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Title:
PEPTIDE MATERIAL, AND PREPARATIONS AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2011/112099
Kind Code:
A1
Abstract:
A peptide preparation and processes for its preparation are described. Also uses thereof are described.

Inventors:
AKSNES ANDERS (NO)
BERGE ROLF KRISTIAN (NO)
SANDNES KJARTAN (NO)
Application Number:
PCT/NO2011/000078
Publication Date:
September 15, 2011
Filing Date:
March 08, 2011
Export Citation:
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Assignee:
MARINE BIOPRODUCTS AS (NO)
AKSNES ANDERS (NO)
BERGE ROLF KRISTIAN (NO)
SANDNES KJARTAN (NO)
International Classes:
A23J1/04; A23J3/34; A23K1/10; A23K1/16; A23L1/305; A61K38/01
Domestic Patent References:
WO2005002605A12005-01-13
WO2006084383A12006-08-17
WO2006005757A22006-01-19
WO2006084351A12006-08-17
WO2009128713A12009-10-22
Foreign References:
FR2927335A12009-08-14
NO2004000202W2004-07-02
NO2004000043W2004-02-12
US5268360A1993-12-07
US4940662A1990-07-10
JPH02113859A1990-04-26
Other References:
GUERARD F ET AL: "Production of tuna waste hydrolysates by a commercial neutral protease preparation", JOURNAL OF MOLECULAR CATALYSIS. B, ENZYMATIC, ELSEVIER, AMSTERDAM, NL, no. 19-20, 2 December 2002 (2002-12-02), pages 489 - 498, XP002293270, ISSN: 1381-1177, DOI: DOI:10.1016/S1381-1177(02)00203-5
LAURA GILMARITN ET AL.: "Production of Cod (Gadus morhua) Muscle Hydrolysates, Influence of Combinations of Commercial Enzyme Preparations", HYDROLYSATE PEPTIDE SIZE RANGE, J. AGRIC. FOOD CHEM., vol. 50, 2002, pages 5417 - 5423
COHEN, MICHAUD, ANAL. BIOCHEM., vol. 211, 1993, pages 279 - 287
BERGE, R. K., FLATMARK, T., OSMUNDSEN, H.: "Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators", EUR J BIOCHEM, vol. 141, 1984, pages 637 - 644
BLIGH, E. G., DYER, W. J.: "A rapid method of total lipid extraction and purification", CAN J BIOCHEM PHYSIOL, vol. 37, 1959, pages 911 - 91
LIE, O., LAMBERTSEN, G.: "Fatty acid composition of glycerophospholipids in seven tissues of cod (Gadus morhua), determined by combined high-performance liquid chromatography and gas chromatography", J CHROMATOGR, vol. 565, 1991, pages 119 - 129
Attorney, Agent or Firm:
ACAPO AS (Bergen, NO)
Download PDF:
Claims:
Claims

1. Peptide preparation, characterized in that said preparation is prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second enzymatic treatment with an enzyme preparation manufactured from a strain of Aspergillus oryzae.

2. Peptide preparation in accordance with claim 1 , wherein said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan. 3. Peptide preparation in accordance with claim 1 , wherein said alkaline protease and neutral protease is a Bacillus protease complex, preferable

Protamex™ from Novozymes AS.

4. Peptide preparation in accordance with any of the preceding claims, wherein said peptide preparation is substantially fat free.

5. Peptide preparation in accordance with any of the preceding claims, wherein said fish is Salmonidae, preferable Atlantic salmon. 6. Peptide preparation in accordance with any of the preceding claims, wherein said fish is Atlantic salmon, and the enzyme of the first enzymatic treatment is the Bacillus protease complex Protamex™ from Novozymes AS and wherein said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan, and wherein fat is separated from the peptide material, preferable the first enzymatic treatment.

7. Peptide preparation in accordance with any of the preceding claims, wherein at least 60% of the peptides have a size of 1000 Daltons or less. 8. Peptide preparation in accordance with any of the preceding claims, wherein at least 35% of the peptides have a size of about 100 - 1.000 Dalton, more preferable that at least 40% of the peptides have a size of about 100 - 1.000 Dalton, and more preferable that at about 45% of the peptides have a size of about 100 - 1.000 Dalton.

9. Peptide preparation in accordance with any of the preceding claims, wherein at least 25% of the peptides have a size of less than about 100 Dalton.

10. Peptide preparation in accordance with any of the preceding claims, wherein

- at least 35% of the peptides have a size of about 100 - 1.000 Dalton, more preferable that at least 40% of the peptides have a size of about 100 - 1.000 Dalton, and more preferable that at about 45% of the peptides have a size of about 100 - 1.000 Dalton, and

- at least 25% of the peptides have a size of less than about 100 Dalton.

11. Peptide preparation in accordance with any of the preceding claims, wherein a fraction of the peptide preparation corresponding to peptide sizes of about 1200 to

200 Daltons has an amino acid composition as indicated in table 4.

12. Peptide preparation in accordance with any of the preceding claims, wherein a fraction of the peptide preparation corresponding to peptide sizes of about 200 to 100 Daltons has an amino acid composition as indicated in table 4.

13. Peptide preparation in accordance with any of the preceding claims, wherein the relative amount of the amino acid arginine in the peptide preparation

corresponding to peptide sizes of about 1200 to 200 Daltons is at least 20 %, preferable at least 40 %, more preferable about 60 % lower than compared to the fish protein hydrolysate.

