NO20100370A1 - Peptide material, feed compositions and preparations, and uses thereof. - Google Patents
Peptide material, feed compositions and preparations, and uses thereof. Download PDFInfo
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- NO20100370A1 NO20100370A1 NO20100370A NO20100370A NO20100370A1 NO 20100370 A1 NO20100370 A1 NO 20100370A1 NO 20100370 A NO20100370 A NO 20100370A NO 20100370 A NO20100370 A NO 20100370A NO 20100370 A1 NO20100370 A1 NO 20100370A1
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- peptide
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- peptide preparation
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Classifications
-
- 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
- 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
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
Description
Peptidmateriale, forsammensetninger og preparater, og anvendelser derav. Peptide material, precompositions and preparations, and applications thereof.
OMRÅDE FOR OPPFINNELSEN. FIELD OF THE INVENTION.
Den foreliggende oppfinnelse vedrører et peptid preparat, forsammensetninger, en fremgangsmåte for fremstilling av et peptidpreparat, og anvendelser derav. The present invention relates to a peptide preparation, precompositions, a method for producing a peptide preparation, and applications thereof.
BAKGRUNN FOR OPPFINNELSEN. BACKGROUND OF THE INVENTION.
Fiskeoppdrettsindustrien har vokst enormt både i Norge og resten av verden i løpet av de siste år, spesielt oppdrett av laks. Det meste av fisken selges til forbrukeren som slaktet hel fisk, men betydelige mengder selges som fileter. Kun 50-70 % av laksen er fileter, mens resten selges som lawerdiprodukter så som fiskemel og fiske ensilasje. 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.
Gjennom enzymatisk behandling av fiskekjøttet og også fiskeskjelettet kan det separeres til en vandig fraksjon som er rik på proteiner, benevnt fiskeproteinhydrolysat (FPH). Den enzymatiske hydrolyseprosess kan kontrolleres, og produktene er reproduserbare og godt definerte. 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.
Et slikt proteinmateriale, det vil si fiskeproteinhydrolysat (FPH) har flere nyttige biologiske effekter, og et slikt materiale kan anvendes som et farmasøytisk materiale og som et emæringsmateriale. Søkers egen patentsøknad PCT/NO04/00202 beskriver at anvendelse av et slikt FPH-materiale senker konsentrasjon av kolesterol og homocystein i plasma, og også senker konsentrasjon av hepatiske triacyl-glyseroler. 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 that the use of such an FPH material lowers the concentration of cholesterol and homocysteine in plasma, and also lowers the concentration of hepatic triacyl-glycerols.
Vi har nå overraskende funnet at vi kan behandle et enzymbehandlet proteinmateriale videre, det vil si behandle det enzymbehandlete proteinmateriale i en sekundær andre behandling og at vi kan oppnå et nytt peptidmateriale som er forskjellig med hensyn til størrelsesfordeling av peptidfragmentene. 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.
Vi har fremstilt flere nye peptidmaterialer og vi har overraskende påvist at de biologiske aktiviteter forandres blant de forskjelllige peptidmaterialer, og også at de nye peptidmaterialer har effekter som er avvikende fra proteinmaterialet som kun har blitt behandlet en gang med ett enzym. Disse nye materialer kan anvendes for bedre å skreddersy materialene for spesifikke ernæringseffekter, medisinske indikasjoner eller sykdommer. 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.
Det spesifikke peptidmaterialet ifølge foreliggende oppfinnelse, benevnt som E2, representerer en forbedring av det kjente fiskeproteinhydrolysat (FPH) som har blitt behandlet med kun en primær enzymbehandling. Vi antar at også enzymbehandlete ikke-fiskeproteinmaterialer kan anvendes som en basis for en andre enzymatisk behandling, og foreliggende oppfinnelse er således ikke begrenset til fiskeproteinmaterialer. The specific peptide material according to the present invention, designated as E2, 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.
Uten å være bundet av teori antar vi at virkningsmekanismen er relatert til størrelses-fordeling av peptidblandingen, og ikke til opprinnelsen av proteinprøven. 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.
Peptidmaterialet i samsvar med foreliggende oppfinnelse er spesielt nyttig som et funksjonelt protein i ernæringsprodukter, spesielt idet det anvendes som et substitutt for naturlig plasma i dyrefor og i for for kjæledyr. Dersom det anvendes i for eller for for kjæledyr, kan ytterligere ingredienser tilsettes produktet så som fett, sukker, salt, smaksmidler, mineraler, etc. Produktet kan deretter formes til klumper som ligner naturlige kjøttklumper i utseende og tekstur. Produktet ifølge oppfinnelsen har de ytterligere fordeler at det enkelt kan formuleres til å inneholde nødvendige ernæringsmidler, at det er enkelt å fordøye av dyrene og velsmakende for dyrene. 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.
Peptidmaterialet i samsvar med foreliggende oppfinnelse kan også anvendes for fremstilling av et nutrasøytisk eller farmasøytisk sammensetning for hindring og/eller behandling av forskjellige sykdommer, som angitt i kravseksjonen. 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.
DETALJERT BESKRIVELSE AV OPPFINNELSEN. DETAILED DESCRIPTION OF THE INVENTION.
Et første aspekt av foreliggende oppfinnelse vedrører et peptidpreparat fremstilt med enzymatisk behandling av et proteinmateriale hvor proteinene kuttes til mindre peptidfragmenter, kjennetegnet ved at størrelsen av peptidfragmentene er fra 0 til 10.000 Dalton, mer fortrinnsvis fra 0 til 1000 Dalton, og mer foretrukket fra 0 til 100 Dalton. 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.
I en foretrukket utførelse er nevnte proteinmateriale etfiskeproteinmateriale, fortrinnsvis et proteinmateriale fra laks. In a preferred embodiment, said protein material is a fish protein material, preferably a protein material from salmon.
Fortrinnsvis, peptidpreparatet er fremstilt med enzymatisk behandling av et enzym med peptidaseaktivitet og/eller proteaseaktivitet, fortrinnsvis et enzym fremstilt fra en stamme av Aspergillus oryzae, og mer fortrinnsvis enzympreparatet Umamizyme tilgjengelig fra Amano Enzyme Inc, Japan Preferably, the peptide preparation is prepared by enzymatic treatment of an enzyme with peptidase activity and/or protease activity, preferably an enzyme prepared from a strain of Aspergillus oryzae, and more preferably the enzyme preparation Umamizyme available from Amano Enzyme Inc, Japan
Mer fortrinnsvis, nevnte enzymatiske behandling involverer også en enzymatisk behandling med et Bacillus proteasekompleks, fortrinnsvis før behandlingen med et enzym med peptidaseaktivitet. More preferably, said enzymatic treatment also involves an enzymatic treatment with a Bacillus protease complex, preferably before the treatment with an enzyme with peptidase activity.
