CN113735965A - Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof - Google Patents

Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof Download PDF

Info

Publication number
CN113735965A
CN113735965A CN202111074561.2A CN202111074561A CN113735965A CN 113735965 A CN113735965 A CN 113735965A CN 202111074561 A CN202111074561 A CN 202111074561A CN 113735965 A CN113735965 A CN 113735965A
Authority
CN
China
Prior art keywords
collagen
sturgeon cartilage
solution
sturgeon
cartilage
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111074561.2A
Other languages
Chinese (zh)
Inventor
侯虎
白雪
李兆霞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ocean University of China
Original Assignee
Ocean University of China
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ocean University of China filed Critical Ocean University of China
Priority to CN202111074561.2A priority Critical patent/CN113735965A/en
Publication of CN113735965A publication Critical patent/CN113735965A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides sturgeon cartilage II type non-denatured collagen and a preparation method and application thereof, which comprises the steps of treating sturgeon cartilage by using alkali liquor, performing homogenization treatment, cleaning by using Tris-HCl buffer solution added with guanidine hydrochloride, performing enzymolysis by using pepsin added in acid liquor, centrifuging enzymolysis liquid, and collecting supernatant; salting out the supernatant; and centrifuging and collecting a precipitate product; and redissolving the precipitated product by using an acid solution, dialyzing the solution, and freeze-drying the dialyzate to obtain the sturgeon cartilage II type non-denatured collagen. The invention provides a method for deep processing and application of sturgeon cartilage resources by taking sturgeon cartilage which is a byproduct of sturgeon processing as a raw material, and enriches collagen products in the market. The collagen II extracted by the invention keeps the triple helix structure and the activity of the collagen, and the application value of the product is greatly improved. The extraction rate of the II type collagen extracted by the invention is high.

Description

Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof
Technical Field
The invention belongs to the technical field of animal collagen extraction, and particularly relates to sturgeon cartilage II type non-denatured collagen and a preparation method and application thereof.
Background
The aquatic organism has abundant cartilage resources in vivo, and compared with terrestrial animals, the aquatic organism collagen has good biochemical characteristics and unique advantages of safety, low allergy, low antigenicity and the like, so that the preparation and application of the aquatic organism collagen II are worthy of attention.
Sturgeon cartilage contains rich active ingredients such as type II collagen and polysaccharide, and has important physiological effects, but is not regarded and utilized. The type II collagen is one of the main components of cartilage matrix, can promote the differentiation of chondrocyte and improve the bone health, and has positive effect on treating rheumatoid arthritis diseases.
Sturgeons are one of the existing freshwater fishes with large body size and long service life, and have high nutritional value and economic value, so that the culture area and culture yield of sturgeons in China are increased year by year. Sturgeons are large in size, bones of other parts are cartilages except a fin which is partially ossified and a real bone of the head, a large amount of by-product fishbones are generated in the deep processing process of the sturgeons, and the sturgeons contain rich collagen and are good raw materials for extracting type II collagen.
At present, the extraction methods of collagen at home and abroad mainly comprise an alkaline method, hot water extraction, a salt method, an acid method, an enzyme method and the like. Some of the methods can cause hydrolysis of peptide bonds of collagen, all amino acids containing hydroxyl and sulfhydryl groups are destroyed, collagen is denatured, and some methods can cause a large amount of collagen to be lost. The reaction condition for producing collagen by the enzyme method is mild, the triple helix structure of the collagen is not damaged, and the method can keep the characteristics and is widely applied. However, the existing enzymolysis technology is carried out at normal temperature and normal pressure, even at high temperature and high pressure, the enzymolysis, concentration and drying processes all need higher temperature, the influence on the activity of collagen is larger, and the product quality is difficult to ensure.
Disclosure of Invention
The invention provides sturgeon cartilage II type non-denatured collagen and a preparation method and application thereof, thereby making up for the defects of the prior art.
