WO2014130007A1 - Compositions protéolytiques permettant une dégradation rapide et poussée des compléments protéiques - Google Patents

Compositions protéolytiques permettant une dégradation rapide et poussée des compléments protéiques Download PDF

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Publication number
WO2014130007A1
WO2014130007A1 PCT/US2013/026657 US2013026657W WO2014130007A1 WO 2014130007 A1 WO2014130007 A1 WO 2014130007A1 US 2013026657 W US2013026657 W US 2013026657W WO 2014130007 A1 WO2014130007 A1 WO 2014130007A1
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protein
supplement
subtilisin carlsberg
proteins
composition
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PCT/US2013/026657
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English (en)
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John Deaton
Hilton Grant DAWSON
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Deerland Enzymes, Inc.
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Priority to PCT/US2013/026657 priority Critical patent/WO2014130007A1/fr
Publication of WO2014130007A1 publication Critical patent/WO2014130007A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
    • 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
    • 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
    • A23L33/185—Vegetable 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
    • A23L33/195—Proteins from microorganisms
    • 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
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • 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)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • 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
    • A61K38/54—Mixtures of enzymes or proenzymes covered by more than a single one of groups A61K38/44 - A61K38/46 or A61K38/51 - A61K38/53
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/332—Promoters of weight control and weight loss

Definitions

  • This invention relates to proteolytic compositions and uses thereof for rapidly and extensively degrading protein supplements.
  • Protein digestion begins in the stomach, where hydrochloric acid converts pepsinogen to the digestive enzyme pepsin. Pepsin degrades proteins into smaller sized peptides. As the food enters the intestine, pancreatic enzymes trypsin and chymotrypsin continue the digestive process ideally reducing the peptides to amino acid or smaller peptides of several amino acids.
  • Some of the best protein sources are often the hardest to digest. If the body's digestive enzymes cannot degrade the protein into amino acids or very small peptides, small amounts of larger protein fragments may be absorbed. The presence of only a slight amount of such protein fragments in the brush border region, mucosal lumen or blood may cause allergy like symptoms.
  • Intestinal toxemia occurs when large particles of undigested food enter the small intestine and colon.
  • the partially-digested food mass is a fertile breeding ground for bacteria and yeast fermentation. These bacteria then produce harmful by-products that damage the intestines, reduce nutrient assimilation, create excess gas and bloating, and lead to persistent diarrhea. Mild to intense stomach pains (the result of muscle cramping and excessive gas) accompany this process.
  • Prolonged intestinal toxemia may be a major contributing factor in the onset of Irritable Bowel Syndrome and Crohn's Disease.
  • Digestive proteases have been supplemented with various proteases such as bromelain, papain or fungal protease. These additional enzymes can improve the rate of digestion by reducing the size of the protein fragments similar to the action of the pancreatic proteases. While these proteases may help, they have been shown to not degrade the various supplemental proteins to small enough fragments for absorption or to affect intolerance
  • Another way of controlling the degree of hydrolysis is to employ proteases which only cleave the protein with specificity.
  • proteases which only cleave the protein with specificity.
  • Dambmann et al. describes the use of Bacillus licheniformis protease, which reportedly has specificity for the hydrophilic amino acids aspartic and glutamic acid and thereby limits the hydrolysis and resulting bitterness of the protein hydrolysates produced with that enzyme. While this method reduces bitterness, it still reportedly requires additional processing before consumption.
  • the composition rapidly and effectively degrades, with or without the action of the animal derived digestive enzymes (e.g., pepsin, trypsin or chymotrypsin) a variety of proteins used in protein supplements to very small molecular weight fragments which can be further degraded by the intestinal peptidase to absorbable free amino acids.
  • the animal derived digestive enzymes e.g., pepsin, trypsin or chymotrypsin
  • the proteolytic composition comprises subtilisin Carlsberg, also known as subtilisin A or bacterial alkaline protease.
  • the proteolytic composition further comprises a bromelain protease. Therefore, the proteolytic composition may be a blend of subtilisin Carlsberg and bromelain protease.