En foretrukket utførelse av foreliggende oppfinnelse vedrører et peptidpreparat hvor minst 60% av peptidene har en størrelse på 1000 Dalton eller mindre. A preferred embodiment of the present invention relates to a peptide preparation in which at least 60% of the peptides have a size of 1000 Daltons or less.
Mer foretrukket, minst 35% av peptidene har en størrelse på ca. 100-1000 Dalton, mer foretrukket at minst 40% av peptidene har en størrelse på ca. 100-1000 Dalton, og mer foretrukket at ca. 45% av peptidene har en størrelse på ca. 100-1000 Dalton. 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. 45% of the peptides have a size of approx. 100-1000 Daltons.
Mer foretrukket, minst 25% av peptidene har en størrelse på mindre enn ca. 100 Dalton. More preferably, at least 25% of the peptides have a size of less than about 100 Daltons.
Mer fortrinnsvis, More preferably,
- minst 35% av peptidene har en størrelse på ca. 100-1000 Dalton, mer fortrinnsvis at minst 40% av peptidene har en størrelse på ca. 100-1000 Dalton, og mer foretrukket at ca. 45% av peptidene haren størrelse på ca. 100-1000 Dalton, og - 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. 45% of the peptides have a size of approx. 100-1000 Daltons, and
- minst 25% av peptidene har en størrelse på mindre enn ca. 100 Dalton. - at least 25% of the peptides have a size of less than approx. 100 Daltons.
Mer foretrukket, en fraksjon av peptidpreparatet korresponderende til peptid-størrelser av ca. 1200 til 200 Dalton har en aminosyresammensetning som angitt i tabell 4. More preferably, 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 4.
Mer foretrukket, en fraksjon av peptidpreparatet korresponderende til peptid-størrelser av ca. 200 til 100 Dalton har en aminosyresammensetning som angitt i tabell 4. More preferably, a fraction of the peptide preparation corresponding to peptide sizes of approx. 200 to 100 Daltons have an amino acid composition as indicated in Table 4.
Mer foretrukket, More preferably,
- en fraksjon av peptidpreparatet korresponderende til peptidstørrelser av ca. 1200 til 200 Dalton har en aminosyresammensetning som angitt i tabell 4, og - en fraksjon av peptidpreparatet korresponderende til peptidstørrelser av ca. 200 til 100 Dalton har en aminosyresammensetning som angitt i tabell 4. - 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 4, and - a fraction of the peptide preparation corresponding to peptide sizes of approx. 200 to 100 Daltons have an amino acid composition as indicated in Table 4.
En utførelse av dette aspekt vedrører et peptidpreparat hvor den relative mengde av aminosyren arginin i peptidpreparatet korresponderende til peptidstørrelser av ca. 1200 til 200 Dalton er minst 20%, fortrinnsvis minst 40%, mer foretrukket ca. 60% lavere enn sammenlignet med fiskeproteinhydrolysatet. An embodiment of this aspect relates to a peptide preparation where 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 20%, preferably at least 40%, more preferably approx. 60% lower than compared to the fish protein hydrolyzate.
En utførelse av dette aspekt vedrører et peptidpreparat hvor den relative mengde av aminosyren arginin i peptidpreparatet korresponderende til peptidstørrelser av ca. 200 til 100 Dalton er minst 20%, fortrinnsvis minst 40%, mer foretrukket ca. 60% høyere enn sammenlignet med fiskeproteinhydrolysatet. An embodiment of this aspect 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 Dalton is at least 20%, preferably at least 40%, more preferably approx. 60% higher than compared to the fish protein hydrolyzate.
En utførelse av dette aspekt vedrører et peptidpreparat hvor den relative mengde av aminosyren tyrosin i peptidpreparatet korresponderende til peptidstørrelser av ca. 1200 til 200 Dalton er 50%, mer foretrukket 100%, mer foretrukket 150% høyere enn sammenlignet med fiskeproteinhydrolysatet. An embodiment of this aspect relates to a peptide preparation where the relative amount of the amino acid tyrosine in the peptide preparation corresponding to peptide sizes of approx. 1200 to 200 Dalton is 50%, more preferably 100%, more preferably 150% higher than compared to the fish protein hydrolyzate.
Et andre aspekt av foreliggende oppfinnelse vedrører en fremgangsmåte for fremstilling av et peptidpreparat, hvor fremgangsmåten omfatter et trinn hvor et protein eller peptidmateriale behandles med et Umamizyme-preparat tilgjengelig fra Amano Enzyme Inc, Japan, for å redusere størrelsen av peptidfragmentene. 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 Umamizyme preparation available from Amano Enzyme Inc, Japan, to reduce the size of the peptide fragments.
I en foretrukket utførelse inkluderer fremgangsmåten en ytterligere enzymatisk behandling av materialet, fortrinnsvis før behandlingen med en sur protease, og fortrinnsvis er den enzymatiske behandling ved et Bacillus proteasekompleks. Behandlingen med Umamizyme-preparatet utføres fortrinnsvis ved en pH av ca. 5 til ca. 9, fortrinnsvis ved en pH av ca. 6 til 8, fortrukket ved en pH av ca. 6-7, eller mer fortrinnsvis ved en pH av ca. 6,5. In a preferred embodiment, the method includes a further enzymatic treatment of the material, preferably before the treatment with an acid protease, and preferably the enzymatic treatment is by a Bacillus protease complex. The treatment with the Umamizyme preparation is preferably carried out at a pH of approx. 5 to approx. 9, preferably at a pH of approx. 6 to 8, preferred at a pH of approx. 6-7, or more preferably at a pH of approx. 6.5.
Et tredje aspekt av foreliggende oppfinnelse vedrører en anvendelse av et peptidpreparat i samsvar med foreliggende oppfinnelse for fremstilling av et farmasøytisk eller nutrasøytisk preparat for hindring og/eller behandling av kardiovaskulære sykdommer. A third aspect of the present invention relates to the use of a peptide preparation in accordance with the present invention for the production of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of cardiovascular diseases.
Nevnte kardiovaskulære sykdommer kan velges blant gruppen som består av aterosklerose, angina, cerebrovaskulær hendelse (slag), cerebrovaskulær sykdom, kongestiv hjertesvikt, koronar arteriesykdom, myokardisk infarkt (hjerteanfall) og perifer vaskulær sykdom. Said cardiovascular diseases may be selected from the group consisting of atherosclerosis, angina, cerebrovascular event (stroke), cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction (heart attack) and peripheral vascular disease.
