Method and composition for stabilizing blood glucose levels

Abstract

To provide compositions and methods useful for regulating hyperglycemia (e.g., reducing, stabilizing, etc.). [Solution] The present invention provides a method for reducing blood glucose, suppressing the rise in blood glucose, and / or lowering the glycemic index of a food in a subject, comprising administering to the subject a composition containing leguminous protein and S53 protease. The present invention provides a composition containing leguminous protein and / or S53 protease for reducing blood glucose, suppressing the rise in blood glucose, and / or lowering the glycemic index of a food in a subject.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 303,112, filed Jan. 26, 2022. The entire disclosure of the provisional application is incorporated herein by reference.

Background Art

[0002] Hyperglycemia is well known to have a great adverse impact on health. Long-term complications due to hyperglycemia can range from cardiovascular diseases, neuropathy, damage to the kidneys and blood vessels to disorders of the bones and joints. The most commonly used method for controlling blood glucose levels is by controlling dietary carbohydrate intake. This is a sound and proven strategy, but individuals need to pay a lot of attention to understand and calculate the carbohydrate intake per meal. Drugs such as insulin are widely used, but many problems can accompany the effective control of blood glucose levels by dosing.

Summary of the Invention

[0003] Despite the increasing understanding of the biology and etiology of diseases associated with or resulting from hyperglycemia, there is still a great burden on patients who cannot regulate their blood glucose levels regardless of any intervention. Both drug therapy (e.g., insulin hormone) and non-drug therapy (e.g., diet monitoring) can be considered substantial interventions that have essential barriers to the effective treatment and management of hyperglycemia-related diseases. Such essential barriers include, for example, compliance with treatment (e.g., due to the complexity of treatment), economic burden and access (e.g., out-of-pocket costs for both insured and uninsured individuals), as well as other patient-related, prescription-related, and prescriber-related factors. The impact of such barriers and the resulting hyperglycemia-related diseases on treatment impose a significant health and economic burden on individuals and health management systems.

[0004] Provided and described herein are compositions and methods useful for regulating hyperglycemia (e.g., reducing, stabilizing, etc.). The methods and compositions provided are based in part on the remarkable discovery that regulation of hyperglycemia can be achieved by administering leguminous proteins (e.g., pea proteins) and S53 family proteases to individuals. In some embodiments, the S53 family protease is procumamorisin. In some embodiments, the S53 protease (e.g., procumamorisin) 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., procumamorisin) 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., procumamorisin) 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 includes the 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 of less than approximately pH 5 (as measured, for example, by protein digestion). In some embodiments, the S53 protease is active at a pH of less than approximately pH 4.5. In some embodiments, the S53 protease is active at a pH of less than approximately pH 5. In some embodiments, the S53 protease is active at a pH of less than approximately pH 4. In some embodiments, the S53 protease is active at a pH of less than approximately pH 3.5. In some embodiments, the S53 protease is active at a pH of less than approximately pH 3.

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

[0007] In some cases, the regulation of protein digestion patterns (e.g., by administration of leguminous proteins and S53 proteases (e.g., procoumamorisin)) increases the concentration of certain food amino acids in the blood. In such cases, the regulation of protein digestion patterns can induce various physiological responses, such as a decrease in blood glucose levels. In some cases, digesting food proteins in the intestines by administering, for example, leguminous proteins and S53 proteases (e.g., procoumamorisin) produces bioactive peptides that have potent biological responses, including antidiabetic effects (e.g., a decrease in blood glucose levels). As described and provided herein, a food composed of protein foods (e.g., leguminous proteins) and acid proteases (e.g., procoumamorisin) can lower blood glucose levels (e.g., when ingested with sugar). Furthermore, in some cases, the addition of protein foods (e.g., leguminous proteins) and acid proteases (e.g., procoumamorisin) lowers the glycemic index of a food (e.g., a food containing sugar).

[0008] Provided and described herein is a method for reducing blood glucose in a subject, the method comprising administering to the subject a composition comprising a leguminous protein and an S53 protease (e.g., procumamorisin). Similarly provided is a method for suppressing an increase in blood glucose in a subject, the method comprising administering to the subject a composition comprising a leguminous protein and an S53 protease (e.g., procumamorisin).

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

[0010] In some embodiments, the S53 protease (e.g., procumamorisin) includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., procumamorisin) includes an active site comprising the 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., procumamorisin) includes an active site comprising 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 1 to 5 amino acid substitutions. In some embodiments, the S53 protease (e.g., procumamorisin) comprises one or more cleavages of SEQ ID NO: 1, wherein the one or more cleavages include N-terminal cleavage, C-terminal cleavage, or cleavage of both the N-terminal and C-terminal.

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

[0012] In some embodiments, the administration is performed after the subject has ingested a food containing sugar. In some embodiments, the rise in blood glucose is suppressed compared to when a composition without the S53 protease (e.g., procumamorisin) is administered. 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 a cardiovascular disease or neuropathy or diabetic nephropathy or retinopathy or cataracts or bone and joint disorders or dental and gingival infections.

[0014] Also provided is a composition used to reduce blood glucose in a subject, the composition comprising a leguminous protein and an S53 protease (e.g., procumamorisin). Further provided is a composition used to suppress the rise in blood glucose in a subject, the composition comprising a leguminous protein and an S53 protease (e.g., procumamorisin).

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

[0016] The novel features of the present invention, along with their specific characteristics, are described in the appended claims. A further understanding of the features and advantages of the present invention can be obtained by referring to the following embodiments for carrying out the invention, which describe exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0017] [Figure 1] This shows a decrease in blood glucose levels after administration of leguminous proteins and S53 proteases (e.g., procumamorisin). [Figure 2] This shows a decrease in blood glucose levels after administration of a low-carbohydrate protein beverage and protease. [Figure 3] This shows a decrease in blood glucose levels after administration of a protein-fruit smoothie and protease. [Figure 4] This shows the proteolytic activity of a typical S53 protease in the low pH range. [Modes for carrying out the invention]

[0018] Dysregulation of blood glucose and / or the presence of hyperglycemia are associated with and / or cause a variety of diseases affecting human health and well-being. Such diseases (e.g., diabetes mellitus) tend to be complex conditions with troublesome associated symptoms 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 individuals. In particular, in some cases, the methods and compositions provided herein are based on the discovery that blood glucose levels are lowered (e.g., reduced or suppressed) by administering leguminous proteins (e.g., pea proteins) and S53 proteases (e.g., procumamorisin). Furthermore, in some cases, the methods and compositions provided herein are useful in inhibiting, suppressing, reducing, and / or preventing increases in blood glucose levels (e.g., blood glucose levels).