14. Peptide preparation in accordance with any of the preceding claims, wherein the relative amount of the amino acid arginine in the peptide preparation

corresponding to peptide sizes of about 200 to 100 Daltons is at least 20 %, preferable at least 40 %, more preferable about 60 % higher than compared to the fish protein hydrolysate.

15. Peptide preparation in accordance with any of the preceding claims, wherein the relative amount of the amino acid tyrosine in the peptide preparation

corresponding to peptide sizes of about 1200 to 200 Daltons is 50%, more preferable 100%, more preferable 150 % higher than compared to the fish protein hydrolysate.

16. Peptide preparation in accordance with any of the preceding claims for the prevention and/or treatment of cardiovascular diseases, wherein said cardiovascular disease preferable is selected from the group consisting of atherosclerosis, angina, cerebrovascular accident (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack) and peripheral vascular disease.

17. Process for the preparation of a peptide preparation, characterized in that said preparation is prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second

enzymatic treatment with an enzyme preparation manufactured from a strain of Aspergillus oryzae. 8. Process in accordance with claim 17, wherein said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan.

19. Process in accordance with claim 17, wherein said alkaline protease and neutral protease is a Bacillus protease complex, preferable Protamex™ from

Novozymes AS 20. Process in accordance with any of the preceding claims, wherein fat is separated from the peptide material after the first enzymatic treatment.

21. Process in accordance with claim 17, wherein said fish is Salmonidae, preferable Atlantic salmon.

22. Process in accordance with any of the claims 17 to 21 , wherein said treatment is conducted at a pH of about 5 to about 9, preferable at a pH at about 6 to 8, preferable at a pH of about 6 to 7, or more preferable at a pH of about 6.5.

23. Use of a peptide preparation as a food product or food supplement or nutraceutical preparation for the prevention and/or treatment of cardiovascular diseases, wherein said preparation is prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second enzymatic treatment with an enzyme preparation

manufactured from a strain of Aspergillus oryzae.

24. Use of a peptide preparation for the manufacturing of a pharmaceutical composition for the prevention and/or treatment of cardiovascular diseases, wherein said preparation is prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second enzymatic treatment with an enzyme preparation manufactured from a strain of Aspergillus oryzae.

25. Use in accordance with claim 23 or 24, wherein said cardiovascular disease is selected from the group consisting of atherosclerosis, angina, cerebrovascular accident (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack) and peripheral vascular disease.

26. Use in accordance with claim 23 or 24, wherein said cardiovascular disease is treated and/or prevented by increasing the level of high-density lipoprotein (HDL) in the blood.

27. Use in accordance with claim 23 or 24, wherein said cardiovascular disease is treated and/or prevented by increasing the ratio of HDL cholesterol to LDL cholesterol in the blood.

28. Use of a peptide preparation according to claim 23 or 24, for the

manufacturing of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of hypocholsterolemia.

29. Use of a peptide preparation according to claim 23 or 24, for the

manufacturing of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of underweight, undernourishment, malnutrition or malnourishment disorders.

30. Use in accordance with claim 29, wherein said disorders are prevented or treated by increasing the body mass index (BMI).

31. Use in accordance with any of the claims 23 to 30, wherein said protein material is a fish protein material. 32. Use in accordance with claim 23 or 24, wherein said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan.

33. Use in accordance with claim 23 or 24, wherein said wherein said alkaline protease and neutral protease is a Bacillus protease complex, preferable

Protamex™ from Novozymes AS.

34. Use in accordance with claim 23 or 24, wherein said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan, and wherein said alkaline protease and neutral protease is a Bacillus protease complex, preferable Protamex™ from Novozymes AS.

35. Use in accordance with claim 23 or 24, wherein peptide preparation is substantially fat free. 36. Feed composition comprising a peptide preparation in accordance with any of the claims 1 to 16, or prepared by a process in accordance with any of the claims 17- 23.

37. Feed composition in accordance with claim 36, for increasing the weight of an animal fed said feed composition.

38. Feed composition in accordance with claim 36, for increasing the weight gain and/or the specific growth rate of an animal, characterized by feeding the animal with a feed composition containing a peptide preparation according to claim 1.

39. Feed composition in accordance with claim 36, wherein said animal is suffering of underweight, undernourishment, malnutrition or malnourishment.

Description:
Title: Peptide material, and preparations and uses thereof

FIELD OF INVENTION

The present invention relates to a peptide preparation, a process for preparation of a peptide preparation, and uses thereof.

BACKGROUND OF THE INVENTION

The fish farming industry has grown enormously both in Norway and world-wild during the recent years, especially the farming of Salmon. Much of the fish are sold to the consumer as gutted, whole fish, but significant amounts are sold as fillets.

Only 50-70 % of the salmon is fillets, whereas the rest is sold as low-valued products such as fish meal and fish ensilage.

Through enzymatic treatment the fish meat and also the fish frames can be separated into an aqueous fraction rich in proteins, called fish protein hydrolysate (FPH). The enzymatic hydrolysing process is highly controllable, and the products are reproducible and well defined.