I en foretrukket utførelse er den kardiovaskulære sykdom behandlet og/eller hindret ved å øke nivået av høytetthet lipoprotein (HDL) i blodet. In a preferred embodiment, the cardiovascular disease is treated and/or prevented by increasing the level of high-density lipoprotein (HDL) in the blood.
I en foretrukket utførelse behandles og/eller hindres den kardiovaskulære sykdom ved å øke forholdet av HDL-kolesterol til LDL-kolesterol i blodet. In a preferred embodiment, the cardiovascular disease is treated and/or prevented by increasing the ratio of HDL cholesterol to LDL cholesterol in the blood.
Et fjerde aspekt av foreliggende oppfinnelse vedrører en anvendelse av et peptidpreparat i samsvar med foreliggende oppfinnelse for fremstilling av et farmasøytisk eller nutrasøytisk preparat for hindring og/eller behandling av hypokolesterolemia. A fourth aspect of the present invention relates to the use of a peptide preparation in accordance with the present invention for the production of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of hypocholesterolemia.
Et femte aspekt av foreliggende oppfinnelse vedrører en anvendelse av et peptidpreparat i samsvar med oppfinnelsen for fremstilling av et farmasøytisk eller nutra-søytisk preparat for hindring og/eller behandling av for lag vekt, underernæring, feilernæring eller underernæringssykdommer, gjenvinning etter sykdom eller kirurgi, fortrinnsvis ved å øke kroppsmasseindeksen (BMI). A fifth aspect of the present invention relates to a use of a peptide preparation in accordance with the invention for the production of a pharmaceutical or nutraceutical preparation for the prevention and/or treatment of overweight, malnutrition, malnourishment or malnutrition diseases, recovery after illness or surgery, preferably by increasing body mass index (BMI).
I foretrekkende utførelser til de tredje til femte aspekter er nevnte proteinmateriale et fiskeproteinmateriale. In preferred embodiments of the third to fifth aspects, said protein material is a fish protein material.
Et sjette aspekt av foreliggende oppfinnelse vedrører et for som i tillegg til kommersielle foringredienser omfatter et peptidpreparat i samsvar med krav 1. A sixth aspect of the present invention relates to a lining which, in addition to commercial pre-ingredients, comprises a peptide preparation in accordance with claim 1.
Et syvende aspekt av foreliggende oppfinnelse vedrører en fremgangsmåte for å øke vektøkningen og/eller den spesifikke vektrate til et dyr, hvor dyret fores en forsammensetning inneholdende et peptidpreparat i samsvar med krav 1. A seventh aspect of the present invention relates to a method for increasing the weight gain and/or the specific weight rate of an animal, where the animal is fed a pre-composition containing a peptide preparation in accordance with claim 1.
DEFINISJONER AV TERMER ANVENDT I SØKNADEN. DEFINITIONS OF TERMS USED IN THE APPLICATION.
Dyr Animals
I denne konteksten inkluderer termen "dyr" pattedyr så som mennesker og husdyr (landbruksdyr), spesielt dyr av økonomisk viktighet så som hønsefugler, bovine, ovine, caprine og porcine dyr, spesielt de som produserer produkter egnet for humant forbruk, så som kjøtt, egg og melk. Videre, termen er tiltenkt å inkludere fisk og skalldyr, så som laks, torsk, Tilapia, skjell og østers. Termen inkluderer også husdyr så som hunder og katter. 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.
Behandling Treatment
I relasjon til de farmasøytiske applikasjoner ifølge oppfinnelsen angir termen "behandling" en reduksjon av alvorligheten av sykdommen. In relation to the pharmaceutical applications of the invention, the term "treatment" denotes a reduction in the severity of the disease.
Hindring Obstacle
Termen "hindring" refererer til å hindre en gitt sykdom, det vil si en forbindelse ifølge foreliggende oppfinnelse administreres før start av tilstand. Dette betyr at forbindelsene ifølge foreliggende oppfinnelse kan anvendes som profylaktiske midler eller som ingredienser i funksjonelle matvarer eller for for å hindre risiko for start av en gitt sykdom. 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 - Enzvmbehandlet fiskeproteinhvdrolvsat FPH - Enzyme-treated fish protein hydrolvsate
FPH-materialet er et proteinhydrolysat resulterende fra en primær enzymatisk behandling av et fiskemateriale med enzymet Protamex™. FPH-materialet inneholder høye andeler av proteiner og peptider og anvendes som en kontroll i den eksperimentelle seksjon. Peptidmaterialet ifølge foreliggende oppfinnelse er forskjellig fra dette FPH-materialet med hensyn til størrelsesfordeling av peptidene og biologisk aktivitet. 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.
Peptidmateriale i samsvar med oppfinnelsen Peptide material in accordance with the invention
Peptidmaterialet i samsvar med foreliggende oppfinnelse er basert på en primær enzymatisk behandlet proteinmateriale, og en sekundær enzymatisk behandling har blitt utført for å redusere størrelsene av peptidfragmentene. 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 Protease
En protease, (også benevnt peptidase eller proteinase) bryter ned proteinene. En protease er et enzym som utfører proteolyse, det vil si, starter proteinkarabolisme ved å hydrolysere peptidbindinger som forbinder aminosyrer sammen i polypeptid-kjeden som danner proteinet. 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.
Umamiz<y>me Umamiz<y>me
Umamizyme, tilgjengelig fra Amano Enzyme Inc., Japan, har høy peptidaseaktivitet og høy proteinaseaktivitet og er i stand til å hydrolysere forskjellige proteiner i høyt utbytte. Umamizyme, available from Amano Enzyme Inc., Japan, has high peptidase activity and high proteinase activity and is capable of hydrolyzing various proteins in high yield.
ADMINISTRERING AV FORBINDELSENE IFØLGE FORELIGGENDE OPPFINNELSE. ADMINISTRATION OF THE COMPOUNDS ACCORDING TO THE PRESENT INVENTION.
Som et farmasøytisk medikament kan materialene ifølge foreliggende oppfinnelse administreres direkte til dyret med enhver egnet teknikk, inkluderende parenteralt, intranasalt, oralt, eller ved absorpsjon gjennom huden. Det kan administreres lokalt eller systemisk. Den spesifikke administrasjonsrute for hvert middel vil avhenge for eksempel av den medisinske historie til dyret. Den foretrukne administrasjonsrute er oralt. 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.
Eksempler på parenteral administrering inkluderer subkutanøs, intramuskulær, intravenøs, intraarteriell og intraperitoneal administrering. Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial and intraperitoneal administration.
