Method of producing microparticles of the type having a crosslinked, aggregated protein matrix by spray drying

Abstract

A method of producing microparticles by spray drying comprises the steps of providing a spray-drying feedstock solution comprising water, a volatile divalent metal salt, weak acid, 5-15% dairy or vegetable protein (w/v) and 1-20% active agent (w/v). The feedstock solution is adjusted to have a pH at which the volatile divalent metal salt is substantially insoluble. The feedstock solution is then spray-dried at an elevated temperature to provide atomised droplets, whereby the volatile divalent metal salt disassociates at the elevated temperature to release divalent metal ions which crosslink and aggregate the protein in the atomised droplets to produce microparticles having a crosslinked aggregated protein matrix and active agent dispersed throughout the matrix.

Claims

1. A method of producing microparticles by spray drying, the method comprising the steps of: providing a food-grade spray-drying feedstock solution comprising water, a volatile divalent metal salt, a weak acid, 5-15% dairy or vegetable protein (w/v) and an active agent, the feedstock solution having a pH at which the volatile divalent metal salt is substantially insoluble; spray drying the feedstock solution at an elevated temperature to provide atomised droplets whereby the volatile divalent metal salt disassociates at the elevated temperature to release divalent metal ions which crosslink and aggregate the protein in the atomised droplets to produce microparticles having a crosslinked aggregated protein matrix and the active agent dispersed throughout the matrix, wherein the active agent is a cellular active agent, the spray-drying feedstock comprises 1-20% of the active agent (w/v), and the solids content of the feedstock is 40-70%.

2. The method according to claim 1 in which the spray-drying feedstock comprises 1-10% hydrocolloid (w/v).

3. The method according to claim 1 in which the spray-drying feedstock comprises 1-3% hydrocolloid (w/v).

4. The method according to claim 2 in which the hydrocolloid is selected from the group consisting of fructooligosaccharide, galactooligosaccharide, carrageenan and guar gum.

5. The method according to claim 4 in which the spray-drying feedstock comprises 1-3% fructooligosaccharide (w/v).

6. The method according to claim 1 in which the volatile divalent metal salt is selected from the group consisting of a divalent metal ion carbonate, a divalent metal ion chloride, a divalent metal ion phosphate, a divalent metal ion citrate, a divalent metal ion ascorbate, a divalent metal ion HMB, and a mixture thereof.

7. The method according to claim 1 in which the weak acid is ascorbic acid or succinic acid or a mixture thereof.

8. The method as claimed in claim 1 in which the protein is a dairy protein selected from the group consisting of UHT milk, milk protein, skim milk powder, and a mixture thereof.

9. The method as claimed in claim 1 in which the protein is a vegetable protein selected from the group consisting of pea protein, rice protein, wheat protein, and a mixture thereof.

10. A preparation of spray-dried microparticles prepared according to the method of claim 1.

11. A method of producing microparticles by spray drying, the method comprising the steps of: providing a food-grade spray-drying feedstock solution comprising water, a volatile divalent metal salt, a weak acid, 5-15% dairy or vegetable protein (w/v), and an active agent, the feedstock solution having a pH at which the volatile divalent metal salt is substantially insoluble; spray drying the feedstock solution at an elevated temperature to provide atomised droplets whereby the volatile divalent metal salt disassociates at the elevated temperature to release divalent metal ions which crosslink and aggregate the protein in the atomised droplets to produce microparticles having a crosslinked aggregated protein matrix and the active agent dispersed throughout the matrix, wherein the active agent is a compound and the spray-drying feedstock comprises 30-60% of the active agent (w/v).

