WO2018145712A1 - Food ingredient comprising 3-(4-hydroxyphenyl)propanoic acid amide and whey protein - Google Patents

Food ingredient comprising 3-(4-hydroxyphenyl)propanoic acid amide and whey protein Download PDF

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Publication number
WO2018145712A1
WO2018145712A1 PCT/DK2018/050026 DK2018050026W WO2018145712A1 WO 2018145712 A1 WO2018145712 A1 WO 2018145712A1 DK 2018050026 W DK2018050026 W DK 2018050026W WO 2018145712 A1 WO2018145712 A1 WO 2018145712A1
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Prior art keywords
whey protein
food product
composition according
composition
hydroxyphenyl
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PCT/DK2018/050026
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French (fr)
Inventor
Jan-Elo Bjarne JØRGENSEN
Martin Jørgensen
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Nmetics Ivs
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    • 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
    • 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
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/03—Products from fruits or vegetables; Preparation or treatment thereof consisting of whole pieces or fragments without mashing the original pieces
    • A23L19/05—Stuffed or cored products; Multilayered or coated products; Binding or compressing of original pieces
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A23L2/39—Dry compositions
    • 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
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • 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
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • A23V2250/00—Food ingredients
    • A23V2250/54—Proteins
    • A23V2250/542—Animal Protein
    • A23V2250/5424—Dairy protein
    • A23V2250/54252—Whey protein

Definitions

  • the present invention relates to a food ingredient comprising 3-(4- hydroxyphenyl)propanoic acid amide and whey protein, preferably non-hydrolyzed whey protein.
  • a food product comprising such ingredient a uses thereof such as a wound healing agent, in the treatment and/or alleviation of PostoOperative Nausea and Vomiting (PONV), a tissue repair agent and a tissue restitution agent e.g. muscle restitution of athletes.
  • PONV PostoOperative Nausea and Vomiting
  • tissue repair agent e.g. muscle restitution of athletes.
  • the repair of cell tissue is a complex process involving several cell biochemical sub-processes. These involve disinfecting and cleaning up the damaged area by infusion of cytokines, migration of cells to cover up the damage as well as contraction and granulation of the new tissue.
  • the cell migration primarily being driven by coordinated assembly and disassembly of actin filaments.
  • improvement of the cells ability to adhere at focal points to the substratum and migrate more efficiently to recover the tissue damage will overall improve efficacy of the cell tissue repair process.
  • Such processes are also relevant after surgery.
  • PA 3-(4-hydroxyphenyl)propanoic acid amide
  • WO2007046721 discloses a method of manufacturing PA, its application in the manufacture of anti-aging compositions.
  • Whey protein has been found to possess beneficial effects on wound healing (Ebaid et al. Whey protein enhances normal inflammatory responses during cutaneous wound healing in diabetic rats). Lipids in Health and Disease 2011, 10: 235).
  • an improved wound healing composition would be advantageous, and in particular a more efficient and/or reliable food ingredient/food product assisting in wound healing would be advantageous.
  • This invention combines the tissue recovering effects by two distinct molecular compounds into a broadly acting nutrition for damaged tissues.
  • the invention combines the overall cell repair stimulation by proteins with the molecular cell repair mechanisms of an aromatic organic compound from the apple plant.
  • the protein source is whey protein (preferably non-hydrolyzed that ensures a neutral taste, avoiding the "chalk-like" sensation in the mouth that hydrolysed proteins sources normally give rise to).
  • the apple compound is Phloretamide (PA) that interacts directly with the cellular organelles to improve the molecular mechanisms of two of the three main phases of the tissue repair process (see examples 4 and 5). Individually, these two compounds addresses several distinct sub-processes of the overall tissue recovery process and together the combination constitute a package that boosts the overall condition of the recovering tissue as well as improving the agility of the repairing cells on a molecular level.
  • these two molecular sources have been combined into a potentially tissue-restoring apple/whey based organic ice cream, optimized for post operational patients.
  • Sensory and intake experiments on post-operational patients further revealed a surprisingly high intake percentage of the whey protein/apple ice cream vehicle (example 1) and that the addition of higher concentrations of phloretamide had no apparent impact on the overall taste (example 2).
  • Examples of compositions according to the present invention are disclosed in example 3.
  • the present invention relates to a composition (such as a food ingredient) comprising 3-(4-hydroxyphenyl)propanoic acid amide (HA) and whey protein. It has been found that 3-(4-hydroxyphenyl)propanoic acid amide has positive effects on would healing and similar processes (documented in example 4 & 5 in different cell migration experiments).
  • Improvement of wound healing may be particular relevant for patients who has e.g. just experienced surgery. After general anaesthesia however, patients often have difficulties swallowing, making it difficult for the patient to obtain the required amount of nutrition through normal eating.
  • Whey protein may be highly concentrated in certain types of frozen confectionary such as ice cream. Ice creme may be easier for patients to eat just after surgery than normal food.
  • an object of the present invention relates to the provision of a
  • composition/food ingredient/food product which would have beneficial effects for patients (such as improved wound healing) who has just had surgery, while maintaining the ability for the patient to eat the product just after surgery.
  • one aspect of the invention relates to a composition
  • a composition comprising
  • composition is preferably used in a food product.
  • the composition is a food ingredient.
  • Another aspect of the present invention relates to a food product comprising the composition according to the invention.
  • Yet another aspect of the present invention is to provide a composition according to the invention and/or food product according to the invention, for use as a wound healing agent.
  • the invention relates to the composition according to the invention and/or food product according to the invention for use in the treatment and/or alleviation of Postoperative nausea and vomiting (PONV).
  • PONV Postoperative nausea and vomiting
  • the invention relates to a composition according to the invention and/or food product according to the invention, for use as a tissue repair agent and/or a tissue restitution agent e.g. muscle restitution of athletes.
  • Still another aspect of the present invention is to provide a process for preparing a composition according to the invention, said process comprising
  • composition (such as food ingredient) comprising
  • PA 3-(4-hydroxyphenyl)propanoic acid amide
  • whey protein 0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 0.1-99% (w/w) whey protein.
  • Figure 1 shows in vitro assays reflecting tissue repair sub-processes in vivo.
  • Figure 2 shows the effect of PA treatment on the migration rate of human fibroblast cells in a bidirectional scratch assay. Fibroblasts were pre-treated with PA (80 ⁇ ) for three days before performing the scratch assay. Migration was quantified by a micrometer scale (see figure 3).
  • Figures 3A + 3B show quantification of results of experiments from figure 2. PA improves tissue migration by about 37% over the untreated.
  • Figure 4 shows the effect of PA treatment on the migration rate of human fibroblast cells in a uni-directional scratch assay. Fibroblasts were pre-treated with PA (80 ⁇ ) for three days before performing the scratch assay.
  • Figure 5 shows quantification of results of experiments from figure 4. After 24 hrs PA enhances migration (tissue repair) in vitro by more than 60% as measured by uni-directional cell migration. Top: cell migration distance. Bottom : Rate of cell migration.
