Hydrolyzed collagen for use in reducing blood glucose

Abstract

To provide a hydrolyzed collagen formulation for use in decreasing blood glucose.SOLUTION: The hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material with a combination of enzymes comprising two or more enzymes selected from the group consisting of a neutral protease, a carboxypeptidase and an aminopeptidase. The hydrolyzed collagen formulation is particularly suitable as a food supplement, such as a food supplement for use in ameliorating hyperglycemia and / or a hyperglycemia risk factor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an active ingredient and its use for ameliorating hyperglycemia and conditions associated with hyperglycemia. The active ingredient of the present invention is suitable as a dietary supplement. [Background technology]

[0002] The human body depends on tight control of its blood glucose levels for its normal function. Disruption of normal blood glucose levels can lead to diseases, particularly metabolic syndrome, cardiovascular disease, neurological damage, and renal failure. Conditions associated with abnormal insulin levels and / or insulin sensitivity (e.g., type 2 diabetes, obesity) are closely linked to the development of hyperglycemia.

[0003] Control of blood glucose involves the interaction of various organs (e.g., pancreas, brain, liver, intestine, adipose, and muscle tissue) and is mediated in part by the release of various hormones. Insulin and glucagon are peptide hormones that work to counteract each other's actions to balance blood glucose levels. In response to high blood glucose levels, pancreatic beta cells secrete insulin into the blood, which then lowers blood glucose levels by promoting glucose absorption from the blood into liver cells, fat cells, and skeletal muscle cells. Glucagon is secreted by pancreatic alpha cells. Glucagon enhances the liver's conversion of stored glycogen into glucose, which is released into the bloodstream.

[0004] First-line treatment for hyperglycemia initially involves lifestyle changes that reduce one or more of the risk factors for hyperglycemia. These typically include lifestyle changes that target insulin resistance, diabetes, obesity, or high blood pressure. Often, lifestyle changes alone are insufficient, necessitating the inclusion of metformin and / or insulin treatment as part of the first-line treatment.

[0005] In many individuals, these first-line treatments do not adequately reduce hyperglycemia. A new class of glucose-lowering drugs is currently being developed and tested as a potential second-line treatment (Davies et al. Diabetes Care 2018;41(12):2669-2701).

[0006] A new class of blood glucose-lowering drugs targets the activity of incretins. Incretins are metabolic hormones that lower blood glucose levels by enhancing insulin secretion and / or inhibiting glucagon secretion in a glucose-dependent manner. Glucagon-like peptide-1 (GLP-1) is considered one of the most important incretins and is therefore an important target in new strategies to improve hyperglycemia. Although GLP-1 receptor agonists have demonstrated clinical efficacy (Trujillo et al. Ther Adv Endocrinol Metab. 2021 Mar 9;12:2042018821997320), they have several limitations that prevent their widespread use. First, they cause several side effects, including pancreatitis, pancreatic cancer, or bone disease (Storgaard et al. Diabetes Obes Metab 2017;19:906-908), as well as gastrointestinal side effects such as nausea, vomiting, and diarrhea. Second, treatment with GLP-1 receptor agonists requires repeated subcutaneous injections, which can lead to patient discomfort and injection site reactions (Trujillo et al. TherAdv Endocrinol Metab. 2021 Mar 9;12:2042018821997320).

[0007] Another example of a new blood glucose-lowering drug is sitagliptin. Sitagliptin is a selective inhibitor of dipeptidyl peptidase IV (DPPIV). DPPIV causes the hydrolysis of incretin hormones. Sitagliptin can thereby increase the plasma concentration of active forms of incretins, such as GLP-1 and gastric inhibitory polypeptide (GIP). This increases insulin release and decreases glucagon levels. However, professional medical practitioners remain hesitant to prescribe sitagliptin to patients. This is largely due to its several side effects and contraindications, including complications such as hyperglycemia, headache, respiratory tract infection, and nasopharyngitis in up to 10% of patients. Collagen peptides have also been suggested to lower blood glucose. JP 2009-235064 A describes a collagen peptide that reduces blood glucose and is obtained by treating starfish with proteases, and JP 2012-116773 A describes a collagen peptide that increases insulin-like growth factor 1 (IGF-1) and can be obtained by treating collagen derived from fish scales and / or fish skin with protease.

[0008] There is an unmet need for active ingredients that ameliorate hyperglycemia more efficiently and / or safely. Such active ingredients can be used in addition to, or as an alternative to, first-line and / or second-line treatments for individuals with (or at risk for) hyperglycemia. The present invention aims to provide such active ingredients. Summary of the Invention

[0009] The present inventors have discovered that enzymatic hydrolysis of collagen-containing starting materials using a combination of two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases produces compositions with potent glucose-lowering activity, which appears to be highest when all three of the enzymes are used in the hydrolysis.

[0010] In one aspect, the present invention relates to hydrolyzed collagen for use in reducing blood glucose, the 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 proteases, carboxypeptidases, and aminopeptidases.

[0011] In one aspect, the present invention relates to hydrolyzed collagen for (further) 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 proteases, carboxypeptidases, and aminopeptidases.

[0012] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) providing 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 proteases, carboxypeptidases, and aminopeptidases; The present invention relates to a method for obtaining hydrolyzed collagen, comprising: The hydrolyzed collagen is preferably for use in reducing blood glucose and / or ameliorating hyperglycemia.

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

[0014] [Figure 1] GLP-1 secretion by STC-1 cells after incubation with 10 mg / ml of various hydrolyzed collagen fractions obtained by various enzymatic treatments. Supernatants were collected after 2 hours of incubation. GLP-1 concentrations were determined by enzyme immunoassay. Results are expressed as the mean (n=3) ± standard deviation. "H080" represents the hydrolyzed collagen of the present invention. [Figure 2] Area under the curve (AUC) of blood glucose concentration in mice after glucose load at time 0 and ingestion of active ingredient / vehicle at time -45 minutes. "H080" represents hydrolyzed collagen of the present invention. [Figure 3] Ratio of insulin / blood glucose concentration in mice at 15 minutes after glucose load at time 0 and ingestion of active ingredient / vehicle at -45 minutes. "H080" represents hydrolyzed collagen of the present invention. [Figure 4] Plasma GLP-1 concentrations in mice 15 and 30 minutes after ingestion of H080 hydrolyzed collagen compared to baseline. "H080" represents hydrolyzed collagen of the present invention. [Figure 5] Plasma GIP concentrations in mice 15 and 30 minutes after ingestion of H080 hydrolyzed collagen compared to baseline. "H080" represents hydrolyzed collagen of the present invention. DETAILED DESCRIPTION OF THE INVENTION

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

[0016] In one aspect, the present invention relates to hydrolyzed collagen for use in ameliorating hyperglycemia, the hydrolyzed collagen being obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases.

[0017] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) providing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes, preferably comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases; The present invention relates to a method for obtaining hydrolyzed collagen, comprising: The hydrolyzed collagen is preferably for use in reducing blood glucose and / or ameliorating hyperglycemia as taught in the present disclosure.

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

[0019] In one aspect, the present invention relates to a method of lowering blood glucose comprising administering to a subject hydrolyzed collagen obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases.

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

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

[0022] In one aspect, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for ameliorating 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, preferably using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases.

[0023] Advantages of this invention The use of hydrolyzed collagen, for example as a food supplement, is generally safe; The inventors have found that the blood glucose lowering effect of the hydrolyzed collagen of the present invention is similar to that of the pharmaceutical compound sitagliptin. Therefore, hydrolyzed collagen is a valuable additive or alternative to glucose-lowering pharmaceutical compounds, which generally cause side effects and have contraindications; The inventors have discovered that the blood glucose-lowering effect of the hydrolyzed collagen of the present invention involves different mechanisms of action, including increasing glucagon-like peptide-1 (GLP-1) and inhibiting dipeptidyl peptidase IV (DPP IV). The different effects of hydrolyzed collagen appear to explain its potent blood glucose-lowering activity in vivo. Furthermore, the existence 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 subject populations and / or at different stages of hyperglycemia). The inventors have established the beneficial effects of hydrolyzed collagen in healthy mice (i.e., showed prevention of hyperglycemia) and in obese mice with hyperglycemia (i.e., showed treatment of hyperglycemia), indicating that the hydrolyzed collagen of the present invention is effective in improving hyperglycemia both as a preventative and therapeutic.

[0024] Blood sugar 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, the combination of enzymes comprising two or more selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases.

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

[0026] In certain preferred embodiments, the enzyme combinations disclosed herein include a neutral protease, a carboxypeptidase, and an 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 blood glucose levels as disclosed in the present disclosure and / or as an active ingredient in improving hyperglycemia as disclosed in the present disclosure.

[0027] As used in this disclosure, the term "blood glucose" (i.e., the concentration of glucose in the blood) may 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" refers to an individual who has not consumed food for 8 hours or more.

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

[0029] The term "blood" may be used interchangeably with the term "plasma" in this disclosure when referring to the concentration (level) of a molecule. For example, "blood glucose" may also be read as "plasma glucose." For example, "blood GLP-1" may also be read as "plasma GLP-1."

