WO2024256662A1 - Nanocapsules pour l'encapsulation et la distribution de composés hydrosolubles - Google Patents

Nanocapsules pour l'encapsulation et la distribution de composés hydrosolubles Download PDF

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WO2024256662A1
WO2024256662A1 PCT/EP2024/066610 EP2024066610W WO2024256662A1 WO 2024256662 A1 WO2024256662 A1 WO 2024256662A1 EP 2024066610 W EP2024066610 W EP 2024066610W WO 2024256662 A1 WO2024256662 A1 WO 2024256662A1
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casein
milk
nanocapsules
cheese
lecithin
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WO2024256662A8 (fr
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Marité Cárdenas GÓMEZ
Rita DEL GIUDICE
Sumiyo KANAFUSA
Johan Larsson
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/05—Treating milk before coagulation; Separating whey from curd
    • A23C19/054—Treating milk before coagulation; Separating whey from curd using additives other than acidifying agents, NaCl, CaCl2, dairy products, proteins, fats, enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A23P10/35—Encapsulation of particles, e.g. foodstuff additives with oils, lipids, monoglycerides or diglycerides

Definitions

  • the present invention relates to dispersions such as nanocapsules and emulsions useful for as carrier for added value compounds, for stabilizing 5 added value compounds and/or for stabilizing food products and/or beverages.
  • the invention also relates to nanocapsules for encapsulation and delivery of added value compounds, such as water-soluble nutrients to cheese and to a method for producing the dispersions or the nanocapsules.
  • the present invention further relates to a method for producing food products, such as a0 fortified cheese and/or a cheese with added functionality or beverages, such as fortified juice or milk.
  • Nanotechnology has increasingly been employed in food science,5 specifically in the development of functional food products where enhanced delivery of nutrients and bioactive compounds is desirable.
  • nanocapsules and emulsions have garnered attention for their ability to encapsulate, protect, and deliver both hydrophilic and hydrophobic compounds. Emulsions further offer the advantage improving the sensory0 qualities of food.
  • Cheese is a dairy product that comprises proteins and fat from milk.
  • the process for cheese making consists of several steps. Briefly, pasteurized milk which contains casein micelles is slowly heated, acidified, and inoculated with a starter culture in the presence of a renneting enzyme under gentle stirring. 0
  • the casein micelles consist of alpha- and beta-caseins enclosed and stabilized by kappa(K)-casein. During this so-called coagulation phase, the hydrophilic and hydrophobic ends of the K-caseins are cleaved by the renneting enzyme leading to coagulation and formation of cheese curd.
  • the cheese curd is cut into small pieces and syneresis (dehydration phase) takes place while the mix containing solid curd and liquid whey is swirled.
  • the whey which is a supernatant rich in water soluble compounds, is then removed from the curd and the curd is pressed overnight at room temperature to obtain the final cheese mass.
  • the so-called ripening phase that goes from two weeks up to two years, the mature cheese is finally formed thanks to slow enzymatic activity of proteases and lipases.
  • Cheese is valued for its portability, long shelf life, and high content of fat, protein, calcium, and phosphorus.
  • Cheese is furthermore one of the best diet sources of calcium, and vitamin B12 is typically abundant in cheese.
  • the nutritional value of cheese could be further improved to include antioxidants, vitamins, and minerals, such as iron.
  • Cheese could also be produced containing enzymes, such as the ones needed to metabolize lactose (beta-galactosidase), and to promote digestion of fat and complex carbohydrates (lipases and amylases).
  • enzymes such as the ones needed to metabolize lactose (beta-galactosidase), and to promote digestion of fat and complex carbohydrates (lipases and amylases).
  • Such compounds are either themselves essential or enable adsorption of essential compounds for human metabolism.
  • their addition to food products is particularly important for those found in insufficient amounts in a normal diet (especially important for vegetarians).
  • Beta-galactosidase supplementation would benefit lactose intolerants whereas lipases and amylases supplementation favour food digestion in general but is particularly beneficial for individuals with exocrine pancreatic insufficiency.
  • Such enrichment with enzymes will give cheese an added functionality.
  • cheese is an animal milk product and, as such, its final nutritional properties mirror those of the type of milk used. Additionally, an important technological consequence of cheese making is that supplementation with water soluble compounds is limited because these components are lost in the whey during the process of cheese making.
  • casein micelles have been used to encapsulate iron and to fortify cheese (Raouche et al. 2009).
  • the iron used in this case is derived from iron-containing salts, which are not as easily up-taken by the body and metabolized. For this reason, the amount of iron used was high and the cheese obtained had a red-brownish color and unpleasant taste.
  • the presence of iron contributed to the oxidation and instability of milk.
  • US 2006/0246180 discloses a process for natural cheese-making.
  • Cheese fortification during this process may occur at three locations: the first fortification being after separation of the curds and whey, the second fortification being supplementing the brine solution with fortificant, and a final fortification being prior to or during the final packaging.
  • a drawback of this process is that the fortificants are applied after rennet addition and curd formation. This results in that the fortificants are poorly incorporated or protected by the curd structure.
  • the present invention aims to solve the problems of the prior art.
  • This invention aims to utilize the unique properties of K-casein and specific phospholipids to develop novel dispersions, such as nanocapsules and emulsions. These systems are designed to enhance the encapsulation efficiency, stability, and delivery of added value compounds in food products, and provide effective delivery systems suitable for a wide range of applications in the food and pharmaceutical industries.
  • the present invention provides dispersions made from K-casein and phospholipids.
  • the dispersions can be useful for multiple purposes including as carrier for added value compounds and/or for stabilizing emulsions.
  • the dispersions of the invention can be particularly useful for delivering added value compounds in vivo, because of their stability under conditions mimicking the gastric environment, but they may also have high utility in manufacture of cheese or cheese analogues.
  • the dispersions can be useful for stabilizing added value compounds in certain environments, for example in milk and other beverages, such as fruit juice.
  • Dispersions comprising a high percentage of K-casein loose colloidal stability in the presence of calcium chloride and precipitate in the presence of chymosin, which render such dispersions particularly useful for production of cheese or cheese analogues, because the dispersion can carry added value compounds, and release them into the cheese during production.
  • a first aspect of the invention relates to a dispersion, said dispersion comprising: a) casein, wherein at least 60% of the casein is K-casein; and b) phospholipids.
  • the dispersions may take different forms.
  • the dispersion may be in the form of nanocapsules or emulsions.
  • Nanocapsules typically comprise a shell of phospholipid and K-casein encapsulating a hydrophilic core.
  • Nanocapsules are typically prepared by preparing a lipid film, preparing an aqueous solution comprising K-casein and optionally hydrophilic added value compounds, and solubilizing the lipid film in the solution.
  • Nanocapsules of the invention are particularly useful as carriers for hydrophilic added value compounds.
  • Emulsions typically comprise oil in water droplets comprising a shell of K-casein and a hydrophobic core.
  • the emulsions comprise further phospholipids.
  • Emulsions may be prepared from a hydrophobic phase optionally comprising an added value compound, and mixing with an aqueous solution comprising K-casein.
  • Emulsions of the invention are particularly useful as stabilizers for food products and/or beverages beverages and/or as carriers for hydrophobic added value compounds.
  • a second aspect of the invention relates to a nanocapsule comprising: a) a shell comprising phospholipid and casein, wherein at least 60% of the casein is K-casein; and b) a hydrophilic core.
  • a third aspect of the invention relates to an emulsion comprising oil in water droplets comprising: a) a shell comprising casein, wherein at least 60% of the casein is K-casein and optionally phopholipid; and b) a hydrophobic core.
  • a fourth aspect of the invention relates to method of producing the nanocapsules as described herein, wherein the method comprises the following steps: a) preparing a lipid film; b) preparing a hydrophilic solution comprising casein, wherein at least 60% of the casein is K-casein and optionally an added value compound; c) solubilizing said lipid film prepared in step a) in the solution prepared in step b); and d) obtaining a solution containing the nanocapsules.
  • a fifth aspect of the invention relates to a method of producing the emulsion as described herein, wherein the method comprises the following steps: a) providing a hydrophobic solution, preferably comprising an oil and/or phospholipid; b) preparing a hydrophilic solution comprising casein, wherein at least 60% of the casein is K-casein; c) Emulsifying said hydrophobic solution with said casein solution; d) thereby obtaining an emulsion.
  • a sixth aspect of the invention relates to a use of the dispersion, the nanocapsules or the emulsion as described herein for the transport and/or encapsulation of added value compound(s).
  • a seventh aspect of the invention relates to a use of the dispersion, the nanocapsules or the emulsion as described herein for enrichment of a food product and/or beverages with an added value compound.
  • An eighth aspect of the invention relates to of the dispersion, the nanocapsules or the emulsion as described herein for increasing the nutritional value in food products, such as cheese, cheese analogues and/or beverages.
  • a ninth aspect of the invention relates to method of producing a food product and/or beverages, wherein the method comprises the following steps: a) Providing a food product, a beverage, food product ingredient or a beverage base; b) contacting said food product, beverage, food product ingredient or beverage base with the dispersion, the nanocapsules or the emulsion as described herein, c) processing the mixture of step b) into a product or a beverage.
  • a tenth aspect of the invention relates to method of producing cheese or cheese analogue, wherein the method comprises the following steps: a) Providing milk or milk analogue comprising the dispersion, the nanocapsules or the emulsion as described herein; b) processing said milk or milk analogue into cheese or cheese analogue.
  • Figure 1 illustrates a scheme for generation of useful sources of K-casein that for example can be used to make nanocapsules and emulsions.
  • Figure 2 shows a multiple sequence alignment of K-casein from different organisms. The alignment has been generated by using CLUSTAL W (1.83).
  • Figure 3 illustrate that capsules can be made using K-casein produced in E. coli
  • E. coli 0.5 mg/mL Lactoferrin, 0.8 mg/mL K-casein.
  • E. coli 4 mg/mL K-casein, 3.5 vol% oil and 15 mg/mL lecithin.
  • Figure 4 illustrates that emulsions can be made using K-casein produced in C. reinhardtii A) Western Blot showing the presence of bovine K-casein in protein extract from C. reinhardtii B) Emulsions made with recombinant K casein produced in microalgae (70 mg/mL algae protein including K-casein)
  • Figure 5 shows an example of a useful scheme for production of K-casein stabilized nanocapsules.
  • the scheme shows a method to produce a nanocapsule for encapsulation and delivery of water-soluble added value compounds to cheese according to an embodiment of the inventive concept.
  • the figure shows a method to produce a nanocapsule for encapsulation and delivery of water-soluble nutrients to cheese.
  • the method comprising the steps: a) Preparing a lipid film by dissolving phospholipids in an organic solvent, such as chloroform or hexane, and evaporating the organic solvent.
  • the phospholipid may be synthetic phospholipid, animal-based phospholipid and/or plant-based phospholipid.
  • phospholipids is lecithin.
  • the lecithin may be both animal lecithin and plant based lecithin.
  • animal lecithin are egg yolk lecithin and milk lecithin.
  • plant based lecithin are soybean lecithin, com lecithin, rapeseed lecithin, sunflower lecithin, cotton seed lecithin and grain lecithin.
  • the lecithin may be pure lecithin or food grade lecithin.
  • the evaporation may be conducted under vacuum using a desiccator or a plate evaporator. It should be noted that any type of solvent evaporator may be used for evaporation of the organic solvent.
  • b) Preparing an aqueous solution comprising at least one water-soluble added value compound.
  • c) Preparing a K-casein solution.
  • d) Solubilizing the lipid film prepared in step a) by adding to and mixing with the solutions prepared in step b).
  • Step e) may occur simultaneously with or after step d)
  • the protein concentration of step b) may be up to 10 mg/mL, preferably from 3 to 6 mg/mL.
  • concentration of K-casein in the K-casein solution of step c) may be up to 20 mg/mL, preferably from 1 to10 mg/mL.
  • the vitamin concentration of step b) may be up to 20 mg/mL, preferably from 1 to 5 mg/mL.
