NUTRITIONAL COMPOSITION, AND METHOD FOR MASKING STRANGE FLAVORS OF LEUCINE IN IT

专利摘要:
compositions to mask the taste of nutrients and methods of making the same. The present invention relates to nutritional compositions and methods of making and using the nutritional compositions. in a general embodiment, the present invention provides nutritional compositions providing a sufficient amount of leucine to improve protein synthesis in humans, while also maintaining a fluid matrix of low viscosity and acceptable organoleptic properties.
公开号:BR112012023025B1
申请号:R112012023025-5
申请日:2011-03-09
公开日:2021-06-01
发明作者:Kevin Burke Miller；Candis Diane Kvamme；Trent Stellingwerff；Lionel Jean René Bovetto
申请人:Société des Produits Nestlé S.A.；
IPC主号:

专利说明:

Background of the Invention
[0001] The present invention relates generally to health and nutrition. More specifically, the present invention relates to nutritional compositions having whey protein micelles and at least one amino acid, and methods for making and using the nutritional compositions to optimize the taste profile and physical properties of the compositions to provide the patient improved health.
[0002] There are currently on the market many types of nutritional compositions. The nutritional compositions can be intended for certain types of consumer, for example, young, elderly, athletic etc., based on the specific ingredients of the nutritional composition. Nutritional compositions can also be formulated based on certain physiological conditions that the nutritional compositions are intended to treat or ameliorate, or can be made based on the desired physical or organoleptic properties of the nutritional compositions.
[0003] One of the goals of nutritional support is to increase the amounts of nutrients offered in nutritional compositions to provide the consumer with a sufficient amount of the nutrient to obtain a specific biological result. However, many nutrients that are used in nutritional compositions to offer the consumer a specific nutritional benefit instead impart an undesirable taste or odor to the composition leaving it unattractive for consumption. As a result, the desired biological result is not obtained when the consumer refuses to ingest the composition due to its poor organoleptic properties. Therefore, it is desired to offer nutritional compositions having increased amounts of nutrients while offering tolerable physical and organoleptic properties. summary
[0004] The invention provides nutritional compositions and methods for making and using the nutritional compositions. In a generic embodiment, the present invention provides nutritional compositions having whey protein micelles and leucine. The nutritional compositions provide a sufficient amount of leucine to improve protein synthesis in humans, while also maintaining a fluid matrix of low viscosity and acceptable organoleptic properties.
[0005] In one embodiment, a nutritional composition is offered that includes whey protein powder comprising whey protein micelles, and leucine, where the total leucine in the composition comprises between 20% and 40% by weight of matter dry.
[0006] In another embodiment, a nutritional composition is offered that includes whey protein powder having whey protein micelles, and added leucine, where the dry weight ratio of added leucine to whey protein micelles of milk ranges from about 1:2 to about 1:3.
[0007] In one embodiment, the dry weight ratio of leucine added to whey protein micelles is about 1:2.6.
[0008] In one embodiment, the whey protein powder includes at least about 20% whey protein micelles. The whey protein can also include at least 50% whey protein micelles. The whey protein can also include at least 80% whey protein micelles.
[0009] In one embodiment, the composition is a powdered composition.
[00010] In one embodiment, the whey protein powder is obtained by spray drying or freeze drying a whey protein micelle concentrate.
[00011] In one embodiment, the whey protein powder has a water-fixing capacity of at least 50%. Whey protein powder can also have a water-fixing capacity of at least 90%. Whey protein powder can also have a water-fixing capacity of at least 100%.
[00012] In one embodiment, the whey protein powder has a glycerol fixing capacity of at least 50%.
[00013] In one embodiment, the whey protein powder has an ethanol fixing capacity of at least 50%.
[00014] In one embodiment, the whey protein powder has an oil-fixing capacity of at least 30%.
[00015] In one embodiment, the whey protein powder is mixed with the leucine.
[00016] In one embodiment, the whey protein powder includes whey protein micelles and leucine in a weight ratio of about 30:1 to about 1:100.
[00017] In one embodiment, whey protein powder is obtained by a spray drying or freeze drying process that is performed with leucine.
[00018] In one embodiment, the whey protein powder has an angle of repose less than 35°.
[00019] In one embodiment, the whey protein powder is prepared to act as a flow agent.
[00020] In one embodiment, the whey protein micelles are less than 1 micron in size.
[00021] In one embodiment, the whey protein micelles are coated with a coating. The coating can be selected from the group consisting of an emulsifier, a protein, a peptide, a protein hydrolyzate, a gum, or combinations thereof. The protein can be selected from the group consisting of protamine, lactoferrin, rice proteins, or combinations thereof. The protein hydrolyzate can be a hydrolyzate selected from the group consisting of protamine, lactoferrin, rice, casein, whey, wheat, soy protein, or combinations thereof. The emulsifier may be selected from the group consisting of sulfated butyl oleate, diacetyl tartaric acid esters of monoglycerides and diglycerides, citric acid esters of monoglycerides, stearoyl lactylates, or combinations thereof.
[00022] In one modality, the composition is a source of complete nutrition. Alternatively, the composition is a source of incomplete nutrition. The composition can also be a tube feed composition, or it can be used for short term administration or for long term administration.
[00023] In one embodiment, the composition may include an antioxidant selected from the group consisting of beta-carotene, vitamin C, vitamin E, selenium, or combinations thereof.
[00024] In one modality, the composition may include a vitamin selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, and vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, folic acid, biotin, or combinations thereof.
[00025] In one embodiment, the composition may include a mineral selected from the group consisting of boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium , zinc, or combinations thereof.
[00026] In yet another modality, a nutritional composition is offered that includes whey protein micelles, leucine, and a liquid, where the total amount of leucine in the composition is less than about 2.5 g per 100 g of the liquid.
[00027] In one embodiment, leucine is present in an amount of about 1 g to about 2 g.
[00028] In yet another embodiment, a nutritional composition is offered that includes whey protein micelles, and leucine, where the total amount of leucine in the composition is less than about 25 g per 1 liter of a liquid.
[00029] In one modality, the liquid is selected from the group consisting of water, water-based beverages, fruit juices, milk, or combinations thereof.
[00030] In one embodiment, leucine is present in an amount of about 24 g.
[00031] In one embodiment, the composition is a tube-fed composition.
[00032] In another embodiment, a nutritional composition is offered that includes whey protein powder comprising whey protein micelles and the added leucine, where the total dry weight of the added leucine ranges from about 30% to about 40% of the total dry weight of the whey protein micelles.
[00033] In one embodiment, the total dry weight of added leucine is about 37% of the total dry weight of the whey protein micelles.
[00034] In one embodiment, the composition is a powdered composition.
[00035] In one embodiment, the whey protein micelles are spherical agglomerates of denatured whey protein. The whey proteins can be arranged in such a way that the hydrophilic parts of the proteins are oriented towards the outside of the cluster and the hydrophobic parts of the protein are oriented towards an inner core of said micelle.
[00036] In yet another embodiment, a process for the production of a whey protein micelle concentrate is offered. The process includes the steps of (a) adjusting the pH of an aqueous solution of whey protein to a value between 3.0 and 8.0, (b) subjecting the aqueous solution to a temperature between 70 and 95°C , (c) concentrating the dispersion obtained in step (b), (d) adding leucine to the dispersion, and (e) spray drying or freeze drying the whey protein micelle concentrate with leucine. Step (a) adjustment is absolutely very accurate so the pH is adjusted to an accuracy of ±0.05 pH units.
[00037] In one embodiment, the mineral content of the whey protein solution is less than 2.5%. Whey protein can also be demineralized.
[00038] In one embodiment, the pH of the whey protein solution is adjusted to a value between 5.8 and 6.6. The pH can also be adjusted to a value between 3.8 and 4.5.
[00039] In one embodiment, the concentration of the whey protein aqueous solution is less than 12%. The concentration can also be less than 4%.
[00040] In one mode, heating is performed for a period of 10 seconds to 2 hours. The aqueous solution can also be heated for a period of 15 minutes. Heating can also be done by microwave.
[00041] In one embodiment, the micelle yield before concentration is at least 35%. The micelle yield before concentration can also be at least 50%. The yield of micelles before concentration can also be at least 80%.
[00042] In one embodiment, micelles have an average size less than 1 micron. Micelles can have an average size of 100-900 nm. The proportion of micelles with an average size between 100 nm and 700 nm can be greater than 80%.
[00043] In one embodiment, concentration is performed by a method selected from the group consisting of evaporation, centrifugation, sedimentation, ultrafiltration, microfiltration, or combinations thereof. Centrifugation can be carried out after acidification to a pH of 4.5. Spontaneous sedimentation can be carried out at a pH of 4.5. Settling time can be longer than 12 hours.
[00044] In yet another modality, a method for preparing a nutritional product is offered. The method includes the steps of (a) adjusting the pH of an aqueous solution of whey protein to a value between 3.0 and 8.0, (b) subjecting the aqueous solution to a temperature between 70 and 95°C , (c) concentrate the dispersion obtained in step (b), (d) add leucine to the dispersion, (e) spray dry or freeze dry the whey protein micelle concentrate with leucine, and (f) add the concentrate of dry whey protein micelles with leucine to a composition for preparing the product.
[00045] In another embodiment, a method for producing a consumable product is offered which includes mixing whey protein micelles, a concentrate thereof or a powder thereof with added leucine to create a mixture and process the mixture to form a consumable product. The total amount of leucine in the consumable product ranges between 20% and 40% by weight of dry matter. Processing may include subjecting mixing to heat, pressure, acidic or basic conditions, shearing, cooling, or combinations thereof.
[00046] In yet another embodiment, a method for producing a consumable product is offered which includes cosecting a solution or concentrate of whey protein micelles with added leucine to form a powder having a dry weight ratio of leucine added to whey protein micelles from about 1:2 to about 1:3 and add the powder to the product. Co-drying is selected from the group consisting of spray drying, freeze drying, or combinations thereof.
[00047] In another embodiment, a method is offered to mask the unpleasant tastes of a nutrient in a composition. The method includes the steps of mixing a powder of whey protein micelles and up to 2.5 g of added leucine to form a mixture and adding the mixture to 100 g of a liquid carrier to form a composition.
[00048] An advantage of the present invention is to offer improved nutritional compositions.
[00049] Another advantage of the present invention is to offer nutritional compositions having increased amounts of nutrients.
[00050] Yet another advantage of the present invention is to offer nutritional compositions that provide acceptable organoleptic properties.
[00051] Yet another advantage of the present invention is to offer nutritional compositions that provide acceptable characteristics.
[00052] Another advantage of the present invention is to offer nutritional compositions with low viscosities.
[00053] An advantage of the present invention is to offer nutritional compositions that stimulate protein synthesis in humans.
[00054] Yet another advantage of the present invention is to offer nutritional compositions that promote muscle development.
[00055] Yet another advantage of the present invention is to offer nutritional compositions that mask the off-tastes of the nutrients in the nutritional composition.
[00056] Another advantage of the present invention is to offer methods for making compositions including increased amounts of nutrients.
[00057] Yet another advantage of the present invention is to offer methods of administering a nutritional composition.
[00058] Additional features and advantages are described in this report, and will become evident from the detailed description that follows. BRIEF DESCRIPTION OF THE FIGURES
[00059] Figure 1 shows a highly schematic structure of a whey protein micelle according to an embodiment of the present invention.
[00060] Figure 2 shows a solubility curve of whey protein micelles at different pH according to an embodiment of the present invention. DETAILED DESCRIPTION
[00061] As used in this report, “about” is understood to refer to numbers within a range of numerals. In addition, it is understood that all numeric ranges in this report include all whole numbers, integers or fractions within that range.
[00062] As used in this report, "effective amount" is preferably an amount that prevents a deficiency, treats a disease or medical condition in an individual, or more generally reduces symptoms, controls disease progression or provides a nutritional benefit, physiological, or medical to the individual. A treatment can be related to the patient or the doctor.
[00063] As used in this report, the terms "treatment", "treat" and "alleviating" preferably refer to both prophylactic and preventive treatment (which prevents and/or reduces the development of a targeted pathological condition or disorder) and to curative, therapeutic, or disease-modifying treatment, including therapeutic measures that cure, reduce, alleviate symptoms, and/or halt the course of a diagnosed pathological condition or disorder; and the treatment of patients who are at risk of contracting a disease or suspected of having a disease, as well as patients who are already ill or who have been diagnosed as having a disease or medical condition. The term does not necessarily mean that the individual is treated until full recovery. The terms "treatment" and "treating" also refer to the maintenance and/or promotion of health in an individual who does not suffer from any disease but who may be susceptible to the development of an unhealthy condition, such as nitrogen imbalance or muscle loss. . The terms “treatment”, “treat” and “relieve” are also intended to include the enhancement or any other type of intensification of one or more of the primary prophylactic or therapeutic measures. The terms "treatment", "treating" and "alleviating" are further intended to include dietary management of a disease or condition or dietary management for the prophylaxis or prevention of a disease or condition.
[00064] As used in this report it is understood that the term "patient" preferably includes an animal, especially a mammal, and more especially a human being who is receiving, or may be benefiting from, or is indicated to receive treatment, as it is here defined.
[00065] As used in this report, "animals" includes, but is not limited to, mammals that include, but are not limited to, rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses , and humans. Where the terms animal or mammal or their plurals are used, we contemplate that they also apply to all animals that are capable of achieving the displayed effect or that are expected to be displayed in the context.
[00066] As used in this report, “mammal” includes, but is not limited to, rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Where the term mammal is used, we contemplate that it also applies to all other animals that are capable of achieving the effect exhibited or expected to be exhibited by the mammal.
[00067] It is understood that the term "protein", "peptide", "oligopeptides" or "polypeptide" as used in this report preferably refers to any composition that includes two or more amino acids joined by a peptide bond (dipeptide, tripeptide, or polypeptide), collagen, precursor, homologue, analog, mimetic, salt, prodrug, metabolite, or fragment thereof or combination. For the sake of clarity, the use of any of the above terms is interchangeable, unless otherwise specified. It will be appreciated that polypeptides (or peptides or proteins or oligopeptides) generally contain amino acids other than the 20 amino acids commonly indicated as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, can be modified in a given polypeptide, either by natural processes such as glycosylation and other post-translational modifications, or by chemical modification techniques that are well known in the literature. Among the known modifications that may be present in the polypeptides of the present invention are, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavanoid or a heme moiety, covalent attachment of a polynucleotide or a polynucleotide derivative, covalently binding a polyphenol, covalently binding a lipid or a lipid derivative, covalently binding phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation , gamma-carboxylation, glycation, glycosylation, glycosylphosphatidyl inositol (GPI) membrane anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, RNA-mediated addition of amino acids of transfer to polypeptides such as arginylation, and ubiquitination. The term "protein" also includes "artificial proteins", and refers to linear or non-linear polypeptides consisting of alternating repeats of a peptide.
