Hydrolyzed collagen for lowering blood sugar

Abstract

This invention relates to hydrolyzed collagen preparations for lowering blood sugar. The hydrolyzed collagen is obtained by enzymatic hydrolysis of collagen-containing materials using an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases. The hydrolyzed collagen preparations are particularly suitable as dietary supplements, for example, for improving hyperglycemia and / or risk factors for hyperglycemia.

Description

Technical Field

[0001] This invention relates to active ingredients for improving hyperglycemia and hyperglycemia-related conditions, and their uses. The active ingredients of this invention are suitable as dietary supplements. Background Technology

[0002] The normal functioning of the human body depends on strict control of its blood sugar levels.

[0003] Disorders of normal blood glucose levels can lead to diseases such as metabolic syndrome, cardiovascular disease, nerve damage, and kidney failure. Conditions associated with abnormal insulin levels and / or insulin sensitivity (such as type 2 diabetes and obesity) are closely related to the occurrence of hyperglycemia.

[0004] Blood glucose regulation involves the interaction of various hormones released from different organs (such as the pancreas, brain, liver, intestines, fat, and muscle tissue).

[0005] Insulin and glucagon are peptide hormones that counteract each other to balance blood sugar levels. In response to high blood sugar levels, pancreatic beta cells secrete insulin into the bloodstream. This subsequently lowers blood sugar levels by promoting the uptake of glucose from the blood into liver, fat, and skeletal muscle cells. Glucagon is secreted by pancreatic alpha cells. Glucagon enhances the liver's conversion of stored glycogen into glucose, releasing it into the bloodstream.

[0006] First-line treatment for high blood sugar initially includes lifestyle modifications that reduce one or more risk factors for high blood sugar. These typically include lifestyle changes targeting insulin resistance, diabetes, obesity, or high blood pressure. Often, lifestyle modifications alone are insufficient and need to include metformin and / or insulin therapy as part of first-line treatment.

[0007] In large individual groups, the aforementioned first-line treatments failed to adequately relieve hyperglycemia. As a possible second-line treatment, new classes of hypoglycemic agents are currently being developed and tested (Davies et al. Diabetes Care 2018; 41(12):2669-2701).

[0008] A new class of hypoglycemic drugs includes those targeting incretin activity. Incretins are metabolic hormones that lower blood glucose levels by enhancing insulin secretion and / or inhibiting glucagon secretion in a glucose-dependent manner. Glucagon-like peptide-1 (GLP-1) is considered one of the most important incretins and is therefore an important target in new strategies for improving hyperglycemia. Although the clinical efficacy of GLP-1 receptor agonists has been demonstrated (Trujillo et al. Ther Adv Endocrinol Metab. 2021 Mar 9; 12:2042018821997320), they have several limitations that hinder their widespread use. First, they cause several side effects, including pancreatitis, pancreatic cancer, or bone disease (Storgaard et al. Diabetes Obes Metab 2017; 19:906-908), as well as gastrointestinal side effects such as nausea, vomiting, and diarrhea. Second, GLP-1 receptor agonist therapy requires repeated subcutaneous injections, which can cause patient discomfort and injection site reactions (Trujillo et al. Ther AdvEndocrinol Metab. 2021 Mar9; 12:2042018821997320).

[0009] Another example of a new blood glucose-lowering drug is sitagliptin. Sitagliptin is a selective inhibitor of dipeptidyl peptidase IV (DPPIV). DPPIV affects the hydrolysis of incretin hormones. By doing so, sitagliptin increases plasma concentrations of active forms of incretin, such as GLP-1 and gastric inhibitory polypeptide (GIP). This increases insulin release and lowers glucagon levels. However, medical professionals remain hesitant to prescribe sitagliptin to patients. This is largely due to its numerous side effects and contraindications; for example, up to 10% of patients experience complications such as hypoglycemia, headache, airway infection, and nasopharyngitis.

[0010] Collagen peptides have also been suggested for lowering blood sugar. JP2009235064 describes a blood sugar-lowering collagen peptide obtained by treating starfish with a protease, and JP2012116773 describes a collagen peptide that increases insulin-like growth factor 1 (IGF-1) by treating collagen derived from fish scales and / or fish skin with a proteolytic enzyme.

[0011] There remains an unmet need for active ingredients that can more effectively and / or more safely improve hyperglycemia. Such active ingredients could be used as an adjunct or alternative to first-line and / or second-line treatment for individuals with hyperglycemia (or at risk of hyperglycemia). This invention aims to provide such active ingredients. Summary of the Invention

[0012] The inventors have discovered that enzymatic hydrolysis of collagen-containing starting materials using a combination of two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases produces a composition with potent hypoglycemic activity. The hypoglycemic activity is highest when all three of the aforementioned enzymes are used for hydrolysis.

[0013] In one aspect, the present invention relates to hydrolyzed collagen for lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0014] In one aspect, the present invention relates to (further) hydrolyzed collagen for improving hyperglycemia or risk factors for hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0015] In one aspect, the present invention relates to a method for obtaining hydrolyzed collagen, comprising:

[0016] a) Provide materials containing collagen for use in liquid formulations; and

[0017] b) subjecting a collagen-containing material to an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases, wherein the hydrolyzed collagen is preferably used to lower blood glucose and / or improve hyperglycemia.

[0018] In one aspect, the present invention relates to hydrolyzed collagen, which can be obtained by methods as disclosed herein, wherein the hydrolyzed collagen is preferably used to lower blood sugar and / or improve hyperglycemia. Detailed Implementation

[0019] Some aspects of the present invention

[0020] In one aspect, the present invention relates to hydrolyzed collagen for lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0021] In one aspect, the present invention relates to hydrolyzed collagen for improving hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0022] In one aspect, the present invention relates to a method for obtaining hydrolyzed collagen, comprising:

[0023] a) Provide materials containing collagen for use in liquid formulations; and

[0024] b) subjecting the collagen-containing material to an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases, wherein the hydrolyzed collagen is preferably used to lower blood glucose and / or improve hyperglycemia, as taught herein.

[0025] In one aspect, the present invention relates to hydrolyzed collagen that can be obtained by methods disclosed herein, wherein the hydrolyzed collagen is preferably used to lower blood sugar and / or improve hyperglycemia, as taught herein.

[0026] In one aspect, the present invention relates to a method for lowering blood sugar, the method comprising administering to a subject hydrolyzed collagen obtained by enzymatic hydrolysis of a collagen-containing material with an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0027] In one aspect, the present invention relates to a method for treating hyperglycemia or risk factors for hyperglycemia, the method comprising administering to a subject hydrolyzed collagen obtained by enzymatic hydrolysis of a collagen-containing material with an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0028] In one aspect, the present invention relates to the use of hydrolyzed collagen in the preparation of a medicament for lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0029] In one aspect, the present invention relates to the use of hydrolyzed collagen in the preparation of a medicament for improving hyperglycemia or risk factors for hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0030] Benefits of the present invention

[0031] - The use of hydrolyzed collagen (e.g., as a dietary supplement) is generally considered safe;

[0032] The inventors have discovered that the hypoglycemic effect of the hydrolyzed collagen of this invention is similar to that of the pharmaceutical compound sitagliptin. Therefore, hydrolyzed collagen is a valuable additive and / or substitute for hypoglycemic drug compounds, as these compounds typically cause side effects and have contraindications; and

[0033] The inventors have discovered that the hypoglycemic effect of the hydrolyzed collagen of this invention comprises different mechanisms of action. These mechanisms include increasing glucagon-like peptide-1 (GLP-1) and inhibiting dipeptidyl peptidase IV (DPP IV). These different mechanisms of action explain the potent hypoglycemic activity of hydrolyzed collagen in vivo. Furthermore, the presence of these different mechanisms suggests that hydrolyzed collagen may (and may also) be suitable for treating the potential effects of hyperglycemia (e.g., in different subject populations and / or at different stages of hyperglycemia);

[0034] The inventors have determined the beneficial effects of hydrolyzed collagen in healthy mice (i.e., showing prevention of hyperglycemia) and obese mice with hyperglycemia (i.e., showing treatment of hyperglycemia). This demonstrates that the hydrolyzed collagen of the present invention is effective in improving hyperglycemia as both a preventative and therapeutic agent.

[0035] Blood sugar and high blood sugar

[0036] In one embodiment, the present invention relates to hydrolyzed collagen for lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, wherein the combination of enzymes comprises two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0037] In a preferred embodiment, the present invention relates to hydrolyzed collagen for lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes comprising neutral protease, carboxypeptidase, and aminopeptidase.

[0038] In a preferred embodiment, the combination of enzymes disclosed herein comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

[0039] Preferably, hydrolyzed collagen as disclosed herein is used as an active ingredient. More preferably, hydrolyzed collagen as disclosed herein is used as an active ingredient for lowering blood sugar as disclosed herein and / or as an active ingredient for improving hyperglycemia as disclosed herein.

[0040] As used herein, the term "blood glucose" (i.e., the level of glucose in the blood) may be used interchangeably with "blood sugar". "Blood glucose" is typically and preferably expressed in millimoles per liter (mmol / L) and / or in milligrams per deciliter (mg / dL). Blood glucose is typically and preferably determined in a fasting individual. In one embodiment, a "fasting individual" means an individual who has not eaten for at least eight hours.

[0041] In one implementation, hydrolyzed collagen (further) as disclosed herein is used to improve hyperglycemia.

[0042] When referring to the molecular level, the term "blood" in this article may be used interchangeably with the term "plasma." For example, "blood glucose" may also be considered "plasma glucose." Similarly, "blood GLP-1" may also be considered "plasma GLP-1."

[0043] The term "hyperglycemia" as used in this article refers to high blood glucose. In this article, the normal blood glucose range for fasting individuals is considered to be 80 to 110 mg / dL. Individuals with blood glucose levels of 126 mg / dL or higher are considered to have hyperglycemia. Individuals with fasting blood glucose levels of 111 to 125 mg / dL are generally considered to have impaired glucose tolerance or prediabetes. In this article, individuals with "impaired glucose tolerance" or "prediabetes" are considered to have hyperglycemia.

[0044] In one implementation, "hyperglycemia" as disclosed herein is acute and is typically characterized by the need for direct administration of insulin.

[0045] In one implementation, "hyperglycemia" as disclosed herein is chronic and / or requires long-term hypoglycemic treatment, with or without combination with lifestyle modifications. "Hyperglycemia" as disclosed herein is preferably chronic hyperglycemia.

[0046] In the context of this invention, a preferred method for measuring hyperglycemia is a hemoglobin A1c (HbA1c) test, which determines glycated hemoglobin. The most widely accepted and / or most accurate methods for measuring HbA1c are known to those skilled in the art; however, preferred analytical methods for measuring HbA1c include affinity chromatography, immunoassay, cation exchange chromatography, and / or capillary electrophoresis.

[0047] In one embodiment, hyperglycemia as disclosed herein includes “postprandial hyperglycemia.” In the context of this invention, “postprandial hyperglycemia” is defined as excessively high postprandial blood glucose levels. The term “postprandial glucose level” (PPG level) as used herein refers to the amount of glucose in the blood after a meal. In healthy or non-diabetic individuals, blood glucose levels peak approximately one hour after the start of a meal and typically do not exceed 140 mg / dL, returning to pre-meal levels within 2 to 3 hours. These time profiles are typically different in diabetic patients or patients with various pathological conditions. In one embodiment, PPG levels are measured 2 hours after the start of a meal; however, another timeframe may be more appropriate, depending on when the peak typically (or is expected) occurs. Acceptable PPG levels, threshold PPG levels defining postprandial hyperglycemia, and optimal methods for measuring PPG levels can vary among medical professionals and practitioners. In the context of this invention, for adults under 50 years of age, a PPG level above 140 mg / dl is considered excessively high (i.e., postprandial hyperglycemia); for individuals aged 50 to 60 years, a PPG level above 150 mg / dl is considered excessively high; and for individuals over 60 years of age, a PPG level above 160 mg / dl is considered excessively high (i.e., postprandial hyperglycemia).

[0048] This invention may relate to non-therapeutic and / or therapeutic uses of hydrolyzed collagen, wherein the distinction is based on different groups of subjects with elevated blood glucose levels and / or those experiencing elevated blood glucose.

[0049] The first group (referred to herein as the "healthy subject group") comprises healthy individuals who have not received therapeutic benefit from treatment using the hydrolyzed collagen of this invention, for example, when their blood sugar levels are not excessively high or severe enough to be expected to cause health problems or (severe) suffering. As a supplement or alternative, blood sugar levels may have a cause or severity that causes the elevation to disappear naturally over time (i.e., it is not chronic). As a supplement or alternative, the blood sugar levels in the healthy subject group may not be excessively high or severe enough that they would not typically seek help from a professional medical practitioner.

