Protease-loaded microcapsules

Abstract

The present invention provides a method for manufacturing a composition comprising protease-loaded microcapsules, the method comprising microencapsulating one or more proteases by complex coacervation with one or more anionic polysaccharides. The present invention further provides a composition comprising protease-loaded microcapsules obtained by or obtainable by said method and uses thereof.

Description

[0001] PROTEASE-LOADED MICROCAPSULES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for manufacturing a composition comprising protease-loaded microcapsules. The present invention also relates to a composition comprising protease-loaded microcapsules obtained by or obtainable by said method and uses thereof.

[0004] BACKGROUND TO THE INVENTION

[0005] Oral administration of proteolytic enzymes has been shown to be associated with beneficial immunomodulatory effects. For example, in a randomized trial of subjects with moderate-to- severe knee osteoarthritis, oral administration of proteolytic enzymes was found to have comparable effectiveness to diclofenac in relieving pain and increasing function (see Ueberall, M.A., et al., 2016. Journal of pain research, 9, pp.941-961).

[0006] Proteolytic enzymes have also been shown as a highly efficient strengthening factor for antibiotic therapy of urogenital chlamydiosis (see Sukhikh, G.T., et al., 1997. International journal of immunotherapy, 13(3-4), pp.131-133); to provide an additional beneficial effect for the patients suffering from cervical syndrome (see Tilscher, H., et al., 1996. Wiener medizinische Wochenschrift, 146(5), pp.91-95); and to decrease incidence of obstructive bronchitis in children with recurrent obstructive bronchitis (see Lanchava, N., et al., 2005. Georgian medical news, 127, pp.50-53).

[0007] Traditionally, proteolytic enzymes are consumed in tablet or capsule forms. However, there is a growing consumer demand for more convenient formats, such as powdered drinks. Powdered formats pose challenges regarding enzyme stability and degradation during storage. Existing microencapsulation methods have only partially addressed these concerns, often failing to preserve enzyme activity and ensure adequate delivery.

[0008] Thus, there is a demand for improved methods for microencapsulating proteases, which are efficient and scalable, and which improve stability, enhance payload efficiency, and extend shelf-life of the protease-loaded microcapsules.

[0009] SUMMARY OF THE INVENTION

[0010] The present inventors have developed an improved method for microencapsulating proteases, employing complex coacervation with anionic polysaccharides. The method of the present invention significantly surpasses traditional encapsulation technologies by achieving near-complete encapsulation efficiency (>90%) and maintaining a high enzyme payload (>60%). This higher payload reduces production costs, for example by requiring less encapsulating material per unit of active enzyme.

[0011] The method of the present invention achieves >90% recovery of protease activity. This high level of activity recovery not only enhances the efficacy of the final product, but also contributes to the process's cost-effectiveness, ensuring that minimal active ingredient is lost during processing. Moreover, through rigorous in vitro simulations of gastrointestinal conditions, the present inventors have demonstrated that the encapsulated proteases retain substantial activity and ensure their targeted release within the gastrointestinal tract, highlighting the potential for improved therapeutic efficacy.

[0012] The method of the present invention involves forming a solid matrix by complex coacervation of a mixture comprising one or more proteases and one or more anionic polysaccharides. The anionic polysaccharides not only protect the proteases from degradation, but also ensure their targeted release within the gastrointestinal tract. The solid matrix may subsequently be filtered out before drying. This reduces the water content significantly before the drying phase, thereby decreasing energy consumption and overall production costs. The present inventors have also shown that incorporating antioxidants directly into the encapsulation matrix can further extend the shelf-life of the proteases.

[0013] Thus, by optimizing the encapsulation efficiency and payload, maximizing enzyme recovery and shelf-life, and simplifying the drying process, the method of the present can be tailored for scalable production and offers substantial improvements over existing microencapsulation technologies. The method of the present invention may allow for the industrial-scale production of more stable and effective protease-based products.

[0014] In one aspect, the present invention provides a method for manufacturing a composition comprising protease-loaded microcapsules, the method comprising: (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides; (b) mixing the first solution and the second solution to provide a mixed solution; and (c) adjusting the pH of the mixed solution to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides.

[0015] Any suitable proteases may be used in the present invention. In some embodiments, the one or more proteases are selected from trypsin, bromelain, papain, pancreatin, chymotrypsin, serratiopeptidase, ficain, nattokinase, or any combination thereof. In some embodiments, the one or more proteases comprise or consist of trypsin and / or bromelain. In some embodiments, the one or more proteases consist of trypsin. In some embodiments, the one or more proteases consist of bromelain.

[0016] Any suitable anionic polysaccharides may be used in the present invention. In some embodiments, the one or more anionic polysaccharides are selected from alginate, pectin, carrageenan, gum arabic, xanthan gum, carboxymethyl cellulose, gellan gum, or any combination thereof. In some embodiments, the one or more anionic polysaccharides consist of alginate.

[0017] The present invention may use any suitable protease:anionic polysaccharide weight ratio. Suitably, the mixed solution has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1. In some embodiments, the mixed solution has a protease:anionic polysaccharide weight ratio of at least 5:2, preferably wherein the mixed solution has a protease:anionic polysaccharide weight ratio of at least 3:1. In some embodiments, the mixed solution has a protease:anionic polysaccharide weight ratio of 9:2 or less, preferably wherein the mixed solution has a protease:anionic polysaccharide weight ratio of 4:1 or less. In some embodiments, the mixed solution has a protease:anionic polysaccharide weight ratio of from 5:2 to 9:2, preferably wherein the mixed solution has a protease:anionic polysaccharide weight ratio of from 3:1 to 4:1.

[0018] In some embodiments, the method further comprises mixing one or more antioxidants with the first solution and the second solution. In some embodiments, the microcapsules further comprise the one or more antioxidants. Any suitable antioxidants may be used in the present invention. In some embodiments, the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, erythorbic acid, sodium erythorbate, glutathione, N-acetylcysteine (NAC), alpha-lipoic acid, or any combination thereof. In some embodiments, the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, or any combination thereof. In some embodiments, the one or more antioxidants consist of ascorbic acid.

[0019] The pH of the mixed solution may be adjusted to a pH of from 2 to 4 to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides. In some embodiments, the pH is adjusted to a pH of from 2.5 to 4.0, preferably wherein the pH is adjusted to a pH of from 3.0 to 4.0. In some embodiments, the pH is adjusted to a pH of from 2.0 to 3.5, preferably wherein the pH is adjusted to a pH of from 2.0 to 3.0. In some embodiments, the pH is adjusted to a pH of from 2.5 to 3.5, preferably wherein the pH is adjusted to a pH of about 3.0. The microcapsules may comprise 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total protease comprised in the mixed solution. The microcapsules may have 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total protease activity of the mixed solution. The microcapsules may have a protease:anionic polysaccharide weight ratio of at least 2:1 , at least 3:1 , or at least 4:1.

[0020] In some embodiments, the method further comprises recovering the microcapsules from the mixed solution. In some embodiments, the microcapsules are recovered from the mixed solution by filtration. In some embodiments, the microcapsules are recovered from the mixed solution using a 25 micron filter. In some embodiments, 50wt% or more, 60wt% or more, 70wt% or more, 80wt% or more, or 90wt% or more of the water in the mixed solution is removed upon recovery of the microcapsules.

[0021] In some embodiments, the method further comprises drying the microcapsules to provide a powder comprising microencapsulated protease. In some embodiments, the microcapsules are dried by freeze-drying. In some embodiments, the microcapsules have a solid content of 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more, prior to drying.

[0022] In some embodiments, the method further comprises milling and / or sieving the powder.

[0023] In another aspect, the present invention provides a composition comprising protease-loaded microcapsules obtained by or obtainable by the method of the present invention.

[0024] In another aspect, the present invention provides a solution comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules.

[0025] Suitably, the solution has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1. In some embodiments, the solution has a protease:anionic polysaccharide weight ratio of at least 5:2, preferably wherein the solution has a protease:anionic polysaccharide weight ratio of at least 3:1. In some embodiments, the solution has a protease:anionic polysaccharide weight ratio of 9:2 or less, preferably wherein the solution has a protease:anionic polysaccharide weight ratio of 4:1 or less. In some embodiments, the solution has a protease:anionic polysaccharide weight ratio of from 5:2 to 9:2, preferably wherein the solution has a protease:anionic polysaccharide weight ratio of from 3:1 to 4:1.

[0026] Suitably, the solution has a pH of from 2 to 4. In some embodiments, the solution has a pH of from 2.5 to 4.0, preferably wherein the solution has a pH of from 3.0 to 4.0. In some embodiments, the solution has a pH of from 2.0 to 3.5, preferably wherein the solution has a pH of from 2.0 to 3.0. In some embodiments, the solution has a pH of from 2.5 to 3.5, preferably wherein the solution has a pH of about 3.0.

[0027] In another aspect, the present invention provides a powder comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules.

[0028] Suitably, the powder has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1. In some embodiments, the powder has a protease:anionic polysaccharide weight ratio of at least 5:2, preferably wherein the powder has a protease:anionic polysaccharide weight ratio of at least 3:1. In some embodiments, the powder has a protease:anionic polysaccharide weight ratio of 9:2 or less, preferably wherein the powder has a protease:anionic polysaccharide weight ratio of 4:1 or less. In some embodiments, the powder has a protease:anionic polysaccharide weight ratio of from 5:2 to 9:2, preferably wherein the powder has a protease:anionic polysaccharide weight ratio of from 3:1 to 4:1.

[0029] Suitably, at least 60%, at least 70%, at least 80%, or at least 90% of the total protease in the powder is in a coacervated form.

[0030] In some embodiments, the powder is a freeze-dried powder. The powder may have a maximum particle size of 800 microns or less, 500 microns or less, or 300 microns or less.

[0031] The powder of the present invention may be shelf-stable. For example, 80% or more of the protease activity may be retained following storage of the powder in paper packaging for 3 months at 40°C and 75% relative humidity. In some embodiments, the microcapsules further comprise one or more antioxidants.

[0032] The powder of the present invention may prevent release of the one or more proteases in the gastric phase and / or release the one or more proteases in the small intestinal phase. For example, 90% or more of the protease activity may be retained following incubation of the powder for 2 hours at 37°C and pH 1 .2-3.0. For example, 90% or more of the protease activity may be released following incubation of the powder for 2 hours at 37°C and pH 6.8.

[0033] In another aspect, the present invention provides a nutritional composition, supplement, or pharmaceutical composition comprising the powder according to the present invention.

[0034] The nutritional composition, supplement, or pharmaceutical composition may be provided in a powder form for reconstitution with liquid. In some embodiments, the nutritional composition, supplement, or pharmaceutical composition further comprises one or more stabilisers, flavourings, sweeteners, and / or colourants.

[0035] In some embodiments, the nutritional composition, supplement, or pharmaceutical composition further comprises one or more flavonoids. In some embodiments, the one or more flavonoids are selected from rutin, quercetin, kaempferol, myricetin, fisetin, catechin, epicatechin, hydroxytyrosol, oleuropein, hesperidin, or any combination thereof. In some embodiments, the one or more flavonoids comprise or consist of rutin.

[0036] In another aspect, the present invention provides a powder according to the present invention or a nutritional composition, supplement, or pharmaceutical composition according to the present invention, for use as a medicament.

[0037] In another aspect, the present invention provides a powder according to the present invention or a nutritional composition, supplement, or pharmaceutical composition according to the present invention, for use in treating and / or preventing joint pain, an inflammatory disease, arthrosis, injuries, back pain, or sciatica, or for use in accelerating healing.

[0038] In another aspect, the present invention provides a powder according to the present invention or a nutritional composition, supplement, or pharmaceutical composition according to the present invention, for use in treating and / or preventing joint pain, such as joint pain in the shoulder, joint pain in the elbow, joint pain in the fingers, joint pain in the knee, menopausal joint pain, or rheumatic pain.

