PLANT PROTEIN-BASED MICROCAPSULES

Abstract

The present invention relates to plant-based microcapsules for the efficient encapsulation and retention of water-soluble ingredients, as well as the efficient co-encapsulation of water-soluble and water-insoluble ingredients. The present invention also relates to compositions comprising the microcapsules, a method of making the microcapsules and compositions, and to uses of the microcapsules and compositions.

Claims

1. A microcapsule comprising: (a) a hydrophilic phase comprising a water-soluble ingredient; (b) a lipophilic phase; and (c) a plant-based protein hydrogel shell.

2. A microcapsule according to claim 1, wherein the hydrophilic phase comprises water.

3. A microcapsule according to claim 1 or claim 2, wherein the water-soluble ingredient is selected from one or more of Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B7, Vitamin B9, Vitamin B12, Vitamin C, panthenol, α-hydroxy acids, water-soluble minerals salts, water-soluble plant extracts and yeasts, enzymes, antibiotics, oligopeptides, proteins and protein hydrolysates.

4. A microcapsule according to any preceding claim, wherein the plant-based protein(s) in the plant-based protein hydrogel shell is obtained from soybean, pea, rice, potato, wheat, corn zein or sorghum; preferably the plant protein(s) is selected from soy protein, pea protein, potato protein, rapeseed protein and/or rice protein.

5. A microcapsule according to any preceding claim, wherein the plant-based protein hydrogel shell is a self-assembled plant-based protein hydrogel shell.

6. A microcapsule according to any preceding claim, wherein the plant-based protein hydrogel shell comprises plant-based proteins having a protein secondary structure with at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet; and/or wherein the plant-based protein hydrogel shell has a storage modulus (G′) at 10 rad/s of greater than 500 Pa, greater than 1000 Pa, greater than 2500 Pa, greater than 3000 Pa, greater than 4000 Pa.

7. A microcapsule according to any preceding claim, wherein the plant-based protein hydrogel shell comprises protein aggregates with a median average length of between 50 to 500 nm or a mean average length of between 50 to 500 nm; or 80% of the aggregates have an average length of between 50 to 500 nm; and/or the aggregates may have a median height of between 5 to 50 nm; or the aggregates may have a mean average height of between 5 to 50 nm; or 80% of the aggregates have an average height of between 5 to 50 nm; preferably, the aggregates have a median average length of between 50 to 500 nm and/or a median average height of between 5 to 50 nm.

8. A microcapsule according to any preceding claim, wherein the hydrophilic phase is dispersed in the lipophilic phase.

9. A microcapsule according to any preceding claim, wherein the hydrophilic phase and the lipophilic phase are encapsulated by the plant-based protein hydrogel shell.

10. A microcapsule according to claim 9, wherein the hydrophilic phase and the lipophilic phase form a water-in-oil emulsion.

11. A microcapsule according to claim 10, having a multicore morphology.

12. A microcapsule according to claim 10, having a single core morphology.

13. A microcapsule according to any preceding claim, wherein the plant-based protein hydrogel shell has a thickness in the range 10 nm to 50,000 μm, preferably in the range 10 μm to 100 μm.

14. A microcapsule according to any preceding claim, wherein the protein content of the plant-based protein hydrogel shell is 5 to 20 g/100 g.

15. A microcapsule according to any preceding claim, wherein the Boisen protein digestibility of the plant-based protein hydrogel shell as measured according to the Boisen protocol is 80 to 100%.

16. A microcapsule according to any preceding claim, wherein the biodegradation percentage based upon O.sub.2 consumption of the plant-based protein hydrogel shell as measured according to ISO-14851 after 28 days is 70 to 100%.

17. A microcapsule according to any preceding claim, wherein the biodegradation percentage based upon CO.sub.2 production of the plant-based protein hydrogel shell as measured according to ISO-14851 after 28 days is 70 to 100%.

18. A microcapsule according to claim 1 or 2, which encapsulates at least one vitamin or mineral.

19. A microcapsule according to claim 18, wherein the at least one vitamin or mineral is selected from Vitamin A, Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B7, Vitamin B9, Vitamin B12, Vitamin C, Vitamin D, Vitamin E, Vitamin K, magnesium, sodium, potassium, zinc, iron, calcium, iodine and phosphorous, or mixtures thereof.

20. A microcapsule according to claim 18 or 19, wherein at least 25%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60% of the at least one vitamin or mineral initially encapsulated remains present inside the microcapsule after incubation in water at 20° C. for 10 days, as determined by HPLC.

21. A composition comprising at least one microcapsule according to any one of claims 1 to 20 and an external phase.

22. A composition according to claim 21, wherein the at least one microcapsule is dispersed in the external phase.

23. A method for preparing a microcapsule according to any preceding claim, comprising: (a) emulsifying a hydrophilic phase comprising a water-soluble ingredient in a first lipophilic phase to give a primary emulsion; (b) re-emulsifying said primary emulsion in a plant-based protein solution comprising one or more plant-based protein(s), wherein said plant-based protein solution is at a temperature above the sol-gel transition temperature of the plant-based protein solution, to give a double emulsion; (c) re-emulsifying said double emulsion in a second lipophilic phase to give a triple emulsion; (d) inducing the plant-based protein(s) in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel shell, wherein said plant-based protein hydrogel shell encapsulates said primary emulsion to form a microcapsule which is suspended in an external phase which is the second lipophilic phase; and (e) washing the microcapsule to remove the second lipophilic phase.

