Methods and compositions for stabilizing blood glucose

Abstract

Provided and described herein are methods and compositions for lowering blood glucose, reducing blood glucose elevation, and / or reducing the glycemic index of a foodstuff.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 303,112, filed January 26, 2022, the entire contents of which are incorporated herein by reference. Background Technology

[0003] High blood glucose levels are well-known to have numerous adverse health effects. Long-term complications of hyperglycemia can range from cardiovascular disease, neuropathy, kidney and vascular damage to bone and joint problems. The most common method for controlling blood glucose levels is by controlling dietary sugar intake. While this is a reasonable and effective strategy, it requires individuals to be very careful about knowing and calculating the amount of carbohydrates they consume at each meal. Medications such as insulin are widely used; however, effectively controlling blood glucose levels through medication can introduce a range of problems. Summary of the Invention

[0004] Despite a growing understanding of the biology and etiology of diseases associated with or caused by high blood glucose levels, individuals who cannot regulate their blood glucose levels independently of any intervention continue to bear a significant burden. Both pharmacological treatments (e.g., insulin) and non-pharmacological treatments (e.g., dietary monitoring) can be considered substantial interventions with inherent barriers to the effective treatment and management of hyperglycemic-related diseases. These inherent barriers include, for example, treatment adherence (e.g., due to the complexity of treatment), economic burden and accessibility (e.g., out-of-pocket costs for insured and uninsured individuals), and other patient-related, prescription-related, and prescribing-related factors. These barriers and their impact on the treatment of hyperglycemic-related diseases impose a significant health and economic burden on individuals and the healthcare system.

[0005] This document provides and describes compositions and methods for regulating (e.g., reducing, stabilizing, etc.) hyperglycemia. The provided methods and compositions are partly based on the surprising finding that hyperglycemia can be regulated by providing an individual with legume proteins (e.g., pea proteins) and S53 family proteases. In some embodiments, the S53 family protease is a pro-Kumamolisin. In some embodiments, the S53 protease (e.g., the pro-Kumamolisin) comprises an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NO:1 and 3-11. In some embodiments, the S53 protease (e.g., the pro-Kumamolisin) comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO:1 and 3-11. In some embodiments, the S53 protease (e.g., the pro-Kumamolisin) comprises an amino acid sequence having at least 98% sequence identity with any one of SEQ ID NO:1 and 3-11. In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465 and A466 of SEQ ID NO:1.

[0006] In some embodiments, the S53 protease is active at a pH less than about pH 5 (e.g., as measured by protein digestion). In some embodiments, the S53 protease is active at a pH less than about pH 4.5. In some embodiments, the S53 protease is active at a pH less than about pH 5. In some embodiments, the S53 protease is active at a pH less than about pH 4. In some embodiments, the S53 protease is active at a pH less than about pH 3. In some embodiments, the S53 protease is active at a pH less than about pH 3.

[0007] In some embodiments, the S53 protease is active in a pH range between about pH 2 and pH 5. In some embodiments, the S53 protease is active in a pH range between about pH 2.5 and pH 4.5. In some embodiments, the S53 protease has at least 50% activity (e.g., relative to its maximum activity) in a pH range between about pH 2.5 and pH 4.5.

[0008] In some instances, modulating protein digestion patterns (e.g., by administering legume protein and S53 protease (e.g., kummolisin precursor)) increases the concentration of certain dietary amino acids in the blood. In such instances, modulating protein digestion patterns can induce various physiological responses, such as the observed decrease in blood glucose levels. In some instances, such as by administering legume protein and S53 protease (e.g., kummolisin precursor) to induce the digestion of dietary protein in the gut, the digestion of which produces bioactive peptides with potent biological responses, including anti-diabetic effects (e.g., lowering blood glucose levels). As described and provided herein, diets consisting of protein foods (e.g., legume protein) and acidic proteases (e.g., kummolisin precursor) can lower blood glucose levels (e.g., when consumed with sugar). Furthermore, in some instances, the addition of protein foods (e.g., legume protein) containing acidic proteases (e.g., kummolisin precursor) can lower the glycemic index of diets (e.g., diets containing sugar).

[0009] This article provides and describes methods for lowering blood glucose in subjects, comprising administering to the subject a composition comprising: legume protein; and S53 protease (e.g., a precursor of Kumamolisin). Methods for reducing elevated blood glucose in subjects are also described and provided, comprising administering to the subject a composition comprising legume protein and S53 protease (e.g., a precursor of Kumamolisin).

[0010] In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:1.

[0011] In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an active site containing 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., a Kumamolisin precursor) comprises an active site containing 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 contains between one and five amino acid substitutions. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises one or more truncations of SEQ ID NO:1, wherein the one or more truncations include an N-terminal truncation, a C-terminal truncation, or both N-terminal and C-terminal truncations.

[0012] In some embodiments, the legume protein is pea protein. In some embodiments, the pea protein is derived from peas. In some embodiments, the peas are common peas, sugar peas, purple peas, or any combination thereof. In some embodiments, the peas are common peas. In some embodiments, the peas are sugar peas. In some embodiments, the peas are purple peas. In some embodiments, the peas are any combination of common peas, sugar peas, and / or purple peas. In some embodiments, the peas are standard peas, commercially available peas, genetically modified peas, or combinations thereof. In some embodiments, the peas are smooth peas, wrinkled peas, or combinations thereof.

[0013] In some embodiments, the administration occurs after the subject has ingested a food containing sugar. In some embodiments, the increase in blood glucose is reduced compared to administration of a composition that does not contain S53 protease (e.g., Kumamoli sin precursor). In some embodiments, the subject self-administers the composition.

[0014] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human suffers from hyperglycemia. In some embodiments, the human suffers from a condition associated with and / or caused by hyperglycemia. In some embodiments, the condition is cardiovascular disease or neuropathy or diabetic nephropathy or retinopathy or cataracts or bone and joint problems or tooth and gum infections.

[0015] A composition for lowering blood glucose in subjects has also been demonstrated, comprising legume protein and S53 protease (e.g., a precursor of Kumamolisin). A further composition for reducing elevated blood glucose in subjects, comprising legume protein and S53 protease (e.g., a precursor of Kumamolisin).