  • the concentration of phospholipids in the solubilized lipid film of step d) may be from 0.1 up to 20 mg/mL, preferably 10 mg/mL.
  • the method may further comprise the steps of: a’) providing a protein powder comprising K-casein and at least one water-soluble added value compound, and b') suspending the protein powder in a buffer solution containing imidazole or Bis-Tris and sodium chloride, wherein steps a' and b' occur before step b).
  • the concentration of imidazole or Bis-Tris in the buffer solution may be between 20 and 25 mM.
  • the concentration of sodium chloride in the buffer solution may be between 20 and 150 mM.
  • step d) a solution comprising nanocapsules and excess of K-caseins and/or phospholipids is obtained.
  • step d) may comprise mixing via vortexing, sonication or homogenization for solubilization of the lipid film.
  • Figure 6 shows an example of a useful scheme for production of K-casein stabilized emulsions.
  • the scheme shows a method to produce an emulsion, which for example can be used for making milk or milk analogues and/or can be used for encapsulation and/or delivery of hydrophobic added value compounds to food products and/or beverages.
  • the method may comprise the steps: a) Preparing a hydrophobic solution, for example comprising an oil comprising phospholipid and optionally at least one hydrophobic added value compound.
  • the oil may be olive oil, rapeseed oil, soybean oil, sunflower oil or com oil.
  • the phospholipid may be synthetic phospholipid, animal-based phospholipid and/or plant-based phospholipid.
  • the lecithin may be both animal lecithin plant-based lecithin.
  • animal lecithin are egg yolk lecithin and milk lecithin.
  • plant-based lecithin are soybean lecithin, corn lecithin, rapeseed lecithin, sunflower lecithin, cotton seed lecithin and grain lecithin.
  • the lecithin may be pure lecithin or food grade lecithin.
  • a solution may be obtained containing emulsions comprising a shell made of phospholipid and k-casein and a core optionally comprising at least one hydrophobic added value compound.
  • the concentration of step a) may be up to 20 mg/mL, preferably from 0.1 to 10 mg/mL.
  • the concentration of oil of step a) may be up to 20 vol %, preferably 0.5- 10 vol %.
  • the concentration of phospholipids of step a) may be up to 20 mg/mL, preferably 10- 15 mg/mL.
  • the concentration of k-casein in the k-casein solution of step b) may be up to 40 mg/mL, preferably from 20 to 40 mg/mL.
  • the method may further comprise the steps of: a’) providing a protein powder comprising k-casein, containing imidazole or BisTris and sodium chloride.
  • concentration of imidazole or Bis-Tris in the buffer solution may be between 10 and 40 mM, preferably 20-25 mM.
  • concentration of sodium chloride in the buffer solution may be between 20 and 150 mM, preferably 20-60 mM.
  • step c) a solution comprising emulsions and excess of k-casein and/or phospholipids is obtained.
  • Step c) may comprise mixing via vortexing, sonication or homogenization.
  • Figure 7 shows examples of useful schemes for methods for various food fortification.
  • the upper part illustrates a method for production of fortified cheese according to an embodiment of the inventive concept.
  • the figure illustrates a method for production of fortified cheese and/or cheese with added functionality.
  • the method comprising the steps of: a) providing nanocapsules comprising a shell comprising phospholipid and K- casein, and a core comprising at least one water-soluble added value compound; b) adding the nanocapsules to milk thus forming fortified milk; c) adding renneting enzymes to the fortified milk thus forming fortified cheese curd and whey; d) separating the fortified cheese curd from the whey; and e) pressing the fortified cheese curd into cheese.
  • the nanocapsules are added with the milk and thereafter renneting enzymes are added to form the cheese curd.
  • the milk used in the method may comprise plant-based milk and/or dairy milk.
  • the milk may comprise between 2.9 and 3.1 % by weight of fat.
  • the milk may be non-homogenized milk.
  • the renneting enzymes such as chymosin, may be added in amount of typically, 0.2 U/mL to the fortified milk.
  • the method may comprise a step of: f) enriching the milk with calcium chloride, wherein the step f) occurs after step b).
  • the method may further comprise a step of: g) acidifying the milk by adding an acid, such as citric acid, or by adding starter, such as lactic acid bacteria, to the milk, wherein the step g) occurs after step b).
  • curd is cut, and further incubation may take place at e.g. 32°C for 15 min.
  • the curd may be cut in 5 minutes intervals.
  • the cheese mass is then separated from whey, e.g. by filtration.
  • Figure 8 illustrates dynamic light scattering (DLS) measurements showing that nanocapsules are a single type of particle that differ from that of its single components: lecithin, casein or the cargo protein lactoferrin. Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein and 10 mg/mL lecithin. The figure illustrates results from DLS showing that nanocapsules are highly efficient in incorporating lactoferrin. Lactoferrin is used to fortify cheese with bioavailable iron.
  • the curve representing nanocapsules made by the method according to the inventive concept is larger than the curve representing free casein, lactoferrin and lecithin liposomes, respectively.
  • Figure 9 illustrates DLS data showing that nanocapsules can be made with plant-based purified lecithin from soy or sunflower, and also with food graded lecithin.
  • lecithin was used in 25 mM Bis-Tris buffer pH 6.7 enriched with 25 mM NaCI.
  • Other phospholipid sources besides natural lecithin of animal or plant origin can be used including purified phospholipid type groups (e.g. phosphatidylcholine) or specific synthetic phospholipids.
  • Figure 10 illustrates DLS measurements showing that nanocapsules are stable at A) pH 4.6 to 6.7 and B) in different salinity between 0 to 100 mM NaCI in imidazole buffer at pH 6.5. Concentrations used are: A) 0.5 mg/ml soy lecithin, B) 1 mg/ml soy lecithin, A+B) 1.36 mg/mL K-casein.
  • Figure 11 illustrates cryogenic transmission electron microscopy images and small angle X ray scattering data and best fit for a nanocapsule according to the inventive concept, showing the shell-core structure of the nanocapsule.
  • the figure shows structural confirmation of the nanocapsule wall being composed of lipid bilayer and K casein.
  • the inner part of the capsule is made of water.
  • SAXS Small Angle Xray Scattering
  • Figure 12 illustrates stability of the nanocapsules upon storage at 4 °C (left panels) and 25 °C (right panels) as measured by DLS (top) and visual inspection (bottom) of the samples.
  • the nanocapsules are stable for at least up to 38 weeks at 4 °C and 5 weeks at 25 °C. Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5.
  • Figure 13 illustrates stability of the nanocapsules upon freeze drying and after resuspension in water as measured by DLS (top) and visual inspection (bottom) of the samples.
  • the samples are stable at least after 7 weeks of resuspension and storage at 4 °C. Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein. No precipitate was observed.
  • Figure 14 illustrates loss of colloidal stability of the nanocapsules in the presence of calcium chloride and precipitation in the present of chymosin as measured by A) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and B) size exclusion chromatography (SEC).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • SEC size exclusion chromatography
  • the figure illustrates the principle of nanocapsule de-stabilization: Colloidal aggregation takes place in presence of calcium and complete precipitation only takes place upon addition of renneting enzyme.
  • the size exclusion chromatography picture shows the partial aggregation of nanocapsules in presence of calcium and their complete aggregation in the presence of renneting enzyme.
  • the SDS-PAGE picture confirms that lactoferrin is eliminated from the soluble fraction upon successive addition of calcium and renneting enzyme to nanocapsules. This picture also confirms the coexistence of K-casein with lactoferrin in the nanocapsules.
  • FIG. 15 shows A) DLS data showing a comparison of nanocapsules formulated with dairy casein mixture and nanocapsules formulated with purified dairy K-casein.
  • Figure 16 illustrate a demonstration of protective effect of lactoferrin during in vitro digestion.
  • A+B Scheme of experiment mimicking the gastric phase and the intestinal phase.
  • C) SDS- PAGE show that proteolytic degradation occurs completely in the first incubation step with pepsin only for free lactoferrin whereas a significant fraction of lactoferrin remains intact even after pepsin and pancreatin incubation when added inside the nanocapsules.
  • Figure 17 illustrate the result from a measurement of encapsulation efficiency for lactoferrin in curd.
  • B Scheme of Lactoferrin delivery to cheese mass thanks to encapsulation.
  • A) SDS-PAGE of supernatant (whey) shows that 49% of the fortified lactoferrin is present in the whey when added inside the capsules while 80% remain in the whey if lactoferrin is added in free form (no capsules added).
  • A) illustrates the result from a measurement of encapsulation efficiency for lactoferrin in curd, wherein free-lactoferrin is used as a control. Curd is formed and lactoferrin concentration is quantified via SDS PAGE measurements in whey. The results, presented in B), show that 51 % vs 20% of added lactoferrin is retained in the curd when added in the nanocapsules vs free in the milk.
  • Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5, added 0.2 mg/mL calcium 5 mg/mL citric acid and 0.2 U/mL enzyme.
  • Figure 18 shows A) comparison of size between lactoferrin nanocapsule and Vitamin B12 nanocapsules measured by DLS.
  • Figure 19 illustrates increased stability of milk when fortified by nanocapsules as demonstrated by A) visual inspection, B) particle size analyser and C) SDS- PAGE. Milk is stable against aggregation or proteolytic degradation at least up to 12 weeks as compared with less than 2 weeks in the absence of nanocapsules. Controls were made with free lactoferrin, liposomes, buffer, or K-casein. Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5. 5 vol % of nanocapsules were added to 3 % fat milk. The temperature of storage was 4°C.
  • Figure 20 Stability of nanocapsules in apple juice up to 6 days as demonstrated by A) visual inspection, B) particle size analyzer and C) SDS- PAGE. Fortification with nanocapsules make the juice cloudy and produce a sediment that can be resuspended upon shaking. Controls were made with free lactoferrin or buffer. SDS-PAGE were run on both the precipitate and the supernatant showing that the cargo protein, lactoferrin, remains inside the capsules even in the sediment. The SDS-PAGE also shows that lactoferrin remains intact when formulated inside of the nanocapsules.
  • Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5. 5 vol % of nanocapsules were added to apple juice. The temperature of storage was 4 °C.
  • Figure 21 Stability of oat milk when fortified with nanocapsules as demonstrated by A) visual inspection, B) particle size analyzer and C) SDS- PAGE. Similar stability was observed at 9 days of storage at 4 °C for oat milk when fortified with free-lactoferrin or nanocapsules. Controls were made with free lactoferrin, buffer, or K-casein. Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5. 5 vol % of nanocapsules were added to oat milk.
  • Figure 22 Soybean milk stability is unchanged when fortified by nanocapsules as demonstrated by A) visual inspection, B) particle size analyzer and C) SDS- PAGE. Controls were made with free lactoferrin, buffer, or K-casein. Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5. 5 vol % of nanocapsules were added to soybean milk. The temperature of storage was 4 °C and samples were monitored for up to 4 weeks.
  • FIG 23 Yogurt stability is unchanged when fortified by nanocapsules as demonstrated by A) visual inspection, B) particle size analyser and C) SDS- PAGE. Controls were made with free lactoferrin, buffer, liposomes or K-casein. Concentrations used are: 2 mg/mL lactoferrin, 2 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5. 5 vol % of nanocapsules were added to yoghurt. The temperature of storage was 4 °C.
  • Figure 24 shows that plant-based emulsions using bovine K-casein purified from dairy milk as emulsifier give similar size distribution as emulsions present in milk.
  • K-casein emulsification properties are suited for a wide range of food-based systems including dairy analogous.
  • the emulsion was prepared by mixing 3.5 vol% vegetable oil containing 15 mg/mL lecithin with 35 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5 and subjecting to homogenization.
  • Figure 25 illustrates stability of the emulsions upon storage at 4 °C (left panels) and 25 °C (right panels) as measured by particle size distribution and visual inspection of the samples.
  • Bovine-K casein stabilized emulsions are stable upon storage at both 4 and 25 °C at least for up to seven weeks and one week, respectively, as measured by particle size analyzer (top) and visual inspection (bottom).
  • the emulsion in this test was prepared by mixing 3.5 vol% oil containing 15 mg/mL lecithin with 35 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5.