24. A method according to claim 23, wherein the plant-based protein solution comprises one or more plant-based protein(s) in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases solubility of the plant-based protein(s), and a second co-solvent decreases solubility of the plant-based protein(s).

25. A method according to claim 24, wherein the first co-solvent is an organic acid; preferably acetic acid and/or an α-hydroxy acid; wherein the α-hydroxy acid may preferably be selected from glycolic acid, lactic acid, malic acid, citric acid and/or tartaric acid; with particularly preferred organic acids being acetic acid and/or lactic acid.

26. A method according to claim 24 or 25, wherein the second co-solvent(s) is an aqueous buffer solution, preferably selected from water, ethanol, methanol, acetone, acetonitrile, dimethylsulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, t-butanol, ethyl acetate, hexafluoroisopropanol, more preferably water and/or ethanol, particularly preferably water.

27. The method according to any one of claims 23 to 26, wherein in step (d) the protein solution is heated to a first temperature above the sol-gel temperature of the one or more plant-based protein(s), then reduced to a second temperature below the sol-gel temperature of the one or more plant-based protein(s) to form the plant-based protein hydrogel shell.

28. A microcapsule prepared according to the method of any one of claims 23 to 27.

29. A method for preparing a composition according to claim 21 or 22, comprising: (a) emulsifying a hydrophilic phase comprising a water-soluble ingredient in a first lipophilic phase to give a primary emulsion; (b) re-emulsifying said primary emulsion in a plant-based protein solution comprising one or more plant-based protein(s), wherein said plant-based protein solution is at a temperature above the sol-gel transition temperature of the plant-based protein, to give a double emulsion; (c) re-emulsifying said double emulsion in a second lipophilic phase to give a triple emulsion; (d) inducing the plant-based protein(s) in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel shell, wherein said plant-based protein hydrogel shell encapsulates said primary emulsion to form a microcapsule which is suspended in an external phase which is the second lipophilic phase.

30. A method according to claim 29, further comprising: (e) washing the microcapsule to remove the second lipophilic phase; and (f) re-suspending the microcapsule in an external aqueous phase.

31. A method according to claim 30, further comprising adding suspending agents to said external aqueous phase.

32. A method according to claim 31, wherein said suspending agents are selected from acacia gum, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, hydrogenated vegetable oil category 1, aluminum magnesium silicate, maltodextrin, carboxymethyl cellulose, polymethacrylate, poly vinyl pyrrolidone, sodium alginate, starch, zein, water-insoluble cross-linked polymers such as cross-linked cellulose, cross-linked starch, cross-linked CMC, cross-linked carboxymethyl starch, cross-linked polyacrylate, and cross-linked polyvinylpyrrolidone, and expanded clays such as bentonite and laponite.

33. A composition prepared according to the method of any one of claims 29 to 32.

34. A food, beverage, cosmetic, home care product, personal care product, pharmaceutical, medical device, biomaterial, or agrochemical incorporating a microcapsule according to any one of claims 1 to 20, or a composition according to any one of claim 21 or 22.

35. Use of a microcapsule according to any one of claims 1 to 20, or a composition according to any one of claim 21 or 22 to produce a food, beverage, cosmetic, home care product, personal care product, pharmaceutical, medical device, biomaterial, or agrochemical.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0196] FIG. 1 shows graphical representations of embodiments of the microcapsules of the present invention.

[0197] FIG. 2 is a photograph of a composition comprising the microcapsules of Example 1 of the present invention.

[0198] FIG. 3 is a photograph of a composition comprising the microcapsules of Example 2 of the present invention.

[0199] FIG. 4 is a plot showing the biodegradation percentage over time of the microcapsules of Example 5 based upon O.sub.2 consumption, compared to a cellulose reference sample.

[0200] FIG. 5 is a plot showing the biodegradation percentage over time of the microcapsules of Example 5 based upon CO.sub.2 production, compared to a cellulose reference sample.

[0201] FIG. 6 shows protein secondary structure analysis of the shell of the microcapsules of Example 5. FIG. 6a is an FTIR spectrum of the dried microcapsules and FIG. 6b is their second derivatives, calculated from the amide I bands in the FTIR spectrum. FIG. 6c is a bar graph providing quantification of the secondary structure content calculated from amide I band in the FTIR spectrum. The indicated error bars are the standard deviation of the average of three different spectra, wherein each is a co-average of 128 scans.