  • Figure 6 illustrates the principle of the Boyden chamber assay for chemotaxic cell migration.
  • FIG. 7 shows the result of three independent Boyden chamber assays. Migrated cells become attached to a polycarbonate membrane at the bottom of the chamber and stained (dots). Three separate experiments are shown. Top:
  • Figure 8 is the quantification of the Boyden chamber assay results. PA enhances chemotactic migration towards chemo-attractants by a factor 2.7 in vitro.
  • Figure 9 illustrates the principle of the free floating collagen lattice assay.
  • Figure 10 shows on example of a free floating collagen lattice assay and the effect or pre-treatment of the embedded fibroblast cells with PA. Top: Control and PA pre-treated. Bottom : Measurement of the collageneous lattices upon contraction.
  • Figure 11 shows the quantitative results of several free floating collagen lattice assays.
  • X-axis Hours.
  • Y-axis Surface area in %.
  • 3-(4-hydroxyphenyl)propanoic acid amide corresponds to a compound of the formula (I)
  • the compound of formula (I), may in here also be denoted as PA.
  • the compound of the formula (I) is also known as (4-hydroxyphenyl)propanoic acid amid, 3-(P- hydroxyphenyl)propionamide or simply phloretamide.
  • PA is naturally present in apples at a concentration around 1 ⁇ /kg (0.0000001%
  • Whey protein is the collection of globular proteins isolated from whey.
  • the protein in cow's milk is 20% whey protein and 80% casein protein.
  • the protein fraction in whey constitutes approximately 10% of the total dry solids in whey.
  • This protein is typically a mixture of beta-lactoglobulin ( ⁇ 65%), alpha-lactalbumin ( ⁇ 25%), bovine serum albumin ( ⁇ 8%), and immunoglobulins. These are soluble in their native forms, independent of pH.
  • the caseino-glycomacropeptide has been removed or substantially removed, such as to a level of less than 2 mg casein/L whey, e.g. less than 1 mg casein/L whey, such as less than 0.5 mg casein/L whey.
  • Whey protein typically comes in four major forms: concentrate (WPC), isolate (WPI), hydrolysate (WPH) and Native Whey. Most whey concentrates and isolates are available as intact proteins, but either can be also hydrolyzed. Hydrolysates have been partially broken down by exposing the protein to heat, acid or enzymes that break apart the bonds linking amino acids.
  • the whey protein according to the present invention is preferably un-hydrolyzed whey protein to improve the taste.
  • the total whey protein content is composed of at least about 60-100% unhydrolyzed intact whey protein. In one embodiment, the total protein content may compose of at least about 90%-100%; at least about 95%- 100%; at least about 95% or at least about 100% unhydrolyzed intact whey.
  • the whey is also preferably of bovine origin and not of human origin.
  • Other sources of whey protein are goat, camel, sheep, and horse.
  • PA 3-(4-hydroxyphenyl)propanoic acid amide
  • composition such as a food product (ice cream) or food ingredient (e.g. dried ingredient)
  • improved wound healing properties which may be beneficial e.g. for patients having trouble eating just after surgery.
  • the invention relates to a composition comprising
  • composition is preferably used in a food product.
  • the composition is a food ingredient.
  • the amount of PA in the composition may vary depending on the final desired use.
  • the amount of PA in the composition is in the range 0.001-1%, such as 0.01-1%, preferably around 0.1-1%.
  • the natural amount of PA in apples are estimated to be around 1 pg/kg (0.0000001 % w/w).
  • the above-indicated values in the compositions according to the invention are substantially higher than what can be found in a natural product (apples), especially in combination with whey proteins.
  • the amount of whey protein in the composition may also vary depending on the final desired use.
  • concentration of whey protein is in the range 1-90%, such as 10-90%, such as 10-70%, such as in the range 20- 50% (w/w).
  • the whey may be provided from different sources.
  • the whey protein is provided from the group consisting of whey protein concentrate (WPC), whey protein isolate (WPI) and native whey.
  • WPC whey protein concentrate
  • WPI whey protein isolate
  • native whey native whey.
  • the way is preferably of bovine origin although other sources may also be used.
  • the whey protein is of non-human origin, such as bovine origin.
  • the composition may also comprise further components.
  • the composition further comprises one or more anti-nausea agents.
  • the one or more anti-nausea agents is selected from the group consisting of ginger, lemon, peppermint, and vitamin B6.
  • the composition according to the invention has a long shelf life and/or is easy to transport.
  • the composition is in a dry state, such as having a moisture content below 10%, such as below 8% or such as below 6%.
  • compositions according to the invention may comprise: - 0.1-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and
  • whey protein such as un-hydrolyzed whey protein.
  • w/w 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 20-70% (w/w) whey protein, such as un-hydrolyzed whey protein.
  • w/w 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 20-40% (w/w) whey protein, such as un-hydrolyzed whey protein.
  • PA 3-(4-hydroxyphenyl)propanoic acid amide
  • compositions according to the invention may be included in a food product as an ingredient.
  • an aspect of the invention relates to a food product comprising the composition according to the invention.
  • the food product may of course further comprise different taste and nutritional components.
  • the food product further comprises fruit, such as extract and/or pulp.
  • said fruit is selected from the group consisting of strawberry, banana, raspberry, apple, lemon, pear, and combination thereof, preferably apple.
  • the food product further comprises probiotics, such as lactic acid bacteria (LAB). LAB's are well known for the health improving effects, e.g. in beverages.
  • the food product further comprises a sweetener, such as sugar.
  • a sweetener such as sugar.
  • sugars are glucose, sucrose etc.
  • the food product is a frozen or cooled/chilled
  • the frozen confectionary is selected from the group consisting of milkshakes, fruit smoothie, ice cream, ice lollies, frozen fruit bar, frozen yogurt.
  • the food product is selected from the group consisting of beverages, such as restituting beverages and sports drinks, nutripharmaceuticals, nutracosmetics, and nutraceuticals.
  • PA 3-(4-hydroxyphenyl)propanoic acid amide
  • whey protein preferably un-hydrolyzed whey protein; - optionally, one or more anti-nausea agents, such as ginger; and
  • compositions, food ingredients, and food products according to the invention may be used for different medical purposes, such a wound healing agent after surgery.
  • the invention relates to a composition according to the invention and/or food product according to the invention, for use as a medicament.
  • the invention relates to a composition according to the invention and/or food product according to the invention, for use as a wound healing agent.
  • the invention relates to the composition according to the invention and/or food product according to the invention for use in the treatment and/or alleviation of Postoperative nausea and vomiting (PONV).
  • PONV Postoperative nausea and vomiting
  • the invention relates to a composition according to the invention and/or food product according to the invention, for use as a tissue repair agent and/or a tissue restitution agent e.g. muscle restitution of athletes.
  • composition according to the invention and/or food product according to the invention is for oral administration.
  • composition and/or food product according to the invention is for administration to the patient within such as 2 hours after awakening after general anesthesia, such as after surgery.
  • composition and/or food product is for administration within 2 hours, such as 1 hour after awakening after general anesthesia, such as within 30 minutes.
