Blood sugar stabilization method and composition

Abstract

Provided and described herein are methods and compositions for reducing blood glucose, inhibiting blood glucose rise, and / or lowering the glycemic index of foods.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 303,112, filed January 26, 2022, the entirety of which is incorporated herein by reference. [Background technology]

[0002] It is well known that hyperglycemia has a great deal of adverse health effects. Long-term complications from hyperglycemia can range from cardiovascular disease, neurological disorders, damage to the kidneys and blood vessels, to bone and joint damage. The most commonly used method to control blood glucose levels is by controlling dietary carbohydrate intake. Although this is a sound and proven strategy, it requires a lot of attention from the individual to understand and calculate the carbohydrate intake at each meal. Although drugs such as insulin are widely used, many problems can accompany the effective control of blood glucose levels with medication. Summary of the Invention

[0003] Despite the increasing understanding of the biology and pathogenesis of diseases associated with or resulting from hyperglycemia, patients who are unable to regulate their blood glucose levels regardless of any intervention still bear a significant burden. With regard to both pharmacological (e.g., insulin hormone) and non-pharmacological (e.g., dietary monitoring) treatments, both can be considered substantial interventions with intrinsic barriers to the effective treatment and management of hyperglycemia-related diseases. Such intrinsic barriers include, for example, adherence to treatment (e.g., due to the complexity of treatment), financial burden and access (e.g., out-of-pocket costs for both insured and uninsured individuals), and other patient-related, prescription-related, and prescriber-related factors. Such barriers and the resulting impact on the treatment of hyperglycemia-related diseases impose a significant health and economic burden on individuals and on the health care system.

[0004] Provided and described herein are compositions and methods useful for modulating (e.g., reducing, stabilizing, etc.) hyperglycemia. The methods and compositions provided are based in part on the surprising discovery that modulating hyperglycemia can be achieved by providing an individual with a legume protein (e.g., pea protein) and an S53 family protease. In some embodiments, the S53 family protease is prokumamolisin. In some embodiments, the S53 protease (e.g., prokumamolisin) comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the S53 protease (e.g., prokumamolisin) comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the S53 protease (e.g., prokumamolisin) comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465, and A466 of SEQ ID NO:1.

[0005] In some embodiments, the S53 protease is active at a pH less than about pH 5 (e.g., as measured by protein digestion). In some embodiments, the S53 protease is active at a pH less than about pH 4.5. In some embodiments, the S53 protease is active at a pH less than about pH 5. In some embodiments, the S53 protease is active at a pH less than about pH 4. In some embodiments, the S53 protease is active at a pH less than about pH 3.5. In some embodiments, the S53 protease is active at a pH less than about pH 3.

[0006] In some embodiments, the S53 protease is active in a pH range of about pH 2 to pH 5. In some embodiments, the S53 protease is active in a pH range of about pH 2.5 to pH 4.5. In some embodiments, the S53 protease is at least 50% active (e.g., relative to its maximum activity) in a pH range of about pH 2.5 to pH 4.5.

[0007] In some cases, the modulation of protein digestion patterns (e.g., by administration of legume proteins and S53 proteases (e.g., prokumamolisin)) increases the concentration of certain food amino acids in the blood. In such cases, the modulation of protein digestion patterns can induce various physiological responses, such as the discovery of lower blood glucose levels. In some cases, digesting food proteins in the intestine, for example, by administration of legume proteins and S53 proteases (e.g., prokumamolisin), produces bioactive peptides with strong biological responses, including antidiabetic effects (e.g., lower blood glucose levels). As described and provided herein, a food composed of a protein food (e.g., legume proteins) and an acid protease (e.g., prokumamolisin) can lower blood glucose levels (e.g., when ingested with sugar). Furthermore, in some cases, the addition of a protein food (e.g., legume proteins) and an acid protease (e.g., prokumamolisin) reduces the glycemic index of a food (e.g., a food containing sugar).

[0008] Provided and described herein are methods of reducing blood glucose in a subject, the methods comprising administering to the subject a composition comprising a legume protein and an S53 protease (e.g., prokumamolisin).Also described and provided are methods of inhibiting an increase in blood glucose in a subject, the methods comprising administering to the subject a composition comprising a legume protein and an S53 protease (e.g., prokumamolisin).

[0009] In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1.

[0010] In some embodiments, the S53 protease (e.g., prokumamolysin) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an active site that includes amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO:1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an active site that includes one or more amino acid substitutions at residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site includes 1-5 amino acid substitutions. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises one or more truncations of SEQ ID NO: 1, the one or more truncations including an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[0011] In some embodiments, the legume protein is a pea protein. In some embodiments, the pea protein is derived from pea. In certain embodiments, the pea is a garden pea, a sugar pea, a field pea, or any combination thereof. In certain embodiments, the pea is a garden pea. In certain embodiments, the pea is a sugar pea. In certain embodiments, the pea is a field pea. In certain embodiments, the pea is any combination of garden pea, sugar pea, and / or field pea. In some embodiments, the pea is a standard pea, a commercial pea, a genetically modified pea, or a combination thereof. In certain embodiments, the pea is a round pea, a wrinkled pea, or a combination thereof.

[0012] In some embodiments, the administration is performed after the subject ingests a food containing sugar.In some embodiments, the increase in blood glucose is suppressed compared to when the composition does not contain the S53 protease (e.g., prokumamolisin).In some embodiments, the subject self-administers the composition.

[0013] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human has hyperglycemia. In some embodiments, the human has a condition related to and / or caused by hyperglycemia. In some embodiments, the condition is cardiovascular disease or neuropathy or diabetic nephropathy or retinopathy or cataract or bone and joint disorder or tooth and gum infection.

[0014] Also provided is a composition for use in reducing blood glucose in a subject, the composition comprising a legume protein and an S53 protease (e.g., prokumamolisin). Also provided is a composition for use in inhibiting an increase in blood glucose in a subject, the composition comprising a legume protein and an S53 protease (e.g., prokumamolisin).

[0015] In some embodiments, provided is a composition comprising a food, a legume protein (e.g., pea protein), and an S53 protease (e.g., prokumamolisin) (e.g., any one of the S53 proteases described herein (e.g., prokumamolisin)). In certain embodiments, the composition comprising the food, the legume protein, and the S53 protease (e.g., prokumamolisin) results in a lower rise in blood glucose after ingestion than a second composition comprising only the food.

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows a reduction in blood glucose following administration of legume proteins and S53 proteases (eg, prokumamolisin). [Diagram 2] 1 shows the reduction in blood glucose following administration of a low carbohydrate protein drink and protease. [Diagram 3] A reduction in blood glucose after administration of protein-fruit smoothie and protease. [Figure 4] 1 shows the proteolytic activity of a representative S53 protease in the low pH range. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The presence of dysregulation of blood glucose and / or high blood glucose levels is associated with and / or contributes to a variety of diseases that affect human health and well-being. Such diseases (e.g., diabetes) tend to be complex conditions with troublesome symptoms associated therewith and are further characterized by glucose dysregulation. In some cases, the methods and compositions provided herein are useful in reducing and / or suppressing blood glucose levels in an individual. In particular, in some cases, the methods and compositions provided herein are based on the discovery that administering a legume protein (e.g., pea protein) and an S53 protease (e.g., prokumamolisin) results in lowering (e.g., decreasing or suppressing) blood glucose levels. Furthermore, in some cases, the methods and compositions provided herein are useful in inhibiting, suppressing, reducing, and / or preventing an increase in blood glucose levels (e.g., the amount of glucose in the blood).

