Methods and compositions for stabilizing blood glucose

Abstract

Methods and compositions for reducing blood glucose, reducing blood glucose elevation, and / or reducing foodstuff glycemic index are provided and described herein.

Description

[0001] Cross-references

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

[0003] It is well known that high blood glucose has a number of adverse health effects. Long-term complications of hyperglycemia can range from cardiovascular disease, neuropathy, kidney and blood vessel damage to bone and joint problems. The most common way to control blood glucose levels is by controlling dietary sugar intake. While this is a reasonable and proven strategy, it requires the individual to be very careful about knowing and calculating the amount of carbohydrates consumed at each meal. Medications such as insulin are widely used, however, effective control of blood glucose levels through medications can come with a number of problems. Summary of the invention

[0004] Despite the increasing understanding of the biology and etiology of diseases associated with or caused by high blood glucose levels, individuals who are unable to regulate blood glucose levels independent of any intervention continue to bear a significant burden. Both pharmacological treatments (e.g., insulin hormones) and non-pharmacological treatments (e.g., dietary monitoring) can be considered substantial interventions with inherent barriers to the effective treatment and management of high blood glucose-related diseases. Such inherent barriers include, for example, treatment adherence (e.g., due to the complexity of treatment), financial burden and access (e.g., out-of-pocket costs for insured and uninsured individuals), and other patient-related, prescription-related, and prescriber-related factors. Such barriers and their impact on the treatment of high blood glucose-related diseases impose significant health and economic burdens on individuals and the healthcare system.

[0005] Compositions and methods that can be used to regulate (e.g., reduce, stabilize, etc.) high blood glucose are provided and described herein. The methods and compositions provided are based in part on the surprising discovery that regulation of high blood glucose can be achieved by providing legume proteins (e.g., pea proteins) and S53 family proteases to individuals. In some embodiments, S53 family protease Kumamolisin precursor (pro-Kumamolisin). In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 1 and 3-11. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1 and 3-11. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 98% sequence identity to 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 3.5. 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 is at least 50% active (e.g., relative to its maximum activity) in a pH range between about pH 2.5 and pH 4.5.

[0008] In some instances, regulating the protein digestion pattern (e.g., by administering bean protein and S53 protease (e.g., Kumamolisin precursor)) increases the concentration of certain dietary amino acids in the blood. In such instances, regulating the protein digestion pattern can induce different physiological responses, such as the reduction in blood glucose levels found. In some instances, digestion of dietary protein in the intestine, such as by administering bean protein and S53 protease (e.g., Kumamolisin precursor)), produces bioactive peptides with effective biological responses, including anti-diabetic effects (e.g., reducing blood glucose levels). As described and provided herein, a food composed of a protein food (e.g., bean protein) and an acidic protease (e.g., Kumamolisin precursor) can reduce blood sugar levels (e.g., when consumed with sugar). In addition, in some instances, the addition of a protein food (e.g., bean protein) with an acidic protease (e.g., Kumamolisin precursor) can reduce the glycemic index of a food (e.g., a food containing sugar).

[0009] Provided herein are methods for reducing blood glucose in a subject, comprising: administering to the subject a composition comprising: a legume protein; and a S53 protease (e.g., a Kumamolisin precursor). Also described and provided are methods for reducing elevated blood glucose in a subject, comprising: administering to the subject a composition comprising a legume protein and a S53 protease (e.g., a Kumamolisin precursor).

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

[0011] In some embodiments, wherein the S53 protease (e.g., Kumamolisin precursor) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an active site comprising amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an active site comprising one or more amino acid substitutions of residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site comprises an amino acid substitution between 1 and 5. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises one or more truncations of SEQ ID NO: 1, wherein the one or more truncations comprise an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[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, field 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 field peas. In some embodiments, the peas are any combination of common peas, sugar peas, and / or field peas. In some embodiments, the peas are standard peas, commercial peas, genetically modified peas, or a combination thereof. In some embodiments, the peas are smooth peas, wrinkled peas, or a combination thereof.

[0013] In some embodiments, the administration occurs after the subject ingests a food material comprising sugar. In some embodiments, relative to administration of a composition not comprising S53 protease (e.g., Kumamoli sin precursor), blood glucose elevation is reduced. 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 disorder associated with and / or caused by hyperglycemia. In some embodiments, the disorder is cardiovascular disease or neuropathy or diabetic nephropathy or retinopathy or cataracts or bone and joint problems or tooth and gum infections.

[0015] Also demonstrated is a composition for reducing blood sugar in a subject, the composition comprising a bean protein and a S53 protease (e.g., a Kumamolisin precursor). Further provided is a composition for reducing elevated blood sugar in a subject, the composition comprising a bean protein and a S53 protease (e.g., a Kumamolisin precursor).

[0016] In some embodiments, a composition is provided, comprising a food material, a legume protein (e.g., pea protein), and a S53 protease (e.g., Kumamolisin precursor) (e.g., any of the S53 proteases (e.g., Kumamolisin precursors) described herein). In certain embodiments, the composition comprising a food material, a legume protein, and a S53 protease (e.g., Kumamolisin precursor) results in a lower increase in blood glucose after consumption than a second composition comprising a single food material. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention may be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings, in which:

[0018] Figure 1 The reduction of blood glucose after administration of legume protein and S53 protease (eg, Kumamolisin precursor) is shown.