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

[0020] In some embodiments, the legume protein is pea protein. Pea generally refers to, and includes, the seeds or outer 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 pea plants. Both intact pea protein and hydrolyzed pea protein sources can be used. In some embodiments, the pea protein is intact legume protein. In some embodiments, the pea protein is hydrolyzed legume protein. In some embodiments, the pea protein is provided in solid form. In some embodiments, the pea protein is provided in liquid form. In some embodiments, the pea protein is a concentrate (e.g., protein material obtained from peas after removal of soluble carbohydrates, ash, and other trace components). In certain embodiments, the pea is garden pea, sugar pea, field pea, or any combination thereof. In certain embodiments, the pea is garden pea. In certain embodiments, the pea is sugar pea. In certain embodiments, the pea is a field pea. In certain embodiments, the pea is any combination of garden peas, sugar peas, and / or field peas. 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] protease The compositions and methods described herein generally utilize acidic proteases. In some embodiments, the acidic protease is a protease of the S53 family. The S53 family of proteases generally refers to and includes a family of serine proteases found in prokaryotes and eukaryotes. In some embodiments, the S53 family of proteases refers to and includes proteases within and / or identified by the MEROPS accession MER0000995 (e.g., sedolisin, sedolisin-b, tripeptidyl peptidase I, coumamorisin, coumamorisin-B, physarolisin, aorsin, physarolisin II, coumamorisin-As, grifolisin, scytalidolisin). In some embodiments, the acidic protease is an S53 protease (e.g., procoumamorisin). Procumamorisin generally refers to, and includes, a heat-stable calcium-dependent endopeptidase derived from the acidic / thermophilic Bacillus (Bacillus novosp. MN-32). In some embodiments, procumamorisin refers to, and includes, the NCBI gene ID: 18765799 (NCBI reference sequences XP_007297753.1, XM_007297691.1~XP_007297753, and / or NW_006763082.1 (137488..139728)).

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

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

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

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

[0026] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 5.

[0027] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 6.

[0028] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 7.

[0029] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 8.

[0030] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 9.

[0031] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 10.

[0032] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 11.

[0033] In some embodiments, the active site of the S53 protease includes the 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 the percentage of identity or similarity between two sequences can be achieved using mathematical algorithms. In some embodiments, a non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, which is 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 in Altschul et al., 1990, J. Mol. Biol. 215:403-410. Alternatively, in some embodiments, PSI-Blast can be used to perform iterative searches to detect distance relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used. In some embodiments, a non-restrictive example of a mathematical algorithm used for sequence comparison 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 described in Torellis and Robotti, 1994, Comput.Appl.Biosci.10:3-5, and FASTA described in Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA85:2444-8. In some embodiments, sequence alignment is performed using the CLUSTAL algorithm described in Higgins et al., 1996, Methods Enzymol.266:383-402.

[0035] In some embodiments, the active site of the S53 protease (e.g., procumamorisin) includes the 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., procumamorisin) includes an active site comprising one or more amino acid substitutions of the 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 1 to 5 amino acid substitutions.

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

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

[0038] Amino acids generally refer to and / or include natural amino acids, non-natural amino acids, amino acid analogs and amino acid mimetics that function similarly to natural amino acids. In this specification, amino acids are generally referred to by their names, widely known three-letter symbols, or single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. As used herein, natural amino acids include and / or include amino acids that are commonly found in nature and have not been manipulated by humans. In some embodiments, naturally occurring substances include, and / or further refer to, 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, nonpolar amino acids can be substituted and exchanged with other nonpolar amino acids, such as alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. In some embodiments, neutrally charged amino acids can be substituted and exchanged with other neutrally charged amino acids, such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In some embodiments, positively charged amino acids can be substituted and exchanged with other positively charged amino acids, such as arginine, lysine, and histidine. In some embodiments, positively charged amino acids can be substituted and exchanged with other positively charged amino acids, such as aspartic acid and glutamic acid. As used herein, peptides include and / or refer to any of a variety of natural or synthetic compounds comprising two or more amino acids linked by a peptide bond that links the carboxyl group of one amino acid to the amino group of another amino acid. Similarly, as used herein, amino acids refer to and / or include natural amino acids, non-natural amino acids, amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Amino acids are generally referred herein by their names, widely known three-letter symbols, or single-letter symbols as recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0040] In some embodiments, the active site includes 1 to 5 amino acid substitutions. In some embodiments, the S53 protease (e.g., procumamorisin) includes one or more cleavages of SEQ ID NO: 1, where the one or more cleavages include N-terminal cleavage, C-terminal cleavage, or cleavage of both the N-terminal and C-terminal.

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

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

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

[0044] In some embodiments, the provided composition comprises a food, a leguminous protein (e.g., pea protein), and an S53 protease (e.g., procumamorisin) (e.g., any one of the S53 proteases described herein (e.g., procumamorisin)). In certain embodiments, the food comprises a sugar. In certain embodiments, the sugar comprises sucrose, lactose, maltose, or another disaccharide, trisaccharide, or polysaccharide containing 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 containing glucose as a monomer. In some cases, the addition of the leguminous protein (e.g., pea protein) and the S53 protease (e.g., procumamorisin) lowers the glycemic index of the composition comprising the food. In certain embodiments, a composition comprising the food, the leguminous protein, and the S53 protease (e.g., procoumamorisin) results in a lower rise in blood glucose after ingestion than a second composition comprising the food alone.

