CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Patent Application No. 62/971,089, filed 6 February 2020, the disclosure of which is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

[0002] Field of the Invention. The present invention is directed towards a proteolytic fungal enzyme food supplement composition comprising a unique combination of at least one acid protease fungal enzyme and at least one semi-alkaline protease fungal enzyme. More particularly, the invention relates to a method of aiding digestion of plant-based proteins using the protease enzyme blend food supplement composition, wherein total or near total digestion of the plant-based protein into amino acids is achieved in vitro using a set of digestive conditions similar to normal gastro-intestinal physiology.

[0003] The proteolytic enzyme food supplement composition of the present invention was designed for use as a tablet, capsule, powder or liquid food supplement, to be taken with plant- based protein-containing foods in order to convert ingested dietary proteins into free amino acids, which then can be used by the body for muscle protein synthesis during anabolic cycles.

SUMMARY OF THE INVENTION

[0004] This invention relates to an enzyme food supplement composition comprising at least one acid protease fungal enzyme and at least one semi-alkaline protease fungal enzyme. This invention further relates to an enzyme food supplement composition comprising at least one acid protease fungal enzyme obtained from Rhizopus niveus or Aspergillus niger var. macrosporus and at least one semi-alkaline protease fungal enzyme obtained from Aspergillus oryzae or any species of Rhizopus including Rhizopus niveus and Rhizopus oryzae.

[0005] This invention still further relates to an enzyme food supplement composition wherein the acid protease fungal enzyme is present in an amount of at least 50 SAP per gram of composition, and the semi-alkaline protease fungal enzyme is present in an amount of at least 25,000 HUT per gram of composition. [0006] In a further embodiment of the present invention, the enzyme food supplement composition further comprises a carrier material, such as a maltodextrin.

[0007] In a still further embodiment, the present invention relates to an enzyme food supplement composition consisting essentially of an acid protease fungal enzyme obtained from Rhizopus niveus or Aspergillus niger var. macrosporus and a semi-alkaline protease fungal enzyme obtained from Aspergillus oryzae or any species of Rhizopus including Rhizopus niveus and Rhizopus oryzae , wherein the acid protease fungal enzyme is present in an amount of at least 50 SAP and the semi-alkaline protease fungal enzyme is present in an amount of at least 25,000 HUT, the amounts being per gram of composition.

[0008] Finally, in a method of use embodiment, the present invention relates to a method of using an enzyme food supplement composition to convert, in the gastrointestinal system of a human being, ingested dietary protein into free amino acids and short chain peptides, wherein the improvement comprises using as the proteolytic fungal enzyme food supplement composition a unique combination of at least one acid protease fungal enzyme and at least one semi-alkaline protease fungal enzyme such that enzymatic activity occurs throughout the gastrointestinal pH spectrum associated with a human digestive system.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0009] Figure 1 is an SDS-PAGE sample at different time intervals of whey protein isolate that was incubated with Digestive Aid 2 and Digestive Aid 1 compared to an undigested lane without any digestive aid added.

[0010] Figure 2 is an SDS-PAGE sample at different time intervals of whey protein isolate without the protease blend compared to an undigested lane of the whey protein isolate without any digestive aid added.

[0011] Figure 3 is an SDS-PAGE sample at different time intervals of a different whey protein isolate that was incubated with Digestive Aid 2 and Digestive Aid 1 compared to an undigested lane without any digestive aid added. [0012] Figure 4 is an SDS-PAGE sample at different time intervals of that second different whey protein isolate without the protease blend compared to an undigested lane of the whey protein isolate without any digestive aid added.

[0013] Figure 5 is an SDS-PAGE sample at different time intervals of soy protein isolate that was incubated with Digestive Aid 2 and Digestive Aid 1 compared to an undigested lane without any digestive aid added.

[0014] Figure 6 is an SDS-PAGE sample at different time intervals of soy protein isolate without the protease blend compared to an undigested lane of the isolate without the digestive aid.

