CN111004320A

CN111004320A - Extraction method and application of type II collagen

Info

Publication number: CN111004320A
Application number: CN201911392203.9A
Authority: CN
Inventors: 张春晖; 郭玉杰; 李侠; 王航; 黄峰; 郑乾坤
Original assignee: DELISI GROUP Ltd; Institute of Food Science and Technology of CAAS
Current assignee: DELISI GROUP Ltd; Institute of Food Science and Technology of CAAS
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-04-14

Abstract

The invention discloses a method for extracting type II collagen, which comprises the following steps: step one, mixing cartilage, pepsin and an acetic acid solution, and treating the mixture for 24-36 minutes by using ultrasonic waves with the power of 900-1000W; step two, centrifuging the mixed solution obtained in the step one, and taking supernatant; regulating the pH value of the supernatant to 7, then adding sodium chloride for salting out and collecting protein precipitate; and step four, dissolving the protein precipitate obtained in the step three by using an acetic acid solution, and dialyzing by using distilled water. The invention also provides the application of the type II collagen. Compared with the prior art, the method provided by the invention has the advantages that the purity and yield of the type II collagen are greatly improved, the obtained type II collagen has a complete structure, and the method has good thermal stability, high foamability and emulsibility, and has good application prospects in the industries of food, medicine, cosmetics and the like.

Description

Extraction method and application of type II collagen

Technical Field

The invention relates to the field of collagen extraction. More specifically, the invention relates to a method for extracting type II collagen and application thereof.

Background

Cartilage is a major by-product of meat processing and can be used as a raw material for collagen extraction. However, the existing extraction method has low yield, the obtained type II collagen has poor thermal stability, weak physical and chemical properties such as foamability, emulsibility and the like, and small application prospect. Therefore, it is desirable to design an extraction method that can overcome the above-mentioned drawbacks to some extent.

Disclosure of Invention

Compared with the prior art, the method greatly improves the purity and yield of the type II collagen, and the obtained type II collagen can be used as a foaming agent and an emulsifying agent for preparing foods, medicines and cosmetics.

To achieve the above-mentioned objects and other advantages of the present invention, there is provided a method for extracting type ii collagen, comprising:

step one, mixing cartilage, pepsin and an acetic acid solution, and treating the mixture for 24-36 minutes by using ultrasonic waves with the power of 900-1000W;

step two, centrifuging the mixed solution obtained in the step one, and taking supernatant;

regulating the pH value of the supernatant to 7, then adding sodium chloride for salting out and collecting protein precipitate;

and step four, dissolving the protein precipitate obtained in the step three by using an acetic acid solution, and dialyzing by using distilled water.

Preferably, the method for extracting type ii collagen further comprises, before the step one:

a, cutting cartilage, putting the cut cartilage into a sodium chloride solution, stirring, and washing away grease and bubbles;

b, sequentially adding a sodium carbonate solution and an ethylene diamine tetraacetic acid solution, and replacing the solvent at intervals;

and c, washing the cartilage obtained in the step b by using distilled water until the pH value of the washed distilled water is 7.

Preferably, in the method for extracting type II collagen, in the first step, an acetic acid solution is added to maintain the pH at 2.8.

Preferably, in the method for extracting type II collagen, in the first step, ultrasonic treatment is performed in an ice bath, wherein the frequency of ultrasonic waves is 20-25kHz, and the amplitude is phi 10.

Preferably, the type II collagen is extracted by adopting a pulse mode in the ultrasonic treatment, and the ratio of the running time to the pause time is 2: 3.

Preferably, the method for extracting type II collagen,

the operation time comprises a first operation time and a second operation time, and the first operation time and the second operation time are alternated;

in the first operation time, the frequency of the ultrasonic wave is 20kHz, and the power is 1000W, and in the second operation time, the frequency of the ultrasonic wave is 25kHz, and the power is 900W;

the pause time comprises a first pause time and a second pause time, and the first pause time and the second pause time are alternately appeared;

microwave with power of 900W is applied to the mixture of cartilage, pepsin and acetic acid solution in the first pause time, and nitrogen is blown into the mixture of cartilage, pepsin and acetic acid solution in the second pause time.