EXAMPLES

Materials

[0202] Pea Protein Isolate (PPI) (80% protein) and lactic acid (80%) were purchased from Cambridge Commodities Ltd. [0203] Calcium chloride dihydrate was purchased from Fisher Scientific. [0204] Miglyol 840 and polyglycerol polyricinoleate (PGPR) were purchased from 101 Oleo. [0205] (1H,1H,2H,2H-Perfluoro-1-octanol) was purchased from Sigma Aldrich. [0206] HFE-7500 3M™ Novec™ Engineered fluid was purchased from Fluorochem. [0207] 008-FluoroSurfactant was purchased from RAN Biotechnologies. [0208] Lactic acid (85%, FG), Pyridoxine hydrochloride (n8%, HPLC) (Vitamin B6) and Polysorbate 80 (FG) were purchased from Sigma-Aldrich. [0209] Sodium tripolyphosphate Alfa Aesar™, sodium hydrogen carbonate Ph.Eur., potassium chloride Ph.Eur. and magnesium sulphate were purchased from Fisher Scientific. [0210] Gellan Gum High Acyl LT100 was purchased from Special Ingredients. [0211] Miglyol 812, Miglyol 829 and Myglyol 840 were purchased from 101 Oleochemical. [0212] Polyglycerol polyricinoleate (PGPR) was purchased from Danisco. [0213] Calcium sulphate dihydrate was purchased from Honeywell.

Example 1—Preparation of Microcapsules

[0214] Preparation of Internal Phase:

[0215] A water-in-oil emulsion was produced by emulsifying 200 μl of a 0.25 M CaCl.sub.2 solution in 800 μl of Miglyol 840 containing 4% (w/w) PGPR by ultrasonication for 30 seconds.

[0216] Preparation of Middle Phase:

[0217] Pea Protein Isolate was added to a 35% (v/v) lactic acid aqueous solution at a final protein concentration of 125 mg/ml. A dispersion of non-soluble protein was obtained. For solubilisation of the protein, the mixture was exposed to ultrasonication for 30 min (using a Bandelin Sonopuls HD 4200 Ultrasonic Homogeniser operating under the following parameters: High-Frequency Power Output=200 W, Frequency=20 KHz, Amplitude=20%). During this process, the sample temperature was kept at 85° C.-90° C. After 30 min, a completely translucent liquid solution was obtained. The dispersed liquid PPI phase (125 mg/ml PPI in 35% v/v acetic acid, kept at 85° C.) was loaded in a 15 ml tube and rapidly placed on a heating block at 85° C. In order to prevent the gelation of the PPI solution during transport into the microfluidic device, a custom-made silicone heater (Holroyd Components), comprising a 1/32″ ID stainless steel tubing was used to maintain the temperature of the PTFE tubing connecting the PPI reservoir and the inlet in the microfluidic device. The silicon heater temperature was controlled by a custom-built temperature controller

[0218] Preparation of External Oil Phase:

[0219] 008-FluoroSurfactant was dissolved in HFE-7500 3M™ Novec™ to a final concentration of 2% (w/w).

[0220] Preparation of Microcapsules:

[0221] Non-planar microfluidic devices (droplet generators) were fabricated using standard soft lithography techniques with a negative master photoresist (SU8 3050), as described in Biomacromolecules 2017, 18, 11, 3642-3651. For the fabrication of a triple emulsion, a tandem emulsification approach was followed. The first non-planar droplet generator device was comprised of an internal channel (50×50 μm), and an external channel (100×100 μm), and was rendered hydrophilic by exposure to oxygen plasma for 500 seconds and 80 W. The second non-planar droplet generator microfluidic device was comprised of an internal channel (200×200 μm) and an external channel (300×300 μm), and was rendered hydrophobic by flushing all channels with a solution of Duxback®. Both devices were connected via a small 1/32″ OD PTFE tubing.

[0222] Droplets of −50 μm diameter were first generated by pumping the internal and middle phase into the hydrophilic device by a pressure-driven system (Elveflow OB1), thus obtaining a double emulsion. The generated droplets were transferred to the hydrophobic device and emulsified again by the outer phase, thus obtaining a triple emulsion.

[0223] Different pressure rates were tested until a uniform and continuous generation of a monodisperse population of microdroplets was achieved. Final pressure rates were 350 mbar for the internal phase, 500 mbar for the middle protein phase and 180 mbar for the external oil phase. The generated droplets were collected in a glass vial, and were kept at 10° C. for 12 h to ensure the gelation process was complete.

[0224] The formed microcapsules were then washed by a standard de-emulsification procedure: the continuous oil phase containing fluorosurfactant was first removed from the vial. For 500 ul of microcapsules, an equal volume of 10% PFO solution in HFE-7500 3M™ Novec™ was added and thoroughly mixed for 30 seconds. The 10% PFO solution in HFE-7500 3M™ Novec™ was then removed and two subsequent oil washes were performed by adding an equal volume of pure HFE-7500 3M™ Novec™. Finally, 2500 ul of a 0.1 M CaCl.sub.2 solution were added to the vial, resulting in the transfer of the microcapsules from the oil to the aqueous phase. The supernatant containing the microcapsules suspension was transferred to a separate vial. FIG. 2 is a photograph of the microcapsule composition of Example 1.

Example 2—Preparation of Microcapsules Containing Vitamin B6

[0225] Preparation of Internal Phase:

[0226] A water-in-oil (W/O) emulsion was produced by probe ultra-sonication. 0.03% gellan gum with 1 M Vitamin B6 solution was emulsified in Miglyol 829 oil with 4 wt % PGPR in a volume ratio of 1 part aqueous to 4 parts oil.

