant (0.25g) until dissolved. The resulting solution was lyophilized to obtain a solid product (fresh GSM whole body extract).

Fresh GSM whole body extract was completely water soluble. 56% of the solids present in the shellfish material were recovered. The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 3 below.

Example 3: Extraction of GSM Whole Body Powder (Extract of GSM Whole Body Powder) PAR36
Freeze-dried powdered green mussel (160 g) was suspended in 600 ml of water. Oxyless U antioxidant (0.25g) was added to adjust the pH to 6.0-8.0. Alcalase (2.5ml) and Enzidase Neutral (2.5m
1) was added and the mixture was heated to 60° C. in a shaking water bath for 3 hours. Enzidase FP (0.125 g) was then added and hydrolysis continued for an additional hour at 60° C. in a shaking water bath.

The hydrolysis mixture was heated at 95° C. for a further 40 min, cooled to 55° C., and the supernatant (590 ml) was collected after subsequent centrifugation at 10000 rpm for 30 min. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through a Seitz 900 filterboard using diatomaceous earth (20 g precoat + 20 g bodyfeed). The filtrate was collected (550ml) and mixed with maltodextrin (27.2g) and Oxyless U antioxidant (0.25g) until dissolved. The resulting solution was lyophilized to obtain a solid product (extract of GSM whole body powder).

The extract of GSM whole body powder was completely water soluble. 66% of the solids present in the starting mussel material were recovered.
The process was repeated replacing Alcalase and Enzidase Neutral with papain (PAR36a).

The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 4 below.

Example 4: Fresh mussel whole body extract (Fresh mussel whole body extract) PAR40
Homogenized meat from mussels (516 g) was suspended in 300 ml of water. Oxyless U antioxidant (0.25g) was added. The pH was adjusted to 6.0-8.0 followed by the addition of Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) and the mixture was heated to 60 °C in a shaking water bath for 3 h. . Enzidase FP (0.125 g) was then added and hydrolysis continued for an additional hour at 60° C. in a shaking water bath.

The hydrolysis mixture was heated at 95° C. for an additional 30 min, cooled to 55° C., and the supernatant (746 ml) was collected after centrifugation at 10000 rpm for 30 min. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through a Seitz 900 filterboard using diatomaceous earth (20 g precoat + 20 g bodyfeed). The filtrate was collected (700ml) and mixed with maltodextrin (27.2g) and Oxyless U antioxidant (0.25g) until dissolved. The resulting solution was freeze-dried to obtain a solid product (fresh mussel whole body extract).

Fresh mussel whole body extract was completely water soluble. 83% of the solids present in the shellfish material were recovered. The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 5 below.

Example 5: Extraction of fresh GSM whole body with defatting step (defatted extract of fresh GSM whole body) PAR41
Homogenized meat from GSM (507 g) was suspended in 400 ml of water. Oxyless U antioxidant (0.25g) was added. The pH was adjusted to 6.5-8.0 followed by the addition of Alcalase (2.5 ml) and Enzidase Neutral (2.5 ml) and the mixture was heated to 60 °C in a shaking water bath for 3 h. . Enzidase FP (0.125 g) was then added and hydrolysis continued for an additional hour at 60° C. in a shaking water bath.

Subsequently, the hydrolyzed material (890 ml) was freeze-dried and 100 g of powder was recovered. The powder was rinsed with cold acetone (3 x 250 ml) to remove lipids. The powder was collected by filtering through paper during rinsing. The final defatted powder was dried under nitrogen.

The defatted powder was resuspended in 400 ml of water and heated at 95°C for 40 minutes, followed by cooling to 55°C. The hydrolyzed aqueous suspension was then centrifuged at 10,000 rpm for 30 minutes, and the supernatant (415 ml) was collected. The pH of the supernatant was adjusted to pH 4 using phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through a Seitz 900 filterboard using diatomaceous earth (20 g precoat + 20 g bodyfeed). The filtrate was collected (375ml) and mixed with maltodextrin (27.2g) and Oxyless U antioxidant (0.25g) until dissolved. The resulting solution was lyophilized to obtain a solid product (defatted extract of fresh GSM whole body).