[00068] It is understood that the nutritional products and compositions preferably further include any number of optional additional ingredients, including conventional food additives, for example one or more, acidulants, additional thickeners, buffers or pH adjusting agents, chelating agents, colorants, emulsifiers, excipients, flavoring agents, minerals, osmotic agents, a pharmaceutically acceptable carrier, preservatives, stabilizers, sugar, sweeteners, texturizers, and/or vitamins. Optional ingredients can be added in any suitable amount.
[00069] For example, the compositions and products of the present invention may also include, for example, antioxidants, vitamins and minerals. As used in this report, it is understood that the term "antioxidant" preferably includes one or more of several substances (such as beta-carotene (a precursor to vitamin A), vitamin C, vitamin E, and selenium) that inhibit the oxidation or reactions promoted by the species. of reactive oxygen (ROS) and other species of radicals and non-radicals. Additionally, antioxidants are molecules capable of reducing or preventing the oxidation of other molecules.
[00070] As used in this report it is understood that the term "vitamin" preferably includes any of the various fat-soluble or water-soluble organic substances essential in minimal amounts for normal body growth and activity and obtained naturally from food sources vegetable and animal or synthetically made, pro-vitamins, derivatives, analogues. Non-limiting examples of vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride ), vitamin B7 (biotin), vitamin B9 (folic acid), and vitamin B12 (various cobalamins; commonly cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, folic acid, and biotin.
[00071] As used in this report, it is understood that the term "minerals" preferably includes boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, and combinations thereof.
[00072] Whey protein micelles are spherical microgels (regular shape close to that of natural casein micelles) monodispersed obtained by self-assembly of native whey proteins during heat treatment at a very specific pH. Whey protein micelles have unique characteristics and properties which include, for example, a narrow size distribution with a diameter between 100 and 900 nm and a polydispersity index below 0.2, a turbidity index measured at 500 nm (between 20 and 50 absorbance units for a 4% protein solution) that is stable for 10 minutes, and a spherical shape in the image seen under a TEM microscope.
[00073] The final architecture of the whey protein micelle aggregates confers properties such as emulsification, micellar casein replacement, bleaching, foaming, texturing and/or loading agents. Whey protein micelles are microgels with a 30% concentration of whey protein with unique physical characteristics (size, filler, density, size distribution) that impart exceptional properties including, for example, stability to addition. of salt, low viscosity at high concentration, gelling between pH 4 and 5 and with high stability to heat treatment used for pasteurization or sterilization.
[00074] Whey protein micelles are obtained by heat treatment of solutions of native whey protein adjusted to a very specific and precise pH at which the net charge (negative or positive), induced this specific aggregation by self-assembly. These aggregates are in a particular organized state that results from a balance between the electrostatic forces of repulsion and attraction associated with hydrophobic interactions and an asymmetric distribution of charges present on the surface of proteins. This phenomenon occurred 0.7 pH unit below and above the isoelectric point (i.e., pH 4.3 and 5.8 for IEP of 5.1) for pure beta-lactoglobulin.
[00075] Micellization does not occur at room temperature because the hydrophobicity of whey proteins is hidden in the structure of native proteins. To induce micellization (formation of a spherical microgel of monodispersed proteins by self-assembly), a conformational modification of the proteins is required. This modification is induced by heat treatment; during the first stage of micelle formation. This self-assembly phenomenon is reversible by acidification to pH 2.0 shortly after the ideal temperature has been reached (i.e., 85°C). This very acidic pH prevents thiol/disulfide exchanges and the unstabilized micellar structure is quickly dismantled. Under normal conditions, without post-acidification at pH 2.0, due to the activation of the thiol by heat treatment, a rapid crosslinking stabilizes the micelle during incubation at a constant temperature (15 minutes at 85°C), and this time of incubation could be extended to up to 45 minutes or 120 minutes. After this incubation, micellization is not spontaneously reversible. A dissociating agent and a reducing agent are needed to recover the individual proteins.
[00076] Whey protein is one of the most abundant natural sources of branched chain amino acids (leucine, isoleucine and valine). As the nutritional profile of whey protein is among the best sources for such amino acids, it is very desirable for use in nutritional compositions. More specifically, whey protein micelles, which are the product of technologies described in patent applications from Nestec SA, allow whey protein to be concentrated beyond the level to which it is typically feasible using methods. traditional processing, and still continue in liquid form. Nestec S.A. pending patent applications describing such whey protein micelle technologies include International Application PCT/EP2007/052877, filed March 26, 2007; International Application PCT/EP2007/052900, filed March 27, 2007; and US Application Serial No. 12/280,244, filed August 21, 2008, the contents of which are incorporated herein by reference in their entirety. A benefit offered by micelles produced by the technologies described in the processes mentioned above is that whey protein can be included in high concentrations, but retain a fluid matrix of low viscosity.
[00077] Additionally, the amino acid profile of the original whey protein is also maintained during the manufacturing processes described in the applications identified above, which offers the same nutritional value as whey. Branched-chain amino acids are those amino acids that have aliphatic side chains that are non-linear. The combination of these three essential amino acids constitutes approximately 1/3 of the skeletal muscle in the human body, and plays an important role in protein synthesis. Branched-chain amino acids can also be used to aid burn victims in recovery, as well as supplementation to increase endurance in athletes.
[00078] As leucine, isoleucine and valine are essential amino acids, these amino acids cannot be synthesized by the body and therefore must be ingested. As a dietary supplement, leucine has been found to reduce muscle tissue breakdown by increasing muscle protein synthesis in elderly rats. Whey protein is among the richest natural sources of leucine (12-15% by weight of total amino acids), including about 1 g of leucine per 10 g of whey protein micelles in whey protein. milk. However, the amount of leucine needed to significantly improve protein synthesis in humans has been reported to be approximately 3 g or more distributed in a portion of a bolus. As a result, it is necessary to provide more than 30 g of whey protein to obtain 3 g of leucine. However, the taste of leucine is typically unpleasant when included in doses that are effective in stimulating protein synthesis in humans. In fact, leucine's sensory properties include a bitter taste in the mouth that is unpleasant for consumers.
[00079] Therefore, oral nutritional products are limited in terms of their ability to deliver effective amounts of branched amino acids because of their flavor profile. Also, whey protein tends to gel when heated under neutral pH conditions. Therefore, applications of branched chain amino acids in beverages are extremely limited. Furthermore, the delivery of branched-chain amino acids via pills and pills is also not convenient as a result of the dose to be administered (3+ g at a time).
[00080] Applicants have surprisingly found that it is possible to combine whey protein micelles with the free amino acid leucine to create compositions (eg a beverage) for the purpose of aiding muscle development. Specifically, the compositions include whey protein micelles and a significant amount of leucine, but have bitter or unpleasant tastes that are typically associated with doses of leucine that are effective in stimulating protein synthesis in humans. Applicants have surprisingly found, therefore, that whey protein micelles can be used as a disguise to mask the bitterness of off-tasting amino acids in beverages and other oral nutritional products. Although the present invention relates to the use of whey protein micelles and leucine, one skilled in the art will readily appreciate that other branched chain amino acids such as isoleucine and valine can also be employed in similar uses.
[00081] In fact, applicants have found that the combination of whey protein micelles and leucine can be incorporated into nutritional compositions (eg beverages) at concentrations of whey protein and supplemental leucine that provide a consumer benefit without the sensory limitations noted above. For example, prior art beverages are limited either by the inclusion of whey protein, which imparts unacceptable viscosity, or leucine, which imparts unacceptable organoleptic properties. At least these two limitations are solved by combining the micellar protein with the controversial nutrient. Without wanting to stick to any theory, we believe that the structure of protein micelles and their interaction with leucine (or other unpleasant nutrients) prevent the consumer's unpleasant perception of bitterness.
Thus, Applicants have surprisingly found that whey protein micelles can act as a masking substance to prevent the unpleasant perception of bitterness from a specific nutrient by masking a bitter taste receptor present on the surface of the tongue. As proposed by Noriao Ishibashi's model, bitterness is an unpleasant taste sensory perception that often induces food rejection. Bitter sensitivity ranges from 1 to 500 depending on each specific person. See Ishibashi, N. et al., A Mechanism for Bitter Taste Sensibility in Peptides, Agr. Biol. Chem., 52, 819-827 (1988).
[00083] In addition to whey protein micelles, the person skilled in the art will realize that the use of micellar casein proteins, as well as any potential plant proteins, can also be used as a protein component that masks bitterness or flavor unpleasantness transmitted to a nutritional composition by leucine or other similar nutrients.
[00084] In one embodiment, whey protein micelles and leucine can be part of a complete nutritional product. As used in this report, "complete nutritional" products are preferably nutritional products that contain sufficient types and levels of macronutrients (proteins, fats and carbohydrates) and sufficient micronutrients to be the sole source of nutrition for the animal to which they are being administered . Patients can receive 100% of their nutritional needs through such complete nutritional compositions.
[00085] Whey protein micelles and leucine can alternatively form part of an incomplete nutritional product. As used in this report, “incomplete nutritional” products are preferably nutritional products that do not contain sufficient levels of macronutrients (protein, fat and carbohydrates) or micronutrients that are sufficient to be the sole source of nutrition for the animal to which they are being administered. Partial or incomplete nutritional compositions can be used as a nutritional supplement.
[00086] Similarly, whey protein micelles and leucine can be included in tube feeding compositions. As used in this report, a "tube feeding" is preferably a complete or incomplete nutritional product that is administered to an animal's gastrointestinal system, as opposed to oral administration, including but not limited to a nasogastric tube, an orogastric tube, a gastric tube, a jejunostomy feeding tube (J-tube), percutaneous endoscopic gastronomy (PEG), an orifice such as an orifice in the chest wall that provides access to the stomach, jejunum, and other suitable access orifices.
[00087] In one embodiment, whey protein micelles and leucine can be used in compositions for short-term administration. As used in this report, "short term administrations" are preferably continuous administrations for less than 6 weeks. Alternatively, whey protein micelles and leucine can be used in compositions for long-term administration. As used in this report, "long-term administrations" are preferably continuous administrations for more than 6 weeks.
[00088] Figure 1 illustrates a schematic representation of the micelles used in the present invention, where the whey proteins are arranged in such a way that the hydrophilic parts of the proteins are oriented towards the outside of the cluster and the hydrophobic parts of the protein are oriented towards the inner “core” of said micelle. The designation “whey protein micelle” is indicative of homology to casein micelles based on the following criteria: shape, size, and whitening properties, but also the whey protein micelle is a spherical protein microgel of whey from denatured whey protein. Both physical and chemical interactions are involved in whey protein microgels or whey protein micelle. In Figure 1, S* indicates accessible thiol/activated thiol from cysteine, and S-S indicates disulfide bonds stabilizing the whey protein micelle. This energy-friendly configuration offers good stability to these structures in a hydrophilic environment. Therefore, micelles consist essentially of spherical agglomerates of denatured whey protein. Micelles are particularly characterized by their spherical and regular shape.
[00089] Due to its dual character (hydrophilic and hydrophobic), this denatured state of the protein seems to allow an interaction with a hydrophobic phase, for example a drop of fat or air, and a hydrophilic phase. Whey protein micelles therefore have perfect emulsifying and foaming properties.
[00090] The micelles of the present invention can have an extremely well-defined size distribution so that more than 80% of the micelles produced will have a size less than 1 micron, preferably between 100 nm and 900 nm, more preferably between 100770 nm, even more preferably between 200 and 400 nm. Without wanting to stick to the theory, we believe that during micelle formation, micelles reach a “maximum” size, due to the total electrostatic charge of the micelles repelling any additional protein molecules, so the micelles can no longer develop. This is the reason for the narrow size distribution observed here.
[00091] As discussed above, the whey protein micelles of the present invention can be produced by the methods described in International Application PCT/EP2007/052877, filed March 26, 2007; in International Application PCT/EP2007/052900, filed March 27, 2007; and US Application Serial No. 12/280,244, filed August 21, 2008, the contents of which are incorporated herein by reference in their entirety. An advantage of using the methods described in such applications is that the whey protein micelles prepared according to them have not been subjected to any mechanical stress that would lead to particle size reduction during their formation, unlike conventional processes. known in the literature. Instead, the methods induce spontaneous micellization of whey proteins during heat treatment in the absence of shear. The person skilled in the art will understand, however, that micelles can be produced by methods other than those described in the applications mentioned above.
[00092] Any commercially available whey protein isolate or concentrate can be used in accordance with the present invention. For example, whey protein obtained by any process for the preparation of whey protein known in the literature, as well as whey protein fractions prepared therefrom or proteins such as β-lactoglobulin, α-lactalbumin and serum albumin. In particular, sweet whey obtained as a by-product in cheese making, acid whey obtained as a by-product in the manufacture of acid casein, native whey obtained by microfiltration of milk or rennet whey obtained as a by-product in rennet casein manufacturing can be used as whey protein. Whey protein can come from a single source or from mixtures of any sources. It is preferable that the whey protein does not undergo any hydrolysis step prior to micelle formation. Therefore, whey protein is not subjected to any treatment prior to micellization. In accordance with the present invention, however, it is important that whey protein, and not hydrolysates thereof, is used in the micelle formation process.
[00093] The present invention is not restricted to whey isolates of bovine origin, but refers to whey isolates from all species of mammalian animals, such as sheep, goats, mares and camels. Also, the process according to the present invention applies to mineralized, demineralized or slightly mineralized whey preparations. By "slightly mineralized" is meant any whey preparation after the removal of free minerals that are dialyzable or diafilterable, but which retain minerals associated with it by natural mineralization after the preparation of the whey protein concentrate or isolate, for example. These “slightly mineralized” whey preparations did not receive any enrichment with any specific minerals.
[00094] Whey proteins are an excellent source of essential amino acids (eg about 45% by weight). Compared to casein (containing 0.3 g cysteine/100 g protein), for example, sweet whey proteins contain 7 times more cysteine, and sour whey contains 10 times more cysteine. Cysteine is the rate-limiting amino acid for the synthesis of glutathione, a tripeptide made from cysteine glutamate and glycine that has important primary functions in defending the body in the event of stress. The need for these amino acids can be increased under stress and in elderly people. Also, oral supplementation of glutathione with whey protein has been shown to increase plasma glutathione levels in HIV-infected patients. See Eur. J. Clin. Invest. 31, 171-178 (2001).
[00095] Other health benefits offered by whey proteins include improved muscle development and build, as well as muscle maintenance in children, adults, or the elderly, improved immune function, increased cognitive function, improved blood glucose control. so that they are suitable for diabetics, weight control and food satiety, anti-inflammatory effects, wound healing and skin repair, lowering blood pressure, etc.
[00096] Additionally, whey proteins have a better protein efficiency ratio (PER = 118) compared for example with casein (PER = 100). PER is a measure of protein quality estimated by determining how much that protein supports weight gain. It can be calculated by the following formula: PER = increase in body weight (g) / protein weight intake (g)

