EP3793528A1 - Cannabinoid-rich gelated protein microparticle - Google Patents

Abstract

A cannabinoid-rich gelated microparticle comprises microemulsified hemp oil dispersed throughout a polymerised protein matrix, in which the microparticle is gastro-resistant and ileal-sensitive, and in which the microemulsified hemp oil is stable to oxidation. The surfactant in the microemulsified hemp oil comprises a polysorbate-type non-ionic surfactant. The protein may be selected from dairy protein or vegetable protein.

Description

TITLE

Cannabinoid-rich gelated protein microparticle

Field of the Invention

The present invention relates to cannabinoid-rich gelated protein microparticles containing hemp oil, and compositions comprising the microparticles. Also contemplated are uses of the microparticles.

Background to the Invention

Hemp oil is recognised for its medicinal and nutritional qualities, partly due to the high amounts of cannabinoids, including cannabidiol (CBD), which have been indicated for numerous conditions including pain associated with multiple sclerosis, inflammatory conditions, and epilepsy. The development of an oral formulation of Hemp Oil /

cannabinoids for mammals is hindered by Hemp Oil’s low oral bioavailability. This means that, when given orally, very little CBD reaches the bloodstream. The reason for this is that Hemp Oil and CBD are highly susceptible to oxidation and enzymatic degradation in the gastrointestinal tract and has very low permeability across the gut wall. A further problem associated with hemp oil products is the small and taste of the oil, and the difficulty providing hemp oil in a form in which it can be easily incorporated into aqueous products.

In her paper titled:“Pea Protein - Volatile Compound Interactions: Effects of Binding, Heat and Extraction on Protein Functionality” MELISSA TIESSEN-DYCK sums up a variety of prior art and published papers relating to pea protein. The paper is a Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In Partial Fulfilment of the Requirements of the Degree of MASTER OF SCIENCE, Department of Food Science, University of Manitoba, Winnipeg.

W002/064109 describes process for extraction of pharmaceutically active cannabinoids from plant material followed by cold filtration, decarboxylation and purification.

W002/092217 described microcapsules containing a mixture of at least one solubilised vegetable protein and a polyelectrolyte with an opposite charge to the protein, that are subjected to coacervation in an aqueous medium. WO2012/038061 describes polymeric nanocapsules containing microemulsions of water in oil and at least one hydrophilic active ingredient dissolved in the aqueous phase.

EP0856355 describes enzymatic crosslinking of protein-encapsulated oil particles by complex coacervation.

US5271961 describes protein microspheres formed by phase separation in a non-solvent followed by solvent removal.

It is an object of the invention to overcome at least one of the above-referenced problems.

Summary of the Invention

The present invention addresses the need for a hemp-based product, including versions which contains high-amounts of cannabinoids, is water dispersible, is taste and small masked, and that provides cannabinoids in highly bioavailable form. These objectives are met by providing phyto-cannabinoid rich hemp oil in a microparticulate form comprising gelated microparticles having a continuous protein matrix and micro-emulsified hemp oil dispersed throughout the matrix. The gelated microparticles are generally created by forming an aqueous micro-emulsion comprising hemp oil, surfactant and protein by high- shear homogenisation, and extruding the micro-emulsion into microdroplets which are cured in a polymerisation bath. The resultant microparticles contain high amounts of cannabinoid (for example more than 40 mg cannabinoid per gram of microparticles), are water-dispersible, and are capable of passing through the human stomach intact (gastro- resistant) and breaking down and releasing the cannabinoid-rich hemp oil in the ileum (ileal-sensitive). In addition, as the gelated protein matrix is substantially impermeable to oxygen, microparticles provide hemp oil in a stabilised form that is resistant to oxidation for up to 18 months,

According to a first aspect of the invention, there is provided a gelated microparticle comprising micro-emulsified hemp oil dispersed throughout a polymerised protein matrix, in which the microparticle is typically gastro-resistant, ileal-sensitive and bioavailable in a mammal. In another aspect, there is provided a gelated microparticle comprising hemp powder dispersed throughout a polymerised protein matrix, in which the microparticle is typically gastro-resistant, ileal-sensitive and bioavailable in a mammal. The hemp powder may be any part of the hemp plant. In one embodiment, the hemp powder is micronized hemp plant material.

In one embodiment, the micro-emulsified hemp oil is stable to oxidation. In one

embodiment, the microparticle is cannabinoid-rich.

In one embodiment, the micronized hemp oil comprises a surfactant. In one embodiment, the surfactant comprises a polysorbate-type non-ionic surfactant.

In one embodiment, the polysorbate-type non-ionic surfactant is TWEEN (RTM)-20.

In one embodiment, the protein is selected from dairy protein and / or vegetable protein.

In one embodiment, the protein is selected from milk protein isolate and / or pea protein.

In one embodiment, the gelated microparticle comprises 10 to 50 % protein, 2 to 6 % surfactant, and 40 to 90% hemp oil (w/w).

In one embodiment, the gelated microparticle comprises 20 to 40 % protein, 2 to 6 % surfactant, and 50 to 90% hemp oil (w/w).

In one embodiment, the gelated microparticle comprises 35 to 40 % protein, 2 to 6 % surfactant, and 55 to 65 % hemp oil (w /w).

In one embodiment, the gelated microparticle comprises the microparticle is dried. In one embodiment, the gelated microparticle is dried to a water activity (Aw) of less than 0.40, 0.30 or preferably less than 0.20. The gelated microparticles may be dried by, for example, spray drying.