[0016] In some embodiments, a composition is provided comprising a food, a legume protein (e.g., pea protein), and an S53 protease (e.g., a precursor of Kumamolisin) (e.g., any of the S53 proteases (e.g., precursors of Kumamolisin) described herein). In some embodiments, the composition comprising a food, a legume protein, and an S53 protease (e.g., a precursor of Kumamolisin) results in a lower post-consumption increase in blood glucose compared to a second composition comprising only a food. Attached Figure Description

[0017] The novel features of the invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the invention can be obtained by referring to the following detailed description and accompanying drawings, which illustrate embodiments utilizing the principles of the invention, in which:

[0018] Figure 1 The study showed a decrease in blood glucose following administration of legume protein and S53 protease (e.g., a precursor to Kumamolisin).

[0019] Figure 2 The study showed a decrease in blood glucose after administration of a low-carbohydrate protein drink and protease.

[0020] Figure 3 The study showed a decrease in blood glucose after administration of protein-fruit smoothie and protease.

[0021] Figure 4 The proteolytic activity of a representative S53 protease in a low pH range is shown. Detailed Implementation

[0022] Blood glucose dysregulation and / or the presence of high blood glucose levels are associated with and / or are a cause of a variety of diseases affecting human health and well-being. Such diseases (e.g., diabetes) are often complex conditions with associated bothersome symptoms and are further characterized by glucose dysregulation. In some instances, the methods and compositions provided herein can be used to lower and / or reduce an individual's blood glucose levels. Notably, in some instances, the methods and compositions provided herein are based on the finding that administration of legume proteins (e.g., pea protein) and S53 protease (e.g., kumamolisin precursor) can lower (e.g., reduce or decrease) blood glucose levels. Furthermore, in some instances, the methods and compositions provided herein can be used to inhibit, reduce, lower, and / or prevent elevations in blood glucose levels (e.g., the amount of glucose in the blood).

[0023] Legume protein

[0024] Legumes are a type of plant belonging to the legume family (Fabaceae). As used herein, legumes generally describe and refer to the fruit or seeds of legume plants. In some embodiments, the compositions and methods utilize legume proteins. Legume proteins generally describe proteins derived from (e.g., obtained from) the fruit or seeds of legume plants. Both whole legume proteins and hydrolyzed legume protein sources can be used. In some embodiments, the legume protein is a whole legume protein. In some embodiments, the legume protein is a hydrolyzed legume protein. In some embodiments, the legume protein is provided in solid form. In some embodiments, the legume protein is provided in liquid form. In some embodiments, the legume protein concentrate (e.g., protein material obtained from peas after removal of soluble carbohydrates, ash, and other trace components) is used.

[0025] In some embodiments, the legume protein is pea protein. Peas generally refer to and include seed pods or pods from plants belonging to the genus *Vaccaria*. Legume protein generally describes proteins derived from (e.g., obtained from) the fruit or seeds of a pea plant. Both whole pea protein and hydrolyzed pea protein sources can be used. In some embodiments, the pea protein is whole 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 removing soluble carbohydrates, ash, and other trace components). In some embodiments, the pea is common pea, sugar pea, purple pea, or any combination thereof. In some embodiments, the pea is common pea. In some embodiments, the pea is sugar pea. In some embodiments, the pea is purple pea. In some embodiments, the pea is any combination of common pea, sugar pea, and / or purple pea. In some embodiments, the peas are standard peas, commercially available peas, genetically modified peas, or combinations thereof. In some embodiments, the peas are smooth peas, wrinkled peas, or combinations thereof.

[0026] protease

[0027] The compositions and methods described herein generally utilize acidic proteases. In some embodiments, the acidic protease is an S53 family protease. The S53 family of proteases generally refers to and includes the family of serine proteases found in prokaryotes and eukaryotes. In some embodiments, the S53 family of proteases refers to and includes proteases identified in and / or by MEROPS Registry Number MER0000995 (e.g., sedolisin, sedolisin-b, tripeptidyl peptidase I, kumamolisin, kumamolisin-B, physarolisin, aorsin, physarolisin II, kumamolisin-As, grifolisin, scytalidolisin, etc.). In some embodiments, the acidic protease is an S53 protease (e.g., a kumamolisin precursor). A kumamolisin precursor generally refers to and includes thermostable calcium-dependent endopeptidases derived from *Bacillus acidophilus* (Bacillus species MN-32). In some implementations, the Kumamolisin precursor refers to and includes NCBI gene ID: 18765799 (NCBI reference sequences XP_007297753.1, XM_007297691.1 to XP_007297753 and / or NW_006763082.1 (137488..139728).

[0028] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:1.

[0029] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:2.

[0030] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:3.

[0031] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:4. In some embodiments, the Kumamolisin precursor comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:4.

[0032] In some embodiments, the S53 protease comprises the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:5.

[0033] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:6.

[0034] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:7.

[0035] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:8.

[0036] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:9.

[0037] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:10.

[0038] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:11.

[0039] In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465 and A466 of SEQ ID NO:1.

[0040] The determination of the percentage of identity or similarity between two sequences can be accomplished using mathematical algorithms. In some embodiments, a non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 90:5873-5877, modified as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 87:2264-2268. This algorithm is incorporated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403-410. Alternatively, in some embodiments, PSI-Blast can be used to perform an iterative search to detect distant relationships between molecules (as above). When using BLAST, Gapped BLAST, and PSI-Blast procedures, the default parameters of each procedure (e.g., XBLAST and NBLAST) can be used. In some implementations, a non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. In some implementations, sequence alignment is performed using the CLUSTAL algorithm as described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0041] In some embodiments, the active site of the S53 protease (e.g., a Kumamolisin precursor) comprises amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an active site in which one or more 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 are substituted. In some implementations, the active site contains between one and five amino acid substitutions.

[0042] In some embodiments, the active site comprises one or more amino acid substitutions. In some embodiments, the active site comprises two or more amino acid substitutions. In some embodiments, the active site comprises three or more amino acid substitutions. In some embodiments, the active site comprises four or more amino acid substitutions.

[0043] In some embodiments, the active site contains one amino acid substitution. In some embodiments, the active site contains two amino acid substitutions. In some embodiments, the active site contains three amino acid substitutions. In some embodiments, the active site contains four amino acid substitutions. In some embodiments, the active site contains five amino acid substitutions.

[0044] Amino acids generally refer to and / or include naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimics that function in a manner similar to naturally occurring amino acids. Amino acids are generally referred to herein by their names (usually known three-letter symbols) or by single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. As used herein, naturally occurring amino acids include and / or refer to amino acids that are generally present in nature and have not been artificially manipulated. In some embodiments, naturally occurring amino acids include and / or further refer to 20 common 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).

