NO20100359A1 - Peptide material and preparations and uses thereof. - Google Patents

Description

Peptide material and preparations and uses thereof.

FIELD OF THE INVENTION.

The present invention relates to a peptide preparation, a method for producing a peptide preparation, and applications thereof.

BACKGROUND OF THE INVENTION.

The fish farming industry has grown enormously both in Norway and the rest of the world in recent years, especially salmon farming. Most of the fish is sold to the consumer who slaughtered whole fish, but significant quantities are sold as fillets. Only 50-70% of the salmon are fillets, while the rest are sold as by-products such as fishmeal and fish silage.

Through enzymatic treatment of the fish meat and also the fish skeleton, it can be separated into an aqueous fraction that is rich in proteins, called fish protein hydrolyzate (FPH). The enzymatic hydrolysis process can be controlled, and the products are reproducible and well defined.

Such a protein material, i.e. fish protein hydrolyzate (FPH) has several useful biological effects, and such a material can be used as a pharmaceutical material and as an embalming material. The applicant's own patent application PCT/NO04/00202 describes the use of such an FPH material lowers the concentration of plasma cholesterol and homocysteine, and also lowers the concentration of hepatic triacylglycerols.

We have now surprisingly found that we can process an enzyme-treated protein material further, i.e. process the enzyme-treated protein material in a secondary second treatment and that we can obtain a new peptide material that is different with respect to the size distribution of the peptide fragments.

We have produced several new peptide materials and we have surprisingly demonstrated 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 that has only been treated once with one enzyme. These new materials can be used to better tailor the materials for specific nutritional effects, medical indications or diseases.

The specific peptide material according to the present invention, designated as E1, represents an improvement of the known fish protein hydrolyzate (FPH) which has been treated with only a primary enzyme treatment. We assume that enzyme-treated non-fish protein materials can also be used as a basis for a second enzymatic treatment, and the present invention is thus not limited to fish protein materials.

Without being bound by theory, we assume 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 particularly useful as a functional protein in nutritional products, especially when it is used as a substitute for natural plasma in animal feed and in feed for pets. If it is used in food or food for pets, additional ingredients can be added to the product such as fat, sugar, salt, flavourings, minerals, etc. The product can then be formed into lumps that resemble natural meat lumps in appearance and texture. The product according to the invention has the further advantages that it can be easily formulated to contain the necessary nutrients, that it is easy for the animals to digest and palatable for the animals.

The peptide material in accordance with the present invention can also be used for the production of a nutraceutical or pharmaceutical composition for the prevention and/or treatment of various diseases, as indicated in the claims section.

DETAILED DESCRIPTION OF THE INVENTION.

A first aspect of the present invention relates to a peptide preparation produced by enzymatic treatment of a protein material where the proteins are cut into smaller peptide fragments, characterized in that the size of the peptide fragments is from 0 to 10,000 Daltons, more preferably from 0 to 1000 Daltons, and more preferably from 0 to 100 Daltons.

A preferred embodiment concerns the use of a fish protein material, preferably a material from salmon.

In a preferred embodiment, the peptide preparation is produced by enzymatic treatment with an acid protease, preferably Acid Protease A.

A further preferred embodiment also involves an enzymatic treatment with a Bacillus protease complex, preferably before the treatment with an acid protease.

A preferred embodiment relates to a peptide preparation in which at least 75% of the peptides have a size of 1000 Daltons or less.

More preferably, at least 35% of the peptides have a size of about 100-1000 Dalton, more preferably that at least 40% of the peptides have a size of approx. 100-1000 Dalton, and more preferably that approx. 50% of the peptides have a size of approx. 100-1000 Daltons.

More preferably, at least 25% of the peptides have a size of less than about 100 Dalton, more preferably that at least 28% of the peptides have a size of less than approx. 100 Daltons.

More preferably,

- at least 35% of the peptides have a size of approx. 100-1000 Dalton, more preferably that at least 40% of the peptides have a size of approx. 100-1000 Dalton, and more preferably that approx. 50% of the peptides have a size of approx. 100-1000 Dalton, and - at least 25% of the peptides have a size of less than approx. 100 Dalton, more preferably that at least 28% of the peptides have a size of less than approx. 100 Dalton.

More preferably, a fraction of the peptide preparation corresponding to peptide size of approx. 1200 to 200 Daltons have an amino acid composition as indicated in Table 3.

