WO2011112101A1 - Matériau peptidique, composition alimentaire, ses préparations et ses utilisations - Google Patents

Matériau peptidique, composition alimentaire, ses préparations et ses utilisations Download PDF

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Publication number
WO2011112101A1
WO2011112101A1 PCT/NO2011/000080 NO2011000080W WO2011112101A1 WO 2011112101 A1 WO2011112101 A1 WO 2011112101A1 NO 2011000080 W NO2011000080 W NO 2011000080W WO 2011112101 A1 WO2011112101 A1 WO 2011112101A1
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Prior art keywords
peptide
accordance
preferable
peptide preparation
amino acid
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PCT/NO2011/000080
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English (en)
Inventor
Anders Aksnes
Rolf Kristian Berge
Kjartan Sandnes
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Marine Boproducts As
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Publication of WO2011112101A1 publication Critical patent/WO2011112101A1/fr

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    • 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)
    • 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
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
    • 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
    • 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
    • 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
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals

Definitions

  • the present invention relates to a peptide preparation, feed compositions, 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 concentration of homocystein in plasma.
  • 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 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/N 004/00043 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 (lie-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 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.
  • JP21 13859 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.
  • the specific peptide material of the present invention 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.
  • a first aspect of the present invention relates to a peptide preparation prepared from a fish material by a first enzymatic treatment with an first 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 a second alkaline protease.
  • said alkaline protease is Proleather FG-F.
  • said first alkaline protease and neutral protease is a Bacillus protease complex, preferable ProtamexTM from Novozymes AS.
  • said peptide preparation is substantially fat free.
  • the peptide can be uses for treatment and/or prevention of hypercholesterolemia, cardiovascular diseases, peripheral vascular diseases, xanthoma, xanthelasma palpabrum, arcus senilis, atherosclerosis, overweight or obesity.
  • said fish is Salmonidae, preferable Atlantic salmon.
  • the enzyme of the first enzymatic treatment is the Bacillus protease complex ProtamexTM from Novozymes AS and wherein the enzyme of the second enzymatic treatment is Proleather FG-F, and wherein fat is separated from the peptide material after the first enzymatic treatment.
  • Preferable, at least 50%, preferable 60% and more preferable 70% 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 least 45 % of the peptides have a size of about 100 - 1.000 Dalton.
  • At least 20% of the peptides have a size of less than about 100 Dalton, more preferable that 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 3.
  • 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 3.
  • 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 5%, more preferable 6%, and more preferable at least 7 % lower than compared to the fish protein hydrolysate, or
  • 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 30%, more preferable 40%, and more preferable at least 50 % lower than compared to the fish protein hydrolysate
  • the relative amount of the amino acid tyrosine is at least 50%, more preferable 71% 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 at least 50%, more preferable 60%, and more preferable at least 70 % 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 200 to 100 Daltons is at least 100%, more preferable 125%, and more preferable at least 150 % higher than compared to the fish protein hydrolysate.
  • a second aspect of the present invention relates to a process for the preparation of a peptide material, wherein said material is prepared from a fish material by a first enzymatic treatment with a first 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 a second alkaline protease.
  • FPH fish protein hydrolysate
  • fat is separated from the peptide material after the first enzymatic treatment.
  • fat is separated from said peptide preparation by decanting.
  • said second alkaline protease is Proleather FG-F.
  • the treatment with said second alkaline protease is conducted at a pH of about 8 to about 12, preferable at a pH at about 9 to 11 , or more preferable at a pH of about 10.
  • a third aspect of the present invention relates to use of a peptide preparation as a food product or food supplement or nutraceutical preparation for the prevention and/or treatment of hypercholesterolemia, cardiovascular diseases, peripheral vascular diseases, xanthoma, xanthelasma palpabrum, arcus senilis,
  • said peptide preparation is prepared from a fish material by a first enzymatic treatment with a first 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 a second alkaline protease.
  • a fourth aspect of the present invention relates to use of a peptide preparation for the manufacturing of a pharmaceutical composition for the prevention and/or treatment hypercholesterolemia, cardiovascular diseases, peripheral vascular diseases, xanthoma, xanthelasma palpabrum, arcus senilis, atherosclerosis, overweight or obesity, wherein said peptide preparation is prepared from a fish material by a first enzymatic treatment with a first 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 a second alkaline protease.
  • Preferable embodiments of the third and fourth aspect of the present invention relate (independently of each feature and aspect) to;
  • said fish is Salmonidae, preferable Atlantic salmon.
  • said second alkaline protease is Proleather FG-F.
  • said wherein said first alkaline protease and neutral protease is a Bacillus protease complex, preferable ProtamexTM from Novozymes AS. - said peptide preparation is substantially fat free.
  • - at least 75% 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 50% of the peptides have a size of about 100 - 1.000 Dalton.