  • the disclosed proteolytic composition may be added to a protein supplement just prior to ingestion. Alternatively, the disclosed proteolytic composition may be ingested before, with, or after ingestion of a proteinaceous material (e.g., food or supplement).
  • the oral dosage form can be a tablet or capsule.
  • the oral dosage form can be a liquid dosage form, e.g., containing a sweetener.
  • the proteinaceous material can be one or more proteins in deyhydrated form, such as a powdered form.
  • the subtilisin Carlsberg and bromelain protease are preferably present in an amount to hydrolyzes at least 90% of the proteins in the proteinaceous material to peptide fragments smaller than 3,000 Daltons within 90 minutes without the action of pepsin, trypsin, or chymotrypsin when placed in an aqueous environment at physiological conditions of the stomach and small intestines.
  • the subtilisin Carlsberg and bromelain protease can be present at a concentration of 2 to 20 mg per gram of protein in the proteinaceous material.
  • the disclosed protein supplement may be suitable for ingestion within thirty minutes after mixture with a beverage. This is due to the unexpected ability of subtilisin Carlsberg and bromelain protease to rapidly and effectively degrade hydrolyze proteins under acidic conditions and physiological temperatures.
  • the proteinaceous material can contain any proteins suitable for use in a protein supplement.
  • the proteins can contain whey protein, soy protein, casein, egg- white protein, hemp seed protein, rice protein, pea protein, or any combination thereof.
  • the protein supplement contains one or more additional proteases to facilitate hydrolysis.
  • the protein supplement is preferably stored in a dehydrated form, e.g., powder. Therefore, the protein supplement may be mixed with any drinkable liquid, such as water, milk, or juice, prior to ingestion.
  • any drinkable liquid such as water, milk, or juice
  • the method involves ingesting a proteolytic composition containing subtilisin Carlsberg and bromelain protease within 30 minutes of ingesting a protein supplement.
  • the proteolytic composition increases hydrolysis of the proteins in the stomach by at least 50%.
  • the proteolytic composition can increase hydrolysis of the protein to peptide fragments smaller than 3,000 Daltons within 90 minutes by at least 50%.
  • composition can also increases the rate of essential amino acid absorption by at least 50%.
  • Figure 1 is a silver stained gel showing whey hydrolyzed under USP physiological conditions (37°C, pH6.8) of the small intestine for 90 minutes with either lOmg proteolytic composition 1 (PCI) / g protein, USP pancreatin standard (1%), or a combination of both and then separated by gel electrophoresis.
  • PCI proteolytic composition 1
  • USP pancreatin standard 1%
  • Figure 2 is a bar graph showing the percentage of usable amino acids 0, 30, 60, or 90 minutes after whey protein was hydrolyzed under USP physiological conditions (37°C, pH6.8) of the small intestine for 90 minutes with either lOmg PCI / g protein or USP pancreatin standard (1%).
  • Figure 3 is a bar graph showing the percentage of usable amino acids 0, 30, 60, or 90 minutes after whey protein was hydrolyzed under USP physiological conditions of the stomach (37°C, no pepsin, pH3.0) for 10 minutes and small intestine (37°C, bile salts, pH6.8) for 90 minutes with either lOmg PCI / g protein or USP pancreatin standard (1%).
  • Figure 4 is a bar graph quantifying discomfort peptides (%) in whey after digestion under USP physiological conditions of the stomach (no pepsin, pH3.0) for 10 minutes and small intestine (bile salts, pH6.8) for 90 minutes with either lOmg PCI / g protein or USP pancreatin standard (1%) or digestive aid at their recommended dosage using Veratox kits from NeogenTM.
  • Figure 5 is a graph showing arginine levels ⁇ g/L) in the blood at various time points (minutes) after consumption of 50g of whey protein with or without PC 1.
  • Figure 6 is a graph showing total leucine levels ⁇ g/L) in the blood after consumption of 50g of whey protein over a 4.5 hour time period with or without PCI .
  • Figure 7 is a graph showing total valine levels ⁇ g/L) in the blood after
  • Figure 8 is a graph showing total threonine levels ⁇ g/L) in the blood after consumption of 50g of whey protein over a 4.5 hour time period with or without PCI .