[0019] Legume Protein Legumes are plants of the Fabaceae family. As used herein, legumes generally refers to and refers to the fruits or seeds of legume plants. In certain embodiments, the compositions and methods utilize legume proteins. Legume proteins generally refer to proteins derived from (e.g., obtained from) the fruits or seeds of legume plants. Both intact legume proteins and hydrolyzed legume protein sources can be used. In some embodiments, the legume protein is an intact legume protein. In some embodiments, the legume protein is a hydrolyzed legume protein. In some embodiments, the legume protein is provided in a solid form. In some embodiments, the legume protein is provided in a liquid form. In some embodiments, the legume protein is a concentrate (e.g., a protein material obtained from peas after removing soluble carbohydrates, ash, and other minor components).

[0020] In some embodiments, the legume protein is pea protein. Pea generally refers to and includes the seeds or husk sheaths of plants belonging to the genus Pisum sativum. Pea protein generally refers to proteins derived from (e.g., obtained from) the fruits or seeds of the pea plant. Both intact pea protein and hydrolyzed pea protein sources can be used. In some embodiments, the pea protein is an intact legume protein. In some embodiments, the pea protein is a hydrolyzed legume protein. In some embodiments, the pea protein is provided in a solid form. In some embodiments, the pea protein is provided in a liquid form. In some embodiments, the pea protein is a concentrate (e.g., a protein material obtained from peas after removal of soluble carbohydrates, ash, and other minor components). In certain embodiments, the pea is a garden pea, a sugar pea, a field pea, or any combination thereof. In certain embodiments, the pea is a garden pea. In certain embodiments, the pea is a sugar pea. In certain embodiments, the pea is a field pea. In certain embodiments, the pea is any combination of garden pea, sugar pea, and / or field pea. In some embodiments, the pea is a standard pea, a commercial pea, a genetically modified pea, or a combination thereof. In certain embodiments, the pea is a round pea, a wrinkled pea, or a combination thereof.

[0021] Proteases The compositions and methods described herein generally utilize acidic proteases. In some embodiments, the acidic protease is an S53 family protease. The S53 family proteases generally refer to and include a family of serine proteases found in prokaryotes and eukaryotes. In some embodiments, the S53 family proteases refer to and include proteases in and / or identified by MEROPS accession MER0000995 (e.g., sedolisin, sedolisin-b, tripeptidyl peptidase I, kumamolisin, kumamolisin-B, physarolisin, aorsin, physarolisin II, kumamolisin-As, grifolisin, scytalidolisin, among others). In some embodiments, the acidic protease is an S53 protease (e.g., prokumamolisin). Prokumamolisin generally refers to and includes a thermostable calcium-dependent endopeptidase from an acidophilic / thermophilic Bacillus (Bacillus novosp. MN-32). In some embodiments, prokumamolisin refers to and includes NCBI Gene ID: 18765799 (NCBI Reference Sequences XP_007297753.1, XM_007297691.1 to XP_007297753, and / or NW_006763082.1 (137488..139728).

[0022] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:1.

[0023] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:2.

[0024] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:3.

[0025] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO:4. In some embodiments, prokumamolisin comprises an amino acid sequence having 95% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or more sequence identity to SEQ ID NO:4.

[0026] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:5.

[0027] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:6.

[0028] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:7.

[0029] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:8.

[0030] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:9.

[0031] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10.

[0032] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:11.

[0033] In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465, and A466 of SEQ ID NO:1.

[0034] The determination of percent identity or similarity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, a non-limiting example of a mathematical algorithm utilized to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. Alternatively, in some embodiments, PSI-Blast can be used to perform an iterative search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) may be used. In some embodiments, a non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. Further algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5, and FASTA, as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. In some embodiments, sequence alignment is performed using the CLUSTAL algorithm as described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0035] In some embodiments, the active site of the S53 protease (e.g., prokumamolysin) comprises amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an active site that comprises one or more amino acid substitutions of residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site comprises between 1 and 5 amino acid substitutions.

[0036] In some embodiments, the active site comprises one or more amino acid substitutions. In some embodiments, the active site comprises two or more amino acid substitutions. In some embodiments, the active site comprises three or more amino acid substitutions. In some embodiments, the active site comprises four or more amino acid substitutions.

[0037] In some embodiments, the active site comprises one amino acid substitution. In some embodiments, the active site comprises two amino acid substitutions. In some embodiments, the active site comprises three amino acid substitutions. In some embodiments, the active site comprises four amino acid substitutions. In some embodiments, the active site comprises five amino acid substitutions.

[0038] Amino acids generally refer to and / or include natural amino acids, unnatural amino acids, amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Amino acids are generally referred to herein by either their name, their commonly known three letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. As used herein, natural amino acids include and / or refer to amino acids that are commonly found in nature and that have not been engineered by man. In some embodiments, naturally occurring includes and / or further refers to the 20 conventional amino acids: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr).

[0039] In some embodiments, a non-polar amino acid can be substituted and replaced with another non-polar amino acid, including alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. In some embodiments, a neutrally charged polar amino acid can be substituted and replaced with another neutrally charged polar amino acid, including serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In some embodiments, a positively charged amino acid can be substituted and replaced with another positively charged amino acid, including arginine, lysine, and histidine. In some embodiments, a negatively charged amino acid can be substituted and replaced with another negatively charged amino acid, including aspartic acid and glutamic acid. As used herein, a peptide includes and / or refers to any of a variety of natural or synthetic compounds that include two or more amino acids joined by a peptide bond that links the carboxyl group of one amino acid to the amino group of another amino acid. Also as used herein, amino acid refers to and / or includes natural amino acids, unnatural amino acids, amino acid analogs and amino acid mimetics that function similarly to the natural amino acids. Amino acids are generally referred to herein by either their name, their commonly known three letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0040] In some embodiments, the active site comprises 1-5 amino acid substitutions. In some embodiments, the S53 protease (e.g., prokumamolisin) comprises one or more truncations of SEQ ID NO:1, the one or more truncations comprising an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[0041] In some embodiments, the S53 protease is active at a pH less than about pH 5 (e.g., as measured by protein digestion). In some embodiments, the S53 protease is active at a pH less than about pH 4.5. In some embodiments, the S53 protease is active at a pH less than about pH 5. In some embodiments, the S53 protease is active at a pH less than about pH 4. In some embodiments, the S53 protease is active at a pH less than about pH 3.5. In some embodiments, the S53 protease is active at a pH less than about pH 3.