More preferably, a fraction of the peptide preparation corresponding to peptide sizes of approx. 200 to 100 Daltons have amino acid composition as indicated in Table 3.

Preferably,

- a fraction of the peptide preparation corresponding to peptide sizes of approx. 1200 to 200 Dalton has an amino acid composition as indicated in table 3, and - a fraction of the peptide preparation corresponding to peptide sizes of approx. 200 to 100 Daltons of an amino acid composition as set forth in Table 3.

An embodiment of this aspect relates to a peptide preparation where the relative amount of the amino acid arginine is at least 5% higher than compared to fish protein hydrolysates.

A preferred embodiment relates to a peptide preparation where the relative amount of the amino acid arginine in the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Dalton is at least 5% higher compared to the fish protein hydrolyzate.

A preferred embodiment relates to a peptide preparation where the relative amount of the amino acid arginine in the peptide preparation corresponding to peptide sizes of approx. 200 to 100 Daltons is at least 5%, more preferably 10%, more preferably 20% and more preferably 30% higher compared to the fish protein hydrolyzate.

A further embodiment of this aspect relates to a peptide preparation where the relative amount of the amino acid tyrosine is 5%, more preferably 10%, more preferably 15% lower than compared to the fish protein hydrolyzate.

A preferred embodiment relates to a peptide preparation where the relative amount of the amino acid tyrosine in the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Dalton is 5%, more preferably 10%, more preferably 15% lower than compared to the fish protein hydrolyzate.

A preferred embodiment relates to a peptide preparation where the relative amount of the amino acid tyrosine in the peptide preparation corresponding to a peptide size of approx. 200 to 100 Dalton is 20%, more preferably 30%, more preferably 40%, more preferably 50% lower than compared to the fish protein hydrolyzate.

A second aspect of the present invention relates to a method for producing a peptide preparation, where the method comprises a step where a protein or peptide material is treated with an acid protease to reduce the sizes of the peptide fragments.

Preferably, said acid protease is Acid Protease A.

Preferably, the method includes a further enzymatic treatment of material, preferably before the treatment with an acid protease, and preferably said further enzymatic treatment is with a Bacillus protease complex.

A third aspect of the present invention relates to the use of a peptide preparation in accordance with the features indicated above, for the production of a pharmaceutical or nutraceutical preparation to prevent and/or treat hypertriglyceridemia, atherosclerosis, heart disease, overweight or obesity.

DEFINITIONS OF TERMS USED IN THE APPLICATION.

Animals

In this context, the term "animal" includes mammals such as humans and livestock (agricultural animals), especially animals of economic importance such as fowl, bovine, ovine, caprine and porcine animals, especially those that produce products suitable for human consumption, such as meat, eggs and milk. Furthermore, 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" denotes a reduction in the severity of the disease.

Obstacle

The term "obstacle" refers to preventing a given disease, that is, a compound of the present invention is administered before the onset of the condition. This means that the compounds according to the present invention can be used as prophylactic agents or as ingredients in functional foods or to prevent the risk of starting a given disease.

FPH - Enzyme-treated fish protein hydrolvsate

The FPH material is a protein hydrolyzate resulting from a primary enzymatic treatment of a fish material with the enzyme Protamex™. The FPH material contains high proportions of proteins and peptides and is used as a control in the experimental section. The peptide material according to the present invention differs from this FPH material with respect to the size distribution of the peptides and biological activity.

Peptide material in accordance with the invention

The peptide material in accordance with the present invention is based on a primary enzymatically treated protein material, and a secondary enzymatic treatment has been carried out to reduce the sizes of the peptide fragments.

Protease

A protease, (also called peptidase or proteinase) breaks down the proteins. A protease is an enzyme that performs proteolysis, that is, starts protein carabolism by hydrolyzing peptide bonds that connect amino acids together in the polypeptide chain that forms the protein.

Acid protease

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

ADMINISTRATION OF THE COMPOUNDS ACCORDING TO THE PRESENT INVENTION.

As a pharmaceutical drug, 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. It can be administered locally or systemically. The specific route of administration for each agent will depend, for example, on the medical history of the animal. The preferred route of administration is orally.