[0030] As used in this disclosure, the term "hyperglycemia" means high blood glucose. For the purposes of this disclosure, the normal blood glucose range in a fasting individual is considered to be 80-110 mg / dL. For the purposes of this disclosure, an individual with a blood glucose concentration of 126 mg / dL or greater is considered to have hyperglycemia. Individuals generally are considered to have impaired glucose tolerance, or pre-diabetes, if they have a fasting blood glucose of 111-125 mg / dL. Individuals with "impaired glucose tolerance" or "pre-diabetes" are considered to have hyperglycemia for the purposes of this disclosure.

[0031] In certain embodiments, the "hyperglycemia" disclosed in this 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 / or requires prolonged hyperglycemia treatment, with or without combined lifestyle changes. The "hyperglycemia" disclosed in the present disclosure is preferably chronic hyperglycemia.

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

[0034] In one embodiment, the hyperglycemia disclosed in the present disclosure also includes "postprandial hyperglycemia." "Postprandial hyperglycemia," in the context of the present invention, is defined as excessively high postprandial glucose. 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, blood glucose levels peak approximately one hour after the start of a meal, typically do not exceed 140 mg / dL, and return to preprandial levels within two to three hours. These time profiles are typically different in diabetic patients or patients suffering from different pathological conditions. In one embodiment, PPG values ​​are measured two hours after the start of a meal, although other time points may be more appropriate depending on when peak values ​​typically occur (or are expected to occur). Acceptable PPG values, the threshold PPG value defining postprandial hyperglycemia, and the optimal method for measuring PPG values ​​may vary among medical professionals. In the context of the present invention, PPG values ​​are considered to be excessively / too high (i.e., postprandial hyperglycemia) when they are above 140 mg / dl for adults under the age of 50, above 150 mg / dl for individuals aged 50-60, and above 160 mg / dl for individuals over 60 years of age.

[0035] The present invention may relate to non-therapeutic and / or therapeutic uses of said hydrolyzed collagen, wherein the difference is based on the concentration of blood glucose and / or on distinct groups of subjects experiencing elevated blood glucose.

[0036] The first group (referred to in this disclosure as the "group of healthy subjects") includes healthy people who will not receive therapeutic benefit from treatment with the hydrolyzed collagen of the present invention, e.g., where blood glucose levels are not excessive or severe enough to be expected to lead to health problems or (severe) suffering. Additionally or alternatively, the blood glucose may have a cause or severity such that the increase in blood glucose will naturally resolve over time (i.e., is not chronic). Additionally or alternatively, the blood glucose levels in the group of healthy subjects may not be too excessive or severe, so that subjects will generally not seek help from a professional medical practitioner.

[0037] The second group (in the present disclosure, the "group of pathological subjects") includes subjects whose increased 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 pathological subjects. The severity of the increase in blood glucose is such that help from a specialized physician is usually sought.

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

[0039] Depending on whether an individual is healthy or suffering from a medical condition (e.g., hyperglycemia, diabetes, obesity), an improvement in postprandial glucose can be considered a non-therapeutic intervention or a therapeutic intervention, respectively. Additionally or alternatively, depending on the amount of glucose in the blood after a meal, an improvement in postprandial glucose can be considered 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 therefore, an improvement in postprandial glucose levels of less than 140 mg / dl can also be considered a non-therapeutic intervention. For example, in an individual suffering from a medical condition (e.g., hyperglycemia, diabetes, obesity), the amount of glucose after a meal can typically be 140 mg / dl or more, and therefore, an improvement in postprandial glucose levels of 140 mg / dl or more can also be considered a therapeutic intervention.

[0040] In one embodiment, the hydrolyzed collagen is for use in the therapeutic reduction of blood glucose, e.g., for use in improving hyperglycemia or risk factors for hyperglycemia, hi one embodiment, the hydrolyzed collagen is for use in the non-therapeutic reduction of blood glucose, e.g., for improving postprandial glucose.

[0041] In one embodiment, the hydrolyzed collagen is for the non-therapeutic improvement of postprandial glucose, where postprandial glucose levels are 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 initiation of the hydrolyzed collagen intervention). In one embodiment, the hydrolyzed collagen is for the therapeutic improvement of postprandial glucose, where postprandial glucose levels are preferably 140 mg / dl or greater, more preferably 160 mg / dl or greater, and even more preferably 180 mg / dl or greater (i.e., before initiation of the hydrolyzed collagen intervention).

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

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

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

[0045] As used in this disclosure, "risk factor for hyperglycemia" means a condition associated with an increased chance of developing hyperglycemia. In an individual having risk factors for hyperglycemia, it is feasible to administer a blood glucose-lowering agent as a prophylactic agent in preventing hyperglycemia. Additionally or alternatively, in an individual having risk factors for hyperglycemia, it is preferably feasible to administer a blood glucose-lowering agent as a prophylactic agent to ameliorate hyperglycemia that may develop. For example, obesity is a risk factor for hyperglycemia. Administration of a blood glucose-lowering agent to an obese individual may prevent hyperglycemia in the obese individual and / or may ameliorate hyperglycemia in the individual if hyperglycemia develops.

[0046] There are several risk factors for developing hyperglycemia. In the context of the present invention, too low insulin levels in the body, vascular 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. Additionally or alternatively, 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] Several hormones, when present in excess, act to increase blood glucose levels (hyperglycemia) and are considered risk factors for hyperglycemia in this disclosure, including an excess of one or more of cortisol, catecholamines, 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 conditions 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 increased insulin concentrations without causing hypoglycemia. This procedure can take 2 hours and preferably includes the following steps (or similar steps): inject insulin at a rate of 1 ml per minute through a peripheral vein; 2 The patient is infused at 10–120 mU per minute. To compensate for the insulin infusion, glucose (20%) is infused to maintain blood glucose levels between 5 mmol / L and 5.5 mmol / L. The glucose infusion rate is determined by checking blood glucose levels 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 glucose infusion rate during the last 30 minutes of the test determines insulin sensitivity. If a high value (≥7.5 mg / min) is required, the patient is insulin sensitive. A low value (≤4.0 mg / min) indicates insulin resistance. Values ​​between 4.0 mg / min and 7.5 mg / min are inconclusive and suggest "impaired glucose tolerance," an early sign of insulin resistance.

[0049] Homeostatic model assessment of insulin resistance (HOMA or HOMA-IR) is an alternative and preferred method for determining and quantifying insulin resistance under fasting steady-state conditions and correlates with absolute criteria (see, e.g., Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985). Diabetologia. 28 (7): 412-9. Doi:10.1007 / BF00280883. PMID 3899825; and / or AS Rudenski; DR Matthews; JC Levy; RC 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 surveys and experimental studies. HOMA(-IR) represents a value that represents an estimate of insulin resistance, which is derived from dividing the insulin and glucose levels in a person's blood. The HOMA(-IR) value can be calculated by the following equation:

[0050]

number

[0051] Other methods for measuring insulin resistance may also be suitable. In some embodiments, insulin sensitivity or resistance is measured using an "insulin-suppression test" (IST). In some embodiments, insulin sensitivity or resistance is measured using a "minimal model analysis of frequently sampled intravenous glucose tolerance test" (FSIVGTT). In some 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 medical professional. In an embodiment, a subject may be diagnosed with metabolic syndrome if they have at least two or at least three of the following traits: Large waist: A waistline that is 35 inches (89 centimeters) or more for women and 40 inches (102 centimeters) or more for men; High triglyceride levels: more than 150 milligrams per deciliter (150 mg / dL) or more than 1.7 millimoles per liter (1.7 mmol / L) of this type of fat found in the blood; Decreased "good" or HDL cholesterol: High-density lipoprotein (HDL) cholesterol 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 of 130 / 85 millimeters of mercury (mm Hg) or more; Elevated fasting blood glucose 100 mg / dL (5.6 mmol / L) or higher

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

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

[0055] As used in this disclosure, the term "obesity" refers to a medical condition characterized by the accumulation of excess body fat to the extent that the excess body fat has adverse health effects. In some cases, obesity is merely a cosmetic concern (requiring non-therapeutic treatment). A professional physician may establish whether a person is obese by one or more means. Generally, a body mass index (BMI) of 30.0 or greater is considered to indicate the presence of obesity. For purposes of this disclosure, a BMI of 25.0 or greater but less than 30 indicates that a person is overweight. As used in this disclosure, "overweight" refers to a non-medical condition characterized by the excessive accumulation of body fat.