[0227] Preparation of Middle Phase:

[0228] 500 ml of a mixture was prepared consisting of 10% (w/v) Pea Protein Isolate in 35% (v/v) lactic acid solution. The mixture was sonicated to disrupt large colloidal aggregates (Hielscher UIP1000hdT (1000 W, 20 kHz)), after which a transparent solution was obtained. The energy applied was 500 kJ over 90 minutes.

[0229] Preparation of Continuous Oil Phase:

[0230] The external continuous oil phase comprised Miglyol 840 containing 2 wt % PGPR.

[0231] Preparation of Microcapsules:

[0232] The internal phase was emulsified into the middle phase by using a membrane emulsification device (AXF-1, Micropore Ltd). The size of the droplets created are approximately 40 μm. The resultant emulsion was continuously pumped into a glass jacketed reactor containing the continuous oil phase at 55° C., and shear was applied by a Heidolph Hei-TORQUE Core overhead stirrer with a three blade marine prop impeller to generate a W/O/W/O triple emulsion. The ratio of double emulsion from the Micropore device to bulk emulsification oil was 1:4.

[0233] The continuous oil phase was maintained at a temperature of 55° C. whilst being stirred for 5 minutes at 800 rpm, after which the reactor was cooled with an ice water jacket to reduce the temperature of the completed triple emulsion to less than 20° C. The formed microcapsules were allowed to fully settle at 3° C. for 3-4 hours before decanting the continuous oil phase. A solution of 0.1 M sodium tripolyphosphate with 4 wt % Polysorbate 80 was then added to the microcapsule slurry. This mixture was left for 18 h with gentle mixing (150 rpm using an overhead stirrer). The capsules were then washed with a hard water solution (containing NaHCO.sub.3 at 192 mg/L, CaSO.sub.4.Math.2H.sub.2O at 120 ml/L, MgSO.sub.4 at 120 mg/L, KCl at 8 mg/L) three times in order to ensure the continuous phase oil was thoroughly removed from the surface of the microcapsules.

[0234] The final microcapsules were then decanted and stored in the fridge (3° C.) in the wash solution made up to a 50 vol % slurry. FIG. 3 is a photograph of the microcapsule composition of Example 2.

[0235] Controlled Release Experiments:

[0236] The retention of pyrirdoxine hydrochloride (Vitamin B6) in the microcapsules was followed over the course of 10 days by quantifying the concentration of non-encapsulated pyrirdoxine hydrochloride using HPLC analysis.

[0237] A 200 μl aliquot of washed microcapsules in a 50 vol % slurry in hard water (containing NaHCO.sub.3 at 192 mg/L, CaSO.sub.4.Math.2H.sub.2O at 120 ml/L, MgSO.sub.4 at 120 mg/L, KCl at 8 mg/L) was added to 800 μl of hard water (solution A). To quantify the total amount of pyridoxine hydrochloride in the sample (i.e. encapsulated and non-encapsulated), a control sample of solution A was exposed to sonication using an ultrasonic homogeniser (Bandelin, HD4200) for 30 seconds at 50% amplitude in order to break all the microcapsules. The sample was then centrifuged at 14000 rpm for 15 min, and a 200 μl aliquot from the supernatant was taken for HPLC analysis to quantify the total amount of pyridoxine hydrochloride in 1 ml of sample.

[0238] To quantify the initially non-encapsulated pyridoxine hydrochloride in solution A, a 200 μl aliquot from the supernatant was taken for HPLC analysis. The amount of encapsulated pyridoxine hydrochloride in solution A could then be determined by subtraction of the measured amount of non-encapsulated pyridoxine hydrochloride from the measured initial total amount of pyridoxine hydrochloride present.

[0239] To quantify the retention of pyridoxine hydrochloride in the microcapsules, a 1 ml sample of solution A was left to incubate at 20° C. for 10 days, after which 200 μl of supernatant were taken for HPLC analysis. This provides a measure of the pyridoxine hydrochloride that has leaked from the microcapsules and is no longer encapsulated. This value is then subtracted from the initial total pyridoxine hydrochloride amount to give the encapsulated amount after a specified period of time. This can then be calculated as a percentage of the initially encapsulated pyridoxine hydrochloride amount.

[0240] HPLC quantification confirmed the retention of more than 60% pyridoxine hydrochloride inside the microcapsules after 10 days. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Pyridoxine Retained hydrochloride Pyridoxine Pyridoxine concentration hydrochloride hydrochloride in solution A concentration concentration supernatant encapsulated (% of initially Sample (ppm) (ppm) encapsulated) Total post 21.4 ± 1.09 n/a n/a sonication Day 0 (encapsulated + non-encapsulated) Day 0 (non-  6.7 ± 0.21 n/a n/a encapsulated) Day 0 (encapsulated) n/a 14.7 ± 1.3 100%  Day 5 (non- 11.2 ± 0.3  10.2 ± 1.4 69% encapsulated) Day 10 (non- 11.5 ± 0.14  9.9 ± 1.2 67% encapsulated)

Example 4: Protein Digestibility Test

[0241] In order to assess the digestibility of the microcapsules of the present invention, the plant-based protein hydrogel shell was subjected to a digestibility test.