[0039] The one or more proteases may be orally administered in any suitable dose. In some embodiments, the one or more proteases are administered in a total daily dose of from 100 mg to 2000 mg, from 200 mg to 1500 mg, from 300 mg to 1200 mg, from 400 mg to 1000 mg, from 500 mg to 900 mg, or from 600 mg to 800 mg.

[0040] In another aspect, the present invention provides use of a powder according to the present invention for preparing a protein hydrolysate.

[0041] In another aspect, the present invention provides a method for preparing a protein hydrolysate, the method comprising hydrolysing a proteinaceous material with the powder according to the present invention. DESCRIPTION OF DRAWINGS

[0042] Figure 1 - Schematic of example lab-scale process for encapsulating trypsin

[0043] Figure 2 - Schematic of example lab-scale process for encapsulating bromelain

[0044] DETAILED DESCRIPTION

[0045] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0046] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0047] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0048] Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0049] Unless otherwise stated, wt% means weight percentage or mass percentage, and is calculated as the mass of a component divided by the total mass of the composition, multiplied by 100%; and w / v means weight by volume and is calculated as the mass of a component divided by the total volume of the composition.

[0050] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0051] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed. All publications mentioned in the specification are herein incorporated by reference.

[0052] Methods for manufacture

[0053] The present invention provides a method for manufacturing a composition comprising protease-loaded microcapsules, the method comprising microencapsulating one or more proteases by complex coacervation with one or more anionic polysaccharides.

[0054] The method of the present invention may comprise the following steps:

[0055] (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides;

[0056] (b) mixing the first solution and the second solution to provide a mixed solution; and

[0057] (c) adjusting the pH of the mixed solution, to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides.

[0058] The first solution may be an aqueous solution comprising the one or more proteases. In some embodiments, the first solution comprises or consists of the one or more proteases dissolved in water (e.g. deionized water). In some embodiments, the first solution consists of the one or more proteases dissolved in deionized water.

[0059] The first solution may comprise the one or more proteases in any suitable amount. Suitably, the first solution comprises the one or more proteases in an amount of at least about 1% w / v, at least about 2% w / v, at least about 3% w / v, at least about 4% w / v, or at least about 5% w / v. Suitably, the first solution comprises the one or more proteases in an amount of about 30% w / v or less, about 25% w / v or less, about 20% w / v or less, about 15% w / v or less, about 10% w / v or less, or about 5% w / v or less. Suitably, the first solution comprises the one or more proteases in an amount of from about 1 % w / v to about 30% w / v, from about 1 % w / v to about 25% w / v, from about 1 % w / v to about 20% w / v, from about 1% w / v to about 15% w / v, or from about 1% w / v to about 10% w / v.

[0060] The second solution may be an aqueous solution comprising the one or more anionic polysaccharides. In some embodiments, the second solution comprises or consists of the one or more anionic polysaccharides dissolved in water (e.g. deionized water). In some embodiments, the second solution consists of the one or more anionic polysaccharides dissolved in deionized water.

[0061] The second solution may comprise the one or more anionic polysaccharides in any suitable amount. Suitably, the second solution comprises the one or more anionic polysaccharides in an amount of at least about 0.1% w / v, at least about 0.2% w / v, at least about 0.3% w / v, at least about 0.4% w / v, at least about 0.5% w / v, at least about 1 % w / v, at least about 1.5% w / v, or at least about 2% w / v. Suitably, the second solution comprises the one or more anionic polysaccharides in an amount of about 10% w / v or less, about 9% w / v or less, about 8% w / v or less, about 7% w / v or less, about 6% w / v or less, about 5% w / v or less, about 4% w / v or less, about 3% w / v or less, about 2% w / v or less, or about 1 % w / v or less. Suitably, the second solution comprises the one or more anionic polysaccharides in an amount of from about 0.1% w / v to about 10% w / v, from about 0.1 % w / v to about 9% w / v, from about 0.1 % w / v to about 8% w / v, from about 0.1 % w / v to about 7% w / v, from about 0.1 % w / v to about 6% w / v, or from about 0.1 % w / v to about 5% w / v.

[0062] In some embodiments, the first solution comprises or consists of the one or more proteases dissolved in water (e.g. deionized water) and the second solution comprises or consists of the one or more anionic polysaccharides dissolved in water (e.g. deionized water). In some embodiments, the first solution consists of the one or more proteases dissolved in deionized water and the second solution consists of the one or more anionic polysaccharides dissolved in deionized water. In some embodiments, the first solution comprises the one or more proteases in an amount of from about 1 % w / v to about 30% w / v and the second solution comprises the one or more anionic polysaccharides in an amount of from about 0.1 % w / v to about 10% w / v.

[0063] The first solution and the second solution may be mixed by any suitable method. For example, by stirring. Suitably, the first solution and the second solution may be mixed by a low-shear, high-flow mixer. The first solution and the second solution may be further mixed with one or more further solutions, for example to introduce additional agents into the mixed solution. For example, the first solution and the second solution may be mixed with: (a) a further solution comprising one or more further proteases; (b) a further solution comprising one or more further anionic polysaccharides; and / or (c) a further solution comprising one or more antioxidants.

[0064] The mixed solution may have any suitable protease:anionic polysaccharide weight ratio. As used herein, the “protease:anionic polysaccharide weight ratio” may refer to the ratio between the total amount of proteases and the total amount of anionic polysaccharides in the mixed solution. Suitably, the mixed solution has a protease:anionic polysaccharide weight ratio of at least about 2:1 , at least about 9:4, at least about 5:2, at least about 11 :4, at least about 3: 1 , at least about 13:4, at least about 7:2, at least about 15:4, or at least about 4:1 . Suitably, the mixed solution has a protease:anionic polysaccharide weight ratio of about 5:1 or less, about 19:4 or less, about 9:2 or less, about 17:4 or less, about 4:1 or less, about 15:4 or less, about 7:2 or less, about 13:4 or less, or about 3:1 or less. Suitably, the mixed solution has a protease:anionic polysaccharide weight ratio of about 2:1 to about 5:1 , about 5:2 to about 9:2, or about 3: 1 to about 4:1.

[0065] In some embodiments, for example when the one or more proteases consist of trypsin, the mixed solution has a trypsimanionic polysaccharide weight ratio of at least about 2:1 , at least about 9:4, at least about 5:2, at least about 11 :4, or at least about 3:1. Suitably, the mixed solution has a trypsin:anionic polysaccharide weight ratio of about 4:1 or less, about 15:4 or less, about 7:2 or less, about 13:4 or less, or about 3:1 or less. Suitably, the mixed solution has a trypsimanionic polysaccharide weight ratio of about 2:1 to about 4:1 , about 5:2 to about 7:2, or about 3:1.

[0066] In some embodiments, for example when the one or more proteases consist of bromelain, the mixed solution has a bromelaimanionic polysaccharide weight ratio of at least about 3:1 , at least about 13:4, at least about 7:2, at least about 15:4, or at least about 4:1. Suitably, the mixed solution has a bromelaimanionic polysaccharide weight ratio of about 5:1 or less, about 19:4 or less, about 9:2 or less, about 17:4 or less, or about 4:1 or less. Suitably, the mixed solution has a bromelaimanionic polysaccharide weight ratio of about 3: 1 to about 5: 1 , about 7:2 to about 8:2, or about 4:1.

[0067] The mixed solution may comprise the one or more proteases and the one or more anionic polysaccharides in any suitable amount. Suitably, the mixed solution comprises the one or more proteases in an amount of at least about 0.5% w / v, at least about 1% w / v, at least about 1.5% w / v, at least about 2% w / v, or at least about 2.5% w / v; and the one or more anionic polysaccharides in an amount of at least about 0.1 % w / v, at least about 0.2% w / v, at least about 0.3% w / v, at least about 0.4% w / v, at least about 0.5% w / v, at least about 1% w / v, at least about 1.5% w / v, or at least about 2% w / v. Suitably, the mixed solution comprises the one or more proteases in an amount of about 20% w / v or less, about 15% w / v or less, about 10% w / v or less, about 5% w / v or less, or about 2.5% w / v or less; and the one or more anionic polysaccharides in an amount of about 10% w / v or less, about 5% w / v or less, about 4% w / v or less, about 3% w / v or less, about 2% w / v or less, or about 1% w / v or less. Suitably, the mixed solution comprises the one or more proteases in an amount of from about 0.5% w / v to about 20% w / v; and the one or more anionic polysaccharides in an amount of from about 0.1% w / v to about 10% w / v.

[0068] The pH of the mixed solution may be adjusted to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides. For example, the pH of the mixed solution may be adjusted to a pH of from 2 to 4 (e.g. about pH 2, about pH 3, or about pH 4) by any suitable method. Suitably, the pH is adjusted to a pH of about 2.0 or higher, about 2.5 or higher, or about 3.0 or higher. Suitably, the pH is adjusted to a pH of about 4.0 or lower, about 3.5 or lower, or about 3.0 or lower. Suitably, the pH is adjusted to a pH of from about 2.0 to about 4.0, from about 2.5 to about 3.5, or about 3.0.

[0069] The present inventors have surprisingly shown that the method of the present invention can achieve near-complete encapsulation efficiency. The microcapsules may therefore have a protease:anionic polysaccharide weight ratio that is similar or identical to that of the mixed solution. Suitably, the microcapsules have a protease:anionic polysaccharide weight ratio of at least about 2:1 , at least about 9:4, at least about 5:2, at least about 11 :4, at least about 3:1 , at least about 13:4, at least about 7:2, at least about 15:4, or at least about 4:1. Suitably, the microcapsules have a protease:anionic polysaccharide weight ratio of about 5:1 or less, about 19:4 or less, about 9:2 or less, about 17:4 or less, about 4:1 or less, about 15:4 or less, about 7:2 or less, about 13:4 or less, or about 3:1 or less. Suitably, the microcapsules have a protease:anionic polysaccharide weight ratio of about 2:1 to about 5:1 , about 5:2 to about 9:2, or about 3: 1 to about 4:1.

[0070] In some embodiments, for example when the one or more proteases consist of trypsin, the microcapsules have a trypsin:anionic polysaccharide weight ratio of at least about 2: 1 , at least about 9:4, at least about 5:2, at least about 11 :4, or at least about 3:1. Suitably, the microcapsules have a trypsin:anionic polysaccharide weight ratio of about 4:1 or less, about 15:4 or less, about 7:2 or less, about 13:4 or less, or about 3:1 or less. Suitably, the microcapsules have a trypsimanionic polysaccharide weight ratio of about 2:1 to about 4:1 , about 5:2 to about 7:2, or about 3: 1.

[0071] In some embodiments, for example when the one or more proteases consist of bromelain, the microcapsules have a bromelaimanionic polysaccharide weight ratio of at least about 3:1 , at least about 13:4, at least about 7:2, at least about 15:4, or at least about 4:1. Suitably, the microcapsules have a bromelaimanionic polysaccharide weight ratio of about 5:1 or less, about 19:4 or less, about 9:2 or less, about 17:4 or less, or about 4:1 or less. Suitably, the microcapsules have a bromelaimanionic polysaccharide weight ratio of about 3:1 to about 5:1 , about 7:2 to about 8:2, or about 4: 1.

[0072] The microcapsules may comprise about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more of the total protease comprised in the mixed solution. In some embodiments, the microcapsules comprise least about 90% of the total protease comprised in the mixed solution. The total protease content may be determined by the Kjeldahl method, for example using a conversion factor of 6.25 to convert nitrogen content to protein content.

[0073] The microcapsules may have about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more of the total protease activity of the mixed solution. In some embodiments, the microcapsules have at least about 90% of the total protease activity of the mixed solution. The total protease activity may be determined by any suitable assay, and expressed as any suitable unit. For example, the “FIP unit” refers to the protease activity units as defined by the Federation Internationale Pharmaceutique (International Union of Biochemistry. Nomenclature Committee: Enzyme Nomenclature. London: Academic Press; 1984).

