CN105906710A - Method for sequentially extracting and fractionally salting out and purifying chicken paw skin collagen protein - Google Patents

Abstract

The invention discloses a method for sequentially extracting and grading salting out to purify chicken paw skin collagen, belonging to the field of food bioutilization. Wash the skin of the chicken feet with the visible fat removed, air-dry at low temperature, and cut into small pieces. Soak in NaCl Tris‑HCl buffer to remove impurities and fat. The pretreated chicken paw skin is soaked in the pre-cooled NaCl neutral salt solution, stirred and centrifuged. The precipitate is soaked in acetic acid to extract acid-soluble collagen, and the precipitate is centrifuged and soaked in an enzyme solution to extract enzyme-soluble collagen. The three-step supernatant is graded salting out, dialysis, and freeze-drying to obtain relatively pure salt-soluble collagen, acid-soluble collagen and enzyme-soluble collagen. The present invention uses a solution to pretreat the raw materials efficiently and economically, uses graded extraction, the utilization rate of raw materials is higher, and the corresponding collagen can be selected according to different application ranges, and the pure type I collagen can be obtained by graded salting out and removed Enzyme residues in collagen.

Description

A method for sequentially extracting and grading salting out to purify chicken paw skin collagen

technical field

The invention belongs to the field of food bioutilization, and specifically relates to a method for sequentially extracting and grading salting out to purify collagen.

Background technique

Broiler chickens are consumed in a large amount worldwide, but their chicken feet, which are by-products of poultry industry processing, are often discarded directly, used to make simple processed meat products such as pickled pepper chicken feet, or used to produce animal feed, and many Western countries never eat chicken feet, which causes a great waste of resources. The skin of chicken feet is rich in collagen, so the development and utilization of this advantage of chicken feet skin to extract collagen can greatly improve the economic added value of chicken feet and increase its comprehensive bioavailability, which is of great significance to the broiler chicken industry. significance.

Type I collagen is the most abundant collagen in the body and widely exists in the skin, tendons and ligaments of animals. Type I collagen is widely used in the fields of medicine, food and biology due to its special structure. Chicken paw skin also contains a lot of type I collagen.

At present, the pretreatment method for extracting collagen from animal skin is generally to remove pigment and miscellaneous proteins by alkali soaking, and then remove fat by ethanol. However, this method directly uses the buffer solution to achieve the purposes of the above solutions, greatly saving time and improving efficiency. The traditional extraction process such as: Chinese patent CN In 105218663 A, bovine Achilles tendon collagen is extracted with pepsin solution dissolved in acetic acid, but the utilization rate of raw materials is not high, and a large amount of raw material residues still contain collagen that has not been dissolved. However, the present invention uses neutral salt, acetic acid, and pepsin to extract sequentially, and the solid after each step is left for the next step of extraction, so that the effect that the collagen in the raw material is almost completely extracted can be achieved, and the collagen extracted according to different extraction methods The characteristics of the protein are used in different places to achieve higher efficiency extraction and more refined collagen use. And the present invention not only obtains pure type I collagen after obtaining the crude collagen extract, but also has no enzyme residue in the collagen extracted by enzyme. The extraction is convenient, the raw material utilization rate is high, the efficiency is high, and active pure type I collagen is obtained.

Contents of the invention

In the present invention, chicken paw skin, a by-product of broiler processing, is used as raw material, extracted from three solutions of neutral salt (NaCl), acetic acid and enzyme (Pepsin), and graded and salted out to obtain salt-soluble collagen (SSC), acid-soluble collagen ( ASC) and enzyme soluble collagen (PSC) three kinds of purified type I collagen.

Therefore, the technical scheme that the present invention adopts is as follows: a kind of method of sequentially extracting and graded salting-out purification chicken paw skin collagen, carries out according to the following steps:

(1) Pretreatment of raw materials

Peel off the skin on the chicken feet, cut off the visible fat, wash with tap water, air dry at low temperature, and cut into small pieces. Soak in NaCl Tris-HCl buffer to remove impurities, pigments and fats, stir, and centrifuge to precipitate.