[0042] In some embodiments, the S53 protease is active in a pH range of about pH 2 to pH 5. In some embodiments, the S53 protease is active in a pH range of about pH 2.5 to pH 4.5. In some embodiments, the S53 protease is at least 50% active (e.g., relative to its maximum activity) in a pH range of about pH 2.5 to pH 4.5.

[0043] The S53 protease (e.g., prokumamolisin) may be administered as part of a composition comprising the S53 protease (e.g., prokumamolisin). The composition may further comprise a legume protein. In some embodiments, provided is a composition comprising the S53 protease (e.g., prokumamolisin). In certain embodiments, the composition further comprises a legume protein (e.g., pea protein).

[0044] In some embodiments, provided is a composition comprising a food product, a legume protein (e.g., pea protein), and an S53 protease (e.g., prokumamolisin) (e.g., any one of the S53 proteases described herein (e.g., prokumamolisin)). In certain embodiments, the food product comprises a sugar. In certain embodiments, the sugar comprises sucrose, lactose, maltose, or another disaccharide, trisaccharide, or polysaccharide comprising glucose as a monomer. In certain embodiments, the sugar comprises sucrose. In certain embodiments, the sugar comprises lactose. In certain embodiments, the sugar comprises maltose. In certain embodiments, the sugar comprises a disaccharide, trisaccharide, or polysaccharide comprising glucose as a monomer. In some cases, the addition of the legume protein (e.g., pea protein) and the S53 protease (e.g., prokumamolisin) reduces the glycemic index of the composition comprising the food product. In certain embodiments, a composition comprising the food, the legume protein, and the S53 protease (e.g., prokumamolisin) produces a lower rise in blood glucose following ingestion than a second composition comprising only the food.

[0045] In some embodiments, the composition comprises from about 5 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition comprises from about 5 grams (g) of legume protein (e.g., pea protein) to about 10 grams (g) of legume protein (e.g., pea protein), from about 5 grams (g) of legume protein (e.g., pea protein) to about 15 grams (g) of legume protein (e.g., pea protein), from about 5 grams (g) of legume protein (e.g., pea protein) to about 20 grams (g) of legume protein (e.g., pea protein), from about 5 grams (g) of legume protein (e.g., pea protein), legume protein (e.g., pea protein) to about 25 grams (g) legume protein (e.g., pea protein), about 5 grams (g) legume protein (e.g., pea protein) to about 30 grams (g) legume protein (e.g., pea protein), about 5 grams (g) legume protein (e.g., pea protein) to about 35 grams (g) legume protein (e.g., pea protein), about 5 grams (g) legume protein (e.g., pea protein) to about 40 grams from about 5 grams (g) of legume protein (e.g., pea protein), from about 5 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), from about 5 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), from about 5 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein) ), about 10 grams (g) of legume protein (e.g., pea protein) to about 15 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 20 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 25 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g.,from about 10 grams (g) of legume protein (e.g., pea protein) to about 30 grams (g) of legume protein (e.g., pea protein), from about 10 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), from about 10 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), from about 10 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 20 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 25 grams (g) of maize legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 30 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) , pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 25 grams (g) of legume protein (e.g., pea protein),About 20 grams (g) of legume protein (e.g., pea protein) to about 30 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein) g of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein) to about 30 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of maize protein (e.g., pea protein) to about 30 grams (g) of maize protein (e.g., pea protein), legume protein (e.g., pea protein) to about 35 grams (g) legume protein (e.g., pea protein), about 25 grams (g) legume protein (e.g., pea protein) to about 40 grams (g) legume protein (e.g., pea protein), about 25 grams (g) legume protein (e.g., pea protein) to about 45 grams (g) legume protein (e.g., pea protein), about 25 grams (g) legume protein (e.g., pea protein) to about 50 grams (g) legume protein quality (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein),from about 45 grams (g) of legume protein (e.g., pea protein), from about 30 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), from about 30 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), from about 35 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein). protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), In some embodiments, the composition comprises about 5 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), or about 50 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g). In some embodiments, the composition comprises about 5 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein),About 20 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein), about 50 grams (g) of legume protein (e.g., pea protein), or about 60 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition comprises at least about 5 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein), or about 50 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition contains at most about 10 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein), about 50 grams (g) of legume protein (e.g., pea protein), or about 60 grams (g) of legume protein (e.g.,Pea protein).

