Hydrolyzed collagen for use in reducing blood glucose

Abstract

The present invention relates to a hydrolyzed collagen preparation for use in reducing blood glucose. The hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes including two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase. The hydrolyzed collagen preparation is suitable, inter alia, as a food supplement, for example as a food supplement for use in improving hyperglycemia and / or risk factors for hyperglycemia.

Description

Technical Field

[0001] The present invention relates to an active ingredient and the use of the active ingredient for improving hyperglycemia and conditions associated with hyperglycemia. The active ingredient of the present invention is suitable as a food supplement.

Background Art

[0002] The human body depends on strict regulation of its blood glucose level for its normal functions. Disruption of normal blood glucose levels can lead to diseases, particularly metabolic syndrome, cardiovascular diseases, nerve damage, and kidney failure. Conditions associated with abnormal insulin levels and / or insulin sensitivity (e.g., type 2 diabetes, obesity) are closely linked to the onset of hyperglycemia.

[0003] The regulation of blood glucose involves the interaction of various organs (e.g., pancreas, brain, liver, intestine, adipose and muscle tissues), which is partly due to the release of various hormones. Insulin and glucagon are peptide hormones that act in opposition to each other's actions to balance blood glucose levels. In response to high blood glucose levels, pancreatic β-cells secrete insulin into the blood. This then lowers blood glucose levels by promoting the uptake of glucose from the blood into liver cells, adipocytes, and skeletal muscle cells. Glucagon is secreted by pancreatic α-cells. Glucagon enhances the conversion of glycogen stored in the liver into glucose, which is released into the bloodstream.

[0004] The first-line treatment for hyperglycemia initially involves lifestyle changes that reduce one or more risk factors for hyperglycemia. These typically include lifestyle changes targeted at insulin resistance, diabetes, obesity, or hypertension. Often, lifestyle changes alone are insufficient, which requires the inclusion of metformin and / or insulin treatment as part of the first-line treatment.

[0005] In the majority of individuals, the above first-line treatment does not sufficiently reduce hyperglycemia. As a potential second-line treatment, a new class of glucose-lowering drugs is currently being developed and tested (Davies et al. Diabetes Care 2018;41(12):2669-2701).

[0006] The new class of blood glucose-lowering drugs includes those that target the activity of incretin. Incretin is a metabolic hormone that enhances insulin secretion and / or inhibits glucagon secretion in a glucose-dependent manner, thereby reducing blood glucose levels. Glucagon-like peptide-1 (GLP-1) is considered one of the most important incretins and is thus an important target in new strategies for improving hyperglycemia. GLP-1 receptor agonists have demonstrated clinical efficacy (Trujillo et al. Ther Adv Endocrinol Metab. 2021 Mar 9;12:2042018821997320), but these GLP-1 receptor agonists have several limitations that prevent their widespread use. First, they cause several side effects, such as pancreatitis, pancreatic cancer, or bone diseases (Storgaard et al. Diabetes Obes Metab 2017;19:906-908), as well as gastrointestinal-related side effects such as nausea, vomiting, and diarrhea. Second, treatment with GLP-1 receptor agonists requires repeated subcutaneous injections, which leads to patient discomfort and injection site reactions (Trujillo et al. Ther Adv Endocrinol Metab. 2021 Mar 9;12:2042018821997320).

[0007] Another example of a new blood glucose-lowering agent is sitagliptin. Sitagliptin is a selective inhibitor of dipeptidyl peptidase IV (DPPIV). DPPIV causes the hydrolysis of incretin hormones. Thereby, sitagliptin can increase the plasma concentration of the active form of incretin, such as GLP-1 and gastric inhibitory polypeptide (GIP). This increases insulin release and decreases glucagon concentration. However, professional medical practitioners are still often hesitant to prescribe sitagliptin to patients. This is mostly due to some of its side effects and contraindications. For example, complications such as hyperglycemia, headache, respiratory infection, and pharyngitis occur in up to 10% of patients. Collagen peptides have also been suggested to lower blood glucose. JP 2009-235064 describes a blood glucose-lowering collagen peptide obtained by treating sea cucumbers with proteases, and JP 2012-116773 describes a collagen peptide that can be obtained by treating collagen derived from fish scales and / or fish skin with proteolytic enzymes and that increases insulin-like growth factor 1 (IGF-1).

[0008] There is an unmet need for an active ingredient that more efficiently and / or more safely improves hyperglycemia. Such an active ingredient can be used in addition to, or as an alternative to, the first and / or second choice treatment for an individual having (or at risk of having) hyperglycemia. SUMMARY OF THE INVENTION

[0009] The inventor of the present application has discovered that the enzymatic hydrolysis of a collagen-containing starting material using a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase produces a composition having a strong glucose-lowering activity. The glucose-lowering activity appears to be highest when all three of the above enzymes are used for hydrolysis.

[0010] In one aspect, the present invention relates to hydrolyzed collagen for use in reducing blood glucose, which is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0011] In one aspect, the present invention relates to hydrolyzed collagen for (further) use in improving hyperglycemia or a risk factor for hyperglycemia, which is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0012] In one aspect, the present invention a) preparing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes comprising a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, relates to a method for obtaining hydrolyzed collagen, wherein the hydrolyzed collagen is preferably for use in reducing blood glucose and / or improving hyperglycemia.

[0013] In one aspect, the present invention relates to hydrolyzed collagen obtainable by the methods disclosed in the present disclosure, said hydrolyzed collagen being for use in reducing blood glucose and / or improving hyperglycemia, preferably.

Brief Description of the Drawings

[0014]

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Modes for Carrying Out the Invention

[0015] ● Aspects of the present invention In one aspect, the present invention relates to hydrolyzed collagen for use in reducing blood glucose, which is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0016] In one aspect, the present invention relates to hydrolyzed collagen for use in improving hyperglycemia, which is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0017] In one aspect, the present invention a) preparing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes, preferably a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, relates to a method for obtaining hydrolyzed collagen, wherein the hydrolyzed collagen is preferably for use in reducing blood glucose and / or improving hyperglycemia as taught in the present disclosure.

[0018] In one aspect, the present invention relates to hydrolyzed collagen obtainable by the method disclosed in the present disclosure, wherein the hydrolyzed collagen is preferably for use in reducing blood glucose and / or improving hyperglycemia as taught in the present disclosure.

[0019] In one aspect, the present invention relates to a method for reducing blood glucose, which comprises administering to a subject a hydrolyzed collagen obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0020] In one aspect, the present invention relates to a method for treating hyperglycemia or a risk factor for hyperglycemia, which comprises administering to a subject a hydrolyzed collagen obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0021] In one aspect, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for reducing blood glucose, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, for the said use.

[0022] In one aspect, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for improving hyperglycemia or a risk factor for hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, for the said use.

[0023] ●Advantages of the present invention · The use of hydrolyzed collagen, for example as a food supplement, is generally safe; · The inventor of the present application found that the blood glucose-lowering effect of the hydrolyzed collagen of the present invention was similar to the blood glucose-lowering effect of the pharmaceutical compound sitagliptin. Therefore, hydrolyzed collagen is a valuable additive or alternative to glucose-lowering pharmaceutical compounds because pharmaceutical compounds generally cause side effects and have contraindications; · The inventor of the present application found that the blood glucose-lowering effect of the hydrolyzed collagen of the present invention involves different mechanisms of action. This mechanism of action includes increasing glucagon-like peptide-1 (GLP-1) and inhibiting dipeptidyl peptidase IV (DPP IV). The different effects of hydrolyzed collagen are considered to explain the strong in vivo blood glucose-lowering activity of hydrolyzed collagen. Furthermore, the presence of different mechanisms of action indicates that hydrolyzed collagen may also be suitable for treating the underlying effects of high blood glucose (e.g., in different populations of subjects and / or at different stages of hyperglycemia). · The inventor of the present application established the beneficial effects of hydrolyzed collagen in healthy mice (i.e., showing prevention of hyperglycemia) and in obese mice with hyperglycemia (i.e., showing treatment of hyperglycemia). This indicates that the hydrolyzed collagen of the present invention is effective in improving hyperglycemia both as a prevention and as a treatment.

[0024] ● Blood Glucose and Hyperglycemia In one embodiment, the present invention relates to hydrolyzed collagen for use in reducing blood glucose, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, and the combination of enzymes includes two or more selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0025] In a preferred embodiment, the present invention relates to hydrolyzed collagen for use in reducing blood glucose, which is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes including neutral protease, carboxypeptidase, and aminopeptidase.

[0026] In a preferred embodiment, the combination of enzymes disclosed in the present disclosure includes neutral protease, carboxypeptidase, and aminopeptidase. Preferably, the hydrolyzed collagen disclosed in the present disclosure is used as an active ingredient. More preferably, the hydrolyzed collagen disclosed in the present disclosure is used as an active ingredient in reducing the blood glucose concentration disclosed in the present disclosure and / or as an active ingredient in improving hyperglycemia disclosed in the present disclosure.

[0027] When used in the present disclosure, the term "blood glucose" (i.e., the glucose concentration in the blood) can be used interchangeably with "blood sugar". "Blood glucose" is typically and preferably expressed as millimoles per liter (mmol / l) and / or milligrams per deciliter (mg / dl). Blood glucose is usually and preferably determined in a fasting individual. In one embodiment, a "fasting individual" means an individual who has not ingested food for 8 hours or more.

[0028] In one embodiment, the hydrolyzed collagen disclosed in the present disclosure is for (further) use in improving hyperglycemia.

[0029] In the present disclosure, the term "blood" can be used interchangeably with the term "plasma" when referring to the concentration of a molecule. For example, "blood glucose" can also be read as "plasma glucose". For example, "blood GLP-1" can also be read as "plasma GLP-1".

[0030] As used in the present disclosure, the term "hyperglycemia" means high blood glucose. In the present disclosure, the normal blood glucose range in a fasting individual is considered to be 80-110 mg / dl. In the present disclosure, an individual having a blood glucose concentration of 126 mg / dl or higher is considered to have hyperglycemia. In general, an individual is considered to have impaired glucose tolerance, or prediabetes, when having a fasting blood glucose of 111-125 mg / dl. An individual having "impaired glucose tolerance" or "prediabetes" is considered to have hyperglycemia in the present disclosure.

[0031] In certain embodiments, the "hyperglycemia" disclosed in the present disclosure is acute and is generally characterized by the need for direct administration of insulin.

[0032] In certain embodiments, the "hyperglycemia" disclosed in the present disclosure is chronic and requires prolonged treatment of hyperglycemia, with and / or without combination with a change in lifestyle. The "hyperglycemia" disclosed in the present disclosure is preferably chronic hyperglycemia.

[0033] In the context of the present invention, the preferred method for measuring hyperglycemia is the hemoglobin A1c (HbA1c) test, which establishes glycated haemoglobin. Those skilled in the art know the most widely recognized and / or most accurate ways to measure HbA1c, but preferred analytical methods used to measure HbA1c include affinity chromatography, immunoassay, cation exchange chromatography, and / or capillary electrophoresis.

[0034] In certain embodiments, hyperglycemia as disclosed in the present disclosure includes "postprandial hyperglycemia." "Postprandial hyperglycemia" is defined as unduly high postprandial glucose in the context of the present invention. As used in the present disclosure, the term "postprandial glucose concentration" (PPG value) refers to the amount of glucose in the blood after a meal. In healthy, or non-diabetic, individuals, the concentration of blood glucose peaks at about 1 hour after the start of a meal, usually does not exceed 140 mg / dl, and returns to the pre-meal concentration within 2 to 3 hours. These time profiles are typically different in diabetic patients or patients suffering from different pathological conditions. In certain embodiments, the PPG value is measured 2 hours after the start of a meal, although depending on at which point the peak value is typically located (or is predicted to be located), other time points may be more appropriate. The acceptable PPG value, the threshold PPG value defining postprandial hyperglycemia, and the optimal method for measuring the PPG value can vary by medical expert physicians. In the context of the present invention, for adults under 50 years of age, the PPG value is considered unduly high / too high (i.e., postprandial hyperglycemia) when it exceeds 140 mg / dl, for individuals between 50 and 60 years of age when the PPG value exceeds 150 mg / dl, and for individuals over 60 years of age when the PPG value exceeds 160 mg / dl.

[0035] The present invention may relate to the non-therapeutic use and / or therapeutic use of the hydrolyzed collagen, where the difference is based on the concentration of blood glucose and / or a separate group of subjects in which an increase in blood glucose is occurring.

