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WO2009128713A1 - Hydrolysats de protéine d'œuf - Google Patents

Hydrolysats de protéine d'œuf Download PDF

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Publication number
WO2009128713A1
WO2009128713A1 PCT/NL2009/050193 NL2009050193W WO2009128713A1 WO 2009128713 A1 WO2009128713 A1 WO 2009128713A1 NL 2009050193 W NL2009050193 W NL 2009050193W WO 2009128713 A1 WO2009128713 A1 WO 2009128713A1
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WIPO (PCT)
Prior art keywords
lysozyme
hydrolysate
dpp
ovomucin
ovotransferrin
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PCT/NL2009/050193
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Inventor
Aart Van Amerongen
Maria Josepha Catharina Beelen - Thomissen
Louise Aleida Maria Van Zeeland-Wolbers
Wiekert Hendrikus Van Gilst
Jan Hendrik Buikema
Joseph Wilhelmus Petrus Maria Nelissen
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NEWTRICIOUS BV
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NEWTRICIOUS BV
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention is in the field of diabetes and other DPP-IV modulated diseases.
  • GLP-I glucagon-like peptide
  • GIP glucose-dependent insulinotropic polypeptide
  • GLP-I and GIP are believed to regulate satiety, lipidemia and osteogenesis. Given these properties, it is not surprising that the enhancement of incretin activity has emerged as a therapeutic goal in the development of new therapies for the management of diabetes, in particular type 2 diabetes mellitus (T2DM). Obesity and diabetes are predicted to reach epidemic proportions throughout the world in the next 20 years and current treatments do not restore normal insulin sensitivity or glucose homeostasis, therein resulting in debilitating diabetic complications and premature death.
  • T2DM type 2 diabetes mellitus
  • the therapeutic potential of native GLP-I is limited by its short physiologic half-life, owing to its rapid inactivation by the enzyme dipeptidyl peptidase DPP-IV and to renal clearance.
  • modulation of DPP-IV activity has been a target and several selective chemical compound inhibitors of DPP-IV have been developed for the treatment of T2DM.
  • DPP-IV inhibitors can increase circulating levels of endogenous GLP-I and GIP and improve glucose homeostasis in human subjects with type 2 diabetes mellitus.
  • DPP-IV inhibition is not expected to increase the level of insulin at inappropriate times, such as between meals, which can lead to excessively low blood sugar (hypoglycemia).
  • DPP-IV is believed to be involved in, among others, obesity, appetite regulation, lipidemia, osteoporosis, neuropeptide metabolism and T-cell activation.
  • WO 2006/068480 describes protein hydrolysates, in particular casein hydrolysates with peptides of 2-8 amino acids long and that comprise proline, that inhibit activity of DPP- IV. Besides the disadvantage of having to process casein hydrolysates to make them enriched in the active peptides, there is an ongoing need for improved and/or alternative ingredients that inhibit DPP-IV.
  • the object of the present invention is to provide inhibitors of DPP-IV which may be effective in the treatment of type 2 diabetes mellitus and other DPP-IV modulated diseases.
  • poorly controlled hyperglycemia is a direct cause of the multiplicity of complications (cataracts, neuropathy, nephropathy, retinopathy, and cardiomyopathy) that characterize advanced type 2 diabetes mellitus. Therefore, there is a continuing need for new oral hypoglycemic agents which control blood glucose levels in patients in need thereof.
  • egg-protein hydrolysates had significant DPP-IV inhibitory activity.
  • hydrolysates selected from the group consisting of ovomucin, ovotransferrin and lysozyme displayed excellent DPP- IV inhibitory activity.
  • the DPP-IV inhibitory activity of ovomucin, ovotransferrin and lysozyme has been compared to that of several other protein hydrolysates, including other egg protein hydrolysates such as ovalbumin and also the DPP-IV inhibitory activity of ovomucin, ovotransferrin and lysozyme has been compared to that of casein hydrolysate as disclosed in WO 2006/068480.
  • hydrolysates of ovomucin, ovotransferrin and lysozyme and in a particular advantageous embodiment hydrolysates of lysozyme can be advantageously used for the treatment and/or prevention of DPP-IV mediated conditions, in particular for the treatment and/or prevention of type 2 diabetes mellitus.
  • the term “food” refers herein to liquid, semi-liquid or solid food products suitable for human and/or animal consumption. Thus beverages are included.