  • composition and/or food product is for administration after awakening after general anesthesia and when the patient is able to swallow.
  • composition according to the invention may be prepared by different processes.
  • the invention relates to a process for preparing a composition according to the invention, said process comprising
  • composition (such as food ingredient) comprising
  • the process further comprises drying said composition, e.g. by spray drying and/or evaporation.
  • the process further comprising mixing said product with a food a food product or food ingredient(s) to provide a food product or food ingredient according to the invention.
  • a specially designed ice cream for adult patients with a high content of non- hydrolyzed whey protein was developed.
  • Apple pulp was used as a major ingredient in the ice creams in order to retain the apple theme referring to the origin of Phloretamide.
  • the ice creams have a texture, which had neither a sticky or greasy feel in the mouth. It is organic, based on birch juice and sugar tongs. The content of natural sugars and minerals promotes ice sweet taste.
  • the apple strain used as a basis is optimized for sour sweet taste ratio in order to stimulate saliva secretion upon surgery.
  • Unhydrolyzed whey protein was provided from cow milk. Whey protein fractions are available from several suppliers. Unhydrolyzed whey protein may be prepared by up-concentrating whey several times using standard membrane technology in the field. To avoid hydrolysis the steps should be performed without any heating steps. Liquefied whey protein and prepared apple pulp all at 2-5 °C are mixed with the remaining ingredients in a mixing pasteurizer. The mixture is cooled to 4°C within 3 hours and the mixture is transferred to an ice cream-machine from there to filling and chock frozen to stock at minimum -18°C. Total of 80 mis pr. ice cream. Recipe I:
  • a collection of five different ice cream recipes were initially sensory taste evaluated by a panel of independent taste evaluators. All five ice creams were based on apple pulp and whey protein. The apple strains used were selected as being organic and with a relatively high content of acid to stimulate saliva production in awakening post operational patients. Granny Smith was selected for the sweater Apple cake ice cream with vanilla and Green Sleeves was selected for the fresh apple ice cream and the ginger added ice cream. Ginger was added to lower nausea upon operation. In the above examples birch sap was used as sweetener, but other sugar sources may just as well be used. These ice creams were to serve as a basis for the final Phloretamide/whey protein combination ice cream.
  • Phloretamide/whey recovery compound combination Further, the cell recovery using protein rich nutrition is established as described above, but protein powder in food often results in a chalk like taste.
  • non-hydrolyzed (un-hydrolyzed) whey protein as a protein tissue recovery booster in an ice cream that has a creamy but neutral taste.
  • Phloretamide (PA) this will constitute a powerful package for cell and tissue recovery, as Phloretamide has been show to aid cell tissue damage recovery in cell in vitro tests addressing cell motility as well as collagen contraction (see examples 4 and 5). Phloretamide together with non-hydrolyzed whey protein in a vehicle of easily consumed and well tasting apple ice cream will constitute an ideal package for nutritioning post operational anaesthetic patients and recovery after physical exercise.
  • Phloretamide and non-hydrolyzed whey protein will address tissue recovery in a range of food vehicles thus being a potential component of a range of tissue recovery food products.
  • Phloretamide (PA) was tested for taste and solubilized at 0.1% (w/w), thus added to 55°C apple pulp. There was no apparent difference in taste upon addition of PA.
  • WO2007046721 discloses one method of manufacturing PA.
  • compositions according to the invention are:
  • Composition 1 Powder mixture for drinks or smoothies
  • Composition 2 Ice cream
  • Aim to study contraction of ECM molecules by fibroblasts
  • Collagen type I from bovine skin can be purchased from IBFB Pharma GmbH, für, Germany and is prepared according to the distributed protocols.
  • Cells are trypsinized, suspended in complete growth medium and carefully mixed in the collagen solution (collagen in 0.9x DMEM, 10 % FCS, 0.2 mM NaOH, prepared at room temperature).
  • Final cell concentrations might range from 0.8xl0 4 to 3xl0 5 cells/ml lattice.
  • Final concentration of collagen might range from 0.3-0.6 mg/ml lattice.
  • the solution is placed onto bacteriological Petri dishes and immediately put back into the incubator.
  • the lattices are carefully detached from the borders of the dishes using a pipette tip. It is important that the Free Floating Collagen Lattices "swim" freely in the medium.
  • Measurement of the diameter is performed at different time points depending on cell type and seeded cell number. Measurement can be done using a scale paper. From the diameter the area of the collagen lattice can be calculated and their size can be compared at different time points.
  • Cells were trypsinized, and resuspended at ⁇ 10.0xl0 5 cells in 2.5 ml of media. Cell starting number has to be adjusted to the cell type and time of growth.
  • Collagen I comes dissolved in 0.6% acetic acid from First Link. Any other rat tail collagen (e.g. Sigma) may be used, but protocol has to be adjusted for acetic acid and collagen stock concentration.
  • rat tail collagen e.g. Sigma
  • the prepared collagen solution (1.5 ml) was gently mixed with the cell solution (2.5 ml) at RT, resulting in a final cell concentration of 2.5 xlO 5 cells/ml and final collagen concentration of 0.72 mg/ml. Again, you may vary the collagen concentration from 0.5 mg/ml (compliant) to ⁇ 2 mg/ml (stiff) by adapting the protocol.
  • the concentration of cells in the suspension should be adjusted so that 50 ⁇ _ contains the desired number of cells for one well. For example, since the exposed filter area for each well is 8mm 2 , a suspension of 8,000 cells in 50 ⁇ _ will yield 1,000 cells/mm 2 . 50,000 cells in 50 ⁇ _ will yield approximately 6,000 cells/mm 2 .
  • the filled chamber is incubated at 37°C in humidified air with 5% CO2. Incubation times vary considerably depending on cell types and chemotactic factors.
  • One good way to determine the optimum incubation time is to use 6 to 12 blind-well chambers (e.g. stock # BW100) set up as negative controls and placed simultaneously in the incubator. Remove one blind-well chamber after a set period (e.g. 30 minutes), and remove the rest sequentially, one every 5 minutes. Stain the filters and examine them to see how long unstimulated cells have taken to migrate through the filter, or, if you are using cellulose nitrate filters, to a specified optimum depth.
  • blind-well chambers e.g. stock # BW100
  • Aim To determine effects of 3-(4-hydroxyphenyf)propanoic acid amide treatments on fibroblasts migration.
  • Figure 1 shows a general overview of which in vivo processes are reflected by the in vitro assays employed in the presented experiments.

Abstract

The present invention relates to a composition comprising 3-(4- hydroxyphenyl)propanoic acid amide (PA) and whey protein. In further aspects, the invention relates to food ingredients and food products comprising said composition and uses such as as a wound healing agent, in the treatment and/or alleviation of PostoOperative Nausea and Vomiting (PONV), a tissue repair agent and a tissue restitution agent e.g. muscle restitution of athletes.