Such a protein material, i.e. the fish protein hydrolysate (FPH) has several beneficial biological effects, and that such a material can be used as a pharmaceutical or nutritional material. Applicant own patent application PCT/NO04/00202 discloses that the use of this FPH material lowers the concentration of plasma cholesterol and homocysteine, and also lowers the concentration of hepatic triacylglycerols. PCT/NO04/00202 describes an enzyme treated fish protein hydrolysate which lowers the concentration of cholesterol in plasma, and triglycerides in the liver. FPH also induces a favourable change in the fatty acid pattern, and lowers the

concentration of homocysteine in plasma. It is also disclosed that this hydrolysate can be used as an anti-atherogenic and cardio protective agent. It is also shown that this enzyme treated fish protein hydrolysate increases the mitochondrial β-oxidation. The hydrolysate is produced from fish flesh remnants on salmon bone frames after filleting, and the enzymatic hydrolysis was performed with Protamex™ at a pH of about 6,5 and at a temperature of 55 ± 2 °C, and with 60 minutes of enzymatic treatment. The resulting material is identical to the material used as control in the experiments of the present invention. The present invention shows improved nutritional and medical effects compared to this (control/reference) material disclosed in PCT/NO04/00202. PCT/NO04/00043 discloses a similar enzyme treated fish protein hydrolysate.

Protamex was used for the enzymatic treatment, and the hydrolysis was run for two hours.

WO06/005757 discloses a process for preparation of material where the ration of the small tripeptides IPP (lle-Pro-Pro) and VPP (Val-Pro-Pro) is at least 5. Two enzymes are used, i.e. an enzyme with a proline specific endoprotease activity or prolyl oligopeptidase activity, and an enzyme which is capable of hydrolyzing the bond at the amino terminal side of a IPP-sequence. US 5,268,360 discloses opioid peptides recovered from hydrolysates of wheat proteins with a combination of action of an acid protease and a neutral or acid protease. Four opioid peptides were identified and they are useful as narcotic and algesic medicines. US 4,940,662 discloses a method of producing a low-molecular weight peptide mixture where a first and second protease in combination with the addition of at least one amino acid pre-formed by esterification of the amino acid with an alcohol. The amino acids are incorporated into proteins by a reverse reaction of proteolysis. WO06/084351 discloses anti-hypertensive tripeptides with ACE inhibitory activities were identified. JP2113859 describes the use of Protease M to prepare a low-molecular peptide material.

WO09/128713 describes an egg-protein hydrolysate with significant DPP-IV inhibitory activity and this hydrolysate can be used for the treatment of diabetes.

The publication of Laura Gilmaritn et al., (Production of Cod (Gadus morhua) Muscle Hydrolysates, Influence of Combinations of Commercial Enzyme Preparations on Hydrolysate Peptide Size Range, J. Agric. Food Chem. 2002, 50, 5417-5423, describes how combination of different enzymes influenced on the degree of hydrolysis and the peptide molecular weight range of the produced peptides.

The fish protein hydrolysate (FPH) provided by the method described in Applicant's own patent application PCT/NO04/00202 is the starting point for the improved processes, products, feed compositions and uses of the present invention, and this fish protein hydrolysate (FPH) has been treated with a second enzyme in order to improve the processes, products, feed compositions and uses thereof.

We have now surprisingly shown that we can treat a primary enzyme treated protein material such as FPH further, i.e. to treat the enzyme treated protein material in a secondary enzymatic treatment, and we obtain a new peptide material that is different with respect to size distribution of the peptide fragments.

We have made several new peptide materials and we have surprisingly shown that the biological activities change among the different peptide materials, and also that the new peptide materials have effects that differ from the protein material only treated once. These new materials can be used to better tailor the materials for specific nutritional effects, and for new and improved medical activity. The specific peptide material of the present invention, termed E1 , represents an improvement above the known fish protein hydrolysate (FPH) material that has been treated with only a primary enzymatic treatment. The FPH material (control) we have used for the experimental testing is from salmon, but since the amino acid composition is fairly similar for all fish species we assume that also other non-salmon enzyme treated protein materials can be used as a basis for a second enzymatic treatment.

Without being bound by theory, we believe that the mechanism of action is related to the size distribution of the peptide mixture, and not to the origin of the protein sample.

The peptide material in accordance with the present invention is especially useful as a functional protein in food products, particularly when used as a substitute for natural plasma in animal feeds and in pet foods. When used in feed or pet foods, additional ingredients may be added to the product such as fats, sugars, salt, flavourings, minerals, etc. The product may then be formed into chunks resembling natural meat chunks in appearance and texture. The product of the invention has the further advantages that this is readily formulated to contain necessary nutrients, is easily digested by the animals and is palatable to the animals. The peptide material in accordance with the invention can also be used for the manufacturing of a nutraceutical or pharmaceutical composition for the prevention and/or treatment of various diseases, as indicated in the claim section.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a peptide preparation prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second enzymatic treatment with an enzyme preparation manufactured from a strain of Aspergillus oryzae.

Preferable, said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan. Preferable, said alkaline protease and neutral protease is a Bacillus protease complex, preferable Protamex™ from Novozymes AS. Preferable, said peptide preparation is substantially fat free.

Preferable, said fish is Salmonidae, preferable Atlantic salmon.

Preferable, said fish is Atlantic salmon, and the enzyme of the first enzymatic treatment is the Bacillus protease complex Protamex™ from Novozymes AS and wherein said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan, and wherein fat is separated from the peptide material, preferable the first enzymatic treatment. Preferable, at least 60% of the peptides have a size of 1000 Daltons or less.