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

[0058] The inventors have found that the hydrolyzed collagen of the present invention has a particularly high ability to stimulate GLP-1 secretion and / or lower blood glucose, and this biological activity appears to be related to the specific molecular weight and / or molecular weight distribution of the hydrolyzed collagen obtainable by hydrolysis with 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: Hydrolysis of gelatin to obtain a hydrolyzed collagen preparation; subjecting the hydrolyzed collagen preparation to simulated gastrointestinal digestion (SGID) to obtain digested hydrolyzed collagen, preferably 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) with the digested hydrolyzed collagen or a blank as a reference, preferably according to the protocol described by Qi et al. (BioProtoc. 2020 Aug 20; 10(16): e3717). Cells are stimulated at a concentration preferably in the range of 1-20 mg / ml, preferably 10 mg / ml. The supernatant is collected 1-3 hours after the start of stimulation and the GLP-1 level in the supernatant is determined, for example, by enzyme immunoassay. A particularly suitable protocol is that described for 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 GLP-1 concentration (level) after stimulation, which is preferably an increase relative to stimulation using a blank reference group (control group).

[0061] Hydrolyzed collagen As used in this disclosure, "hydrolyzed collagen" refers to a mixture of short chains of amino acid residues derived from a collagen-containing starting material that has native (full-length) collagen, typically by a hydrolysis step, including 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 in the present invention may include hydrolyzed or partially hydrolyzed collagen. The term "collagen hydrolysate" can be used interchangeably with, and is synonymous with, the terms "hydrolyzed collagen" or "collagen peptides."

[0062] Hydrolyzed collagen may be produced in a single step from a collagen-containing starting material (e.g., animal connective tissue) or through an intermediate gelatin stage, in which type A and / or type B gelatin can be used. As used in this disclosure, hydrolyzed collagen may refer to hydrolyzed gelatin obtained by hydrolysis of gelatin obtained from collagen. The terms "hydrolyzed collagen" and "hydrolyzed gelatin" can be used interchangeably in this 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 and is synonymous with the term "hydrolyzed gelatin." In a preferred embodiment, the collagen-containing material is gelatin. In this 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. Additionally or alternatively, the hydrolyzed collagen disclosed in the present disclosure is preferably derived from an animal raw material containing various collagen types, for example, two or more of type I collagen, type II collagen, and type III collagen. Additionally or alternatively, 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 this disclosure may be derived from any one or more animals or animal species, such as bovine species, pig species, and fish species. In this disclosure, "animal" may refer to any animal capable of providing connective tissue that can be used to prepare hydrolyzed collagen.

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

[0065] In various embodiments, the collagen disclosed herein is a mixture of collagens from various sources, such as collagens from multiple animal species and / or collagens from different tissues. For example, the collagen disclosed herein is a mixture of two or more collagens selected from the group consisting of fish collagen, porcine collagen, and bovine collagen. Additionally or alternatively, the collagen may be a mixture of collagens selected from collagens derived from skin, cartilage, bone, and / or connective tissue. As used herein, the term "hide" refers to the outer covering of a large animal, such as a bovine group or any other large animal. As used herein, the term "skin" refers to the outer covering of a small animal, such as a deer, goat, sheep, etc. The terms "skin" and "hide" can be used interchangeably in this 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 connective tissue from the corpus callosum, skin, antler, process (e.g., knob), horn, head, brain, neck, ear, eye, nose, tongue, lips, mouth, esophagus, trachea, limbs, feet, toe, palm, claw, bone, cartilage, bone marrow, joint, membrane, hind, ligament, tendon, rib, diaphragm, muscle, skeletal muscle, smooth muscle, intestine, blood vessels, bladder, stomach, aorta, heart, liver, kidney, chest, lung, spleen, pancreas, egg, sperm, testes, ovary, nerve, gallbladder, and belly.

[0067] In some embodiments, the collagen taught in the present disclosure is derived from skin and / or dermal connective tissue. In some preferred embodiments, the collagen taught in the present disclosure is derived from cartilage. In some preferred embodiments, the collagen taught in the present disclosure is derived from bone. The collagen taught in 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 a preferred embodiment, 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 a preferred embodiment, the collagen disclosed in the present disclosure is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen.

[0069] ●Enzymatic hydrolysis In one embodiment, the present invention provides a) providing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes, preferably an enzyme combination comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases; The present invention relates to a method for obtaining hydrolyzed collagen, comprising:

[0070] In one preferred embodiment, the present invention provides a) providing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes comprising a neutral protease, a carboxypeptidase, and an aminopeptidase; The present invention relates to a method for obtaining hydrolyzed collagen, comprising:

[0071] Preferably, step b) of the method disclosed in the present disclosure is or comprises 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. Additionally 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 preparations disclosed in the present disclosure are preferably aqueous preparations. In certain preferred embodiments, the liquid preparations disclosed in the present disclosure are or comprise water, and more preferably, the liquid preparations are or comprise distilled and / or demineralized water.

[0073] As part of the methods disclosed herein, the collagen-containing material may be provided in the liquid preparation in an amount of 10 wt.% (weight %) 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. Additionally 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 one embodiment, the collagen-containing material is provided in the liquid preparations disclosed herein in an amount of 20-50% by weight, preferably 30-40% by weight.

[0075] In some embodiments, the enzyme combinations disclosed herein are mixtures of said enzymes. In certain embodiments, the enzyme combinations disclosed herein comprise one or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases.

[0076] In certain embodiments, the enzyme combinations disclosed herein comprise two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases. In some embodiments, the enzyme combinations disclosed herein include a neutral protease, a carboxypeptidase, and an aminopeptidase. In some embodiments, the enzyme combinations disclosed herein comprise a neutral protease and a carboxypeptidase. In some embodiments, the enzyme combinations disclosed herein comprise a neutral protease and an aminopeptidase. In some embodiments, the enzyme combinations disclosed herein comprise a carboxypeptidase and an aminopeptidase.

[0077] In certain preferred embodiments, the enzyme combinations disclosed herein include a neutral protease and one or both of a carboxypeptidase and an aminopeptidase.

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

[0079] As used herein, "serine protease" refers to an enzyme that cleaves peptide bonds in proteins or peptides, where serine serves as the nucleophilic amino acid in the active site. The serine proteases disclosed herein may be chymotrypsin-like (trypsin-like) serine proteases. Additionally or alternatively, the serine proteases disclosed herein may be subtilisin-like serine proteases.

[0080] As used in this disclosure, an "aspartic protease" is a catalytic form of a protease enzyme that uses an activated water molecule bound to one or more aspartic acid residues for protein or peptide catalysis.

[0081] As used herein, "cysteine ​​protease" refers to a protease that has a catalytic mechanism that involves a nucleophilic cysteine ​​thiol in a catalytic triad or dyad for protein or peptide catalysis. "Cysteine ​​protease" can be used interchangeably with "thiol protease" in this disclosure. As used herein, "metalloprotease" refers to a protease that involves a metal, such as zinc or cobalt, in its catalytic mechanism. In this disclosure, the metalloprotease is preferably a zinc-dependent protease.

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

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

[0084] In some preferred embodiments, the neutral protease disclosed in this disclosure is a serine protease. In some preferred embodiments, the neutral protease disclosed in this disclosure is an aspartic acid protease. In some preferred embodiments, the neutral protease disclosed in this disclosure is a cysteine ​​protease. In some preferred embodiments, the neutral protease disclosed in this disclosure is a metalloprotease. In some preferred embodiments, the aminopeptidase disclosed in this disclosure is a leucine aminopeptidase.

[0085] An "enzyme" in the context of the present invention may be a commercially available enzyme. In some embodiments, the enzyme combinations disclosed in this disclosure are commercially available mixtures of enzymes. In some embodiments, the enzyme combinations disclosed in this disclosure are 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 a microorganism. In some embodiments, the enzymes disclosed herein (e.g., one or more enzymes in an enzyme combination) are derived from a microorganism of the genus Aspergillus, preferably Aspergillus oryzae and / or Aspergillus melleus. In some embodiments, the enzymes disclosed herein (e.g., one or more enzymes in an enzyme combination) are derived from a microorganism of the genus Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus aminolicuefaciens).

[0087] In one embodiment, an enzyme combination is used that includes no more than one enzyme derived from Bacillus sp., thereby excluding blends of endoproteases derived from Bacillus sp., such as the commercially available Protamex. In some embodiments, the enzymes disclosed herein (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 certain embodiments, one or more of the neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably from Aspergillus oryzae, hi certain preferred embodiments, the 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 (e.g., a commercially available mixture available from Sumizyme, which includes enzyme(s) from Aspergillus oryzae), or the proteases may be derived from separate sources (e.g., different microorganisms or different strains thereof). For example, the neutral protease may be derived from Bacillus, the carboxypeptidase may be derived from Aspergillus, and the aminopeptidase may be derived from Lactobacillus.

[0090] In certain embodiments, the enzymes disclosed in the present disclosure (eg, one or more enzymes in an enzyme combination) are not derived from plants (eg, bromelain, papain).

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

[0092] In a preferred embodiment, the amount of carboxypeptidase used in the enzymatic hydrolysis, preferably in step b) of the method disclosed herein, is defined by an enzymatic activity of 0.001-500 U / g, preferably 0.01-50 U / g, more preferably 0.1-5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation.

