阅读说明：本技术 高纯度未变性胶原蛋白及其制造方法 (High-purity undenatured collagen and method for producing same ) 是由 李杰翰 洪雅萍 廖耿毅 许智尧 林美吟 苏育森 萧述文 于 2018-10-16 设计创作，主要内容包括：本发明提供一种高纯度未变性胶原蛋白及其制造方法,其利用酸及非胃蛋白酶(non-pepsin protease)水解鸡胸骨及粗滤后,所述滤液无需经离心回溶处理,直接进行加盐处理及精滤处理,以在较短的制程时间内获得高纯度未变性胶原蛋白,其中高纯度未变性胶原蛋白的总胶原蛋白含量为60％以上,且含有至少50％的未变性第二型胶原蛋白。(The invention provides a high-purity undenatured collagen and a preparation method thereof, which utilizes acid and non-pepsin (non-pepsin protease) to hydrolyze chicken sternum and perform rough filtration, and the filtrate is directly subjected to salt treatment and fine filtration without centrifugal redissolution treatment so as to obtain the high-purity undenatured collagen in a shorter process time, wherein the total collagen content of the high-purity undenatured collagen is more than 60 percent, and the high-purity undenatured collagen contains at least 50 percent of undenatured second type collagen.)

1. A method for producing high-purity undenatured collagen, comprising:

providing a chicken breast bone;

acid hydrolyzing the chicken sternum at 0 ℃ to 7 ℃ for at least 30 days to form an acid hydrolysate;

adding non-pepsin into the acid hydrolysate for enzymatic hydrolysis to form an enzymatic hydrolysate, wherein the weight ratio of the non-pepsin to the chicken breast bone is 1: 60 to 1: 100, respectively;

subjecting the enzymatic hydrolysate to a coarse filtration treatment to form a filtrate;

performing salting treatment on the filtrate to form a salt solution; and

after the salt adding treatment, the salt solution is directly subjected to fine filtration treatment to obtain collagen,

the collagen comprises at least 50% undenatured type II collagen.

2. The method for producing high-purity undenatured collagen according to claim 1, further comprising:

after obtaining the collagen, the collagen is subjected to low-temperature drying treatment.

3. The method for producing high-purity undenatured collagen according to claim 1, wherein the step of acid hydrolysis is performed using acetic acid having a pH of 1.5 to 2.

4. The method for producing a high-purity undenatured collagen according to claim 1, wherein the enzymatic hydrolysis is carried out at 12 ℃ to 36 ℃ for 8 to 72 hours.

5. The method for producing a high-purity undenatured collagen according to claim 1, wherein the salt concentration of the salting treatment is 0.7M to 1.1M.

6. The method for producing high-purity undenatured collagen according to claim 1, wherein the fine filtration treatment comprises:

diluting the salt solution to at least 300 liters of diluent solution; and

passing the dilution through a plurality of ceramic membranes at a flow rate of 30 liters to 50 liters per minute to remove impurities having a molecular weight of 1 to 300 kD.

7. The method for producing high-purity undenatured collagen according to claim 6, wherein said fine filtration treatment comprises a plurality of cycles of performing said dilution step and said step of passing said ceramic membrane for 4 to 8 days.

8. The method for producing a high-purity undenatured collagen according to claim 1, wherein said production method does not comprise a centrifugal precipitation treatment and a redissolution treatment before said fine filtration.

9. A high-purity undenatured collagen produced using the method for producing high-purity undenatured collagen according to any one of claims 1 to 8, wherein the high-purity undenatured collagen has a total collagen content of at least 60%, and at least 50% of the high-purity undenatured collagen is undenatured type ii collagen.

10. The high purity native collagen according to claim 9, wherein said high purity native collagen has an ash content of less than 1.5%.

Technical Field

The present invention relates to a collagen and a method for producing the same, and more particularly, to a high-purity undenatured collagen having a high content of undenatured type II collagen, and a method for producing the same.

