Method for forming a composition containing microencapsulated probiotics in a denatured plant protein matrix.

Abstract

This invention provides a method for producing microencapsulated activators that are robust, protect encapsulated probiotic bacteria, and have storage stability. [Solution] The method comprises the steps of preparing a protein suspension containing denatured plant protein, preparing a suspension of hydrated probiotics, mixing the protein suspension and the hydrated probiotics suspension to encapsulate the probiotics in a denatured pea protein matrix, and polymerizing the denatured plant protein matrix with calcium salts. The mixing step may include extruding the protein suspension and the hydrated probiotics suspension to form microdroplets, and the polymerization step includes curing the extruded microdroplets in a curing bath containing calcium citrate buffer having a pH of 6.5 and a molar concentration of 0.05 to 0.15 M.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a method for forming a composition comprising microencapsulated probiotics in a denatured plant protein matrix. The present invention also relates to a method for forming a denatured plant protein suspension. The present invention also provides products and compositions formed according to the method of the present invention. [Background technology]

[0002] Background of the Invention To produce robust, encapsulated microencapsulated activators that protect the encapsulated probiotic bacteria and offer storage stability, it is necessary to use protein suspensions with a high solids content and a high proportion of protein in solution. International Publication No. 2008 / 056344 (WO2008 / 056344) (Patent Document 1) describes the encapsulation of probiotic bacteria into a denatured whey protein matrix using a low-temperature gelling process in which probiotic bacteria are mixed with a denatured whey suspension, extruded into microdroplets, and cured in an acidic bath at pH 4.6. Depending on the probiotic, adding probiotics to whey protein using the low-temperature gelling method often results in side effects such as solidification and a decrease in cell count. Furthermore, the pH required to produce low-temperature gelled whey protein, such as pH 4.6, is often detrimental to the survival of probiotics. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2008 / 056344 [Non-patent literature]

[0004] [Non-Patent Document 1] A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation product, Minekus, M., Smeets-Peeters M, Bernalier A, Marol-Bonnin S, Havenaar R, Marteau P, Alric M, Fonty G, Huis in't Veld JH, Applied Microbiology Biotechnology.1999 Dec;53(1):108-14 [Non-Patent Document 2] A standardized static in vitro digestion method suitable for food - an international consensus, Minekus, A. et al., Food Function, 2014, 5, 1113) [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The object of the present invention is to overcome at least one of the above problems. [Means for solving the problem]

[0006] Summary of the Invention This objective is achieved by providing a method for encapsulating probiotics using plant proteins as an encapsulation matrix. The applicant has found that using plant proteins such as denatured pea protein and denatured mung bean protein as microencapsulation matrices for probiotic bacteria results in greater bacterial survival during the encapsulation process, with cell viability exceeding 75-80%, compared to 40-50% in dairy-based encapsulation systems such as WPI or MPC. Without being bound by theory, the increased viability is thought to be due to the polymerization of plant proteins at pH 6-7, in contrast to the pH of whey protein at 4.6. A further advantage of the present invention is that the use of plant proteins as an encapsulation matrix makes the product suitable for vegetarians and vegans. This disclosure describes numerous methods for encapsulating probiotics using denatured pea protein and denatured mung bean protein (as examples of plant proteins), all of which utilize calcium salt chelating agents that allow the polymerization of denatured plant proteins at a slightly acidic pH, which does not have a harmful effect on the survival or metabolism of the probiotics. The examples provided herein illustrate encapsulated probiotics produced by extrusion into a gelling bath and gel immobilization and drying (e.g., freeze-drying or vacuum drying). The present invention also describes a method for producing a suspension of plant protein (generally denatured pea protein or denatured mung bean protein) obtained by hydrating the protein at pH 7-8, allowing the protein suspension to stand, and a two-step heating step typically carried out under high pressure. Embodiments of this process have been found to produce a suspension of plant protein with a high solids content (e.g., up to 15%), a high proportion of soluble protein (e.g., 90% or more), and a pH that is neither acidic nor alkaline, making this suspension ideal for microencapsulating pH-sensitive drugs such as probiotics.Furthermore, an alternative heat treatment (ultra-high temperature (UHT) denaturation) is employed to denature plant proteins, which results in a more efficient encapsulation process due to a reduction in the viscosity of the protein solution. The method of this disclosure can also be applied to encapsulate plant extracts, vitamins, minerals, marine bioactive substances, and other activators such as amino acids.

[0007] In a first embodiment, the present invention provides a method for forming microparticles containing an activator such as a probiotic encapsulated in a denatured plant protein matrix, the method comprising: A step of preparing a protein suspension containing denatured plant proteins, The step of mixing this protein suspension with the activator to form a mixture, A step of processing this mixture to form microparticles containing an encapsulated activator in a denatured plant protein matrix, wherein the processing step includes polymerizing the denatured plant protein matrix with a calcium salt or spray englobing it on a fluidized bed dryer; and Step to dry the fine particles Includes.

[0008] In any embodiment, the method includes the step of preparing a suspension of hydrated probiotics, the mixing step of mixing a protein suspension with the suspension of hydrated probiotics.

[0009] In any embodiment, the plant protein is either a plant protein isolate or comprises a plant protein isolate.

[0010] In any embodiment, the plant protein is either pea protein or mung bean protein, or comprises pea protein or mung bean protein.

[0011] In any embodiment, a simple carbohydrate is added to the protein suspension. Examples include monosaccharides and / or disaccharides. The simple carbohydrate is added to the suspension before encapsulation to form a charged slurry, enhancing the compatibility between the denatured plant protein suspension and the probiotic cells, resulting in a stable equilibrium of the cells in the protein matrix during encapsulation, and creating a charged slurry that interacts with the charged cell membranes of each probiotic. The simple carbohydrate also helps to cause instantaneous chelation / polymerization through the interaction between the charged simple carbohydrate and the denatured plant protein after heat treatment. This charged interaction encapsulates the sensitive bacteria and forms a strong lattice for encapsulation.

[0012] In any embodiment, the processing step includes extruding the protein suspension and the activator suspension to form microdroplets, and the polymerization step includes curing the extruded microdroplets in a curing bath containing a chelating agent such as calcium salt to form microcapsules.

[0013] In any embodiment, the curing bath contains a calcium citrate buffer.

[0014] In any embodiment, the curing bath contains a calcium citrate buffer having a pH of 5 - 6.5.

[0015] In any embodiment, the curing bath contains a calcium citrate buffer having a molar concentration of 0.05 - 0.15 M.

[0016] In any embodiment, the curing bath contains a calcium citrate buffer having a pH of 5.2 - 6.1.

[0017] In any embodiment, the curing bath contains a calcium citrate buffer having a molar concentration of approximately 0.1 M.

[0018] In any embodiment, the curing bath contains a calcium chloride buffer solution.

[0019] In any embodiment, the curing bath contains a calcium chloride buffer solution having a pH of 4.3 to 5.

[0020] In any embodiment, the curing bath contains a calcium chloride buffer solution having a molar concentration of 0.05 to 0.15 M, or about 0.1 M.

[0021] In any embodiment, the curing bath contains a surfactant, particularly a nonionic surfactant, particularly a polysorbate-type nonionic surfactant. In any embodiment, the curing bath contains 0.01 to 0.1, particularly about 0.05% surfactant.

[0022] In any embodiment, the curing bath contains chitosan. In any embodiment, the curing bath contains 0.01 to 0.1, particularly about 0.05% chitosan.

[0023] In any embodiment, the processing step includes adding a calcium salt buffer solution to gel the mixture, drying the gelled mixture by lyophilization or vacuum drying, and reducing the size of the dried gelled mixture to provide microparticles.

[0024] In any embodiment, the calcium salt buffer solution contains a calcium citrate buffer having a pH of 5 to 6.5.

[0025] In any embodiment, the calcium salt buffer solution contains a calcium citrate buffer solution having a molar concentration of 0.05 to 0.15 M.