[0093] In a preferred embodiment, the amount of aminopeptidase used in the enzymatic hydrolysis, preferably in step b) of the method disclosed herein, is defined by an enzymatic activity of 0.002-500 U / g, preferably 0.02-50 U / g, more preferably 0.2-5 U / g, where the weight in g is the total weight of the collagen-containing material and the liquid preparation.

[0094] The time period for which the collagen-containing material is exposed to an enzyme 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 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. Additionally or alternatively, the time period for which the collagen-containing material is exposed to an enzyme disclosed herein, preferably to one or more of the combinations of enzymes disclosed herein (e.g., 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 an enzyme disclosed herein, preferably one or more of a combination of enzymes disclosed herein, for 60 to 180 minutes (e.g., in step b of the method).

[0096] The temperature at which the collagen-containing material is exposed to the enzymes disclosed herein, preferably to one or more of the combinations of enzymes disclosed herein (e.g., 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. Additionally or alternatively, the temperature at which the collagen-containing material is exposed to the enzymes disclosed herein, preferably to one or more of the combinations of enzymes disclosed herein (e.g., 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] As used in this disclosure, the term "temperature" preferably refers to the temperature of the 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 an enzyme disclosed herein, preferably one or more of a combination of enzymes disclosed herein, at a temperature between 30°C and 60°C (e.g., in step b of the method).

[0099] The use of the term "enzyme combination" disclosed in the present disclosure does not exclude the collagen-containing material being exposed to the enzymes (parts of the enzyme combination) sequentially. This means that the collagen-containing material is not necessarily exposed to all enzymes in the enzyme combination at the same time. In some embodiments, the enzymatic hydrolysis disclosed in the present disclosure (e.g., the hydrolysis in step b of the method) comprises two or more sequential hydrolysis steps, in which the collagen-containing material is preferably exposed to different enzymes and / or different enzyme mixtures. In some preferred embodiments, the collagen-containing material is sequentially exposed to two enzymes in the enzyme combination, preferably two selected from a neutral protease, a carboxypeptidase, and an aminopeptidase. In an embodiment, the enzymatic hydrolysis, preferably the enzymatic hydrolysis in step b of the method disclosed herein, comprises two or more hydrolysis steps, preferably two hydrolysis steps, wherein: The collagen-containing material is exposed to a neutral protease in one step and to a carboxypeptidase and / or an aminopeptidase in another step; The collagen-containing material is exposed to a carboxypeptidase in one step and to a neutral protease and / or an aminopeptidase in another step; The collagen-containing material is exposed to an aminopeptidase in one step and to a neutral protease and / or a carboxypeptidase in another step. In certain embodiments, the enzymatic hydrolysis disclosed herein comprises three hydrolysis steps, in which the collagen-containing material is preferably exposed independently to a neutral protease, a carboxypeptidase, and an aminopeptidase in each of the three hydrolysis steps.

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

[0101] If there is more than one hydrolysis step, the "time" that the hydrolyzed collagen is exposed to the enzyme preferably refers to the total time. For example, if the hydrolyzed collagen is exposed to enzyme A for 60 minutes in the first hydrolysis step and 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 that can be obtained by enzymatic hydrolysis In one aspect, the present invention relates to hydrolyzed collagen obtainable by the methods disclosed in the present disclosure. In the present disclosure, "hydrolyzed collagen" preferably means hydrolyzed collagen obtainable by the methods disclosed in the present disclosure. In certain preferred embodiments, the hydrolyzed collagen disclosed herein, e.g., collagen obtainable by the methods taught herein, is derived from collagen-containing material derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendons, ligaments, and connective tissue. In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure, e.g., collagen obtainable by the methods 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 certain preferred embodiments, the hydrolyzed collagen disclosed in the present disclosure, e.g., collagen obtainable by the methods taught in the present disclosure, is derived from a collagen-containing material, wherein the collagen-containing material is gelatin.

[0104] The hydrolyzed collagen disclosed herein, e.g., collagen obtainable by the methods taught herein, preferably has a (weight average) molecular weight of 1000 to 7000 Da, which 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, e.g., collagen obtainable by the methods taught in the present disclosure, may have a (weight average) molecular weight of 1000 Da or more (e.g., 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. Additionally or alternatively, the hydrolyzed collagen, e.g., collagen obtainable by the methods 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 weights disclosed in this disclosure are preferably weight average molecular weights. Molecular weight appears to play a role in the ability of the hydrolyzed collagen of the present invention to stimulate GLP-1 secretion and / or reduce blood glucose. In one embodiment, the hydrolyzed collagen has a weight average molecular weight of 1500 to 4500 Da, preferably 2000 to 4000 Da, more preferably 2500 to 3500 Da, and even more preferably 2750 to 3250 Da.

[0107] In one embodiment, the hydrolyzed collagen has a number average molecular weight (Mn) of 500 to 3500 Da, preferably 1000 to 3000 Da, more preferably 1500 to 2500 Da, and even more preferably 1750 to 2250 Da. In one embodiment, the hydrolyzed collagen has a weight average molecular weight of 2000 to 4000 Da, preferably 2500 to 3500 Da, and a number average molecular weight of 1000 to 3000 Da, preferably 1500 to 2500 Da.

[0108] Additionally or alternatively, molecular weight distribution appears to play a role in the ability of the hydrolyzed collagen of the present invention to stimulate GLP-1 secretion and / or reduce blood glucose. Compared to other hydrolyzed collagens, e.g., hydrolyzed collagens that are not obtained by enzymatic hydrolysis using a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases, the hydrolyzed collagen of the present invention exhibits, inter alia: a relatively high proportion of collagen peptides in the range of 2000-5000 Da (e.g., 35% or more, 40% or more, or even 45% or more); and / or a relatively low proportion of collagen peptides in the range below 1000 Da (e.g., 15% or less, 12% or less, or even 10% or less); and / or A relatively low percentage of collagen peptides in the range above 10,000 Da (e.g., less than 5%, less than 2%, or even less than 1%) may have.

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

[0111] In a preferred embodiment, the hydrolyzed collagen contains 5 to 25 wt %, preferably 10 to 20 wt %, of collagen peptides having a molecular weight within the range of 5,000 Da to 10,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

[0112] In a preferred embodiment, the hydrolyzed collagen contains 0 to 10 wt %, preferably 0.1 to 5 wt %, and more preferably 0.2 to 2 wt %, of collagen peptides having a molecular weight of more than 10,000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen. The preferred way to measure 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] An Agilent HPLC 1260 Infinity Series (G1316A, G1329B, G1311C, G1315D) with a TSK Gel SWXL precolumn and a G2000SWXL column (Toso Biosciences) was used. Analysis was performed using WinGPC software (PSS). The eluent was 100 mM phosphate buffer, pH 5.3. The sample was eluted from the column (e.g., 0.5 mL / min, isocratic) and monitored by UV detection (e.g., 214 nm, analysis time: 40 min per injection + 180 min equilibration). Calibration was performed using a Narrow Calibration Standard (Low FILK).

[0114] The hydrolyzed collagen, such as that obtainable by the methods taught in this disclosure, preferably has a polydispersity of 1.2 to 1.8, more preferably 1.3 to 1.7, and even more preferably 1.4 to 1.6, which appears to play a role in the high glucose-lowering 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. Additionally or alternatively, 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] As used in this disclosure, "polydispersity" refers to 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 certain preferred embodiments, 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) a meal, within 180 minutes (before or after) a meal, within 120 minutes (before or after) a meal, within 60 minutes (before or after) a meal, within 30 minutes (before or after) a meal, or within 15 minutes (before or after) a meal. In a preferred embodiment, the hydrolyzed collagen disclosed in the present disclosure is administered within 60 minutes (before or after) a meal.

[0118] In certain other embodiments, the daily dose of hydrolyzed collagen disclosed herein 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 daily dose is the amount by total dry weight administered to a subject per day. Additionally or alternatively, in certain other embodiments, the daily dose of hydrolyzed collagen disclosed herein is 200g or less, 190g or less, 180g or less, 170g or less, 160g or less, 150g or less, 140g or less, 130g or less, 120g or less, 110g or less, 100g or less, 95g or less, 90g or less, 85g or less, 80g or less, 75g or less, 70g or less, 65g or less, 60g or less, 55g or less, 50g or less, 45g or less, 40g or less, 35g or less, 30g or less, 25g or less, 20g or less, 15g or less, 10g or less, 5g or less, or 1g or less, where daily dose is the amount by total dry weight administered to a subject per day.

[0119] In a preferred embodiment, the use of hydrolyzed collagen disclosed in the present disclosure comprises 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, wherein the daily dose is the amount by total dry weight administered to a subject per day. In a preferred embodiment, the use of hydrolyzed collagen disclosed in the present disclosure comprises administering a daily dose of said hydrolyzed collagen in an amount of 1 g to 100 g, preferably 2 g to 50 g, more preferably 5 g to 25 g, said amount being the amount by dry weight of hydrolyzed collagen.