Preferable, at least 35% of the peptides have a size of about 100 - 1.000 Dalton, more preferable that at least 40% of the peptides have a size of about 100 - 1.000 Dalton, and more preferable that at about 45% of the peptides have a size of about 00 - .000 Dalton.

Preferable, at least 25% of the peptides have a size of less than about 100 Dalton.

Preferable,

- at least 35% of the peptides have a size of about 100 - 1.000 Dalton, more preferable that at least 40% of the peptides have a size of about 100 - 1.000 Dalton, and more preferable that at about 45% of the peptides have a size of about 100 - 1.000 Dalton, and

- at least 25% of the peptides have a size of less than about 100 Dalton.

Preferable, a fraction of the peptide preparation corresponding to peptide sizes of about 1200 to 200 Daltons has an amino acid composition as indicated in table 4.

Preferable, a fraction of the peptide preparation corresponding to peptide sizes of about 200 to 100 Daltons has an amino acid composition as indicated in table 4. Preferable, the relative amount of the amino acid arginine in the peptide preparation corresponding to peptide sizes of about 1200 to 200 Daltons is at least 20 %, preferable at least 40 %, more preferable about 60 % lower than compared to the fish protein hydrolysate.

Preferable, the relative amount of the amino acid arginine in the peptide preparation corresponding to peptide sizes of about 200 to 100 Daltons is at least 20 %, preferable at least 40 %, more preferable about 60 % higher than compared to the fish protein hydrolysate.

Preferable, the relative amount of the amino acid tyrosine in the peptide preparation corresponding to peptide sizes of about 1200 to 200 Daltons is 50%, more preferable 100%, more preferable 150 % higher than compared to the fish protein hydrolysate.

Preferable, the peptide preparation is used for the prevention and/or treatment of cardiovascular diseases, wherein said cardiovascular disease preferable is selected from the group consisting of atherosclerosis, angina, cerebrovascular accident (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack) and peripheral vascular disease.

A second aspect of the present invention relates to a process for the preparation of a peptide preparation, wherein said preparation is prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second enzymatic treatment with an enzyme preparation

manufactured from a strain of Aspergillus oryzae.

Preferable, said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan.

Preferable, said alkaline protease and neutral protease is a Bacillus protease complex, preferable Protamex™ from Novozymes AS Preferably, fat is separated from the peptide material after the first enzymatic treatment. Preferable, said fish is Salmonidae, preferable Atlantic salmon.

Preferably, said treatment is conducted at a pH of about 5 to about 9, preferable at a pH at about 6 to 8, preferable at a pH of about 6 to 7, or more preferable at a pH of about 6.5.

A third aspect of the present invention relates to a use of a peptide preparation as a food product or food supplement or nutraceutical preparation for the prevention and/or treatment of cardiovascular diseases, wherein said preparation is prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second enzymatic treatment with an enzyme preparation manufactured from a strain of Aspergillus oryzae. A fourth aspect of the present invention relates to a use of a peptide preparation for the manufacturing of a pharmaceutical composition for the prevention and/or treatment of cardiovascular diseases, wherein said preparation is prepared from a fish material by a first enzymatic treatment with an alkaline protease and a neutral protease resulting in a fish protein hydrolysate (FPH), and thereafter said fish protein hydrolysate (FPH) is subjected to a second enzymatic treatment with an enzyme preparation manufactured from a strain of Aspergillus oryzae.

Preferable embodiments (independently of each feature and aspect) of the third and fourth aspect of the present invention are given below;

Preferable, said cardiovascular disease is selected from the group consisting of atherosclerosis, angina, cerebrovascular accident (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack) and peripheral vascular disease.

Preferable, said cardiovascular disease is treated and/or prevented by increasing the level of high-density lipoprotein (HDL) in the blood.

Preferable, said cardiovascular disease is treated and/or prevented by increasing the ratio of HDL cholesterol to LDL cholesterol in the blood. Preferable, the peptide preparation can be used for the manufacturing of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of hypocholsterolemia. Preferable, the peptide preparation can be used for the manufacturing of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of underweight, undernourishment, malnutrition or malnourishment disorders.

Preferably, said disorders are prevented or treated by increasing the body mass index (BMI).

Preferable, said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan. Preferable, said alkaline protease and neutral protease is a Bacillus protease complex, preferable Protamex™ from Novozymes AS.

Preferable, said peptide preparation is substantially fat free. A fifth aspect of the present invention relates to a feed composition comprising a peptide preparation in accordance with any of the claims 1 to 16, or prepared by a process in accordance with any of the claims 17-23.

Preferable, said feed composition is used for increasing the weight of an animal fed said feed composition.

Preferable, said feed composition is used for increasing the weight gain and/or the specific growth rate of an animal, characterized by feeding the animal with a feed composition containing a peptide preparation according to claim 1.

Preferable, said animal is suffering of underweight, undernourishment, malnutrition or malnourishment. DEFINITIONS OF TERMS USED IN THE APPLICATION Animals

In this context the term "animals" include mammals such as humans and farm (agricultural) animals, especially the animals of economic importance such as gallinaceous birds, bovine, ovine caprine and porcine mammals, especially those that produce products suitable for the human consumption, such as meat, eggs and milk. Further, the term is intended to include fish and shellfish, such as salmon, cod, Tilapia, clams and oysters. The term also includes domestic animals such as dogs and cats.

