Hydrolyzed collagen for reducing blood glucose

Abstract

The present invention relates to hydrolyzed collagen formulations for reducing blood glucose. The hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material with an enzyme combination comprising two or more enzymes selected from the group consisting of a neutral protease, a carboxypeptidase and an aminopeptidase. The hydrolyzed collagen formulations are particularly suitable as dietary supplements, for example for ameliorating hyperglycemia and / or risk factors of hyperglycemia.

Description

Technical Field

[0001] The present invention relates to an active ingredient for improving hyperglycemia and disorders associated with hyperglycemia and uses thereof. The active ingredient of the present invention is suitable as a dietary supplement. Background Art

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

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

[0004] Regulation of blood glucose involves the interaction of different organs (eg, pancreas, brain, liver, intestine, adipose and muscle tissue) in part through the release of multiple hormones.

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

[0006] First-line treatment for hyperglycemia initially includes lifestyle changes that can mitigate one or more risk factors for hyperglycemia. These typically include lifestyle changes for insulin resistance, diabetes, obesity, or hypertension. Often, lifestyle changes alone are not enough and need to include metformin and / or insulin therapy as part of the first-line treatment.

[0007] In large groups of individuals, the above first-line treatments are unable to adequately relieve hyperglycemia. As possible second-line treatments, new classes of glucose-lowering drugs are currently being developed and tested (Davies et al. Diabetes Care 2018; 41(12): 2669-2701).

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

[0009] Another example of a new hypoglycemic drug is Sitagliptin. Sitagliptin is a selective inhibitor of dipeptidyl peptidase IV (DPPIV). DPPIV affects the hydrolysis of incretin hormones. By doing so, sitagliptin can increase the plasma concentration of active forms of incretins (e.g., GLP-1 and gastric inhibitory polypeptide (GIP)). This increases the release of insulin and reduces glucagon concentrations. However, professional medical practitioners remain hesitant to prescribe sitagliptin to patients. This is largely due to its several side effects and contraindications, for example, up to 10% of patients experience complications such as hypoglycemia, headache, airway infection, and nasopharyngitis.

[0010] Collagen peptides have also been suggested for lowering blood sugar. JP2009235064 describes hypoglycemic collagen peptides obtained by treating starfish with proteases, and JP2012116773 describes collagen peptides that increase insulin-like growth factor 1 (IGF-1) obtainable by treating fish scale and / or fish skin derived collagen with proteolytic enzymes.

[0011] There is still an unmet need for active ingredients that are more effective and / or safer to improve hyperglycemia. Such active ingredients can be used to add or replace the first-line and / or second-line treatment of individuals with hyperglycemia (or at risk of hyperglycemia). The present invention aims to provide such active ingredients. Summary of the invention

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

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

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

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

[0016] a) providing a collagen-containing material in a liquid formulation; and

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

[0018] In one aspect, the present invention relates to hydrolyzed collagen obtainable by the method as disclosed herein, wherein the hydrolyzed collagen is preferably used for lowering blood sugar and / or improving hyperglycemia. DETAILED DESCRIPTION

[0019] Some aspects of the invention

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

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

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

[0023] a) providing a collagen-containing material in a liquid formulation; and

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

[0025] In one aspect, the present invention relates to hydrolyzed collagen obtainable by a method as disclosed herein, wherein the hydrolyzed collagen is preferably used for lowering blood sugar and / or ameliorating hyperglycemia as taught herein.

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

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

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

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

[0030] Benefits of the Invention

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

[0032] - The inventors have found that the hypoglycemic effect of the hydrolyzed collagen of the present invention is similar to that of the pharmaceutical compound sitagliptin. Therefore, the hydrolyzed collagen is a valuable addition and / or alternative to hypoglycemic pharmaceutical compounds, which often cause side effects and have contraindications; and

[0033] - The inventors have found that the hypoglycemic effect of the hydrolyzed collagen of the present invention comprises different mechanisms of action. The mechanisms of action include increasing glucagon-like peptide-1 (GLP-1) and inhibiting dipeptidyl peptidase IV (DPP IV). The different effects of hydrolyzed collagen appear to explain the potent hypoglycemic activity of hydrolyzed collagen in vivo. In addition, the presence of different mechanisms of action indicates that hydrolyzed collagen may (also) be suitable for treating the potential effects of hyperglycemia (e.g., in different subject groups and / or at different stages of hyperglycemia);

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

[0035] Blood sugar and hyperglycemia

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

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

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

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

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

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

[0042] When referring to molecular levels, the term "blood" herein may be used interchangeably with the term "plasma". For example, "blood glucose" may also be considered "plasma glucose". For example, "blood GLP-1" may also be considered "plasma GLP-1".

[0043] As used herein, the term "hyperglycemia" means high blood glucose. In this article, it is believed that the normal blood glucose range in fasting individuals is 80 to 110 mg / dl. In this article, it is believed that individuals with blood glucose levels of 126 mg / dl or higher suffer from hyperglycemia. It is believed that individuals with fasting blood glucose of 111 to 125 mg / dL typically suffer from impaired glucose tolerance or prediabetes. In this article, it is believed that individuals suffering from "impaired glucose tolerance" or "prediabetes" suffer from hyperglycemia.

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

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

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

[0047] In one embodiment, hyperglycemia as disclosed herein includes "postprandial hyperglycemia". In the context of the present invention, "postprandial hyperglycemia" is defined as excessive postprandial blood sugar. The term "postprandial blood sugar level" (postprandial glucose level, PPG level) used herein refers to the amount of glucose in the blood after a meal. In healthy or non-diabetic individuals, blood sugar levels reach a peak about one hour after the start of a meal, and usually do not exceed 140 mg / dl, and return to pre-meal levels within 2 to 3 hours. These time spectra are usually different in diabetic patients or patients with different pathological conditions. In one embodiment, the PPG level is measured 2 hours after the start of the meal, however, another time may be more appropriate, depending on when the peak is usually (or expected) located. Acceptable PPG levels, threshold PPG levels for defining postprandial hyperglycemia, and the best method for measuring PPG levels may vary between medical professionals and medical practitioners. In the context of the present invention, PPG levels are considered to be excessive / too high (i.e., postprandial hyperglycemia) when PPG levels are above 140 mg / dl for adults under 50 years of age, above 150 mg / dl for individuals between 50 and 60 years of age, and above 160 mg / dl for individuals over 60 years of age.

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

[0049] The first group (herein "healthy subject group") comprises healthy individuals who do not obtain therapeutic benefit from treatment with the hydrolyzed collagen of the present invention, for example when the blood sugar level is not so high or severe as to be expected to cause health problems or (serious) distress. In addition or alternatively, the blood sugar may have a cause or severity such that the increase in blood sugar disappears naturally over time (i.e. it is not chronic). In addition or alternatively, the blood sugar level in the healthy subject group may not be so high or severe that they would not normally seek help from a professional medical practitioner.