[0120] In certain other embodiments, the unit dose of hydrolyzed collagen disclosed herein 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, wherein said amount is by dry weight of hydrolyzed collagen. Additionally or alternatively, in certain other embodiments, the unit dose of hydrolyzed collagen disclosed herein is 200g or less, 190g or less, 180g or less, 170g or less, 160g or less, 150g or less, 140g or less, 130g or less, 120g or less, 110g or less, 100g or less, 95g or less, 90g or less, 85g or less, 80g or less, 75g or less, 70g or less, 65g or less, or less than 60g, 55g, 50g, 45g, 40g, 35g, 30g, 25g, 20g, 15g, 14g, 13g, 12g, 11g, 10g, 9g, 8g, 7g, 6g, 5g, 4g, 3g, 2g, or 1g, wherein said amount is by dry weight of hydrolyzed collagen.

[0121] In some embodiments, the hydrolyzed collagen disclosed herein is administered in a unit dose 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, wherein said amount is the amount of hydrolyzed collagen by dry weight.

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

[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 be the same or different amounts. The daily dose of hydrolyzed collagen disclosed herein is preferably administered as two unit doses, more preferably two unit doses each representing 30-70%, preferably 40-60%, of the daily dose amount.

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

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

[0126] In one embodiment, the daily dose of hydrolyzed collagen is administered as two or more unit doses, wherein: each unit dose is in an amount of 5 to 25 g, preferably 5 to 15 g, said amount being the amount by dry weight of hydrolyzed collagen; and / or The two unit doses are administered at least 6 hours apart, preferably at least 8 hours apart, more preferably at least 12 hours apart from each other.

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

[0128] Hydrolyzed collagen preparation The hydrolyzed collagen disclosed in the present disclosure may be provided in a food preparation, a dietary preparation, a food supplement preparation, a dietary supplement preparation, or a pharmaceutical preparation, preferably in a dietary supplement preparation. The hydrolyzed collagen disclosed in the present disclosure may be provided in a solid dosage form such as a capsule, tablet, or powder, preferably in powder form. The hydrolyzed collagen disclosed herein may be provided in preparations such as a drinkable solution or suspension, a beverage such as beer, a syrup, an artificially flavored beverage, a carbonated beverage, a (water-soluble) powdered mixture, a (water-soluble) paste, a (water-soluble) powder, a (water-soluble) tablet, a (water-soluble) pill, a (water-soluble) dragee, a (water-soluble) caplet, a (water-soluble) sachet, or a (water-soluble) capsule. Additionally or alternatively, the hydrolyzed collagen taught herein may be present in functional foods, including, for example, juices, shakes, dairy drinks, yogurt, yogurt drinks, desserts, energy bars, nutrition bars, slimming bars, or confectioneries such as gummies or center-filled gummies.

[0129] Use of hydrolyzed collagen in treatment methods In one embodiment, the present invention relates to a method of treatment, preferably a therapeutic method of treatment, for lowering blood glucose. In one embodiment, the present invention relates to a method of treatment, preferably a therapeutic method of treatment, for ameliorating hyperglycemia. In one embodiment, the present invention relates to methods of treatment, preferably therapeutic methods of treatment, for improving risk factors for hyperglycemia.

[0130] The treatment methods disclosed herein may be used in subjects experiencing high blood sugar (e.g., hyperglycemia), and the treatment methods may be preventative in nature. The treatment methods may include one or more features of the embodiments disclosed herein for the use of hydrolyzed collagen in reducing blood glucose and / or ameliorating (risk factors for) hyperglycemia, including the type of hydrolyzed collagen for use disclosed herein, the preparation of hydrolyzed collagen for use disclosed herein, the timing of administration or ingestion of hydrolyzed collagen for use disclosed herein, and the mode of administration (e.g., dosing regimen, unit dose, daily dose) of administration or ingestion of hydrolyzed collagen for use disclosed herein.

[0131] Use of hydrolyzed collagen for the preparation of medicines In one embodiment, 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 enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases. In one embodiment, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for ameliorating hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes, preferably a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases. In one embodiment, the present invention relates to the use of hydrolyzed collagen for the manufacture of a medicament for improving risk factors 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 enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases.

[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 for the use of hydrolyzed collagen in reducing blood glucose and / or ameliorating (risk factors for) hyperglycemia, 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 administration mode (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 definition As used in this disclosure, the terms "comprise" or "comprises" and their conjugations refer to the context in which the term is used in its open-ended sense, meaning that the items listed after the word are included, but not excluding anything not specifically mentioned. This also encompasses the more restrictive verbs "consist essentially of" and "consist only of." The recitation of an element with the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element is present. The indefinite article "a" or "an" therefore normally means "at least one".

[0134] The terms "increase" and "increased level" and "decrease" and "decreased level" (or "reduction" and "reduced level") preferably refer to a change of at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher or lower, respectively, than the corresponding concentration in the subject or reference. Additionally or alternatively, a concentration in a sample may be said to be increased or decreased if it is statistically significantly increased or decreased compared to the concentration in the subject or reference, regardless of the magnitude of the change. 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 per day. Breakfast, lunch, and dinner are considered to be major meals. Additionally or alternatively, a meal may be considered to include a reasonably 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 a human. The terms "administer" or "administration," as used in this disclosure, relate to the act of giving an agent to a subject who will ingest it. A subject who ingests an agent may administer the agent to themselves. In such cases, the term "administer to" is to be interpreted as "taken by," "used by," or "consumed by."

[0136] The term "unit dose", as used in this disclosure, relates to an amount or unit of, for example, a compound, substance, active ingredient, or composition used at one time. The unit dose may, for example, be in a ready-to-administer prepared form (e.g., a packaged formulation). The unit dose may, for example, be identifiable from the product packaging or label. The total daily dose may be divided into multiple unit doses. The term "active ingredient," as used in this disclosure, 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 merely confers a physical function. The biological response may be a direct or indirect response, and is preferably measured in cells or tissues.

[0137] ● Section In this disclosure, clauses are embodiments of the present invention. Features of clauses (embodiments) may be combined in this disclosure. Section 1: 1. Hydrolyzed collagen for use in reducing blood glucose, the 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 proteases, carboxypeptidases, and aminopeptidases. Section 2: Item 1. The hydrolyzed collagen according to item 1, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase. Section 3: Item 1 or 2. The hydrolyzed collagen according to item 1 or 2 for use in improving hyperglycemia. Section 4: Item 1 or 2. The hydrolyzed collagen according to item 1 or 2 for use in improving risk factors for hyperglycemia. Section 5: Item 5. The hydrolyzed collagen according to item 4, wherein the risk factors for hyperglycemia are 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: Item 11. The hydrolyzed collagen of any one of the preceding clauses for use in improving postprandial glucose. Section 7: Increases blood glucagon-like peptide-1; -Decreasing blood glucagon; ·Increasing blood insulin; Increases the blood insulin / glucose ratio Item 11. The hydrolyzed collagen of any one of the preceding items for use in one or more selected from the group consisting of:

[0138] Section 8: Item 11. The hydrolyzed collagen of 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, said amounts being by dry weight. Section 9: Item 11. The hydrolyzed collagen of any one of the preceding clauses, wherein the hydrolyzed collagen is administered orally, preferably as a dietary supplement. Section 10: Item 11. The hydrolyzed collagen of any one of the preceding clauses, wherein the hydrolyzed collagen is administered within 60 minutes of a meal. Section 11: Item 11. The hydrolyzed collagen of any one of the preceding items, 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. Section 12: Item 11. The hydrolyzed collagen of any one of the preceding items, wherein the collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen. Section 13: Item 11. The hydrolyzed collagen of any one of the preceding clauses, wherein the collagen-containing material is gelatin. Section 14: Item 10. The hydrolyzed collagen of any one of the preceding items, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity index of 1.2 to 1.8. Section 15: a) providing 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 proteases, carboxypeptidases, and aminopeptidases; A method for obtaining hydrolyzed collagen, comprising: Section 16: Item 16. The method of item 15, wherein the combination of enzymes comprises a neutral protease, a carboxypeptidase, and an aminopeptidase. Section 17: 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 proteases, aspartic acid proteases, cysteine ​​proteases, and metalloproteases. Section 18: Item 18. The method according to any one of Items 15 to 17, wherein the aminopeptidase is leucine aminopeptidase. Section 19: Item 19. The method according to any one of Items 15 to 18, wherein one or more of the 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 an amount of neutral protease defined by an enzymatic activity of 0.2 to 25,000 U / g, preferably 2 to 2,500 U / g, more preferably 20 to 250 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation.

[0139] Section 21: 21. The method according to any one of items 15 to 20, wherein the collagen-containing material is exposed to an amount of carboxypeptidase defined by an enzymatic activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation. Section 22: 22. The method according to any one of items 15 to 21, wherein the collagen-containing material is exposed to an amount of aminopeptidase defined by an enzymatic activity of 0.002 to 500 U / g, preferably 0.02 to 50 U / g, more preferably 0.2 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation. Section 23: 23. The method of any one of items 15 to 22, wherein the collagen-containing material is exposed to one or more enzymes in the enzyme combination for 60 to 180 minutes. Section 24: 24. The method of any one of items 15 to 23, wherein the collagen-containing material is exposed to one or more enzymes in the enzyme combination at 30°C to 60°C. Section 25: 25. The method of any one of items 15 to 24, wherein the collagen-containing material is exposed to one or more enzymes in the enzyme combination at a pH of 5 to 8.