[0074] In some embodiments, for example when the one or more proteases comprise or consist of trypsin, the microcapsules have about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more of the total trypsin activity of the mixed solution. In some embodiments, the microcapsules have at least about 90% of the total trypsin activity of the mixed solution. Suitably, trypsin activity is determined using the TAME method.

[0075] In some embodiments, for example when the one or more proteases comprise or consist of bromelain, the microcapsules have about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more of the total bromelain activity of the mixed solution. In some embodiments, the microcapsules have at least about 90% of the total bromelain activity of the mixed solution. Suitably, bromelain activity is determined using the Z- RR-pNA method.

[0076] Proteases

[0077] The method of the present invention may be used to microencapsulate one or more proteases.

[0078] Proteases (also known as peptidases or proteinases) are enzymes that catalyse proteolysis, breaking down proteins into smaller polypeptides or amino acids. Proteases catalyse this reaction by cleaving the peptide bonds within proteins by hydrolysis. Proteases can be classified into broad groups including: serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases. Sources of proteases include plants, animals, and microbes. Suitably, the one or more proteases are derived from plants and / or animals. Suitably, the one or more proteases are selected from the group consisting of: trypsin, bromelain, papain, pancreatin, chymotrypsin, serratiopeptidase, ficain, nattokinase, or any combination thereof. Suitably, the one or more proteases are selected from the group consisting of: trypsin, bromelain, papain, and chymotrypsin, or any combination thereof.

[0079] In some embodiments, the one or more proteases comprise or consist of trypsin and / or bromelain. In some embodiments, the one or more proteases comprise or consist of trypsin or bromelain. In some embodiments, the one or more proteases consist of trypsin or bromelain.

[0080] In some embodiments, the one or more proteases comprise or consist of trypsin. In some embodiments, the one or more proteases consist of trypsin. Trypsin (EC 3.4.21.4) is a serine protease found in the digestive system of many vertebrates. Trypsin is available in high quantity in pancreases (e.g. in porcine, bovine, or ovine pancreas) and can be purified by methods known in the art.

[0081] In some embodiments, the one or more proteases comprise or consist of bromelain. In some embodiments, the one or more proteases consist of bromelain. Bromelain is an enzyme extract usually derived from the stems of pineapples, although it exists in all parts of the fresh pineapple. As used herein, the term “bromelain” may refer to either of two protease enzymes extracted from the plants of the family Bromeliaceae, or it may refer to a combination of those enzymes along with other compounds produced in an extract. Bromelain enzymes are called fruit bromelain (EC 3.4.22.33) and stem bromelain (EC 3.4.22.32). Sources of bromelain are known in the art (see e.g. Arshad, Z.I.M., et al., 2014. Applied microbiology and biotechnology, 98(17), pp.7283-7297). Suitably, the bromelain is stem bromelain.

[0082] Anionic polysaccharides

[0083] The method of the present invention uses one or more anionic polysaccharides to microencapsulate the one or proteases.

[0084] Polysaccharides are long-chain polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkages. As used herein, an “anionic polysaccharide” may refer to a negatively charged polysaccharide, for example composed of carboxylated sugars (COO- )•

[0085] Suitably, the one or more anionic polysaccharides are selected from the group consisting of: alginate, pectin, carrageenan, gum arabic, xanthan gum, carboxymethyl cellulose, gellan gum, or any combination thereof. In some embodiments, the one or more anionic polysaccharides comprise or consist of alginate. Alginate is a naturally occurring anionic polymer typically obtained from brown algae (Phaeophyceae) by treatment with aqueous alkali solutions. The extract is filtered, and either sodium or calcium chloride is added to the filtrate in order to precipitate alginate. This alginate salt can be transformed into alginic acid by treatment with dilute HCI. After further purification and conversion, water-soluble sodium alginate power is produced. The alginate may be provided in the form of an alginate salt (e.g. sodium alginate or calcium alginate) or alginic acid (se e.g. Lee, K.Y. and Mooney, D.J., 2012. Progress in polymer science, 37(1), pp.106- 126).

[0086] In some embodiments, the alginate is in the form of an alginate salt. In some embodiments, the alginate is in the form of sodium alginate or calcium alginate. In some embodiments, the alginate is in the form of sodium alginate.

[0087] Suitably, the alginate has a viscosity (measured at 1 % w / v in water at 20°C) of about 500 mPa s or less, about 400 mPa s or less, about 300 mPa s or less, or about 200 mPa s or less. In some embodiments, the alginate has a viscosity (measured at 1% w / v in water at 20°C) of about 200 mPa s or less. Suitably, the alginate has a viscosity (measured at 1% w / v in water at 20°C) of about 1 mPa s or more. Suitably, the alginate has a viscosity (measured at 1% w / v in water at 20°C) of from about 1 mPa s to about 500 mPa s, from about 1 mPa s to about 400 mPa s, from about 1 mPa s to about 300 mPa s, or from about 1 mPa s to about 200 mPa s. In some embodiments, the alginate has a viscosity (measured at 1% w / v in water at 20°C) of from about 1 mPa s to about 200 mPa s.

[0088] Antioxidants

[0089] The method of the present invention may further comprise mixing one or more antioxidants with the first solution and the second solution and / or the mixed solution.

[0090] The present inventors have surprisingly shown that incorporating antioxidants during the encapsulation process can further extend the shelf-life of the proteases, in particular those prone to oxidation, such as cysteine proteases (e.g. bromelain).

[0091] In some embodiments, for example when the one or more proteases comprise or consist of bromelain, the method further comprises incorporating antioxidants. In some embodiments, the method further comprises mixing one or more antioxidants with the first solution and the second solution. In some embodiments, the mixed solution further comprises one or more antioxidants. In some embodiments, the microcapsules further comprise one or more antioxidants. Antioxidants may include any substance that reduces oxidation and suitable antioxidants for use in the present invention will be well known in the art. Suitably, the one or more antioxidants are selected from the group consisting of: ascorbic acid, sodium ascorbate, calcium ascorbate, erythorbic acid, sodium erythorbate, glutathione, N-acetylcysteine (NAC), alpha-lipoic acid, or any combination thereof.

[0092] In some embodiments, the one or more antioxidants are selected from the group consisting of: ascorbic acid, sodium ascorbate, calcium ascorbate, or any combination thereof. In some embodiments, the one or more antioxidants comprise or consist of ascorbic acid. In some embodiments, the one or more antioxidants consist of ascorbic acid. Any suitable source of ascorbic acid may be used. For example, sources include E300, ascorbic acid; E301 , sodium ascorbate; E302, calcium ascorbate; E303, potassium ascorbate; E304, fatty acid esters of ascorbic acid (ascorbyl palmitate and ascorbyl stearate) (see e.g. Varvara, M., et al., 2016. Italian journal of food safety, 5(1), 4313).

[0093] In other embodiments, for example when the one or more proteases comprise or consist of trypsin, the method does not further comprise incorporating antioxidants. In some embodiments, the method does not comprise mixing one or more antioxidants with the first solution and the second solution. In some embodiments, the mixed solution does not comprise one or more antioxidants. In some embodiments, the microcapsules do not comprise one or more antioxidants. pH modulators

[0094] The pH of the mixed solution may be adjusted by the addition of one or more pH modulators.

[0095] Suitable pH modulators for use in the present invention will be well known in the art. For example, strong organic or inorganic acids may be used to adjust the pH. Suitably, the one or more pH modulators are selected from the group consisting of: hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, tartaric acid, or any combination thereof.

[0096] In some embodiments, the pH is adjusted using one or more inorganic acids. In some embodiments, the pH is adjusted using hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, or any combination thereof. In some embodiments, the pH is adjusted using hydrochloric acid.

[0097] In some embodiments, the one or more pH modulators are introduced under gentle stirring. Additional steps

[0098] The method of the present invention may comprise any additional steps. For example, the method of the present invention may further comprise one or more of the following steps: (d) recovering the microcapsules from the mixed solution; (e) drying the microcapsules to provide a powder; and / or (f) milling and / or sieving the powder.

[0099] In some embodiments, the method of the present invention comprises:

[0100] (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides;

[0101] (b) mixing the first solution and the second solution to provide a mixed solution;

[0102] (c) adjusting the pH of the mixed solution, to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides; and

[0103] (d) recovering the microcapsules from the mixed solution.

[0104] In some embodiments, the method of the present invention comprises:

[0105] (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides;

[0106] (b) mixing the first solution and the second solution to provide a mixed solution;

[0107] (c) adjusting the pH of the mixed solution, to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides;

[0108] (d) recovering the microcapsules from the mixed solution; and

[0109] (e) drying the microcapsules to provide a powder.

[0110] In some embodiments, the method of the present invention comprises:

[0111] (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides;

[0112] (b) mixing the first solution and the second solution to provide a mixed solution;

[0113] (c) adjusting the pH of the mixed solution, to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides;

[0114] (d) recovering the microcapsules from the mixed solution; (e) drying the microcapsules to provide a powder; and

[0115] (f) milling and / or sieving the powder.

[0116] Suitably, the mixed solution is allowed to react for several minutes, to ensure complete complexation, before recovery of the microcapsules. In some embodiments, the mixed solution is allowed to react for at least about one minute, at least about five minutes, or at least about ten minutes, following pH adjustment. The mixed solution may be allowed to react whilst standing or with gentle mixing (e.g. stirring).

[0117] In some embodiments, the mixed solution is concentrated prior to recovery and / or drying. Any suitable method may be used to concentrate the mixed solution, such as evaporation or membrane filtration. In some embodiments, the mixed solution is not concentrated prior to recovery and / or drying. In some embodiments, the mixed solution is concentrated to a solid content of about 5 wt% or more, about 10 wt% or more, about 15 wt% or more, or about 20 wt% or more, prior to recovery and / or drying.

[0118] In some embodiments, the method of the present invention comprises recovering the microcapsules from the mixed solution. The microcapsules may be recovered by any suitable method, for example by filtration. In some embodiments, the microcapsules are recovered from the mixed solution by filtration, to provide a filtride comprising the microcapsules.

[0119] Any suitable filter pore size may be used. Suitably, the microcapsules are recovered from the mixed solution using a filter having a pore size of from about 1 micron to about 100 microns, from about 5 micron to about 50 microns, or from about 10 microns to about 25 microns. In some embodiments, the microcapsules are recovered from the mixed solution using a 5 micron filter, a 10 micron filter, a 20 micron filter, a 25 micron filter, or a 50 micron filter. In some embodiments, the microcapsules are recovered from the mixed solution using a 25 micron filter.

[0120] The filtration step can allow for the removal of a large amount of water, leading to improved drying efficiency. Suitably, about 80 wt% or more, about 85 wt% or more, or about 90 wt% or more, or about 95 wt% or more of the water in the mixed solution is removed by filtration. The filtride may comprise about 20 wt% or less, about 15 wt% or less, about 10 wt% or less, or about 5 wt% or less of the water in the mixed solution. In some embodiments, the filtride has a water content of about 50 wt% or less, about 40 wt% or less, about 30 wt% or less, about 20 wt% or less, or about 10 wt% or less.

[0121] In some embodiments, the method of the present invention comprises drying the microcapsules (e.g. in the form of a filtride or mixed solution) to provide a powder. In some embodiments, the method of the present invention comprises drying the microcapsules (e.g. in the form of a filtride or mixed solution) to provide a powder. In some embodiments, the microcapsules (e.g. in the form of a filtride or mixed solution) have a solid content of about 10 wt% or more, about 20 wt% or more, about 30 wt% or more, about 40 wt% or more, or about 50 wt% or more, prior to drying.