[0019] Figure 2 The reduction in blood glucose following administration of a low carbohydrate protein drink and protease is shown.

[0020] Figure 3 Shown is the reduction in blood glucose after administration of protein-fruit smoothie and protease.

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

[0022] Blood glucose imbalance and / or the presence of high blood glucose sugar levels are associated with and / or are the cause of various diseases that affect human health and well-being. Such diseases (e.g., diabetes) are often complex illnesses with annoying symptoms associated therewith, and are further characterized by glucose imbalance. In some examples, the methods and compositions provided herein can be used to reduce and / or reduce individual blood glucose levels. It is noteworthy that, in some examples, the methods and compositions provided herein are based on the following discovery, i.e., the administration of legume proteins (e.g., pea proteins) and S53 proteases (e.g., Kumamolisin precursors) can reduce (e.g., reduce or reduce) blood glucose levels. In addition, in some examples, the methods and compositions provided herein can be used to suppress, reduce, reduce and / or prevent blood glucose levels (e.g., the amount of glucose in the blood) from increasing.

[0023] Bean Protein

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

[0025] In some embodiments, legume protein is pea protein. Peas generally refer to and include seed pods from seeds or pods of plants belonging to the genus Pisum. Legume protein generally describes proteins derived from (e.g., obtained from) fruits or seeds of pea plants. Both complete pea protein and hydrolyzed pea protein sources can be used. In some embodiments, pea protein is complete legume protein. In some embodiments, pea protein is hydrolyzed legume protein. In some embodiments, pea protein is provided in solid form. In some embodiments, pea protein is provided in liquid form. In some embodiments, pea protein concentrate (e.g., protein material obtained from peas after removing soluble carbohydrates, ash and other trace components). In certain embodiments, peas are garden peas, sugar peas, field peas, or any combination thereof. In certain embodiments, peas are garden peas. In certain embodiments, peas are sugar peas. In certain embodiments, peas are field peas. In certain embodiments, peas are any combination of garden peas, sugar peas and / or field peas. In some embodiments, the peas are standard peas, commercial peas, genetically modified peas, or a combination thereof. In certain embodiments, the peas are smooth peas, wrinkled peas, or a combination thereof.

[0026] Protease

[0027] Compositions and methods described herein generally utilize acidic proteases. In some embodiments, acidic proteases are S53 family proteases. S53 family proteases generally refer to and are included in the serine protease family found in prokaryotes and eukaryotes. In some embodiments, S53 family proteases refer to and include the proteases (e.g., sedolisin, sedolisin-b, tripeptidyl peptidase I, kumamolisin, kumamolisin-B, physarolisin, aorsin, physarolisin II, kumamolisin-As, grifolisin, scytalidolisin, etc.) in and / or identified by MEROPS accession number MER0000995. In some embodiments, acidic proteases are S53 proteases (e.g., Kumamolisin precursor). Kumamolisin precursors generally refer to and include the heat-stable calcium-dependent endopeptidase derived from acidic / thermophilic bacillus (new species of bacillus. MN-32). In some embodiments, 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 an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 80% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 85% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 90% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 95% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 97% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 98% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 99% sequence identity to SEQ ID NO: 1.

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

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

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

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

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

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

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

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

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

[0038] In some embodiments, the S53 protease comprises an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 80% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 85% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 90% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 95% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 97% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 98% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to 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 identity percentage or similarity percentage between two sequences can be completed using a mathematical algorithm. In some embodiments, a non-limiting example of a mathematical algorithm for comparing two sequences is Karlin and Altschul, 1993, Proc.Natl.Acad.Sci.USA 90:5873-5877, modified as Karlin and Altschul, 1990, Proc.Natl.Acad.Sci.USA 87:2264-2268 algorithm. This algorithm is incorporated into Altschul et al., 1990, J.Mol.Biol.215:403-410 NBLAST and XBLAST programs. Alternatively, in some embodiments, PSI-Blast can be used to perform an iterative search (the same) for the distant relationship (distant relationship) between detection molecules. When using BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. In some embodiments, 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 embodiments, 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., 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., Kumamolisin precursor) comprises an active site comprising one or more amino acid substitutions of residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site comprises between 1 and 5 amino acid substitutions.

[0042] In some embodiments, the active site comprises between 1 or more amino acid substitutions. In some embodiments, the active site comprises between 2 or more amino acid substitutions. In some embodiments, the active site comprises between 3 or more amino acid substitutions. In some embodiments, the active site comprises between 4 or more amino acid substitutions.

[0043] In some embodiments, the active site comprises 1 amino acid substitution. In some embodiments, the active site comprises 2 amino acid substitutions. In some embodiments, the active site comprises 3 amino acid substitutions. In some embodiments, the active site comprises 4 amino acid substitutions. In some embodiments, the active site comprises 5 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 mimetics that function in a manner similar to naturally occurring amino acids. Amino acids generally may be referred to herein by their names (commonly known three-letter symbols) or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. As used herein, naturally occurring amino acids include and / or refer to amino acids that are generally found in nature and are not artificially manipulated. In some embodiments, naturally occurring includes and / or further refers to the 20 conventional amino acids: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp) and tyrosine (Y or Tyr).