Treatment

In relation to the pharmaceutical applications of the invention the term "treatment" refers to a reduction of the severity of the disease and/or to reduce symptoms of the disease.

Prevention

The tem "prevention" refers to the preventing of a given disease, i.e. a compound of the present invention is administered prior to the onset of the condition. This means that the compounds of the present invention can be used as prophylactic agents or as ingredients in functional foods or feed in order to prevent the risk or onset of a given disease, or to inhibit deterioration of a given disease. FPH - Enzyme treated fish protein hydrolvsate

We have throughout the specification and claims used the term "FPH" (or PFH material) for the specific fish protein prepared by one enzymatic treatment, i.e. the protein hydrolysate resulting from a primary enzymatic treatment of a fish material with the enzyme mixture Protamex™. The Protamex mixture contains an alkaline protease and a neutral protease. The FPH material contains high proportions of proteins and peptides and is used as a control in the experimental section. The peptide material of the present invention is different (due to the second enzymatic treatment) from this FPH material with regard to size distribution of the peptides, and has improved biological activities. Peptide material according to the invention

The peptide material according to the invention is based on a primary enzymatic treated protein material, and a secondary enzymatic treatment has been conducted in order to reduce the sizes of the peptide fragments.

Protease

A protease (also termed peptidase or proteinase) breaks down proteins. A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein.

Acid protease

An acid protease is a proteolytic enzyme with an pH optimum for activity below pH 5. One variant of the enzyme may be produced by fungus for Aspergillus niger.

ADMINISTRATION OF THE COMPOUNDS OF THE PRESENT INVENTION As a pharmaceutical medicament the materials of the present invention may be administered directly to the animal by any suitable technique, including parenterally, intranasally, orally, or by absorption through the skin. They can be administered locally or systemically. The specific route of administration of each agent will depend, e.g., on the medical history of the animal. The preferred administration route is orally.

Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration

If given continuously, the compounds of the present invention are each typically administered by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The key factor in selecting an appropriate dose is the result obtained, as measured by decreases in total body weight or ratio of fat to lean mass, or by other criteria for measuring control or prevention of obesity or prevention of obesity-related

conditions, as are deemed appropriate by the practitioner. For parenteral administration, in one embodiment, the compounds of the present invention are formulated generally by mixing each at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a

pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

Generally, the formulations are prepared by contacting the compounds of the present invention each uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier may suitably contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as

phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or non-ionic surfactants such as polysorbates, poloxamers, or PEG.

For oral pharmacological compositions such carrier material as, for example, water, gelatine, gums, lactose, starches, magnesium-stearate, talc, oils, polyalkene glycol, petroleum jelly and the like may be used. Such pharmaceutical preparation may be in unit dosage form and may additionally contain other therapeutically valuable substances or conventional pharmaceutical adjuvants such as preservatives, stabilising agents, emulsifiers, buffers and the like. The pharmaceutical preparations may be in conventional liquid forms such as tablets, capsules, dragees, ampoules and the like, in conventional dosage forms, such as dry ampulles, and as

suppositories and the like. In addition, the compounds of the present invention are appropriately administered in combination with other treatments for combatting or preventing a specific disease.

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.

A preferred embodiment of the present invention relates to a nutritional composition comprising the peptide material of the present invention formulated in any conventional way to a feed or food product.

FIGURE LEGENDS

Figure 1 shows the control (FPH) and various filtering fractions after treatment with the enzyme preparation Acid protease A at pH 3 as described in example 1. The X- axis shows the logMW for the peptides.

Figure 2 shows the effect of the peptide preparation E1 on weight gain relative to control (FPH).

Figure 3 shows the effect of the peptide preparation E1 on specific growth rate relative to control (FPH).

Figure 4 shows the effect of the peptide preparation E1 on the concentration of plasma TAG relative to control (FPH).

Figure 5 shows the correlation of weight gain and Plasma TAG relative as an effect of the peptide preparation E1 relative to control (FPH).

EXPERIMENTAL SECTION

Example 1

First enzymatic treatment - preparation of the fish protein hvdrolvsate (FPH) material used as a control in example 2

The control material is the enzyme treated fish protein hydrolyzate (FPH) and this material has several beneficial biological effects (as shown in PCT/NO04/00202) . FPH was produced from fish flesh remnants on salmon bone frames after filleting. Frames without heads from freshly filleted Atlantic salmon (Salmo salar, L.) were taken directly from the production line and frozen at -20 ± 2 °C: Within a week the frozen frames were used in the enzymatic hydrolyzing process. The enzymatic hydrolysis was performed with Protamex™ at a pH of about 6.5 and at temperature of 55 ± 2 °C. Protamex™ (E.C. 3.4.21.62/3.4.24.28) is a Bacillus protease complex from Novozymes AS (Bagsvaerd, Denmark) containing an alkaline protease and a neutral protease, and fulfils the purity demands for food-grad enzymes. The ratio of salmon frames to water was 1.14. An enzyme to substrate ratio of 1 1.1 AU/kg crude protein was used in the hydrolysis. After 60 min of enzymatic treatment the temperature was elevated to 98 °C, which was reached after 105 min.