In one embodiment, the gelated microparticle comprises more than 8% cannabidiol (CBD) (w/w). In one embodiment, the gelated microparticle comprises more than 12% cannabidiol (CBD) (w/w). In one embodiment, the gelated microparticle comprises more than 15% cannabidiol (CBD) (w/w). In one embodiment, the gelated microparticle comprises more than 20% cannabidiol (CBD) (w/w). In one embodiment, the gelated microparticle comprises more than 30% cannabidiol (CBD) (w/w). In one embodiment, the gelated microparticle comprises more than 40% cannabidiol (CBD) (w/w). In one embodiment, the gelated microparticle comprises more than 50% cannabidiol (CBD) (w/w).

In one embodiment, the hemp oil comprises less than 0.20% tetrahydrocannabinol (THC) (w/w).

In one embodiment, the gelated microparticle comprises less than 0.10%

tetrahydrocannabinol (THC) (w/w).

In one embodiment, the hemp oil is provided in the form of hemp plant material (i.e. hemp seeds or plant matter). In one embodiment, the hemp plant material is powdered hemp plant material. The term“hemp powder” should be understood to mean any part of the hemp plant, typically the above-the-ground parts, in a powdered form.

According to a further aspect of the invention, there is provided a powder composition comprising a multiplicity of dried gelated microparticles according to the invention.

According to a further aspect of the invention, there is provided composition suitable for oral administration to a human comprising a multiplicity of microencapsulates according to the invention.

In one embodiment, the composition is selected from a food product, a beverage, a food ingredient, a nutritional supplement, an infant formula or oral dosage pharmaceutical.

In one embodiment, the gelated microparticles have an average dimension of about 10 to about 250 microns, about 50 to about 200 microns, about 50 to about 150 microns, about 90 to about 200 microns, about 80 to about 120 microns.

The invention also provides a gelated micropartice of the invention, or a composition of the invention, for use in a method selected from:

improving antioxidant status

improving cardiovascular health; treatment of cardiovascular disease;

reducing inflammation;

treatment of an inflammatory condition.

Also contemplated is a method of making a gelated microparticles of the invention. The method comprises the steps of:

homogenising hemp oil, protein and optionally a surfactant under high shear conditions to form an aqueous micro-emulsion

extruding the aqueous microemulsion through a device to form microdroplets; and curing the microdroplets in a polymerisation bath to provide gelated microparticles.

In one embodiment, the hemp oil is provided in the form of hemp powder, typically micronized hemp powder. In one embodiment, the hemp powder is mixed with a protein solution/suspension in the absence of added surfactant.

In one embodiment, the homogenisation step employs a high shear rotor-stator homogeniser. Examples include UltraTurrax (RTM) homogenisers.

In one embodiment, the homogenisation step comprises mixing surfactant and protein under high shear conditions to produce a pre-mixture in which the surfactant is dissolved, and then addition of the oil to the pre-mixture under high shear conditions.

In one embodiment, the polymerisation bath is an acidification bath.

In one embodiment, the acidification bath comprises an acetate buffer.

In one embodiment, the polymerisation bath is agitated to create a vortex during the curing step.

In one embodiment, the surfactant comprises a non-ionic surfactant, typically a

polysorbate-type non-ionic surfactant.

In one embodiment, the polysorbate-type non-ionic surfactant is TWEEN (RTM)-20.

In one embodiment, the protein is selected from milk protein or vegetable protein. In one embodiment, the aqueous microemulsion is de-gassed prior to the extrusion step.

In one embodiment, the gelated microparticles are dried by spray-drying.

In one embodiment, a hygroscopicity aid is added to the gelated microparticles at the drying stage. The hygroscopicity aid minimises moisture adsorption, provides excellent dispersibility, and enhances rheological behaviour to enable the final powder to be used in various product applications.

In one embodiment, the dried microparticles are sieved after drying.

In another aspect, there is provided a gelated microparticle formed according to a method of the invention.

In one aspect, the present invention provides a hemp derivative rich, -or cannabinoid rich powder that is water-dispersible, taste-masked; stomach resistant, ileal released, and highly bioavailable. It has reduced aroma and taste of the encapsulated material relative to un-encapsulated form. It has significantly improved bio-availability profile compared to the same in its un-encapsulated form. It is a dry stable powder with versatility for human supplement and food applications, free from primary and secondary oxidation, and a product that is protected from environmental conditions for manufacture and has an increased shelf life compared to non-encapsulated forms. The active ingredients are all derived from or extracted, from European industrial hemp varieties, using any known conventional industrial extraction method, such as SC-CC>2 or ethanol extraction. The level of processing behind the product to be encapsulated, can be fully tailored to suit the need of the client and the end use. It provides a simple and natural hemp derivative or hemp oil vehicle, that is fully plant-based using two macro-components and relevant processing aids. It has a significantly higher bio-availability degree, compared to regular cannabinoid rich extracts and hempseed oil-based extracts.

In one embodiment of the microparticle, composition or method of the invention, the surfactant comprises of a concentration between 1-20% from one or more of the following: a. Lecithin b. Vegetable Glycerin

c. Hemp wax

d. Beeswax

e. Candelilla wax

f. Coconut oil

g. Turkey-red oil

h. Acasia tree sap as gel, powder or liquid.

i. Ethanol

j. Any non-ionic surfactant

In one embodiment, of the cannabinoid-rich gelated microparticle of the invention, the core material, instead of hemp oil, comprises of micro emulsified powdered hemp, made from any of the, above the ground, parts of the hemp plant, dispersed throughout a polymerized protein matrix, in which the microparticle is gastro-resistant and small intestine-sensitive in which the microemulsified hemp powder is stable to oxidation.