[0045] In some embodiments, non-polar amino acids can be replaced and substituted by another non-polar amino acid, wherein non-polar amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine. In some embodiments, electrically neutral polar amino acids can be replaced and substituted by another electrically neutral polar amino acid, wherein electrically neutral polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine. In some embodiments, positively charged amino acids can be replaced and substituted by another positively charged amino acid, wherein positively charged amino acids include arginine, lysine and histidine. In some embodiments, negatively charged amino acids can be replaced and substituted by another negatively charged amino acid, wherein negatively charged amino acids include aspartic acid and glutamic acid. As used herein, peptides include and / or refer to any of the various natural or synthetic compounds containing two or more amino acids connected by a peptide bond connecting the carboxyl group of an amino acid to the amino group of another amino acid. As also used herein, amino acids refer to and / or include naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Amino acids may generally be referred to herein by their names (commonly known three letter symbols) or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0046] In some embodiments, the active site comprises between 1 and 5 amino acid substitutions. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises one or more truncations of SEQ ID NO: 1, wherein the one or more truncations comprise an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[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 3.5. 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 is at least 50% active (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., Kumamolisin precursor) can be administered as part of a composition comprising S53 protease (e.g., Kumamolisin precursor). The composition may further comprise legume protein. In some embodiments, a composition comprising S53 protease (e.g., Kumamolisin precursor) is provided. In certain embodiments, the composition further comprises legume protein (e.g., pea protein).

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

[0051] In some embodiments, the composition comprises about 5 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition comprises about 5 grams (g) of legume protein (e.g., pea protein) to about 10 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 15 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 20 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 25 grams (g) of legume protein (e.g., pea protein), about 5 grams (g) of legume protein (e.g., pea protein) to about 30 grams (g) of legume protein (e.g., pea protein). 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) of legume protein (e.g., pea protein) to about ) 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 (e.g., pea protein) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 2 ...5 grams (g) of legume protein (e.g., pea protein), about 10 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 10 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein),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 (e.g., pea protein), protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 25 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 30 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein) to about (g) legume protein (e.g., pea protein) to about 45 grams (g) legume protein (e.g., pea protein), about 20 grams (g) legume protein (e.g., pea protein) to about 50 grams (g) legume protein (e.g., pea protein), about 20 grams (g) legume protein (e.g., pea protein) to about 60 grams (g) legume protein (e.g., pea protein), about 25 grams (g) legume protein (e.g., pea protein) to about 30 grams (g) legume protein (e.g., pea protein), about 25 grams (g) legume protein (e.g., pea protein) to about 35 grams (g) legume protein (e.g., pea protein), about 25 grams (g) legume protein (e.g., pea protein) to about ) to about 40 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein) to about 45 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein) to about 50 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein) to about 60 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 35 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 40 grams (g) of legume protein (e.g.,pea protein), about 30 grams (g) of legume protein (e.g., pea protein) to about 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) to about The invention relates to a method of manufacturing a protein composition of the present invention that is at least 50 grams (g) of legume protein (e.g., pea protein) and at least 60 grams (g) of legume protein (e.g., pea protein), at least 40 grams (g) of legume protein (e.g., pea protein) and at least 45 grams (g) of legume protein (e.g., pea protein), at least 40 grams (g) of legume protein (e.g., pea protein) and at least 50 grams (g) of legume protein (e.g., pea protein), at least 40 grams (g) of legume protein (e.g., pea protein) and at least 60 grams (g) of legume protein (e.g., pea protein), at least 45 grams (g) of legume protein (e.g., pea protein) and at least 50 grams (g) of legume protein (e.g., pea protein) and at least 60 grams (g) of legume protein (e.g., pea protein), or at least 50 grams (g) of legume protein (e.g., pea protein) and at least 60 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition comprises about 5 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g., pea protein), about 30 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein), about 50 grams (g) of legume protein (e.g., pea protein), or about 60 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition comprises at least about 5 grams (g) of legume protein (e.g., pea protein), about 10 grams (g) of legume protein (e.g., pea protein), about 15 grams (g) of legume protein (e.g., pea protein), about 20 grams (g) of legume protein (e.g., pea protein), about 25 grams (g) of legume protein (e.g.,pea protein), about 30 grams (g) of legume protein (e.g., pea protein), about 35 grams (g) of legume protein (e.g., pea protein), about 40 grams (g) of legume protein (e.g., pea protein), about 45 grams (g) of legume protein (e.g., pea protein), or about 50 grams (g) of legume protein (e.g., pea protein). In some embodiments, the composition 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 milligrams (mg) of Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor. In some embodiments, the composition comprises about 50 milligrams (mg) of Kumamolisin precursor to about 100 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 200 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 300 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 400 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 5 ... 00 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 750 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 1,000 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 1,250 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor to about 1,500 milligrams (mg) of Kumamolisin precursor, about 50 milligrams (mg) of Kumamolisin precursor Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 200 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 300 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 400 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 500 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 750 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 1,000 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 1,250 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 1,500 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 300 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 400 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 500 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 750 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 1,000 milligrams (mg) of Kumamolisin precursor milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 1,250 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 1,500 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor to about 400 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor to about 500 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor to about 750 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor to about 1,000 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor to about 1,250 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor to about 1,500 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor to about 2, 000 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor to about 500 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor to about 750 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor to about 1,000 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor to about 1,250 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor to about 1,500 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor to about 750 milligrams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor to about 1,000 milligrams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor to about 1,250 milligrams (mg) of Kumamolisin precursor grams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor to about 1,500 milligrams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor, about 750 milligrams (mg) of Kumamolisin precursor to about 1,000 milligrams (mg) of Kumamolisin precursor, about 750 milligrams (mg) of Kumamolisin precursor to about 1,250 milligrams (mg) of Kumamolisin precursor g) Kumamolisin precursor, about 750 milligrams (mg) Kumamolisin precursor to about 1,500 milligrams (mg) Kumamolisin precursor, about 750 milligrams (mg) Kumamolisin precursor to about 2,000 milligrams (mg) Kumamolisin precursor, about 1,000 milligrams (mg) Kumamolisin precursor to about 1,250 milligrams (mg) Kumamolisin precursor, about 1,000 milligrams (mg) Kumamolisin precursor to about 1,500 milligrams (mg) mg) of Kumamolisin precursor, about 1,000 milligrams (mg) of Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor, about 1,250 milligrams (mg) of Kumamolisin precursor to about 1,500 milligrams (mg) of Kumamolisin precursor, about 1,250 milligrams (mg) of Kumamolisin precursor to about 2,000 milligrams (mg) of Kumamolisin precursor, or about 1,500 milligrams (mg) of Kumamolisin precursor to about 2,In some embodiments, the composition comprises about 50 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor, about 750 milligrams (mg) of Kumamolisin precursor, about 1,000 milligrams (mg) of Kumamolisin precursor, about 1,250 milligrams (mg) of Kumamolisin precursor, about 1,500 milligrams (mg) of Kumamolisin precursor, or about 2,000 milligrams (mg) of Kumamolisin precursor. In some embodiments, the composition comprises at least about 50 milligrams (mg) of Kumamolisin precursor, about 100 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor, about 750 milligrams (mg) of Kumamolisin precursor, about 1,000 milligrams (mg) of Kumamolisin precursor, about 1,250 milligrams (mg) of Kumamolisin precursor, or about 1,500 milligrams (mg) of Kumamolisin precursor. In some embodiments, the composition comprises up to about 100 milligrams (mg) of Kumamolisin precursor, about 200 milligrams (mg) of Kumamolisin precursor, about 300 milligrams (mg) of Kumamolisin precursor, about 400 milligrams (mg) of Kumamolisin precursor, about 500 milligrams (mg) of Kumamolisin precursor, about 750 milligrams (mg) of Kumamolisin precursor, about 1,000 milligrams (mg) of Kumamolisin precursor, about 1,250 milligrams (mg) of Kumamolisin precursor, about 1,500 milligrams (mg) of Kumamolisin precursor, or about 2,000 milligrams (mg) of Kumamolisin precursor.