  • Figure 9 is a graph showing total arginine levels ⁇ g/L) in the blood after consumption of 50g of whey protein over a 4.5 hour time period with or without PCI .
  • Figure 10 is a graph showing total glutamine levels ⁇ g/L) in the blood after consumption of 50g of whey protein over a 4.5 hour time period with or without PCI .
  • Figure 11 is a graph showing total phenylalanine levels ⁇ g/L) in the blood after consumption of 50g of whey protein over a 4.5 hour time period with or without PCI .
  • Figure 12 is a graph showing total methionine levels ⁇ g/L) in the blood after consumption of 50g of whey protein over a 4.5 hour time period with or without PCI .
  • Figure 13 is a graph showing total amino acid levels ⁇ g/L) in the blood after consumption of 50g of whey protein over a 4.5 hour time period with or without PCI .
  • proteolytic compositions that exhibit the unique ability to rapidly degrade many proteins extensively to very small peptide fragments (e.g., less than 3000 Daltons) under normal physiological conditions (i.e. pH 6.0-8.0 and 37°C).
  • This protease can be used for pretreatment of proteins prior to ingestion.
  • it may also be consumed with the protein supplement.
  • the problem of bitterness is eliminated.
  • the disclosed proteolytic compositions produces very extensive hydrolysis on most of the protein sources used for protein supplementation under physiological conditions of the stomach.
  • the proteolytic composition comprises subtilisin Carlsberg, also known as subtilisin A or bacterial alkaline protease.
  • the subtilisin Carlsberg protease can be an enzyme having the Enzyme Commission number EC 3.4.21.62.
  • the subtilisin Carlsberg protease may be derived from a proteolytic enzyme preparation obtained from Bacillus licheniformis .
  • the proteolytic composition further comprises a bromelain protease.
  • the bromelain protease can be an enzyme having the Enzyme Commission number EC 3.4.22.32 or EC 3.4.22.33.
  • the bromelain protease may be derived from a extract of plants of the family Bromeliaceae (e.g., pineapples).
  • the proteolytic composition may be a blend of subtilisin Carlsberg and bromelain protease.
  • the subtilisin Carlsberg is present in the blend at a concentration that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times higher than that of the bromelain protease.
  • the ratio of subtilisin Carlsberg to bromelain protease can be at least 2: 1 to about 15: 1, including about 4:1 to about 9: 1.
  • the disclosed proteolytic composition may be added to a protein supplement just prior to ingestion. Alternatively, the disclosed proteolytic composition may be ingested before, with, or after ingestion of a proteinaceous material (e.g., food or dietary supplement).
  • a forumulation comprising the disclosed proteolytic composition in an oral dosage form.
  • the oral dosage form can be a tablet or capsule.
  • the oral dosage form can be a liquid dosage form, e.g., containing a sweetener.
  • the product When provided in this form, the product would be used as a protein digestive aid, by consuming the oral dosage form along with proteinaceous matter.
  • Oral dosage forms are either solid, gel or liquid.
  • the solid dosage forms are tablets, pills, capsules, granules, and bulk powders.
  • Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or
  • oral tablets include compressed, chewable lozenges and tablets which may be sugar-coated or film-coated.
  • the oral dosage form is not enteric- coated so that the enzyme is able to begin hydrolyzing the proteinaceous matter in the stomach.
  • the oral dosage forms can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an emetic coating; and a film coating.
  • binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses,
  • Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.
  • Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.
  • Glidants include, but are not limited to, colloidal silicon dioxide.
  • Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and
  • Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate.
  • Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.
  • Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.
  • Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates.
  • Film coatings include hydroxy ethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and
  • effervescent preparations reconstituted from effervescent granules.
  • Aqueous solutions include, for example, elixirs and syrups.
  • Emulsions are either oil-in- water or water- in-oil.
  • Elixirs are clear, sweetened, hydroalcoholic preparations.
  • Nutritionally acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative.
  • An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid.
  • Nutritionally acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use nutritionally acceptable suspending agents and preservatives.