  • Figure 26 illustrates stability of the emulsions upon freeze drying and after resuspension in water and storage at 4 °C, as measured by particle size distribution (top) and visual inspection (bottom panels) of the samples.
  • the samples are stable at least after seven weeks of re-suspension and storage at 4 °C.
  • the emulsion was prepared by mixing 3.5 vol% vegetable oil containing 15 mg/mL lecithin with 35 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5.
  • Figure 27 illustrates that K-casein in the emulsion surface can be cleaved by chymosin inducing a precipitate that mirrors the dairy cheese curd as shown by A) visual observation and B) SDS-PAGE.
  • the emulsion was prepared by mixing 3.5 vol% oil containing 15 mg/mL lecithin with 35 mg/mL K-casein in 20 mM imidazole, 20 mM NaCI buffer pH 6.5. 0.2 mg/mL calcium chloride, 5 mg/mL citric acid and 0.2 U/mL enzyme were added.
  • Figure 28 Stability of milk enriched with emulsions of the invention remains unchanged upon storage 4 °C as shown by A) visual inspection, B) particle size distribution and C) SDS-PAGE. Milk enriched with a mixture of caseins looses its stability after 3 weeks. Controls were made with free lecithin in oil, buffer, or K-casein. 5 vol % of emulsion was added to 3% fat milk.
  • the term "dispersion” refers to a heterogeneous system having a dispersed phase and a dispersion medium.
  • the dispersed phase may be particles, which for example may be nanocapsules or droplets. Therefore, a dispersion can describes a homogeneous mixture where the dispersed particles will not settle if the solution is left undisturbed for a prolonged period of time or a heterogeneous dispersion of larger particles in a medium where the particles will settle out of the mixture if left undisturbed for a prolonged period of time.
  • a dispersion can describe a heterogeneous mixture where the dispersed particles, for example the nanoparticles or the droplets have at least in one direction a dimension roughly in the nm to pm range.
  • dispersions herein are suspensions of nanocapsules and emulsions, such as oil-in-water emulsions.
  • emulsion shall mean a composition, usually liquid, comprising two or more immiscible phases in which a first phase (the “dispersed phase”) is dispersed in a second phase (the “continuous phase”).
  • the emulsions of the inventions are preferably oil-in-water emulsions (oil dispersed in water) comprising oil droplets dispersed in an aqueous phase. Said droplet typically contain a shell and a hydrophobic core.
  • a nanocapsule according to the invention is a small sphere with a uniform membrane around it (a compartment formed by the membrane).
  • the material inside the nanocapsule (entrapped in the nanocapsule) is referred to as the core, internal phase, or fill, whereas the membrane is sometimes called a shell, coating, or wall.
  • the nanocapsules of the invention typically have diameters between 10 and 200 nanometers. The diameter of the nanocapsule is measured in aqueous phase after homogenization is complete. The diameter of the capsule may change depending on the water activity of the surrounding chemical environment.
  • the nanocapsule can be an artificial compartment whose delimiting borders restrict the exchange of the components enclosed therein. Thus, added value compounds contained in the hydrophilic core of nanocapsules may be completely enclosed by the shell and prohibited from interacting with the surroundings of the nanocapsule as long as the nanocapsule is intact.
  • hydrophilic and “water-soluble” are used interchangeably throughout the description.
  • hydrophobic and “oil-soluble” are used interchangeably throughout the description.
  • An “added value compound” according to the invention may be any compound, which is desirable to encapsulate and/or transport with the dispersions of the invention.
  • added value compounds are food supplements, nutrients, substances with beneficial nutritional and/or physiological effect or therapeutically active substances.
  • the present disclosure concerns a dispersion, said dispersion comprising: a) casein, wherein at least 60% of the casein is K-casein; and b) phospholipids.
  • the invention further relates to methods of producing said dispersions and to methods of producing food products and/or beverage comprising said dispersions.
  • the present disclosure concerns a nanocapsule comprising a shell comprising phospholipid and casein, wherein at least 60% of the casein is K-casein and a hydrophilic core.
  • the dispersion is such a nanocapsule or a composition comprising such nanocapsules in an aqueous solution. Nanocapsules can be the dispersed phase of a suspension.
  • Nanocapsules can be used to deliver hydrophilic added value compounds to hydrophobic environments. This can have high utility in manufacture of cheese or cheese analogues, since the hydrophilic added value compound can be delivered to the cheese or cheese analogue instead of remaining in the whey.
  • the nanocapsules are prepared by a method as described in the following chapter “Method of producing nanocapsules”.
  • the nanocapsule comprises K- casein as described in the chapter “casein”.
  • the nanocapsules of the invention comprise K-casein. Whereas the nanocapsules may contain small amounts of other caseins, it is important that the vast majority of caseins in the nanocapsules are K-casein.
  • the nanocapsules comprise phospholipids as described in the chapter “phospholipids”.
  • the hydrophilic core is enclosed by a hydrophobic shell, wherein the hydrophobic shell contains phospholipids and K-casein.
  • the hydrophilic core preferably comprises one or more added value compounds, for example hydrophilic added value compounds, which may be any of the added value compounds described in the chapter “Added value compounds”.
  • the hydrophobic shell substantially encloses or encapsulates the hydrophilic core.
  • Emulsions can be used to deliver hydrophobic added value compounds to hydrophilic environments. Furthermore, emulsions of this invention can be used to formulate dairy analogues with increased stability of said food products or beverages, and thus do not necessarily comprise any added value compound.
  • the present disclosure concerns an emulsion comprising oil in water droplets comprising a shell comprising casein, wherein at least 60% of the casein is K-casein and a hydrophobic core.
  • the shell further comprises phospholipids.
  • the dispersion is an emulsion comprising oil in water droplets comprising a shell comprising casein, wherein at least 60% of the casein is K-casein and a hydrophobic core.
  • the emulsions are preferably prepared by a method as described in the following chapter “Method of producing emulsions”.
  • the emulsion comprises K-casein, preferably as described in the chapter “K-casein”.
  • the emulsion comprises phospholipids, for example as described in the chapter “phospholipids”.
  • the hydrophobic core comprises phospholipids mixed with oil. In some embodiments of this invention, other emulsifiers than phospholipids can be mixed with oil. In addition, the hydrophobic core may comprise one or more added value compound, such as any of the hydrophobic added value compounds described in the chapter “Added value compounds”. K-casein
  • Casein is a family of related phosphoproteins (aS1 , aS2, (3, K) that are commonly found in mammalian milk, constituting about 80% of the proteins in cow's milk. Casein is a mix of the different phosphoproteins aS1 -, aS2-, (3-, and K-casein, which naturally occur in bovine casein in a percentage ratio of approximately 40: 10:40: 10. K-casein is split by chymosin as found in rennet into an insoluble peptide and water-soluble glycomacropeptide.
  • the present invention discloses that dispersions comprising a high percentage of K-casein, in contrast to casein mix, loose the colloidal stability in the presence of calcium chloride and precipitate in the present of chymosin. This is rendering the dispersions particularly useful for production of cheese or cheese analogues.
  • the K-casein is purified from milk, such as from bovine milk, goat milk, sheep milk, camel milk, donkey milk, horse milk, reindeer milk, yak milk or human milk.
  • the K-casein is recombinant K- casein.
  • recombinant refers to said K-casein being produced by recombinant methods.
  • recombinant K-casein is produced by a host comprising a heterologous nucleic acid encoding K-casein.
  • Said heterologous nucleic acid may be comprised within a vector.
  • recombinant host cells express genes that are not found in identical form within the native (non-recombinant) form of the cell.
  • the K-casein is recombinant K-casein produced by a host cell or host organism comprising a heterologous nucleic acid encoding K-casein.
  • the host cell or host organism is yeast, bacteria, fungi, algae, plants, insect or mammalian. In some embodiments of this invention, the host cell is a bacterial cell, such as E. coli or B. subtilis.
  • the host organism is an algae, such as C. reinhardtii or C. vulgaris.
  • the host cell is a yeast cell, such as a yeast cell of the genus Saccharomyces or Pichia.
  • the host cells are transfected, transformed or transduced with a nucleic acid encoding K-casein or a functional homologue of said K-casein.
  • the K-casein is a K-casein of SEQ ID NO: 1 or a functional homologue of said K-casein sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.
  • the K-casein is a K- casein of SEQ ID NO: 2 or a functional homologue of said K-casein sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.
  • the K-casein is a K-casein of SEQ ID NO: 3 or a functional homologue of said K- casein sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.
  • a functional homologue of an amino acid sequence, refers to a polypeptide comprising said amino acid sequence with the proviso that one or more amino acids are substituted, deleted, added, and/or inserted, and which polypeptide has (qualitatively) the same functionality.
  • a functional homologue shares at least at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said amino acid sequence.
  • sequence identity describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment.
  • sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.
  • the Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position in a reference sequence.
  • sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • At least 70%, such as at least 80%, such as at least 90% of the caseins in the dispersion are K-caseins.
  • at most 40%, such as at most 30%, such as at most 20%, such as at most 10%, such as at most 5%, such as at most 1 %, such as at most 0,1 % of the caseins in the dispersion are aS1 -casein.
  • at most 40%, such as at most 30%, such as at most 20%, such as at most 10%, such as at most 5%, such as at most 1 %, such as at most 0,1 % of the caseins in the dispersion are aS2-casein.
  • At most 40%, such as at most 30%, such as at most 20%, such as at most 10%, such as at most 5%, such as at most 1 %, such as at most 0,1 % of the caseins in the dispersion are [3-casein.
  • at most 40%, such as at most 30%, such as at most 20%, such as at most 10%, such as at most 5%, such as at most 1 %, such as at most 0,1 % of the caseins in the dispersion are aS1 -casein, aS2-casein and [3-casein.
  • the casein of the dispersion does not comprise aS1 -casein, aS2-casein and/or [3-casein. In some embodiments of this invention, the casein of the dispersion does not comprise any of aS1 -casein, aS2-casein and [3-casein.
  • the shell of the nanocapsule comprises kappacasein, or K -casein, which is a monomeric milk protein that belongs to the group caseins.
  • the K-casein may comprise animal-free casein, recombinant casein, and milk casein.
  • the milk casein may originate from dairy milk.
  • Caseins are a family of phosphoproteins that form soluble aggregates known as "casein micelles" in milk. In these casein micelles, the K-casein is located primarily on the surface, stabilizing the micelle structure.
  • K-casein in the shell of the nanocapsule is its ability, prior to addition of renneting enzymes, to stabilize the nanocapsule in the milk and to destabilize the nanocapsule in the milk after addition of renneting enzymes.
  • the renneting enzymes cut the hydrophilic parts of the K-casein, causing the nanocapsules to coagulate and be a part of the curd formation. Colloidal aggregation may take place in presence of calcium and complete precipitation may take place upon addition of the renneting enzyme.
  • the dispersions of the invention comprise phospholipids. Many different phospholipids may be comprised within the dispersions of the invention, however, preferred phospholipids are lecithins.
  • the term Lecithin as used herein describes amphiphilic substances, comprising a mixture of phospholipids. Lecithin can be extracted chemically from natural sources using solvents such as hexane, ethanol, acetone, petroleum ether or benzene. Common sources include egg yolk, marine foods, soybeans, milk, rapeseed, cottonseed, and sunflower oil.
  • the phospholipid is a lecithin, which is pure or food grade lecithin.
  • food grade means and includes, lecithin that is commonly consumed as a food or employed as a food additive.
  • food grade can be understood as materials that are approved by the Regulatory authorities for use in pharmaceutical, food, nutritional, and/or dietetic applications.
  • pure grade means and includes, lecithin that is purified, but not pure enough to be offered for food, drug, or medicinal use of any kind.
  • the phospholipids are one or more phospholipids selected from the group consisting of synthetic phospholipids, animal-based phospholipids and plant-based phospholipids.
  • the phospholipids are a mixture of glycerophospholipids, for example phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, or phosphatidic acid.