Preferably, the extraction method of the type II collagen comprises the step of adding chicken breast cartilage into 0.5M acetic acid solution according to the mass-volume ratio of 1: 6.

Preferably, the extraction method of the type II collagen is to add sodium chloride until the concentration of the sodium chloride is 5M.

The invention also provides the application of the extraction method of the type II collagen in preparing the foaming agent.

The invention also provides the application of the extraction method of the type II collagen, which is used for preparing the emulsifier.

The invention at least comprises the following beneficial effects:

compared with the prior art, the extraction method has higher yield and purity, and the obtained type II collagen has higher thermal stability, better water retention and stronger oil absorption, and has application prospect as a foaming agent and an emulsifier.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a flow chart of a method for extracting collagen type II from chicken sternum cartilage through non-ultrasonic treatment and ultrasonic treatment;

FIG. 2 is the type II collagen yield based on wet weight, dry weight and protein weight of the present invention;

FIG. 3 is an ultraviolet absorption spectrum of type II collagen according to the present invention;

FIG. 4 is a circular dichroism spectrum of type II collagen of the present invention;

FIG. 5 is a Fourier transform infrared spectrum of type II collagen according to the present invention;

FIG. 6 is a differential scanning calorimetry measurement of type II collagen in accordance with the present invention;

FIG. 7 is a microstructure of type II collagen according to the present invention;

FIG. 8 is a graph comparing the effect of sonication time on (A) foaming power (FA) and (B) Foaming Stability (FS);

FIG. 9 is a graph comparing the effect of sonication time on (A) Emulsion Activity Index (EAI) and (B) Emulsion Stability Index (ESI).

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The invention provides a method for extracting type II collagen, which comprises the following steps:

In the technical scheme, the ultrasonic power is preferably 950W, the ultrasonic treatment is preferably 36 minutes, the centrifugation is preferably performed by using an electromagnetic stirrer, the centrifugation time is preferably at least 1 hour, and the centrifugation condition is preferably 13000 g. The pH of the supernatant is preferably adjusted to 7 with sodium hydroxide solution. And then, separating out type II collagen by a conventional salting-out method. The precipitate is preferably dissolved with 0.1M acetic acid. Dialysis is preferably performed at 4 ℃. According to the technical scheme, the yield and the physicochemical property of the type II collagen can be improved through the ultrasonic treatment process.

In another embodiment, the method for extracting type ii collagen further comprises, before the step one:

and c, washing the cartilage obtained in the step b by using distilled water until the pH value of the washed distilled water is 7. The technical scheme is a pretreatment step, preferably, 20 percent (w/v) NaCl solution is adopted, and 0.5M Na is added according to the proportion of 1:10(w/v)₂CO₃Removing pigment, adding at a ratio of 1:10(w/v)Into 0.3M EDTA-Na₂And keeping for 24 hours to remove mineral ions in the chicken breast cartilage.

In another embodiment, in the first step of the method for extracting type II collagen, an acetic acid solution is added to maintain the pH at 2.8.

In another technical scheme, the method for extracting type II collagen comprises the step one of performing ultrasonic treatment in an ice bath, wherein the frequency of ultrasonic is 20-25kHz, and the amplitude is phi 10.

In another technical scheme, the ultrasonic treatment adopts a pulse mode, and the ratio of the running time to the pause time is 2: 3.

In another technical scheme, the method for extracting the type II collagen,

The technical scheme is further improved on the basis of pulse mode ultrasonic treatment, namely that low-frequency and high-power ultrasonic waves and high-frequency and low-power ultrasonic waves are respectively applied in two running times, and microwaves and nitrogen gas are respectively applied and blown in two pause times. The technical scheme can enhance the ultrasonic treatment process in the pulse mode and further improve the yield and the physical and chemical properties of the type II collagen.