[0045] In some embodiments, the composition contains about 5 grams (g) to about 60 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition contains about 5 grams (g) to about 10 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) to about 15 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) to about 20 grams (g) of legume protein (e.g., pea protein), and about 5 grams (g) of Leguminous protein (e.g., pea protein) - approximately 25 grams (g) of leguminous protein (e.g., pea protein), approximately 5 grams (g) of leguminous protein (e.g., pea protein) - approximately 30 grams (g) of leguminous protein (e.g., pea protein), approximately 5 grams (g) of leguminous protein (e.g., pea protein) - approximately 35 grams (g) of leguminous protein (e.g., pea protein), approximately 5 grams (g) of leguminous protein (e.g., pea protein) - approximately 40 grams 1 gram of legume protein (e.g., pea protein), approximately 5 grams of legume protein (e.g., pea protein) ~ approximately 45 grams of legume protein (e.g., pea protein), approximately 5 grams of legume protein (e.g., pea protein) ~ approximately 50 grams of legume protein (e.g., pea protein), approximately 5 grams of legume protein (e.g., pea protein) ~ approximately 60 grams of legume protein (e.g., pea protein) ), approximately 10 grams (g) of legume protein (e.g., pea protein) to approximately 15 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein) to approximately 20 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein) to approximately 25 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g.,Pea protein) ~ approximately 30 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein) ~ approximately 35 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein) ~ approximately 40 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein) ~ approximately 45 grams (g) of legume protein (e.g., pea protein), Approximately 10 grams (g) of legume protein (e.g., pea protein) to approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein) to approximately 60 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein) to approximately 20 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein) to approximately 25 grams (g) of Leguminous protein (e.g., pea protein), approximately 15 grams (g) of leguminous protein (e.g., pea protein) ~ approximately 30 grams (g) of leguminous protein (e.g., pea protein), approximately 15 grams (g) of leguminous protein (e.g., pea protein) ~ approximately 35 grams (g) of leguminous protein (e.g., pea protein), approximately 15 grams (g) of leguminous protein (e.g., pea protein) ~ approximately 40 grams (g) of leguminous protein (e.g., pea protein), approximately 15 grams (g) of leguminous protein (e.g., , pea protein) ~ approximately 45 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein) ~ approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein) ~ approximately 60 grams (g) of legume protein (e.g., pea protein), approximately 20 grams (g) of legume protein (e.g., pea protein) ~ approximately 25 grams (g) of legume protein (e.g., pea protein),Approximately 20 grams (g) of legume protein (e.g., pea protein) to approximately 30 grams (g) of legume protein (e.g., pea protein), approximately 20 grams (g) of legume protein (e.g., pea protein) to approximately 35 grams (g) of legume protein (e.g., pea protein), approximately 20 grams (g) of legume protein (e.g., pea protein) to approximately 40 grams (g) of legume protein (e.g., pea protein), approximately 20 grams (g) of legume protein (e.g., pea protein) to approximately 45 grams 1 gram of legume protein (e.g., pea protein), approximately 20 grams (g) of legume protein (e.g., pea protein) ~ approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 20 grams (g) of legume protein (e.g., pea protein) ~ approximately 60 grams (g) of legume protein (e.g., pea protein), approximately 25 grams (g) of legume protein (e.g., pea protein) ~ approximately 30 grams (g) of legume protein (e.g., pea protein), approximately 25 grams (g) of Leguminous protein (e.g., pea protein) - approximately 35 grams (g) of leguminous protein (e.g., pea protein), approximately 25 grams (g) of leguminous protein (e.g., pea protein) - approximately 40 grams (g) of leguminous protein (e.g., pea protein), approximately 25 grams (g) of leguminous protein (e.g., pea protein) - approximately 45 grams (g) of leguminous protein (e.g., pea protein), approximately 25 grams (g) of leguminous protein (e.g., pea protein) - approximately 50 grams (g) of leguminous protein Quality (e.g., pea protein), approximately 25 grams (g) of legume protein (e.g., pea protein) ~ approximately 60 grams (g) of legume protein (e.g., pea protein), approximately 30 grams (g) of legume protein (e.g., pea protein) ~ approximately 35 grams (g) of legume protein (e.g., pea protein), approximately 30 grams (g) of legume protein (e.g., pea protein) ~ approximately 40 grams (g) of legume protein (e.g., pea protein), approximately 30 grams (g) of legume protein (e.g.,Pea protein) ~ approximately 45 grams (g) of legume protein (e.g., pea protein), approximately 30 grams (g) of legume protein (e.g., pea protein), approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 30 grams (g) of legume protein (e.g., pea protein), approximately 60 grams (g) of legume protein (e.g., pea protein), approximately 35 grams (g) of legume protein (e.g., pea protein), approximately 40 grams (g) of legume protein (e.g., pea protein), approximately 35 grams (g) of legume protein (e.g., pea protein) to approximately 45 grams (g) of legume protein (e.g., pea protein), approximately 35 grams (g) of legume protein (e.g., pea protein) to approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 35 grams (g) of legume protein (e.g., pea protein) to approximately 60 grams (g) of legume protein Bean protein (e.g., pea protein), approximately 40 grams (g) of legume protein (e.g., pea protein) ~ approximately 45 grams (g) of legume protein (e.g., pea protein), approximately 40 grams (g) of legume protein (e.g., pea protein) ~ approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 40 grams (g) of legume protein (e.g., pea protein) ~ approximately 60 grams (g) of legume protein (e.g., pea protein) The composition includes approximately 45 grams (g) of legume protein (e.g., pea protein) to approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 45 grams (g) of legume protein (e.g., pea protein) to approximately 60 grams (g) of legume protein (e.g., pea protein), or approximately 50 grams (g) of legume protein (e.g., pea protein) to approximately 60 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition includes approximately 5 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein),Contains approximately 20 grams (g) of legume protein (e.g., pea protein), approximately 25 grams (g) of legume protein (e.g., pea protein), approximately 30 grams (g) of legume protein (e.g., pea protein), approximately 35 grams (g) of legume protein (e.g., pea protein), approximately 40 grams (g) of legume protein (e.g., pea protein), approximately 45 grams (g) of legume protein (e.g., pea protein), approximately 50 grams (g) of legume protein (e.g., pea protein), or approximately 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 may contain 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.,Contains pea protein.