[0036] The first group (in the present disclosure, the "group of healthy subjects") includes healthy people who do not receive a therapeutic benefit from treatment with the hydrolyzed collagen of the present invention. For example, the blood glucose concentration is not excessive or severe such that it is not expected to lead to health problems or (severe) suffering. In addition to or instead of this, the blood glucose may have a cause or severity such that an increase in blood glucose naturally disappears over time (i.e., is not chronic). In addition to or instead of this, the blood glucose concentration in the group of healthy subjects may not be too excessive or severe such that the subject does not generally seek help from a professional medical practitioner.

[0037] The second group (in the present disclosure, the "group of diseased subjects") includes subjects in whom an increase in blood glucose concentration is of a pathological nature, which means that the increased blood glucose concentration typically leads to severe symptoms of pain and (severe) suffering and / or may lead to (severe) health and psychological risks. In addition, the increased blood glucose may be chronic in the group of diseased subjects. The severity of the increase in blood glucose is such that help from a professional medical practitioner is generally required.

[0038] A professional medical practitioner can typically determine on a case-by-case basis whether a change in blood glucose concentration requires a therapeutic or non-therapeutic intervention (i.e., whether the person belongs to the group of healthy subjects or the group of diseased subjects). The professional medical practitioner may, for example, establish whether the change in blood glucose may lead to (severe) health and psychological risks and / or whether it is a chronic change in blood glucose.

[0039] Depending on whether an individual is healthy or has a medical condition (e.g., hyperglycemia, diabetes, obesity) occurring, the improvement of postprandial glucose can be considered to be a non-therapeutic intervention or a therapeutic intervention, respectively. In addition to or instead of this, depending on the amount of glucose in the blood after a meal, the improvement of postprandial glucose can be considered to be a non-therapeutic intervention or a therapeutic intervention. For example, in a healthy individual, the amount of glucose after a meal typically does not exceed 140 mg / dl, and thus, the improvement of postprandial glucose concentration below 140 mg / dl can also be considered to be a non-therapeutic intervention. For example, in an individual with a medical condition (e.g., hyperglycemia, diabetes, obesity) occurring, the amount of glucose after a meal can typically be 140 mg / dl or more, and thus, the improvement of postprandial glucose concentration of 140 mg / dl or more can also be considered to be a therapeutic intervention.

[0040] In certain embodiments, the hydrolyzed collagen is for use in the therapeutic reduction of blood glucose, for example, for use in improving hyperglycemia or risk factors for hyperglycemia. In certain embodiments, the hydrolyzed collagen is for the non-therapeutic reduction of blood glucose, for example, for the non-therapeutic reduction of blood glucose in the improvement of postprandial glucose.

[0041] In certain embodiments, the hydrolyzed collagen is for the non-therapeutic improvement of postprandial glucose, where the postprandial glucose concentration is preferably less than 140 mg / dl, more preferably less than 120 mg / dl, and even more preferably less than 100 mg / dl (i.e., before the start of the intervention with the hydrolyzed collagen). In certain embodiments, the hydrolyzed collagen is for the therapeutic improvement of postprandial glucose, where the postprandial glucose concentration is preferably 140 mg / dl or more, more preferably 160 mg / dl or more, and even more preferably 180 mg / dl or more (i.e., before the start of the intervention with the hydrolyzed collagen).

[0042] In one embodiment, the hydrolyzed collagen disclosed in the present disclosure is for (further) use in improving the risk factors for hyperglycemia.

[0043] The beneficial effects of the hydrolyzed collagen of the present invention were demonstrated in healthy mice induced to have hyperglycemia after receiving the hydrolyzed collagen. The beneficial effects of the hydrolyzed collagen of the present invention were also demonstrated in obese mice with hyperglycemia administered the hydrolyzed collagen. This indicates that the hydrolyzed collagen of the present invention is effective in both the prevention and treatment of hyperglycemia.

[0044] In the context of the present invention, the term "ameliorate" encompasses both "prevent" and "treat" a condition. The term "prevent" means to cause a subject not to develop a condition (e.g., hyperglycemia or a risk factor for hyperglycemia). An intervention is considered a form of "prevention" in the present disclosure if the condition is delayed, its severity is reduced, and / or its occurrence is decreased, even if the condition is not completely prevented from occurring. As used in the present disclosure, "preventing" or "prevention" by an intervention encompasses situations where a subject has previously experienced a condition (e.g., hyperglycemia or a risk factor for hyperglycemia), but the intervention prevents recurrence of the condition. "Preventing" or "prevention" may have therapeutic and / or non-therapeutic effects. If "preventing" or "prevention" is of a therapeutic nature, it may also be directed at the symptoms of a disease or condition and / or the underlying pathology. "Preventing" or "prevention" can also be defined by any standard technique as any delay, change in severity, and / or change in incidence compared to a subject or a reference, such delay or change being, for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or a delay or change between them compared to the subject or the reference. In the context of the present invention, "treating" means that, where a condition (e.g., hyperglycemia or a risk factor for hyperglycemia) already exists, an intervention reduces and / or cures the said condition. "Treating" may have therapeutic and / or non-therapeutic effects. If "treating" is of a therapeutic nature, it may also be directed at the symptoms of a disease or condition and / or the underlying pathology. "Treating" can also be defined by any standard technique as any reduction in the severity, incidence, and / or frequency of a condition compared to a subject or a reference, such reduction being, for example, at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, or a reduction between them compared to the subject or the reference.As used in the present disclosure, "ameliorate" includes "cure". The term "ameliorate" can be used interchangeably with "reduce" or "decrease" in the present disclosure.

[0045] As used in the present disclosure, "risk factor for hyperglycemia" means a condition associated with an increased chance of developing hyperglycemia. In an individual having a risk factor for hyperglycemia, it is feasible to administer a blood glucose reducing agent as a prophylactic agent in the prevention of hyperglycemia. In addition to or instead of this, in an individual having a risk factor for hyperglycemia, it is preferably feasible to administer a blood glucose reducing agent as a prophylactic agent for ameliorating the hyperglycemia that may develop. For example, obesity is a risk factor for hyperglycemia. Administration of a blood glucose reducing agent to an obese individual can prevent hyperglycemia in the obese individual and / or can ameliorate the hyperglycemia in the individual if hyperglycemia has developed.

[0046] There are several risk factors for developing hyperglycemia. In the context of the present invention, an insulin level that is too low in the body, the vessels of insulin action, and / or insulin resistance are considered to be risk factors for hyperglycemia. In the present disclosure, type 2 diabetes is considered to be a risk factor for hyperglycemia. In addition to or instead of this, one or more of the following conditions are considered to be risk factors for developing hyperglycemia in the present disclosure: gestational diabetes, high body mass index (BMI), obesity, and hyperglucagonemia.

[0047] Some hormones act to increase blood glucose concentration hyperglycemia when present in excess, and are considered to be risk factors for hyperglycemia in the present disclosure, and this includes an excess of one or more of cortisol, catecholamine, growth hormone, glucagon, and thyroid hormone.

[0048] In a preferred embodiment, the risk factor for hyperglycemia disclosed in the present disclosure is insulin resistance. As used in the present disclosure, the term "insulin resistance" encompasses all states diagnosed as "insulin resistance" by a specialized physician. As used in the present disclosure, the term "insulin resistance" preferably refers to peripheral insulin resistance and / or hepatic insulin resistance. Insulin resistance is preferably diagnosed by the absolute criterion of the "hyperinsulinemic euglycemic 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 the increased insulin concentration without causing hypoglycemia. This procedure can take 2 hours and preferably includes the following steps (or similar steps): Through the peripheral vein, insulin is injected at 10-120 mU per minute. To compensate for the insulin injection, glucose (20%) is injected to maintain the blood glucose level between 5 mmol / L and 5.5 mmol / L. The rate of glucose injection is determined by checking the blood glucose level every 5-10 minutes (Muniyappa R, Lee S, Chen H, Quon MJ, January 2008, American Journal of Physiology. Endocrinology and Metabolism. 294 (1): E15-26). The rate of glucose injection during the last 30 minutes of the test determines insulin sensitivity. If a high value (7.5 mg / min or more) is required, the patient is insulin sensitive. A low value (4.0 mg / min or less) indicates insulin resistance. A value between 4.0 mg / min and 7.5 mg / min is inconclusive and suggests "impaired glucose tolerance", an early sign of insulin resistance. 2 Inject at 10-120 mU per minute. To compensate for the insulin injection, glucose (20%) is injected to maintain the blood glucose level between 5 mmol / L and 5.5 mmol / L. The rate of glucose injection is determined by checking the blood glucose level every 5-10 minutes (Muniyappa R, Lee S, Chen H, Quon MJ, January 2008, American Journal of Physiology. Endocrinology and Metabolism. 294 (1): E15-26). The rate of glucose injection during the last 30 minutes of the test determines insulin sensitivity. If a high value (7.5 mg / min or more) is required, the patient is insulin sensitive. A low value (4.0 mg / min or less) indicates insulin resistance. A value between 4.0 mg / min and 7.5 mg / min is inconclusive and suggests "impaired glucose tolerance", an early sign of insulin resistance.

[0049] The homeostatic model assessment (HOMA or HOMA-IR) for insulin resistance is an alternative and preferred method for determining and quantifying insulin resistance under fasting steady-state conditions and correlates with an absolute reference (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 A. S. Rudenski; D. R. Matthews; J. C. Levy; R. C. Turner (1991) Metabolism. 40 (9): 908-917). HOMA(-IR) scores outside the reference range may indicate insulin resistance. HOMA(-IR) has been widely applied in epidemiological investigations and experimental studies. HOMA(-IR) represents a value that estimates insulin resistance, which is derived from dividing the insulin concentration and glucose concentration in a person's blood. The HOMA(-IR) value can be calculated by the following equation.

[0050]

Number

[0051] Other methods may also be suitable for measuring insulin resistance. In certain embodiments, insulin sensitivity or resistance is measured using an "insulin-suppression test" (IST). In certain embodiments, insulin sensitivity or resistance is measured using a "minimal model analysis of frequently sampled intravenous glucose tolerance test" (FSIVGTT). In certain embodiments, insulin sensitivity or resistance is measured using an "oral glucose tolerance test" (OGTT).

[0052] In a preferred embodiment, the "risk factor for hyperglycemia" disclosed in the present disclosure is metabolic syndrome. As used in the present disclosure, the term "metabolic syndrome" encompasses all conditions diagnosed as "metabolic syndrome" by a professional physician. In one embodiment, a subject can be diagnosed with metabolic syndrome if the subject has at least two, or at least three, of the following traits: · A waistline that is 35 inches (89 centimeters) or more for women and 40 inches (102 centimeters) or more for men; · High triglyceride concentration, where this type of fat is found in the blood at 150 milligrams per deciliter (150 mg / dL) or more, or 1.7 millimoles per liter (1.7 mmol / L) or more; · Reduced "good" or HDL cholesterol, where the high-density lipoprotein (HDL) cholesterol is less than 40 mg / dL (1.04 mmol / L) in men or less than 50 mg / dL (1.3 mmol / L) in women; · Increased blood pressure, 130 / 85 millimeters of mercury (mm Hg) or more; · Elevated fasting blood glucose, 100 mg / dL (5.6 mmol / L) or more

[0053] In a preferred embodiment, the risk factor for hyperglycemia disclosed in the present disclosure is type 2 diabetes. The term "type 2 diabetes" can be used interchangeably with "type 2 diabetes mellitus" in the present disclosure. In the context of the present invention, a patient with type 2 diabetes mellitus is preferably characterized by high blood glucose, insulin resistance, and a relative deficiency of insulin.

[0054] In a preferred embodiment, the risk factor for hyperglycemia disclosed in the present disclosure is obesity. In a preferred embodiment, the risk factor for hyperglycemia disclosed in the present disclosure is being overweight.

[0055] As used herein, the term "obesity" means a medical condition in which excessive body fat has accumulated to an extent that it has an adverse effect on health. In some cases, obesity is merely a cosmetic concern (requiring non-therapeutic treatment). A professional physician can determine whether a person is obese by one or more means. Generally, a body mass index (BMI) of 30.0 or higher is considered to indicate the presence of obesity. In the present disclosure, a BMI of 25.0 or higher but less than 30 indicates that a person is overweight. As used in the present disclosure, "overweight" refers to a non-medical condition characterized by excessive accumulation of body fat.