  • Nutritionally acceptable substances used in non- effervescent granules, to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents.
  • Nutritionally acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.
  • Solvents include glycerin, sorbitol, ethyl alcohol and syrup.
  • preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol.
  • non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil.
  • emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.
  • Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia.
  • Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.
  • Organic acids include citric and tartaric acid.
  • Sources of carbon dioxide include sodium bicarbonate and sodium carbonate.
  • Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof.
  • Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.
  • the disclosed proteolytic composition may mixed with a proteinaceous material prior to ingestion. Therefore, also provided is a protein supplement comprising a proteinaceous material and the disclosed proteolytic composition .
  • a protein supplement comprising a proteinaceous material and the disclosed proteolytic composition .
  • examples of products containing proteinaceous material that can be combined with the proteolytic composition include protein supplements, protein drink mixes, and meal replacements. Therefore, the proteinaceous material can contain any proteins suitable for use in a protein supplement.
  • the proteins can contain whey protein, soy protein, casein, egg-white protein, hemp seed protein, rice protein, pea protein, or any combination thereof.
  • the proteinaceous material is preferably in deyhydrated form, such as a powdered form, prior to hydration and ingestion to prevent premature hydrolysis, which can cause a bitter taste.
  • the disclosed protein supplement may be suitable for ingestion within 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 minutes after mixture with an aqueous solution. This is due to the unexpected ability of the disclosed proteolytic composition to rapidly and effectively hydro lyze proteins under acidic conditions and physiological temperatures. Also unexpected are organoleptic qualities of the formulation when used within about 30 minutes.
  • the protein supplement is preferably stored in a dehydrated form, e.g., powder.
  • the protein supplement may be mixed (i.e., hydrated) with a nutritionally acceptable aqueous solvent prior to ingestion.
  • the protein supplement may be mixed with water, milk, or juice.
  • the resulting hydrated product is preferably a drinkable liquid (e.g., protein shake).
  • the composition may include one or more of various enzymes, vitamins, minerals, antioxidants, essential oils, herbs, or extracts, in various combinations. Additional components or additives, such as suitable standardizing ingredients and/or processing aids, can be included in the composition to provide acceptable physical and mechanical properties such as flow, compaction and compressibility, and concentration.
  • the protein supplement contains one or more additional proteases to facilitate hydrolysis.
  • suitable proteases include, but are not limited to, a-Amino-Acyl-Peptide Hydrolases (EC 3.4.1), Peptidyl-Amino-Acid Hydrolases (EC 3.4.2), Dipeptide Hydrolases (EC 3.4.3), Peptidyl Peptide Hydrolases (EC 3.4.4), Aminopeptidases (EC 3.4.11), Peptidylamino-Acid Hydrolases or Acylamino- Acid Hydrolases (EC 3.4.12), Dipeptidases (EC 3.4.13), Dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), Peptidyl-dipeptidases (EC 3.4.15), Serine-type carboxypeptidases (EC 3.4.16), Metallocarboxypeptidases (EC 3.4.17), Cysteine-type carboxypeptidases (EC 3.4.18),
  • the protein supplement further contains papain trypsin chymotrypsin pepsin,
  • aspergillopepsin fungal protease, bacterial neutral protease, or any combination thereof.
  • Subtilisin Carlsberg and bromelain protease are therefore preferably present in an amount to hydrolyzes at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the proteins in the proteinaceous material to peptide fragments smaller than 3,000 Daltons, 2,500 Daltons, 2,000 Daltons, 1,500 Daltons, 1,000 Daltons, or 500 Daltson within 90 minutes, 60 minutes, 45 minutes, or 30 minutes without the action of pepsin, trypsin, or chymotrypsin when placed in an aqueous environment at physiological conditions of the stomach and small intestines (e.g., 37°C, pH6.8).
  • pepsin, trypsin, or chymotrypsin when placed in an aqueous environment at physiological conditions of the stomach and small intestines (e.g., 37°C, pH6.8).