The sturgeon cartilage II type non-denatured collagen provided by the invention is prepared by the following steps:
treating sturgeon cartilage with alkali liquor, homogenizing, washing with Tris-HCl buffer solution containing guanidine hydrochloride, performing enzymolysis with pepsin in acid liquor, centrifuging the enzymolysis solution, and collecting supernatant; salting out the supernatant; and centrifuging and collecting a precipitate product; redissolving the precipitate with acid solution, dialyzing the solution, and freeze-drying the dialyzate to obtain sturgeon cartilage II type non-denatured collagen;
as a specific description of the examples, wherein the alkali solution is a NaOH solution of 0.02mol/L to 0.1 mol/L;
wherein the Tris-HCl buffer solution added with the guanidine hydrochloride is 0.05mol/L Tris-HCl buffer solution added with 1mol/L guanidine hydrochloride, and the pH value is 7.5;
the enzymolysis is carried out under the conditions that the liquid-material ratio is 1:10-1:20(w/v), the concentration of acetic acid is 0.5mol/L, the enzyme adding amount is 0.3-0.5% of the mass of the substrate, and the enzymolysis time is 48 h;
preferably, the liquid-to-feed ratio is 15:1, the acetic acid concentration is 0.5mol/L, and the enzyme addition amount is 0.4%.
The salting-out is carried out, wherein NaCl is added to the salt until the final concentration is 0.9-1.2 mol/L;
the centrifugation is carried out under the conditions of 4 ℃, the rotating speed is 9000g, and the time is 20-30 min;
the invention provides a method for deep processing and application of sturgeon cartilage resources by taking sturgeon cartilage which is a byproduct of sturgeon processing as a raw material, and enriches collagen products in the market. The collagen II extracted by the invention keeps the triple helix structure and the activity of the collagen, and the application value of the product is greatly improved. The extraction rate of the II type collagen extracted by the invention is high.
Drawings
FIG. 1 is a three-dimensional surface graph showing the influence of enzyme addition on extraction yield;
FIG. 2 is a three-dimensional surface graph of the effect of liquid-to-material ratio on extraction yield;
FIG. 3 is a three-dimensional surface plot of the effect of acetic acid concentration on extraction yield;
FIG. 4 is a UV-VIS spectrum of CII of sturgeon cartilage;
FIG. 5 is a Fourier transform infrared spectrum of CII of sturgeon cartilage;
FIG. 6 is a circular dichroism spectrum of CII of sturgeon cartilage;
FIG. 7 is an X-ray diffraction pattern of CII of sturgeon cartilage.
Detailed Description
The specific detection method adopted in the embodiment of the invention is described as follows:
1. analysis of amino acid composition
A sample is taken and placed in an ampoule bottle, 10mL of 6mol/L hydrochloric acid (containing 5 per mill of mercaptoethanol) solution is added, nitrogen is filled, and the bottle mouth is sealed by an alcohol spray gun. Hydrolyzing at 110 deg.C for 22h, and cooling. Adding ultrapure water to the solution until the volume is 50 mL. Evaporating 1mL of hydrolysate to dryness, adding 3-5 drops of ultrapure water, continuing to evaporate to dryness, repeating for 3 times to remove acid, dissolving in 2mL of 0.02mol/L hydrochloric acid, passing through a 0.22 μm water system film, and analyzing by an automatic amino acid analyzer.
2. Ultraviolet-visible absorption spectroscopy
0.25mg/mL collagen sample solution was prepared with 0.5mol/L acetic acid solution. The baseline was a 0.5mol/L acetic acid solution. Scanning and analyzing the sample by an ultraviolet spectrophotometer (UV-2102 PC); scanning range: 190 and 400nm, at medium speed.
3. Fourier transform Infrared Spectroscopy (FTIR) analysis
Drying the spectrally pure potassium bromide to constant weight, grinding and mixing the spectrally pure potassium bromide and a proper amount of sample in a mortar, and pressing the mixture into uniform and transparent sheets. Resolution ratio: 2cm-1The scanning range is as follows: 4000-500cm-1The number of scanning times: 32. the peak and wavelength values were analyzed using OMNIC 9.0 software.
