Method for producing collagen peptides from bone, and produced collagen peptides

Abstract

The present invention relates to a method for producing collagen peptides from bone, the method comprising the steps of: a) providing vertebrate bone; b) mechanically crushing the bone to a particle size of less than 1000 μm, preferably less than 500 μm, more preferably less than 300 μm, at a temperature of less than 70° C. during crushing; c) heating the crushed bone in an aqueous suspension to a temperature greater than 100° C., preferably greater than 120° C., more preferably greater than 130° C., for a period of 1 to 30 minutes, preferably 2 to 10 minutes, more preferably 4 to 8 minutes; d) adding one or more proteases to the suspension to obtain an aqueous solution of collagen peptides; and e) separating the aqueous solution of collagen peptides from the crushed bone, wherein the method does not involve maceration of the bone with an acid or liming of the bone with a base, and wherein the bone provided in step a) has not been subjected to maceration or liming. The present invention further relates to collagen peptides produced by this method.

Description

Technical Field

[0001] The present invention relates to a method for producing collagen peptides from bone.

[0002] The present invention further relates to collagen peptides produced by this method.

Background Art

[0003] Collagen peptides are produced by hydrolysis of collagen, an animal structural protein, specifically by enzymatic hydrolysis. Thus, another name is collagen hydrolysis product or hydrolyzed collagen. When animal bone is the starting material, this is specifically type I collagen.

[0004] Collagen peptides are used in various ways, not only for their physiological effects in food supplements or so-called functional foods, but also from the perspective of food processing, for example as emulsifiers, stabilizers, binders, etc., specifically in the food industry. The characteristic properties of collagen peptides are that they are soluble even in cold water and have a very slight gel-forming ability. This differentiates collagen peptides from gelatin, which is a denatured collagen that is only slightly hydrolyzed. Collagen peptides have a molecular weight of less than 25,000 Da, actually usually less than 10,000 Da, while the molecular weight of gelatin is significantly higher.

[0005] Collagen peptides are normally produced together with gelatin as an intermediate product (see, for example, R Schrieber and H Gareis: Gelatin Handbook, 2007, Section 2.2.11). In the prior art, the production of gelatin from bones is, in part, an essential step, including the demineralization (maceration) of bones in a strongly acidic medium and subsequent treatment in a strong alkaline medium (liming) in a multi-step procedure so that gelatin can be extracted in multiple steps at a high temperature (typically between 50 and 100 °C) later (see Gelatin Handbook, Section 2.2.5).

[0006] During maceration, coarsely ground bones are treated in a countercurrent process with dilute hydrochloric acid for a period of approximately one week to elute the mineral components (calcium carbonate and calcium phosphate) from the bone tissue (see Gelatin Handbook, Section 2.2.1.1). The product obtained from this process is called ossein. In addition to the chemicals, a cost factor associated with maceration is the cooling required by the exothermic reaction between hydrochloric acid and calcium minerals. A further drawback is the high chloride load in the wastewater.

[0007] The subsequent liming of ossein is necessary to enable the effective extraction of gelatin. Typically, the liming process involves treatment with a calcium hydroxide suspension (pH value higher than 12) for a period of several months (see Gelatin Handbook, Section 2.2.4.1). The treatment time can be shortened by using a stronger alkali (for example, to 2 or 3 days when sodium hydroxide is used), but this results in a reduction in yield.

[0008] The method described above yields B-type bone gelatin characterized by an isoelectric point (IEP) of less than 5.6, typically in the range of 4.8 to 5.5. This IEP corresponds to the pH value at which the polypeptide chains of the gelatin (or collagen peptides produced therefrom) have a neutral overall charge. The relatively low IEP of B-type gelatin results from the fact that, during the liming step, almost all of the asparagine and glutamine amino acids are converted to aspartic acid and glutamic acid, respectively.

[0009] At the same gel strength, B-type gelatin has a significantly higher viscosity than A-type gelatin and is therefore preferred for most applications. For this reason, the production of A-type bone gelatin, where ossein is extracted in an acidic medium without liming, plays only a secondary role. A-type gelatin has an IEP greater than 6 and, in the case of A-type bone gelatin, typically has an IEP in the range between 6 and 8 (in the case of porcine skin gelatin, in the range between 8 and 9).

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0010] It is an object of the present invention to propose an alternative method for producing collagen peptides in which the disadvantages of the method described above, which uses B-type bone gelatin as an intermediate product, can be avoided, either in whole or in part.