[0015] Figure 7 is an SDS-PAGE sample at different time intervals of pea protein isolate that was incubated with Digestive Aid 2 and Digestive Aid 1 compared to an undigested lane without the digestive aid.

[0016] Figure 8 is an SDS-PAGE sample at different time intervals of pea protein isolate compared to an undigested lane of the isolate without the digestive aid.

[0017] Figure 9 is an SDS-PAGE sample at different time intervals of 2 mg/mL hemp protein that was incubated with Digestive Aid 2 in artificial gastric fluid at pH 1.2 and 37 °C.

[0018] Figure 10 is an SDS-PAGE sample at different time intervals of 5 mg/mL hemp protein that was incubated with Digestive Aid 2 in artificial gastric fluid at pH 1.2 and 37 °C.

[0019] Figure 11 is an SDS-PAGE sample at different time intervals of 5 mg/mL hemp protein that was incubated with Digestive Aid 2 in artificial gastric fluid at pH 1.2 and 50 °C.

[0020] Figure 12 is an SDS-PAGE sample at different time intervals of 5 mg/mL hemp protein that was incubated with Digestive Aid 2 in artificial gastric fluid at pH 4.0 and 50 °C.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention is based on the surprising discovery that the proteolytic enzyme food supplement composition in accordance with this invention includes at least one acid protease fungal enzyme and at least one semi-alkaline protease fungal enzyme. Both of these protease fungal enzymes are essential ingredients of the food supplement compositions of the present invention. Moreover, the inventors have found that the use of this blend of proteases from fungal sources provides optimum results in the digestion of plant-based proteins.

[0022] The proteolytic food supplement composition is preferably a combination of an acid protease enzyme obtained from Rhizopus niveus or Aspergillus niger var. macrosporus and a neutral or semi-alkaline protease enzyme obtained from Aspergillus oryzae or any species of Rhizopus. In one embodiment, the acid protease fungal enzyme is present in an amount of at least 50 SAP and the semi-alkaline protease fungal enzyme is present in an amount of at least 25,000 HUT, the amounts being per gram of composition.

[0023] For the present application, the term "composition" refers to a product that treats, improves, promotes, increases, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or prevents a particular condition associated with a natural state, biological process or disease or disorder. For example, a composition improves the digestion of plant-based proteins, and the like in a subject. The term composition includes, but is not limited to, pharmaceutical (i.e., drug), over-the counter (OTC), cosmetic, food, food ingredient or dietary supplement compositions that include an effective amount of the protease enzyme blend. Exemplary compositions include powder, or as an emulsion, tablets, or granules. Compositions can also include beverages, for example, beverages infused with an effective amount of the protease blend. Non-limiting examples of food compositions containing an effective amount of protease blend include protein powders, protein bars, and protein beverages.

[0024] As used herein, the term "effective amount" or "therapeutically effective amount" of a pure compound, composition, and/or active agent or ingredient, or a combination thereof refers to an amount effective at dosages and for periods of time sufficient to achieve a desired result. For example, the "effective amount" or "therapeutically effective amount" refers to that amount of a pure compound, composition, extract, protease enzyme blend, component of the protease enzyme blend, and/or active agent or ingredient, or a combination thereof of this invention which, when administered to a subject ( e.g ., mammal, such as a human), is sufficient to effect treatment, such as improving the digestion of plant-based proteins, and the like in a subject. The amount of a composition, protease enzyme blend, component of the protease enzyme blend, and/or active agent or ingredient of this disclosure that constitutes an "effective amount" or "therapeutically effective treatment" will vary depending on the amount or concentration of the protease enzyme blend ingested, the manner of administration, the duration of treatment, or the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0025] The term "pharmaceutically acceptable" means those drugs, medicaments, extracts or inert ingredients, which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, and the like, commensurate with a reasonable benefit/risk ratio.

[0026] The terms "administer", "administered", "administers", and "administering" are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intra-arterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In preferred embodiments, oral routes of administering a composition are suitable.