Dersom de gis kontinuerlig administreres forbindelsene ifølge foreliggende oppfinnelse typisk med 1-4 injeksjoner per dag eller med kontinuerlig subkutanøse infu- sjoner, for eksempel ved anvendelse av en minipumpe. En intravenøs poseløsning kan også benyttes. Nøkkelfaktoren for å velge en egnet dosering er resultatet som skal oppnås, som målt med reduksjon i totalt kroppsfett eller forholdet av fett til mager masse, eller med andre kriterier for å måle kontroll eller hindring av obesitet eller for å hindre obesitets relaterte tilstander, slik det er hensiktsmessig av den praktiserende. 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 administrering, i en utførelse, formuleres forbindelsene ifølge foreliggende oppfinnelse generelt ved å blande hver i en ønsket grad av enhet, i en enhetsdoserings injiserbar form (løsning, suspensjon eller emulsjon), med en farma-søytisk akseptabel bærer, det vil si en som er ikke-toksisk til mottakeren i de doseringer og konsentrasjoner som benyttes og som er kompatibel med de andre ingredienser i formuleringen. 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.
Generelt, formuleringene fremstilles ved å sette forbindelsene ifølge foreliggende oppfinnelse hver uniformt og intimt i kontakt med væskebærere eller finfordelte faststoffbærere, eller begge deler. Deretter, dersom nødvendig, formes produktene til den ønskete formulering. Fortrinnsvis er bæreren en parenteral bærer, mer fortrinnsvis en løsning som er isoton med blodet til mottakeren. Eksempler på slike bærervehikler inkluderer vann, saltløsning, Ringers løsning og dekstrose løsning. Ikke-vandige vehikler så som faste oljer og etyl oleat er også nyttige heri og likeledes liposomer. 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.
Bæreren kan hensiktsmessig inneholde mindre mengder av tilsetningsstoffer så som substanser som forsterker isotonisitet og kjemisk stabilitet. Slike materialer er ikke-toksiske til mottakerne i de doseringer og konsentrasjoner som benyttes, og inkluderer buffere så som fosfat, sitrat, sukinat, eddiksyre, og andre organiske syrer eller deres salter; antioksidanter så som askorbinsyre, immunglobuliner; hydrofile poly-merer så som polyvinyl polidon; aminosyrer, så som glysin, glutamisk syre, aspargin-syre eller arginin; monosakkarider, disakkarider og andre karbohydrater inkluderende cellulose eller dets derivater, glukose, mannose eller dekstriner, kela-terende midler så som EDTA; sukkeralkoholer så som mannitol eller sorbitol; motion så som natrium; og/eller ikke-ioniske surfaktanter så som polysorbater, poloks-amerer eller PEG. 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 orale farmakologiske sammensetninger kan slike bærer materialer være for eksempel vann, gelatin, gummi, laktose, stivelser, magnesium stearat, talg, oljer, polyalkenglykol, petroleum jelly og lignende benyttes. Slike farmasøytiske preparater kan være i enhets doseringsform og kan ytterligere inneholde andre terapeutiske verdifulle substanser eller konvensjonelle farmasøytiske adjuvanter så som konser-verende midler, stabiliserende midler, emulsifiserende midler, buffere og lignende. De farmasøytiske preparater kan være i konvensjonelle væskeformer så som tablet-ter, kapsler, drageer, ampuller og lignende, i konvensjonelle doseringsformer så som tørrampuller, og som stikkpiller og lignende. 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.
I tillegg, forbindelsen ifølge foreliggende oppfinnelse administreres hensiktsmessig i kombinasjon med andre behandlinger for å bekjempe eller hindre spesifikk sykdom. In addition, the compound of the present invention is conveniently administered in combination with other treatments to combat or prevent specific disease.
Oppfinnelsen vil forstås bedre med referanse til de påfølgende eksempler. Disse skal imidlertid ikke vurderes som begrensende for rammen av oppfinnelsen. 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.
En foretrukket utførelse av foreliggende oppfinnelse vedrører en ernæringssammen-setning omfattende peptidmaterialet ifølge foreliggende oppfinnelse formulert på enhver konvensjonell måte til et for eller matprodukt. 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.
FIGURER FIGURES
Figur 1 viser kontrollen (FPH) og forskjellige filtreringsfraksjoner etter behandling med enzympreparatet Umamizyme som beskrevet i eksempel 1. X-aksen viser logMW for peptidene. Figur 2 viser effekten av peptidpreparatet E2 på vektøkning i forhold til kontroll. Figur 3 viser effekten av peptidpreparatet E2 på spesifikk vekstrate relativ til kontroll. Figure 1 shows the control (FPH) and different filtration fractions after treatment with the enzyme preparation Umamizyme as described in example 1. The X-axis shows the logMW for the peptides. Figure 2 shows the effect of the peptide preparation E2 on weight gain compared to control. Figure 3 shows the effect of the peptide preparation E2 on specific growth rate relative to control.
Figur 4 viser effekten av peptidpreparatet E2 på plasmanivå av kolesterol. Figure 4 shows the effect of the peptide preparation E2 on the plasma level of cholesterol.
Figur 5 viser effekten av peptidpreparatet E2 på plasmanivå av HDL-kolesterol relativ til kontroll. Figur 6 viser effekten av peptidpreparatet E2 på forholdet HDL-kolesterol: LDL-kolesterol i plasma relativ til kontroll. Figure 5 shows the effect of the peptide preparation E2 on the plasma level of HDL-cholesterol relative to control. Figure 6 shows the effect of the peptide preparation E2 on the ratio HDL-cholesterol: LDL-cholesterol in plasma relative to control.
EKSPERIMENTELL SEKSJON EXPERIMENTAL SECTION
Eksempel 1 Example 1
Primær enzymatisk behandling, fremstilling av hvdrolvsat materialet anvendt som kontroll i eksempel 2. Primary enzymatic treatment, preparation of the hvdrolvsat material used as a control in example 2.
Kontrollmaterialet er et enzymbehandlet fiskeproteinhydrolysat (FPH) og har flere nyttige biologiske effekter. The control material is an enzyme-treated fish protein hydrolyzate (FPH) and has several useful biological effects.
FPH ble produsert fra fiskekjøttrester av laksebenskjeletter etter filetering. Skjelet-tene uten hoder fra fersk filetert Atlantisk laks { Salmo salar, L.) ble tatt direkte fra produksjonslinjen og frosset ved -20 ± 2 °C: Innen en uke ble de frosne kroppene anvendt i den enzymatiske hydrolyseprosess. 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.