[0050] The second group (referred to as the "pathological subject group" in this article) includes subjects whose elevated blood glucose levels are pathological in nature. This means that the elevated blood glucose levels typically lead to severe pain symptoms and (severe) suffering, and / or may result in (serious) health and psychological risks. Additionally, in the pathological subject group, the elevated blood glucose may be chronic. The severity of the elevated blood glucose often necessitates seeking help from a professional medical practitioner.

[0051] Medical professionals are typically able to determine, on a case-to-case basis, whether changes in blood glucose levels require therapeutic or non-therapeutic intervention (i.e., whether the individual falls into a healthy or pathological group). For example, they can determine whether changes in blood glucose levels may lead to (serious) health and psychological risks and / or whether they are chronic changes in blood glucose.

[0052] Depending on whether an individual is healthy or has a medical condition (e.g., hyperglycemia, diabetes, obesity), improvements in postprandial blood glucose can be considered either non-therapeutic or therapeutic interventions. Alternatively, improvements in postprandial blood glucose can be considered either non-therapeutic or therapeutic interventions based on the amount of glucose in the blood after a meal. For example, in healthy individuals, postprandial glucose levels typically do not exceed 140 mg / dL, so improvements in postprandial blood glucose levels below 140 mg / dL can be considered non-therapeutic interventions. Conversely, in individuals with medical conditions (e.g., hyperglycemia, diabetes, obesity), postprandial glucose levels can typically be 140 mg / dL or higher, so improvements in postprandial blood glucose levels of 140 mg / dL or higher can be considered therapeutic interventions.

[0053] In one embodiment, hydrolyzed collagen is used for therapeutically lowering blood glucose, such as to improve hyperglycemia or risk factors for hyperglycemia. In another embodiment, hydrolyzed collagen is used for non-therapeuticly lowering blood glucose, such as to improve postprandial blood glucose.

[0054] In one embodiment, hydrolyzed collagen is used for non-therapeutic improvement of postprandial blood glucose, wherein the postprandial blood glucose level is preferably below 140 mg / dL, more preferably below 120 mg / dL, and even more preferably below 100 mg / dL (i.e., before initiating hydrolyzed collagen intervention). In one embodiment, hydrolyzed collagen is used for therapeutic improvement of postprandial blood glucose, wherein the postprandial blood glucose level is preferably at least 140 mg / dL, more preferably at least 160 mg / dL, and even more preferably at least 180 mg / dL (i.e., before initiating hydrolyzed collagen intervention).

[0055] In one implementation, hydrolyzed collagen (further) as disclosed herein is used to improve risk factors for high blood sugar.

[0056] The beneficial effects of the hydrolyzed collagen of this invention were demonstrated in healthy mice in which hyperglycemia was induced after administration of hydrolyzed collagen. The beneficial effects of the hydrolyzed collagen of this invention were also demonstrated in obese mice with hyperglycemia after administration of hydrolyzed collagen. This indicates that the hydrolyzed collagen of this invention is effective in both the prevention and treatment of hyperglycemia.

[0057] In the context of this invention, the term “improvement” encompasses both “prevention” and “treatment” of a condition. The term “prevention” means ensuring that a subject does not develop a condition (e.g., hyperglycemia or a risk factor for hyperglycemia). An intervention is also considered a form of “prevention” herein when the condition is delayed, its severity reduced, and / or its incidence decreased, or even when the condition is not completely prevented from occurring. As used herein, “prevention” by intervention or its variations cover situations where the subject has previously experienced a condition (e.g., hyperglycemia or a risk factor for hyperglycemia) but the intervention prevents its recurrence. “Prevention” or its variations may have therapeutic and / or non-therapeutic effects. If “prevention” or its variations are therapeutic in nature, they may also target the symptoms of a disease or condition and / or its underlying pathological condition. “Prevention” or its variations may be defined by any change in delay, severity, and / or incidence, such as a change of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control or reference, as measured by any standard technique. In the context of this invention, "treatment" means, once a symptom has been present, an intervention that reduces and / or cures the symptom (e.g., hyperglycemia or risk factors for hyperglycemia). "Treatment" can have therapeutic and / or non-therapeutic effects. If "treatment" is inherently therapeutic, it can target the symptoms of a disease or symptom and / or its underlying pathological condition. For example, "treatment" can be any reduction in the severity, incidence, and / or frequency of a symptom, such as a reduction of at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to a control or reference, as measured by any standard technique. The term "improvement" as used herein also encompasses "cure." The term "improvement" as used herein may be used interchangeably with "reduction" or "reduction."

[0058] As used in this article, "risk factors for hyperglycemia" refers to conditions associated with an increased chance of developing hyperglycemia. In individuals with risk factors for hyperglycemia, administering a hypoglycemic agent as a preventative measure against hyperglycemia is feasible. As a supplement or alternative, in individuals with risk factors for hyperglycemia, it is preferably feasible to administer a hypoglycemic agent as a preventative measure to improve the likelihood of developing hyperglycemia. For example, obesity is a risk factor for hyperglycemia. Administering a hypoglycemic agent to an obese individual may prevent hyperglycemia in said obese individual and / or improve hyperglycemia in said individual (if it occurs).

[0059] Several risk factors may exist for hyperglycemia. In the context of this invention, low insulin levels, insulin deficiency, and / or insulin resistance are considered risk factors for hyperglycemia. In this disclosure, type 2 diabetes is considered a risk factor for hyperglycemia. Alternatively, one or more of the following conditions are considered risk factors for hyperglycemia herein: gestational diabetes, high body mass index (BMI), obesity, and hyperglycemia.

[0060] Hyperglycemia occurs when several hormones are present in excess and are considered risk factors for hyperglycemia in this paper, including excesses of one or more of the following: cortisol, catecholamines, growth hormone, glucagon, and thyroid hormones.

[0061] In a preferred embodiment, the risk factor for hyperglycemia as disclosed herein is insulin resistance. The term “insulin resistance” as used herein encompasses all conditions diagnosed as “insulin resistance” by a professional medical practitioner. The term “insulin resistance” as used herein preferably refers to peripheral insulin resistance and / or hepatic insulin resistance. Insulin resistance is preferably diagnosed by the gold standard, which is the “hyperinsulinemic-uglycemic clamp” (DeFronzo RA, Tobin JD, Andres R, Am J Physiol. 1979 Sep; 237(3): E214-23). ​​This method measures the amount of glucose required to compensate for elevated insulin levels without causing hypoglycemia. The procedure may take up to two hours and preferably includes the following steps (or similar steps): via a peripheral vein, at a rate of 10 to 120 mU / m³. 2 Insulin infusion. To compensate for the insulin infusion, glucose (20%) was infused to maintain blood glucose levels at 5 to 5.5 mmol / L. The glucose infusion rate was determined by checking blood glucose levels every five to ten minutes (Muniyappa R, Lee S, Chen H, Quon MJ, January 2008, American Journal of Physiology. Endocrinology and Metabolism. 294(1):E15–26). The glucose infusion rate during the last 30 minutes of the test determined insulin sensitivity. If a high level (7.5 mg / min or higher) was required, the patient was insulin sensitive. A low level (4.0 mg / min or lower) indicated insulin resistance. Levels between 4.0 mg / min and 7.5 mg / min were not definitive and indicated “impaired glucose tolerance,” an early sign of insulin resistance.

[0062] Homeostatic model assessment (HOMA or homeostatic model assessment for insulin resistance, HOMA-IR) is an alternative preferred method for determining and quantifying insulin resistance under fasting homeostatic conditions and is associated with the gold standard (see, for example, Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985). Diabetologia. 28(7):412–9. Doi:10.1007 / BF00280883.PMID 3899825; and / or AS Rudenski; DR Matthews; J Clevy; R Cturner (1991) Metabolism. 40(9):908–917). HOMA(-IR) scores deviating from the reference range can indicate insulin resistance. HOMA(-IR) has been widely used in epidemiological and experimental studies. HOMA(-IR) represents an estimate of insulin resistance, obtained by dividing an individual's blood insulin and glucose levels. The HOMA(-IR) value can be calculated using the following formula:

[0063]

[0064] Where H is the HOMA(-IR) value expressed in mg / dL, glucose represents the fasting glucose level in the blood expressed in mmol / L, and insulin represents the fasting insulin level in the blood expressed in mIU / L. IU (related to enzyme activity) is an abbreviation for International Units, also known as enzyme units. Methods for quantifying these levels are familiar to those skilled in the art. Enzyme activity is the amount of substrate converted per unit time.

[0065] 1 IU equals 1 μmol of substrate turnover per minute. For individuals with normal insulin sensitivity, HOMA(-IR) can be equal to 1. The upper limit of normal HOMA(-IR) is generally considered to be 2.0, although normal HOMA(-IR) can vary depending on the characteristics of a subgroup. In this disclosure, healthy subjects are considered to have HOMA-IR values ​​below 2.0 mg / dL, preferably below 1.9, 1.8, or 1.7 mg / dL, more preferably below 1.6, 1.5, 1.4, 1.3, 1.2, or 1.1 mg / dL, and most preferably below 1.0. In this disclosure, a fasting serum insulin level greater than 25 mU / L or 174 pmol / L is considered to indicate insulin resistance.

[0066] Other methods can also be applied to measure insulin resistance. In one implementation, the insulin-suppression test (IST) is used to measure insulin sensitivity or insulin resistance. In another implementation, the frequently sampled intravenous glucose tolerance test (FSIVGTT) is used to measure insulin sensitivity or insulin resistance. In yet another implementation, the oral glucose tolerance test (OGTT) is used to measure insulin sensitivity or insulin resistance.

[0067] In a preferred embodiment, the "risk factor for hyperglycemia" disclosed herein is metabolic syndrome. The term "metabolic syndrome" as used herein encompasses all conditions diagnosed as "metabolic syndrome" by a professional medical practitioner. In one embodiment, a person may be diagnosed with metabolic syndrome if they possess at least two or at least three of the following characteristics:

[0068] - Large waist circumference - Women are measured to have a waist circumference of at least 35 inches (89 cm) and men are measured to have a waist circumference of at least 40 inches (102 cm);

[0069] - High triglyceride levels - This type of fat present in the blood reaches 150 mg / dL or 1.7 mmol / L or higher;

[0070] - Lowered "beneficial" or HDL cholesterol - High-density lipoprotein (HDL) cholesterol below 40 mg / dL (1.04 mmol / L) for men or below 50 mg / dL (1.3 mmol / L) for women;

[0071] Elevated blood pressure – 130 / 85 mmHg or higher;

[0072] - Elevated fasting blood glucose - 100 mg / dL (5.6 mmol / L) or higher.

[0073] In a preferred embodiment, the risk factor for hyperglycemia as disclosed herein is type 2 diabetes. The term "type 2 diabetes" as used herein may be used interchangeably with "type 2 diabetes mellitus". In the context of this invention, a patient with type 2 diabetes is preferably characterized by hyperglycemia, insulin resistance, and a relative deficiency of insulin.

[0074] In a preferred embodiment, as disclosed herein, the risk factor for high blood sugar is obesity.

[0075] In a preferred embodiment, as disclosed herein, the risk factor for hyperglycemia is being overweight.

[0076] As used in this article, the term "obesity" refers to a medical condition in which excessive body fat accumulates to a degree that may have adverse health effects. In some cases, obesity is merely a cosmetic issue (requiring non-therapeutic treatment). Professional medical practitioners can determine whether an individual is obese through one or more methods.

[0077] Generally, a body mass index (BMI) of 30.0 or higher is considered obese. In this disclosure, a BMI of 25.0 to <30 indicates an individual is overweight. As used herein, "overweight" refers to a non-medical condition characterized by excessive accumulation of body fat.

[0078] The inventors have discovered that the hydrolyzed collagen of this invention can have one or more of the following effects, for example, as a mechanism of action to lower blood sugar and / or improve hyperglycemia:

[0079] - Increase the level of one or more incretins in the body, for example by increasing the secretion of one or more incretins by gastrointestinal cells;

[0080] - Reduce the level of one or more inhibitors of incretins (e.g., DPP-IV) in the body, for example by reducing the secretion of incretins by gastrointestinal cells;

[0081] - Increase blood GLP-1 levels, for example by increasing the secretion of GLP-1 by gastrointestinal cells;

[0082] - Reduce blood glucagon levels, for example by reducing glucagon secretion from pancreatic (α) cells;

[0083] - Increase blood insulin levels, for example by increasing insulin secretion from pancreatic (β) cells;

[0084] - Reduces glucose absorption in the small intestine; and

[0085] - Increases the blood insulin / glucose ratio.