Background

The second type collagen is the main structural component of cartilage, and is mixed with other types of collagen, proteoglycan and other components in cartilage to support tissue and absorb impact. Type II collagen has good biocompatibility, and thus is often used in the fields of food, medical treatment, beauty treatment, and the like.

Recent studies have indicated that Undenatured type II collagen has significant therapeutic effects on rheumatoid arthritis. Although many products of undenatured type II collagen are available, their solubility in various pH solutions is poor, the amount of undenatured type II collagen is low, and the total collagen content in the product is also low.

In addition, in conventional processes, the native second type collagen is often inactivated by degradation due to heat or chemical treatment (e.g., enzymatic treatment). Furthermore, the enzymes or other reagents used in conventional processes are generally non-food grade, which results in limited application of the collagen products and longer processing time. In particular, pepsin, which is commonly used for purifying collagen, is not currently included in food grade enzymes at home due to its complex and undefined origin.

Therefore, there is a need to provide a high-purity undenatured collagen and a method for producing the same, which can solve the above problems.

Disclosure of Invention

One aspect of the present invention is to provide a method for producing high-purity undenatured collagen, which can produce collagen having a high total collagen content (corresponding to purity) and a high undenatured second-type collagen content (corresponding to activity).

Another aspect of the present invention is to provide a high-purity undenatured collagen produced by the above-described production method.

According to the above aspect of the present invention, a method for producing high-purity undenatured collagen is provided. In some embodiments, the chicken sternum is provided first. The chicken sternum is then subjected to acid hydrolysis to form an acid hydrolysate. The acid treatment is carried out at 0 ℃ to 7 ℃ for at least 30 days. Then, non-pepsin is added to the acid hydrolysate for enzymatic hydrolysis to form an enzymatic hydrolysate. The weight ratio of the non-pepsin to the chicken breast bone is 1: 60 to 1: 100. thereafter, the enzymatic hydrolysate is subjected to a coarse filtration treatment to form a filtrate. Then, the filtrate is subjected to a salt-adding treatment to form a salt solution. And after the salt adding treatment, directly performing fine filtration treatment on the salt solution by using ceramic membranes with different apertures to obtain the high-purity unmodified collagen. The collagen comprises at least 50% undenatured type II collagen.

According to some embodiments of the present invention, after obtaining the collagen, the collagen may be subjected to a low temperature drying process.

According to some embodiments of the invention, the step of acid hydrolysis is performed using acetic acid at pH 1.5 to 2.

According to some embodiments of the invention, the enzymatic hydrolysis is performed at 12 ℃ to 36 ℃ for 8 to 72 hours.

According to some embodiments of the invention, the salting treatment has a salt concentration of 0.7M to 1.1M.

According to some embodiments of the present invention, the fine filtration treatment comprises diluting the salt solution to at least 300 liters of diluent and passing the salt solution through a plurality of ceramic membranes at a flow rate of 30 liters to 50 liters per minute to remove impurities having a molecular weight of 1 to 300 kD.

According to some embodiments of the invention, the fine filtration treatment comprises performing a plurality of cycles of the dilution step and the step of passing through a plurality of ceramic membranes for 4 to 8 days.

According to some embodiments of the present invention, the manufacturing method does not include a centrifugal precipitation treatment and a redissolution treatment before the fine filtration treatment.

According to the above aspect of the present invention, a high-purity undenatured collagen is provided. In some embodiments, the high purity undenatured collagen is produced using the above described manufacturing methods. The high purity undenatured collagen has a total collagen content of at least 60% and at least 50% of the high purity undenatured collagen is undenatured type II collagen.

According to some embodiments of the invention, ash in the high purity undenatured collagen is less than 1.5%.

Drawings

In order to make the aforementioned and other objects, features, and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail as follows:

FIG. 1 is a schematic flow diagram illustrating a method for producing high purity undenatured collagen according to some embodiments of the present invention.