[0045] In some embodiments, a nonpolar amino acid may be substituted and replaced by another nonpolar amino acid, wherein the nonpolar amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. In some embodiments, an electrically neutral polar amino acid may be substituted and replaced by another electrically neutral polar amino acid, wherein the electrically neutral polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In some embodiments, a positively charged amino acid may be substituted and replaced by another positively charged amino acid, wherein the positively charged amino acid includes arginine, lysine, and histidine. In some embodiments, a negatively charged amino acid may be substituted and replaced by another negatively charged amino acid, wherein the negatively charged amino acid includes aspartic acid and glutamic acid. As used herein, a peptide includes and / or refers to any of a variety of natural or synthetic compounds containing two or more amino acids linked by peptide bonds that link the carboxyl group of one amino acid to the amino group of another amino acid. As used herein, amino acids refer to and / or include naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Amino acids may generally be referred to herein by their name (usually known three-letter symbol) or by a single-letter symbol recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature.

[0046] In some embodiments, the active site comprises one or more amino acid substitutions. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises one or more truncations of SEQ ID NO:1, wherein the one or more truncations include an N-terminal truncation, a C-terminal truncation, or both an N-terminal and C-terminal truncation.

[0047] In some embodiments, the S53 protease is active at a pH less than about pH 5 (e.g., as measured by protein digestion). In some embodiments, the S53 protease is active at a pH less than about pH 4.5. In some embodiments, the S53 protease is active at a pH less than about pH 5. In some embodiments, the S53 protease is active at a pH less than about pH 4. In some embodiments, the S53 protease is active at a pH less than about pH 3. In some embodiments, the S53 protease is active at a pH less than about pH 3.

[0048] In some embodiments, the S53 protease is active in a pH range between about pH 2 and pH 5. In some embodiments, the S53 protease is active in a pH range between about pH 2.5 and pH 4.5. In some embodiments, the S53 protease has at least 50% activity (e.g., relative to its maximum activity) in a pH range between about pH 2.5 and pH 4.5.

[0049] S53 protease (e.g., a kumamolisin precursor) can be administered as part of a composition containing S53 protease (e.g., a kumamolisin precursor). The composition may further contain legume protein. In some embodiments, a composition containing S53 protease (e.g., a kumamolisin precursor) is provided. In some embodiments, the composition further contains legume protein (e.g., pea protein).

[0050] In some embodiments, a composition is provided comprising a food, a legume protein (e.g., pea protein), and an S53 protease (e.g., a kumamolisin precursor) (e.g., any of the S53 proteases (e.g., kumamolisin precursors) described herein). In some embodiments, the food comprises a sugar. In some embodiments, the sugar comprises sucrose, lactose, maltose, or other disaccharides, trisaccharides, or polysaccharides containing glucose as a monomer. In some embodiments, the sugar comprises sucrose. In some embodiments, the sugar comprises lactose. In some embodiments, the sugar comprises maltose. In some embodiments, the sugar comprises a disaccharide, trisaccharide, or polysaccharide containing glucose as a monomer. In some examples, the addition of a legume protein (e.g., pea protein) and an S53 protease (e.g., a kumamolisin precursor) reduces the glycemic index of the composition comprising the food. In some embodiments, the composition comprising the food, legume protein, and an S53 protease (e.g., a kumamolisin precursor) results in a lower post-consumption blood glucose rise than a second composition comprising only the food.

[0051] In some embodiments, the composition comprises about 5 g of legume protein (e.g., pea protein) to about 60 g of legume protein (e.g., pea protein). In some embodiments, the composition comprises about 5 g of legume protein (e.g., pea protein) to about 10 g of legume protein (e.g., pea protein), about 5 g of legume protein (e.g., pea protein) to about 15 g of legume protein (e.g., pea protein), about 5 g of legume protein (e.g., pea protein) to about 20 g of legume protein (e.g., pea protein), about 5 g of legume protein (e.g., pea protein) to about 25 g of legume protein (e.g., pea protein), and about 5 g of legume protein (e.g., pea protein) to about 30 g of legume protein (e.g., pea protein). White protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) Legume protein (e.g., pea protein) to about 15 grams (g) legume protein (e.g., pea protein), about 10 grams (g) legume protein (e.g., pea protein) to about 20 grams (g) legume protein (e.g., pea protein), about 10 grams (g) legume protein (e.g., pea protein) to about 25 grams (g) legume protein (e.g., pea protein), about 10 grams (g) legume protein (e.g., pea protein) to about 30 grams (g) legume protein (e.g., pea protein), about 10 grams (g) legume protein (e.g., pea protein) to about 35 grams (g) legume protein (e.g., pea protein), about 10 grams (g) legume protein The protein content (e.g., pea protein) is 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), approximately 50 grams (g) of legume protein (e.g., pea protein), approximately 10 grams (g) of legume protein (e.g., pea protein), approximately 60 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein), approximately 20 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein), approximately 15 grams (g) of legume protein (e.g., pea protein),Pea protein) to about 25 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 30 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein White protein (e.g., pea protein), about 15 g (g) of legume protein (e.g., pea protein) to about 60 g (g) of legume protein (e.g., pea protein), about 20 g (g) of legume protein (e.g., pea protein) to about 25 g (g) of legume protein (e.g., pea protein), about 20 g (g) of legume protein (e.g., pea protein) to about 30 g (g) of legume protein (e.g., pea protein), about 20 g (g) of legume protein (e.g., pea protein) to about 35 g (g) of legume protein (e.g., pea protein), about 20 g (g) of legume protein (e.g., pea protein) to about 40 g (g) of legume protein (e.g., pea protein), about 20 g (g) Legume protein (e.g., pea protein) to about 45 g (g) Legume protein (e.g., pea protein), about 20 g (g) Legume protein (e.g., pea protein) to about 50 g (g) Legume protein (e.g., pea protein), about 20 g (g) Legume protein (e.g., pea protein) to about 60 g (g) Legume protein (e.g., pea protein), about 25 g (g) Legume protein (e.g., pea protein) to about 30 g (g) Legume protein (e.g., pea protein), about 25 g (g) Legume protein (e.g., pea protein) to about 35 g (g) Legume protein (e.g., pea protein), about 25 g (g) Legume protein (e.g., pea protein) From approximately 40 grams (g) of legume protein (e.g., pea protein), from approximately 25 grams (g) of legume protein (e.g., pea protein), from approximately 45 grams (g) of legume protein (e.g., pea protein), from approximately 25 grams (g) of legume protein (e.g., pea protein), from approximately 50 grams (g) of legume protein (e.g., pea protein), from approximately 25 grams (g) of legume protein (e.g., pea protein), from approximately 60 grams (g) of legume protein (e.g., pea protein), from approximately 30 grams (g) of legume protein (e.g., pea protein), from approximately 35 grams (g) of legume protein (e.g., pea protein), from approximately 30 grams (g) of legume protein (e.g., pea protein), from approximately 40 grams (g) of legume protein (e.g., pea protein).Pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein) The protein content is approximately 60 g (g) of legume protein (e.g., pea protein), approximately 40 g (g) of legume protein (e.g., pea protein) to approximately 45 g (g) of legume protein (e.g., pea protein), approximately 40 g (g) of legume protein (e.g., pea protein) to approximately 50 g (g) of legume protein (e.g., pea protein), approximately 40 g (g) of legume protein (e.g., pea protein) to approximately 60 g (g) of legume protein (e.g., pea protein), approximately 45 g (g) of legume protein (e.g., pea protein) to approximately 50 g (g) of legume protein (e.g., pea protein), approximately 45 g (g) of legume protein (e.g., pea protein) to approximately 60 g (g) of legume protein (e.g., pea protein), or approximately 50 g (g) of legume protein (e.g., pea protein) to approximately 60 g (g) of legume protein (e.g., pea protein). In some embodiments, the composition comprises about 5 g of legume protein (e.g., pea protein), about 10 g of legume protein (e.g., pea protein), about 15 g of legume protein (e.g., pea protein), about 20 g of legume protein (e.g., pea protein), about 25 g of legume protein (e.g., pea protein), about 30 g of legume protein (e.g., pea protein), about 35 g of legume protein (e.g., pea protein), about 40 g of legume protein (e.g., pea protein), about 45 g of legume protein (e.g., pea protein), about 50 g of legume protein (e.g., pea protein), or about 60 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), or 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 comprises up to about 10 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein), about 50 grams (g) of legume protein (e.g., pea protein), or about 60 grams (g) of legume protein (e.g., pea protein).