[0053] method

[0054] As described herein, in certain examples, providing legume protein (e.g., pea protein) and S53 protease (e.g., Kumamolisin precursor) protease reduces (e.g., lowers or decreases) blood glucose levels. Thus, provided herein is a method of reducing blood glucose in a subject, comprising: administering a composition comprising legume protein and S53 protease (e.g., Kumamolisin precursor) to the subject. (e.g., wherein legume protein and S53 protease (e.g., Kumamolisin precursor) are administered to the subject). Further provided are legume protein and S53 protease (e.g., Kumamolisin precursor) for use in a method of reducing blood glucose in a subject (e.g., wherein legume protein and S53 protease (e.g., Kumamolisin precursor) are administered to the subject).

[0055] Also provided is a method for reducing elevated blood glucose in a subject, comprising: administering to the subject a composition comprising a bean protein and an S53 protease (e.g., a Kumamolisin precursor). Further provided is the use of a bean protein and an S53 protease (e.g., a Kumamolisin precursor) in a method for reducing elevated blood glucose in a subject (e.g., wherein the bean protein and the S53 protease (e.g., a Kumamolisin precursor) are administered to the subject). In addition, also provided is a bean protein and an S53 protease (e.g., a Kumamolisin precursor) for use in a method for reducing elevated blood glucose in a subject (e.g., wherein the bean protein and the S53 protease (e.g., a Kumamolisin precursor) are administered to the subject).

[0056] In certain examples, providing legume protein (e.g., pea protein) and S53 protease (e.g., Kumamolisin precursor) can stabilize (e.g., reduce or prevent the range or degree of variation) blood glucose levels associated with food intake. In some embodiments, provided herein is a method of reducing blood glucose variation (positive or negative) in a subject, comprising: administering to the subject a composition comprising 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).