Description

FOOD INGREDIENT COMPRISING 3-(4-HYDROXYPHENYL)PROPANOIC ACID AMIDE AND WHEY PROTEIN
Technical field of the invention
The present invention relates to a food ingredient comprising 3-(4- hydroxyphenyl)propanoic acid amide and whey protein, preferably non-hydrolyzed whey protein. In particular the present invention relates to a food product comprising such ingredient a uses thereof such as a wound healing agent, in the treatment and/or alleviation of PostoOperative Nausea and Vomiting (PONV), a tissue repair agent and a tissue restitution agent e.g. muscle restitution of athletes.
Background of the invention
The repair of cell tissue is a complex process involving several cell biochemical sub-processes. These involve disinfecting and cleaning up the damaged area by infusion of cytokines, migration of cells to cover up the damage as well as contraction and granulation of the new tissue. The cell migration primarily being driven by coordinated assembly and disassembly of actin filaments. Thus, improvement of the cells ability to adhere at focal points to the substratum and migrate more efficiently to recover the tissue damage will overall improve efficacy of the cell tissue repair process. Such processes are also relevant after surgery.
3-(4-hydroxyphenyl)propanoic acid amide (PA) was originally detected as a compound in apple tree sap. This compound was purified, identified and subsequently synthesized chemically.
WO2007046721 discloses a method of manufacturing PA, its application in the manufacture of anti-aging compositions. Whey protein has been found to possess beneficial effects on wound healing (Ebaid et al. Whey protein enhances normal inflammatory responses during cutaneous wound healing in diabetic rats). Lipids in Health and Disease 2011, 10: 235). Hence, an improved wound healing composition would be advantageous, and in particular a more efficient and/or reliable food ingredient/food product assisting in wound healing would be advantageous.
Summary of the invention
This invention combines the tissue recovering effects by two distinct molecular compounds into a broadly acting nutrition for damaged tissues. Thus, the invention combines the overall cell repair stimulation by proteins with the molecular cell repair mechanisms of an aromatic organic compound from the apple plant.
The protein source is whey protein (preferably non-hydrolyzed that ensures a neutral taste, avoiding the "chalk-like" sensation in the mouth that hydrolysed proteins sources normally give rise to). The apple compound is Phloretamide (PA) that interacts directly with the cellular organelles to improve the molecular mechanisms of two of the three main phases of the tissue repair process (see examples 4 and 5). Individually, these two compounds addresses several distinct sub-processes of the overall tissue recovery process and together the combination constitute a package that boosts the overall condition of the recovering tissue as well as improving the agility of the repairing cells on a molecular level.
As a non-limiting example, these two molecular sources have been combined into a potentially tissue-restoring apple/whey based organic ice cream, optimized for post operational patients. Sensory and intake experiments on post-operational patients further revealed a surprisingly high intake percentage of the whey protein/apple ice cream vehicle (example 1) and that the addition of higher concentrations of phloretamide had no apparent impact on the overall taste (example 2). Examples of compositions according to the present invention are disclosed in example 3.
Thus, the present invention relates to a composition (such as a food ingredient) comprising 3-(4-hydroxyphenyl)propanoic acid amide (HA) and whey protein. It has been found that 3-(4-hydroxyphenyl)propanoic acid amide has positive effects on would healing and similar processes (documented in example 4 & 5 in different cell migration experiments).
Improvement of wound healing may be particular relevant for patients who has e.g. just experienced surgery. After general anaesthesia however, patients often have difficulties swallowing, making it difficult for the patient to obtain the required amount of nutrition through normal eating.
Whey protein may be highly concentrated in certain types of frozen confectionary such as ice cream. Ice creme may be easier for patients to eat just after surgery than normal food.
Thus, an object of the present invention relates to the provision of a
composition/food ingredient/food product, which would have beneficial effects for patients (such as improved wound healing) who has just had surgery, while maintaining the ability for the patient to eat the product just after surgery.
In particular, it is an object of the present invention to provide an ice creme that solves the above mentioned problems of the prior art with eating problems and wound healing for patients who had surgery such as under general anaesthesia.
Thus, one aspect of the invention relates to a composition comprising
0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 0.1-99% (w/w) whey protein, preferably un-hydrolyzed whey protein. The composition is preferably used in a food product. Thus, in an embodiment the composition is a food ingredient.
Another aspect of the present invention relates to a food product comprising the composition according to the invention.
Yet another aspect of the present invention is to provide a composition according to the invention and/or food product according to the invention, for use as a wound healing agent. In yet another aspect, the invention relates to the composition according to the invention and/or food product according to the invention for use in the treatment and/or alleviation of Postoperative nausea and vomiting (PONV). In yet an aspect, the invention relates to a composition according to the invention and/or food product according to the invention, for use as a tissue repair agent and/or a tissue restitution agent e.g. muscle restitution of athletes.
Still another aspect of the present invention is to provide a process for preparing a composition according to the invention, said process comprising
a) providing a source of 3-(4-hydroxyphenyl)propanoic acid amide; b) providing a source of whey protein;
c) mixing the sources from a) and b); and
d) providing a composition (such as food ingredient) comprising
0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 0.1-99% (w/w) whey protein.
Brief description of the figures
Figure 1 shows in vitro assays reflecting tissue repair sub-processes in vivo.
Figure 2 shows the effect of PA treatment on the migration rate of human fibroblast cells in a bidirectional scratch assay. Fibroblasts were pre-treated with PA (80 μΜ) for three days before performing the scratch assay. Migration was quantified by a micrometer scale (see figure 3).
Figures 3A + 3B show quantification of results of experiments from figure 2. PA improves tissue migration by about 37% over the untreated.
Figure 4 shows the effect of PA treatment on the migration rate of human fibroblast cells in a uni-directional scratch assay. Fibroblasts were pre-treated with PA (80 μΜ) for three days before performing the scratch assay.
Figure 5 shows quantification of results of experiments from figure 4. After 24 hrs PA enhances migration (tissue repair) in vitro by more than 60% as measured by uni-directional cell migration. Top: cell migration distance. Bottom : Rate of cell migration.
Figure 6 illustrates the principle of the Boyden chamber assay for chemotaxic cell migration.
Figure 7 shows the result of three independent Boyden chamber assays. Migrated cells become attached to a polycarbonate membrane at the bottom of the chamber and stained (dots). Three separate experiments are shown. Top:
Controls. Bottom : PA treated.
Figure 8 is the quantification of the Boyden chamber assay results. PA enhances chemotactic migration towards chemo-attractants by a factor 2.7 in vitro. Figure 9 illustrates the principle of the free floating collagen lattice assay.
Figure 10 shows on example of a free floating collagen lattice assay and the effect or pre-treatment of the embedded fibroblast cells with PA. Top: Control and PA pre-treated. Bottom : Measurement of the collageneous lattices upon contraction.
Figure 11 shows the quantitative results of several free floating collagen lattice assays. X-axis: Hours. Y-axis: Surface area in %.
Graphs show the extent of collagen lattice contraction by PA from two
independent experiments. PA improves collagen contraction by 85%.
The present invention will now be described in more detail in the following.