[0086] In a preferred embodiment, the hydrolyzed collagen (further) as disclosed herein is selected from one or more of the following:

[0087] - Improves blood GLP-1, with a preference for increasing blood GLP-1;

[0088] - Improves blood glucagon levels, with a preference for lowering blood glucagon levels;

[0089] - Improves blood insulin levels, with a preference for increasing blood insulin levels;

[0090] - Improve the blood insulin / glucose ratio, preferably increase the blood insulin / glucose ratio;

[0091] - Improve the level of DPP-IV in the body, preferably reduce the level of DPP-IV in the body, for example by reducing the secretion of DPP-IV by gastrointestinal cells;

[0092] - Improve the level of one or more incretins in the body, preferably by increasing the level of one or more incretins in the blood, for example by increasing the secretion of one or more incretins by gastrointestinal cells;

[0093] - Improve glucose absorption in the small intestine, preferably reduce glucose absorption in the small intestine.

[0094] The inventors have discovered that the hydrolyzed collagen of this invention has a particularly high ability to stimulate GLP-1 secretion and / or lower blood glucose. The bioactivity is shown to be related to the specific molecular weight and / or molecular weight distribution of the hydrolyzed collagen, which can be obtained by hydrolysis with a specific combination of enzymes.

[0095] In a preferred embodiment, hydrolyzed collagen can stimulate GLP-1 secretion. The ability to stimulate GLP-1 secretion is preferably demonstrated in STC-1 cells. The ability to stimulate GLP-1 secretion is preferably confirmed using the following protocol:

[0096] - Hydrolyze gelatin to obtain hydrolyzed collagen preparations;

[0097] - The hydrolyzed collagen preparation is subjected to simulated gastrointestinal digestion (SGID) to obtain digested hydrolyzed collagen, preferably according to the scheme described in Songet et al. (Food Funct. 2020 Jun 24; 11(6): 5553-5564);

[0098] - Stimulate cells with digested hydrolyzed collagen or with a blank as a reference (e.g., STC-1 cells), preferably according to the protocol described by Qi et al. (Bio Protoc. 2020 Aug 20; 10(16):e3717). Cells are preferably stimulated at a concentration in the range of 1 mg / ml to 20 mg / ml, and more preferably at a concentration of 10 mg / ml;

[0099] - Collect the supernatant 1 to 3 hours after the start of stimulation and determine the GLP-1 level in the supernatant, for example by enzyme immunoassay.

[0100] A particularly suitable approach is the approach for stimulating STC-1 cells described in Example 1 of this disclosure.

[0101] The ability to stimulate GLP-1 secretion is preferably defined as an increase of at least 100 times, preferably 500 times, more preferably 1000 times, even more preferably 2000 times, and most preferably 5000 times, of GLP-1 levels after stimulation, preferably relative to stimulation with a blank reference group (control group).

[0102] Hydrolyzed collagen

[0103] The term "hydrolyzed collagen" as used in this article refers to a mixture of short-chain amino acids derived from collagen-containing starting materials that produce natural (full-length) collagen. It is typically obtained through hydrolysis steps, including enzymatic hydrolysis (also known as enzymatic hydrolyzation). The degree of hydrolysis generally affects the average molecular weight of the final product.

[0104] Hydrolyzed collagen typically has a molecular weight of 1 to 10 kDa. The hydrolyzed collagen taught in this invention may comprise hydrolyzed or partially hydrolyzed collagen. The term "collagen hydrolysate" is used interchangeably and synonymously with the terms "hydrolyzed collagen" or "collagen peptides".

[0105] Hydrolyzed collagen can be produced in a one-step process from collagen-containing starting materials (e.g., animal connective tissue) or via an intermediate gelatin stage, where type A and / or type B gelatin can be used. Therefore, as used herein, hydrolyzed collagen can refer to hydrolyzed gelatin, which is obtained by hydrolyzing gelatin derived from collagen. In this disclosure, the terms "hydrolyzed collagen" and "hydrolyzed gelatin" are used interchangeably. Hydrolyzed gelatin is preferably obtained by enzymatic or chemical hydrolysis of gelatin. Hydrolyzed gelatin can be obtained from type A gelatin, type B gelatin, or mixtures thereof. The term "hydrolyzed collagen" is used interchangeably with and synonymously with the term "hydrolyzed gelatin."

[0106] In a preferred embodiment, the collagen-containing material is gelatin.

[0107] The collagen mentioned in this article may be selected from one or more of type I collagen, type II collagen, type III collagen, type V collagen, and type X collagen.

[0108] Preferably, the hydrolyzed collagen disclosed herein is derived from one or more types of collagen selected from type I, type II, and type III collagen. Alternatively, the hydrolyzed collagen disclosed herein is preferably derived from animal raw materials containing different collagen types (e.g., two or more of type I, type II, and type III collagen). Alternatively, the hydrolyzed collagen disclosed herein may be a mixture containing two or more of type I, type II, and type III collagen.

[0109] The hydrolyzed collagen disclosed herein is preferably derived from type I collagen and / or type II collagen.

[0110] The collagen disclosed herein may be derived from any one or more animals or animal species, such as bovine, swine, and fish species. "Animal" as used herein may refer to any animal capable of providing connective tissue, which may be used to prepare hydrolyzed collagen.

[0111] In one embodiment, the collagen as taught herein is derived from bovine collagen. In one embodiment, the collagen as taught herein is derived from swine collagen. In one embodiment, the collagen as taught herein is derived from fish collagen.

[0112] In several embodiments, the collagen disclosed herein is a mixture of collagen from different sources (e.g., collagen derived from multiple animal species and / or collagen derived from different tissues). For example, the collagen disclosed herein may be a mixture of two or more collagens selected from fish collagen, porcine collagen, and bovine collagen. As a supplement or alternative, the collagen may be a mixture of collagens selected from skin, cartilage, bone, and / or connective tissue sources.

[0113] As used herein, the term "hide" refers to the outer covering of large animals (such as cattle or any other large animal). The term "skin" refers to the outer covering of small animals such as deer, goats, and sheep. The terms "skin" and "hide" are used interchangeably and can refer to the outer covering of an animal regardless of size.

[0114] As disclosed herein, “connective tissue” can be selected from one or more types of connective tissue, including: corpus callosum, skin, antlers, horns (e.g., camel humps), head, brain, neck, ears, eyes, nose, tongue, lips, mouth, esophagus, trachea, limbs, feet, toes, palms, claws, bones, cartilage, bone marrow, joints, membranes, hind feet, ligaments, tendons, ribs, diaphragm, muscles, skeletal muscles, smooth muscles, intestines, blood vessels, bladder, stomach, aorta, heart, liver, kidneys, chest, lungs, spleen, pancreas, ovaries, sperm, testes, ovaries, nerves, gallbladder, and abdomen.

[0115] In one embodiment, the collagen as taught herein is derived from skin and / or skin connective tissue. In a preferred embodiment, the collagen as taught herein is derived from cartilage. In a preferred embodiment, the collagen as taught herein is derived from bone. The collagen disclosed herein may be a mixture of two or more collagens selected from skin collagen, cartilage collagen, and bone collagen.

[0116] In a preferred embodiment, the collagen disclosed herein is derived from one or more tissues selected from skin, squamous cells, cartilage, bone, tendons, ligaments, and connective tissue.

[0117] In a preferred embodiment, the collagen disclosed herein is one or more types selected from the following: porcine collagen, bovine collagen, and fish collagen.

[0118] Enzymatic hydrolysis

[0119] In one embodiment, the present invention relates to a method for obtaining hydrolyzed collagen, comprising:

[0120] a) Provide materials containing collagen for use in liquid formulations; and

[0121] b) subjecting the collagen-containing material to an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0122] In a preferred embodiment, the present invention relates to a method for obtaining hydrolyzed collagen, comprising:

[0123] a) Provide materials containing collagen for use in liquid formulations; and

[0124] b) Subject collagen-containing materials to a combination of enzymes including neutral protease, carboxypeptidase, and aminopeptidase.

[0125] Preferably, step b) of the method disclosed herein is an enzymatic hydrolysis step or covers enzymatic hydrolysis steps.

[0126] As used herein, "liquid formulation" refers to a formulation in which the collagen-containing material is at least partially soluble. Alternatively, "liquid formulation" refers to a formulation in which the collagen-containing material remains at least partially soluble after undergoing a dissolution step. Liquid formulations disclosed herein are preferably aqueous formulations. In a preferred embodiment, the liquid formulation disclosed herein is water or contains water, more preferably distilled water and / or demineralized water or contains distilled water and / or demineralized water.

[0127] As part of the method disclosed herein, collagen-containing material may be provided in liquid formulations in amounts of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65% by weight. As a supplement or alternative, collagen-containing material may be provided in liquid formulations in amounts not exceeding 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10% by weight.

[0128] In one embodiment, the collagen-containing material is provided in an amount of 20% to 50% by weight, preferably 30% to 40% by weight, in a liquid formulation as disclosed herein.

[0129] In one embodiment, the combination of enzymes as disclosed herein is a mixture of the enzymes.

[0130] In one embodiment, the combination of enzymes disclosed herein comprises one or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0131] In one embodiment, the combination of enzymes disclosed herein comprises two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0132] In one embodiment, the combination of enzymes disclosed herein comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

[0133] In one embodiment, the combination of enzymes disclosed herein comprises a neutral protease and a carboxypeptidase. In one embodiment, the combination of enzymes disclosed herein comprises a neutral protease and an aminopeptidase. In one embodiment, the combination of enzymes disclosed herein comprises a carboxypeptidase and an aminopeptidase.

[0134] In a preferred embodiment, the enzyme combination as disclosed herein comprises one or both of carboxypeptidase and aminopeptidase, as well as a neutral protease.

[0135] As used herein, the term "neutral protease" refers to a class of proteases that can catalyze the hydrolysis of peptide bonds in proteins in neutral, weakly acidic, or weakly alkaline environments. In this disclosure, the optimal pH for neutral proteases in enzymatic hydrolysis is considered to be between 6.0 and 7.5. Based on their catalytic mechanisms and the functional groups at their active sites, neutral proteases can be classified into four main classes: serine proteases, aspartic proteases, cysteine ​​proteases, and metalloproteinases.

[0136] As used herein, "serine protease" refers to an enzyme that cleaves peptide bonds in proteins or peptides, where serine acts as a nucleophilic amino acid at the active site. Serine proteases disclosed herein may be chymotrypsin-like (trypsin-like) serine proteases. Alternatively, serine proteases disclosed herein may be subtilisin-like serine proteases.

[0137] As used in this article, "aspartic protease" refers to a catalytic protease that utilizes an activated water molecule bound to one or more aspartic residues used to catalyze proteins or peptides.

[0138] As used herein, "cysteine ​​protease" refers to a protease having a catalytic mechanism involving a nucleophilic cysteine ​​sulfhydryl group in a catalytic triplet or dummy for catalyzing a protein or peptide. The term "cysteine ​​protease" may be used interchangeably with "thiol protease."

[0139] As used herein, "metalloproteinase" refers to a protease whose catalytic mechanism involves a metal (e.g., zinc or cobalt). In this disclosure, the metalloproteinase is preferably a zinc-dependent protease.

[0140] As used in this article, the term "carboxypeptidase" refers to a protease that hydrolyzes peptide bonds located at the carboxyl terminus (C-terminus) of a protein or peptide.

[0141] Carboxypeptides are typically classified into several families based on their active site mechanism. In one embodiment, the carboxypeptide described herein is a metallocarboxypeptide, i.e., a carboxypeptide that uses a metal at its active site for its catalytic mechanism.

[0142] As used herein, the term "aminopeptidase" refers to a protease that cleaves the peptide bond at the N-terminus of a protein or peptide. In this disclosure, the aminopeptidase is preferably a leucine aminopeptidase. As used herein, the term "leucine aminopeptidase" refers to an aminopeptidase that preferentially catalyzes the hydrolysis of leucine residues located at the N-terminus of peptides and proteins.

[0143] In a preferred embodiment, the neutral protease disclosed herein is a serine protease. In a preferred embodiment, the neutral protease disclosed herein is an aspartic protease. In a preferred embodiment, the neutral protease disclosed herein is a cysteine ​​protease. In a preferred embodiment, the neutral protease disclosed herein is a metalloproteinase. In a preferred embodiment, the aminopeptidase disclosed herein is a leucine aminopeptidase.

[0144] In the context of this invention, "enzyme" can refer to a commercially available enzyme. In one embodiment, the combination of enzymes disclosed herein is a commercially available mixture of enzymes. In one embodiment, the combination of enzymes disclosed herein is commercially available as "Sumizyme," preferably "Sumizyme FP-G."