[0050] The second group (herein "pathological subject group") comprises subjects in which the increase in blood glucose level is of a pathological nature, meaning that the increase in blood glucose level often results in severe pain symptoms and (severe) distress, and / or can result in (severe) health and psychological risks. Additionally, in the pathological subject group, the increase in blood glucose may be chronic. The severity of the increase in blood glucose is such that help from a professional medical practitioner is often sought.

[0051] A professional medical practitioner is usually able to determine on a case-to-case basis whether a change in blood glucose level requires therapeutic intervention or non-therapeutic intervention (i.e., whether the individual falls into the group of healthy subjects or the group of pathological subjects). For example, a professional medical practitioner can determine whether a change in blood glucose may lead to (serious) health and psychological risks and / or whether it is a chronic change in blood glucose.

[0052] Depending on whether the individual is healthy or experiences a medical condition (e.g., hyperglycemia, diabetes, obesity), the improvement of postprandial blood glucose can be considered to be a non-therapeutic intervention or a therapeutic intervention, respectively. In addition or alternatively, according to the amount of glucose in the blood after a meal, the improvement of postprandial blood glucose can be considered to be a non-therapeutic intervention or a therapeutic intervention. For example, in healthy individuals, the amount of glucose after a meal is usually no more than 140 mg / dl, so it can be considered that the improvement of postprandial blood glucose levels below 140 mg / dl is a non-therapeutic intervention. For example, in individuals experiencing medical conditions (e.g., hyperglycemia, diabetes, obesity), the amount of glucose after a meal can usually be 140 mg / dl or higher, so it can be considered that the improvement of postprandial blood glucose levels of 140 mg / dl or higher is a therapeutic intervention.

[0053] In one embodiment, hydrolyzed collagen is used for therapeutic lowering of blood sugar, such as for improving hyperglycemia or risk factors of hyperglycemia. In one embodiment, hydrolyzed collagen is used for non-therapeutic lowering of blood sugar, such as improving postprandial blood sugar.

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

[0055] In one embodiment, the hydrolyzed collagen as disclosed herein is (further) used to improve risk factors for hyperglycemia.

[0056] The beneficial effects of the hydrolyzed collagen of the present invention were confirmed in healthy mice in which hyperglycemia was induced after receiving the hydrolyzed collagen. The beneficial effects of the hydrolyzed collagen of the present invention were also confirmed in obese mice with hyperglycemia to which the hydrolyzed collagen was administered. This shows that the hydrolyzed collagen of the present invention is effective in both the prevention and treatment of hyperglycemia.

[0057] In the context of the present invention, the term "improvement" encompasses both "prevention" and "treatment" of a condition. The term "prevention" means ensuring that a subject does not develop a condition (e.g., hyperglycemia or risk factors for hyperglycemia). When a condition is delayed, the severity is reduced and / or the incidence is reduced, even when the condition is not completely prevented from occurring, intervention is also considered a form of "prevention" herein. "Prevention" or its variants used herein by intervention encompass situations in which the subject has previously experienced a condition (e.g., hyperglycemia or risk factors for hyperglycemia) but the intervention prevents the condition from recurring. "Prevention" or its variants may have therapeutic and / or non-therapeutic effects. If "prevention" or its variants are therapeutic in nature, it may also be directed to the symptoms of a disease or condition and / or its underlying pathological conditions. "Prevention" or its variants may be defined by any delay, change in severity and / or change in incidence, such as a change of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to a control or reference, as measured by any standard technique. In the context of the present invention, "treatment" means intervention to alleviate and / or cure a condition (e.g., hyperglycemia or risk factors for hyperglycemia) once the condition already exists. "Treatment" may have therapeutic and / or non-therapeutic effects. If "treatment" is therapeutic in nature, it may be directed to the symptoms of a disease or condition and / or its underlying pathological conditions. For example, "treatment" may be any reduction in the severity, incidence, and / or frequency of a condition, such as a reduction of at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to a control or reference, as measured by any standard technique. "Improvement" as used herein also encompasses "cure." The term "improvement" herein may be used interchangeably with "reduction" or "reduction."

[0058] "Risk factors for hyperglycemia" used herein means a condition associated with an increased chance of hyperglycemia. In individuals with risk factors for hyperglycemia, it is feasible to use hypoglycemic agents as prophylaxis in the prevention of hyperglycemia. As a supplement or alternative, in individuals with risk factors for hyperglycemia, it is preferably feasible to use hypoglycemic agents as prevention to improve hyperglycemia that may occur. For example, obesity is a risk factor for hyperglycemia. Using hypoglycemic agents to obese individuals can prevent hyperglycemia in the obese individuals and / or improve hyperglycemia in the individuals if it occurs.

[0059] There may be several risk factors for the occurrence of hyperglycemia. In the context of the present invention, low levels of insulin in the body, defective insulin action and / or insulin resistance are considered to be risk factors for hyperglycemia. In the present disclosure, type 2 diabetes is considered to be a risk factor for hyperglycemia. In addition or in lieu thereof, one or more of the following conditions are considered herein as risk factors for the occurrence of hyperglycemia: gestational diabetes, high body mass index (BMI), obesity and hyperglucagonemia.

[0060] Several hormones, when present in excess, function to raise blood sugar levels to hyperglycemia and are considered herein as risk factors for hyperglycemia, including excess of one or more of the following: Cortisol, catecholamines, growth hormone, glucagon, and thyroid hormone.

[0061] In a preferred embodiment, the risk factor for hyperglycemia as disclosed herein is insulin resistance. The term "insulin resistance" as used herein encompasses all conditions diagnosed as "insulin resistance" by professional medical practitioners. The term "insulin resistance" as used herein preferably refers to peripheral insulin resistance and / or hepatic insulin resistance. Insulin resistance is preferably diagnosed by the gold standard, which is the "hyperinsulinemiceuglycemic 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 levels without causing hypoglycemia. The process can take two hours and preferably includes the following steps (or similar steps): via a peripheral vein, at 10 to 120 mU / m per minute. 2 Insulin is infused. To compensate for the insulin infusion, glucose (20%) is infused to maintain blood glucose levels of 5 to 5.5 mmol / L. The rate of glucose infusion is determined by checking blood glucose levels every five to ten minutes (Muniyappa R, Lee S, Chen H, Quon MJ, January 2008, American Journal of Physiology. Endocrinology and Metabolism. 294 (1): E15–26). The glucose infusion rate during the last 30 minutes of the test determines insulin sensitivity. If a high level (7.5 mg / min or higher) is required, the patient is sensitive to insulin. Low levels (4.0 mg / min or lower) indicate insulin resistance. Levels between 4.0 mg / min and 7.5 mg / min are not definitive and indicate "impaired glucose tolerance," which is an early sign of insulin resistance.

