Methods for forming compositions containing probiotics microencapsulated in modified plant protein matrices

Abstract

The method of forming a composition comprising a probiotic encapsulated in a modified plant protein matrix includes the steps of preparing a protein suspension comprising modified plant protein, preparing a suspension of hydrated probiotic, mixing the protein suspension and the suspension of hydrated probiotic to encapsulate the probiotic in the modified pea protein matrix, and polymerizing the modified plant protein matrix with a calcium salt. The mixing step can include extruding the protein suspension and the suspension of hydrated probiotic to form microdroplets, and the polymerization step includes hardening the extruded microdroplets in a hardening bath comprising a calcium citrate buffer having a pH of 6.5 and a molarity of 0.05-0.15 M. The mixing step can also include mixing the protein suspension and the probiotic suspension to form a mixture, adding a calcium salt buffer to the mixture to gel the mixture, and drying the gelled mixture by freeze drying or vacuum drying.

Description

[Technical field]

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

[0002] 2. Background of the Invention To produce a microencapsulated active agent that is robust, protects the encapsulated probiotic bacteria, and is shelf-stable, it is necessary to use a protein suspension with a high solids content and a high percentage of protein in solution. International Publication No. WO2008 / 056344 (Patent Document 1) describes the encapsulation of probiotic bacteria in a modified whey protein matrix using a low-temperature gelation process in which the probiotic bacteria are mixed with a modified whey suspension, extruded into microdroplets, and hardened in an acid bath at pH 4.6. Depending on the probiotic, the addition of probiotics to whey protein by the low-temperature gelation method often results in the side effect of solidification and reduced cell count. Furthermore, the pH required to create low-temperature gelling whey protein is often detrimental to the survival of probiotics, such as pH 4.6. [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) Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention 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 the encapsulation matrix. The applicant has discovered that using plant proteins such as modified pea protein and modified mung bean (mung) protein as a microencapsulation matrix for probiotic bacteria results in more bacteria surviving the encapsulation process, for example 75-80% or more cell viability compared to 40-50% in dairy-based encapsulation systems such as WPI or MPC. Without being bound by theory, it is believed that the increased viability is due to the polymerization of plant proteins at pH 6-7 as opposed to whey protein at pH 4.6. A further advantage of the present invention is that the use of plant proteins as the encapsulation matrix allows the product to be used by vegetarians and vegans. The present disclosure describes a number of methods for encapsulating probiotics using modified pea protein and modified mung bean protein (as examples of plant proteins), all of which use calcium salt chelating agents that can polymerize the modified plant proteins at a slightly acidic pH, a pH that does not adversely affect the survival or metabolism of the probiotics. The examples provided herein show encapsulated probiotics produced by extrusion into a gelling bath, gel fixation and drying (e.g. freeze-drying or vacuum drying). The invention also describes a method for producing a suspension of a plant protein (commonly a modified pea protein or modified mung bean protein) obtained by hydration of the protein at pH 7-8, settling of the protein suspension, and a two-stage heating step typically carried out under high pressure. An embodiment of this process has been found to produce suspensions of plant proteins with high solids content (e.g. up to 15% solids), a high percentage of soluble protein (e.g. 90% or more), and a pH that is not acidic or alkaline, making them ideal for microencapsulating pH-sensitive agents such as probiotics.Additionally, 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 the reduced viscosity of the protein solution. The methods of the present disclosure can also be applied to encapsulate other active agents such as plant extracts, vitamins, minerals, marine bioactives and amino acids.

[0007] In a first aspect, the present invention provides a method of forming a microparticle comprising an active agent, such as a probiotic, encapsulated in a modified plant protein matrix, the method comprising: preparing a protein suspension comprising a denatured plant protein; mixing the protein suspension with an active agent to form a mixture; treating the mixture to form microparticles comprising an active agent encapsulated in a modified plant protein matrix, said treating step comprising polymerizing the modified plant protein matrix with a calcium salt or spray englobing on a fluid bed dryer; and Drying the microparticles Includes.

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

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

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

[0011] In either 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 to enhance the compatibility of the modified plant protein suspension with the probiotic cells, provide a stable equilibrium of the cells in the protein matrix during encapsulation, and create a charged slurry that interacts with the respective charged cell membranes of the probiotics. The simple carbohydrate also helps in the interaction between the charged simple carbohydrate and the modified plant protein after heat treatment to cause instant chelation / polymerization. This charge interaction forms a strong lattice to encase and encapsulate sensitive bacteria.

[0012] In either embodiment, the processing step involves extruding the protein suspension and the active agent suspension to form microdroplets, and the polymerization step involves hardening the extruded microdroplets in a hardening bath containing a chelating agent such as a calcium salt to form microcapsules.

[0013] In either embodiment, the hardening bath includes a calcium citrate buffer.

[0014] In either embodiment, the hardening bath comprises a calcium citrate buffer having a pH of 5 to 6.5.

[0015] In either embodiment, the hardening bath contains a calcium citrate buffer having a molar concentration of 0.05 to 0.15M.

[0016] In either embodiment, the hardening bath comprises a calcium citrate buffer having a pH of 5.2 to 6.1.

[0017] In either embodiment, the hardening bath comprises a calcium citrate buffer having a molarity of about 0.1 M.

[0018] In either embodiment, the hardening bath includes a calcium chloride buffer.

[0019] In either embodiment, the hardening bath comprises a calcium chloride buffer having a pH of 4.3 to 5.

[0020] In either embodiment, the hardening bath comprises a calcium chloride buffer having a molar concentration of 0.05 to 0.15M, or about 0.1M.

[0021] In an optional embodiment, the hardening bath comprises a surfactant, particularly a non-ionic surfactant, particularly a polysorbate-type non-ionic surfactant. In an optional embodiment, the hardening bath comprises 0.01 to 0.1, particularly about 0.05% of a surfactant.

[0022] In an optional embodiment, the hardening bath comprises chitosan. In an optional embodiment, the hardening bath comprises 0.01 to 0.1, particularly about 0.05% chitosan.

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

[0024] In either embodiment, the calcium salt buffer comprises a calcium citrate buffer having a pH of 5 to 6.5.

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

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

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

[0028] In any 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 comprises 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 comprises mixing the protein suspension with the suspension of active agent in a dry weight ratio of denatured plant protein to active agent of 1:5 to 1:25 or 1:8 to 1:20.