MEANS FOR SOLVING THE PROBLEMS

[0011] This object is achieved by the following steps: a) providing bones of a vertebrate; b) mechanically crushing the bones at a temperature below 70°C to a particle size of less than 1000 μm, preferably less than 500 μm, more preferably less than 300 μm during crushing; c) heating the crushed bones in an aqueous suspension to a temperature higher than 100°C, preferably higher than 120°C, more preferably higher than 130°C for a period of 1 to 30 minutes, preferably 2 to 10 minutes, more preferably 4 to 8 minutes; d) adding one or more proteases to the suspension to obtain an aqueous solution of collagen peptides; e) separating an aqueous solution of collagen peptides from the crushed bone, which is realized by the method of the type described at the beginning, comprising The method does not include maceration of bone with acid or liming of bone with base, and the bone provided in step a) has not been subjected to maceration or liming.

[0012] In the context of the present invention, surprisingly, it has been found that collagen peptides can be produced by direct enzymatic treatment of bone material with proteases without using an indirect method by means of producing bone gelatin. Thus, the method according to the present invention clearly does not require maceration and / or liming of bone, and as a result, the entire period of the method until collagen peptides are obtained is dramatically reduced. In extreme cases, maceration and liming take several months or at least several days, while the method according to the present invention can be carried out within two or three hours. The energy requirement and wastewater pollution are also significantly less in the method according to the present invention than in the prior art.

[0013] In the context of this specification, the term "maceration" is understood to mean treatment with an acid having a pH value of less than 1, and the term "liming" is understood to mean treatment with a base having a pH value of more than 12.

[0014] As starting materials for the method according to the present invention, in principle, any vertebrate bone, thus also bones of birds or fish, etc., may be used. However, preferably, the method is carried out on mammalian bones, in particular bovine bones.

[0015] It is preferred if the bone is purified before being crushed, in particular before being defatted. Purifying the starting material is advantageous for the efficient implementation of enzymatic hydrolysis, thereby enabling the production of high-quality collagen peptides.

[0016] Preferably, the purification of the bone comprises treatment with one or more enzymes, preferably protease and / or lipase. Lipase helps with defatting, and non-collagenous proteins can be degraded and removed using protease. Before the bone is appropriately crushed, the protease hydrolyzes the collagen to an extent that it can be ignored.

[0017] Before crushing, the bone preferably has a fat content of less than 4% by weight, preferably less than 1% by weight, more preferably less than 0.5% by weight.

[0018] Regarding the removal of non-collagenous proteins, it is advantageous if the bone has a collagen content of at least 55%, preferably 70 - 90%, based on the total protein amount before crushing. In this case, the collagen amount is determined from the hydroxyproline amount multiplied by a factor of 7.3, and the total protein amount is determined from the Kjeldahl nitrogen amount multiplied by a factor of 6.25. The said factors take into account the proportions of hydroxyproline and nitrogen in collagen and differ from the corresponding proportions in the whole protein.

[0019] Preferably, the mechanical crushing of the purified bone to give a particle size of less than 1000 μm is an essential feature of the method according to the invention. The small particle size enables direct enzymatic hydrolysis of the collagen in the bone material without the need for pre-treatment known from the prior art such as maceration or liming. The mechanical crushing may include dry or wet grinding of the bone, and wet grinding in an aqueous suspension is preferred. During crushing, the temperature is maintained below 70°C to avoid local overheating of the material.

[0020] For the purpose of preparation for enzymatic hydrolysis, the crushed bone is heated to a temperature higher than 100°C in an aqueous suspension, and a period of at most 30 minutes is sufficient for this preliminary heat treatment. During this time, the collagen is denatured and becomes available for enzymatic hydrolysis. In this case, the amount of crushed bone by weight in the aqueous suspension is preferably 0.05 - 0.5 kg / l, preferably 0.1 - 0.3 kg / l, more preferably 0.15 - 0.2 kg / l.

[0021] In some cases, the preliminary heat treatment of the crushed bone may be further accelerated and / or enhanced by additional energy input by means of cavitation, for example by ultrasonic or high-pressure homogenizer. Another possibility is to apply an alternating current electric field to the suspension.

[0022] A further advantage of the method according to the invention consists in the fact that the isoelectric point of the generated collagen peptides can be influenced by a simple technique by adjusting the appropriate pH value during the heating of the crushed bone in the aqueous suspension. Depending on the field of application, collagen peptides with a high or low IEP may be preferred, and the difference in properties is not as pronounced in this case as that between type A and type B gelatin.

[0023] In order to obtain collagen peptides with a high IEP exceeding 5.6, before heating, the pH value of the aqueous suspension is adjusted to a range of 5-7, preferably 6-7. Typically, a high IEP is generated even without adjusting the pH value.

[0024] In order to obtain collagen peptides with a low IEP less than 5.6, before heating, the pH value of the aqueous suspension is adjusted to a range of 7-9, preferably 7.9-8.6.