[0056] The inventors of the present application have found that the hydrolyzed collagen of the present invention may have one or more of the following actions as a mechanism of action in, for example, reducing blood glucose and / or improving hyperglycemia: · Increasing the concentration (level) of one or more incretins in the body, which is, for example, by increasing the secretion of one or more incretins by gastrointestinal cells; · Decreasing the concentration (level) of one or more incretin inhibitors (e.g., DPP-IV) in the body, which is, for example, by decreasing the secretion of one or more incretin inhibitors by gastrointestinal cells; · Increasing blood GLP-1, which is, for example, by increasing the secretion of GLP-1 by gastrointestinal cells; · Decreasing blood glucagon, which is, for example, by decreasing the secretion of glucagon by pancreatic (alpha) cells; · Increasing blood insulin, which is, for example, by increasing the secretion of insulin by pancreatic (beta) cells; · Decreasing the absorption of glucose in the small intestine; and · Increasing the blood insulin / glucose ratio.

[0057] In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure is · Improving blood GLP-1, which preferably means increasing blood GLP-1; · Improving blood glucagon, which preferably means decreasing blood glucagon; · Improving blood insulin, which preferably means increasing blood insulin; · Improving the blood insulin / glucose ratio, which preferably means increasing the blood insulin / glucose ratio; · Improving the concentration (level) of DPP-IV in the body, which preferably means decreasing the concentration of DPP-IV in the body, which is achieved, for example, by decreasing the secretion of DPP-IV by gastrointestinal cells; · Improving the concentration (level) of one or more incretins in the body, which preferably means increasing the concentration of said one or more incretins in the body, which is achieved, for example, by increasing the secretion of said one or more incretins by gastrointestinal cells; · Improving glucose absorption in the small intestine, which preferably means decreasing glucose absorption by the small intestine, It is for (further) use in one or more selected from the group consisting of.

[0058] The inventor of the present application has found that the hydrolyzed collagen of the present invention has a particularly high ability to stimulate GLP-1 secretion and / or lower blood glucose. This biological activity appears to be related to the specific molecular weight and / or molecular weight distribution of the hydrolyzed collagen that can be obtained by hydrolysis using the specific combination of enzymes.

[0059] In a preferred embodiment, the hydrolyzed collagen is capable of stimulating GLP-1 secretion. The ability to stimulate GLP-1 secretion is preferably present in STC-1 cells. The ability to stimulate GLP-1 secretion is preferably demonstrated using the following protocol: · Hydrolyze gelatin to obtain a hydrolyzed collagen preparation; · Perform simulated gastrointestinal digestion (SGID) on the hydrolyzed collagen preparation to obtain digested hydrolyzed collagen, which is preferably carried out according to the protocol described in Song et al. (Food Funct. 2020 Jun 24;11(6):5553-5564); · Stimulate cells (e.g., STC-1 cells) using the digested hydrolyzed collagen or using a blank as a reference, which is preferably carried out according to the protocol described in Qi et al. (Bio Protoc. 2020 Aug 20; 10(16): e3717). The cells are preferably stimulated at a concentration within the range of 1 to 20 mg / ml, preferably at a concentration of 10 mg / ml. · Recover the supernatant 1 to 3 hours after the start of stimulation and determine the GLP-1 level in the supernatant. This is carried out, for example, by enzyme immunoassay. A particularly preferred protocol is the protocol described for the stimulation of STC-1 cells in Example 1 of the present disclosure.

[0060] The ability to stimulate GLP-1 secretion is preferably defined by at least a 100-fold increase, preferably at least a 500-fold increase, more preferably at least a 1000-fold increase, even more preferably at least a 2000-fold increase, and most preferably at least a 5000-fold increase in the GLP-1 level after stimulation, which is preferably an increase relative to stimulation using a blank reference group (control group).

[0061] ● Hydrolyzed collagen As used herein, "hydrolyzed collagen" is a mixture of short chains of amino acid residues derived from a collagen-containing starting material having untreated (native) (full-length) collagen, generally by a hydrolysis step, which may include enzymatic hydrolysis (also called enzymatic hydrolyzation). The degree of hydrolysis usually affects the average molecular weight of the final product. Hydrolyzed collagen typically has a molecular weight of 1 to 10 kDa. The hydrolyzed collagen taught by the present invention may contain hydrolyzed or partially hydrolyzed collagen. The term "collagen hydrolyzate" can be used interchangeably with the term "hydrolyzed collagen" or "collagen peptide" and is synonymous with them.

[0062] Hydrolyzed collagen may be produced from a collagen-containing starting material (e.g., animal connective tissue) in a one-step process or via an intermediate gelatin stage, where type A and / or type B gelatin can be used. When used in the present disclosure, hydrolyzed collagen may thus refer to hydrolyzed gelatin obtained by hydrolysis of gelatin obtained from collagen. The terms "hydrolyzed collagen" and "hydrolyzed gelatin" can be used interchangeably in the present disclosure. Hydrolyzed gelatin is preferably obtained by enzymatic or chemical hydrolysis of gelatin. Hydrolyzed gelatin may be obtained from type A gelatin, type B gelatin, or a mixture thereof. The term "hydrolyzed collagen" can be used interchangeably with the term "hydrolyzed gelatin" and is synonymous with them. In a preferred embodiment, the collagen-containing material is gelatin. In the present disclosure, the collagen may be derived from one or more selected from the group consisting of type I collagen, type II collagen, type III collagen, type V collagen, and type X collagen. Preferably, the collagen disclosed in the present disclosure is derived from one or more types of collagen selected from the group consisting of type I collagen, type II collagen, and type III collagen. In addition to or instead of this, the hydrolyzed collagen disclosed in the present disclosure is preferably derived from an animal raw material containing various collagen types, and the various collagen types are, for example, two or more of type I collagen, type II collagen, and type III collagen. In addition to or instead of this, the hydrolyzed collagen disclosed in the present disclosure may be a mixture containing two or more of type I collagen, type II collagen, and type III collagen.

[0063] The hydrolyzed collagen disclosed in the present disclosure is preferably derived from type I collagen and / or type II collagen. The collagen disclosed in the present disclosure may be derived from any one or more animals or animal species, such as bovine species, pig species, and fish species. In the present disclosure, the term "animal" may refer to any animal capable of providing connective tissue that can be used to prepare hydrolyzed collagen.

[0064] In certain embodiments, the collagen taught by the present disclosure is derived from cows. In certain embodiments, the collagen taught by the present disclosure is derived from pigs. In certain embodiments, the collagen taught by the present disclosure is derived from fish.

[0065] In various embodiments, the collagen disclosed in the present disclosure is a mixture of collagens from various sources, for example, collagens derived from multiple animal species and / or collagens derived from different tissues. For example, the collagen disclosed in the present disclosure is a mixture of two or more collagens selected from the group consisting of fish collagen, pig collagen, and bovine collagen. In addition to or instead of this, the collagen may be a mixture of collagens selected from collagens derived from skin, cartilage, bone, and / or connective tissue. As used in the present disclosure, the term "hide" means the outer covering of a large animal such as a bovine group or any other large animal. As used in the present disclosure, the term "skin" means the outer covering of a small animal such as a deer, goat, sheep, etc. The terms "skin" and "hide" can be used interchangeably in the present disclosure and can refer to the outer covering of an animal regardless of size.

[0066] The "connective tissue" disclosed in the present disclosure may be one or more types selected from the group consisting of the corpus callosum, skin, antler, protrusions (e.g., warts), horn, head, brain, neck, ear, eye, nose, tongue, lip, mouth, esophagus, trachea, limbs, feet, toes, palm, claws, bone, cartilage, bone marrow, joints, membranes, hind, ligaments, tendons, rib bones, diaphragm, muscles, skeletal muscles, smooth muscles, intestines, blood vessels, bladder, stomach, aorta, heart, liver, kidneys, chest, lungs, spleen, pancreas, eggs, sperm, testes, ovaries, nerves, gallbladder, and connective tissue derived from the belly.

[0067] In certain embodiments, the collagen taught by the present disclosure is derived from skin and / or skin connective tissue. In certain preferred embodiments, the collagen taught by the present disclosure is derived from cartilage. In certain preferred embodiments, the collagen taught by the present disclosure is derived from bone. The collagen taught by the present disclosure may be a mixture of two or more collagens selected from the group consisting of skin collagen, cartilage collagen, and bone collagen.

[0068] In certain preferred embodiments, the collagen disclosed in the present disclosure is derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendons, ligaments, and connective tissue. In certain preferred embodiments, the collagen disclosed in the present disclosure is one or more types selected from the group consisting of porcine collagen, bovine collagen, and fish collagen.

[0069] ● Enzymatic hydrolysis In certain embodiments, the present invention a) preparing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes, preferably a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, relates to a method for obtaining hydrolyzed collagen.

[0070] In a preferred embodiment, the present invention relates to a) providing a collagen-containing material in a liquid preparation; and b) subjecting the collagen-containing material to a combination of enzymes comprising neutral protease, carboxypeptidase, and aminopeptidase to obtain a hydrolyzed collagen.

[0071] Preferably, step b) of the method disclosed in the present disclosure is an enzymatic hydrolysis step or includes an enzymatic hydrolysis step.

[0072] As used in the present disclosure, a "liquid preparation" is a preparation in which the collagen-containing material is at least partially soluble. In addition or alternatively, a "liquid preparation" is a preparation in which the collagen-containing material remains at least partially soluble after being subjected to a dissolution step. The liquid preparation disclosed in the present disclosure is preferably an aqueous preparation. In a preferred embodiment, the liquid preparation disclosed in the present disclosure is water or contains water, more preferably, the liquid preparation is distilled water and / or demineralized water, or contains distilled water and / or demineralized water.

[0073] As part of the method disclosed in the present disclosure, the collagen-containing material may be provided in the liquid preparation in an amount of 10 wt.% (weight percent) or more, 15 wt.% or more, 20 wt.% or more, 25 wt.% or more, 30 wt.% or more, 35 wt.% or more, 40 wt.% or more, 45 wt.% or more, 50 wt.% or more, 55 wt.% or more, 60 wt.% or more, or 65 wt.% or more. In addition or alternatively, the collagen-containing material may be provided in the liquid preparation in an amount of 60 wt.% or less, 55 wt.% or less, 50 wt.% or less, 45 wt.% or less, 40 wt.% or less, 35 wt.% or less, 30 wt.% or less, 25 wt.% or less, 20 wt.% or less, 15 wt.% or less, or 10 wt.% or less.

[0074] In certain embodiments, the collagen-containing material is provided in an amount of 20 to 50% by weight, preferably in an amount of 30 to 40% by weight, in the liquid preparation disclosed in the present disclosure.

[0075] In certain embodiments, the combination of enzymes disclosed in the present disclosure is a mixture of said enzymes. In certain embodiments, the combination of enzymes disclosed in the present disclosure comprises one or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase.

[0076] In certain embodiments, the combination of enzymes disclosed in the present disclosure comprises two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase. In certain embodiments, the combination of enzymes disclosed in the present disclosure comprises neutral protease, carboxypeptidase, and aminopeptidase. In certain embodiments, the combination of enzymes disclosed in the present disclosure comprises neutral protease and carboxypeptidase. In certain embodiments, the combination of enzymes disclosed in the present disclosure comprises neutral protease and aminopeptidase. In certain embodiments, the combination of enzymes disclosed in the present disclosure comprises carboxypeptidase and aminopeptidase.

[0077] In certain preferred embodiments, the combination of enzymes disclosed in the present disclosure comprises neutral protease and one or both of carboxypeptidase and aminopeptidase.

[0078] As used in the present disclosure, the term "neutral protease" refers to a class of proteases that function to catalyze in a neutral, weakly acidic, or weakly alkaline environment and catalyze the hydrolysis of peptide bonds in proteins. In the present disclosure, the optimal pH for neutral protease in enzymatic hydrolysis is considered to be 6.0 to 7.5. Neutral proteases can be divided into four main categories: serine proteases, aspartic proteases, cysteine proteases, and metalloproteases, based on their catalytic mechanism and the functional groups at the active site.

[0079] As used in the present disclosure, "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. The serine protease disclosed in the present disclosure may be a chymotrypsin-like (trypsin-like) serine protease. In addition to or instead of this, the serine protease disclosed in the present disclosure may be a subtilisin-like serine protease.

[0080] As used in the present disclosure, "aspartic protease" refers to a catalytic type of protease enzyme that uses activated water molecules bound to one or more aspartic acid residues for the catalysis of proteins or peptides.