[0052] In some embodiments, the composition comprises about 50 mg of kummolisin precursor to about 2,000 mg of kummolisin precursor. In some embodiments, the composition comprises about 50 mg of kummolisin precursor to about 100 mg of kummolisin precursor, about 50 mg of kummolisin precursor to about 200 mg of kummolisin precursor, about 50 mg of kummolisin precursor to about 300 mg of kummolisin precursor, about 50 mg of kummolisin precursor to about 400 mg of kummolisin precursor, or about 50 mg of kummolisin precursor to about 5... 0.00 mg Kumolisin precursor, about 50 mg Kumolisin precursor to about 750 mg Kumolisin precursor, about 50 mg Kumolisin precursor to about 1,000 mg Kumolisin precursor, about 50 mg Kumolisin precursor to about 1,250 mg Kumolisin precursor, about 50 mg Kumolisin precursor to about 1,500 mg Kumolisin precursor, about 50 mg Kumolisin precursor Kumamolisin precursor up to 2,000 mg; Kumamolisin precursor up to 200 mg; Kumamolisin precursor up to 300 mg; Kumamolisin precursor up to 400 mg; Kumamolisin precursor up to 500 mg. Prodrug, approximately 100 mg Kumamolisin prodrug to approximately 750 mg Kumamolisin prodrug, approximately 100 mg Kumamolisin prodrug to approximately 1,000 mg Kumamolisin prodrug, approximately 100 mg Kumamolisin prodrug to approximately 1,250 mg Kumamolisin prodrug, approximately 100 mg Kumamolisin prodrug to approximately 1,500 mg Kumamolisin prodrug, approximately 100 mg Kumamolisin prodrug to approximately 2,000 mg Kumamolisin precursor, about 200 mg Kumamolisin precursor to about 300 mg Kumamolisin precursor, about 200 mg Kumamolisin precursor to about 400 mg Kumamolisin precursor, about 200 mg Kumamolisin precursor to about 500 mg Kumamolisin precursor, about 200 mg Kumamolisin precursor to about 750 mg Kumamolisin precursor, about 200 mg Kumamolisin precursor to about 1,000 mg Kumamolisin precursor mg kumamolisin precursor, about 200 mg kumamolisin precursor to about 1,250 mg kumamolisin precursor, about 200 mg kumamolisin precursor to about 1,500 mg kumamolisin precursor, about 200 mg kumamolisin precursor to about 2,000 mg kumamolisin precursor, about 300 mg kumamolisin precursor to about 400 mg kumamolisin precursor, about 300 mg kumamolisin precursor to about 500 mg kumamolisin precursor mg kumamolisin precursor, about 300 mg kumamolisin precursor to about 750 mg kumamolisin precursor, about 300 mg kumamolisin precursor to about 1,000 mg kumamolisin precursor, about 300 mg kumamolisin precursor to about 1,250 mg kumamolisin precursor, about 300 mg kumamolisin precursor to about 1,500 mg kumamolisin precursor, about 300 mg kumamolisin precursor to about 2, 000 mg Kumamolisin precursor, about 400 mg Kumamolisin precursor to about 500 mg Kumamolisin precursor, about 400 mg Kumamolisin precursor to about 750 mg Kumamolisin precursor, about 400 mg Kumamolisin precursor to about 1,000 mg Kumamolisin precursor, about 400 mg Kumamolisin precursor to about 1,250 mg Kumamolisin precursor, about 400 mg Kumamolisin precursor to about 1,500 mg Kumamolisin precursor, approximately 400 mg Kumamolisin precursor to approximately 2,000 mg Kumamolisin precursor, approximately 500 mg Kumamolisin precursor to approximately 750 mg Kumamolisin precursor, approximately 500 mg Kumamolisin precursor to approximately 1,000 mg Kumamolisin precursor, approximately 500 mg Kumamolisin precursor to approximately 1,250 mg 500 mg Kumamolisin precursor, approximately 1,500 mg Kumamolisin precursor, approximately 2,000 mg Kumamolisin precursor, approximately 750 mg Kumamolisin precursor, approximately 1,000 mg Kumamolisin precursor, approximately 750 mg Kumamolisin precursor, approximately 1,250 mg Kumamolisin precursor g) Kumamolisin precursor, approximately 750 mg Kumamolisin precursor to approximately 1,500 mg Kumamolisin precursor, approximately 750 mg Kumamolisin precursor to approximately 2,000 mg Kumamolisin precursor, approximately 1,000 mg Kumamolisin precursor to approximately 1,250 mg Kumamolisin precursor, approximately 1,000 mg Kumamolisin precursor to approximately 1,500 mg ( approximately 1,000 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, or approximately 1,50 ... approximately 1,250 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, or approximately 1,500 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 1,250 mg of kumamolisin precursor, approximately 2,000 mg of kumamolisin precursor, approximately 1,250 mg of kumamol000 mg of kummolisin precursor. In some embodiments, the composition comprises about 50 mg of kummolisin precursor, about 100 mg of kummolisin precursor, about 200 mg of kummolisin precursor, about 300 mg of kummolisin precursor, about 400 mg of kummolisin precursor, about 500 mg of kummolisin precursor, about 750 mg of kummolisin precursor, about 1,000 mg of kummolisin precursor, about 1,250 mg of kummolisin precursor, about 1,500 mg of kummolisin precursor, or about 2,000 mg of kummolisin precursor. In some embodiments, the composition comprises at least about 50 mg of kummolisin precursor, about 100 mg of kummolisin precursor, about 200 mg of kummolisin precursor, about 300 mg of kummolisin precursor, about 400 mg of kummolisin precursor, about 500 mg of kummolisin precursor, about 750 mg of kummolisin precursor, about 1,000 mg of kummolisin precursor, about 1,250 mg of kummolisin precursor, or about 1,500 mg of kummolisin precursor. In some embodiments, the composition comprises up to about 100 mg of kummolisen prodrug, about 200 mg of kummolisen prodrug, about 300 mg of kummolisen prodrug, about 400 mg of kummolisen prodrug, about 500 mg of kummolisen prodrug, about 750 mg of kummolisen prodrug, about 1,000 mg of kummolisen prodrug, about 1,250 mg of kummolisen prodrug, about 1,500 mg of kummolisen prodrug, or about 2,000 mg of kummolisen prodrug.