12. The method according to claim 11 in which the solids content of the feedstock is 50-80%.

13. A method of producing microparticles by spray drying, the method comprising the steps of: providing a food-grade spray-drying feedstock solution comprising water, a volatile divalent metal salt, a weak acid, 5-15% dairy or vegetable protein (w/v), and an active agent, the feedstock solution having a pH at which the volatile divalent metal salt is substantially insoluble; spray drying the feedstock solution at an elevated temperature to provide atomised droplets whereby the volatile divalent metal salt disassociates at the elevated temperature to release divalent metal ions which crosslink and aggregate the protein in the atomised droplets to produce microparticles having a crosslinked aggregated protein matrix and the active agent dispersed throughout the matrix, wherein the spray-drying feedstock solution is prepared by the steps of: preparing an aqueous solution of the weak acid; preparing an aqueous dispersion of the volatile divalent metal salt; mixing the aqueous solution and the aqueous dispersion to provide a weak acid/volatile divalent metal salt dispersion and adjusting the pH such that the volatile divalent metal salt is substantially insoluble in the dispersion; preparing an aqueous dispersion of the protein; admixing the active agent with the aqueous dispersion of the protein to provide an active agent/protein dispersion; and admixing the active agent/protein dispersion and the weak acid/volatile divalent metal salt dispersion at a ratio of 1.0:1.5 to 1.5:1.0 to form the spray-drying feedstock solution.

14. The method as claimed in claim 13 in which the spray drying feedstock solution further comprises a hydrocolloid, wherein a dispersion of the hydrocolloid is added to the aqueous solution of the weak acid or added to the active agent/protein dispersion.

15. The method as claimed in claim 13 in which the aqueous solution of the weak acid has a weak acid concentration of 0.2 M-2.2 M.

16. The method as claimed in claim 13 in which the aqueous dispersion of the volatile divalent metal salt has a volatile divalent metal salt concentration of 0.2 M-2.2 M.

17. The method as claimed in claim 13 in which the aqueous dispersion of the protein has a protein concentration of 4.0-15.0 (w/v).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Schematic of a standard Spray drier.

(2) FIG. 2. Illustration of Mug Ring in a dry chamber.

(3) FIG. 3. List of divalent salt with potential use in the presented invention.

(4) FIG. 4. Calibration range for the use of a weak acid and a relevant calcium salt (single sources or mixtures of relevant chloride, carbonate or citrate salts).

(5) FIG. 5. Size distribution of spray-dried native protein (Skim Milk Protein) showing the small particle size (COMPARATIVE).

(6) FIG. 6. SEM image of native protein (Skim Milk Protein) spray-dried in the absence of the Acid/base mix (COMPARATIVE).

(7) FIG. 7. Size distribution of pea protein microparticles spray-dried according to the method of the invention in the presence of a calcium salt mixture (3 different calcium salt sources).

(8) FIG. 8. SEM images illustrating the pea protein microparticles of the invention of FIG. 7.

(9) FIG. 9. Size distribution of microparticles spray dried according to the invention, and which employ two protein sources (Whey Protein Isolate and Pea Protein isolate) and one calcium salt (calcium triphosphate).

(10) FIG. 10. SEM image illustrating the pea protein/WPI microparticles of the invention of FIG. 9.

(11) FIG. 11. Size distribution of microparticles spray dried according to the invention, and which employ one protein source (Whey Protein Isolate) and one calcium salt (calcium carbonate).

(12) FIG. 12. SEM image illustrating the WPI microparticles of the invention of FIG. 11.

(13) FIG. 13. Survival of a probiotic stain (Lb. acidophilus) in the presence of succinic acid and ascorbic acid as the weak acid in the mix. Image of the particulate network after drying with entrapped probiotic cells.

(14) FIG. 14. Percent insoluble generated in the presence/absence of EDTA.