[0027] As used herein, the term "subject" or "individual" includes mammals to which a composition may be administered. Non-limiting examples of mammals include humans, non human primates, canines, felines, equines, bovines, rodents (including transgenic and non- transgenic mice) or the like. In some embodiments, the subject is a non-human mammal, and in some embodiments, the subject is human.

[0028] Unless indicated otherwise, all proportions and percentages recited throughout this disclosure are by weight.

[0029] An acid protease fungal enzyme is defined as an enzyme, which is derived from a fungal source and is capable of breaking down proteins and their degradation products, polypeptides and peptides, by hydrolysis, and is active in a pH environment ranging from a pH 1.0 to a pH of 6.0, with the optimum pH being around 2.0 to 4.0.

[0030] The acid protease fungal enzymes that can be utilized include those obtained from Rhizopus niveus and Aspergillus niger var. macrosporus. These enzymes can be obtained by culturing the organism, then extracting and purifying the enzyme by known and conventional techniques. Optionally, the acid protease fungal enzymes can be purchased from, for example, Bio-Cat, Inc., Industrial Drive, Louisa, Va. 23093, or Amano International Enzyme Company, Inc., 250 East Zion Crossroads, Troy, Va. 22974.

[0031] The composition can include one or more acceptable carriers. The carrier can aid in enabling incorporation of the protease enzyme into a composition having a suitable form for administration to a subject. A wide number of acceptable carriers are known in the art, and the carrier can be any suitable carrier. The carrier is preferable suitable for administration to animals, including humans, and can be able to act as a carrier without substantially affecting the desired activity of the plant extract and/or any active ingredient. The carrier can be chosen based upon the desired administration route and dosage form of the composition.

[0032] Suitable dosage forms include liquid and solid forms. In one embodiment, the composition is in the form of a gel, a syrup, a slurry, or a suspension. In another embodiment, the composition is in a liquid dosage form such as a drink shot or a liquid concentrate. In a further embodiment, the composition is present in a solid dosage form, such as a tablet, a pill, a capsule, a dragee, or a powder. When in liquid or solid dosage form, the composition can be in a food delivery form suitable for incorporation into food for delivery. Examples of suitable carriers for use in solid forms (particularly tablet and capsule forms) include, but are not limited to, organic and inorganic inert carrier materials such as gelatin, starch, magnesium stearate, talc, gums, silicon dioxide, stearic acid, cellulose, maltodextrin, and the like. The carrier can be substantially inert.

[0033] As an example, silicified microcrystalline cellulose can be used as a carrier or binder. Silicified microcrystalline cellulose is a physical mixture of microcrystalline cellulose and colloidal silicon dioxide. One such suitable form of silicified microcrystalline cellulose is ProSolv SMCC ^® 90, available from Penwest Pharmaceutical Co., Patterson, N.J. Silicon dioxide, in addition to that provided by the silicified microcrystalline cellulose, may be added to the composition as a processing aid. For example, silicon dioxide can be included as a glidant to improve the flow of powder during compression in the manufacturing of solid dosage units, such as tablet. [0034] In another embodiment, the carrier is at least a functional carrier such as buckwheat or spelt. By the addition of functional carriers into the composition, additional benefits may be provided such as lower glycemic index compared to standard carriers such as those mentioned above. Further, functional carriers can be allergen free ( e.g ., buckwheat), and by adding them into the production process, the botanical extracts of the invention may benefit from the flavonoids of these functional carriers, such as rutin and quercetin. Further, the high fiber content of these functional carriers may also facilitate and regulate intestinal transit. Finally, the added mineral benefit of selenium found in spelt may aid in metabolism.

[0035] The protease enzyme blend digestive aid composition can include other inert ingredients, such as lubricants and/or glidants. Lubricants aid in the handling of tablets during manufacturing, such as during ejection from dies. Glidants improve powder flow during tablet compression. Stearic acid is an example of an acceptable lubricant/glidant.