[0053] method

[0054] As described herein, in some instances, providing legume protein (e.g., pea protein) and S53 protease (e.g., kumamolisin precursor) can lower (e.g., reduce or decrease) blood glucose levels. Therefore, this document provides a method for lowering blood glucose in a subject, comprising: administering to the subject a composition containing legume protein and S53 protease (e.g., kumamolisin precursor). (e.g., wherein legume protein and S53 protease (e.g., kumamolisin precursor) are administered to the subject). Further, a method for lowering blood glucose in a subject using legume protein and S53 protease (e.g., kumamolisin precursor) is provided (e.g., wherein legume protein and S53 protease (e.g., kumamolisin precursor) are administered to the subject).

[0055] A method for reducing elevated blood glucose levels in a subject is also provided, comprising administering to the subject a composition containing legume protein and S53 protease (e.g., a precursor of kummolidinin). Further, the use of legume protein and S53 protease (e.g., a precursor of kummolidinin) in the method for reducing elevated blood glucose levels in a subject is provided (e.g., wherein legume protein and S53 protease (e.g., a precursor of kummolidinin) are administered to the subject). Additionally, a method for using legume protein and S53 protease (e.g., a precursor of kummolidinin) to reduce elevated blood glucose levels in a subject is provided (e.g., wherein legume protein and S53 protease (e.g., a precursor of kummolidinin) are administered to the subject).

[0056] In some instances, providing legume protein (e.g., pea protein) and S53 protease (e.g., kumamolisin precursor) can stabilize (e.g., reduce or prevent the range or extent of changes in) blood glucose levels associated with food intake. In some embodiments, this document provides a method for reducing changes (positive or negative) in blood glucose in a subject, comprising administering to the subject a composition containing legume protein and S53 protease (e.g., wherein the legume protein and S53 protease (e.g., kumamolisin precursor) are administered to the subject.

[0057] In some embodiments, administration of legume protein and S53 protease (e.g., a kummolisin precursor) may reduce the degree or amount of increase in blood glucose after food intake. In some examples, the degree or amount of reduction in the increase in blood glucose after food intake can be compared to the increase in blood glucose after food intake before or without the administration of legume protein and S53 protease (e.g., a kummolisin precursor). In some embodiments, administration of legume protein and S53 protease (e.g., a kummolisin precursor) may reduce the degree or amount of decrease in blood glucose after food intake. In some examples, the degree or amount of reduction in the decrease in blood glucose after food intake can be compared to the decrease in blood glucose after food intake before or without the administration of legume protein and S53 protease (e.g., a kummolisin precursor). In some embodiments, administration of legume protein and S53 protease (e.g., kummolisin precursor) reduces changes in blood glucose levels (e.g., increases or decreases) after food intake. In some embodiments, the reduction in changes in blood glucose levels (e.g., increases or decreases) after food intake when legume protein and / or S53 protease (e.g., kummolisin precursor) are administered is compared to changes in blood glucose levels after food intake before administration of legume protein and / or S53 protease (e.g., kummolisin precursor) or in the absence of administration of legume protein and / or S53 protease (e.g., kummolisin precursor).

[0058] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:1.

[0059] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:2. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:2.

[0060] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:3. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:3.

[0061] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:4. In some embodiments, the Kumamolisin precursor comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:4. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:4.

[0062] In some embodiments, the S53 protease comprises the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:5. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:5.

[0063] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:6. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:6.

[0064] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:7. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:7.

[0065] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:8. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:8.

[0066] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:9. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:9.

[0067] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:10. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:10.

[0068] In some embodiments, the S53 protease comprises the amino acid sequence listed in SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 80% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 85% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 90% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 95% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 97% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 98% sequence identity with SEQ ID NO:11. In some embodiments, the S53 protease comprises an amino acid sequence having equal or greater than 99% sequence identity with SEQ ID NO:11.

[0069] In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465 and A466 of SEQ ID NO:1.

[0070] Any S53 protease described herein (e.g., a Kumamolisin precursor) can be used in the provided methods. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:1.

[0071] In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an active site containing 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., a Kumamolisin precursor) comprises an active site containing 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 contains between one and five amino acid substitutions. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises one or more truncations of SEQ ID NO:1, wherein the one or more truncations include an N-terminal truncation, a C-terminal truncation, or both N-terminal and C-terminal truncations.