In one embodiment, the composition of the invention comprises a cannabinoid-rich gelated microparticle according to the invention, which is carrying any quantifiable level of a food or feed ingredient, a nutritional supplement, a functional food an active pharmaceutical ingredient (API) or a human or animal drug,

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

Figure 1 (L-R): A Hemp oil (88%) dark green to brown colour oil with slight odour and dry resultant microparticulated generated using a highly concentrated oil material.

Figure 2 (L-R): A, B Microscope images of hemp oil gelated micro-particulates in wet (A) and dry (B) phase.

Figure 3: (L-R): Micro-emulsion comprising hemp oil, surfactant and protein at low and high loading concentrations. No significant visible difference for reproducible processing. Figure 4 (L-R) A, B: Hemp Oil micro-emulsion degassed prior to micro-encapsulation (A) and microscopic visualisation (B) of homogenous, stable micro-emulsion (magnification x200).

Figure 5 (L-R) A, B, C: Dry gelated micro-particulates with different concentrations and particle sizes. Images illustrate the free-flowing nature of the material to enable a range of product applications.

Figure 6 (T- B): A, B; Microscopy image of dry gelated hemp oil micro-particulates at 40x (A) and 100x (B) magnification.

Figure 7. Microscope image of hemp Oil micro-particulates after salivary digestion (microparticulate diameter approx. 180 urn) and no evidence of disturbance / digestion. Magnification x200.

Figure 8 (L-R): A, B, C. Microscope image of hemp Oil microparticulates after 1 hour (A), 2 hour (B) stomach incubation (37DegC; pH 1.8) and nno evidence of damage or release of oil. Images show the environmental digesta surrounding the microparticulates. Image C shows the free hemp oil after 2 stomach incubation. Magification x 40.

Figure 9: lmage_Hemp Oil Microparticulates after 2 hour gastric / stomach incubation. Microparticulates were not dissolved and remain intact during this step and demonstrate the robustbness of the Hemp Oil Microparticulates and resistance to stomach digestion.

Figure 10 (L-R: A, B): Free hemp oil remains floating on surface of gastric fluid (A) and flotation Activity (FA) is lost relatively fast during gastric incubation (B), which has a detrimental effect on Hemp oil functionality. It is clear that gastric protection is needed via microparticulates to maintain Hemp oil functionality.

Figure 11 : Hemp Oil Microparticulates 15 minutes after intestinal incubation (15 minutes, 37 DegC pH 7.2). Magification x 40.

Figure 12: Hemp Oil Micro-particulates 30 minutes after intestinal Incubation (30 minutes, 37 DegC pH 7.2). Magification x 40. Figure 13: Hemp Oil Microparticulates after 60 min Intestinal Incubation (60 minutes, 37 DegC pH 7.2). Magification x 40.

Figure 14: Hemp Oil Microparticulates after 90 min intestinal digestion. Microparticulates are visibly digested and oil is liberated.

Figure 15: (T - B) A, B): Hemp Oil CBD Content Calibration Histogram (A) and standard cureve (B) for quantification of dose response.

Figure 16: Quantification and Calibration of Hemp Oil CBD Content prior to In Vitro Digestion with relevant compensation for background / baseline values.

Figure 17: Quantification of Hemp Oil CBD Content after In Vitro Digestion demonstrating free CBD content after intestinal digestion. This demonstrates a highly efficient process.

Figure 18A: Ussing Chamber analysis from isolated rat colonic mucosae (n=5). Percent TEER as a function of time and cannabinoid release. Different treatments are presented with different line colours: Control Mannitol (Grey Line / Diamond emblem/top line); Free Hemp Oil / CBD 10 mg dose of each (Green Line / Triangle Emblem/second line); Micro- encapsulated Cannabinoid-Rich Hemp Oil / Cannabinoid-CBD at 10 mg each (Red Line / Square Emblem/third line); Micro-encapsulated Cannabinoid-Rich Hemp Oil / Cannabinoid- CBD at 5 mg dose each (Blue Line/ circle emblem/bottom line). Data presented in Figure 18A shows validates the efficiency of the testing method and demonstrates the effect of micro-encapsulation on Cannabinoid-Rich Hemp oil bioavailability and demonstrates the reproducibility of the data and analysis.

Figure 18B: Ussing Chamber analysis from isolated rat colonic mucosae (n=5) for 190 min incubation time. Percent TEER as a function of time (190 min) and cannabinoid release. Different treatments are presented with different line colours: Free Hemp Oil / CBD 10 mg dose of each (Black line/diamond/top line); Free Hemp Oil / CBD 10 mg dose of each in the presence of protein suspension (Green Line/triangle/second line); Micro-encapsulated Cannabinoid-Rich Hemp Oil / Cannabinoid-CBD at 10 mg each (Red Line/square/third line); Micro-encapsulated Cannabinoid-Rich Hemp Oil / Cannabinoid-CBD at 5 mg dose each (Blue Line/circle/bottom line). Figure 19: Figure 19A, B and C show the emulsion preparation prior to micro- encapsulation.