  • the phospholipids or the lecithin are derived from one or more source selected from the group consisting of egg yolk, marine foods, soybeans, milk, rapeseed, cottonseed, and sunflower oil.
  • the phospholipids comprise or consist of lecithin, for example dihexanoyl-L-alpha-lecithin, dioctanoyl-L-alpha-lecithin, didecanoyl-L-alpha-lecithin, didodecanoyl-L-alpha- lecithin, ditetradecanoyl-L-alpha-lecithin, dihexadecanoyl-L-alpha-lecithin, dioctadecanoyl-L-alpha-lecithin, dioleoyl-Lalpha-lecithin, dilinoleoyl-L-alpha- lecithin, or alpha-palmitol.
  • lecithin for example dihexanoyl-L-alpha-lecithin, dioctanoyl-L-alpha-lecithin, didecanoyl-L-alpha-lecithin,
  • the phospholipids are acetone-insoluble phosphatides.
  • An advantage with phospholipids in the shell is the self-assembling properties of phospholipids.
  • Phospholipids are amphiphilic lipids with hydrophobic fatty acid chains and hydrophilic moieties.
  • the phospholipid may comprise synthetic phospholipid, animal-based phospholipid and/or plantbased phospholipid.
  • the lecithin may be pure lecithin or food grade lecithin.
  • the dispersions of this invention can comprise an added value compound, but can also show a technical effect such as increasing the stability of food products or beverages without any added value compound.
  • the added value compound may any compound, which is beneficial to add to a food product or a beverage, for example, the added value compound may be a food supplement, a nutrient, substances with beneficial nutritional and/or physiological effect or therapeutically active substances.
  • the added value compound may for example be antibodies or active fragments thereof, peptides, lipids, protein drugs, protein conjugate drugs, enzymes, oligonucleotides, ribozymes or genetic material.
  • the hydrophobic core comprises an oil-soluble added value compound. In some embodiments of this invention, the hydrophobic core is a hydrophobic core as described in the chapter “emulsions”.
  • At least one added value compound is a food supplement.
  • the term "food supplement” in the context of the present invention is interchangeable with the terms food additive, a dietary supplement and nutritional supplement.
  • the term can be understood in a broad sense as any ingestible ingredient capable of supplementing the user's diet.
  • the food supplement can be understood as a product to achieve specific benefits. Examples of the benefits include, but are not limited to adding [3-galactosidase to milk products to provide milk products for lactose intolerant subjects, adding iron carrying proteins to milk or plantbased products to supplement the diet of vegetarians and vegans or adding vitamins to food products to support the immune system.
  • At least one added value compound is a vitamin, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E or vitamin K.
  • at least one added value compound is an antioxidant, such as vitamin A, vitamin C, vitamin E, xanthophylls, such as zeaxanthin or lutein, polyphenols or flavonoids such as resveratrol or quercetin.
  • at least one added value compound is a protein, such as an iron carrying protein, such as hemoglobin or lactoferrin.
  • at least one added-value compound is an enzyme, such as amylase or [3-galactosidase.
  • at least one added value compound is a fatty acid, such as omega 3 fatty acid or omega 6 fatty acid.
  • hydrophilic added value compounds include but are not limited to water soluble vitamins such as Vitamin C and B, iron carrying proteins such as hemoglobin and lactoferrin, enzymes such as amylase and [3- galactosidase, water soluble antioxidants, such as polyphenols and flavonoids and therapeutical components and drugs.
  • hydrophobic added value compounds include, but are not limited to oil soluble vitamins such as Vitamin A, D, and K, antioxidants, such as Vitamin A, vitamin E and xanthophylls, omega 3 and 6 fatty acids, other kinds of essential fats and therapeutical components and drugs.
  • Non-limiting examples of functional proteins are those for iron supplementation, such as hemoglobin or lactoferrin.
  • Cheese fortification with iron salts to increase the amount of iron in the food product, is known.
  • iron salts would be used, very high doses would be required because iron has low bioavailability during digestion, which further leads to digestive problems.
  • the iron salts also tend to add a metallic flavor and unwanted color to the final product. This is avoided by the use of lactoferrin. Since iron is incorporated in a metabolizable way, i.e. bound to a protein that favors the uptake, there is no need to use high iron quantities to fortify the cheese. This allows the fortification of cheese in a way that has little impact on its quality, in particular its taste.
  • the digestive enzyme may give the cheese an added functionality, apart from enhancing its nutritional properties.
  • the digestive enzyme may be betagalactosidase or amylase. Beta-galactosidase metabolizes lactose. Adding beta-galactosidase to the cheese is thus beneficial for persons who suffer from lactose intolerance.
  • Amylase breaks complex sugars.
  • the vitamin may be Vitamin B complex.
  • the antioxidant may be glutathione.
  • the water-soluble added value compound may further comprise a mineral, such as magnesium salts.
  • the present disclosure concerns a method of producing the nanocapsules as described herein, wherein the method comprises the following steps: a) preparing a lipid film; b) preparing a hydrophilic solution comprising casein, wherein at least 60% of the casein is K-casein and optionally an added value compound; c) solubilizing said lipid film prepared in step a) in the solution prepared in step b); and d) obtaining a solution containing the nanocapsules.
  • lipid film refers to lipid in a film form.
  • lipid refers to a variety of organic compounds. Typically, the lipids include long chain fatty acids and derivatives or analogs thereof.
  • Lipids are, for example, 1 ) simple lipid (which is ester of fatty acid and an alcohol and may also be referred to as neutral lipid; for example, fat and oil (triacylglycerol), wax (fatty acid ester of higher alcohol), sterolester, fatty acid esters of vitamins, and the like); 2) complex lipid (compounds which have ester bond or amide bond and have a polar group such as phosphoric acid, saccharide, sulfuric acid, amine and the like besides fatty acid and alcohol, including glycerophospholipid, sphingophospholipid, glyceroglycolipid, sphingoglycolipid, lipid having C-P bond, sulfolipid, and the like); 3) derived lipid (compounds produced
  • any lipid can be used, however, preferably lecithin is used to prepare the lipid film of step a).
  • the lipid film of step a) is prepared by dissolving phospholipids, for example lecithin in an organic solvent, and subsequently removing said organic solvent, for example evaporating the organic solvent.
  • the lipid film may essentially consist of phospholipids.
  • the organic solvent in step a) is chloroform or hexane.
  • the lipid film in step a) is prepared by dissolving lecithin.
  • hydrophilic means water soluble. Hydrophilic components are those which are soluble in water at 25 °C at a concentration of 1 weight part hydrophilic component to 9 weight parts water.
  • the hydrophilic solution of step b) is prepared by a method comprising resuspending K-casein in an aqueous buffer.
  • the hydrophilic solution of step b) comprises an added value compound.
  • the added value compound can be an added value compound as described in the chapter “added value compound”.
  • the solution in step b) may be prepared by resuspending an added value compound in a buffer and mixing said buffer with a solution comprising said K-casein.
  • the concentration of K-casein in the aqueous solution of step b) is at least 0.5 mg/ml, such as at least 1 mg/ml, such as at least 1 .5 mg/ml, such as at least 2 mg/ml, such as at least 2.5 mg/ml, such as at least 3 mg/ml, such as at least 4 mg/ml, such as at least 5 mg/ml.
  • the concentration of K-casein in the aqueous solution of step b) is at most 20 mg/ml, such as at most 10 mg/ml, such as at most 8 mg/ml, such as at most 6 mg/ml, such as at most 4 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml.
  • the concentration of K-casein in the aqueous solution of step b) is between 1 mg/ml and 10 mg/ml.
  • the solubilization of the lipid film in the hydrophilic solution is achieved by mixing.
  • the solution comprises phospholipids at a concentration of at least 0.1 mg/ml, such as at least 0.5 mg/ml, such as at least 1 mg/ml, such as at least 2 mg/ml, such as at least 5 mg/ml, such as at least 8 mg/ml, such as at least 10 mg/ml, such as at least 10 mg/ml of the total solution.
  • the solution in step d) of the method comprises phospholipids at a concentration of at most 20 mg/ml, such as at most 10 mg/ml, such as at most 8 mg/ml, such as at most 5 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml, such as at most 0.5 mg/ml, such as at most 0.1 mg/ml of the total solution. In some embodiments of this invention, in step d) of the method the solution comprises phospholipids at a concentration of between 5 mg/ml and 10 mg/ml.
  • step c) of the method the lipid film is solubilized by mixing the lipid film of step a) with the solution of step b), wherein the mixing is performed by one or more of vortexing, bath sonication, tip sonication and/or homogenization.
  • step d) of the method a solution containing nanocapsules comprising a shell made of phospholipid and K-casein and a core comprising at least one water-soluble added value compound is obtained.
  • the nanocapsules may be further purified, for example via size exclusion chromatography.
  • the nanocapsules can be eluted from the column at a flow rate of at least 0.1 ml/min, such as at least 0.25 ml/min, such as at least 0.5 ml/min or at a flow rate of at most 1 ml/min, such as at most 0.75 ml/min, such as at most 0.5 ml/min.
  • the nanocapsules can be eluted in a buffer comprising sodium chloride and imidazole and/or Bis-Tris.
  • the phospholipid is preferably as described in chapter “phospholipids”.
  • the added value compound is preferably as described in chapter “added value compound”.
  • the hydrophilic solution of step b) comprises the added value compound at a concentration of at least 1 mg/ml, such as at least 1.5 mg/ml, such as at least 2 mg/ml, such as at least 3 mg/ml, such as at least 4 mg/ml, such as at least 5 mg/ml, such as at least 6 mg/ml.
  • the hydrophilic solution of step b) comprises the added value compound at a concentration of at most 10 mg/ml, such as at most 8 mg/ml, such as at most 6 mg/ml, such as at most 5 mg/ml, such as at most 4 mg/ml, such as at most 3 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml total protein.
  • the hydrophilic solution of step b) comprises the added value compound at a concentration between 1 mg/ml and 6 mg/ml total protein.
  • K-casein is preferably as defined in chapter “K-casein”
  • the solution in step b) of the method comprises a buffer, for example a buffer comprising sodium chloride and imidazole and/or Bis-Tris.
  • the buffer can have a pH of at least 5, such as at least 5.5, such as at least 6, such as at least 6.5 or a pH of at most 7, such as at most 6.5, such as at most 6, such as at most 5.5, such as at most
  • the buffer has a pH of between 5 and 6.5.
  • the sodium chloride concentration in the buffer can be at least 20 mM, such as at least 40 mM, such as at least 60 mM, such as at least 80mM, such as at least 100 mM, such as at least 120 mM, such as at least 140 mM, such as at least 150 mM or at most 150 mM, such as at most 140 mM, such as at most 120 mM, such as at most 100 mM, such as at most 80 mM, such as at most 60 mM, such as at most 40 mM, such as at most 20 mM.
  • the imidazole concentration in the buffer can be at least 10 mM, such as at least 20 mM, such as at least 30 mM, such as at least 40 mM, or at most 40 mM, such as at most 30 mM, such as at most 20 mM, such as at most 10 mM.
  • the buffer can also comprise Bis-Tris instead of imidazole.
  • the Bis-Tris concentration in the buffer can be at least 10 mM, such as at least 20 mM, such as at least 30 mM, such as at least 40 mM or at most 40 mM, such as at most 30 mM, such as at most 20 mM, such as at most 10 mM.
  • the present disclosure concerns a method of producing the emulsion as described herein, wherein the method comprises the following steps: a) providing a hydrophobic solution, preferably comprising an oil; b) preparing a hydrophilic solution comprising casein, wherein at least 60% of the casein is K-casein; c) Emulsifying said hydrophobic solution with said casein solution; d) thereby obtaining an emulsion.
  • the hydrophobic solution in step a) comprises oil and a phospholipid, such as lecithin.
  • the oil may be any oil, preferably a food grade oil.
  • the oil may be olive oil, rapeseed oil, soybean oil, sunflower oil or corn oil.
  • the concentration of the oil is at least 0.5 vol%, such as at least 5 vol%, such as at least 10 vol%, such as at least 15 vol%, such as at least 20 vol% of the hydrophobic solution.