[00097] To produce whey protein micelles according to methods disclosed in the patent applications of Nestec SA mentioned above, whey proteins can be present in an aqueous solution in an amount of 0.1% by weight to 12.0% by weight, preferably in an amount of 0.1% by weight to 8% by weight, more preferably in an amount of 0.2% by weight to 7.0% by weight, even more preferably in an amount of 0.5% by weight to 6.0% by weight, more preferably in an amount of 1.0% by weight to 4.0% by weight based on the total weight of the solution.
[00098] The aqueous solution of the whey protein preparation as presented before the micellization step can also comprise additional compounds, such as by-products of the respective whey production processes, other proteins, gums or carbohydrates. The solution may also contain other food ingredients (fat, carbohydrates, plant extracts, etc.). The amount of such additional compounds preferably should not exceed 50% by weight, preferably 20% by weight, and more preferably not exceed 10% by weight of the total weight of the solution.
[00099] Whey protein can be used in purified form or conversely in the form of a crude product. According to an embodiment, the content of divalent cations in the whey protein for the preparation of the whey protein micelle concentrate may be less than 2.5%, more preferably less than 2%, even more preferably less at 0.2%. In one embodiment, whey proteins are completely demineralized.
[000100] According to the present invention, pH and ionic strength are important. Therefore, for heavily dialyzed samples that are virtually devoid of or depleted of free cations such as K, Na, Mg, it has been found that when the heat treatment is carried out for a period of time 10 seconds to 2 hours at a pH below 5, 4, a rennet is obtained, whereas at a pH greater than 6.8, a soluble whey protein results. Therefore, only in this very narrow pH range whey protein micelles having a diameter smaller than 1 μm will be obtained. These micelles will have an overall negative charge. The same form of micelle can also be obtained symmetrically below isoelectric pH, i.e. from 3.5 to 5.0, more preferably 3.8 to 4.5, resulting in positively charged micelles.
[000101] Therefore, according to a modality, to obtain positively charged micelles, the micelles of whey proteins must be micellized in a salt-free solution at a pH value adjusted between 3.8 and 4.5 depending on the mineral content of the protein source.
[000102] In one modality, the micelles obtained will have a total negative charge. Therefore, in one embodiment, the pH is adjusted in a range of 6.3 to 9.0, to a content of divalent cations of between 0.2% and 2.5% in the whey protein powder.
[000103] More specifically, to obtain negatively charged micelles, the pH is adjusted in a range from 5.6 to 6.4, more preferably from 5.8 to 6.0 for a content of divalent cations (for example, less than 0.2% of the initial whey protein powder). The pH can be increased up to 8.4 depending on the mineral content of the whey protein source (concentrate or isolate). In particular, the pH can vary between 7.5 and 8.4, preferably 7.6 and 8.0 to obtain negatively charged micelles in the presence of large amounts of free minerals and the pH can vary between 6.4 and 7, 4, preferably 6.6 and 7.2 to obtain negatively charged micelles in the presence of moderate amounts of free minerals. As a general rule, the higher the calcium and/or magnesium content of the starting whey protein powder, the higher the micellization pH.
[000104] To standardize the formation conditions of whey protein micelles, it is more preferable to demineralize by any of the known demineralization techniques (dialysis, ultrafiltration, reverse osmosis, ion exchange chromatography etc.), any source of protein of liquid native whey with a protein concentration ranging from that of sweet whey, milk microfiltration permeate or acid whey (protein content 0.9%) to that of a concentrate at a protein content 30%. Dialysis can be done against water (distilled, deionized or soft), or as this will only allow the removal of the weakly bound ions in whey proteins, it is more preferable to do dialysis against an acid at a pH below 4, 0 (organic or inorganic) to better control the ionic composition of whey proteins. Thereby, the pH of whey protein micelle formation will be below pH 7.0, more preferably comprised between 5.8 and 6.6.
[000105] Before heating the aqueous solution of whey protein, the pH is usually adjusted by adding an acid, which is preferably food grade, such as, for example, hydrochloric acid, phosphoric acid, acetic acid, citric acid, glycolic acid or lactic acid. When the mineral content is high, the pH is usually adjusted by adding an alkaline solution, which is preferably food grade, such as sodium hydroxide, potassium hydroxide or ammonium hydroxide.
[000106] Alternatively, if a pH adjustment step is not desired, it is possible to adjust the ionic strength of the whey protein preparation while the pH is kept constant. Then, the ionic strength can be adjusted by organic or inorganic ions in such a way as to allow micellization at a constant pH value equal to 7. In one embodiment, micelles can be formed at a constant pH value equal to 7 in step that the ionic strength is varied by the addition of 70-80 mM arginine HCl.
[000107] A buffer can be further added to the whey protein aqueous solution to avoid a substantial change in the pH value during the heat treatment of the whey protein. In principle, the buffer can be selected from any food-grade buffer system, ie, acetic acid and its salts, such as, for example, sodium acetate or potassium acetate, phosphoric acid and its salts, for example, NaH2PO4, Na2HPO4, KH2PO4, K2HPO4, or citric acid and its salts, etc.
[000108] Adjusting the pH and/or ionic strength of the aqueous solution, according to the present invention, results in a controlled process producing micelles having a size between 100 nm-900 nm, preferably between 100 nm-700 nm, more preferably still between 200 nm-400 nm. Preferably, the proportion of micelles with an average size comprised between 100-700 nm is greater than 80% when carrying out the process of the invention.
[000109] To obtain regular sized micelles, it is also important that the whey protein does not undergo any hydrolysis step before the formation of micelles.
[000110] In a second step of the process, the initial whey protein aqueous solution is then subjected to heat treatment. In that case, it has been found that to obtain whey protein micelles, it is important that the temperature is in the range from about 70°C to less than 95°C, preferably from 80°C to about 90°C, more preferably from about 82°C to about 89°C, even more preferably from about 84°C to about 87°C, most preferably about 85°C. It has also been found that, on an industrial scale, it is important that the temperature is preferably below 95°C, more preferably between 80°C and 90°C, most preferably about 85°C.
[000111] Once the desired temperature is reached, it is held at that temperature for a minimum of 10 seconds and a maximum of 2 hours. Preferably, the period of time during which the aqueous whey protein solution is kept at the desired temperature ranges from 12 to 25 minutes, more preferably from 12 to 20 minutes, or more preferably it is about 15 minutes .
[000112] The heat treatment can also be done in a microwave oven or any similar equipment that allows microwave heating with a time/quantity ratio of 10 s/10 mL for a heated 4% by weight protein solution in a 1500 W device to its boiling point (98°C at an altitude of 833 m). A continuous process can also be used by adding 8 or more magnetrons around a glass tube potentially elongated by a containment tube to increase incubation time.
[000113] Turbidity measurements are an indication of micelle formation. According to the present invention, turbidity measured by absorbance at 500 nm is at least 3 absorbance units for a 1% protein solution but can reach 16 absorbance units when the micellization yield is above 80%.
[000114] To further illustrate the effect of micelle formation from a physicochemical point of view, a 1% by weight BIPRO® dispersion was heated for 15 minutes at 85°C at a pH of 6.0 and 6, 8 in MiIIIQ water. The hydrodynamic diameter of the aggregates obtained after heat treatment was measured by dynamic light scattering. The apparent molecular weight of the aggregates was determined by static light scattering using the so-called Debye plot. Surface hydrophobicity was examined using the hydrophobic ANS probe and free accessible thiol groups by the DTNB method using cysteine as the standard amino acid. Finally, aggregate morphology was studied by negative TEM staining. The results are shown in table 1. TABLE 1 Physicochemical properties of whey protein aggregates obtained by heat treatment (85°C, 15 min) of a 1% by weight protein dispersion at pH 6.0 or 6.8.