[0122] The microcapsules (e.g. in the form of a filtride or mixed solution) may be dried by any suitable method, for example by freeze-drying or spray-drying. In some embodiments, the microcapsules (e.g. in the form of a filtride or mixed solution) are dried by freeze-drying or spray-drying.

[0123] In preferred embodiments, the microcapsules (e.g. in the form of a filtride) are dried by freeze- drying. In some embodiments, the microcapsules (e.g. in the form of a filtride) are not dried by spray-drying. In some embodiments, the method of the present invention comprises: (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides; (b) mixing the first solution and the second solution to provide a mixed solution; (c) adjusting the pH of the mixed solution, to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides; (d) recovering the microcapsules from the mixed solution; and (e) freeze-drying the microcapsules to provide a powder.

[0124] In other embodiments, the microcapsules (e.g. in the form of a mixed solution) are dried by spray-drying. In some embodiments, the method of the present invention comprises: (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides; (b) mixing the first solution and the second solution to provide a mixed solution; (c) adjusting the pH of the mixed solution, to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides; (d) concentrating the mixed solution; and (e) spray-drying the microcapsules to provide a powder.

[0125] In some embodiments, the method of the present invention comprises milling and / or sieving the powder. In some embodiments, the method of the present invention comprises milling and sieving the powder. The powder may be milled and / or sieved to have a uniform particle size. Suitably, the powder has a maximum particle size of about 800 microns or less, about 700 microns or less, about 600 microns or less, about 500 microns or less, about 400 microns or less, or about 300 microns or less. Suitably, the powder has a maximum particle size of about 100 microns or more. Suitably, the particle size is determined by static light scattering.

[0126] In some embodiments, the method of the present invention comprises: (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides; (b) mixing the first solution and the second solution to provide a mixed solution; (c) adjusting the pH of the mixed solution, to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides; (d) recovering the microcapsules from the mixed solution; (e) freeze-drying the microcapsules to provide a powder; and (f) milling and / or sieving the powder.

[0127] Microcapsule compositions

[0128] The present invention provides a composition comprising protease-loaded microcapsules obtained by or obtainable by the method of the present invention.

[0129] The composition may be provided in any suitable form, for example in solid (e.g. powder), liquid or semi-liquid form. In some embodiments, the composition is in liquid form (e.g. in the form of a solution). In other embodiments, the composition is in solid form (e.g. in the form of a powder).

[0130] Solutions

[0131] The present invention provides a solution comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules.

[0132] The solution may be obtained or obtainable by steps (a)-(c) of the method of the present invention and may have any features described above in the section entitled “Methods for manufacture”, such as a protease:anionic polysaccharide weight ratio of from about 2:1 to about 5: 1 and / or a pH of from about 2 to about 4.

[0133] Other suitable protease:anionic polysaccharide weight ratios and pH levels are described above. Suitable proteases are described above in the section entitled “Proteases”. Suitable anionic polysaccharides are described above in the section entitled “Anionic polysaccharides”. In some embodiments, the microcapsules further comprise one or more antioxidants. Suitable antioxidants are described above in the section entitled “Antioxidants”.

[0134] Powders

[0135] The present invention provides a powder comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules.

[0136] The powder may be obtained or obtainable by steps (a)-(f) of the method of the present invention and may have any features described above in the section entitled “Methods for manufacture”, such as a protease:anionic polysaccharide weight ratio of from about 2:1 to about 5:1.

[0137] Other suitable protease:anionic polysaccharide weight ratios are described above. Suitable proteases are described above in the section entitled “Proteases”. Suitable anionic polysaccharides are described above in the section entitled “Anionic polysaccharides”. In some embodiments, the microcapsules further comprise one or more antioxidants. Suitable antioxidants are described above in the section entitled “Antioxidants”.

[0138] In some embodiments, the powder is a freeze-dried or spray-dried powder. In preferred embodiments, the powder is a freeze-dried powder. In some embodiments, the powder is not a spray-dried powder. A freeze-dried powder can be distinguished from a spray-dried powder by characteristics of the powder (e.g. by the particle size distribution, particle morphology, etc.), see e.g. Dolly, P., et al., 2011. Journal of microencapsulation, 28(6), pp.568-574.

[0139] In some embodiments, the powder has a uniform particle size. In some embodiments, the powder has a maximum particle size of about 800 microns or less, about 700 microns or less, about 600 microns or less, about 500 microns or less, about 400 microns or less, or about 300 microns or less. In some embodiments, the powder has a maximum particle size of about 100 microns or more. Suitably, the particle size is determined by static light scattering.

[0140] In some embodiments, the powder has a highly porous surface. In some embodiments, the powder has irregular particle shapes. In some embodiments, the powder does not have spherical or substantially spherical particles.

[0141] Percentage of coacervated protease

[0142] The powder of the present invention comprises coacervated protease. As used herein, protease which is in a “coacervated” form (i.e. “coacervated protease”) may refer to protease which is reversibly aggregated with the one or more anionic polysaccharides.

[0143] In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the total protease in the powder is in a coacervated form.

[0144] The percentage of coacervated protease in the powder may be determined by any suitable method (see e.g. Blocher McTigue, W.C. and Perry, S.L., 2020. Small, 16(27), p.1907671). For example, the percentage of coacervated protease in the powder may be determined using the following equation: % coacervated protease = 100

[0145] The amount of soluble protease in the powder may, for example, be determined by: dispersing the powder in water at about pH 3 to release the soluble protease; centrifuging the suspension to remove the coacervated protease; and determining the amount of soluble protease in the supernatant (e.g. by an activity assay or protein assay).

[0146] The total amount of protease in the powder may, for example, be determined by: dispersing the powder in water at about pH 8 to release the soluble protease and coacervated protease; and determining the total amount of protease in the solution (e.g. by an activity assay or protein assay).

[0147] The amount of protease may be determined by any suitable assay. For example, the assays described in the examples herein. Suitably, trypsin activity is determined using the TAME method. Suitably, bromelain activity is determined using the Z-RR-pNA method. The protein content may be determined by the Kjeldahl method or by the Bradford method.

[0148] In some embodiments, the percentage of coacervated protease in the powder is determined by a method described in the examples.

[0149] In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the total protein in the powder is in a coacervated form.

[0150] The percentage of coacervated protein in the powder may be determined by any suitable method (see e.g. Blocher McTigue, W.C. and Perry, S.L., 2020. Small, 16(27), p.1907671). For example, the percentage of coacervated protein in the powder may be determined using the following equation:

[0151] % coacervated protein = 100

[0152] The amount of soluble protein in the powder may, for example, be determined by: dispersing the powder in water at about pH 3 to release the soluble protein; centrifuging the suspension to remove the coacervated protein; and determining the amount of soluble protein in the supernatant. The total amount of protein in the powder may, for example, be determined by: dispersing the powder in water at about pH 8 to release the soluble protein and coacervated protein; and determining the total amount of protein in the solution.

[0153] The protein content may be determined by the Kjeldahl method or by the Bradford method.

[0154] In some embodiments, the percentage of coacervated protein in the powder is determined by a method described in the examples.

[0155] Shelf-stability

[0156] The powder of the present invention may be shelf-stable. As used herein, a “shelf-stable” product can be safely stored at room temperature. In some embodiments, the powder is shelfstable in paper packaging at room temperature for at least about 1 month, at least about 2 months, or at least about 3 months.

[0157] Any suitable method or conditions may be used to assess whether the powder is shelf-stable. Suitably, the powder is considered shelf-stable if about 80% or more of the protease activity is retained. Suitably, the powder is stored in paper packaging. Suitably, the temperature for assessing shelf stability is from about 22°C to about 40°C. Suitably, the relative humidity for assessing shelf stability is from about 15% to about 85% relative humidity. Suitably, the duration for assessing shelf stability is from about 1 month to about 6 months.

[0158] In some embodiments, about 80% or more of the protease activity is retained following storage of the powder in paper packaging for about 3 months at about 22°C to about 40°C and about 15% to about 85% relative humidity.

[0159] In some embodiments, about 80% or more of the protease activity is retained following storage of the powder in paper packaging for about 3 months at about 40°C and about 75% relative humidity.

[0160] Release profile

[0161] The powder of the present invention may selectively release the one or more proteases in the gastrointestinal tract. For example, the powder of the present invention may prevent release of the one or more proteases in the gastric phase and release the one or more proteases in the small intestinal phase.

[0162] The “gastric phase” may refer to the period in which the powder is in the stomach following oral administration. The “small intestinal phase” may refer to the period in which the powder is in the small intestine following oral administration. In vitro assays to simulate the gastric phase and small intestinal phase are known in the art (see e.g. Brodkorb, A., et al., Nature Protocols, 2019. 14(4): p. 991-1014) and may be used to determine the kinetic profile of the powder of the present invention. In the context of an in vitro assay, the “gastric phase” may refer to gastric conditions for about 2 hours. Suitably, the gastric phase may comprise a pH of about 1.2 to about 3.0 for about 2 hours at about 37°C. In the context of an in vitro assay, the “small intestinal phase” may refer to small intestinal conditions for about 2 hours. Suitably, the small intestine phase may comprise a pH of about 6.8 for about 2 hours at about 37°C. Suitably, a standardized enzyme and bile liquid mix may be added in the small intestine phase. Suitably, the gastric phase and / or small intestinal phase may be simulated as described in the examples.

[0163] Suitably, protease activity is not substantially released in the gastric phase. In this context, “not substantially released” may mean that about 10% or less, about 5% or less, or about 1% or less of the protease activity present in the powder is released. Suitably, protease activity is not released in the gastric phase. In this context, “not released” may mean that protease activity is not detectable in the gastric phase (e.g. that protease activity is not detected or that protease activity is not significantly greater than background protease activity).

[0164] The powder of the present invention may not release the one or more proteases under gastric conditions. In some embodiments, about 80% or more, about 90% or more, or about 95% or more of the protease activity is retained following incubation of the powder for about 2 hours at about 37°C and about pH 1.2 to about 3.0.

[0165] Suitably, protease activity is substantially released in the small intestinal phase. In this context, “substantially released” may mean that at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of protease activity present in the powder is released. Suitably, protease activity is fully released in the small intestinal phase. In this context, “fully released” may mean about 90% or more of protease activity present in the powder is released. The powder of the present invention may release the one or more proteases under small intestinal conditions. In some embodiments, about 80% or more, about 90% or more, or about 95% or more of the protease activity is released following incubation of the powder for about 2 hours at about 37°C and about pH 6.8.

[0166] The protease activity may be determined by any suitable assay. For example, the assays described in the example. Suitably, trypsin activity is determined using the TAME method. Suitably, bromelain activity is determined using the Z-RR-pNA method.

[0167] Nutritional, supplement, or pharmaceutical composition

[0168] The present invention provides a nutritional composition, supplement, or pharmaceutical composition comprising the protease-loaded microcapsules obtained by or obtainable by the method of the present invention.

[0169] The protease-loaded microcapsules may be comprised in the nutritional composition, supplement, or pharmaceutical composition in any suitable form, for example in the form of a powder (e.g. a freeze-dried powder).

[0170] In some embodiments, the composition is a nutritional composition. As used herein, a “nutritional composition” may mean a composition which nourishes a subject. This nutritional composition is usually to be taken orally, and typically includes a lipid or fat source and a protein source. Suitable nutritional compositions include breakfast cereals, breakfast drinks, frozen meals, etc.

[0171] In some embodiments, the composition is a supplement. As used herein, a "supplement" or “dietary supplement” may be used to complement the nutrition of a subject (it is typically used as such but it might also be added to any kind of compositions intended to be ingested by the subject). When the composition is a supplement, it can be provided in the form of unit doses. Supplements are typically present in the form of a liquid, a gel, a powder, a tablet or capsule.