[0026] In any embodiment, the calcium salt buffer solution is added to the mixture at a volume ratio of 1:100 to 1:300.

[0027] In any embodiment, the protein suspension contains 10-15%, 11-15%, 12-15%, 13-15%, or 14-15% (by weight) of denatured plant protein.

[0028] In each embodiment, at least 90%, 91%, 92%, 93%, 94%, or 95% of the protein in the protein suspension is solubilized.

[0029] In any embodiment, the protein suspension contains 0.5–12.0%, 1.0–6.0%, 1.0–3.0%, or about 2% simple sugar (w / v).

[0030] In any embodiment, the mixing step includes mixing the protein suspension and the activator suspension in a dry weight ratio of denatured plant protein to activator of 1:5 to 1:25 or 1:8 to 1:20.

[0031] In any embodiment, the activator is a probiotic, and the suspension of the hydrated probiotic is suspended in 0.05–0.15 M, 0.08–0.12 M, or about 0.1 M phosphate buffer.

[0032] In each embodiment, the simple carbohydrate comprises maltodextrin and glucose. GLUCIDEX is a commercially available product containing maltodextrin and glucose.

[0033] In any embodiment, the method includes forming fine particles by spray engroving, in which the mixing and processing steps are carried out on a fluidized bed dryer.

[0034] In any embodiment, the method includes adding a carrier material and an activator to the floor of a fluidized bed dryer, fluidizing the carrier material and activator, spraying a first coating material onto the fluidized carrier material and activator to produce fine particles having an activator and carrier trapped within the shell of the first coating material, and drying the fine particles.

[0035] In each embodiment, a simple sugar is sprayed into the fluidized bed dryer before the first coating material is sprayed into the fluidized bed dryer, the simple sugar forms granules, and the first coating material coats these granules.

[0036] In any embodiment, the first coating material is selected from denatured plant proteins, oils, and simple sugars such as maltodextrin.

[0037] In any embodiment, the method comprises spraying a second coating material onto the shell of a first coating material, wherein at least one of the first and second coating materials is a denatured plant protein.

[0038] In any embodiment, the method includes spraying a chelated salt onto a denatured plant protein coating.

[0039] The following particles are suspected: Fine particles 1 Core of activator (such as probiotics) and carrier material Polymerized denatured plant protein coating fine particles 2 Activators (such as probiotics), simple sugars, and core of carrier material Coating of non-polymerizable drying-denatured plant proteins fine particles 3 Core of activator (such as probiotics) and carrier material Polymerized denatured plant protein inner coating Non-polymerizable drying-denatured plant protein outer layer An intermediate coating of oil. fine particles 4 Core of activator (such as probiotics) and carrier material Polymerized denatured plant protein inner coating Polymerized denatured plant protein outer coating The first intermediate coating of oil. A second intermediate coating for non-polymerizable, dehydration-denatured plant proteins.

[0040] In one embodiment, spray engroving includes the following steps. (a) Adding a carrier material (e.g., native protein), an activator (e.g., probiotics), and optionally a simple sugar to the floor of a fluidized bed drying chamber. (b) A step of fluidizing the carrier material and the active substance and heating it to form a first fluidized powder, (c) A step of spraying a denatured protein suspension onto a fluid bed at high pressure (e.g., 2-4.5 Bar) to provide a second fluidized powder. (d) A step of drying the second fluidized powder on a fluidized bed to reduce the moisture content of the second fluidized powder (for example, reducing the moisture content by at least 30%, 40%, or 50%), (e) A step of forming a third fluid powder by spraying an englobing component onto a second fluid powder, wherein the englobing component is selected from chelate salts (e.g., calcium salts or magnesium salts); edible oils; and simple sugars. (f) A step of drying the third fluidized powder on a fluidized bed to further reduce the moisture content of the fluidized powder (for example, to further reduce the moisture content by at least 10%, 20%, or 25%), (g) A step of spraying a denatured protein suspension onto a fluid bed under high pressure (e.g., 2-4.5 Bar) to provide a fourth fluidized powder containing fine particles, and Optionally, the fourth fluidized powder is further dried to typically have a moisture content of less than 10%, less than 8%, or less than 5%.

[0041] In any embodiment, the method comprises at least two or three rounds of steps (f) and (g).

[0042] In any embodiment, if the edible oil component is edible oil, the method includes an additional step of spraying a chelated salt onto the third fluidized powder before the second drying step. The method may include at least two or three rounds of spraying edible oil and then spraying a chelated salt.

[0043] Generally, the spraying step mentioned above is top spraying, where, for example, the components are sprayed from above the fluidized bed.

[0044] In any embodiment, the method includes the following additional steps: (h) The step of bottom spraying the denatured protein suspension onto the fourth fluidized mixture to form a fifth fluidized powder. (i) A step of drying the fifth fluidized powder on a fluidized bed to further reduce the moisture content of the fluidized powder (for example, further reducing the moisture content by at least 10%, 20%, or 25%), (j) A step of forming a sixth fluid powder by bottom spraying an engroving component onto a fifth fluid powder, wherein the engroving component is selected from a chelated salt (e.g., a calcium salt or a magnesium salt) and an edible oil. (k) drying the sixth fluidized powder on a fluidized bed to further reduce the moisture content of the fluidized powder (for example, further reducing the moisture content by at least 10%, 20%, or 25%), and (l) Optionally, further dry the fourth fluidized powder to a moisture content of less than 10%, less than 8%, or less than 5%.

[0045] In any embodiment, the method includes steps (j) and (k) of at least two or three rounds.

[0046] In any embodiment, if the engroving component of step (j) is edible oil, the method includes an additional step of spraying a chelated salt onto the sixth fluidized powder before step (k). The method may include at least two or three rounds of spraying edible oil, and then spraying the chelated salt.

[0047] In all embodiments, the fluidized bed is 110-300 m 3 It becomes fluid due to the air currents of time.

[0048] In any embodiment, the fluidized bed chamber is heated during all or part of the method, typically to 35–45°C.

[0049] In all embodiments, the protein solution is sprayed onto the fluid bed at a high spray nozzle pressure, for example, 2 to 4.5 Bar.

[0050] In each embodiment, the simple sugar is added to the second fluidized powder in an amount of 10-20% of the total dry solids content.

[0051] In each embodiment, the simple sugar is maltodextrin or contains maltodextrin.

[0052] In all embodiments, the chelated salt is added as a 0.2-0.4 M solution.

[0053] In each embodiment, edible oil is added to the second fluidized powder in an amount of 5-15% of the total dry solids content.

[0054] In any embodiment, step (a) includes adding the carrier material and the activator to the floor of the fluidized bed drying chamber in a weight ratio of 1:5 to 5:1.

[0055] The carrier material is generally a native protein, but may also be a carbohydrate such as maltodextrin or starch, a powder of prebiotic fiber, or any colloid.

[0056] In another embodiment, the spray engroving is The step of fluidizing carrier materials such as native proteins, activators (such as probiotics), and chelate salts on the fluidized bed of a fluidized bed drying chamber. Steps include spraying edible oil into the fluid bed drying chamber, Steps include spraying a suspension of denatured proteins into a fluid bed drying chamber, Includes, As a result, the chelated salt reacts with the denatured plant protein to polymerize the protein, forming aggregated microparticles having a polymerized denatured protein coating and a core containing an activator and a carrier material.

[0057] In all embodiments, the carrier material is added to the fluidized bed first, and the activator and chelating salt are added to the fluidized bed after the carrier material.

[0058] In all embodiments, the carrier material is fluidized at a high temperature (e.g., 30-40°C) before adding the activator and chelate salt.

[0059] In each embodiment, the carrier material, activator, and chelate salt are fluidized at a high temperature (e.g., 30-40°C) before the addition of edible oil to form a fluidized mixture.

[0060] In all embodiments, simple sugars are sprayed onto the fluidized mixture before the edible oil to promote granulation of the mixture.

