HYDROLYZED COLLAGEN FOR USE IN REDUCING BLOOD GLUCOSE

Abstract

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

Claims

1. Hydrolyzed collagen for use in ameliorating hyperglycemia or a risk factor for hyperglycemia, wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material with a combination of enzymes comprising a neutral protease and one or both of a carboxypeptidase, and an aminopeptidase.

2. Hydrolyzed collagen for use according to claim 1, wherein the combination of enzymes comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

3. Hydrolyzed collagen for use according to claim 1, wherein the risk factor for hyperglycemia is one or more selected from the group consisting of insulin resistance, type 2 diabetes, gestational diabetes, high body mass index, obesity, and hyperglucagonemia.2 diabetes, gestational diabetes, high body mass index, obesity, and hyperglucagonemia.

4. Hydrolyzed collagen for use according to claim 1, for use in one or more selected from the group consisting of: increasing blood glucagon-like peptide-1; decreasing blood glucagon; increasing blood insulin; increasing blood insulin/glucose ratio.

5. Hydrolyzed collagen for use according to claim 1, wherein the hydrolyzed collagen is administered at a daily dose in an amount of 1-100 gram, wherein the amount is the dry weight amount, preferably as a food supplement.

6-9. (canceled)

10. Hydrolyzed collagen for use according to claim 1, wherein the collagen-containing material is gelatin.

11. (canceled)

12. Use of hydrolyzed collagen for non-therapeutic lowering of blood glucose, wherein the hydrolyzed collagen is as defined by claim 1.

13. (canceled)

14. Use according to claim 12 or 13, wherein the hydrolyzed collagen is administered at a daily dose in an amount of 1-100 gram, wherein the amount is the dry weight amount.

15. Method for obtaining hydrolyzed collagen, comprising: a) providing a collagen-containing material in a liquid formulation; and b) subjecting the collagen-containing material to a combination of enzymes comprising a neutral protease and one or both of a carboxypeptidase, and an aminopeptidase, optionally wherein the combination of enzymes comprises a neutral protease, a carboxypeptidase, and an aminopeptidase.

16. (canceled)

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

18. (canceled)

19. Method according to claim 15, wherein one or more of the neutral protease, carboxypeptidase, and aminopeptidase are derived from a microorganism of the Aspergillus genus, preferably from Aspergillus oryzae.

20. Method according to claim 15, wherein the collagen-containing material is subjected to an amount of neutral protease defined by an enzyme activity of 0.2-25000 U/g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation, and/or wherein the collagen-containing material is subjected to an amount of carboxypeptidase defined by an enzyme activity of 0.001-500 U/g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation, and/or wherein the collagen-containing material is subjected to an amount of aminopeptidase defined by an enzyme activity of 0.002-500 U/g, wherein the weight in g is the total weight of the collagen-containing material and the liquid formulation.

21-22. (canceled)

23. Method according to claim 15, wherein the collagen-containing material is subjected to one or more enzymes in the combination of enzymes for 60-180 min.

24-25. (canceled)

26. Method according to claim 15, wherein the collagen-containing material is subjected successively to two or more enzymes in the combination of enzymes, and/or wherein the collagen-containing material is subjected simultaneously to two or more enzymes in the combination of enzymes, preferably wherein the collagen-containing material is subjected to a mixture comprising the combination of enzymes.

27-32. (canceled)

33. Hydrolyzed collagen, wherein the hydrolyzed collagen: has a weight average molecular weight of 2000-5000 Da, preferably 2500-4500 Da; and comprises 35-60% by weight of collagen peptides with a molecular weight in the range of 2000-5000 Da, calculated on total weight of collagen peptides in the hydrolyzed collagen.

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

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

36. Hydrolyzed collagen according to claim 35, wherein the hydrolyzed collagen has a polydispersity of 1.2-1.8.

37. Hydrolyzed collagen according to claim 35, wherein the hydrolyzed collagen is capable of stimulating glucagon-like peptide-1 secretion.

38-39. (canceled)

40. Use of hydrolyzed collagen according to claim 33 in non-therapeutic lowering of blood glucose wherein the hydrolyzed collagen is obtained by enzymatic hydrolysis of a collagen-containing material with a combination of enzymes comprising two or more enzymes selected from the group consisting of a neutral protease, a carboxypeptidase, and an aminopeptidase.