Detailed description of the invention
Definitions
Prior to discussing the present invention in further details, the following terms and conventions will first be defined : 3-(4-hydroxyphenyl)propanoic acid amide
In the present context, 3-(4-hydroxyphenyl)propanoic acid amide corresponds to a compound of the formula (I)
Figure imgf000008_0001
The compound of formula (I), may in here also be denoted as PA. The compound of the formula (I) is also known as (4-hydroxyphenyl)propanoic acid amid, 3-(P- hydroxyphenyl)propionamide or simply phloretamide.
PA is naturally present in apples at a concentration around 1 μς/kg (0.0000001%
The taste of PA in high concentrations is considered to be bitter, which makes it less suitable in consumables unless the taste is compensated for. In low
concentrations however this is not considered relevant. Whey protein
Whey protein is the collection of globular proteins isolated from whey. The protein in cow's milk is 20% whey protein and 80% casein protein. The protein fraction in whey constitutes approximately 10% of the total dry solids in whey. This protein is typically a mixture of beta-lactoglobulin (~65%), alpha-lactalbumin (~25%), bovine serum albumin (~8%), and immunoglobulins. These are soluble in their native forms, independent of pH. Thus, the caseino-glycomacropeptide has been removed or substantially removed, such as to a level of less than 2 mg casein/L whey, e.g. less than 1 mg casein/L whey, such as less than 0.5 mg casein/L whey.
Whey protein typically comes in four major forms: concentrate (WPC), isolate (WPI), hydrolysate (WPH) and Native Whey. Most whey concentrates and isolates are available as intact proteins, but either can be also hydrolyzed. Hydrolysates have been partially broken down by exposing the protein to heat, acid or enzymes that break apart the bonds linking amino acids. The whey protein according to the present invention is preferably un-hydrolyzed whey protein to improve the taste. Thus, in an embodiment, the total whey protein content is composed of at least about 60-100% unhydrolyzed intact whey protein. In one embodiment, the total protein content may compose of at least about 90%-100%; at least about 95%- 100%; at least about 95% or at least about 100% unhydrolyzed intact whey.
The whey is also preferably of bovine origin and not of human origin. Other sources of whey protein are goat, camel, sheep, and horse. Composition
As mentioned above, it has been found that 3-(4-hydroxyphenyl)propanoic acid amide (PA) has wound healing properties. Thus, combining PA with other components known to have wound healing properties, may results in a
composition (such as a food product (ice cream) or food ingredient (e.g. dried ingredient)), having improved wound healing properties, which may be beneficial e.g. for patients having trouble eating just after surgery. Thus, in a first aspect, the invention relates to a composition comprising
0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 0.1-99% (w/w) whey protein, preferably un-hydrolyzed whey protein. The composition is preferably used in a food product. Thus, in an embodiment the composition is a food ingredient.
The amount of PA in the composition may vary depending on the final desired use. Thus, in an embodiment the amount of PA in the composition is in the range 0.001-1%, such as 0.01-1%, preferably around 0.1-1%. It is again noted that the natural amount of PA in apples are estimated to be around 1 pg/kg (0.0000001 % w/w). Thus, the above-indicated values in the compositions according to the invention are substantially higher than what can be found in a natural product (apples), especially in combination with whey proteins.
The amount of whey protein in the composition may also vary depending on the final desired use. Thus, in an embodiment the concentration of whey protein is in the range 1-90%, such as 10-90%, such as 10-70%, such as in the range 20- 50% (w/w). The whey may be provided from different sources. Thus, in an embodiment the whey protein is provided from the group consisting of whey protein concentrate (WPC), whey protein isolate (WPI) and native whey. The way is preferably of bovine origin although other sources may also be used. Thus, in an embodiment, the whey protein is of non-human origin, such as bovine origin.
To optimize the composition even further for use for hospitalized subjects, the composition may also comprise further components. Thus, in an embodiment, the composition further comprises one or more anti-nausea agents. In yet an embodiment the one or more anti-nausea agents is selected from the group consisting of ginger, lemon, peppermint, and vitamin B6.
It may be advantageous if the composition according to the invention has a long shelf life and/or is easy to transport. Thus, in a further embodiment the composition is in a dry state, such as having a moisture content below 10%, such as below 8% or such as below 6%.
Preferred compositions according to the invention may comprise: - 0.1-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and
5-80% (w/w) whey protein, such as un-hydrolyzed whey protein. or
0.1-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 20-70% (w/w) whey protein, such as un-hydrolyzed whey protein. or
0.1-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 20-40% (w/w) whey protein, such as un-hydrolyzed whey protein.
Food products
The compositions according to the invention may be included in a food product as an ingredient. Thus, an aspect of the invention relates to a food product comprising the composition according to the invention.
The food product may of course further comprise different taste and nutritional components. Thus, in an embodiment, the food product further comprises fruit, such as extract and/or pulp. In yet an embodiment, said fruit is selected from the group consisting of strawberry, banana, raspberry, apple, lemon, pear, and combination thereof, preferably apple. In another embodiment, the food product further comprises probiotics, such as lactic acid bacteria (LAB). LAB's are well known for the health improving effects, e.g. in beverages.
In another embodiment, the food product further comprises a sweetener, such as sugar. Examples of sugars are glucose, sucrose etc.
In yet an embodiment, the food product is a frozen or cooled/chilled
confectionary. In yet a further embodiment, the frozen confectionary is selected from the group consisting of milkshakes, fruit smoothie, ice cream, ice lollies, frozen fruit bar, frozen yogurt.
In another embodiment, the food product is selected from the group consisting of beverages, such as restituting beverages and sports drinks, nutripharmaceuticals, nutracosmetics, and nutraceuticals.
In a preferred embodiment the food product comprises
0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA);
0.1-99% (w/w) whey protein, preferably un-hydrolyzed whey protein; - optionally, one or more anti-nausea agents, such as ginger; and
- optionally, fruit extract.
Medical uses
The compositions, food ingredients, and food products according to the invention, may be used for different medical purposes, such a wound healing agent after surgery. Thus, in yet an aspect the invention relates to a composition according to the invention and/or food product according to the invention, for use as a medicament. In yet a further aspect the invention relates to a composition according to the invention and/or food product according to the invention, for use as a wound healing agent. In yet another aspect, the invention relates to the composition according to the invention and/or food product according to the invention for use in the treatment and/or alleviation of Postoperative nausea and vomiting (PONV).
In yet an aspect, the invention relates to a composition according to the invention and/or food product according to the invention, for use as a tissue repair agent and/or a tissue restitution agent e.g. muscle restitution of athletes.
In an embodiment, the composition according to the invention and/or food product according to the invention is for oral administration.