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

[0063]

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

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

[0066] Other methods may also be applicable to measuring insulin resistance. In one embodiment, insulin sensitivity or insulin resistance is measured using an "insulin-suppression test" (IST). In one embodiment, insulin sensitivity or insulin resistance is measured using a "frequently sampled intravenous glucose tolerance test" (FSIVGTT). In one embodiment, insulin sensitivity or insulin resistance is measured using an "oral glucose tolerance test" (OGTT).

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

[0068] - Large waist - a waist measuring at least 35 inches (89 cm) for women and at least 40 inches (102 cm) for men;

[0069] - High triglyceride levels — 150 milligrams per deciliter (mg / dL), or 1.7 millimoles per liter (mmol / L), or higher of this type of fat in the blood;

[0070] - Reduced "good" or HDL cholesterol - high-density lipoprotein (HDL) cholesterol less than 40 mg / dL (1.04 mmol / L) for men or less than 50 mg / dL (1.3 mmol / L) for women;

[0071] Elevated blood pressure—130 / 85 millimeters of mercury (mm Hg) or higher;

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

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

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

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

[0076] The term "obesity" as used herein means a medical condition in which excess body fat accumulates to an extent that may have adverse effects on health. In some cases, obesity is merely a cosmetic problem (requiring non-therapeutic treatment). A professional medical practitioner can determine whether an individual is obese in one or more ways.

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

[0078] The present inventors have found that the hydrolyzed collagen of the present invention may have one or more of the following effects, for example, as a mechanism of action in lowering blood sugar and / or improving hyperglycemia:

[0079] - 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;

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

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

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

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

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

[0085] - Improves blood insulin / glucose ratio.

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

[0087] - Improve blood GLP-1, preferably increase blood GLP-1;

[0088] - Improve blood glucagon, preferably reduce blood glucagon;

[0089] - Improve blood insulin, preferably increase blood insulin;

[0090] - improving the blood insulin / glucose ratio, preferably increasing the blood insulin / glucose ratio;

[0091] - improving the level of DPP-IV in the body, preferably reducing the level of DPP-IV in the body, for example by reducing the secretion of DPP-IV by gastrointestinal cells 4;

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

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

[0094] 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. The biological activity appears to be associated with a specific molecular weight and / or molecular weight distribution of the hydrolyzed collagen, such as can be obtained by hydrolysis with a specific combination of enzymes.

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

[0096] - hydrolyzing gelatin to obtain a hydrolyzed collagen preparation;

[0097] - Subjecting the hydrolyzed collagen preparation to simulated gastrointestinal digestion (SGID) to obtain digested hydrolyzed collagen, preferably according to the protocol described by Song et al. (Food Funct. 2020 Jun 24; 11(6): 5553-5564);

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

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

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

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

[0102] Hydrolyzed collagen

[0103] As used herein, "hydrolyzed collagen" is a mixture of short-chain amino acids derived from a collagen-containing starting material having native (full-length) collagen, typically obtained by a hydrolysis step, including enzymatic hydrolysis (also known as enzymatic hydrolyzation). The degree of hydrolysis typically has an effect on the average molecular weight of the final product.

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

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

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

[0107] The collagen herein may be one or more selected from type I collagen, type II collagen, type III collagen, type V collagen, and type X collagen.

[0108] Preferably, the hydrolyzed collagen as disclosed herein is derived from one or more types of collagen selected from type I collagen, type II collagen and type III collagen. In addition or as an alternative, the hydrolyzed collagen as disclosed herein is preferably derived from an animal raw material comprising different collagen types (e.g., type I collagen, type II collagen and type III collagen, two or more). In addition or as an alternative, the hydrolyzed collagen as disclosed herein can be a mixture comprising two or more of type I, type II and type III collagen.

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

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

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

[0112] In various embodiments, collagen as disclosed herein is a mixture of collagen from different sources (e.g., collagen derived from a variety of animal species and / or collagen derived from different tissues). For example, collagen as disclosed herein can be a mixture of two or more collagens selected from fish collagen, porcine collagen, and bovine collagen. In addition or in lieu thereof, collagen can be a mixture of collagen selected from skin, cartilage, bone, and / or connective tissue derived collagen.

[0113] As used herein, the term "hide" means the outer covering of a large animal, such as from cattle or any other large animal. As used herein, the term "skin" means the outer covering of a small animal, such as deer, goats, sheep, etc. The terms "skin" and "hide" are used interchangeably herein and may refer to the outer covering of an animal regardless of size.

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

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

[0116] In a preferred embodiment, the collagen as disclosed herein is derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendon, ligament and connective tissue.

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

[0118] Enzymatic hydrolysis

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

[0120] a) providing a collagen-containing material in a liquid formulation; and

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

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

[0123] a) providing a collagen-containing material in a liquid formulation; and

[0124] b) Subjecting the collagen-containing material to a combination of enzymes comprising a neutral protease, a carboxypeptidase and an aminopeptidase.

[0125] Preferably, step b) of the process as disclosed herein is or encompasses a step of enzymatic hydrolysis.

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

[0127] As part of the method as disclosed herein, the material containing collagen can be provided in a liquid preparation in an amount of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 % by weight (wt %). In addition or as an alternative, the material containing collagen can be provided in a liquid preparation in an amount of no more than 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10 % by weight.

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

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

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

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

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

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

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

[0135] The term "neutral protease" as used herein means a class of proteases that can catalyze the hydrolysis of peptide bonds of proteins in a neutral, weakly acidic or weakly alkaline environment. In the present disclosure, it is believed that the optimal pH of neutral proteases in enzymatic hydrolysis is 6.0 to 7.5. Based on the catalytic mechanism and the functional group of the active site, neutral proteases can be divided into four major categories: serine proteases, aspartic acid proteases, cysteine ​​proteases and metalloproteases.

[0136] "Serine protease" as used herein means an enzyme that cuts a peptide bond in a protein or peptide, wherein serine is used as a nucleophilic amino acid in the active site. As disclosed herein, a serine protease may be a chymotrypsin-like (trypsin-like) serine protease. In addition or as an alternative, as disclosed herein, a serine protease may be a subtilisin-like serine protease.

[0137] As used herein, "aspartic protease" refers to a catalytic protease that utilizes activated water molecules bound to one or more aspartic acid residues of a protein or peptide for catalysis.

[0138] As used herein, "cysteine ​​protease" means a protease having a catalytic mechanism involving a nucleophilic cysteine ​​thiol group in the catalytic triad or dyad for catalysis of a protein or peptide. "Cysteine ​​protease" is used interchangeably with "thiol protease" herein.

[0139] As used herein, "metalloprotease" means a protease whose catalytic mechanism involves a metal, such as zinc or cobalt. In the present disclosure, the metalloprotease is preferably a zinc-dependent protease.

[0140] As used herein, the term "carboxypeptidase" means a protease that hydrolyzes peptide bonds at the carboxyl terminus (C-terminus) of a protein or peptide.