[0242] Sample Preparation:

[0243] Empty microcapsules having only a plant-based protein hydrogel shell were prepared as follows:

[0244] 400 ml of a mixture was prepared consisting of 12.5% (w/v) Pea Protein Isolate in 35% (v/v) lactic acid solution. The mixture was sonicated to disrupt large colloidal aggregates (Hielscher UIP500hdT (500 W, 20 kHz)), after which a transparent solution was obtained. The energy applied was 100 kJ over 15 minutes.

[0245] This solution was poured into 1 L of 85° C. Miglyol 840 oil (with 2 wt % PGPR) in a 2 l vessel, whilst the Miglyol was being stirred at 1100 rpm with an overhead stirrer. After 1 min of stirring at 1100 rpm, the speed of stirring was turned down to 500 rpm, and the mixture was cooled to 20° C. by surrounding the container with ice water.

[0246] After cooling, stirring was ceased, and the mixture was stored at 3° C. for 18 h to enable the spheres formed by the emulsification process to settle. The supernatant oil was then poured off. The spheres were then washed with a series of aqueous solutions: 0.1 M CaCl.sub.2) plus 0.5 wt % Polysorbate 80; 0.1 M CaCl.sub.2; 0.1 M Na Citrate and 20 mM sodium citrate buffer. The wash steps removed the residual oil and raised the pH to 5.4. The process yielded spheres with a mean diameter of 83 micron.

[0247] Test Method and Results:

[0248] Protein digestibility was measured by following the Boisen protocol as detailed in Animal Feed Science and Technology, 51, pp. 29-43 (1995). The measured Boisen Protein digestibility was 100.8±2.0%. The control sample (commercial Pea Protein Isolate, ProEarth, from Cambridge Commodities Ltd) had a Boisen Protein digestibility of 99.7±2.0%.

[0249] The results therefore show that the plant-based protein hydrogel shell of the microcapsules of the present invention has a digestibility that is comparable to pure pea protein. The microcapsules of the present invention therefore have a useful application in food and beverage products.

Example 5: Biodegradability Test

[0250] In order to assess the biodegradability of the microcapsules of the present invention, the plant-based protein hydrogel shell was subjected to a biodegradability test.

[0251] Sample Preparation:

[0252] Empty microcapsules having only a plant-based protein hydrogel shell were prepared as described in Example 4. Following this preparation, the microcapsule slurry was poured into a tray and dried in an oven at 85° C. for 3 to 4 h until the dried material reached a water activity of 0.5.

[0253] Test Method:

[0254] The dried protein microcapsules were then subjected to an aqueous aerobic biodegradability test in fresh water according to the ISO-14851 standard, using cellulose as a reference standard.

[0255] Results:

[0256] The amount of biodegradation based on O.sub.2 consumption is expressed as the ratio of the Biochemical Oxygen Demand (BOD, corrected for the control) to the Theoretical Oxygen Demand (ThOD) of the used test material.

[0257] Table 2 shows the ThOD (theoretical oxygen demand), net O.sub.2 consumption and the biodegradation percentage of the reference test and the tested microcapsules. An overview of the evolution of the biodegradation percentage (based on O.sub.2 consumption) of the two different materials is given in FIG. 4. No intermediate correction is made for the oxygen consumption by nitrification.

[0258] After 28 days, as measured by O.sub.2 consumption, the reference sample cellulose reached a biodegradation percentage of 82.4%. The biodegradation of the tested microcapsules proceeded further, with an absolute biodegradation of 87.5% having been measured.

TABLE-US-00002 TABLE 2 [AVG = average biodegradation, SD = standard deviation and REL = relative biodegradation] ThOD (mg/g test Net O.sub.2 Biodegradation (%) Test series item) (mg/l) AVG SD REL Cellulose 1135 93.6 82.4 1.4 100.0 (reference) Microcapsules 1409 123.1 87.5 3.2 106.1 of Example 5

[0259] The biodegradation based on CO.sub.2 production is calculated as the percentage of solid carbon of the test material which has been converted to CO.sub.2.

[0260] The CO.sub.2 evolved was determined by titration. Table 3 shows the TOC (total organic carbon content), net CO.sub.2 production and the biodegradation percentage of the reference test and the tested microcapsules. An overview of the evolution of the biodegradation percentages (based on CO.sub.2 production) of the two different materials is given in FIG. 5.

[0261] After 28 days, as measured by CO.sub.2 production, the reference sample cellulose had reached a biodegradation percentage of 84.2%. The biodegradation of the tested microcapsules proceeded with an average absolute biodegradation of 82.4% having been measured. On a relative basis, compared to cellulose, a biodegradation of 98.0% was calculated for the tested microcapsules. The 90% biodegradability requirement stipulated in the ISO-14851 standard was therefore met.

TABLE-US-00003 TABLE 3 [AVG = average, SD = standard deviation and REL = relative biodegradation] TOC Net CO.sub.2 Biodegradation (%) (%) (mg) AVG SD REL Cellulose (reference) 43.6 33.7 84.2 4.0 101.0 Microcapsules 49.3 37.2 82.4 2.1 98.0 of Example 5

[0262] The results show that the plant-based protein hydrogel shell of the microcapsules of the present invention easily achieves the biodegradability requirements under fresh water conditions for microplastics, as stipulated by ISO-14851. Accordingly, the microcapsules of the present invention represent environmentally-friendly encapsulation technology, which is particularly suited to use in home care and personal care applications, as if the microcapsules ultimately end up in waterways/the sea they will fully biodegrade in a short amount of time.