[0036] The digestive aid composition can be made in solid dosage form, such as tablets and capsules. This form provides a product that can be easily transported by an individual to a place of eating, such as a restaurant, and taken prior to, during, or after consumption of a foodstuff.

The composition can be formulated into dosage units containing suitable amounts of the plant extract and/or active ingredient that permit an individual to determine an appropriate number of units to take based upon appropriate parameters, such as body weight, foodstuff size, or carbohydrate (e.g., sugar) content.

[0037] In one embodiment, the protease enzyme blend is present in the digestive aid composition in a therapeutically effective amount, such as an amount of about 1.0 mg/mL or greater, preferably from about 1.0 mg/mL to about 20.0 mg/mL, more preferably from about 1.0 mg/mL to about 15.0 mg/mL, even more preferably from about 1.0 mg/mL to about 10 mg/mL. The composition can be administered, for example, in a dosage of from about 1.0 mg/mL to about 20.0 mg/mL per day of the protease enzyme blend, based on amount of plant-based protein ingested. Preferably, the protease blend digestive aid is present in an amount of about 1.0 wt% to about 10.0 wt% based on total weight percent of the plant-based protein. The composition can be administered as a single dose, or in multiple doses. In one example, the compound is administered in up to three doses per day. For example, the compound may be administered prior to a meal, during a meal, or after a meal. In one embodiment, the composition is a protease enzyme blend supplement in a therapeutically effective amount.

[0038] The dosage can be chosen to provide a level of digestive effect in a single unit that may be effective for some individuals and/or certain plant-based proteins, while also allowing for relatively simple dosage increases to provide other levels of digestive effects that can be effective for other individuals and/or other foodstuffs.

[0039] The digestive aid composition can be in a form adapted for oral ingestion. This form can be configured as a single dosage form intended to provide a specified dose of the digestive aid. For example, the single dosage form can be a powder, a pill, a tablet, a capsule, or a drink shot. The single dosage form can include, for example, from about 1.0 mg/mL to about 20.0 mg/mL of the protease enzyme blend. In another embodiment, the digestive aid composition can be blended with a plant-based protein powder prior to ingestion, wherein the digestive aid composition in present in the protein powder in an amount of about 1.0 wt% to about 10.0 wt%.

EXAMPLES

Examples - Materials and Protein Separation

[0040] Two different concentrations of a digestive aid composition were assessed against various plant-based proteins to determine the efficacy of the digestive aid composition in breaking down the plant-based protein under artificial conditions that approximate those in the gastrointestinal tract. The first digestive aid composition comprised an acid protease fungal enzyme obtained from Rhizopus niveus or Aspergillus niger var. macrosporus and a semi- alkaline protease fungal enzyme obtained from Aspergillus oryzae or any species of Rhizopus including Rhizopus niveus and Rhizopus oryzae , wherein the acid protease fungal enzyme is present in an amount of at least 50 SAP and the semi-alkaline protease fungal enzyme is present in an amount of at least 25,000 HUT, the amounts being per gram of composition, and a carrier. In this digestive aid composition, the protease blend comprised about 15 wt% with the remainder carrier (“Digestive Aid 1”). In the second digestive aid composition, the protease blend comprised about 75 wt% with the remainder carrier (“Digestive Aid 2”). Total weight of each digestive aid was equivalent. Accordingly, the Digestive Aid 2 contained about five times the amount of protease blend compared to Digestive Aid 1. Digestive Aid 1 and Digestive Aid 2 are commercially available from Innophos, LLC, Cranbury, New Jersey, as AMINOGEN® protease blend and AMINOGEN® Advanced protease blend, respectively.