[0141] Carboxypeptidases are generally classified into one of several families based on their active site mechanism. In one embodiment, the carboxypeptidase herein is a metallocarboxypeptidase, ie a carboxypeptidase that uses a metal in the active site for the catalytic mechanism.

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

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

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

[0145] In one embodiment, an enzyme (eg, one or more enzymes of a combination of enzymes) as disclosed herein is derived from a microorganism.

[0146] In one embodiment, an enzyme as disclosed herein (eg, one or more enzymes of a combination of enzymes) is derived from a microorganism of the genus Aspergillus, preferably from Aspergillus oryzae and / or Aspergillus melleus.

[0147] In one embodiment, an enzyme as disclosed herein (e.g., one or more enzymes of a combination of enzymes) is derived from a microorganism of the genus Bacillus (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens).

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

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

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

[0151] The protease used in the present invention can be a mixture of enzymes (e.g., a commercial mixture obtained from Sumizyme comprising an enzyme from Aspergillus oryzae), or the protease can be derived from different sources (e.g., from different microorganisms or strains thereof). For example, the neutral protease can be derived from Bacillus, the carboxypeptidase from Aspergillus, and the aminopeptidase from Lactobacillus.

[0152] In one embodiment, an enzyme as disclosed herein (eg, one or more of the combination of enzymes) is not derived from a plant (eg, bromelain, papain).

[0153] In a preferred embodiment, the amount of neutral protease used in the enzymatic hydrolysis, preferably in step b) of the process as disclosed herein, is defined by an enzyme activity of 0.2 to 25000 U / g, preferably 2 to 2500 U / g, more preferably 20 to 250 U / g, wherein the weight in g is the total weight of the collagen-containing material and the liquid preparation.

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

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

[0156] The time for which the collagen-containing material is subjected (e.g. in step b of the method) to an enzyme as disclosed herein (preferably one or more of a combination of enzymes as disclosed herein) may be at least

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

[0158] Additionally or alternatively, the collagen-containing material may be subjected (e.g. in step b of the method) to an enzyme as disclosed herein (preferably one or more of a combination of enzymes as disclosed herein) for a period not exceeding

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

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

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

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

[0163] Additionally or alternatively, the temperature at which the collagen-containing material is subjected (e.g. in step b of the method) to an enzyme as disclosed herein (preferably one or more of a combination of enzymes as disclosed herein) may not exceed

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

[0165] As used herein, "temperature" preferably means the temperature of the liquid formulation (including the collagen-containing material therein), preferably the temperature in which the collagen-containing material is dissolved.

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

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

[0168] In a preferred embodiment, the collagen-containing material is subjected successively to two enzymes of the combination of enzymes, preferably successively to two selected from the group consisting of neutral proteases, carboxypeptidases and aminopeptidases.

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

[0170] - subjecting the collagen-containing material to a neutral protease in one step and to a carboxypeptidase and / or an aminopeptidase in another step;

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

[0172] - Subjecting the collagen-containing material to an aminopeptidase in one step and to a neutral protease and / or a carboxypeptidase in another step.

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

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

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

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

[0177] Products obtainable by enzymatic hydrolysis.

[0178] In one aspect, the present invention relates to hydrolyzed collagen obtainable by a process as disclosed herein.

[0179] In the present disclosure, "hydrolyzed collagen" preferably means hydrolyzed collagen obtainable by the method as disclosed herein.

[0180] In a preferred embodiment, the hydrolyzed collagen disclosed herein (e.g., obtainable by the methods taught herein) is derived from a collagen-containing material derived from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendon, ligament, and connective tissue.

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

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

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

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

[0185] The average molecular weight as disclosed herein is preferably a weight average molecular weight.

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

[0187] In one embodiment, the weight average molecular weight of the hydrolyzed collagen is from 1500 to 4500 Da, preferably from 2000 to 4000 Da, more preferably from 2500 to 3500 Da, even more preferably from 2750 to 3250 Da.

[0188] In one embodiment, the number average (M) of hydrolyzed collagen n ) has a molecular weight of 500 to 3500 Da, preferably 1000 to 3000 Da, more preferably 1500 to 2500 Da, even more preferably 1750 to 2250 Da.

[0189] 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.

[0190] Additionally or alternatively, it is shown that molecular weight distribution plays a role in the ability of the hydrolyzed collagen of the present invention to stimulate GLP-1 secretion and / or lower blood glucose. Compared to other hydrolyzed collagens, for example, hydrolyzed collagens not obtained by enzymatic hydrolysis with an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases, the hydrolyzed collagen of the present invention may have:

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

[0192] - a relatively low fraction of collagen peptides falling within the range below 1000 Da (e.g. no more than 15%, or no more than 12%, or even no more than 10%); and / or

[0193] - A relatively low fraction of collagen peptides falling within the range above 10000 Da (eg no more than 5%, or no more than 2%, or even no more than 1%).

[0194] In a preferred embodiment, the hydrolyzed collagen comprises 1 wt% to 20 wt%, preferably 2 wt% to 12 wt%, of collagen peptides having a molecular weight below 1000 Da, based on the total weight of collagen peptides in the hydrolyzed collagen.

[0195] In a preferred embodiment, the hydrolyzed collagen comprises 20 to 40 wt%, preferably 25 to 35 wt%, of collagen peptides having a molecular weight in the range of 1000 to 2000 Da, based on the total weight of collagen peptides in the hydrolyzed collagen.

[0196] Preferably, the hydrolyzed collagen comprises less than 35 wt%, more preferably less than 32.5 wt%, even more preferably less than 30 wt% of collagen peptides having a molecular weight in the range of 1000 to 2000 Da, based on the total weight of collagen peptides in the hydrolyzed collagen.

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

[0198] Preferably, the hydrolyzed collagen comprises more than 40 wt%, more preferably more than 42.5 wt%, even more preferably more than 45 wt% of collagen peptides having a molecular weight in the range of 2000 to 5000 Da, based on the total weight of collagen peptides in the hydrolyzed collagen.

[0199] In a preferred embodiment, the hydrolyzed collagen comprises 5 to 25 wt%, preferably 10 to 20 wt%, of collagen peptides having a molecular weight in the range of 5000 to 10000 Da, based on the total weight of collagen peptides in the hydrolyzed collagen.

[0200] In a preferred embodiment, the hydrolyzed collagen contains 0 wt % to 10 wt %, preferably 0.1 wt % to 5 wt %, more preferably 0.2 wt % to 2 wt % of collagen peptides with a molecular weight higher than 10000 Da, based on the total weight of collagen peptides in the hydrolyzed collagen.

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

[0202] Agilent HPLC, 1260 Infinity series (G1316A, G1329B, G1311C, G1315D) with TSKgel SWXL precolumn and G2000SWXL column (Tosoh Bioscience) was used. Analysis was performed using WinGPC software (PSS). The eluent was 100 mM phosphate buffer at pH 5.3.