  • - at least 25% of the peptides have a size of less than about 100 Dalton, more preferable that at least 28% 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 50% of the peptides have a size of about 100 -
  • - at least 25% of the peptides have a size of less than about 100 Dalton, more preferable that at least 28% of the peptides have a size of less than about 100 Dalton.
  • 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 3.
  • the relative amount of the amino acid arginine is at least 5 % higher 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 1200 to 200 Daltons is at least 5 % higher 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 5%, more preferable 10%, more preferable 20% and more preferable 30 % higher than compared to the fish protein hydrolysate.
  • the relative amount of the amino acid tyrosine is 5%, more preferable 10%, more preferable 15 % lower 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 5%, more preferable 10%, more preferable 15 % lower 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 200 to 100 Daltons is 20 %, more preferable 30%, more preferable 40 %, more preferable 50% lower than compared to the fish protein hydrolysate.
  • 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 17, or prepared by process in accordance with any of the claims 18 to 22.
  • the feed composition is used for lowering the weight of an animal fed said feed composition.
  • the feed composition is used wherein said animal is an overweight obese animal.
  • the feed composition is uses wherein said animal is suffering of overweight, overnourishment, malnutrition or malnourishment.
  • said feed is a pet feed.
  • 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 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, i.e. FPH, and thereafter has a secondary enzymatic treatment been conducted in order to further reduce the sizes of the peptide fragments.
  • FPH primary enzymatic treated protein material
  • 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.
  • Proleather FG-F is a alkaline proteinase available from Amano Enzyme Inc., is fermented by a unique fermentation process of a selected strain of Bacillus subtilis.
  • 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 conditions, as are deemed appropriate by the practitioner.
  • 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
  • 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.
  • buffers such as phosphate, citrate
  • 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.
  • 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
  • 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 Proleather FG-F as described in example 2.
  • the X-axis shows the logMW for the peptides.
  • Figure 2 shows the effect of the peptide preparation E3 on weight gain relative to control (FPH).
  • Figure 3 shows the effect of the peptide preparation E3 on specific growth rate relative to control (FPH).
  • Figure 4 shows the effect of the peptide preparation E2 on the plasma level of cholesterol relative to control.
  • 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.
  • ProtamexTM 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 11.1 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.
  • 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.
  • the solution was adjusted to pH 10 by adding 23.5 litre of a 10 % (w/v) solution of NaOH.
  • the temperature during hydrolysis was 50 - 60 °C, but dropped to 38 °C over night.
  • the enzyme reaction in the vessel was going on for 18.5 hours.
  • the FPH solution was treated with Proleather FG-F which is a proteolytic alkaline enzyme preparation manufactured by a unique fermentation process with a selected strain of Bacillus subtilis, and is available from Amano Enzyme Inc. 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.
  • Proleather FG-F is a proteolytic alkaline enzyme preparation manufactured by a unique fermentation process with a selected strain of Bacillus subtilis, and is available from Amano Enzyme Inc. 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.
  • Figure 1 shows the Peptide distribution of the control sample (FPH) and filtering fractions after treatment with Proleather FG-F as the second enzymatic treatment.
  • the peptide distribution was determined as described in the Rubin report no 4617/115, 2004.
  • the enzyme treated solution termed "E3" 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 00 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.
  • 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 enzyme Umamizyme.
  • the secondary enzyme treated preparations (example 2), i.e. the E3 peptide 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 3 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 (Proleather FG-F) hydrolysate contain a higher level of amino acids present in smaller peptides (Fraction 2 and fraction 3) as compared to the control (FPH).
  • Table 4 shows the amount of the various amino acids in fraction 2 and 3. Table 4.
  • Wistar rats Eighty male Wistar rats ( ollegaard 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 .
  • 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 transesterificatibn.
  • 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 130°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 mL/min).
  • 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 E3 material of the present invention reduces the weight gain with about 35%. Also the specific growth rate is significantly reduced. It is thus anticipated that the E3 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 27.0 g for the control and treatment groups, respectively, and the final weight (after feeding) were 35.1 and 31.9, respectively.
  • the peptides of the present invention have thus a general weight lowering effect of about 9.2 %.
  • the peptide material of the present invention decreases the level of cholesterol in plasma, and it is thus anticipated that the present invention can be used for the treatment and/or prevention of hypercholesterolemia, i.e. situations with high levels of cholesterol (higher levels than normal).
  • Hypercholesterolemia (literally: high blood cholesterol) is the presence of high levels of cholesterol in the blood. It is not a disease but a metabolic derangement that can contribute to many forms of disease, most notably cardiovascular disease. Some types of hypercholesterolemia lead to specific physical findings: xanthoma
  • xanthelasma palpabrum yellow patches around the eyelids
  • arcus senilis white discoloration of the peripheral cornea