[0081] As used in the present disclosure, "cysteine protease" refers to a protease having a catalytic mechanism that involves a nucleophilic cysteine thiol in the catalytic triad or dyad for the catalysis of proteins or peptides. "Cysteine protease" can be used interchangeably with "thiol protease" in the present disclosure. As used in the present disclosure, "metalloprotease" refers to a protease in which a metal such as zinc or cobalt is involved in its catalytic mechanism. In the present disclosure, the metalloprotease is preferably a zinc-dependent protease.

[0082] As used in the present disclosure, the term "carboxypeptidase" means a protease enzyme that hydrolyzes peptide bonds at the carboxyl terminus (C-terminus) of a protein or peptide. Carboxypeptidases are typically classified into one of several families based on the mechanism of their active sites. In certain embodiments, the carboxypeptidases in the present disclosure are metallocarboxypeptidases, i.e., carboxypeptidases that use a metal in the active site for the catalytic mechanism.

[0083] As used in the present disclosure, the term "aminopeptidase" means a protease enzyme that hydrolyzes peptide bonds at the N-terminus of a protein or peptide. In the present disclosure, the aminopeptidase is preferably leucine aminopeptidase. As used in the present disclosure, the term "leucine aminopeptidase" means an aminopeptidase that preferentially catalyzes the hydrolysis of leucine residues at the N-terminus of a peptide or protein.

[0084] In certain preferred embodiments, the neutral protease disclosed in the present disclosure is a serine protease. In certain preferred embodiments, the neutral protease disclosed in the present disclosure is an aspartic protease. In certain preferred embodiments, the neutral protease disclosed in the present disclosure is a cysteine protease. In certain preferred embodiments, the neutral protease disclosed in the present disclosure is a metalloprotease. In certain preferred embodiments, the aminopeptidase disclosed in the present disclosure is leucine aminopeptidase.

[0085] The "enzyme" in the context of the present invention may be a commercially available enzyme. In certain embodiments, the combination of enzymes disclosed in the present disclosure is a commercially available mixture of enzymes. In certain embodiments, the combination of enzymes disclosed in the present disclosure is commercially available as "Sumizyme", preferably "Sumizyme FP-G".

[0086] In certain embodiments, the enzymes disclosed in the present disclosure (e.g., one or more enzymes in an enzyme combination) are derived from microorganisms. In certain embodiments, the enzymes disclosed in the present disclosure (e.g., one or more enzymes in an enzyme combination) are derived from microorganisms of the genus Aspergillus, preferably Aspergillus oryzae and / or Aspergillus melleus. In certain embodiments, the enzymes disclosed in the present disclosure (e.g., one or more enzymes in an enzyme combination) are derived from microorganisms of the genus Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus amylolquefaciens).

[0087] In certain embodiments, an enzyme combination containing one or fewer enzymes derived from Bacillus sp. is used, thereby eliminating blends of endoproteases derived from Bacillus sp., such as commercially available Protamex. In certain embodiments, the enzymes disclosed in the present disclosure (e.g., one or more enzymes in an enzyme combination) are derived from lactic acid bacteria, preferably from one or more genera selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella.

[0088] In one embodiment, one or more of neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably from Aspergillus oryzae. In a preferred embodiment, neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably from Aspergillus oryzae.

[0089] The protease used in the present invention may be a mixture of enzymes (for example, a commercially available mixture obtainable from Sumizyme containing enzymes (plural) derived from Aspergillus oryzae), and the protease(s) may be derived from separate sources (for example, different microorganisms or different strains thereof). For example, the neutral protease may be derived from the genus Bacillus, the carboxypeptidase may be derived from the genus Aspergillus, and the aminopeptidase may be derived from the genus Lactobacillus.

[0090] In one embodiment, the enzyme(s) (for example, one or more enzymes in the enzyme combination) disclosed in the present disclosure are not those derived from plants (for example, bromelain, papain).

[0091] In a preferred embodiment, in enzymatic hydrolysis, preferably in step b) of the method disclosed in the present disclosure, the amount of neutral protease used is defined by an enzyme activity of 0.2 to 25000 U / g, preferably 2 to 2500 U / g, more preferably 20 to 250 U / g, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation.

[0092] In a preferred embodiment, in enzymatic hydrolysis, preferably in step b) of the method disclosed in the present disclosure, the amount of carboxypeptidase used is 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation.

[0093] In a preferred embodiment, in enzymatic hydrolysis, preferably in step b) of the method disclosed in the present disclosure, the amount of aminopeptidase used is 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation.

[0094] The time for which the collagen-containing material is exposed to the enzyme disclosed in the present disclosure, preferably one or more of the combinations of enzymes disclosed in the present disclosure (e.g., exposed in step b of the method), may be 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, 90 minutes or more, 100 minutes or more, 110 minutes or more, 120 minutes or more, 130 minutes or more, 140 minutes or more, 150 minutes or more, 160 minutes or more, 170 minutes or more, 180 minutes or more, 190 minutes or more, 200 minutes or more, 210 minutes or more, 220 minutes or more, 230 minutes or more, 240 minutes or more, 300 minutes or more, or 360 minutes or more. In addition to or instead of this, the time for which the collagen-containing material is exposed to the enzyme disclosed in the present disclosure, preferably one or more of the combinations of enzymes disclosed in the present disclosure (e.g., exposed in step b of the method), may be 360 minutes or less, 300 minutes or less, 240 minutes or less, 230 minutes or less, 220 minutes or less, 210 minutes or less, 200 minutes or less, 190 minutes or less, 180 minutes or less, 170 minutes or less, 160 minutes or less, 150 minutes or less, 140 minutes or less, 130 minutes or less, 120 minutes or less, 110 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, or 10 minutes or less.

[0095] In a preferred embodiment, the collagen-containing material is exposed to the enzyme disclosed in the present disclosure, preferably one or more of the combinations of enzymes disclosed in the present disclosure, (e.g., in step b of the method) for 60 to 180 minutes.

[0096] The temperature at which the collagen-containing material is exposed to the enzymes disclosed in the present disclosure, preferably to one or more of the combinations of enzymes disclosed in the present disclosure (e.g., exposed in step b of the method), may be 15 °C or higher, 20 °C or higher, 25 °C or higher, 30 °C or higher, 35 °C or higher, 40 °C or higher, 45 °C or higher, 50 °C or higher, 55 °C or higher, 60 °C or higher, 65 °C or higher, 70 °C or higher, 75 °C or higher, or 80 °C or higher. In addition to or instead of this, the temperature at which the collagen-containing material is exposed to the enzymes disclosed in the present disclosure, preferably to one or more of the combinations of enzymes disclosed in the present disclosure (e.g., exposed in step b of the method), may be 80 °C or lower, 75 °C or lower, 70 °C or lower, 65 °C or lower, 60 °C or lower, 55 °C or lower, 50 °C or lower, 45 °C or lower, 40 °C or lower, 35 °C or lower, 30 °C or lower, 25 °C or lower, 20 °C or lower, or 15 °C or lower.

[0097] When used in the present disclosure, the term "temperature" preferably means the temperature of a liquid preparation in which the collagen-containing material is contained, preferably in which the collagen-containing material is dissolved.

[0098] In a preferred embodiment, the collagen-containing material is exposed to the enzymes disclosed in the present disclosure, preferably to one or more of the combinations of enzymes disclosed in the present disclosure, (e.g., in step b of the method) at a temperature of 30 °C to 60 °C.

[0099] The use of the term "combination of enzymes" disclosed in the present disclosure does not exclude the collagen-containing material being exposed to the enzymes (part of the combination of enzymes) sequentially. This means that the collagen-containing material is not necessarily exposed to all of the enzymes in the combination of enzymes simultaneously. In certain embodiments, the enzymatic hydrolysis disclosed in the present disclosure (for example, the hydrolysis in step b of the method) includes two or more hydrolysis steps that are sequential, in which the collagen-containing material is preferably exposed to different enzymes and / or different enzyme mixtures. In certain preferred embodiments, the collagen-containing material is sequentially exposed to two enzymes in the combination of enzymes, preferably sequentially exposed to two selected from neutral protease, carboxypeptidase, and aminopeptidase. In certain embodiments, the enzymatic hydrolysis, preferably the enzymatic hydrolysis in step b of the method disclosed in the present disclosure, includes two or more hydrolysis steps, preferably two hydrolysis steps, where · the collagen-containing material is exposed to neutral protease in one step and to carboxypeptidase and / or aminopeptidase in another step; · the collagen-containing material is exposed to carboxypeptidase in one step and to neutral protease and / or aminopeptidase in another step; · the collagen-containing material is exposed to aminopeptidase in one step and to neutral protease and / or carboxypeptidase in another step. In certain embodiments, the enzymatic hydrolysis disclosed in the present disclosure includes three hydrolysis steps, in which the collagen-containing material is preferably exposed to neutral protease, carboxypeptidase, and aminopeptidase independently in each of the three hydrolysis steps.

[0100] In a preferred embodiment, the collagen-containing material is exposed to all of the enzymes (in the combination of enzymes disclosed in the present disclosure) simultaneously. In a preferred embodiment, the collagen-containing material is exposed to a mixture containing two or more of neutral protease, carboxypeptidase, and aminopeptidase, and the mixture preferably contains all three of neutral protease, carboxypeptidase, and aminopeptidase. Here, the collagen-containing material is more preferably exposed to the mixture at 30-60 °C and pH 5-8 for 60-180 minutes.

[0101] If there are two or more hydrolysis steps, the "time" during which the hydrolyzed collagen is exposed to the enzyme preferably means the total time. For example, if the hydrolyzed collagen is exposed to enzyme A for 60 minutes in the first hydrolysis step and then exposed to enzyme A for 60 minutes in the second hydrolysis step, the hydrolyzed collagen is considered to be exposed to enzyme A for 120 minutes.

[0102] ● Products obtainable by enzymatic hydrolysis In one aspect, the present invention relates to hydrolyzed collagen obtainable by the method disclosed in the present disclosure. In the present disclosure, "hydrolyzed collagen" preferably means hydrolyzed collagen obtainable by the method disclosed in the present disclosure. In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure, for example, collagen obtainable by the method taught in the present disclosure, is derived from a collagen-containing material derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendon, ligament, and connective tissue. In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure, for example, collagen obtainable by the method taught in the present disclosure, is derived from a collagen-containing material in which the collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen.

[0103] In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure, for example, the collagen obtainable by the method taught in the present disclosure, is derived from a collagen-containing material that is gelatin.

[0104] The hydrolyzed collagen disclosed in the present disclosure, for example, the collagen obtainable by the method taught in the present disclosure, preferably has a (weight average) molecular weight of 1000 to 7000 Da. This molecular weight appears to play a role in the high glucose-lowering effect of the hydrolyzed collagen of the present invention.

[0105] The hydrolyzed collagen disclosed in the present disclosure, for example, the collagen obtainable by the method taught in the present disclosure, may have a (weight average) molecular weight of 1000 Da or more (for example, 1200 Da or more, 1400 Da or more, 1600 Da or more, or 1800 Da or more), or 1500 Da or more, or 2000 Da or more, or 2500 Da or more, or 3000 Da or more, or 3500 Da or more, or 4000 Da or more, or 4500 Da or more, or 5000 Da or more, or 6000 Da or more, or 6500 Da or more, or 7000 Da or more. In addition to or instead of this, the hydrolyzed collagen, for example, the collagen obtainable by the method taught in the present disclosure, may have a (weight average) molecular weight of 7000 Da or less, or 6500 Da or less, or 6000 Da or less, or 5500 Da or less, or 5000 Da or less, or 4500 Da or less, or 4000 Da or less, or 3500 Da or less, or 3000 Da or less, or 2500 Da or less, or 2000 Da or less, or 1500 Da or less, or 1000 Da or less.

[0106] The average molecular weight disclosed in the present disclosure is preferably the weight average molecular weight. In the ability of the hydrolyzed collagen of the present invention to stimulate GLP-1 secretion and / or reduce blood glucose, the molecular weight appears to play a role. 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.

[0107] In one embodiment, the hydrolyzed collagen has a number average molecular weight (Mn) of 500 to 3,500 Da, preferably 1,000 to 3,000 Da, more preferably 1,500 to 2,500 Da, and even more preferably 1,750 to 2,250 Da. 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.

[0108] In addition to or instead of this, in the ability of the hydrolyzed collagen of the present invention to stimulate GLP-1 secretion and / or reduce blood glucose, the molecular weight distribution appears to play a role. Compared to other hydrolyzed collagens - for example, hydrolyzed collagens obtained by enzymatic hydrolysis using a combination of two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase - the hydrolyzed collagen of the present invention, in particular, · A relatively high proportion of collagen peptides in the range of 2,000 to 5,000 Da (for example, 35% or more, 40% or more, or even 45% or more); and / or · A relatively low proportion of collagen peptides in the range of less than 1,000 Da (for example, 15% or less, 12% or less, or even 10% or less); and / or · A relatively low proportion of collagen peptides in the range of more than 10,000 Da (for example, 5% or less, 2% or less, or even 1% or less) may have.