  • the concentration of the oil is at most 20 vol%, such as at most 15 vol%, such as at most 10 vol%, such as most 5 vol% of the hydrophobic solution.
  • the concentration of the oil is between 0.5 vol% and 10 vol% of the hydrophobic solution.
  • the concentration of the phospholipids in the hydrophobic solution in step a) of the method of producing emulsions is at least 1 mg/ml, such as at least 5 mg/ml, such as at least 10 mg/ml, such as at least 15 mg/ml.
  • the concentration of the phospholipids in the hydrophobic solution in step a) of the method of producing emulsions is at most 20 mg/ml, such as at most 15 mg/ml, such as at most 10 mg/ml, such as at most 5 mg/ml.
  • the concentration of the phospholipids in the hydrophobic solution in step a) of the method of producing emulsions is between 10 mg/ml and 15 mg/ml.
  • the hydrophobic solution of step a) comprises an added value compound.
  • the concentration of K-casein in the solution of step b) is at least 10 mg/ml, such as at least 15 mg/ml, such as at least 20 mg/ml, such as at least 25 mg/ml, such as at least 30 mg/ml, such as at least 35 mg/ml.
  • the concentration of K-casein in the solution of step b) is at most 40 mg/ml, such as at most 35 mg/ml, such as at most 30 mg/ml, such as at most 25 mg/ml, such as at most 20 mg/ml, such as at most 15 mg/ml, such as at most 10 mg/ml.
  • the concentration of K-casein in the solution of step b) is between 20 mg/ml and 40 mg/ml.
  • step d) of the method an emulsion is preferably obtained.
  • step c) of the method the emulsifying is achieved by mixing.
  • the mixing is performed by homogenization.
  • Homogenization refers to any process for altering particle or droplet size (e.g., reducing size and/or creating size uniformity) in a fluid under conditions of pressure, shear, and/or stress.
  • the term “homogenization” is intended to include the many and varied homogenization processes that involve the use of ultrasonic, pressure, and/or mechanical forces to homogenize a fluid. Examples of such homogenization techniques include, but are not limited to, two-stage homogenization, high- pressure homogenization (also known as micronization), very high pressure homogenization (VPH), rotator-stator homogenization, blade homogenization, high shear mixers, sonication, high shear impellers, milling, and the like.
  • the phospholipid is preferably as described in chapter “phospholipids”.
  • the added value compound is preferably as defined in chapter “added value compound”.
  • the hydrophobic solution of step a) comprises the added value compound at a concentration of at least 0.1 mg/ml, such as at least 0.5 mg/ml, such as at least 1 mg/ml, such as at least 1 .5 mg/ml, such as at least 2 mg/ml, such as at least 3 mg/ml, such as at least 4 mg/ml, such as at least 5 mg/ml.
  • the hydrophobic solution of step a) comprises the added value compound at a concentration of at most 20 mg/ml, such as at most 10 mg/ml, such as at most 5 mg/ml, such as at most 4 mg/ml, such as at most 3 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml, such as at most 0.5 mg/ml .
  • the hydrophobic solution of step a) comprises the added value compound at a concentration between 0.1 mg/ml and 10 mg/ml.
  • K-casein is preferably as described in chapter “K-casein”.
  • the solution in step b) comprises a buffer.
  • the buffer can comprise sodium chloride and imidazole and/or BisTris.
  • the buffer can have a pH of at least 5, such as at least 5.5, such as at least 6, such as at least 6.5 or of at most 7, such as at most 6.5, such as at most 6, such as at most 5.5, such as at most 5.
  • the present invention provides dispersions, for example nanocapsules or emulsions, which can be used in production of or as an ingredient in food products and/or beverages. For example, they can be used to fortify food products and/or beverages, to increase the stability of the food products/beverages, to transfer added value compounds to food products/beverages, or to enhance stability of added value compounds in food products or beverages.
  • the dispersions of the invention are particularly useful for delivering added value compounds in vivo, because of their stability under conditions mimicking the gastric environment. Even in conditions where the nanoparticles of the invention sediment, the cargo is in general not released and the nanocapsules can be resuspended by shaking.
  • the dispersions of the invention and in particular, the nanocapsules of the invention show high stability under conditions mimicking the gastric environment, and therefore they are believed to be particularly useful for transporting added value compounds to the intestines, thereby enabling increased in vivo accessibility of the added value compounds.
  • the dispersion of the invention, and in particular, the nanocapsules of the invention show a high stability also under conditions mimicking the proteolytic activity of the intestinal environment.
  • the content of the dispersions may still be released in the intestines due to the presence of other components of the intestinal environment e.g. the presence of bile acids.
  • dispersions of the invention and in particular the nanocapsules of the invention have high utility in manufacture of cheese or cheese analogues or mixtures thereof.
  • dispersions of the invention and in particular the nanocapsules of the invention have high utility in manufacture of acidic beverages, such as juice.
  • the dispersion when mixed with food products and/or beverages increases the stability of said food products and/or beverages.
  • stability refers to storage stability (e.g., storage stability at room temperature or at 4 °C).
  • stability may describe the lack of aggregation or proteolytic degradation of said food products and/or beverages.
  • the dispersion when mixed with milk increases the stability of said milk, such as decreases the aggregation and proteolytic degradation.
  • the nanocapsules increase the stability of milk up to 12 weeks at 4 °C.
  • the dispersion, the nanocapsules or the emulsion as described herein are used for the transport of added value compound(s).
  • transport describes the ability of the dispersion, to deliver the added value compound into food products and/or beverages.
  • hydrophilic added value compounds are transported into a hydrophobic environment.
  • hydrophobic added value compounds are transported into a hydrophilic environment.
  • hydrophilic added value compounds may also be transported into a hydrophilic environment, and hydrophobic added value compounds may also be transported into a hydrophobic environment.
  • transport can also describe the process of delivering added value compounds to a location of interest in vivo. Since the dispersions or the nanocapsules of the invention have high stability under conditions mimicking the gastric environment, they can be used to transport added value compounds, such as food supplements and/or therapeutically active substrates into the body, without being digested in the stomach.
  • the dispersion, the nanocapsules or the emulsion as described herein are used for enrichment of a food products and/or beverages with an added value compound.
  • the food products and/or beverages are dairy products or dairy alternative analogues or mixtures thereof.
  • the plant-based dairy alternative analogues are plant-base, such as made of soy or other legumes, almond, cashew, coconut, hemp, quinoa and/or oat.
  • the food products and/or beverages are milk, milk analogues, yoghurt, yoghurt analogues, cheese, cheese analogues, juice or mixtures of any of the aforementioned.
  • the milk analogues can be plant based or a mix of dairy and plant-based milk.
  • the yoghurt analogues can be plant based or a mix of dairy and plant-based yoghurt.
  • the cheese analogues can be plant based or a mix of dairy and plantbased cheese.
  • the dispersion, the nanocapsules or the emulsion as described herein are used for increasing the nutritional value in food products, such as in cheese, cheese analogues or mixtures thereof and/or in beverages.
  • the term "nutritional value” is associated with the quantity of available micronutrients, like iron, vitamins, antioxidants and other food supplements in the food product.
  • the amount of nutrients in the food product is increased.
  • the term “increased” means an increase of at least 1 %, preferably of at least 5%, even more preferably of at least 10% and most preferably of at least 20% of the food supplement when compared to the food product without added dispersion.
  • the present disclosure concerns a method of producing a food product and/or beverages, wherein the method comprises the following steps: a) Providing a food product, a beverage, food product ingredient or a beverage base; b) contacting said food product, beverage, food product ingredient or beverage base with the dispersion, the nanocapsules or the emulsion as described herein, c) processing the mixture of step b) into a product or a beverage.
  • the food product or the beverage is as described above.
  • the food product is cheese, a cheese analogue or mixtures thereof.
  • the dispersion, the nanocapsule and/or the emulsion are added in step b) to the food product, beverage, food product ingredient or beverage base at a concentration of at least 3 vol%, such as at least 4 vol%, such as at least 5 vol% of the food product, beverage, food product ingredient or beverage base.
  • the dispersion, the nanocapsule and/or the emulsion are added in step b) to the food product, beverage, food product ingredient or beverage base at a concentration of at most 50 vol%, such as at most 40 vol%, such as at most 30 vol%, such as at most 10 vol%, such as at most 8 vol%, such as at most 6 vol%, such as at most 5 vol% of the food product, beverage, food product ingredient or beverage base.
  • the dispersion, the nanocapsule and/or the emulsion are added in step b) to the food product, beverage, food product ingredient or beverage base at a concentration of 5 vol% of the food product, beverage, food product ingredient or beverage base.
  • beverage base means an intermediate beverage product which, when mixed with an appropriate amount of water or other suitable liquid or semi-liquid and/or a sweetening agent, forms a beverage syrup or alternatively a beverage.
  • food product ingredient includes a formulation which is or can be added to food products or foodstuffs, for example, as a nutritional supplement.
  • the term food ingredient as used here also refers to formulations which can be used at low levels in a wide variety of products that require stabilizing.
  • the dispersion of this invention has a high utility in manufacture of cheese or cheese analogues or mixtures thereof. Accordingly, whereas this section refers to “cheese”, the methods disclosed herein are equally applicable to cheese analogues or mixtures thereof.
  • the cheese analogues can be plant based or a mix of dairy and plant-based cheese.
  • the dispersion of this invention can be used in different methods of cheese making. When added value compounds are delivered using the dispersions or the nanocapsules of the invention during cheese manufacturing, the added value compounds can will typically be delivered to the cheese or cheese analogue, rather than remaining in the whey. In other words, the cheese or cheese analogue will have a higher level of the added value compounds, whereas the waste product, the whey will have a lower level. This is in particular the case for hydrophilic added value compounds.
  • the present disclosure concerns a method of producing cheese or cheese analogues, wherein the method comprises the following steps: a) Providing milk or milk analogue or mixture thereof comprising the dispersion, the nanocapsules or the emulsion as described herein; b) processing said milk or milk analogue into cheese.
  • the provided milk in the method of producing cheese in step a) is non-homogenized milk.
  • the milk or milk analogue of step a) of the method of producing cheese or cheese analogue comprises at least 2.5% fat, such as at least 2.7% fat, such as at least 2.9% fat, such as at least 3% fat, such as at least 3.1 % fat.
  • the milk or milk analogue of step a) of the method of producing cheese or cheese analogue comprises at most 4.8% fat, such as at most 4% fat, such as at most 3.5% fat, such as at most 3% fat, such as at most 2.5% fat.
  • the milk or milk analogue of step a) of the method of producing cheese or cheese analogue comprises between 2.9% and 3.1 % fat.
  • calcium chloride is added in the method of producing cheese or cheese analogue at a concentration of at least 0.2 mg/ml, such as at least 0.5 mg/ml, such as at least 1 mg/ml, such as at least 2 mg/ml, such as at least 5 mg/ml, such as at least 8 mg/ml, such as at least 10 mg/ml.
  • calcium chloride is added in the method of producing cheese or cheese analogue at a concentration of at most 10 mg/ml, such as at most 8 mg/ml, such as at most 5 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml, such as at most 0.5 mg/ml, such as at most 0.2 mg/ml.
  • calcium chloride is added in the method of producing cheese or cheese analogue at a concentration of between 0.2 mg/ml and 10 mg/ml.
  • the milk or milk analogue is acidified by adding citric acid in the method of producing cheese or cheese analogue.
  • the citric acid is added at a concentration of at least 1 mg/ml, such as at least 2 mg/ml, such as at least 3 mg/ml, such as at least 4 mg/ml, such as at least 5mg/ml.
  • the citric acid is added at a concentration of at most 5 mg/ml, such as at most 4 mg/ml, such as at most 3 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml.
  • the milk or milk analogue is equilibrated in the method of producing cheese or cheese analogue at a temperature of at least 30 °C, such as at least 31 °C, such as at most 32 °C. In some embodiments of this invention, the milk or milk analogue is equilibrated in the method of producing cheese or cheese analogue at a temperature of at most 34 °C, such as at most 33°C, such as at most 32 °C. In some embodiments of this invention, the milk or milk analogue is equilibrated in the method of producing cheese or cheese analogue at a temperature of 32 °C.