[0046] In some embodiments, the composition comprises about 50 milligrams (mg) of prokumamolisin to about 2,000 milligrams (mg) of prokumamolisin. In some embodiments, the composition comprises about 50 milligrams (mg) of prokumamolisin to about 100 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin to about 200 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin to about 300 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin to about 400 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin ~ about 500 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin to about 750 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin to about 1,000 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin to about 1,250 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin to about 1,500 milligrams (mg) of prokumamolisin, about 50 milligrams (mg) of prokumamolisin about 100 milligrams (mg) to about 200 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) to about 300 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) to about 400 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) to about 500 milligrams (mg) of prokumamolisin sine, about 100 milligrams (mg) of prokumamolisin to about 750 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) of prokumamolisin to about 1,000 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) of prokumamolisin to about 1,250 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) of prokumamolisin to about 1,500 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) of prokumamolisin to about 2,000 milligrams (mg) of prokumamolisin, about 200 milligrams (mg) of prokumamolisin to about 300 milligrams (mg) of prokumamolisin, about 200 milligrams (mg) of prokumamolisin to about 400 milligrams (mg) of prokumamolisin, about 200 milligrams (mg) of prokumamolisin to about 500 milligrams (mg) of prokumamolisin, about 200 milligrams (mg) of prokumamolisin to about 750 milligrams (mg) of prokumamolisin, about 200 milligrams (mg) of prokumamolisin to about 1,000 milligrams (mg) of prokumamolisin Kumamolysin, about 200 milligrams (mg) of prokumamolysin to about 1,250 milligrams (mg) of prokumamolysin, about 200 milligrams (mg) of prokumamolysin to about 1,500 milligrams (mg) of prokumamolysin, about 200 milligrams (mg) of prokumamolysin to about 2,000 milligrams (mg) of prokumamolysin, about 300 milligrams (mg) of prokumamolysin to about 400 milligrams (mg) of prokumamolysin, about 300 milligrams (mg) of prokumamolysin to about 500 milligrams (mg) of prokumamolysin, about 300 milligrams (mg) of prokumamolysin 100 milligrams (mg) of prokumamolisin to about 750 milligrams (mg) of prokumamolisin, about 300 milligrams (mg) of prokumamolisin to about 1,000 milligrams (mg) of prokumamolisin, about 300 milligrams (mg) of prokumamolisin to about 1,250 milligrams (mg) of prokumamolisin, about 300 milligrams (mg) of prokumamolisin to about 1,500 milligrams (mg) of prokumamolisin, about 300 milligrams (mg) of prokumamolisin to about 2,000 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin Prokumamolisin to about 500 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin to about 750 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin to about 1,000 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin to about 1,250 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin to about 1,500 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin to about 2,000 milligrams (mg) of prokumamolisin, about 500 milligrams (mg) of prokumamolisin to about 750 milligrams (mg) of prokumamolisin, about 500 milligrams (mg) of prokumamolisin to about 1,000 milligrams (mg) of prokumamolisin, about 500 milligrams (mg) of prokumamolisin to about 1,250 milligrams (mg) of prokumamolisin, about 500 milligrams (mg) of prokumamolisin to about 1 ,500 milligrams (mg) of prokumamolisin, about 500 milligrams (mg) of prokumamolisin to about 2,000 milligrams (mg) of prokumamolisin, about 750 milligrams (mg) of prokumamolisin to about 1,000 milligrams (mg) of prokumamolisin, about 750 milligrams (mg) of prokumamolisin to about 1,250 milligrams (mg) of prokumamolisin, about 750 milligrams (mg) of prokumamolisin ~ about 1,500 milligrams (mg) of prokumamolisin, about 750 milligrams (mg) of prokumamolisin to about 2,000 milligrams (mg) of prokumamolisin, about 1,000 milligrams (mg) of prokumamolisin to about 1,250 milligrams (mg) of prokumamolisin, about 1,000 milligrams (mg) of prokumamolisin to about 1,500 milligrams (mg) of prokumamolisin, about 1,000 milligrams (mg) of prokumamolisin from about 1,250 milligrams (mg) to about 1,500 milligrams (mg) of prokumamolisin, from about 1,250 milligrams (mg) to about 2,000 milligrams (mg) of prokumamolisin, or from about 1,500 milligrams (mg) to about 2,000 milligrams (mg) of prokumamolisin. In some embodiments, the composition comprises about 50 milligrams (mg) of prokumamolysin, about 100 milligrams (mg) of prokumamolysin, about 200 milligrams (mg) of prokumamolysin, about 300 milligrams (mg) of prokumamolysin, about 400 milligrams (mg) of prokumamolysin, about 500 milligrams (mg) of prokumamolysin, about 750 milligrams (mg) of prokumamolysin, about 1,000 milligrams (mg) of prokumamolysin, about 1,250 milligrams (mg) of prokumamolysin, about 1,500 milligrams (mg) of prokumamolysin, about 1,600 milligrams (mg) of prokumamolysin, about 1,700 milligrams (mg) of prokumamolysin, about 1,800 milligrams (mg) of prokumamolysin, about 1,900 milligrams (mg) of prokumamolysin, about 2,000 milligrams (mg) of prokumamolysin, about 2,100 milligrams (mg) of prokumamolysin, about 2,200 milligrams (mg) of prokumamolysin, about 2,300 milligrams (mg) of prokumamolysin, about 2,400 milligrams (mg) of prokumamolysin, about 2,500 milligrams (mg) of prokumamolysin, about 2,600 milligrams (mg) of prokumamolysin, about 2,700 milligrams (mg) of prokumamolysin, about 2,800 milligrams (mg) of prokumamolysin, about 2,900 milligrams (mg)Contains 500 milligrams (mg) of prokumamolisin, or about 2,000 milligrams (mg) of prokumamolisin. In some embodiments, the composition contains at least about 50 milligrams (mg) of prokumamolisin, about 100 milligrams (mg) of prokumamolisin, about 200 milligrams (mg) of prokumamolisin, about 300 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin, about 500 milligrams (mg) of prokumamolisin, about 750 milligrams (mg) of prokumamolisin, about 1,000 milligrams (mg) of prokumamolisin, about 1,250 milligrams (mg) of prokumamolisin, or about 1,500 milligrams (mg) of prokumamolisin. In some embodiments, the composition contains at most about 100 milligrams (mg) of prokumamolisin, about 200 milligrams (mg) of prokumamolisin, about 300 milligrams (mg) of prokumamolisin, about 400 milligrams (mg) of prokumamolisin, about 500 milligrams (mg) of prokumamolisin, about 750 milligrams (mg) of prokumamolisin, about 1,000 milligrams (mg) of prokumamolisin, about 1,250 milligrams (mg) of prokumamolisin, about 1,500 milligrams (mg) of prokumamolisin, or about 2,000 milligrams (mg) of prokumamolisin.

[0047] method As described herein, in some cases, blood glucose levels are lowered (e.g., reduced or inhibited) by providing a legume protein (e.g., pea protein) and an S53 protease (e.g., prokumamolisin) protease. Accordingly, provided herein is a method of reducing blood glucose in a subject, the method comprising administering to the subject a composition comprising a legume protein and an S53 protease (e.g., prokumamolisin) (e.g., the legume protein and the S53 protease (e.g., prokumamolisin) are administered to the subject). Also provided is a legume protein and an S53 protease (e.g., prokumamolisin) for use in a method of reducing blood glucose in a subject (e.g., the legume protein and the S53 protease (e.g., prokumamolisin) are administered to the subject).

[0048] Also provided is a method of inhibiting an increase in blood glucose in a subject, the method comprising administering to the subject a composition comprising a legume protein and an S53 protease (e.g., prokumamolisin). Also provided is the use of a legume protein and an S53 protease (e.g., prokumamolisin) in a method of inhibiting an increase in blood glucose in a subject (e.g., the legume protein and the S53 protease (e.g., prokumamolisin) are administered to the subject). Also provided is a legume protein and an S53 protease (e.g., prokumamolisin) for use in a method of inhibiting an increase in blood glucose in a subject (e.g., the legume protein and the S53 protease (e.g., prokumamolisin) are administered to the subject).

[0049] In some cases, providing a legume protein (e.g., pea protein) and an S53 protease (e.g., prokumamolisin) stabilizes blood glucose levels (e.g., reduces or prevents the extent or magnitude of changes) associated with ingestion of a food. In some embodiments, provided herein are methods of inhibiting changes (positive or negative) in blood glucose in a subject, the methods comprising administering to the subject a composition comprising a legume protein and an S53 protease (e.g., prokumamolisin) (e.g., the legume protein and the S53 protease (e.g., prokumamolisin) are administered to the subject).

[0050] In certain embodiments, administration of the legume protein and the S53 protease (e.g., prokumamolisin) suppresses the degree or amount of blood glucose rise after ingestion of food. In some cases, the suppression of the degree or amount of blood glucose rise after ingestion of food can be compared to the increase in blood glucose after ingestion of food before or without administration of the legume protein and the S53 protease (e.g., prokumamolisin). In certain embodiments, administration of the legume protein and the S53 protease (e.g., prokumamolisin) suppresses the degree or amount of blood glucose decrease after ingestion of food. In some cases, the suppression of the degree or amount of blood glucose decrease after ingestion of food can be compared to the decrease in blood glucose after ingestion of food before or without administration of the legume protein and the S53 protease (e.g., prokumamolisin). In some embodiments, administration of the legume protein and the S53 protease (e.g., prokumamolisin) suppresses the change (e.g., increase or decrease) in blood glucose after ingestion of food. In certain embodiments, the inhibition of the change (e.g., increase or decrease) in blood glucose rise after ingestion of food when the legume protein and the S53 protease (e.g., prokumamolisin) are administered is compared to the change in blood glucose after ingestion of food before or without administration of the legume protein and / or the S53 protease (e.g., prokumamolisin).