Description

FIGURE LEGENDS

[0325] FIG. 1. GLP-1 secretion by STC-1 cells following incubation with 10 mg/ml of different hydrolyzed collagen fractions, as obtained by the different enzymatic treatments. Supernatants were collected after 2 h of incubation. GLP-1 levels were determined by enzyme immunoassay. Results are expressed in mean (n=3)standard deviation. H080 denotes the hydrolyzed collagen of the invention.

[0326] FIG. 2. Area Under the Curve (AUC) of blood glucose levels in mice after glucose load at time 0 and intake of the active ingredient/vehicle at time-45 min. H080 denotes the hydrolyzed collagen of the invention.

[0327] FIG. 3. Ratio of insulin/blood glucose levels in mice at time 15 min, after glucose load at time 0 and intake of the product/drug/vehicle at time-45 min. H080 denotes the hydrolyzed collagen of the invention.

[0328] FIG. 4. Plasma GLP-1 levels in mice after 15 and 30 minutes after intake of H080 hydrolyzed collagen, compared to baseline. H080 denotes the hydrolyzed collagen of the invention.

[0329] FIG. 5. Plasma GIP levels in mice at 15 and 30 minutes after intake of H080 hydrolyzed collagen, compared to baseline. H080 denotes the hydrolyzed collagen of the invention.

EXPERIMENTAL SECTION

Example 1

[0330] Example 1 shows the effect of a gelatin hydrolysate obtained by enzymatic hydrolysis with a combination of enzymes chosen from a neutral protease, a carboxypeptidase and an aminopeptidase on increasing in vitro GLP-1 and lowering in vivo blood glucose. Study design

[0331] Fourteen different enzyme (mixtures) were used in the hydrolysis of gelatin into hydrolyzed collagen formulations. The hydrolyzed collagen formulations obtained thereby were subjected to simulated gastrointestinal digestion (SGID) to obtain bioactive hydrolyzed collagen fractions as would be expected after in vivo ingestion and digestion (Song et al. Food Funct. 2020 Jun. 24; 11(6):5553-5564).

[0332] The hydrolyzed collagen fractions were screened in vitro for their ability to enhance GLP-1 secretion by STC-1 cells. STC-1 cells are an intestinal secretin tumor cells and are reported to be a predictive cell model to study hormonal secretion mechanisms in the gastrointestinal tract (Qi et al. Bio Protoc. 2020 Aug. 20; 10(16): e3717). GLP-1 acts as a main determinant of blood glucose homeostasis, mostly by regulating gastric emptying, enhancing pancreatic insulin secretion, and suppressing pancreatic glucagon secretion. The GLP-1 secretion by STC-1 cells was therefore the preferred outcome parameter determine how the hydrolyzed collagen fractions may ameliorate glucose levels in the body.

[0333] The most promising hydrolyzed collagen fraction identified in vitro was further tested for its glucose-lowering activity in mice. The glucose-lowering activity was established based on the changes in plasma GLP-1, GIP, glucose, and insulin. The efficacy of the hydrolyzed collagen fraction was compared to that of the glucose-lowering medicine Sitagliptin.

Methods

Enzymatic Hydrolysis of Collagen

[0334] Gelatin powder was dissolved in demi water by heating and stirring to provide a 35 wt. % gelatin solution. The temperature of the gelatin solution was adjusted to 40-55 C. The pH of the gelatin was adjusted to pH 5.5-8.0. When the appropriate temperature and pH was reached, the enzymes (mixtures) as shown in in Table 1 were added and hydrolysis was carried out under stirring. For the H080 condition, a commercially available enzyme mixture was used (Sumizyme FP-G obtainable from Takabio, Japan), which comprises a mixture of a neutral protease, a carboxypeptidase, and an aminopeptidase, derived from Aspergillus oryzae. The temperature was kept constant. After 120 min, enzymatic hydrolysis was terminated by heat inactivation of the enzymes. Subsequently, the solution was cooled down to 55 C. The hydrolyzed gelatin solution was subjected to purification, filtration and sterilization. A final hydrolyzed gelatin powder was obtained by spray drying.