[0145] In one embodiment, the enzymes disclosed herein (e.g., one or more enzymes in a combination of enzymes) are derived from microorganisms.

[0146] In one embodiment, the enzymes disclosed herein (e.g., one or more of a combination of enzymes) are derived from microorganisms of the genus Aspergillus, preferably Aspergillus oryzae and / or Aspergillus melleus.

[0147] In one embodiment, the enzymes disclosed herein (e.g., one or more of the combinations of enzymes) are derived from microorganisms of the genus Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens).

[0148] In one embodiment, a combination of enzymes containing no more than one enzyme derived from the genus Bacillus sp. is used, thereby excluding blends of endopeptides from the genus Bacillus, such as commercially available Protamex.

[0149] In one embodiment, the enzymes disclosed herein (e.g., one or more of a combination of enzymes) are derived from lactic acid bacteria, preferably selected from one or more genera including: Lactobacillus, Leuconostoc, Pediococcus, Lactococcus and Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus and Weissella.

[0150] In one embodiment, one or more of the neutral protease, carboxypeptidase, and aminopeptidase are derived from microorganisms of the genus *Aspergillus*, preferably from *Aspergillus oryzae*. In a preferred embodiment, the neutral protease, carboxypeptidase, and aminopeptidase are derived from microorganisms of the genus *Aspergillus*, preferably from *Aspergillus oryzae*.

[0151] The proteases used in this invention can be mixtures of enzymes (e.g., commercial mixtures obtained from Sumizyme containing enzymes from Aspergillus oryzae), or the proteases can be derived from different sources (e.g., from different microorganisms or strains thereof). For example, neutral proteases can be derived from Bacillus, carboxypeptidases from Aspergillus, and aminopeptidases from Lactobacillus.

[0152] In one embodiment, the enzymes disclosed herein (e.g., one or more of a combination of enzymes) are not derived from plants (e.g., bromelain, papain).

[0153] In a preferred embodiment, during enzymatic hydrolysis, the amount of neutral protease used in step b) of the method disclosed herein is limited by an enzyme activity of 0.2 to 25,000 U / g, preferably 2 to 2,500 U / g, and more preferably 20 to 250 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0154] In a preferred embodiment, during enzymatic hydrolysis, the amount of carboxypeptidase used in step b) of the method disclosed herein is limited by an enzyme activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, and more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0155] In a preferred embodiment, during enzymatic hydrolysis, the amount of aminopeptidase used in step b) of the method disclosed herein is limited by an enzyme activity of 0.002 to 500 U / g, preferably 0.02 to 50 U / g, more preferably 0.2 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0156] The time that collagen-containing materials are subjected to (e.g., in step b of the method) with enzymes such as those disclosed herein (preferably one or more combinations of enzymes such as those disclosed herein) can be at least

[0157] 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 300, or 360 minutes.

[0158] As a supplement or alternative, the time that collagen-containing materials are subjected to (e.g., in step b of the method) with enzymes such as those disclosed herein (preferably one or more combinations of enzymes such as those disclosed herein) may not exceed [a certain amount].

[0159] 360, 300, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 90, 80, 70, 60, 50, 40, 30, 20, or 10 minutes.

[0160] In a preferred embodiment, the collagen-containing material is subjected (e.g., in step b of the method) to an enzyme as disclosed herein (preferably one or more combinations of enzymes as disclosed herein) for 60 to 180 minutes.

[0161] The temperature at which the collagen-containing material is subjected (e.g., in step b of the method) to an enzyme such as those disclosed herein (preferably one or more combinations of enzymes disclosed herein) can be at least

[0162] 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, or 80℃.

[0163] As a supplement or alternative, the temperature at which collagen-containing materials are subjected (e.g., in step b of the method) to enzymes such as those disclosed herein (preferably one or more combinations of enzymes such as those disclosed herein) may not exceed [a certain temperature].

[0164] 80℃, 75℃, 70℃, 65℃, 60℃, 55℃, 50℃, 45℃, 40℃, 35℃, 30℃, 25℃, 20℃, or 15℃.

[0165] As used herein, “temperature” preferably refers to the temperature of a liquid preparation (which contains collagen-containing materials), preferably in which collagen-containing materials are dissolved.

[0166] In a preferred embodiment, the collagen-containing material is subjected (e.g., in step b of the method) to one or more enzymes as disclosed herein, preferably combinations of enzymes as disclosed herein, at a temperature of 30 to 60°C.

[0167] The use of the term "enzyme combination" as disclosed herein does not preclude subjecting collagen-containing materials to enzymes (part of an enzyme combination) in a sequential manner. This means that collagen-containing materials are not necessarily subjected to all the enzymes in the enzyme combination simultaneously. In one embodiment, an enzymatic hydrolysis as disclosed herein (e.g., in step b of the method) comprises two or more sequential hydrolysis steps, wherein the collagen-containing materials are preferably subjected to different enzymes and / or different mixtures of enzymes.

[0168] In a preferred embodiment, the collagen-containing material is subjected to two enzymes in a combination of enzymes, preferably two selected from neutral protease, carboxypeptidase, and aminopeptidase.

[0169] In one embodiment, the enzymatic hydrolysis (preferably in step b of the method disclosed herein) involves two or more hydrolysis steps, preferably two steps, wherein:

[0170] - Subject collagen-containing materials to neutral proteases in one step and to carboxypeptidase and / or aminopeptidase in another step;

[0171] - The collagen-containing material is subjected to carboxypeptidase in one step and to neutral protease and / or aminopeptidase in another step.

[0172] - The collagen-containing material is subjected to aminopeptidase in one step and to neutral protease and / or carboxypeptidase in another step.

[0173] In one embodiment, the enzymatic hydrolysis as disclosed herein comprises three hydrolysis steps, wherein the collagen-containing material is preferably subjected to neutral protease, carboxypeptidase, and aminopeptidase independently in each of the three hydrolysis steps.

[0174] In a preferred embodiment, the collagen-containing material is subjected to all enzymes simultaneously (such as the combination of enzymes disclosed herein).

[0175] In a preferred embodiment, the collagen-containing material is subjected to a mixture of two or more of a neutral protease, a carboxypeptidase, and an aminopeptidase, preferably a mixture of all three of the neutral protease, carboxypeptidase, and aminopeptidase, wherein the collagen-containing material is more preferably subjected to the mixture at 30 to 60°C and at pH 5 to 8 for 60 to 180 minutes.

[0176] If there are two or more hydrolysis steps, the “time” for which the hydrolyzed collagen is subjected to the enzyme preferably refers to the total time. For example, if the hydrolyzed collagen is subjected to enzyme A for 60 minutes in the first hydrolysis step and 60 minutes in the second hydrolysis step, then it is considered herein that the hydrolyzed collagen is subjected to enzyme A for 120 minutes.

[0177] Products that can be obtained through enzymatic hydrolysis.

[0178] In one aspect, the present invention relates to hydrolyzed collagen that can be obtained by methods disclosed herein.

[0179] In this disclosure, "hydrolyzed collagen" preferably means hydrolyzed collagen that can be obtained by the methods disclosed herein.

[0180] In a preferred embodiment, the hydrolyzed collagen disclosed herein (e.g., obtainable by the methods taught herein) is derived from a collagen-containing material derived from one or more tissues selected from skin, squamous cells, cartilage, bone, tendons, ligaments, and connective tissue.

[0181] In a preferred embodiment, the hydrolyzed collagen as disclosed herein (e.g., obtainable by the methods taught herein) is derived from a collagen-containing material, wherein the collagen is one or more types selected from: porcine collagen, bovine collagen, and fish collagen.

[0182] In a preferred embodiment, the hydrolyzed collagen as disclosed herein (e.g., obtainable by the methods taught herein) is derived from a collagen-containing material, wherein the collagen-containing material is gelatin.

[0183] The hydrolyzed collagen disclosed herein (e.g., obtainable by the methods taught herein) preferably has a (weight-average) molecular weight of 1000 to 7000 Da. This molecular weight is shown to play a role in the hyperglycemic effect of the hydrolyzed collagen of the present invention.

[0184] The (weight-average) molecular weight of hydrolyzed collagen (e.g., obtainable by the methods taught herein) may be at least 1000 Da (e.g., at least 1200 Da, 1400 Da, 1600 Da, or 1800 Da), or at least 1500 Da, or at least 2000 Da, or at least 2500 Da, or at least 3000 Da, or at least 3500 Da, or at least 4000 Da, or at least 4500 Da, or at least 5000 Da, or at least 6000 Da, or at least 6500 Da, or at least 7000 Da. As a supplement or alternative, the (weight-average) molecular weight of hydrolyzed collagen (e.g., obtainable by the methods taught herein) may be no more than 7000 Da, or no more than 6500 Da, or no more than 6000 Da, or no more than 5500 Da, or no more than 5000 Da, or no more than 4500 Da, or no more than 4000 Da, or no more than 3500 Da, or no more than 3000 Da, or no more than 2500 Da, or no more than 2000 Da, or no more than 1500 Da, or no more than 1000 Da.

[0185] The average molecular weight disclosed herein is preferably the weight-average molecular weight.

[0186] This demonstrates that molecular weight plays a role in the ability of the hydrolyzed collagen of the present invention to stimulate GLP-1 secretion and / or lower blood sugar.

[0187] In one embodiment, the hydrolyzed collagen has a weight-average molecular weight of 1,500 to 4,500 Da, preferably 2,000 to 4,000 Da, more preferably 2,500 to 3,500 Da, and even more preferably 2,750 to 3,250 Da.

[0188] In one implementation, the number-mean (M) of hydrolyzed collagen is... n The molecular weight is 500 to 3500 Da, preferably 1000 to 3000 Da, more preferably 1500 to 2500 Da, and even more preferably 1750 to 2250 Da.

[0189] In one embodiment, the hydrolyzed collagen has a weight-average molecular weight of 2,000 to 4,000 Da, preferably 2,500 to 3,500 Da, and a number-average molecular weight of 1,000 to 3,000 Da, preferably 1,500 to 2,500 Da.

[0190] As a supplement or alternative, molecular weight distribution is shown to play a role in the ability of the hydrolyzed collagen of the present invention to stimulate GLP-1 secretion and / or lower blood glucose. Compared to other hydrolyzed collagens—for example, those not obtained by enzymatic hydrolysis using an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases—the hydrolyzed collagen of the present invention particularly possesses:

[0191] - Relatively high fractions of collagen peptides falling within the range of 2000 to 5000 Da (e.g., at least 35%, or at least 40%, or even at least 45%); and / or

[0192] - Relatively low fractions of collagen peptides falling below 1000 Da (e.g., no more than 15%, or no more than 12%, or even no more than 10%); and / or

[0193] - Relatively low fractions of collagen peptides that fall within the range above 10,000 Da (e.g., no more than 5%, or no more than 2%, or even no more than 1%).

[0194] In a preferred embodiment, the hydrolyzed collagen contains 1% to 20% by weight, preferably 2% to 12% by weight, of collagen peptides with a molecular weight of less than 1000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0195] In a preferred embodiment, the hydrolyzed collagen comprises 20% to 40% by weight, preferably 25% to 35% by weight, of collagen peptides with a molecular weight in the range of 1,000 to 2,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0196] Preferably, the hydrolyzed collagen contains less than 35% by weight, more preferably less than 32.5% by weight, and even more preferably less than 30% by weight of collagen peptides with a molecular weight in the range of 1,000 to 2,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0197] In a preferred embodiment, the hydrolyzed collagen comprises 35% to 60% by weight, preferably 40% to 55% by weight, of collagen peptides with a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0198] Preferably, the hydrolyzed collagen contains more than 40% by weight, more preferably more than 42.5% by weight, and even more preferably more than 45% by weight of collagen peptides with a molecular weight in the range of 2,000 to 5,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0199] In a preferred embodiment, the hydrolyzed collagen contains 5% to 25% by weight, preferably 10% to 20% by weight, of collagen peptides with a molecular weight in the range of 5,000 to 10,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0200] In a preferred embodiment, the hydrolyzed collagen contains 0% to 10% by weight, preferably 0.1% to 5% by weight, more preferably 0.2% to 2% by weight of collagen peptides with a molecular weight greater than 10,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0201] The preferred method for measuring the molecular weight and molecular weight distribution of hydrolyzed collagen is high-performance size exclusion chromatography (HPSEC). The following are preferred HPSEC protocols:

[0202] An Agilent HPLC system with TSKgel SWXL pre-columns and G2000 SWXL columns (Tosoh Bioscience), 1260 Infinity series (G1316A, G1329B, G1311C, G1315D), was used. Analysis was performed using WinGPC software (PSS). The eluent was 100 mM phosphate buffer at pH 5.3.