[0051] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:1.

[0052] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:2.

[0053] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:3.

[0054] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO:4. In some embodiments, prokumamolisin comprises an amino acid sequence having 95% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or more sequence identity to SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or more sequence identity to SEQ ID NO:4.

[0055] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:5.

[0056] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:6.

[0057] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:7.

[0058] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:8.

[0059] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:9.

[0060] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10.

[0061] In some embodiments, the S53 protease comprises an amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:11.

[0062] In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465, and A466 of SEQ ID NO:1.

[0063] Any of the S53 proteases (e.g., prokumamolysin) described herein can be used in the methods provided. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:1.

[0064] In some embodiments, the S53 protease (e.g., prokumamolysin) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an active site that comprises amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an active site that includes one or more amino acid substitutions at residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site includes 1-5 amino acid substitutions. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises one or more truncations of SEQ ID NO: 1, the one or more truncations including an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[0065] In some embodiments, the dosage of the S53 protease (e.g., prokumamolysin) comprises about 50 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the dosage of the S53 protease (e.g., prokumamolysin) comprises about 50 milligrams (mg) to about 100 milligrams (mg), about 50 milligrams (mg) to about 150 milligrams (mg), about 50 milligrams (mg) to about 200 milligrams (mg), about 50 milligrams (mg) to about 300 milligrams (mg), about 50 milligrams (mg) to about 400 milligrams (mg), about 50 milligrams (mg) to about 500 milligrams (mg), about 50 milligrams (mg) to about 700 milligrams (mg), or about 50 milligrams (mg) to about 800 milligrams (mg). 50 milligrams (mg), about 50 milligrams (mg) to about 1,000 milligrams (mg), about 50 milligrams (mg) to about 1,500 milligrams (mg), about 100 milligrams (mg) to about 150 milligrams (mg), about 100 milligrams (mg) to about 200 milligrams (mg), about 100 milligrams (mg) to about 300 milligrams (mg), about 100 milligrams (mg) to about 400 milligrams (mg), about 100 milligrams (mg) to about 500 milligrams (mg), about 100 milligrams (mg) to about 750 milligrams (mg), about 100 milligrams (mg) to about 1,000 milligrams (mg), about 100 milligrams (mg) to about 1,500 milligrams (mg), about 150 milligrams (mg) to about 200 milligrams (mg), about 150 milligrams (mg) to about 300 milligrams (mg), about 150 milligrams (mg) to about 400 milligrams (mg), about 150 milligrams (mg) to about 500 milligrams (mg), about 150 milligrams (mg) to about 750 milligrams (mg), About 150 milligrams (mg) to about 1,000 milligrams (mg), about 150 milligrams (mg) to about 1,500 milligrams (mg), about 200 milligrams (mg) to about 300 milligrams (mg), about 200 milligrams (mg) to about 400 milligrams (mg), about 200 milligrams (mg) to about 500 milligrams (mg), about 200 milligrams (mg) to about 750 milligrams (mg), about 200 milligrams (mg) to about 1,000 milligrams (mg), about 200 milligrams (mg) to about 1,500 milligrams (mg), about 300 milligrams (mg) to about 400 milligrams (mg), about 300 milligrams (mg) to about 500 milligrams (mg), about 300 milligrams (mg) to about 750 milligrams (mg), about 300 milligrams (mg) to about 1,000 milligrams (mg), about 300 milligrams (mg) to about 1,500 milligrams (mg), about 400 milligrams (mg) to about 500 milligrams (mg), about 400 milligrams (mg) to about 750 milligrams (mg), about 400 milligrams (mg) to about 1 The range of effective doses includes from about 1,000 milligrams (mg), about 400 milligrams (mg) to about 1,500 milligrams (mg), about 500 milligrams (mg) to about 750 milligrams (mg), about 500 milligrams (mg) to about 1,000 milligrams (mg), about 500 milligrams (mg) to about 1,500 milligrams (mg), about 750 milligrams (mg) to about 1,000 milligrams (mg), about 750 milligrams (mg) to about 1,500 milligrams (mg), or about 1,000 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the dosage of the S53 protease (e.g., prokumamolisin) comprises about 50 milligrams (mg), about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), about 1,000 milligrams (mg), or about 1,500 milligrams (mg). In some embodiments, the dosage of the S53 protease (e.g., prokumamolisin) comprises at least about 50 milligrams (mg), about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), or about 1,000 milligrams (mg). In some embodiments, the dosage of the S53 protease (e.g., prokumamolisin) is at most about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), about 1,000 milligrams (mg), or about 1,000 milligrams (mg).Contains 500 milligrams (mg).

[0066] In some embodiments, the dosage of the legume protein comprises about 5 grams (g) to about 50 grams (g). In some embodiments, the dosage of the legume protein comprises about 5 grams (g) to about 10 grams (g), about 5 grams (g) to about 15 grams (g), about 5 grams (g) to about 20 grams (g), about 5 grams (g) to about 25 grams (g), about 5 grams (g) to about 30 grams (g), about 5 grams (g) to about 35 grams (g), about 5 grams (g) to about 40 grams (g), about 5 grams (g) to about 45 grams (g), about 5 grams (g) to about 50 grams (g), about 10 grams (g) to about 15 grams (g), about 10 ... grams (g), about 10 grams (g) to about 25 grams (g), about 10 grams (g) to about 30 grams (g), about 10 grams (g) to about 35 grams (g), about 10 grams (g) to about 40 grams (g), about 10 grams (g) to about 45 grams (g), about 10 grams (g) to about 50 grams (g), about 15 grams (g) to about 20 grams (g), about 15 grams (g) to about 25 grams (g), about 15 grams (g) to about 30 grams (g), about 15 grams (g) to about 35 grams (g), about 15 grams (g) to about 40 grams (g), about 15 grams Lamb (g) to about 45 grams (g), about 15 grams (g) to about 50 grams (g), about 20 grams (g) to about 25 grams (g), about 20 grams (g) to about 30 grams (g), about 20 grams (g) to about 35 grams (g), about 20 grams (g) to about 40 grams (g), about 20 grams (g) to about 45 grams (g), about 20 grams (g) to about 50 grams (g), about 25 grams (g) to about 30 grams (g), about 25 grams (g) to about 35 grams (g), about 25 grams (g) to about 40 grams (g), about 25 grams (g) to about 45 grams (g)

[0043] The range of dosages includes about 25 grams (g) to about 50 grams (g), about 30 grams (g) to about 35 grams (g), about 30 grams (g) to about 40 grams (g), about 30 grams (g) to about 45 grams (g), about 30 grams (g) to about 50 grams (g), about 35 grams (g) to about 40 grams (g), about 35 grams (g) to about 45 grams (g), about 35 grams (g) to about 50 grams (g), about 40 grams (g) to about 45 grams (g), about 40 grams (g) to about 50 grams (g), or about 45 grams (g) to about 50 grams (g).In some embodiments, the dosage of legume protein comprises about 5 grams (g), about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), about 45 grams (g), or about 50 grams (g). In some embodiments, the dosage of legume protein comprises at least about 5 grams (g), about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), or about 45 grams (g). In some embodiments, the dosage of legume protein comprises at most about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), about 45 grams (g), or about 50 grams (g).