4. Circular Dichroism (CD) analysis
A sample of CII was dissolved with 0.5mol/L acetic acid to give a 0.25mg/mL solution. Centrifuging at 9000 Xg and 4 deg.C for 20min, collecting supernatant, and performing circular dichroism. Scanning speed: 50 nm/min; scanning interval: 0.1 nm; scanning wavelength: 190-300 nm. A0.5 mol/L acetic acid solution was used to set a baseline.
5. X-ray diffraction analysis
The sample was subjected to X-ray diffraction analysis by an X-ray diffractometer. Ray source: cu target Ka radiation, radiation wavelength:
Figure BDA0003261763170000041
voltage: 40kVCurrent: 40 mA. Scanning angle: 5 ° -50 ° (2 θ), scanning speed: 4 deg./min.
6. Peptide mass fingerprinting
(1) Trypsin solution: taking a proper amount of mass spectrum pure trypsin, dissolving in 0.1mol/L NH4HCO3The final concentration of the solution was 1 mg/mL.
(2) Treatment of collagen samples: the mixture was dissolved in acetic acid (0.1mol/L) to give a 10mg/mL CII sample solution. With 0.1mol/L NH4HCO3The solutions (1:1, v: v) were mixed well, reacted in a 50 ℃ water bath for 3 hours, and then centrifuged at 9000 Xg for 20min to obtain a supernatant. Then evenly mixing with pancreatin solution at a ratio of 25:1(v: v), and carrying out enzymolysis for 20h at 37 ℃. By passing
Figure BDA0003261763170000051
Desalting with micro-chromatographic column, and passing through membrane.
Data analysis was performed using MaxQuant (version 1.5.0.12) and uniprot's collagen database (uniport-collagen. fasta) was selected for data retrieval.
Liquid phase conditions: column C18(2.1 mm. times.100 mm, 1.7 μm); the column temperature is 40 ℃; mobile phase: mobile phase A: 0.1% formic acid-acetonitrile, mobile phase B: 0.1% formic acid-water flow rate 0.25 mL/min; sample introduction amount: 10 mu L of the solution; elution gradient: 0-39min, 5% -80% A; 42min, 80% A; 42.5min, 80% -5% A; 46min, 5% A.
Mass spectrum conditions: mass spectrum scanning range: 350-1500 Da; reaction mode: a positive ion; atomizing gas GS 1: 35 psi; supplemental heater GS 2: 45 psi; air pressure of an air curtain: 35 psi; spraying voltage: 5500 eV; ion source temperature: 500 ℃; cluster-splitting voltage: 100V; collision energy: 20 eV; signal strength threshold: 2e4。
The present invention will be described in detail below with reference to examples and the accompanying drawings.
Example 1
After thawing, the sturgeon cartilage was washed with pre-cooled distilled water and then cut into pieces of 5mm × 5mm × 5 mm. The prepared cartilage was mixed with 0.02mol/L NaOH solution (1:10, w/v), stirred continuously for 48h, the alkali solution was replaced every 16h, and then washed with pre-cooled deionized water until the pH was neutral. Then soaked in 1mol/L guanidine hydrochloride-0.05 mol/L Tris-HCl (pH 7.5, 1:10, w/v) buffer and stirred for 24 h. After repeatedly washing with pre-cooled distilled water, the sample was placed in 0.5mol/L acetic acid containing 0.7% pepsin at a ratio of 1:20, stirring is continued for 48 h. The mixture was centrifuged at 9000 Xg for 30min at 4 ℃ to obtain a supernatant. Finely ground sodium chloride powder was added to the supernatant to a final salt concentration of 0.9mol/L to precipitate collagen. The precipitate was collected by centrifugation at 9000 Xg at 4 ℃ for 30min and dissolved in 0.5mol/L acetic acid solution. Dialyzing with 0.1mol/L acetic acid to prevent the dialysis bag from bursting, dialyzing with distilled water for 12h, and replacing distilled water every 6h until the conductivity of the dialysate is unchanged. The dialyzate was lyophilized by freeze-drying to obtain Type II Collagen (CII) with a yield of 67.86.