[0109] In a preferred embodiment, the hydrolyzed collagen contains 1 to 20% by weight, preferably 2 to 12% by weight, of collagen peptides having a molecular weight of less than 1000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen. In a preferred embodiment, the hydrolyzed collagen contains 20 to 40% by weight, preferably 25 to 35% by weight, of collagen peptides having a molecular weight in the range of 1000 Da to 2000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen. 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 having a molecular weight in the range of 1000 Da to 2000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen.

[0110] In a preferred embodiment, the hydrolyzed collagen contains 35 to 60% by weight, preferably 40 to 55% by weight, of collagen peptides having a molecular weight in the range of 2000 Da to 5000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen. 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 having a molecular weight in the range of 2000 Da to 5000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen.

[0111] In a preferred embodiment, the hydrolyzed collagen contains 5 to 25% by weight, preferably 10 to 20% by weight, of collagen peptides having a molecular weight in the range of 5000 Da to 10000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen.

[0112] In a preferred embodiment, the hydrolyzed collagen contains, in terms of calculation based on the total weight of collagen peptides in the hydrolyzed collagen, 0 to 10% by weight, preferably 0.1 to 5% by weight, more preferably 0.2 to 2% by weight of collagen peptides having a molecular weight exceeding 10,000 Da. A preferred method for measuring the molecular weight and molecular weight distribution of hydrolyzed collagen is by high performance size extrusion chromatography (HPSEC). The following protocol is a preferred HPSEC protocol.

[0113] Use an Agilent HPLC 1260 Infinity series (G1316A, G1329B, G1311C, G1315D) with a TSKgel SWXL precolumn and a G2000SWXL column (Tosoh Bioscience). The analysis is performed using WinGPC software (PSS). The eluent is 100 mM phosphate buffer pH 5.3. Elute the sample from the column (e.g., at 0.5 mL / min, isocratic) and monitor by UV detection (e.g., at 214 nm, analysis time: 40 minutes per injection + 180 minutes for equilibration). Calibration is performed with a Narrow Calibration Standard (Low FILK).

[0114] The hydrolyzed collagen, for example, the collagen obtainable by the method taught in the present disclosure, preferably has a polydispersity of 1.2 to 1.8, more preferably a polydispersity of 1.3 to 1.7, and even more preferably a polydispersity of 1.4 to 1.6. This polydispersity appears to play a role in the high glucose-reducing effect of the hydrolyzed collagen of the present invention.

[0115] The polydispersity of the hydrolyzed collagen may be 1.85 or less, or 1.75 or less, or 1.70 or less, or 1.65 or less, or 1.60 or less, or 1.55 or less. In addition to or instead of this, the polydispersity of the hydrolyzed collagen may be 1.20 or more, or 1.25 or more, or 1.30 or more, or 1.35 or more, or 1.40 or more, or 1.45 or more, or 1.50 or more, or 1.55 or more.

[0116] When used in the present disclosure, "polydispersity" means Mw / Mn, where Mw is the weight average molecular weight (Da) and Mn is the number average molecular weight (Da). Mw is given by the following equation.

Number

Number

[0117] ● Administration of hydrolyzed collagen In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure is administered orally. To improve postprandial glucose levels, the hydrolyzed collagen disclosed in the present disclosure is preferably administered during or around a meal. In the context of the present invention, the hydrolyzed collagen is preferably administered within 240 minutes (before or after), within 180 minutes (before or after), within 120 minutes (before or after), within 60 minutes (before or after), within 30 minutes (before or after), or within 15 minutes (before or after) of a meal. In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure is administered within 60 minutes (before or after) of a meal. ​

[0118] In another embodiment, the daily dosage of the hydrolyzed collagen disclosed in the present disclosure is 1 g or more, 5 g or more, 10 g or more, 15 g or more, 20 g or more, 25 g or more, 30 g or more, 35 g or more, 40 g or more, 45 g or more, 50 g or more, 55 g or more, 60 g or more, 65 g or more, 70 g or more, 75 g or more, 80 g or more, 85 g or more, 90 g or more, 95 g or more, 100 g or more, 110 g or more, 120 g or more, 130 g or more, 140 g or more, 150 g or more, 160 g or more, 170 g or more, 180 g or more, 190 g or more, or 200 g or more, where the daily dosage is the amount by the total dry weight administered to the subject per day. In addition to or instead of this, in another embodiment, the daily dosage of the hydrolyzed collagen disclosed in the present disclosure is 200 g or less, 190 g or less, 180 g or less, 170 g or less, 160 g or less, 150 g or less, 140 g or less, 130 g or less, 120 g or less, 110 g or less, 100 g or less, 95 g or less, 90 g or less, 85 g or less, 80 g or less, 75 g or less, 70 g or less, 65 g or less, 60 g or less, 55 g or less, 50 g or less, 45 g or less, 40 g or less, 35 g or less, 30 g or less, 25 g or less, 20 g or less, 15 g or less, 10 g or less, 5 g or less, or 1 g or less, where the daily dosage is the amount by the total dry weight administered to the subject per day.

[0119] In a preferred embodiment, the use of the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen in an amount of 1 g to 200 g, or 1 g to 100 g, or 2 g to 50 g, or 5 g to 25 g, where the daily dosage is the amount by the total dry weight administered to the subject per day. In a preferred embodiment, the use of the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen in a daily dosage in an amount of 1 g to 100 g, preferably 2 g to 50 g, more preferably 5 g to 25 g, where the amount is the amount in terms of the dry weight of the hydrolyzed collagen.

[0120] In another embodiment, the unit dose of the hydrolyzed collagen disclosed in the present disclosure is an amount of 1 g or more, 2 g or more, 3 g or more, 4 g or more, 5 g or more, 6 g or more, 7 g or more, 8 g or more, 9 g or more, 10 g or more, 11 g or more, 12 g or more, 13 g or more, 14 g or more, 15 g or more, 20 g or more, 25 g or more, 30 g or more, 35 g or more, 40 g or more, 45 g or more, 50 g or more, 55 g or more, 60 g or more, 65 g or more, 70 g or more, 75 g or more, 80 g or more, 85 g or more, 90 g or more, 95 g or more, 100 g or more, 110 g or more, 120 g or more, 130 g or more, 140 g or more, 150 g or more, 160 g or more, 170 g or more, 180 g or more, 190, or 200 g or more, where the amount is the amount in dry weight of the hydrolyzed collagen. In addition to or instead of this, in another embodiment, the unit dose of the hydrolyzed collagen disclosed in the present disclosure is an amount of 200 g or less, 190 g or less, 180 g or less, 170 g or less, 160 g or less, 150 g or less, 140 g or less, 130 g or less, 120 g or less, 110 g or less, 100 g or less, 95 g or less, 90 g or less, 85 g or less, 80 g or less, 75 g or less, 70 g or less, 65 g or less, 60 g or less, 55 g or less, 50 g or less, 45 g or less, 40 g or less, 35 g or less, 30 g or less, 25 g or less, 20 g or less, 15 g or less, 14 g or less, 13 g or less, 12 g or less, 11 g or less, 10 g or less, 9 g or less, 8 g or less, 7 g or less, 6 g or less, 5 g or less, 4 g or less, 3 g or less, 2 g or less, or 1 g or less, where the amount is the amount in dry weight of the hydrolyzed collagen.

[0121] In one embodiment, the hydrolyzed collagen disclosed in the present disclosure is administered in a unit dose of an amount of 0.5 g to 200 g, or 1 g to 100 g, or 2 g to 50 g, or 5 g to 25 g, or 5 g to 15 g, where the amount is the amount in dry weight of the hydrolyzed collagen.

[0122] In one embodiment, the use of the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen in a unit dose of an amount of 2 g to 50 g, preferably 5 g to 25 g, more preferably 5 g to 15 g, where the amount is the amount in dry weight of the hydrolyzed collagen.

[0123] The daily dose of hydrolyzed collagen may be administered as a single unit dose or as two, three, four, or more unit doses. The two or more unit doses may have the same or different amounts. The daily dose of hydrolyzed collagen disclosed in the present disclosure is preferably administered as two unit doses, more preferably as two unit doses each corresponding to 30-70%, preferably 40-60% of the amount of the daily dose.

[0124] In certain embodiments, two or more unit doses disclosed in the present disclosure are administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours apart from each other.

[0125] In a preferred embodiment, the dosing regimen for hydrolyzed collagen disclosed in the present disclosure includes administering the daily dose of hydrolyzed collagen as two unit doses, where: · Each unit dose is in an amount of 5-25 g, preferably 5-15 g, and said amount is the amount in the dry weight of hydrolyzed collagen; and / or · The two unit doses are administered at least 6 hours, preferably at least 8 hours, more preferably at least 12 hours apart from each other.

[0126] In certain embodiments, the daily dose of hydrolyzed collagen is administered as two or more unit doses, where: · Each unit dose is in an amount of 5-25 g, preferably 5-15 g, and said amount is the amount in the dry weight of hydrolyzed collagen; and / or · The two unit doses are administered at least 6 hours, preferably at least 8 hours, more preferably at least 12 hours apart from each other.

[0127] In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure is administered with food. In certain embodiments, the dosing regimen for the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen one or more times per week. In certain embodiments, the dosing regimen for the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen daily or every other day. In certain embodiments, the hydrolyzed collagen is administered daily or every other day. In certain embodiments, the dosing regimen for the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen one or more times per day. In certain embodiments, the hydrolyzed collagen is administered one or more times per day. In certain embodiments, the dosing regimen for the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen for two or more consecutive days, preferably four or more consecutive days, more preferably seven or more consecutive days. In certain embodiments, the dosing regimen for the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen for two or more consecutive weeks, preferably four or more consecutive weeks, more preferably eight or more consecutive weeks. In certain embodiments, the dosing regimen for the hydrolyzed collagen disclosed in the present disclosure includes administering the hydrolyzed collagen for one or more weeks, preferably two or more consecutive weeks, more preferably four or more consecutive weeks.

[0128] ● Preparation of hydrolyzed collagen The hydrolyzed collagen disclosed in the present disclosure may be provided in a food preparation, a diet preparation, a food supplement preparation, a diet supplement preparation, or a pharmaceutical preparation, preferably in a food supplement preparation. The hydrolyzed collagen disclosed in the present disclosure may be provided in a solid dosage form such as a capsule, a tablet, or a powder, preferably in the form of a powder. The hydrolyzed collagen disclosed in the present disclosure may be provided in preparations such as a drinkable solution or suspension, beverages such as beer, syrups, artificially flavored beverages, carbonated beverages, (water-soluble) powdered mixtures, (water-soluble) pastes, (water-soluble) powders, (water-soluble) tablets, (water-soluble) pills, (water-soluble) dragees, (water-soluble) caplets, (water-soluble) sachets, or (water-soluble) capsules. In addition to or in place of this, the hydrolyzed collagen taught in the present disclosure may be present in functional foods, such as juices, shakes, milk beverages, yogurts, yogurt beverages, desserts, energy bars, nutritional bars, slimming bars, or confectioneries such as gummies or center-filled gummies.

[0129] ▲Use of Hydrolyzed Collagen in Treatment Methods In certain embodiments, the present invention relates to a treatment method for reducing blood glucose, preferably a therapeutic treatment method. In certain embodiments, the present invention relates to a treatment method for improving hyperglycemia, preferably a therapeutic treatment method. In certain embodiments, the present invention relates to a treatment method for improving risk factors for hyperglycemia, preferably a therapeutic treatment method.

[0130] The treatment method disclosed in the present disclosure may be used in a subject in which high blood glucose (e.g., hyperglycemia) has occurred, where the treatment method may be prophylactic in nature. The treatment method may include one or more features of the embodiments disclosed in the present disclosure regarding the use of hydrolyzed collagen in reducing blood glucose and / or improving (risk factors for) hyperglycemia, including the type of hydrolyzed collagen for use disclosed in the present disclosure, the preparation of hydrolyzed collagen for use disclosed in the present disclosure, the timing of administration or ingestion of hydrolyzed collagen for use disclosed in the present disclosure, and the mode of administration of hydrolyzed collagen for use disclosed in the present disclosure (e.g., dosing regimen, unit dose, daily dose).