Den enzymatiske hydrolyse ble utført med Protamex™ ved en pH på ca. 6,5 og ved en temperatur på 55 ± 2 °C. Protamex™ (E.C. 3.4.21.62/3.4.24.28) er et Bacillus protease kompleks fra Novozymes AS (Bagsvaerd, Denmark) og tilfredsstiller ren-hetskravene for ernæringsgraderte enzymer. Forholdet av laksekropper til vann var 1,14. Et enzym til substratforhold på 11,1 AU/kg ubearbeidet protein ble anvendt i hydrolysene. Etter 60 minutters enzymatisk behandling ble temperaturen økt til 98 °C, som ble nådd etter 105 minutter. 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.
Store ben ble tilbakeholdt i hydrolysetanken, mens små ben ble fjernet med filtrering av hydrolysatet gjennom et gitter. Deretter ble de uløselige fraksjoner fjernet i en tofase separator (Westfalia, Germany, SC.35-26-177, 15 kW, 7200 rpm), før den resulterende blanding ble separert i en trefase separator (Westfalia, Germany, SB-7-36-+76, 4 kW, 8520 rpm) til lakseolje, emulsjonsfraksjon og en vandig fraksjon. Den vandige fraksjon ble konsentrert i en 4-trinns fallende film evaporator (APV Anhydro) til en pasta med ca. 30 % tørrstoff. Ytterligere evaporering ble utført i en laboratorium-evaporator ved 70 °C inntil tørrstoffinnholdet var ca. 55%. 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. 30% dry matter. Further evaporation was carried out in a laboratory evaporator at 70 °C until the solids content was approx. 55%.
Det evaporerte hydrolysat benevnes som fiskeproteinhydrolysat (FPH), det vil si kontrollprøvene i eksempel 2, og inneholder ca. 83 % protein, 10 % aske og ca. 2 % lipider, basert på tørrvekt. Aminosyresammensetningen er gitt i tabell 1. 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.
Eksempel 2 Example 2
Sekundær enzymatisk behandling - Enzymatisk behandling av FPH ( kontroll) med Umamizyme ved pH 6, 5 Secondary enzymatic treatment - Enzymatic treatment of FPH (control) with Umamizyme at pH 6.5
Substratprøven (FPH) (pasta) ble oppløst i preoppvarmet springvann (50°C) til 10% tørrsubstans. The substrate sample (FPH) (paste) was dissolved in preheated tap water (50°C) to 10% dry matter.
FPH-løsningen ble behandlet med Umamizyme som er et proteolytisk enzympreparat for proteinhydrolyse som er rik i aminosyrer. Enzympreparatet fremstilles med en unik fermenteringsprosess med en utvalgt stamme av Aspergillus oryzae, og er tilgjengelig fra Amano Enzyme Inc., Japan. The FPH solution was treated with Umamizyme which is a proteolytic enzyme preparation for protein hydrolysis which is rich in amino acids. The enzyme preparation is produced by a unique fermentation process with a selected strain of Aspergillus oryzae, and is available from Amano Enzyme Inc., Japan.
pH under hydrolysen var 6,5, og varigheten var 2,75 timer. Temperaturen ble holdt konstant ved 40°C. The pH during the hydrolysis was 6.5, and the duration was 2.75 hours. The temperature was kept constant at 40°C.
Ingen spesifikk inaktivering av enzymene ble utført etter inkubering. Inaktiveringen av enzymene ble utført i filtreringstrinnene og under evaporering av de aktuelle fraksjonene. I disse prosesseringstrinn overstiger temperaturen flere ganger inaktiveringstemperaturen av enzymene inkludert i forsøket. No specific inactivation of the enzymes was performed after incubation. The inactivation of the enzymes was carried out in the filtration steps and during evaporation of the relevant fractions. In these processing steps, the temperature exceeds several times the inactivation temperature of the enzymes included in the experiment.
Figur 1 viser peptidfordelingen av kontrollprøven (FPH) og filtreringsfraksjoner etter behandling med Umamizyme. Peptidfordelingen ble bestemt som beskrevet i Rubin-rapporten nr. 4617/115, 2004. Figure 1 shows the peptide distribution of the control sample (FPH) and filtration fractions after treatment with Umamizyme. Peptide distribution was determined as described in Rubin Report No. 4617/115, 2004.
Fraksjonering med filtrering Fractionation with filtration
Den enzymbehandlete løsning E2 ble foredlet med mikrofiltrering og ultrafiltrering i løsning med ca. 10% tørr substans ved 50-60X. Filtreringene ble utført i en filtreringsenhet (Membralox SD 3-A modules M-3P1940, Pall,USA) med keramiske membraner med 100 nm og 20 nm porestørrelse (Membralox EP1940, Pall, USA). Kun permeatene ble samlet for evaluering av bioaktive peptider, selv om mindre prøver av retentater ble samlet for analytiske formål for å kunne ha informasjon om utbytte og utførelse under de spesifikke filtreringstrinn. The enzyme-treated solution E2 was refined with microfiltration and ultrafiltration in solution with approx. 10% dry substance at 50-60X. 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 EP1940, Pall, USA). Only the permeates were collected for evaluation of bioactive peptides, although smaller samples of retentates were collected for analytical purposes in order to have information on yield and performance during the specific filtration steps.
Detaljert informasjon om utførelse under mikro- og ultrafiltreringen er gitt i tabell 2, nedenfor. Detailed information on performance during the micro- and ultrafiltration is given in Table 2, below.
Permeatet etter ultrafiltreringstrinnet ble anvendt for de biologiske eksperimenter beskrevet i eksempel 3, nedenfor. The permeate after the ultrafiltration step was used for the biological experiments described in Example 3, below.
Peptidstørrelsesfordeling Peptide size distribution
Ca. 2 ml av den primære og sekundære enzymatisk behandlete løsning E2 eller en kontrollprøve (nullprøve) av FPH ble tilsatt 20 ml buffer, blandet, sentrifugert og filtrert før applisering til HPLC for separasjon med størrelseseksklusjonskromatografi (Rubin-rapporten 4617/115, 204; http://www.rubin.no/files/documents/4617-115_peptidstorrelse_hydrolysat2.pdf). About. 2 ml of the primary and secondary enzymatically treated solution E2 or a control sample (blank sample) of FPH was added to 20 ml of buffer, mixed, centrifuged and filtered before application to HPLC for separation by size exclusion chromatography (Rubin report 4617/115, 204; http: //www.rubin.no/files/documents/4617-115_peptidstorrelse_hydrolysat2.pdf).