[0172] In some embodiments, the composition is a pharmaceutical composition. Pharmaceutical compositions include, for example, drops, syrups, powder, tablet or capsule products intended to treat or prevent an adverse medical condition in a subject in need thereof.

[0173] The nutritional composition, supplement, or pharmaceutical composition may be in solid (e.g. powder), liquid or semi-liquid form. In preferred embodiments, the nutritional composition, supplement, or pharmaceutical composition is in a powder form for reconstitution with liquid, for example an aqueous medium (e.g. water), prior to administration. In other embodiments, the nutritional composition, supplement, or pharmaceutical composition is in a liquid form ready for administration (e.g. ready- to-d rink).

[0174] The nutritional composition, supplement, or pharmaceutical composition may comprise any suitable amount of proteases (and optionally other agents). Example dosages are provided in the section below entitled “Therapeutic uses”. Each dose of the nutritional composition, supplement, or pharmaceutical composition (e.g. when reconstituted with liquid) may have a volume of from about 1 mL to about 500 mL, from about 5 mL to about 250 mL, or from about 10 mL to about 200 mL.

[0175] Flavonoids

[0176] The nutritional composition, supplement, or pharmaceutical composition of the present invention may further comprise one or more flavonoid.

[0177] Flavonoids (also known as bioflavonoids) are a class of polyphenolic secondary metabolites found in plants which may include flavonoids, isoflavonoids and neoflavonoids. Flavonoids, isoflavonoids and neoflavonoids are natural products derived from 2-phenylchromen-4-one (flavone), 3-phenylchromen-4-one and 4-phenylcoumarin, respectively (see IIIPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997)). Flavonoids can be classified into different broad groups including anthocyanidins, chaicones, flavonols, flavanones, flavan-3-ols, flavanonols, flavones, and isoflavonoids. Any flavonoid which is suitable for oral administration may be used in the present invention (see e.g. Ververidis, F., et al., 2007. Biotechnology Journal: Healthcare Nutrition Technology, 2(10), pp.1214-1234).

[0178] Flavonoids can be found in plants in glycoside-bound and free aglycone forms. Suitably, the one or more flavonoids are in either form. In some embodiments, the one or more flavonoids are one or more flavonoid glycosides.

[0179] Suitably, the one or more flavonoids are derived from plants. Suitably, the one or more flavonoids are selected from the group consisting of: rutin, quercetin, kaempferol, myricetin, fisetin, catechin, epicatechin, hydroxytyrosol, oleuropein, hesperidin, or any combination thereof.

[0180] Suitably, the one or more flavonoids comprises or consists of one or more flavonol, in glycoside-bound or free aglycone form, optionally a flavonol glycoside.

[0181] Suitably, the one or more flavonoids comprises or consists of quercetin, in glycoside-bound or free aglycone form, optionally a quercetin glycoside. In some embodiments, the one or more flavonoids comprise or consist of rutin. In some embodiments, the one or more flavonoids consist of rutin. Rutin (also called rutoside, quercetin-3-rutinoside, sophorin, 3,3',4',5,7-pentahydroxyflavone-3-rhamnoglucoside) is a flavonol, abundantly found in plants, such as passion flower, buckwheat, tea, and apple. Chemically it is a glycoside comprising of flavonolic aglycone quercetin along with disaccharide rutinose (see e.g. Ganeshpurkar, A. and Saluja, A.K., 2017. Saudi pharmaceutical journal, 25(2), pp.149-164).

[0182] Other components

[0183] The nutritional composition, supplement, or pharmaceutical composition of the present invention may comprise one or more further components such as bulking agents, preservatives, stabilisers, binders, emulsifiers, solubilising agents (e.g. oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, dispersing agents, wetting agents, processing aids, flowing agents, weighting agents, taste-masking agents, flavourings, sweeteners, and colourants.

[0184] In some embodiments, the nutritional composition, supplement, or pharmaceutical composition further comprises one or more stabilisers, flavourings, sweeteners, and / or colourants.

[0185] The list of known flavouring agents includes thousands of molecular compounds which are well known in the art. Sweeteners can include, but are not limited to, sugar alcohols such as maltitol, xylitol, sorbitol, erythritol, mannitol, isomalt, lactitol, hydrogenated starch hydrolysates, and the like, alone or in combination. Colorants include any substance that imparts colour to the composition.

[0186] Therapeutic uses

[0187] The present invention provides for the use of the microcapsule composition (e.g. powder), nutritional composition, supplement, or pharmaceutical composition of the present invention in therapy.

[0188] Oral administration of proteases may be associated with beneficial immunomodulatory effects. One possible mechanism is by regulation of T cell activation thresholds. At sites of inflammation, with vascular permeability locally increased, serum protein exudate can enter the interstitial compartment. Thus, trypsin and other proteases, along with complement components and IgM, are delivered and enriched at the site of inflammation. Proteases, such as trypsin, may cleave antigens which promote T-cell activation, thereby modulating the immune response (see e.g. Lehmann, P.V., 1996. Nephrology Dialysis Transplantation, 11(6), pp.953-955). Further, oral administration of rutin has been explored for a number of pharmacological effects, including immunomodulatory effects (see e.g. Ganeshpurkar, A. and Saluja, A.K., 2017. Saudi pharmaceutical journal, 25(2), pp.149-164).

[0189] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use as a medicament.

[0190] In one aspect, the present invention provides a method of treatment comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0191] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0192] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in treating and / or preventing joint pain, an inflammatory disease, arthrosis, injuries, back pain, or sciatica, or for use in accelerating healing.

[0193] In one aspect, the present invention provides a method of treating and / or preventing joint pain, an inflammatory disease, arthrosis, injuries, back pain, or sciatica, or of accelerating healing, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0194] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for treating and / or preventing joint pain, an inflammatory disease, arthrosis, injuries, back pain, or sciatica, or for accelerating healing, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0195] The subject administered the microcapsule composition, nutritional composition, supplement, or pharmaceutical composition of the present invention may be any subject in need thereof. For example, the subject may have, or may be at risk of, any of the diseases or disorders described herein. The subject may be a human. The subject may be an adult. The microcapsule composition, nutritional composition, supplement, or pharmaceutical composition of the present invention may be orally administered, for example in any suitable regime, in order to deliver an effective amount of the one or more active agents.

[0196] Suitably, the microcapsule composition, nutritional composition, supplement, or pharmaceutical composition of the present invention is administered once, twice, or three times daily. Suitably, the microcapsule composition, nutritional composition, supplement, or pharmaceutical composition of the present invention is administered in a fasting state (e.g. at least about 45 minutes before a meal). Suitably, the microcapsule composition, nutritional composition, supplement, or pharmaceutical composition of the present invention is administered for at least 15 days, for at least 30 days, for at least 45 days, or for at least 60 days.

[0197] Suitably, the one or more proteases are administered in a total daily dose of at least about 100 mg, at least about 200 mg, at least about 300 mg, at least about 400 mg, at least about 500 mg, or at least about 600 mg. Suitably, the one or more proteases are administered in a total daily dose of about 2000 mg or less, about 1500 mg or less, about 1200 mg or less, about 1000 mg or less, about 900 mg or less, or about 800 mg or less. Suitably, the one or more proteases are administered in a total daily dose of from about 100 mg to about 2000 mg, from about 200 mg to about 1500 mg, from about 300 mg to about 1200 mg, from about 400 mg to about 1000 mg, from about 500 mg to about 900 mg, or from about 600 mg to about 800 mg.

[0198] Suitably, the one or more proteases are administered in a total daily dose of at least about 2000 FIP units, at least about 4000 FIP units, at least about 6000 FIP units, at least about 8000 FIP units, or at least about 10000 FIP units. Suitably, the one or more proteases are administered in a total daily dose of about 30000 FIP units or less, about 25000 FIP units or less, about 20000 FIP units or less, about 15000 FIP units or less, or about 12000 FIP units or less. Suitably, the one or more proteases are administered in a total daily dose of from about 2000 FIP units to about 30000 FIP units, from about 4000 FIP units to about 25000 FIP units, from about 6000 FIP units to about 20000 FIP units, from about 8000 FIP units to about 15000 FIP units, or from about 10000 FIP units to about 12000 FIP units.

[0199] Suitably, trypsin is administered in a daily dose of at least about 50 mg, at least about 100 mg, at least about 150 mg, at least about 175 mg, or at least about 200 mg. Suitably, trypsin is administered in a daily dose of about 500 mg or less, about 400 mg or less, about 350 mg or less, about 300 mg or less, or about 250 mg or less. Suitably, trypsin is administered in a daily dose of from about 50 mg to about 500 mg, from about 100 mg to about 400 mg, from about 150 mg to about 350 mg, from about 175 mg to about 300 mg, or from about 200 mg to about 250 mg.

[0200] Suitably, trypsin is administered in a daily dose of at least about 2000 FIP units, at least about 3000 FIP units, at least about 4000 FIP units, at least about 5000 FIP units, at least about 6000 FIP units or at least about 7000 FIP units. Suitably, trypsin is administered in a daily dose of about 20000 FIP units or less, about 15000 FIP units or less, about 12000 FIP units or less, about 11000 FIP units or less, about 10000 FIP units or less, or about 9000 FIP units or less. Suitably, trypsin is administered in a daily dose of from about 2000 FIP units to about 20000 FIP units, from about 3000 FIP units to about 15000 FIP units, from about 4000 FIP units to about 12000 FIP units, from about 5000 FIP units to about 11000 FIP units, from about 6000 FIP units to about 10000 FIP units, or from about 7000 FIP units to about 9000 FIP units.

[0201] Suitably, bromelain is administered in a daily dose of at least about 100 mg, at least about 200 mg, at least about 300 mg, at least about 350 mg, at least about 400 mg, or at least about 450 mg. Suitably, bromelain is administered in a daily dose of about 1000 mg or less, about 900 mg or less, about 800 mg or less, about 700 mg or less, about 600 mg or less, or about 500 mg or less. Suitably, bromelain is administered in a daily dose of from about 100 mg to about 1000 mg, from about 200 mg to about 900 mg, from about 300 mg to about 800 mg, from about 350 mg to about 700 mg, from about 400 mg to about 600 mg, or from about 450 mg to about 500 mg.

[0202] Suitably, bromelain is administered in a daily dose of at least about 500 FIP units, at least about 1000 FIP units, at least about 1500 FIP units, at least about 2000 FIP units, or at least about 2500 FIP units. Suitably, bromelain is administered in a daily dose of about 10000 FIP units or less, about 8000 FIP units or less, about 6000 FIP units or less, about 4000 FIP units or less, or about 3000 FIP units or less. Suitably, bromelain is administered in a daily dose of from about 500 FIP units to about 10000 FIP units, from about 1000 FIP units to about 8000 FIP units, from about 1500 FIP units to about 6000 FIP units, from about 2000 FIP units to about 4000 FIP units, or from about 2500 FIP units to about 3000 FIP units.

[0203] Suitably, one or more flavonoids are administered in a total daily dose of at least about 100 mg, at least about 200 mg, at least about 300 mg, at least about 400 mg, or at least about 500 mg. Suitably, one or more flavonoids are administered in a total daily dose of about 1200 mg or less, about 1000 mg or less, about 900 mg or less, about 800 mg or less, or about 700 mg or less. Suitably, one or more flavonoids are administered in a total daily dose of from about 100 mg to about 1200 mg, from about 200 mg to about 1000 mg, from about 300 mg to about 900 mg, from about 400 mg to about 800 mg, from about 500 mg to about 700 mg. Suitably, rutin is administered in a total daily dose of at least about 100 mg, at least about 200 mg, at least about 300 mg, at least about 400 mg, or at least about 500 mg. Suitably, rutin is administered in a total daily dose of about 1200 mg or less, about 1000 mg or less, about 900 mg or less, about 800 mg or less, or about 700 mg or less. Suitably, rutin is administered in a total daily dose of from about 100 mg to about 1200 mg, from about 200 mg to about 1000 mg, from about 300 mg to about 900 mg, from about 400 mg to about 800 mg, from about 500 mg to about 700 mg.