[0031] In either embodiment, the active agent is a probiotic and the suspension of hydrated probiotic is suspended in 0.05-0.15M, 0.08-0.12M, or about 0.1M phosphate buffer.

[0032] In either embodiment, the simple carbohydrate comprises maltodextrin and glucose. GLUCIDEX is a commercial product that contains maltodextrin and glucose.

[0033] In either embodiment, the method includes forming microparticles by spray englobing, where the mixing and processing steps are carried out on a fluidized bed dryer.

[0034] In either embodiment, the method includes adding a support material and an active agent to the bed of a fluid bed dryer, fluidizing the support material and active agent, spraying the fluidized support material and active agent with a first coating material to produce microparticles having the active agent and support entrapped within a shell of the first coating material, and drying the microparticles.

[0035] In either embodiment, a simple sugar is sprayed into the fluid bed dryer prior to spraying the first coating material into the fluid bed dryer, the simple sugar forming granules which the first coating material coats.

[0036] In either embodiment, the first coating material is selected from modified vegetable proteins, oils, and simple sugars such as maltodextrin.

[0037] In either embodiment, the method includes spraying a second coating material onto the shell of the first coating material, where at least one of the first and second coating materials is a modified vegetable protein.

[0038] In either embodiment, the method includes spraying the modified vegetable protein coating with a chelating salt.

[0039] The following particles are envisaged: Fine particles 1 A core of active agents (e.g. probiotics) and carrier material Polymerized modified plant protein coating fine particles 2 A core of active agents (such as probiotics), simple sugars, and carrier materials Non-polymerizable dry modified plant protein coatings fine particles 3 A core of active agents (e.g. probiotics) and carrier material Polymerized modified plant protein inner coating Non-polymerizable dry denatured vegetable protein envelope Intermediate coating of oil. fine particles 4 A core of active agents (e.g. probiotics) and carrier material Polymerized modified plant protein inner coating Polymerized modified vegetable protein outer coating First intermediate coating of oil. A second intermediate coating of non-polymerizable dry modified vegetable protein.

[0040] In one embodiment, the spray engroving comprises the following steps. (a) adding carrier material (e.g., native protein), active agent (e.g., probiotic), and optionally simple sugars to the bed of a fluidized bed drying chamber; (b) fluidizing and heating the support material and the active material to form a first fluidized powder; (c) spraying the denatured protein suspension onto a fluidized bed at high pressure (e.g., 2-4.5 Bar) to provide a second fluidized powder; (d) drying the second fluidized powder on the fluidized bed to reduce the moisture content of the second fluidized powder (e.g., to reduce the moisture content by at least 30%, 40%, or 50%); (e) spraying an englobing component onto the second fluidized powder to form a third fluidized powder, the englobing component being selected from a chelating salt (e.g., a calcium salt or a magnesium salt); an edible oil; and a simple sugar; (f) drying the third fluidized powder on a fluidized bed to further reduce the moisture content of the fluidized powder (e.g., to further reduce the moisture content by at least 10%, 20%, or 25%); (g) spraying the denatured protein suspension onto a fluidized bed at high pressure (e.g., 2-4.5 Bar) to provide a fourth fluidized powder comprising microparticles; and Optionally, further drying the fourth fluidized powder, typically to a moisture content of less than 10%, less than 8%, or less than 5%.

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

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

[0043] Generally, the spraying steps referred to above are top spraying, eg, the components are sprayed from above the fluidized bed.

[0044] In either embodiment, the method includes the additional steps of: (h) bottom spraying the denatured protein suspension into the fourth fluidized mixture to form a fifth fluidized powder; (i) drying the fifth fluidized powder on a fluidized bed to further reduce the moisture content of the fluidized powder (e.g., to further reduce the moisture content by at least 10%, 20%, or 25%); (j) bottom spraying an engrobing component onto the fifth fluidized powder to form a sixth fluidized powder, the engrobing component being selected from a chelating salt (e.g., a calcium salt or a magnesium salt) and an edible oil; (k) drying the sixth fluidized powder on the fluidized bed to further reduce the moisture content of the fluidized powder (e.g., to further reduce the moisture content by at least 10%, 20%, or 25%); and (l) Optionally, further drying the fourth fluidized powder, typically to a moisture content of less than 10%, less than 8%, or less than 5%.

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

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

[0047] In any embodiment, the fluidized bed is 110 to 300 m 3 / Fluidized by the currents of time.

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

[0049] In either embodiment, the protein solution is sprayed into the fluidized bed at high spray nozzle pressure, for example 2-4.5 Bar.

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

[0051] In either embodiment, the simple sugar is or includes maltodextrin.

[0052] In either embodiment, the chelating salt is added as a 0.2-0.4M solution.

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

[0054] In either embodiment, step (a) involves adding the support material and the active agent to the bed of a 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 prebiotic fiber powder, or any colloid.

[0056] In another embodiment, the spray engraving comprises: fluidizing the carrier material, such as native protein, active agents (such as probiotics), and chelating salts, on a fluidized bed in a fluidized bed drying chamber; spraying edible oil into a fluid bed drying chamber; spraying the suspension of denatured protein into a fluid bed drying chamber; Including, This causes the chelating salt to react with the denatured plant protein to polymerize the protein and form agglomerated microparticles having a coating of polymerized denatured protein and a core containing the active agent and carrier material.

[0057] In either embodiment, the support material is added to the fluidized bed first, and the active agent and chelating salt are added to the fluidized bed after the support material.

[0058] In either embodiment, the support material is fluidized at an elevated temperature (eg, 30-40° C.) prior to addition of the active agent and chelating salt.

[0059] In either embodiment, the carrier material, active agent, and chelating salt are fluidized at an elevated temperature (eg, 30-40° C.) prior to the addition of the edible oil to form a fluidized mixture.

[0060] In either embodiment, a simple sugar is sprayed onto the fluidized mixture prior to the edible oil to aid in granulation of the mixture.

[0061] In either embodiment, the simple sugars are sprayed into the fluid bed chamber at a high flow rate (eg, 23-30 RPM), low air flow (eg, 150 RPM or greater), and low spray nozzle pressure (eg, less than 1.5 Bar).

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

[0063] In either embodiment, the suspension of modified plant protein is sprayed into a fluid bed drying chamber at high flow rates (e.g., 23-30 RPM), high airflow (e.g., >150 RPM), and high spray nozzle pressures (e.g., >2.5 Bar).