[0072] In some embodiments, the amount of S53 protease (e.g., a kumamolisin precursor) applied includes from about 50 mg to about 1,500 mg. In some embodiments, the amount of S53 protease (e.g., a kumamolisin precursor) applied includes from about 50 mg to about 100 mg, from about 50 mg to about 150 mg, from about 50 mg to about 200 mg, from about 50 mg to about 300 mg, from about 50 mg to about 400 mg, from about 50 mg to about 500 mg, from about 50 mg to about 750 mg, from about 50 mg to about 1,000 mg, and from about 50 mg to about 1,500 mg. 0 mg, about 100 mg to about 150 mg, about 100 mg to about 200 mg, about 100 mg to about 300 mg, about 100 mg to about 400 mg, about 100 mg to about 500 mg, about 100 mg to about 750 mg, about 100 mg to about 1,000 mg, about 100 mg to about 1,500 mg, about 150 mg to about 200 mg, about 150 mg to about 300 mg g (mg), about 150 mg to about 400 mg, about 150 mg to about 500 mg, about 150 mg to about 750 mg, about 150 mg to about 1,000 mg, about 150 mg to about 1,500 mg, about 200 mg to about 300 mg, about 200 mg to about 400 mg, about 200 mg to about 500 mg, about 200 mg to about 750 mg, about 200 mg to about 1,000 mg g (mg), about 200 mg to about 1,500 mg, about 300 mg to about 400 mg, about 300 mg to about 500 mg, about 300 mg to about 750 mg, about 300 mg to about 1,000 mg, about 300 mg to about 1,500 mg, about 400 mg to about 500 mg, about 400 mg to about 750 mg, about 400 mg to about 1,000 mg, about 400 mg to about 1,The amounts of the S53 protease (e.g., a Kumamolisin precursor) applied include about 500 mg, about 500 mg to about 750 mg, about 500 mg to about 1,000 mg, about 500 mg to about 1,500 mg, about 750 mg to about 1,000 mg, or about 1,000 mg to about 1,500 mg. In some embodiments, the amounts of the S53 protease (e.g., a Kumamolisin precursor) applied include about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, about 1,000 mg, or about 1,500 mg. In some embodiments, the amount of S53 protease (e.g., a kumamolisin precursor) applied includes at least about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, or about 1,000 mg. In some embodiments, the amount of S53 protease (e.g., a kumamolisin precursor) applied includes up to about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, about 1,000 mg, or about 1,500 mg.

[0073] In some embodiments, the amount of legume protein applied includes from about 5 grams (g) to about 50 grams (g). In some embodiments, the amount of legume protein applied includes from about 5 grams (g) to about 10 grams (g), from about 5 grams (g) to about 15 grams (g), from about 5 grams (g) to about 20 grams (g), from about 5 grams (g) to about 25 grams (g), from about 5 grams (g) to about 30 grams (g), from about 5 grams (g) to about 35 grams (g), from about 5 grams (g) to about 40 grams (g), from about 5 grams (g) to about 45 grams (g), from about 5 grams (g) to about 50 grams (g), from about 10 grams (g) to about 15 grams (g), and from about 10 grams (g) to about 20 grams (g). about 10 grams to about 25 grams, about 10 grams to about 30 grams, about 10 grams to about 35 grams, about 10 grams to about 40 grams, about 10 grams to about 45 grams, about 10 grams to about 50 grams, about 15 grams to about 20 grams, about 15 grams to about 25 grams, about 15 grams to about 30 grams, about 15 grams to about 35 grams, about 15 grams to about 40 grams, about 1 5 grams (g) to about 45 grams (g), about 15 grams (g) to about 50 grams (g), about 20 grams (g) to about 25 grams (g), about 20 grams (g) to about 30 grams (g), about 20 grams (g) to about 35 grams (g), about 20 grams (g) to about 40 grams (g), about 20 grams (g) to about 45 grams (g), about 20 grams (g) to about 50 grams (g), about 25 grams (g) to about 30 grams (g), about 25 grams (g) to about 35 grams (g), about 25 grams (g) to about 40 grams (g), about 25 grams (g) to about 45 grams (g), about 25 grams (g) to about 50 grams (g), about 30 grams (g) to about 35 grams (g), about 30 grams (g) to about 40 grams (g), about 30 grams (g) to about 45 grams (g), about 30 grams (g) to about 50 grams (g), about 35 grams (g) to about 45 grams (g), about 35 grams (g) to about 50 grams (g), about 40 grams (g) to about 45 grams (g), about 40 grams (g) to about 50 grams (g), or about 45 grams (g) to about 50 grams (g). In some embodiments, the amount of legume protein applied 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 implementations, the amount of legume protein applied 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 implementations, the amount of legume protein applied includes up to 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).

[0074] In some embodiments, the legume protein is pea protein. In some embodiments, the pea is the whole pea or a component thereof. In some embodiments, the pea is common pea, sugar pea, purple pea, or any combination thereof. In some embodiments, the pea is common pea. In some embodiments, the pea is sugar pea. In some embodiments, the pea is purple pea. In some embodiments, the pea is any combination of common pea, sugar pea, and / or purple pea. In some embodiments, the pea is standard pea, commercially available pea, genetically modified pea, or a combination thereof. In some embodiments, the pea is smooth pea, wrinkled pea, or a combination thereof.

[0075] In some embodiments, a composition comprising legume protein is applied. In some embodiments, a composition comprising S53 protease (e.g., a precursor of Kumamolisin) is applied. In some embodiments, a composition comprising both legume protein and S53 protease (e.g., a precursor of Kumamolisin) is applied.

[0076] In some embodiments, the administration occurs after the subject has ingested a food containing sugar. In some embodiments, the sugar includes sucrose, lactose, maltose, or other disaccharides, trisaccharides, or polysaccharides containing glucose as a monomer. In some embodiments, the sugar includes sucrose. In some embodiments, the sugar includes lactose. In some embodiments, the sugar includes maltose. In some embodiments, the sugar includes disaccharides, trisaccharides, or polysaccharides containing glucose as a monomer.

[0077] In some embodiments, the increase in blood glucose is reduced compared to administration of a composition that does not contain the S53 protease (e.g., a Kumamolisin precursor). 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 suffers from hyperglycemia. In some embodiments, the human suffers from a condition associated with and / or caused by hyperglycemia. In some embodiments, the condition is cardiovascular disease, neuropathy, diabetic nephropathy, retinopathy, cataracts, bone and joint problems, or dental and gingival infections. In some embodiments, the condition is cardiovascular disease. In some embodiments, the condition is neuropathy. In some embodiments, the condition is diabetic nephropathy. In some embodiments, the condition is retinopathy. In some embodiments, the condition is cataracts. In some embodiments, the condition is bone and joint problems. In some embodiments, the condition is dental infections. In some embodiments, the condition is gingival infections.

[0078] A method for reducing the glycemic index of a diet is further provided, comprising: providing the diet with legume protein and S53 protease (e.g., a kumamolisin precursor). The use of legume protein and S53 protease (e.g., a kumamolisin precursor) for reducing the glycemic index of a diet is further provided. Additionally, legume protein and S53 protease (e.g., a kumamolisin precursor) for reducing the glycemic index of a diet are also provided.