(2) Extraction of salt-soluble collagen

The pretreated chicken paw skin is soaked in the precooled NaCl Tris-HCl buffer solution, stirred, extracted, and the centrifuged supernatant is the crude salt-soluble collagen extract. The centrifuged pellet was washed with distilled water.

(3) Acid-soluble collagen extraction

After salt extraction, the precipitate obtained by centrifugation is soaked in pre-cooled acetic acid solution, stirred, extracted, and the centrifuged supernatant is the crude acid-soluble collagen extract. The centrifuged pellet was washed with distilled water.

(4) Enzyme-soluble collagen extraction

The precipitate obtained after acid extraction is soaked in pre-cooled pepsin solution, stirred, extracted, and the centrifuged supernatant is the crude enzyme-soluble collagen extract.

(5) Purification of collagen

First add HCl to the crude salt-soluble collagen and stir, then slowly add NaCl while stirring until it is completely dissolved, conduct the first salting out overnight, centrifuge, precipitate the crude salt-soluble collagen, and dissolve it in acetic acid.

Add NaCl directly to the crude acid-soluble collagen extract, carry out the first salting-out overnight as in the previous step, and centrifuge to collect the crude acid-soluble collagen, which is dissolved in acetic acid.

Add NaCl directly to the enzyme-soluble collagen crude extract, carry out the first salting out overnight as in the previous step, centrifuge, and dissolve the precipitated acid-soluble collagen crude product with NaCl Tris-HCl buffer solution, and dissolve the collagen in a neutral environment Inactivates residual pepsin.

The above three dissolved collagen solutions were centrifuged to take the supernatant, and NaCl was added again for the second salting out, and the precipitate was collected by centrifugation and dissolved in an appropriate amount of acetic acid, dialyzed with distilled water, and vacuum freeze-dried to obtain a relatively pure collagen freeze-dried product .

Step (1) Peel off the chicken paw skin with a scalpel, remove visible fat by hand, cut the chicken paw skin into 1cm×1cm squares, rinse with tap water, drain and air-dry at 20°C, and store at -20°C for the next step. The ratio of chicken feet skin to salt solution is 1g:20mL, the Tris-HCl buffer is 0.05M, the pH is adjusted to 7.5, and 20g NaCl is added to 100mL of buffer. Centrifugation was performed at 10000 g for 20 min. This operation is repeated until no visible fat and pigment are left.

In step (2), the Tris-HCl buffer solution is the same as the previous step, adding NaCl to the buffer solution to 0.45mol/L, extracting for 48 hours, and centrifuging at 17000g for 30 minutes. After centrifugation, the precipitate was washed with distilled water until the pH was 7.0.

The acid extraction solution in step (3) is 0.5mol/L acetic acid, extracted for 48 hours, and centrifuged at 17000g for 30 minutes.

In step (4), the enzyme solution is 0.5 mol/L acetic acid and the pepsin ratio is 0.1 g: 100 L. The extraction was performed for 48 hours, and the centrifugal condition was 17000g for 30 minutes.

In step (5), add HCl to the crude salt-soluble collagen extract to 0.01mol/L, add NaCl to 0.9mol/L for the first salting-out of the three collagen crude extracts, and conduct the first centrifugation at 2500g 30min. The precipitate obtained from salt extraction and acid extraction was dissolved in 0.5mol/L acetic acid, and the Tris-HCl buffer solution for dissolving the precipitate obtained from enzyme extraction was 0.05mol/L to adjust the pH to 7.5, and NaCl was added to 1.0mol/L. The centrifugation condition is 2500g for 30min, and NaCl is added to the supernatant to 2.4mol/L for the second salting out. After stirring, centrifuge at 17,000 g for 30 min, collect the precipitates obtained by the three extraction methods, dissolve them in 0.5 mol/L acetic acid, and dialyze with distilled water until no white precipitate is detected in the dialyzed external fluid with 0.01 mol/L AgNO ₃ . Afterwards, the collagen solution is poured into a petri dish for vacuum freeze-drying to obtain a pure white dry collagen product in the form of a fibrous sponge.

All operations such as impurity removal, extraction, centrifugation, and purification in the above technical schemes are carried out at 4°C.