Samling av peptidfraksjoner av spesifikk lengde og analyser av disse for å karakterisere den generelle aminosyreprofil av den spesifiserte peptidfraksjon oppnådd etter enzymbehandling med de forskjellige protolytiske enzymer ble evaluert. Følgende fraksjoner ble samlet etter størrelseseksklusjonskromatografi med frem gangsmåten beskrevet i Rubin-rapporten 4617/115, 204). Eluert væske ble samlet ved spesifikke elueringstider for å karakterisere peptidene som foreligger i det opp-finneriske produkt. Under gis elueringstidene for prøver samlet for å analyseres for aminosyreprofil: Fraksjon 1: 18-22 min (korresponderende til peptider av ca. 8500 - 1200 Dalton<*>) Fraksjon 2: 22-26 min (korresponderende til peptider av ca. 1200 - 200 Dalton<*>) Fraksjon 3: 26-30 min (korresponderende til peptider av ca. 200 - 100 Dalton<*>) Fraksjon 4: 30-34 min (korresponderende til peptider av ca. <100 Dalton<*>) 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 procedure 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<*>)
Fraksjon 5: 34-40 min Fraction 5: 34-40 min
Størrelsen av peptidene beregnes i samsvar med korrelasjonen mellom elueringstid og logMW beskrevet i Rubin-rapporten 4617/115, 2004. The size of the peptides is calculated according to the correlation between elution time and logMW described in the Rubin report 4617/115, 2004.
Utbytter Dividends
Utbyttene i permeatet etter ultrafiltrering ble analysert med forskjellen mellom innledende tørrstoff og retentatet etter mikro- og ultrafiltrering: Utbytte = 100 - ((tørrstoff ved start hydrolysat - (mikrofiltrat retentat tørrstoff + ultrafiltrerings retentat tørrstoff))/ (tørrstoff ved start hydrolysat)<*>100)). 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)).
Et utbytte på 66 % ble oppnådd på E2-materialet i samsvar med foreliggende oppfinnelse. A yield of 66% was obtained on the E2 material in accordance with the present invention.
Peptidstørrelsesfordeling: Peptide size distribution:
Fig 1 viser peptidfordelingen oppnådd i forskjellige filtreringsfraksjoner etter enzymatisk behandling med enzymet Umamizyme. Fig 1 shows the peptide distribution obtained in different filtration fractions after enzymatic treatment with the enzyme Umamizyme.
Som det fremgår av resultatene over, har den enzymatiske behandling signifikant redusert mengden av de største peptider og i varierende grad forandret forholdet av de andre peptidfraksjoner. Det skal bemerkes at foreliggende enzymstudier er preliminære i den betydning av enzymbetingelsene og utbyttet ikke er optimalisert. Aminosvreanalvse av samlete fraksjoner etter størrelseseksklusionskromatografi. De sekundære enzymbehandlete preparater (eksempel 2) det vil si E2-materialet i samsvar med foreliggende oppfinnelse, og det primære enzymbehandlete preparat (eksempel 1) det vil si FPH (kontroll), ble samlet og analysert for totalt innhold av aminosyrer. Kun fraksjon 1, 2 og 3 ble analysert. 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 E2 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.
Fraksjon 1: 18-22 min (korresponderende til ca. 8500 - 1200 Dalton<*>Fraction 1: 18-22 min (corresponding to approx. 8500 - 1200 Dalton<*>
Fraksjon 2: 22-26 min (korresponderende til ca. 1200 - 200 Dalton<*>Fraction 2: 22-26 min (corresponding to approx. 1200 - 200 Dalton<*>
Fraksjon 3: 26-30 min (korresponderende til ca. 200 - 100 Dalton<*>Fraction 3: 26-30 min (corresponding to approx. 200 - 100 Dalton<*>
Tabell 3 viser den relative mengde av totale aminosyrer detektert i de forskjellige fraksjoner. Generelt, hovedpeptidene forekom i fraksjoner 2 og fraksjoner 3 for enzymene som viser det høyeste utbyttet i enzymhydrolysene. Table 3 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.
Tabell 3 viser at hydrolysatet som er behandlet med et andre enzym (Umamizyme) inneholder et høyere nivå av aminosyrer foreliggende i mindre peptider (fraksjon 2 og fraksjon 3) sammenlignet med kontroll (FPH). Table 3 shows that the hydrolyzate treated with a second enzyme (Umamizyme) contains a higher level of amino acids present in smaller peptides (fraction 2 and fraction 3) compared to control (FPH).
Tabell 4, nedenfor, viser mengden av de forskjellige aminosyrer i fraksjon 2 og 3. Table 4, below, shows the amount of the different amino acids in fractions 2 and 3.
Aminosyreanalyse ble analysert etter hydrolyse i 6 M i 221 ved 110 °C med HPLC ved anvendelse av en fluoressens teknikk for deteksjon (Cohen and Michaud, Anal. Biochem. 1993, 211, 279-287). 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).
Eksempel 3 Example 3
Biologisk aktivitet av enzympreparatene Biological activity of the enzyme preparations
Dyr og dyrehold Animals and animal husbandry
80 hannkjønn Wistar rotter (Mollegaard og Blomholtgaard, Danmark) 12 uker gamle, ble huset individuelt i bur av typen Makrolon III i et åpent system. De ble holdt under standard laboratoriebetingelser med temperatur 22±1 °C, mørke/lys-sykluser av 12/121, relativ fuktighet 55±5 % og 20 luftforandringer per time. 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.
Rottene har fri aksess til intervensjonsdiettene på dager 1-29. Alle rotter ble drept på dag 30. På dag 30 ble alle rottene anestetisert ved inhalering av Sevoflurane 2 % i et anestesikammer. Torakotomi, hjertepunktuering og blodtømming ble utført. 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.
Prøvesamling og analyser av biokjemiske parametere under foringsperioden. Sample collection and analyzes of biochemical parameters during the feeding period.
Kroppsvekt og forinntak. (dag 0-7-14-21, 23-29) Body weight and intake. (days 0-7-14-21, 23-29)
Blodprøver (fra lår, plasma) ble opptatt i en heparininneholdende Vacutainer, plas-sert på is i 10 minutter, og sentrifugert ved 2000 rpm i 10 min ved 4 °C. (Dag 0-22-30 og plasma ble lagret ved -80 °C). 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).
Urinprøver for målinger av protein, isoprostaner, natrium og kreatinin (dag 0-22-30), urin ble lagret ved -80 °C. Urine samples for measurements of protein, isoprostanes, sodium and creatinine (day 0-22-30), urine was stored at -80 °C.