[0025] After the preliminary heat treatment, the aqueous suspension is cooled to a temperature within the range of 40-60 °C before the addition of one or more proteases. The optimum activity values of the proteases typically used for the enzymatic hydrolysis of collagen are present in this temperature range.

[0026] Preferably, the one or more proteases added after heating the aqueous suspension are selected from microbial endoproteases, preferably serine proteases of a specific Bacillus subtilis. The use of such enzymes for the hydrolysis of collagen is known from the prior art. A frequently used protease is, for example, subtilisin.

[0027] Typically, one or more proteases are added in an amount of 0.01 to 0.5% by weight, preferably 0.02 to 0.2% by weight, more preferably 0.03 to 0.1% by weight, based on the dry weight of the crushed bone.

[0028] After the addition of one or more proteases, the enzymatic reaction is carried out for a period of preferably 0.5 to 4 hours, more preferably 1 to 3 hours.

[0029] In a preferred embodiment of the present invention, once the aqueous solution of collagen peptides is separated, the crushed bone is subjected to steps c) to e) for a further period of time. This double heating and treatment with protease of the crushed bone can increase the yield of collagen peptides.

[0030] Preferably, separating the aqueous solution of collagen peptides from the crushed bone includes filtration, specifically membrane filtration. This can remove even the smallest particles of the crushed bone and other solids.

[0031] After filtration, the aqueous solution of collagen peptides may preferably undergo an ion exchange procedure, specifically salt removal.

[0032] According to a preferred embodiment, the method according to the present invention further includes drying the aqueous solution of collagen peptides to obtain collagen peptide powder, specifically by spray drying. The aqueous solution may be concentrated in advance using an evaporator.

[0033] The present invention also relates to collagen peptides produced by the method according to the present invention.

[0034] The collagen peptides according to the present invention typically have a weight average molecular weight of less than 25,000 Da, preferably less than 10,000 Da, more preferably less than 5,000 Da. A weight average molecular weight of 500 to 5,000 Da, specifically 2,000 to 4,000 Da, is particularly suitable.

[0035] In a preferred embodiment of the present invention, the collagen peptide has a high isoelectric point exceeding 5.6, specifically exceeding 6.0.

[0036] According to a further aspect of the present invention, the collagen peptide has a low isoelectric point of less than 5.6, specifically 5.2 - 5.6.

[0037] These and further advantages of the present invention will become apparent from the examples described below.

Brief Description of the Drawings

[0038]

Figure 1

Examples

[0039] Example 1: Generation of Collagen Peptides from Bone on a Laboratory Scale Bovine bone was pre - purified by hot water and defleshing and degreasing with protease assistance, and then ground into bone powder with particle sizes of d50 < 350 μm and d90 < 700 μm. The bone powder was then mixed with an equal mass of water and heated to 120 - 130 °C in a microwave oven for approximately 1 minute while stirring. After cooling to less than 100 °C (approximately 20 minutes), 0.1 wt% (relative to the mass of the bone powder) of the protease subtilisin was added, and the suspension was stirred at 60 °C. Due to the enzymatic reaction involving the formation of soluble collagen peptides, as shown in Table 1, the concentration of the aqueous phase increased over time. The concentration was measured using a refractometer calibrated in Brix units for measuring sucrose.

Table 1

[0040] After the deposition of the bone powder, the supernatant was filtered and the salt was removed therefrom. The collagen peptide was concentrated and dried. The IEP of the collagen peptide was 6.23.

[0041] The distribution of the molecular charge of these collagen peptides according to the invention can be determined by isoelectric focusing electrophoresis. Figure 1 shows the corresponding chromatogram, where the pH value of the gel is shown on the left and three tracks are assigned as follows: Track 1: Marker peptide Track 2: Collagen peptides from type B bone gelatin of the prior art (including maceration and liming) Track 3: Collagen peptides according to the invention, according to the previous example

[0042] The charge of the molecules is generally similar in both samples, and in this case the collagen peptides according to the invention also show a band of negative, i.e., alkaline, molecules. The pH value during denaturation determines the position of the band and thus the isoelectric point of the collagen peptide.

[0043] Example 2: Production of collagen peptides from bone on a pilot scale An aqueous suspension of 15% by weight of purified bone (d90 < 700 μm) from bovine bone meal was placed in a stirred vessel, and the bone was pre-purified as in Example 1. The ratio of total protein to collagen was 1.7 (normalized to the low fat content of the dry mass). The pH value of the suspension was adjusted to 6.5.