[0064] In either embodiment, the active agent is added in the form of an oil-in-water nanoemulsion, where the active agent is contained in the oil phase of the oil-in-water nanoemulsion, which is particularly appropriate when the active agent is hydrophobic.

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

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

[0067] In either embodiment, the oil is an edible oil, such as a high oleic oil.

[0068] In either embodiment, the bed is fluidized with air at 30-40° C. and high airflow (approximately 30-55 RPM).

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

[0070] In either embodiment, the native and denatured proteins are protein isolates.

[0071] In either embodiment, the suspension of denatured plant protein is a denatured plant protein suspension of the present invention.

[0072] In either embodiment, the agglomerated microparticles are gastroresistant and ileal sensitive, meaning that they pass the human stomach intact and break down in the ileum releasing the active agent.

[0073] In any embodiment, the protein suspension comprises: hydrating the vegetable protein in an aqueous solvent having a pH of 7-8, with stirring and optionally with heating; Allowing the aqueous suspension of plant protein to stand; Optionally, adjusting the pH of the aqueous suspension to 7-8; heating the settled aqueous suspension, optionally under pressure; and Cooling the heated aqueous suspension The method is formed by a process comprising:

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

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

[0076] In any embodiment, the heating step comprises: preheating the aqueous suspension at a first heating temperature of 60 to 70° C. (e.g., about 65° C.) under pressure for a first heating time (e.g., 5 seconds to 15 minutes); and Heating the preheated aqueous suspension under pressure at a second heating temperature of 90 to 100°C (e.g., about 95°C) for a second heating time (2 seconds to 3 minutes). Includes.

[0077] In any embodiment, the heating step includes heating the suspension of plant proteins to achieve a heat treatment of F0=3. This results in 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 process. The term "F0" is defined as the number of equivalent minutes of steam sterilization of the load (product) at 250°F (121°C).

[0078] The ultra-high temperature denaturation process serves three purposes when denaturing plant proteins: (a) it kills bacterial spores in the protein suspension, ensuring the product is of food grade quality and safe for human consumption; (b) this high temperature denaturation is necessary to provide the relevant functionality for polymerizing the plant proteins; and (c) it creates a viscosity that allows for better flow rates during the encapsulation process, resulting in higher efficiency in the encapsulation process and higher throughput during encapsulation. The ultra-high temperature denaturation process has been found to reduce the viscosity of the plant protein suspension 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 either embodiment, the heating step comprises ultra-high temperature treatment (UHT) of the suspension of plant protein.

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

[0081] In either embodiment, the aqueous solvent has a pH of about pH 7.5 (eg, 7.3 to 7.7).

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

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

[0084] In either embodiment, the chilled protein suspension is allowed to settle at room temperature for at least 2 hours.

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

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

[0087] In another aspect, the invention provides a composition comprising an active agent, such as a probiotic, encapsulated in a modified plant protein matrix formed according to the methods of the invention. The composition is generally a particle, e.g., a microparticle, which may be a granule.

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

[0089] In either embodiment, the microparticles are gastroresistant and ileal sensitive, meaning that they pass through the human stomach intact and degrade in the ileum releasing the probiotic payload and allowing subsequent colonization.

[0090] In another aspect, the present invention provides a method of forming a denatured plant protein suspension comprising the steps of: hydrating the plant protein in an aqueous solvent having a pH of 6-8 or 7-8, with stirring and optionally with heating; Allowing the aqueous suspension of plant protein to stand; adjusting the pH of the aqueous suspension to 6-8 or 7-8, if necessary; heating the stationary aqueous suspension; and Cooling the heated aqueous suspension.

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

[0092] In either embodiment, the settled aqueous suspension is subjected to ultra-high temperature treatment (UHT).

[0093] In either embodiment, the aqueous suspension that has been allowed to stand is heated to about 138.5° C. for about 3 to 4 seconds.

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

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

[0096] In any embodiment, the heating step comprises: preheating the aqueous suspension at a first heating temperature of 60 to 70° C. (e.g., about 65° C.) under pressure for a first heating time (e.g., 5 seconds to 15 minutes); and Heating the preheated aqueous suspension under pressure at a second heating temperature of 90 to 100°C (e.g., about 95°C) for a second heating time (2 seconds to 3 minutes). Includes.

[0097] In either embodiment, the aqueous solvent has a pH of about pH 7.5 (eg, 7.3 to 7.7).

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

[0099] In either embodiment, the plant protein is a plant protein isolate.

[0100] In either embodiment, the plant protein is pea protein.

[0101] In either embodiment, the plant protein is mung bean protein.

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

[0103] In either embodiment, the method includes the step of adding a simple carbohydrate to the protein suspension to provide a protein suspension containing typically 0.5-5.0%, 1-3% or about 2% simple carbohydrate (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 from the same source (e.g., both pea protein). In any embodiment, the native protein and the denatured protein are protein isolates.

[0105] In either embodiment, the suspension of denatured plant protein is a denatured plant protein suspension of the present invention.

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

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

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

[0109] In another aspect, the present invention provides compositions of microparticles, wherein the microparticles comprise an active agent entrapped within modified plant protein structures, the modified plant protein structures typically comprising polymerized modified plant proteins.

[0110] In either embodiment, the active agent is entrapped within a carrier, which may be an edible oil droplet with the active agent entrapped within the edible droplet, or a carrier powder such as a native protein or sugar, such as maltodextrin or starch.

[0111] In either embodiment, the polymerized modified plant protein structure is a polymerized modified plant protein matrix and the oil droplets are distributed throughout the matrix.

[0112] In either embodiment, the microparticles have a core-shell structure with a shell of modified vegetable protein and a core that typically contains the active agent in a carrier, which may be a powder (as described above), edible oil droplets, or a solution / suspension of the active agent.

[0113] In either embodiment, the modified plant protein shell is polymerized.

[0114] In either embodiment, the microparticles have two or more shells, at least one shell being a shell of modified vegetable protein and at least one shell being selected from a shell of oil, and a shell of non-polymeric modified vegetable protein.

[0115] In either embodiment, the microparticles are granules.

[0116] In another aspect, the present invention provides a food, beverage or dietary supplement in any form comprising the composition of the present invention.

[0117] In any embodiment, the beverage, food or dietary supplement is shelf stable.

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

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

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

[0121] In any embodiment, the method provides for a percent viability of the probiotic of greater than 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0122] In either embodiment, the agglomerated microparticles are gastroresistant and ileal sensitive, meaning that they pass the human stomach intact and degrade in the ileum releasing the probiotic payload.