[0140] Section 26: Item 26. The method of any one of items 15 to 25, wherein the collagen-containing material is sequentially exposed to two or more enzymes in the enzyme combination. Section 27: Item 27. The method of any one of items 15 to 26, wherein the collagen-containing material is simultaneously exposed to two or more enzymes in the enzyme combination. Section 28: 28. The method of claim 27, wherein the collagen-containing material is exposed to a mixture containing the combination of enzymes. Section 29: Item 29. The method according to any one of items 15 to 28, wherein the collagen-containing material is provided in the liquid preparation in an amount of 20 to 50% by weight, preferably 30 to 40% by weight. Section 30: Item 30. The method according to any one of Items 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. Section 31: Item 31. The method according to any one of Items 15 to 30, wherein the collagen is one or more types selected from the group consisting of porcine collagen, bovine collagen, and fish collagen. Section 32: Item 32. The method according to any one of Items 15 to 31, wherein the collagen-containing material is gelatin. Section 33: Item 33. Hydrolyzed collagen obtainable by the method according to any one of Items 15 to 32. Section 34: Item 34. The hydrolyzed collagen according to Item 33, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity index of 1.2 to 1.8. Section 35: Item 35. The hydrolyzed collagen according to Item 33 or 34, for use as defined in any one of Items 1 to 14.

[0141] Section 36: 1. A hydrolyzed collagen for use in improving hyperglycemia or risk factors for hyperglycemia, the hydrolyzed collagen being obtained by enzymatic hydrolysis of a collagen-containing material with a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases. Section 37: Item 37. The hydrolyzed collagen of item 36, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase. Section 38: Item 38. The hydrolyzed collagen according to Items 36 to 37, wherein the risk factors for hyperglycemia are one or more selected from the group consisting of insulin resistance, type 2 diabetes, gestational diabetes, high body mass index (BMI), obesity, and hyperglucagonemia. Section 39: Increases blood glucagon-like peptide-1; -Decreasing blood glucagon; ·Increasing blood insulin; Increases the blood insulin / glucose ratio Item 39. 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] Section 40: 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 1 to 100 grams, preferably 2 to 50 g, more preferably 5 to 25 g, said amount being the amount by dry weight. Section 41: Item 42. The hydrolyzed collagen according to any one of items 36 to 41, wherein the hydrolyzed collagen is administered orally, preferably as a food supplement. Section 42: Item 42. The hydrolyzed collagen according to any one of Items 36 to 41, wherein the hydrolyzed collagen is administered within 60 minutes after a meal. Section 43: Item 43. The hydrolyzed collagen according to any one of Items 36 to 42, 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. Section 44: Item 44. The hydrolyzed collagen according to any one of Items 36 to 43, wherein the collagen is one or more types selected from the group consisting of porcine collagen, bovine collagen, and fish collagen. Section 45: Item 45. The hydrolyzed collagen according to any one of Items 36 to 44, wherein the collagen-containing material is gelatin. Section 46: Item 46. The hydrolyzed collagen according to any one of Items 36 to 45, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity index of 1.2 to 1.8.

[0143] Section 47: 12. Use of hydrolyzed collagen for non-therapeutic lowering of blood glucose, wherein the hydrolyzed collagen is defined by any one of items 1 to 11. Section 48: Item 48. The use according to item 47, wherein the use is in improving postprandial glucose. Section 49: Item 49. The use of item 47 or 48, wherein the hydrolyzed collagen is administered as defined in any one of items 40 to 42. Section 50: a) providing 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 proteases, carboxypeptidases, and aminopeptidases; A method for obtaining hydrolyzed collagen, comprising: Section 51: 51. The method of claim 50, wherein the combination of enzymes comprises a neutral protease, a carboxypeptidase, and an aminopeptidase. Section 52: 52. The method of claim 50 or 51, wherein the neutral protease is one or more selected from the group consisting of serine proteases, aspartic proteases, cysteine ​​proteases, and metalloproteases. Section 53: 53. The method according to any one of Items 50 to 52, wherein the aminopeptidase is leucine aminopeptidase. Section 54: 54. The method according to any one of Items 50 to 53, wherein one or more of the neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably Aspergillus oryzae. Item 55: The method of any one of items 50 to 54, wherein the collagen-containing material is exposed to an amount of neutral protease defined by an enzymatic activity of 0.2 to 25000 U / g, preferably 2 to 2500 U / g, more preferably 20 to 250 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation.

[0144] Section 56: 56. The method according to any one of items 50 to 55, wherein the collagen-containing material is exposed to an amount of carboxypeptidase defined by an enzymatic activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation. Section 57: 57. The method according to any one of items 50 to 56, wherein the collagen-containing material is exposed to an amount of carboxypeptidase defined by an enzymatic activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation. Section 58: 58. The method according to any one of items 50 to 57, wherein the collagen-containing material is exposed to an amount of aminopeptidase defined by an enzymatic activity of 0.002 to 500 U / g, preferably 0.02 to 50 U / g, more preferably 0.2 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation. Section 59: 59. The method of any one of items 50 to 58, wherein the collagen-containing material is exposed to one or more enzymes in the enzyme combination for 60 to 180 minutes. Section 60: 60. The method of any one of items 50 to 59, wherein the collagen-containing material is exposed to one or more enzymes in the enzyme combination at 30°C to 60°C. Section 61: 61. The method of any one of items 50 to 60, wherein the collagen-containing material is exposed to one or more enzymes in the enzyme combination at a pH between 5 and 8.

[0145] Section 62: 62. The method of any one of 50 to 61, wherein the collagen-containing material is sequentially exposed to two or more enzymes in the enzyme combination. Section 63: Item 63. The method of any one of items 50 to 62, wherein the collagen-containing material is exposed to two or more enzymes in the enzyme combination simultaneously. Section 64: 64. The method of claim 63, wherein the collagen-containing material is exposed to a mixture containing the combination of enzymes. Section 65: 65. The method according to any one of items 50 to 64, wherein the collagen-containing material is provided in the liquid preparation in an amount of 20 to 50% by weight, preferably 30 to 40% by weight. Section 66: Item 66. The method according to any one of Items 50 to 65, 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. Section 67: Item 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, scales, cartilage, bone, tendon, ligament, and connective tissue. Section 68: Item 68. The method according to any one of Items 50 to 67, wherein the collagen-containing material is gelatin.

[0146] Section 69: Item 69. Hydrolyzed collagen obtainable by the method according to any one of items 50 to 68, wherein the hydrolyzed collagen is has a weight average molecular weight of 2000 to 4000 Da, preferably 2500 to 3500 Da; and The collagen peptide having a molecular weight in the range of 2000 to 5000 Da is contained in an amount of 35 to 60% by weight calculated based on the total weight of the collagen peptide in the hydrolyzed collagen. The hydrolyzed collagen. Section 70: Item 70. The hydrolyzed collagen according to Item 69, comprising 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 collagen peptides in the hydrolyzed collagen. Section 71: The hydrolyzed collagen is calculated based on the total weight of collagen peptides in the hydrolyzed collagen, 1 to 20% by weight of collagen peptides with a molecular weight of less than 1000 Da, and / or 20 to 40% by weight of collagen peptides having a molecular weight in the range of 1000 Da to 2000 Da, and / or 5 to 25% by weight of collagen peptides having a molecular weight in the range of 5,000 Da to 10,000 Da, and / or Collagen peptides with a molecular weight of over 10,000 Da (0-10% by weight) Item 71. The hydrolyzed collagen according to Item 69 or 70, comprising:

[0147] Section 72: Item 72. The hydrolyzed collagen according to any one of Items 69 to 71, wherein the hydrolyzed collagen has a polydispersity of 1.2 to 1.8. Section 73: Item 73. The hydrolyzed collagen according to any one of Items 69 to 72, wherein the hydrolyzed collagen is capable of stimulating glucagon-like peptide-1 secretion. Section 74: 74. The hydrolyzed collagen of any one of items 69 to 73 for use in improving hyperglycemia or risk factors for hyperglycemia as defined in any one of items 36 to 46. Section 75: 74. Use of hydrolyzed collagen according to any one of paragraphs 69 to 73 in non-therapeutic lowering of blood glucose as defined in any one of paragraphs 47 to 49.

[0148] ●●Experimental section Example 1 Example 1 shows the effect of gelatine hydrolysates obtained by enzymatic hydrolysis using a combination of enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases in increasing GLP-1 in vitro and lowering blood glucose in vivo.