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

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

[0205] The polydispersity of hydrolyzed collagen (e.g. obtainable by the method taught herein) is preferably 1.2 to 1.8, more preferably 1.3 to 1.7, even more preferably 1.4 to 1.6. This polydispersity shows that the hydrolyzed collagen of the present invention plays a role in high glucose-lowering effect.

[0206] The polydispersity of the hydrolyzed collagen may be no more than 1.85, or no more than 1.75, or no more than 1.70, or no more than 1.65, or no more than 1.60, or no more than 1.55. In addition or alternatively, the polydispersity of the hydrolyzed collagen may be at least 1.20, or at least 1.25, or at least 1.30, or at least 1.35, or at least 1.40, or at least 1.45, or at least 1.50, or at least 1.55.

[0207] As used herein, "polydispersity" means M w / M n , where M w is the weight average molecular weight (Da), and M n is the number average molecular weight (Da).

[0208] M wGiven by:

[0209]

[0210] M n Given by:

[0211]

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

[0213] Administration of hydrolyzed collagen

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

[0215] In order to improve postprandial blood sugar level, the hydrolyzed collagen as disclosed herein is preferably used during meals or before and after meals. In the context of the present invention, the hydrolyzed collagen is preferably used no more than 240, 180, 120, 60, 30 or 15 minutes away from meals (before or after). In a preferred embodiment, the hydrolyzed collagen as disclosed herein is used no more than 60 minutes away from meals (before or after).

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

[0217] 1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190,or 200,

[0218] All in g, wherein the daily dose is the total dry weight administered to the subject per day. In addition or alternatively, in some different embodiments, the daily dose of the hydrolyzed collagen as disclosed herein does not exceed

[0219] 200,190,180,170,160,150,140,130,120,110,100,95,90,85,80,75,70,65,60,55,50,45,40,35,30,25,20,15,10,5,or1,

[0220] All are in grams, where the daily dose is the total dry weight administered to a subject per day.

[0221] In a preferred embodiment, the use of hydrolyzed collagen as disclosed herein comprises administering 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 total dry weight administered to the subject per day.

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

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

[0224] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190,or 200,

[0225] All in g, wherein the amount is the dry weight of the hydrolyzed collagen. In addition or alternatively, in some different embodiments, the amount of a unit dose of the hydrolyzed collagen disclosed herein does not exceed

[0226] 200,190,180,170,160,150,140,130,120,110,100,95,90,85,80,75,70,65,60,55,50,45,40,35,30,25,20,15,14,13,12,11,10,9,8,7,6,5,4,3,2,or1,

[0227] All are in g, where the amounts stated are the dry weight of the hydrolyzed collagen.

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

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

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

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

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

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

[0234] - The two unit doses are administered at least 6 hours, preferably 8 hours, more preferably 12 hours apart from each other.

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

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

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

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

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

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

[0241] In one embodiment, the hydrolyzed collagen is administered daily or every other day.

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

[0243] In one embodiment, the hydrolyzed collagen is administered at least once a day.

[0244] In one embodiment, the dosage regimen of hydrolyzed collagen as disclosed herein comprises administering hydrolyzed collagen for at least 2 consecutive days, preferably for at least 4 consecutive days, more preferably for at least 7 consecutive days.

[0245] In one embodiment, the dosage regimen of hydrolyzed collagen as disclosed herein comprises administering hydrolyzed collagen for at least 2 consecutive weeks, preferably for at least 4 consecutive weeks, more preferably for at least 8 consecutive weeks.

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

[0247] Preparation of hydrolyzed collagen

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

[0249] The hydrolyzed collagen as disclosed herein may be provided in a solid dosage form, such as a capsule, a tablet or a powder, preferably a powder.

[0250] The hydrolyzed collagen as disclosed herein can be provided in the form of a preparation 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. In addition or in lieu thereof, the hydrolyzed collagen as taught herein can be present in functional foods such as juices, milkshakes, dairy drinks, yogurt, yogurt drinks, desserts, energy bars, nutrition bars, slimming bars, or confectionery such as soft candies or center-filled soft candies.

[0251] Use of hydrolyzed collagen in therapeutic treatments

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

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

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

[0255] The therapeutic methods as disclosed herein can be used for subjects experiencing hyperglycemia (e.g., hyperglycemia), wherein the therapeutic methods can be preventive in nature. The therapeutic methods may include one or more features of the embodiments of the use of hydrolyzed collagen described herein in reducing blood sugar and / or improving (risk factors for) hyperglycemia, including the type of hydrolyzed collagen used for the use as disclosed herein, the preparation of the hydrolyzed collagen used for the use as disclosed herein, the time of administration or intake of the hydrolyzed collagen used for the use as disclosed herein, and the dosage (e.g., dosage regimen, unit dose, daily dose) of the hydrolyzed collagen used for the use as disclosed herein or the intake.

[0256] Use of hydrolyzed collagen in preparing medicines

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

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

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

[0260] The use of hydrolyzed collagen for the preparation of a medicament as disclosed herein may include one or more features of the embodiments described herein for the use of hydrolyzed collagen in lowering blood sugar and / or improving (risk factors for) hyperglycemia, including the type of hydrolyzed collagen for the use disclosed herein, the formulation of the hydrolyzed collagen for the use disclosed herein, the time of administration or ingestion of the hydrolyzed collagen for the use disclosed herein, and the dose (e.g., dosage regimen, unit dose, daily dose, daily amount) of the hydrolyzed collagen for the use disclosed herein.

[0261] General Definition

[0262] - As used herein, the terms "comprises / comprising" or "to comprise / including" and variations thereof refer to instances where the terms are used in their non-limiting sense to mean including items following the word, but not excluding items not specifically mentioned. It also encompasses the more restrictive verbs "consisting essentially of" and "consisting of.

[0263] Unless the context clearly requires the presence of one and only one element, the reference to an element by a noun without a quantifier does not exclude the possibility that more than one element is present. Thus, a noun without a quantifier generally means "at least one".

[0264] - The terms "increase" and "increased levels" and the terms "decrease" and "decreased levels" (or "reduction" and "reduced levels") preferably refer to a change of at least 5%, for example 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% above or below the corresponding level of a control or reference, respectively. Additionally or alternatively, the level in a sample may be increased or decreased when there is a statistically significant increase or decrease compared to the level in a control or reference, regardless of the degree of change.

[0265] - The term "meal" as used herein refers to an eating occasion that occurs at a specific time, and includes prepared foods. Meals occur on a daily basis, usually several times a day. Breakfast, lunch and dinner are considered to be major meals. In addition or alternatively, a meal may be considered to contain relatively large amounts of food (e.g. at least 500 to 1000 kcal per meal).

[0266] - The term "subject" or "individual" as used herein refers to any animal (eg, mammal), preferably including humans.