The advantages of the present invention are:

(1) Countries around the world consume a large amount of chicken every day, but chicken feet, a by-product of the broiler industry, are directly regarded as processing waste in many countries, and are usually processed into feed or fertilizer, or even directly buried as garbage. Only some Asian countries can eat chicken feet, and they only carry out simple processing, which has caused a great waste of resources and pollution to the environment. However, the present invention uses chicken paw skin as a raw material to produce collagen with higher nutritional value and use value, which greatly improves its added value and increases its comprehensive bioavailability.

(2) The extraction of traditional collagen requires multi-step operations during the pretreatment of raw materials. Various solutions are used to remove impurities, pigments and fats from the raw materials, which is time-consuming and wastes a lot of solutions, and after each step must be used Rinse the raw material repeatedly with distilled water to remove the solution residue from the previous step. However, the present invention directly uses the buffer solution of neutral salt during pretreatment to complete the above cumbersome operations, saves time and effort, has high efficiency, and is easy to operate.

(3) Contain a large amount of collagen protein in the chicken paw skin, the collagen protein in the chicken paw skin is difficult to be extracted completely only with single method such as hot water, salt, acid etc. traditionally, just exist raw material utilization rate like this, economic benefit is not high And the extracted collagen is impure. And the present invention utilizes fractional extraction method, will use neutral salt, acid and enzyme successively to extract the collagen in chicken paw skin almost completely. And the structural classification of collagen proposed according to the three methods can be utilized in the fields of food and cosmetics.

(4) During the extraction of enzyme-soluble collagen, the crude collagen in the purification step is dissolved in the Tris-HCl neutral buffer of NaCl, and the enzyme is inactivated while the collagen is purified, so as to avoid collagen in the subsequent steps of dialysis, freeze-drying and storage. The pepsin in the protein is still active, which can enzymatically hydrolyze the collagen into small molecular peptides, which will affect the biological activity of the collagen.

Description of drawings

Figure 1: Flow chart of chicken paw skin collagen extraction method.

Figure 2: SDS-PAGE profiles of chicken paw skin collagen extracted by different solutions.

Figure 3: Diagram of rheological properties of collagen extracted from different solutions: dynamic modulus (G') from dynamic frequency sweep experiments.

Specific examples

The present invention is elaborated below in conjunction with example:

The rheological measurement of three kinds of solutions extracting collagen among the present invention is by the following method, and collagen is dissolved in 0.5M acetic acid and prepares 1.0% (w/w) solution, with diameter 40mm grinding tool, grinding tool and sample platform distance 1000 μ m, sweep mode, the temperature is 25°C, the frequency is 0.1-10HZ, the continuous stress is 0.5%, and the dynamic modulus G' value is measured by oscillating rheology.

Example 1 :

Wash the skin of the chicken feet with the visible fat removed, dry in low temperature air, and cut into small pieces (about 1cm×1cm). Weigh 100g and soak in 20L 20% (w/v) NaCl Tris-HCl (0.05mol/L, pH7.5) buffer to remove foreign protein, pigment and fat, stir for 48h, centrifuge at 10000g for 20min, collect the precipitate, distilled water Wash to pH 7.0.

Soak the pretreated chicken paw skin in 8L pre-cooled 0.45mol/L NaCl Tris-HCl (0.05mol/L, pH7.5) buffer solution, stir for 48h, centrifuge at 17000g for 30min, and the supernatant is the crude salt-soluble collagen Extraction. The resulting precipitate after centrifugation was washed with distilled water to pH 7.0.

After salt extraction, the precipitate obtained by centrifugation was soaked in 8L pre-cooled 0.5mol/L acetic acid solution, stirred for 48h, and centrifuged at 17000g for 30min. The supernatant was the crude acid-soluble collagen extract.

The precipitate obtained after acid extraction was soaked in 8L pre-cooled pepsin solution (8g pepsin was added to 8L acetic acid), stirred for 48h, centrifuged at 17000g for 30min, and the supernatant was the crude enzyme-soluble collagen extract.

Add HCl to the crude salt-soluble collagen extract to 0.01mol/L, then slowly add NaCl to 0.9mol/L while stirring for the first salting out overnight, centrifuge at 2500g for 30min, precipitate the crude salt-soluble collagen, dissolve in 8L 0.5mol/L acetic acid.