[0149] ●Exam design A mixture of 14 different enzymes was used to hydrolyze gelatin into a hydrolyzed collagen preparation, which underwent simulated gastrointestinal digestion (SGID) to yield a bioactive hydrolyzed collagen fraction similar to that 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 cellular model for studying hormone secretion mechanisms in the gastrointestinal tract (Qi et al. Bio Protoc. 2020 Aug 20; 10(16): e3717). GLP-1 acts as a primary determinant of blood glucose homeostasis, in large part by regulating gastric emptying, enhancing pancreatic insulin secretion, and suppressing pancreatic glucagon secretion. GLP-1 secretion by STC-1 cells was therefore a desirable outcome parameter to determine how hydrolyzed collagen can improve glucose levels in the body. The most promising hydrolyzed collagen fractions identified in vitro were further tested for their glucose-lowering activity in mice. Glucose-lowering activity was established based on changes in plasma GLP-1, GIP, glucose, and insulin. The efficacy of the hydrolyzed collagen fractions was compared to that of the glucose-lowering drug sitagliptin.

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

[0152] Table 1. Overview 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, pH 7.4). Supernatants were collected after 2 hours of incubation. GLP-1 concentrations were determined by enzyme immunoassay and expressed in pg / ml. Data are presented as mean (n=3) ± standard deviation.

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

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

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

[0157] Plasma GLP-1 and GIP concentrations were measured one week after the OGTT test. To do so, mice were fasted for 6 hours and then treated with sitagliptin (400 μg / mouse) to prevent the degradation of GLP-1 and GIP by dipeptidyl peptidase IV (DPP-IV). Thirty minutes later, mice were administered 4 g of vehicle or test substance H080 per kg of body weight. 15 or 30 minutes after administration of the active ingredient, mice were anesthetized, and blood was collected from the portal vein. Plasma GLP-1 and GIP concentrations were measured by ELISA.

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

[0159] Following a similar procedure as described for healthy mice, studies were also carried out in obese mice with naturally occurring elevated blood glucose, with similar results.

[0160] ●Results In vitro GLP-1 secretion FIG. 1 shows GLP-1 secretion in STC-1 cells after incubation with 10 mg / ml of hydrolyzed collagen obtained by hydrolysis with the enzymes (mixtures) listed in Table 1 under optimal treatment conditions. Hydrolyzed collagen, designated H080, induced the greatest GLP-1 secretion. Compared to the blank, H080 induced a 5,800-fold increase in GLP-1 secretion. The H080-mediated increase in GLP-1 was more than 2.5-fold greater than the GLP-1 increase mediated by the other enzymes (mixtures) in the comparative study.

[0161] The data show that the combination of a neutral protease (e.g., a serine protease), a carboxypeptidase, and an aminopeptidase (e.g., a leucine aminopeptidase) induces the highest GLP-1 secretion, far higher than other enzymes and enzyme combinations. It has been found that the mere combination of two or more enzymes (e.g., the combination of two neutral proteases in "CH3") is not sufficient to further enhance GLP-1 secretion compared to the use of only one enzyme (e.g., neutral proteases in "CH1" and "CH2", among others).

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

[0163] The results shown in Figure 1 were reproduced with different batches of gelatin. These results indicate that hydrolysis of collagen using a combination of two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases is important for obtaining hydrolyzed collagen with strong glucose-reducing potential, such as GLP-1-induced glucose reduction.

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

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

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

[0167] Figure 5 shows plasma GIP concentrations after control treatment or treatment with 4 g / kg H080. Treatment with H080 reduced plasma GIP concentrations, and this effect was maximal 30 minutes after H080 ingestion.

[0168] In the healthy mouse model, the mice are administered with the active ingredient before glucose loading. Therefore, the healthy mice are particularly suitable for studying the prevention of hyperglycemia. Obese mice, which naturally have high blood glucose levels, were also tested. The methodology is similar to that described for healthy mice. Because obese mice already have increased blood glucose levels 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 with the active ingredient.

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

[0170] Combination of enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases in glucose-lowering activity The present inventors have discovered that high glucose-lowering activity (e.g., based on GLP-1 secretion levels) can be achieved when two or more enzymes selected from neutral proteases, carboxypeptidases, and aminopeptidases are used in the enzymatic hydrolysis of collagen-containing materials.

[0171] As shown in Table 2, the use of neutral protease in the enzymatic hydrolysis of gelatin leads to gelatin hydrolysates with limited, but glucose-reducing activity. Combining neutral protease with carboxypeptidase alone or with 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 collagen hydrolysis and the effect of hydrolysates in reducing glucose [Table 2]

[0173] Characterization of hydrolyzed collagen with high glucose-lowering activity Hydrolyzed collagens with high glucose-lowering activity (e.g., Groups III-IV in Table 2) are characterized by a weight-average molecular weight of 1000-7000 Da as determined by HPSEC and a polydispersity index (weight-average molecular weight / number-average molecular weight) of 1.2-1.8 (an average of about 1.56 is measured over 100 measurements).

[0174] Example 2 Example 2 shows the effect of process conditions on obtaining hydrolyzed collagen for lowering blood glucose. ●Method Gelatine hydrolysates were prepared 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 was tested, covering those typically used for enzymatic hydrolysis. The gelatin concentration in the starting solution was varied between 10% and 50% by weight. 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 effect of gelatin concentration, pH, and temperature of enzymatic hydrolysis on obtaining gelatin hydrolysate fractions for lowering blood glucose in preliminary studies. GLP-1 secretion was tested using the same method as in Example 1. Table 4 shows the effect of gelatin concentration, pH, and temperature of enzymatic hydrolysis on obtaining gelatin hydrolysate fractions for lowering blood glucose, based on further refined and more extensive testing. GLP-1 secretion was tested using the same procedure as in Example 1.

[0176] Table 3. Influence of gelatin concentration, pH, and temperature of enzymatic hydrolysis on obtaining gelatin hydrolysate fractions for lowering blood glucose in preliminary studies. [Table 3]

[0177] Table 4. Effect of gelatin concentration, pH, and temperature of enzymatic hydrolysis on obtaining gelatin hydrolysate fractions for lowering blood glucose, based on further refined and more extensive testing. +: 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 (wt % of gelatin), pH, and temperature. With respect to precursor concentration (% by weight of gelatin), good results were obtained throughout the range tested of 10% to 50% by weight, with an apparent optimum at concentrations of 20% to 35% by weight. With regard to pH, good results were obtained over the entire pH range tested, from pH 4.0 to pH 10.0, with an apparent optimum at pH 5.5 to pH 6.5. Good results were obtained over the entire temperature range tested, from 20°C to 60°C, with an apparent optimum at 45°C to 50°C.

[0179] These results suggest that hydrolysis performed at (excessively) high temperatures (e.g., temperatures above 70°C) may not lead to hydrolyzed collagen products suitable for enhancing GLP-1 secretion, which is believed to be a result of enzyme denaturation and subsequent impaired reaction kinetics.

[0180] A preferred combination of gelatin concentration, pH, and temperature may lead to the best activity in enhancing GLP-1 secretion by STC-1 cells. As shown in Table 5, a combination of gelatin concentration (30% to 40% by weight), pH (6 to 7.5), and temperature (40°C to 55°C) leads 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 a combination of neutral proteases, carboxypeptidases, and aminopeptidases, where enzymatic hydrolysis was performed 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 with various enzymes (mixtures) in relation to the ability of the 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 procedures used for assessing hydrolysis and GLP-1 secretion were the same as those described in Example 1. "H080" 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. 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 there was a 1000-fold or greater increase in GLP-1 secretion compared to stimulation with the blank control. As can be seen from Table 6, "H080" induced approximately 6000-fold increased GLP-1 secretion compared to stimulation with the blank control. "H080" is considered to be a "very high" GLP-1 secretion. Hydrolyzed collagens from groups "CH11-CH19" - that is, obtained by hydrolysis with either neutral protease, alkaline protease, or acid protease - show no / little 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 mean average molecular weight of approximately 2000 Da). "H080" is further characterized by a high proportion of collagen peptides (i.e., approximately 47.2%) that fall within the 2 kDa to 5 kDa range. "H080" is further characterized by a high stimulatory activity on GLP-1 secretion (i.e., an approximately 6000-fold increase compared to the blank control group). The "CH11-CH19" groups lack one or more of these characteristics.