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

If they are given continuously, the compounds according to the present invention are typically administered with 1-4 injections per day or with continuous subcutaneous infusions, for example by using a mini pump. An intravenous bag solution can also be used. The key factor in choosing a suitable dosage is the result to be achieved, as measured by reduction in total body fat or the ratio of fat to lean mass, or by other criteria to measure the control or prevention of obesity or to prevent obesity-related conditions, such as is appropriate by the practitioner.

For parenteral administration, in one embodiment, the compounds of the present invention are generally formulated by mixing each in a desired degree of unity, in a unit dosage injectable form (solution, suspension or emulsion), with a pharmaceutically acceptable carrier, that is, a which is non-toxic to the recipient in the dosages and concentrations used and which is compatible with the other ingredients in the formulation.

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

The carrier can appropriately contain smaller amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to the recipients in the dosages and concentrations used, 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 polyvinyl polydone; 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; exercise such as sodium; and/or non-ionic surfactants such as polysorbates, poloxamers or PEG.

For oral pharmacological compositions, such carrier materials can be, for example, water, gelatin, gum, lactose, starches, magnesium stearate, tallow, oils, polyalkene glycol, petroleum jelly and the like used. Such pharmaceutical preparations may be in unit dosage form and may further contain other therapeutically valuable substances or conventional pharmaceutical adjuvants such as preservatives, stabilizing agents, emulsifying agents, buffers and the like. The pharmaceutical preparations can be in conventional liquid forms such as tablets, capsules, dragees, ampoules and the like, in conventional dosage forms such as dry ampoules, and as suppositories and the like.

In addition, the compound of the present invention is conveniently administered in combination with other treatments to combat or prevent specific disease.

The invention will be better understood with reference to the following examples. However, these should not be considered as limiting the scope of the invention.

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

FIGURE EXPLANATIONS

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

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

Figure 3 shows the effect of the peptide preparation E1 for specific growth rate relative to control. Figure 4 shows the effect of the peptide preparation E1 on the concentration of plasma TAG relative to control. Figure 5 shows correlation of weight gain and relative plasma TAG as an effect of the peptide preparation E1 relative to control.

EXPERIMENTAL SECTION

Example 1

Primary enzymatic treatment, preparation of the hvdrolvsat material used as a control in example 2.

The control material is an enzyme-treated fish protein hydrolyzate (FPH) and has several useful biological effects.

FPH was produced from fish meat residues of salmon bone skeletons after filleting. The headless skeletons 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 bodies were used in the enzymatic hydrolysis process.

The enzymatic hydrolysis was carried out with Protamex™ at a pH of approx. 6.5 and at a 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) and meets the purity requirements for nutritionally graded enzymes. The ratio of salmon carcasses to water was 1.14. An enzyme to substrate ratio of 11.1 AU/kg raw protein was used in the hydrolyses. After 60 minutes of enzymatic treatment, the temperature was increased to 98 °C, which was reached after 105 minutes.

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

The evaporated hydrolyzate is referred to as fish protein hydrolyzate (FPH), i.e. the control samples in example 2, and contains approx. 83% protein, 10% ash and approx. 2% lipids, based on dry weight. The amino acid composition is given in Table 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. The pH was adjusted to the expected optimal pH for the enzyme using concentrated (47%) HCl. No pretreatment of the sample (eg filtration) was performed.

Enzyme reactions

No specific inactivation of the enzyme was performed after incubation. The inactivation of the enzymes was carried out during the filtration step and during evaporation of the relevant fractions. In these processing steps, the temperature exceeded several times the inactivation temperature of the enzymes included in the experiment.

The pH was reduced to 3 by adding 4.0 liters of concentrated (37%) HCI to 36.8 kg of hydrolyzate (60% solids). The hydrolyzate solution was treated with Acid Protease A, which is an acidic proteolytic enzyme preparation available from Amano Enzyme Inc. The enzyme preparation is produced by a unique fermentation process of a selected strain of Aspergillus niger. Acid protease A is a faint yellow powder and soluble in water, stable in the acidic pH range from 3.0 to 6.0, with an optimal pH around 2.5. The enzyme preparation is non-pathogenic and can be applied within the pharmaceutical industry, and the nutrition and food industry.

The hydrolyzate solution was incubated with the enzyme preparation for 20 hours. The temperature was maintained at 38-48 °C during working hours but was reduced to 23 °C overnight.

Figure 1 shows the peptide distribution of the control sample (FPH) and filtration fractions after treatment with Acid Protease A, at pH 3. The peptide distribution was determined as described in Rubin report number 4617/115, 2004.