[0079] Any S53 protease described herein (e.g., a Kumamolisin precursor) can be used in the provided methods. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NO:1 and 3-11. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO:1 and 3-11. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an amino acid sequence having at least 98% sequence identity with any one of SEQ ID NO:1 and 3-11. In some embodiments, the active site of the S53 protease comprises amino acid residues E266, F295 or A295, S316, W317, G318, A349, A350 or S350, G351, D352, S353 or D353 or A353 or N353, D367 or E367, G462, G463, T464, S465 and A466 of SEQ ID NO:1.

[0080] In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises an active site containing 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., a Kumamolisin precursor) comprises an active site containing 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 contains between one and five amino acid substitutions. In some embodiments, the S53 protease (e.g., a Kumamolisin precursor) comprises one or more truncations of SEQ ID NO:1, wherein the one or more truncations include an N-terminal truncation, a C-terminal truncation, or both N-terminal and C-terminal truncations.

[0081] In some embodiments, the amount of the administered kumamolisin precursor provided includes about 50 mg to about 1,500 mg. In some embodiments, the amount of the administered kumamolisin precursor provided includes about 50 mg to about 100 mg, about 50 mg to about 150 mg, about 50 mg to about 200 mg, about 50 mg to about 300 mg, about 50 mg to about 400 mg, about 50 mg to about 500 mg, about 50 mg to about 750 mg, about 50 mg to about 1,000 mg, about 50 mg to about 1,500 mg, and about 100 mg. 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 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 (mg) to about 400 mg, about 300 mg to about 500 mg, about 300 mg to about 750 mg, about 300 mg to about 1,000 mg, about 300 mg to about 1,500 mg, about 400 mg to about 500 mg, about 400 mg to about 750 mg, about 400 mg to about 1,000 mg, about 400 mg to about 1,500 mg, about 500 mg to about 750 mg, about 500 mg to about 1,The amounts of the administered Kumamolisin precursor provided include about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, about 1,000 mg, or about 1,500 mg. In some embodiments, the amount of the administered kumamolisin precursor provided includes at least about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, or about 1,000 mg. In some embodiments, the amount of the administered kumamolisin precursor provided includes up to about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, about 1,000 mg, or about 1,500 mg.

[0082] In some embodiments, the amount of applied legume protein provided includes about 5 grams (g) to about 60 grams (g). In some embodiments, the amount of applied legume protein provided 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 5 grams (g) to about 60 grams (g), about 10 grams (g) to about 15 grams (g), about 10 grams (g) to about 20 grams (g), about 10 grams (g) to about 25 grams (g), about... 10g to about 30g, about 10g to about 35g, about 10g to about 40g, about 10g to about 45g, about 10g to about 50g, about 10g to about 60g, about 15g to about 20g, about 15g to about 25g, about 15g to about 30g, about 15g to about 35g, about 15g to about 40g, about 15g to about 45g, about 15g to about 50g, about 15g to about 60g, about 20g to about 25g, about 20g to about 30g, about 20g to about 35g, about 20g to about 40g, about 20g to about 45g, about 20g to about 50g, about 20g to about 60g, about 25g to about 30g, about 25g to about 35g, about 25g to about 40g, about 25g to about 45g, about 25g to about 50g, about 25g to about 60g, about 30g to about 35g, about 30 grams (g) to about 40 grams (g), about 30 grams (g) to about 45 grams (g), about 30 grams (g) to about 50 grams (g), about 30 grams (g) to about 60 grams (g), about 35 grams (g) to about 40 grams (g), about 35 grams (g) to about 45 grams (g), about 35 grams (g) to about 50 grams (g), about 35 grams (g) to about 60 grams (g), about 40 grams (g) to about 45 grams (g), about 40 grams (g) to about 50 grams (g), about 40 grams (g) to about 60 grams (g), about 45 grams (g) to about 50 grams (g), about 45 grams (g) to about 60 grams (g), or about 50 grams (g) to about 60 grams (g).In some embodiments, the amount of applied legume 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 amount of applied legume 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 implementations, the amount of applied legume protein provided includes up to 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).

[0083] In some embodiments, the legume protein is pea protein. In some embodiments, the pea is the whole pea or a component thereof. In some embodiments, the pea is common pea, sugar pea, purple pea, or any combination thereof. In some embodiments, the pea is common pea. In some embodiments, the pea is sugar pea. In some embodiments, the pea is purple pea. In some embodiments, the pea is any combination of common pea, sugar pea, and / or purple pea. In some embodiments, the pea is standard pea, commercially available pea, genetically modified pea, or a combination thereof. In some embodiments, the pea is smooth pea, wrinkled pea, or a combination thereof.

[0084] In some embodiments, a composition comprising legume protein is added. In some embodiments, a composition comprising S53 protease (e.g., a precursor of Kumamolisin) is added. In some embodiments, a composition comprising both legume protein and S53 protease (e.g., a precursor of Kumamolisin) is added.

[0085] In some embodiments, the food includes sugar. In some embodiments, the sugar includes sucrose, lactose, maltose, or other disaccharides, trisaccharides, or polysaccharides containing glucose as a monomer. In some embodiments, the disease is a cardiovascular disease. In some embodiments, the disease is a neuropathy. In some embodiments, the disease is diabetic nephropathy. In some embodiments, the disease is retinopathy. In some embodiments, the disease is cataracts. In some embodiments, the disease is a bone and joint problem. In some embodiments, the disease is a dental infection. In some embodiments, the disease is a gingival infection.

[0086] In some embodiments, the method further includes administering a low-carbohydrate diet. In some embodiments, the low-carbohydrate diet comprises a diet in which less than 65% of total daily energy intake (e.g., total daily calorie intake) comes from carbohydrates. In some embodiments, the low-carbohydrate diet comprises a diet in which less than 60% of total daily energy intake (e.g., total daily calorie intake) comes from carbohydrates. In some embodiments, the low-carbohydrate diet comprises a diet in which less than 50% of total daily energy intake (e.g., total daily calorie intake) comes from carbohydrates. In some embodiments, the low-carbohydrate diet comprises a diet in which less than 45% of total daily energy intake (e.g., total daily calorie intake) comes from carbohydrates. In some embodiments, the low-carbohydrate diet comprises a diet in which less than 40% of total daily energy intake (e.g., total daily calorie intake) comes from carbohydrates. In some embodiments, the low-carbohydrate diet comprises a diet in which less than 35% of total daily energy intake (e.g., total daily calorie intake) comes from carbohydrates. In some embodiments, the low-carbohydrate diet comprises a diet in which less than 30% of total daily energy intake (e.g., total daily calorie intake) comes from carbohydrates. In some embodiments, the method further includes reducing calorie intake from carbohydrates (e.g., total daily calorie intake). In some embodiments, reducing calorie intake from carbohydrates (e.g., total daily calorie intake) includes reducing the amount (e.g., grams or calories) of carbohydrate intake, as measured by or compared to carbohydrate intake prior to administration of the protease or a composition containing the protease to the subject.