[0061] In each embodiment, the simple sugar is sprayed into the fluidized bed chamber at a high flow rate (e.g., 23-30 RPM), low airflow (e.g., 150 RPM or higher), and low spray nozzle pressure (e.g., less than 1.5 Bar).

[0062] In any embodiment, the oil is sprayed into the fluidized bed chamber at a low flow rate (e.g., 18-22 RPM), high airflow (e.g., greater than 150 RMP), and low spray nozzle pressure (e.g., less than 1.5 Bar).

[0063] In each embodiment, the suspension of denatured plant proteins is sprayed into a fluidized bed drying chamber at a high flow rate (e.g., 23–30 RPM), high airflow (e.g., >150 RMP), and high spray nozzle pressure (e.g., >2.5 Bar).

[0064] In all embodiments, the activator is added in the form of an oil-in-water nanoemulsion, and the activator is contained in the oil phase of the oil-in-water nanoemulsion. This is particularly suitable when the activator is hydrophobic.

[0065] In each embodiment, the oil-in-water nanoemulsion is the oil-in-water nanoemulsion of the present invention.

[0066] In each embodiment, the carrier material is typically a native protein having a water content of about 5%, 4%, 3%, or 2% or less.

[0067] In all embodiments, the oil is an edible oil, such as a high-oleic acid oil.

[0068] In all embodiments, the floor is fluidized with air at 30-40°C and a high airflow rate (approximately 30-55 RPM).

[0069] In each embodiment, the native protein and the denatured protein are plant proteins, such as pea protein or mung bean protein.

[0070] In all embodiments, the native protein and the denatured protein are protein isolates.

[0071] In all embodiments, the suspension of denatured plant proteins is the denatured plant protein suspension of the present invention.

[0072] In all embodiments, the aggregated microparticles are gastric-resistant and ileum-sensitive. This means that the microparticles pass through the human stomach intact and are broken down in the ileum, releasing the activator.

[0073] In each embodiment, the protein suspension is A step in which plant proteins are hydrated in an aqueous solvent with a pH of 7-8 while stirring and optionally heating. Steps include: allowing the aqueous suspension of plant protein to stand; If necessary, adjust the pH of the aqueous suspension to 7-8. The steps include: optionally heating a standing aqueous suspension under pressure, and Step of cooling the heated aqueous suspension. It is formed by a method that includes the following.

[0074] In any embodiment, the aqueous suspension of plant protein is allowed to stand for at least 30, 60, or 90 minutes.

[0075] In all embodiments, the hydration step is carried out at a temperature of 25-40°C, 30-40°C, and ideally about 35°C.

[0076] In any embodiment, the heating step is The steps include preheating a standing aqueous suspension under pressure at a first heating temperature of 60-70°C (e.g., approximately 65°C) for a first heating time (e.g., 5 seconds to 15 minutes), and The step involves heating the preheated aqueous suspension under pressure at a second heating temperature of 90-100°C (e.g., approximately 95°C) for a second heating time (2 seconds to 3 minutes). Includes.

[0077] In any embodiment, the heating step includes heating the plant protein suspension to achieve a heat treatment of F0=3. This causes denaturation of the plant proteins (e.g., to at least 80% denaturation) and kills bacterial spores in the suspension. The heat treatment also reduces the viscosity of the protein suspension, making it easier to handle. The term "F0" is defined as the number of equivalent minutes for steam sterilization of the feed (load) (product) at 250°F (121°C).

[0078] The ultra-high temperature denaturation process serves three purposes when denaturation denatures plant proteins: (a) killing bacterial spores in the protein suspension, ensuring the product is food-grade quality and safe for human consumption; (b) this high-temperature denaturation is necessary to provide the relevant functionality for polymerizing the plant protein; and (c) generating viscosity that allows for better flow rates during the encapsulation process, resulting in higher efficiency and higher throughput during encapsulation. The ultra-high temperature denaturation process has been found to reduce the viscosity of plant protein suspensions from 300-550 cP at 21+ / -1°C to 50-120 cP at 21+ / -1°C. This step also helps ensure that the encapsulated material is clean from contaminants and acceptable for commercial use and human consumption when the microparticles of the present invention fortify beverages.

[0079] In each embodiment, the heating step includes ultra-high temperature (UHT) treatment of the plant protein suspension.

[0080] In each embodiment, the heating step includes heating the suspension to about 138.5°C for about 3 to 4 seconds.

[0081] In all embodiments, the aqueous solvent has a pH of approximately 7.5 (e.g., 7.3-7.7).

[0082] In all embodiments, the heating step is performed at a pressure of 1.3 to 1.6 Bar.

[0083] In any embodiment, the pea protein is either a pea protein isolate or a mung bean protein isolate, or comprises a pea protein isolate or a mung bean protein isolate.

[0084] In all embodiments, the cooled protein suspension is allowed to settle at room temperature for at least 2 hours.

[0085] In any embodiment, the method includes the step of adding simple carbohydrates to a protein suspension to provide a protein suspension containing typically 0.5–5.0%, 1–3%, or about 2% simple carbohydrates (w / v).

[0086] In any embodiment, the method has a % probiotic survival rate greater than 50%, 55%, 60%, 65%, 70%, or 75%. The % probiotic survival rate as applied to the method of the present invention refers to the percentage of probiotic bacteria that survive the encapsulation step. The method for calculating the % probiotic survival rate is shown below.

[0087] In another embodiment, the present invention provides a composition comprising an activator, such as a probiotic encapsulated in a denatured plant protein matrix, formed according to the method of the present invention. The composition is generally in the form of particles, such as microparticles, which may be granules.

[0088] In any embodiment, the microparticles can have an average size of 50-700, 200-700, 300-700, 400-700, 500-600, 50-400, 50-300, 50-200, 50-150, 50-100, 20-100, 20-50, 100-500, 100-400, 100-300, or 100-200 (in microns, μm). When formed by extrusion into a bath, the microparticles typically have an average size of 5000-600 μm. When using a single nozzle, the microparticles have a continuous denatured protein matrix and probiotic bacteria distributed throughout the matrix (these are called microbeads). When a concentric nozzle is employed, the microparticles have a core-shell morphology, where the shell consists of denatured protein and the core consists of probiotic bacteria (these are called microcapsules). When formed by gel immobilization, microparticles typically have an average dimension of 50–150 μm. When formed by spray immobilization, fine particles typically have an average dimension Dv of 50–500, 100–500, 200–500, and 300–400 μm.

[0089] In all embodiments, the microparticles are gastric-tolerant and ileum-sensitive. This means that the microparticles pass through the human stomach intact, break down in the ileum to release a probiotic payload, and enable subsequent colonization.

[0090] In another embodiment, the present invention provides a method for forming a denatured plant protein suspension, comprising the following steps: A step of hydrating plant proteins in an aqueous solvent with a pH of 6-8 or 7-8, while stirring and optionally heating. Steps include: allowing the aqueous suspension of plant protein to stand; If necessary, adjust the pH of the aqueous suspension to 6-8 or 7-8. The steps include: heating the standing aqueous suspension, and A step of cooling a heated aqueous suspension.

[0091] In each embodiment, the standing aqueous suspension is heated to achieve a heat treatment of F0=3.

[0092] In all embodiments, the standing aqueous suspension is subjected to ultra-high temperature (UHT) treatment.

[0093] In all embodiments, the standing aqueous suspension is heated to approximately 138.5°C for approximately 3-4 seconds.

[0094] In any embodiment, the aqueous suspension of plant protein is allowed to stand for at least 30, 60, or 90 minutes.

[0095] In all embodiments, the hydration step is carried out at a temperature of 25-40°C, 30-40°C, and ideally about 35°C.

[0096] In any embodiment, the heating step is The steps include preheating a standing aqueous suspension under pressure at a first heating temperature of 60-70°C (e.g., approximately 65°C) for a first heating time (e.g., 5 seconds to 15 minutes), and The step involves heating the preheated aqueous suspension under pressure at a second heating temperature of 90-100°C (e.g., approximately 95°C) for a second heating time (2 seconds to 3 minutes). Includes.