[0123] Further aspects and preferred embodiments of the invention are defined and described in the other claims set out below. [Brief description of the drawings]

[0124] [Figure 1] Figure 1 is an image of probiotic modified pea protein microcapsules formed according to the method of Example 20 and imaged prior to the drying step. This microscopic image represents polymerized wet capsules. The average diameter is 500-600um. [Diagram 2] Figure 2 is an image of probiotic modified pea protein microcapsules formed according to the method of Example 20 and imaged after the drying step. The microscope image shows individual microcapsules with a free-flowing form. Average diameter 485-550um. [Figure 3A] FIG. 3A is a summary of probiotic viability for probiotic encapsulation using pea protein versus whey (milk) protein and a single nozzle encapsulator. [Figure 3B]Summary of probiotic viability for probiotic encapsulation using different plant protein encapsulation matrices and single nozzle extrusion technology. [Figure 4] Figure 4 shows a comparison of particle size of probiotic encapsulation systems produced using pea protein and a single nozzle encapsulation device. [Figure 5A] Figure 5A is a probiotic release rate profile of encapsulated probiotic cells in a pea protein encapsulation system produced using a single nozzle encapsulation extrusion technique. The data represents cell release as a function of in vitro human digestion time and GI section. [Figure 5B] Figure 5B is a probiotic release rate profile of encapsulated probiotic cells in an ultra-denatured pea protein encapsulation system produced using a single nozzle encapsulation / extrusion technique. The data represents cell release as a function of in vitro human digestion time and GI section. [Figure 6] Figure 6: HPLC protein digestion as a function of GI section. The HPLC data shows the onset of protein degradation in the intestine, which indicates controlled cell release. The data represents the digestion profile of cell release from pea protein encapsulation systems produced using extrusion, single nozzle encapsulation technology. [Figure 7] Figure 7 is an HPLC chromatogram for quantification of protein degradation in human in vitro digestion assay. The data shows the onset of protein digestion in the intestine after 45 minutes (black) and 60 minutes (pink), indicating controlled cell release due to sustained and controlled digestion of plant protein microcapsules. The data represents the digestion profile of cell release from a plant protein encapsulation system produced using extrusion, single nozzle encapsulation technology. [Figure 8]FIG. 8: Micrographs of bifidobacteria (lactic acid bacteria) encapsulated in plant proteins produced by single nozzle / extrusion technology: (A) mung bean protein, (B) broad bean protein, (C) chickpea protein. [Figure 9A] FIG. 9 depicts the pH profile of plant proteins before and after UHT plant protein denaturation: (A) Flax seed (Linseed) protein isolate and (B) Pumpkin seed protein isolate. [Figure 9B] FIG. 9 depicts the pH profile of plant proteins before and after UHT plant protein denaturation: (A) Flax seed (Linseed) protein isolate and (B) Pumpkin seed protein isolate. [Figure 10A] Figure 10 depicts the pH profile of plant proteins before and after UHT plant protein denaturation: (A) Fava (Fava / Broad) protein isolate and (B) mung bean protein isolate. [Figure 10B] Figure 10 depicts the pH profile of plant proteins before and after UHT plant protein denaturation: (A) Fava (Fava / Broad) protein isolate and (B) mung bean protein isolate. [Figure 11] Figure 11 represents the pH profile of the pea protein hydrolysates before and after the UHT pea protein denaturation procedure, confirming the denaturation of the partially hydrolyzed pea proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0125] Detailed Description of the Invention All publications, patents, patent applications and other references mentioned in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

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

[0127] As used herein, the use of the singular includes the plural, and vice versa, unless the context requires otherwise. The term "a" or "an" when used in reference to an entity is intended to refer to one or more of that entity. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0128] As used herein, the term "comprising" or variations thereof, such as "comprises" or "comprising," should be read to indicate the inclusion of any implied integer (e.g., a feature, element, property, quality, method / process step, or limitation) or group of integers (e.g., a feature, element, property, quality, method / process step, or limitation) but not the exclusion of other integers or groups of integers. Thus, as used herein, the term "comprising" is inclusive or open-ended and does not exclude additional, implied integers or method / process steps.

[0129] As used herein, 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 with 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, and disaccharides such as sucrose, lactose and maltose, and mixtures thereof. In one embodiment, the simple carbohydrate comprises GLUCIDEX.

[0131] The term "probiotic" as used herein refers to live beneficial bacteria and / or yeasts that live naturally in the body. Probiotics help the body stay healthy and functioning well, including fighting off overgrowth of bad bacteria and helping you feel better. The probiotics used in the present invention may consist of a combination of different probiotics. There are many types of bacteria that are considered probiotics, but there are two specific types of bacteria that are common probiotics found in stores. These include Lactobacillus and Bifidobacterium.

[0132] As used herein, the term "% probiotic survival" as applied to compositions produced according to the methods of the present invention means the % 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 viable cells input to the step and comparing it to the number of cells in the output product.

[0133] As used herein, the term "active agent" refers to any ingredient suitable for delivery to the small intestine or ileum of a mammal, but typically refers to an ingredient that is sensitive to external conditions, such as heat, pH, moisture, pressure, chemical stress or enzymes. Thus, the active ingredient may be sensitive to pH, enzymes (i.e., protease enzymes), high pressure, moisture, high shear, and temperature abuse during storage. In a particularly preferred embodiment of the present invention, the active agent comprises a probiotic or bioactive agent selected from, for example, caffeine, fat-soluble vitamins (A, D, E, K), water-soluble vitamins (B and C), hyaluronic acid, melatonin, and marine bioactive agents such as fucoidan and laminarin, green algae, brown algae and red algae extracts.

[0134] "Gastro-resistant" means that the microparticles are able to survive intact for at least 60 minutes in a simulated gastric digestion model described in Minekus et al., 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 Document 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 Document 2)).

[0135] "Ileum-sensitive" means that the microparticle is capable of releasing its contents in vivo in the ileum (proximal or distal) of a mammal. EXAMPLES

[0136] Example The present invention will now be described with reference to specific examples, which are merely exemplary and for purposes of illustration only, and are not intended to limit in any way the scope of the claims or the described invention, and which constitute the best modes currently contemplated for carrying out the invention.