[0041] The protein samples utilized were whey protein isolate (Hilmar™ 9010 instantized whey protein isolate, Hilmar Ingredients, Hilmar, California), a second whey protein isolate (Provon® whey protein isolate, Glanbia Nutritional, Carlsbad, California), soy protein isolate (Soy Protein Isolate ISP660, Pingdingshantianjing Plant Albumen Co., Ltd., Henan Province, China), pea protein blend (Proteins & Greens, Vega, Burnaby, Canada), and hemp protein (Hemp Pro 70, commercially available from Manitoba Harvest, Manitoba, Canada). The digestive aids were dissolved in artificial gastric fluid, pH 1.2 or pH 4 (adjusted using NaOH), as were the protein samples (specified in the Results section below). Digestion experiments were incubated at 37°C or 50°C, treated with EDTA-NaOH to neutralize and frozen at -20°C. Once sample collection was completed for each time course, all samples were thawed and further processed simultaneously.

[0042] SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was utilized to separate, identify, and isolate protein molecules. Proteins were dissolved in buffer with a reducing agent (b-mercaptoethanol), denaturing agent (SDS), and loading dye (bromophenol blue) and boiled to denature. The protein samples were then loaded into the stacking gel of a polyacrylamide SDS-PAGE gel and subjected to constant voltage to cause the proteins to migrate toward the bottom of the gel. Larger proteins stay near the top and smaller proteins migrate farther, thus separating them based on size. Very small peptides were generally not visible unless they are in exceedingly high amounts. Once the gel was run to the desired extent, it was stained in Coomassie Blue and thoroughly de-stained to visualize the proteins.

[0043] In particular, 37.5 pg of protein digestion sample was mixed 1:1 with SDS Sample Buffer (0.1 M Tris, pH 6.8, 20% glycerol, 4% SDS, 0.05% bromophenol blue, 10% b-mercaptoethanol), boiled for 5 minutes, and loaded into the stacking gel of a 10-20% gradient polyacrylamide 1- mm thick SDS-PAGE gel. The gel was run at 85 V for 120 minutes before being stained with 0.25% Coomassie G-250 in 45% MeOH, 10% glacial acetic acid overnight at 4°C. The gel was then de-stained in 45% MeOH, 10% glacial acetic acid, replacing de- stain solution every 90 minutes for 4.5 hours, nutating at room temperature, before the gel image was captured on a fluorescent light box.

Results -

[0044] Example 1 - Digestion of Whey Protein Isolate with Protease Blend

[0045] 1 mg/mL whey protein isolate (Hilmar™ 9010 instantized whey protein isolate, Hi 1 mar Ingredients, Hilmar, California) was digested under two different conditions - with Digestive Aid 2 at a final concentration of 1%, and with Digestive Aid 1 at a final concentration of 5%, both in artificial gastric fluid at 50 °C and pH of 4.0.

[0046] The digestion results are illustrated in Figure 1. Referring to Figure 1 it is seen that in both conditions there was significant digestion by 46.5 hours, as evidenced by the disappearance of the lactalbumin band at 13 kDa and the partial fading of the lactoglobulin band at 18 kDa, when compared to the undigested lanes. There was no observable or noteworthy difference between the Whey Protein digestion rate with Digestive Aid 2 at 1% or Digestive Aid 1 at 5%, except perhaps a lighter lactoglobulin (18 kDa) band at 52.5 hours in the 5% Digestive Aid 1 condition, but that may have been due to variation in gel loading or sample dilution. There was no difference between the intensities of the lactoglobulin bands between the two conditions at 72.5 hours of digestion.

[0047] Example 2 - Digestion of Whey Protein Isolate Under Ideal Conditions

[0048] From Example 1 it is seen that 1 mg/mF whey protein samples digested fully using 5% Digestive Aid 2 within 72 hours at 50°C in artificial gastric fluid, pH 4. We digested this same whey protein sample of Example 1 under the conditions described above while collecting samples at regular intervals to observe protein digestion. As shown in Figure 2, compared to the undigested lane, there was significant digestion of the whey protein after 5 hours, as evidenced by the disappearance of the lactalbumin band at 13 kDa. Even the lactoglobulin band at 18 kDa was completely gone by 52 hours, indicating complete digestion.

[0049] Example 3 - Digestion of Whey Protein Isolate with Protease Blend [0050] 1 mg/mL whey protein isolate (Provon® whey protein isolate, Glanbia Nutritional, Carlsbad, California) was digested under the same two conditions as the previous whey sample - with Digestive Aid 2 at a final concentration of 1 % and with the Digestive Aid 1 at a final concentration of 5%.