  • “Functional food” refers to a food product which comprises one or more active ingredients, especially one or more egg protein hydrolysates according to the invention.
  • Food supplement refers to supplements suitable for human and/or animal consumption which comprise a suitable amount of one or more bioactive protein hydrolysates according to the invention as functional ingredient. Supplements may be in the form of pills, sachets, powders and the like.
  • medicaments or pharmaceutical compositions means one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the medicaments or pharmaceutical compositions of the present invention encompass any composition made by admixing a hydrolysate of the present invention and a pharmaceutically acceptable carrier.
  • Subjects means any member of the class mammalia, including without limitation humans, non-human primates, farm animals, domestic animals and laboratory animals.
  • Food-grade refers to components which are considered as not harmful, when ingested by a human or animal subject. Food grade components should preferably have a GRAS status. "Effective dose” or “effective amount” refers to a dose sufficient to result in the therapeutic or prophylactic effect in vivo.
  • DPP-IV inhibitor or "DPP-IV inhibitory activity” refers herein to the ability of a protein hydrolysate to significantly inhibit DPP-IV (dipeptidyl peptidase-IV) activity in vitro and/or in vivo.
  • IC50 refers to the concentration at which 50% of enzyme activity is inhibited.
  • “Ovomucin” is a sialo-protein found in egg white reported to play a role in bio- protection (anti-viral and anti-tumour activities).
  • Oxotransferrin is a glycoprotein of egg white albumen; transferrins are iron binding proteins and acute phase reactants of animal serum.
  • “Lysozyme” is an enzyme which catalyzes hydrolysis of 1,4-beta- linkages between N- acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins.
  • Lysozyme include 1,4-N-acetylmuramidase, L-7001, N,O-diacetylmuramidase, PRl-
  • Lysozyme Globulin Gl, Globulin G, Lysozyme g, Mucopeptide N- acetylmuramoylhydrolase, Mucopeptide glucohydrolase and Muramidase.
  • This catalytic activity is non- specifically targeted to the bacterial cell membranes, in particular Gram positive bacteria, and related with general non-specific organism defence.
  • Lysozyme is present in the mucosal secretion such as saliva and tears and in the lysomes of phagocytic cells. In high concentration, about 3% from all proteins, lysozyme is present in chicken egg-white.
  • Hydrolysate of ovotransferrin is herein also collectively referred to as "hydrolysate selected from the group consisting of ovomucin, ovotransferrin and lysozyme” or “hydrolysate of ovomucin, ovotransferrin and/or lysozyme".
  • Eggs refer herein preferably to chicken eggs, although eggs from other birds may also be used.
  • Egg protein hydrolysates is used herein as a general term to refer to protein hydrolysates (prepared in vitro) of whole eggs, egg fractions (e.g. egg white or egg yolk) or of substantially pure egg proteins, especially lipovitellin, ovomucin, lysozyme, ovalbumin and ovotransferrin.
  • Metabloic Syndrome refers to multiple interrelated clinical disorders, including obesity, insulin resistance and hyperinsulinemia, glucose intolerance, hypertension and dyslipidemia (hypertriglyceridemia and low HDL cholesterol levels) as described e.g. in Moller and Kaufman (Annual Rev. of Medicine VoI 56, 45-62). Metabloic syndrome is also typed "Syndrome X”.
  • an "active fragment” of a peptide or protein refers to a protein part which is shorter than the full-length protein or protein variant (e.g. obtained by enzymatic hydrolysis or in vivo synthesis) and which shows DPP-IV inhibitory activity.
  • the term “comprising” is to be interpreted as specifying the presence of the stated parts, steps or components, but does not exclude the presence of one or more additional parts, steps or components.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • the invention thus concerns a method for the treatment and/or prevention of type 2 diabetes mellitus, said method comprising administering a hydrolysate selected from the group consisting of ovomucin, ovotransferrin and lysozyme, or a composition comprising a hydrolysate selected from the group consisting of ovomucin, ovotransferrin and lysozyme, in an effective amount to a subject in need thereof.
  • the invention can be worded as the use of a hydrolysate selected from the group consisting of ovomucin, ovotransferrin and lysozyme for the prepration of a composition for the treatment and/or prevention of type 2 diabetes mellitus.
  • the invention can also be worded as a hydrolysate selected from the group consisting of ovomucin, ovotransferrin and lysozyme for the treatment and/or prevention of type 2 diabetes mellitus.