[0067] In some embodiments, the legume protein is a pea protein. In some embodiments, the pea is a whole pea or a component thereof. In certain embodiments, the pea is a garden pea, a sugar pea, a field pea, or any combination thereof. In certain embodiments, the pea is a garden pea. In certain embodiments, the pea is a sugar pea. In certain embodiments, the pea is a field pea. In certain embodiments, the pea is any combination of garden pea, sugar pea, and / or field pea. In some embodiments, the pea is a standard pea, a commercial pea, a genetically modified pea, or a combination thereof. In certain embodiments, the pea is a round pea, a wrinkled pea, or a combination thereof.

[0068] In some embodiments, a composition comprising the legume protein is administered. In some embodiments, a composition comprising the S53 protease (e.g., prokumamolysin) is administered. In some embodiments, a composition comprising the legume protein and the S53 protease (e.g., prokumamolysin) is administered.

[0069] In some embodiments, the administration is performed after the subject ingests a food product that contains sugar.In some embodiments, the sugar comprises sucrose, lactose, maltose, or another disaccharide, trisaccharide, or polysaccharide that contains glucose as a monomer.In certain embodiments, the sugar comprises sucrose.In certain embodiments, the sugar comprises lactose.In certain embodiments, the sugar comprises maltose.In certain embodiments, the sugar comprises a disaccharide, trisaccharide, or polysaccharide that contains glucose as a monomer.

[0070] In some embodiments, the blood glucose rise is suppressed compared to administering a composition that does not include the S53 protease (e.g., prokumamolisin). In some embodiments, the subject self-administers the composition. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human has hyperglycemia. In some embodiments, the human has a condition associated with and / or caused by hyperglycemia. In some embodiments, the condition is cardiovascular disease or neuropathy or diabetic nephropathy or retinopathy or cataract or bone and joint disorder or tooth and gum infection. In certain embodiments, the condition is cardiovascular disease. In certain embodiments, the condition is neuropathy. In certain embodiments, the condition is diabetic nephropathy. In certain embodiments, the condition is retinopathy. In certain embodiments, the condition is cataract. In certain embodiments, the condition is bone and joint disorder. In certain embodiments, the condition is a tooth infection. In certain embodiments, the condition is a gum infection.

[0071] Also provided is a method of reducing the glycemic index of a food product, the method comprising providing said food product with a legume protein and an S53 protease (e.g., prokumamolisin). Also provided is the use of a legume protein and an S53 protease (e.g., prokumamolisin) to reduce the glycemic index of a food product. Also provided is a legume protein and an S53 protease (e.g., prokumamolisin) for use in reducing the glycemic index of a food product.

[0072] Any of the S53 proteases (e.g., prokumamolysin) described herein can be used in the methods provided. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465, and A466 of SEQ ID NO:1.

[0073] In some embodiments, the S53 protease (e.g., prokumamolysin) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an active site that comprises amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises an active site that includes one or more amino acid substitutions at residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site includes 1-5 amino acid substitutions. In some embodiments, the S53 protease (e.g., prokumamolysin) comprises one or more truncations of SEQ ID NO: 1, the one or more truncations including an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[0074] In some embodiments, the dosage of procoumamolysin provided includes about 50 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the dosage of procoumamolysin provided includes about 50 milligrams (mg) to about 100 milligrams (mg), about 50 milligrams (mg) to about 150 milligrams (mg), about 50 milligrams (mg) to about 200 milligrams (mg), about 50 milligrams (mg) to about 300 milligrams (mg), about 50 milligrams (mg) to about 400 milligrams (mg), about 50 milligrams (mg) to about 500 milligrams (mg), about 50 milligrams (mg) to about 750 milligrams (mg). g), about 50 milligrams (mg) to about 1,000 milligrams (mg), about 50 milligrams (mg) to about 1,500 milligrams (mg), about 100 milligrams (mg) to about 150 milligrams (mg), about 100 milligrams (mg) to about 200 milligrams (mg), about 100 milligrams (mg) to about 300 milligrams (mg), about 100 milligrams (mg) to about 400 milligrams (mg), about 100 milligrams (mg) to about 500 milligrams (mg), about 100 milligrams (mg) to about 750 milligrams (mg), about 100 milligrams (mg) to about 1,000 milligrams (mg), about 100 milligrams (mg) to about 1,500 milligrams (mg), about 150 milligrams (mg) to about 200 milligrams (mg), about 150 milligrams (mg) to about 300 milligrams (mg), about 150 milligrams (mg) to about 400 milligrams (mg), about 150 milligrams (mg) to about 500 milligrams (mg), about 150 milligrams (mg) to about 750 milligrams (mg), about 15 0 milligrams (mg) to about 1,000 milligrams (mg), about 150 milligrams (mg) to about 1,500 milligrams (mg), about 200 milligrams (mg) to about 300 milligrams (mg), about 200 milligrams (mg) to about 400 milligrams (mg), about 200 milligrams (mg) to about 500 milligrams (mg), about 200 milligrams (mg) to about 750 milligrams (mg), about 200 milligrams (mg) to about 1,000 milligrams (mg), about 200 milligrams (mg) to about 1,500 milligrams (mg), about 300 milligrams (mg) to about 400 milligrams (mg), about 300 milligrams (mg) to about 500 milligrams (mg), about 300 milligrams (mg) to about 750 milligrams (mg), about 300 milligrams (mg) to about 1,000 milligrams (mg), about 300 milligrams (mg) to about 1,500 milligrams (mg), about 400 milligrams (mg) to about 500 milligrams (mg), about 400 milligrams (mg) to about 750 milligrams (mg), about 400 milligrams (mg) to about 1 The range of effective doses includes from about 1,000 milligrams (mg), about 400 milligrams (mg) to about 1,500 milligrams (mg), about 500 milligrams (mg) to about 750 milligrams (mg), about 500 milligrams (mg) to about 1,000 milligrams (mg), about 500 milligrams (mg) to about 1,500 milligrams (mg), about 750 milligrams (mg) to about 1,000 milligrams (mg), about 750 milligrams (mg) to about 1,500 milligrams (mg), or about 1,000 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the dosage of prokumamolisin provided comprises about 50 milligrams (mg), about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), about 1,000 milligrams (mg), or about 1,500 milligrams (mg). In some embodiments, the dosage of prokumamolisin provided comprises at least about 50 milligrams (mg), about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), or about 1,000 milligrams (mg). In some embodiments, the dosage of prokumamolisin provided comprises at most about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), about 1,000 milligrams (mg), or about 1,500 milligrams (mg).