Example 2
After thawing, the sturgeon cartilage was washed with pre-cooled distilled water and then cut into 6mm × 5mm × 5mm pieces. The prepared cartilage was mixed with 0.05mol/L NaOH solution (1:15, w/v), stirred continuously for 36h, the alkali solution was replaced every 12h, and then washed with pre-cooled deionized water until the pH was neutral. Then soaked in 1mol/L guanidine hydrochloride-0.05 mol/L Tris-HCl (pH 7.5, 1:10, w/v) buffer and stirred for 24 h. After repeatedly washing with precooled distilled water, the sample is placed in 0.9mol/L ethyl acetate containing 0.1% pepsin, and the material-liquid ratio is 1: stirring is continued for 48h 10. The mixture was centrifuged at 9000 Xg for 25min at 4 ℃ to obtain a supernatant. Finely ground sodium chloride powder was added to the supernatant to a final salt concentration of 1mol/L to precipitate collagen. The precipitate was collected by centrifugation at 9000 Xg, 4 ℃ for 25min and dissolved in 0.5mol/L acetic acid solution. Dialyzing with 0.1mol/L acetic acid to prevent the dialysis bag from bursting, dialyzing with distilled water for 18h, and replacing distilled water every 6h until the conductivity of the dialysate is unchanged. The dialyzate was lyophilized by freeze-drying to obtain Type II Collagen (CII) with a yield of 52.79.
Example 3
After thawing, the sturgeon cartilage was washed with pre-cooled distilled water and then cut into pieces of 5mm × 4mm × 4 mm. The prepared cartilage was mixed with 0.1mol/L NaOH solution (1:20, w/v), stirred continuously for 24h, the alkali solution was replaced every 8h, and then washed with pre-cooled deionized water until the pH was neutral. Then soaked in 1mol/L guanidine hydrochloride-0.05 mol/L Tris-HCl (pH 7.5, 1:10, w/v) buffer and stirred for 24 h. After repeatedly washing with pre-cooled distilled water, the sample was placed in 0.5mol/L acetic acid containing 0.4% pepsin at a ratio of 1:15, stirring is continued for 48 h. The mixture was centrifuged at 9000 Xg for 20min at 4 ℃ to obtain a supernatant. Finely ground sodium chloride powder was added to the supernatant to a final salt concentration of 1.2mol/L to precipitate collagen. The precipitate was collected by centrifugation at 9000 Xg at 4 ℃ for 20min and dissolved in 0.5mol/L acetic acid solution. Dialyzing with 0.1mol/L acetic acid to prevent the dialysis bag from bursting, dialyzing with distilled water for 24h, and replacing distilled water every 6h until the conductivity of the dialysate is unchanged. The dialyzate was lyophilized by freeze-drying to obtain Type II Collagen (CII) with a yield of 76.09.
The collagen II obtained by the extraction methods in the three embodiments has higher quality, and the triple helix structure and the biological activity are kept. The process was optimized for optimal conditions.
Example 4: optimization of extraction condition of non-denatured II type collagen of sturgeon cartilage
First, response surface experimental design
In order to further optimize the extraction process of the sturgeon cartilage type II collagen, a response surface test is designed through Box-Benhnken on the basis of a laboratory prophase preparation process, as shown in Table 1, 3 factors of enzyme addition amount (%), liquid-material ratio and acetic acid concentration (mol/L) are A, B, C correspondingly, the high and low levels of the factors are represented by 1, 0 and-1, and the extraction rate is used as a response value. The experiment result is analyzed by using Design-expert software 8.0.6 version, and the extraction process for extracting sturgeon cartilage type II Collagen (CII) by a pepsin method is optimized.