  • the hydrolysate selected from the group consisting of ovomucin, ovotransferrin and lysozyme, or the composition comprising the hydrolysate selected from the group consisting of ovomucin, ovotransferrin and lysozyme may be in the form of a medicament, pharmaceutical composition, food product, functional food or food supplement or otherwise indicated nutritional compositions.
  • JP 5344863 discloses compositions comprising hydrolysates of rice, legumes and egg protein in a specif c ratio to provide sufficient protein nutrients to subjects with diabetes.
  • the balanced composition of hydrolysates and free amino acids that is described in JP 5344863 is to normalize a diabetic's amino acid imbalance in the blood.
  • the present invention thus preferably does not concern a composition containing a total nitrogen source composition (proteins and free amino acids) consisting of (i) 21-26 wt.% of pepsin hydrolysate of rice protein, (ii) 21-26 wt.% of pepsin hydrolysate of bean, preferably soy bean, protein and (iii) 8.2-6.8 wt.% of pepsin hydrolysate of egg protein, based on total nitrogen source.
  • a total nitrogen source composition proteins and free amino acids
  • Egg white is the clear liquid (also called the albumen or the glair/glaire) contained within an egg. It consists mainly of about 15% proteins dissolved in water. Egg white contains approximately 40 different proteins including ovalbumin (ca. 54 wt%; for nourishment; blocks digestive enzymes), ovotransferrin (ca. 12 wt%; binds iron), ovomucoid (ca. 11%; blocks digestive enzymes), globulins (ca. 8 wt%; plugs defects in membranes, shell), lysozyme (ca. 3.5 wt%; enzyme that digests bacterial cell walls), ovomucin (ca. 1.5 wt%; thickens egg white; inhibits viruses), others (ca 10 wt%; bind vitamins, block digestive enzymes).
  • Lysozyme is also present in other sources besides eggs. However, the concentration of lysozyme in egg white is high (3-4%) as compared to other sources of lysozyme. Thus ovotransferrin, ovomucin and lysozyme as well are most conveniently isolated from chicken eggs.
  • the lysozyme is chicken egg lysozyme, and preferably the hydrolysate of lysome is obtained by hydrolysis of chicken egg lysozyme. Ovotransferrin, ovomucin and lysozyme may be extracted from eggs and subsequently purified.
  • the egg white fraction may be separated using known methods and independently ovotransferrin, ovomucin and lysozyme may be purified by cation and anion exchange chromatography, gel permeation chromatography, affinity chromatography and other methods known in the art.
  • a process used routinely for lysozyme purification is Cation Exchange Chromatography. Lysozyme is bound to a cation exchanger at suitable pH (e.g. pH 9). This may be done in a stirred tank reactor or in a chromatography column. After elution from the adsorption particles by salt, lysozyme is pure enough for food applications.
  • lysozyme is purified using Cation Exchange Chromatography. Further purification steps such as membrane ultrafiltration and diafiltration may be applied, also for obtaining ovotransferrin and ovomucin.
  • suitable lysozyme sources are commercially available protein compositions or egg fraction compositions, such as egg white powder.
  • lysozyme may for example be obtained from Belovo (100% pure protein), ovotransferrin may be obtained from Sigma- Aldrich (81.3% pure protein) or from Belovo (89.5% protein), ovomucin may be obtained from Belovo. Ovotransferrin, ovomucin and lysozyme may also be produced using recombinant DNA technology as known in the art. It is envisaged that as a starting material whole egg or egg white may be used that is enriched in more lysozyme, via for example appropriate diet of the egg producing animal(s) or even via transgenic animal(s).
  • hydrolysate of ovotransferrin refers to a mixture of peptides, which are shorter than the full length ovotransferrin, ovomucin and lysozyme and which are derived from the hydroysis of ovotransferrin, ovomucin or lysozyme.
  • the hydrolysate has a minimal degree of hydrolysis.
  • Degree of hydrolysis means the percentage of hydrolysed peptide bonds of the total amount of peptide bonds in the full length lysozyme.
  • the degree of hydrolysis of ovotransferrin, ovomucin or lysozyme ranges from at least 5% to at most 75%, preferably the degree of hydrolysis is at least 10% to at most 35%.