TABLE-US-00001 TABLE 1 Overview of enzyme (mixtures) used for hydrolysis of collagen Group Activity of enzyme(s) name Enzym 1 Enzym 2 Enzym 3 Cleavage site Blank CH1 Neutral protease Nonterminal (serine protease) amino acids CH2 Neutral protease Nonterminal (serine protease) amino acids CH3 Neutral protease Neutral Nonterminal (serine protease) protease amino acids (metallo- protease) CH4 Neutral protease Nonterminal (Serine protease) amino acids CH5 Neutral protease Nonterminal (Metalloprotease) amino acids CH6 Neutral protease Nonterminal (Serine protease) amino acids CH7 Neutral protease Nonterminal (Serine protease) amino acids CH8 Neutral protease Nonterminal (Cysteine amino acids protease) CH9 Neutral protease Nonterminal (Serine protease) amino acids CH10 Collagenase Nonterminal + terminal amino acids H080 Neutral protease Carboxy- Leucine Nonterminal + (50.000 U/g) peptidase aminopeptidase terminal amino (300-700 (500-1200 U/g) acids U/g)

GLP-1 Secretion by STC-1 Cells

[0335] STC-1 cells were incubated with different final concentrations (0.2, 0.5 and 1% dry matter in Hepes buffer at pH 7.4) of the different hydrolyzed collagen obtained after simulated gastrointestinal digestion (SGID). Supernatants were collected after 2 h of incubation. GLP-1 levels were determined by enzyme immunoassay and expressed in pg/ml. Data are expressed in mean (n=3)standard deviation.

[0336] Cells were stimulated with 2, 5, or 10 mg/ml of enzyme (mixture). For all enzyme (mixtures), a dose-dependent increase was seen in GLP-1 secretion, with highest GLP-1 secretion for the 10 mg/ml condition. Hence, FIG. 1 shows the results for the 10 mg/ml concentration for comparison of the enzyme (mixtures).

In Vivo Mouse Model

[0337] The following mouse model was used to for determining Area Under the Curve (AUC) of blood glucose levels, insulin/blood glucose, plasma GLP-1 levels, and Plasma GIP levels. C57BL6/J Mice (male, 23-25 g, 8-week old) were randomized into the treatment groups (n=10 mice/group) according to their body weight.

[0338] Mice were fasted for 6 hours, after which an oral glucose tolerance test (OGTT) was performed. Mice were treated with either vehicle, test item H080 at 3 different concentrations (40 mg/kg, 400 mg/kg and 4 g/kg), and Sitagliptin as a positive control. 45 minutes after receiving the active ingredients, mice received an oral glucose load (2 g/kg of body weight). Blood glucose was measured in blood collected at time 45, 0, 15, 30, 60, 90 and 120 minutes after glucose load. Insulin was determined (by enzyme-linked immunosorbent assay, ELISA) in plasma collected 45 min before (45 min) and 15 minutes after (+15 min) the glucose load. From this, the ratio insulin/glucose could be determined.

[0339] The 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 g/mouse) to avoid the degradations of GLP-1 and GIP by the dipeptidyl peptidase 4 (DPP-IV). After 30 min, mice received vehicle or test item H080 in an amount of 4 g/kg body weight. 15 minutes or 30 minutes after receiving the active ingredients, mice were anesthetized and blood was sampled from the portal vein. Plasma GLP-1 and GIP levels were measured by ELISA.

[0340] The model in healthy mice demonstrates both prevention and treatment of high blood glucose (and other parameters related to hyperglycemia) following treatment with active ingredients.

[0341] Following a similar methodology as described for the healthy mice, a study was also conducted in obese mice which have naturally developed high blood glucose with a similar result.

Results

In Vitro GLP-1 Secretion

[0342] FIG. 1 shows the GLP-1 secretion in STC-1 cells following incubation with 10 mg/ml of hydrolyzed collagen, as obtained by hydrolysis with the enzyme (mixtures) provided in Table 1 under optimal processing conditions.

[0343] The hydrolyzed collagen denoted as H080 induced the largest GLP-1 secretion. As compared to the blank, H080 induced a 5800-fold increase in GLP-1 secretion. The increase in GLP-1 mediated by H080 was at least 2.5-fold larger than that mediated by the other enzyme (mixtures) in the comparative test.

[0344] The data indicate that the combination of a neutral protease (e.g. serine protease), a carboxypeptidase and an aminopeptidase (e.g. leucine aminopeptidase) induces by far the highest GLP-1 secretion as compared to other enzymes and enzyme combinations.

[0345] It was found that the mere combination of two or more enzymes (e.g. the combination of two neutral proteases in CH3) is not sufficient to further promote the GLP-1 secretion as compared to the use of only one enzyme (e.g. a neutral protease in CH1 or CH2, among others).