[0263] Capsule Shell FTIR Analysis:

[0264] Structural analysis was also performed on the dried empty microcapsules. Structural analysis was performed using an FTIR-Equinox 55 spectrometer (Bruker). The samples were used without further pre-treatment and were loaded into the FTIR holder. The atmospheric compensation spectrum was subtracted from the original FTIR spectra and a secondary derivative was applied for further analysis. Each FTIR measurement was repeated 3 times. The sensitivity of the instrument was detected to be 5%. To resolve the transformation of the native structure of Pea Protein Isolate into supramolecular aggregates, vibrational changes in amide I, which is strictly correlated with protein secondary structure, were followed.

[0265] The results are shown in FIG. 6. FTIR analysis of the capsule shell showed 46% of intermolecular β-sheets. This high level of n-sheets means that the shell will be stable at high temperatures enabling any vitamin encapsulated therein to be protected from degradation during processing into the finished product, particularly where high temperature processing is required, for example, pasteurisation.

Clauses

[0266] 1. A microcapsule comprising: [0267] (a) a hydrophilic phase comprising a water-soluble ingredient; [0268] (b) a lipophilic phase; and [0269] (c) a plant-based protein hydrogel shell. [0270] 2. A microcapsule according to clause 1, wherein the hydrophilic phase comprises water. [0271] 3. A microcapsule according to clause 1 or clause 2, wherein the water-soluble ingredient is selected from one or more of Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B7, Vitamin B9, Vitamin B12, Vitamin C, panthenol, α-hydroxy acids, water-soluble minerals salts, water-soluble plant extracts and yeasts, enzymes, antibiotics, oligopeptides, proteins and protein hydrolysates. [0272] 4. A microcapsule according to any one of clauses 1 to 3, wherein the lipophilic phase comprises an oil. [0273] 5. A microcapsule according to any one of clauses 1 to 4, wherein the lipophilic phase further comprises an oil-soluble surfactant. [0274] 6. A microcapsule according to clause 5, wherein the oil-soluble surfactant is selected from sucrose fatty acid esters such as sucrose stearic acid ester, sucrose palmitic acid ester, sucrose oleic acid ester, sucrose lauric acid ester, sucrose behenic acid ester, and sucrose erucic acid ester; sorbitan fatty acid esters such as sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, and sorbitan sesquioleate; glyceryl fatty acid esters such as glycerol monostearate and glycerol monooleate; and polyglyceryl fatty acid esters such as diglyceryl tetraisostearate, diglyceryl diisostearate, and diglyceryl monoisostearate. [0275] 7. A microcapsule according to clause 5 or clause 6, wherein the concentration of the oil-soluble surfactant in the lipophilic phase is in the range 0.01% w/w to 10% w/w. [0276] 8. A microcapsule according to any one of clauses 1 to 7, wherein the lipophilic phase further comprises an oil-soluble ingredient. [0277] 9. A microcapsule according to clause 8, wherein the oil-soluble ingredient is selected from one or more of: a fatty acid; a triglyceride or a mixture thereof; Omega-3 fatty acids, such as a-linolenic acid (18: 3n3), octadecatetraenoic acid (18: 4n3), eicosapentaenoic acid (20: 5n3) (EPA) and docosahexaenoic acid (22: 6n3) (DHA), and derivatives thereof and mixtures thereof; fat-soluble vitamins, such as Vitamin A, Vitamin D, Vitamin E and Vitamin K; Antioxidants, such as tocopheryl and ascorbyl derivatives; retinoids or retinols; essential oils; bioflavinoids, terpenoids; synthetics of bioflavonoids and terpenoids and the like. [0278] 10. A microcapsule according to any one of clauses 1 to 9, wherein the plant-based protein(s) in the plant-based protein hydrogel shell is obtained from soybean, pea, rice, potato, wheat, corn zein or sorghum; preferably the plant protein(s) is selected from soy protein, pea protein, potato protein, rapeseed protein and/or rice protein. [0279] 11. A microcapsule according to any one of clauses 1 to 10, wherein the plant-based protein hydrogel shell is a self-assembled plant-based protein hydrogel shell. [0280] 12. A microcapsule according to any one of clauses 1 to 11, wherein the plant-based protein hydrogel shell comprises plant-based proteins having a protein secondary structure with at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet. [0281] 13. A microcapsule according to any one of clauses 1 to 12, wherein the plant-based protein hydrogel shell has a storage modulus (G′) at 10 rad/s of greater than 500 Pa, greater than 1000 Pa, greater than 2500 Pa, greater than 3000 Pa, greater than 4000 Pa. [0282] 14. A microcapsule according to any one of clauses 1 to 13, wherein the plant-based protein hydrogel shell comprises protein aggregates with a median average length of between 50 to 500 nm or a mean average length of between 50 to 500 nm; or 80% of the aggregates have an average length of between 50 to 500 nm; [0283] and/or the aggregates may have a median height of between 5 to 50 nm; or the aggregates may have a mean average height of between 5 to 50 nm; or 80% of the aggregates have an average height of between 5 to 50 nm; [0284] preferably, the aggregates have a median average length of between 50 to 500 nm and/or a median average height of between 5 to 50 nm. [0285] 15. A microcapsule according to any one of clauses 1 to 14, wherein the hydrophilic phase is dispersed in the lipophilic phase. [0286] 16. A microcapsule according to any one of clauses 1 to 15, wherein the hydrophilic phase and the lipophilic phase