[0186] Table 6. Summary of enzymes (mixtures) used for collagen hydrolysis, molecular weight and molecular weight distribution of the resulting hydrolyzed collagen, and their ability to stimulate GLP-1 secretion by STC-1 cells. [Table 6] Aspects of the present disclosure also include aspects described in the following sections. <1> 1. A hydrolyzed collagen for use in improving hyperglycemia or risk factors for hyperglycemia, the hydrolyzed collagen being obtained by enzymatic hydrolysis of a collagen-containing material with a combination of enzymes comprising two or more enzymes selected from the group consisting of neutral proteases, carboxypeptidases, and aminopeptidases. <2> the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase; <1> The hydrolyzed collagen according to claim 1. <3> Item 11. The hydrolyzed collagen of any one of the preceding items, wherein the risk factors for hyperglycemia are 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> Increases blood glucagon-like peptide-1; -Decreasing blood glucagon; ·Increasing blood insulin; Increases the blood insulin / glucose ratio 10. The hydrolyzed collagen of any one of the preceding claims for use in one or more selected from the group consisting of: <5> 7. The hydrolyzed collagen of any one of the preceding clauses, 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, said amounts being by dry weight. <6> Item 11. The hydrolyzed collagen of any one of the preceding clauses, wherein the hydrolyzed collagen is administered orally, preferably as a dietary supplement. <7> Item 11. The hydrolyzed collagen of any one of the preceding paragraphs, wherein the hydrolyzed collagen is administered within 60 minutes of a meal. <8> Item 11. The hydrolyzed collagen of any one of the preceding items, 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. <9> Item 10. The hydrolyzed collagen of any one of the preceding items, wherein the collagen is one or more selected from the group consisting of porcine collagen, bovine collagen, and fish collagen. <10> Item 11. The hydrolyzed collagen of any one of the preceding items, wherein the collagen-containing material is gelatin. <11> Item 10. The hydrolyzed collagen of any one of the preceding items, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity index of 1.2 to 1.8. <12> 1. Use of hydrolyzed collagen for non-therapeutic lowering of blood glucose, said hydrolyzed collagen comprising: <1> ~ <11> The above use is as defined in any one of the above. <13> Use in improving postprandial glucose. <12> Use as described in. <14> The hydrolyzed collagen <5> ~ <7> administered as specified in one of the following: <12> or <13> Use as described in. <15> a) providing 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 proteases, carboxypeptidases, and aminopeptidases; A method for obtaining hydrolyzed collagen, comprising: <16> the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase; <15> The method described below. <17> The neutral protease is one or more selected from the group consisting of serine proteases, aspartic proteases, cysteine ​​proteases, and metalloproteases. <15> or <16> The method described below. <18> the aminopeptidase is leucine aminopeptidase; <15> ~ <17> The method according to any one of the above. <19> one or more of the neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the genus Aspergillus, preferably Aspergillus oryzae; <15> ~ <18> The method according to any one of the above. <20> the collagen-containing material is exposed to an amount of neutral protease defined by an enzymatic activity of 0.2 to 25000 U / g, preferably 2 to 2500 U / g, more preferably 20 to 250 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation; <15> ~ <19> The method according to any one of the above. <21> the collagen-containing material is exposed to an amount of carboxypeptidase defined by an enzymatic activity of 0.001 to 500 U / g, preferably 0.01 to 50 U / g, more preferably 0.1 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation; <15> ~ <20> The method according to any one of the above. <22> the collagen-containing material is exposed to an amount of aminopeptidase defined by an enzymatic activity of 0.002 to 500 U / g, preferably 0.02 to 50 U / g, more preferably 0.2 to 5 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation; <15> ~ <21> The method according to any one of the above. <23> The collagen-containing material is exposed to one or more enzymes in the enzyme combination for 60 to 180 minutes. <15> ~ <22> The method according to any one of the above. <24> The collagen-containing material is exposed to one or more enzymes in the enzyme combination at a temperature between 30°C and 60°C. <15> ~ <22> The method according to any one of the above. <25> The collagen-containing material is exposed to one or more enzymes in the enzyme combination at a pH between 5 and 8. <15> ~ <24> The method according to any one of the above. <26> the collagen-containing material is sequentially exposed to two or more enzymes in the enzyme combination; <15> ~ <25> The method according to any one of the above. <27> The collagen-containing material is simultaneously exposed to two or more enzymes in the enzyme combination. <15> ~ <26> The method according to any one of the above. <28> The collagen-containing material is exposed to a mixture containing the enzyme combination. <27> The method described below. <29> The collagen-containing material is provided in the liquid preparation in an amount of 20 to 50% by weight, preferably 30 to 40% by weight. <15> ~ <28> The method according to any one of the above. <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; <15> ~ <29> The method according to any one of the above. <31> The collagen is one or more types selected from the group consisting of porcine collagen, bovine collagen, and fish collagen. <15> ~ <30> The method according to any one of the above. <32> The collagen-containing material is gelatin. <15> ~ <31> The method according to any one of the above. <33> <15> ~ <32> 1. A hydrolyzed collagen obtainable by any one of the methods described above, wherein the hydrolyzed collagen comprises: has a weight average molecular weight of 2000 to 4000 Da, preferably 2500 to 3500 Da; and The collagen peptide having a molecular weight in the range of 2000 to 5000 Da is contained in an amount of 35 to 60% by weight calculated based on the total weight of the collagen peptide in the hydrolyzed collagen. The hydrolyzed collagen. <34> The collagen peptide having a molecular weight in the range of 2000 to 5000 Da is contained in an amount of more than 40% by weight, calculated based on the total weight of the collagen peptide in the hydrolyzed collagen. <33> The hydrolyzed collagen according to claim 1. <35> The hydrolyzed collagen is calculated based on the total weight of collagen peptides in the hydrolyzed collagen, 1 to 20% by weight of collagen peptides with a molecular weight of less than 1000 Da, and / or 20 to 40% by weight of collagen peptides having a molecular weight in the range of 1000 Da to 2000 Da, and / or 5 to 25% by weight of collagen peptides having a molecular weight in the range of 5,000 Da to 10,000 Da, and / or Collagen peptides with a molecular weight of over 10,000 Da (0-10% by weight) include, <33> or <34> The hydrolyzed collagen according to claim 1. <36> The hydrolyzed collagen has a polydispersity of 1.2 to 1.8. <33> ~ <35> 1. The hydrolyzed collagen according to claim 1 . <37> The hydrolyzed collagen is capable of stimulating glucagon-like peptide-1 secretion. <33> ~ <36> 1. The hydrolyzed collagen according to claim 1 . <38> <1> ~ <11> for use in improving hyperglycemia or a risk factor for hyperglycemia as defined in any one of <33> ~ <37> 1. The hydrolyzed collagen according to claim 1 , <39> <12> ~ <14> In non-therapeutic lowering of blood glucose as defined in any one of <33> ~ <37> 10. Use of hydrolyzed collagen according to any one of the preceding claims.

Claims

1. 1. Hydrolyzed collagen obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes including a neutral protease and one or both of a carboxypeptidase and an aminopeptidase, said hydrolyzed collagen comprising: having a weight average molecular weight of 2000 to 4500 Da; and - Contains 35 to 60 wt% of collagen peptides having a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen; The hydrolyzed collagen.

2. 2. The hydrolyzed collagen of claim 1, wherein the hydrolyzed collagen is capable of stimulating glucagon-like peptide-1 secretion.

3. -Increasing glucagon-like peptide-1 in the blood; - Decreasing blood glucagon; -Increasing blood insulin; -Increasing the blood insulin / glucose ratio - improving postprandial blood glucose levels; - Improve postprandial blood glucose peaks, Improve postprandial glucose 3. The hydrolyzed collagen of claim 1 or claim 2, wherein one or more of the following are possible:

4. 3. The hydrolyzed collagen of claim 1 or claim 2, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material using a combination of enzymes including neutral proteases, carboxypeptidases, and aminopeptidases.

5. 3. The hydrolyzed collagen according to claim 1, comprising more than 40% by weight of collagen peptides having a molecular weight in the range of 2000 to 5000 Da, calculated relative to the total weight of collagen peptides in the hydrolyzed collagen.

6. The hydrolyzed collagen is calculated based on the total weight of collagen peptides in the hydrolyzed collagen, 1 to 20% by weight of collagen peptides with a molecular weight of less than 1000 Da, and / or 20-40% by weight of collagen peptides having a molecular weight in the range of 1000 Da to 2000 Da, and / or 5 to 25% by weight of collagen peptides having a molecular weight in the range of 5,000 Da to 10,000 Da, and / or 0 to 10% by weight of collagen peptides with a molecular weight of more than 10,000 Da 3. The hydrolyzed collagen of claim 1 or claim 2, comprising:

7. 3. A dietary supplement preparation comprising the hydrolyzed collagen of claim 1 or claim 2.

8. 3. An agent comprising the hydrolyzed collagen of claim 1 or claim 2, for use in non-therapeutic lowering of blood glucose.

9. 3. A method for treating hyperglycemia comprising administering to a subject a therapeutically effective amount of hydrolyzed collagen ....

10. a) providing a collagen-containing material in a liquid preparation; and b) exposing the collagen-containing material to a combination of enzymes including a neutral protease and one or both of a carboxypeptidase and an aminopeptidase; 1. A method for obtaining hydrolyzed collagen, comprising: Step b) is a step of preparing a hydrolyzed collagen product comprising: having a weight average molecular weight of 2000 to 4500 Da; and - Contains 35 to 60% by weight of collagen peptides with a molecular weight in the range of 2000 to 5000 Da, calculated based on the total weight of collagen peptides in the hydrolyzed collagen. This will be done until The method.

11. The method of claim 10, wherein the resulting hydrolyzed collagen is capable of stimulating glucagon-like peptide-1 secretion.

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