[0203] The sample is eluted from the column (e.g., 0.5 mL / min, isocratic) and monitored with a UV detector (e.g., 214 nm, analysis time: 40 min + 180 min equilibration per injection).

[0204] Calibration is performed using narrow calibration standards (low FILK).

[0205] The polydispersity of the hydrolyzed collagen (e.g., obtainable by the methods taught herein) is preferably 1.2 to 1.8, more preferably 1.3 to 1.7, and even more preferably 1.4 to 1.6. This polydispersity indicates that the hydrolyzed collagen of the present invention plays a role in the hyperglycemic effect.

[0206] The polydispersity of hydrolyzed collagen may not exceed 1.85, or 1.75, or 1.70, or 1.65, or 1.60, or 1.55. Alternatively, the polydispersity of hydrolyzed collagen may be at least 1.20, or at least 1.25, or at least 1.30, or at least 1.35, or at least 1.40, or at least 1.45, or at least 1.50, or at least 1.55.

[0207] The term "polydispersity" used in this article refers to M w / M n M w It is the weight-average molecular weight (Da), and M n It is the number-average molecular weight (Da).

[0208] M wIt is given by the following formula:

[0209]

[0210] M n It is given by the following formula:

[0211]

[0212] Where N i It is a weight of M i The number of molecules.

[0213] Application of hydrolyzed collagen

[0214] In a preferred embodiment, the hydrolyzed collagen, as disclosed herein, is administered orally.

[0215] To improve postprandial blood glucose levels, hydrolyzed collagen, as disclosed herein, is preferably administered during or before meals. In the context of this invention, hydrolyzed collagen is preferably administered no more than 240, 180, 120, 60, 30, or 15 minutes after a meal (before or after). In a preferred embodiment, hydrolyzed collagen, as disclosed herein, is administered no more than 60 minutes after a meal (before or after).

[0216] In some different implementations, such as those disclosed herein, the daily dose of hydrolyzed collagen is at least

[0217] 1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190,or200

[0218] All figures are in grams, where the daily dose is the total dry weight applied to the subject daily. As a supplement or alternative, in some different embodiments, the daily dose of hydrolyzed collagen, as disclosed herein, does not exceed [a certain value].

[0219] 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1

[0220] All figures are in grams, where the daily dose is the total dry weight applied to the subject each day.

[0221] In a preferred embodiment, the use of hydrolyzed collagen as disclosed herein includes applying hydrolyzed collagen in amounts of 1 g to 200 g, or 1 g to 100 g, or 2 g to 50 g, or 5 g to 25 g, wherein the daily dose is the total dry weight applied to the subject daily.

[0222] In a preferred embodiment, the use of hydrolyzed collagen as disclosed herein comprises administering hydrolyzed collagen at a daily dose of 1 g to 100 g, preferably 2 g to 50 g, more preferably 5 g to 25 g, wherein the amount is the dry weight of the hydrolyzed collagen.

[0223] In some different embodiments, the amount of hydrolyzed collagen per unit dose, as disclosed herein, is at least

[0224] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190,or200

[0225] All amounts are expressed in grams, where the quantity refers to the dry weight of the hydrolyzed collagen. As a supplement or alternative, in some different embodiments, the amount of a unit dose of hydrolyzed collagen as disclosed herein does not exceed [amount not specified].

[0226] 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1

[0227] All figures are in grams, where the amount refers to the dry weight of hydrolyzed collagen.

[0228] In one embodiment, the hydrolyzed collagen as disclosed herein is administered in unit doses of amounts ranging from 0.5g to 200g, or 1g to 100g, or 2g to 50g, or 5g to 25g, or 5g to 15g, wherein the amount is the dry weight of the hydrolyzed collagen.

[0229] In one embodiment, the use of hydrolyzed collagen as disclosed herein comprises applying a unit dose of hydrolyzed collagen in an amount of 2g to 50g, preferably 5g to 25g, more preferably 5g to 15g, wherein the amount is the dry weight of the hydrolyzed collagen.

[0230] The daily dose of hydrolyzed collagen can be administered as a single unit dose, or as two, three, four, or more unit doses. The amounts of the two or more unit doses can be the same or different. The daily dose of hydrolyzed collagen as disclosed herein is preferably administered as two unit doses, more preferably as two unit doses each corresponding to an amount of 30% to 70%, preferably 40% to 60% of the daily dose.

[0231] In one embodiment, two or more unit doses, as disclosed herein, are administered at intervals of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours from each other.

[0232] In a preferred embodiment, the dosage regimen of hydrolyzed collagen as disclosed herein comprises administering a daily dose of hydrolyzed collagen as two unit doses, wherein:

[0233] - Each unit dose is 5 to 25 g, preferably 5 to 15 g, wherein the amount is the dry weight of the hydrolyzed collagen; and / or

[0234] - Two unit doses are administered at least 6 hours apart, preferably 8 hours, and more preferably 12 hours apart.

[0235] In a preferred embodiment, the daily dose of hydrolyzed collagen is administered as two or more unit doses, wherein:

[0236] - Each unit dose is 5 to 25 g, preferably 5 to 15 g, wherein the amount is the dry weight of the hydrolyzed collagen; and / or

[0237] - Two or more unit doses are administered at least 6 hours apart, preferably 8 hours, and more preferably 12 hours apart.

[0238] In a preferred embodiment, the hydrolyzed collagen, as disclosed herein, is administered with meals.

[0239] In one embodiment, the dosage regimen of hydrolyzed collagen, as disclosed herein, comprises administering hydrolyzed collagen at least once a week.

[0240] In one embodiment, the dosage regimen of hydrolyzed collagen as disclosed herein involves administering hydrolyzed collagen daily or every other day.

[0241] In one implementation, hydrolyzed collagen is applied daily or every other day.

[0242] In one embodiment, the dosage regimen of hydrolyzed collagen, as disclosed herein, comprises administering hydrolyzed collagen at least once daily.

[0243] In one implementation scheme, hydrolyzed collagen is applied at least once daily.

[0244] In one embodiment, the dosage regimen of hydrolyzed collagen as disclosed herein comprises administering hydrolyzed collagen for at least two consecutive days, preferably for at least four consecutive days, and more preferably for at least seven consecutive days.

[0245] In one embodiment, the dosage regimen of hydrolyzed collagen as disclosed herein comprises administering hydrolyzed collagen for at least two consecutive weeks, preferably for at least four consecutive weeks, and more preferably for at least eight consecutive weeks.

[0246] In one embodiment, the dosage regimen of hydrolyzed collagen as disclosed herein comprises administering hydrolyzed collagen for at least one week, preferably for at least two consecutive weeks, and more preferably for at least four consecutive weeks.

[0247] Hydrolyzed collagen preparations

[0248] The hydrolyzed collagen disclosed herein may be provided as a dietary preparation, feed preparation, dietary supplement preparation, feed supplement preparation or pharmaceutical preparation, preferably as a dietary supplement preparation.

[0249] Hydrolyzed collagen, as disclosed herein, can be provided in solid dosage forms, such as capsules, tablets, or powders, preferably in powder form.

[0250] Hydrolyzed collagen as disclosed herein may be provided in formulations such as: drinkable solutions or suspensions, beverages such as beer, syrups, artificially flavored beverages, carbonated beverages, (water-soluble) powder mixtures, (water-soluble) pastes, (water-soluble) powders, (water-soluble) tablets, (water-soluble) pills, (water-soluble) sugar-coated pills, (water-soluble) capsules, (water-soluble) sachets, or (water-soluble) capsules. As a supplement or alternative, hydrolyzed collagen as taught herein may be present in functional foods such as juices, smoothies, dairy beverages, yogurt, yogurt drinks, desserts, energy bars, nutrition bars, slimming bars, or confectionery such as gummies or filled gummies.

[0251] Uses of hydrolyzed collagen in treatment

[0252] In one embodiment, the present invention relates to a treatment method for lowering blood glucose, preferably a therapeutic treatment method.

[0253] In one embodiment, the present invention relates to a treatment method for improving hyperglycemia, preferably a therapeutic treatment method.

[0254] In one embodiment, the present invention relates to a treatment method for improving risk factors for hyperglycemia, preferably a therapeutic treatment method.

[0255] The treatments disclosed herein may be used for subjects experiencing hyperglycemia (e.g., hyperglycemia), and the treatments may be preventative in nature. The treatments may include one or more features of embodiments described herein for the use of hydrolyzed collagen in lowering blood glucose and / or improving hyperglycemia (a risk factor), including the type of hydrolyzed collagen used for the purpose disclosed herein, the formulation of the hydrolyzed collagen used for the purpose disclosed herein, the timing of administration or ingestion of the hydrolyzed collagen used for the purpose disclosed herein, and the dosage (e.g., dosing regimen, unit dose, daily dose) of the hydrolyzed collagen used for the purpose disclosed herein.

[0256] Uses of hydrolyzed collagen in pharmaceutical preparation

[0257] In one embodiment, the present invention relates to the use of hydrolyzed collagen in the preparation of a medicament for lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0258] In one embodiment, the present invention relates to the use of hydrolyzed collagen in the preparation of a medicament for improving hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0259] In one embodiment, the present invention relates to the use of hydrolyzed collagen in the preparation of a medicament for improving risk factors of hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination preferably comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0260] The use of hydrolyzed collagen in the preparation of medicaments as disclosed herein may include one or more features of embodiments of the use of hydrolyzed collagen in lowering blood glucose and / or improving hyperglycemia (a risk factor), including the type of hydrolyzed collagen for the use disclosed herein, the formulation of the hydrolyzed collagen for the use disclosed herein, the timing of administration or intake of the hydrolyzed collagen for the use disclosed herein, and the dosage (e.g., dosing regimen, unit dose, daily dose, daily amount) of the hydrolyzed collagen for the use disclosed herein.

[0261] General definition

[0262] - As used herein, the terms “include / encompass” or “include / encompass” and their variations mean that the term is used in its non-limiting sense to mean that the items following the word are included, but not excluded, from being specifically mentioned. It also covers the more restrictive verbs “consistently of” and “comprises of”.

[0263] Unless the context explicitly requires the existence of one and only one element, mentioning an element through a noun without a quantifier does not preclude the possibility that there is more than one element. Therefore, a noun without a quantifier usually means "at least one / kind".

[0264] The terms “increase” and “increased level” and the terms “decrease” and “decreased level” (or “reduced” and “reduced level”) preferably refer to changes that are at least 5% higher or lower than the corresponding level of the control or reference, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. Alternatively, the level in the sample may be increased or decreased, regardless of the degree of change, when it is statistically significant compared to the level in the control or reference.

[0265] - As used in this article, the term "meal" refers to a specific occasion of eating that occurs at a particular time and includes prepared foods. Meals occur on a daily basis, typically several times a day. Breakfast, lunch, and dinner are considered the main meals. As a supplement or alternative, a meal can be considered to contain a relatively large amount of food (e.g., at least 500 to 1000 kcal per meal).

[0266] -The term “object” or “individual” as used herein refers to any animal (e.g., a mammal), preferably including humans.

[0267] - The term "application" or its variations as used herein refers to the act of providing a medicine to an object that consumes it. An object consuming a medicine may apply the medicine to himself / herself / itself. In such cases, the term "application to" can be interpreted as "being taken," "being used," or "being consumed."

[0268] - The term "unit dose" as used herein refers to an amount or unit of a compound, substance, active ingredient, or composition, for example, for immediate use. For example, a unit dose can be a pre-prepared form ready for administration (e.g., a pre-packaged dose). For example, a unit dose can also be identified from product packaging or labeling. Total daily doses can be divided into multiple unit doses.

[0269] -As used herein, the term "active ingredient" means any substance, compound, or composition that induces a measurable biological response in vitro and / or in vivo, as opposed to a substance, compound, or composition that provides only a physiological function. The biological response can be a direct or indirect response, preferably a direct or indirect response measurable in cells or organs.

[0270] Terms and Conditions

[0271] In this document, the clauses are some embodiments of the present invention. The features of the clauses (implementations) herein can be combined.

[0272] Clause 1: Hydrolyzed collagen for lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of collagen-containing material using an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0273] Clause 2: Hydrolyzed collagen used according to Clause 1, wherein the enzyme combination comprises neutral protease, carboxypeptidase and aminopeptidase.

[0274] Clause 3: Hydrolyzed collagen used as described in Clause 1 or 2 for improving high blood sugar.

[0275] Clause 4: Hydrolyzed collagen used as described in Clause 1 or 2, for improving risk factors for high blood sugar.