Wherein the description of the symbols of the drawings is as follows:

100: a method;

110: extracting the chicken breast bone;

120: acid hydrolyzing chicken sternum to form an acid hydrolysate;

130: adding non-pepsin to the acid hydrolysate for enzymatic hydrolysis to form an enzymatic hydrolysate;

140: subjecting the enzymatic hydrolysate to a coarse filtration treatment to form a filtrate;

150: performing salting treatment on the filtrate to form a salt solution;

160: after the salt treatment, the salt solution is directly subjected to fine filtration treatment to obtain high-purity unmodified collagen.

Detailed Description

The present invention provides a method for producing high-purity undenatured collagen, and more particularly, to a method for producing high-purity undenatured food-grade collagen, which comprises hydrolyzing chicken sternum with an acid and a non-pepsin (non-pepsin protease), coarsely filtering the hydrolyzed product, and then directly subjecting the filtrate to a salt treatment and a fine filtration treatment without centrifugation of the filtrate and redissolution of the precipitate, thereby obtaining high-purity undenatured collagen in a short process time.

The food-grade collagen protein of the present invention is herein described as being made using food-grade reagents. The food grade specification is defined by the food additive license of food and drug administration of Taiwan health and welfare department.

Referring now to fig. 1, a schematic flow diagram 100 of a method for producing high purity native collagen according to some embodiments of the present invention is shown.

As shown in fig. 1, in step 110 of method 100, a chicken sternum is first provided. In one embodiment, at least 20 kilograms of chicken sternum are provided. In a preferred embodiment, the amount of chicken breast bone used is 20 kg to 40 kg. Generally speaking, chicken sternums are suitable for the production of undenatured type II collagen due to the high content of type II collagen in chicken sternum, ranging from 45% to 70% by weight of the total protein in chicken sternum.

In some embodiments, the chicken sternum has been first fleshed, chopped, and cleaned, as described in step 110. In some instances, the washing process may be performed, for example, using Ethylenediaminetetraacetic acid (EDTA).

Next, as shown in step 120, the chicken sternum is subjected to acid hydrolysis to form an acid hydrolysate. The acid hydrolysis may be performed at 0 ℃ to 7 ℃ for at least 30 days. In some embodiments, the acid hydrolysis is performed using acetic acid at a pH of 1.5 to 2. If the time for acid hydrolysis is insufficient, the subsequent enzymatic hydrolysis efficiency will be poor. Furthermore, since acid hydrolysis is carried out for a long time, if the temperature is too high, the chicken breast bone is easy to deteriorate, thereby reducing the content of the undenatured second type collagen.

Then, as shown in step 130, non-pepsin is added to the acid hydrolysate for enzymatic hydrolysis to form an enzymatic hydrolysate. In one example, the enzymatic hydrolysis is carried out at a pH of 1.9 to 6. In one embodiment, the enzymatic hydrolysis is carried out at 12 ℃ to 36 ℃ for 8 to 72 hours. If the pH, temperature or time range is exceeded, the enzymatic hydrolysis effect is not good. In one embodiment, a step of adjusting the pH value may be further included between step 120 and step 130, so that the pH value of the acid hydrolysate may fall within an appropriate range of the enzymatic hydrolysis step. In some embodiments, the weight ratio of non-pepsin to chicken sternum is 1: 60 to 1: 100. in particular, the non-pepsin used in the present invention is a food grade enzyme, which may include, but is not limited to, protease m (proteasem), papain, bromelain, other suitable enzymes, or a combination of the above enzymes. In some embodiments, the present invention does not include the use of pepsin for enzymatic hydrolysis. In one embodiment, the enzymatic hydrolysis is carried out in an acetic acid solution.