In another embodiment, the composition and/or food product according to the invention is for administration to the patient within such as 2 hours after awakening after general anesthesia, such as after surgery. In a further embodiment, the composition and/or food product is for administration within 2 hours, such as 1 hour after awakening after general anesthesia, such as within 30 minutes. In yet a further embodiment, the composition and/or food product is for administration after awakening after general anesthesia and when the patient is able to swallow. Process for preparing composition
The composition according to the invention may be prepared by different processes. Thus, in a further aspect, the invention relates to a process for preparing a composition according to the invention, said process comprising
a) providing a source of 3-(4-hydroxyphenyl)propanoic acid amide; b) providing a source of whey protein;
c) mixing the sources from a) and b); and
d) providing a composition (such as food ingredient) comprising
0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and - 0.1-99% (w/w) whey protein. In an embodiment, the process further comprises drying said composition, e.g. by spray drying and/or evaporation. In an embodiment, the process further comprising mixing said product with a food a food product or food ingredient(s) to provide a food product or food ingredient according to the invention.
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
The invention will now be described in further details in the following non-limiting examples.
Examples
Example 1
Aim
Optimization of apple ice cream for patients.
A specially designed ice cream for adult patients with a high content of non- hydrolyzed whey protein was developed. Apple pulp was used as a major ingredient in the ice creams in order to retain the apple theme referring to the origin of Phloretamide. The ice creams have a texture, which had neither a sticky or greasy feel in the mouth. It is organic, based on birch juice and sugar tongs. The content of natural sugars and minerals promotes ice sweet taste. The apple strain used as a basis is optimized for sour sweet taste ratio in order to stimulate saliva secretion upon surgery.
Materials and methods
Unhydrolyzed whey protein was provided from cow milk. Whey protein fractions are available from several suppliers. Unhydrolyzed whey protein may be prepared by up-concentrating whey several times using standard membrane technology in the field. To avoid hydrolysis the steps should be performed without any heating steps. Liquefied whey protein and prepared apple pulp all at 2-5 °C are mixed with the remaining ingredients in a mixing pasteurizer. The mixture is cooled to 4°C within 3 hours and the mixture is transferred to an ice cream-machine from there to filling and chock frozen to stock at minimum -18°C. Total of 80 mis pr. ice cream. Recipe I:
Ingredient Amount % by weight
Apple pulp (Green Sleeves) 37.5
Un-hydrolyzed Whey protein 27.5
Vanilla 0.0006
Cane Sugar 17.5
Egg 6
Cream 9
Birch sap 1.5
Sugar Tongs 1
Naturally present Phloretamide (estimated) <0.0000001
Recipe II
Ingredient Amount % by weight
Apple pulp (Granny Smith) 37.5
Un-hydrolyzed Whey protein 27.5
Cane Sugar 17.5
Egg 6
Cream 9
Birch sap 1.5
Sugar Tongs 1
Naturally present Phloretamide (estimated) <0.0000001
Recipe III
Ingredient Amount % by weight Apple pulp (Granny Smith) 37.5
Un-hydrolyzed Whey protein 27.5
Cane Sugar 17.5
Egg 6
Cream 9
Birch sap 1.5
Sugar Tongs 1
Ginger 0.5
Naturally present Phloretamide (estimated) <0.0000001
Results
Three different ice cream recipes (see above) were developed and tested for intake by post-operational patients:
A collection of five different ice cream recipes were initially sensory taste evaluated by a panel of independent taste evaluators. All five ice creams were based on apple pulp and whey protein. The apple strains used were selected as being organic and with a relatively high content of acid to stimulate saliva production in awakening post operational patients. Granny Smith was selected for the sweater Apple cake ice cream with vanilla and Green Sleeves was selected for the fresh apple ice cream and the ginger added ice cream. Ginger was added to lower nausea upon operation. In the above examples birch sap was used as sweetener, but other sugar sources may just as well be used. These ice creams were to serve as a basis for the final Phloretamide/whey protein combination ice cream.
From the five recipes, three ice creams (see above) were chosen for an intake test in the anaesthetics department at Svendborg Hospital Department of Odense University Hospital (OUH). The cohort of test persons of 100 patients was selected through random statistical parameters. The patients gave the various ice creams scores and were tested for intake of ice cream in %.
Very surprisingly the intake for almost all patients were 92% - thus way above normal for protein ice creams, which is normally around 60% in similar tests with alternative protein ice cream recipes. 82% ranged the ice creams within the primary two scores out of five (see table below).
Figure imgf000016_0001
Conclusion
Accordingly, the apple ice creams will constitute a great vehicle for
Phloretamide/whey recovery compound combination. Further, the cell recovery using protein rich nutrition is established as described above, but protein powder in food often results in a chalk like taste. Here we use non-hydrolyzed (un-hydrolyzed) whey protein as a protein tissue recovery booster in an ice cream that has a creamy but neutral taste. In combination with
Phloretamide (PA) this will constitute a powerful package for cell and tissue recovery, as Phloretamide has been show to aid cell tissue damage recovery in cell in vitro tests addressing cell motility as well as collagen contraction (see examples 4 and 5). Phloretamide together with non-hydrolyzed whey protein in a vehicle of easily consumed and well tasting apple ice cream will constitute an ideal package for nutritioning post operational anaesthetic patients and recovery after physical exercise.
The general combination of Phloretamide and non-hydrolyzed whey protein will address tissue recovery in a range of food vehicles thus being a potential component of a range of tissue recovery food products.
Example 2
Aim To evaluate the taste of the ice creams of example 1, when phloretamide is added.
Methods
Phloretamide (PA) was tested for taste and solubilized at 0.1% (w/w), thus added to 55°C apple pulp. There was no apparent difference in taste upon addition of PA.
Conclusion
Addition of phloretamide to the compositions of example 1 did not change the overall taste impression.
Example 3
Preparation of food ingredients
WO2007046721 discloses one method of manufacturing PA.
Examples of compositions according to the invention are:
Composition 1 : Powder mixture for drinks or smoothies
Figure imgf000017_0001
Composition 2: Ice cream
Ingredient Amount % by weight
PA 0.1
Whey protein 10
Apple pulp 39.6
Cane sugar 13.4
Pasteurized yolk 4.8
Egg white powder 1.2
Inulin 0.8
Sugar tongs 0.01
Probiotic bacteria 0.01
Lemon juice 11.4 Composition 3: Protein bar
Figure imgf000018_0001
Example 4 - Methods:
Distinct mechanism of fibroblasts in dermis equivalents
Free Floating Collagen Lattice
Aim : to study contraction of ECM molecules by fibroblasts
Protocol:
Collagen type I from bovine skin can be purchased from IBFB Pharma GmbH, Leipzig, Germany and is prepared according to the distributed protocols.
Cells are trypsinized, suspended in complete growth medium and carefully mixed in the collagen solution (collagen in 0.9x DMEM, 10 % FCS, 0.2 mM NaOH, prepared at room temperature). Final cell concentrations might range from 0.8xl04 to 3xl05 cells/ml lattice. Final concentration of collagen might range from 0.3-0.6 mg/ml lattice.
After mixing, the solution is placed onto bacteriological Petri dishes and immediately put back into the incubator.
Half an hour after polymerization in the incubator, the lattices are carefully detached from the borders of the dishes using a pipette tip. It is important that the Free Floating Collagen Lattices "swim" freely in the medium.