[0267] - As used herein, the term "administering" or variations thereof relates to the act of providing a drug to a subject consuming the drug. A subject consuming a certain drug may administer the drug to himself / herself / itself. In such cases, the term "administering to" may be interpreted as "taken by", "used by" or "consumed by".

[0268] - The term "unit dose" as used herein relates to an amount or unit of a compound, substance, active ingredient or composition, e.g. for immediate use. For example, a unit dose may be in a pre-prepared form (e.g. a pre-packaged dose) ready for administration. For example, a unit dose may (also) be identifiable from the product packaging or label. The total daily dose may be divided into a plurality of unit doses.

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

[0270] Terms

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

[0272] Item 1: Hydrolyzed collagen for use in lowering blood sugar, wherein the hydrolyzed collagen is obtained by enzymatically hydrolyzing a collagen-containing material with an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases.

[0273] Item 2: The hydrolyzed collagen for use according to Item 1, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase and an aminopeptidase.

[0274] Item 3: The hydrolyzed collagen used according to Item 1 or 2 for improving hyperglycemia.

[0275] Item 4: The hydrolyzed collagen used according to Item 1 or 2 for improving risk factors for hyperglycemia.

[0276] Item 5: The hydrolyzed collagen for use 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.

[0277] Item 6: The hydrolyzed collagen used according to any one of the preceding items for improving postprandial blood sugar.

[0278] Item 7: The hydrolyzed collagen used according to any one of the preceding items, for use in one or more selected from the following:

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

[0280] - Decreases blood glucagon;

[0281] - Increase blood insulin;

[0282] - Improves blood insulin / glucose ratio.

[0283] Clause 8: Hydrolyzed collagen for use according to any one of the preceding clauses, wherein the hydrolyzed collagen is administered in a daily dose in an amount of 1 to 100 g, preferably 2 to 50 g, more preferably 5 to 25 g, wherein the amount is by dry weight.

[0284] Item 9: Hydrolyzed collagen for use according to any one of the preceding items, wherein the hydrolyzed collagen is administered orally, preferably as a dietary supplement.

[0285] Clause 10: Hydrolyzed collagen for use according to any one of the preceding clauses, wherein the hydrolyzed collagen is administered no more than 60 minutes apart from a meal.

[0286] Clause 11: Hydrolyzed collagen for use according to any one of the preceding clauses, 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.

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

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

[0289] Item 14: The hydrolyzed collagen for use according to any one of the preceding items, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity of 1.2 to 1.8.

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

[0291] a) providing a collagen-containing material in a liquid formulation; and

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

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

[0294] Clause 17: The method according to clause 15 or 16, wherein the neutral protease is one or more selected from the group consisting of a serine protease, an aspartic acid protease, a cysteine ​​protease and a metalloprotease.

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

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

[0297] Item 20: A method according to any one of items 15 to 19, wherein the collagen-containing material is subjected to an amount of neutral protease defined by an enzyme activity of 0.2 to 25000 U / g, 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.

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

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

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

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

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

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

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

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

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

[0307] Clause 30: A method according to any one of clauses 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.

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

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

[0310] Item 33: Hydrolyzed collagen obtainable by the process according to any one of items 15 to 32.

[0311] Item 34: Hydrolyzed collagen according to Item 33, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity of 1.2 to 1.8.

[0312] Item 35: Hydrolyzed collagen according to Item 33 or 34, for use as defined in any one of Items 1 to 14.

[0313] Item 36: Hydrolyzed collagen for use in improving hyperglycemia or risk factors of hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatically hydrolyzing a collagen-containing material with an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases.

[0314] Item 37: The hydrolyzed collagen for use according to Item 36, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase and an aminopeptidase.

[0315] Clause 38: Hydrolyzed collagen for use according to clauses 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, obesity and hyperglucagonemia.

[0316] Clause 39: The hydrolyzed collagen according to any one of clauses 36 to 38, for use in one or more selected from the following:

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

[0318] - Decreases blood glucagon;

[0319] - Increase blood insulin;

[0320] - Improves blood insulin / glucose ratio.

[0321] Item 40: Hydrolyzed collagen for use according to any one of items 36 to 39, wherein the hydrolyzed collagen is administered in a daily dose in an amount of 1 to 100 g, preferably 2 to 50 g, more preferably 5 to 25 g, wherein the amount is by dry weight.

[0322] Item 41 : Hydrolyzed collagen for use according to any one of items 36 to 41 , wherein the hydrolyzed collagen is administered orally, preferably as a dietary supplement.

[0323] Item 42: Hydrolyzed collagen for use according to any one of items 36 to 41, wherein the hydrolyzed collagen is administered no more than 60 minutes apart from a meal.

[0324] Item 43: Hydrolyzed collagen for use 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.

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

[0326] Clause 45: Hydrolyzed collagen for use according to any one of clauses 36 to 44, wherein the collagen-containing material is gelatin.

[0327] Item 46: The hydrolyzed collagen for use 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 of 1.2 to 1.8.

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

[0329] Clause 48: The use according to Clause 47, wherein the use is for improving postprandial blood sugar.

[0330] Clause 49: Use according to clause 47 or 48, wherein the hydrolyzed collagen is administered as defined in any one of clauses 40 to 42.

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

[0332] a) providing a collagen-containing material in a liquid formulation; and

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

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

[0335] Clause 52: The method according to clause 50 or 51, wherein the neutral protease is one or more selected from the group consisting of a serine protease, an aspartic acid protease, a cysteine ​​protease and a metalloprotease.

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

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

[0338] Item 55: A method according to any one of items 50 to 54, wherein the collagen-containing material is subjected to an amount of neutral protease defined by an enzyme activity of 0.2 to 25000 U / g, 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.

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

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

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

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

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

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

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

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

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

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

[0349] Clause 66: A method according to any one of clauses 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.

[0350] Clause 67: A method according to any one of clauses 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.

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

[0352] Clause 69: Hydrolyzed collagen obtainable by the process according to any one of clauses 50 to 68, wherein the hydrolyzed collagen:

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

[0354] - comprising 35% to 60% by weight of collagen peptides having a molecular weight ranging from 2000 to 5000 Da, calculated on the total weight of collagen peptides in the hydrolyzed collagen.

[0355] Clause 70: Hydrolyzed collagen according to clause 69, wherein the hydrolyzed collagen comprises more than 40% by weight of collagen peptides having a molecular weight in the range of 2000 to 5000 Da, calculated on the total weight of collagen peptides in the hydrolyzed collagen.

[0356] Clause 71: Hydrolyzed collagen according to clause 69 or 70, wherein the hydrolyzed collagen comprises:

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

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

[0359] - 5% to 25% by weight of collagen peptides having a molecular weight of 5000 to 10000 Da; and / or

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

[0361] Clause 72: The hydrolyzed collagen according to any one of clauses 69 to 71, wherein the polydispersity of the hydrolyzed collagen is from 1.2 to 1.8.