  • subtilisin Carlsberg and bromelain protease can be present in an amount to hydrolyzes at least 90% of the proteins in the proteinaceous material to peptide fragments smaller than 3,000 Daltons within 90 minutes in an aqueous environment at 37°C and a pH of about 4.5 to 8.0, including about pH 6.8.
  • the subtilisin Carlsberg and bromelain protease is present at a concentration of about 1 to 30 mg per gram of protein in the proteinaceous material, including about 5 to 20 mg per gram of protein, or about 10 to 15 mg per gram of protein.
  • a method for increasing hydrolysis of proteins in a food or protein supplement after ingestion by a subject involves ingesting the disclosed proteolytic composition within 10, 15, 30, 45, or 60 minutes of ingesting a proteinaceous food or protein supplement.
  • the disclosed proteolytic composition increases hydrolysis of the proteins in the stomach by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99.9 %.
  • Antibody testing of hydro lyzed material using a VERATOX quantitative microwell assay available from Neogen Corporation, Lansing, Michigan 48912 USA, has shown reduction in allergen levels for soy, whey, egg and gliadin below detectable levels.
  • the disclosed proteolytic composition can also increase the rate of essential amino acid absorption by at least 50%>.
  • Example 1 PCI with Milk Protein Whey in a Sports Protein Drink: A Controlled Study to Evaluate Efficacy
  • Whey protein is one of the richest sources of all of the nine essential amino acids which the body is unable to produce. However, whey protein must first be processed into a useable form for building and recovery of muscle (peptides and amino acids) and it must do so in less than two hours to be absorbed. This is the time from the stomach through the small intestine in which digestion/absorption primarily occurs (Kim SK.
  • the in vitro conditions were designed to mimic the processes that occur in the digestive tract when whey is consumed, including buffering capacity of the drink, pH of the stomach and small intestine, salt conditions, bile salts and transient time.
  • Drinks show a transient time of approximately 30 minutes in the stomach and less than two hours in the small intestine (Kim SK. 1968. Am. J. Roentgenol 104:522-24).
  • the body produces proteases of its own including pepsin in the stomach and pancreatic enzymes in the small intestine.
  • Most protein drinks buffer the stomach between 4-6 pH, which greatly reduces the acidity needed to unfold/hydrolyze the proteins and the efficiency of pepsin (Fordtran, J. S., et al. 1973. J. Clin. Invest.
  • PCI protease formulation
  • PDCAAS is a method of evaluating the protein quality based on both the amino acid requirements of humans and their ability to digest it.
  • the formula for calculating the PDCAAS percentage is: (mg of limiting amino acid in 1 g of test protein / mg of same amino acid in 1 g of reference protein) x fecal true digestibility percentage (Schaafsma G. 2000. J. Nutr. 130[7], 1865S-7S), which means that the protein can be broken down in the large intestine where very little absorption (less than 10%) occurs.
  • Whey also has a high Biological Value (BV), which is a measure of the proportion of absorbed protein from a food which becomes incorporated into the proteins of the organism's body. Biological value is determined based on this formula:
  • Nr nitrogen incorporated into the body on the test diet.
  • Nr is problematic. While both PDCAA and BV give a measurement of the nutritional quality of the protein, they have limits to how much of the protein is actually absorbed and used for protein synthesis. The best way to determine if a whey supplement is being processed so the body can use it is by conducting in vitro and in vivo tests. The following is an example of the testing of whey protein supplement and whether or not it is processed enough to provide benefit to the user.
  • Protein must be broken down small enough to pass through the small intestine, absorbed and re-assembled into muscle protein.
  • active people require, many turn to protein supplements.
  • These products are usually in the form of protein products rich in essential amino acids (because of cost and concentration) such as whey, soy, and/or egg. Some of these products can have a suggested serving size as high as 50g of protein, requiring large amounts of processing by the body before they can be absorbed and used for protein synthesis.
  • PCI protease supplement
  • Control Groups Before the study, all participants reported after an overnight fast for Day 1 of the study. On Day 1 , control samples were collected after all participants ingested one 50g, pre -measured packet of whey protein isolate without PCI . The entire contents of each individual serving packet were emptied into 0.5 L of distilled water, vigorously shaken and consumed. Blood samples were collected at 0 hr (baseline, immediately prior to ingestion) 0.5 hr, 1.5 hr, 2.5 hr, 3.5 hr and 4.5 hr. The blood samples were tested for amino acids that are essential to the body and those that play a significant role in muscle protein synthesis (12 total).