[0137] Example 1 Preparation of pea protein solutions for denaturation - Hydrate pea protein isolate at 14% (w / v) in water or phosphate buffer 0.1 M pH 7.5. Hydrate for 45 minutes at -30°C. - Use a jacketed vessel or hot plate at 30°C and stir at 450 RPM. - Allow to stand for a minimum of 90 minutes as a required settling step. - Check the pH of the water and make sure it is pH 7.5. If it is not pH 7.5, adjust it with 1N HCl / NaOH. - Heat treat as follows: Preheat to 65℃, min. 2 sec / max. 15 min · Final heating 95.5℃, minimum 2 seconds / maximum 3 minutes · Cooling temperature 22~25℃ · Flow rate 1.5L~20L / min Pressure 1.3~1.6bar - Allow the material to sit at room temperature for 4 hours before using. - This material is called "dPPI."

[0138] Example 2 Preparation of hyper-denatured pea protein - Hydrate pea protein 14% (w / v) in water or phosphate buffer 0.1M pH 7.5. Hydrate at -30°C for 45 minutes. - Use a jacketed vessel and stir at 450 rpm. - Allow to stand for a minimum of 90 minutes as a required settling step. - Check the pH of the water and make sure it is pH 7.5. If it is not pH 7.5, adjust it with 1N HCl / NaOH. - Heat treatment as follows: Preheat 60~98℃ for 2 seconds Final temperature 126~144.5℃ Hold time - minimum 2 seconds for each step a Cooling temperature 22~25℃ Flow rate: min. 1.45L~2.0L / min Pressure 1.0~6.0bar - Allow to sit at room temperature for 2 hours before use. - This material is called "udPPI".

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

[0140] Example 4 Probiotic Hydration - Make a stock solution of probiotics. Hydrate in phosphate buffer pH 0.1M. - Add probiotics (liquid or powder) to the phosphate buffer solution. - Resuscitate the cells by stirring at 200-450 RPM for 10-45 minutes at RT. - If necessary, Ultraturrax cell suspension (9,000-11,000 RPM, 30-90 seconds).

[0141] Example 5 Immobilization of probiotics onto PPI using modified PPI - Using a dPPI prepared as in Example 1 and a hydrated probiotic prepared as in Example 4, add the probiotic suspension to the dPPI solution at 410 RPM to provide a 9:1 or 18:1 ratio of PPI to probiotic suspension. - Add 2% glusidex / simple carbohydrates to the protein solution prior to encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at RT using gentle agitation to avoid air pockets. - After all probiotics have been added, 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 during pH adjustment at 450 RPM and RT. - Maintain stirring for a minimum of 15 minutes and transfer to a drying chamber. - The solution will visually appear to be more viscous. - Drying (freeze-drying or vacuum drying) can be carried out as follows: - Freeze-dried (see Table 1)

[0142] [Table 1]

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

[0144] After drying, the dried product can be size reduced into particles using a high shear solids 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, add the probiotic suspension to the dMBP solution at 410 RPM to provide a 9:1 or 18:1 ratio of dMBP to probiotic suspension. - Add 2% glucidex / simple carbohydrates to the protein solution prior to encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at RT with gentle stirring to avoid air pockets. - After all probiotics have been added, 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 and RT during pH adjustment. - Maintain stirring for a minimum of 15 minutes and transfer to a drying chamber. - The solution will visually appear to be more viscous. - Drying (freeze-drying or vacuum drying) can be carried out as follows: - Freeze-dried (see Table 1) - Vacuum drying: Set the product temperature to 25°C. A vacuum is created in the chamber. Once full vacuum (i.e. less than 10mBar) is achieved, dry for 24 hours. After drying, the dried product can be size reduced into particles using a high shear solids separator.

[0146] Example 7 Microbeads containing probiotics encapsulated in modified mung bean protein - single nozzle extrusion - Prepare a suspension of dMBP using dMBP prepared according to example 3 and stir at 250 RPM at RT. - Add 2% glucidex / simple carbohydrates to the protein solution prior to encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at room temperature with gentle stirring to prevent air pockets. - The hydrated probiotic suspension of Example 4 is added to the dMBP solution and stirred at RT. -Add probiotic suspension to protein in a ratio of 9:1 or 18:1 (protein:probiotic). - Stir the protein-probiotic suspension at 400-450 RPM for 10 minutes.

[0147] Preparation of the hardening bath - Prepare calcium citrate buffer 0.1M and adjust the pH to 5.2 (range 5.2-6.1). Add 0.05% polysorbate 20% as a surfactant. - An alternative hardening bath is calcium chloride buffer 0.1M, pH adjusted to 4.8 (range 4.3-5.0), with 0.05% polysorbate 20% added as a surfactant. - An alternative hardening bath is calcium citrate buffer 0.1M 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 through 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 with a neutralizing solution of pH 6.1 (to prevent microcapsules from sticking during drying and optimize yield). - The microcapsules are loaded into an associated drying chamber. Drying can be carried out as follows: Freeze-dry for 48 hours (see Table 1) Fluidized bed drying: 37℃, 4 to 6 hours Vacuum drying: less than 10mBar, 37℃ for at least 24-48 hours

[0148] Example 8 Microbeads containing probiotics encapsulated in modified plant proteins - Single nozzle extrusion - A suspension of modified protein is prepared using modified vegetable protein prepared according to Example 1 and stirred at 250 RPM and RT. - Adding 2% to 4% simple carbohydrates to the protein solution before encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at room temperature with gentle stirring to prevent air pockets. - Add the hydrated probiotic suspension in denatured aqueous protein solution and stir at RT. -Add probiotic suspension to protein in a ratio of 9:1 or 18:1 (protein:probiotic). - Stir the protein-probiotic suspension at 400-450 RPM for 10 minutes.