[0087] In some embodiments, the amount of carbohydrates (grams or calories) is reduced by about 10% to about 65%. In some 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 some 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 some implementations, the amount of carbohydrates (grams or calories) is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, 60%, or about 65%.

[0088] Carbohydrates generally refer to and include those with the general formula (CH2O). n Aldehydes or ketones (where n is 3-30) substituted with multiple hydroxyl groups, 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. Carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Carbohydrate derivatives or substituted carbohydrates may be deoxygenated at any corresponding C position and / or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonyl, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, aminosulfonyl, carbonylalkoxy, carboxamide, phosphonyl, phosphonoid, phosphoryl, phosphoester, thioether, oxime, hydrazine, carbamoyl, phosphate, phosphonate / phosphonate group or any other feasible functional group.

[0089] Throughout this application, various embodiments may be presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of this disclosure. Therefore, the scope description should be considered as having specifically disclosed all possible sub-scopes and the individual values ​​within those scopes. For example, a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within those scopes, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope.

[0090] Unless the context clearly specifies otherwise, as used in this specification and claims, the singular forms “a / an” and “the” include plural references. For example, the term “a sample” includes multiple samples, including mixtures thereof.

[0091] The terms “determine,” “measure,” “evaluate,” “assess,” “determine,” and “analyze” are often used interchangeably in this document to refer to forms of measurement. These terms include determining the presence of an element (e.g., detection). These terms can include quantitative, qualitative, or both quantitative and qualitative determinations. Assessments can be relative or absolute. “Detecting the presence of…” can include determining the quantity of a substance present, in addition to determining its presence in context.

[0092] The terms “subject,” “individual,” or “patient” are used interchangeably throughout this document. A “subject” can be a biological entity containing expressed genetic material. A biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a tissue, cell, or progeny of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. A mammal can be a human. A subject may be diagnosed or suspected of being at high risk of having a disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk of having a disease.

[0093] As used herein, the term "about" for a number means that number plus or minus 10% of that number. The term "about" for a range means that range minus 10% of its lowest value and plus 10% of its highest value.

[0094] The section headings used in this article are for organizational purposes only and are not intended to limit the topics described.

[0095] As used herein, the terms “comprising” (and any form of “comprising”, such as “comprise” and “comprises”), “having” (and any form of “having”, such as “have” and “has”), “including” (and any form of “including”, such as “include” and “includes”) or “containing” (and any form of “containing”, such as “contain” and “contains”) are inclusive or open-ended and do not exclude other unmentioned elements or processing steps. Also as used herein, in any example or embodiment described herein, “comprising / including” may be replaced with “substantially constitutes…” and / or “consisting of…” as used herein in any example or embodiment described.

[0096] Example

[0097] Example 1 - Lowering blood glucose using legume protein and kumamolisin precursor

[0098] Protease production

[0099] The DNA sequence of the acidic protease of interest (SEQ ID NO:2) was cloned into the expression vector pET29b(+) for protease production in *E. coli*. The completed DNA construct was converted into an expression strain of *E. coli* (BL21) and grown in baffled shake flasks at 37°C for 4–6 h in Terrific Broth until the cell density (measured using OD 600) reached 0.6. The cultures were then induced with 0.5 mM IPTG for protease expression. After induction, the cultures were grown at 30°C for 12 h and then harvested. The harvested cells were lysed using sonication, and the protease was purified from the cell lysates using IMAC chromatography.

[0100] Application of Kumamolisin precursor in pea protein beverages

[0101] 30 grams of pea protein were dissolved in 330 mL of water. Then, 14 grams of sucrose were dissolved, followed by 200 mg of acidic protease (SEQ ID NO. 1). The protein drink from this study can be consumed as is or prepared with other commonly found ingredients in protein shakes. Figure 1 As shown, adding protease can reduce the peak blood glucose level after consumption by more than half. Figure 1 The study presents a comparison of post-consumption blood glucose levels resulting from the intake of (i) a protein beverage containing kummolisin precursor and pea protein and (ii) a protein beverage without kummolisin precursor and pea protein.

[0102] Example 2 - Activity of S53 protease at low pH

[0103] Generally, the S53 proteases described and used herein should be capable of completely digesting proteins (e.g., legume proteins) in the acidic environment of the stomach. Therefore, the S53 proteases used and described herein should be active throughout the entire postprandial pH range of the gastric environment. pH distribution data for 10 representative S53 proteases (covering SEQ ID NO: 1 and 3-11) were generated. The representative S53 proteases exhibited optimal activity (e.g., 100% or substantially active) in a pH range between 2.5 and 4.5. Figure 4 The proteolytic activity of S53 protease 1-10 (P1-P10) is shown, wherein S53 protease 1-10 is in the pH range of 2 to 5.

[0104] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions can be made by those skilled in the art without departing from the scope of this disclosure. It should be understood that various alternatives to the embodiments described herein can be employed in the practice of this invention. This means that the appended claims define the scope of the embodiments disclosed herein, and the methods and structures within the scope of these claims and their equivalents are therefore covered.

[0105] sequence

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

Claims

1. Use of S53 family protease in the preparation of a medicament for reducing elevated blood glucose levels in subjects after consuming a composition containing legume protein, wherein the legume protein is pea protein, and wherein the amino acid sequence of the S53 family protease is SEQ ID NO:

1.

2. The use as described in claim 1, wherein the composition further comprises sugar.

3. The use as described in claim 1, wherein the composition further comprises fruit.

4. The use as described in claim 1, wherein the S53 family protease is used in conjunction with the composition.

5. The use as claimed in claim 1, wherein the S53 family protease is used after the composition.

6. The use as claimed in claim 1, wherein the S53 family protease is active in a pH range between pH 2 and pH 5.

7. The use as claimed in claim 1, wherein the pea protein is derived from peas.

8. The use as claimed in claim 1, wherein the increase in blood glucose is reduced relative to consuming a composition that does not contain the S53 family proteases.

9. The use as claimed in claim 1, wherein the drug is for self-administration by the subject.

10. The use as claimed in claim 1, wherein the subject is a mammal.

11. The use as claimed in claim 1, wherein the subject is a human.

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