The defatted extract of fresh GSM whole body was completely water soluble. 55% of the solids present in the shellfish material were recovered. The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 6 below.

Example 6: Factory-scale extraction of GSM supercritical residue (factory GSM residue extract) PAR43
Oxyless U antioxidant (80g) was added to cold (19°C) water (500L). Papain (200 g) was stirred therein followed by the residue (50.0 kg) resulting from the supercritical CO ₂ extraction of freeze-dried green mussel. The mixture was heated to 55°C and allowed to digest for 2 hours. Enzidase FP (160 g) was then added and hydrolysis continued for an additional 1.5 hours at 55°C.

The hydrolysis mixture was heated for an additional 20 min at 80-88°C, cooled to 60°C, and then centrifuged (twice) through a liquid-sludge separator before collecting the supernatant (450 L). . The pH of the supernatant was adjusted to 4 using phosphoric acid. Activated carbon was added (200g). The supernatant was filtered through a filter press using diatomaceous earth (6.0 kg precoat + 6.0 kg bodyfeed). The filtrate was collected (500 L) and mixed with maltodextrin (7.0 kg) and Oxyless U antioxidant (80 g) until dissolved. The resulting solution was freeze-dried to obtain 24.0 kg of solid (GSM residue extract) excluding additives.

The GSM residue extract was completely water soluble and had a pleasant fresh sea flavor. 50% of the solids present in the starting supercritical _CO2 residue were recovered.
The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 7 below.

Example 7: Fresh Copacopa Whole Body Extract (Fresh Copacopa Whole Body Extract) PAR45
Homogenized Copacopa meat (501 g) was suspended in 400 ml of water. Oxyless U antioxidant (0.25g) was added. pH (6) was not adjusted. Papain (5ml) was added and the mixture was heated to 55°C in a shaking water bath for 2 hours. Enzidase FP (0.5 g) was then added and hydrolysis continued for an additional 1.5 hours at 55° C. in a shaking water bath.

The hydrolysis mixture was heated at 95° C. for an additional 30 min, cooled to 55° C., and the supernatant (720 ml) was collected after centrifugation at 10000 rpm for 30 min. The pH of the supernatant was adjusted to 4 using phosphoric acid. Activated carbon was added (2g). The supernatant was filtered through a Seitz 900 filterboard using diatomaceous earth (20 g Precoat (Celite HyFlo) + 20 g Body Feed (Celite 545)). The filtrate was collected (575ml) and mixed with maltodextrin and Oxyless U antioxidant. The resulting solution was lyophilized to obtain a solid product (fresh whole body extract of Copacopa).

Fresh Copacopa whole body extract was completely water soluble. 55% of the solids present in the Copacopa feedstock were recovered.
The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 8 below.

Example 8: Extraction of GSM residue powder (pilot scale) PAR58
Oxyless U (24g) was added to 150L of cold water. Papain (500 mL) (Zymus Enzidase Papain 6000 L) was added with continuous stirring. GSM supercritical residue (15.0 kg) was added and the suspension was heated at 50° C. for 15 hours. Enzidase FP (50g) was added and hydrolysis continued for an additional 2 hours at 55°C.

The suspension was then heated at 80° C. for 20 minutes and then cooled.
The suspension was centrifuged through a liquid-sludge separator and 60 g of activated carbon was added during the process. The supernatant was collected again and its pH was changed to 4 using phosphoric acid. The supernatant was centrifuged through a liquid sludge separator. The final supernatant was collected (95L). Maltodextrin (2.4 kg) and Oxyless U (24 g) were added and mixed until dissolved.

Subsequently, the water-soluble extract of the present invention was freeze-dried to obtain a water-soluble solid product. 69% of the solids present in the mussel residue were recovered. The flavor was pleasant (tasty).
The procedure was repeated using the same enzyme system (500 mL of papain, digested for 21 hours at 50°C, followed by hydrolysis with 45 g of Enzidase FP for an additional 2 hours at 55°C) (PAR63).

83% of the solids present in the supercritical GSM residue were recovered. The product was water soluble and had a pleasant delicious flavor.
The molecular weight profile of the product as analyzed by SE-HPLC is shown in Table 9 below.