[0346] It was found that the mere combination of an endoprotease (i.e. enzyme cleaving at nonterminal amino acids) and an exoprotease (i.e. enzyme cleaving at terminal amino acids) is not sufficient to further promote the GLP-1 secretion as compared to the use of only an endoprotease. For example, CH12 comprises both endopeptidase and exopeptidase activity, but could not further promote GLP-1 secretion as compared to enzymes comprising only endopeptidase activity.

[0347] The results shown in FIG. 1 were reproduced with different batches of gelatin.

[0348] The results show that hydrolysis of collagen with a combination of two or more enzymes selected from a neutral protease, a carboxypeptidase, and an aminopeptidase is important for obtaining hydrolyzed collagen with strong potency in lowering glucose, such as induced by GLP-1.

In Vivo Glucose-Lowering Activity

[0349] FIG. 2 shows the blood glucose levels in mice following treatment with 40 mg/kg H080, 400 mg H080, 4 g/kg H080, and 400 g/mouse Sitagliptin. A dose-dependent decrease in blood glucose was seen following treatment with H080. The highest dose of H080 had a similar blood glucose-lowering effect as the drug Sitagliptin. This shows that the hydrolyzed collagen may be provided as a safe alternative to pharmaceuticals drugs which are often associated with side effects.

[0350] FIG. 3 shows the ratio of insulin/blood glucose in mice following treatment with 40 mg/kg H080, 400 mg H080, 4 g/kg H080, and 400 g/mouse Sitagliptin. H080 mediated a significant increase in the insulin/glucose ratio. A dose-dependent increase in insulin/glucose ratio was seen following treatment with H080.

[0351] FIG. 4 shows the plasma GLP-1 levels following control treatment or following treatment with 4 g/kg H080. Treatment with H080 increased the plasma GLP-1 levels, wherein the effect was largest 30 min after intake of H080.

[0352] FIG. 5 shows the plasma GIP levels following control treatment or following treatment with 4 g/kg H080. Treatment with H080 decreased the plasma GIP levels, wherein the effect was largest 30 min after intake of H080.

[0353] In the healthy mouse model, mice receive active ingredients before the glucose load. The healthy mouse is therefore particularly suitable to study the prevention of hyperglycemia. A study was also conducted in obese mice which have naturally developed high blood glucose. The methodology was similar as described for the healthy mice. Since the obese mice already have an increased blood glucose when receiving the active ingredients, an improvement in blood glucose (and other parameters related to hyperglycemia) in the obese mouse model further strengthens the treatment of disease by active ingredients.

[0354] Similar effects on the parameters studied in FIGS. 1-5 were observed in obese mice with high blood glucose. This further strengthens effective treatment of hyperglycemia (and related parameters) by H080.

Combination of Enzymes Chosen from a Neutral Protease, Carboxypeptidase and Aminopeptidase on Glucose-Lowering Activity

[0355] The inventors found that high glucose-lowering activity (e.g. based on the GLP-1 secretion level) can be achieved when at least two enzymes are used in the enzymatic hydrolysis of the collagen-containing material, wherein the two enzymes are chosen from a neutral protease, a carboxypeptidase and an aminopeptidase.

[0356] As illustrated in Table 2, the use of a neutral protease in enzymatic hydrolysis of gelatin leads to a gelatin hydrolysate that has a glucose-lowering activity albeit limited. A combination of a neutral protease with a carboxypeptidase alone or an aminopeptidase alone provides a stronger glucose-lowering activity relative to the blank or the use of a neutral protease alone. The combination of all three enzymes leads to a gelatin hydrolysate with the highest glucose-lowering activity.

TABLE-US-00002 TABLE 2 Enzyme(s) used for hydrolysis of collagen and the effect of the hydrolysate on the lowering of glucose. Glucose- lowering Group Enzyme(s) activity I Blank Very low/absent II 1. Neutral protease (from Aspergillus) Low III 1. Neutral protease (from Aspergillus) + High 2. Carboxypeptidase (from Aspergillus) IV 1. Neutral protease (from Aspergillus) + High 2. Aminopeptidase (from Aspergillus) V 1. Neutral protease (from Aspergillus) + Very high 2. Carboxypeptidase (from Aspergillus) + 3. Aminopeptidase (from Aspergillus) VI 1. Neutral protease (from Bacillus) + Very high 2. Carboxypeptidase (from Aspergillus) + 3. Aminopeptidase (from Lactobacillus)
Characterization of Hydrolyzed Collagen with High Glucose-Lowering Activity

[0357] The hydrolyzed collagen with high glucose-lowering activity (e.g. group III-IV in Table 2) were characterized by a weight-average molecular weight of 1000-7000 Da and a polydispersity (weight average molecular weight/number average molecular weight) of 1.2-1.8 (average of 1.56 was measured for >100 measurements), as determined by HPSEC.

