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WO2011112099A1 - Matériau peptidique, ses préparations et ses utilisations - Google Patents

Matériau peptidique, ses préparations et ses utilisations Download PDF

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Publication number
WO2011112099A1
WO2011112099A1 PCT/NO2011/000078 NO2011000078W WO2011112099A1 WO 2011112099 A1 WO2011112099 A1 WO 2011112099A1 NO 2011000078 W NO2011000078 W NO 2011000078W WO 2011112099 A1 WO2011112099 A1 WO 2011112099A1
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WIPO (PCT)
Prior art keywords
accordance
preparation
peptide
preferable
enzyme
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PCT/NO2011/000078
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English (en)
Inventor
Anders Aksnes
Rolf Kristian Berge
Kjartan Sandnes
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Marine Bioproducts AS
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Marine Bioproducts AS
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Publication of WO2011112099A1 publication Critical patent/WO2011112099A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/22—Animal feeding-stuffs from material of animal origin from fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21026—Prolyl oligopeptidase (3.4.21.26), i.e. proline-specific endopeptidase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a peptide preparation, a process for preparation of a peptide preparation, and uses thereof.
  • 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
  • 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 ⁇ -oxidation.
  • the hydrolysate is produced from fish flesh remnants on salmon bone frames after filleting, and the enzymatic hydrolysis was performed with ProtamexTM 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 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.
  • E1 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.
  • FPH fish protein hydrolysate
  • 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.
  • 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.
  • 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.
  • 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.
  • said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan.
  • said alkaline protease and neutral protease is a Bacillus protease complex, preferable ProtamexTM from Novozymes AS.
  • said peptide preparation is substantially fat free.
  • said fish is Salmonidae, preferable Atlantic salmon.
  • said fish is Atlantic salmon
  • the enzyme of the first enzymatic treatment is the Bacillus protease complex ProtamexTM 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.
  • at least 60% of the peptides have a size of 1000 Daltons or less.
  • 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.
  • At least 25% of the peptides have a size of less than about 100 Dalton.
  • - 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan.
  • said alkaline protease and neutral protease is a Bacillus protease complex, preferable ProtamexTM from Novozymes AS
  • fat is separated from the peptide material after the first enzymatic treatment.
  • said fish is Salmonidae, preferable Atlantic salmon.
  • 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.
  • FPH fish protein hydrolysate
  • 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.
  • FPH fish protein hydrolysate
  • 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.
  • said cardiovascular disease is treated and/or prevented by increasing the level of high-density lipoprotein (HDL) in the blood.
  • HDL high-density lipoprotein
  • said cardiovascular disease is treated and/or prevented by increasing the ratio of HDL cholesterol to LDL cholesterol in the blood.
  • the peptide preparation can be used for the manufacturing of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of hypocholsterolemia.
  • 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.
  • said disorders are prevented or treated by increasing the body mass index (BMI).
  • BMI body mass index
  • said enzyme from a strain of Aspergillus oryzae is Umamizyme available from Amano Enzyme Inc., Japan.
  • said alkaline protease and neutral protease is a Bacillus protease complex, preferable ProtamexTM from Novozymes AS.
  • 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.
  • said feed composition is used for increasing the weight of an animal fed said feed composition.
  • 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.
  • said animal is suffering of underweight, undernourishment, malnutrition or malnourishment.
  • mammals 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.
  • 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 refers to a reduction of the severity of the disease and/or to reduce symptoms of the disease.
  • 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.
  • 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 FPH - Enzyme treated fish protein hydrolvsate
  • FPH FPH
  • 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 ProtamexTM.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • parenteral administration examples include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • the compounds of the present invention are appropriately administered in combination with other treatments for combatting or preventing a specific disease.
  • a preferred embodiment of the present invention relates to a nutritional composition
  • a nutritional composition comprising the peptide material of the present invention formulated in any conventional way to a feed or food product.
  • 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).
  • 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 ProtamexTM at a pH of about 6.5 and at temperature of 55 ⁇ 2 °C. ProtamexTM (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.
  • 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.
  • FPH fish protein hydrolyzate
  • 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 ProtamexTM.
  • 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.
  • 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.
  • 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
  • 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.
  • Fig 1 shows the peptide distribution obtained in the various filtering fractions after enzymatic treatment with the enzymes Acid protease A at pH 3.
  • 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.
  • 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 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.
  • the rats will have free access to the intervention diets on day 1 -29. All rats are killed on day 30 .
  • 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).
  • DPA docosapentaenoic acid
  • 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,
  • the fatty acids were identified by retention time using standard mixtures of methyl esters (Nu- Chek-Prep, Elyian, MN, USA).
  • the fatty acid composition 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.
  • 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 0.20 ⁇ (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.
  • 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.
  • 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.
  • the E1 material of the present invention reduces the weight gain with about 33%. Also the specific growth rate is significantly reduced.
  • 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.
  • 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
  • E1 material 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.
  • the present inventive E1 material can be used in the prevention and/or treatment of hypertriglyceridemia, atherosclerosis, heart diseases and stroke.

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Abstract

La présente invention concerne une préparation peptidique et les procédés pour sa préparation, ainsi que ses utilisations.
PCT/NO2011/000078 2010-03-08 2011-03-08 Matériau peptidique, ses préparations et ses utilisations Ceased WO2011112099A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103923963A (zh) * 2014-04-10 2014-07-16 江南大学 一种制备鱼皮胶原蛋白及ace抑制肽的方法
CN105624250A (zh) * 2016-03-09 2016-06-01 广东海洋大学 一种酶解-发酵偶联的制备水产蛋白活性肽方法
WO2017103618A1 (fr) * 2015-12-18 2017-06-22 Bergen Teknologioverføring As Procédé d'hydrolyse d'arêtes de poissons, produit ainsi obtenu et son utilisation pour améliorer la pigmentation de la chair d'un salmonidé

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103923963A (zh) * 2014-04-10 2014-07-16 江南大学 一种制备鱼皮胶原蛋白及ace抑制肽的方法
WO2017103618A1 (fr) * 2015-12-18 2017-06-22 Bergen Teknologioverføring As Procédé d'hydrolyse d'arêtes de poissons, produit ainsi obtenu et son utilisation pour améliorer la pigmentation de la chair d'un salmonidé
CN105624250A (zh) * 2016-03-09 2016-06-01 广东海洋大学 一种酶解-发酵偶联的制备水产蛋白活性肽方法

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