Fractionation with filtration

The enzyme-treated solution E1 was refined with microfiltration and ultrafiltration in solution with approx. 10% solids at 50-60 °C. The filtrations were carried out in a filtration unit (Membralox SD 3-A modules M-3P1940, Pall.USA) with ceramic membranes with 100 nm and 20 nm pore size (Membralox EP 1940, Pall, USA). Only the permeates were collected for evaluation of bioactive peptides, although small samples of the retentates were collected for analytical purposes to provide information on yield and performance during the specific filtration steps.

Detailed information on performance during micro and ultrafiltration is given in Table 2, below.

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 to 20 ml of buffer, mixed, centrifuged and filtered before application to HPLC for separation by size exclusion chromatography as described in Rubin report 4617/115, 204;

http://www.rubin.no/files/documents/4617-115_peptidstorrelse_hydrolysat2.pdf.

Collection of peptide fractions of specific length and analyzes of these to characterize the general amino acid profile of the specified peptide fraction obtained after enzyme treatment with the various protolytic enzymes were evaluated. The following fractions were collected after size exclusion chromatography using the method described in the Rubin report 4617/115, 204). Eluted liquid was collected at specific elution times to characterize the peptides present in the inventive product. Below are the elution times for samples collected to be analyzed for amino acid profile: Fraction 1: 18-22 min (corresponding to peptides of approx. 8500 - 1200 Dalton<*>) Fraction 2: 22-26 min (corresponding to peptides of approx. 1200 - 200 Dalton<*>) Fraction 3: 26-30 min (corresponding to peptides of approx. 200 - 100 Dalton<*>) Fraction 4: 30-34 min (corresponding to peptides of approx. <100 Dalton<*>)

Fraction 5: 34-40 min

The size of the peptides is calculated according to the correlation between elution time and logMW described in the Rubin report 4617/115, 2004.

Dividends

The yields in the permeate after ultrafiltration were analyzed with the difference between the initial dry matter and the retentate after micro- and ultrafiltration: Yield = 100 - (dry matter at initial hydrolyzate - (microfiltrate retentate dry matter + ultrafiltration retentate dry matter))/ (dry matter at start hydrolyzate)<*>100)).

A yield of 79% was obtained on the E1 material in accordance with the present invention.

Peptide size distribution:

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

As can be seen from the results above, the enzymatic treatment significantly reduced the quantity of the largest peptides and to varying degrees changed the ratio of the other peptide fractions. It should be noted that the present enzyme studies are preliminary in the sense of the enzyme conditions and the yield has not been optimized.

Amino acid analysis of pooled fractions by size exclusion chromatography. The secondary enzyme-treated preparations (example 2), i.e. the E1 material in accordance with the present invention, and the primary enzyme-treated preparation (example 1), i.e. FPH (control), were collected and analyzed for total content of amino acids. Only fractions 1, 2 and 3 were analyzed.

Fraction 1: 18-22 min (corresponding to approx. 8500 - 1200 Dalton<*>

Fraction 2: 22-26 min (corresponding to approx. 1200 - 200 Dalton<*>

Fraction 3: 26-30 min (corresponding to approx. 200 - 100 Dalton<*>

Table 2 shows the relative amount of total amino acids detected in the different fractions. In general, the main peptides occurred in fractions 2 and fractions 3 for the enzymes showing the highest yield in the enzyme hydrolyses.

Table 3 shows that the hydrolyzate treated with a second enzyme (acid protease A) contains a higher level of amino acids present in smaller peptides (fraction 2 and fraction 3) compared to control (FPH).

Table 3, below, shows the relative amount of the different amino acids in fractions 2 and 3.

Amino acid analysis was analyzed after hydrolysis in 6 M in 221 at 110°C by HPLC using a fluorescence detection technique (Cohen and Michaud, Anal. Biochem. 1993, 211, 279-287).

Example 3

Biological activity of the enzyme preparations

Animals and animal husbandry

80 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 12/121, relative humidity 55±5% and 20 air changes per hour.

The rats have free access to the intervention diets on days 1-29. All rats were killed on day 30. On day 30, all rats were anesthetized by inhalation of Sevoflurane 2% in an anesthesia chamber. Thoracotomy, cardiac puncture and blood drainage were performed.