Large bones were retained in the hydrolysing tank, while small bones were removed by filtering the hydrolysate through a mesh. Thereafter the insoluble fraction was removed in a two-phase separator (Westfalia, Germany, SC.35-26-177, 15 kW, 7200 rpm), before the remaining mixture was separated in a three-phase separator (Westfalia, Germany, SB-7-36-+76, 4 kW, 8520 rpm) into salmon oil, emulsion fraction and aqueous fraction. The aqueous fraction was concentrated in a 4 stage falling film evaporator (APV Anhydro) to a paste with about 60% dry matter.

The evaporated hydrolysate is termed fish protein hydrolyzate (FPH), i.e. the control sample of example 2, and contains about 83 % protein, 10 % ash and about 2 % lipids, based on dry weight. The amino acid compositions are given in table 1. Table 1

Total amino acids in the control sample, i.e. the hydrolysate obtained by hydrolyses of salmon frames with Protamex™.

Component Control FPH

Amino acids (g kg " crude protein)

Arg 59.4+0.7

His 39±1

He 27.5±0.4

Leu 56.4±0.1

Lys 63.7+0.3

Met 22+1

Phe 26.9+0.7

Thr 39+1

Trp 5.3+0.1

Val 35.5+0.4

Ala 74+1

Asn + Asp 73+3

Cysteine (total) 6.1+0.7

Gin + Glu 116±5

Gly 89+3

OH - Pro 20.7+0.7

Pro 47+1

Ser 37+2

Tyr 21±2

Tau 6.2+.1

Example 2

Secondary enzymatic treatment - Enzymatic treatment of FPH (control) with Acid Protease A at pH 3

Dilution of samples:

The substrate sample (FPH) (paste) was dissolved in preheated tap water (50 °C) to 10% dry matter. pH was adjusted to expected optimal pH for the enzyme using concentrated (37%) HCI. No pre-treatment of the sample (for instance filtration) was carried out.

Enzyme reactions

No specific inactivation of the enzymes was carried out after incubation. The inactivation of enzymes was done during the filtration steps and during evaporation of the fractions of interest. In these processing steps the temperature several times exceeded the inactivation temperature of the enzymes included in the study.

The pH was reduced to 3 by adding 4.0 litre of concentrated (37%) HCI to 36,8 kg hydrolysate (60% dm). The hydrolysate solution was treated by Acid Protease A, which is an acid proteolytic enzyme preparation available from Amano Enzyme Inc. The enzyme preparation is manufactured by an unique fermentation process of a selected strain of Aspergillus niger. Acid protease A is a slightly yellowish powder and soluble in water, stable in acid range of pH 3.0 to 6.0, with an optimum pH around 2.5. The enzyme preparation is non-pathogenic and applicable to all pharmaceutical, food and feed industries.

The hydrolysate solution was incubated with the enzyme preparation for 20 hours. The temperature was kept at 38 to 48 °C during working hours but was reduced to 23 °C over night.

Figure 1 shows the Peptide distribution of the control sample (FPH) and filtering fractions after treatment with Acid Protease A, at pH 3. The peptide distribution was determined as described in the Rubin report no 4617/1 15, 2004.

Fractionation by Filtration

The enzyme treated solution E1 was refined by icrofiltration and Ultrafiltration in solution with about 10% dry matter at 50 -60 °C. The filtrations were carried out in filtration unit (Membralox SD 3-A modules M-3P1940, Pall, USA) with ceramic membranes with 100 nm and 20 nm pore size (Membralox EP1940, Pall, USA). Only the permeates were collected for evaluation of bioactive peptides, although small samples of the retentates were collected for analytical purposes to have information about yield and performance during the specific filtration steps. Detailed information about performance during micro- and ultra-filtration are given in table 2, below. Table 2

Preparation of the peptide material according to the invention

The permeate after the ultrafiltration step was used for the biological experiments described in example 3, below.

Peptide size distribution

About 2 ml of the primary and secondary enzymatically treated solution E1 or a control sample (zero-sample) of FPH was added 20 ml buffer, mixed, centrifuged and filtered before application to the HPLC for separation by size exclusion chromatography as described in the Rubin report 4617/115, 204;

http://www.rubin.no/files/documents/4617-115_peptidstorre lse_hydrolysat2.pdf.

Collection of peptide fractions of specific length and analyses of these to

characterise the general amino acid profile of the specific peptide fraction obtained after enzyme treatment of the various proteolytic enzymes were evaluated. The following fractions were collected after size-exclusion chromatography by the method described in the Rubin report 4617/115, 204). Eluted liquid was collected at specific elution times to characterise the peptides present in the invented product. Below is given the elution time for samples collected to be analysed for amino acid profile: Fraction 1 : 18-22 min (corresponding to peptides of about 8500 - 1200 Daltons * )

Fraction 2: 22-26 min (corresponding to peptides of about 1200 - 200 Daltons * )

Fraction 3: 26-30 min (corresponding to peptides of about 200 - 100 Daltons * )

Fraction 4: 30-34 min (corresponding to peptides of about <100 Daltons * ) Fraction 5: 34-40 min

The size of the peptides are calculated in accordance with the correlation between elution time and logMW described in the Rubin report 4617/115, 2004.

Yields

The yields in permeate after ultra filtration were analysed by the difference between initial dry matter and retentates after micro- and ultra-filtration:

Yield = 100 - ((Dry matter of start hydrolysate - (micro-filtrate retentate dry matter + ultrafiltration retentate dry matter))/ (dry matter of start hydrolysate) *100)).