  • Test group Following five days and an overnight fast, the participants returned to the laboratory for Day 2 of the study. Each participant received 500mg PCI, pre -blended in 50g whey powder isolate. The entire contents of each individual serving packet containing PCI were emptied into 0.5 L of distilled water, vigorously shaken and consumed. Blood samples were collected at 0 hr (baseline, immediately prior to ingestion) 0.5 hr, 1.5 hr, 2.5 hr, 3.5 hr and 4.5 hr. The blood samples were tested for twelve amino acids that are essential to the body and those that play a significant role in muscle protein synthesis. Following the study, the control data from each participant was compared to the data from the corresponding patient in either of the two test groups.
  • Sample collection Whole blood samples (approximately 5 mL) were collected by a phlebotomist from multiple venous punctures, and transferred to plain Vacutainer® tubes. Serum was prepared by centrifugation and stored in 200 aliquots at -20°C until needed for analyses.
  • Analytical analyses All serum samples were submitted to the laboratory blind to remove any analytical bias. Amino acid analyses consist of quantification of twelve individual serum amino acids for each patient at each time point. Amino acid analyses consisted of quantification of twelve individual serum amino acids for each patient over the 270 minute time period. Analyses were performed on an AA analyzer using ion exchange chromatography and a post column derivatazation with ninhydrin and UV detection.
  • amino acid quantity was calculated from area under the curve (AUC) over the entire time period and compared (270 minutes) instead of individual time points. Quantification was done versus reference standard mixtures and control mixtures of known quantities of all twelve amino acids (Sigma, St. Louis, MO). Amino acid levels are reported in ⁇ g/L. Total Serum Amino Acids (TSAA) levels were reported as the total sum of all twelve amino acids. The percent AUC was reported as the amount each amino acid contributed to the total. AUC was calculated using the
  • BCAA branch chain amino acids
  • the first is the branch chain amino acids (BCAA).
  • BCAA is a combination of essential branched chain amino acids such as leucine, isoleucine, and valine that are of special importance for bodybuilders and athletes because these amino acids are used by the body to build up protein for muscle synthesis, muscle repair, etc (Blomstrand, E., et al. 2006. J. Nutr. 136, 269-73). Because BCAAs are essential amino acids, not produced by the body, they must be acquired from the diet or from nutritional supplements. BCAA is metabolized in the muscle rather than the liver; consequently the effect of these branched chain amino acids is much quicker and efficient than of any other amino acid.
  • BCAAs are digested, protein breaks down into individual amino acids that can either be used to build new proteins or used as energy for the body. If the diet is balanced, branched chain amino acids will be used for protein synthesis, essential for endurance athletes and strenuous workouts. BCAAs are also used to reduce fatigue in both anaerobic and endurance sports.
  • leucine Because of its anticatabolic properties and vital role in protein synthesis, leucine is considered to be one of the most critical BCAAs. Leucine is the strongest of the BCAAs and is responsible for the regulation of blood-sugar levels, the growth and repair of tissues in skin, bones and of course skeletal muscle. It's a strong potentiator to Human Growth Hormone. It helps in healing wounds, regulating energy and assists in the preventing the breakdown of muscle tissue. Levels increased by 5%.
  • Valine contributes to repair and growth of muscle tissue, as commonly attributed to BCAAs. It is not processed by the liver; rather actively taken up by muscle. It maintains the nitrogen balance and preserves the use of glucose. Levels increased by 11%.
  • Threonine is an essential amino acid that is never manufactured within the body. Since its main sources are animal (dairy and meat) this doesn't bode well to vegans. It's found in heart, skeletal muscle and nerve tissue in the central nervous system. Threonine is used to form the body's two most important binding substances, collagen and elastin. Threonine is involved in liver functioning, lipotropic functions (when combined with aspartic acid and methionine) and in the maintenance of the immune system by helping in the production of antibodies and promoting growth and activity of the thymus. Perhaps its most useful property is that it allows better absorption of other nutrients; therefore protein sources containing threonine are more bioavailable than others. Levels increased by 6%.