  • a hydrolysate of ovotransferrin, ovomucin or lysozyme can be obtained by any means known in the art. This includes methods of chemical hydrolysis or enzymatic hydrolysis or fermentative processes such as fermentation by bacteria or other microorganisms (yeast, or fungi) or combinations thereof. It is preferred to prepare hydrolysates of ovotransferrin, ovomucin or lysozyme using enzymatic hydrolysis.
  • the enzyme suitable for hydrolysis may in principle be any food grade protease or protease mixture from a variety of sources, such as plants, animals, or microorganisms such as fungi or bacteria. Suitable proteases are commercially available.
  • Newlase F comprises an acid protease derived from Rhizopus niveus
  • Promod 278P comprises proteinase from Carica papaya (papain) and proteinases and peptidases from Bacillus subtilis
  • Umamizyme comprises protease derived from Aspergillus oryzae
  • Alcalase comprises a Bacillus licheniformi protease mix with as the main enzyme an endoproteinase.
  • a skilled person can determine the suitability by making a hydrolysate as described and testing the DPP-IV inhibitory activity using an in vitro assay as described elsewhere herein.
  • particular enzymes e.g. Peptidase R
  • examples of using combinations of enzymes for this purpose are shown herein.
  • the hydrolysate of ovotransferrin, ovomucin or lysozyme is obtained by hydrolysis with an enzyme selected from the group consisting of Newlase F, Promod 278P, Umamizyme, Alcalase and pepsin, which also herein is referred to as Newlase F, Promod 278P, Umamizyme, Alcalase and pepsin hydrolysates, respectively.
  • the present invention concerns a protein hydrolysate obtainable by protease hydrolysis of ovotransferrin, ovomucin or lysozyme, characterized in that said hydrolysate has DPP- IV inhibiting activity.
  • the hydrolysate is lysozyme hydrolysate.
  • DPP-IV inhibiting activity depended on the protease that was used to prepare the enzyme hydrolysate. Without being bound by theory, this suggests that by selecting the appropriate combinations of protein and protease the specificity and the strength of DPP-IV inhibiting activity can be increased which may be independent from the degree of hydrolysis. Rather it is considered that certain DPP-IV inhibiting peptides are released from the native proteins upon hydrolysis by a specific protease.
  • the protein hydrolysate of ovotransferrin is obtainable by hydrolysis with Umamizyme
  • the protein hydrolysate of ovomucin is obtainable by hydrolysis with Newlase F
  • the protein hydrolysate of lysozyme is obtainable by hydrolysis with Newlase F.
  • hydrolysis is carried out in water instead of buffer solutions, especially for large scale production for human and/or animal consumption, as high amounts of salts are undesirable.
  • hydrolysis is optimized by varying the amount of enzyme used, the pH, the temperature and/or the incubation times.
  • An example of a suitable hydrolysis protocol is provided in the Examples.
  • adaptation to large scale production, e.g. 500, 1000, 5000 liter or more, of raw starting material and/or protein hydrolysate production method is a matter of routine experimentation to a person skilled in the art.
  • the invention concerns a method for making a protein hydrolysate with DPP-IV inhibiting activity comprising the steps of
  • the target protein is selected from the group consisting of ovotransferrin, ovomucin and lysozyme, preferably substantially pure ovotransferrin, ovomucin and lysozyme
  • the enzyme to be used preferably comprises an enzyme selected from the group consisting of Newlase F, Promod 278P, Alcalase, Umamizyme and pepsin.
  • compositions especially food and/or food supplement compositions comprising a suitable amount of ovotransferrin, ovomucin or lysozyme hydrolysate according to the invention.
  • the present invention also concerns the use of a hydrolysate of ovotransferrin, ovomucin or lysozyme for the preparation of a food product or food supplement with DPP-IV inhibiting activity.
  • the present invetion concerns the use of a hydrolysate of ovotransferrin, ovomucin or lysozyme for the preparation of a food product or food supplement for the treatment of type 2 diabetes mellitus.
  • the food product or food supplement comprises at least 10 wt.%, based on total protein, including free amino acids, of a hydrolysate according to the present invention. In one embodiment, the food product or food supplement comprises at least 10 wt.%, based on total protein including free amino acids, of ovotransferrin, ovomucin and/or lysozyme. In one embodiment, the food product or food supplement comprises at least 5 wt.%, based on total protein, including free amino acids of lysozyme, preferably at least 10 wt.%, more preferably at least 20 wt.%.
  • hydrolysates of ovotransferrin, ovomucin or lysozyme according to the present invention can also suitably be used for the treatment and/or prevention of other DPP-IV linked or modulated diseases.