[0097] In all embodiments, the aqueous solvent has a pH of approximately 7.5 (e.g., 7.3-7.7).

[0098] In all embodiments, the heating step is performed at a pressure of 1.3 to 1.6 Bar.

[0099] In all embodiments, the plant protein is a plant protein isolate.

[0100] In all embodiments, the plant protein is pea protein.

[0101] In all embodiments, the plant protein is mung bean protein.

[0102] In any embodiment, the cooled protein suspension is allowed to settle at room temperature for at least 1, 2, 3, 4, or 5 hours.

[0103] In any embodiment, the method includes the step of adding simple carbohydrates to a protein suspension to provide a protein suspension containing typically 0.5–5.0%, 1–3%, or about 2% simple carbohydrates (w / v).

[0104] In any embodiment, the native protein and the denatured protein are plant proteins, such as pea protein or mung bean protein. In any embodiment, the native protein and the denatured protein are derived from the same source (e.g., both are pea protein). In any embodiment, the native protein and the denatured protein are protein isolates.

[0105] In all embodiments, the suspension of denatured plant proteins is the denatured plant protein suspension of the present invention.

[0106] In any embodiment, the denatured plant protein suspension contains 10-15%, 11-15%, 12-15%, 13-15%, or 14-15% (by weight) of denatured plant protein.

[0107] In each embodiment, at least 90%, 91%, 92%, 93%, 94%, or 95% of the protein in the denatured plant protein suspension is solubilized.

[0108] In another embodiment, the present invention provides a denatured plant protein suspension formed according to the method of the present invention.

[0109] In another embodiment, the present invention provides a composition of microparticles comprising an activator encapsulated within a denatured plant protein structure, the denatured plant protein structure typically comprising a polymerized denatured plant protein.

[0110] In all embodiments, the activator is encapsulated within the carrier. The carrier may be an edible oil droplet in which the activator is encapsulated within an edible droplet, a native protein, or a carrier powder such as maltodextrin or a sugar such as starch.

[0111] In each embodiment, the polymerization-modified plant protein structure is a polymerization-modified plant protein matrix, and the oil droplets are distributed through the matrix.

[0112] In any embodiment, the microparticles have a core-shell structure comprising a shell of denatured plant protein and a core typically containing an activator in a carrier. The carrier may be a powder (as described above), an edible oil droplet, or a solution / suspension of the activator.

[0113] In all embodiments, the shell of the denatured plant protein is polymerized.

[0114] In any embodiment, the fine particles have two or more shells, at least one of which is a shell of a denatured plant protein, and at least one of which is selected from an oil shell and a shell of a non-polymerizable denatured plant protein.

[0115] In all embodiments, the fine particles are granules.

[0116] In another embodiment, the present invention provides any form of food, beverage or nutritional supplement comprising the composition of the present invention.

[0117] In any embodiment, the beverage, food, or nutritional supplement is storage-stable.

[0118] In each embodiment, the food or beverage is pasteurized or treated with UHT.

[0119] In all embodiments, the beverage is either a carbonated beverage or a non-carbonated acidic beverage.

[0120] In each embodiment, the beverage is a fermented beverage (e.g., kombucha).

[0121] In any embodiment, the method achieves a probiotic % survival rate of 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0122] In all embodiments, the aggregated microparticles are gastric-resistant and ileum-sensitive. This means that the microparticles pass through the human stomach intact and are broken down in the ileum to release the probiotic payload.

[0123] Other aspects and preferred embodiments of the present invention are defined and described in the other claims presented below. [Brief explanation of the drawing]

[0124] [Figure 1] Figure 1 shows an image of probiotic-modified pea protein microcapsules formed according to the method of Example 20 and imaged before the drying step. This microscopic image represents the polymerized wet capsules. The average diameter is 500–600 μm. [Figure 2] Figure 2 shows images of probiotic-modified pea protein microcapsules formed according to the method of Example 20 and imaged after the drying step. The microscopic images show individual microcapsules in a free-flowing state. Average diameter 485–550 μm. [Figure 3A] Figure 3A summarizes the probiotic survival rates of pea protein versus whey (milk) protein and probiotics encapsulated using a single-nozzle encapsulation device. [Figure 3B]This is a summary of probiotic survival rates for probiotics encapsulated using various plant protein encapsulation matrices and single-nozzle extrusion technology. [Figure 4] Figure 4 shows a comparison of particle sizes between pea protein and a probiotic encapsulation system produced using a single-nozzle encapsulation device. [Figure 5A] Figure 5A shows the probiotic release rate profile of encapsulated probiotic cells in a pea protein encapsulation system manufactured using single-nozzle encapsulation extrusion technology. The data represent cell release as a function of in vitro human digestion time and GI section. [Figure 5B] Figure 5B shows the probiotic release rate profile of encapsulated probiotic cells in a hyperdenatured pea protein encapsulation system manufactured using single-nozzle encapsulation / extrusion technology. The data represent cell release as a function of in vitro human digestion time and GI section. [Figure 6] Figure 6 shows HPLC protein digestion as a function of the GI section. The HPLC data indicate the initiation of protein degradation in the intestine, which leads to controlled cell release. This data represents the digestion profile of cell release from a pea protein encapsulation system manufactured using extrusion, single-nozzle encapsulation technology. [Figure 7] Figure 7 shows an HPLC chromatogram for the quantification of proteolysis in human in vitro digestive analysis. The data shows the initiation of protein digestion in the intestine at 45 minutes (black) and 60 minutes (pink), which indicates controlled cellular release due to sustained and controlled digestion of plant protein microcapsules. This data represents the digestive profile of cellular release from plant protein encapsulation systems manufactured using extrusion, single-nozzle encapsulation technology. [Figure 8]Figure 8 shows micrographs of Bifidobacteria (lactic acid bacteria) encapsulated in plant proteins produced by single-nozzle / extrusion technology: (A) mung bean protein, (B) broad bean protein, and (C) chickpea protein. [Figure 9A] Figure 9 shows the pH characteristics of plant proteins before and after UHT denaturation, with (A) flaxseed (linseed) protein isolate and (B) pumpkin seed protein isolate. [Figure 9B] Figure 9 shows the pH characteristics of plant proteins before and after UHT denaturation, with (A) flaxseed (linseed) protein isolate and (B) pumpkin seed protein isolate. [Figure 10A] Figure 10 shows the pH characteristics of plant proteins before and after UHT denaturation. (A) is a broad bean (Fava (Fava / Broad)) protein isolate and (B) is a mung bean protein isolate. [Figure 10B] Figure 10 shows the pH characteristics of plant proteins before and after UHT denaturation. (A) is a broad bean (Fava (Fava / Broad)) protein isolate and (B) is a mung bean protein isolate. [Figure 11] Figure 11 shows the pH characteristics of pea protein hydrolysates before and after the UHT pea protein denaturation procedure. This allows us to confirm the denaturation of partially hydrolyzed pea protein. [Modes for carrying out the invention]

[0125] Detailed description of the invention All publications, patents, patent applications, and other documents described herein are incorporated herein by reference in their entirety for any purpose as if their entire contents were included, as is specifically and individually indicated to be incorporated by reference in their entirety.

[0126] As used herein, unless otherwise indicated, the following terms are intended to have the following meanings in addition to the broader (or narrower) meanings that such terms may enjoy in the art:

[0127] Unless otherwise specified in the context, the singular form in this specification includes the plural form, and vice versa. The term "a" or "an" used in relation to an entity refers to one or more of those entities. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification.

[0128] Where used herein, the term “comprising,” or its variations such as “comprises,” or “comprising,” should be read as indicating that it includes any implicit integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature, element, characteristic, property, method / process step, or limitation), but does not exclude other integers or groups of integers. Therefore, where used herein, the term “comprising” is comprehensive or open-ended and does not exclude additional, implicit integers or methods / process steps.