In another technical scheme, the extraction method of the type II collagen comprises the step of adding chicken breast cartilage into 0.5M acetic acid solution according to the mass-volume ratio of 1: 6.

In another technical scheme, the method for extracting the type II collagen comprises the step of adding sodium chloride until the concentration of the sodium chloride is 5M.

The invention also provides the application of the II type collagen obtained based on the extraction method, which is used for preparing the foaming agent.

The invention also provides the application of the type II collagen obtained based on the extraction method, which is used for preparing the emulsifier.

The extraction method of the present invention is specifically described below by taking chicken breast cartilage as an example, and compared with the prior art (referred to herein as "non-sonication").

1. Raw materials and chemical reagents:

chicken breast cartilage, large-scale industrial limited of Henan, China, samples were stored at-20 ℃; pepsin, sigma aldrich, usa; the chemical reagents (including acetic acid, sodium chloride and hydroxyproline standard) are all analytical pure

2. Pretreatment:

a solution containing 20% (w/v) NaCl was prepared, followed by the addition of chicken breast cartilage fragments, the pH was maintained at 7.5 by the addition of 0.05M hydrochloric acid at a ratio of 1:10(w/v), stirred overnight with a magnetic stirrer, and washed with distilled water to remove grease and air bubbles. 0.5M Na was added in a ratio of 1:10(w/v)₂CO₃Removing the pigment. Subsequently, 0.3M EDTA-Na was added at a ratio of 1:10(w/v) at pH 7.4₂And keeping for 24h to remove mineral ions in the chicken breast cartilage (the solvent is replaced every 8 h). The splits were washed with distilled water to pH 7.0. The pretreated chicken breast cartilage fragments are uniformly mixed by a mixer and stored at the temperature of minus 20 ℃ for later use.

The results of the analysis of the main composition of the chicken breast cartilage material and the treated chicken breast cartilage are shown in Table 1, and the water content in the material was the highest at 74.32%. In contrast, the fat, ash, protein and carbohydrate content were 0.17%, 2.30%, 12.70% and 10.51%, respectively.

In the pretreated chicken breast cartilage, the water content was significantly increased to 81.76%. However, the fat, ash and carbohydrate content are significantly reduced, respectively: 0.10%, 0.43%, 4.57%, the protein content is 13.53%. These results show that: the pretreatment has obvious influence on chicken breast cartilage raw materials, can effectively remove non-collagen and pigment, and is beneficial to efficient extraction and further analysis of collagen.

Table 1 composition of Chicken Sternal Cartilage (CSC).

Values are expressed as mean ± SD (n ═ 3), and different letters (a, b) within the same row indicate significant sample differences (independent sample t-test).

3. The extraction method comprises the following steps:

non-ultrasonic treatment:

the prior art methods for extracting collagen type II are collectively referred to herein as non-sonicated (UPSCII0, sonication for 0min) collagen type II extraction methods. The pretreated chicken breast cartilage was added to 0.5M acetic acid at a ratio of 1:6(w/v), pepsin was added, the pH was adjusted with acetic acid to maintain it at 2.8, the mixture was stirred with an electromagnetic stirrer at 4 ℃ for 96 hours, and the sample was centrifuged at 13,000g for 1 hour. By salting out, 10ml of 1M hydrochloric acid was added to 1L of the supernatant, 10M NaOH was added to adjust the pH to 7.0, and then NaCl powder was slowly added to the supernatant while stirring until the concentration became 5M, and the rate was kept constant. The resulting precipitate was centrifuged at 13,000g for 1h, and the solid particles were collected and dissolved in 0.1M acetic acid. The samples were dialyzed against distilled water at 4 ℃ and then lyophilized. Then storing at-20 ℃ for later use.