[000115] From table 1, it is clear that whey protein micelles that were formed at a pH of 6.0 allow the protein to decrease its specific ANS surface hydrophobicity by a factor of 2 compared to the whey protein. un-mycelized whey heated under the same conditions but at a pH of 6.8. Micelle formation can also be seen in the very high molecular weight of 27 x 106 g.mol-1 compared to 0.64 X 106 g.mol-1 for the non-micelized protein, indicating a very condensing state of matter inside. of the micelle (low amount of water). Interestingly, the Ç potential of micelles is even more negative than that of non-micelized proteins even though the latter were formed at a more basic pH than micelles. This is the result of a more hydrophilic surface of the micelles being exposed to the solvent. Finally, it should be noted that the thiol reactivity of micelles is much lower than that of non-micelized protein because of the different pH of the heat treatment.
[000116] It has been found that the yield of conversion of native whey protein into micelles decreases when the initial protein concentration is increased before pH adjustment and heat treatment. For example, when starting from a PROLACTA® 90 whey protein isolate (Lactalis lot 673), the whey protein micelle formation yield drops by 85% (when starting from 4% protein ) to 50% (when starting with 12% protein). To maximize the formation of whey protein micelles (>85% of the initial protein content), it is best to start with an aqueous solution of whey protein having a protein concentration of less than 12%, preferably less than 6%. Depending on the intended end applications, the protein concentration is adjusted prior to heat treatment to control the optimal yield of whey protein micelles. Whey protein micelles obtained according to the methods of the patent applications of Nestec SA mentioned above should have an average size of less than 1 µm, preferably from 100 nm to 900 nm, more preferably from 100 nm to 700 nm , most preferably from 200 nm to 400 nm.
[000117] Depending on the desired application, the micelle yield before concentration is at least 35%, preferably at least 50%, more preferably at least 80% and the content of soluble aggregates or residual soluble proteins is preferably less than 20%. The average size of micelles is characterized by a polydispersity index of less than 0.200. It was observed that whey protein micelles could form aggregates around pH 4.5, but with no signs of macroscopic phase separation after at least 12 hours at 4°C.
[000118] The purity of the whey protein micelles produced according to the methods of the patent applications of Nestec S.A. mentioned above can be obtained by determining the amount of residual soluble proteins. Micelles are eliminated by centrifugation at 20°C and 26900 g for 15 minutes. The supernatant is used to determine the amount of protein in the quartz cuvettes at 280 nm (length of light path 1 cm). Values are expressed as a percentage of the initial value before heat treatment.
[000119] Proportion of micelles = (Amount of starting proteins - amount of soluble proteins) / Amount of starting proteins
[000120] An advantage of the methods described in this invention is that the whey protein micelles prepared according to them were not subjected to any mechanical stress that would lead to particle size reduction during its formation, unlike conventional processes . This method induces spontaneous micellization of whey proteins during heat treatment in the absence of shear.
[000121] Whey protein micelles can be used as such in any composition, such as nutritional compositions, cosmetic compositions, pharmaceutical compositions etc. According to the present invention, whey protein micelles are used in consumable products. In addition, whey protein micelles can be mixed with an active component. Said component can be selected from coffee, caffeine, green tea extracts, plant extracts, vitamins, minerals, bioactive agents, salt, sugar, sweeteners, aroma, fatty acids, oils, protein hydrolysates, peptides, amino acids, etc., or combinations thereof.
[000122] In addition, the whey protein micelles (pure or mixed with active components) of the present invention can be coated with an emulsifier such as phospholipids, for example, or with other coating agents such as a protein, a peptide, a protein hydrolyzate or a gum such as acacia gum to modulate the functionality and taste of whey protein micelles. When a protein is used as a coating agent, it can be selected from any proteins having an isoelectric point significantly higher or lower than that of whey protein. These are, for example, protamine, lactoferrin and some rice proteins. When a protein hydrolyzate is used as a coating agent, it is preferably a protein hydrolyzate such as protamine, lactoferrin, rice, casein, whey, wheat, soy protein, or combinations thereof. In one embodiment, the coating is an emulsifier selected from sulfated butyl oleate, diacetyl tartaric acid esters of monoglycerides and diglycerides, citric acid esters of monoglycerides, stearoyl lactylates, or combinations thereof. In one embodiment, the coating is sulfated butyl oleate. Coating can be done by any of the methods known in the literature. In addition, spray drying, as already described in this report, can also result in a coating of the whey protein micelles.
[000123] Whey protein micelles have been shown to be perfectly suitable for use as emulsifier, fat replacer, micellar casein replacer or foaming agent, as they are able to stabilize fat and/or air in a system watery for a prolonged period. In fact, whey protein micelles can be used as an emulsifying agent, for which the material is perfectly suited, as it has a neutral taste and no unpleasant taste is created by using such material. They can also be used as a micellar casein substitute.
[000124] Furthermore, the present whey protein micelles can function as a bleaching agent, so that with a compound several functions are fulfilled. As whey is an abundantly available material, its use reduces the cost of a product that requires an emulsifying agent, a filler, a bleaching agent or a foaming agent, while increasing its nutritional value.
[000125] Therefore, whey protein micelles obtainable by the methods described in this invention can be used for the preparation of any type of consumable product that requires stabilization of an emulsion or a foam, such as for example, present in mousses or ice creams, in coffee-flavored cream substitutes, or also low-fat or essentially fat-free dairy products, or where it has application as a micellar casein substitute.
[000126] By “consumable” is meant any food product in any form, including beverages, soups, semi-solid foods, etc., which can be consumed by a human or an animal. Examples of products where the present whey protein micelles can be applied are, for example, dairy products, mayonnaise, salad dressings, pasteurized UHT milk, sweet condensed milk, yogurt, fermented milks, sauces, sauces with fat content reduced such as bechamel sauce for example, fermented milk-based products, milk chocolate, white chocolate, dark chocolate, mousses, foams, emulsions, ice cream, fermented cereal-based products, milk-based powders, infant formula, dietary booster, animal feed, tablets, liquid bacterial suspensions, dry oral supplement, wet oral supplement, energy nutrition bars, pastes, fruit drinks, coffee drinks, etc.
[000127] The compositions and nutritional products of the present invention can be powder or liquid compositions. When the compositions are liquid, whey protein micelles and other powdered ingredients such as, for example, active ingredients, functional ingredients, leucine, etc., can be added to a reconstitution liquid to form a composition or product liquid nutritional. The reconstitution liquid can be any consumable liquid including, but not limited to, water, deionized water, carbonated water, fruit juices, milk, syrups, and other water-based beverages such as tea. In one embodiment, powdered whey protein micelles and leucine can also be added to foods such as eggs to form an emulsion. The person skilled in the art will appreciate that any type of food and/or liquid can be used as a base or carrier for the whey and leucine protein micelles.
[000128] Therefore, a consumable product comprising whey protein micelles forms part of the present invention, as already discussed above. By "whey protein micelles" is meant spherical agglomerates of denatured whey protein. Preferably, the whey protein is not hydrolyzed prior to micelle formation, so that spherical micelles of regular shape are obtained. In micelles, the whey protein is arranged in such a way that the hydrophilic parts of the proteins are oriented towards the outside of the cluster and the hydrophobic parts of the protein are oriented towards an inner core of said micelle. Typically, whey protein micelles are less than 1 micron in size.
[000129] According to an embodiment, and as discussed above, the consumable product comprises whey protein micelles and an additional nutrient such as, for example, an amino acid. Non-limiting examples of amino acids include HIsoleucineH, HAlanineH, HLeucineH, HAsparagineH, HLysinH, HAspartateH, HMethionineH, HCysteineH, HFenylalanineH, HGlutamatoH, HTreoninH, HGlutamineH, HGlutamine HHHHHH,HHHH, CitrulineH, combinations of the same.
[000130] In one embodiment, the consumable product comprises whey protein micelles and leucine in an amount sufficient to stimulate protein synthesis in humans while avoiding an increase in viscosity due to whey protein or too little organoleptic properties due to the large amounts of leucine present in the composition. Generally, the amount of leucine present in nutritional compositions or products will depend on the final volume of the compositions or products, as well as the fact that the solubility limit of leucine at 25°C is 2.426 g per 100 g net, and the fact of which 10 g of whey protein micelles inherently include about 1 g of leucine. Based on this information, it is possible to achieve a large amount of leucine in a nutritional composition without experiencing few organoleptic properties.
[000131] For example, a dry weight ratio of added leucine to whey protein micelles in the present compositions can range from about 1:2 to about 1:3. In one embodiment, the dry weight ratio of added leucine to whey protein micelles is about 1:2.6. Alternatively, a liter of a nutritional composition can contain up to about 25 g of total leucine. In one embodiment, a liter of a nutritional composition can contain about 24 g of leucine. In another example, 100 g of a liquid can contain up to about 2.5 g of leucine. In one embodiment, 100 g of a liquid can contain about 1 to about 2 g of leucine, or about 1 g to about 3 g of leucine, or about 2.462 g of leucine. In an example where the composition is a powder composition, the composition may include a percentage by weight of total dry matter leucine between about 20% and about 40%. In one embodiment, the percentage by weight of total dry matter of leucine in a powder composition is about 37%. Additionally, the total dry weight of added leucine can range from about 30% to about 40% of the total dry weight of the whey protein micelles. In one embodiment, the total dry weight of added leucine can be about 37% of the total dry weight of the whey protein micelles. The person skilled in the art will be able to adjust these amounts of leucine based on the portion of the nutritional composition or product being served.
[000132] For example, nutritional compositions and products can also be offered in a variety of serving sizes as long as the amounts of whey protein and leucine micelles are appropriately sized. For example, and as discussed above, the solubility limit of leucine at 25°C is about 2.426 g per 100 g of liquid. Using this information, a 250 mL serving, which can contain from about 3 to 6 g of leucine by dry weight or have a leucine to whey protein micelles ratio of about 2.6, can be changed. to increase or decrease the amount of composition or product offered to the patient. For example, the serving can also be a serving (eg 80-100 mL), a can (eg 120 or 250 or 375 mL), a bag (eg 1 liter or 1.5 to 2 liters) , or a powder in a module for conventional supplemental diet products or enteral products.
[000133] In a specific example, a 250 mL portion of a nutritional composition may include, by weight of dry matter, 10.1 g of whey protein micelles, 3.8 g of added leucine, and 55, 6g of other ingredients to create a dry matter composition of about 69.5g. In this example, the total amount of leucine in the product is about 4.8 g (1 g from the whey protein micelles and 3.8 g from added leucine), and the whey protein micelles are about 14.5% of the total dry volume and the added leucine is about 5.5% of the total dry volume. The percentage by total dry weight of leucine in this example is 6.9% (4.8 g total leucine / 69.5 g total dry matter).
[000134] As discussed above, whey protein micelles can be used in nutritional compositions to mask the off-tastes of the nutrients to trick the bitter taste receptors on the tongue. In fact, it is believed that the structure of protein micelles and their interaction with leucine (or other unpleasant-tasting nutrients) prevent the consumer's unpleasant perception of bitterness. Therefore, in addition to compositions containing whey protein micelles, the present invention also includes methods for making and using such compositions.
[000135] According to another embodiment, the consumable product comprises whey protein micelles which are soluble in the product and have a pH below 4. A solubility curve for whey protein micelles is shown in the figure 2. As shown in Figure 2, whey protein micelles are more soluble and stable at a pH below 4.0 and at a pH above 5.5. In addition, whey protein micelles could be used in the critical solubility region, pH 4.5 to pH 5.0, for gels or pliable protein texture. By "soluble" it is meant that the micelles do not aggregate or coagulate to form insoluble aggregates of whey protein micelles. In other words, the whey protein micelles are dispersed in the product. This has the advantage that acidic products can comprise the whey protein micelles according to the invention without presenting stability problems.
[000136] Similarly, products having a salt content above 0.01%, or even above 0.1%, or even above 1% and comprising soluble whey protein micelles also form part of the invention. The stability of whey protein micelles in salty or acidic food matrices is of considerable advantage.
[000137] For example, consumable products such as mayonnaise, low-fat or non-fat mayonnaise, a sauce such as a bechamel sauce, a hollandaise sauce, tartar sauce, pasta sauce, a white sauce, a sauce pepper, sauce with chunks, sauce for baking dishes such as salmon with cream gratin, a soup, a creamy soup such as cream of mushroom soup, cream of asparagus soup, cream of broccoli soup, a Thai soup, a vegetable soup, a salad cream, a topping, a candy cream, pastes, thick sauces, salads etc. which comprise whey protein micelles can be produced. The presence of whey protein micelles gives the products all the advantages described in the present application, such as protein enrichment, whitening/opacifying effect, fat reduction, improved creamy texture and pleasant taste, etc.
[000138] A mayonnaise-like acidic product comprising soluble whey protein micelles is a product according to the invention. By mayonnaise type is meant any sauce of condiments having the texture and appearance of mayonnaise. It can be traditional mayonnaise, salad mayonnaise, salad cream, paste, thick sauce, etc. Typically, the pH of the mayonnaise-like product of the invention ranges between 2 and 6, preferably between 2.5 and 4.5. The product may also comprise salt in an amount of 0-3%, preferably between 0.1 and 2.5%, more preferably between 0.1 and 1.5%. The product may comprise less than 50% fat, 50-70% fat or more than 70% fat. Preferably, the product does not comprise fats. The product may or may not be based on an emulsion.
[000139] Other ingredients present in the mayonnaise-like product of the invention may include egg-derived products (e.g. egg yolk, chicken egg white, chicken egg products etc.), sugars, seasonings, spices, herbs fruits and vegetables including fruit and vegetable juices, mustard, dairy products, water, emulsifiers, thickeners, etc.
[000140] According to another embodiment, a soup or sauce-like product comprising soluble whey protein micelles and having a salt content between 0.01-3%, preferably 0.1-2.5% is offered. The soup or sauce type product can also be acidic, for example in tomato soups or sauces, etc. Typically the soup or sauce type product is salty although in some cases it can be sweet (eg Polish soups). Typically, the soup or sauce type product of the invention comprises a flavoring base and thickening agents. The flavor base may comprise flavors, flavor enhancers, spices etc., or combinations thereof. Thickening agents can be selected from starches, gums, flours etc., or combinations thereof. In addition, the soup or sauce product may comprise other ingredients selected from fat, cream, creamer, oil, emulsifiers, vegetables, legumes, garnishes, pasta, meat, savory dumplings, dairy products, or combinations thereof. Preferably, the soup or sauce type product is fat-free or low in fat. Examples of such sauce-like products are bechamel-type sauce, hollandaise-type sauce, white sauce, noodle sauce, diced sauce, sauce for baking dishes such as cream gratin salmon, pepper sauce, tartar sauce etc. Soup-like products can include creamy soups such as cream soups of asparagus, broccoli, mushrooms, Thai soups, vegetable soups etc.
[000141] The products described above can be offered as "ready to eat" products, i.e., they can be consumed as such without adding other ingredients such as water, for example. Alternatively, they can be products reconstituted from a dehydrated mixture.
[000142] The products described in this invention can be produced by mixing whey protein micelles, a concentrate thereof or a powder thereof with other ingredients and processing the mixture. Processing can involve any processing step known in the literature used in the production of food products. These steps can be subjecting the mixture to heat, pressure, acidic or basic conditions, cold etc.