  • Arginine has amazing nitrogen retention ability. Nitrogen is one of the key elements in muscle protein synthesis. Some plants can absorb nitrogen, but mammals have to make do with what we make our. Arginine enhances the immune system and stimulates the size and activity of the Thymus gland, which makes it a prime choice for anyone in less than optimal health, such as people recovering from injury and HIV patients. Arginine is also a precursor of very important molecules such as creatine and gamma amino butric acid (GAB A, a neurotransmitter in the brain). The hormonal release properties include releasing insulin from the pancreas and a massive stimulator in the manufacture of GH (Growth Hormone) from the anterior pituitary. It increases blood flow.
  • GH Crowth Hormone
  • Glutamine is a non-essential amino acid that is present in the body in large amounts. At some times it forms 60 percent of the total amino acid pool. Because it passes through the blood-brain barrier rather easily, it's often called "brain-food". In the brain it converts to glutamic acid, which is essential for brain functioning and increase GABA (gamma-amino-butyric-acid, another popular supplemented amino) needed for mental activities. It is used in synthesis of muscle tissue. It is a nontoxic nitrogen carrier. Perhaps most importantly, it balances the acid/ alkaline level, so it reduces lactic acid. Levels increased by 28%.
  • Phenylalanine the major precursor of tyrosine, enhances learning, memory, mood and alertness. Is a major element in the production of collagen and suppresses appetite levels increased by 9%.
  • Methionine is the precursor of cystine and creatine, and may increase antioxidant levels (glutathione) and reduce blood cholesterol levels. It also helps remove toxic wastes from the liver and assists in the regeneration of liver and kidney tissue. Levels increased by 6%.
  • Peptide discomfort is caused by the presence of specific sequences of amino acids, called epitopes, in the native protein.
  • Epitopes are areas on the protein surface which are recognized by the immune system and identified as having between 8-16 amino acids. It is thought that a discomfort peptide has a minimum of 14 amino acid residues. Trypsin digestion of ⁇ -lactoglobulin has shown that there are many epitopes spread over its surface.
  • the molecular weight of a protein also determines whether it will act as a potentially discomfort material or not.
  • Potential discomfort material consists of components with the ability to stimulate antibody production and a component with at least two antibody binding sites. The probability that a component will meet these criteria increases above a molecular weight of 3,000 Daltons (Rosendal A, et al.

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Abstract

La présente invention concerne des compositions protéolytiques et des procédés permettant une hydrolyse rapide et efficace des protéines que l'on trouve dans les compléments protéiques utilisés en nutrition sportive et dans les substituts de repas, ladite hydrolyse donnant des fragments de très faible poids moléculaire dans les conditions physiologiques normales régnant au sein de l'estomac et de l'intestin grêle. En conséquence, à la différence d'autres compositions à base de protéases, les compositions protéolytiques selon l'invention peuvent être consommées en même temps que les protéines (par exemple en même temps que le complément protéique), ce qui élimine le problème d'amertume associé à la prédigestion des protéines.