[0046] In some embodiments, the composition comprises about 50 milligrams (mg) to about 2,000 milligrams (mg) of procomamorisin. In some embodiments, the composition comprises about 50 milligrams (mg) to about 100 milligrams (mg) of procomamorisin, about 50 milligrams (mg) to about 200 milligrams (mg) of procomamorisin, about 50 milligrams (mg) to about 300 milligrams (mg) of procomamorisin, about 50 milligrams (mg) to about 400 milligrams (mg) of procomamorisin, and about 50 milligrams (mg) of procomamorisin. ~Approximately 500 mg (mg) of procomamorisin, approximately 50 mg (mg) of procomamorisin ~Approximately 750 mg (mg) of procomamorisin, approximately 50 mg (mg) of procomamorisin ~Approximately 1,000 mg (mg) of procomamorisin, approximately 50 mg (mg) of procomamorisin ~Approximately 1,250 mg (mg) of procomamorisin, approximately 50 mg (mg) of procomamorisin ~Approximately 1,500 mg (mg) of procomamorisin, approximately 50 mg ( Procomamorisin (mg) ~ approximately 2,000 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin ~ approximately 200 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin ~ approximately 300 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin ~ approximately 400 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin ~ approximately 500 mg (mg) of procomamorisin Procomamorisin, approximately 100 mg (mg) of procomamorisin to approximately 750 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin to approximately 1,000 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin to approximately 1,250 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin to approximately 1,500 mg (mg) of procomamorisin, approximately 100 mg (mg) of procomamorisin to approximately 2,000 mg procoumamorisin, approximately 200 mg procoumamorisin to approximately 300 mg procoumamorisin, approximately 200 mg procoumamorisin to approximately 400 mg procoumamorisin, approximately 200 mg procoumamorisin to approximately 500 mg procoumamorisin, approximately 200 mg procoumamorisin to approximately 750 mg procoumamorisin, approximately 200 mg procoumamorisin to approximately 1,000 mg procoumamorisin Coumamorisin, approximately 200 mg (mg) of procoumamorisin to approximately 1,250 mg (mg) of procoumamorisin, approximately 200 mg (mg) of procoumamorisin to approximately 1,500 mg (mg) of procoumamorisin, approximately 200 mg (mg) of procoumamorisin to approximately 2,000 mg (mg) of procoumamorisin, approximately 300 mg (mg) of procoumamorisin to approximately 400 mg (mg) of procoumamorisin, approximately 300 mg (mg) of procoumamorisin to approximately 500 mg (mg) of procoumamorisin, approximately 300 mg 1 mg of procomamorisin ~ approximately 750 mg of procomamorisin, approximately 300 mg of procomamorisin ~ approximately 1,000 mg of procomamorisin, approximately 300 mg of procomamorisin ~ approximately 1,250 mg of procomamorisin, approximately 300 mg of procomamorisin ~ approximately 1,500 mg of procomamorisin, approximately 300 mg of procomamorisin ~ approximately 2,000 mg of procomamorisin, approximately 400 mg of Procomamorisin ~ approximately 500 mg (mg) of procomamorisin, approximately 400 mg (mg) of procomamorisin ~ approximately 750 mg (mg) of procomamorisin, approximately 400 mg (mg) of procomamorisin ~ approximately 1,000 mg (mg) of procomamorisin, approximately 400 mg (mg) of procomamorisin ~ approximately 1,250 mg (mg) of procomamorisin, approximately 400 mg (mg) of procomamorisin ~ approximately 1,500 mg (mg) of procomamorisin, approximately 400 mg (mg) of procomamorisin ~ approximately 2,000 mg procomamorisin, approximately 500 mg procomamorisin to approximately 750 mg procomamorisin, approximately 500 mg procomamorisin to approximately 1,000 mg procomamorisin, approximately 500 mg procomamorisin to approximately 1,250 mg procomamorisin, approximately 500 mg procomamorisin to approximately 1 500 mg (mg) of procomamorisin, approximately 500 mg (mg) of procomamorisin to approximately 2,000 mg (mg) of procomamorisin, approximately 750 mg (mg) of procomamorisin to approximately 1,000 mg (mg) of procomamorisin, approximately 750 mg (mg) of procomamorisin to approximately 1,250 mg (mg) of procomamorisin, approximately 750 mg (mg) of procomamorisin ~Approximately 1,500 mg (mg) of procomamorisin, approximately 750 mg (mg) of procomamorisin, ~Approximately 2,000 mg (mg) of procomamorisin, approximately 1,000 mg (mg) of procomamorisin, ~Approximately 1,250 mg (mg) of procomamorisin, approximately 1,000 mg (mg) of procomamorisin, ~Approximately 1,500 mg (mg) of procomamorisin, approximately 1,000 mg (mg) of procomamorisin Contains procomamorisin ~ approximately 2,000 milligrams (mg) of procomamorisin, approximately 1,250 milligrams (mg) of procomamorisin ~ approximately 1,500 milligrams (mg) of procomamorisin, approximately 1,250 milligrams (mg) of procomamorisin ~ approximately 2,000 milligrams (mg) of procomamorisin, or approximately 1,500 milligrams (mg) of procomamorisin ~ approximately 2,000 milligrams (mg) of procomamorisin. In some embodiments, the composition contains about 50 mg of procomamorisin, about 100 mg of procomamorisin, about 200 mg of procomamorisin, about 300 mg of procomamorisin, about 400 mg of procomamorisin, about 500 mg of procomamorisin, about 750 mg of procomamorisin, about 1,000 mg of procomamorisin, about 1,250 mg of procomamorisin, and about 1,The composition contains 500 milligrams (mg) of procomamorisin, or about 2,000 milligrams (mg) of procomamorisin. In some embodiments, the composition contains at least about 50 milligrams (mg) of procomamorisin, about 100 milligrams (mg) of procomamorisin, about 200 milligrams (mg) of procomamorisin, about 300 milligrams (mg) of procomamorisin, about 400 milligrams (mg) of procomamorisin, about 500 milligrams (mg) of procomamorisin, about 750 milligrams (mg) of procomamorisin, about 1,000 milligrams (mg) of procomamorisin, about 1,250 milligrams (mg) of procomamorisin, or about 1,500 milligrams (mg) of procomamorisin. In some embodiments, the composition comprises at most about 100 milligrams (mg) of procomamorisin, about 200 milligrams (mg) of procomamorisin, about 300 milligrams (mg) of procomamorisin, about 400 milligrams (mg) of procomamorisin, about 500 milligrams (mg) of procomamorisin, about 750 milligrams (mg) of procomamorisin, about 1,000 milligrams (mg) of procomamorisin, about 1,250 milligrams (mg) of procomamorisin, about 1,500 milligrams (mg) of procomamorisin, or about 2,000 milligrams (mg) of procomamorisin.