The method comprises the following steps:

ultrasonic extracting collagen with ultrasonic homogenizer with probe, adding pretreated chicken breast cartilage sample into 0.5M acetic acid at a ratio of 1:6(w/v), and mixing. The sample with the volume of 300ml can bear the maximum output power 950W of the ultrasonic homogenizer, the frequency is 20-25kHz, and the amplitude is phi 10. The samples were sonicated in glass beakers fitted with ice sleeves. The selected ultrasonic pulse modes are running for 2s and pausing for 3s, the processing time is respectively 6,12,24 and 36min, and the ultrasonic pulse modes are sequentially represented by UPSCII6, UPSCII12, UPSCII24 and UPSCII 36. In the ultrasonic treatment process, a high-grade titanium alloy probe with the diameter of 6mm is soaked in a sample solution, and the depth is 3cm from the bottom. After the ultrasonic treatment, the pH of the solution was maintained at 2.8 with acetic acid, and then the ultrasonic treated samples were stirred in an electromagnetic stirrer at 4 ℃ for 96h, with the stirring speed being kept constant among the different samples. The subsequent steps were performed according to non-sonication of type II collagen.

4. Yield of

Reasonable sonication time was beneficial for collagen extraction (figure 2). The yields of type II collagen were 1.730g, 2.395g, 2.861g, 3.177g and 3.367g, respectively, at different sonication times (0, 6,12,24,36 min). The results show that the amount of type II collagen obtained increases significantly with the duration of the sonication. And calculating the yield of the type II collagen according to the protein content of the pretreated chicken breast cartilage. When the yield is expressed as protein weight, the yield of type II collagen extracted by the ultrasonic treatment is higher than that extracted by the wet weight method and the dry weight method. The results show that: when calculated by the three methods, the yield of UPSCII36 group is the greatest relative to the non-sonicated group, and there is a rule of UPSCII36> UPSCII24> UPSCII12> UPSCII6> UPSCII 0.

5. Measurement by ultraviolet absorption method

CONH in the polypeptide chain of collagen₂Functional groups such as-COOH and-C ═ O can produce an ultraviolet absorption effect. Therefore, the ultraviolet absorption spectrum is used for judging the quality of the collagen and determining the concentration of the collagen, and the maximum absorption wavelength of the type II collagen standard is between 217.8 and 218.6 nm. The maximum absorption of the non-sonicated type II collagen was between 216.9-217.9nm, while the maximum absorption of the sonicated type II collagen was between 217.4nm and 218.1nm (FIG. 3). An increase in collagen concentration typically causes an upward shift in the uv absorbance peak. The type II collagen obtained by ultrasonic treatment and non-ultrasonic treatment has similar absorption peaks with the standard substance. The method can efficiently extract the type II collagen from the chicken breast cartilage. Non-ultrasonic treated type II collagen and ultrasonic treated type II collagenNo maximum absorption peak appears at 280nm of the protein, which indicates that the extracted type II collagen has higher purity and has no non-collagen interference.

6. Determination of amino acid content

The physicochemical properties of collagen are directly influenced by the amino acid distribution and the crosslinking ability, and the amino acid content in collagen is expressed as the number of residues per 1000 amino acid residues. In the invention, the total amino acid content of the extraction method of the type II collagen increases along with the presentation of ultrasonic time: UPSCII0< UPSCII6< UPSCII12< UPSCII24 ≧ UPSCII36 (Table 2). The extracted type II collagen is rich in glycine, glutamic acid, alanine, proline and hydroxyproline. The glycine content in UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 was 223.62, 281.16, 293.40, 312.87 and 302.88 residues/1000 amino acid residues in this order, indicating that the extracted type II collagen is most abundant in glycine content. The proline content of UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 is 108.96, 115.94, 124.44, 107.37 and 126.44 residues/1000 amino acid residues in sequence, and the hydroxyproline content is 119.40, 118.31, 117.84, 125.31 and 142.74 residues/1000 amino acid residues in sequence. Compared with collagen extracted by non-ultrasonic treatment, the ultrasonic treatment extraction obviously improves the content of proline and hydroxyproline in the II type collagen. The content of proline and hydroxyproline is positively correlated with the heat stability of type II collagen, so that the collagen extracted by the ultrasonic method has good heat stability and structural integrity.