[0075] In some embodiments, the dosage of legume protein provided includes from about 5 grams (g) to about 60 grams (g). In some embodiments, the dosage of legume protein provided includes from about 5 grams (g) to about 10 grams (g), from about 5 grams (g) to about 15 grams (g), from about 5 grams (g) to about 20 grams (g), from about 5 grams (g) to about 25 grams (g), from about 5 grams (g) to about 30 grams (g), from about 5 grams (g) to about 35 grams (g), from about 5 grams (g) to about 40 grams (g), from about 5 grams (g) to about 45 grams (g), from about 5 grams (g) to about 50 grams (g), from about 5 grams (g) to about 60 grams (g), from about 10 grams (g) to about 15 grams (g). (g), about 10 grams (g) to about 20 grams (g), about 10 grams (g) to about 25 grams (g), about 10 grams (g) to about 30 grams (g), about 10 grams (g) to about 35 grams (g), about 10 grams (g) to about 40 grams (g), about 10 grams (g) to about 45 grams (g), about 10 grams (g) to about 50 grams (g), about 10 grams (g) to about 60 grams (g), about 15 grams (g) to about 20 grams (g), about 15 grams (g) to about 25 grams (g), about 15 grams (g) to about 30 grams (g), about 15 grams (g) to About 35 grams (g), about 15 grams (g) to about 40 grams (g), about 15 grams (g) to about 45 grams (g), about 15 grams (g) to about 50 grams (g), about 15 grams (g) to about 60 grams (g), about 20 grams (g) to about 25 grams (g), about 20 grams (g) to about 30 grams (g), about 20 grams (g) to about 35 grams (g), about 20 grams (g) to about 40 grams (g), about 20 grams (g) to about 45 grams (g), about 20 grams (g) to about 50 grams (g), about 20 grams (g) to about 60 grams (g), about 25 grams g to about 30 g, about 25 g to about 35 g, about 25 g to about 40 g, about 25 g to about 45 g, about 25 g to about 50 g, about 25 g to about 60 g, about 30 g to about 35 g, about 30 g to about 40 g, about 30 g to about 45 g, about 30 g to about 50 g, about 30 g to about 60 g, about 35 g to about 40 g,Including about 35 grams (g) to about 45 grams (g), about 35 grams (g) to about 50 grams (g), about 35 grams (g) to about 60 grams (g), about 40 grams (g) to about 45 grams (g), about 40 grams (g) to about 50 grams (g), about 40 grams (g) to about 60 grams (g), about 45 grams (g) to about 50 grams (g), about 45 grams (g) to about 60 grams (g), or about 50 grams (g) to about 60 grams (g). In some embodiments, the dosage of legume protein provided comprises about 5 grams (g), about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), about 45 grams (g), about 50 grams (g), or about 60 grams (g). In some embodiments, the dosage of legume protein provided comprises at least about 5 grams (g), about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), about 45 grams (g), or about 50 grams (g). In some embodiments, the dosage of legume protein provided comprises at most about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), about 45 grams (g), about 50 grams (g), or about 60 grams (g).

[0076] In some embodiments, the legume protein is a pea protein. In some embodiments, the pea is a whole pea or a component thereof. In certain embodiments, the pea is a garden pea, a sugar pea, a field pea, or any combination thereof. In certain embodiments, the pea is a garden pea. In certain embodiments, the pea is a sugar pea. In certain embodiments, the pea is a field pea. In certain embodiments, the pea is any combination of garden pea, sugar pea, and / or field pea. In some embodiments, the pea is a standard pea, a commercial pea, a genetically modified pea, or a combination thereof. In certain embodiments, the pea is a round pea, a wrinkled pea, or a combination thereof.

[0077] In some embodiments, a composition comprising the legume protein is added. In some embodiments, a composition comprising the S53 protease (e.g., prokumamolisin) is added. In some embodiments, a composition comprising the legume protein and the S53 protease (e.g., prokumamolisin) is added.

[0078] In some embodiments, the food product comprises a sugar. In some embodiments, the sugar comprises sucrose, lactose, maltose, or another disaccharide, trisaccharide, or polysaccharide that comprises glucose as a monomer. In certain embodiments, the condition is a cardiovascular disease. In certain embodiments, the condition is a neuropathy. In certain embodiments, the condition is diabetic nephropathy. In certain embodiments, the condition is a retinopathy. In certain embodiments, the condition is a cataract. In certain embodiments, the condition is a bone and joint disorder. In certain embodiments, the condition is a dental infection. In certain embodiments, the condition is a gum infection.

[0079] In some embodiments, the method further comprises administering a low-carbohydrate diet. In certain embodiments, the low-carbohydrate diet comprises a diet in which less than 65% of total energy intake per day (e.g., total caloric intake per day) is derived from carbohydrates. In certain embodiments, the low-carbohydrate diet comprises a diet in which less than 60% of total energy intake per day (e.g., total caloric intake per day) is derived from carbohydrates. In certain embodiments, the low-carbohydrate diet comprises a diet in which less than 50% of total energy intake per day (e.g., total caloric intake per day) is derived from carbohydrates. In certain embodiments, the low-carbohydrate diet comprises a diet in which less than 45% of total energy intake per day (e.g., total caloric intake per day) is derived from carbohydrates. In certain embodiments, the low-carbohydrate diet comprises a diet in which less than 40% of total energy intake per day (e.g., total caloric intake per day) is derived from carbohydrates. In certain embodiments, the low-carbohydrate diet comprises a diet in which less than 35% of total energy intake per day (e.g., total caloric intake per day) is derived from carbohydrates. In certain embodiments, the low-carbohydrate diet comprises a diet in which less than 30% of total energy intake per day (e.g., total caloric intake per day) is derived from carbohydrates. In some embodiments, the method further comprises reducing the caloric intake from carbohydrates (e.g., total caloric intake per day). In certain embodiments, reducing the caloric intake from carbohydrates (e.g., total caloric intake per day) comprises reducing the carbohydrate intake (e.g., grams or calories) as measured by or compared to the carbohydrate intake before administering the protease or a composition comprising the protease to the subject.

[0080] In certain embodiments, the amount of carbohydrates (grams or calories) is reduced by about 10% to about 65%. In certain embodiments, the amount of carbohydrates (grams or calories) is reduced by about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 65%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 65%, about 30% to about 40%, about 30% to about 50%, about 30% to about 65%, about 40% to about 50%, about 40% to about 65%, or about 50% to about 65%. In certain embodiments, the amount of carbohydrates (grams or calories) is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, or about 60%. In certain embodiments, the amount of carbohydrates (grams or calories) is reduced by at least about 10%, about 20%, about 30%, about 40%, 50%, 60%, or about 65%.

[0081] Carbohydrates are generally multi-hydroxyl substituted carbohydrates of the general formula (CHO) n(n is 3-30), and their oligomers and polymers. Carbohydrates may be substituted or deoxygenated at one or more positions. Carbohydrates include and / or encompass monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Carbohydrates further include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. The carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates can be deoxygenated at any corresponding C-position and / or substituted with one or more moieties, such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group.