Measurement of the diameter is performed at different time points depending on cell type and seeded cell number. Measurement can be done using a scale paper. From the diameter the area of the collagen lattice can be calculated and their size can be compared at different time points.
Production of collagen lattices:
•Estimated time for 20 lattices = 1 h conduction plus 1 h until medium addition. •Per assay, 20 empty 35 mm cell culture dishes were pre-warmed to 37°C. The type of plastic dish used may considerably influence attachment strength of the gels.
•Cells were trypsinized, and resuspended at ~10.0xl05 cells in 2.5 ml of media. Cell starting number has to be adjusted to the cell type and time of growth.
»Cell suspension was stored on ice during preparation of collagen solution.
•Collagen solution was prepared on ice from cold and sterile components:
Figure imgf000019_0001
Total : 1500 μΙ
"Collagen I comes dissolved in 0.6% acetic acid from First Link. Any other rat tail collagen (e.g. Sigma) may be used, but protocol has to be adjusted for acetic acid and collagen stock concentration.
•NaOH was added drop wise (ca. 8-9 drops from a 100 μΙ) yellow pipette tip to neutralize the acetic acid. At pH 7.4 phenol in medium turns from yellow to pink and collagen starts to polymerize, work rapidly to prevent formation of collagen aggregates or gelling.
•The prepared collagen solution (1.5 ml) was gently mixed with the cell solution (2.5 ml) at RT, resulting in a final cell concentration of 2.5 xlO5 cells/ml and final collagen concentration of 0.72 mg/ml. Again, you may vary the collagen concentration from 0.5 mg/ml (compliant) to ~2 mg/ml (stiff) by adapting the protocol.
•200 μΙ of collagen/cell mixture were slowly pipetted as a drop onto the center of one pre-warmed 35 mm cell culture dish and polymerized at 37°C for 60 min. A void shaking of the dish to prevent outflow of the droplet and to keep nice circular drop morphology.
•After 60-180 min, 2 ml of media were added and the lattices were incubated for up to 5 days without medium change.
Boyden Chamber:
To study chemotaxis-directed mobility of fibroblast cells toward a chemical bait Neuro Probe AP48
48-well Microchemotaxis Chamber Protocol
NOTE: The following directions assume that you are working with polycarbonate filters.
Preparing the Chamber
1. Adjust a variable-volume micropipette with a 1mm tip so that the ejected liquid fills a bottom well. The well will hold 25 to 26 μΙ_. A slight positive meniscus should form when the well is filled; this prevents air bubbles from being trapped when the filter is applied.
2. Orient the bottom plate on a flat surface so that the NP trademark is at the upper left. Warm chemo-attractants or control reagents to about 37°C and de-gas them by vortex or vacuum. Fill the bottom wells, completing the 48 wells in no more than 5 minutes, to prevent excessive evaporation.
3. Cut 1mm off the corner of a filter membrane and orient it with the cut
corner at the upper left. Lift the filter by the ends with two forceps, hold it evenly over the filled wells, and lower it onto them, allowing the middle portion of the filter to make contact first. The filter position can be adjusted at this point if necessary, but note that too much movement will cause contamination between wells.
4. Apply the silicone gasket with its cut corner at the upper left, then the top plate, aligning its NP trademark with the trademark on the bottom plate. Push the top plate down firmly and do not let go; this helps prevent air bubbles from being drawn in and trapped in the bottom wells. With your free hand, apply and tighten the thumb nuts until finger tight. Do not use pliers or other tools to tighten them.
Preparing and Adding Responding Cells
1. In the upper wells the concentration of cells in the suspension should be adjusted so that 50μΙ_ contains the desired number of cells for one well. For example, since the exposed filter area for each well is 8mm2, a suspension of 8,000 cells in 50μΙ_ will yield 1,000 cells/mm2. 50,000 cells in 50μΙ_ will yield approximately 6,000 cells/mm2.
2. Pipette cell suspension into each upper well, adjusting the volume so that the filled wells have a slight positive meniscus. Hold the pipette at a steep angle so that the end of the pipette tip rests against the wall of the chamber just above the filter, and the side of the tip rests against the top rim of the well. Eject the fluid with a rapid motion to dislodge air in the bottom of the well.
Check for trapped bubbles in the upper wells. One easy way to do this is to look at the reflections of overhead lights in the meniscuses: a well with an abnormally large positive meniscus usually has a trapped air bubble. To remove any bubbles, suck the well completely dry with a suction line and disposable pipette tip, then refill it.
For most chemotaxis assays the filled chamber is incubated at 37°C in humidified air with 5% CO2. Incubation times vary considerably depending on cell types and chemotactic factors. One good way to determine the optimum incubation time is to use 6 to 12 blind-well chambers (e.g. stock # BW100) set up as negative controls and placed simultaneously in the incubator. Remove one blind-well chamber after a set period (e.g. 30 minutes), and remove the rest sequentially, one every 5 minutes. Stain the filters and examine them to see how long unstimulated cells have taken to migrate through the filter, or, if you are using cellulose nitrate filters, to a specified optimum depth.
Staining Polycarbonate Filters
1. Aspirate fluid from the top wells or empty them by shaking the chamber over a sink or container.
2. Remove the thumbnuts while holding down the top plate, and invert the entire chamber onto a paper towel. Grasp the four corners of the top plate (now on the bottom) and push down evenly so that it stays level as it drops to the table. If the gasket should hang up on the post hardware, carefully push it down evenly onto the plate. Take care not to touch the filter, which should be stuck to the gasket. Immerse the remaining plate (with stud hardware in place) in cool distilled water.
3. The migrated cells are now facing up on the filter; this side of the filter is henceforth referred to as the cell side. Lift up one end of the filter with forceps and catch 1mm of the edge in the large filter clamp. Lift the filter and quickly attach the small filter clamp to the edge of the free end.
Keeping the cell side up, wet the underside (non-migrated cell side) of the filter in a dish containing PBS. Do not let the PBS wash over the cell side of the filter. Holding the filter by the large clamp, with the small clamp attached to the other end and hanging free, wipe the cells off the non-migrated cell side of the filter by drawing the filter up over the wiper blade. The blade should first contact the filter just below the jaws of the wide clamp. Use only gentle pressure against the blade, and maintain an angle of about 30° from the vertical for the portion of the filter above the wiper. It is important to complete the wiping carefully and quickly so that the cells will not dry on the filter; drying takes place in 10 to 20 seconds, and will prevent complete removal of the non-migrated cells.
Clean the wiper with a Q-Tip, again wet the underside of the filter in PBS, and repeat Step 3. Clean the wiper again, then wet the filter a third time in PBS, and repeat Step 3.
For granulocytes and monocytes, carefully immerse the filter in methanol, then place the filter cell-side up on a disposable lint-free towel for air- drying. Rinse all chamber components in cool distilled water. For other kinds of cells, consult the literature for staining techniques.
When the filter is dry, clamp the edge of one end with a large filter clamp, weight the other end with a small filter clamp, and stain in Diff-Quik® or equivalent dye, according to the manufacturer's instructions. To avoid contaminating the chamber components with stain, it is convenient to have two sets of filter clamps, one for removing the filter from the gasket, and one for staining.