[0057] In certain embodiments, the administration of bean protein and S53 protease (e.g., Kumamolisin precursor) can reduce the degree or amount of blood glucose rise after ingestion of food. In certain examples, the degree or amount of blood glucose rise after reducing the intake of food can be compared with the increase of blood glucose after ingestion of food before the administration of bean protein and S53 protease (e.g., Kumamolisin precursor) or without the administration of bean protein and S53 protease (e.g., Kumamolisin precursor). In certain embodiments, the administration of bean protein and S53 protease (e.g., Kumamolisin precursor) can reduce the degree or amount of blood glucose decline after ingestion of food. In certain examples, the degree or amount of blood glucose decline after reducing the intake of food can be compared with the decline of blood glucose after ingestion of food before the administration of bean protein and S53 protease (e.g., Kumamolisin precursor) or without the administration of bean protein and S53 protease (e.g., Kumamolisin precursor). In some embodiments, administration of bean protein and S53 protease (e.g., Kumamolisin precursor) can reduce changes in blood glucose (e.g., increases or decreases) after ingestion of a food. In certain embodiments, the reduction in changes in blood glucose (e.g., increases or decreases) after ingestion of a food when bean protein and S53 protease (e.g., Kumamolisin precursor) are compared to changes in blood glucose after ingestion of a food before or without administration of bean protein and / or S53 protease (e.g., Kumamolisin precursor).

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

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

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

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

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

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

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

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

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

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

[0068] In some embodiments, the S53 protease comprises an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 80% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 85% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 90% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 95% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 97% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to or greater than 98% sequence identity with SEQ ID NO: 11. In some embodiments, the S53 protease comprises an amino acid sequence having equal to 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., Kumamolisin precursor) can be used in the provided methods. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1.

[0071] In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an active site comprising amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an active site comprising one or more amino acid substitutions of residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site comprises an amino acid substitution between 1 and 5. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises one or more truncations of SEQ ID NO: 1, wherein the one or more truncations comprise an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[0072] In some embodiments, the amount of S53 protease (e.g., Kumamolisin precursor) administered includes about 50 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the amount of S53 protease (e.g., Kumamolisin precursor) administered includes about 50 milligrams (mg) to about 100 milligrams (mg), about 50 milligrams (mg) to about 150 milligrams (mg), about 50 milligrams (mg) to about 200 milligrams (mg), about 50 milligrams (mg) to about 300 milligrams (mg), about 50 milligrams (mg) to about 400 milligrams (mg), about 50 milligrams (mg) to about 500 milligrams (mg), about 50 milligrams (mg) to about 750 milligrams (mg), about 50 milligrams (mg) to about 1,000 milligrams (mg), about 50 milligrams (mg) to about 1,50 0 milligrams (mg), about 100 milligrams (mg) to about 150 milligrams (mg), about 100 milligrams (mg) to about 200 milligrams (mg), about 100 milligrams (mg) to about 300 milligrams (mg), about 100 milligrams (mg) to about 400 milligrams (mg), about 100 milligrams (mg) to about 500 milligrams (mg), about 100 milligrams (mg) to about 750 milligrams (mg), about 100 milligrams (mg) to about 1,000 milligrams (mg), about 100 milligrams (mg) to about 1,500 milligrams (mg), about 150 milligrams (mg) to about 200 milligrams (mg), about 15 ... grams (mg), about 150 milligrams (mg) to about 400 milligrams (mg), about 150 milligrams (mg) to about 500 milligrams (mg), about 150 milligrams (mg) to about 750 milligrams (mg), about 150 milligrams (mg) to about 1,000 milligrams (mg), about 150 milligrams (mg) to about 1,500 milligrams (mg), about 200 milligrams (mg) to about 300 milligrams (mg), about 200 milligrams (mg) to about 400 milligrams (mg), about 200 milligrams (mg) to about 500 milligrams (mg), about 200 milligrams (mg) to about 750 milligrams (mg), about 20 ...). grams (mg), about 200 milligrams (mg) to about 1,500 milligrams (mg), about 300 milligrams (mg) to about 400 milligrams (mg), about 300 milligrams (mg) to about 500 milligrams (mg), about 300 milligrams (mg) to about 750 milligrams (mg), about 300 milligrams (mg) to about 1,000 milligrams (mg), about 300 milligrams (mg) to about 1,500 milligrams (mg), about 400 milligrams (mg) to about 500 milligrams (mg), about 400 milligrams (mg) to about 750 milligrams (mg), about 400 milligrams (mg) to about 1,000 milligrams (mg), about 400 milligrams (mg) to about 1,In some embodiments, the amount of S53 protease (e.g., Kumamolisin precursor) administered includes 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., Kumamolisin precursor) administered includes 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., Kumamolisin precursor) administered includes at least about 50 milligrams (mg), about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), or about 1,000 milligrams (mg). In some embodiments, the amount of S53 protease (e.g., Kumamolisin precursor) administered includes at most about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), about 1,000 milligrams (mg), or about 1,500 milligrams (mg).