[0047] method As described herein, in some cases, blood glucose levels are lowered (e.g., reduced or suppressed) by administering a leguminous protein (e.g., pea protein) and an S53 protease (e.g., procomamorisin). Provided herein is a method for reducing blood glucose in a subject, the method comprising administering to the subject a composition comprising a leguminous protein and an S53 protease (e.g., procomamorisin) (e.g., the leguminous protein and the S53 protease (e.g., procomamorisin) are administered to the subject). Further provided are leguminous proteins and an S53 protease (e.g., procomamorisin) used in a method for reducing blood glucose in a subject (e.g., the leguminous protein and the S53 protease (e.g., procomamorisin) are administered to the subject).

[0048] Also provided is a method for suppressing 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., procomamorisin). Further provided is the use of a legume protein and an S53 protease (e.g., procomamorisin) in a method for suppressing an increase in blood glucose in a subject (e.g., the legume protein and the S53 protease (e.g., procomamorisin) are administered to the subject). Further provided is a legume protein and an S53 protease (e.g., procomamorisin) used in a method for suppressing an increase in blood glucose in a subject (e.g., the legume protein and the S53 protease (e.g., procomamorisin) are administered to the subject).

[0049] In some cases, administering a leguminous protein (e.g., pea protein) and an S53 protease (e.g., procumamorisin) stabilizes blood glucose levels associated with food intake (e.g., the range or degree of change is reduced or prevented). In some embodiments, the foregoing provides a method for suppressing changes (positive or negative) in blood glucose in a subject, the method comprising administering to the subject a composition comprising a leguminous protein and an S53 protease (e.g., procumamorisin) (e.g., the leguminous protein and the S53 protease (e.g., procumamorisin) are administered to the subject).

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

[0051] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 1.

[0052] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 2. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 2. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 2. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 2. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 2. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 2. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 2. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 2.

[0053] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 3. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 3. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 3. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 3. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 3. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 3. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 3. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 3.

[0054] In some embodiments, the S53 protease contains the amino acid sequence described in SEQ ID NO: 4. In some embodiments, the S53 protease contains an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 4. In some embodiments, the S53 protease contains an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 4. In some embodiments, the S53 protease contains an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 4. In some embodiments, procumamorisin contains an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 4. In some embodiments, the S53 protease contains an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 4. In some embodiments, the S53 protease contains an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 4. In some embodiments, the S53 protease contains an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 4.

[0055] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 5. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 5.

[0056] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 6. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 6.

[0057] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 7. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 7.

[0058] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 8. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 8.

[0059] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 9. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 9.

[0060] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 10. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 10.

[0061] In some embodiments, the S53 protease includes the amino acid sequence described in SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 90% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease includes an amino acid sequence having 99% or more sequence identity with SEQ ID NO: 11.

[0062] In some embodiments, the active site of the S53 protease includes the 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 described herein (e.g., procumamorisin) can be used in the provided method. In some embodiments, the S53 protease (e.g., procumamorisin) includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., procumamorisin) includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., procumamorisin) includes an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 1.

[0064] In some embodiments, the S53 protease (e.g., procumamorisin) contains the amino acids of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., procumamorisin) contains an active site comprising the 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., procumamorisin) includes an active site comprising 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 1 to 5 amino acid substitutions. In some embodiments, the S53 protease (e.g., procumamorisin) comprises one or more cleavages of SEQ ID NO: 1, wherein the one or more cleavages include N-terminal cleavage, C-terminal cleavage, or cleavage of both the N-terminal and C-terminal.

[0065] In some embodiments, the dosage of the S53 protease (e.g., procumamorisin) ranges from about 50 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the dosage of the S53 protease (e.g., procumamorisin) ranges from 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), and about 50 milligrams (mg) to about 7 50 milligrams (mg), approximately 50 milligrams (mg) to approximately 1,000 milligrams (mg), approximately 50 milligrams (mg) to approximately 1,500 milligrams (mg), approximately 100 milligrams (mg) to approximately 150 milligrams (mg), approximately 100 milligrams (mg) to approximately 200 milligrams (mg), approximately 100 milligrams (mg) to approximately 300 milligrams (mg), approximately 100 milligrams (mg) to approximately 400 milligrams (mg), approximately 100 milligrams (mg) to approximately 500 milligrams (mg), approximately 100 milligrams (mg) ~ approximately 750 mg (mg), approximately 100 mg (mg) ~ approximately 1,000 mg (mg), approximately 100 mg (mg) ~ approximately 1,500 mg (mg), approximately 150 mg (mg) ~ approximately 200 mg (mg), approximately 150 mg (mg) ~ approximately 300 mg (mg), approximately 150 mg (mg) ~ approximately 400 mg (mg), approximately 150 mg (mg) ~ approximately 500 mg (mg), approximately 150 mg (mg) ~ approximately 750 mg (mg), Approximately 150 mg to approximately 1,000 mg, approximately 150 mg to approximately 1,500 mg, approximately 200 mg to approximately 300 mg, approximately 200 mg to approximately 400 mg, approximately 200 mg to approximately 500 mg, approximately 200 mg to approximately 750 mg, approximately 200 mg to approximately 1,000 mg, approximately 200 mg to approximately 1,500 mg, approximately 300 mg to approximately 400 mg, approximately 300 mg to approximately 500 mg, approximately 300 mg to approximately 750 mg, approximately 300 mg to approximately 1,000 mg, approximately 300 mg to approximately 1,500 mg, approximately 400 mg to approximately 500 mg, approximately 400 mg to approximately 750 mg, approximately 400 mg to approximately 1 Contains 1,000 milligrams (mg), approximately 400 milligrams (mg) to approximately 1,500 milligrams (mg), approximately 500 milligrams (mg) to approximately 750 milligrams (mg), approximately 500 milligrams (mg) to approximately 1,000 milligrams (mg), approximately 500 milligrams (mg) to approximately 1,500 milligrams (mg), approximately 750 milligrams (mg) to approximately 1,000 milligrams (mg), approximately 750 milligrams (mg) to approximately 1,500 milligrams (mg), or approximately 1,000 milligrams (mg) to approximately 1,500 milligrams (mg). In some embodiments, the dosage of the S53 protease (e.g., procumamoricin) includes 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., procumamoricin) includes 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 dose of the S53 protease (e.g., procumamoricin) 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,Contains 500 milligrams (mg).