DETAILED DESCRIPTION OF THE INVENTION

(15) This invention relates to the making of microparticles in a spray dryer using a liquid feedstock comprising protein and a crosslinking agent in an inactive precursor form. The liquid feedstock comprises a crosslinking agent in the form of a volatile divalent metal salt, and a weak acid that maintains the pH of the feedstock prior to atomisation above the pH at which the volatile divalent metal salt solubilises. The feedstock typically dispersion remains dispersible, flowable to enable extrusion through a nozzle or rotating disk. Generally, no cross-linking or protein aggregation occurs before atomisation in the drying chamber due to sequential addition of native protein, a weak acid and a salt of a volatile base. The volatile divalent metal salt comprises a divalent metal cation (i.e. calcium, zinc, magnesium or potassium) capable of crosslinking and aggregating the protein, and a volatile anion (i.e. chloride, citrate, carbonate, sulfate, gluconate), that is sensitive to high heat. Thus, at relatively basic pH the salt is insoluble in the feedstock dispersion and unavailable to react with the native protein. However, upon heating of the atomised droplets in the drying chamber, four steps spontaneously occur: i) the heat of the drying chamber partially hydrolyses the protein; ii) the salt disassociates releasing and vaporising the volatile base i.e. anions; iii) evaporation of the volatile base reduces the pH of the dispersion; iv) the development of an acidic environment results in the solubilisation (bioavailability) of divalent metal cations iv) the bioavailability of divalent cations activates the crosslinking and subsequent aggregation reaction of partially hydrolysed protein. Without being bound by theory, the affinity of calcium ions for the protein is a higher attractive force relative the weak acid present. Hence the formation of a calcium ascorbate or calcium acetate is not a likely occurrence due to the high availability of protein aggregates and the significantly lower concentration of weak acid. Hence, the calcium will always be attracted and available for protein aggregation, crosslinking and polymerisation.

(16) The release of the cross-linking agent, the generation of an acidic environment and the availability of partially hydrolysed protein, provides optimum conditions for the crosslinking and aggregation in the drying chamber of a standard spray-drier. Its activation of protein aggregation as a result of calcium availability is a consequence of protein-protein interaction via calcium (divalent cation). This results in disulfide bond formation during the spray drying. The use of a mixture of calcium salts sources i.e. calcium triphosphate, calcium chloride, calcium carbonate, can provide for a better and enhanced aggregation reaction. When two different protein sources are admixed prior to spray drying i.e. milk protein+pea protein or whey protein+sodium casein, the result is polymerisation of the proteins in the presence of divalent cations with the formation of ionic and disulfide links.

(17) Thus, the present invention illustrates a crosslinking and agglomeration reaction between a protein and a divalent salt for the stabilisation of bioactive compounds with efficient atomisation characteristics for the generation of a flowable, stable, wettable and commercial powders suitable for a platform of food and therapeutic applications. In essence, the presented invention illustrates that the protein matrix material and crosslinking agent must be i) combined and introduced to the drying chamber as single liquid feedstock. The feedstock dispersion must remain soluble, flowable and suitable for extrusion through a nozzle or rotating disk. No cross-linking agglomeration reaction is should occur before atomisation in the drying chamber i.e. divalent ions must not be available for interaction. For this reason, a volatile base and weak acid are introduced sequentially into the feedstock in order to maintain a flowable dispersion. Upon introduction of the liquid feedstock into the dryer chamber, high heat conditions will catalyse the evaporation of the volatile base, leading to a significant pH reduction and concomitant release of (crosslinking) divalent cations, which will be present in a bioavailable, soluble form. The bioavailability of divalent cations will spontaneously activate the aggregation of protein molecules; the latter of which are hydrolysed as a result of high heat in the drying chamber. Hence, the release of cross-linking ions and the availability of partially hydrolysed protein, provides optimum conditions for a protein crosslinking and subsequent aggregation in the drying chamber.

(18) Materials Required

(19) Polymer:

(20) Hydrocolloid or protein i.e. UHT milk, skim milk powder SMP, soy, milk or vegetable protein, FOS, GOS, carrageenan, alginate or hydrocolloid mixtures thereof

(21) Salt:

(22) Divalent salt i.e. Calcium, Magnesium, zinc, or mixtures thereof

(23) Acid

(24) Weak Acid i.e. ascorbic acid, succinic acid, acetic acid, or mixtures thereof

(25) Succinic Acid:

(26) Succinic acid (IUPAC name is butanedioic acid is a diprotic, dicarboxylic acid with chemical formula C.sub.4H.sub.6O.sub.4 and structural formula HOOC—(CH.sub.2).sub.2—COOH. It is a white, odourless solid. Succinate plays a role in the citric acid cycle, an energy-yielding process. Succinic acid is used in the food and beverage industry, primarily as an acidity regulator. It is also sold as a food additive and dietary supplement, and is generally recognized as safe for those uses by the U.S. Food and Drug Administration. As an excipient in pharmaceutical products it is used to control acidity and, more rarely, in effervescent tablets. At the level succinic acid occurs naturally in foods, there is no evidence that it is hazardous to man or animals. Moreover, experimental animals tolerate succinic acid in amounts equivalent to several g per kg of body weight. By contrast, a reasonable average daily intake of succinic acid added to foods is estimated to be less than 0.01 mg per day, a dosage that is orders of magnitude less than that required to elicit toxic signs in experimental animals. Based on these considerations, the Select Committee concludes that: There is no evidence in the available information on succinic acid that demonstrates, or suggests reasonable ground to suspect, a hazard to the public when it is used at levels that are now current or that might reasonably be expected in the future. Ascorbic Acid: Ascorbic acid is a six carbon compound related to glucose. It is found naturally in citrus fruits and many vegetables. Ascorbic acid is an essential nutrient in human diets, and necessary to maintain connective tissue and bone. Its biologically active form, vitamin C, functions as a reducing agent and coenzyme in several metabolic pathways. Vitamin C is considered an antioxidant. It is a white to slightly yellow crystalline powder that gradually darkens on exposure to light. Solubility in water is approx. 80% at 100 Deg C. and 40% at 45 Deg C. Manufacture: The classical Reichstein-Grussner synthesis starts with reduction of D-glucose to D-sorbitol by hydrogenation over a nickel catalyst. The microbiological oxidation of D-sorbitol to L-sorbose is carried out with Acetobacter xylinum. On treatment of L-sorbose with acetone at low temperature in the presence of sulfuric acid, 2,3:4,6-di-O-isopropylidene-alpha-L-sorbofuranose formed. The di-O-isopropylidenyl protection of the hydroxyl-groups at C-2, C-3 and C-4, C-6 allows high-yield oxidation to di-O-isopropylidene-2-ketogulonic acid, without over-oxidation or other side reactions. The oxidation is carried out with potassium permanganate in alkaline solution. Treatment of/di-O-isopropylidene-2-ketogulonic acid/with hot water affords 2-keto-L-gulonic acid, which is converted to L-ascorbic acid by heating in water at 100 deg C. (20% yield) or by esterification and treatment with sodium methoxide in methanol followed by acidification with hydrogen chloride, yielding ca. 70% of/L-ascorbic acid/. The overall yield of ascorbic acid from D-glucose is 15-18%.

(27) Analytical Method for Detection of Ascorbic Acid:

(28) AOAC Method 967.21. Vitamin C (Ascorbic Acid) in Vitamin Preparations and Juices. 2,4-Dichloroindophenol Titrimetric Method. Ascorbic acid reduces oxidation-reduction indicator dye, 2,4-dichloroindophenol, to colorless solution. At end point, excess unreduced dye is rose pink in acid solution. Vitamin is extracted and titration is performed in presence of HPO3-HOAc or HPO3-HOAc—H2SO4 solution to maintain proper acidity for reaction and to avoid autoxidation of ascorbic acid at high pH. (Reference: Association of Official Analytical Chemists. Official Methods of Analysis. 15th ed. and Supplements. Washington, D.C.: Association of Analytical Chemists, 1990, p. 1059).