[0073] In some embodiments, the amount of legume protein administered includes about 5 grams (g) to about 50 grams (g). In some embodiments, the amount of legume protein administered includes about 5 grams (g) to about 10 grams (g), about 5 grams (g) to about 15 grams (g), about 5 grams (g) to about 20 grams (g), about 5 grams (g) to about 25 grams (g), about 5 grams (g) to about 30 grams (g), about 5 grams (g) to about 35 grams (g), about 5 grams (g) to about 40 grams (g), about 5 grams (g) to about 45 grams (g), about 5 grams (g) to about 50 grams (g), about 10 grams (g) to about 15 grams (g), about 10 grams (g) to about 20 gram (g), about 10 grams (g) to about 25 grams (g), about 10 grams (g) to about 30 grams (g), about 10 grams (g) to about 35 grams (g), about 10 grams (g) to about 40 grams (g), about 10 grams (g) to about 45 grams (g), about 10 grams (g) to about 50 grams (g), about 15 grams (g) to about 20 grams (g), about 15 grams (g) to about 25 grams (g), about 15 grams (g) to about 30 grams (g), about 15 grams (g) to about 35 grams (g), about 15 grams (g) to about 40 grams (g), about 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 In some embodiments, the amount of legume protein administered includes about 5 g, about 10 g, about 15 g, about 20 g, about 25 g, about 30 g, about 35 g, about 40 g, about 30 g, about 45 g, about 30 g to about 50 g, about 35 g to about 40 g, about 35 g to about 45 g, about 35 g to about 50 g, about 40 g to about 45 g, about 40 g to about 50 g, or about 45 g to about 50 g. In some embodiments, the amount of legume protein administered includes about 5 g, about 10 g, about 15 g, about 20 g, about 25 g, about 30 g, about 35 g, about 40 g, about 45 g, or about 50 g. In some embodiments, the amount of pulse protein administered includes at least about 5 grams (g), about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), or about 45 grams (g).In some embodiments, the amount of pulse protein administered 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 whole pea or a component thereof. In some embodiments, the pea is common pea, sugar pea, field 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 field pea. In some embodiments, the pea is any combination of common pea, sugar pea, and / or field pea. In some embodiments, the pea is standard pea, commercial pea, genetically modified pea, or a combination thereof. In some embodiments, the pea is smooth pea, wrinkled pea, or a combination thereof.

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

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

[0077] In some embodiments, relative to the use of a composition that does not include S53 protease (e.g., Kumamolisin precursor), blood sugar elevation is reduced. 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 an illness associated with hyperglycemia and / or caused by hyperglycemia. In some embodiments, the illness is cardiovascular disease or neuropathy or diabetic nephropathy or retinopathy or cataract or bone and joint problems or tooth and gum infection. In certain embodiments, the illness is cardiovascular disease. In certain embodiments, the illness is neuropathy. In certain embodiments, the illness is diabetic nephropathy. In certain embodiments, the illness is retinopathy. In certain embodiments, the illness is cataract. In certain embodiments, the illness is bone and joint problems. In certain embodiments, the illness is a tooth infection. In certain embodiments, the illness is a gum infection.

[0078] Further provided is a method for reducing the glycemic index of a food, comprising: providing a legume protein and an S53 protease (e.g., a Kumamolisin precursor) to the food. Further provided is the use of the legume protein and the S53 protease (e.g., a Kumamolisin precursor) for reducing the glycemic index of a food. In addition, legume protein and an S53 protease (e.g., a Kumamolisin precursor) for reducing the glycemic index of a food are also provided.

[0079] Any S53 protease described herein (e.g., Kumamolisin precursor) can be used in the provided methods. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NO: 1 and 3-11. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1 and 3-11. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an amino acid sequence having at least 98% sequence identity to 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., Kumamolisin precursor) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an active site comprising amino acid residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises an active site comprising one or more amino acid substitutions of residues E266, F295, S316, W317, G318, A349, A350, G351, D352, S353, D367, G462, G463, T464, S465, and A466 of SEQ ID NO: 1. In some embodiments, the active site comprises an amino acid substitution between 1 and 5. In some embodiments, the S53 protease (e.g., Kumamolisin precursor) comprises one or more truncations of SEQ ID NO: 1, wherein the one or more truncations comprise an N-terminal truncation, a C-terminal truncation, or both an N-terminal and a C-terminal truncation.