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

[0363] Clause 74: Hydrolyzed collagen according to any one of clauses 69 to 73, for use in improving hyperglycemia or a risk factor for hyperglycemia as defined in any one of clauses 36 to 46.

[0364] Clause 75: Use of hydrolyzed collagen according to any one of clauses 69 to 73 for non-therapeutic lowering of blood sugar as defined in any one of clauses 47 to 49 BRIEF DESCRIPTION OF THE DRAWINGS

[0365] Figure 1 . GLP-1 secretion of STC-1 cells after incubation with 10 mg / ml of different hydrolyzed collagen fractions (as obtained by different enzymatic treatments). The supernatants were collected after 2 hours of incubation. GLP-1 levels were determined by enzyme immunoassay. The results are expressed as mean (n=3) ± standard deviation. "H080" represents the hydrolyzed collagen of the present invention.

[0366] Figure 2 . Area Under the Curve (AUC) of blood glucose levels in mice after glucose loading at time 0 and active ingredient / vehicle intake at time -45 minutes. "H080" indicates hydrolyzed collagen of the present invention.

[0367] Figure 3 . Ratio of insulin / blood glucose levels in mice at time 15 minutes after glucose load at time 0 and product / drug / vehicle ingestion at time -45 minutes. "H080" indicates hydrolyzed collagen of the present invention.

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

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

[0370] Experimental Section

[0371] Example 1

[0372] Example 1 shows the effects of gelatin hydrolysate obtained by enzymatic hydrolysis with a combination of enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases on increasing GLP-1 in vitro and lowering blood glucose in vivo.

[0373] Study Design

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

[0375] The hydrolyzed collagen fractions were screened in vitro for their ability to enhance the secretion of GLP-1 by STC-1 cells. STC-1 cells are secretin tumor cells and are reported to be predictive cell models for studying the mechanism of hormone secretion in the gastrointestinal tract (Qi et al. Bio Protoc. 2020Aug 20; 10(16): e3717). GLP-1 acts as a major determinant of blood glucose homeostasis mainly by regulating gastric emptying, enhancing pancreatic insulin secretion, and inhibiting pancreatic glucagon secretion. Therefore, the secretion of GLP-1 by STC-1 cells is a preferred outcome parameter for determining how the hydrolyzed collagen fractions can improve glucose levels in vivo.

[0376] The most promising hydrolyzed collagen fractions identified in vitro were further tested for their glucose-lowering activity in mice. The glucose-lowering activity was determined based on changes in GLP-1, GIP, glucose, and insulin in plasma. The efficacy of the hydrolyzed collagen fractions was compared with that of the glucose-lowering drug sitagliptin.

[0377] method

[0378] Enzymatic hydrolysis of collagen

[0379] Gelatin powder is dissolved in demineralized water by heating and stirring to provide a 35% by weight gelatin solution. The temperature of the gelatin solution is adjusted to 40 to 55°C. The pH of the gelatin is adjusted to pH5.5 to 8.0. When the appropriate temperature and pH are reached, the enzyme (mixture) as shown in Table 1 is added and hydrolyzed under stirring. For the "H080" condition, a commercially available enzyme mixture (Sumizyme FP-G available from Takabio, Japan) is used, which comprises a mixture of neutral protease, carboxypeptidase and aminopeptidase derived from Aspergillus oryzae. Keep the temperature constant. After 120 minutes, the enzymatic hydrolysis is terminated by thermal inactivation of the enzyme. Subsequently, the solution is cooled to 55°C. The hydrolyzed gelatin solution is purified, filtered and sterilized. The final hydrolyzed gelatin powder is obtained by spray drying.

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

[0381]

[0382]

[0383] STC-1 cells secrete GLP-1

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

[0385] Cells were stimulated with 2 mg / ml, 5 mg / ml or 10 mg / ml of the enzyme (mixture). For all enzymes (mixtures), a dose-dependent increase in GLP-1 secretion was observed, with the 10 mg / ml condition producing the highest GLP-1 secretion. Figure 1 Results for a 10 mg / ml concentration of comparative enzyme (mix) are shown.

[0386] In vivo mouse model

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

[0388] Mice were fasted for 6 hours, and then an oral glucose tolerance test (OGTT) was performed. Mice were treated with vehicle, 3 different concentrations of test article H080 (40mg / kg, 400mg / kg and 4g / kg) and sitagliptin as a positive control. 45 minutes after receiving the active ingredient, mice received oral glucose load (2g / kg body weight). Blood glucose was measured in the blood collected at -45, 0, 15, 30, 60, 90 and 120 minutes (after glucose load). Insulin was determined in the plasma collected 45 minutes (-45 minutes) before glucose load and 15 minutes (+15 minutes) after glucose load (by enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA)). Thus, the ratio of insulin / glucose can be determined.

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

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

[0391] Following a similar approach as described for healthy mice, studies were also performed in obese mice with naturally occurring hyperglycemia, with similar results.

[0392] result

[0393] In vitro GLP-1 secretion

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

[0395] The hydrolyzed collagen denoted as H080 induced the greatest secretion of GLP-1. H080 induced a 5800-fold increase in GLP-1 secretion compared to blank. In comparative tests, the increase in GLP-1 mediated by H080 was at least 2.5 times greater than the increase in GLP-1 mediated by other enzymes (mixtures).

[0396] The data indicate that a combination of a neutral protease (eg, a serine protease), a carboxypeptidase, and an aminopeptidase (eg, a leucine aminopeptidase) induced by far the highest GLP-1 secretion compared to other enzymes and combinations of enzymes.

[0397] It was found that the combination of only two or more enzymes (e.g., a combination of two neutral proteases in "CH3") was not sufficient to further promote GLP-1 secretion compared to using only one enzyme (e.g., a neutral protease in "CH1" or "CH2", etc.).

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

[0399] Reproduced with different batches of gelatin Figure 1 The results shown in .

[0400] The results indicate that hydrolyzing collagen with a combination of two or more enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases is important for obtaining hydrolyzed collagen with a strong glucose-lowering effect (eg, induced by GLP-1).

[0401] In vivo hypoglycemic activity

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

[0403] Figure 3 Shown are the insulin / glucose ratios in mice after treatment with 40 mg / kg H080, 400 mg H080, 4 g / kg H080, and 400 μg / mouse of 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.

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

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

[0406] In the healthy mouse model, mice receive active ingredients before glucose load. Therefore, healthy mice are particularly suitable for studying the prevention of hyperglycemia. Also studied in obese mice suffering from spontaneous hyperglycemia. The method is similar to the method described for healthy mice. Since obese mice have increased blood sugar when receiving active ingredients, the improvement of blood sugar (and other parameters related to hyperglycemia) in the obese mouse model further strengthens the treatment of the disease by active ingredients.