[0149] Preparation of the hardening bath - Prepare calcium citrate buffer 0.1M and adjust the pH to 5.2 (range 5.2-6.1). Add 0.05% polysorbate 20% as a surfactant. - Adjust the pH to 4.8 (range 4.3-5.0) with calcium cation buffer 0.1 M. Add 0.05% polysorbate 20% as a surfactant. - An alternative hardening bath is calcium citrate buffer 0.1M 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 through a single nozzle extruder and polymerized in a calcium hardening bath. - Polymerization occurs in a calcium buffer bath at room temperature. - The microcapsules are collected and washed with a neutralizing solution of pH 6.1. - The microcapsules are loaded into an associated drying chamber. - Drying can be carried out as follows: Freeze-dry for 48 hours (see Table 1) Fluidized bed drying: 37℃, 4 to 6 hours Vacuum drying: less than 10mBar, 37℃ for 24-48 hours

[0150] Example 9 Microbeads containing probiotics encapsulated in modified mung bean protein - Dual (concentric) nozzle extrusion - Prepare a suspension of dMBP using dMBP prepared according to Example 3 and stir at 250 RPM and RT. - Add 2% glucidex / simple carbohydrates to the protein solution prior to encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at room temperature with gentle stirring to prevent air pockets. Prepare a calcium bath according to Example 7. - The dMBP / Glusidex suspension is extruded through the outer nozzle and the probiotic suspension through the inner nozzle. - Polymerization occurs in a calcium buffer bath at room temperature. - The microcapsules are collected and washed with a neutralizing solution of pH 6.1. - Loading the microcapsules into an associated drying chamber. Drying can be carried out as follows: Freeze-dry for 48 hours (see Table 1) Fluidized bed drying: 37℃, 4 to 6 hours Vacuum drying: less than 10mBar, 37℃ for 24-48 hours

[0151] Example 10 Microbeads containing probiotics encapsulated in modified plant proteins - Dual (concentric) nozzle extrusion - A suspension of dPP is prepared using modified plant proteins prepared as in Example 1 and stirred at 250 RPM and RT. - Add 2%-4% glucidex / simple carbohydrates to the protein solution before encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at room temperature with gentle stirring to prevent air pockets. Prepare a calcium bath according to Example 7. - The dPP / Glucidex suspension is extruded through the outer nozzle and the probiotic suspension through the inner nozzle. - Polymerization occurs in a calcium buffer bath at room temperature. - The microcapsules are collected and washed with a neutralizing solution of pH 6.1. - The microcapsules are loaded into an associated drying chamber. Drying can be carried out as follows: Freeze-dry for 48 hours (see Table 1) Fluidized bed drying: 37℃, 4 to 6 hours Vacuum drying: less than 10mBar, 37℃ for 24-48 hours

[0152] Example 11 Probiotic spray englobing with modified pea protein dPPI - Prepare dry ingredients and load into chamber (proteins, probiotics, simple sugars) - Prepare a denatured protein suspension. - Start the fluidization process and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be kept between 2~4.5Bar and the airflow of the nozzle pressure should be adjusted accordingly. - Apply an intermediate drying step to reduce the moisture content to 50%. - Start the spray englove process again using maltodextrin (15% w / v solution). - Apply an intermediate drying step to reduce the moisture content to 25%. - Start the spray engrobing process again with the denatured protein and perform 2-3 cycles of spray engrobing with the denatured protein and a simple carbohydrate solution (maltodextrin). - A final post-drying step is carried out to achieve a final moisture content of less than 5%.

[0153] Example 12 Probiotic spray englobing using modified pea protein dPPI and calcium cation crosslinker - Prepare dry ingredients (proteins, probiotics, simple sugars) and load into the chamber. - Prepare a denatured protein suspension and a calcium cation crosslinker. - Start the fluidization process and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be kept between 2~4.5Bar and the airflow of the nozzle pressure should be adjusted accordingly. - An intermediate drying step reduces the moisture content to 50%. - Restart the spray engraving process with calcium chloride (0.2-0.4M). - An intermediate drying step is carried out to reduce the moisture content to 25%. - Start spray engrobing with denatured protein again and perform 2-3 cycles of spray engrobing with denatured protein and calcium crosslinker. - A final post-drying step is carried out to achieve a final moisture content of less than 5%.

[0154] Example 13 Probiotic Spray Englobing with Modified Pea Protein, High Oleic Coconut Oil, and Calcium Cationic Crosslinker - Prepare dry ingredients (proteins, probiotics, simple sugars) and load into the chamber. -Prepare a modified pea protein suspension and a calcium cation crosslinker. - Start the fluidization process and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidize with air flow for hours. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be maintained between 2~4.5Bar, and the airflow of the nozzle pressure should be adjusted accordingly. - An intermediate drying step reduces the moisture content to 50%. -Restart the spray engraving process with high oleic coconut oil. - An intermediate drying step is carried out to reduce the moisture content to 25%. - Restart the spray engraving process with calcium chloride (0.2-0.4M). - An intermediate drying step is carried out to reduce the moisture content to 25%. -Start the spray engrobing process again with the denatured protein and perform 2-3 cycles of spray engrobing with the denatured protein and calcium or magnesium cation crosslinker. - A final post-drying step is carried out to achieve a final moisture content of less than 5%.

[0155] Example 14 Probiotic spray engrobing using modified mung bean protein and calcium cation crosslinker - Prepare dry ingredients (proteins, probiotics, simple sugars) and load into the chamber. -Prepare a modified mung bean protein suspension and a calcium cation crosslinker. - Start the fluidization process and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be kept between 2~4.5Bar, and the airflow of the nozzle pressure should be adjusted accordingly. - Apply an intermediate drying step to reduce the moisture content to 50%. - Start the spray engraving process again using calcium chloride (0.2-0.4M). - An intermediate drying step is carried out to reduce the moisture content to 25%. - Start spray engrobing with the denatured protein again and perform 2-3 cycles of spray engrobing with the denatured protein and calcium or magnesium cation crosslinker. - A final post-drying step is carried out to achieve a final moisture content of less than 5%.

[0156] Example 15 Probiotic Spray Englobing with Modified Mung Bean Protein, High Oleic Sunflower Oil, and Calcium Cationic Crosslinker - Prepare the dry ingredients (proteins, probiotics, simple sugars) and introduce them into the chamber. -Prepare a modified mung bean protein suspension and a calcium cation crosslinker. - Start the fluidization process and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, the spray engraving process using modified vegetable proteins is started. - The nozzle pressure should be maintained between 2~4.5Bar, and the airflow of the nozzle pressure should be adjusted accordingly. - 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 carried out to reduce the moisture content to 25%. - Restart the spray engraving process with calcium chloride (0.2-0.4M). - An intermediate drying step is carried out to reduce the moisture content to 25%. - Start spray engrobing with the denatured protein again and perform 2-3 cycles of spray engrobing with the denatured protein and calcium or magnesium cation crosslinker. - A final post-drying step is carried out to achieve a final moisture content of less than 5%.