In addition, the contents of methionine, histidine and tyrosine in the collagen extracted by ultrasonic treatment are low. This is due to the removal of non-collagenous proteins using a combination of pepsin digestion and ultrasound. Different ultrasonic times affect the physicochemical properties of the collagen type II from chicken breast cartilage, and have important significance for the preservation and modification of the collagen type II.

Table 2 amino acid profile (residues/1000 amino acid residues) of chicken sternal cartilage type ii collagen at different sonication times for UPSCII0, UPSCII6, UPSCII12, UPSCII24, UPSCII 36.

The numerical values are expressed as mean ± standard deviation (n ═ 3), and the differences are marked by different letters in the same row; (p < 0.05).

7. Determination of the Secondary Structure of type II collagen

Circular dichroism analysis

Circular dichroism can detect the secondary structure of protein. The circular dichroism spectrum of collagen has positive band and negative band, the wavelengths of adult Carnis Pseudosciaenae bands are 197nm and 199nm, chicken bone is 221nm and 197nm, and eel skin collagen is 230nm and 204 nm. In this study, circular dichroism spectra of UPSCII0, UPSCII6, UPSCII12, UPSCII24, and UPSCII36 showed maximum degrees of rotation (positive bands) at wavelengths of 221.69nm, 221.65nm, 221.77nm, 221.89nm, and 221.89nm, and minimum degrees of rotation (negative bands) at wavelengths of 198.32nm, 198.96nm, 199.08nm, 204.05nm, and 209.73nm, respectively. The intersection point (zero band) is located approximately at 214.74nm wavelength (FIG. 4). This indicates that the extracted protein has a triple helix structure and has the typical characteristics of collagen. The triple helix structure of the sonicated chicken breast cartilage type II soluble collagen was not completely hydrolyzed by pepsin and was not destroyed by sonication at different times.

However, the sonicated type II collagen has a higher amino acid content and its triple helix structure is more stable due to intramolecular and intermolecular crosslinks. The results show that the triple helix structure of collagen treated with ultrasound at different times (0, 6,12 min) remains stable after pepsin digestion, whereas the triple helix structure of collagen treated with ultrasound at 24min and 36min is partially denatured, probably as a result of the synergistic effect of pepsin digestion and prolonged ultrasound treatment.

Fourier transform infrared spectroscopy

By Fourier transform infrared spectroscopyWhen analyzing type II collagen, the method mainly comprises the following absorption peaks: amide a, amide B, amide I, amide II, and amide III (fig. 5). The absorption peaks of amide A of UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 were at 3307.71cm^-1,3307.93cm^-1,3301.71cm^-1,3300.93cm^-1And 3311.04cm^-1. Wave number of the protein compared with that of other proteins (3400-3440 cm)^-1) The wave number of these proteins decreased slightly. This may be due to the formation of hydrogen bonds between the N-H group and other functional groups. The positions of the B peaks of UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 amide were respectively reduced to 2923.79cm^-1And 2933.90cm^-1. These peaks occur due to asymmetric stretching of-CH 2, -CH3, and-C-H. The increase of the absorption peaks of amide a and amide B may be associated with the increase of the protein content and the cross-linking of hydrogen bonds within the collagen molecule.

The amide I peaks of UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 were at 1633.74cm^-1、1629.86cm^-1、1636.85cm^-1、1633.96cm^-1And 1637.63cm^-1To (3). The absorption peaks of amide II UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 were at 1550.54cm^-1、1546.65cm^-1、1543.54cm^-1、1549.76cm^-1And 1547.43cm^-1Here, N-H bending couples C-N tensile vibrations. The absorption peaks of amide III UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 were respectively at 1452.56cm^-1、1452.56cm^-1、1452.56cm^-1、1453.56cm^-1And 1456.45cm^-1Here, the characteristic absorption peak of the triple helical structure of collagen is shown. The subtle differences in the absorption peaks are due to the sonication time and pepsin digestion during the extraction process. When pepsin destroys the telopeptide of procollagen, the ultrasonication of the extracted type II collagen also causes a partial secondary structural transformation. Located at 1200cm^-1And 1400cm^-1The peak for regional collagen may be at 1396.57cm^-1Is in the form of-CH₂Bending, -CO-O-C asymmetric stretching; stretching of-CN and-NH at 1236.39cm^-1Deformation of the (c). Fourier transform infrared spectroscopy studies show that it is non-ultrasonicAnd the type II collagen extracted by ultrasonic treatment maintains a stable triple-helical structure.