(29) General Methodology Spray drying of an aqueous dispersion containing a hydrocolloid mixture containing sufficient protein to allow a crosslinking and subsequent aggregation reaction (UHT milk, skim milk powder, vegetable protein, or mixtures thereof). A divalent salt (Calcium, Magnesium, zinc) is introduced that is soluble at acidic pH only. A weak acid (ascorbic acid, succinic acid or acetic acid) is introduced at a pH (preferably above pH 5, ideally above pH 7) just above the pike with a volatile base (calcium carbonate, calcium chloride, etc.; See FIG. 4). Under these conditions, the divalent salt is insoluble and ions are not available for cross-linking and aggregation with the protein molecules from the hydrocolloid component of the mixture. when a hydrocolloid such as FOS or GOS is employed, the hydrocolloid can be admixed to the weak acid, or added to the liquid feedstock at any point but typically just prior to the preheating stage. The solution (liquid feedstock) in this fluid state is preheated and pumped through the nozzle of the single/multi-stage spray drier, where it is effectively atomised. Inlet and outlet temperatures are maintained in accordance with standard commercially viable conditions to allow appropriate atomisation. i.e. 180 DegC inlet and 85-90 Dec outlet temperature Upon atomisation at these temperatures the volatile base is vaporised and the protein content enters into a hydrolysed state. Upon evaporation of the base, the pH is reduced (hydrogen ions are released into solution) and divalent ions are released and enter a bioavailable state. Calcium/the relevant divalent cations is readily available for crosslinking and aggregation with the protein/hydrocolloid source, mixture thereof. The crosslinking and subsequent aggregation reaction is initiated as a result of the temperature of the drier. i.e. high temperature permits evaporation of the volatile base, and concomitant hydrolysis of the protein source and bioavailability of the divalent cations. These three conditions accelerate the cross-linking aggregation and entrapment of an active compound. Spray drying is the method of choice as it the industry standard for dehydration of materials. Furthermore, it prevents multiplication of contaminants because powders are easy to handle in the field and the method is well documented an accepted across all industrial disciplines.

(30) Method 1: Whey protein isolate (WPI) or concentrate (WPC) at a total protein content of 8.0% w/v (protein basis) in water The protein dispersion is centrifuged at 10,000 rpm; 16 Dig Celsius; 45 minutes Supernatant is agitated overnight at 4° C. at pH 7.5 The protein dispersion is filtered (0.2 micron) at room temperature A solution (8.0% w/v) of ascorbic acid is prepared in water and agitated at room temperature before addition of 2% FOS A calcium β-hydroxy-β-methylbutyrate (CaHMB) solution (0.4% (w/v) is prepared and dispersed in water. The pH is adjusted to 7.5 using 4M NaOH. This represents a Asc/CaHMB mix. The pellet of cells/powdered bioactive material is resuspended in the protein dispersion Cell concentrations is approx. 1×10.sup.11 CFU/mL. Bioactive material can be dispersed at max 50% solid content. The protein dispersion with cells/active is then mixed with Ascorbic-Calcium solution at ratio 1:1 (v/v) Agitation is then performed at 65° C. to pre-heat for the drier At this point the solution is fluid and call the feedstock Spray dry the suspension using a single-stage drier Standard inlet and outlet temperatures will apply i.e. inlet 180° C. and outlet 85-90° C. Delivery of suspension via peristaltic pump was fixed to 600 mL/hr (Bench top) or 20 L per hour (pilot scale). Nozzle atomization is utilized as per standard industry practice. Material is dried to a Aw of 0.2 and storage at refrigerated temperatures in hermetically sealed drums/foil bags.

(31) Method 2 Pea protein isolate (PPI) prepared at a total protein content of 9.0% w/v (protein basis) in water The protein dispersion is centrifuged at 10,000 rpm; 16 Dig Celsius; 45 minutes Supernatant is agitated overnight at 4° C. at pH 7.2 The protein dispersion is filtered (0.2 micron) at room temperature A solution (12.0% w/v) of ascorbic acid is prepared in water and agitated at room temperature before addition of 2% FOS (w/v) A calcium carbonate (or calcium ascorbate or calcium citrate or CaHMB) (0.4% (w/v) is prepared and dispersed in water. Ascorbic acid and Calcium source is admixed At this point, ascorbic acid concentrations are optimum to permit the natural and adequate neutral pH for the feedstock i.e. neutral pH to avoid maintain divalent ions in an ‘Unavailable state”. This represents a Asc/Ca mix. The pellet of cells/powdered bioactive material is re-suspended in the protein dispersion Cell concentrations is approx. 1×10.sup.11 CFU/mL. Bioactive material can also be dispersed at max 50% solid content. The protein dispersion with cells/active is then mixed with Ascorbic-Calcium dispersion at ratio 1:1 (v/v) Agitation is then performed at 65° C. to pre-heat for the drier At this point the solution is fluid and classified as feedstock Spray dry the suspension using a single stage drier Standard inlet and outlet temperatures will apply i.e. inlet 180° C. and outlet 85-90° C. Delivery of suspension via peristaltic pump was fixed to 600 mL/hr (Bench top) or 20 L per hour (pilot scale). Nozzle atomization was used as per standard industry procedure. Material is dried to a Aw of 0.2 and storage at refrigerated temperatures in hermetically sealed drums/foil bags.