[0157] Example 16 Probiotic Spray Englobing with Modified Mung Bean Protein, High Oleic Coconut Oil, and Calcium Cationic Crosslinker - Prepare dry ingredients (proteins, probiotics, simple sugars) and load into the chamber. -Prepare a modified mung bean protein suspension and a calcium cation crosslinker. - Start the fluidization process and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be maintained between 2~4.5Bar, and the airflow of the nozzle pressure should be adjusted accordingly. - Apply an intermediate drying step to reduce the moisture content to 50%. -Restart the spray engraving process with high oleic coconut oil. - An intermediate drying step is carried out to reduce the moisture content to 25%. - Restart the spray engraving process using high oleic sunflower oil. - An intermediate drying step is carried out to reduce the moisture content to 25%. - Restart the spray engraving process with calcium chloride (0.2-0.4M). - An intermediate drying step is carried out to reduce the moisture content to 25%. - Start spray engrobing with the denatured protein again and perform 2-3 cycles of spray engrobing with the denatured protein and calcium cation crosslinker. - A final post-drying step is carried out to achieve a final moisture content of less than 5%.

[0158] Example 17 Probiotic Spray Engroving with Modified Pea Protein and High Oleic Coconut Oil - Prepare dry ingredients (proteins, probiotics) and fill the chamber. -Prepare a modified mung bean protein suspension and a calcium cation crosslinker. - Start the fluidization process and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be maintained between 2~4.5Bar, and the airflow of the nozzle pressure should be adjusted accordingly. - Apply an intermediate drying step to reduce the moisture content to 50%. -Restart the spray engraving process with high oleic coconut oil. - An intermediate drying step is carried out to reduce the moisture content to 25%. - Restart the spray englove process using a simple carbohydrate (maltodextrin). - An intermediate drying step is carried out to reduce the moisture content to 25%. - Start the spray engrobing process again with the denatured protein and perform 2-3 cycles of spray engrobing with the denatured protein and simple carbohydrate solutions. - A final post-drying step is carried out to achieve a final moisture content of less than 5%.

[0159] Example 18 Probiotic spray engloved using modified mung bean protein and calcium cation crosslinker with optimized coating layer - Prepare dry ingredients (proteins, probiotics) and fill the chamber. -Prepare a modified mung bean protein suspension and a calcium cation crosslinker. - Start the fluidization process by spraying from above and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be maintained between 2~4.5Bar, and the airflow of the nozzle pressure should be adjusted accordingly. - Apply an intermediate drying step to reduce the moisture content to 50%. - Start the spray engraving process again using calcium chloride (0.2-0.4M). - An intermediate drying step is carried out to reduce the moisture content to 25%. - Start the spray engrobing process again with the denatured protein and perform 2-3 cycles of spray engrobing with the denatured protein and magnesium cation crosslinker. - A final post-drying step is carried out to achieve a final moisture content of less than 5%. - Start bottom spray engrooving with modified mung bean protein. - Using nozzle pressure above 2 Bar. - Maintain accelerator air pressure above 1.5 Bar during spray engroving. - Keep air flow in balance (approx. 90~170m 3 / time). - Maintain product temperature between 37-45°C. - Apply intermittent post-drying at 45-55°C. - At least two cycles of calcium cross-linked spray engrobing steps followed by intermittent drying are performed to achieve a 25% reduction in moisture content. - A final post-drying step is carried out to ensure a final moisture content of less than 5%.

[0160] Example 19 Probiotic spray globules using modified pea protein, high oleic coconut oil and calcium cation crosslinker with an optimized coating layer - Prepare dry ingredients (proteins, probiotics) and fill the chamber. -Prepare a modified mung bean protein suspension and a calcium cation crosslinker. - Start the fluidization process by spraying from above and begin heating (35-45°C). - 110~300m at set air inlet temperature 3 / Fluidized by the air flow of time. - Once the product temperature has equilibrated, begin the spray engraving process with the denatured protein. - The nozzle pressure should be maintained between 2~4.5Bar, and the airflow of the nozzle pressure should be adjusted accordingly. - Apply an intermediate drying step to reduce the moisture content to 50%. -Restart the spray engraving process with high oleic coconut oil. - An intermediate drying step is carried out to reduce the moisture content to 25%. - Restart the spray engraving process with calcium chloride (0.2-0.4M). - An intermediate drying step is carried out to reduce the moisture content to 25%. - Start the spray engrobing process again with the denatured protein and perform 2-3 cycles of spray engrobing with the denatured protein and magnesium cation crosslinker. - A final post-drying step is carried out to achieve a final moisture content of less than 5%. - Start bottom spraying with denatured protein. - Using nozzle pressure above 2 Bar. - Maintain accelerator air pressure above 1.5 Bar during spray engroving. - Keep air flow in balance (approx. 90~170m 3 / time). - Maintain product temperature between 37-45°C. - Apply intermittent post-drying at 45-55°C. - Spray englove process using high oleic coconut oil. - An intermediate drying step is carried out to reduce the moisture content to 25%. - At least two cycles of calcium cross-linking spray engraving steps followed by intermittent drying are performed to achieve a 25% reduction in moisture content. - Final post-drying is carried out to a final moisture content of less than 5%.

[0161] Example 20 Microbeads containing probiotics encapsulated in modified PPI (dPPI) - Single nozzle extrusion - Prepare a suspension of dPPI using dPPI prepared according to Example 1 and stir at 250 RPM at RT. - Add 2% glucidex / simple carbohydrates to the protein solution prior to encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at room temperature with gentle stirring to prevent air pockets. - The hydrated probiotic suspension of Example 2 is added to the dPPI solution, which is stirred at RT. -Add probiotic suspension to protein in a ratio of 9:1 or 18:1 (protein:probiotic). - Stir the protein-probiotic suspension at 400-450 RPM for 10 minutes.