[000143] In another aspect, the invention also offers dehydrated products such as instant soups, sauces, condiments, pre-cooked soups etc., which can be easily reconstituted with water or other liquid to make them suitable for consumption.
[000144] Typically, the dehydrated product of the invention comprises whey protein micelle powder and dry food ingredients. Whey protein micelle powder is described in the present application. It may consist of spray dried whey protein micelles. Alternatively, the whey protein micelle powder comprises additional ingredients that can be selected from soluble or insoluble salts, probiotic bacteria, dyes, sugars, maltodextrins, fats, oils, fatty acids, emulsifiers, sweeteners, flavor, plant extracts, binders, bioactive agents, caffeine, vitamins, minerals, drugs, milk, milk protein, skimmed milk powder, micellar casein, caseinate, vegetable protein, protein hydrolysates such as wheat gluten hydrolysate, peptides, amino acids, polyphenols, pigments , yeast extracts, monosodium glutamate, etc., or combinations thereof.
[000145] When the whey protein micelle powder comprises other ingredients, the ratio of whey protein micelles to the additional ingredient preferably ranges from 1:1 to 1:100.
[000146] The dry food ingredients present in the dehydrated products of the invention are selected from carbohydrates, protein sources, starches, fibers, fat, flavorings, spices, salts, etc., or combinations thereof.
[000147] The ratio of whey protein micelle powder to other dry ingredients typically ranges from 1:1 to 1:10, preferably 1:1 to 1:5, most preferably 1:3.
[000148] Such dehydrated product can be produced by mixing a whey protein micelle powder with other dry ingredients or codrying a whey protein micelle solution or concentrate with other ingredients. Typically, this is achieved by spray drying. Furthermore, the present whey protein micelles can be used alone or together with other active materials such as polysaccharides (for example acacia gum or carrageenans) to stabilize matrices and for example milk foam matrices. Due to their neutral taste, their whitening power and their stability after heat treatment, the present whey protein micelles can be used to increase the whiteness and flavor of skim milk.
[000149] Just as the whitening power of dairy systems can be increased for the same total protein content, the fat content in a food matrix can be reduced. This aspect represents a particular advantage of the present whey protein micelles as it allows to produce products with a low fat content, for example to add a creamer substitute without adding additional fat derived from milk as such.
[000150] In one embodiment, the whey protein micelle dispersion obtained after heat treatment is concentrated to produce a whey protein micelle concentrate. The concentration step can be carried out by evaporation, centrifugation, sedimentation, ultrafiltration and/or microfiltration. Evaporation can be carried out on the micelle dispersion by introducing it into a vacuum evaporator having a temperature between 50°C and 85°C. Centrifugation can be carried out with a high acceleration rate (more than 2000 g) or with a low acceleration rate (less than 500 g) after acidification of the whey protein micelle dispersion to a pH of less than 5, preferably 4.5. Spontaneous sedimentation can also be performed on the dispersion of whey protein micelles by acidification. Preferably the pH is 4.5 and the settling time is greater than 12 hours.
[000151] In one embodiment, the concentration of the whey protein micelles can be obtained by microfiltration of the micelle dispersion. This enrichment technique not only allows for concentrating whey protein micelles by solvent removal but also allows for the removal of non-micelized protein (such as native proteins or soluble aggregates). Therefore, the final product consists only of micelles, as verified by transmission electron microscopy. In this case, the concentration factor obtainable after the initial permeate flux rate through the membrane has dropped to 20% of its initial value.
[000152] Whey protein concentrate will have a protein concentration of at least 12%. Furthermore, the concentrate will contain at least 50% of the protein in the form of micelles.
[000153] It is interesting to note that the concentrate, if adjusted to have a protein content of 10%, is able to withstand a subsequent heat treatment at 85°C for 15 minutes at a pH of 7.0 in the presence, for example, of up to 0, 15 M sodium chloride. By way of comparison, a native whey protein dispersion (PROLACTA® 90, lot 500658 from Lactalis) forms a gel in the presence of 0.1 M sodium chloride at a protein concentration of only 4%.
[000154] The micelles used in the present invention also have the advantage that the high stability of the micellar structure is preserved during the concentration step. Furthermore, micelles according to the present invention have a protein efficiency ratio (PER) equivalent to that of the starting whey protein of at least 100, preferably at least 110, which makes them important nutrient ingredients.
[000155] The enrichment of whey protein micelles offers the exceptional advantages that products enriched with proteins can be obtained at concentrations never reached before. Furthermore, as the concentrate can function as a fat substitute while maintaining the desired structural, textural and organoleptic properties, a wider range of low-fat products is possible.
[000156] Additionally, it has the cost advantage that a smaller amount of concentrate is needed to obtain the desired effects.
[000157] Whey protein micelle concentrate (resulting from evaporation or microfiltration) can be used in liquid form as a dispersion or in a semi-solid form, or in a dry form. It can be used in a wide variety of applications such as those described above regarding whey protein micelle applications. For example, 20% protein concentrate obtained by evaporation has a creamy semi-solid texture and can be textured into a spreadable texture by acidification using lactic acid. This liquid, creamy, pasty texture can be used to prepare acidic, sweet, savory, aromatic, protein-rich consumables.
[000158] Whey protein micelle concentrate in any form can be mixed with 5% acidic fruit base and 5% sucrose to obtain an acidic fruit based drink enriched with whey protein and stable. It can also be used in the manufacture of dairy products, ice cream, or used as a coffee whitener, among other uses.
[000159] Other applications including skin care and mouth care, such as toothpaste, chewing gum, or gum cleaning agent, for example.
[000160] The whitening power of concentrate in any form is tremendously increased compared to unconcentrated micelles or native protein powders. For example, the whitening power of 4 mL of a 15% whey protein micelle concentrate is equivalent to 0.3% titanium oxide in 100 mL of a 2% cup of instant coffee. Interestingly, it is possible to disperse soluble coffee and sucrose in a whey protein micelle concentrate so as to obtain a 3-in-1 concentrate having a total solids concentration of 60% non-fat.
[000161] The concentrate can be used as such or diluted depending on the application. For example, whey protein micelle concentrate in liquid or dry form can be diluted to obtain a protein content of 9% as in condensed and sweet milk. Milk minerals, lactose and sucrose can be added so that the final product has a similar nutritional profile compared to milk, but only whey protein as the protein source. This whey protein-based mixture is more stable to the Maillard reaction than sweet condensed milk (based on the rate of development of a brown color) when incubated for 2 hours at 98°C (boiling water temperature at an altitude of 833 m).
[000162] The dry form of whey protein concentrate obtainable by the method described in this invention can be obtained by any known technique, such as spray drying, freeze drying, roller drying etc. Therefore, the whey protein concentrate of the present invention can be spray dried with or without the addition of other ingredients and can be used as a delivery system or as a building block to be used in a wide range of processes, eg production of consumables, cosmetic applications etc.
[000163] In one embodiment, a powder is obtained by spray drying without adding any other ingredients, and has an average particle diameter greater than 1 micron due to micelle aggregation that occurs during spray drying. A typical volume mean diameter (D43) of the powders of the invention ranges between 45 and 55 microns, preferably 51 microns. The average surface diameter (D32) of the powders of the present invention preferably ranges between 3 and 4 microns, more preferably it equals 3.8 microns. The moisture content of the powders obtained after spray drying is preferably less than 10%, more preferably less than 4%.
[000164] Such whey protein micelle powder may comprise at least 90% whey protein, of which at least 20%, preferably more than 50%, more preferably more than 80% are in micellar form .
[000165] In addition, the whey protein micelle powder used in the present invention has a high binding capacity for solvents such as water, glycerol, ethanol, oil, organic solvents etc. The water fixing capacity of the powders is at least 50%, preferably at least 90%, more preferably about 100%. For solvents such as glycerol and ethanol, the fixing capacity is at least 50%. For oil, the holding capacity is at least 30%. This property of the whey protein micelle powders of the present invention allows them to be sprayed or mixed with functional ingredients such as coffee, caffeine, green tea extracts, plant extracts, vitamins, minerals, bioactive agents, salt, sugar, sweeteners, aroma, fatty acids, oils, protein hydrolysates, peptides, amino acids, etc., or combinations thereof.
[000166] Functional ingredients can be included in the powder in an amount of 0.1-50%. Therefore, the powder can act as a carrier for these functional ingredients. This has the advantage that, for example, the bitterness perception of caffeine is reduced when mixed in the powders of the present invention and used in caffeinated nutritional bars for example. Additional ingredients can be mixed with the whey protein micelle concentrate prior to spray drying. These comprise soluble or non-soluble salts, peptides, protein hydrolysates (eg wheat gluten hydrolyzate), probiotic bacteria, dyes, sugars, maltodextrins, fats, emulsifiers, sweeteners, flavor, plant extracts, binders, bioactive agents, caffeine , vitamins, minerals, drugs, milk, milk proteins, skimmed milk powder, micellar casein, caseinate, vegetable protein, amino acids, polyphenols, pigment, etc., and combinations thereof. The mixed whey protein micelle powders comprise whey protein micelles and at least one additional ingredient in a weight ratio ranging from 1:1 to 1:100.
[000167] This spray drying results in powders consisting of whey protein micelles agglomerated or coated with an additional ingredient. Preferably, the weight ratio of whey protein micelles to additional ingredient is 1:1. This can further facilitate the solubilization of such powders and can be particularly interesting in the production of dehydrated food products such as soups, sauces, etc. comprising whey protein micelles.
[000168] The whey protein micelle powders used in the present invention are characterized by an internal structure composed mainly of hollow spheres but also collapsed spheres. The structure of the hollow spheres can be easily explained by the formation of the vapor drop within the WPM concentrate drop during spray drying. When the steam drop comes out of the WPM drop due to a temperature above 100°C, it becomes a hollow sphere. The “bone shape” is due to a combination of water evaporation from the drop and the external pressure inside the drop.
[000169] The internal structure of the spherical hollow spheres was investigated by SEM after sectioning the particle close to its diameter. The particle wall thickness was about 5 µm and appeared very smooth, while the internal structure had a more grainy appearance. Further amplification showed that this granularity was actually due to the presence of the initial WPM that was fused to form the internal matrix of the powder particle. Interestingly, the spherical shape of the micelles was maintained during spray drying as well as the homogeneous particle size distribution.
Therefore, in microscopic terms, whey protein micelle powders are characterized by a unique granular morphology of hollow or collapsed spheres containing individualized intact whey protein micelles.
[000171] Whey protein micelle powders are characterized by a very high flowability, which offers advantages that were not previously possible. For example, these powders behave almost like liquids and have the advantages of easy usability and transferability. The angle of repose of such powders is preferably less than 35°, more preferably less than 30°. This low angle of repose allows the powders of the present invention to be used as flow agents in food applications, for example.
[000172] A very important aspect of these powders, mixed or "pure" is that the basic micellar structure of the whey proteins is conserved. Furthermore, the micellar structure can be easily reconstituted in solvents. It has already been shown that powders obtained from whey protein micelle concentrate can be easily redispersed in water at room temperature or at 50°C. The size and structure of the whey protein micelles are completely preserved in relation to the starting concentrate. For example, in one embodiment, whey protein concentrate that has been spray-dried to a 20% protein concentration has been redispersed in deionized water at 50°C at a 4% protein concentration. The structure of micelles was examined by TEM. The diameter of the micelles was found to be 315 nm by dynamic light scattering with a polydispersity index of 0.2.
[000173] The fact that whey protein micelles and only a small aggregate fraction were observed in solution after reconstitution and homogenization at 250 bars of the spray-dried or freeze-dried powder confirms that whey protein micelles milk are physically stable in spray drying and freeze drying.
[000174] The powders of the present invention can be used in a wide range of applications, such as all those described above in relation to whey protein micelles and concentrates thereof. For example, protein enriched consumables such as chocolate, energy nutrition bars, dehydrated culinary products, chewing gum etc. can be easily produced using the micelle concentrate powders. Due to their high stability during processing, the powders of the present invention can also be further coated with emulsifiers, gums, proteins, peptides, protein hydrolysates, for example. This can be advantageous for modulating the functionality and flavor of these powders.
[000175] The invention is further defined with reference to the following examples describing in detail the preparation of micelles used in the present invention. The invention described and claimed in this report is not limited in scope by the specific embodiments described herein, as these embodiments are illustrative of various aspects of the invention. All equivalent embodiments are considered within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described in this report will become apparent to those skilled in the art from the foregoing description. Such modifications are also considered within the scope of the appended claims. Example 1 β-Lactoglobulin mycelization by pH adjustment
[000176] β-Lactoglobulin (lot JE002-8-922, 13-12-2000) was purchased from Davisco (Le Sueur, MN, USA). The protein was purified from sweet whey by ultrafiltration and ion exchange chromatography. The composition of the powder is 89.7% protein, 8.85% moisture, 1.36% ash (0.079% Ca2+, 0.013% Mg2+, 0.097% K+, 0.576% Na+, 0.050% Cl -). All other reagents used were analytical grade (Merck Darmstadt, Germany).
[000177] The protein solution was prepared at a concentration of 0.2% by solvation of β-lactoglobulin in MilliQ® water (Millipore), and stirring at 20°C for 2 hours. Then the pH of the aliquots was adjusted to 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7 .0 by addition of HCl. The solutions were filled into 20 ml glass vials (Agilent Technologies) and sealed with aluminum caps containing a silicon/PTFE seal. The solutions were heated at 85°C for 15 minutes (time to reach temperature 2.30 - 3.00 min). After heat treatment, the samples were cooled in ice water to 20°C. The visual appearance of the products indicates that the ideal micellization pH is 5.8. Example 2 Whey Protein Isolate Micelization
[000178] Whey protein isolate (WPI) (BIPRO®, Batch JE032-1-420) was purchased from Davisco (Le Sueur, MN, USA). The composition of the powder is shown in table 2.
[000179] The protein solution was prepared at a concentration of 3.4% protein by solvating whey protein powder in MilliQ® water (Millipore), and stirring at 20°C for 2 hours. The initial pH was 7.2. Then the pH of the aliquots was adjusted to 5.6, 5.8, 6.0, 6.2, 6.4 and 6.6 by addition of 0.1 N HCl.
[000180] The solutions were filled into 20 mL glass vials (Agilent Technologies) and sealed with aluminum caps containing a silicon/PTFE seal. The solutions were heated at 85°C for 15 minutes (time to reach temperature 2.30 - 2.50 min). After heat treatment, the samples were cooled in ice water to 20°C.
[000181] The turbidity of the heated whey proteins was determined at 500 nm and 25°C, the samples were diluted to enable measurement in the range of 0.1-3 Abs units (Spectrophotometer Uvikon 810, Kontron Instrument). Values were calculated for the initial 3.4% protein concentration.
[000182] The micellization pH was considered achieved with the stability (variation less than 5% of the initial value) of the absorbance measured at 500 nm in an interval of 10 minutes for the same sample. For this product, the ideal pH for micellization was between 6.0 and 6.2. At this pH adjusted before heat treatment, the stable turbidity was 21 and the residual soluble protein assessed by absorbance at 280 nm after centrifugation was 1.9%. We can conclude that 45% of the initial proteins were transformed into micelles at pH 6.0. TABLE 2 WPI composition and sample characteristics after micellization