(32) Method 3 Pea protein isolate (PPI) or milk protein isolate (WPI) prepared at a total protein content of 9.5% w/v (protein basis) in water The protein dispersion is centrifuged at 10,000 rpm; 16° C.; 45 minutes Supernatant is agitated overnight at 4° C. at pH 7.5 The protein dispersion is filtered (0.2 micron) at room temperature A solution (12.0% w/v) of ascorbic acid is prepared in water and agitated at room temperature before addition of 2% FOS (w/v) A calcium triphosphate, calcium chloride, calcium carbonate solution totally (1.8% (w/v) is prepared and dispersed in water. Ascorbic acid and Calcium mix is admixed At this point, ascorbic acid concentrations are optimum to permit the natural and adequate neutral pH for the feedstock i.e. neutral pH to avoid maintain divalent ions in an ‘Unavailable state”. This represents a Asc/Ca mix. The pellet of cells/powdered bioactive material is resuspended in the protein dispersion Cell concentrations is approx. 1×10.sup.11 CFU/mL. Bioactive material can also be dispersed at max 50% solid content. The protein dispersion with cells/active is then mixed with Ascorbic-Calcium dispersion at ratio 1:1 (v/v) Agitation is then performed at 65° C. to pre-heat for the drier At this point the solution is fluid and classified as feedstock Spray dry the suspension using a single stage drier Standard inlet and outlet temperatures will apply i.e. inlet 180° C. and outlet 85-90° C. Delivery of suspension via peristaltic pump was fixed to 600 mL/hr (Bench top) or 20 L per hour (pilot scale). Nozzle atomization was used as per standard industry procedure. Material is dried to a Aw of 0.2 and storage at refrigerated temperatures in hermetically sealed drums/foil bags.

(33) Method 4 Whey Protein (WPI/WPC) and pea protein isolate (PPI) prepared at a total protein content of 15.0% w/v (protein basis) in water The protein dispersion is centrifuged at 10,000 rpm; 16° C.; 45 minutes Supernatant is agitated overnight at 4° C. at pH 7.2 The protein dispersion is filtered (0.2 micron) at room temperature A solution (12.0% w/v) of succinic acid is prepared in water and agitated at room temperature A calcium triphosphate (0.5% (w/v) is prepared and dispersed in water. Succinic acid and Calcium source is admixed At this point, succinic acid concentrations are optimum to permit the natural and adequate neutral pH for the feedstock i.e. neutral pH to avoid maintain divalent ions in an ‘Unavailable state”. This represents a Succ/Ca mix. The pellet of cells/powdered bioactive material is resuspended in the protein dispersion Cell concentrations is approx. 1×10.sup.11 CFU/mL. Bioactive material can also be dispersed at max 50% solid content. The protein dispersion with cells/active is then mixed with Ascorbic-Calcium dispersion at ratio 1:1 (v/v) Agitation is then performed at 65° C. to pre-heat for the drier At this point the solution is fluid and classified as feedstock Spray dry the suspension using a single stage drier Standard inlet and outlet temperatures will apply i.e. inlet 180° C. and outlet 85-90° C. Delivery of suspension via peristaltic pump was fixed to 600 mL/hr (Bench top) or 20 L per hour (pilot scale). Nozzle atomization was used as per standard industry procedure. Material is dried to a Aw of 0.2 and storage at refrigerated temperatures in hermetically sealed drums/foil bags