[0276] Clause 5: Hydrolyzed collagen used according to Clause 4, wherein the risk factors for hyperglycemia are selected from one or more of the following: insulin resistance, type 2 diabetes, gestational diabetes, high body mass index, obesity, and hyperglycemia.

[0277] Clause 6: Hydrolyzed collagen used in accordance with any of the preceding clauses, for improving postprandial blood glucose.

[0278] Clause 7: Hydrolyzed collagen used according to any of the preceding clauses, wherein it is selected from one or more of the following:

[0279] - Increase blood glucagon-like peptide-1;

[0280] - Lowers blood glucagon levels;

[0281] - Increase blood insulin levels;

[0282] - Increases the blood insulin / glucose ratio.

[0283] Clause 8: Hydrolyzed collagen used according to any of the preceding clauses, wherein the hydrolyzed collagen is administered at a daily dose of 1 to 100 grams, preferably 2 to 50 grams, more preferably 5 to 25 grams, wherein the amount is dry weight.

[0284] Clause 9: Hydrolyzed collagen used according to any of the preceding clauses, wherein the hydrolyzed collagen is administered orally, preferably as a dietary supplement.

[0285] Clause 10: Hydrolyzed collagen used according to any of the preceding clauses, wherein the hydrolyzed collagen is applied no more than 60 minutes apart from meals.

[0286] Clause 11: Hydrolyzed collagen for any of the preceding clauses, wherein the collagen-containing material is derived from one or more tissues selected from: skin, squamous cells, cartilage, bone, tendons, ligaments and connective tissue.

[0287] Clause 12: Hydrolyzed collagen used according to any of the preceding clauses, wherein the collagen is one or more types selected from: porcine collagen, bovine collagen and fish collagen.

[0288] Clause 13: Hydrolyzed collagen used according to any of the preceding clauses, wherein the collagen-containing material is gelatin.

[0289] Clause 14: Hydrolyzed collagen for any of the preceding clauses, wherein the average molecular weight of the hydrolyzed collagen is 1,000 to 7,000 Da and / or the polydispersity is 1.2 to 1.8.

[0290] Clause 15: A method for obtaining hydrolyzed collagen, comprising

[0291] a) Provide materials containing collagen for use in liquid formulations; and

[0292] b) subjecting the collagen-containing material to an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0293] Clause 16: The method according to Clause 15, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

[0294] Clause 17: The method according to Clause 15 or 16, wherein the neutral protease is selected from one or more of the following: serine protease, aspartic protease, cysteine ​​protease, and metalloproteinase.

[0295] Clause 18: The method according to any one of Clauses 15 to 17, wherein the aminopeptidase is a leucine aminopeptidase.

[0296] Clause 19: The method according to any one of Clauses 15 to 18, wherein one or more of the neutral protease, carboxypeptidase and aminopeptidase are derived from Aspergillus, preferably from Aspergillus oryzae.

[0297] Clause 20: The method according to any one of Clauses 15 to 19, wherein the collagen-containing material is subjected to a neutral protease of an amount defined by an enzyme activity of 0.2 to 25,000 U / g, preferably 2 to 2,500 U / g, more preferably 20 to 250 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0298] Clause 21: The method according to any one of Clauses 15 to 20, wherein the collagen-containing material is subjected to an amount of carboxypeptidase defined by an enzyme activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0299] Clause 22: The method according to any one of Clauses 15 to 21, wherein the collagen-containing material is subjected to an amount of aminopeptidase defined by an enzyme activity of 0.002 to 500 U / g, preferably 0.02 to 50 U / g, more preferably 0.2 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0300] Clause 23: The method according to any one of Clauses 15 to 22, wherein the collagen-containing material is subjected to one or more enzymes from the enzyme combination for 60 to 180 minutes.

[0301] Clause 24: The method according to any one of Clauses 15 to 23, wherein the collagen-containing material is subjected to one or more enzymes of the enzyme combination at a temperature of 30 to 60°C.

[0302] Clause 25: The method according to any one of Clauses 15 to 24, wherein the collagen-containing material is subjected to one or more enzymes in the enzyme combination at pH 5 to 8.

[0303] Clause 26: The method according to any one of Clauses 15 to 25, wherein the collagen-containing material is continuously subjected to two or more enzymes from the enzyme combination.

[0304] Clause 27: The method according to any one of Clauses 15 to 26, wherein the collagen-containing material is subjected to two or more enzymes in the enzyme combination simultaneously.

[0305] Clause 28: The method according to Clause 27, wherein the collagen-containing material is subjected to a mixture comprising the enzyme combination.

[0306] Clause 29: The method according to any one of Clauses 15 to 28, wherein the collagen-containing material is provided in a liquid formulation in an amount of 20% to 50% by weight, preferably 30% to 40% by weight.

[0307] Clause 30: The method according to any one of Clauses 15 to 29, wherein the collagen-containing material is derived from one or more tissues selected from: skin, squamous cells, cartilage, bone, tendon, ligament and connective tissue.

[0308] Clause 31: The method according to any one of Clauses 15 to 30, wherein the collagen is one or more types selected from the group consisting of porcine collagen, bovine collagen and fish collagen.

[0309] Clause 32: The method according to any one of Clauses 15 to 31, wherein the collagen-containing material is gelatin.

[0310] Clause 33: Hydrolyzed collagen that can be obtained by any of the methods described in Clauses 15 to 32.

[0311] Clause 34: Hydrolyzed collagen as described in Clause 33, wherein the average molecular weight of the hydrolyzed collagen is 1,000 to 7,000 Da and / or the polydispersity is 1.2 to 1.8.

[0312] Clause 35: Hydrolyzed collagen as described in Clause 33 or 34, used for the purposes defined in any of Clauses 1 to 14.

[0313] Clause 36: Hydrolyzed collagen for improving hyperglycemia or risk factors for hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of collagen-containing material using an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0314] Clause 37: Hydrolyzed collagen used according to Clause 36, wherein the enzyme combination comprises neutral protease, carboxypeptidase, and aminopeptidase.

[0315] Clause 38: Hydrolyzed collagen used according to Clauses 36 to 37, wherein the risk factors for hyperglycemia are selected from one or more of the following: insulin resistance, type 2 diabetes, gestational diabetes, high body mass index, obesity, and hyperglycemia.

[0316] Clause 39: Hydrolyzed collagen used in any of Clauses 36 to 38, wherein the collagen is selected from one or more of the following:

[0317] - Increase blood glucagon-like peptide-1;

[0318] - Lowers blood glucagon levels;

[0319] - Increase blood insulin levels;

[0320] - Increases the blood insulin / glucose ratio.

[0321] Clause 40: Hydrolyzed collagen for any of the applications described in Clauses 36 to 39, wherein the hydrolyzed collagen is administered at a daily dose of 1 to 100 grams, preferably 2 to 50 grams, more preferably 5 to 25 grams, wherein the amount is dry weight.

[0322] Clause 41: Hydrolyzed collagen for any of the applications described in Clauses 36 to 41, wherein the hydrolyzed collagen is administered orally, preferably as a dietary supplement.

[0323] Clause 42: Hydrolyzed collagen used according to any one of Clauses 36 to 41, wherein the hydrolyzed collagen is applied no more than 60 minutes apart from a meal.

[0324] Clause 43: Hydrolyzed collagen for any of the applications described in Clauses 36 to 42, wherein the collagen-containing material is derived from one or more tissues selected from: skin, squamous cells, cartilage, bone, tendons, ligaments, and connective tissue.

[0325] Clause 44: Hydrolyzed collagen used in any of Clauses 36 to 43, wherein the collagen is one or more types selected from the group consisting of porcine collagen, bovine collagen, and fish collagen.

[0326] Clause 45: Hydrolyzed collagen for any of the applications described in Clauses 36 to 44, wherein the collagen-containing material is gelatin.

[0327] Clause 46: Hydrolyzed collagen for any of the applications described in Clauses 36 to 45, wherein the average molecular weight of the hydrolyzed collagen is 1,000 to 7,000 Da and / or the polydispersity is 1.2 to 1.8.

[0328] Clause 47: Use of hydrolyzed collagen for non-therapeutic purposes of lowering blood sugar, wherein the hydrolyzed collagen is defined in any one of Clauses 1 to 11.

[0329] Clause 48: Use as described in Clause 47, wherein said use is for improving postprandial blood glucose.

[0330] Clause 49: The use as described in Clause 47 or 48, wherein the hydrolyzed collagen is applied as defined in any one of Clauses 40 to 42.

[0331] Clause 50: A method for obtaining hydrolyzed collagen, comprising

[0332] a) Provide materials containing collagen for use in liquid formulations; and

[0333] b) subjecting the collagen-containing material to an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0334] Clause 51: The method according to Clause 50, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

[0335] Clause 52: The method according to Clause 50 or 51, wherein the neutral protease is selected from one or more of the following: serine protease, aspartic protease, cysteine ​​protease, and metalloproteinase.

[0336] Clause 53: The method according to any one of Clauses 50 to 52, wherein the aminopeptidase is a leucine aminopeptidase.

[0337] Clause 54: The method according to any one of Clauses 50 to 53, wherein one or more of the neutral protease, carboxypeptidase and aminopeptidase are derived from Aspergillus, preferably from Aspergillus oryzae.

[0338] Clause 55: The method according to any one of Clauses 50 to 54, wherein the collagen-containing material is subjected to a neutral protease of an amount defined by an enzyme activity of 0.2 to 25,000 U / g, preferably 2 to 2,500 U / g, more preferably 20 to 250 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0339] Clause 56: The method according to any one of Clauses 50 to 55, wherein the collagen-containing material is subjected to an amount of carboxypeptidase defined by an enzyme activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0340] Clause 57: The method according to any one of Clauses 50 to 56, wherein the collagen-containing material is subjected to an amount of carboxypeptidase defined by an enzyme activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0341] Clause 58: The method according to any one of Clauses 50 to 57, wherein the collagen-containing material is subjected to an amount of aminopeptidase defined by an enzyme activity of 0.002 to 500 U / g, preferably 0.02 to 50 U / g, more preferably 0.2 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

[0342] Clause 59: The method according to any one of Clauses 50 to 58, wherein the collagen-containing material is subjected to one or more enzymes in the enzyme combination for 60 to 180 minutes.

[0343] Clause 60: The method according to any one of Clauses 50 to 59, wherein the collagen-containing material is subjected to one or more enzymes in the enzyme combination at 30 to 60°C.

[0344] Clause 61: The method according to any one of Clauses 50 to 60, wherein the collagen-containing material is subjected to one or more enzymes in the enzyme combination at pH 5 to 8.

[0345] Clause 62: The method according to any one of Clauses 50 to 61, wherein the collagen-containing material is continuously subjected to two or more enzymes from the enzyme combination.

[0346] Clause 63: The method according to any one of Clauses 50 to 62, wherein the collagen-containing material is subjected simultaneously to two or more enzymes from the enzyme combination.

[0347] Clause 64: The method according to Clause 63, wherein the collagen-containing material is subjected to a mixture comprising the enzyme combination.

[0348] Clause 65: The method according to any one of Clauses 50 to 64, wherein the collagen-containing material is provided in a liquid formulation in an amount of 20% to 50% by weight, preferably 30% to 40% by weight.

[0349] Clause 66: The method according to any one of Clauses 50 to 65, wherein the collagen-containing material is derived from one or more tissues selected from: skin, squamous cells, cartilage, bone, tendon, ligament and connective tissue.

[0350] Clause 67: The method according to any one of Clauses 50 to 66, wherein the collagen-containing material is derived from one or more tissues selected from: skin, squamous cells, cartilage, bone, tendon, ligament and connective tissue.

[0351] Clause 68: The method according to any one of Clauses 50 to 67, wherein the collagen-containing material is gelatin.

[0352] Clause 69: Hydrolyzed collagen that can be obtained by any one of the methods described in Clauses 50 to 68, wherein the hydrolyzed collagen:

[0353] - Weight-average molecular weight of 2000 to 4000 Da, preferably 2500 to 3500 Da; and

[0354] - Contains 35% to 60% by weight of collagen peptides with a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0355] Clause 70: Hydrolyzed collagen as described in Clause 69, wherein the hydrolyzed collagen comprises more than 40% by weight of collagen peptides with a molecular weight in the range of 2,000 to 5,000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen.

[0356] Clause 71: Hydrolyzed collagen as described in Clause 69 or 70, wherein said hydrolyzed collagen comprises:

[0357] -1% to 20% by weight of collagen peptides with a molecular weight of less than 1000 Da; and / or

[0358] -20% to 40% by weight of collagen peptides with a molecular weight of 1000 to 2000 Da; and / or

[0359] -5% to 25% by weight of collagen peptides with a molecular weight of 5,000 to 10,000 Da; and / or

[0360] -0% to 10% by weight of collagen peptides with a molecular weight greater than 10,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0361] Clause 72: Hydrolyzed collagen according to any one of Clauses 69 to 71, wherein the polydispersity of said hydrolyzed collagen is 1.2 to 1.8.