EXAMPLE 3 Microscopic observation of micelles Production of micelles:
[000183] A protein solution was prepared at a concentration of 2% protein by solvation of whey protein powder (WPI 90 batch 989/2, Lactalis, Retier, France) in MilliQ® water (Millipore), and stirred at 20°C for 2 hours. Then, the pH of the aliquots was adjusted using 0.1N HCl or 0.1N NaOH.
[000184] The solutions were filled into 20 mL glass vials (Agilent Technologies) and sealed with aluminum caps containing a silicon/PTFE seal. The solutions were heated at 85°C for 15 minutes (time to reach temperature 2.30-2.50 min).
[000185] After heat treatment, the samples were cooled in ice water to 20°C. For this product, the ideal pH for micellization was 7.4. Microscopic observations:
[000186] Samples of liquid micelles were encapsulated in agar gel tubes. Fixation was achieved by immersion in a 2.5% glutaraldehyde solution in 0.1M cacodylate buffer, pH 7.4 and post-fixation was achieved with 2% osmium tetroxide in the same buffer, both solutions containing 0 .04% ruthenium red. After dehydration in a graded series of ethanol (70, 80, 90, 96, 100% ethanol), the samples were embedded in Spurr resin (Spurr/ethanol 1:1, 2:1, 100%). After resin polymerization (70°C, 48 hours), semi-thin and ultra-thin sections were cut with a Leica ultracut UCT ultramicrotome. The ultrathin sections, stained with aqueous uranyl acetate and lead citrate, were examined by transmission electron microscopy (Philips CM12, 80 kV).
[000187] The micelles obtained showed a spherical shape with a diameter of 200 nm, measured by TEM. Particle Size Distribution
[000188] Size distributions based on micelle intensity were measured for micelles obtained by heat treatment of a 1% by weight dispersion of β-lactoglobulin for 15 minutes at 85°C at pH 4.25 (positively charged at a zeta potential of about +25 mV) and at a pH 6.0 (negatively charged with a zeta potential of about -30 mV). The Z-average hydrodynamic diameter of the micelles was 229.3 mm at pH 4.25 and 227.2 mm at pH 6.0. Aggregations of β-LG and whey protein were performed using dynamic light scattering. A Nanosizer ZS apparatus (Malvern Instruments, UK) equipped with a laser emitting at 633 nm and with a power of 4.0 mW was used. The instrument was used in the backscatter configuration, where detection is done at a scattering angle of 173°C. This allows for a considerable reduction in the multiple scattering signals found in turbid samples. The samples were placed in a square quartz cell (Helima, path length 1 cm). The length of the light beam path was automatically regulated by the device, depending on the turbidity (attenuation) of the sample. The autocorrelation function was calculated from the fluctuation of the scattered intensity. The results indicate that the average particle is characterized by a very narrow polydispersity index (<0.200). Example 4 Mycelization of a β-lactoglobulin at a constant pH
[000189] The method described in example 1 was repeated using a 2% aqueous solution of β-lactoglobulin. The pH of this solution was adjusted to 7.0 after addition of arginine HCl solutions to obtain a final salt concentration ranging from 5 to 200 mM and a final β-lactoglobulin concentration of 1%. Subsequent heat treatment (80°C, 10 min, heating for about 2 min) was carried out to produce micelles.
[000190] The results indicate that only in the ionic strength range of about 50 to 70 mM, it is possible to observe a substantial turbidity, indicating the presence of whey protein micelles. Example 5 Preparation of a bleaching agent
[000191] Native whey proteins (WPI 95 lot 848, Lactalis; 8% by weight aqueous solution) were treated according to Example 2. The luminosity (L) of the resulting product was measured in the trans-reflectance mode using A MacBeth CE-XTH D65 10° SCE apparatus equipped with a 2 mm measuring cell is used. The resulting luminosity was L = 74.8, which could be compared to the value of L = 74.5 for whole milk. Example 6 Preparation of a coffee flavored cream substitute
[000192] Native whey proteins (BIPRO®, lot JE 032-1420, 0.5% by weight aqueous solution) were mixed at 50°C with 10% by weight of partially hydrogenated palm oil, 14% in weight of maltodextrin (DE 21) and in the presence of 50 mM phosphate-citrate buffer adjusted to micellization pH pH 6.0 for these BIPRO®. The mixture was homogenized at 400/50 bars using a Rannie homogenizer and subsequently heat treated for 15 minutes at 85°C.
[000193] The obtained emulsion showed a high stability for a time period of at least one month under the storage conditions at 4°C and gave a whiteness of L = 78 compared to a liquid reference cream substitute (flavored cream substitute coffee, Emmi, Switzerland) having a fat content of 15% and a luminosity of L = 75.9. Example 7 Preparation of an aqueous foam
[000194] Native β-lactoglobulin (Biopure, Davisco, lot JE 002-8922, 2% by weight aqueous solution) was mixed with a 120 mM arginine HCl solution so that the final β-lactoglobulin concentration was 1% by weight and that of arginine HCl 60 mM. The pH was then adjusted to 7.0 by addition of 1N HCl. The mixture was then heat treated at 80°C for 10 minutes so that 90% of the starting β-lactoglobulin was converted to micelles having a z-average diameter of 130 nm. In this case, the micelle diameter was determined using a Nanosizer ZS apparatus (Malvern Instruments, UK). The sample was poured into a quartz cuvette and the scattered light variations were automatically recorded. The autocorrelation function obtained was fitted using the cumulative method so that the diffusion coefficient of the particles could be calculated and then the z-average hydrodynamic diameter using the Stokes-Einstein law. For this measurement, the refractive index of the solvent was considered equal to 1.33 and that of the micelles, equal to 1.45. A 50 mL volume of the resulting dispersion of β-lactoglobulin micelles is then foamed by nitrogen spray through a glass frit generating 12-16 µm bubbles to produce a foam volume of 180 cm3 using the Foamscan™ apparatus. (ITConcept) standardized. The volume stability of the foam was then monitored over time at 26°C using image analysis and compared with the stability of the foam obtained with β-lactoglobulin treated under the same conditions, but without arginine HCl, where no formed micelles. In fact, the volume stability of the foam is greatly improved by the presence of β-lactoglobulin micelles. Example 8 Fermented Whey Dairy Product - Fermentation Assays Whey Protein Isolate Material (WPI) (BIPRO®) was purchased from Davisco (Le Sueur, MN, USA) (protein concentration 92.7% )
[000195] Spray dried whey permeate (Variolac 836): Lactose concentration: 83% - Minerals: 8%. Lactic Acid 50% Edible Lactose (Lactalis) Deionized Water Method
[000196] The BIPRO® powder was dissolved in deionized water to have a protein concentration of 4.6%, ie for 3 liters of solution 154.5 g of powdered WPI and 2845.5 g of water. Hydration time was 3 hours. After hydration, this solution was divided into 200 mL samples to prepare the different assays: TABLE 3