  • the present hydrolysates of lysozyme can suitably be used for the treatment and or prevention of risk factors of the metabolic syndrome such as obesity, insulin resistance and hyperinsulinemia, glucose intolerance, hypertension and dyslipidemia (hypertriglyceridemia and low HDL cholesterol levels).
  • the present hydrolysates of ovotransferrin, ovomucin or lysozyme are for or are used for the preparation of a food product or food supplement for lowering of blood glucose levels and/or increasing of blood insulin levels. In one embodiment the present hydrolysates of ovotransferrin, ovomucin or lysozyme are for or are used for the preparation of a food product or food supplement for management of diabetes and/or for management of diabetic symptoms.
  • One embodiment of the present invention thus also concerns the prevention of diabetes, in particular type 2 diabetes mellitus and/or insuline resistance and/or diabetic symptoms, said method of prevention comprising administering a suitable amount of a hydrolysates of ovotransferrin, ovomucin or lysozyme.
  • Food supplements and food products comprising a hydrolysate of ovotransferrin, ovomucin or lysozyme according to the invention may be made as known in the art.
  • Food supplements may for example be in the form of any dosage form such as tablets, pills, powder sachets, gels, capsules, and the like. Intake by the subject is preferably oral.
  • Food products may be in the form of drinks including fruit flavoured drinks or dairy type drinks (e.g. 100ml bottles, 150ml solutions), solid or semi-solid foods, such as snacks, desserts such as puddings, custards or spoonable yoghurts, sauces, whole meals, etc.
  • the food product is a product which is consumed on a regular basis, preferably daily, such as staple foods (e.g. bread, noodles, soft drinks, dairy products such as cheese, yoghurt, fermented dairy products, milk, butter etc.).
  • staple foods e.g. bread, noodles, soft drinks, dairy products such as cheese, yoghurt, fermented dairy products, milk, butter etc.
  • the ovotransferrin, ovomucin or lysozyme hydrolysate may thus be added to a food base or may be incorporated into the food product during its production process.
  • any existing food or food supplement products comprising a hydrolysate according to the invention are included herein.
  • the food product is a drink or beverage, preferably based on a fruit juice or vegetable juice, although milk based drinks are also included.
  • Food products according to the present invention may thus comprise or may be typed as skimmed milk, semi- skimmed milk, buttermilk, fermented milk, especially yoghurt, soy drink or soy milk, fermented soy milk, fruit juices, fruit purees, syrups, vegetable juices, vegetable purees and combinations thereof.
  • the food product or supplement is a fruit or vegetgable juice, fruit or vegetable puree or dairy product or combination thereof.
  • the drink or beverage may be made in daily dosage volumes of 50ml, 100ml, 150ml, 200ml or more.
  • the food supplement or food product may further comprise additional food-grade ingredients, such as but not limited to flavorings, vitamins, minerals, stabilizers, emulsifiers, other biologically active ingredients, food bases/nutrients such as a protein, carbohydrate and/or fat component, and the like.
  • additional food-grade ingredients such as but not limited to flavorings, vitamins, minerals, stabilizers, emulsifiers, other biologically active ingredients, food bases/nutrients such as a protein, carbohydrate and/or fat component, and the like.
  • the ovotransferrin, ovomucin and/or lysozyme hydrolysate may be added at any stage during the normal production process of the food product/food supplement.
  • the food product/supplement may also comprise other inactive ingredients and carriers, such as e.g. glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, e.g. carboxymethylcellulose (CMC), magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. It may also comprise water, electrolytes, essential and non-essential amino acids, trace elements, minerals, fiber, sweeteners, flavorings, colorants, emulsifiers and stabilizers (such as soy lecithin, citric acid, esters of mono- or di-glycerides), preservatives, binders, fragrances, and the like.
  • CMC carboxymethylcellulose
  • the effective dose which needs to be added depends on a number of factors, such as the subject (e.g. human or animal), the dosage form (daily, several times a day, weekly) and product composition and/or texture.
  • the daily effective dose of ovotransferrin, ovomucin and/or lysozyme hydrolysate will range between about 10 mg/kg body weight to 100 mg/kg, 500 mg/kg to 1000 mg/kg body weight, or more. It is within the realm of a skilled person to determine the effective dose using routine experimentation.