The approximate composition of PAR58 was compared to that of the GSM residue starting material. The samples were dried at 103° C. for 16 hours (gravimetrically). AOAC 945.15, 19th edition. The ash content was determined by ignition in a muffle furnace at 600° C. for 6 hours (gravimetry). AOAC 942.05, 19th edition. Total nitrogen was determined using Dumas combustion. AOAC 992.
15, 19th edition. Total protein was calculated based on total nitrogen x 6.25. AOAC 992.15, 19th edition. Total protein values are also given based on a factor of 6.75 (which is more appropriate for highly hydrolyzed proteins where significant water is attached to the peptide bonds). Lipids are described by Blight, E. G. , & Dyer, W. J. (1959), “A rapid method of total lipid extraction and purification”, Can. J. Biochem. and Physiol. , Vol. 37(8), pp. 911-917.

Table 10 below shows the results.

Values are g/100g of freeze-dried material without any additives.
6.3 Anti-inflammatory properties of the water-soluble extract of the invention Example 9: Inhibition of TLR4 (LPS)-stimulated IL-1β and TNFα secretion in monocytic THP-1 cells by the water-soluble extract of the invention IL-1β and TNFα is an important mediator of inflammatory responses. They initiate a cascade of events that modulate the body's natural defenses against invading microorganisms and trauma. Under normal conditions, these proinflammatory cytokines are localized and short-lived. However, under chronic inflammatory conditions their expression is prolonged and, in certain circumstances, spread throughout the body. This perpetuates the pro-inflammatory response and is the basis for abnormal recruitment and activation of immune cells.

Therefore, by limiting IL-1β and TNFα expression and biological activity in chronic inflammatory conditions, chronic inflammation is suppressed. Additionally, various immune cells are known to express IL-1β and TNFα during chronic inflammation. However, the triggers for their expression are highly diverse and they are the result of aberrant immune cell signaling to the invading microorganism.

As part of their natural defense system, humans have developed a complex array of cell surface markers, including a group known as Toll-like receptors (TLRs) that recognize foreign substances and trigger appropriate cellular defense processes.

Bacterial ligands that activate TLR4 in monocytic immune cells can be utilized to upregulate the expression of IL-1β and TNFα to suppress the expression of these proinflammatory cytokines in chronic inflammatory conditions. The efficiency of mussel extract was explored.
Method: The monocytic cell line THP-1 (2 x ¹⁰ cells/mL) grown in RPMI (Roswell Park Memorial Institute) medium containing 10% FBS (fetal bovine serum) was used as a mammalian cell line. Under culture conditions, TLR4-lipopolysaccharide [LPS] (0.5-50 ng/mL) was incubated for 6 hours. Supernatants were collected and measured for IL-1β and TNFα production using commercially available ELISAs. Optimal doses of TLR4 (approximately 70% of maximal bioactivity) were used to examine the effects of mussel extracts on IL-1β and TNFα secretion. Dexamethasone (0.001-10 μM or 0-3.925 μg/mL), a known immunosuppressant, was used to validate this bioassay.

The efficacy of mussel extract in attenuating IL-1β and TNFα expression in a chronic inflammatory scenario was evaluated. THP-1 cells were first stimulated with optimal doses of TLR4 for 30 minutes to induce upregulation of IL-1β and TNFα expression. Mussel extract (usually 1-100 μg/mL) was then added and incubated with the cells for an additional 6 hours. Subsequently, the culture medium was collected (by centrifuging the cells) and measured for IL-1β and TNFα secretion by ELISA. Data were calculated as pg of cytokine/mL and presented as percent inhibition of TLR4-stimulated IL-1β or TNFα secretion.

The results are shown in FIGS. 1 to 18. All tested water-soluble extracts of the invention showed anti-inflammatory activity (Figures 1-14).
Figures 15-18 are the results of control experiments in which unhydrolyzed mussel material was tested in the assay described above.

Figures 15 and 16 show the effect of GSM residue powder and GSM systemic powder on inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells.
Figures 17 and 18 show the effect of the same materials on the inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells.