Place the wet filter cell-side up on a 50 x 75mm microscope slide to dry. When the filter is dry, center it on the slide and place a drop of immersion oil on it. Rub the oil over the filter with a smooth, blunt instrument to remove all bubbles and wrinkles. The filter is now ready for counting.
Example 5 - Uni and Bi Directional Cell Migration, Boyden chamber and free floating lattices
Aim: To determine effects of 3-(4-hydroxyphenyf)propanoic acid amide treatments on fibroblasts migration.
Introduction Delicate cell monolayers at confluency are mechanically disrupted leaving an area devoid of cells. This procedure is accomplished by a "scrape" made on the cells and later advancement of the adjacent cells into the open area by cell mechanical migration, which is monitored by microscopy. Depending on the cell type, the covering process can take from many hours to less than a day, which is entirely dependent on the type of cells and extent of the scrape. The rate or the extent of migration of the cells (often termed as repopulation rate) can be calculated by a series of photomicrographs.
Protocol
· Plate fibroblasts at a density lxlO4 in 35mm tissue culture plates and incubate for three days using novel test compounds with a control at 37° C ,5% C02.
• At confluency, displace a group of cells within the monolayer by making one streak using a sterile pipette tip.
· Photograph under phase contrast microscopy sequentially at an interval of 19 hrs post scraping.
• Using an ocular micrometer measure the scrape size at each time point.
• Evaluate statistical significance by Student's t test.
• Observe significant differences in scrape repairin PA pre-treatment on fibroblasts.
Results
Figure 1 shows a general overview of which in vivo processes are reflected by the in vitro assays employed in the presented experiments.
The results of the bi-directional scratch assays presented in figures 2 and 3, clearly show improved cell migration when PA is added.
The results of the uni-directional scratch assays presented in figures 4 and 5, clearly show improved cell migration when PA is added.
The results of the chemotactic migration assays (Boyden Chamber assay) presented in figures 7 and 8, clearly show improved cell migration when PA is added. The Boyden Chamber assay is schematically illustrated in figure 6. The results of the free-floating collagen lattice assays presented in figures 10 and 11, clearly show improved cell migration/motility when PA is added. The free- floating collagen lattice assay is schematically illustrated in figure 9. Conclusion
The results obtained by employing this collection of cellular migration assays clearly demonstrate a significant improvement of cell motility in PA-treated cells, indicating that PA potentially strongly improve human cell's ability to repair imposed tissue damage.

Claims

Claims
1. A composition comprising
0.0001 - 1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 0.1 - 99% (w/w) whey protein.
2. The composition according to claim 1, wherein the composition is a food ingredient.
3. The composition according to claim 1 or 2, wherein the amount of PA in the composition is in the range 0.001 - 1%, such as 0.01 - 1%, preferably around 0.1 - 1%.
4. The composition according to any of the preceding claims, wherein the concentration of whey protein is in the range 1-90%, such as 10-90%, such as 10-70%, such as in the range 20-50% (w/w).
5. The composition according to any of the preceding claims, wherein the whey protein is provided from the group consisting of whey protein concentrate (WPC), whey protein isolate (WPI) and native whey.
6. The composition according to any of the preceding claims, wherein the whey protein is un-hydrolyzed whey protein or substantially un-hydrolyzed whey protein.
7. The composition according to any of the preceding claims, comprising one or more anti-nausea agents.
8. The composition according to any of the preceding claims, wherein the one or more anti-nausea agents is selected from the group consisting of ginger, lemon, peppermint, and vitamin B6.
9. The composition according to any of the preceding claims, wherein the composition is in a dry state, such as having a moisture content below 10%, such as below 8% or such as below 6%.
10. The composition according to any of the preceding claims, comprising
0.1-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 5-80% (w/w) whey protein.
11. The composition according to any of the preceding claims, comprising
0.1-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 20-70% (w/w) whey protein.
12. The composition according to any of the preceding claims, comprising
0.1-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 20-40% (w/w) whey protein.
13. A food product comprising the composition according to any of the preceding claims.
14. The food product according to claim 13, further comprising fruit, such as extract and/or pulp.
15. The food product according to claim 13 or 14, wherein said fruit is selected from the group consisting of strawberry, banana, raspberry, apple, lemon, pear, and combination thereof, preferably apple.
16. The food product according to any of claims 13-15, further comprising probiotics, such as lactic acid bacteria (LAB).
17. The food product according to any of claims 13-16, wherein the food product is a frozen or cooled/chilled confectionary.
18. The food product according to claim 17, wherein the frozen confectionary is selected from the group consisting of milkshakes, fruit smoothie, ice cream, ice lollies, frozen fruit bar, frozen yogurt.
19. The food product according to any of claims 13-16, wherein the food product is selected from the group consisting of beverages, such as restituting beverages and sports drinks, nutripharmaceuticals, nutracosmetics, and nutraceuticals.
5 20. The food product according to any of claims 13-19 comprising :
0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA);
0.1-99% (w/w) whey protein;
- optionally, one or more anti-nausea agents, such as ginger; and
- optionally, fruit extract.
10
21. The composition according to any of claims 1-12, and/or food product according to any of claims 13-20, for use as a medicament.
22. The composition according to any of claims 1-12, and/or food product 15 according to any of claims 13-20, for use as a wound healing agent.
23. The composition according to any of claims 1-12, and/or food product according to any of claims 13-20, for use in the treatment and/or alleviation of Postoperative nausea and vomiting (PONV).
20
24. The composition according to any of claims 1-12, and/or food product according to any of claims 13-20, for use as a tissue repair agent and/or, a tissue restitution agent e.g. muscle restitution of athletes.
25 25. The composition according to any of claims 1-12 and/or food product
according to any of claims 13-20, for oral administration.
26. The composition according to any of claims 1-12 and/or food product according to any of claims 13-20, for administered to the patient within such as 2
30 hours after awakening after general anesthesia, such as after surgery.
27. The composition according to claim 26 and/or food product according to claim 26, for administration within 2 hours, such as 1 hour after awakening after general anesthesia, such as within 30 minutes.
35
28. The composition according to claim 26 or 27 and/or food product according to claim 26 or 27, for administration after awakening after general anesthesia and when the patient is able to swallow.
5 29. A process for preparing composition according to any of claims 1-12, said process comprising
a) providing a source of 3-(4-hydroxyphenyl)propanoic acid amide; b) providing a source of whey protein;
c) mixing the sources from a) and b); and
10 d) providing a composition comprising
0.0001-1% (w/w) 3-(4-hydroxyphenyl)propanoic acid amide (PA); and 0.1-99% (w/w) whey protein.
15 30. The process according to claim 29, wherein the process further comprising drying said product, e.g. by spray drying or evaporation.
PCT/DK2018/050026 2017-02-07 2018-02-06 Food ingredient comprising 3-(4-hydroxyphenyl)propanoic acid amide and whey protein WO2018145712A1 (en)

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