Sample collection and analyzes of biochemical parameters during the feeding period.

Body weight and intake. (days 0-7-14-21, 23-29)

Blood samples (from thigh, plasma) were collected in 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 was stored at -80 °C).

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

Stool samples were collected on day 29 and kept below 20 °C until analysis for GMP. Briefly, 1 g of the fecal material is diluted in 4 ml of extraction buffer and thoroughly homogenized using an Ultra Turrax (20,000 rpm) before centrifugation at 45,000 g for 20 minutes. The upper halves of the supernatant are carefully harvested and run on a standard 1-step enzyme-linked immunosorbent assay (ELISA) as described previously (10).

Whole blood was spun, plasma withdrawn, and the sample was stored temporarily on ice, and transferred to a -80 °C freezer.

Fatty acids in plasma and in rectal samples were analyzed as previously described. The same method was used for analysis of the fatty acid profile in plasma and homogenized biopsy samples taken from the rectal mucosa. Plasma lipid/fatty acids, triglycerides, cholesterol, LDL cholesterol, HDL cholesterol, EPA, DHA, docosapentanoic acid (DPA, C22:5n-3), AA, total n-3 and total n-6 PUFAs were analyzed.

Preparation of subcellular fractions.

Livers from the rats were homogenized 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 homogenizer. 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 1000 x g for 10 min to separate the post-nuclear from 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-microchondrial fraction at 23500 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 resulting supernatant was collected as the cytosolic fraction. The procedure was carried out at 0-4 °C, and the fractions were stored at - 80 °C. Protein was analyzed using the BioRad protein analysis kit (BioRad, Heraules, CA) and bovine serum albumin as a standard.

Lipid analyses

Lipids in whole liver and heparinized plasma were measured in the Tecnicon Axon system (Miles, Tarrytown, NY), with the Bayer triglyceride and cholesterol enzymatic kit (Bayer, Terrytown, NY) and the PAP 150 phospholipid enzymatic kit (bioMérieux, 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% BF3 in methanol (v/v). The fatty acid composition of total lipids from liver and plasma was analyzed using the 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, ramp 25 °C min"<1>to 160 °C and hold at 160 °C for 28 min , increasing at 25 °C min"<1>to 190 °C and holding at 190 °C for 17 min, increasing at 25 °C min"<1>to 220 °C and holding at 220 °C for 9 min) equipped with a 50 m CP-sil 88 (Chrompack, Middelburg, The Netherlands) fumed 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) coupled to the GLC.

Lipids were extracted from plasma triacylglycerol-enriched lipoprotein fraction using a mixture of chloroform and methanol, and separated by thin-layer chromatography on silica gel plates (0.25 mm 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 an internal standard. BF3 methanol was added to the samples for transesterification. To remove neutral sterols and unsaponified material, extracts of fatty acyl methyl esters were heated in 0.5 mol/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 vapor 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 0.20 µm (Supelco). Initial temperature was 130 °C, heating 1.4 °C/min to final temperature 214 °C. The injector temperature was 235 °C. Detector temperature was 235 °C, using hydrogen (25 ml/min), air (350 ml/min) and nitrogen as make-up gas (30 ml/min). The samples were run at constant flow using hydrogen as carrier gas (1.6 ml/min). The split 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 performed with Chrom-Card A/D 1.0 chromatography station (Carlo Erba Instruments) based on heneicosanoic acid as an internal standard.

Isolation of plasma triacvlglvserol-rich lipoprotein fraction

Plasma triacylglycerol-rich lipoprotein fraction was prepared by ultracentrifugation of 3 ml of plasma with a density of 1.063 g/ml for 19 h at 105000 x g at 15 °C. The tubes were divided, and the liquid 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 7.5, saturated with nitrogen.

Results of biological activity.

The biological experimental data for enzyme preparation E1 are shown in Table 4 below and given in Figures 2 to 5.

Compared to the control material, E1 material according to the present invention reduces the increase in weight by approx. 33%. The specific growth rate is also significantly reduced. It is thus considered that the E1 material according to the present invention can be used as a weight-reducing agent, for example to prevent or treat an overweight or obese condition.

Table 4 also shows that the plasma level of triacylglycerols (TAGs) is significantly reduced for the E1 material compared to control (FPH). High levels of triglycerides in the bloodstream have been linked to atherosclerosis, and the risk of heart disease and stroke. It is thus considered that the present inventive E1 material can be used to prevent and/or treat hypertriglyceridemia, atherosclerosis, heart diseases and strokes.