[000197] For each solution, 50% lactic acid was added to adjust the pH before heating.
[000198] The samples were heated with the double boiler to 85°C and kept at this temperature for 15 minutes. After heating, the solutions were cooled to 40°C and inoculated with Lactobacillus bulgaricus and Streptococcus thermophilus. The samples were incubated for 5 hours and 30 minutes in a steam chamber at 41°C before being placed in a 6°C cold chamber. The results are shown in table 4. TABLE 4

EXAMPLE 9 Low Fat Whey Protein Reinforced Ice Cream Whey Protein Isolate Material (WPI, PROLACTA® 90 from Lactalis, Retiers, France) with a protein content of 90% Skimmed milk powder with a 35% protein content Sucrose Maltodextrins DE39 Anhydrous milk fat Emulsifier Deionized water Edible hydrochloric acid 1M Method
[000199] Using an 80 L double jacketed tank, PROLACTA® 90 powder was dispersed at 50°C in deionized water at a protein concentration of 9.67% by weight with gentle agitation to avoid foaming, ie, 3.3 kg of PROLACTA® 90 was dispersed in 31.05 kg of deionized water. After 1 hour of dispersion, the pH of the dispersion was adjusted to the micellization pH by addition of HCl. The temperature of the dispersion was increased to 85°C and held for 15 minutes to generate the whey protein micelles. After 15 minutes, the temperature was lowered to 50°C and additional ingredients were sequentially added to the micelle dispersion (i.e., skim milk powder, DE39 maltodextrins, sucrose, emulsifier and anhydrous milk fat). The final blend amount was 50 kg with a total solids content of 39.5% and a fat content of 5% by weight. After 30 minutes of hydration, the mixture was homogenized in two stages (80/20 bars) and pasteurized (86°C/30s) before being aged for one night. The next day, the ice cream mix was frozen in excess of 100% using a Hoyer MF50 appliance and hardened at -40°C before being stored at -20°C. The final ice cream contained 8% by weight protein (20% casein, 80% whey protein) and 5% by weight fat based on the ice cream mix. Example 10 Whey protein micelle powder obtained by spray drying Whey protein isolate (WPI, PROLACTA® 90 from Lactalis, Retiers, France) with a protein content of 90% Edible lactose Maltodextrins DE39 Water deionized 1M Edible Hydrochloric Acid Method
[000200] Using a 100 L double jacketed tank, the PROLACTA® 90 powder was dispersed at 50°C in deionized water at a protein concentration of 10% by weight with gentle agitation to avoid foaming, ie, 11 kg of PROLACTA® 90 was dispersed in 89 kg of deionized water. After 1 hour of dispersion, the pH of the dispersion was adjusted to the micellization pH (about 6.3 in that case) by addition of HCl. The temperature of the dispersion was increased to 85°C and held for 15 minutes to generate the whey protein micelles. After 15 minutes, the temperature was lowered to 50°C and the 10% by weight whey protein micelle dispersion was divided into two 50 kg batches. In a first trial, 20 kg of lactose were dispersed in 50 kg of micelle dispersion at 50°C and stirred for 30 minutes. Similarly, 20 kg of DE39 maltodextrins were added to the remaining 50 kg of the whey protein micelle dispersion.
[000201] The two mixtures were then spray dried in a NIRO SD6.3N tower at a flow rate of 15 L/h. The inlet air temperature was 140°C and the outlet air temperature was 80°C. The water content of the obtained powders was less than 5%. Whey protein micelle size was determined in the presence of lactose and maltodextrin (DE39) in water using dynamic light scattering and then spray drying. The total protein concentration was set to 0.4% by weight by diluting the dispersion before spray drying or reconstituting the powder to be in the diluted viscosity regime for whey protein micelles. A Nanosizer ZS apparatus (Malvern Instruments) was used and the mean diameter of micelles was calculated from 20 measurements.
[000202] The particle diameter determined for the whey protein micelles in the presence of lactose and maltodextrins (DE39) was 310.4 nm and 306.6, respectively. After reconstitution of the powders, the respective diameters found were 265.3 nm and 268.5, respectively. These measurements confirm that the whey protein micelles were physically stable to spray drying. The results were corroborated by observations made by TEM microscopy of 0.1% by weight whey protein micelle dispersions in water using negative staining in the presence of 1% phosphotungstic acid at pH 7. A transmission electron microscope Philips CM12 operating at 80 kV was used. Whey protein micelles were observed in solution before spray drying and after reconstitution of the spray dried powder. It was not possible to detect any difference in morphology and structure. Example 11 Concentration by evaporation
[000203] A PROLACTA® 90 whey protein isolate from Lactalis (batch 500648) was reconstituted at 15°C in soft water at a protein concentration of 4% to achieve a final batch size of 2500 kg. The pH was adjusted by adding 1M hydrochloric acid so that the final pH value was 5.90. The whey protein dispersion was pumped through the APV plate mixing heat exchanger at a flow rate of 500 l/h. Preheating to 60°C was followed by heat treatment at 85°C for 15 minutes. The formation of whey protein micelles was verified by measuring particle size using dynamic light scattering as well as a turbidity measurement at 500 nm. The 4% whey protein micelle dispersion obtained was characterized by a hydrodynamic particle radius of 250 nm, a polydispersity index of 0.13 and a turbidity of 80. The whey protein micelle dispersion it was then used to feed a Scheffers evaporator at a flow rate of 500 l/h. The temperature and vacuum in the evaporator were adapted so that about 500 kg of whey protein micelle concentrate having a protein concentration of 20% was produced and cooled to 4°C. Example 12 Enrichment by microfiltration
[000204] A PROLACTA® 90 whey protein isolate from Lactalis (batch 500648) was reconstituted at 15°C in soft water at a protein concentration of 4% to achieve a final batch size of 2500 kg. The pH was adjusted by adding 1M hydrochloric acid so that the final pH value was 5.90. The whey protein dispersion was pumped through the APV plate mixing heat exchanger at a flow rate of 500 L/h. Preheating to 60°C was followed by heat treatment at 85°C for 15 minutes.
[000205] The formation of whey protein micelles was verified by measuring the particle size using dynamic light scattering as well as a turbidity measurement at 500 nm. The 4% whey protein micelle dispersion obtained was characterized by a hydrodynamic particle radius of 260 nm, a polydispersity index of 0.07 and a turbidity of 80. The form of protein micelles was also verified by TEM, and micelle structures with an average diameter of 150-200 nm were clearly visible. The whey protein micelle dispersion could be cooled to 4°C for storage or was directly fed to a filtration unit equipped with a 6.8 m2 Carbosep M14 membrane at a flow rate of 180 L/ H. In this case, the whey protein micelle concentration was carried out at 10°C until the permeate flow rate reached 70 L/h. In this case, the final whey protein concentrate contained 20% protein. The structure of the micelles in the concentrate was verified by TEM, and clearly there was no significant change compared to the 4% whey protein dispersion prior to microfiltration. Although the concentration of the whey protein micelles was carried out at 10°C in the present example, the concentration could also be carried out at 55°C, or at a temperature from about 60°C to about 63°C. Example 13 Whey protein micelle powder comprising at least 90% whey protein
[000206] 200 kg of a whey protein micelle concentrate obtained by microfiltration at 20% protein (see example above) were injected into a NIRO SD6.3N tower using an atomizing nozzle (0 = 0, 5 mm, spray angle = 65°, pressure = 40 bars) at a product flow rate of 25 kg/h. The inlet temperature of the product was 150°C and the outlet temperature was 75°C. The airflow in the tower was 150 m3/h. The moisture content in the powder was less than 4% and the powder was characterized by very high flowability. Scanning electron microscopy of the powder showed very spherical particles having an apparent diameter ranging from 10 to 100 µm. Example 14 Mixed powder of whey protein micelles
[000207] 20 kg of a whey protein micelle concentrate were mixed with 1.7 kg of maltodextrins with an DE of 39 so that the final ratio of whey protein micelles to powder is 70 /30. This mixture was injected into a NIRO SD6.3N tower using an atomizing nozzle (0 = 0.5 mm, spray angle = 65°, pressure = 40 bars) at a product flow rate of 25 kg/h. The inlet temperature of the product was 150°C and the outlet temperature was 75°C. The airflow in the tower was 150 m3/h. The moisture content in the powder was less than 4% and the powder was characterized by very high flowability.
[000208] The powders of examples 13 and 14, when reconstituted in water, comprise essentially micelles having the same structure and morphology as the whey protein micelle concentrate. Example 15 Whey protein micelle powder obtained by freeze drying Material Whey protein micelle concentrate at 20% protein produced by microfiltration in Example 12 with a protein content of 90% Method
[000209] 100 g of whey protein micelle concentrate were introduced into plastic beaker and frozen at -25°C for one week. This beaker was then placed in a Virtis laboratory freeze dryer equipped with a vacuum pump. The sample was held for 7 days until the pressure in the lyophilizer was constant at about 30 mbars. About 20 g of freeze-dried whey protein micelles were recovered Example 16 Whey protein enriched dark chocolate without sucrose Material
METHOD
[000210] Cocoa liquor is mixed with cocoa butter, butter fat, whey protein micelle powder, sucralose, vanillin and lecithin. This mixture is then shelled overnight at 65°C until a homogeneous paste is obtained. This chocolate mass is then molded into chocolate shapes and cooled. Dark chocolate is characterized by a final whey protein content of 45-50%. Example 17 White chocolate enriched with whey protein Material
METHOD 1
[000211] Whey protein micelles, whey powder, sucrose and vanillin are mixed and ground until the desired particle size distribution is obtained. This mixture is then shelled overnight at 65°C with cocoa butter, anhydrous milk fat and lecithin until a homogeneous paste is obtained. This chocolate mass is then molded into chocolate shapes and cooled. This white chocolate is characterized by a final whey protein content of 20%. Method 2
[000212] Whey protein micelles, whey powder, sucrose and vanillin are mixed and ground until the desired particle size distribution is obtained. This mixture is then shelled overnight at 65°C with cocoa butter, anhydrous milk fat and lecithin until a homogeneous paste is obtained. This chocolate mass is then molded into chocolate shapes and cooled. This white chocolate is characterized by a final whey protein content of 30%. Method 3
[000213] Whey protein micelles, sucrose and vanillin are mixed and ground until the desired particle size distribution is obtained. This mixture is then shelled overnight at 65°C with cocoa butter, anhydrous milk fat and lecithin until a homogeneous paste is obtained. This chocolate mass is then molded into chocolate shapes and cooled. This white chocolate is characterized by a final whey protein content of 30-35%. Example 18 Aqueous dispersion of whey protein micelles coated with sulfated butyl oleate (SBO) or any other negatively charged emulsifier Material Whey protein (WPM) micelle powder from Example 13 with a protein content of 90% SBO Hydrochloric Acid (1M) Method
[000214] The WPM powder described in example 13 is dispersed in MiIIIiQ water to reach a final protein concentration of 0.1% by weight. This dispersion is filtered through 0.45 µm filters to remove possible WPM aggregates. The pH of this WPM dispersion was lowered to 3.0 by addition of 1 M hydrochloric acid. A 1% by weight SBO dispersion is prepared at pH 3.0.
[000215] The hydrodynamic radius and the zeta potential of these WPM were determined using the Nanosizer ZS device (Malvern Instruments Ltd.). The diameter was equal to 250 nm and the electrophoretic mobility was +2.5 μm.cm.V-1.s-1. The hydrodynamic radius and electrophoretic mobility of the SBO dispersion at pH 3.0 are 4 nm and -1.5/-2.0 μm.cm.V-1.s-1, respectively.
[000216] After performing this preliminary characterization, the SBO dispersion is used to titrate the WPM one, while the evolution of the hydrodynamic radius and the electrophoretic mobility of the mixture occurs. The hydrodynamic radius was found to be constant and equal to about 250-300 nm until a WPM/SBO mixing ratio by weight of 5:1 was reached. At this point, the hydrodynamic radius dramatically shifts to 20000 nm and precipitation of WPM SBO complexes occurs. With one more addition of SBO, greater than a mixing ratio of 5:1, the hydrodynamic radius progressively decreased until reaching 250 nm, as initially found for WPM, stabilizing from a ratio of 4:1 onwards. Monitoring the electrophoretic mobility of the mixture showed that it decreased with the addition of SBO, reaching a value of zero for a mixing ratio of 5:1. It then continued to fall with the addition of SBO, starting to stabilize at -3.0 μm.cm.V’1.s’1 from 4:1 ratio onwards.
[000217] The explanation for these results is that the positively charged WPMs are, in a first step, electrostatically charged with the negative head of the SBO until the complete neutralization of the charge is reached (mixing ratio 5:1). At this point, the hydrophobic tails of the SBO are capable of self-association, leading to excessive aggregation with very large hydrodynamic diameter and precipitation of complexes. With yet another addition of SBO, the hydrophobic tails further combine to form a double coating, exposing their negative head to the solvent. This leads to negatively charged WPM with a double coating of SBO comparable to an integral protein core liposome.
[000218] Similar results have been obtained with other acidic food grade emulsifiers such as DATEM, CITREM, SSL (from Danisco) in aqueous solution at pH 4.2 where they are mainly ionized in their anionic form (COO chemical functions). Example 19 Protein-enriched béchamel-type sauce Material Mixed powder of whey protein micelles from Example 14 with a protein content of 70% Butter Flour Skimmed milk Salt Method
[000219] 30 g of mixed whey protein powder are dispersed in 1 liter of skim milk under heating. 30 g of butter and 80 g of flour are then added along with 2.85 g of salt. The mixture is then boiled to produce a bechamel-like sauce having a whey protein content of about 3 g/100 g. Example 20 Whey Protein Enriched Base for an Energetic Nutrition Bar Materials
METHOD
[000220] Brown rice syrup is mixed with maltitol and glycerol at 25°C. The whey protein micelle powder is then added and mixing is carried out for 10 minutes. A base enriched with whey protein for an energy nutritious bar is then obtained and can be mixed with other ingredients (minerals, vitamins, flavors). This preparation contains more protein than milk (38%). Example 21
[000221] Determination of angle of repose for spray dried whey protein micelle powder, mixed whey protein micelle powder, whey protein isolate powder and calorific value skim milk powder Low Material Example 12 Whey Protein Micelles Powder with a Protein Content of 90% (3.5% Moisture) Example 13 Whey Protein Micelles Mixed Powder with a Protein Content of 90% (moisture 3.5%) PROLACTA® 90 Whey Protein Isolate Powder (lot 500658 from Lactalis, France; moisture 4%) Low calorific skim milk powder (lot 334314 from Emmi, Switzerland; moisture 3, 5%) Measuring device described to measure the angle of repose for powders according to ISO 4324 Method
[000222] The powder is placed in a funnel with a rod 99 mm in diameter and the powder is forced out with the stirrer. The powder falls into a transparent plastic container with a diameter of 100 mm and a height of 25 mm. The angle of repose, Φ, is measured by the following equation: Angle of repose Φ = ARCTAN (2h/100)
[000223] Where h is the maximum height of the powder cone that can be obtained, the entire surface of the plastic container being covered with powder. Angle of repose test results (values are mean of 3 measurements and standard deviation is indicated).