[0051] Compared to the undigested lane, in both conditions there was significant digestion by 52.5 hours, as evidenced by the disappearance of the lactalbumin band at 13 kDa at 5.5 hours and the partial fading of the lactoglobulin band at 18 kDa shown in Figure 4. There was no difference between the Whey Protein Isolate digestion rate with 5X Digestive Aid 1 at 1% or IX Digestive Aid 1 at 5%.

[0052] Example 4 - Digestion of Whey Protein Isolate Under Ideal Conditions

[0053] Using the same digestion conditions as in Example 3, the whey protein isolate of Example 3 was digested while collecting samples at regular intervals to observe protein digestion. Similar to the whey protein isolate of Example 1, there was significant digestion of the whey protein after 5 hours as shown in Figure 4, as evidenced by the disappearance of the lactalbumin band at 13 kDa and the fading of the lactoglobulin band at 18 kDa compared to the undigested lane. The lactoglobulin band was completely gone by 52 hours, indicating complete digestion.

[0054] From the above Examples 1-4 is it seen that the rates of digestion for the two whey protein isolates were approximately the same, as observed by the disappearance of protein bands over time. The lactalbumin (13 kDa) band had disappeared by 5 to 5.5 hours in both conditions for both protein isolates, and the lactoglobulin bands (18 kDa) in the two conditions remained near identical over the time course, with the possible exception of the 52.5-hour time point in the whey protein isolate of Example 1. At 72.5 hours, though, the bands for that protein look near identical, so this may have been due to variation in loading or dilution. The two digestion time courses looked near identical, with neither condition being more efficient than the other for both protein isolates

[0055] 1 mg/mL of each whey protein isolate was digested with Digestive Aid 2 at a final concentration of 5% to observe complete digestion of the protein sample. From Examples 2 and 4 it is seen that by 52 hours, both the lactoglobulin and lactalbumin bands had been completely digested.

[0056] Example 5 - Digestion of Soy Protein Isolate with Protease Blend

[0057] 50 mg/mL of soy protein isolate (Soy Protein Isolate ISP660. Pingdingshantianjing Plant Albumen Co., Ltd., Henan Province, China) was digested under two different conditions - with Digestive Aid 2 at a final concentration of 1% (Figure 5, left), and with Digestive Aid 1 at a final concentration of 5% (Figure 5, right). Referring to Figure 5, in both conditions there was significant digestion by five hours compared to the undigested lane, as evidenced by the disappearance of the high molecular weight bands. By 23 hours of digestion, the band at 15 kDa had digested further. The two prominent bands at 19 kDa and 11 kDa were also digesting over the time course, but because there was a high amount of those proteins in the protein sample, it was hard to observe complete digestion. There was no difference in soy protein digestion rate with Digestive Aid 2 at 1% or Digestive Aid 1 at 5%.

[0058] Example 6 - Digestion of Soy Protein Isolate Under Ideal Conditions

[0059] From Example 5, it is seen that 50 mg/mL soy protein did not fully digest after 6 hours with Digestive Aid 2 at 5% final concentration. In this Example, the soy protein concentration was reduced to 10 mg/mL and the incubation time of the digestion reaction was increased to 24 hours. As shown in Figure 6, there was significant digestion of the soy protein after one hour compared to the undigested lane, as evidenced by the disappearance of high molecular weight bands, and the fading of bands at 19 kDa and 11 kDa. The band at 15 kDa disappeared by 3 hours of digestion, and the two most prominent proteins were almost fully digested by 24.5 hours.