[0129] In this specification, the term "plant protein" refers to protein preparations obtained from plant sources. Examples include plant protein powders, plant protein concentrates, and plant protein isolates. Plant protein preparations can be obtained from peas, zein, barley, spelt, soybeans, seaweed, hemp, seiten (wheat gluten), chickpeas, tofu, lentils, mung beans, quinoa, and other plant sources. Plant proteins having an isoelectric point of pH 5, 5.5, or 6 or higher are preferred.

[0130] As used herein, the term "simple carbohydrate" includes monosaccharides such as glucose, fructose, and sucrose, as well as disaccharides such as sucrose, lactose, and maltose, and mixtures thereof. In one embodiment, the simple carbohydrate includes GLUCIDEX.

[0131] As used herein, the term “probiotic” refers to live, beneficial bacteria and / or yeasts that naturally inhabit the body. Probiotics help keep the body healthy and functioning properly, such as by combating an overgrowth of harmful bacteria and helping to improve mood. The probiotics used in this invention may consist of combinations of different probiotics. While there are many types of bacteria that can be considered probiotics, there are two specific types of bacteria that are commonly found as probiotics in stores. These include Lactobacillus and Bifidobacterium.

[0132] As used herein, the term “% probiotic survival” applied to compositions produced according to the method of the present invention means the percentage of viable cells that survive the encapsulation step (including the drying step, if the step includes a drying step). Probiotic survival can be measured by measuring the number of viable cells introduced into the step and comparing it to the number of cells in the output product.

[0133] As used herein, the term “activator” refers to any component suitable for delivery to the small intestine or ileum of mammals, but typically means a component that is sensitive to external conditions such as heat, pH, moisture, pressure, chemical stress, or enzymes. Thus, active components may be sensitive to pH, enzymes (i.e., protease enzymes), high pressure, moisture, high shear, and temperature disturbances during storage. In one particularly preferred embodiment of the present invention, the activator includes probiotics, or bioactivators selected from, for example, caffeine, fat-soluble vitamins (A, D, E, K), water-soluble vitamins (B and C), hyaluronic acid, melatonin, and marine bioactivators such as fucoidan and laminarin, as well as extracts of green algae, brown algae, and red algae.

[0134] "Gastric tolerance" means that the microparticles can remain intact for at least 60 minutes in a digestion model of a stimulated stomach described by Minekus et al. in 1999 and 2014 (A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation product, Minekus, M., Smeets-Peeters M, Bernalier A, Marol-Bonnin S, Havenaar R, Marteau P, Alric M, Fonty G, Huis in't Veld JH, Applied Microbiology Biotechnology. 1999 Dec;53(1):108-14 (Non-Patent Literature 1)), and (Minekus et al., 2014, A standardised static in vitro digestion method suitable for food - an international consensus, Minekus, A. et al., Food Function, 2014, 5, 1113) (Non-Patent Literature 2)).

[0135] "Ileal sensitivity" means that the microparticles can release their contents in vivo in the mammalian ileum (proximal or distal). [Examples]

[0136] Example Next, the present invention will be described with reference to specific examples. These are merely illustrative and for illustrative purposes only. They do not limit in any way the claims or the scope of the described invention. These examples constitute the best possible mode currently considered for carrying out the present invention.

[0137] Example 1 Preparation of denaturing pea protein solution - Hydrate 14% (w / v) pea protein isolate in water or 0.1 M phosphate buffer pH 7.5. - Hydrate at 30°C for 45 minutes. - Use a jacketed container or a 30°C hot plate and stir at 450 RPM. - As a necessary settling step, leave it for at least 90 minutes. - Check the pH of the water and confirm that it is pH 7.5. If it is not pH 7.5, adjust it using 1N HCl / NaOH. - Heat treatment is performed as follows: Preheat to 65°C for a minimum of 2 seconds and a maximum of 15 minutes. · Final heating 95.5℃, minimum 2 seconds / maximum 3 minutes · Cooling temperature 22~25℃ · Flow rate 1.5L~20L / min • Pressure 1.3~1.6 bar - Allow the materials to settle at room temperature for 4 hours before use. - This material is called "dPPI".

[0138] Example 2 Preparation of hyperdenatured pea protein - Hydrate 14% (w / v) pea protein in water or 0.1M phosphate buffer pH 7.5. - Hydrate at 30°C for 45 minutes. - Use a jacketed container and stir at 450 revolutions per minute. - As a necessary settling step, leave it for at least 90 minutes. - Check the pH of the water and confirm that it is pH 7.5. If it is not pH 7.5, adjust it using 1N HCl / NaOH. - Heat treatment as follows: Preheat to 60-98°C for 2 seconds. Final temperature: 126-144.5°C Holding time - Minimum 2 seconds per step a Cooling temperature 22~25℃ Flow rate: min. 1.45L~2.0L / min Pressure 1.0~6.0 bar - Allow to settle at room temperature for 2 hours before use. - This material is called "udPPI".

[0139] Example 3 Preparation of hyperdenatured mung bean protein - Hydrate 10% (w / v) mung bean protein isolate in water or 0.1 M phosphate buffer pH 7.5. - Hydrate at 30°C for 90 minutes. - Use a jacketed container and stir at 450 revolutions per minute. - A minimum of 90 minutes of rest is required. - Check the pH of the water and confirm that it is pH 7.5. If it is not pH 7.5, adjust it using 1N HCl / NaOH. - Heat treatment as follows: Preheat to 60-90°C Final heating: 120-144°C for 2 seconds Cooling temperature 22-25°C, 5 seconds Flow rate 1.5L~2.0L / min Pressure 1.0~6.0 bar - Allow the materials to settle at room temperature for 2 hours before use. - This material is called "udMBP".

[0140] Example 4 Probiotic hydration - Prepare a stock solution of probiotics. Hydrate with phosphate buffer pH 0.1M. - Add the probiotics (liquid or powder) to the phosphate buffer. - Stir the mixture at 200-450 RPM for 10-45 minutes using RT to revive the cells. - If necessary, Ultraturrax cell suspension (9,000-11,000 RPM, 30-90 seconds).

[0141] Example 5 Immobilization of probiotics to PPIs using modified PPIs - Using dPPI prepared as in Example 1 and hydrated probiotics prepared as in Example 4, the probiotic suspension is added to the dPPI solution at 410 RPM to provide a PPI to probiotic suspension in a ratio of 9:1 or 18:1. - Before encapsulation, add 2% glucidex / simple carbohydrate to the protein solution (this imparts charge to the denatured protein solution and creates a matrix compatible with probiotics). - To avoid the formation of air pockets, hydrate at RT for 2 hours with gentle stirring. - After adding all the probiotics, add 0.1M / pH6.1 calcium citrate or calcium chloride 2 minutes later. - Add calcium citrate until the buffer / suspension ratio is 1:200. - Maintain a high level of agitation at 450 RPM, RT during pH adjustment. - Maintain stirring for at least 15 minutes, then transfer to a drying chamber. - The solution will appear more viscous to the naked eye. - Drying (freeze-drying or vacuum drying) can be performed as follows: - Freeze-dried (see Table 1)

[0142] [Table 1]

[0143] - Vacuum drying: Set the product temperature to 25°C. The chamber containing the product is evacuated. Once a complete vacuum (i.e., less than 10 mBar) is achieved, dry for 24 hours.

[0144] After drying, the dried product can be reduced to a granular form using a high-shear solid separator.