[0407] In obese mice with hyperglycemia, Figures 1 to 5 This further reinforces the effective treatment of hyperglycemia (and related parameters) by H080. The hypoglycemic activity of the combination of enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases

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

[0409] As shown in Table 2, the use of neutral protease in the enzymatic hydrolysis of gelatin produced gelatin hydrolysates with glucose-lowering activity, although the activity was limited. Relative to blank or neutral protease alone, the combination of neutral protease with either carboxypeptidase alone or aminopeptidase alone provided stronger glucose-lowering activity. The combination of all three enzymes produced gelatin hydrolysates with the highest glucose-lowering activity.

[0410] Table 2: Enzymes used for collagen hydrolysis and the effects of hydrolysates on hypoglycemic effect.

[0411]

[0412] Characterization of hydrolyzed collagen with high glucose-lowering activity

[0413] The hydrolyzed collagen with high hypoglycemic activity (e.g., Groups III to IV in Table 2) is characterized by a weight average molecular weight of 1000 to 7000 Da and a polydispersity (weight average molecular weight / number average molecular weight) of 1.2 to 1.8 (the average value measured in >100 measurements is about 1.56), as determined by HPSEC.

[0414] Example 2

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

[0416] method

[0417] As described in Example 1, hydrolysis was performed using a combination of neutral protease (50.000 U / g), aminopeptidase (500 to 1200 U / g), and carboxypeptidase (300 to 700 U / g) to produce gelatin hydrolysate.

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

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

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

[0421] Table 4 shows the effects of gelatin concentration, enzymatic hydrolysis temperature and pH in obtaining gelatin hydrolysate fractions for lowering blood glucose based on more detailed, extensive testing. The same method as in Example 1 was used to test GLP-1 secretion.

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

[0423]

[0424]

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

[0426]

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

[0428] With regard to precursor concentration (gelatin wt %), satisfactory results were obtained over the entire range of 10 wt % to 50 wt % tested, with an apparent optimum at concentrations of 20 wt % to 35 wt %.

[0429] With regard to pH, satisfactory results were obtained over the entire range of pH 4.0 to 10.0 tested, with an apparent optimum at pH 5.5 to 6.5.

[0430] With regard to temperature, satisfactory results were obtained over the entire range of 20 to 60°C tested, with the optimum clearly being achieved at temperatures of 45 to 50°C.

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

[0432] The combination of preferred gelatin concentration, pH and temperature can produce the best activity of enhancing the secretion of GLP-1 by STC-1 cells. As shown in Table 5, the combination of gelatin concentration (30% to 40% by weight), pH (6 to 7.5) and temperature (40 to 55° C.) produces unexpectedly high activity of gelatin hydrolysate in enhancing the secretion of GLP-1 by STC-1 cells. GLP-1 secretion was tested using the same method as in Example 1.

[0433] Table 5. Stimulation of GLP-1 secretion from STC-1 cells with hydrolyzed collagen obtained by a combination of neutral proteases, carboxypeptidases and aminopeptidases, wherein the enzymatic hydrolysis was performed under different process conditions.

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

[0435] Example 3

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

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

[0438] "H080" was produced by hydrolysis using a combination of neutral protease (50.000 U / g), aminopeptidase (500 to 1200 U / g) and carboxypeptidase (300 to 700 U / g) as described in Example 1. Collagen hydrolysates "CH11 to CH19" were obtained by hydrolysis with neutral, alkaline or acidic proteases.

[0439] GLP-1 secretion was considered "average" if it was increased at least 1000-fold compared to stimulation by vehicle control.

[0440] As can be seen from Table 6, "H080" induced an increase in GLP-1 secretion of about 6000 times compared to the stimulation of the blank control and is considered to be a "very high" GLP-1 secretion. Hydrolyzed collagen according to the group "CH11 to CH19" - i.e. obtained by hydrolysis with neutral, alkaline or acidic proteases - showed no / little or only average GLP-1 secretion.

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

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

[0443]

Claims

1. Hydrolyzed collagen for improving hyperglycemia or risk factors of hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatically hydrolyzing a material containing collagen with an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases.

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

3. The hydrolyzed collagen for use according to any one of the preceding claims, 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.

4. The hydrolyzed collagen according to any one of the preceding claims, for use in one or more selected from the following: - Increases blood glucagon-like peptide-1; - Decreases blood glucagon; - Increase blood insulin; - Improves blood insulin / glucose ratio.

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

6. Hydrolyzed collagen for use according to any one of the preceding claims, wherein the hydrolyzed collagen is administered orally, preferably as a dietary supplement.

7. The hydrolyzed collagen for use according to any one of the preceding claims, wherein the hydrolyzed collagen is administered no more than 60 minutes apart from a meal.

8. Hydrolyzed collagen for use according to any one of the preceding claims, wherein the collagen-containing material originates from one or more tissues selected from the group consisting of skin, scales, cartilage, bone, tendon, ligament and connective tissue.

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

10. Hydrolyzed collagen for use according to any one of the preceding claims, wherein the collagen-containing material is gelatin.

11. Hydrolyzed collagen for use according to any one of the preceding claims, wherein the hydrolyzed collagen has an average molecular weight of 1000 to 7000 Da and / or a polydispersity of 1.2 to 1.

8.

12. Use of hydrolyzed collagen for non-therapeutic lowering of blood sugar, wherein the hydrolyzed collagen is as defined in any one of claims 1 to 11.

13. The use according to claim 12, wherein the use is for improving postprandial blood sugar.

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

15. A method for obtaining hydrolyzed collagen, comprising: a) providing a collagen-containing material in a liquid formulation; as well as b) subjecting the collagen-containing material to an enzyme combination comprising two or more enzymes selected from neutral proteases, carboxypeptidases and aminopeptidases.

16. The method of claim 15, wherein the enzyme combination comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

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

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

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

20. The method according to any one of claims 15 to 19, wherein the collagen-containing material is subjected to an amount of neutral protease defined by an enzyme activity of 0.2 to 25000 U / g, 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.

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

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

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

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

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

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

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

28. The method of claim 27, wherein the collagen-containing material is subjected to a mixture comprising the enzyme combination.

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

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

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

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

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

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

35. The hydrolyzed collagen according to claim 33 or 34, wherein the hydrolyzed collagen comprises: - 1 to 20% by weight of collagen peptides with a molecular weight below 1000 Da; and / or - 20% to 40% by weight of collagen peptides having a molecular weight of 1000 to 2000 Da; and / or - 5% to 25% by weight of collagen peptides having a molecular weight of 5000 to 10000 Da; and / or - 0 to 10% by weight of collagen peptides with a molecular weight greater than 10,000 Da, Calculated based on the total weight of collagen peptides in the hydrolyzed collagen.

36. The hydrolyzed collagen according to any one of claims 33 to 35, wherein the polydispersity of the hydrolyzed collagen is from 1.2 to 1.

8.

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

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

39. Use of hydrolyzed collagen according to any one of claims 33 to 37 for non-therapeutic lowering of blood sugar as defined in any one of claims 12 to 14.

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