  • a food or food supplement composition comprising a dose of Ig, 3g, 1Og, 20g or more lysoszyme hydrolysate is provided. Such a composition is suitable for daily intake.
  • a coating may be added which changes the place and/or time of release in vivo of the hydrolysate of ovotransferrin, ovomucin and/or lysozyme.
  • Slow release formulations are known in the art.
  • the present food products or food supplements may be labeled as having a blood glucose lowering effect and/or blood insulin increasing effect and may be ingested as a treatment, by subjects having been diagnosed with diabetes, in parrticular type 2 diabetes mellitus.
  • the present food products or food supplements may be ingested prophylactically by subjects at risk of developing diabetes.
  • the present food supplements and food products comprise ovotransferrin, ovomucin and/or lysozyme hydrolysate according to the invention in an effective amount.
  • Significant DPP-IV inhibitory activity is defined as 3 mg/ml hydrolysate (or less) being able to inhibit about 50% (or more) of DPP-IV activity (the IC50 value), using the in vitro assay as described in the examples.
  • the IC50 value of a hydrolysate is below 2 mg/ml and, more preferably below 1 mg/ml.
  • a significant effect on type 2 diabetes mellitus can be regarded as the ability of the hydrolysate to lower blood glucose levels and/or increase blood insulin levels in vivo, as may be determined for example in test animals following oral ingestion or in human trials.
  • Leucine is dissolved in 1% SDS.
  • the stock solution is diluted 2Ox, 1Ox, 4x en 2x with
  • Hydrolysed protein is diluted in 1% SDS to a concentration of 0,5 mg/ml in duplo
  • a calibration line of the leucine standard is made. This calibration line is used to convert absorption values of sample to NH 2 -equivalents. Correct mmol NH 2 equivalents in sample for mmol eq. in non hydrolysed sample.
  • the protein concentration of the sample was 0,5 mg/ml, so 15 ⁇ l contains 7,5 ⁇ g protein.
  • htot-erwteiwit 8300 meq/kg (value depends on protein)
  • DPP-IV activity can be determined by measuring the increase in absorption at 385 nm using Gly-Pro-p-nitroanilide as DPP-IV substrate. A decrease in DPP-IV activity is a measure for the inhibition.
  • DPP-IV is diluted with Tris buffer (pH8.0) to 1 Unit/ml and finally 100 times diluted with Tris buffer •
  • the hydrolysis samples (10 mg/ml) are dissolved in Tris buffer (pH 8.0)
  • Example 3 Hydrolysis in water and optimization of the hydrolysis protocol Hydrolysis was performed in water in view of commercial applications, where the use of buffers is less desired. Consumer products should not contain additional salt components if not explicitly necessary and, therefore, the optimal buffer conditions for each enzyme was replaced by water. The pH of the solution during the reaction was adjusted by addition of NaOH or HCl.
  • Hydrolysates were dissolved in water (5% w/v) and heated at 9O 0 C for 20 minutes.
  • a taste panel consisting of six persons scored bitterness on a scale of 0 (not bitter) to 4 (bitter). Each person tasted 32 hydrolysates by sipping a sample from a plastic spoon at the back of the tongue. In between samples the mouth was rinsed with pure water.
  • Table 1 Influence of combinations of proteases on bitterness of final hydrolysates Lysozyme hydrolysate (6 hours hydrolysis)
  • Egg proteins egg white protein, lysozyme, ovomucin, ovotransferrin and ovalbumin
  • casein were hydrolysed by four proteases / protease mixtures (Newlase F, Promod 278P, Alcalase and Umamizyme and pepsin for egg white protein, lysozyme). Incubation times varied from 30 minutes till 12 hours and samples were taken after various time points. For final hydrolysates the degree of hydrolysis was determined and the DPP-IV inhibiting activity was assessed with the assay as described herein.
  • Protein hydrolysates with IC50 values of less than 2 mg/ml are regarded as particularly advantageous having the most significant in vitro and as (potentially) having most significant in vivo DPP-IV inhibitory activity.
  • Pepsin hydrolysate of egg white protein gave no measurable DPP-IV inhibitin actibvity (IC50 > 5mg/ml).