form a water-in-oil emulsion. [0287] 17. A microcapsule according to any one of clauses 1 to 15, wherein the hydrophilic phase and the lipophilic phase are encapsulated by the plant-based protein hydrogel shell. [0288] 18. A microcapsule according to clause 16, wherein the water-in-oil emulsion is encapsulated by the plant-based protein hydrogel shell. [0289] 19. A microcapsule according to any one of clauses 1 to 18, wherein the plant-based protein hydrogel shell has a thickness in the range 10 nm to 50,000 μm, preferably in the range 10 μm to 100 μm. [0290] 20. A microcapsule according to any one of clauses 1 to 19, wherein the microcapsule has a size of less than 1 mm in the largest dimension, preferably less than 900 μm. [0291] 21. A microcapsule according to any one of clauses 1 to 20, wherein the microcapsule releases the water-soluble ingredient and/or the oil-soluble ingredient to a surface upon application of pressure to the microcapsule. [0292] 22. A microcapsule according to clause 21, wherein said surface is a bio-surface. [0293] 23. A microcapsule according to clause 22, wherein said bio-surface is selected from hair, skin and teeth. [0294] 24. A microcapsule according to clause 21, wherein said surface is a textile. [0295] 25. A microcapsule according to any one of clauses 1 to 20, wherein the microcapsule releases the water-soluble ingredient and/or the oil-soluble ingredient as a result of enzymatic degradation of the plant-based protein hydrogel shell. [0296] 26. A microcapsule according to clause 25, wherein said enzymatic degradation occurs in the digestive system of a human or animal. [0297] 27. A composition comprising at least one microcapsule according to any one of clauses 1 to 26 and an external phase. [0298] 28. A composition according to clause 27, wherein the at least one microcapsule is dispersed in the external phase. [0299] 29. A composition according to clause 27 or clause 28, wherein the external phase is an external aqueous phase. [0300] 30. A composition according to clause 29, wherein the external aqueous phase is an aqueous salt solution. [0301] 31. A composition according to clause 29 or clause 30, wherein the external aqueous phase is a continuous external aqueous phase. [0302] 32. A composition according to clause 27 or clause 28, wherein the external phase is an external lipophilic phase. [0303] 33. A method for preparing a microcapsule according to any one of clauses 1 to 26, comprising: [0304] (a) emulsifying a hydrophilic phase comprising a water-soluble ingredient in a first lipophilic phase to give a primary emulsion; [0305] (b) re-emulsifying said primary emulsion in a plant-based protein solution comprising one or more plant-based protein(s), wherein said plant-based protein solution is at a temperature above the sol-gel transition temperature of the plant-based protein solution, to give a double emulsion; [0306] (c) re-emulsifying said double emulsion in a second lipophilic phase to give a triple emulsion; [0307] (d) inducing the plant-based protein(s) in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel shell, wherein said plant-based protein hydrogel shell encapsulates said primary emulsion to form a microcapsule which is suspended in an external phase which is the second lipophilic phase; and [0308] (e) washing the microcapsule to remove the second lipophilic phase. [0309] 34. A method according to clause 33, wherein the primary emulsion has a diameter of less than or equal to 5 μm. [0310] 35. A method according to clause 33 or clause 34, wherein the double emulsion has a diameter of less than or equal to 100 μm. [0311] 36. A method according to any one of clauses 33 to 35, wherein the triple emulsion has a diameter of less than or equal to 200 μm. [0312] 37. A method according to any one of clauses 33 to 36, wherein the first and second lipophilic phases are the same or different. [0313] 38. A method according to any one of clause 33 to 37, wherein the first and/or second lipophilic phase further comprises an oil-soluble surfactant. [0314] 39. A method according to clause 38, wherein the oil-soluble surfactants in the first and second lipophilic phase are the same or different. [0315] 40. A method according to any one of clauses 33 to 39, wherein the plant-based protein solution comprises one or more plant-based protein(s) in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases solubility of the plant-based protein(s), and a second co-solvent decreases solubility of the plant-based protein(s). [0316] 41. A method according to clause 40, wherein the first co-solvent is an organic acid; preferably acetic acid and/or an α-hydroxy acid; wherein the α-hydroxy acid may preferably be selected from glycolic acid, lactic acid, malic acid, citric acid and/or tartaric acid; with particularly preferred organic acids being acetic acid and/or lactic acid. [0317] 42. A method according to clause 40 or 41, wherein the second co-solvent(s) is an aqueous buffer solution, preferably selected from water, ethanol, methanol, acetone, acetonitrile, dimethylsulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, t-butanol, ethyl acetate, hexafluoroisopropanol, more preferably water and/or ethanol, particularly preferably water. [0318] 43. A method according to any one of clauses 40 to 42, wherein said solvent system comprises a co-solvent ratio of about 20-80% v/v, preferably about 20-60% v/v, about 25-55% v/v, about 30-50% v/v, about 20%, about 30%, about 40% about 50% or about 60% v/v, most preferably about 30-50% v/v. [0319] 44. The method according to any one of clauses 33 to 43, wherein in step (d) the protein