[0204] In some embodiments, the one or more proteases are administered in a total daily dose of from about 100 mg to about 2000 mg; and one or more flavonoids are administered in a total daily dose of from about 100 mg to about 1200 mg.

[0205] In some embodiments, the one or more proteases are administered in a total daily dose of from about 2000 FIP units to about 30000 FIP units; and one or more flavonoids are administered in a total daily dose of from about 100 mg to about 1200 mg.

[0206] In some embodiments, trypsin is administered in a daily dose of from about 50 mg to about 500 mg; bromelain is administered in a daily dose of from about 100 mg to about 1000 mg; and rutin is administered in a total daily dose of from about 100 mg to about 1200 mg.

[0207] In some embodiments, trypsin is administered in a daily dose of from about 2000 FIP units to about 20000 FIP units; bromelain is administered in a daily dose of from about 500 FIP units to about 10000 FIP units; and rutin is administered in a total daily dose of from about 100 mg to about 1200 mg

[0208] Joint pain

[0209] Oral administration of proteases may be associated with beneficial effects in treating and / or preventing joint pain (see e.g. Wald, M. and Rovensky, J., 2017. Systemic Enzyme Therapy in Comprehensive Treatment of Degenerative Rheumatic Diseases in the Elderly. In Gerontorheumatology (pp. 363-366). Springer, Cham).

[0210] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in treating and / or preventing joint pain.

[0211] In one aspect, the present invention provides a method of treating and / or preventing joint pain, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof. In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for treating and / or preventing joint pain, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0212] As used herein, “joint pain” or “arthralgia” may refer to aching or pain in the joints (e.g. without inflammation). The causes of arthralgia are varied and range, from a joints perspective, from degenerative and destructive processes such as osteoarthritis and sports injuries to inflammation of tissues surrounding the joints, such as bursitis. Affected joints may include those in knees, elbows, shoulders, hips, wrists, hands, and feet.

[0213] In some embodiments, the joint pain is joint pain in the elbow, joint pain in the fingers, joint pain in the knee, menopausal joint pain, or rheumatic pain.

[0214] Inflammatory disease

[0215] Oral administration of proteases may be associated with beneficial effects in treating and / or preventing inflammatory disease. For example, the oral administration of proteolytic enzymes has been shown to be a safe and effective option to treat acute thrombophlebitis in the absence of compression treatment (Baumueller, M. and Rau, S., 2018 Journal of Phlebology and Lymphology, 11 (1), pp. 7-12). An enzyme combination of proteolytic enzymes has been shown to decrease and normalize the biomarkers of inflammation in vestibulodynia and painful bladder syndrome (Murina, F., et al, V., 2013. Open Journal of Obstetrics and Gynecology, 3(4A), 33232).

[0216] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in treating and / or preventing an inflammatory disease.

[0217] In one aspect, the present invention provides a method of treating and / or preventing an inflammatory disease, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0218] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for treating and / or preventing an inflammatory disease, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention. As used herein, an “inflammatory disease” may refer to a disease in which the immune system attacks the body's own tissues, resulting in inflammation. Exemplary inflammatory diseases include encephalitis, myelitis, arachnoiditis, neuritis, dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis, otitis externa, otitis media, labyrinthitis, mastoiditis, carditis, endocarditis, myocarditis, pericarditis, vasculitis, arteritis, phlebitis, capillaritis, sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritis, mediastinitis, stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis / parotitis, cheilitis, pulpitis, gnathitis, esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, caecitis, appendicitis, proctitis, hepatitis, ascending cholangitis, cholecystitis, pancreatitis, peritonitis, dermatitis, folliculitis, cellulitis, hidradenitis, arthritis, dermatomyositis, myositis, synovitis / tenosynovitis, bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, tendinitis, panniculitis, osteochondritis, spondylitis, periostitis, chondritis, nephritis, ureteritis, cystitis, urethritis, oophoritis, salpingitis, endometritis, parametritis, cervicitis, vaginitis, vulvitis, mastitis, orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, posthitis, balanoposthitis, chorioamnionitis, funisitis, omphalitis, insulitis, hypophysitis, thyroiditis, parathyroiditis, adrenalitis, lymphangitis, and lymphadenitis.

[0219] In some embodiments, the inflammatory disease is selected from phlebitis, prostate inflammation, or cystitis.

[0220] Arthrosis

[0221] Oral administration of proteases may be associated with beneficial effects in treating and / or preventing joint arthrosis. For example, in a randomized trial of subjects with moderate-to- severe knee osteoarthritis, oral administration of proteolytic enzymes was found to have comparable effectiveness to diclofenac in relieving pain and increasing function (see Ueberall, M.A., et al., 2016. Journal of pain research, 9, pp.941-961).

[0222] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in treating and / or preventing arthrosis.

[0223] In one aspect, the present invention provides a method of treating and / or preventing arthrosis, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0224] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for treating and / or preventing arthrosis, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0225] As used herein, “arthrosis” may include arthritis and other similar disorders. The term arthritis may include osteoarthritis, rheumatoid arthritis, gout and pseudo-gout, septic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, Still's disease, and psoriatic arthritis.

[0226] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in treating and / or preventing osteoarthritis.

[0227] In one aspect, the present invention provides a method of treating and / or preventing osteoarthritis, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0228] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for treating and / or preventing osteoarthritis, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0229] Osteoarthritis is a type of degenerative joint disease that results from breakdown of joint cartilage and underlying bone. Osteoarthritis can affect the hands, lower back, neck, and weight-bearing joints such as knees, hips, and feet.

[0230] In some embodiments, the osteoarthritis is osteoarthritis in the hip, osteoarthritis in the knee, arthrosis in the metatarsophalangeal joint of the big toe, osteoarthritis in the finger, osteoarthritis in the foot, osteoarthritis of the ankle, Heberden and Bouchard nodes.

[0231] In some embodiments, the osteoarthritis is osteoarthritis in the knee.

[0232] Injuries

[0233] Oral administration of proteases may be associated with beneficial effects in treating and / or preventing injuries. For example, there are reports that enzyme products have proven effective in treating sport injuries such as sprains, contusions and compression injuries (see Pdttgen, K., Nutrition as a form of treatment. Sports Medicine Newspaper 2017).

[0234] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in treating and / or preventing injuries. In one aspect, the present invention provides a method of treating and / or preventing injuries, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0235] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for treating and / or preventing injuries, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0236] In some embodiments, the injuries are selected from sports injuries, muscle soreness, strains, overload damage, sprains, or bruises.

[0237] Back pain and sciatica

[0238] Oral administration of proteases may be associated with beneficial effects in treating and / or preventing back pain and sciatica.

[0239] In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in treating and / or preventing back pain or sciatica.

[0240] In one aspect, the present invention provides a method of treating and / or preventing back pain or sciatica, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0241] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for treating and / or preventing back pain or sciatica, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0242] In some embodiments, the back pain is selected from upper back pain, disc prolapse, or lumbago.

[0243] Healing

[0244] Oral administration of proteases may be associated with beneficial effects in accelerating healing. For example, the percentage of patients with a severe Caesarean section scars scar was lower in a group treated with proteolytic enzymes (see Dosedla, E., et al., 2016. Ceska gynekologie, 81 (3), pp.202-207). In one aspect, the present invention provides a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention for use in accelerating healing.

[0245] In one aspect, the present invention provides a method of accelerating healing, the method comprising administering a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0246] In one aspect, the present invention provides use of protease-loaded microcapsules in the manufacture of a medicament for accelerating healing, preferably wherein the medicament is in the form of a microcapsule composition, nutritional composition, supplement, or pharmaceutical composition according to the present invention.

[0247] In some embodiments, the protease-loaded microcapsules, microcapsule composition, nutritional composition, supplement, or pharmaceutical composition accelerates wound healing or healing after operation

[0248] Industrial uses

[0249] The present invention provides for the use of the microcapsule composition of the present invention in industrial processes. For example, the microcapsule composition of the present invention may be used to prepare protein hydrolysates.

[0250] In one aspect, the present invention provides a method for preparing a protein hydrolysate, the method comprising hydrolysing a proteinaceous material with the microcapsule composition of the present invention.

[0251] The proteinaceous material may be a vegetable protein (e.g. soy protein, grain proteins, rape seed protein, alfalfa protein, pea protein, fabaceous bean protein, cotton seed protein or sesame seed protein) or animal protein (e.g. milk protein, whey protein, casein, meat protein, fish protein, blood protein, egg white or gelatin).

[0252] Suitable incubation conditions (e.g. amount of protease, pH, temperature, duration, etc.) will be known to the skilled person and will depend on the proteases encapsulated in the microcapsule composition. Suitably, the hydrolysis is carried out at a pH of from about 5 to about 9. In some embodiments, the microcapsule composition is mixed with the proteinaceous material in a solution having a pH of from 2 to 4, and the pH of the solution is subsequently raised to release the protease activity. Suitably, the hydrolysis is carried out at a temperature of from about 20°C to about 70°C. Suitably, the hydrolysis is carried out for a duration of from about 1 hour to about to about 24 hours. EXAMPLES

[0253] The invention will now be further described by way of examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0254] Example 1 - Microencapsulation of bromelain and trypsin by complex coacervation

[0255] Materials and methods

[0256] The following microencapsulation method was carried out:

[0257] 1. Enzyme and Alginate Preparation: Trypsin and bromelain were dissolved in deionized water at concentrations of 5% (w / v), respectively. Separately, a 2% (w / v) solution of sodium alginate was prepared in deionized water.

[0258] 2. Complex Coacervation Process: The alginate-bromelain mixture was subjected to complex coacervation by dropwise addition of 0.1 M hydrochloric acid under gentle stirring. The process was carried out to pH of 3. The mixture was allowed to react for 10 minutes to ensure complete complexation, forming alginate-bromelain microcapsules.

[0259] 3. Microcapsule Recovery: The formed microcapsules were filtered through a 25pm mesh bag and the residual water was compressed out.

[0260] 4. Drying of Microcapsules: The microcapsules were freeze-dried using a lyophilizer until the product thermometer reached room temperature.

[0261] 5. Incorporation of Antioxidants: Antioxidants, specifically ascorbic acid, were integrated into the alginate-bromelain mixture prior to coacervation to extend shelf-life and protect against oxidation.

[0262] 6. Final Particle Formation: Following the drying phase, the microcapsules underwent a gentle milling process utilizing a standard laboratory mixer to achieve a uniform particle size. Subsequently, the milled microcapsules were subjected to a sieving procedure, employing a sieve with a mesh size of 300 micrometers (pm). This step ensured the removal of any oversized particles, achieving a consistent particle size distribution. Characterization of microcapsules

[0263] Encapsulation Efficiency: To monitor the encapsulation process, three key parameters were evaluated: total solids, total protein, and total enzyme activity. Encapsulation efficiency was quantified by establishing the ratio of successfully encapsulated protein to the initial total protein content. The measurement of total protein was derived from the quantification of total nitrogen. Moreover, enzyme activity assays were utilized to systematically evaluate any loss in enzymatic function that might occur during the encapsulation process, providing a comprehensive assessment of the procedure's efficacy.

[0264] Shelf-life Study: Accelerated shelf-life testing was conducted by storing the microcapsule powder in a paper bag at 40°C and 75% relative humidity (RH) for three months. The enzymatic activity of bromelain was measured at predetermined intervals using a suitable assay to evaluate stability.