Table 10 below shows the effect of various GSM products on the inhibition of IL-1β in TLR4 (LPS) stimulated monocytic THP-1 cells when applied at 100 μg/mL in the assay. The lyophilized product was resuspended in either phosphate buffered saline (PBS) or dimethyl sulfoxide (DMSO) prior to addition to the assay.

In each column, the results are expressed in comparison to the effect of the soluble extract (highest inhibition), which was assigned a value of 1.

Table 11 below shows the effect of various GSM products on the inhibition of TNFα in TLR4 (LPS) stimulated monocytic THP-1 cells when applied at 100 μg/mL in the assay. The lyophilized product was resuspended in either phosphate buffered saline (PBS) or dimethyl sulfoxide (DMSO) prior to addition to the assay.

In each column, the results are expressed in comparison to the effect of the soluble extract (highest inhibition), which was assigned a value of 1.

6.4 Analysis of Water-Soluble Extracts of the Invention Example 10: LC-MS Analysis Method for GSM Residues, Whole Body Powder and Water-Soluble Extracts of the Invention Each sample was assigned and vortexed using 0.5% formic acid. Although mixed in an aqueous solution to dissolve the polar components, some samples retained some undissolved solids, as shown in Table 12 below.

The LC-MS system consisted of a Thermo Electron Corporation (San Jose, CA, USA) Finnigan Surveyor MS pump, a Thermo Accela Open autosampler (PAL HTC-xt with DLW), and a Finnigan detector. Surveyor PDA, and ThermaSphere TS- It consisted of a 130 column heater (Phenomenex, Torrance, CA, USA).

A 2 μL aliquot of each prepared extract was subjected to aqueous normal phase chromatography (Cogent™ Diamond-Hydride™, 4 μm, 100 Å, 150 x 2.1 mm, MicroSolv) maintained at 30°C at a flow rate of 200 μl/min. Technologies Corporation, USA) using a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A gradient was applied: 0-2 min, 5% A; 30-39.5 min, 90% A; 40-45 min, 5% A.

Data were collected using electrospray ionization (ESI) in negative and positive modes for precursor masses, product ions for MS in the range m/z 100-2000 amu, and API-MS (LTQ, 2D linear ion trap, Thermo- Finnigan, San Jose, CA, USA).

The results are shown in FIGS. 19 to 24. The LS-MS chromatograms of the aqueous extracts of the present invention (Figs. 19 and 22) show that the LC-MS chromatograms of the starting mussel material (Figs. shows a more complex composition with peaks of
Example 11: Solid Phase Microextraction-GC-MS Analysis of Water-Soluble GSM Extracts Volatile organic compounds in mussel extracts PAR58 and PAR63 were determined according to the modified method of Tuckey, Day and Miller (Tuckey, N.P.L.). , Day, J.R., and Miller, M.R. (2013) New Zealand Greenshell™ mussel (Perna canaliculus) during refrigerated storage using solid-phase microextraction gas chromatography-mass spectrometry. ) Determination of volatile compounds in Food
Chemistry, Vol. 136(1), pp. 218-223) and analyzed by SPME-GC-MS. A sample of the lyophilized extract (1.20 g) was placed in a 20 mL glass vial fitted with a self-sealing septum. For each extract, two vials were prepared and each vial was analyzed in duplicate. The vials were incubated at 40°C for 4 min with agitation, and then the volatiles were removed for 20 min using an 85 μm carboxene poly(dimethylsiloxane) coated SPME fiber (Supelco, Bellefonte, PA, USA). It was adsorbed. Blank fiber samples were run prior to and between each analytical run to ensure a clean baseline. Adsorbed volatile substances were analyzed by GC-MS. The GC-MS system consisted of a GC-2010 gas chromatograph coupled to a GCMS-QP2010 mass spectrometry unit (Shimadzu, Kyoto, Japan) and also equipped with an AOC-5000 automatic injector (PAL, CTC Analytics, Zwingen, Switzerland). was. Conditions for GC-MS analysis were as follows: injection temperature 250 °C; splitless mode; 2 min desorption in the injector; Rtx-5Sil MS capillary column - 30 m x 0.25 mm ID x 0.25 μm film thickness ( Restek, Bellefonte, PA, USA); Furnace temperature program: Start at 50°C, hold for 1 minute, increase at 10°C/min to 160°C, increase at 40°C/min to 250°C, hold at 250°C for 1 minute; He Carrier gas, flow rate 1.90 mL/min; MS detector temperature, 260°C. Peaks were identified by comparison with a mass spectral database (Wiley 7.0 Library, Wiley, New York, NY, USA). The results are shown below in Tables 13 and 14.