[0362] Clause 73: Hydrolyzed collagen according to any one of Clauses 69 to 72, wherein the hydrolyzed collagen is capable of stimulating the secretion of glucagon-like peptide-1.

[0363] Clause 74: Hydrolyzed collagen pursuant to any one of Clauses 69 to 73, used for improving hyperglycemia or risk factors for hyperglycemia as defined in any one of Clauses 36 to 46.

[0364] Clause 75: Use of hydrolyzed collagen according to any one of Clauses 69 to 73 in non-therapeutic hypoglycemic agents as defined in any one of Clauses 47 to 49 Attached Figure Description

[0365] Figure 1 GLP-1 secretion from STC-1 cells was determined after incubation with different fractions of hydrolyzed collagen (e.g., obtained through different enzymatic treatments) at 10 mg / ml. The supernatant was collected after 2 hours of incubation. GLP-1 levels were determined by enzyme immunoassay. Results are expressed as mean (n=3) ± standard deviation. “H080” indicates the hydrolyzed collagen of this invention.

[0366] Figure 2 The area under the curve (AUC) of blood glucose levels in mice after glucose loading at time 0 and after intake of the active ingredient / carrier at time -45 minutes. "H080" indicates the hydrolyzed collagen of this invention.

[0367] Figure 3 The ratio of insulin to blood glucose levels in mice at time 15 minutes after glucose loading at time 0 and product / drug / carrier intake at time -45 minutes. "H080" indicates the hydrolyzed collagen of this invention.

[0368] Figure 4 Plasma GLP-1 levels in mice 15 and 30 minutes after ingestion of H080 hydrolyzed collagen, compared to baseline. "H080" represents the hydrolyzed collagen of this invention.

[0369] Figure 5 Plasma GIP levels in mice at 15 and 30 minutes after ingestion of H080 hydrolyzed collagen, compared to baseline. "H080" represents the hydrolyzed collagen of this invention.

[0370] Experimental Section

[0371] Example 1

[0372] Example 1 illustrates the effect of gelatin hydrolysate obtained by enzymatic hydrolysis using a combination of enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases on increasing GLP-1 levels in vitro and decreasing blood glucose levels in vivo.

[0373] Research Design

[0374] Fourteen different enzymes (mixtures) were used to hydrolyze gelatin into hydrolyzed collagen formulations. The resulting hydrolyzed collagen formulations were subjected to simulated gastrointestinal digestion (SGID) to obtain bioactive hydrolyzed collagen fractions, as expected after in vivo uptake and digestion (Song et al. Food Funct. 2020 Jun 24; 11(6): 5553-5564).

[0375] Hydrolyzed collagen fractions were screened in vitro to enhance the ability of STC-1 cells to secrete GLP-1. STC-1 cells are incretin tumor cells and have been reported as a predictive cell model for studying hormone secretion mechanisms in the gastrointestinal tract (Qi et al. Bio Protoc. 2020 Aug 20; 10(16):e3717). GLP-1 plays a major role in glycemic homeostasis by regulating gastric emptying, enhancing pancreatic insulin secretion, and inhibiting pancreatic glucagon secretion. Therefore, GLP-1 secretion by STC-1 cells is a preferred outcome parameter for determining how hydrolyzed collagen fractions can improve glucose levels in vivo.

[0376] The most promising hydrolyzed collagen fraction identified in vitro was further tested for its hypoglycemic activity in mice. Hypoglycemic activity was determined based on changes in plasma GLP-1, GIP, glucose, and insulin levels. The potency of the hydrolyzed collagen fraction was compared with that of the hypoglycemic drug sitagliptin.

[0377] method

[0378] Enzymatic hydrolysis of collagen

[0379] Gelatin powder was dissolved in demineralized water by heating and stirring to provide a 35% by weight gelatin solution. The temperature of the gelatin solution was adjusted to 40 to 55°C. The pH of the gelatin was adjusted to pH 5.5 to 8.0. When the appropriate temperature and pH were reached, the enzyme (mixture) as shown in Table 1 was added, and hydrolysis was carried out under stirring. For “H080” conditions, a commercially available enzyme mixture (Sumizyme FP-G, available from Takabio, Japan) containing a mixture of neutral protease, carboxypeptidase, and aminopeptidase derived from Aspergillus oryzae was used. The temperature was kept constant. After 120 minutes, the enzymatic hydrolysis was terminated by thermal inactivation of the enzyme. The solution was then cooled to 55°C. The hydrolyzed gelatin solution was purified, filtered, and sterilized. The final hydrolyzed gelatin powder was obtained by spray drying.

[0380] Table 1. Overview of enzymes (mixture) used for collagen hydrolysis

[0381]

[0382]

[0383] STC-1 cells secrete GLP-1

[0384] STC-1 cells were incubated with different final concentrations (0.2%, 0.5%, and 1% dry matter in Hepes buffer at pH 7.4) of hydrolyzed collagen obtained after simulated gastrointestinal digestion (SGID). Supernatants were collected after 2 hours of incubation. GLP-1 levels were determined by enzyme immunoassay and expressed as pg / ml. Data are presented as mean (n=3) ± standard deviation.

[0385] Cells were stimulated with enzymes (mixtures) at concentrations of 2 mg / ml, 5 mg / ml, or 10 mg / ml. A dose-dependent increase in GLP-1 secretion was observed for all enzymes (mixtures), with the highest GLP-1 secretion observed at the 10 mg / ml condition. Therefore, Figure 1 The results are shown for comparing enzyme (mixture) concentrations of 10 mg / ml.

[0386] In vivo mouse model

[0387] The following mouse model was used to determine the area under the curve (AUC) for blood glucose level, insulin / blood glucose ratio, plasma GLP-1 level, and plasma GIP level. C57BL6 / J mice (male, 23-25g, 8 weeks old) were randomized to treatment groups (n = 10 mice / group) based on their body weight.

[0388] Mice were fasted for 6 hours, followed by an oral glucose tolerance test (OGTT). Mice were treated with a carrier, three different concentrations (40 mg / kg, 400 mg / kg, and 4 g / kg) of test sample H080, and sitagliptin as a positive control. Forty-five minutes after receiving the active ingredient, mice received an oral glucose load (2 g / kg body weight). Blood glucose levels were measured in blood collected at -45, 0, 15, 30, 60, 90, and 120 minutes (after the glucose load). Insulin was determined in plasma collected 45 minutes before (-45 minutes) and 15 minutes after (+15 minutes) after the glucose load (by enzyme-linked immunosorbent assay, ELISA). This determined the insulin / glucose ratio.

[0389] Plasma GLP-1 and GIP levels were assessed one week after the OGTT test. For this purpose, mice were fasted for 6 hours and treated with sitagliptin (400 μg / mouse) to prevent GLP-1 and GIP from being degraded by dipeptidyl peptidase 4 (DPP-IV). Thirty minutes later, mice received either the carrier or test product H080 at a dose of 4 g / kg body weight. Mice were anesthetized and blood was collected from the portal vein 15 or 30 minutes after administration of the active ingredient. Plasma GLP-1 and GIP levels were measured by ELISA.

[0390] Models in healthy mice have demonstrated both prevention and treatment of hyperglycemia (and other parameters associated with hyperglycemia) after treatment with the active ingredient.

[0391] Similar studies were also conducted in obese mice with naturally occurring hyperglycemia, using a similar method as described for healthy mice, with similar results.

[0392] result

[0393] In vitro GLP-1 secretion

[0394] Figure 1 GLP-1 secretion in STC-1 cells is shown after incubation with 10 mg / ml hydrolyzed collagen (such as that obtained by hydrolysis with the enzymes (mixture) provided in Table 1 under optimal process conditions).

[0395] Hydrolyzed collagen, denoted as H080, induced the greatest GLP-1 secretion. Compared to the control, H080 induced a 5800-fold increase in GLP-1 secretion. In comparative tests, the increase in GLP-1 mediated by H080 was at least 2.5 times greater than the increase in GLP-1 mediated by other enzymes (a mixture).

[0396] Data indicate that, compared to other enzymes and combinations of enzymes, the combination of neutral proteases (e.g., serine proteases), carboxypeptidases, and aminopeptidases (e.g., leucine aminopeptidases) induces the highest GLP-1 secretion to date.

[0397] It was found that, compared with using only one enzyme (e.g., a neutral protease in “CH1” or “CH2”), a combination of two or more enzymes (e.g., a combination of two neutral proteases in “CH3”) was insufficient to further promote GLP-1 secretion.

[0398] It was found that, compared with the use of endopeptidase alone, the combination of only endopeptidase (i.e., enzymes that cleave at non-terminal amino acids) and exopeptidase (i.e., enzymes that cleave at terminal amino acids) was insufficient to further promote GLP-1 secretion. For example, "CH12" contains both endopeptidase and exopeptidase activities, but it cannot further promote GLP-1 secretion compared with enzymes containing only endopeptidase activity.

[0399] Reproduced using different batches of gelatin Figure 1 The results are shown in the figure.

[0400] The results showed that hydrolyzing collagen with a combination of two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases is important for obtaining hydrolyzed collagen with strong hypoglycemic effects (e.g., induced by GLP-1).

[0401] In vivo hypoglycemic activity

[0402] Figure 2 The study presents blood glucose levels in mice after treatment with 40 mg / kg H080, 400 mg H080, 4 g / kg H080, and 400 μg / mouse of sitagliptin. A dose-dependent decrease in blood glucose was observed after treatment with H080. The highest dose of H080 had a similar hypoglycemic effect to the drug sitagliptin. This suggests that hydrolyzed collagen may be provided as a safe alternative to pharmaceutically available drugs that are often associated with side effects.

[0403] Figure 3 The insulin / glucose ratio in mice after treatment with 40 mg / kg H080, 400 mg H080, 4 g / kg H080, and 400 μg / mouse of sitagliptin is shown. H080 mediated a significant increase in the insulin / glucose ratio. A dose-dependent increase in the insulin / glucose ratio was observed after treatment with H080.

[0404] Figure 4 Plasma GLP-1 levels are shown after control treatment or treatment with 4 g / kg H080. Treatment with H080 increased plasma GLP-1 levels, with the maximum effect observed 30 minutes after H080 ingestion.

[0405] Figure 5 Plasma GIP levels are shown after control treatment or treatment with 4 g / kg H080. Treatment with H080 reduced plasma GIP levels, with the maximum effect observed 30 minutes after H080 ingestion.

[0406] In healthy mouse models, mice received the active ingredient prior to a glucose load. Therefore, healthy mice are particularly well-suited for studying the prevention of hyperglycemia. Studies were also conducted in obese mice with naturally occurring hyperglycemia. The method was similar to that used in healthy mice. Since obese mice already had elevated blood glucose levels upon receiving the active ingredient, the improvement in blood glucose (and other parameters associated with hyperglycemia) in the obese mouse model further enhanced the therapeutic effect of the active ingredient on the disease.

[0407] An effect was observed in obese mice with hyperglycemia. Figures 1 to 5 Similar effects to the parameters studied in [the study]. This further enhances the effectiveness of H080 in treating hyperglycemia (and related parameters). The hypoglycemic activity of a combination of enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0408] The inventors have discovered that high hypoglycemic activity (e.g., based on GLP-1 secretion levels) can be achieved when at least two enzymes are used in the enzymatic hydrolysis of collagen-containing materials, wherein the two enzymes are selected from neutral proteases, carboxypeptidases, and aminopeptidases.

[0409] As shown in Table 2, the use of neutral protease in the enzymatic hydrolysis of gelatin produced gelatin hydrolysates with hypoglycemic activity, although this activity was limited. Compared to the blank or the use of neutral protease alone, combinations of neutral protease with either a carboxypeptidase or an aminopeptidase alone provided stronger hypoglycemic activity. Combinations of all three enzymes produced gelatin hydrolysates with the highest hypoglycemic activity.

[0410] Table 2: Enzymes used for collagen hydrolysis and the effects of hydrolysates on blood sugar control.

[0411]

[0412] Characterization of hydrolyzed collagen with high hypoglycemic activity

[0413] Hydrolyzed collagen with high hypoglycemic activity (e.g., groups III to IV in Table 2) is characterized by a weight-average molecular weight of 1,000 to 7,000 Da and a polydispersity (weight-average molecular weight / number-average molecular weight) of 1.2 to 1.8 (average of about 1.56 measured in >100 measurements), as determined by HPSEC.