[0265] Gastric stability assessment:

[0266] Simulation of Gastrointestinal Conditions: The study utilized an in vitro digestion model, adhering to the INFOGEST 2.0 standardized protocol, to simulate human gastrointestinal conditions accurately (see Brodkorb, A., et al., Nature Protocols, 2019. 14(4): p. 991-1014). This method was chosen to mimic the fasted state's oral, gastric, and intestinal phases:

[0267] • Preparation of Simulated Digestive Fluids:

[0268] Simulated Salivary Fluid (SSF): Prepared according to INFOGEST guidelines, adjusting for enzyme activity and electrolyte composition to reflect fasted state conditions. Simulated Gastric Fluid (SGF): Composed of pepsin with a pH adjusted to 1.2-3.0 to replicate stomach acidity. Simulated Intestinal Fluid (SIF): Contains pancreatin and bile salts, adjusted to a pH of 6.8 to mimic the small intestine environment.

[0269] • Encapsulation and Enzyme Preparation: Microencapsulated and non-encapsulated forms of trypsin and bromelain were prepared in separate solutions. The concentration of enzymes was measured prior to digestion simulation to establish baseline activity levels.

[0270] • Digestive Process Simulation:

[0271] Oral Phase: Enzyme solutions were mixed with SSF for 2 minutes at 37°C to simulate the oral digestion. Gastric Phase: The mixture was then incubated with SGF at 37°C for 2 hours, simulating gastric digestion. Samples were taken at 0, 30, 60, and 120 minutes to assess enzyme stability. Intestinal Phase: SIF was added, and the incubation continued for a further 2 hours at 37°C, simulating intestinal digestion. Enzyme activity was measured at intervals to evaluate release and activity post-gastric phase.

[0272] Enzyme activity and protein assays

[0273] Trypsin activity using pNA derivative colorimetric assay: Ac-Arg-pNA is utilized as synthetic substrates for trypsin. The assay's core involves measuring the absorbance at 405 nm post-hydrolytic cleavage of the pNA segment, using the Gallery™ Plus automated analyzer for peptidase activity determination. The procedure includes combining 120 pL of Bis-Tris propane buffer (pH 8.2, 100 mM) and 60 pL of the protease solution in cuvettes, followed by substrate addition after a 15-minute pre-incubation at 37°C. Absorbance is kinetically measured at 405 nm at intervals.

[0274] Bromelain activity assay: The Bromelain Activity Assay quantifies enzyme activity by hydrolyzing Z-Arg-Arg-pNA 2HCI, releasing p-nitroaniline detectable at 405 nm, while selectively inhibiting trypsin to isolate bromelain's activity. Equipment includes a microplate reader for absorbance measurement and a vibrating plate for thorough mixing. The reagents: a specialized buffer at pH 7.15, combining Tris(hydroxymethyl)aminomethane with L-cysteine, potassium chloride, Titriplex III, and Tween 20 for optimal reaction conditions; a pipetting solution created from soybean trypsin inhibitor and the substrate Z-Arg-Arg-pNA 2HCI, ensuring precise initiation of the enzymatic reaction; and a standard solution for calibration.

[0275] Protein quantification assay: The protein content was determined by the Kjeldahl method, using a conversion factor of 6.25 to convert nitrogen content to protein content. This method involves digestion of the sample to convert organic nitrogen to ammonia, followed by distillation and titration to quantify the ammonia, indicative of the total nitrogen present.

[0276] Results

[0277] Encapsulation Efficiency and Payload Efficiency

[0278] The microencapsulation process demonstrated remarkable efficiency in encapsulating trypsin and bromelain (see Table 1). The encapsulation efficiency exceeded 90% for trypsin and ranged between 65-70% for bromelain. Notably, the total enzyme activity retained within the microcapsules was significantly preserved, showcasing the method's ability to maintain enzymatic function post-encapsulation. The lower total solids observed in bromelain may be due to its initial content, where only about 67% is protein, with the remainder being non-protein substances that are removed during encapsulation. This discrepancy in total solids before and after encapsulation highlights the dual role of the process in purifying the enzyme while maintaining its activity, despite the addition of alginate as a polymer.

[0279] Table 1. Encapsulation Efficiency and Activity Retention of Trypsin and Bromelain.

[0280] The encapsulation method demonstrated a remarkable efficacy in payload delivery of proteases. The process achieves a substantial payload of approximately 70%. This increased payload capacity ensures a higher concentration of active enzyme per unit volume of the encapsulated material, which allows for maintaining therapeutic efficacy, while reducing the dosage volume required.

[0281] Percentage of Coacervate

[0282] The percentage of coacervated protease was determined for microencapsulated and nonencapsulated forms of trypsin and bromelain using the following equation:

[0283] % coacervated protease = 100

[0284] For both the microencapsulated enzymes, -100% of the total protease was in a coacervated form (see Table 2A and 2B).

[0285] For the microencapsulated and non-encapsulated bromelain, the amount of protease was determined by measuring the total nitrogen using the Kjeldahl method. The amount of soluble protease was determined by making a 2% solution of the powder at pH 3 to release the soluble protease, allowing the powder to settle, and measuring the total nitrogen in the supernatant. The total amount of protease was determined by measuring the total nitrogen in the powder directly.

[0286] For the microencapsulated and non-encapsulated trypsin, the amount of soluble protease was determined by mixing 0.5 g solid in 100g buffer at pH 3 to release the soluble protease and measuring the trypsin activity. The total amount of protease was determined by mixing 0.5 g solid in 100g buffer at pH 8 to release the soluble protease and coacervated protease and measuring the trypsin activity. The variation in enzyme activity tests for analytical analysis is typically around 10%.

[0287] Table 2A. Percentage of coacervate in bromelain samples - according to total nitrogen (Kjeldahl method)

[0288] Table 2B. Percentage of coacervate in trypsin samples - according to trypsin activity

[0289] Shelf-stability study

[0290] Accelerated shelf stability tests conducted at 40°C and 75% relative humidity revealed enhanced stability for encapsulated enzymes compared to their free forms.

[0291] Encapsulated trypsin exhibited a remarkable preservation of activity, maintaining 94% of its original activity over three months, compared to only 75% in its free form (see Table 3). Similarly, encapsulated bromelain showed substantial stability improvements, with a maximum of 84% activity retention, compared to only 50% in its free form (see Table 4). These results underscore the protective capacity of the encapsulation matrix against environmental stressors. Table 3. Shelf-Life Stability of Encapsulated Enzymes at Accelerated Conditions.

[0292] The integration of ascorbic acid as an antioxidant during the encapsulation process was suitable for enhancing bromelain's shelf-life. Encapsulated bromelain, with ascorbic acid mixed in during the encapsulation, showed improved stability results. Conversely, mixing ascorbic acid post-encapsulation resulted in diminished protective effects, emphasizing that incorporating antioxidants during the encapsulation phase provides optimal stability (see Table 4).

[0293] Table 4. Stability of Bromelain: Encapsulated with and without Antioxidants.

[0294] Gastric and Intestinal Release Profiles

[0295] The gastric stability of trypsin and bromelain was assessed at a gastric pH of 3, simulating the stomach's acidic environment. The activity recovery after 30 minutes in a simulated small intestine environment revealed marked differences in stability between free and encapsulated forms of the enzymes (see Table 5).

[0296] Free trypsin showed an 80% activity recovery, whereas encapsulated trypsin demonstrated full activity recovery, indicating complete protection against gastric degradation.

[0297] In the case of bromelain, the free form showed no activity recovery, signifying complete deactivation in gastric conditions. However, encapsulated bromelain exhibited full activity recovery, highlighting the encapsulation's effectiveness in safeguarding bromelain's functional integrity through the gastric phase.

[0298] These results emphasize the potential of microencapsulation technology to significantly enhance the gastric stability of sensitive enzymes, ensuring their bioavailability and therapeutic efficacy upon reaching the small intestine.

[0299] Table 5. Gastric and Intestinal Release Profiles of Encapsulated Enzymes.

[0300] Discussion

[0301] The utilization of complex coacervation, optimized for proteases, demonstrated exceptional encapsulation efficiency and payload optimization, far exceeding traditional methods. This success can be attributed to the selection of a biocompatible anionic polysaccharide, which not only protects the enzymes from degradation but also ensures their targeted release within the gastrointestinal tract.

[0302] One of the most notable findings from this research is the enhanced shelf stability of the encapsulated enzymes, particularly when antioxidants are incorporated during the encapsulation process. Furthermore, the study's results on the gastric and intestinal release profiles of the encapsulated enzymes offer promising insights into their potential for improved therapeutic efficacy and bioavailability.

[0303] The synergy between high payload efficiency and enhanced shelf stability positions this encapsulation method as a robust solution for providing proteases in a commercially viable and therapeutically effective manner.

[0304] EMBODIMENTS

[0305] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras). 1. A method for manufacturing a composition comprising protease-loaded microcapsules, the method comprising:

[0306] (a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides;

[0307] (b) mixing the first solution and the second solution to provide a mixed solution, wherein the mixed solution has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1 ; and

[0308] (c) adjusting the pH of the mixed solution to a pH of from 2 to 4 to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides.

[0309] 2. The method according to para 1 , wherein the one or more proteases are selected from trypsin, bromelain, papain, pancreatin, chymotrypsin, serratiopeptidase, ficain, nattokinase, or any combination thereof.

[0310] 3. The method according to para 1 or 2, wherein the one or more proteases comprise or consist of trypsin and / or bromelain.

[0311] 4. The method according to any of paras 1-3, wherein the one or more proteases consist of trypsin.

[0312] 5. The method according to any of paras 1-3, wherein the one or more proteases consist of bromelain.

[0313] 6. The method according to any preceding para, wherein the first solution comprises the one or more proteases in an amount of from 1 % w / v to 30% w / v, from 1 % w / v to 20% w / v, or from 1 % w / v to 10% w / v.

[0314] 7. The method according to any preceding para, wherein the first solution comprises the one or more proteases dissolved in water.

[0315] 8. The method according to any preceding para, wherein the one or more anionic polysaccharides are selected from alginate, pectin, carrageenan, gum arabic, xanthan gum, carboxymethyl cellulose, gellan gum, or any combination thereof.

[0316] 9. The method according to any preceding para, wherein the one or more anionic polysaccharides consist of alginate. 10. The method according to any preceding para, wherein the second solution comprises the one or more anionic polysaccharides in an amount of from 0.1% w / v to 10% w / v, or from 0.1 % w / v to 5% w / v.

[0317] 11. The method according to any preceding para, wherein the second solution comprises the one or more anionic polysaccharides dissolved in water.

[0318] 12. The method according to any preceding para, wherein the mixed solution has a protease:anionic polysaccharide weight ratio of at least 5:2, preferably wherein the mixed solution has a protease:anionic polysaccharide weight ratio of at least 3:1.

[0319] 13. The method according to any preceding para, wherein the mixed solution has a protease:anionic polysaccharide weight ratio of 9:2 or less, preferably wherein the mixed solution has a protease:anionic polysaccharide weight ratio of 4:1 or less.

[0320] 14. The method according to any preceding para, wherein the mixed solution has a protease:anionic polysaccharide weight ratio of from 5:2 to 9:2, preferably wherein the mixed solution has a protease:anionic polysaccharide weight ratio of from 3:1 to 4:1.

[0321] 15. The method according to any preceding para, wherein the method further comprises mixing one or more antioxidants with the first solution and the second solution, and wherein the microcapsules further comprise the one or more antioxidants.

[0322] 16. The method according to para 15, wherein the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, erythorbic acid, sodium erythorbate, glutathione, N-acetylcysteine (NAC), alpha-lipoic acid, or any combination thereof.

[0323] 17. The method according to para 15 or 16, wherein the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, or any combination thereof, preferably wherein the one or more antioxidants consist of ascorbic acid.

[0324] 18. The method according to any preceding para, wherein the pH is adjusted to a pH of from 2.5 to 4.0, preferably wherein the pH is adjusted to a pH of from 3.0 to 4.0.