本発明の態様には、以下のものも含まれる。
態様１
水溶性イガイ抽出物。
態様２
固体を基準にして少なくとも約４５重量％のペプチドを含む、態様１に記載の水溶性イガイ抽出物。
態様３
固体を基準にして約４５重量％～約６５重量％のペプチドを含む、態様１に記載の水溶性イガイ抽出物。
態様４
ペプチドの９０重量％超が、５ｋＤａ未満である、態様１から３のいずれか一項に記載の水溶性イガイ抽出物。
態様５
固体を基準にして約５重量％以下の脂質を含む、態様１から４のいずれか一項に記載の水溶性イガイ抽出物。
態様６
前記イガイが、ミドリイガイ（ＧＳＭ）、ムラサキイガイおよびリブ状イガイを含む群から選択される、態様１から５のいずれか一項に記載の水溶性イガイ抽出物。
態様７
水溶性イガイ抽出物を生成する方法であって、
（ａ）１つまたは複数のタンパク質分解酵素を使用して、イガイ材料の水性懸濁液を加水分解するステップと、
（ｂ）加水分解混合物中の該酵素を変性させるステップと、
（ｃ）不溶性材料を加水分解混合物から除去して、水溶性イガイ抽出物をもたらすステップと
を含む方法。
態様８
前記タンパク質分解酵素が、わずかに酸性からほぼ中性のｐＨ最適値を有する、態様７に記載の方法。
態様９
前記タンパク質分解酵素が、パパイン、Ａｌｃａｌａｓｅ、Ｅｎｚｉｄａｓｅ ＦＰおよびＥｎｚｉｄａｓｅ Ｎｅｕｔｒａｌを含む群から選択される、態様７に記載の方法。態様１０
前記イガイ材料が、新鮮なイガイ全身、イガイ粉末およびイガイ超臨界残留物を含む群から選択される、態様７から９のいずれか一項に記載の方法。
態様１１
前記イガイが、ミドリイガイ、ムラサキイガイおよびリブ状イガイを含む群から選択される、態様７から１０のいずれか一項に記載の方法。
態様１２
態様７から１１のいずれか一項に記載の方法によって生成される水溶性イガイ抽出物。態様１３
態様１から６のいずれか一項に記載の水溶性イガイ抽出物および１つまたは複数の薬学的に許容される賦形剤を含む医薬組成物。
態様１４
態様１から６のいずれか一項に記載の水溶性イガイ抽出物および１つまたは複数の摂取可能な賦形剤を含む栄養補助食品組成物。
態様１５
食品組成物、食品添加物組成物、健康補助食品または医療食品である、態様１４に記載の栄養補助食品組成物。
態様１６
必要とする対象において炎症を低減させる方法であって、該対象に、治療有効量の態様１から６のいずれか一項に記載の水溶性イガイ抽出物を投与することを含む方法。
態様１７
必要とする対象において炎症と関連する状態を予防、治療または管理する方法であって、該対象に、治療有効量の態様１から６のいずれか一項に記載の水溶性イガイ抽出物を投与することを含む方法。
態様１８
前記炎症と関連する状態が、喘息、脳炎、クローン病および潰瘍性大腸炎を含む炎症性腸疾患、慢性閉塞性肺疾患（ＣＯＰＤ）、アレルギー性疾患、線維症、若年性関節炎、乾癬性関節炎、関節リウマチおよび変形性関節症を含む関節炎、乾癬、多発性筋痛、腱炎、滑液包炎、喉頭炎、歯肉炎、胃炎、耳炎、セリアック病、憩室炎、アテローム性動脈硬化症、心臓病、肥満症、糖尿病、癌ならびにアルツハイマー病を含む群から選択される、態様１７に記載の方法。