  • the milk or milk analogue is equilibrated in the method of producing cheese or cheese analogue for at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes. In some embodiments of this invention, the milk or milk analogue is equilibrated in the method of producing cheese or cheese analogue for at most 40 minutes, such as at most 30 minutes, such as at most 20 minutes. In some embodiments of this invention, the milk or milk analogue is equilibrated in the method of producing cheese or cheese analogue for 20 minutes.
  • the concentration of the chymosin enzyme in the milk or milk analogue is at least 0.05 ll/rnl, such as at least 0.1 ll/rnl, such as at least 0.15 ll/rnl, such as at least 0.2 ll/rnl. In some embodiments of this invention, the concentration of the chymosin enzyme in the milk or milk analogue is at most 0.4 ll/rnl, such as at least 0.3 ll/rnl, such as at least 0.2 ll/rnl. In some embodiments of this invention, the concentration of the chymosin enzyme in the milk or milk analogue is between 0.2 ll/rnl and 0.4 U/ml.
  • curd is cut during the incubation process.
  • step b) comprises the steps of
  • the cheese mass or cheese analogue mass and the whey are separated in step i) by filtration.
  • the present invention also relates to a nanocapsule for encapsulation and delivery of water-soluble added value compounds to cheese.
  • the nanocapsule comprises: a shell comprising phospholipid and K-casein, and a core comprising at least one water-soluble added value compound.
  • the present invention is based on the understanding that a nanocapsule which comprises phospholipid and K-casein in the shell provides a stable container for delivery of added value compounds to cheese, which container destabilizes and coagulates upon addition of a renneting enzyme.
  • the nanocapsules are used to incorporate water-soluble molecules with the objectives to increase the nutritional value of the cheese, and to add a health promoting functionality.
  • the present inventive concept further relates to a method to produce a nanocapsule for encapsulation and delivery of water-soluble added value compounds to cheese.
  • the method comprising the steps: a) preparing a lipid film by dissolving phospholipids in an organic solvent and evaporating the organic solvent; b) preparing an aqueous solution comprising at least one water-soluble added value compound; c) preparing a K-casein solution, d) solubilizing said lipid film prepared in step a) by adding to and mixing with said solutions prepared in step b), and e) adding the K-casein solution obtained in step c) thus obtaining a solution containing nanocapsules comprising a shell made of phospholipid and K-casein and a core comprising at least one water-soluble added value compound, wherein step e) occurs simultaneously with or after step d).
  • the solvent used in step a) may be an organic solvent, such as chloroform or hexane.
  • step a) may be conducted under vacuum by use of desiccator or a plate evaporator. It should be noted that any type of solvent evaporator may be used for evaporation of the organic solvent.
  • a protein solution comprising at least one water-soluble added value compound and K- casein is prepared.
  • the protein solution may typically be an aqueous protein solution.
  • This protein solution may be obtained by mixing a solution comprising at least one water-soluble added value compound with a K-casein solution.
  • the solution comprising the water-soluble added value compound and the K-casein solution, respectively, may be aqueous solutions.
  • the protein concentration of step b) may be up to 10 mg/mL, preferably from 1 to 6 mg/mL.
  • the concentration of K-casein in the K-casein solution of step c) may be up to 20 mg/mL, preferably from 1 to 10 mg/mL.
  • the concentration of phospholipids in the solubilized lipid film of step d) may be from 0.1 to 20 mg/mL, preferably from 5 to 10 mg/mL.
  • the preparation in step b) and/or step c) may comprise the steps of: a’) providing a powder comprising K-casein and/or at least one water-soluble added value compound, and b') suspending said powder in a buffer solution containing imidazole or Bis-Tris (2-bis(2-hydroxyethyl)amino-2(hydroxymethyl)-1 ,3-propanediol, bis(2hydroxyethyl)amino-tris(hydroxymethyl)methane, 2,2-bis(hydroxymethyl)- 2,2',2"-nitrilotriethanol) and sodium chloride.
  • the concentration of imidazole or Bis-Tris in the buffer solution may be between 10 and 40 mM.
  • the concentration of sodium chloride in the buffer solution may be between 20 and 150 mM.
  • the buffer solution may have a pH of from 4.5 to 7, preferably from 5.5 to 6.7.
  • step d) a solution comprising nanocapsules and excess of K-caseins and/or phospholipids is obtained.
  • step d) may comprise mixing via vortexing, extrusion, homogenization and/or sonication for solubilization of the lipid film.
  • the present inventive concept further relates to a method for production of fortified cheese and/or cheese with added functionality.
  • the method comprising the steps of: a) providing the nanocapsule according to the inventive concept; b) adding the nanocapsule to milk thus forming fortified milk or milk with added functionality; c) adding renneting enzymes to the fortified milk or milk with added functionality, thus forming fortified cheese curd and whey; d) separating the fortified or functional cheese curd from the whey; and e) pressing the fortified or functional cheese curd into cheese.
  • the process consists of using the self-assembly properties of K-casein and combining it with the self-assembly properties of phospholipids to create nanocontainers with the capacity to hold e.g. enzymes and other components of high nutritional value.
  • the nanocapsules are added to the milk, which comprises casein micelles, to provide fortified milk or milk with added functionality.
  • renneting enzymes such as chymosin
  • the soluble part of the K-casein present in the nanocapsules and in the casein micelles, respectively, is cleaved and a cheese curd is formed.
  • the nanocapsules thus coagulate together with the casein micelles in the milk and form a cheese curd with incorporated and protected water-soluble nutrients.
  • the milk used in step b) may comprise plant-based milk and/or dairy milk or mixtures thereof.
  • the milk may comprise between 2.5 and 4.8% by weight, preferably between 2.9 and 3.1 % by weight of fat.
  • the milk may be non-homogenized milk.
  • renneting enzymes may be added to the fortified milk.
  • the method may further comprise the step of: f) enriching the milk with calcium chloride, wherein the step f) occurs after step b).
  • step f) occurs after step b).
  • 0.2 to 10 mg/mL of calcium chloride may be added to the milk.
  • the method may further comprise the step of: g) acidifying the milk by adding an acid, such as citric acid, or by adding starter, such as lactic acid bacteria, to the milk, wherein the step g) occurs after step b).
  • an acid such as citric acid
  • starter such as lactic acid bacteria
  • Starter is used for production of acids and flavour.
  • concentration of starter may be 10 4 to 10 8 Colony Forming Unit/mL.
  • step f) and g), respectively, occur after step b) is that addition of calcium chloride and acidification may induce colloidal aggregation of the casein micelles. If the aggregation takes place before the addition of the nanocapsules, it is believed that the nanocapsules may be poorly incorporated into the cheese curd.
  • the present inventive concept further relates to a fortified cheese and/or a cheese with added functionality obtained by the method.
  • a result of the method may thus be a fortified cheese or a cheese with added-value or added functionality, the cheese being provided with good structure, taste, and visual appearance.
  • a nanocapsule for encapsulation and delivery of water-soluble added value compounds to cheese comprising: a shell comprising phospholipid and K-casein, and a core comprising at least one water-soluble added value compound.
  • said phospholipid comprises synthetic phospholipid, animal-based phospholipid and/or plantbased phospholipid.
  • said water-soluble added value compound comprises a compound selected from the group consisting of a functional protein, a digestive enzyme, an antioxidant, and a vitamin.
  • a method to produce a nanocapsule for encapsulation and delivery of water-soluble added value compounds to cheese comprising the steps: a) preparing a lipid film by dissolving phospholipids in an organic solvent and evaporating the organic solvent; b) preparing an aqueous solution comprising at least one water-soluble added value compound; c) preparing a K-casein solution, d) solubilizing said lipid film prepared in step a) by adding to and mixing with said solutions prepared in step b), and e) adding said K-casein solution obtained in step c) thus obtaining a solution containing nanocapsules comprising a shell made of phospholipid and K-casein and a core comprising at least one water-soluble added value compound, wherein step e) occurs simultaneously with or after step d).
  • step a) is an organic solvent, such as chloroform or hexane.
  • step a) is conducted under vacuum by use of a desiccator or a plate evaporator.
  • said preparation in said step b) and/or step c) comprises the steps of: a’) providing a powder comprising K-casein and/or at least one water- soluble added value compound, and b') suspending said powder in a buffer solution containing imidazole or bis tris (2-bis(2-hydroxyethyl)amino-2(hydroxymethyl)-1 ,3-propanediol, bis(2hydroxyethyl)amino-tris(hydroxymethyl)methane, 2,2-bis(hydroxymethyl)- 2,2',2"-nitrilotriethanol) and sodium chloride.
  • step d) comprises mixing via vortexing, homogenization, extrusion and/or sonication for solubilization of the lipid film.
  • a method for production of fortified cheese and/or cheese with added functionality comprising the steps of: a) providing said nanocapsule according to embodiment 1 ; b) adding said nanocapsule to milk thus forming fortified milk or milk with added functionality; c) adding renneting enzymes to said fortified milk or milk with added functionality, thus forming fortified cheese curd and whey; d) separating said fortified or functional cheese curd from said whey; and e) pressing said fortified or functional cheese curd into cheese.
  • a method according to embodiment 11 wherein said milk comprises plant-based milk and/or dairy milk.
  • an acid such as citric acid
  • starter such as lactic acid bacteria
  • Example 1 Nanocapsules and emulsions can be made using K-casein recombinant produced in E. coli
  • a codon optimized sequence encoding K-casein from donkey of SEQ ID NO:3 (Equus asinus africanus - Uniprot accession number5 F0V6V5, 23 May 2024) was cloned in pET30a plasmid.
  • the recombinant plasmid was used for the expression of the protein in E. coli.
  • Bacterial cells were grown at 37 °C in Terrific Broth (12 g/L tryptone, 24 g/L yeast extract, 0.5% glycerol, 89 mM phosphate buffer) supplemented with 30 pg/mL kanamycin.
  • Protein expression was induced by adding 1 mM isopropyl-beta-0 thiogalactopyranoside (IPTG) when the bacterial cultures reached an optical density at 600 nm of 0.5 - 0.7.
  • IPTG isopropyl-beta-0 thiogalactopyranoside
  • K-casein was performed at 37 °C for 5 hours.
  • cells were harvested by centrifugation and lysed in lysis buffer (20 mM imidazole, pH 6.5, 20 mM NaCI) by sonication (10 % amplitude, 30 min total time, 2 sec on, 2 sec off, on ice).
  • Donkey K-casein was purified from bacterial cell lysate by cationic exchange chromatography (CIEX) on an Akta Pure FPLC system equipped with a Hi-Trap SP column (Cytiva).
  • CIEX cationic exchange chromatography
  • the CIEX method for purification employed a wash/equilibration buffer (also known as buffer A: 20 mM imidazole, pH 6.5, 20 mM NaCI) and an elution buffer (also known as buffer B: 20 mM imidazole, pH 6.5, 1 M NaCI), according to the following steps:
  • Donkey K-casein peak eluted at 60 % of buffer B. Protein purity was analyzed by stain-free SDS-PAGE and protein concentration determined by using a NanoDrop.
  • a lipid film was prepared by dissolving pure soy lecithin in hexane. Hexane was evaporated overnight using a desiccator under vacuum. The lipid film was stored at -20 °C until use.
  • Lactoferrin 0.5 mg/mL Lactoferrin was mixed with 0.8 mg/mL recombinant donkey K-casein.
  • the aqueous solution for resuspension was a buffer containing 20 mM imidazole, 60 mM sodium chloride and pH 6.5.
  • the lipid film was solubilized by addition of 10 mg/mL lactoferrin solution to the film.
  • gentle mixing via vortexing and then tip sonication (Sonifier S-450D, 400 Watts, Branson Ultrasonics, Connecticut, USA) was performed for complete resuspension.
  • the sample was sonicated for 1 .5 min.