Figure 20 shows the micro-encapsulation (under magnification) particles generated using the invention presented. Images endorse the robust process and illustrate the reproducible particle size achieved using the process.

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and general preferences

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or

"comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps. As used herein, the term“disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.

As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a human) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term“therapy”.

Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a human) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term“prophylaxis”.

As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a human without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from human to human, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.

“Microparticle”: means a particle having an average dimension in the range of 20-250 microns, preferably 80-120 microns as determined using a method of laser diffractometery (Mastersizer (RTM) 2000, Stable Micro Systems, Surrey, UK). This method determines the diameter, mean size distribution and D (v, 0.9) (size at which the cumulative volume reaches 90% of the total volume), of micro-encapsulates with diameters in the range of 0.2-2000 pm. For microencapsulate size analysis, micro-encapsulate batches were re- suspended in Milli-Q (RTM) water and size distribution is calculated based on the light intensity distribution data of scattered light. Measurement of microencapsulate size is performed at 25°C and six runs are performed for each replicate batch (Doherty et al., 201 11 ) (Development and characterisation of whey protein micro-beads as potential matrices for probiotic protection, S.B. Doherty, V.L. Gee, R.P. Ross, C. Stanton, G.F.

Fitzgerald, A. Brodkorb, Food Hydrocolloids Volume 25, Issue 6, August 2011 , Pages 1604-1617). Preferably, the microencapsulate is substantially spherical as shown in the attached figures. The microparticle comprises microemulsified hemp derivative or hemp oil dispersed throughout a polymerised protein matrix. The hemp derivative may be powdered hemp plant matter.

“Microemulsified hemp oil” means droplets of hemp oil dispersed through the protein matrix of the microparticle. The droplets comprise a suitable surfactant, for example a

polysorbate-type non-ionic surfactant. The droplets typically have an average dimension as measured by electron microscopy of less than 20 or 10 microns, and ideally about 5 microns. The small droplet size is achieved by forming the microemulsion using high shear homogenisation.

“Hemp derivative” generally means a derivative product formed from any part of the hemp plant. In one embodiment, the hemp derivative is selected from hemp oil, powdered hemp plant matter, resinous hemp extract, cannabinoid fractions or hemp flour. In one embodiment, the hemp derivative may be any part selected from the hemp plant, for example special cuttings, flowers or flower parts, leaves or leaf parts, tops of plant whole plants or other parts or size selections, extracts or such part or isolated parts or fractions such as pure CBD, In one embodiment,“hemp fraction” or“hemp derivative” consists of powdered hemp leaves and or powdered hemp flowers.

“Hemp powder” should be understood to mean any“hemp derivative” or“hemp fraction” of the hemp plant, including seeds, typically the above-the-ground parts of the hemp plant, in a powder form. In one embodiment, the hemp powder is micronized hemp powder, for example micronized hemp flowers and leaves. In another the hemp powder is micronized hemp stems and or stalks)

“Cannabinoid rich” as applied to a microparticle means a microparticle containing at least 30 mg cannabidiol (CBD) per gram of dried microparticles having a water activity (Aw) of 0.20. In one embodiment, the microparticle contains at least 35 mg, 40 mg, 45 mg, or 50 mg CBD per gram of dried microparticles having a water activity (Aw) of 0.20.

“Gelated microparticle” means a microparticle that is initially formed/extruded as a liquid microdroplet which is immediately cured in a polymerisation bath to form the gelated microparticle.

“Suitable for delivery intact to the human lower intestine via an oral route” means that the microparticle when delivered orally is capable of surviving gastric transit and being delivered to the lower intestine substantially intact.

“Ileal-sensitive”: means that the microencapsulates are capable of releasing their contents in vivo in the ileum of a human.

In one embodiment, the microparticles of the invention comprise a monodispersed matrix. “Monodispersed matrix” means that the components of the microparticle are homogenously mixed in a single phase. This is distinct from microcapsules having a core-shell

morphology. In a monodispersed matrix, all of the components are exposed on the surface and available to interact with their environment to provide a functional benefit.

“Gastro-resistant”: means that the microencapsulates can survive intact for at least 60 minutes in the simulated stomach digestion model described in Minekus et al., 1999 and 2014 (A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation product, Minekus, M., Smeets-Peeters M, Bernalier A, Marol-Bonnin S, Havenaar R, Marteau P, Alric M, Fonty G, Huis in't Veld JH, Applied Microbiology Biotechnology. 1999 Dec;53 (1 ): 108-14) and (Minekus et al., 2014, A standardised static in vitro digestion method suitable for food - an international consensus, Minekus, A. ef al., Food Function, 2014, 5, 1 113). “Protein” means any protein susceptible to thermal or enzymatic denaturation, for example dairy protein or vegetable protein, or a mixture of dairy protein or vegetable protein.

Preferably, the protein is a globular protein.

“Denatured”: means partially or fully denatured. Preferably at least 90%, 95% or 99% of the protein is denatured. A method of determining the % of denatured protein is provided below.

Denatured protein was removed by acid precipitation using an acetic acid buffer pH 4.6 followed by centrifugation at 20,000g for 20 minutes. Ju and Kilara (1998) claimed that at pH 4.6 some native whey proteins formed aggregates. This did not occur in this presented invention as no protein precipitated when the protein was unheated, (TO), sample was adjusted to pH 4.6. (Ju, Z. Y., & Kilara, A. (1998). Gelation of Ph-Aggregated Whey Protein Isolate Solution Induced by Heat, Calcium Salt and Acidulant. Journal of

Agricultural and Food Chemistry, 46(5), 1830-1835).