Slowly add NaCl to 0.9mol/L to the acid-soluble collagen crude extract, carry out the first salting out overnight, centrifuge at 2500g for 30min, and the collected precipitate is acid-soluble collagen crude product, which is dissolved in 8L 0.5mol/L acetic acid.

Slowly add NaCl to 0.9mol/L to the enzyme-soluble collagen crude extract while stirring, carry out the first salting-out overnight, centrifuge at 2500g for 30min, and obtain the precipitated acid-soluble collagen crude product, and use 8L of NaCl (1.0mol/L) Dissolve in Tris-HCl (0.05mol/L, pH7.5) buffer.

The above three dissolved collagen solutions were centrifuged at 2500g for 30min to take the supernatant, and NaCl was added again to 2.4mol/L for the second salting out, and the precipitate was collected by centrifugation at 17000g for 30min and dissolved in 500mL of acetic acid (0.5mol/L). Put the solution into a dialysis bag with a molecular weight cut-off of 14KDa, dialyze with distilled water for 72 hours, and vacuum freeze-dry to obtain a collagen freeze-dried product. Calculated according to the ratio of hydroxyproline in the crude collagen extract and the raw material, the yield is PSC>ASC>SSC, which are (49.10±1.95)%, (14.49±0.90)% and (1.13±0.31)% in turn. The collagen obtained by SDS-PAGE analysis was pure type I collagen. The three collagens were dissolved in acetic acid to measure their rheological properties. As shown in Figure 3, the three collagen solutions mainly exhibited elastic behavior, while the mechanical loss trend was PSC>ASC>SSC.

Example 2 :

Rinse the skin of the chicken feet with the visible fat removed, drain, and cut into small pieces (about 1cm×1cm). Weigh 100g and soak in 25L 20% (w/v) NaCl Tris-HCl (0.05mol/L, pH7.5) buffer to remove foreign protein, pigment and fat, stir for 72h, centrifuge at 10000g for 20min, collect the precipitate, distilled water Wash to pH 7.0.

Soak the pretreated chicken paw skin in 10L pre-cooled 0.45mol/L NaCl Tris-HCl (0.05mol/L, pH7.5) buffer solution, stir for 72h, centrifuge at 17000g for 30min, and the supernatant is the crude salt-soluble collagen Extraction. The resulting precipitate after centrifugation was washed with distilled water to pH 7.0.

After salt extraction, the precipitate obtained by centrifugation was soaked in 10L of pre-cooled 0.5mol/L acetic acid solution, stirred for 72h, centrifuged at 17000g for 30min, and the supernatant was the crude acid-soluble collagen extract.

The precipitate obtained after acid extraction was soaked in 10L of pre-cooled pepsin solution (10g of pepsin in 10L of acetic acid), stirred for 72h, centrifuged at 17000g for 30min, and the supernatant was the crude enzyme-soluble collagen extract.

Add HCl to the crude salt-soluble collagen extract to 0.01mol/L, then slowly add NaCl to 0.9mol/L while stirring for the first salting out overnight, centrifuge at 2500g for 30min, precipitate the crude salt-soluble collagen, dissolve in 10L 0.5mol/L acetic acid.

Slowly add NaCl to 0.9mol/L to the acid-soluble collagen crude extract, carry out the first salting out overnight, centrifuge at 2500g for 30min, and the collected precipitate is acid-soluble collagen crude product, which is dissolved in 10L 0.5mol/L acetic acid

Slowly add NaCl to 0.9mol/L to the enzymatically soluble collagen crude extract while stirring, carry out the first salting-out overnight, centrifuge at 2500g for 30min, and the obtained precipitated acid-soluble collagen crude product is washed with 10L of NaCl (1.0mol/L) Dissolve in Tris-HCl (0.05mol/L, pH7.5) buffer.

The above three dissolved collagen solutions were centrifuged at 2500g for 30min to take the supernatant, and NaCl was added again to 2.4mol/L for the second salting out, and the precipitate was collected by centrifugation at 17000g for 30min and dissolved in 500mL of acetic acid (0.5mol/L). Distilled water was dialyzed for 72 hours, and vacuum freeze-dried to obtain a collagen freeze-dried product. According to the proportion of hydroxyproline in the crude collagen extract and the raw material, the yield was PSC>ASC>SSC, which were (50.60±2.21)%, (16.01±0.03)% and (1.28±0.97)%. Compared with Case 1, it shows that the yield will increase appropriately when the solid-liquid ratio becomes higher during extraction. The collagen obtained by SDS-PAGE analysis was pure type I collagen.