[0414] Example 2

[0415] Example 2 illustrates the effect of process conditions on obtaining hydrolyzed collagen for lowering blood sugar.

[0416] method

[0417] As described in Example 1, gelatin hydrolysate was produced by hydrolysis using a combination of neutral protease (50,000 U / g), aminopeptidase (500 to 1200 U / g), and carboxypeptidase (300 to 700 U / g).

[0418] A wide range of working conditions were tested, covering those commonly used in enzymatic hydrolysis.

[0419] The concentration of gelatin in the initial solution varied between 10% by weight and 50% by weight. Enzymes were used at a concentration of 2000 ppm for hydrolysis. The pH during enzymatic hydrolysis varied between pH 4.0 and 10.0. The temperature during enzymatic hydrolysis varied between 20 and 70°C.

[0420] Table 3 shows the effects of gelatin concentration, enzymatic hydrolysis temperature, and pH on obtaining gelatin hydrolysate fractions for lowering blood sugar in preliminary tests. GLP-1 secretion was tested using the same method as in Example 1.

[0421] Table 4 illustrates the effects of gelatin concentration, enzymatic hydrolysis temperature, and pH on obtaining gelatin hydrolysate fractions for lowering blood sugar, based on a more detailed and extensive set of tests. GLP-1 secretion was tested using the same methods as in Example 1.

[0422] Table 3. Effects of gelatin concentration, enzymatic hydrolysis temperature, and pH on obtaining gelatin hydrolysate fractions for lowering blood sugar in preliminary tests.

[0423]

[0424]

[0425] Table 4. Effects of gelatin concentration, enzymatic hydrolysis temperature, and pH on obtaining gelatin hydrolysate fractions for lowering blood sugar, based on more detailed and extensive testing. +: Less suitable; ++: More suitable; +++: Best suited

[0426]

[0427] As shown in Tables 3 and 4 together, it was found that hydrolyzed collagen suitable for enhancing GLP-1 secretion can be obtained within a wide range of precursor concentrations (gelatin weight%), pH values, and temperatures.

[0428] Regarding the precursor concentration (gelatin weight %), satisfactory results were obtained across the tested range of 10% to 50% by weight, with the optimal concentrations being clearly found at 20% to 35% by weight.

[0429] Regarding pH, satisfactory results were obtained across the entire tested pH range of 4.0 to 10.0, with the optimal results clearly observed at pH 5.5 to 6.5.

[0430] Regarding temperature, satisfactory results were obtained across the entire tested range of 20 to 60°C, with the optimal temperature range of 45 to 50°C being clearly optimal.

[0431] The results indicate that hydrolysis at (excessively) high temperatures (e.g., 70°C or higher) may not produce hydrolyzed collagen products suitable for enhancing GLP-1-secreted collagen. This is thought to be a result of enzyme denaturation and subsequent impaired reaction rate.

[0432] Preferred combinations of gelatin concentration, pH, and temperature yielded optimal activity in enhancing GLP-1 secretion from STC-1 cells. As shown in Table 5, combinations of gelatin concentration (30% to 40% by weight), pH (6 to 7.5), and temperature (40 to 55°C) produced unexpectedly high activity of gelatin hydrolysates in enhancing GLP-1 secretion from STC-1 cells. GLP-1 secretion was tested using the same method as in Example 1.

[0433] Table 5. Stimulation of STC-1 cells to secrete GLP-1 with hydrolyzed collagen obtained from a combination of neutral protease, carboxypeptidase and aminopeptidase, wherein enzymatic hydrolysis was carried out under different process conditions.

[0434] Process conditions GLP-1 level 30% to 40% gelatin, pH 6 to 7.5, 40 to 55°C 3471pg / ml 30% to 40% gelatin, pH 7.5 to 8.5, 55 to 65°C 346pg / ml

[0435] Example 3

[0436] Example 3 illustrates the molecular weight and molecular weight distribution of hydrolyzed collagen obtained by hydrolysis with different enzymes (mixtures), which are related to the ability of hydrolyzed collagen to stimulate STC-1 cells to secrete GLP-1.

[0437] Table 6 shows the molecular weight and molecular weight distribution of hydrolyzed collagen preparations obtained with different enzymes, as well as their varying abilities to stimulate GLP-1 secretion in STC-1 cells. The methods used for hydrolysis and evaluation of GLP-1 secretion are the same as those described in Example 1.

[0438] "H080" is produced by hydrolysis using a combination of neutral protease (50,000 U / g), aminopeptidase (500 to 1200 U / g), and carboxypeptidase (300 to 700 U / g) as described in Example 1. Collagen hydrolysates "CH11 to CH19" are obtained by hydrolysis using neutral, alkaline, or acidic proteases.

[0439] GLP-1 secretion is considered "average" if it is at least 1000-fold higher than that of a blank control.

[0440] As can be seen from Table 6, compared with the blank control stimulation, "H080" induced an increase in GLP-1 secretion of approximately 6000-fold, which is considered "very high" GLP-1 secretion. Groups "CH11 to CH19"—that is, hydrolyzed collagen obtained by hydrolysis with neutral, alkaline, or acidic proteases—showed no / very little or only average GLP-1 secretion.

[0441] As can be seen from Table 6, “H080” is characterized by a weight-average molecular weight of approximately 3000 Da (or a mean average molecular weight of approximately 2000 Da). Furthermore, “H080” is characterized by a high fraction (approximately 47.2%) of collagen peptides falling within the 2kDa to 5kDa range. Additionally, “H080” is characterized by high stimulatory activity against GLP-1 secretion (i.e., an increase of approximately 6000-fold compared to the blank control group). The “CH11 to CH19” groups lack one or more of these characteristics.

[0442] Table 6. An overview of the enzymes (mixture) used to hydrolyze collagen, the molecular weight and molecular weight distribution of the resulting hydrolyzed collagen, and their ability to stimulate GLP-1 secretion in STC-1 cells.

[0443]

Claims

1. Hydrolyzed collagen, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of collagen-containing material using an enzyme combination comprising one or both of carboxypeptidase and aminopeptidase and a neutral protease. The hydrolyzed collagen - Weight-average molecular weight is 2000-4500 Da; and - Contains 35% to 60% by weight collagen peptides with a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen; and The hydrolyzed collagen mentioned therein can stimulate the secretion of glucagon-like peptide-1.

2. The hydrolyzed collagen according to claim 1, wherein the enzyme combination comprises neutral protease, carboxypeptidase, and aminopeptidase.

3. The hydrolyzed collagen according to claim 1 or 2, wherein the collagen-containing material is derived from one or more tissues selected from: skin, squamous cells, cartilage, bone, tendon, and ligament.

4. The hydrolyzed collagen according to claim 1 or 2, wherein the collagen is one or more types selected from: porcine collagen, bovine collagen and fish collagen.

5. The hydrolyzed collagen according to claim 1 or 2, wherein the collagen-containing material is gelatin.

6. The hydrolyzed collagen according to claim 1, wherein the hydrolyzed collagen comprises more than 40% by weight of collagen peptides with a molecular weight of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

7. The hydrolyzed collagen according to any one of claims 1 or 6, wherein the hydrolyzed collagen comprises: -1% to 20% by weight of collagen peptides with a molecular weight of less than 1000 Da; and / or -20% to 40% by weight of collagen peptides with a molecular weight of 1000 to 2000 Da; and / or -5% to 25% by weight of collagen peptides with a molecular weight of 5,000 to 10,000 Da; and / or -0% to 10% by weight of collagen peptides with a molecular weight greater than 10,000 Da. Calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

8. The hydrolyzed collagen according to claim 1, 2 or 6, wherein the polydispersity of the hydrolyzed collagen is 1.2 to 1.

8.

9. A method for obtaining hydrolyzed collagen capable of stimulating the secretion of glucagon-like peptide-1, the method comprising: a) Provide collagen-containing materials for liquid formulations; as well as b) subjecting the collagen-containing material to an enzyme combination comprising one or both of carboxypeptidase and aminopeptidase, and a neutral protease. Step b) is performed until the hydrolyzed collagen is obtained. - Weight-average molecular weight is 2000-4500 Da; and - Contains 35% to 60% by weight of collagen peptides with a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

10. The method of claim 9, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

11. The method according to claim 9 or 10, wherein the neutral protease is selected from one or more of the following: serine protease, aspartic protease, cysteine ​​protease, and metalloproteinase.

12. The method according to claim 9 or 10, wherein the aminopeptidase is a leucine aminopeptidase.

13. The method according to claim 9 or 10, wherein one or more of the neutral protease, carboxypeptidase and aminopeptidase are derived from microorganisms of the genus Aspergillus.

14. The method of claim 13, wherein one or more of the neutral protease, carboxypeptidase, and aminopeptidase are derived from Aspergillus oryzae.

15. The method according to claim 9 or 10, wherein the collagen-containing material is subjected to an amount of neutral protease defined by an enzyme activity of 0.2 to 25000 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

16. The method according to claim 9 or 10, wherein the collagen-containing material is subjected to an amount of carboxypeptidase defined by an enzyme activity of 0.001 to 500 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

17. The method according to claim 9 or 10, wherein the collagen-containing material is subjected to an amount of aminopeptidase defined by an enzyme activity of 0.002 to 500 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

18. The method of claim 9 or 10, wherein the collagen-containing material is subjected to one or more enzymes in the enzyme combination for 60 to 180 minutes.

19. The method according to claim 9 or 10, wherein the collagen-containing material is subjected to one or more enzymes in the enzyme combination at a temperature of 30 to 60°C.

20. The method of claim 9 or 10, wherein the collagen-containing material is subjected to one or more enzymes from the enzyme combination at pH 5 to 8.

21. The method of claim 9 or 10, wherein the collagen-containing material is continuously subjected to two or more enzymes from the enzyme combination.

22. The method of claim 9 or 10, wherein the collagen-containing material is subjected simultaneously to two or more enzymes from the enzyme combination.

23. The method of claim 22, wherein the collagen-containing material is subjected to a mixture comprising the enzyme combination.

24. The method of claim 9 or 10, wherein the collagen-containing material is provided in a liquid formulation in an amount of 20 wt.% to 50 wt.%.

25. The method according to claim 9 or 10, wherein the collagen-containing material is derived from one or more tissues selected from skin, squamous cells, cartilage, bone, tendons, and ligaments.

26. The method according to claim 9 or 10, wherein the collagen is one or more types selected from: porcine collagen, bovine collagen, and fish collagen.

27. The method according to claim 9 or 10, wherein the collagen-containing material is gelatin.

28. Use of hydrolyzed collagen in the preparation of a medicament for improving hyperglycemia or risk factors for hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using an enzyme combination comprising one or both of carboxypeptidase and aminopeptidase and a neutral protease. The hydrolyzed collagen - Weight-average molecular weight is 2000-4500 Da; and - Contains 35% to 60% by weight collagen peptides with a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen; and The hydrolyzed collagen mentioned therein can stimulate the secretion of glucagon-like peptide-1.

29. The use according to claim 28, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

30. The use according to claim 28 or 29, wherein the risk factor for hyperglycemia is selected from one or more of the following: insulin resistance, type 2 diabetes, gestational diabetes, high body mass index, and hyperglycemia.

31. The use according to claim 28 or 29, wherein the risk factor for said hyperglycemia is obesity.

32. The use according to claim 28 or 29, wherein the hydrolyzed collagen is selected from one or more of the following: - Increase blood glucagon-like peptide-1; - Lowers blood glucagon levels; - Increase blood insulin levels; - Increases the blood insulin / glucose ratio.

33. The use according to claim 28 or 29, wherein the hydrolyzed collagen is administered at a daily dose of 1 to 100 grams, wherein the amount is dry weight.

34. The use according to claim 33, wherein the hydrolyzed collagen is administered at a daily dose of 5 to 25 g, wherein the amount is dry weight.

35. The use according to claim 28 or 29, wherein the hydrolyzed collagen is administered orally.

36. The use according to claim 28 or 29, wherein the hydrolyzed collagen is administered as a dietary supplement.

37. The use according to claim 28 or 29, wherein the hydrolyzed collagen is applied no more than 60 minutes apart from meals.

38. The use according to claim 28 or 29, wherein the collagen-containing material is derived from one or more tissues selected from skin, squamous cells, cartilage, bone, tendons, and ligaments.

39. The use according to claim 28 or 29, wherein the collagen is one or more types selected from: porcine collagen, bovine collagen and fish collagen.

40. The use according to claim 28 or 29, wherein the collagen-containing material is gelatin.

41. The use according to claim 28 or 29, wherein the polydispersity of the hydrolyzed collagen is 1.2 to 1.8.

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