[0082] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0083] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0084] The terms "identifying," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present (e.g., detecting). These terms can include quantitative, qualitative, or quantitative and qualitative determination. Evaluating can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent, depending on the context.

[0085] The terms "subject", "individual", or "patient" are often used interchangeably herein. A "subject" may be a biological entity that contains expressed genetic material. The biological entity may be, for example, a plant, an animal, or a microorganism, including bacteria, viruses, fungi, and protozoa. The subject may be tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject may be a mammal. The mammal may be a human. The subject may have been diagnosed or may be suspected of being at high risk for developing a disease. In some cases, the subject has not necessarily been diagnosed or is not suspected of being at high risk for developing a disease.

[0086] As used herein, the term "about" refers to a number that is within the range of that number plus or minus 10%. The term "about" refers to a range from the minimum value minus 10% to the maximum value plus 10%.

[0087] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0088] As used herein, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of comprising, e.g., "include" and "includes" or "containing" (and any form of comprising, e.g., "contain" and "contains") are inclusive or open ended and do not exclude further, unrecited elements or process steps. In any example or embodiment described herein, "comprising" as similarly used herein may be replaced with "consisting essentially of" and / or "consisting of" as used herein in any example or embodiment described. EXAMPLES

[0089] Example 1 - Lowering blood glucose using legume proteins and prokumamolisin Protease production The DNA sequence of the acid protease of interest (SEQ ID NO:2) was cloned into the expression vector pET29b(+) for protease production in E. coli. The completed DNA construct was transformed into an expression strain of E. coli (BL21) and grown in Terrific Broth at 37°C for 4-6 hours using baffled shake flasks until the cell density (measured using OD600) reached 0.6. The culture was then induced with 0.5 mM IPTG for protease expression. After induction, the culture was grown at 30°C for 12 hours and then harvested. The harvested cells were lysed using sonication and the protease was purified from the cell lysate using IMAC chromatography.

[0090] Application of prokumamolisin to pea protein drinks 30 grams of pea protein were dissolved in 330 mL of water. Then 14 grams of sucrose were dissolved followed by 200 mg of acid protease (SEQ ID NO: 1). The protein drink of this study can be consumed as is or formulated with ingredients found in other commonly used protein shakes. As shown in FIG. 1, the addition of protease can reduce the post-ingestion blood glucose spike by more than half at the blood glucose peak. FIG. 1 shows a comparison of the post-ingestion blood glucose levels resulting from the ingestion of (i) a protein drink containing prokumamolisin and pea protein versus (ii) a protein drink without prokumamolisin and pea protein.

[0091] Example 2 - Activity of S53 protease at low pH In general, the S53 proteases described and used herein should have the ability to completely digest proteins (e.g., legume proteins) in the acidic environment of the stomach. Thus, the S53 proteases used and described herein should have activity over the entire postprandial pH range of the stomach environment. pH profile data was generated for ten representative S53 proteases (including SEQ ID NOs: 1 and 3-11). The representative S53 proteases exhibited optimal activity (e.g., 100% or substantially active) over the entire range of pH 2.5-4.5. Figure 6 shows the proteolytic activity of S53 proteases 1-10 (P1-P10) over pH 2-5.

[0092] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It will be understood that various alternatives to the embodiments described herein may be employed in the practice of the present invention. It is intended that the following claims define the scope of the embodiments disclosed herein, and that methods and structures falling within the scope of these claims and their equivalents are covered thereby.

[0093] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

Claims

1. 1. A method of reducing blood glucose in a subject, comprising: For the subject, Legume proteins, and S53 family proteases administering a composition comprising The administering reduces blood glucose in the subject.

2. A method for suppressing an increase in blood glucose in a subject, comprising: For the subject, Legume proteins, and S53 family proteases administering a composition comprising The method, wherein the administration suppresses an increase in blood glucose in the subject.

3. 3. The method of claim 1 or 2, wherein the S53 family protease comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 and 3-11.

4. 3. The method of claim 1 or 2, wherein the S53 family protease comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 and 3-11.

5. 3. The method of claim 1 or 2, wherein the S53 family protease comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1 and 3-11.

6. 3. The method of claim 1 or 2, wherein the S53 family protease comprises an amino acid sequence of any one of SEQ ID NOs: 1 and 3-11.

7. 7. The method of any one of claims 1 to 6, wherein the S53 family protease comprises an active site comprising amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO:

1.

8. 7. The method of any one of claims 1 to 6, wherein the S53 family protease comprises an active site that comprises one or more amino acid substitutions of residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO:

1.

9. The method of claim 8, wherein the active site comprises 1 to 5 amino acid substitutions.

10. 7. The method of any one of claims 1 to 6, wherein the S53 family protease comprises one or more truncations of SEQ ID NO:1, wherein the one or more truncations comprise an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

11. 11. The method of any one of claims 1 to 10, wherein the S53 family protease is active in the pH range from about pH 2 to about pH 5.

12. The method of any one of claims 1 to 10, wherein the S53 family protease is active at a pH below pH 5.

13. The method according to any one of claims 1 to 12, wherein the legume protein is a pea protein.

14. 14. The method of claim 13, wherein the pea protein is derived from pea.

15. 14. The method of claim 13, wherein the pea is a whole pea or a component thereof.

16. 16. The method of claim 13 or 15, wherein the pea is a standard pea, a commercial pea, a genetically modified pea, or a combination thereof.

17. 16. The method of claim 13 or 15, wherein the peas are round peas, wrinkled peas, or a combination thereof.

18. 16. The method of claim 13 or 15, wherein the pea is garden pea, sugar pea, field pea, or any combination thereof.

19. 10. The method of claim 1, wherein said administering occurs after said subject has ingested a food product containing sugar.

20. 3. The method of claim 2, wherein the rise in blood glucose is suppressed compared to administration of a composition that does not contain the S53 family protease.

21. The method of any one of claims 1 to 20, wherein the subject self-administers the composition.

22. The method according to any one of claims 1 to 21, wherein the subject is a mammal.

23. 23. The method of claim 22, wherein the mammal is a human.

24. 24. The method of claim 23, wherein the human has hyperglycemia.

25. 23. The method of claim 22, wherein the human has a condition associated with and / or caused by hyperglycemia.

26. 26. The method of claim 25, wherein the condition is selected from the group consisting of cardiovascular disease, neuropathy, diabetic nephropathy, retinopathy, cataracts, bone and joint disorders, dental infections, and gum infections.

27. 27. The method of any one of claims 1 to 26, further comprising administering a low or reduced carbohydrate diet.

28. 28. The method of any one of claims 1 to 27, further comprising reducing caloric intake from carbohydrates.

29. 30. The method of claim 28, wherein the amount comprises total grams of carbohydrates or calories from carbohydrates.

30. 13. A composition for use in reducing blood glucose in a subject, the composition comprising a legume protein and a protease of the S53 family.

31. A composition for use in inhibiting an increase in blood glucose in a subject, the composition comprising a legume protein and a protease of the S53 family.

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