[0081] In some embodiments, the amount of Kumamolisin precursor administered provided includes about 50 milligrams (mg) to about 1,500 milligrams (mg). In some embodiments, the amount of Kumamolisin precursor administered provided includes about 50 milligrams (mg) to about 100 milligrams (mg), about 50 milligrams (mg) to about 150 milligrams (mg), about 50 milligrams (mg) to about 200 milligrams (mg), about 50 milligrams (mg) to about 300 milligrams (mg), about 50 milligrams (mg) to about 400 milligrams (mg), about 50 milligrams (mg) to about 500 milligrams (mg), about 50 milligrams (mg) to about 750 milligrams (mg), about 50 milligrams (mg) to about 1,000 milligrams (mg), about 50 milligrams (mg) to about 1,500 milligrams (mg), about 100 milligrams (mg) ) to about 150 milligrams (mg), about 100 milligrams (mg) to about 200 milligrams (mg), about 100 milligrams (mg) to about 300 milligrams (mg), about 100 milligrams (mg) to about 400 milligrams (mg), about 100 milligrams (mg) to about 500 milligrams (mg), about 100 milligrams (mg) to about 750 milligrams (mg), about 100 milligrams (mg) to about 1,000 milligrams (mg), about 100 milligrams (mg) to about 1,500 milligrams (mg), about 150 milligrams (mg) to about 200 milligrams (mg), about 150 milligrams (mg) to about 300 milligrams (mg), about 150 milligrams (mg) to about 400 milligrams (mg) , about 150 milligrams (mg) to about 500 milligrams (mg), about 150 milligrams (mg) to about 750 milligrams (mg), about 150 milligrams (mg) to about 1,000 milligrams (mg), about 150 milligrams (mg) to about 1,500 milligrams (mg), about 200 milligrams (mg) to about 300 milligrams (mg), about 200 milligrams (mg) to about 400 milligrams (mg), about 200 milligrams (mg) to about 500 milligrams (mg), about 200 milligrams (mg) to about 750 milligrams (mg), about 200 milligrams (mg) to about 1,000 milligrams (mg), about 200 milligrams (mg) to about 1,500 milligrams (mg), about 300 milligrams (mg) (mg) to about 400 milligrams (mg), about 300 milligrams (mg) to about 500 milligrams (mg), about 300 milligrams (mg) to about 750 milligrams (mg), about 300 milligrams (mg) to about 1,000 milligrams (mg), about 300 milligrams (mg) to about 1,500 milligrams (mg), about 400 milligrams (mg) to about 500 milligrams (mg), about 400 milligrams (mg) to about 750 milligrams (mg), about 400 milligrams (mg) to about 1,000 milligrams (mg), about 400 milligrams (mg) to about 1,500 milligrams (mg), about 500 milligrams (mg) to about 750 milligrams (mg), about 500 milligrams (mg) to about 1,In some embodiments, the amount of Kumamolisin precursor administered provided includes 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, about 750 mg, about 1,500 mg, or about 1,000 mg to about 1,500 mg. In some embodiments, the amount of Kumamolisin precursor administered provided includes 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 Kumamolisin precursor provided for administration includes at least about 50 milligrams (mg), about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), or about 1,000 milligrams (mg). In some embodiments, the amount of Kumamolisin precursor provided for administration includes at most about 100 milligrams (mg), about 150 milligrams (mg), about 200 milligrams (mg), about 300 milligrams (mg), about 400 milligrams (mg), about 500 milligrams (mg), about 750 milligrams (mg), about 1,000 milligrams (mg), or about 1,500 milligrams (mg).

[0082] In some embodiments, the amount of legume protein administered provided includes about 5 grams (g) to about 60 grams (g). In some embodiments, the amount of legume protein administered 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 10 grams (g) to about 30 grams (g), about 10 grams (g) to about 35 grams (g), about 10 grams (g) to about 40 grams (g), about 10 grams (g) to about 45 grams (g), about 10 grams (g) to about 50 grams (g), about 10 grams (g) to about 60 grams (g), about 15 grams (g) to about 20 grams (g), about 15 grams (g) to about 25 grams (g), about 15 grams (g) to about 30 grams (g), about 15 grams (g) to about 35 grams (g), about 15 grams (g) to about 40 grams (g), about 15 grams (g) to about 45 grams (g), about 15 grams (g) to about 50 grams (g), about 15 grams (g) to about 60 grams (g), about 20 grams (g) to about 25 grams (g), about 20 grams (g) to about 30 grams (g), about 20 grams (g) to about 35 grams (g), about 20 grams (g) to about 40 grams (g), about 20 grams (g) to about 45 grams (g), about 20 grams (g) to about 50 grams (g), about 20 grams (g) to about 60 grams (g), about 25 grams (g) to about 30 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 25 grams (g) to about 60 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 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 soy protein provided for administration 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 soy protein provided for administration includes at least about 5 grams (g), about 10 grams (g), about 15 grams (g), about 20 grams (g), about 25 grams (g), about 30 grams (g), about 35 grams (g), about 40 grams (g), about 45 grams (g), or about 50 grams (g). In some embodiments, the amount of pulse protein administered 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 whole pea or a component thereof. In some embodiments, the pea is common pea, sugar pea, field 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 field pea. In some embodiments, the pea is any combination of common pea, sugar pea, and / or field pea. In some embodiments, the pea is standard pea, commercial pea, genetically modified pea, or a combination thereof. In 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., Kumamolisin precursor) is added. In some embodiments, a composition comprising legume protein and S53 protease (e.g., Kumamolisin precursor) is added.

[0085] In some embodiments, the foodstuff comprises sugar. In some embodiments, the sugar comprises sucrose, lactose, maltose or other disaccharides, trisaccharides or polysaccharides containing glucose as a monomer. In certain embodiments, the disorder is cardiovascular disease. In certain embodiments, the disorder is neuropathy. In certain embodiments, the disorder is diabetic nephropathy. In certain embodiments, the disorder is retinopathy. In certain embodiments, the disorder is cataract. In certain embodiments, the disorder is bone and joint problems. In certain embodiments, the disorder is tooth infection. In certain embodiments, the disorder is gum infection.

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

[0087] In certain embodiments, the amount of carbohydrate (gram or calorie) is reduced by about 10% to about 65%.In certain embodiments, the amount of carbohydrate (gram or calorie) is reduced by about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 65%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 65%, about 30% to about 40%, about 30% to about 50%, about 30% to about 65%, about 40% to about 50%, about 40% to about 65% or about 50% to about 65%.In certain embodiments, the amount of carbohydrate (gram or calorie) is reduced by about 10%, about 20%, about 30%, about 40%, about 50% or about 60%. In certain embodiments, the amount of carbohydrate (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 carbohydrates having the general formula (CH2O) n (wherein n is 3-30) substituted with multiple hydroxyl groups of aldehyde or ketone compounds, and their oligomers and polymers. Carbohydrates can 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. The carbohydrate derivative or substituted carbohydrate can be deoxygenated at any corresponding C position and / or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, sulfhydryl, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carbonylalkoxy, carboxamido, phosphono, phosphinyl, phosphoryl, phosphino, thioester, thioether, oxime, hydrazine, carbamoyl, phosphate, phosphonate / phosphonate (phosphonato) or any other feasible functional group.