Example 2

[0358] Example 2 shows the influence of the process conditions on obtaining hydrolyzed collagen for lowering blood glucose.

Methods

[0359] Gelatin hydrolysate was produced by hydrolysis using a combination of a neutral protease (50.000 U/g), an aminopeptidase (500-1200 U/g) and a carboxypeptidase (300-700 U/g) as described in Example 1.

[0360] A broad range of working conditions was tested, covering the conditions typically used in enzymatic hydrolysis.

[0361] The concentration of gelatin in the start solution was varied between 10-50 wt. %. The enzymes were used in a concentration of 2000 ppm for hydrolysis. The pH during enzymatic hydrolysis was varied between pH 4.0-10.0. The temperature during enzymatic hydrolysis was varied between 20-70 C.

[0362] Table 3 shows the influence of the gelatin concentration, pH, and temperature of enzymatic hydrolysis in obtaining gelatin hydrolysate fractions for lowering blood glucose in a preliminary test. The GLP-1 secretion was tested using the same methodology as in Example 1.

[0363] Table 4 shows the influence of the gelatin concentration, pH, and temperature of enzymatic hydrolysis in obtaining gelatin hydrolysate fractions for lowering blood glucose based on further elaborated, more extensive tests. The GLP-1 secretion was tested using the same methodology as in Example 1.

TABLE-US-00003 TABLE 3 Influence of the gelatin concentration, pH, and temperature of enzymatic hydrolysis in obtaining gelatin hydrolysate fractions for lowering blood glucose in a preliminary test. Suitable as blood glucose-lowering Process condition active ingredient Gelatin wt. % 10% No 20% Yes 30% Yes 40% Yes 50% No pH pH 4 No pH 5 Yes pH 6 Yes pH 8 Yes pH 10 No Temperature 20 C. No 30 C. Yes 40 C. Yes 50 C. Yes 60 C. Yes 70 C. No

TABLE-US-00004 TABLE 4 Influence of the gelatin concentration, pH, and temperature of enzymatic hydrolysis in obtaining gelatin hydrolysate fractions for lowering blood glucose based on further elaborated, more extensive tests. Suitable as blood glucose-lowering Process condition active ingredient Gelatin wt. % 10% ++ 20% +++ 30% +++ 35% +++ 40% ++ 45% + 50% + pH pH 4.0 + pH 4.5 + pH 5.0 ++ pH 5.5 +++ pH 6.0 +++ pH 6.5 +++ pH 7.0 ++ pH 7.5 ++ pH 8.0 + pH 9.0 + pH 10.0 + Temperature 20 C. + 30 C. + 35 C. + 40 C. + 45 C. +++ 50 C. +++ 55 C. ++ 60 C. + +: less suitable; ++: more suitable; +++: most suitable

[0364] As shown in Tables 3 and 4 collectively, it is found that a hydrolyzed collagen suitable for enhancing GLP-1 secretion can be achieved over a broad range of values for the precursor concentration (gelatin wt. %), pH, and temperature.

[0365] Regarding the precursor concentration (gelatin wt. %), satisfactory results were obtained over the entire range of 10-50 wt. % tested, with apparently an optimum at a concentration of 20-35 wt. %.

[0366] Regarding the pH, satisfactory results were obtained over the entire range of pH 4.0-10.0 tested, with apparently an optimum at pH 5.5-6.5.

[0367] Regarding the temperature, satisfactory results were obtained over the entire range of 20-60 C. tested, with apparently an optimum at 45-50 C.

[0368] The results show that hydrolysis performed at (excessive) high temperatures (e.g. at a temperature of 70 C. or higher) may not lead to a hydrolyzed collagen product suitable for enhancing GLP-1 secretion collagen. It is considered that this is the result of enzyme denaturation and subsequently impaired reaction rate.