Example 3 :

Wash the skin of the chicken feet with the visible fat removed, drain, and cut into small pieces (about 1cm×1cm). Weigh 100g and soak in 15L 20% (w/v) NaCl Tris-HCl (0.05mol/L, pH7.5) buffer to remove foreign protein, pigment and fat, stir for 48h, centrifuge at 10000g for 20min, collect the precipitate, distilled water Wash to pH 7.0.

Soak the chicken feet skin after pretreatment in 6L precooled 0.45mol/L NaCl Tris-HCl (0.05mol/L, pH7.5) buffer solution, stirred for 48h, centrifuged at 17000g for 30min, and the supernatant was the crude salt-soluble collagen extract. The resulting precipitate after centrifugation was washed with distilled water to pH 7.0.

After salt extraction, the precipitate obtained by centrifugation was soaked in 6L pre-cooled 0.5mol/L acetic acid solution, stirred for 48h, and centrifuged at 17000g for 30min. The supernatant was the crude acid-soluble collagen extract.

The precipitate obtained after acid extraction was soaked in 6L pre-cooled pepsin solution (6g pepsin was added to 6L acetic acid), stirred for 48h, centrifuged at 17000g for 30min, and the supernatant was the crude enzyme-soluble collagen extract.

Add HCl to the crude salt-soluble collagen extract to 0.01mol/L, then slowly add NaCl to 0.9mol/L while stirring for the first salting out overnight, centrifuge at 2500g for 30min, precipitate the crude salt-soluble collagen, dissolve at 6L 0.5mol/L acetic acid.

Slowly add NaCl to 0.9mol/L to the crude acid-soluble collagen extract while stirring, carry out the first salting out overnight, centrifuge at 2500g for 30min, the collected precipitate is the crude acid-soluble collagen, dissolve in 6L 0.5mol/L acetic acid.

Slowly add NaCl to 0.9mol/L to the enzyme-soluble collagen crude extract while stirring, carry out the first salting-out overnight, centrifuge at 2500g for 30min, and obtain precipitated acid-soluble collagen crude product, use 6L of NaCl (1.0mol/L) Dissolve in Tris-Hcl (0.05mol/L, pH7.5) buffer.

The above three dissolved collagen solutions were centrifuged at 2500g for 30min to take the supernatant, and NaCl was added again to 2.4mol/L for the second salting out, and the precipitate was collected by centrifugation at 17000g for 30min and dissolved in 500mL of acetic acid (0.5mol/L). Put the solution into a dialysis bag with a molecular weight cut-off of 14KDa, dialyze with distilled water for 72 hours, and vacuum freeze-dry to obtain a collagen freeze-dried product. According to the ratio of hydroxyproline in the crude collagen extract and the raw material, the yield is PSC>ASC>SSC, which are (47.78±0.20)%, (13.11±1.65)% and (0.78±0.33)%. The collagen obtained by SDS-PAGE analysis was pure type I collagen.

All operations such as impurity removal, extraction, centrifugation, and purification in the above examples were carried out at 4°C. The process of sequentially extracting chicken paw skin collagen and grading and salting out is shown in the flow chart. The purity of the extracted collagen can be identified by SDS-PAGE. The proteins obtained by the three methods are all relatively pure type Ⅰ collagen. It can be seen that SSC, ASC and PSC all have three clear peptide chains, namely α ₁ (Ⅰ) and α ₂ (Ⅱ) two α chains, α A dimer of β and a trimeric γ chain of α.