  • nanocapsules and emulsions can be produced using recombinant K-casein from donkey ( Figure 3).
  • Example 2 Nanocapsules and emulsions can be made using K-casein recombinant produced in C. reinhardtii
  • a codon optimized sequence encoding K- casein from bovine of SEQ ID NO:2 (Bos taurus- Uniprot accession number P02668, 23 May 2024) was cloned in pChlamy _4 vector, which allows the constitutive expression of the recombinant protein in the intracellular compartment of transformed cells.
  • the recombinant vector was introduced in the expression host - Chlamydomonas reinhardtii by electroporation.
  • Cells were grown in Tris Acetate Phosphate medium (also known as TAP medium) supplemented with 2.5 pg/ml zeocin, at 26 °C with light source set at 50 pE m- 2 s-1.
  • recombinant microalgae were harvested, lysed in lysis buffer (20 mM imidazole, pH 6.5, 20 mM NaCI) by sonication (20 % amplitude, 30 min total time, 30 sec on, 30 sec off, on ice), and bovine K- casein protein levels were determined by stain-free SDS-PAGE followed by Western blot developed by using a polyclonal rabbit anti-bovine K-casein primary antibody (AA 111-170, antibodies-online) and a polyclonal HRP- conjugated anti-rabbit secondary antibody (31460, Invitrogen).
  • the emulsion was produced as described in example 1 , with the exception that 70 mg/mL algae protein including bovine K-casein was used instead of donkey K-casein. Sample were homogenized 15000 rpm, 15 min, 25 °C.
  • Example 3 Nanocapsules are a single type of particle
  • a lipid film was prepared by dissolving pure soy lecithin in hexane. Hexane was evaporated overnight using a desiccator under vacuum. The lipid film was stored at -20 °C until use. 2 mg/mL lactoferrin was mixed with 2 mg/mL K-casein from bovine milk. The aqueous solution for resuspension was a buffer containing 20 mM imidazole, 20 mM sodium chloride and pH 6.5.
  • the lipid film was solubilized by addition of 10 mg/ml lactoferrin solution to the film.
  • gentle mixing via vortexing and then tip sonication (Sonifier S-450D, 400 Watts, Branson Ultrasonics, Connecticut, USA) was performed for complete resuspension.
  • the sample was sonicated.
  • DLS Dynamic light scattering
  • nanocapsules are a single type of particle that differ from that of its single components ( Figure 8).
  • Example 4 Nanocapsules made with plant-based purified lecithin
  • nanocapsules can be made from different sources of lecithin.
  • the nanocapsules were prepared as described in example 3, with the exception that 0.2 mg/mL lecithin from either soy or sunflowers were dissolved in chloroform.
  • the lecithin was either foodgrade lecithin or purified lecithin.
  • 1.36 mg/mL K-casein from bovine milk without encapsulated protein in 25 mM Bis-Tris buffer, 25 mM sodium chloride and pH 6.7 were used. The sample was sonicated at 80 % amplitude, 5 min, 0.5 sec on, 0.5 sec off, 25 °C.
  • nanocapsules can be made with plant-based purified lecithin, and also with food graded lecithin from soy lecithin and sunflower lecithin ( Figure 9).
  • Example 5 Stability of nanocapsules
  • the nanocapsules were prepared as described in example 3, with the exception that either 0.5 mg/ml lecithin for the stability test in different pH or 1 mg/mL lecithin for the stability test in different salinity conditions from soy were dissolved in chloroform. Furthermore, 1.36 mg/mL K-casein from bovine milk without encapsulated protein either 25 mM Bis-Tris buffer, 25 mM sodium chloride and pH 4.6-6.7 were used for the stability test in different pH or 20 mM imidazole buffer, 0-100 mM sodium chloride, pH 6.5 were used for the stability test in different salinity conditions. The sample was sonicated at 80 % amplitude, 5 min, 0.5 sec on, 0.5 sec off, 25 °C.
  • nanocapsules are stable at pH 4.6 to 6.7 and in salinity conditions of between 20 to 100 mM NaCI in imidazole buffer at pH 6.5 ( Figure 10).
  • nanocapsules were prepared as described in example 3, with the exception that 3 mg/mL pure soy lecithin dissolved in chloroform and 1.36 mg/mL K-casein from bovine milk without encapsulated protein in 20 mM imidazole buffer, 60 mM sodium chloride and pH 6.5 was used. The sample was sonicated at 80 % amplitude, 5 min, 0.5 sec on, 0.5 sec off, 25 °C.
  • SAXS Small Angle Xray Scattering
  • Cryo-TEM was performed at University of Copenhagen, Denmark, on FEI Tecnai G2 20 TWIN is a 200 kV Transmission Electron Microscope.
  • the nanocapsules have a core-shell structure, in which the core is water filled and the shell is composed of phospholipids and K-casein ( Figure 11 ).
  • Example 7 Stability of the nanocapsules upon storage
  • the nanocapsules were prepared as described in example 3. DLS was performed as described in example 1 .
  • nanocapsules are stable for at least up to 38 weeks at 4 °C and 5 weeks at 25 °C ( Figure 12).
  • the nanocapsules were prepared as described in example 3. DLS was performed as described in example 1 .
  • the nanocapsules were prepared as described in example 3. To test the colloidal stability of the nanocapsules either 10 mg/mL calcium chloride or 10 mg/mL calcium chloride and 0.4 U/mL chymosin was added. DLS was performed as described in example 1. Furthermore, SDS-PAGE was performed.
  • the colloidal stability of the nanocapsules is decreased in the presence of calcium chloride and the nanocapsule fully precipitate in presence of calcium chloride and chymosin.
  • nanocapsules comprising casein mixture with nanocapsules comprising K-casein.
  • the nanocapsules were prepared as described in example 3 or in case of the nanocapsules comprising the casein mixture, 2 mg/ml dairy casein in 20 mM imidazole, 20 mM NaCI, 5.5 mM NaOH buffer, pH 6.5 was used.
  • 2 mg/ml dairy casein in 20 mM imidazole, 20 mM NaCI, 5.5 mM NaOH buffer, pH 6.5 was used.
  • To test the colloidal stability of the nanocapsules either 10 mg/mL calcium chloride or 10 mg/mL calcium chloride and 0.4 U/mL chymosin was added. DLS was performed as described in example 1. Furthermore, SDS-PAGE was performed.
  • K-casein is not functional anymore as the nanocapsules do not loose colloidal stability in the presence of calcium chloride or chymosin ( Figure 15).
  • K-casein is needed to achieve the effect of the nanocapsules of this invention, thus to loose colloidal stability in the presence of calcium chloride or chymosin.
  • the protein cargo is aqainst :ic degradation during in vitro digestion
  • Aim The protein cargo is aqainst :ic degradation during in vitro digestion
  • the nanocapsules were prepared as described in example 3. To mimic the gastric phase, 1.7 mg/mL pepsin was added to the nanocapsules and incubated for 1 hour at 37 °C under shaking at 300 rpm. To mimic the proteolytic activity of the intestinal phase, 0.4 mg/mL pancreatin was added to the gastric phase mixture. The intestinal phase mixture was incubated for 2 hours at 37 °C. Free lactoferrin was used as a control. SDS-PAGE was performed.
  • the inventors of the present disclosure have shown that the protein cargo, lactoferrin, is protected against proteolytic degradation during in vitro digestion, when encapsulated in the nanocapsules.
  • the nanocapsules were prepared as described in example 3.
  • the nanocapsules were added to 2.9-3.1 % fat non-homogenized milk.
  • the milk was further enriched with 0.2 mg/mL calcium chloride and acidified by adding 5 mg/mL citric acid. Then, the milk was equilibrated at 32 °C for 20 minutes and 0.2 U/mL chymosin enzyme was added. Incubation took place at 32 °C for 15 minutes. The curd was cut, and further incubation takes place at 32 °C for 15 min. During the incubation process, the curd was cut in 5 minutes intervals. The cheese mass was then separated from whey by filtration. The concentration of lactoferrin was determined via SDS-PAGE.
  • the inventors of the present disclosure have shown that the encapsulation of lactoferrin in the nanocapsules leads to delivery of lactoferrin into cheese mass.
  • nanocapsules were prepared as described in example 3, except that 2 mg/ml vitamin B12 instead of lactoferrin was added. DLS as described in example 3 was performed.
  • the nanocapsules were added to 2.9-3.1 % fat non-homogenized milk.
  • the milk was further enriched with 0.2 mg/mL calcium chloride and acidified by adding 5 mg/mL citric acid. Then, the milk was equilibrated at 32 °C for 20 minutes and 0.2 U/mL chymosin enzyme was added. Incubation took place at 32 °C for 15 minutes. The curd was cut, and further incubation takes place at 32 °C for 15 min. During the incubation process, the curd was cut in 5 minutes intervals. The cheese mass was then separated from whey by filtration.
  • vitamin B12 can be incorporated in nanocapsules.
  • the vitamin B12 nanocapsules are smaller compared to lactoferrin nanocaspules.
  • cheese fortified with vitamin B12 nanocapsules changes colour (Figure 18).
  • the inventors of the present disclosure have shown that the encapsulation of vitamin B12 in the nanocapsules leads to delivery of vitamin B12 into cheese mass.
  • Example 14 Stability of milk when fortified by nanocapsules
  • the nanocapsules were prepared as described in example 3. For the enrichment of milk, the nanocapsules at a concentration of 5 vol %, were added to the 3% fat milk. Gentle mixing via ten times inversion was performed for complete dispersion. Milk was stored at 4 °C for stability assay. The stability assay was performed by visual inspection, particle size analyser and SDS- PAGE. Controls were made with free lactoferrin, liposomes, buffer, or K-casein.
  • the particle size distribution was measured by using a LS230 laser diffraction particle size analyzer (Beckman Coulter, Inc., California, USA). A refractive index (Rl) 1 .46 for fat such as milk fat and olive oil and 1.33 for water were used to calculate the droplet size. The droplet diameter profile of the samples was measured at least three times.
  • Milk comprising nanocapsules is stable against aggregation or proteolytic degradation for at least up to 12 weeks as compared with less than 2 weeks in the absence of nanocapsules (Figure 19).
  • nanocapsules can increase the stability of milk.
  • Example 15 Stability of apple juice and protein cargo when fortified by nanocapsules
  • the nanocapsules were prepared as described in example 3.
  • the nanocapsules were added to the apple juice.
  • Gentle mixing via ten times inversion was performed for complete dispersion.
  • Apple juice was stored at 4°C for stability assay.
  • the stability assay was performed by visual inspection, particle size analyser as described in example 14 and SDS-PAGE. Controls were made with free lactoferrin or buffer.
  • Example 16 Stability of oat milk when fortified with nanocapsules
  • the nanocapsules were prepared as described in example 3. For the enrichment of oat milk, the nanocapsules at a concentration of 5 vol %, were added to the oat milk. Gentle mixing via ten times inversion was performed for complete dispersion. Milk was stored at 4°C for stability assay. The stability assay was performed by visual inspection, particle size analyser as described in example 14 and SDS-PAGE. Controls were made with free lactoferrin or buffer.
  • Example 17 Stability of soybean milk when fortified with nanocapsules
  • the nanocapsules were prepared as described in example 3. For the enrichment of soybean milk, the nanocapsules at a concentration of 5 vol %, were added to the soybean milk. Gentle mixing via ten times inversion was performed for complete dispersion. Milk was stored at 4°C for stability assay. The stability assay was performed by visual inspection, particle size analyser as described in example 14 and SDS-PAGE. Controls were made with free lactoferrin or buffer.
  • the nanocapsules were prepared as described in example 3. For the enrichment of yoghurt, the nanocapsules at a concentration of 5 vol %, were added to the yoghurt. Gentle mixing via ten times inversion was performed for complete dispersion. Yoghurt was stored at 4°C for stability assay. The stability assay was performed by visual inspection, particle size analyser as described in example 14 and SDS-PAGE. Controls were made with free lactoferrin or buffer.
  • the particle size distribution of the emulsions was measured by using a LS230 laser diffraction particle size analyzer (Beckman Coulter, Inc., California, USA).