[0066] In some embodiments, the dosage of the legume protein includes about 5 grams (g) to about 50 grams (g). In some embodiments, the dosage of the legume protein includes 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), and about 10 grams (g) to about 20 grams (g). Grams (g), approximately 10 grams (g) to approximately 25 grams (g), approximately 10 grams (g) to approximately 30 grams (g), approximately 10 grams (g) to approximately 35 grams (g), approximately 10 grams (g) to approximately 40 grams (g), approximately 10 grams (g) to approximately 45 grams (g), approximately 10 grams (g) to approximately 50 grams (g), approximately 15 grams (g) to approximately 20 grams (g), approximately 15 grams (g) to approximately 25 grams (g), approximately 15 grams (g) to approximately 30 grams (g), approximately 15 grams (g) to approximately 35 grams (g), approximately 15 grams (g) to approximately 40 grams (g), approximately 15 grams Lamb (g) ~ approx. 45 grams (g), approx. 15 grams (g) ~ approx. 50 grams (g), approx. 20 grams (g) ~ approx. 25 grams (g), approx. 20 grams (g) ~ approx. 30 grams (g), approx. 20 grams (g) ~ approx. 35 grams (g), approx. 20 grams (g) ~ approx. 40 grams (g), approx. 20 grams (g) ~ approx. 45 grams (g), approx. 20 grams (g) ~ approx. 50 grams (g), approx. 25 grams (g) ~ approx. 30 grams (g), approx. 25 grams (g) ~ approx. 35 grams (g), approx. 25 grams (g) ~ approx. 40 grams (g), approx. 25 grams (g) ~ approx. 45 grams (g) Contains 1g of sugar, approximately 25g to 50g, approximately 30g to 35g, approximately 30g to 40g, approximately 30g to 45g, approximately 30g to 50g, approximately 35g to 40g, approximately 35g to 45g, approximately 35g to 50g, approximately 40g to 45g, approximately 40g to 50g, or approximately 45g to 50g.In some embodiments, the dosage of the legume protein includes 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 the legume protein includes 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 the legume protein includes 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 pea protein. In some embodiments, the pea is the whole pea or its components. In certain embodiments, the pea is garden pea, sugar pea, field pea, or any combination thereof. In certain embodiments, the pea is garden pea. In certain embodiments, the pea is sugar pea. In certain embodiments, the pea is 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 standard pea, commercial pea, genetically modified pea, or a combination thereof. In certain embodiments, the pea is round pea, wrinkled pea, or a combination thereof.

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

[0069] In some embodiments, the administration is performed after the subject has ingested a food containing sugar. In some embodiments, the sugar includes sucrose, lactose, maltose, or another disaccharide, trisaccharide, or polysaccharide containing glucose as a monomer. In certain embodiments, the sugar includes sucrose. In certain embodiments, the sugar includes lactose. In certain embodiments, the sugar includes maltose. In certain embodiments, the sugar includes a disaccharide, trisaccharide, or polysaccharide containing glucose as a monomer.

[0070] In some embodiments, the increase in blood glucose is suppressed compared to when a composition without the S53 protease (e.g., procumamorisin) is administered. 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 related to and / or caused by hyperglycemia. In some embodiments, the condition is a cardiovascular disease or neuropathy or diabetic nephropathy or retinopathy or cataract or bone and joint disorder or dental and gingival infection. 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 retinopathy. In certain embodiments, the condition is 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 gingival infection.

[0071] Furthermore, a method for lowering the glycemic index of a food is provided, the method comprising supplying the food with a leguminous protein and an S53 protease (e.g., procomamorisin). Furthermore, the use of a leguminous protein and an S53 protease (e.g., procomamorisin) for lowering the glycemic index of a food is provided. Furthermore, leguminous protein and an S53 protease (e.g., procomamorisin) used for lowering the glycemic index of a food is also provided.

[0072] Any of the S53 proteases described herein (e.g., procumamorisin) can be used in the methods provided. In some embodiments, the S53 protease (e.g., procumamorisin) comprises an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the S53 protease (e.g., procumamorisin) comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the S53 protease (e.g., procumamorisin) comprises an amino acid sequence having at least 98% sequence identity with any one of SEQ ID NOs: 1 and 3-11. In some embodiments, the active site of the S53 protease includes the 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., procumamorisin) contains the amino acids of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., procumamorisin) contains an active site comprising the 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., procumamorisin) includes an active site comprising 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 1 to 5 amino acid substitutions. In some embodiments, the S53 protease (e.g., procumamorisin) comprises one or more cleavages of SEQ ID NO: 1, wherein the one or more cleavages include N-terminal cleavage, C-terminal cleavage, or cleavage of both the N-terminal and C-terminal.