[0145] Example 6 Immobilization of probiotics onto plant proteins using modified mung beans - Using dMBP prepared as in Example 3 and hydrated probiotics prepared as in Example 4, the probiotic suspension is added to the dMBP solution at 410 RPM to provide a dMBP to probiotic suspension in a ratio of 9:1 or 18:1. - Before encapsulation, add 2% glucidex / simple carbohydrate to the protein solution (this imparts charge to the denatured protein solution and creates a matrix compatible with the probiotics). - To avoid the formation of air pockets, hydrate with RT for 2 hours while gently stirring. - After adding all the probiotics, add 0.1M / pH6.1 calcium citrate or calcium chloride 2 minutes later. - Add calcium citrate until the buffer / suspension ratio is 1:200. - Maintain a high level of agitation at 450 RPM, RT during pH adjustment. - Maintain stirring for at least 15 minutes, then transfer to a drying chamber. - The solution will appear more viscous to the naked eye. - Drying (freeze-drying or vacuum drying) can be performed as follows: - Freeze-dried (see Table 1) - Vacuum drying: Set the product temperature to 25°C. Create a vacuum inside the chamber. Once a complete vacuum (i.e., less than 10 mBar) is achieved, dry for 24 hours. After drying, the dried product can be reduced to particulate size using a high-shear solid separator.

[0146] Example 7 Microbeads containing probiotics encapsulated in denatured mung bean protein - single nozzle extrusion - Using the dMBP prepared according to Example 3, prepare a suspension of dMBP and stir at 250 RPM, RT. - Before encapsulation, add 2% glucidex / simple carbohydrate to the protein solution (this charges the denatured protein solution and forms a matrix compatible with the probiotics). - To prevent the formation of air pockets, hydrate at room temperature for 2 hours while gently stirring. - Add the hydrated probiotic suspension from Example 4 to the dMBP solution and stir with RT. - Add the probiotic suspension to the protein in a 9:1 or 18:1 ratio (protein:probiotics). - Stir the protein-probiotic suspension at 400-450 RPM for 10 minutes.

[0147] Preparation of the curing bath - Prepare a 0.1M calcium citrate buffer and adjust the pH to 5.2 (range 5.2-6.1). Add 20% 0.05% polysorbate as a surfactant. - For the alternative curing bath, use 0.1M calcium chloride buffer and adjust the pH to 4.8 (range 4.3-5.0). Add 20% 0.05% polysorbate as a surfactant. - The alternative curing bath is a 0.1M calcium citrate buffer containing 0.05% chitosan calcium (chelating agent) and 0.05% polysorbate 20% (surfactant). - Once the probiotics are fully hydrated, the protein-probiotic solution is extruded at room temperature using a single-nozzle extruder and polymerized in a calcium-hardening bath. Polymerization occurs in a calcium buffer bath at room temperature. - Collect the microcapsules and wash them with a pH 6.1 neutralizing solution (to prevent microcapsule adhesion during drying and optimize yield). - Fill the associated drying chamber with microcapsules. - Drying can be done as follows: ○ 48 hours of freeze-drying (see Table 1) ○ Drying of fluidized bed: 37℃, 4-6 hours ○ Vacuum drying: Less than 10 mBar, at 37°C for a minimum of 24-48 hours

[0148] Example 8 Microbeads containing probiotics encapsulated in denatured plant proteins - single nozzle extrusion - Using the denatured plant protein prepared according to Example 1, prepare a suspension of denatured protein and stir at 250 RPM, RT. - Add 2% to 4% simple carbohydrates to the protein solution before encapsulation (this imparts charge to the denatured protein solution, forming a matrix compatible with probiotics). - To prevent the formation of air pockets, hydrate at room temperature for 2 hours while gently stirring. - Add the hydrated probiotic suspension of denatured protein aqueous solution and stir with RT. - Add the probiotic suspension to the protein in a 9:1 or 18:1 ratio (protein:probiotics). - Stir the protein-probiotic suspension at 400-450 RPM for 10 minutes.

[0149] Preparation of the curing bath - Prepare a 0.1M calcium citrate buffer solution and adjust the pH to 5.2 (range 5.2-6.1). Add 20% 0.05% polysorbate as a surfactant. - Adjust the pH to 4.8 (range 4.3-5.0) with 0.1M calcium cation buffer. Add 20% 0.05% polysorbate as a surfactant. - The alternative curing bath is a 0.1M calcium citrate buffer containing 0.05% chitosan calcium (chelating agent) and 0.05% polysorbate 20% (surfactant). - Once the probiotics are fully hydrated, the protein-probiotic solution is extruded at room temperature using a single-nozzle extruder and polymerized in a calcium hardening bath. Polymerization occurs in a calcium buffer bath at room temperature. - Collect the microcapsules and wash them with a pH 6.1 neutralizing solution. - Fill the associated drying chamber with microcapsules. - Drying can be done as follows: ○ 48 hours of freeze-drying (see Table 1) ○ Drying of fluidized bed: 37℃, 4-6 hours 〇 Vacuum drying: Less than 10 mBar, 24-48 hours at 37°C

[0150] Example 9 Microbeads containing probiotics encapsulated in denatured mung bean protein - Dual (concentric) nozzle extrusion - Using the dMBP prepared according to Example 3, prepare a suspension of dMBP and stir at 250 RPM, RT. - Before encapsulation, add 2% glucidex / simple carbohydrate to the protein solution (this imparts a charge to the denatured protein solution, forming a matrix compatible with the probiotics). - To prevent the formation of air pockets, hydrate at room temperature for 2 hours while gently stirring. Prepare the calcium bath according to Example 7. - The dMBP / glucidex suspension is extruded from the outer nozzle, and the probiotic suspension is extruded from the inner nozzle. Polymerization occurs in a calcium buffer bath at room temperature. - Collect the microcapsules and wash them with a pH 6.1 neutralizing solution. - Load the microcapsules into the associated drying chamber. - Drying can be done as follows: ○ 48 hours of freeze-drying (see Table 1) ○ Drying of fluidized bed: 37℃, 4-6 hours 〇 Vacuum drying: Less than 10 mBar, 24-48 hours at 37°C

[0151] Example 10 Microbeads containing probiotics encapsulated in denatured plant proteins - Dual (concentric) nozzle extrusion - Using denatured plant proteins prepared in the same manner as in Example 1, a suspension of dPP was prepared and stirred at 250 RPM, RT. - Add 2% to 4% of glucidex / simple carbohydrates to the protein solution before encapsulation (this imparts a charge to the denatured protein solution, forming a matrix compatible with probiotics). - To prevent the formation of air pockets, hydrate at room temperature for 2 hours while gently stirring. Prepare the calcium bath according to Example 7. - The dPP / glucidex suspension is dispensed from the outer nozzle, and the probiotic suspension is dispensed from the inner nozzle. Polymerization occurs in a calcium buffer bath at room temperature. - Collect the microcapsules and wash them with a pH 6.1 neutralizing solution. - Fill the associated drying chamber with microcapsules. - Drying can be done as follows: ○ 48 hours of freeze-drying (see Table 1) ○ Drying of fluidized bed: 37℃, 4-6 hours 〇 Vacuum drying: Less than 10 mBar, 24-48 hours at 37°C

[0152] Example 11 Probiotic spray engroving using denatured pea protein dPPI - Prepare the dried materials and fill the chamber (proteins, probiotics, simple sugars). - Prepare a denatured protein suspension. - Start the fluidization process and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3 It becomes fluid due to the airflow over time. - Once the product temperature has reached equilibrium, the spray engroving process is initiated using denatured proteins. - The nozzle pressure should be maintained between 2 and 4.5 Bar, and the airflow of the nozzle pressure should be adjusted as needed. - Apply an intermediate drying step to reduce the moisture content to 50%. - Restart the spray engroving process using maltodextrin (15% w / v solution). - Apply an intermediate drying step to reduce the moisture content to 25%. - Restart the spray engroving process using denatured protein, and perform 2-3 cycles of spray engroving using denatured protein and a simple carbohydrate solution (maltodextrin). - A final post-drying step is performed to reduce the final moisture content to less than 5%.