solution is heated to a first temperature above the sol-gel temperature of the one or more plant-based protein(s), then reduced to a second temperature below the sol-gel temperature of the one or more plant-based protein(s) to form the plant-based protein hydrogel shell. [0320] 45. The method according to any one of clauses 33 to 44 wherein said microcapsule is formed using a microfluidic device. [0321] 46. A method according to any one of clauses 33 to 45, further comprising drying the microcapsule to form a dry powder. [0322] 47. A method according to clause 46, wherein said drying is selected from spray drying, fluid bed drying and/or tray drying. [0323] 48. A microcapsule prepared according to the method of any one of clauses 33 to 47. [0324] 49. A method for preparing a composition according to any one of clauses 27 to 32, comprising: [0325] (a) emulsifying a hydrophilic phase comprising a water-soluble ingredient in a first lipophilic phase to give a primary emulsion; [0326] (b) re-emulsifying said primary emulsion in a plant-based protein solution comprising one or more plant-based protein(s), wherein said plant-based protein solution is at a temperature above the sol-gel transition temperature of the plant-based protein, to give a double emulsion; [0327] (c) re-emulsifying said double emulsion in a second lipophilic phase to give a triple emulsion; [0328] (d) inducing the plant-based protein(s) in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel shell, wherein said plant-based protein hydrogel shell encapsulates said primary emulsion to form a microcapsule which is suspended in an external phase which is the second lipophilic phase. [0329] 50. A method according to clause 49, wherein the primary emulsion has a diameter of less than or equal to 5 μm. [0330] 51. A method according to clause 49 or clause 50, wherein the double emulsion has a diameter of less than or equal to 100 μm. [0331] 52. A method according to any one of clauses 49 to 51, wherein the triple emulsion has a diameter of less than or equal to 200 μm. [0332] 53. A method according to any one of clauses 49 to 52, wherein the first and second lipophilic phases are the same or different. [0333] 54. A method according to any one of clauses 49 to 53, wherein the first and/or second lipophilic phase further comprises an oil-soluble surfactant. [0334] 55. A method according to clause 54, wherein the oil-soluble surfactants in the first and second lipophilic phase are the same or different. [0335] 56. A method according to any one of clauses 49 to 55, further comprising: [0336] (e) washing the microcapsule to remove the second lipophilic phase; and [0337] (f) re-suspending the microcapsule in an external aqueous phase. [0338] 57. A method according to clause 56, further comprising adding suspending agents to said external aqueous phase. [0339] 58. A method according to clause 57, wherein said suspending agents are selected from acacia gum, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, hydrogenated vegetable oil category 1, aluminum magnesium silicate, maltodextrin, carboxymethyl cellulose, polymethacrylate, poly vinyl pyrrolidone, sodium alginate, starch, zein, water-insoluble cross-linked polymers such as cross-linked cellulose, cross-linked starch, cross-linked CMC, cross-linked carboxymethyl starch, cross-linked polyacrylate, and cross-linked polyvinylpyrrolidone, and expanded clays such as bentonite and laponite. [0340] 59. A method according to any one of clauses 49 to 58, wherein the plant-based protein solution comprises one or more plant-based protein(s) in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases solubility of the plant-based protein(s), and a second co-solvent decreases solubility of the plant-based protein(s). [0341] 60. A method according to clause 59, wherein the first co-solvent is an organic acid; preferably acetic acid and/or an α-hydroxy acid; wherein the α-hydroxy acid may preferably be selected from glycolic acid, lactic acid, malic acid, citric acid and/or tartaric acid; with particularly preferred organic acids being acetic acid and/or lactic acid. [0342] 61. A method according to clause 59 or clause 60, wherein the second co-solvent(s) is an aqueous buffer solution, preferably selected from water, ethanol, methanol, acetone, acetonitrile, dimethylsulfoxide, dimethylformamide, formamide, 2-propanol, 1-butanol, 1-propanol, hexanol, t-butanol, ethyl acetate, hexafluoroisopropanol, more preferably water and/or ethanol, particularly preferably water. [0343] 62. A method according to any one of clauses 59 to 61, wherein said solvent system comprises a co-solvent ratio of about 20-80% v/v, preferably about 20-60% v/v, about 25-55% v/v, about 30-50% v/v, about 20%, about 30%, about 40% about 50% or about 60% v/v, most preferably about 30-50% v/v. [0344] 63. The method according to any one of clauses 49 to 62, wherein in step (d) the protein solution is heated to a first temperature above the sol-gel temperature of the one or more plant-based protein(s), then reduced to a second temperature below the sol-gel temperature of the one or more plant-based protein(s) to form the plant-based protein hydrogel shell. [0345] 64. The method according to any one of clauses 49 to 63 wherein said microcapsule is formed using a microfluidic device. [0346] 65. A composition prepared according to the method of any one of clauses 49 to 64. [0347] 66. A food, beverage, cosmetic, pharmaceutical, medical device, biomaterial, or agrochemical incorporating a microcapsule according to any one of clauses 1 to 26, or a composition according to any one of clauses 27 to 32.