[0325] 19. The method according to any of paras 1 to 17, wherein the pH is adjusted to a pH of from 2.0 to 3.5, preferably wherein the pH is adjusted to a pH of from 2.0 to 3.0.

[0326] 20. The method according to any of paras 1 to 17, wherein the pH is adjusted to a pH of from 2.5 to 3.5, preferably wherein the pH is adjusted to a pH of about 3. 21. The method according to any preceding para, wherein the pH is adjusted using one or more acids selected from hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, tartaric acid, or any combination thereof.

[0327] 22. The method according to any preceding para, wherein the pH is adjusted using one or more inorganic acids, preferably wherein the one or more inorganic acids are selected from hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid, or any combination thereof.

[0328] 23. The method according to any preceding para, wherein the microcapsules comprise 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total protease comprised in the mixed solution and / or wherein the microcapsules have 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total protease activity of the mixed solution.

[0329] 24. The method according to any preceding para, wherein the microcapsules have a protease:anionic polysaccharide weight ratio of at least 2:1 , at least 3:1 , or at least 4:1.

[0330] 25. The method according to any preceding para, wherein the method further comprises recovering the microcapsules from the mixed solution.

[0331] 26. The method according to any preceding para, wherein the microcapsules are recovered from the mixed solution by filtration, optionally using a 25 micron filter, preferably wherein 50wt% or more, 60wt% or more, 70wt% or more, 80wt% or more, or 90wt% or more of the water in the mixed solution is removed by filtration.

[0332] 27. The method according to any preceding para, wherein the method further comprises drying the microcapsules to provide a powder comprising microencapsulated protease, preferably wherein the microcapsules are dried by freeze-drying.

[0333] 28. The method according to para 27, wherein the microcapsules have a solid content of 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more, prior to drying.

[0334] 29. The method according to para 27 or 28, wherein the method further comprises milling and / or sieving the powder.

[0335] 30. A composition comprising protease-loaded microcapsules obtained by or obtainable by the method according to any of paras 1-29.

[0336] 31. A solution comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules, wherein the solution has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1 , and wherein the solution has a pH of from 2 to 4.

[0337] 32. A powder comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules, and wherein the powder has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1.

[0338] 33. The powder according to para 32, wherein at least 60%, at least 70%, at least 80%, or at least 90% of the total protease in the powder is in a coacervated form.

[0339] 34. The powder according to para 32 or 33, wherein the one or more proteases are selected from trypsin, bromelain, papain, pancreatin, chymotrypsin, serratiopeptidase, ficain, nattokinase, or any combination thereof, preferably wherein the one or more proteases comprise or consist of trypsin and / or bromelain.

[0340] 35. The powder according to any of paras 32-34, wherein the one or more proteases consist of trypsin.

[0341] 36. The powder according to any of paras 32-34, wherein the one or more proteases consist of bromelain.

[0342] 37. The powder according to any of paras 32-36, wherein the one or more anionic polysaccharides are selected from alginate, pectin, carrageenan, gum arabic, xanthan gum, carboxymethyl cellulose, gellan gum, or any combination thereof.

[0343] 38. The powder according to any of paras 32-37, wherein the one or more anionic polysaccharides consist of alginate.

[0344] 39. The powder according to any of paras 32-38, wherein the powder has a protease:anionic polysaccharide weight ratio of at least 5:2, preferably wherein the powder has a protease:anionic polysaccharide weight ratio of at least 3:1 .

[0345] 40. The powder according to any of paras 32-39, wherein the powder has a protease:anionic polysaccharide weight ratio of 9:2 or less, preferably wherein the powder has a protease:anionic polysaccharide weight ratio of 4:1 or less.

[0346] 41. The powder according to any of paras 32-40, wherein the powder has a protease:anionic polysaccharide weight ratio of from 5:2 to 9:2, preferably wherein the powder has a protease:anionic polysaccharide weight ratio of from 3:1 to 4:1. 42. The powder according to any of paras 32-41 , wherein the microcapsules further comprise one or more antioxidants.

[0347] 43. The powder according to para 42, wherein the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, erythorbic acid, sodium erythorbate, or any combination thereof, preferably wherein the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, or any combination thereof.

[0348] 44. The powder according to any of paras 32-43, wherein the powder is a freeze-dried powder.

[0349] 45. The powder according to any of paras 32-44, wherein the powder has a maximum particle size of 800 microns or less, 500 microns or less, or 300 microns or less.

[0350] 46. The powder according to any of paras 32-45, wherein the powder is shelf-stable, optionally wherein 80% or more of the protease activity is retained following storage of the powder in paper packaging for 3 months at 22-40°C (e.g. 40°C) and 15%-85% (e.g. 75%) relative humidity.

[0351] 47. The powder according to any of paras 32-46, wherein the protease is not released under gastric conditions, optionally wherein 90% or more of the protease activity is retained following incubation of the powder for 2 hours at 37°C and pH 1.2-3.0.

[0352] 48. The powder according to any of paras 32-47, wherein the protease is released under small intestinal conditions, optionally wherein 90% or more of the protease activity is released following incubation of the powder for 2 hours at 37°C and pH 6.8.

[0353] 49. The powder according to any of paras 32-48, wherein the powder is obtained by or obtainable by the method according to para 28 or 29.

[0354] 50. A nutritional composition, supplement, or pharmaceutical composition comprising the powder according to any of paras 32-49.

[0355] 51. The nutritional composition, supplement, or pharmaceutical composition according to para 50, wherein the nutritional composition, supplement, or pharmaceutical composition is provided in a powder form for reconstitution with liquid.

[0356] 52. The nutritional composition, supplement, or pharmaceutical composition according to para 50 or 51 , wherein the nutritional composition, supplement, or pharmaceutical composition further comprises one or more flavonoids. 53. The nutritional composition, supplement, or pharmaceutical composition according to para 52, wherein the one or more flavonoids are selected from rutin, quercetin, kaempferol, myricetin, fisetin, catechin, epicatechin, hydroxytyrosol, oleuropein, hesperidin, or any combination thereof.

[0357] 54. The nutritional composition, supplement, or pharmaceutical composition according to para 52 or 53, wherein the one or more flavonoids comprise or consist of rutin.

[0358] 55. The nutritional composition, supplement, or pharmaceutical composition according to any of paras 50-54, wherein the nutritional composition, supplement, or pharmaceutical composition further comprises one or more stabilisers, flavourings, sweeteners, and / or colourants.

[0359] 56. A powder according to any of paras 32-49 or a nutritional composition, supplement, or pharmaceutical composition according to any of paras 50-55, for use as a medicament.

[0360] 57. A powder according to any of paras 32-49 or a nutritional composition, supplement, or pharmaceutical composition according to any of paras 50-55, for use in treating and / or preventing joint pain, an inflammatory disease, arthrosis, injuries, back pain, or sciatica, or for use in accelerating healing.

[0361] 58. A powder according to any of paras 32-49 or a nutritional composition, supplement, or pharmaceutical composition according to any of paras 50-55, for use in treating and / or preventing joint pain, such as joint pain in the shoulder, joint pain in the elbow, joint pain in the fingers, joint pain in the knee, menopausal joint pain, or rheumatic pain.

[0362] 59. The powder, nutritional composition, supplement, or pharmaceutical composition for use according to any of paras 56-58, wherein the one or more proteases are administered in a total daily dose of from 100 mg to 2000 mg, from 200 mg to 1500 mg, from 300 mg to 1200 mg, from 400 mg to 1000 mg, from 500 mg to 900 mg, or from 600 mg to 800 mg.

[0363] 60. Use of a powder according to any of paras 32-49 for preparing a protein hydrolysate.

[0364] 61. A method for preparing a protein hydrolysate, the method comprising hydrolysing a proteinaceous material with the powder according to any of paras 32-49.

[0365] 62. The method according to para 61 , wherein the powder is mixed with the proteinaceous material in a solution having a pH of from 2 to 4, and the pH of the solution is subsequently raised to release the protease activity from the powder.

Claims

CLAIMS1. A method for manufacturing a composition comprising protease-loaded microcapsules, the method comprising:(a) providing a first solution comprising one or more proteases and a second solution comprising one or more anionic polysaccharides;(b) mixing the first solution and the second solution to provide a mixed solution, wherein the mixed solution has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1 ; and(c) adjusting the pH of the mixed solution to a pH of from 2 to 4 to form microcapsules comprising the one or more proteases and the one or more anionic polysaccharides.

2. The method according to claim 1 , wherein the one or more proteases are selected from trypsin, bromelain, papain, pancreatin, chymotrypsin, serratiopeptidase, ficain, nattokinase, or any combination thereof, preferably wherein the one or more proteases comprise or consist of trypsin and / or bromelain.

3. The method according to claim 1 or 2, wherein the one or more anionic polysaccharides are selected from alginate, pectin, carrageenan, gum arabic, xanthan gum, carboxymethyl cellulose, gellan gum, or any combination thereof, preferably wherein the one or more anionic polysaccharides consist of alginate.

4. The method according to any preceding claim, wherein the method further comprises mixing one or more antioxidants with the first solution and the second solution, and wherein the microcapsules further comprise the one or more antioxidants, optionally wherein the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, erythorbic acid, sodium erythorbate, glutathione, N-acetylcysteine (NAC), alpha-lipoic acid, or any combination thereof, preferably wherein the one or more antioxidants are selected from ascorbic acid, sodium ascorbate, calcium ascorbate, or any combination thereof, more preferably wherein the one or more antioxidants consist of ascorbic acid.

5. The method according to any preceding claim, wherein the method further comprises recovering the microcapsules from the mixed solution, preferably wherein the microcapsules are recovered from the mixed solution by filtration.

6. The method according to any preceding claim, wherein the method further comprises drying the microcapsules to provide a powder comprising microencapsulated protease, preferablywherein the microcapsules are dried by freeze-drying, optionally wherein the method further comprises milling and / or sieving the powder.

7. A composition comprising protease-loaded microcapsules obtained by or obtainable by the method according to any of claims 1-6.

8. A solution comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules, wherein the solution has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1 , and wherein the solution has a pH of from 2 to 4.

9. A powder comprising one or more proteases and one or more anionic polysaccharides, wherein the one or more proteases and the one or more anionic polysaccharides are in the form of microcapsules, wherein the powder has a protease:anionic polysaccharide weight ratio of from 2:1 to 5:1 , and wherein at least 60% of the total protease in the powder is in a coacervated form.

10. The powder according to claim 9, wherein the powder is a freeze-dried powder.

11. The powder according to claim 9 or 10, wherein the powder is shelf-stable, optionally wherein 80% or more of the protease activity is retained following storage of the powder in paper packaging for under accelerated conditions such as 40°C / 75% relative humidity or at least 12 months under more humidity-controlled conditions at room temperature.

12. The powder according to any of claims 9-11 , wherein (a) the protease is not released under gastric conditions, optionally wherein 90% or more of the protease activity is retained following incubation of the powder for 2 hours at 37°C and pH 1.2-3.0, and / or (b) the protease is released under small intestinal conditions, optionally wherein 90% or more of the protease activity is released following incubation of the powder for 2 hours at 37°C and pH 6.8.

13. A nutritional composition, supplement, or pharmaceutical composition comprising the powder according to any of claims 9-12, preferably wherein the nutritional composition, supplement, or pharmaceutical composition is provided in a powder form for reconstitution with liquid.

14. A powder according to any of claims 9-12 or a nutritional composition, supplement, or pharmaceutical composition according to claim 13, for use as a medicament, preferably for use in treating and / or preventing joint pain, an inflammatory disease, arthrosis, injuries, back pain, or sciatica, or for use in accelerating healing.

15. Use of a powder according to any of claims 9-12 for preparing a protein hydrolysate.

16. A method for preparing a protein hydrolysate, the method comprising hydrolysing a proteinaceous material with the powder according to any of claims 9-12.

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