Thereafter, as shown in step 140, the enzymatic hydrolysate is subjected to a coarse filtration treatment to form a filtrate. The rough filtration treatment can be performed by using mesh screens with different pore sizes, for example, to filter out solid impurities such as broken chicken bones remaining in the enzymatic hydrolysate, and to retain the filtrate for the subsequent process steps. In other embodiments, the coarse filtration may be performed using a mesh screen having a different pore size, such as 60 mesh (mesh), 300 mesh, or 500 mesh. The pore size and the number of the mesh are not particularly limited in the present invention, and can be adjusted according to the conditions of the enzymatic hydrolysate.

Next, the filtrate is salted to form a salt solution, as shown in step 150. In some embodiments, the salting treatment is performed at a salt concentration of 0.7M to 1.1M. In some embodiments, the salting process may, for example, add sodium chloride to the above clarified solution and stir until the sodium chloride is completely dissolved.

Specifically, the salt solution contains a crude collagen product aggregated or precipitated by isoelectric point equilibrium and a salt solution. The present invention does not include centrifugation to separate the crude collagen product from the salt solution, but rather, the salt solution is directly subjected to a subsequent fine filtration (as shown in step 160) to reduce the loss of collagen and thereby increase the total collagen content and the undenatured second type collagen content. In one example, the amount of chicken breast bone treated is at least 20 kg, so the amount of salt solution produced by salting treatment is also considerable (e.g., a total weight of 140 kg or more). If the salt solution is centrifuged, it takes a lot of time because of the limitation of the size of the centrifuge and the need to re-dissolve the precipitate after centrifugation.

Then, as shown in step 160, after the salt-adding treatment in step 150, the salt solution is directly subjected to a fine filtration treatment to obtain the high-purity non-denatured collagen of the present invention. In some embodiments, the fine filtration process comprises diluting the salt solution to a dilution of at least 300 liters and passing the dilution through a plurality of ceramic membranes at a flow rate of 30 liters to 50 liters per minute to remove impurities (e.g., salts, ash) having a molecular weight of 1 to 300 kD. In some embodiments, the fine filtration process dilutes the salt solution to a dilution of 300 liters to 500 liters. In some embodiments, the fine filtration treatment cycle is performed (i.e., repeated through the ceramic membrane described above) for 4 to 8 days. The circulation fine filtration treatment comprises a plurality of circulations of the two steps of dilution and removal of water containing impurities through a ceramic membrane, so as to improve the impurity removal rate.

In some embodiments, each of the plurality of ceramic membranes may be a tubular ceramic membrane comprising alumina, zirconia, and/or titania. In one particular example, the tubular ceramic membrane may have a length of, for example, 120 cm and a cross-sectional area of, for example, 10mm to 25 mm. In some instances, a plurality of tubular ceramic membranes (e.g., 3) are arranged in series so that the diluent can pass through them sequentially. The invention keeps the high purity and the high content of the unmodified second type collagen of the collagen by adjusting the time, equipment (such as the specification or configuration of a ceramic membrane), the flow rate of the fine filtration treatment and the like of the fine filtration treatment.

In some embodiments, the manufacturing method of the present invention further comprises subjecting the high-purity undenatured collagen to a low-temperature drying process after the high-purity undenatured collagen is formed. The high-purity undenatured collagen after low-temperature drying treatment is in a sponge or sheet shape, so that the storage is convenient and the storage time is prolonged. The low temperature drying process referred to herein may be, for example, a drying process below 40 ℃, and may include, but is not limited to, oven drying or lyophilization.

The high-purity undenatured collagen of the present invention has a total collagen content (corresponding to collagen purity) of 60% or more, and at least 50% of the high-purity undenatured collagen is undenatured type II collagen (corresponding to an active ingredient). In some embodiments, the ash in the high purity undenatured collagen is less than 1.5%. In some examples, undenatured type two collagen comprises greater than 70% by weight of the total collagen content.

The following examples and comparative examples are provided to illustrate the production method of high-purity undenatured collagen according to the present invention.