Aspects of the invention also include the following.
Aspect 1
Water-soluble mussel extract.
Aspect 2
The water-soluble mussel extract of embodiment 1, comprising at least about 45% by weight peptides on solids.
Aspect 3
The water-soluble mussel extract of embodiment 1, comprising from about 45% to about 65% by weight peptides on a solids basis.
Aspect 4
Water-soluble mussel extract according to any one of aspects 1 to 3, wherein more than 90% by weight of the peptides are less than 5 kDa.
Aspect 5
5. The water-soluble mussel extract according to any one of aspects 1 to 4, comprising no more than about 5% by weight lipids on solids.
Aspect 6
Water-soluble mussel extract according to any one of aspects 1 to 5, wherein the mussel is selected from the group comprising green mussel (GSM), purple mussel and ribbed mussel.
Aspect 7
A method of producing a water-soluble mussel extract, the method comprising:
(a) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes;
(b) denaturing the enzyme in the hydrolysis mixture;
(c) removing insoluble materials from the hydrolysis mixture to yield a water-soluble mussel extract.
Aspect 8
8. The method of embodiment 7, wherein the proteolytic enzyme has a slightly acidic to about neutral pH optimum.
Aspect 9
8. The method according to aspect 7, wherein the proteolytic enzyme is selected from the group comprising papain, Alcalase, Enzidase FP and Enzidase Neutral. Aspect 10
10. A method according to any one of aspects 7 to 9, wherein the mussel material is selected from the group comprising fresh whole mussel, mussel powder and mussel supercritical residue.
Aspect 11
11. The method according to any one of aspects 7 to 10, wherein the mussel is selected from the group comprising green mussel, purple mussel and ribbed mussel.
Aspect 12
A water-soluble mussel extract produced by the method according to any one of aspects 7 to 11. Aspect 13
A pharmaceutical composition comprising a water-soluble mussel extract according to any one of aspects 1 to 6 and one or more pharmaceutically acceptable excipients.
Aspect 14
A dietary supplement composition comprising a water-soluble mussel extract according to any one of aspects 1 to 6 and one or more ingestible excipients.
Aspect 15
The nutritional supplement composition according to aspect 14, which is a food composition, a food additive composition, a health supplement, or a medical food.
Aspect 16
7. A method of reducing inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract according to any one of aspects 1 to 6.
Aspect 17
A method of preventing, treating or managing a condition associated with inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a water-soluble mussel extract according to any one of aspects 1 to 6. Methods that include.
Aspect 18
The condition associated with inflammation is asthma, encephalitis, inflammatory bowel disease including Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergic disease, fibrosis, juvenile arthritis, psoriatic arthritis, Arthritis including rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendonitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis, heart 18. The method according to aspect 17, wherein the method is selected from the group comprising: disease, obesity, diabetes, cancer and Alzheimer's disease.

7. Industrial Applicability The water-soluble mussel extracts of the present invention and compositions containing them can be added to foods or health supplements to enhance health benefits. Their water solubility and pleasant flavor allow them to be incorporated directly into a wide range of consumable products. The products are likely to exist in the form of tablets, capsules, beverage health "shots," premixed soup tonics, and as components of functional foods, such as tasty health food bars.

They can also be used as medicines to help prevent, treat or manage conditions associated with inflammation.

Claims (20)