(34) Method 5: Whey Protein (WPI/WPC) and pea protein isolate (PPI) prepared at a total protein content of 15.0% w/v (protein basis) in water The protein dispersion is centrifuged at 10,000 rpm; 16° C.; 45 minutes Supernatant is agitated overnight at 4° C. at pH 7.2 The protein dispersion is filtered (0.2 micron) at room temperature A solution (12.0% w/v) of ascorbic acid is prepared in water and agitated at room temperature A calcium triphosphate, calcium chloride, calcium carbonate solution totally (1.8% (w/v) is prepared and dispersed in water. Ascorbic acid and Calcium mix is admixed At this point, ascorbic acid concentrations are optimum to permit the natural and adequate neutral pH for the feedstock i.e. neutral pH to avoid maintain divalent ions in an ‘Unavailable state”. This represents a Asc/Ca mix. The pellet of cells/powdered bioactive material is resuspended in the protein dispersion Cell concentrations is approx. 1×10.sup.11 CFU/mL. Bioactive material can also be dispersed at max 50% solid content. The protein dispersion with cells/active is then mixed with Ascorbic-Calcium dispersion at ratio 1:1 (v/v) Agitation is then performed at 65° C. to pre-heat for the drier At this point the solution is fluid and classified as feedstock Spray dry the suspension using a single stage drier Standard inlet and outlet temperatures will apply i.e. inlet 180° C. and outlet 85-90° C. Delivery of suspension via peristaltic pump was fixed to 600 mL/hr (Bench top) or 20 L per hour (pilot scale). Nozzle atomization was used as per standard industry procedure. Material is dried to a Aw of 0.2 and storage at refrigerated temperatures in hermetically sealed drums/foil bags

(35) Method 6: Generation of Stable Bioactive Vitamin C Whey Protein (WPI/WPC) and pea protein isolate (PPI) prepared at a total protein content of 2.0% w/v (protein basis) in water The protein dispersion is centrifuged at 10,000 rpm; 16° C.; 45 minutes Supernatant is agitated overnight at 4° C. at pH 7.2 A solution (3.5.0% w/v) of citrus fibre is prepared in water and agitated at room temperature Fructo-oligosaccahride (FOS) is prepared at 2.0% (W/v) concentration The protein and fibre dispersions are filtered (0.2 micron) at room temperature Protein, FOS and citrus fibre is admixed. A solution (82.0% w/v) of ascorbic acid is prepared in water and agitated at room temperature A calcium triphosphate or calcium chloride solution total 1.8% (w/v) is prepared and dispersed in water. Ascorbic Acid and Calcium Mix is Admixed

(36) At this point, ascorbic acid concentrations are optimum to permit the natural and adequate neutral pH for the feedstock i.e. neutral pH to avoid maintain divalent ions in an ‘Unavailable state”. The protein, FOS, fibre suspension is then mixed with Ascorbic-Calcium dispersion at ratio 1:1 (v/v) Agitation is then performed at 65 Degrees Celsius to pre-heat for the drier At this point the solution is fluid and classified as feedstock Spray dry the suspension using a single stage drier Standard inlet and outlet temperatures will apply i.e. inlet 180 Dec and outlet 85-90 DEgC Delivery of suspension via peristaltic pump was fixed to 600 mL/hr (Bench top) or 20 L per hour (pilot scale) Nozzle atomization was used as per standard industry pro

(37) Results:

(38) Sequential Formulation:

(39) Formulation optimisation of a weak acid and the salt of a volatile base system where the salt is insoluble at neutral pH values. Ascorbic acid and calcium carbonate were screened at various concentration as illustrated in FIG. 4. Aliquots of 0.2M of the calcium salt was added to ascorbic acid to determine the appropriate concentration to make an feasible mix for drying.

(40) It is evident that a minimum quantity of calcium is required to react with the protein content present in the mix. It is also evident that a minimum quantity of weak acid (ascorbic acid is required) to raise the pH to the relevant level above the pKa of the calcium carbonate. Prior to spray drying the optimum ascorbic/succinic acid concentrations of 0.2 M-2.2 M worked well for introduction to calcium carbonate in the range of 0.1 M-2.0 M. Outside these ranges the mixture becomes turbid and there are precise ratios that are required to maintain the appropriate neutral pH, while also having adequate calcium available for crosslinking and aggregation. When the proposed formulation were applied to a spray-drier, the particle size is again significantly change as a result of the hydrolysis of the protein source with concomitant cross-linking and aggregation reaction with the available calcium ions.