[0153] Example 12 Probiotic spray engroving using denatured pea protein dPPI and calcium cation crosslinking agent - Prepare the dried materials (proteins, probiotics, simple sugars) and fill them into the chamber. - Prepare a denatured protein suspension and a calcium cation crosslinking agent. - Start the fluidization process and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3 It becomes fluid due to the airflow over time. - Once the product temperature has reached equilibrium, the spray engroving process is initiated using denatured proteins. - The nozzle pressure should be maintained between 2 and 4.5 Bar, and the airflow of the nozzle pressure should be adjusted as needed. - Reduce moisture content by 50% during the intermediate drying step. - Restart the spray engroving process using calcium chloride (0.2-0.4M). - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart spray engroving with denatured protein, and perform 2-3 cycles of spray engroving with denatured protein and calcium crosslinking agent. - A final post-drying step is performed to reduce the final moisture content to less than 5%.

[0154] Example 13 Probiotic spray engraving using denatured pea protein, high-oleic coconut oil, and calcium cation crosslinking agent. - Prepare the dried materials (proteins, probiotics, simple sugars) and fill them into the chamber. - Prepare a denatured pea protein suspension and a calcium cation crosslinking agent. - Start the fluidization process and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3 It is made to flow by the air current over time. - Once the product temperature has reached equilibrium, the spray engroving process is initiated using denatured proteins. - The nozzle pressure should be maintained between 2 and 4.5 Bar, and the nozzle pressure airflow should be adjusted as needed. - Reduce moisture content by 50% during the intermediate drying step. - Restart the spray engroving process using high-oleic coconut oil. - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engroving process using calcium chloride (0.2-0.4M). - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engroving process using denatured protein, and perform 2-3 cycles of spray engroving with denatured protein and calcium or magnesium cation crosslinking agent. - A final post-drying step is performed to reduce the final moisture content to less than 5%.

[0155] Example 14 Probiotic spray engroving using denatured mung bean protein and calcium cation crosslinking agent - Prepare the dried materials (proteins, probiotics, simple sugars) and fill them into the chamber. - Prepare a denatured mung bean protein suspension and a calcium cation crosslinking agent. - Start the fluidization process and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3 It becomes fluid due to the airflow over time. - Once the product temperature has reached equilibrium, the spray engroving process is initiated using denatured proteins. - The nozzle pressure should be maintained between 2 and 4.5 Bar, and the airflow of the nozzle pressure should be adjusted as needed. - Apply an intermediate drying step to reduce the moisture content by 50%. - Restart the spray engroving process using calcium chloride (0.2-0.4M). - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart spray engroving with denatured protein, and perform 2-3 cycles of spray engroving with denatured protein and calcium or magnesium cation crosslinking agent. - A final post-drying step is performed to reduce the final moisture content to less than 5%.

[0156] Example 15 Probiotic spray engroving using denatured mung bean protein, high-oleic sunflower oil, and calcium cation crosslinking agent. - Prepare the dried materials (proteins, probiotics, simple sugars) and introduce them into the chamber. - Prepare a denatured mung bean protein suspension and a calcium cation crosslinking agent. - Start the fluidization process and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3 It becomes fluid due to the airflow over time. - Once the product temperature has equalized, the spray engroving process using denatured plant proteins will be initiated. - The nozzle pressure should be maintained between 2 and 4.5 Bar, and the nozzle pressure airflow should be adjusted as needed. - Apply an intermediate drying step to reduce the moisture content to 50%. - Restart the spray engraving process using high-oleic sunflower oil. - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engroving process using calcium chloride (0.2-0.4M). - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart spray engroving with denatured protein, and perform 2-3 cycles of spray engroving with denatured protein and calcium or magnesium cation crosslinking agent. - A final post-drying step is performed to reduce the final moisture content to less than 5%.

[0157] Example 16 Probiotic spray engroving using denatured mung bean protein, high oleic coconut oil, and calcium cation crosslinking agent. - Prepare the dried materials (proteins, probiotics, simple sugars) and fill them into the chamber. - Prepare a denatured mung bean protein suspension and a calcium cation crosslinking agent. - Start the fluidization process and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3 It becomes fluid due to the airflow over time. - Once the product temperature has reached equilibrium, the spray engroving process is initiated using denatured proteins. - The nozzle pressure should be maintained between 2 and 4.5 Bar, and the nozzle pressure airflow should be adjusted as needed. - Apply an intermediate drying step to reduce the moisture content to 50%. - Restart the spray engroving process using high-oleic coconut oil. - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engraving process using high-oleic sunflower oil. - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engroving process using calcium chloride (0.2-0.4M). - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart spray engroving with denatured protein, and perform 2-3 cycles of spray engroving with denatured protein and calcium cation crosslinking agent. - A final post-drying step is performed to reduce the final moisture content to less than 5%.

[0158] Example 17 Probiotic Plain Glove Bing with Modified Pea Protein and High Oleic Acid Coconut Oil - Prepare the dried materials (proteins, probiotics) and fill the chamber with them. - Prepare a denatured mung bean protein suspension and a calcium cation crosslinking agent. - Start the fluidization process and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3Fluidize with an air flow over time. - Once the product temperature has equilibrated, start the spray englobing process using the denatured protein. - The nozzle pressure should be maintained between 2 - 4.5 Bar and the air flow for the nozzle pressure should be adjusted as appropriate. - Apply an intermediate drying step to reduce the moisture to 50%. - Restart the spray englobing process using high oleic coconut oil. - Conduct an intermediate drying step to reduce the moisture to 25%. - Restart the spray englobing process using simple carbohydrates (maltodextrin). - Conduct an intermediate drying step to reduce the moisture to 25%. - Restart the spray englobing process using the denatured protein and perform 2 - 3 cycles of spray englobing using the denatured protein and simple carbohydrate solution. - Conduct a final post - drying step to bring the final moisture content below 5%.

[0159] Example 18 Probiotic spray englobing using a denatured mung bean protein and a calcium cation cross - linker with an optimized coating layer - Prepare the dry materials (protein, probiotic) and fill the chamber. - Prepare the denatured mung bean protein suspension and the calcium cation cross - linker. - Start the fluidization process by spraying from above and start heating (35 - 45°C). - At the set air inlet temperature, fluidize with an air flow of 110 - 300 m 3 / hour. - Once the product temperature has equilibrated, start the spray englobing process using the denatured protein. - The nozzle pressure should be maintained between 2 - 4.5 Bar and the air flow for the nozzle pressure should be adjusted as appropriate. - Apply an intermediate drying step to reduce the moisture content to 50%. - Restart the spray engroving process using calcium chloride (0.2-0.4M). - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engroving process using denatured protein, and perform 2-3 cycles of spray engroving using denatured protein and magnesium cation crosslinking agent. - A final post-drying step is performed to reduce the final moisture content to less than 5%. - Initiate bottom spray engroving using denatured mung bean protein. - Use a nozzle pressure exceeding 2 Bar. - Maintain accelerator air pressure above 1.5 Bar during spray engine groving. - Maintain airflow equilibrium (approximately 90-170m) 3 / time). - Maintain the product temperature at 37-45°C. - Apply intermittent post-drying at 45-55°C. - To achieve a 25% reduction in moisture content, perform at least two cycles of the calcium cross-linked spray englobing step followed by intermittent drying. - Perform the final post-drying step so that the final moisture content is less than 5%.

[0160] Example 19 Probiotic spray grovings with an optimized coating layer, containing denatured pea protein, high-oleic coconut oil, and calcium cation crosslinking agent. - Prepare the dried materials (proteins, probiotics) and fill the chamber with them. - Prepare a denatured mung bean protein suspension and a calcium cation crosslinking agent. - Start the fluidization process by spraying from above, and begin heating (35-45°C). - At the set air inlet temperature, 110-300m 3It becomes fluid due to the airflow over time. - Once the product temperature has reached equilibrium, the spray engroving process is initiated using denatured proteins. - The nozzle pressure should be maintained between 2 and 4.5 Bar, and the nozzle pressure airflow should be adjusted as needed. - Apply an intermediate drying step to reduce the moisture content to 50%. - Restart the spray engroving process using high-oleic coconut oil. - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engroving process using calcium chloride (0.2-0.4M). - An intermediate drying step is performed to reduce the moisture content to 25%. - Restart the spray engroving process using denatured protein, and perform 2-3 cycles of spray engroving using denatured protein and magnesium cation crosslinking agent. - A final post-drying step is performed to reduce the final moisture content to less than 5%. - Initiate bottom spray using denatured proteins. - Use a nozzle pressure exceeding 2 Bar. - Maintain accelerator air pressure above 1.5 Bar during spray engine groving. - Maintain airflow equilibrium (approximately 90-170m) 3 / time). - Maintain the product temperature at 37-45°C. - Apply intermittent post-drying at 45-55°C. - Apply a spray engraving process using high-oleic coconut oil. - An intermediate drying step is performed to reduce the moisture content to 25%. - To achieve a 25% reduction in moisture content, perform at least two cycles of the calcium cross-linked spray engroving step followed by intermittent drying. - Perform a final post-drying to reduce the final moisture content to less than 5%.