The supernatant was diluted and the native protein concentration was determined using reverse phase HPLC using a Source and 5RPC column (Amersham Biosciences UK limited). The HPLC system consisted of a Waters 2695 separation module with a Waters 2487 dual wavelength absorbance detector. The data were acquired and processed using Waters empower software (Milford, MA, USA).

The protein denaturation rate is related to the reaction order by the following equation: dC / dt = kCn... where k is the reaction rate, n is the reaction order and C the concentration of native protein. This equation can be integrated to give

(Ct/C0)1-n = 1 +(n-1 )kt (for n>1 ),

Where Ct is the native b-Lg concentration at time t, CO is the initial b-Lg concentration. Further rearranging gives:

Ct/CO = [1 + (n-1 )kt] 1/1 -n

The natural log of this equation was taken:

In (Ct/CO) = [1/1 -n)] In [ 1 +(n-1 ) kt] (Equation 1 )

Logging decay data such as this equalises the data per unit time and reduces error when solving the equations. The reaction orders and rates were determined by fitting the experimental data to Equation 1. All experimental points were included in the curve fit; this is reasonable considering the rapid heating-up time of the protein solution. Introducing a lag-time to the data did not significantly alter the results obtained. The data were also fitted to the first order decay equation

In (Ct / CO) = -kt (Equation 2) to rule out the possibility of first order kinetics.

“Hydrolysed” means any protein that has been hydrolysed to at least partially break up the protein into smaller peptide or polypeptides. It can be fully or partially hydrolysed. The degree of hydrolysis (%DH) may be variable and can be determined by routine

experimentation. Typically, the protein is hydrolysed to a degree of from 10% to 99%, typically, from 15% to 95%, typically from 20% to 65%, suitably 45%. Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate, and may be determined using the OPA spectrophotometric assay, which involve the using N- acetyl-L-Cysteine (NAC) as the thiol reagent. One means to determine %DH is using a method of hydrolysis of proteins performed at high temperatures and for short times with reduced racemization in order to determine the enantiomers of D and L amino acids, Csapo, J. et al., Acta Univ, Sapientiae, Alimentaria, 2008; pg 31-48. Methods of hydrolysis are known to a person skilled in the art and include thermal and proteolytic hydrolysis.

“Dairy protein” means any protein source isolated from expressed from the mammary glands of a female mammal. Dairy proteins include Milk protein concentrate or isolates (MPC or MPI) - Milk protein concentrates are produced by ultrafiltration (UF) of milk - or whey protein concentrates or isolates or caseinates. The product in liquid form is generally referred to as UF milk while the dry form is known as MPC. This product contains unaltered forms of both casein and whey protein. The level of protein, lactose and mineral present vary depending on the degree of protein concentration.

“Pea protein” should be understood to mean protein obtained from pea, typically total pea protein. Preferably the pea protein is pea protein isolate (PPI), pea protein concentrate (PPC), or a combination of either. Typically, the liquid core comprises 6-8% pea protein, ideally 6.6-7.5% (w/v). Typically the solvent for the pea protein has a pH of greater than 10 or 10.5. Ideally, the pea protein is solubilised in an alkali solvent.

“Hemp oil” refers to an oil or extract fraction derived from any part of the hemp plant of the Cannabis Sativa L. family. It generally contains less than 0.2% tetrahydrocannabinol (THC) (w/w) and 2 - 96 % of cannabidiol (CBD). The hemp oil employed in the process and products of the present invention may be obtained from hemp seeds, or from hemp plant matter, or both. The process of the invention provides hemp oil in a microemulsion form in a gelated protein matrix, in which the cannabinoid content content of the oil is high, for example more than 40 or 50 mg/ g microparticles.

“Cannabidiol” or“CBD” refers to a specific cannabinoid present in hemp oil. It is described in Mechoulam et al (Journal of Clinical Pharmacology, (2002) 42 (1 1 Suppl)). Rustichelli et al 1998 describes how direct gas chromatography (GC) analysis can only determine the total cannabinoid content of plant tissue extracts. This is due to the acidic cannabinoid compounds being converted to neutral cannabinoids by high temperatures when injected into a GC system. High performance liquid chromatography (HPLC) can detect both the acidic and neutral forms of cannabinoids. This paper outlines a room temperature method of analysis with a mobile phase of methanol/ water in the ration of 80:20 (v/v). The flow rate was set to 1.0ml/L min ¹ and the injection volume was 20.0mI_. A mass spectrophotometer (MS) was also used as a method of detection. The mass range of m/z 45-700 was scanned once per second. The following parameters were set on the MS; the electron impact (El) mode was enabled, ionization energy 70eV; ion source temperature 250°C, filament current 200mA, conversion dynode power -15kV and electron multiplier voltage 1500V. To accurately and directly measure the presence of CBD (Cannabidiol), CBD-A (Cannabidiol - A), THC (Tetrahydrocannabinol), CBCh (Cannabichromene) and total cannabinoids by using HPLC-MS. A HPLC -UV with an ultra violet detector array set at 220nm with 0.04 absorbance full scale is commonly used to measure cannabinoid content. The Equipment needed included HPLC Agilent Infinity 1260 (Column: Zorbax (RTM) Eclypse C18, 5pm, 250x4.6mm (Agilent); solvent system: Methanol, H2O, acetic acid. Isocratic

Detector: Diode array -220nm. HPLC /DAD method can also be used (References: De Backer, B., Debrus, B., Lebrun, P., Thenunis, L., Dubois, N., Decock, L., Verstraete, A., Hubert, P. and Charlier, C. (2009) Innovative development and validation of an HPLC/DAD method for the qualitative and quantitative determination of major cannabinoids in cannabis plant material. Journal of Chromatography B, 877(2009) 4115-4124 / Rustichelli, C., Ferioli, V., Baraldi, M., Zanoli, P. and Gamberini, G. (1998) Analysis of Cannabinoids in Fiber Hemp Plant Varieties (Cannabis Sativa L.) by High-Performance Liquid Chromatography. Chromatographia Vol. 47, No. ¾).