(41) When resultant powders were assessed under Light Microscope, the following changes were observed in contrast to the raw materials. They physiochemical properties of the suspension and related dry powder illustrate the occurrence of the cross-linking reaction between the partially hydrolysed protein and the solubilised Ca.sup.2+ ions. The pH of the suspension in a stepwise manner is illustrated as follows:

(42) TABLE-US-00001 Initial weak Acid/Calcium Salt Mix pH 4.1-5.0 Protein hydrocolloid suspension + Mix (T0) pH 7.0-.7.5 After Spray Drying pH 4.5-5.0

(43) Determination of Size Distribution

(44) The presented invention was tested in a standard spray drying process and the effect of the feedstock formulation (protein+weak acid+salt of a volatile base) was investigated as a function of particle size. Firstly, it was important to investigate the effect of spray drying the protein only in the absence of the acid base mixture and FIG. 5 illustrates the size distribution a native protein source, after a spray drying process. The size distribution is relatively low i.e. 12.2 microns±1.53 microns with a narrow size distribution. This illustrates the expected size range that a native protein would generate after heat processing through a spray drier. Microscopy illustrated no change in the particle size of native protein (FIG. 6) where the average particle size is expected to be 12.5 microns.

(45) FIG. 7 illustrates the size distribution of the mix when the calcium is sourced from several salts i.e. chloride, carbonate, phosphate. The size distribution is significantly greater than native protein after drying; which endorses the aggregation of protein particles, as evidenced in FIG. 6. Scanning Electron Microscope images illustrate a change in particle morphology as a result of drying in the presence of a calcium salt mixture. In FIG. 8 the presence is aggregated protein is shown with a large particle size. Individual aggregated particulates are 103 microns±1.5 microns with a narrow distribution for individual protein particles. This illustrates a stable drying process and efficient atomisation process.

(46) The use of two protein sources and once calcium source further illustrates the aggregation process (FIGS. 9 and 10) with the generation of aggregates with diameters of approx. 56.34 microns. SEM endorses the completion of the protein aggregation reaction. Most importantly the use of i) one/two protein sources and ii) a single/cocktail of calcium salt source will dictate the final particle size of the dried powder. This final particle size will be further dictate the production application for the ingredient. FIGS. 10 and 11 further illustrate the aggregation reaction when one protein and one calcium salt is utilised. Particles are generated with a narrow size distribution i.e. 63.6 microns±1.9 microns with specific product applications.

(47) The particle remain below 10 microns in diameter which further provides for a stable dispersion of the powder in aqueous solutions. In essence, the particles a stable dispersion of soluble aggregates for the protection of a specific entrapped bioactive.

(48) The type of weak acid utilised has no significant impact upon the survival of specific bioactives. FIG. 12 illustrates that the use of succinic acid or ascorbic acid has not effect on the viability of probiotic bacteria during the drying process. The pH of the final product will dictate the type of weak acid to be utilised.

(49) The use of calcium as a crosslinking/aggregation agent is further endorsed in FIG. 13 where EDTA is utilised to bind the protein and sedimentation reactions are also performed. EDTA was utilised in this experiment in order to verify if the calcium was in a bound or free state. The assay was performed as follows: Calcium Carbonate was admixed with EDTA in a 2:1 ratio to ensure the excess of EDTA in the reaction mixture Adjust the pH to 8.5 and 10 in order to accelerate the binding capacity of EDTA Several treatment were prepared as outlined in Table 2 All samples were Incubated for 24 h at room temperature Following incubation, pH values were recorded All samples were sedimentation of insolubles were measured Insoluble matter was calculated on a percent basis

(50) It is evident that the spray dried powder with the full reaction mixture generated a higher pelleted material relative the pre-dried mixture. This endorses the fact that the drying process stimulates the aggregation of proteins, which is demonstrated via the presence of higher pellet percent after sedimentation tests i.e. before spray drying the mixture illustrates 9.83% aggregated material; while after spray-drying of the mixture 22.8% sedimentation is generated.