8. Determination of thermal stability

In the differential scanning calorimetry for determining the thermal stability of type II collagen, two endothermic peaks were observed in the non-ultrasonically extracted type II collagen, which are respectively indicated by thermal degradation temperature (Td) and melting temperature (Tm) (FIG. 6). In this study, the Tm values in the UPSCII0, UPSCII6, UPSCII12, UPSCII24 and UPSCII36 groups were 50.91 ℃, 55.90 ℃, 57.54 ℃, 59.17 ℃ and 54.18 ℃, respectively, and the Td values were 44.83 ℃, 46.99 ℃, 52.21 ℃, 53.51 ℃ and 44.97 ℃, respectively. Compared with non-ultrasonic treatment, the ultrasonic treatment collagen has higher thermal stability, and Tm is obviously increased along with the increase of ultrasonic treatment time. The results indicate that the difference in thermal stability of collagen is related to the physicochemical transition during sonication, which is due to hydration. Sonication for a long time (36min) changed the partial covalent cross-linking of collagen, so the Tm of UPSCII36 was 54.18 ℃ and the Tm of UPSCII24 was 59.17 ℃.

Td values of collagens from different sources vary widely, for example, calf collagen, brown bamboo shark collagen, eel skin collagen, pig skin collagen, silver shark collagen, whale shark collagen, chicken joint collagen and chicken cartilage collagen are 40.8 ℃, 36.7 ℃, 35.0 ℃, 37.0 ℃, 30.0 ℃, 31.0 ℃, 47.0 ℃ and 43.8 ℃ respectively. The thermal stability of collagen is determined by the amino acid composition, and the amino acid content determines the structure of the protein, the interchain hydrogen bonds of the triple helix structure, and the thermal denaturation of the protein. The ultrasonic treated collagen has high thermal stability, and the Td value is related to the amino acid spectrum and corresponds to the content of hydroxyproline and proline. In addition, the Td value also indicates that the protein helical structure is transformed from unfolding to random coiling, thereby changing the physicochemical properties of the protein. The heat stability of the ultrasonically treated type II collagen has potential application value to the production of biomedical, nutritional food, cosmetics and biomaterial products.

9. Microstructure measurement results

The experimentally extracted type II collagen is a loose sponge sheet. The microstructure of type II collagen of the sonicated chicken breast cartilage (FIGS. 7A, 7B, 7C, 7D, 7E) was observed by Scanning Electron Microscopy (SEM) to find that (UPSCII0) was a multilayer aggregate structure with few reticular fibers and a small number of fibrous filaments, and the helical fibers were destroyed by pepsin. The surface structure of the spheres is associated with the dehydration of collagen. Whereas UPSCII6, UPSCII12, UPSCII24, and UPSCII36 have a large number of porous, loose cross-sections and have an interconnected fiber network pore structure. With the prolonged sonication time, the pores and fiber-like structural network of collagen become clearer. Extending the sonication time (UPSCII36) affects the length and structure of the fibers. Compared with the conventional method, the microstructure of the marine collagen extracted by ultrasonic has the characteristics of looseness, porosity and uniformity. The microstructural changes are more related to the functional properties of collagen. The microstructure of the type II collagen after ultrasonic treatment shows that the collagen is in the form of porous, loose and long fiber network due to the mechanical oscillation and cavitation of ultrasonic waves. The study suggests that the sonication time may cause significant physical changes in the microstructure of type II collagen in chicken breast cartilage. These structural changes may further be related to functional characteristics.