[0074] In some embodiments, the dosage of procomamoricin provided ranges from about 50 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the dosage of procomamoricin provided ranges from 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), and about 50 milligrams (mg) to about 750 milligrams (mg). g), approximately 50 mg to approximately 1,000 mg, approximately 50 mg to approximately 1,500 mg, approximately 100 mg to approximately 150 mg, approximately 100 mg to approximately 200 mg, approximately 100 mg to approximately 300 mg, approximately 100 mg to approximately 400 mg, approximately 100 mg to approximately 500 mg, approximately 100 mg to approximately 750 mg, approximately 100 mg to approximately 1,000 mg, approximately 100 mg to approximately 1,500 mg, approximately 150 mg to approximately 200 mg, approximately 150 mg to approximately 300 mg, approximately 150 mg to approximately 400 mg, approximately 150 mg to approximately 500 mg, approximately 150 mg to approximately 750 mg, approximately 15 0 mg to approximately 1,000 mg, approximately 150 mg to approximately 1,500 mg, approximately 200 mg to approximately 300 mg, approximately 200 mg to approximately 400 mg, approximately 200 mg to approximately 500 mg, approximately 200 mg to approximately 750 mg, approximately 200 mg to approximately 1,000 mg, approximately 200 mg to approximately 1,500 mg, approximately 300 mg to approximately 400 mg, approximately 300 mg to approximately 500 mg, approximately 300 mg to approximately 750 mg, approximately 300 mg to approximately 1,000 mg, approximately 300 mg to approximately 1,500 mg, approximately 400 mg to approximately 500 mg, approximately 400 mg to approximately 750 mg, approximately 400 mg to approximately 1 Contains 1,000 milligrams (mg), approximately 400 milligrams (mg) to approximately 1,500 milligrams (mg), approximately 500 milligrams (mg) to approximately 750 milligrams (mg), approximately 500 milligrams (mg) to approximately 1,000 milligrams (mg), approximately 500 milligrams (mg) to approximately 1,500 milligrams (mg), approximately 750 milligrams (mg) to approximately 1,000 milligrams (mg), approximately 750 milligrams (mg) to approximately 1,500 milligrams (mg), or approximately 1,000 milligrams (mg) to approximately 1,500 milligrams (mg). In some embodiments, the doses of procoumamoricin provided include 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 doses of procoumamoricin provided include 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 dose of procumamoricin provided includes 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 leguminous protein provided includes approximately 5 grams (g) to approximately 60 grams (g). In some embodiments, the dosage of leguminous protein provided includes approximately 5 grams (g) to approximately 10 grams (g), approximately 5 grams (g) to approximately 15 grams (g), approximately 5 grams (g) to approximately 20 grams (g), approximately 5 grams (g) to approximately 25 grams (g), approximately 5 grams (g) to approximately 30 grams (g), approximately 5 grams (g) to approximately 35 grams (g), approximately 5 grams (g) to approximately 40 grams (g), approximately 5 grams (g) to approximately 45 grams (g), approximately 5 grams (g) to approximately 50 grams (g), approximately 5 grams (g) to approximately 60 grams (g), and approximately 10 grams (g) to approximately 15 grams (g). 10 grams (g), approximately 10 grams (g) to approximately 20 grams (g), approximately 10 grams (g) to approximately 25 grams (g), approximately 10 grams (g) to approximately 30 grams (g), approximately 10 grams (g) to approximately 35 grams (g), approximately 10 grams (g) to approximately 40 grams (g), approximately 10 grams (g) to approximately 45 grams (g), approximately 10 grams (g) to approximately 50 grams (g), approximately 10 grams (g) to approximately 60 grams (g), approximately 15 grams (g) to approximately 20 grams (g), approximately 15 grams (g) to approximately 25 grams (g), approximately 15 grams (g) to approximately 30 grams (g), approximately 15 grams (g) to Approximately 35 grams (g), approximately 15 grams (g) to approximately 40 grams (g), approximately 15 grams (g) to approximately 45 grams (g), approximately 15 grams (g) to approximately 50 grams (g), approximately 15 grams (g) to approximately 60 grams (g), approximately 20 grams (g) to approximately 25 grams (g), approximately 20 grams (g) to approximately 30 grams (g), approximately 20 grams (g) to approximately 35 grams (g), approximately 20 grams (g) to approximately 40 grams (g), approximately 20 grams (g) to approximately 45 grams (g), approximately 20 grams (g) to approximately 50 grams (g), approximately 20 grams (g) to approximately 60 grams (g), approximately 25 grams 10g (g) ~ approx. 30g (g), approx. 25g (g) ~ approx. 35g (g), approx. 25g (g) ~ approx. 40g (g), approx. 25g (g) ~ approx. 45g (g), approx. 25g (g) ~ approx. 50g (g), approx. 25g (g) ~ approx. 60g (g), approx. 30g (g) ~ approx. 35g (g), approx. 30g (g) ~ approx. 40g (g), approx. 30g (g) ~ approx. 45g (g), approx. 30g (g) ~ approx. 50g (g), approx. 30g (g) ~ approx. 60g (g), approx. 35g (g) ~ approx. 40g (g),Contains approximately 35 grams to 45 grams, approximately 35 grams to 50 grams, approximately 35 grams to 60 grams, approximately 40 grams to 45 grams, approximately 40 grams to 50 grams, approximately 40 grams to 60 grams, approximately 45 grams to 50 grams, approximately 45 grams to 60 grams, or approximately 50 grams to 60 grams. In some embodiments, the dosage of leguminous protein provided includes 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 leguminous protein provided includes 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 leguminous protein provided includes 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 pea protein. In some embodiments, the pea is the whole pea or its components. In certain embodiments, the pea is garden pea, sugar pea, field pea, or any combination thereof. In certain embodiments, the pea is garden pea. In certain embodiments, the pea is sugar pea. In certain embodiments, the pea is 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 standard pea, commercial pea, genetically modified pea, or a combination thereof. In certain embodiments, the pea is round pea, wrinkled pea, or a combination thereof.

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

[0078] In some embodiments, the food contains sugar. In some embodiments, the sugar contains sucrose, lactose, maltose, or another disaccharide, trisaccharide, or polysaccharide containing glucose as a monomer. 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 cataracts. In certain embodiments, the condition is bone and joint disorders. In certain embodiments, the condition is a dental infection. In certain embodiments, the condition is a gingival infection.