PCT/US2013/026657 2013-02-19 2013-02-19 Compositions protéolytiques permettant une dégradation rapide et poussée des compléments protéiques WO2014130007A1 (fr)

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WO2016115249A1 (fr) * 2015-01-16 2016-07-21 General Mills, Inc. Procédé in vitro pour estimer la digestibilité d'une protéine in vivo
WO2016156077A1 (fr) * 2015-03-30 2016-10-06 Nestec S.A. Hydrolysats de protéine à base de lait et compositions fabriquées avec ces derniers
WO2017121859A1 (fr) * 2016-01-13 2017-07-20 Wolfgang Priemer Composition contenant des acides aminés
CN107455746A (zh) * 2017-09-12 2017-12-12 安徽养生天下生物科技有限公司 一种综合酵素代餐包及其制备方法
CN111000254A (zh) * 2019-12-30 2020-04-14 湖南点道生物科技有限公司 一种复合蛋白质组件及其制备方法
US20220088151A1 (en) * 2019-02-07 2022-03-24 Bright Lifecare Pvt. Ltd Proteolytic enzyme composition
CN114395600A (zh) * 2022-01-14 2022-04-26 琛蓝(美国)营养制品股份有限公司 一种多功能豌豆肽的制备方法及其应用

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WO2010109257A1 (fr) * 2009-03-23 2010-09-30 Carlo Ghisalberti Composition destinée au traitement de l'intolérance aux protéines
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WO2011063394A2 (fr) * 2009-11-23 2011-05-26 Olmstead Stephen F Compositions et méthodes comprenant une peptidase serratia destinées à l'inhibition et au traitement de biofilms liés à certaines pathologies

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WO2004014153A1 (fr) * 2002-07-25 2004-02-19 Krueger Sabine Produit alimentaire dietetique
US20110110910A1 (en) * 2005-06-24 2011-05-12 Novozymes A/S Proteases for Pharmaceutical Use
US20100143320A1 (en) * 2007-06-18 2010-06-10 Et & Ds Company Ltd. Dietary supplement
WO2009100456A2 (fr) * 2008-02-08 2009-08-13 Prothera, Inc. Inhibition et traitement de biofilms gastro-intestinaux
WO2010109257A1 (fr) * 2009-03-23 2010-09-30 Carlo Ghisalberti Composition destinée au traitement de l'intolérance aux protéines
WO2011063394A2 (fr) * 2009-11-23 2011-05-26 Olmstead Stephen F Compositions et méthodes comprenant une peptidase serratia destinées à l'inhibition et au traitement de biofilms liés à certaines pathologies

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US9738920B2 (en) 2015-01-16 2017-08-22 General Mills, Inc. In vitro method for estimating in vivo protein digestibility
WO2016115249A1 (fr) * 2015-01-16 2016-07-21 General Mills, Inc. Procédé in vitro pour estimer la digestibilité d'une protéine in vivo
AU2016239617B2 (en) * 2015-03-30 2020-08-13 Société des Produits Nestlé S.A. Milk-based protein hydrolysates and compositions made thereof
WO2016156077A1 (fr) * 2015-03-30 2016-10-06 Nestec S.A. Hydrolysats de protéine à base de lait et compositions fabriquées avec ces derniers
AU2016239617B9 (en) * 2015-03-30 2020-09-24 Société des Produits Nestlé S.A. Milk-based protein hydrolysates and compositions made thereof
US10798963B2 (en) 2015-03-30 2020-10-13 Societe Des Produits Nestle S.A. Milk-based protein hydrolysates and compositions made thereof
EP3804532A1 (fr) * 2015-03-30 2021-04-14 Société des Produits Nestlé S.A. Hydrolysats de protéine à base de lait et compositions fabriquées par ce procédé
AU2020267141B2 (en) * 2015-03-30 2022-07-28 Société des Produits Nestlé S.A. Milk-based protein hydrolysates and compositions made thereof
WO2017121859A1 (fr) * 2016-01-13 2017-07-20 Wolfgang Priemer Composition contenant des acides aminés
US10842164B2 (en) 2016-01-13 2020-11-24 Wolfgang Priemer Composition containing amino acids and process for producing same
CN107455746A (zh) * 2017-09-12 2017-12-12 安徽养生天下生物科技有限公司 一种综合酵素代餐包及其制备方法
US20220088151A1 (en) * 2019-02-07 2022-03-24 Bright Lifecare Pvt. Ltd Proteolytic enzyme composition
CN111000254A (zh) * 2019-12-30 2020-04-14 湖南点道生物科技有限公司 一种复合蛋白质组件及其制备方法
CN114395600A (zh) * 2022-01-14 2022-04-26 琛蓝(美国)营养制品股份有限公司 一种多功能豌豆肽的制备方法及其应用
CN114395600B (zh) * 2022-01-14 2023-12-12 琛蓝(美国)营养制品股份有限公司 一种多功能豌豆肽的制备方法及其应用

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