[0162] Preparation of the hardening bath - Prepare calcium citrate buffer 0.1 M and adjust the pH to 5.2 (range 5.2-6.1). Add 0.05 (w / w)% polysorbate 20% as a surfactant. - An alternative hardening bath is calcium chloride buffer 0.1M, adjusted to pH 4.8 (range 4.3-5.0), with 0.05% polysorbate 20% added as a surfactant. - An alternative hardening bath is calcium citrate buffer 0.1M containing 0.05% chitosan calcium (chelating agent) and 0.05% (w / aw) polysorbate 20% (surfactant). - Once the probiotics are fully hydrated, the protein-probiotic solution is extruded at room temperature through a single nozzle extruder and polymerized in a calcium hardening bath. - Polymerization occurs in a calcium buffer bath at room temperature. - The microcapsules are collected and washed with a neutralizing solution of pH 6.1. - The microcapsules are loaded into an associated drying chamber. Drying can be carried out as follows: Freeze-dry for 48 hours (see Table 1) Fluidized bed drying: 37℃, 4 to 6 hours Vacuum drying: less than 10mBar, 37℃ for 24-48 hours

[0163] Example 21 Microbeads containing probiotics encapsulated in modified PPI - Dual (concentric) nozzle extrusion - Prepare a suspension of dPPI using dPPI prepared as in Example 1 and stir at 250 RPM at RT. - Add 2% glucidex / simple carbohydrates to the protein solution prior to encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at room temperature with gentle stirring to prevent air pockets. Prepare a calcium bath according to Example 7. - The dPPI / Glusidex suspension is extruded through the outer nozzle and the probiotic suspension through the inner nozzle. - Polymerization occurs in a calcium buffer bath at room temperature. - The microcapsules are collected and washed with a neutralizing solution of pH 6.1. - The microcapsules are loaded into an associated drying chamber. Drying can be carried out as follows: Freeze-dry for 48 hours (see Table 1) Fluidized bed drying: 37℃, 4 to 6 hours Vacuum drying: less than 10mBar, 37℃ for 24-48 hours

[0164] Example 22 Spray engraving process of probiotics, bioactive substances in modified PPI - Prepare a suspension of dPPI using dPPI prepared as in Example 1 and stir at 250 RPM at RT. - Add 2% glucidex / glucose / simple carbohydrates to the protein solution prior to encapsulation (this imparts a charge to the denatured protein solution, creating a compatible matrix for probiotics). - Hydrate for 2 hours at room temperature with gentle stirring to avoid creating air pockets. -Add dry pea protein powder (native powder, moisture approximately 2%) to the fluid bed chamber. - 37℃, fluidized with high airflow -Add probiotic cultures to the powder in a 5:1 or 10:1 ratio. - 15 Add calcium citrate or calcium chloride to the chamber and fluidize. - Spray 10% Glusidex solution onto the material at a high flow rate (20-30RPM), high airflow (>150RPM) and low nozzle pressure (<1.5bar) to promote agglomeration. - Atomize coconut oil / sunflower oil / high oleic oil into powder with low flow rate (18-22RPM), high airflow (150RPM or higher) and high nozzle pressure (2.5bar or higher). - The dPPI-Glusidex solution is sprayed into the chamber at a high flow rate (25-30 RPM), high airflow (>150 RPM), and high nozzle pressure (>2.5 bar). - Dry after so that Aw<0.15.

[0165] equivalent product The foregoing description details the presently preferred embodiments of the present invention. Numerous modifications and variations in its practice are anticipated to occur to those skilled in the art upon consideration of these descriptions. Such modifications and variations are intended to be encompassed within the scope of the claims appended hereto.

Claims

1. 1. A method for forming microparticles comprising probiotics encapsulated in a modified plant protein matrix, comprising: adding the carrier material and probiotics to the bed of a fluid bed dryer; fluidizing the carrier material and probiotics; spraying a first coating material onto the fluidized carrier material and probiotics to produce microparticles having the probiotics and carrier entrapped within a shell of the first coating material; and Drying the microparticles wherein the first coating material comprises a modified vegetable protein.

2. 10. The method of claim 1, wherein 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 producing granules comprising the carrier material, the probiotic, and the simple sugar, and the first coating material coating the granules.

3. The method of claim 1 , wherein the first coating material comprises an oil or a simple sugar such as maltodextrin.

4. 10. The method of claim 1, wherein the method comprises spraying a second coating material onto the shell of the first coating material, the second coating material comprising a modified vegetable protein.

5. 5. The method of claim 4, wherein the method comprises spraying the second coating material with a chelating salt.

6. the method is a spray engroving process; (a) adding the carrier material and probiotics to the bed of a fluidized bed drying chamber; (b) fluidizing and heating the carrier material and probiotics to form a first fluidized powder. (c) spraying the first coating material comprising a denatured protein onto the fluidized bed at high pressure to provide a second fluidized powder; (d) drying the second fluidized powder on the fluidized bed to reduce the moisture content of the second fluidized powder; (e) spraying an englobing component onto the second fluidized powder to form a third fluidized powder, wherein the englobing component is selected from a chelating salt, an edible oil, and a simple sugar; (f) drying the third fluidized powder on the fluidized bed to further reduce the moisture content of the fluidized powder; (g) spraying the denatured protein suspension onto the fluidized bed at high pressure to provide a fourth fluidized powder comprising microparticles; and Optionally, further drying the fourth fluidized powder, typically to a moisture content of less than 10%, less than 8%, or less than 5%. The method of claim 1 , comprising:

7. 7. The method of claim 6, wherein the method comprises at least two or three rounds of steps (f) and (g).

8. the method is a spray engroving process; fluidizing the carrier material, probiotics, and chelating salt on a fluidized bed in a fluidized bed drying chamber; spraying edible oil into the fluidized bed drying chamber; spraying the first coating material comprising a modified plant protein into a fluidized bed drying chamber. Including, 10. The method of claim 1, whereby the chelating salt reacts with the denatured plant protein to polymerize the protein and form agglomerated microparticles having a coating of polymerized denatured protein and a core comprising the active agent and carrier material.

9. 10. The method of claim 8, wherein the carrier material is added to the fluidized bed first, and the probiotic and chelating salt are added to the fluidized bed after the carrier material.

10. 9. The method of claim 8, wherein the carrier material, probiotics and chelating salt are fluidized at 30-40°C before adding edible oil to form a fluidized mixture.

11. 10. The method of claim 8, wherein a simple sugar is sprayed into the fluidized bed before the edible oil to promote granulation of the mixture.

12. 10. Microparticles obtainable by the method of claim 1, wherein said microparticles are gastroresistant and ileal sensitive.

13. 1. A microparticle comprising a probiotic encapsulated within a polymerized vegetable protein matrix, the microparticle having a core-shell structure, the core comprising the probiotic and a carrier material selected from native proteins and sugars, and the shell comprising the polymerized vegetable protein matrix.

14. 14. The microparticle of claim 13, wherein the polymerized plant protein matrix comprises a simple carbohydrate.

15. 14. The microparticle of claim 13, comprising a coating of edible oil.

16. 14. Microparticles according to claim 12 or 13, having a mean dimension Dv of 200 to 700 μm.

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