  • IC50 > 5mg/ml
  • Table 2 DPP-IV inhibition: IC50
  • Example 6 Food supplements and food products
  • Tablets or sachets with powder containing an egg protein (lysozyme, ovomucin, or ovotransferrin) hydrolysate according to the present invention with the following composition:
  • Example 7 Animal in vivo experiments The in vivo bioactivity of Lysozym/Alcalase in inhibiting metabolic syndrome-related enzymes including DPP-IV and associated modulation of metabolic products (glucose, insuline and GLP-I), was evaluated in treatment experiments in the ZDF (Zucker Diabetic Fatty) rat model of type 2 diabetes, and compared to that of the commercially available DPP-IV-inhibitor Vildagliptin (Galvus® Novartis) as a positive control.
  • the test compounds were administered orally and blood samples were collected immediately before (i.e. baseline pre-treatment values) and at regular time points after onset of treatment.
  • ZDF rats that underwent the same experimental procedures but without receiving any active treatment (i.e. vehicle) served as a (time-)control group.
  • the blood samples were analysed for DPP-IV activity, glucose levels, insulin levels, and GLP-I levels using commercially available kits.
  • Lysozyme/Alcalase and Vildagliptin were administered by means of one oral gavage of a single dose.
  • the acute treatment effects (measured over 6 hours) were analysed by comparison of the relative changes in blood enzyme activity in rats treated with Lysozyme/Alcalase and Vildagliptin versus those that remained untreated.

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Abstract

La présente invention porte sur des hydrolysats de protéine d'œuf avec une activité inhibitrice de DPP-IV qui sont particulièrement appropriés pour le traitement du diabète. Il est particulièrement avantageux d'utiliser un hydrolysat de lysozyme pour le traitement du diabète.
PCT/NL2009/050193 2008-04-14 2009-04-14 Hydrolysats de protéine d'œuf Ceased WO2009128713A1 (fr)

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WO2011112101A1 (fr) 2010-03-08 2011-09-15 Marine Boproducts As Matériau peptidique, composition alimentaire, ses préparations et ses utilisations
WO2011112099A1 (fr) 2010-03-08 2011-09-15 Marine Bioproducts As Matériau peptidique, ses préparations et ses utilisations
WO2011112100A1 (fr) 2010-03-08 2011-09-15 Marine Bioproducts As Matériau peptidique, composition alimentaire, ses préparations et ses utilisations
WO2015082741A1 (fr) * 2013-12-04 2015-06-11 Consejo Superior De Investigaciones Cientificas (Csic) Utilisation de produits bioactifs multifonctionnels dérivés de l'hydrolyse enzymatique de protéines de blanc d'oeuf pour le traitement du syndrome metabolique
US10653613B2 (en) 2013-12-18 2020-05-19 Dsm Ip Assets B.V. Compositions comprising lysozyme hydrolysate for use in keratin-containing tissue
CN112638180A (zh) * 2018-07-31 2021-04-09 纽崔瑟斯私人有限公司 用于短期肾功能的蛋白质水解产物
CN117467732A (zh) * 2023-11-09 2024-01-30 厦门元之道生物科技有限公司 一种白蛋白活性肽的制备方法及活性多肽

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011112101A1 (fr) 2010-03-08 2011-09-15 Marine Boproducts As Matériau peptidique, composition alimentaire, ses préparations et ses utilisations
WO2011112099A1 (fr) 2010-03-08 2011-09-15 Marine Bioproducts As Matériau peptidique, ses préparations et ses utilisations
WO2011112100A1 (fr) 2010-03-08 2011-09-15 Marine Bioproducts As Matériau peptidique, composition alimentaire, ses préparations et ses utilisations
WO2015082741A1 (fr) * 2013-12-04 2015-06-11 Consejo Superior De Investigaciones Cientificas (Csic) Utilisation de produits bioactifs multifonctionnels dérivés de l'hydrolyse enzymatique de protéines de blanc d'oeuf pour le traitement du syndrome metabolique
US10653613B2 (en) 2013-12-18 2020-05-19 Dsm Ip Assets B.V. Compositions comprising lysozyme hydrolysate for use in keratin-containing tissue
EP3082837B1 (fr) * 2013-12-18 2020-09-09 DSM IP Assets B.V. Composition contenant hydrolysate de lysozyme pour l'emploi sur un tissu contenant de la keratine
CN112638180A (zh) * 2018-07-31 2021-04-09 纽崔瑟斯私人有限公司 用于短期肾功能的蛋白质水解产物
CN117467732A (zh) * 2023-11-09 2024-01-30 厦门元之道生物科技有限公司 一种白蛋白活性肽的制备方法及活性多肽

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