Avføringsprøver ble samlet på dag 29 og holdt under 20 °C inntil analyse for GMP. Kort forklart, 1 g av det føkale materiale fortynnes i 4 ml ekstraheringsbuffer og homogeniseres grundig ved anvendelse av en Ultra Turrax (20,000 rpm) før sentrifugering ved 45,000g i 20 minutter. De øvre halvdeler av supernatanten høstes forsiktig og kjøres på en standard 1-trinns enzymkoplet immunosorbentanalyse (ELISA) som beskrevet tidligere (10). 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).
Helblod ble spunnet, plasma tatt ut, og prøven ble lagret midlertidig på is, og overført til -80 °C fryser. Whole blood was spun, plasma withdrawn, and the sample was stored temporarily on ice, and transferred to a -80 °C freezer.
Fettsyrer i plasma og i rektale prøver ble analysert som tidligere beskrevet. Den samme metode ble anvendt for analyse av fettsyreprofil i plasma og homogeniserte biopsiprøver tatt fra rektal mukosa. Plasma lipid/fettsyrer, triglyserider, kolesterol, LDL kolesterol, HDL kolesterol, EPA, DHA, docosapentanoisk syre (DPA, C22:5n-3), AA, total n-3 og total n-6 PUFAer ble analysert. 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.
Fremstilling av subcellulære fraksjoner. Preparation of subcellular fractions.
Lever fra rottene ble homogenisert individuelt i iskald sukroseløsning (0,25 mol/l sukrose i 10 mmol/l HEPES-buffer pH 7,4 og 1 mmol/l EDTA) ved anvendelse av en Potter-Elvehjem homogeniserer. De subcellulære fraksjoner ble isolert som beskrevet i 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). Kort forklart, homogenatet ble sentrifugert ved 1000 x g i 10 min for å separere post-nukleære fra nukleær fraksjon. En mitokondriell-anriket fraksjon ble fremstilt fra den post nukleære fraksjon ved 10000 x g i 10 min. En peroksisom anriket fraksjon ble fremstilt med sentrifugering av den post-mikrokondrielle fraksjon ved 23500 x g i 30 min. En mikrosomal anriket fraksjon ble isolert fra den post-peroksisomal fraksjon ved 100000 x g i 75 min. Den resulterende supernatant ble samlet som den cytosoliske fraksjon. Prosedyren ble utført ved 0-4 °C, og fraksjonene ble lagret ved - 80 °C. Protein ble analysert ved anvendelse av BioRad protein analyse kit (BioRad, Heraules, CA) og bovin serum albumin som standard. 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.
Lipidanalvser Lipid analysis
Lipider i hellever og heparinisert plasma ble målt i Tecnicon Axon system (Miles, Tarrytown, NY), med Bayer triglyserid og kolesterol enzymatiske kit (Bayer, Terrytown, NY) og PAP 150 fosfolipid enzymatisk kit (bioMérieux, Lyon, France). Leverlipider ble først ekstrahert i samsvar med 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. 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.
Fettsvresammensetning Fatty acid composition
Fettsyrer ble ekstrahert fra prøvene med 2:1 kloroform: metanol (v/v) (35). Prøvene ble filtrert, forsåpet og esterifisert i 12 % BF3i metanol (v/v). Fettsyresammensetningen total lipider fra lever og plasma ble analysert ved anvendelse av metodene beskrevet av Lie og 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). Fettsyre metylestere ble separert ved anvendelse av en Carlo Erba gasskromatograf ("kald på kolonne" -injeksjon, 69 °C i 20 s, øker med 25 °C min"<1>til 160 °C og holdes ved 160 °C i 28 min, øker med 25 °C min"<1>til 190 °C og holdes ved 190 °C i 17 min, øker ved 25 °C min"<1>til 220 °C og holdes ved 220 °C i 9 min) utstyrt med en 50 m CP-sil 88 (Chrompack, Middelburg, The Netherlands) brent silica kappilærkolonne (i.d. 0,32 mm). Fettsyrene ble identifisert med retensjonstid ved anvendelse av standard blandinger av metylestere (Nu-Chek-Prep, Elyian, MN, USA). Fettsyresammensetningen (vekt prosentandel) ble beregnet ved anvendelse av en integrator (Turbochrom Navigator, Version 4,0) koplet til GLC. 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.
Lipider ble ekstrahert fra plasma triacylglyserol-anriket lipoproteinfraksjon ved anvendelse av en blanding av kloroform og metanol, og separert med tynnsjikt-kromatografi på silicagelplater (0.25mm Silica gel 60, Merck) utviklet i heksan-dietyleter-eddiksyre (80:20:1, v/v/v) og visualisert ved anvendelse av Rhodamine 6G (0,05 % i metanol, Sigma) og UV-lys. Flekkene ble skrapet av, og overført til rør inneholdende heneicosanoisk syre (21:0) som indre standard. BF3-metanol ble tilsatt til prøvene for transesterifisering. For å fjerne nøytrale steroler og ikke-forsåpet materiale, ble ekstrakter av fettacylmetylestere oppvarmet i 0,5mol/l KOH i etanol-vann-løsning (9:1). Gjenvunnet fettsyrer ble deretter reesterifisert ved anvendelse av BF3-metanol. Metylesterene ble analysert på en GC8000Top gasskromatograf (Carlo Erba Instrument), utstyrt med en flammeioniserings detektor (FID), programmerbar temperatur av dampinjektor, AS 800 autosampler (Carlo Erba Instrument) og en kappelær kolonne (60m x 0,25mm) inneholdende en sterkt polar SP 2340 fase med filmtykkelse 0,20^m (Supelco). Innledende temperatur var 130 °C, oppvarming 1,4 °C/min til final temperatur 214 °C. Injektortemperaturen var 235 °C. Detektor-temperatur var 235 °C, ved anvendelse av hydrogen (25ml/min), luft (350 ml/min) og nitrogen som make-up gass (30 ml/min). Prøvene ble kjørt med konstant strømning ved anvendelse av hydrogen som bærergass (1,6 ml/min). Splittingsforholdet var 20:1. Metylesterene ble positivt identifisert med sammenligning til kjente standard (Larodan Fine Chemicals, Malmo, Sweden) og verifisert med massespektrometri. Kvantifisering av fettsyrene ble utført med Chrom-Card A/D 1,0 kromatografistasjon (Carlo Erba Instruments) basert på heneicosanoisk syre som en indre standard. 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.
Isolering av plasma triacvlglvserol- rik lipoproteinfraksion Isolation of plasma triacvlglvserol-rich lipoprotein fraction
Plasma triacylglyserol-rik lipoproteinfraksjon ble fremstilt med ultrasentrifugering av 3 ml plasma med en tetthet på 1,063 g/ml i 19 t ved 105000 x g ved 15 °C. Rørene ble fordelt, og den flytende fraksjon i topp 1 ml av hvert rør ble høstet. Fraksjonen ble deretter dialysert mot 150 mmol/l natriumklorid, 16 mmol/l natriumfosfat og 4 mmol/l kaliumfosfat, pH 7,5, mettet med nitrogen. 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.