  • a refractive index (Rl) 1 .46 for fat such as milk fat and olive oil and 1 .33 for water were used to calculate the droplet size.
  • the droplet diameter profile of the samples was measured at least three times.
  • K-casein-based emulsions using bovine K-casein purified from dairy milk as emulsifier give similar droplet size distribution as milk emulsion ( Figure 24).
  • K-casein emulsification is suited for a wide range of beverages including milk mimics.
  • Example 20 Stability of emulsions
  • the emulsions were prepared as described in example 19.
  • the droplet size distribution was measured as described in example 19.
  • bovine K-casein stabilized emulsions are stable upon storage at both 4 and 25 °C for at least up to 7 weeks and 1 week respectively (Figure 25).
  • the emulsions were prepared as described in example 19.
  • the droplet size distribution was measured as described in example 19.
  • bovine K-casein stabilized emulsions are stable upon freeze drying at least after 7 weeks of resuspension and storage at 4 °C ( Figure 26).
  • the emulsions were prepared as described in example 19. 0.2 mg/mL calcium chloride, 5 mg/mL citric acid and 0.2 U/mL enzyme were added. SDS- PAGE was performed.
  • K-casein in the emulsion surface can be cleaved by chymosin inducing a precipitate that mirrors the dairy cheese curd ( Figure 27). of milk emulsions
  • the emulsions were prepared as described in example 19. Controls were made with free lecithin in oil, buffer, or K-casein. The K-casein stabilized emulsion was added at a concentration of 5 vol %, to the milk. Gentle mixing via ten times inversion was performed for complete dispersion. Milk was stored at 4 °C for stability assay. The droplet size distribution was measured as described in example 19. SDS-PAGE was performed.
  • SEQ ID NO: 1 Bovine K-casein
  • SEQ ID NO: 2 Recombinant bovine K-casein
  • SEQ ID NO: 3 Recombinant donkey K-casein
  • the invention may further be defined by the following items:
  • a dispersion said dispersion comprising: a) casein, wherein at least 60% of the casein is K-casein; and b) phospholipids.
  • the dispersion according to item 1 wherein the dispersion is a nanocapsule comprising: a) a shell comprising phospholipid and casein, wherein at least 60% of the casein is K-casein; and b) a hydrophilic core.
  • a nanocapsule comprising: a) a shell comprising phospholipid and casein, wherein at least 60% of the casein is K-casein; and b) a hydrophilic core.
  • the delivering composition is an emulsion comprising oil in water droplets comprising: a) a shell comprising casein, wherein at least 60% of the casein is K-casein; and b) a hydrophobic core.
  • An emulsion comprising oil in water droplets comprising: a) a shell comprising casein, wherein at least 60% of the casein is K-casein; and b) a hydrophobic core.
  • the shell further comprises phospholipids.
  • the K-casein is recombinant K-casein produced by a host cell or host organism comprising a heterologous nucleic acid encoding K-casein.
  • K-casein is a K-casein of SEQ ID NO: 1 or a functional homologue of said K-casein sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.
  • K- casein is a K-casein of SEQ ID NO: 2 or a functional homologue of said K- casein sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.
  • K- casein is a K-casein of SEQ ID NO: 3 or a functional homologue of said K- casein sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.
  • the dispersion according to any one of the preceding items wherein at least 70%, such as at least 80%, such as at least 90% of the caseins are K-caseins.
  • the dispersion according to any one of the preceding items, wherein at most 40%, such as at most 30%, such as at most 20%, such as at most 10%, such as at most 5%, such as at most 1 %, such as at most 0,1 % of the caseins are aS2-casein.
  • the dispersion according to any one of the preceding items, wherein at most 40%, such as at most 30%, such as at most 20%, such as at most 10%, such as at most 5%, such as at most 1 %, such as at most 0,1 % of the caseins are [3-casein.
  • the dispersion according to any one of the preceding items, wherein at most 40%, such as at most 30%, such as at most 20%, such as at most 10%, such as at most 5%, such as at most 1 %, such as at most 0,1 % of the caseins are aS1 -casein, aS2-casein and [3-casein.
  • the phospholipids are one or more phospholipids selected from the group consisting of synthetic phospholipids, animal-based phospholipids and plant-based phospholipids.
  • the phospholipids are a mixture of glycerophospholipids, for example phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, or phosphatidic acid.
  • the phospholipids comprise or consist of lecithin, for example dihexanoyl-L- alpha-lecithin, dioctanoyl-L-alpha-lecithin, didecanoyl-L-alpha-lecithin, didodecanoyl-L-alpha-lecithin, ditetradecanoyl-L-alpha-lecithin, dihexadecanoyl-L-alpha-lecithin, dioctadecanoyl-L-alpha-lecithin, dioleoyl- Lalpha-lecithin, dilinoleoyl-L-alpha-lecithin, or alpha-palmitol.
  • lecithin for example dihexanoyl-L- alpha-lecithin, dioctanoyl-L-alpha-lecithin, didecanoyl-L-alpha-lecithin,
  • At least one added value compound is an antioxidant, such as vitamin A, vitamin C, vitamin E, xanthophylls, such as zeaxanthin or lutein, polyphenols or flavonoids such as resveratrol or quercetin.
  • an antioxidant such as vitamin A, vitamin C, vitamin E, xanthophylls, such as zeaxanthin or lutein, polyphenols or flavonoids such as resveratrol or quercetin.
  • At least one added value compound is a protein, such as an iron carrying protein, such as hemoglobin or lactoferrin.
  • At least one added-value compound is an enzyme, such as amylase or [3-galactosidase.
  • At least one added value compound is a fatty acid, such as omega 3 fatty acid or omega 6 fatty acid.
  • step a) The method according to item 43, wherein the lipid film of step a) is prepared by dissolving phospholipids, for example lecithin in an organic solvent, and subsequently removing said organic solvent, for example evaporating the organic solvent.
  • step b) comprises an added value compound.
  • step b) The method according to any one of items 43 to 47, wherein the solution in step b) is prepared by resuspending an added value compound in a buffer and mixing said buffer with a solution comprising said caseins.
  • the hydrophilic solution of step b) is prepared by a method comprising resuspending K-casein in an aqueous buffer.
  • the concentration of K-casein in the aqueous solution of step b) is at least 0.5 mg/ml, such as at least 1 mg/ml, such as at least 1 .5 mg/ml, such as at least 2 mg/ml, such as at least 2.5 mg/ml, such as at least 3 mg/ml, such as at least 4 mg/ml, such as at least 5 mg/ml, such as at least 10 mg/ml.
  • the method according to item 49, wherein the concentration of K-casein in the aqueous solution of step b) is at most 20 mg/ml, such as at most 15 mg/ml, such as at most 10 mg/ml, such as at most 5 mg/ml, such as at most 3 mg/ml, such as at most 2.5 mg/ml, such as at most 2 mg/ml, such as at most 1.5 mg/ml, such as at most 1 mg/ml, such as at most 0.5 mg/ml.
  • step d) of the method comprises phospholipids at a concentration of at least 0.1 mg/ml, such as at least 0.5 mg/ml, such as at least 1 mg/ml, such as at least 2 mg/ml, such as at least 5 mg/ml, such as at least 8 mg/ml, such as at least 10 mg/ml, such as at least 10 mg/ml of the total solution.
  • step d) of the method comprises phospholipids at a concentration of at most 20 mg/ml, such as at most 10 mg/ml, such as at most 8 mg/ml, such as at most 5 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml, such as at most 0.5 mg/ml, such as at most 0.1 mg/ml of the total solution.
  • step c) the lipid film is solubilized by mixing the lipid film of step a) with the solution of step b), wherein the mixing is performed by one or more of the methods of a group comprising vortexing, homogenization, bath sonication and tip sonication.
  • step d) a solution containing nanocapsules comprising a shell made of phospholipid and K-casein and a core comprising at least one water-soluble added value compound is obtained.
  • step d) The method according to any one of items 43 to 56, wherein in step d) the nanocapsules are eluted in a buffer.
  • hydrophobic solution in step a) comprises oil and a phospholipid, such as lecithin.
  • oil olive oil, rapeseed oil, soybean oil, sunflower oil or com oil.
  • the concentration of the oil is at least 0.5 vol%, such as at least 1 vol%, such as at least 5 vol%, such as at least 10 vol%, such as at least 15 vol%, such as at least 20 vol% of the hydrophobic solution.
  • step a) comprises an added value compound.
  • the concentration of K-casein in the solution of step b) is at least 10 mg/ml, such as at least 15 mg/ml, such as at least 20 mg/ml, such as at least 25 mg/ml, such as at least 30 mg/ml, such as at least 35 mg/ml.
  • the concentration of K-casein in the solution of step b) is at most 40 mg/ml, such as at most 35 mg/ml, such as at most 30 mg/ml, such as at most 25 mg/ml, such as at most 20 mg/ml, such as at most 15 mg/ml, such as at most 10 mg/ml.
  • step d) an emulsion is obtained.
  • the hydrophilic solution of step b) or to the hydrophobic solution of step a) comprises the added value compound at a concentration of at least 0.1 mg/ml, such as at least 0.5 mg/ml, such as at least 1 mg/ml, such as at least 1 .5 mg/ml, such as at least 2 mg/ml, such as at least 3 mg/ml, such as at least 4 mg/ml, such as at least 5 mg/ml.
  • the hydrophilic solution of step b) or to the hydrophobic solution of step a) comprises the added value compound at a concentration of at most 20 mg/ml, such as at most 10 mg/ml, such as at most 5 mg/ml, such as at most 4 mg/ml, such as at most 3 mg/ml, such as at most 2 mg/ml, such as at most 1 mg/ml, such as at most 0.5 mg/ml total protein.
  • step 73 The method according to item 43 to 72, wherein the hydrophilic solution of step b) or to the hydrophobic solution of step a) comprises the added value compound at a concentration between 0.1 mg/ml and 10 mg/ml.
  • step b) comprises a buffer.
  • a method of producing a food product and/or beverages comprising the following steps: a) Providing a food product, a beverage, food product ingredient or a beverage base; b) contacting said food product, beverage, food product ingredient or beverage base with the dispersion, the nanocapsules or the emulsion according to any one of the items 1 to 42, c) processing the mixture of step b) into a product or a beverage.
  • step b) The method according to item 83, wherein the dispersion, the nanocapsule and/or the emulsion are added to said in step b) to the food product, beverage, food product ingredient or beverage base at a concentration of at least 3 vol%, such as at least 4 vol%, such as at least 5 vol% of the food product, beverage, food product ingredient or beverage base .
  • the method according to item 83 wherein the dispersion, the nanocapsule and/or the emulsion are added in step b) to the food product, beverage, food product ingredient or beverage base at a concentration of at most 50 vol%, such as at most 40 vol%, such as at most 30 vol%, such as at most 10 vol%, such as at most 8 vol%, such as at most 6 vol%, such as at most 5 vol% of the food product, beverage, food product ingredient or beverage base.
  • the method according to item 83 wherein the dispersion, the nanocapsule and/or the emulsion are added in step b) to the food product, beverage, food product ingredient or beverage base at a concentration of 5 vol% of the food product, beverage, food product ingredient or beverage base.
  • a method of producing cheese comprising the following steps: a) Providing milk or milk analogue comprising the dispersion, the nanocapsules or the emulsion according to any one of the items 1 to 42; b) processing said milk into cheese or cheese analogue.
  • the method according to item 89 wherein the provided milk in the method of producing cheese in step a) is non-homogenized or homogenized milk.
  • the method according to any one of items 89 to 90, wherein step b) comprises the steps of

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Abstract

La présente invention concerne une dispersion pour l'encapsulation et l'administration de composés à valeur ajoutée. Les dispersions comprennent : de la caséine, au moins 60% de la caséine étant de la κ-caséine; et des phospholipides.
PCT/EP2024/066610 2023-06-16 2024-06-14 Nanocapsules pour l'encapsulation et la distribution de composés hydrosolubles Ceased WO2024256662A1 (fr)

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