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

[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 (in 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 composed of molecules substituted with multiple hydroxyl groups, with the general formula (CH2O) nThis refers to and includes aldehyde or ketone compounds (where n is 3 to 30), as well as 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. Examples of such carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Carbohydrate derivatives or substituted carbohydrates can be deoxygenated at any corresponding C position and / or substituted with one or more moieties, e.g., hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamide, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oxyimino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group.

[0082] Throughout this application, various embodiments may be presented in scope form. It should be understood that scope form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of this disclosure. Therefore, scope descriptions should be considered to specifically disclose all possible sub-scopes and the individual numerical values ​​within those scopes. For example, a scope description such as 1-6 should be considered to specifically disclose sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numerical values ​​within those scopes, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0083] As used herein and in the claims, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. For example, the term "a sample" includes multiple samples, including mixtures thereof.

[0084] The terms “identifying,” “measuring,” “evaluating,” “assessing,” and “analyzing” are often used synonymously in this specification to refer to forms of measurement. These terms include identifying whether an element is present or not (e.g., detection). These terms may include quantitative, qualitative, or both quantitative and qualitative identification. Assessment may be relative or absolute. “Detecting the presence of ~” may, depending on the context, include identifying the quantity of something that is present, in addition to identifying whether it is present or not.

[0085] The terms “subject,” “individual,” or “patient” are often used synonymously herein. “Subject” may be a biological entity containing expressed genetic material. Such biological entities may be plants, animals, or microorganisms, including, for example, bacteria, viruses, fungi, and protozoa. Such subject may be tissues, cells, and their offspring obtained in vivo or cultured in vitro. Such subject may be a mammal. Such mammal may be a human. Such subject may be diagnosed or suspected of being at high risk of developing a disease. In some cases, such subject may not necessarily be diagnosed or suspected of being at high risk of developing the disease.

[0086] As used herein, the term "approximately" refers to a value plus or minus 10%. The term "approximately" 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 should not be construed as limiting the subjects described.

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

[0089] Example 1 - Lowering blood glucose levels using leguminous protein and procumamorisin Protease production The DNA sequence of the target acidic protease (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 E. coli expression strain (BL21) and grown in Terrific Broth at 37°C for 4-6 hours using a baffled shaking flask 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 lysates using IMAC chromatography.

[0090] Application of Procomamorisin to Pea Protein Drinks 30 grams of pea protein were dissolved in 330 mL of water. Then, 14 grams of sucrose was dissolved, followed by 200 mg of acid protease (SEQ ID NO: 1). The protein beverage used in this study can be consumed as is or formulated with other ingredients found in commonly used protein shakes. As shown in Figure 1, the addition of protease can reduce the rapid rise in blood glucose after ingestion by more than half at the peak blood glucose level. Figure 1 shows a comparison of post-ingestion blood glucose levels resulting from the consumption of a protein beverage containing procoumamorisin and pea protein, and (ii) a protein beverage without procoumamorisin and pea protein.

[0091] Example 2 - Activity of S53 protease at low pH In general, 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. Therefore, the S53 proteases used and described herein should be active across the entire postprandial pH range of the stomach environment. pH profile data was generated for 10 representative S53 proteases (including SEQ ID NOs: 1 and 3-11). Representative S53 proteases showed optimal activity (e.g., 100% or substantially active) across the entire pH range of 2.5-4.5. Figure 6 shows the proteolytic activity of S53 proteases 1-10 (P1-P10) across pH 2-5.

[0092] Preferred embodiments of the Disclosure have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. Herein, numerous variations, modifications, and substitutions will be conceivable without departing from the Disclosure. It should be understood that various alternative means to those described herein may be employed in the practice of the Invention. The following claims define the scope of the embodiments disclosed herein, and the methods and structures included in these claims, as well as their equivalents, are intended to be encompassed 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. A method for reducing blood glucose in a subject, For the aforementioned target, Leguminous proteins, and S53 family proteases The method includes administering a composition containing the following: The method wherein the blood glucose level in the subject decreases by administering the aforementioned drug.

2. A method for suppressing the rise in blood glucose in a subject, For the aforementioned target, Leguminous proteins, and S53 family proteases The method includes administering a composition containing the following: The method wherein, by administering the above-mentioned substance, the rise in blood glucose in the subject is suppressed.

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

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

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

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

7. The method according to any one of claims 1 to 6, wherein the S53 family protease comprises an active site including the 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. The method according to any one of claims 1 to 6, wherein the S53 family protease comprises an active site comprising 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 according to claim 8, wherein the active site comprises 1 to 5 amino acid substitutions.

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

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

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

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

14. The method according to claim 13, wherein the pea protein is derived from peas.

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

16. The method according to claim 13 or 15, wherein the pea is a standard pea, a commercialized pea, a genetically modified pea, or a combination thereof.

17. The method according to claim 13 or 15, wherein the pea is a round pea, a wrinkled pea, or a combination thereof.

18. The method according to claim 13 or 15, wherein the peas are garden peas, sugar peas, field peas, or any combination thereof.

19. The method according to claim 1, wherein the administration is performed after the subject has ingested a food containing sugar.

20. The method according to claim 2, wherein the increase in blood glucose is suppressed compared to the case in which a composition not containing the S53 family protease is administered.

21. The method according to 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. The method according to claim 22, wherein the mammal is a human.

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

25. The method according to claim 22, wherein the human has a condition related to and / or caused by hyperglycemia.

26. The method according to 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 gingival infections.

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

28. Furthermore, the method according to any one of claims 1 to 27, comprising reducing the amount of calories obtained from carbohydrates.

29. The method according to claim 28, wherein the quantity includes the total grams of carbohydrates or the calories from carbohydrates.

30. A composition used to reduce blood glucose in a subject, comprising a leguminous protein and a protease of the S53 family.

31. A composition used to suppress the rise in blood glucose in a subject, comprising a leguminous protein and a protease of the S53 family.

Images