[0060] Example 7 - Digestion of Pea Protein Blend with Protease Blend

[0061] 50 mg/mL of a commercially available pea protein blend (Proteins & Greens, Vega, Burnaby, Canada) was digested under two different conditions - with Digestive Aid 2 at a final concentration of 1% and with Digestive Aid 1 at a final concentration of 5%. In both conditions there was significant digestion by one hour compared to the undigested lane, as evidenced by the disappearance of the high molecular weight bands. By 6 hours of digestion, most low molecular weight bands had been digested. By 24 hours, there was near-complete digestion of the pea protein blend. There was no difference in pea protein digestion rate between Digestive Aid 2 at 1% or Digestive Aid 1 at 5%.

[0062] Example 8 - Digestion of Pea Protein Blend Under Ideal Conditions

[0063] From Example 7 it is seen that 50 mg/mL pea protein samples digested using 5% Digestive Aid 2 within 24 hours when digested at 37°C in artificial gastric fluid, pH 4. We digested this same pea protein sample under those conditions, while collecting samples at regular intervals to observe protein digestion. As shown in Figure 8, compared to the undigested lane, there was significant digestion of the pea protein after one hour, as evidenced by the disappearance of almost all high molecular weight bands. Even the low molecular weight bands (less than 15 kDa) were gone by eight hours, indicating complete protein digestion.

[0064] From Examples 7 and 8 it is seen that the rates of digestion in digesting Pea Protein Blend with Digestive Aid 2 at a final concentration of 1%, or Digestive Aid 1 at a final concentration of 5% were approximately the same, as observed by the disappearance of protein bands over time. The 15, 13, and 8 kDa bands were still prominent after one hour of digestion but faded over the six-hour time course and were completely gone at 24 hours of digestion. The two digestion time courses looked near identical, with neither condition being more efficient than the other.

[0065] Example 9 - Hemp Protein Digestion Condition Optimization

[0066] Hemp protein (Hemp Pro 70, commercially available from Manitoba Harvest, Manitoba, Canada) was digested in artificial gastric fluid, pH 1.2, at 2 mg/mL and digesting with 5% Aminogen 5X at 37°C (Figure 31). The hemp protein was partially soluble in the artificial gastric fluid at pH 1.2. The concentration of the protein was increased to 5 mg/mL revealing two main protein bands at 35 kDa and 21 kDa (Figure 2), with some smaller peptides visible below the 10 kDa ladder band. Under these conditions, there was partial digestion at 6 hours, with complete digestion at 24 hours. To determine whether conditions from the other proteins would be more effective, both the pH of the artificial gastric fluid was increased to pH 4 and the digestion temperature increased to 50°C (Figure 3). Figure 3 illustrates that the hemp protein was not soluble under these conditions, so we returned to. To visualize protein digestion, we decreased the time points to 1-7 hours and a 24-hour timepoint (Figure 4) using artificial gastric fluid at pH 1.2 and 37°C. Partial digestion was observed after 2 hours, with complete digestion at 24 hours.

[0067] Optimal conditions for digestion with Digestive Aid 2 were used to determine whether complete or near-complete digestion of proteins from various sources was obtainable. For whey proteins, those conditions were - 1 mg/mL protein, artificial gastric fluid, pH 4, 50°C, and 72- hour digestion time course. For the soy protein, the optimal conditions were - 10 mg/mL protein, artificial gastric fluid, pH 4, 37°C, and 72-hour digestion time course. For the pea protein blend, the optimal conditions were - 50 mg/mL, artificial gastric fluid, pH 4, 37°C, and a 24-hour time course. For the hemp protein sample, after testing two different buffers with artificial gastric fluid at two different pH values, two temperatures, three time courses, and two different protein concentrations, it was determined that the optimum condition for hemp protein digestion by the protease blend was 5 mg/mL hemp protein digested with 5% Digestive Aid 2 in artificial gastric fluid (60 mM HC1, 34 mM NaCl, pH 1.2) at 37°C for up to 24 hours. From the above it is seen that the digestive aid of the present invention is able to digest all plant-based protein samples under the conditions described, indicating that there is no specificity for plant- based proteins digested by the digestive aid of the present invention, and it is likely that plant- based proteins from any source can be digested fully.

[0068] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention as embodied in the attached claims.