[0131] ▲Use of hydrolyzed collagen for the preparation of a medicament In certain embodiments, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for reducing blood glucose, wherein said hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, and comprising two or more enzymes. In certain embodiments, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for improving hyperglycemia, wherein said hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, and comprising two or more enzymes. In certain embodiments, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for improving risk factors for hyperglycemia, wherein said hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, and comprising two or more enzymes.

[0132] The use of hydrolyzed collagen for the manufacture of a medicament disclosed in the present disclosure may include one or more features of the embodiments disclosed in the present disclosure regarding the use of hydrolyzed collagen in reducing blood glucose and / or improving hyperglycemia (risk factors), including the type of hydrolyzed collagen for the use disclosed in the present disclosure, the preparation of hydrolyzed collagen for the use disclosed in the present disclosure, the timing of administration or ingestion of hydrolyzed collagen for the use disclosed in the present disclosure, and the mode of administration (e.g., dosing regimen, unit dose, daily dose) of administration or ingestion of hydrolyzed collagen for the use disclosed in the present disclosure.

[0133] ●General definitions · As used in this disclosure, the terms "comprising" or "include" and their conjugations are used in their non-limiting sense to mean that the terms include what is recited following this word, without excluding what is not particularly recited. This includes the more limiting verbs "consisting essentially of" and "consisting of only". · The recitation of an element by the indefinite article "a" or "an" does not exclude the possibility that there are two or more elements, unless the context clearly requires that there be only one element. The indefinite article "a" or "an" thus usually means "at least one".

[0134] · The terms "increase" and "increased level", and the terms "decrease" and "decreased level" (or "decrease" and "reduced level") preferably refer to a change of at least 5%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher or lower than the corresponding level in the subject or reference, respectively. In addition to or instead of this, the level in the sample may be said to be increasing or decreasing, regardless of the magnitude of the change, if it is statistically significantly increased or decreased compared to the level in the subject or reference. · The term "meal" as used in this disclosure refers to an eating occasion that occurs at a point in time and includes prepared food. Meals occur on a daily basis and typically occur several times a day. Breakfast, lunch, and dinner are considered the main meals. In addition to or instead of this, a meal may be considered to include a reasonable large amount of food (i.e., at least 500 - 1000 kcal per meal).

[0135] · The term "subject" or "individual" as used in this disclosure refers to any animal (e.g., a mammal), preferably including humans. · As used in the present disclosure, the term "administer" or "administration" relates to the act of giving an agent to a subject who ingests it. A subject who ingests an agent may administer the agent to himself / herself. In such a case, the term "administer to" is construed as "taken by", "used by", or "consumed by".

[0136] · As used in the present disclosure, the term "unit dose" relates to the amount or unit of, for example, a compound, substance, active ingredient, or composition used once. A unit dose may be, for example, a ready-to-administer prepared form (e.g., a packaged preparation). A unit dose may be, for example, identifiable from a product package or label. The total daily dose may be divided into a plurality of unit doses. · As used in the present disclosure, 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 only confers a physical function. The biological response may be a direct or indirect response, preferably measured in cells or tissues.

[0137] ● Clause In the present disclosure, a clause is an embodiment of the present invention. The features of clauses (embodiments) may be combined in the present disclosure. Clause 1: Hydrolyzed collagen for use in reducing blood glucose, obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase. Clause 2: The hydrolyzed collagen according to Clause 1, wherein the combination of enzymes comprises neutral protease, carboxypeptidase, and aminopeptidase. Clause 3: The hydrolyzed collagen according to claim 1 or 2 for use in improving hyperglycemia. Item 4: The hydrolyzed collagen according to claim 1 or 2 for use in improving risk factors for hyperglycemia. Item 5: The hydrolyzed collagen according to claim 4, wherein the risk factor for hyperglycemia is one or more selected from the group consisting of insulin resistance, type 2 diabetes, gestational diabetes, high body mass index, obesity, and hyperglucagonemia. Item 6: The hydrolyzed collagen according to any one of the preceding items for use in improving postprandial glucose. Item 7: · Increasing glucagon-like peptide-1 in the blood; · Decreasing glucagon in the blood; · Increasing insulin in the blood; · Increasing the blood insulin / glucose ratio The hydrolyzed collagen according to any one of the preceding items for use in one or more selected from the group consisting of.

[0138] Item 8: The hydrolyzed collagen according to any one of the preceding items, wherein the hydrolyzed collagen is administered in a daily dose of 1 to 100 grams, preferably 2 to 50 g, more preferably 5 to 25 g, and the amount is based on dry weight. Item 9: The hydrolyzed collagen according to any one of the preceding items, wherein the hydrolyzed collagen is administered orally, preferably as a food supplement. Item 10: The hydrolyzed collagen according to any one of the preceding items, wherein the hydrolyzed collagen is administered within 60 minutes of the time difference from a meal. Item 11: The hydrolyzed collagen according to any one of the preceding paragraphs, wherein the collagen-containing material is derived from one or more tissues selected from the group consisting of skin, scale, cartilage, bone, tendon, ligament, and connective tissue. Item 12: The hydrolyzed collagen according to any one of the preceding paragraphs, wherein the collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen. Item 13: The hydrolyzed collagen according to any one of the preceding paragraphs, wherein the collagen-containing material is gelatin. Item 14: The hydrolyzed collagen according to any one of the preceding paragraphs, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity of 1.2 to 1.8. Item 15: a) preparing a collagen-containing material in a liquid preparation; and b) subjecting the collagen-containing material to a combination of enzymes including two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, A method for obtaining hydrolyzed collagen, comprising: Item 16: The method according to item 15, wherein the combination of enzymes includes neutral protease, carboxypeptidase, and aminopeptidase. Item 17: The method according to item 15 or 16, wherein the neutral protease is one or more selected from the group consisting of serine protease, aspartic protease, cysteine protease, and metalloprotease. Item 18: The method according to any one of items 15 to 17, wherein the aminopeptidase is leucine aminopeptidase. Item 19: The method according to any one of items 15 to 18, wherein one or more of neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably Aspergillus oryzae. Item 20: The method according to any one of items 15 to 19, wherein the collagen-containing material is exposed to a neutral protease in an amount defined by an enzyme activity of 0.2 to 25000 U / g, preferably 2 to 2500 U / g, more preferably 20 to 250 U / g, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation.

[0139] Item 21: The method according to any one of items 15 to 20, wherein the collagen-containing material is exposed to a carboxypeptidase in an amount 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation. Item 22: The method according to any one of items 15 to 21, wherein the collagen-containing material is exposed to an aminopeptidase in an amount 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation. Item 23: The method according to any one of items 15 to 22, wherein the collagen-containing material is exposed to one or more enzymes in the combination of enzymes for 60 to 180 minutes. Item 24: The method according to any one of items 15 to 23, wherein the collagen-containing material is exposed to one or more enzymes in the combination of enzymes at 30°C to 60°C. Item 25: The method according to any one of items 15 to 24, wherein the collagen-containing material is exposed to one or more enzymes in the combination of enzymes at pH 5 to pH 8.

[0140] Item 26: The collagen-containing material is subjected to two or more enzymes in the enzyme combination sequentially, and the method according to any one of items 15 to 25. Item 27: The collagen-containing material is subjected to two or more enzymes in the enzyme combination simultaneously, and the method according to any one of items 15 to 26. Item 28: The collagen-containing material is subjected to a mixture containing the enzyme combination, and the method according to item 27. Item 29: The collagen-containing material is provided in an amount of 20 to 50% by weight, preferably 30 to 40% by weight, in a liquid preparation, and the method according to any one of items 15 to 28. Item 30: The collagen-containing material is derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendon, ligament, and connective tissue, and the method according to any one of items 15 to 29. Item 31: The collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen, and the method according to any one of items 15 to 30. Item 32: The collagen-containing material is gelatin, and the method according to any one of items 15 to 31. Item 33: Hydrolyzed collagen obtainable by the method according to any one of items 15 to 32. Item 34: The hydrolyzed collagen having an average molecular weight of 1000 to 7000 Da and / or a polydispersity of 1.2 to 1.8, and the hydrolyzed collagen according to item 33. Item 35: The hydrolyzed collagen according to item 33 or item 34 for use as defined in any one of items 1 to 14.

[0141] Item 36: Hydrolyzed collagen for use in improving hyperglycemia or risk factors for hyperglycemia, said hydrolyzed collagen being obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, said hydrolyzed collagen. Item 37: The hydrolyzed collagen according to item 36, wherein the combination of enzymes comprises neutral protease, carboxypeptidase, and aminopeptidase. Item 38: The hydrolyzed collagen according to items 36 to 37, wherein the risk factor for hyperglycemia is one or more selected from the group consisting of insulin resistance, type 2 diabetes, gestational diabetes, high body mass index (BMI), obesity, and hyperglucagonemia. Item 39: · Increasing glucagon-like peptide-1 in the blood; · Decreasing glucagon in the blood; · Increasing insulin in the blood; · Increasing the blood insulin / glucose ratio The hydrolyzed collagen according to any one of items 36 to 38 for use in one or more selected from the group consisting of.

[0142] Item 40: The hydrolyzed collagen according to any one of items 36 to 39, wherein the hydrolyzed collagen is administered in a daily dose of an amount of 1 to 100 grams, preferably 2 to 50 g, more preferably 5 to 25 g, said amount being the amount in dry weight. Item 41: The hydrolyzed collagen according to any one of items 36 to 41, wherein the hydrolyzed collagen is administered orally, preferably as a food supplement. Item 42: The hydrolyzed collagen according to any one of items 36 to 41, wherein the hydrolyzed collagen is administered within 60 minutes of the time difference from a meal. Item 43: The hydrolyzed collagen according to any one of paragraphs 36 to 42, wherein the collagen-containing material is derived from one or more tissues selected from the group consisting of skin, scale, cartilage, bone, tendon, ligament, and connective tissue. Paragraph 44: The hydrolyzed collagen according to any one of paragraphs 36 to 43, wherein the collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen. Paragraph 45: The hydrolyzed collagen according to any one of paragraphs 36 to 44, wherein the collagen-containing material is gelatin. Paragraph 46: The hydrolyzed collagen according to any one of paragraphs 36 to 45, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity of 1.2 to 1.8.

[0143] Paragraph 47: Use of hydrolyzed collagen for non-therapeutically reducing blood glucose, wherein the hydrolyzed collagen is as defined by any one of paragraphs 1 to 11. Paragraph 48: The use according to paragraph 47, wherein the use is for improving postprandial glucose. Paragraph 49: The use according to paragraph 47 or paragraph 48, wherein the hydrolyzed collagen is administered as defined by any one of paragraphs 40 to 42. Paragraph 50: a) preparing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, A method for obtaining hydrolyzed collagen, comprising the steps of: Paragraph 51: The method according to paragraph 50, wherein the combination of enzymes comprises neutral protease, carboxypeptidase, and aminopeptidase. Paragraph 52: The method according to claim 50 or 51, wherein the neutral protease is one or more selected from the group consisting of serine protease, aspartic protease, cysteine protease, and metalloprotease. Claim 53: The method according to any one of claims 50 to 52, wherein the aminopeptidase is leucine aminopeptidase. Claim 54: The method according to any one of claims 50 to 53, wherein one or more of neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably Aspergillus oryzae. Claim 55: The collagen-containing material is exposed to a neutral protease in an amount defined by an enzyme activity of 0.2 to 25000 U / g, preferably 2 to 2500 U / g, more preferably 20 to 250 U / g, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation. The method according to any one of claims 50 to 54.

[0144] Claim 56: The collagen-containing material is exposed to a carboxypeptidase in an amount 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation. The method according to any one of claims 50 to 55. Claim 57: The collagen-containing material is exposed to a carboxypeptidase in an amount 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation. The method according to any one of claims 50 to 56. Claim 58: The collagen-containing material is exposed to aminopeptidase in an amount 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation, according to the method of any one of paragraphs 50 to 57. Paragraph 59: The collagen-containing material is exposed to one or more enzymes in the combination of enzymes for 60 to 180 minutes, according to the method of any one of paragraphs 50 to 58. Paragraph 60: The collagen-containing material is exposed to one or more enzymes in the combination of enzymes at 30°C to 60°C, according to the method of any one of paragraphs 50 to 59. Paragraph 61: The collagen-containing material is exposed to one or more enzymes in the combination of enzymes at pH 5 to pH 8, according to the method of any one of paragraphs 50 to 60.