A yield of 79% was obtained for the E1 material according to the present invention.

Peptide size distribution:

Fig 1 shows the peptide distribution obtained in the various filtering fractions after enzymatic treatment with the enzymes Acid protease A at pH 3.

As can be seen from the above results, the enzymatic treatment has significantly reduced the amount of the largest peptides and to varying degree changed the proportion of the other peptide fractions. It should be kept in mind that the present enzyme studies are preliminary in the sense that the enzyme conditions and yield are not optimized. Amino acid analyses of collected fractions after size exclusion chromatography

The secondary enzyme treated preparations (example 2), i.e. the E1 material according to the present invention, and the primary enzyme treated preparation (example 1), i.e. the FPH (control), were collected and analysed for total content of amino acids. Only fraction 1 , 2 and 3 were analysed.

Fraction 1 : 18-22 min (corresponding to about 8500 - 1200 Daltons*

Fraction 2: 22-26 min (corresponding to about 1200 - 200 Daltons *

Fraction 3: 26-30 min (corresponding to about 200 - 100 Daltons *

Table 2 shows the relative amount of total amino acids detected in the various fractions. Generally the main peptides occurred in Fractions 2 and Fractions 3 for enzymes showing the highest yield in the enzyme hydrolyses.

Table 2

Relative amount of total amino acids in the size-fractions

after specific enzyme treatment

Table 3 show that the secondary enzyme treated (Acid Protease A) hydrolysate contain a higher level of amino acids present in smaller peptides (Fraction 2 and fraction 3) as compared to the control (FPH). Table 3, below, shows the relative amount of the various amino acids in fraction 2 and 3.

Table 3.

Relative amino acid content in Fraction 2 and Fraction 3 after enzyme treatments.

Amino acid analyses were analysed after hydrolysis in 6M for 22h at 110 °C by HPLC using a fluorescence technique for detection (Cohen and Michaud, Anal. Biochem. 1993, 2 1 , 279-287). Example 3

Biological activity of the enzyme preparations Animals and Husbandry

Eighty male Wistar rats (Mollegaard and Blomholtgaard, Denmark) 12 weeks old, were housed individually in Makrolon III cages in an open system. They were kept under standard laboratory conditions with temperature 22±1 °C, dark/light cycles of 12/12 h, relative humidity 55±5% and 20 air changes per hour.

The rats will have free access to the intervention diets on day 1 -29. All rats are killed on day 30 .

On day 30 all rats are anesthetized by inhalation of Sevoflurane 2 % in an anesthesia chamber. Thoracotomy, cardiac puncture, and exsanguination will be performed.

Sample Collection and Analysis of Biochemical Parameters during feeding period

Body weight and food intake (day 0-7-14-21 , 23-29). Blood samples (from leg, plasma) were drawn into a heparin-containing Vacutainer, placed on ice for 10 minutes, and centrifuged at 2000 rpm for 10 minutes at 4 °C. (day 0-22-30, and plasma were stored at -80°C).

Urine samples for measurements of protein, isoprostanes, sodium and creatinine (day 0-22-30, urine were stored at -80°C.

Stools were collected on day 29 and kept below -20°C until analysis for GMP. Briefly, 1 g of fecal material is diluted in 4 ml_ of extraction buffer and thoroughly

homogenized using an Ultra Turrax (20,000 rpm) before centnfugation at 45,000g for 20 minutes. The upper halves of the supematants are carefully harvested and run on a standard -step enzyme-linked immunosorbent assay (ELISA) as described previously (10).

Whole blood were spun, plasma taken out, and the sample were stored stored temporary on ice, and transferred to -80°C freezer. Fatty acids in plasma and rectal specimen were analysed as previously described. The same method were used for analysis of fatty acid profile in plasma and homogenized biopsy samples taken from rectal mucosa. The plasma lipid/fatty acids Triglycerides, Cholesterol, LDL-cholesterol, HDL-cholesterol, EPA, DHA,

docosapentaenoic acid (DPA, C22:5n-3), AA, total n-3 and total n-6 PUFAs were analysed

Preparation of subcellular fractions

Livers from the rats were homogenised individually in ice-cold sucrose-solution (0.25 mol/L sucrose in 10 mmol/L HEPES buffer pH 7.4 and 1 mmol/L EDTA) using a Potter-Elvehjem homogeniser. The subcellular fractions were isolated as described in Berge, R. K. et al (Berge, R. K., Flatmark, T. & Osmundsen, H. (1984),

Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators. Eur J Biochem 141 : 637-644). Briefly, the homogenate was centrifuged at 1 000 x g for 10 min to separate the post- nuclear from the nuclear fraction. A mitochondrial-enriched fraction was prepared from the post-nuclear fraction at 10 000 x g for 10 min. A peroxisome-enriched fraction was prepared by centrifugation of the post-mitochondrial fraction at 23 500 x g for 30 min. A microsomal-enriched fraction was isolated from the post-peroxisomal fraction at 100 000 x g for 75 min. The remaining supernatant was collected as the cytosolic fraction. The procedure was performed at 0-4°C, and the fractions were stored at -80°C. Protein was assayed using the BioRad protein assay kit (BioRad, Heraules, CA) and bovine serum albumin as standard. Lipid analysis