10. Measurement of functional Properties of type II collagen

Water binding capacity measurement results:

water binding is one of the important functional properties of proteins. In this study, the WHC values of UPSCII0, UPSCII6, UPSCII12, UPSCII24, and UPSCII36 were 562.19%, 566.11%, 576.29%, 595.11%, and 572.23%, respectively. The WHC of collagen was significantly increased after sonication, higher than that of distilled water (539.07%), but lower than that of glycerol (623.90%). WHC values can fluctuate due to various factors such as ionic strength, protein concentration, and binding capacity. In this study, the ultrasonic treatment increased the water retention capacity and surface hydrophobicity of collagen. The extraction method of the ultrasonic treatment type II collagen has good application prospect in the industries of medicine, food, cosmetics and the like.

TABLE 3 Water Holding Capacity (WHC), Water Absorption Capacity (WAC) and Oil Absorption Capacity (OAC) of type II collagen

Comparison of non-sonicated collagen UPSCII0 with sonicated collagen UPSCII6, UPSCII12, UPSCII24, UPSCII 36. Distilled water is used as a negative control, and glycerol is used as a positive control. Values are expressed as mean ± SD (n ═ 3), and different letters (a-e) in the same column indicate that the difference is statistically significant (p <0.05)

Water absorption measurement results:

the WAC values of UPSCII0, UPSCII6, UPSCII12, UPSCII24, and UPSCII36 treated collagen types ii were 101.39%, 124.06%, 144.77%, 150.34%, and 130.90%, respectively (table 3). The WAC increased significantly with increasing sonication time. The high hydrophilic group and water form hydrogen bonds, and the water absorption and water retention capacity is good. However, after the sonication time exceeded 36 minutes, the WAC value decreased compared to other sonicated collagen samples. The type II collagen extracted by ultrasonic treatment is a suitable water replenishing material.

Oil absorption measurement results:

the OAC values of collagen type ii extracted at different sonication times were 172.93%, 177.33%, 191.47%, 194.93% and 130.90%, respectively (see table 3), probably due to differences in source (raw material), type of protein, degree of proline hydrolysis, processing method, non-polar side chains and oil type. The results indicate that the different values of OAC of the sonicated type ii collagen are an important property of different product ingredients, which is related to the flavor and texture retention of products in the meat and confectionery industries.

Measurement results of foamability:

foaming capacity (FA) and Foaming Stability (FS) are functional properties of proteins. The FA values of the II type collagen under the conditions of

pH

4,7 and 10 are 139.7 percent, 130.7 percent and 137.8 percent of UPSCII0 respectively at different ultrasonic treatment times; UPSCII6, 149.3%, 135.7% and 151.0%; 160.8 percent of UPSCII12, 151.3 percent of UPSCII and 166.5 percent of UPSCII; UPSCII24: 169.4%, 158.6%, 173.1%; UPSCII36 of 150.0%, 130.5% and 144.5%. The results show that the FA values at pH 10 and pH 4 were significantly increased (p <0.05) compared to pH 7, and were more affected by sonication and pH (fig. 7A and 7B).

The FS value is also influenced by pH and ultrasonic treatment time, and the FS values of the II type collagen at different ultrasonic treatment time under the condition of

pH

4,7 and 10 are respectively UPSCII0: 126.6%, 124.0% and 121.1%; UPSCII6: 132.9%, 113.9%, 142.7%; UPSCII12, 137.5%, 129.9%, 142.7%; UPSCII24: 154.9%, 128.7%, 153.9% and UPSCII36: 135.9%, 116.9%, 129.5%. At

pH

4,10, the FS value of UPSCII24 was significantly higher than UPSCII0, UPSCII6, UPSCII12, and UPSCII36 (fig. 7A).