“Microemulsion” means a hemp oil emulsion that is formed between hemp oil, an aqueous protein phase and a surfactant by homogenisation under high shear conditions, typically using an Ultraturrax homogeniser with a hemp oil droplet size of less than 20 microns and preferably less than 10 microns. “Non-ionic surfactant” refers to a surfactant having covalently bonded oxygen-containing hydrophilic groups, which are bonded to hydrophobic parent structures. Examples include Triton X-100, and fatty acid esters of glycerol or sorbitol.

“Polysorbate-type non-ionic surfactant” refers to a class of non-ionic surfactants derived from ethoxylated sorbitan esterified with fatty acids. Common brand names for

polysorbates include TWEEN (RTM), SCATTICS (RTM), ALKEST (RTM) and CANCAREL (RTM). In one embodiment, the Polysorbate-type non-ionic surfactant is a TWEEN (RTM), for example one of TWEEN (RTM) 20, TWEEN (RTM) 40, TWEEN (RTM) 60 or TWEEN (RTM) 80.

“Polymerisation bath” means a bath of liquid configured to polymerise the microdroplets of microemulsion. In one embodiment, the polymerisation is acidification bath comprising an acidic buffer, for example an acetate salt buffer. Typically, the acidification bath has a pH of less than 5, for example 3.5 to 4.7, 3.8 to 4.6, or 3.8 to 4.4. The acidification bath typically has a buffer concentration of 0.1 M to 0.8M, preferably 0.3M to 0.7M, and more preferably 0.4M to 0.6M. In one embodiment, the acidification bath comprises surfactant. In one embodiment, the acidification bath comprises 0.01 to 1.1% surfactant or co-surfactant or both. In one embodiment, the surfactant is a hydrophilic surfactant. In one embodiment, the surfactant is a TWEEN (RTM) (TWEEN (RTM) -20 or TWEEN (RTM)-80) surfactant.

“Particle Size” means the size of a spherical particle expressed as the diameter measurement. For non-spherical particles, the size can be represented as an apparent diameter.

“Size distribution” relates to the fact that droplets and particles that are produced in a spray dryer are never of one particular size. Any nozzle will produce both large and small droplets. The dryer must operate so that it is able to dry the largest droplet without scorching the smallest one. Size distributions can be represented by a cumulative distribution curve. Particle size distributions were measured with a Nicomp model 380 ZLS particle size system (Nicomp PSS, Santa Barbara, CA, USA). Measurements were taken at 23 °C using a 635 nm source and a scattering angle of 90°. Samples were prepared for measurement by dilution in deionized water. Calorimetry data were collected with a TA Instruments model Q20 differential scanning calorimeter (TA Instruments, New Castle, DE, USA). The instrument was calibrated with an Indium standard. Aluminium sample pans were used and samples were scanned at 5 °C/min over the temperature range of 25 °C to 90 °C.

“Water activity (Aw)” The water activity (Aw) of a food / ingredient is the ratio between the vapour pressure of the ingredient itself, when in a completely undisturbed balance with the surrounding air media, and the vapour pressure of distilled water under identical conditions. The most common method used to measure water activity is the Equilibrium Relative Humidity equation (ERH), which is expressed in percentage or as the water activity expressed as a decimal. A portion of the total water content present in food is strongly bound to specific sites and does not act as a solvent. These sites include the hydroxyl groups of polysaccharides, the carbonyl and amino groups of proteins, and others on which water can be held by hydrogen bonding, by ion-dipole bonds, or by other strong

interactions. This binding action is referred to as the sorption behavior of the food. The most successful method for studying the sorption properties of water in food products has been the preparation of "Sorption Isotherms," or curves relating the partial pressure of water in the food to its water content at constant temperature. The same practice is followed to study curves relating water activity under equilibrium conditions to water content. Food of known moisture content is allowed to come to equilibrium with a small headspace in a tight enclosure and partial pressure of water activity is measured manometrically, or relative humidity is measured using a hydrometer. Water activity is equal to equilibrium relative humidity divided by 100:

(Aw = ERH/100)

...where ERH is the equilibrium relative humidity (%).

Relative humidity sensors of great variety are available for this purpose, including electric hygrometers, dewpoint cells, psychrometers, and others.