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Abstract

La présente invention concerne une préparation peptidique et les procédés pour sa préparation, ainsi que ses utilisations, et une composition alimentaire contenant ladite préparation.
PCT/NO2011/000080 2010-03-08 2011-03-08 Matériau peptidique, composition alimentaire, ses préparations et ses utilisations WO2011112101A1 (fr)

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US10226422B2 (en) 2013-01-23 2019-03-12 Bottled Science Limited Skin enhancing beverage composition

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CN114480533B (zh) * 2022-01-20 2024-02-06 大连民族大学 一种复合菌发酵法制备鱼骨多肽的方法及发酵液

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US4940662A (en) 1982-03-06 1990-07-10 Terumo Kabushiki Kaisha Low-molecular weight peptide mixture and method of producing same
WO2005002605A1 (fr) * 2003-07-04 2005-01-13 Thia Medica As Hydrolysat proteique de poisson
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US4940662A (en) 1982-03-06 1990-07-10 Terumo Kabushiki Kaisha Low-molecular weight peptide mixture and method of producing same
JPH02113859A (ja) 1988-10-24 1990-04-26 Nitto Denko Corp タンパク質加水分解物の製造方法
WO2005002605A1 (fr) * 2003-07-04 2005-01-13 Thia Medica As Hydrolysat proteique de poisson
WO2006005757A2 (fr) 2004-07-12 2006-01-19 Dsm Ip Assets B.V. Hydrolysats proteiques abaissant la pression sanguine
WO2006084383A1 (fr) * 2005-02-14 2006-08-17 Ocean Nutrition Canada Limited Supplement dietetique antidiabetique et antihypertensif
WO2006084351A1 (fr) 2005-02-14 2006-08-17 Ocean Nutrition Canada Limited Complement dietetique anti-hypertensif derive d'hydrolysats de proteines de saumon ou d'oncorhynchus
FR2927335A1 (fr) * 2008-02-12 2009-08-14 Cie Des Peches Saint Malo Sant Hydrolysat de proteines de poissons presentant une activite satietogene, compositions nutraceutiques et pharmacologiques comprenant un tel hydrolysat et procede d'obtention
WO2009128713A1 (fr) 2008-04-14 2009-10-22 Newtricious B.V. Hydrolysats de protéine d'œuf

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Publication number Priority date Publication date Assignee Title
US10226422B2 (en) 2013-01-23 2019-03-12 Bottled Science Limited Skin enhancing beverage composition

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