Claims (42)

1. Peptide preparation produced by enzymatic treatment of a protein material where the proteins are cut into smaller peptide fragments, characterized in that the size of the peptide fragments is from 0 to 10,000 Daltons, more preferably from 0 to 10,000 Daltons, and more preferably from 0 to 1,000 Daltons, and more preferably from 0 to 100 Daltons. 2. Peptide preparation in accordance with claim 1, characterized in that said protein material is a fish protein material. 3. Peptide preparation in accordance with claim 2, characterized in that said fish protein material is a protein material from salmon. 4. Peptide preparation in accordance with one of the preceding claims, characterized in that the peptide preparation is produced by enzymatic treatment with an acid protease. 5. Peptide preparation in accordance with claim 4, characterized in that said acid protease is Acid Protease A. 6. Peptide preparation in accordance with claim 4 or 5, characterized in that said enzymatic treatment also involves an enzymatic treatment with a Bacillus protease complex, preferably before the treatment with an acidic protease. 7. Peptide preparation in accordance with one of the preceding claims, characterized in that at least 75% of the peptides have a size of 1000 Daltons or less. 8. Peptide preparation in accordance with one of the preceding claims, characterized in that at least 35% of the peptides have a size of approx. 100-1000 Dalton, more preferably that at least 70% of the peptides have a size of approx. 100-1000 Dalton, and more preferably that approx. 50% of the peptides have a size of approx. 100-1000 Daltons. 9. Peptide preparation in accordance with one of the preceding claims, characterized in that at least 25% of the peptides have a size of less than approx. 100 Dalton, more preferably that at least 28 5 of the peptides have a size of less than approx. 100 Daltons. 10. Peptide preparation in accordance with one of the preceding claims, characterized in that - at least 35% of the peptides have a size of approx. 100-1000 Dalton, more preferably that at least 40% of the peptides have a size of approx. 100-1000 Dalton, and more preferably that approx. 50% of the peptides have a size of approx. 100-1000 Dalton, and - at least 25% of the peptides have a size of less than approx. 100 Dalton, more preferably that at least 28% of the peptides have a size of less than approx. 100 Daltons. 11. Peptide preparation in accordance with one of the preceding claims, characterized in that a fraction of the peptide preparation corresponding to peptide sizes of approx. 1200 to 200 Daltons have an amino acid composition as indicated in Table 3. 12. Peptide preparation in accordance with one of the preceding claims, characterized in that a fraction of the peptide preparation corresponding to a peptide size of approx. 200 to 100 Daltons have an amino acid composition as indicated in Table 3. 13. Peptide preparation in accordance with one of the preceding claims, characterized in that: - a fraction of the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Dalton has an amino acid composition as indicated in table 3, and - a fraction of the peptide preparation corresponding to a peptide size of approx. 200 to 100 Daltons have an amino acid composition as indicated in Table 3. 14. Peptide preparation in accordance with one of the preceding claims, characterized in that the relative amount of the amino acid arginine is at least 5% higher compared to the fish protein hydrolyzate. 15. Peptide preparation in accordance with claim 14, characterized in that the relative amount of the amino acid arginine in the peptide preparation corresponding to peptide sizes of approx. 1200 to 200 Dalton is at least 5% higher than compared to the fish protein hydrolyzate. 16. Peptide preparation in accordance with claim 14, characterized in that the relative amount of the amino acid arginine in the peptide preparation corresponding to a peptide size of approx. 200 to 100 Daltons is at least 5%, more preferably 10%, more preferably 20% and more preferably 30% higher than compared to the fish protein hydrolyzate. 17. Peptide preparation in accordance with one of the preceding claims, characterized in that the relative amount of the amino acid tyrosine is 5%, more preferably 10%, more preferably 15% lower than compared to the fish protein hydrolyzate. 18. Peptide preparation in accordance with claim 17, characterized in that the relative amount of the amino acid tyrosine in the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Dalton is 5%, more preferably 10%, more preferably 15% lower than compared to the fish protein hydrolyzate. 19. Peptide preparation in accordance with claim 17, characterized in that the relative amount of the amino acid tyrosine in the peptide preparation corresponding to peptide sizes of approx. 200 to 100 Dalton is 20%, more preferably 30%, more preferably 40%, more preferably 50% lower than compared to the fish protein hydrolyzate. 