[0145] Paragraph 62: The collagen-containing material is sequentially exposed to two or more enzymes in the combination of enzymes, according to the method of any one of paragraphs 50 to 61. Paragraph 63: The collagen-containing material is simultaneously exposed to two or more enzymes in the combination of enzymes, according to the method of any one of paragraphs 50 to 62. Paragraph 64: The collagen-containing material is exposed to a mixture containing the combination of enzymes, according to the method described in paragraph 63. Paragraph 65: The collagen-containing material is provided in an amount of 20 to 50% by weight, preferably 30 to 40% by weight, in the liquid preparation, according to the method of any one of paragraphs 50 to 64. Paragraph 66: The collagen-containing material is derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendon, ligament, and connective tissue, according to the method of any one of paragraphs 50 to 65. Paragraph 67: The method according to any one of items 50 to 66, wherein the collagen-containing material is derived from one or more tissues selected from the group consisting of skin, scale, cartilage, bone, tendon, ligament, and connective tissue. Item 68: The method according to any one of items 50 to 67, wherein the collagen-containing material is gelatin.

[0146] Item 69: Hydrolyzed collagen obtainable by the method according to any one of items 50 to 68, wherein the hydrolyzed collagen · has a weight average molecular weight of 2,000 to 4,000 Da, preferably 2,500 to 3,500 Da; and · contains 35 to 60% by weight of collagen peptides having 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. The hydrolyzed collagen. Item 70: The hydrolyzed collagen according to item 69, which contains more than 40% by weight of collagen peptides having 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. Item 71: The hydrolyzed collagen contains, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen, · 1 to 20% by weight of collagen peptides having a molecular weight of less than 1,000 Da, and / or · 20 to 40% by weight of collagen peptides having a molecular weight in the range of 1,000 to 2,000 Da, and / or · 5 to 25% by weight of collagen peptides having a molecular weight in the range of 5,000 to 10,000 Da, and / or · 0 to 10% by weight of collagen peptides having a molecular weight of more than 10,000 Da The hydrolyzed collagen according to item 69 or item 70.

[0147] Item 72: The hydrolyzed collagen is the hydrolyzed collagen according to any one of items 69 to 71, having a polydispersity of 1.2 to 1.8. Item 73: The hydrolyzed collagen is the hydrolyzed collagen according to any one of items 69 to 72, which is capable of stimulating glucagon-like peptide-1 secretion. Item 74: Use of the hydrolyzed collagen according to any one of items 69 to 73 for improving hyperglycemia or a risk factor for hyperglycemia as defined in any one of items 36 to 46. Item 75: Use of the hydrolyzed collagen according to any one of items 69 to 73 for non-therapeutically reducing blood glucose as defined in any one of items 47 to 49.

[0148] ●● Experimental section ●● Example 1 Example 1 shows the effect of a gelatin hydrolysate obtained by enzymatic hydrolysis using a combination of enzymes selected from neutral protease, carboxypeptidase, and aminopeptidase in increasing GLP-1 in vitro and reducing blood glucose in vivo.

[0149] ● Test design In the hydrolysis of gelatin to a hydrolyzed collagen preparation, 14 different enzymes (mixtures) were used. Simulated gastrointestinal digestion (SGID) was performed on the resulting hydrolyzed collagen preparations to obtain bioactive hydrolyzed collagen fractions as predicted after in vivo ingestion and digestion (Song et al. Food Funct. 2020 Jun 24;11(6):5553-5564).

[0150] These hydrolyzed collagen fractions were screened in vitro for their ability to enhance GLP-1 secretion by STC-1 cells. STC-1 cells are intestinal secretin tumor cells and have been reported to be a predictive cell model for studying the hormonal secretion mechanism in the gastrointestinal tract (Qi et al. Bio Protoc. 2020 Aug 20; 10(16): e3717). GLP-1 acts as a major determinant of blood glucose homeostasis, mostly by controlling gastric emptying, enhancing pancreatic insulin secretion, and suppressing pancreatic glucagon secretion. The secretion of GLP-1 by STC-1 cells was, therefore, a favorable outcome parameter for determining how hydrolyzed collagen could improve glucose concentration in the body. The most promising hydrolyzed collagen fraction identified in vitro was further tested for its glucose-lowering activity in mice. The glucose-lowering activity was established based on changes in GLP-1, GIP, glucose, and insulin in plasma. The effectiveness of the hydrolyzed collagen fraction was compared with that of sitagliptin, a glucose-lowering drug.

[0151] ● Method ▲ Enzymatic hydrolysis of collagen Gelatin powder was dissolved in demi-water by heating and stirring to obtain a 35 wt% gelatin solution. The temperature of this gelatin solution was adjusted to 40 - 55 °C. The pH of the gelatin was adjusted to pH 5.5 - pH 8.0. Once the appropriate temperature and pH were reached, the enzyme(s) shown in Table 1 was added and hydrolysis was carried out with stirring. For the "H080" condition, a commercially available enzyme mixture containing a mixture of neutral protease, carboxypeptidase, and aminopeptidase derived from Aspergillus oryzae was used (Sumizyme FP-G available from Takabio in Japan). The temperature was maintained constant. After 120 minutes, the enzyme was heat inactivated to terminate the enzymatic hydrolysis. Next, this solution was cooled to 55 °C. The hydrolyzed gelatin solution was purified, filtered, and sterilized. The final hydrolyzed gelatin powder was obtained by spray drying.

[0152] Table 1. Outline of enzymes (mixtures) used for collagen hydrolysis

Table 1

[0153] ▲GLP-1 secretion by STC-1 cells STC-1 cells were incubated with various hydrolyzed collagens obtained after simulated gastrointestinal digestion (SGID) at various final concentrations (0.2%, 0.5%, and 1% dry matter in Hepes buffer at pH 7.4). After 2 hours of incubation, the supernatant was collected. The GLP-1 concentration was determined by enzyme immunoassay and expressed in pg / ml. The data were expressed as mean (n = 3) ± standard deviation.

[0154] The cells were stimulated with 2 mg / ml, 5 mg / ml, or 10 mg / ml of the enzyme(s) (mixture). For all the enzyme(s) (mixture), a dose-dependent increase in GLP-1 secretion was observed, with the highest GLP-1 secretion at the 10 mg / ml condition. Therefore, Figure 1 shows the results for the comparison of the enzyme(s) (mixture) at a concentration of 10 mg / ml.

[0155] ▲In vivo mouse model The following mouse model was used to determine the area under the curve (AUC) of blood glucose concentration, insulin / blood glucose, plasma GLP-1 concentration, and plasma GIP concentration. C57BL6 / J mice (male, 23 - 25 g, 8 weeks old) were randomly assigned to treatment groups (n = 10 mice / group) according to their body weight.

[0156] The mice were fasted for 6 hours and then an oral glucose tolerance test (OGTT) was performed. The mice were treated with vehicle, test article H080 at three different concentrations (40 mg / kg, 400 mg / kg, and 4 g / kg), and sitagliptin as a positive control. Forty-five minutes after administration of the active ingredient, the mice received an oral glucose load (2 g per kg body weight). Blood glucose was measured in blood samples collected at -45 minutes, 0 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after glucose load. Insulin was determined (by enzyme-linked immunosorbent assay ELISA) in plasma samples collected 45 minutes before (-45 minutes) and 15 minutes after (+15 minutes) glucose load. From this, the insulin / glucose ratio could be determined.

[0157] The plasma GLP-1 concentration and GIP concentration were measured 1 week after the OGTT test. For this, the mice were fasted for 6 hours and then treated with sitagliptin (400 μg / mouse) to avoid degradation of GLP-1 and GIP by dipeptidyl peptidase 4 (DPP-IV). Thirty minutes later, the mice received vehicle or test article H080 at 4 g per kg body weight. Fifteen minutes or 30 minutes after administration of the active ingredient, the mice were anesthetized and blood was collected from the portal vein. The plasma GLP-1 concentration and GIP concentration were measured by ELISA.

[0158] The model in healthy mice demonstrates both the prevention and treatment of high blood glucose (and other parameters related to hyperglycemia) after treatment with the active ingredient.

[0159] Using a method similar to that described for healthy mice, tests were also conducted in obese mice with naturally occurring high blood glucose, and similar results were obtained.

[0160] ● Results ▲ GLP-1 secretion in vitro Figure 1 shows GLP-1 secretion in STC-1 cells after incubation with 10 mg / ml of hydrolyzed collagen obtained by hydrolysis under optimal treatment conditions with the enzyme (mixture) described in Table 1. The hydrolyzed collagen labeled H080 induced the greatest GLP-1 secretion. Compared to the blank, H080 induced a 5800-fold increase in GLP-1 secretion. The increase in GLP-1 mediated by H080 was more than 2.5 times greater than the increase in GLP-1 mediated by other enzymes (mixtures) in the comparative test.

[0161] The data indicate that a combination of neutral protease (e.g., serine protease), carboxypeptidase, and aminopeptidase (e.g., leucine aminopeptidase) induces a much higher, optimal GLP-1 secretion compared to other enzymes and enzyme combinations. It was found that a mere combination of two or more enzymes (e.g., the combination of two neutral proteases in "CH3") is not sufficient to further promote GLP-1 secretion compared to the use of only one enzyme (e.g., neutral protease especially in "CH1" and "CH2").

[0162] It was found that a mere combination of endoprotease (i.e., an enzyme that cleaves at non-terminal amino acid sites) and exoprotease (i.e., an enzyme that cleaves at terminal amino acid sites) is not sufficient to further promote GLP-1 secretion compared to the use of only endoprotease. For example, "CH12" contains both endopeptidase activity and exopeptidase activity, but was unable to further promote GLP-1 secretion compared to an enzyme containing only endopeptidase activity.

[0163] The results shown in Fig. 1 were reproduced with gelatin from different batches. These results indicate that the hydrolysis of collagen using a combination of two or more enzymes selected from neutral protease, carboxypeptidase, and aminopeptidase is important for obtaining hydrolyzed collagen having a strong ability to reduce glucose, such as the glucose reduction induced by GLP-1.

[0164] ▲In vivo glucose-lowering activity Fig. 2 shows the blood glucose concentrations in mice after treatment with 40 mg / Kg of H080, 400 mg of H080, 4 g / kg of H080, and 400 μg / mouse of sitagliptin. After treatment with H080, a dose-dependent decrease in blood glucose was observed. The maximum dose of H080 had a blood glucose-lowering effect similar to that of the sitagliptin drug. This indicates that the hydrolyzed collagen can be provided as a safe alternative to pharmaceuticals often associated with side effects.

[0165] Fig. 3 shows the insulin / blood glucose ratio in mice after treatment with 40 mg / Kg of H080, 400 mg of H080, 4 g / kg of H080, and 400 μg / mouse of sitagliptin. H080 mediated a significant increase in the insulin / glucose ratio. After treatment with H080, a dose-dependent increase in the insulin / glucose ratio was observed.

[0166] Fig. 4 shows the plasma GLP-1 concentration after control treatment or treatment with 4 g / kg of H080. Treatment with H080 increased the plasma GLP-1 concentration, and this effect was maximal 30 minutes after ingestion of H080.

[0167] Fig. 5 shows the plasma GIP concentration after control treatment or treatment with 4 g / kg of H080. Treatment with H080 decreased the plasma GIP concentration, and this effect was maximal 30 minutes after ingestion of H080.

[0168] In the model of healthy mice, the mice are administered with the active ingredient before glucose loading. Therefore, the healthy mice are particularly suitable for the study of the prevention of hyperglycemia. Tests were also conducted on obese mice with naturally occurring high blood glucose. The method is similar to that described for healthy mice. Since obese mice already have an increased blood glucose concentration when administered with the active ingredient, the improvement of blood glucose (and other parameters related to hyperglycemia) in the obese mouse model further supports the treatment of the disease by the active ingredient.

[0169] Similar effects on the parameters tested in Figures 1 - 5 were observed in obese mice with high blood glucose. This further supports the effective treatment of hyperglycemia (and related parameters) by H080.

[0170] ▲Combination of enzymes selected from neutral protease, carboxypeptidase, and aminopeptidase in glucose - reducing activity The inventors of the present application have found that when two or more enzymes selected from neutral protease, carboxypeptidase, and aminopeptidase are used in the enzymatic hydrolysis of collagen - containing materials, high glucose - reducing activity (e.g., based on GLP - 1 secretion level) can be achieved.