[0044] The suspension was pumped into a heat exchanger and thus the suspension was heated to 130 °C. This temperature was maintained for approximately 6 minutes. Then the suspension was cooled to approximately 60 °C by the heat exchanger and recovered in the stirred vessel. An amount of protease subtilisin of 0.05% by weight (relative to the dry bone mass) was added. After a reaction time of 2 hours, the enzymatic hydrolysis was terminated by heating the suspension to 85 °C for 5 minutes.

[0045] The aqueous phase was separated using decanter centrifugation and recovered in a vessel. The solid phase was treated a second time in the same manner as described previously for bone meal (formation of the suspension, preliminary heat treatment, cooling, enzymatic hydrolysis and separation of the aqueous phase by decanting).

[0046] The aqueous phases (collagen peptide solutions) from the two routes were combined, filtered for further purification, subjected to salt removal, and concentrated and dried by appropriate methods.

[0047] The yield from this method is approximately 16 - 19% by weight of collagen peptides with respect to the bone mass used.

[0048] The quality of the collagen peptides may be evaluated at wavelengths of 450 nm and 620 nm using, for example, the high transmittance value of an aqueous solution having a concentration of 20% by weight. The measured values and each quality criterion are shown in Table 2.

Table 2

[0049] The weight average molecular weight of the collagen peptides according to Example 2 is in the region of 3000 ± 500 Da. The selection of the enzyme, the amount of the enzyme, and the reaction time can be used to affect the molecular weight distribution of the collagen peptides according to the present invention.

Claims

1. The following steps: a) providing a bone of a vertebrate; and b) mechanically crushing the bone to a particle size of less than 1000 μm at a temperature of less than 70 °C during crushing; b1) preparing an aqueous suspension containing the crushed bone and adjusting the pH value of the aqueous suspension to a range of 5 to 7 to obtain a collagen peptide having an isoelectric point of more than 5.6, or adjusting the pH value of the aqueous suspension to a range of 7 to 9 to obtain a collagen peptide having an isoelectric point of less than 5.6; c) heating the aqueous suspension to a temperature higher than 100 °C for a period of 1 to 30 minutes; d) adding one or more proteases to the suspension to obtain an aqueous solution of collagen peptide; e) separating the aqueous solution of collagen peptide from the crushed bone, A method for producing collagen peptide from bone, comprising: The method does not include maceration of the bone with an acid or liming of the bone with a base, and the bone provided in step a) has not been subjected to maceration or liming.

2. The method according to claim 1, wherein the bone is derived from a mammal.

3. The method according to claim 1 or 2, wherein the bone is purified before being crushed.

4. The method according to claim 3, wherein the purification of the bone includes treatment with one or more enzymes.

5. The method according to any one of claims 1 to 4, wherein before crushing, the bone has a fat content of less than 4% by weight.

6. Before crushing, the bone has a collagen content of at least 55% based on the total protein content, the collagen content is determined from the hydroxyproline content multiplied by a factor of 7.3, and the total protein content is determined from the Kjeldahl nitrogen content multiplied by a factor of 6.

25. The method according to any one of claims 1 to 5.

7. The method according to any one of claims 1 to 6, wherein the mechanical crushing includes dry crushing or wet crushing of the bone.

8. The method according to any one of claims 1 to 7, wherein the heating in the aqueous suspension is carried out with an amount of crushed bone by weight of 0.05 to 0.5 kg / l.

9. In step b1), in order to obtain a collagen peptide having an isoelectric point exceeding 5.6, the pH value of the aqueous suspension is adjusted to the range of 6 to 7, or in order to obtain a collagen peptide having an isoelectric point less than 5.6, the pH value of the aqueous suspension is adjusted to the range of 7.9 to 8.

6. The method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 9, wherein the aqueous suspension is cooled to a temperature within the range of 40 to 60 °C before the addition of the one or more proteases.

11. The method according to any one of claims 1 to 10, wherein the one or more proteases added after heating the aqueous suspension are selected from microbial endoproteases.

12. The method according to any one of claims 1 to 11, wherein the one or more proteases are added in an amount of 0.01 to 0.5% by weight based on the dry mass of the crushed bone.

13. The method according to any one of claims 1 to 12, wherein after the addition of the one or more proteases, the enzymatic reaction by the protease is carried out for a period of 0.5 to 4 hours.

14. The method according to any one of claims 1 to 13, wherein once the aqueous solution of the collagen peptide is separated, the crushed bone is subjected to steps c) to e) for a further period of time.

15. The method according to any one of claims 1 to 14, wherein separating the aqueous solution of the collagen peptide includes filtration.

16. The method according to any one of claims 1 to 15, further comprising drying the aqueous solution of the collagen peptide to obtain a collagen peptide powder.

Images