Claims (6)

1. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that carry out as steps described below:

(1) pretreatment of raw material

Peel the skin on chicken feet, excise visible fat, tap water wash clean, low-temperature air-drying, be cut into small pieces；It is immersed in the Tris-HCl buffer of NaCl removing foreign protein, pigment and fat, stirring, takes centrifuged deposit；

(2) salt soluble collagen extracts

The chicken feet skin of pretreatment is soaked in the Tris-HCl buffer of the NaCl of pre-cooling, stirring, extracts, centrifuged supernatant i.e. salt soluble collagen crude extract；Centrifugation distilled water flushing；

(3) acid-soluble collagen protein extracts

Salt is centrifuged gained precipitation and is soaked in the acetic acid solution of pre-cooling after putting forward, stirring is extracted, centrifuged supernatant the most acid-soluble collagen protein crude extract；Centrifugation distilled water flushing；

(4) pepsin-solubilized collagen extracts

Acid puies forward rear gained precipitation and is soaked in the pepsin solution of pre-cooling, and stirring is extracted, centrifuged supernatant i.e. pepsin-solubilized collagen crude extract；

(5) purification of collagen protein

First adding HCl stirring in salt soluble collagen crude extract, addition NaCl is until it is completely dissolved the most while stirring, carries out saltouing overnight for the first time, centrifugal, and precipitation i.e. salt soluble collagen crude product is dissolved in acetic acid；

Directly adding NaCl in acid-soluble collagen protein crude extract, ibid step operation carries out saltouing overnight for the first time, centrifugal, and the precipitation the most acid-soluble collagen protein crude product of collection is dissolved in acetic acid；

Pepsin-solubilized collagen crude extract directly adds NaCl, ibid step operation carries out saltouing overnight for the first time, centrifugal, gained precipitated acid soluble collagen crude product, with the Tris-HCl buffer solution of NaCl, while dissolving collagen protein, neutral environment makes pepsin residual inactivation；

Collagen solution after above-mentioned three kinds of dissolvings, centrifuging and taking supernatant, again add NaCl and carry out pickle change, centrifugal collecting precipitation is dissolved in appropriate acetic acid, and distilled water is dialysed, the collagen protein dried frozen aquatic products that vacuum lyophilization must be purer.

A kind of extraction successively the most according to claim 1 and the method for salt fractionation purification chicken feet collagen, it is characterized in that step (1) scalpel peels off chicken feet skin, manual excision visible fat, chicken feet skin is cut into the square of 1cm × 1cm, tap water rinses, draining and air-dry at latter 20 DEG C ,-20 DEG C of preservations are to treat that next step uses；Chicken feet skin and saline solution are 0.05M than for 1g:20mL, Tris-HCl buffer, and adjusting pH is 7.5, adds 20g NaCl in 100mL buffer；It is centrifuged and carries out 20min for 10000g；This operation is repeated up to without visible fat and pigment.

3. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that in step (2), Tris-HCl buffer ibid walks the same terms, adds NaCl to 0.45mol/L in buffer, extracts 48h, and centrifugal condition is that 17000g carries out 30min；Centrifuged deposit distilled water flushing is 7.0 to pH.

4. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that in step (3), acid extracting solution is 0.5mol/L acetic acid, extracts 48h, and centrifugal condition is that 17000g carries out 30min.

5. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that in step (4), enzymatic solution is to add pepsin ratio in 0.5mol/L acetic acid for 0.1g:100L；Extracting 48h, centrifugal condition is that 17000g carries out 30min.

6. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterized in that in step (5), salt soluble collagen crude extract adds HCl to 0.01mol/L, three kinds of collagen protein crude extracts are saltoutd for the first time and are added NaCl to 0.9mol/L, and centrifugal condition is that 2500g carries out 30min for the first time；Salt carries and carries gained with acid and be precipitated and dissolved in 0.5mol/L acetic acid, and it is that 0.05mol/L adjusts pH to be 7.5 that lyase carries the Tris-HCl buffer of gained precipitation, adds NaCl to 1.0mol/L；Centrifugal condition is that 2500g carries out 30min, adds NaCl to 2.4mol/L and carry out pickle change in supernatant；After stirring, 17000g is centrifuged 30min, collects three kinds of extracting method gained precipitations, is dissolved in 0.5mol/L acetic acid, and distilled water is dialysed, until using 0.01mol/L AgNO₃Till detection extracellular fluid dialysis is without white precipitate；Carry out vacuum lyophilization during collagen solution pours culture dish into afterwards, i.e. obtain the collagen protein sterling of white dried in fiber sponge shape.