[0089] Throughout the application, various embodiments may be presented in a range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as a rigid limitation to the scope of the present disclosure. Therefore, the description of the scope should be considered to have specifically disclosed all possible subranges and each numerical value in the scope. For example, the description of a scope such as from 1 to 6 should be considered to have a specifically disclosed subrange, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and each numeral in the scope, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the scope, this applies.

[0090] As used in this specification and claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0091] The terms "determine," "measure," "evaluate," "assess," "measure," and "analyze" are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present (e.g., detecting). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Assessments may be relative or absolute. "Detecting the presence of..." may include determining the amount of a substance present in addition to determining whether a substance is present, depending on the context.

[0092] The terms "subject", "individual" or "patient" are often used interchangeably herein. A "subject" can be a biological entity containing expressed genetic material. A biological entity can be a plant, an animal or a microorganism, including, for example, bacteria, viruses, fungi and protozoa. A subject can be a tissue, a cell and its 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 for a disease. In some cases, a subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0093] As used herein, the term "about" a number refers to the number plus or minus 10% of the number. The term "about" a range refers to the range minus 10% of its lower limit and plus 10% of its upper limit.

[0094] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0095] As used herein, the words "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 additional, unrecited elements or processing steps. Also as used herein, "comprising / including" in any example or embodiment described herein can be replaced with "consisting essentially of" and / or "consisting of" as used in any example or embodiment described herein.

[0096] Example

[0097] Example 1 - Lowering Blood Glucose Using Legume Protein and Kumamolisin Precursor

[0098] Protease production

[0099] The DNA sequence (SEQ ID NO:2) of the acid protease of interest is cloned into the expression vector pET29b (+) for protease production in Escherichia coli. The completed DNA construct is converted into the expression strain of Escherichia coli (BL21), and a baffled shake flask is used to grow 4-6 hours at 37 degrees Celsius in Terrific Broth until the cell density (using OD 600 measurement) reaches 0.6. The culture is then induced with 0.5mM IPTG for protease expression. After induction, the culture is grown at 30 degrees Celsius for 12 hours, and then harvested. The cells harvested using ultrasonic lysis are used, and the IMAC chromatography is used to purify protease from the cell lysate.

[0100] Application of Kumamolisin precursor in pea protein beverage

[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 acid protease (SEQ ID NO. 1). The protein beverage of this study can be consumed as is, or formulated with other common ingredients found in protein shakes. Figure 1 As shown, the addition of protease can reduce the post-meal blood glucose spike during the blood glucose peak period by more than half. Figure 1 Shown is a comparison of post-consumption blood glucose levels resulting from ingestion of (i) a protein beverage comprising kumamolisin precursor and pea protein, compared to (ii) a protein beverage without kumamolisin precursor and pea protein.

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

[0103] Generally speaking, the S53 proteases described and used herein should have the ability to thoroughly digest proteins (e.g., legume proteins) in the acidic environment of the stomach. Therefore, the S53 proteases used and described herein should be active in the entire postprandial pH range of the gastric environment. pH distribution data for 10 representative S53 proteases (covering SEQ ID NOs: 1 and 3-11) were generated. Representative S53 proteases exhibited optimal activity (e.g., 100% or substantially active) in a pH range between 2.5 and 4.5. FIG. 6 shows the proteolytic activity of S53 proteases 1-10 (P1-P10), wherein S53 proteases 1-10 are in the pH range of 2 to 5.

[0104] Although 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. Without departing from the present disclosure, those skilled in the art may also make many changes, modifications and substitutions. It should be understood that various alternatives to the embodiments described herein may be adopted when practicing the present invention. This means that the appended claims define the scope of the embodiments disclosed herein and that methods and structures within the scope of these claims and their equivalents are therefore encompassed.

[0105] sequence

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Claims

1. A method of lowering blood glucose in a subject, comprising: administering to the subject a composition comprising: legume protein; as well as S53 family proteases, wherein said administering lowers blood glucose in said subject.

2. A method of reducing elevated blood glucose in a subject, comprising: administering to the subject a composition comprising: legume protein; as well as S53 family proteases, wherein said administering reduces elevation of blood glucose in said subject.

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

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

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

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

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

1.

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

1.

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

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

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

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

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

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

15. The method of claim 13, wherein the peas are whole peas or parts thereof.

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

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

18. The method of claim 13 or 15, wherein the pea is Common pea, Sugar pea, Field pea, or any combination thereof.

19. The method of claim 1, wherein the administering occurs after the subject ingests a food material comprising sugar.

20. The method of claim 2, wherein the blood glucose elevation is reduced relative to administration of a composition not comprising the S53 family protease.

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

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

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

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

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

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

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

28. The method of any one of claims 1 to 27, wherein the method further comprises reducing caloric intake obtained from carbohydrates.

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

30. A composition for lowering blood sugar in a subject, the composition comprising legume protein and an S53 family protease.

31. A composition for reducing elevated blood sugar in a subject, the composition comprising legume protein and an S53 family protease.

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