The above shows that the influence of pH and ultrasonic treatment time on chicken breast cartilage type II collagen FA and FS is significant (P < 0.05). The FA and FS values also increase significantly with increasing treatment time, since the charge density increases and the protein unfolding and rearrangement at the interface increases under ultrasound. In an air-water interface induced by the alkali liquor, the development of protein and the change of interface protein formation show hydrophobicity, and the closure of bubbles is enhanced. The effect of sonication on the liquid is mainly cavitation induced by ultrasound, which in turn induces a physical force. The interaction of protein and water is important for the formation and stabilization of foam, and the extraction method of the ultrasonic-treated type II collagen in chicken breast cartilage can be used as a foaming agent in food.

Results of the emulsifiability measurement:

both sonication and pH had a significant effect on the Emulsifying Activity Index (EAI) and Emulsion Stability Index (ESI) of type II collagen extracted from chicken breast cartilage (p)<0.05). The emulsifiability can be changed due to the change of the ultrasonic cavitation intensity, and the pH value is an important factor influencing the functional characteristics of the protein. Comparison of EAI values at different sonication times at

pH

4,7,10, respectively, indicated UPSCII0:32.71m²/g,25.62m²/g,43.51m²/g；UPSCII6:36.52m²/g,32.53m²/g,53.25m²/g；UPSCII12:48.92m²/g,42.17m²/g,65.29m²/g；UPSCII24:61.04m²/g,51.92m²/g,71.80m²/g；UPSCII36:46.48m²/g,41.20m²/g,62.90m²G (Figure 8A, and 8B). EAI values increased with increasing sonication time at

pH

4 and 10 compared to pH 7. The results indicate the potential for protein absorption in both the aqueous and oil phases. The extraction method of the ultrasonic treated type II collagen has high surface hydrophobicity, can improve the emulsifying activity and increase the molecular degree of freedom and the surface hydrophobicity.

The ESI values of collagen can be used to estimate the ability of the protein in water to enter the oil-water interface after emulsification. This indicates its resistance to changes in pH and physicochemical properties during sonication. When the pH is 4,7 and 10, the ESI values of different ultrasonic treatment time are respectively UPSCII0:37.60min,22.25min and 44.40min, UPSCII6:43.98min,35.36min and 53.16 min; UPSCII12 at 50.10min,43.62min and 68.75 min; UPSCII24:64.77min,44.97min and 74.01min, UPSCII36:51.27min,33.25min and 55.09 min. Compared with the emulsifying property of collagen extracted from marine organisms, the method has the following discovery: at pH 4 and pH 10, the ESI values of sonicated collagen were higher than non-sonicated collagen compared to pH 7, but prolonged sonication (>24 minutes) reduced the ESI values of collagen, which could alter the lipophilic-hydrophilic arrangement, molecular size and length of collagen. The emulsifying property enables the extraction method of the type II collagen in the chicken breast cartilage to be used as an emulsifying agent for industrial production, and the application of ultrasonic waves has great influence on the emulsifying property.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. The application, modification and variation of the method for extracting type II collagen of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. The method for extracting type II collagen is characterized by comprising the following steps:

2. The method of claim 1 further comprising, prior to step one:

3. The method of extracting type II collagen according to claim 1, wherein in the first step, an acetic acid solution is added to maintain the pH at 2.8.

4. The method for extracting type II collagen according to claim 1, wherein in the first step, the ultrasonic treatment is performed in an ice bath, and the ultrasonic wave has a frequency of 20 to 25kHz and an amplitude of φ 10.

5. The method of claim 4 in which the sonication is performed in a pulsed mode with a ratio of run time to dwell time of 2: 3.

6. The method of claim 5 wherein the operating time comprises a first operating time and a second operating time, the first operating time alternating with the second operating time;

7. The method of claim 6 in which the chicken breast cartilage is added to 0.5M acetic acid solution at a mass to volume ratio of 1: 6.

8. The method of claim 7 in which sodium chloride is added to a concentration of 5M.

9. Use of type II collagen obtained by the method according to claims 1 to 8 for the preparation of a foaming agent.

10. Use of type II collagen obtained by the method according to claims 1 to 8 for the preparation of an emulsifier.