[000224] The angle of repose results clearly show that whey protein micelle powder, pure or mixed with maltodextrins, exhibits a significantly smaller angle than the initial whey protein powder or even milk in skimmed powder. An angle of repose less than 35° is characteristic of very good flowable powders. Example 22 Recipes for Hollandaise Sauce and Mayonnaise Product Comprising Whey Protein Micelles Hollandaise Sauce and Mayonnaise Sauce Hollandaise Sauce
mayonnaise sauce

[000225] Using whey protein micelles, it has been possible to obtain fat-free products that have high acid and salt content. The advantage of having whey protein micelles is that the whitening effect is provided and at the same time the micelles are stable to the culinary matrix and the treatment process. Whey protein micelles further simulate the presence of fat due to their emulsifying properties. Example 23
[000226] Co-drying by spraying WPM with wheat gluten hydrolyzate (WGH) 2.2 kg of WPM powder were dispersed in 45.8 kg of demineralized water at 25°C. After 15 minutes of stirring, the WPM dispersion was homogenized at 250/50 bars using a NIRO-SOAVI homogenizer at a flow rate of 50 kg.h-1. The WGH (wheat gluten hydrolyzate) powder (2 kg) (available commercially or by traditional methods known in the literature) was then dispersed into the WPM dispersion (48 kg) so that the final solids content of the dispersions was 8 % and the weight ratio of WPM to WGH was 1:1. The final WPM content of the spray-dried powder was thus around 50%. Example 24 Soup enriched with whey protein
[000227] Using a whey protein micelle powder according to the invention, a dry mix (28 g) was prepared using the following ingredients: Broccoli cream soup

[000228] The product was reconstituted by adding the dry mixture to 250 ml of cold or hot water and boiling. The soup obtained had a creamy texture and a whey protein content of 4-6 g/100 g. Example 25 Low-fat whey protein-based cream soup Asparagus cream soup (29 g)

[000229] THE PRODUCT IS RECONSTITUTED BY ADDING THE DRY MIXTURE TO 500 ML OF COLD OR HOT AND BOILED WATER TO PRODUCE A SOUP WITH A CREAMY TEXTURE AND APPEARANCE AND A REDUCED FAT CONTENT. Example 26 Spray codrying of whey protein micelles with a soup base
[000230] A dispersion of whey protein micelles was reconstituted by mixing 1.6 kg of whey protein micelles in 43.7 kg of demineralized water. After 15 minutes of stirring, the WPM dispersion was homogenized at 250/50 bars using a NIRO-SOAVI homogenizer at a flow rate of 50 kg.h-1. Then 4.7 kg of soup base was added to the WPM dispersion. The final solids content of the dispersion was 12.6%. The dispersion was spray dried. The product temperature at the outlet of the spray dryer was 75°C and the final moisture content was 3.5%. The final concentration of WPM in the powder was around 23%. A typical dehydrated soup base obtainable by the process described is given:
Example 27 White sauce comprising whey protein micelles
[000231] Using a WPM powder according to the invention, a dry mix (35 g) was prepared using the following ingredients:

[000232] THE PRODUCT WAS RECONSTITUTED BY ADDING THE DRY MIXTURE TO 500 ML OF COLD AND BOILED WATER TO PRODUCE A WHITE SAUCE. Example 28
[000233] Various concentrations of leucine have been included in different compositions. Sensory testing was performed by the applicants to determine the ability of 26 participants to detect the presence of leucine at each of the concentrations of the different compositions. Initially, applicants found that 23 out of 26 participants were able to detect 2 g of leucine supplementing a flavored oral nutritional product with 99.9% confidence. Participants then described the leucine-containing product as “very bitter” and “sourder than other samples”. Participants also said that the product containing the leucine "tasted older and less fresh" than other samples, that the product "appeared to burn a little", and that the product "[had] a bad taste".
[000234] Different compositions containing whey protein micelles and leucine were then prepared and another sensory test was conducted by the applicants using the compositions with whey protein micelles and leucine. Sensory evaluation of internal panelists was conducted using a new nutritional product developed by the applicants which comprised whey protein micelles and supplemental leucine. Leucine concentrations in the examined products included 0.5, 1.0, 2.0 and 3.0 g of L-leucine per serving. The results of this panel suggested that WPM was able to block the perceived bitterness of leucine at a concentration of 1.0 and 2.0 g of leucine, but that 0.5 and 3.0 g of leucine were slightly more easily perceived by the panelists. Therefore, Applicants have surprisingly found that protein micelles have the ability to mask the portion of the nutrient (e.g., leucine) that imparts bitterness to the composition.
[000235] It is to be understood that various changes and modifications to the preferred embodiments described herein will be apparent to those skilled in the art. Such alterations and modifications can be made without departing from the spirit and scope of the present matter and without diminishing the intended advantages. It is, therefore, intended that such changes and modifications are covered by the appended claims.

权利要求:
Claims (13)
[0001]
1. Nutritional composition, characterized in that it comprises whey protein powder comprising whey protein micelles and leucine, the total amount of leucine in the composition comprising between 20% and 40% by weight of dry matter, and the dry weight ratio of added leucine to whey protein micelles being 1:2 to 1:3.
[0002]
2. Composition according to claim 1, characterized in that the whey protein powder comprises at least 20% to 80% of whey protein micelles, or at least 50% of protein micelles of whey.
[0003]
3. Composition according to claim 1, characterized in that the whey protein powder has a water binding capacity of at least 50% to 100%.
[0004]
4. Composition according to claim 1, characterized in that the whey protein powder is obtained by a spray drying or freeze drying process, which is carried out with the whey protein micelles and leucine .
[0005]
5. Composition according to claim 1, characterized in that it further comprises a liquid, the total amount of leucine in the composition being less than 2.5 g per 100 g of liquid, and the liquid being selected from the group consisting of water, water-based beverages, fruit juice, milk and combinations thereof.
[0006]
6. Composition according to any one of claims 1 to 5, characterized in that it further comprises at least one of antioxidants, vitamins, minerals, phytonutrients, prebiotics or probiotics.
[0007]
7. A method of masking off-flavours of leucine in a composition, characterized in that it comprises mixing a whey protein micelle powder and added leucine to form a whey protein powder; the total amount of leucine in the composition being between 20% and 40% by weight of dry matter.
[0008]
8. Method according to claim 7, characterized in that the whey protein powder comprises at least 20% to 80% whey protein micelles, or at least 50% protein micelles of whey.
[0009]
9. Method according to claim 7, characterized in that the whey protein powder comprises a water binding capacity of at least 50% to 100%.
[0010]
10. Method according to claim 7, characterized in that the dry weight ratio of leucine added to whey protein micelles is from 1:2 to 1:3.
[0011]
11. Method according to claim 7, characterized in that the whey protein powder is obtained by a process of spray drying or freeze drying.
[0012]
12. Method according to claim 7, characterized in that it further comprises a liquid, the total amount of leucine in the composition being less than 2.5 g per 100 g of liquid, and the liquid being selected from the group consisting of water, water-based beverages, fruit juice, milk and combinations thereof.
[0013]
13. Method according to any one of claims 7 to 12, characterized in that the composition further comprises at least one of antioxidants, vitamins, minerals, phytonutrients, prebiotics or probiotics.

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JP6240653B2|2017-11-29|

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BR112012023025A2|2020-09-08|

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法律状态:
2020-09-24| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-11-10| B25A| Requested transfer of rights approved|Owner name: SOCIETE DES PRODUITS NESTLE S.A. (CH) |

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2021-01-26| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-04-20| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-20| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: A23L 1/22 , A23C 21/08 , A23J 3/08 , A23L 1/305 , A61K 31/198 Ipc: A23J 3/14 (2006.01), A23J 3/08 (2006.01), ... |

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2021-06-01| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 09/03/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US31334810P| true| 2010-03-12|2010-03-12|

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US61/313,348|2010-03-12|

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US201161447148P| true| 2011-02-28|2011-02-28|

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PCT/US2011/027714|WO2011112695A1|2010-03-12|2011-03-09|Compositions for masking the flavor of nutrients and methods for making same|

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