Resultater av biologisk aktivitet. Results of biological activity.
De biologiske eksperimentelle data på enzympreparat E2 er vist i tabell 5 nedenfor og i de medfølgende figurer 2-6. The biological experimental data on enzyme preparation E2 are shown in table 5 below and in the accompanying figures 2-6.
Tabell 5 Table 5
Et sammendrag av biologiske aktiviteter for E2-materialet, dvs. materiale i samsvar med foreliggende oppfinnelse hvor kontrollmaterialet har blitt behandlet med Umamizyme. A summary of biological activities for the E2 material, i.e. material in accordance with the present invention where the control material has been treated with Umamizyme.
Sammenlignet med kontrollmaterialet øker E2-materialet ifølge foreliggende oppfinnelse vektøkningen med ca. 30%. Videre, den spesifikke vekstrate er også signifikant øket. Compared to the control material, the E2 material according to the present invention increases the weight gain by approx. 30%. Furthermore, the specific growth rate is also significantly increased.
Det vurderes således at E2-materialet ifølge foreliggende oppfinnelse kan anvendes som et vekstøkende middel, for eksempel for å hindre eller behandle undervekt, underernæringsforstyrrelse eller -situasjon, eller i foringsregimer for å øke vekt og/eller veksten av de dyr som mates. It is thus considered that the E2 material according to the present invention can be used as a growth-enhancing agent, for example to prevent or treat underweight, malnutrition disorder or situation, or in feeding regimes to increase the weight and/or growth of the animals that are fed.
Vi har også vist at peptidmaterialet ifølge foreliggende oppfinnelse øker nivået av kolesterol i plasma, og det vurderes således at foreliggende oppfinnelse kan anvendes for behandling og/eller hindring av hypokolesterolemia, dvs. situasjoner med avvikende lave nivåer av kolesterol. We have also shown that the peptide material according to the present invention increases the level of cholesterol in plasma, and it is thus considered that the present invention can be used for the treatment and/or prevention of hypocholesterolemia, i.e. situations with aberrantly low levels of cholesterol.
Vi har også vist at nivåene av høytetthet lipoprotein (HDL) i plasma øker. Høytetthet lipoprotein (HDL) er én av fem hovedgrupper av lipoproteiner (kylomikroner, VLDL, IDL, LDL, HDL) som muliggjør at lipider så som kolesterol og triglyserider kan transporteres i den vannbaserte blodstrøm. I friske individer bæres ca. 30% av kolesterolet i blodet med HDL. Det antas at HDL kan fjerne kolesterol fra ateroma i arterier og transportere det tilbake til lever for utskilling og reanvendelse, som er hovedårsaken til at HDL-bundet kolesterol noen ganger benevnes som "godt kolesterol", eller HDL-C. Et høyt nivå av HDL-C synes å beskytte mot kardiovaskulære sykdommer. Epidemiologiske studier har vist at høye konsentrasjoner av HDL (over 60 mg/dl) har beskyttende verdi mot kardiovaskulære sykdommer så som ischemisk slag og myokardisk infarkt. Kardiovaskulær sykdom (CVD) er en avvikende funksjon av hjertet eller blodkar. Det kan forårsake et øket risiko for hjerteanfall, hjertesvikt, brå død, slag og hjerterytmeproblemer, og således resultere i redusert livskvalitet og redusert livsforventning. We have also shown that the levels of high-density lipoprotein (HDL) in plasma increase. High-density lipoprotein (HDL) is one of five main groups of lipoproteins (chylomicrons, VLDL, IDL, LDL, HDL) which enable lipids such as cholesterol and triglycerides to be transported in the water-based blood stream. In healthy individuals, approx. 30% of cholesterol in the blood with HDL. It is believed that HDL can remove cholesterol from atheroma in arteries and transport it back to the liver for excretion and reuse, which is the main reason why HDL-bound cholesterol is sometimes referred to as "good cholesterol", or HDL-C. A high level of HDL-C appears to protect against cardiovascular disease. Epidemiological studies have shown that high concentrations of HDL (over 60 mg/dl) have protective value against cardiovascular diseases such as ischemic stroke and myocardial infarction. Cardiovascular disease (CVD) is an abnormal function of the heart or blood vessels. It can cause an increased risk of heart attack, heart failure, sudden death, stroke and heart rhythm problems, and thus result in reduced quality of life and reduced life expectancy.
Claims (30)
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| US3697285A (en) | 1969-10-10 | 1972-10-10 | Rohm & Haas | Fish protein solubilization using alkaline bacterial protease |
| JPS58152498A (en) | 1982-03-06 | 1983-09-10 | Terumo Corp | Production of low-molecular peptide mixture |
| JPH02113859A (en) | 1988-10-24 | 1990-04-26 | Nitto Denko Corp | Production of protein hydrolyzate |
| DK46793D0 (en) | 1993-04-26 | 1993-04-26 | Novo Nordisk As | ENZYME |
| NO322425B1 (en) * | 2003-07-04 | 2006-10-02 | Berge Biomed As | Use of a hydrolyzate of proteinaceous fish material for the preparation of a pharmaceutical agent for the treatment and / or prevention of pathologically high levels of tracylglycerols, hypercholesterolemia, hyperhomocysteinemia, or pathologically low levels of beta-oxidation in an animal or human. |
| WO2006005757A2 (en) | 2004-07-12 | 2006-01-19 | Dsm Ip Assets B.V. | Blood pressure lowering oligopeptides |
| CA2639880A1 (en) * | 2005-02-14 | 2006-08-17 | Ocean Nutrition Canada Limited | Anti-diabetic or anti-hypertensive dietary supplement |
| US7179793B2 (en) | 2005-02-14 | 2007-02-20 | Ocean Nutrition Canada Limited | Anti-hypertensive dietary supplement |
| FR2927335B1 (en) * | 2008-02-12 | 2012-04-20 | Cie Des Peches Saint Malo Sante | FISH PROTEIN HYDROLYSAT HAVING SATIETOGENIC ACTIVITY, NUTRACEUTICAL AND PHARMACOLOGICAL COMPOSITIONS COMPRISING SUCH HYDROLYSAT AND PROCESS FOR OBTAINING SAME |
| WO2009128713A1 (en) | 2008-04-14 | 2009-10-22 | Newtricious B.V. | Egg protein hydrolysates |
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