20. Method for producing a peptide preparation, characterized in that the method comprises a step where a protein or peptide material is treated with an acid protease to reduce the size of the peptide fragments. 21. method in accordance with claim 20, characterized in that said acid protease is Acid Protease A. 22. Method according to one of claims 20 to 21, characterized in that the method includes a further enzymatic treatment of the material, preferably before the treatment with an acid protease. 23. Method in accordance with claim 22, characterized in that said further enzymatic treatment is with a Bacillus protease complex. 24. Method in accordance with one of claims 20-23, characterized in that said treatment with an acidic protease is carried out at a pH of approx. 2 to approx. 5, preferably at a pH of approx. 2.5 to 4.5, preferably at a pH of approx. 3 to 4, or more preferably at a pH of approx. 3. 25. Use of a peptide preparation according to claim 1, for the production of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of hypertriglyceridemia, atherosclerosis, heart disease, overweight or obesity. 26. Use in accordance with claim 25, where said protein material is a fish protein material. 27. Use in accordance with claim 25, where the peptide preparation is prepared by enzymatic treatment with an acid protease. 28. Use in accordance with claim 27, wherein said acid protease is Acid Protease A. 29. Use in accordance with claim 28, where said enzymatic treatment also involves an enzymatic treatment with a Bacillus protease complex, preferably before the treatment with an acid protease. 30. Use in accordance with claim 25, wherein at least 75% of the peptides have a size of 1000 Daltons or less. 31. Use in accordance with claim 25, where at least 35% of the peptides have a size of approx. 100-1000 Dalton, more preferably that at least 40% of the peptides have a size of approx. 100-1000 Dalton, and more preferably that approx. 50% of the peptides have a size of approx. 100-1000 Daltons. 32. Use in accordance with claim 25, where at least 25 5 of the peptides have a size of less than approx. 100 Dalton, more preferably that at least 28% of the peptides have a size of less than approx. 100 Daltons. 33. Use in accordance with claim 25, where - at least 35% of the peptides have a size of approx. 100-1000 Dalton, more preferably that at least 40 5 of the peptides have a size of approx. 100-1000 Dalton, and more preferably that approx. 50 5 of the peptides have a size of approx. 100-1000 Dalton, and - at least 25% of the peptides have a size of less than approx. 100 Dalton, more preferably that at least 28 5 of the peptides have a size of less than approx. 100 Daltons. 34. Use in accordance with claim 25, where a fraction of the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Daltons have an amino acid composition as indicated in Table 3. 35. Use in accordance with claim 25, where a fraction of the peptide preparation corresponds to peptide sizes of approx. 200 to 100 Daltons of an amino acid composition as set forth in Table 3. 36. Use in accordance with claim 25, where: - a fraction of the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Dalton has an amino acid composition as indicated in table 3, and - a fraction of the peptide preparation corresponding to a peptide size of approx. 200 to 100 Daltons have an amino acid composition as indicated in Table 3. 37. Use in accordance with one of the preceding claims, where the relative amount of the amino acid arginine is at least 5% higher than compared to the fish protein hydrolyzate. 38. Use in accordance with claim 37, where the relative amount of the amino acid arginine in the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Dalton is at least 5% higher than compared to the fish protein hydrolyzate. 39. Use in accordance with claim 37, where the relative amount of the amino acid arginine in the peptide preparation corresponding to a peptide size of approx. 200 to 100 Daltons is at least 5%, more preferably 10%, more preferably 20% and more preferably 30% higher than compared to the fish protein hydrolyzate. 40 . Use in accordance with one of the preceding claims, where the relative amount of the amino acid tyrosine is 5%, more preferably 10%, more preferably 15% lower than compared to the fish protein hydrolyzate. 41. Use in accordance with claim 40, where the relative amount of the amino acid tyrosine in the peptide preparation corresponding to a peptide size of approx. 1200 to 200 Dalton is 5%, more preferably 10%, more preferably 15% lower than compared to the fish protein hydrolyzate. 42. Use in accordance with claim 40, where the relative amount of the amino acid tyrosine in the peptide preparation corresponding to a peptide size of approx. 200 to 100 Dalton is 20%, more preferably 30%, more preferably 40%, more preferably 50% lower than compared to the fish protein hydrolyzate.