[0161] Example 20 Microbeads - single nozzle extrusion containing probiotics encapsulated in modified PPI (dPPI) - Using the dPPI prepared according to Example 1, prepare a suspension of dPPI and stir at 250 RPM, RT. - Before encapsulation, add 2% glucidex / simple carbohydrate to the protein solution (this imparts a charge to the modified protein solution and forms a matrix compatible with probiotics). - To prevent the generation of air pockets, hydrate at room temperature for 2 hours with gentle stirring. - Add the hydrated probiotic suspension of Example 2 to the dPPI solution and stir this at RT. - Add the probiotic suspension to the protein at a ratio of 9:1 or 18:1 (protein:probiotic ratio). - Stir the protein - probiotic suspension at 400 - 450 RPM for 10 minutes.

[0162] Preparation of the hardening bath - Prepare 0.1M calcium citrate buffer and adjust the pH to 5.2 (range is 5.2 - 6.1). Add 0.05 (w / w)% polysorbate 20% as a surfactant. - An alternative hardening bath is 0.1M calcium chloride buffer with the pH adjusted to 4.8 (range is 4.3 - 5.0). Add 0.05% polysorbate 20% as a surfactant. - An alternative hardening bath is 0.1M calcium citrate buffer containing 0.05% calcium chitosan (chelating agent) and 0.05% (w / aw) polysorbate 20% (surfactant). - Once the probiotics are fully hydrated, extrude the protein - probiotic solution at room temperature with a single - nozzle extruder and polymerize in a calcium hardening bath. - Polymerization occurs in a calcium buffer bath at room temperature. - Collect the microcapsules and wash with a neutralizing solution of pH 6.1. - Fill the associated drying chamber with microcapsules. - Drying can be done as follows: ○ 48 hours of freeze-drying (see Table 1) ○ Drying of fluidized bed: 37℃, 4-6 hours 〇 Vacuum drying: Less than 10 mBar, 24-48 hours at 37°C

[0163] Example 21 Microbeads containing probiotics encapsulated in denatured PPI - Dual (concentric) nozzle extrusion - Prepare a dPPI suspension using dPPI prepared in the same manner as in Example 1, and stir at 250 RPM, RT. - Before encapsulation, add 2% glucidex / simple carbohydrate to the protein solution (this imparts a charge to the denatured protein solution, forming a matrix compatible with the probiotics). - To prevent the formation of air pockets, hydrate at room temperature for 2 hours while gently stirring. Prepare the calcium bath according to Example 7. - The dPPI / glucidex suspension is extruded from the outer nozzle, and the probiotic suspension is extruded from the inner nozzle. Polymerization occurs in a calcium buffer bath at room temperature. - Collect the microcapsules and wash them with a pH 6.1 neutralizing solution. - Fill the associated drying chamber with microcapsules. - Drying can be done as follows: ○ 48 hours of freeze-drying (see Table 1) ○ Drying of fluidized bed: 37℃, 4-6 hours 〇 Vacuum drying: Less than 10 mBar, 24-48 hours at 37°C

[0164] Example 22 Probiotics in denatured PPIs: Spray engroving process of bioactive substances - Using dPPI prepared in the same manner as in Example 1, a suspension of dPPI was prepared and stirred at 250 RPM, RT. - Before encapsulation, add 2% Glucidex / glucose / simple carbohydrate to the protein solution (this imparts charge to the denatured protein solution and creates a matrix compatible with probiotics). - Hydrate at room temperature for 2 hours, stirring gently to avoid creating air pockets. - Add dried pea protein powder (native powder, approximately 2% moisture) to the fluidized bed chamber. - Fluidize at 37℃ with high airflow. - Add the probiotic culture to the powder in a 5:1 or 10:1 ratio. - 15. Add calcium citrate or calcium chloride to the chamber and allow it to fluidize. - Spray a 10% glucidex solution onto the material at a high flow rate (20-30 RPM), high airflow (>150 RPM), and low nozzle pressure (<1.5 bar) to promote aggregation. - Spray coconut oil / sunflower oil / high oleic acid oil onto the powder at a low flow rate (18-22 RPM), high airflow (150 RPM or higher), and high nozzle pressure (2.5 bar or higher). - Spray the dPPI-glucidex solution into the chamber at a high flow rate (25-30 RPM), high airflow (>150 RPM), and high nozzle pressure (>2.5 bar). - Dry afterwards so that Aw < 0.15.

[0165] equivalent product The foregoing description details currently preferred embodiments of the present invention. Those skilled in the art will anticipate that numerous modifications and variations will arise in its implementation. These modifications and variations are intended to be covered within the claims appended herein.

Claims

1. A method for forming a denatured plant protein suspension, A step of hydrating plant proteins in an aqueous solvent with a pH of 6-8 or 7-8, while stirring and optionally heating. Steps include: allowing the aqueous suspension of plant protein to stand; If necessary, adjust the pH of the aqueous suspension to 6-8 or 7-8. The steps include heating the standing aqueous suspension to denature the plant protein, and The step of cooling the heated aqueous suspension. A method comprising the step of heating the standing aqueous suspension, wherein the step of subjecting the standing aqueous suspension to ultra-high temperature treatment (UHT).

2. The method according to claim 1, wherein the standing aqueous suspension is heated to achieve a heat treatment of F0 = 3.

3. The method according to claim 1, wherein the standing aqueous suspension is heated to approximately 138.5°C for approximately 3 to 4 seconds.

4. The method according to claim 1, wherein the aqueous suspension of the plant protein is allowed to stand for at least 30 minutes.

5. The method according to claim 1, wherein the hydration step is carried out at a temperature of 30 to 40°C.

6. The aforementioned heating step is, Under pressure, preheat the standing aqueous suspension at a first heating temperature of 60-70°C for a first heating time, and The step of heating the preheated aqueous suspension under pressure at a second heating temperature of 90 to 100°C for a second heating time. The method according to claim 1, including the method described in claim 1.

7. The method according to claim 6, wherein the heating step is performed under high pressure.

8. The method according to claim 1, wherein the aqueous solvent has a pH of 7.3 to 7.

7.

9. The method according to claim 1, wherein the plant protein is a plant protein isolate.

10. The method according to claim 1, wherein the plant protein is pea protein or mung bean protein.

11. The method according to claim 1, wherein the cooled protein suspension is allowed to settle at room temperature for at least one hour.

12. The method according to claim 1, comprising the step of adding a simple carbohydrate to the aqueous suspension.

13. The method according to claim 12, wherein a sufficient amount of simple carbohydrates is added to the aqueous suspension to provide an aqueous suspension containing 0.5 to 5.0% simple carbohydrates ( / v).

14. The method according to claim 1, wherein the aqueous suspension of the plant protein contains 10-15% (by weight) of denatured plant protein.

15. The method according to claim 1, wherein the aqueous suspension of the plant protein contains 13-15% (by weight) of denatured plant protein.

16. A denatured plant protein suspension formed by the method described in claim 1.

17. A method for encapsulating probiotics, using the denatured plant protein suspension described in claim 16 as the encapsulation matrix.

Images