[0369] The combination of preferred gelatin concentration, pH, and temperature may lead to best activity in enhancing GLP-1 secretion by STC-1 cells. As shown in Table 5, the combination of gelatin concentration of (30-40 wt. %), pH (6-7.5), and temperature (40-55 C.) leads to a surprisingly high activity of the gelatin hydrolysate in enhancing GLP-1 secretion by STC-1 cells. The GLP-1 secretion was tested using the same methodology as in Example 1.

TABLE-US-00005 TABLE 5 GLP-1 secretion by STC-1 cells stimulated with hydrolyzed collagen obtained by a combination of a neutral protease, carboxypeptidase and an aminopeptidase, wherein enzymatic hydrolysis was performed at different process conditions. Process conditions GLP-1 level 30-40 wt. % gelatin, pH 6-7.5, 40-55 C. 3471 pg/ml 30-40 wt. % gelatin, pH 7.5-8.5, 55-65 C. 346 pg/ml

Example 3

[0370] Example 3 shows the molecular weight and molecular weight distribution of hydrolyzed collagen obtained by hydrolysis with different enzymes (mixtures), in relationship to the ability of the hydrolyzed collagens in stimulating GLP-1 secretion by STC-1 cells.

[0371] Table 6 shows the molecular weight and molecular weight distribution of hydrolyzed collagen formulations obtained with different enzymes, with their different abilities to stimulate GLP-1 secretion in STC-1 cells. The methodology used for hydrolysis and to evaluate GLP-1 secretion was the same as described in Example 1.

[0372] H080 was produced by hydrolysis using a combination of a neutral protease (50.000 U/g), an aminopeptidase (500-1200 U/g) and a carboxypeptidase (300-700 U/g) as described in Example 1. The collagen hydrolysates CH11-CH19 were obtained by hydrolysis with either a neutral, alkaline or acid protease.

[0373] An average GLP-1 secretion was considered if the GLP-1 secretion was at least 1000-fold increased compared to stimulation with a blank control.

[0374] As can be seen from Table 6, H080 induced 6000-fold increased GLP-1 secretion compared to stimulation with a blank control and is considered as very high GLP-1 secretion. Hydrolyzed collagens according to groups CH11-CH19i.e. obtained by hydrolysis with either a neutral, alkaline or acid protease-show no/little or only average GLP-1 secretion.

[0375] As can be seen from Table 6, H080 is characterized by a weight average molecular weight of 3000 Da (or mean average molecular weight of 2000 Da). H080 is furthermore characterized by a high fraction of collagen peptides (i.e. 47.2%) falling in the 2-5 kDa range. H080 is furthermore characterized by high stimulatory activity on GLP-1 secretion (i.e. 6000-fold increase compared to the blank control group). The CH11-CH19 groups lack one or more of these features.

TABLE-US-00006 TABLE 6 Overview of enzyme (mixtures) used for hydrolysis of collagen, molecular weight and molecular weight distribution of the obtained hydrolyzed collagen, and their ability to stimulate GLP-1 secretion in STC-1 cells. GLP-1 secretion Molecular weight distribution (%) Relative Mn Mw <1 1-2 2-5 5-10 >10 Group name to blank (Da) (Da) Pd kDa kDa kDa kDa kDa CH11 No/low 1391 2007 1.4 18.4 46.8 30.3 4.2 0.3 Neutral protease (serine protease) CH12 No/low 2131 3972 1.9 7.9 24.9 40.2 21.1 5.9 Neutral protease (serine protease) CH13 No/low 3842 7764 2.0 3 8.8 26.9 33.9 27.5 Neutral protease (metallo protease) CH14 Average 1664 2602 1.6 13.3 36 40 9.8 0.9 Alkaline protease (metallo protease) CH15 No/low 2652 6078 2.3 5.5 14.9 36.3 25.3 18 Alkaline protease (serine protease) CH16 No/low 3289 6681 2.0 2.9 12 31.7 30.8 21.7 Alkaline protease (serine-/metallo protease) CH17 No/low 1119 1484 1.3 27 55.8 16.8 0.4 0.1 Alkaline protease (collagenase) CH18 Average 1425 2055 1.4 17.5 47.8 30.5 3 1.2 Alkaline protease (serine protease) CH19 No/low 1333 1852 1.4 20.5 47.2 30 2.4 0 Acid protease (cysteine protease) H080 Very high 1933 3063 1.6 8.7 28.8 47.2 14.4 0.9 Neutral protease + Carboxypeptidase + Leucine aminopeptidase