Lipids in whole liver and heparinised plasma were measured in the Tecnicon Axon system (Miles, Tarrytown, NY), with the Bayer triglyceride and cholesterol enzymatic kits (Bayer, Terrytown, NY) and the PAP 150 phospholipid enzymatic kit (bioMerieux, Lyon, France). Liver lipids were first extracted according to Bligh and Dyer (Bligh, E. G. & Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-91. Fatty acid composition

Fatty acids were extracted from the samples with 2:1 chloroform: methanol (v/v) (35). The samples were filtered, saponified and esterified in 12% BF 3 in methanol (v/v). Fatty acid composition of total lipids from liver and plasma was analysed using methods described by Lie and Lambertsen (Lie, O. & Lambertsen, G. (1991 ) Fatty acid composition of glycerophospholipids in seven tissues of cod (Gadus morhua), determined by combined high-performance liquid chromatography and gas chromatography. J Chromatogr 565: 119-129). Fatty acid methyl esters were separated using a Carlo Erba gas chromatograph ('cold on column' injection, 69°C for 20 s, increase at 25°C min 1 to 160°C and hold at 160°C for 28 min, increase at 25°C min "1 to 190°C and hold at 190°C for 17 min, increase at 25°C min 1 to 220°C and hold at 220°C for 9 min) equipped with a 50 m CP-sil 88 (Chrompack,

Middelburg, The Netherlands) fused silica capillary column (i.d. 0.32 mm). The fatty acids were identified by retention time using standard mixtures of methyl esters (Nu- Chek-Prep, Elyian, MN, USA). The fatty acid composition (weight percentage) was calculated using an integrator (Turbochrom Navigator, Version 4.0) connected to the GLC.

Lipids were extracted from plasma triacylglycerol-rich lipoprotein fraction using a mixture of chloroform and methanol, and separated by thin layer chromatography on silica gel plates (0.25mm Silica gel 60, Merck) developed in hexane-diethyl ether- acetic acid (80:20:1 , v/v/v) and visualized using Rhodamine 6G (0.05% in methanol, Sigma) and UV light. The spots were scraped off and transferred to tubes containing heneicosanoic acid (21 :0) as internal standard. BF 3 -methanol was added to the samples for transesterification. To remove neutral sterols and non-saponifiable material, extracts of fatty acyl methyl esters were heated in 0.5mol/L KOH in ethanol- water solution (9:1). Recovered fatty acids were then re-esterified using BF 3 - methanol. The methyl esters were analyzed on a GC8000Top gas chromatograph (Carlo Erba Instrument), equipped with a flame ionization detector (FID),

programmable temperature of vaporization injector, AS 800 autosampler (Carlo Erba Instrument) and a capillary column (60m x 0.25mm) containing a highly polar SP 2340 phase with film thickness 0.20μητι (Supelco). The initial temperature was 30°C, heating 1.4°C/min to final temperature 214°C. The injector temperature was 235°C. The detector temperature was 235°C, using hydrogen (25ml_/min), air (350 mL/min) and nitrogen as make-up gas (30mL/min). The samples were run with constant flow using hydrogen as a carrier gas (1.6 mUmin). The splitting ratio was 20:1. The methyl esters were positively identified by comparison to known standards (Larodan Fine Chemicals, Malmo, Sweden) and verified by mass spectrometry. Quantification of the fatty acids was made with Chrom-Card A D 1.0 chromatography station (Carlo Erba Instruments) based on heneicosanoic acid as an internal standard.

Isolation of plasma triacylglvcerol-rich lipoprotein fraction

Plasma triacylglycerol-rich lipoprotein fraction was prepared by ultracentrifugation of 3 mL plasma at a density of 1.063 g/mL for 19 hr at 105 000 x g at 15°C. The tubes were sliced, and the floating fraction in the top 1 mL of each tube was harvested. The fraction was then dialyzed against 150 mmol/L sodium chloride, 16 mmol/L sodium phosphate and 4 mmol/L potassium phosphate, pH 7.4, saturated with nitrogen.

Results of biological activity

The biological experimental data for the enzyme preparation E1 are shown in the table 4 below and the enclosed figures 2 to 5.

Table 4

A summary of biological activities for the E1 material, i.e. the material according to the invention where the control material has been treated with the Acid protease A, at pH 3

Compared to the control material, the E1 material of the present invention reduces the weight gain with about 33%. Also the specific growth rate is significantly reduced.

It is thus anticipated that the E1 material of the present invention can be used as a weight lowering agent, for instance for the prevention or treatment of an overweight or obese condition.

The initial weight of the rats where (average) 27.3 g and 27.9 g for the control and treatment groups, respectively, and the final weight (after feeding) were 35.1 and 33.1 , respectively. This shows that the weight of the E3 fed rats were lowered by 2.6 grams (corrected for difference in initial weight) compared to the control group. The peptides of the present invention have thus a general weight lowering effect of about 5.9 %. The table 4 also shows that the plasma level of triacylglycerols (TAGs) are

significantly reduced for the E1 material compared to the control (FPH). High levels of triglycerides in the bloodstream have been linked to atherosclerosis, and the risk of heart disease and stroke. We thus anticipate that the present inventive E1 material can be used in the prevention and/or treatment of hypertriglyceridemia, atherosclerosis, heart diseases and stroke.