A water-soluble mussel extract comprising peptides , the peptides comprising peptides from 2 to 5 kDa, peptides from 1 to 2 kDa, peptides less than 1 kDa, and peptides greater than 5 kDa, the peptides comprising about 90% by weight of the peptides. a water-soluble mussel extract having a molecular weight of less than 5 kDa, said extract being prepared by a method comprising hydrolyzing an aqueous suspension of mussel material with one or more proteolytic enzymes. wherein the proteolytic enzymes include papain, subtilisin, endo/exopeptidases from selected strains of Aspergillus oryzae var. and non-genetically modified Bacillus amyloliquefaciens. An extract selected from the group consisting of metalloneutral endopeptidases derived from controlled fermentation of a strain. The water-soluble mussel extract according to claim 1,
(a) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes, wherein the proteolytic enzymes include papain, subtilisin, selected strains of Aspergillus oryzae var. and a metalloneutral endopeptidase derived from controlled fermentation of a non-genetically modified strain of Bacillus amyloliquefaciens;
(b) denaturing the enzyme in the hydrolysis mixture;
(c) removing insoluble materials from said hydrolysis mixture, resulting in a water-soluble mussel extract. Water-soluble mussel extract according to claim 1 or 2, wherein the extract has anti-inflammatory activity. 4. A water-soluble mussel extract according to any one of claims 1 to 3, comprising at least about 45% by weight peptides on solids. 5. A water-soluble mussel extract according to any one of claims 1 to 4, comprising from about 45% to about 65% by weight of peptides, based on solids. 6. A water-soluble mussel extract according to any one of claims 1 to 5, comprising no more than about 5% by weight of lipids on solids. Water-soluble mussel extract according to any one of claims 1 to 6, wherein the mussel is selected from the group comprising green mussel (GSM), purple mussel and ribbed mussel. Water-soluble mussel extract according to any one of claims 1 to 7, further comprising carbohydrates and lipids. A method of producing a water-soluble mussel extract, the method comprising:
(a) hydrolyzing an aqueous suspension of mussel material using one or more proteolytic enzymes, wherein the proteolytic enzymes include papain, subtilisin, Aspergillus oryzae var. selected from the group consisting of endo/exopeptidases derived from selected strains and metalloneutral endopeptidases derived from controlled fermentation of non-genetically modified strains of Bacillus amyloliquefaciens;
(b) denaturing the enzyme in the hydrolysis mixture;
(c) removing insoluble materials from the hydrolysis mixture to yield a water-soluble mussel extract. 10. The method of claim 9, wherein the mussel material is selected from the group comprising fresh whole mussel, mussel powder and mussel supercritical residue. 11. The method according to claim 9 or 10, wherein the mussel is selected from the group comprising green mussel, purple mussel and ribbed mussel. A pharmaceutical composition comprising a water-soluble mussel extract according to any one of claims 1 to 8 and one or more pharmaceutically acceptable excipients. A nutraceutical composition comprising a water-soluble mussel extract according to any one of claims 1 to 8 and one or more ingestible excipients. The nutritional supplement composition according to claim 13, which is a food composition, a food additive composition, a health supplement, or a medical food. Use of a water-soluble mussel extract according to any one of claims 1 to 8 for the manufacture of a medicament for reducing inflammation. 9. Use of a water-soluble mussel extract according to any one of claims 1 to 8 for the manufacture of a medicament for preventing, treating or managing conditions associated with inflammation. The condition associated with inflammation is asthma, encephalitis, inflammatory bowel disease including Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergic disease, fibrosis, juvenile arthritis, psoriatic arthritis, Arthritis, including rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendinitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis, heart 17. The use according to claim 16, selected from the group comprising diseases, obesity, diabetes, cancer and Alzheimer's disease. A pharmaceutical or nutritional supplement composition for reducing inflammation, comprising a water-soluble mussel extract according to any one of claims 1 to 8. A pharmaceutical or nutraceutical composition for preventing, treating or managing conditions associated with inflammation, comprising a water-soluble mussel extract according to any one of claims 1 to 8. The condition associated with inflammation is asthma, encephalitis, inflammatory bowel disease including Crohn's disease and ulcerative colitis, chronic obstructive pulmonary disease (COPD), allergic disease, fibrosis, juvenile arthritis, psoriatic arthritis, Arthritis, including rheumatoid arthritis and osteoarthritis, psoriasis, polymyalgia, tendinitis, bursitis, laryngitis, gingivitis, gastritis, otitis, celiac disease, diverticulitis, atherosclerosis, heart 20. A pharmaceutical composition or nutritional supplement composition according to claim 19, selected from the group comprising diseases such as obesity, diabetes, cancer and Alzheimer's disease.

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