[0171] As shown in Table 2, the use of neutral protease in the enzymatic hydrolysis of gelatin, although limited, leads to gelatin hydrolysates with glucose - reducing activity. The combination of neutral protease with carboxypeptidase alone or aminopeptidase alone gives stronger glucose - reducing activity compared to the blank or the use of neutral protease alone. The combination of all three enzymes leads to gelatin hydrolysates with the highest glucose - reducing activity.

[0172] Table 2: Enzymes used in the hydrolysis of collagen and the effect of the hydrolysate in reducing glucose

Table 2

[0173] ▲Characterization of Hydrolyzed Collagen with High Glucose-Lowering Activity Hydrolyzed collagen having high glucose-lowering activity (for example, groups III-IV in Table 2) is characterized by a weight-average molecular weight of 1000-7000 Da and a polydispersity (weight-average molecular weight / number-average molecular weight) of 1.2-1.8 (an average of about 1.56 is measured for more than 100 measurements) as determined by HPSEC.

[0174] ●●Example 2 Example 2 shows the influence of process conditions for obtaining hydrolyzed collagen for reducing blood glucose. ●Method The gelatin hydrolysate was produced by hydrolysis using a combination of neutral protease (50,000 U / g), aminopeptidase (500-1200 U / g), and carboxypeptidase (300-700 U / g) as described in Example 1. A wide range of working conditions were tested, which covered the conditions typically used for enzymatic hydrolysis. The concentration of gelatin in the starting solution was varied between 10 wt% and 50 wt%. The enzyme was used at a concentration of 2000 ppm for hydrolysis. The pH during enzymatic hydrolysis was varied between pH 4.0 and pH 10.0. The temperature during enzymatic hydrolysis was varied between 20°C and 70°C.

[0175] Table 3 shows the influence exerted by the gelatin concentration, pH, and temperature of enzymatic hydrolysis in obtaining a gelatin hydrolysate fraction for reducing blood glucose in preliminary tests. GLP-1 secretion was tested using the same method as in Example 1. Table 4 shows the effects of gelatin concentration, pH, and temperature of enzymatic hydrolysis on obtaining a gelatin hydrolysate fraction for reducing blood glucose, based on a more refined and broader range of tests. GLP-1 secretion was tested using the same method as in Example 1.

[0176] Table 3. Effects of gelatin concentration, pH, and temperature of enzymatic hydrolysis on obtaining a gelatin hydrolysate fraction for reducing blood glucose in preliminary tests

Table 3

[0177] Table 4. Effects of gelatin concentration, pH, and temperature of enzymatic hydrolysis on obtaining a gelatin hydrolysate fraction for reducing blood glucose, based on a more refined and broader range of tests +: less suitable; ++: more suitable; +++: most suitable

Table 4

[0178] As summarized in Tables 3 and 4, it can be seen that it is possible to achieve hydrolyzed collagen suitable for enhancing GLP-1 secretion over a wide range of values for precursor concentration (weight % of gelatin), pH, and temperature. For the precursor concentration (weight % of gelatin), good results were obtained over the entire range of 10 wt% to 50 wt% tested, and it appears that the optimum is at a concentration of 20 wt% to 35 wt%. For pH, good results were obtained over the entire range of pH 4.0 to pH 10.0 tested, and it appears that the optimum is at pH 5.5 to pH 6.5. For temperature, good results were obtained over the entire range of 20°C to 60°C tested, and it appears that the optimum is at 45°C to 50°C.

[0179] These results suggest that hydrolysis carried out at (excessively) high temperatures (e.g., temperatures above 70 °C) may not lead to a hydrolyzed collagen product suitable for enhancing GLP-1 secretion. This is thought to be the result of enzyme denaturation and subsequent impaired reaction rates.

[0180] Preferred combinations of gelatin concentration, pH, and temperature can lead to optimal activity in enhancing GLP-1 secretion by STC-1 cells. As shown in Table 5, combinations of gelatin concentration (30 wt% - 40 wt%), pH (6 - 7.5), and temperature (40 °C - 55 °C) lead to surprisingly high activity of gelatin hydrolysates in enhancing GLP-1 secretion by STC-1 cells. GLP-1 secretion is tested using the same method as in Example 1.

[0181] Table 5. GLP-1 secretion by STC-1 cells stimulated with hydrolyzed collagen obtained by combinations of neutral protease, carboxypeptidase, and aminopeptidase, where enzymatic hydrolysis was carried out under various process conditions

Table 5

[0182] ●●Example 3 Example 3 shows the molecular weight and molecular weight distribution of hydrolyzed collagen obtained by hydrolysis using various enzymes (mixtures) in relation to the ability of said hydrolyzed collagen to stimulate GLP-1 secretion by STC-1 cells.

[0183] Table 6 shows the molecular weight and molecular weight distribution of hydrolyzed collagen preparations obtained using various enzymes, along with their different abilities to stimulate GLP-1 secretion in STC-1 cells. The methods used for the evaluation of hydrolysis and GLP-1 secretion are the same as those described in Example 1. 「H080」 was produced by hydrolysis using the combination of neutral protease (50,000 U / g), aminopeptidase (500 - 1200 U / g), and carboxypeptidase (300 - 700 U / g) as described in Example 1. The collagen hydrolysates “CH11 - CH19” were obtained by hydrolysis using either neutral protease, alkaline protease, or acid protease.

[0184] “Average” GLP-1 secretion was considered if GLP-1 secretion increased by more than 1000-fold compared to the stimulation using the blank control. As can be seen from Table 6, “H080” induced a GLP-1 secretion that increased by approximately 6000-fold compared to the stimulation using the blank control. “H080” is considered to have “very high” GLP-1 secretion. The hydrolyzed collagen related to the group “CH11 - CH19” - that is, obtained by hydrolysis using either neutral protease, alkaline protease, or acid protease - showed no / minimal or only average GLP-1 secretion.

[0185] As can be seen from Table 6, “H080” is characterized by a weight average molecular weight of approximately 3000 Da (or a simple average molecular weight of approximately 2000 Da). “H080” is further characterized by a high proportion (i.e., approximately 47.2%) of collagen peptides belonging to the range of 2 kDa - 5 kDa. “H080” is further characterized by a high stimulatory activity on GLP-1 secretion (i.e., an increase of approximately 6000-fold compared to the blank control group). The group “CH11 - CH19” lacks one or more of these characteristics.

[0186] Table 6. Summary of the enzyme(s) (mixture) used for the hydrolysis of collagen, the molecular weight and molecular weight distribution of the obtained hydrolyzed collagen, and their ability to stimulate GLP-1 secretion by STC-1 cells

Table 6

Claims

1. Hydrolyzed collagen for use in improving hyperglycemia or risk factors for hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase, said hydrolyzed collagen.

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

3. The hydrolyzed collagen according to any one of the preceding claims, wherein the risk factor for hyperglycemia is one or more selected from the group consisting of insulin resistance, type 2 diabetes, gestational diabetes, high body mass index (BMI), obesity, and hyperglucagonemia.

4. - Increasing glucagon-like peptide-1 in the blood; - Decreasing glucagon in the blood; - Increasing insulin in the blood; - Increasing the insulin / glucose ratio in the blood The hydrolyzed collagen according to any one of the preceding claims, for use in one or more selected from the group consisting of.

5. The hydrolyzed collagen according to any one of the preceding claims, wherein the hydrolyzed collagen is administered in a daily dose of an amount of 1 to 100 grams, preferably 2 to 50 g, more preferably 5 to 25 g, said amount being the amount in dry weight.

6. The hydrolyzed collagen according to any one of the preceding claims, wherein the hydrolyzed collagen is administered orally, preferably as a food supplement.

7. The hydrolyzed collagen according to any one of the preceding claims, wherein the hydrolyzed collagen is administered within 60 minutes of the time difference from a meal.

8. The hydrolyzed collagen according to any one of the preceding claims, wherein the collagen-containing material is derived from one or more tissues selected from the group consisting of skin, scale, cartilage, bone, tendon, ligament, and connective tissue.

9. The hydrolyzed collagen according to any one of the preceding claims, wherein the collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen.

10. The hydrolyzed collagen according to any one of the preceding claims, wherein the collagen-containing material is gelatin.

11. The hydrolyzed collagen according to any one of the preceding claims, wherein the hydrolyzed collagen has an average molecular weight of 1,000 to 7,000 Da and / or a polydispersity of 1.2 to 1.

8.

12. Use of hydrolyzed collagen for non-therapeutically reducing blood glucose, wherein the hydrolyzed collagen is as defined in any one of claims 1 to 11.

13. The use according to claim 12, which is use in improving postprandial glucose.

14. The use according to claim 12 or claim 13, wherein the hydrolyzed collagen is administered as defined in any one of claims 5 to 7.

15. a) preparing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral protease, carboxypeptidase, and aminopeptidase. A method for obtaining hydrolyzed collagen, comprising the above steps.

16. The method according to claim 15, wherein the combination of enzymes comprises neutral protease, carboxypeptidase, and aminopeptidase.

17. The method according to claim 15 or claim 16, wherein the neutral protease is one or more selected from the group consisting of serine protease, aspartic protease, cysteine protease, and metalloprotease.

18. The method according to any one of claims 15 to 17, wherein the aminopeptidase is leucine aminopeptidase.

19. The method according to any one of claims 15 to 18, wherein one or more of neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably Aspergillus oryzae.

20. The method according to any one of claims 15 to 19, wherein the collagen-containing material is exposed to neutral protease in 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation.

21. The method according to any one of claims 15 to 20, wherein the collagen-containing material is exposed to carboxypeptidase in an amount 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation.

22. The method according to any one of claims 15 to 21, wherein the collagen-containing material is exposed to aminopeptidase in an amount 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, where the weight represented by g is the total weight of the collagen-containing material and the liquid preparation.

23. The method according to any one of claims 15 to 22, wherein the collagen-containing material is exposed to one or more enzymes in the combination of enzymes for 60 to 180 minutes.

24. The method according to any one of claims 15 to 22, wherein the collagen-containing material is exposed to one or more enzymes in the combination of enzymes at 30°C to 60°C.

25. The method according to any one of claims 15 to 24, wherein the collagen-containing material is exposed to one or more enzymes in the combination of enzymes at pH 5 to pH 8.

26. The method according to any one of claims 15 to 25, wherein the collagen-containing material is sequentially exposed to two or more enzymes in the combination of enzymes.

27. The method according to any one of claims 15 to 26, wherein the collagen-containing material is simultaneously exposed to two or more enzymes in the combination of enzymes.

28. The method according to claim 27, wherein the collagen-containing material is exposed to a mixture containing the combination of enzymes.

29. The method according to any one of claims 15 to 28, wherein the collagen-containing material is provided in an amount of 20 to 50% by weight, preferably 30 to 40% by weight, in the liquid preparation.

30. The method according to any one of claims 15 to 29, wherein the collagen-containing material is derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendon, ligament, and connective tissue.

31. The method according to any one of claims 15 to 30, wherein the collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen.

32. The method according to any one of claims 15 to 31, wherein the collagen-containing material is gelatin.

33. Hydrolyzed collagen obtainable by the method according to any one of claims 15 to 32, wherein the hydrolyzed collagen ・ has a weight average molecular weight of 2000 to 4000 Da, preferably 2500 to 3500 Da; and ・ contains 35 to 60% by weight of collagen peptides having a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen. The hydrolyzed collagen.

34. The hydrolyzed collagen according to claim 33, which contains more than 40% by weight of collagen peptides having a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen.

35. The hydrolyzed collagen, calculated based on the total weight of the collagen peptides in the hydrolyzed collagen, ・ contains 1 to 20% by weight of collagen peptides having a molecular weight of less than 1000 Da, and / or ・ contains 20 to 40% by weight of collagen peptides having a molecular weight in the range of 1000 Da to 2000 Da, and / or ・ contains 5 to 25% by weight of collagen peptides having a molecular weight in the range of 5000 Da to 10000 Da, and / or ・ contains 0 to 10% by weight of collagen peptides having a molecular weight of more than 10000 Da The hydrolyzed collagen according to claim 33 or claim 34.

36. The hydrolyzed collagen according to any one of claims 33 to 35, which has a polydispersity of 1.2 to 1.

8.

37. The hydrolyzed collagen according to any one of claims 33 to 36, which is capable of stimulating glucagon-like peptide-1 secretion.

38. The hydrolyzed collagen according to any one of claims 33 to 37, for use in improving hyperglycemia or a risk factor for hyperglycemia as defined in any one of claims 1 to 11.

39. Use of hydrolyzed collagen according to any one of claims 33 to 37 in non-therapeutically reducing blood glucose as defined in any one of claims 12 to 14.

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