“Water dispersible” as applied to the gelated microparticles of the invention means that microparticulates do not sediment in an aqueous solution and sedimentation measured no more than 1 % of the hydrated material content. Gelated microparticles will remained in a suspended state in an aqueous solution. “Stable to oxidation” as applied to the microemulsified hemp oil in the gelated microparticles means that the hemp oil is stable as per measurements for primary and secondary oxidation. This testing is conducted using Peroxide value (AOCS Cd 8b-90) and Anisidine value (AOCS Cd 18-90) testing. Acceptable ranges for primary (PoV) and secondary (AnV) oxidation would be 3-9 meq and 15-17 units, respectively. Values above these ranges would be consider oxidsed and unstable.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Materials

Table 1 Sample preparation 1 :

Formulation preparation

TWEEN (RTM) 20 was mixed with 1 1 % milk protein isolate until the TWEEN (RTM) 20 was completely dissolved (Table 1 ). Oil was added dropwise to homogenise the suspension. Micro-Encapsulation

The suspension was placed on the micro-encapsulator and set up. Some oil was visible on the surface of emulsion. Micro-encapsulation the settings were adjusted to improve bead chain.

Washing step

The encapsulation dish was removed from the stir plate and the 0.5M sodium acetate waste was poured off the beads. The beads were rinsed in water before and sieved.

Drying

The wet beads were further dried for 2- 4 hours, with the option of using a drying aid, depending on the origin of the oil extract/ distillate (Figure 2).

Sample preparation 2:

Formulation preparation

Polysorbate 80 was mixed with 15% PPI (pea protein isolate) until the dispersion was completely dissolved. Oil was added dropwise to homogenise the suspension.

Micro-Encapsulation

The suspension was micro-encapsulated and polymerised using sodium acetate. Micro- encapsulation the settings were adjusted to improve bead chain.

Washing step

The encapsulation dish was removed from the stir plate and sodium acetate waste was poured off the beads. The beads were rinsed in water before and sieved.

Drying

The wet beads were further dried for 2- 4 hours, with the option of using a drying aid, depending on the origin of the oil extract/ distillate.

CBD Encapsulation Efficiency

EE: (Encapsulation efficiency) is defined as the amount of CBD loaded into the

microparticle. The Encapsulation Efficiency (EE) is calculated as follows by determining the free CBD concentration, and the total amount of CBD (Initial CBD concentration, raw material).

EE% = 100 - [(Free CBD cone. / Initial CBD cone.) x 100]

Protocol

• For determining CBD concentration, the encapsulated samples were dissolved in MilliQ H20. Raw samples were managed in similar technique and dilutions were accounted to equilibrate the systems.

• Total content of CBD, was calculated using the double layer of the ANABIO matrix divided by methanol (Bio - informatic calculation as per GOED - ANABIO method)

• Free CBD concentration was calculated using 100 ppm aliquots of the samples were prepared, filtered (0.22 urn) on all samples.

• Total content of CBD, 100 mg / ml solutions were prepared in methanol, sonicated for 3 minutes, and filtered (0.22 urn), on all samples.

• To determine CBD in ANABIO micro-capsule form, the micro-capsule was removed /extracted (as per the ANABIO methodology provided) and the contents were dissolved in 10 ml. H20, diluted x100, filtered (0.22um) - to determine the free bioavailable (non-bound) CBD concentration.

• In order to determine the initial CBD content, the aqueous solution is diluted x2 in methanol and sonicated for 3 minutes. Aliquots (200 pi) were further diluted with methanol to x50 volume.

• The 100x volume methanol solution was sonicated for 3 minutes and filtered (0.22 urn).

Determination of CBD

Method:

Liquid-chromatographical separation, and photodiode-detection using a device of HPLC- PDA (Shimadzu Corp.). For HPLC separation we used Zorbax (RTM) Eclypse C18, 5 pm, 250 x 4.6 mm (Agilent) A: Acetic Acid (>99.5% for HPLC, Sigma-Aldrich) B: methanol, with eluent composition, with elution of isocratic and gradient.

Results

Microparticles Sample A (80% Oil Loading)

EE: 95.16% (± 0.352 %) Particle size approx. 36 microns (± 1.292 um)

Microparticles Sample B (60% Oil Loading)

EE: 99.076% (± 0.537 %)

Particle size approx. 63 microns (± 4.972 um)

Figure 18B shows the superior improvement in CBD permeation in the body as a result of invention presented.

Figure 18 demonstrates that the micro-encapsulated Cannabinoid-Rich Hemp Oil has a greater permeation capacity relative to free Hemp Oil. Digestion of 5 mg dose of micro- encapsulated Hemp Oil illustrated a greatest reduction in % TEER compared to micro- encapsulated Hemp Oil 10 mg doses of micro-encapsulated Hemp Oil. Hence, reduction in %TEER is dependent on the i) presence of micro-encapsulated Hemp Oil and ii) concentration of micro-encapsulated cannabinoids.

Figure 18 shows that free Hemp Oil have an expected permeability across the gut epithelial tissue; however, micro-encapsulated Hemp Oil provides a greater permeability and bioavailability of its cannabinoids measured by CBD via the use of micro-encapsulation technology and the use of a lower doses (5 mg) concentration of micro-encapsulated Cannabinoid-Rich Hemp Oil. It is important to note that the permeability after 120 min is approx similar between the two doses (5 mg vs. 10 mg CBD) of micro-encapsulated Cannabinoid-Rich Hemp Oil, which both show an above 80 % bioavailability, which is significantly higher than inhalation by smoking which only show approx. 31 % bioavailability respectively (Ohlsson A, et al. Biomed Environ Mass Spectrom. 1986 Feb;13(2):77-83.) Data shows that delivery of 5 mg micro-encapsulated Hemp cannabinoid powder provides a significantly better permeation and bioavailability relative to 10 mg dose post digestion. This enhance kinetic response is generated as a result of micro-encapsulation. Hence, this proves that protein is the ideal chaperon for cannabinoid-rich hemp oil; however, micro- encapsulation is necessary to provide a potential health effects.

Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1. A cannabinoid-rich gelated microparticle comprising microemulsified hemp oil dispersed throughout a polymerised protein matrix, in which the microparticle is gastro-resistant and ileal-sensitive, and in which the microemulsified hemp oil is stable to oxidation.

2. A cannabinoid-rich gelated microparticle as claimed in Claim 1 in which the surfactant in the microemulsified hemp oil comprises a polysorbate-type non-ionic surfactant.

3. A cannabinoid-rich gelated microparticle as claimed in Claim 1 or 2 comprising less than 12% cannabidiol (CBD).

4. A cannabinoid-rich gelated microparticle as claimed in any of Claims 1 to 3, in which the protein is selected from dairy protein or vegetable protein.

5. A cannabinoid-rich gelated microparticle as claimed in Claim 4, in which the protein is selected from milk protein isolate or pea protein isolate.

6. A cannabinoid-rich gelated microparticle as claimed in any preceding Claim comprising 10 to 50 % protein, 2 to 6 % surfactant, and 40 to 90% hemp oil (w/w).

7. A cannabinoid-rich gelated microparticle as claimed in any preceding Claim comprising 20 to 40 % protein, 2 to 6 % surfactant, and 50 to 90% hemp oil (w/w).

8. A cannabinoid-rich gelated microparticle as claimed in any preceding Claim comprising 10 to 30 % protein, 2 to 6 % surfactant, and 70-80 % hemp oil (w/w).

9. A cannabinoid-rich gelated microparticle as claimed in any preceding Claim in which the microparticle is dried.

10. A cannabinoid-rich gelated microparticle as claimed in any preceding Claim comprising more than 40 mg cannabinoid / g microparticles.

1 1. A cannabinoid-rich gelated microparticle as claimed in any preceding Claim comprising more than 50 mg cannabinoid / g microparticles.

12. A cannabinoid-rich gelated microparticle comprising hemp derivative dispersed throughout a polymerised protein matrix, in which the microparticle is gastro-resistant and ileal-sensitive.

13. A powder composition comprising a multiplicity of dried cannabinoid-rich gelated microparticles of any of Claims 1 to 12.

14. A composition suitable for oral administration to a human comprising a multiplicity of dried cannabinoid-rich gelated microparticles according to any of Claims 1 to 12.

15. A composition according to Claim 14 selected from a food product, a beverage, a food ingredient, a nutritional supplement, an infant formula, or oral dosage pharmaceutical.

16. A method of making cannabinoid-rich gelated microparticles of any of Claims 1 to 1 1 , the method comprising the steps of:

homogenising hemp oil, protein and a surfactant under high shear conditions to form an aqueous microemulsion

extruding the aqueous microemulsion through a device to form microdroplets; and curing the microdroplets in a polymerisation bath to provide gelated microparticles.

17. A method according to Claim 16, in which the homogenisation step employs a high shear rotor-stator homogeniser.

18. A method according to Claim 16 or 17, in which the homogenisation step comprises mixing surfactant and protein under high shear conditions to produce a pre-mixture in which the surfactant is dissolved, and then addition of the oil to the pre-mixture under high shear conditions.

19. A method according to any of Claims 16 to 18, in which the polymerisation bath is an acidification bath.

20. A method according to Claim 19, in which the acidification bath comprises an acetate buffer.

21. A method according to any of Claims 16 to 20, in which the polymerisation bath is agitated in a vortex during the curing step.

22. A method according to any of Claims 16 to 21 , in which the surfactant comprises a polysorbate-type non-ionic surfactant.

23. A method according to any of Claims 16 to 22, in which the cannabinoid-rich gelated microparticle comprises less than 12% cannabidiol (CBD).

24. A method according to any of Claims 16 to 23, in which the protein is selected from milk protein or vegetable protein.

25. A method according to any of Claims 16 to 24, in which the aqueous microemulsion is de-gassed prior to the extrusion step.

26. A method according to any of Claims 16 to 25, in which the gelated microparticles are spray- dried.

27. A method according to Claim 26, in which a hygroscopicity aid is added to the gelated microparticles for the drying.

28. A microparticle, composition of method according to any of Claims 1 to 21 , in which the surfactant comprises of a concentration between 1-20% from one or more of the following: a. Lecithin

b. Vegetable Glycerin

c. Hemp wax

d. Beeswax

e. Candelilla wax

f. Coconut oil

g. Turkey-red oil

h. Acasia tree sap as gell, powder or liquid.

i. Ethanol

j. Any non-ionic surfactant

29. A cannabinoid-rich gelated microparticle according to any preceding claim in which the core material, instead of hemp oil, comprises of micro emulsified powdered hemp, made from any of the, above the ground, parts of the hemp plant, dispersed throughout a polymerized protein matrix, in which the microparticle is gastro-resistant and small intestine-sensitive in which the microemulsified hemp powder is stable to oxidation.

30. Any composition suitable for oral administration to a human or an animal, suitable for ileal release and uptake in a human or an animal, comprising a cannabinoid-rich gelated microparticle according to claims 1-5, 9-14 and 28 or 29, which is carrying any quantifiable level of a food or feed ingredient, a nutritional supplement, a functional food an active pharmaceutical ingredient (API) or a human or animal drug,