TABLE 1 Box-Behnken design test factors and horizon table
Figure BDA0003261763170000081
The collagen type II extraction rate was calculated as follows:
Y(％)＝E/S×100
in the formula: y is extraction rate,%; e is the content of collagen in the extracting solution; the S content is 60.07% (the content of collagen in the sturgeon cartilage).
The experimental design for optimizing the response surface of the extraction process of the non-denatured type II collagen of the sturgeon cartilage of the present invention is shown in the following tables 2 and 3.
TABLE 2 CII extraction response surface test design and result table
Figure BDA0003261763170000082
Figure BDA0003261763170000091
Software regression analysis is performed on the results in table 2 to obtain a quadratic regression equation: the extraction rate is 74.39-1.06A +9.04B +2.07C-2.64AB-3.07AC +3.07BC-4.43A2-8.38B2-12.74C2。
Table 3: CII extraction rate response surface experiment regression equation variance analysis table
Figure BDA0003261763170000092
Figure BDA0003261763170000101
The variance results of the extraction rate regression equation are shown in table 3. The influence of each variable in the regression equation on the response value, P (Pro), was analyzed by F-test>F) The smaller the value, the more significant the influence of the corresponding variable. As can be seen from Table 3, the designed model showed a high degree of significance (P)<0.0001). Mismatching error value P0.5674>0.05, showing that the regression equation has better fitting property with the experiment. Analysis of the significance of the regression equation coefficients, factor BThe linear and square effects, the square effect of the factor C, show a very significant influence on the CII extraction rate (P)<0.01). Comparing the F values to obtain that the influence sequence of all the factors on the extraction rate is as follows: b (liquid material ratio)>C (acetic acid concentration)>A (enzyme addition amount) indicates that the model is properly selected. From the magnitude of the P value of the simulation term being greater than 0.05, the simulation first term B, C and the simulation second terms AB, AC, BC, A2、B2、C2Are all significant and consistent with the analysis of variance results. Predicting the regression equation in the set factor level by using Design-export 8.0.6 software, and determining the optimal extraction process as follows: liquid-material ratio of 15:1, acetic acid concentration: 0.27mol/L, 0.4 percent of enzyme adding amount and 76.09 percent of the highest extraction rate.
The experimental conditions in example 3 are closest to the optimal conditions described above: liquid-material ratio of 15:1, acetic acid concentration: 0.5mol/L and 0.4 percent of enzyme, 3 groups of parallel experiments are carried out to verify that the final extraction rate of the CII is 75.8 +/-1.97 percent, and is consistent with the predicted result, and the extraction rate is higher than that of the CII in example 1 and example 2.
Example 5: sturgeon cartilage type ii collagen prepared in example 3 was analyzed.
Analysis of amino acid composition
TABLE 4 CII amino acid composition of sturgeon cartilage (residue/1000 residues)
Figure BDA0003261763170000111
The amino acid composition of CII is shown in table 4. CII has a high glycine content (328 residues/1000 residues) of about 33% of the total content, followed by alanine, proline and hydroxyproline. Glycine is the most predominant amino acid in collagen, and all members of the collagen family have the following domains: the proline-rich tripeptide Gly-X-Y repeats, and these domains are involved in the formation of triple helices, where X is usually proline and Y is mainly hydroxyproline. This result is consistent with the characteristics of collagen.
In addition, the imino acid (proline and hydroxyproline) content of CII is 20.1%, which is similar to the content in brown bamboo shark cartilage collagen (20.3-20.4%) and black sharkskin cartilage collagen (19.6-19.7%), is significantly higher than the content of silver fin shark cartilage collagen (15.1-15.6%), but is significantly lower than the content of bovine skin collagen (21.6%) and porcine skin collagen (22%). This result highlights the conclusion drawn by Foegeing et al: collagen derived from fish has a lower imino acid content than that of mammalian collagen.
It was found that the stability of the triple helix structure of collagen is related to the content of imino acids (Pro and Hyp) and the degree of hydroxylation, proline hydroxylation being important for collagen stability, since hydroxyproline can stabilize the triple helix of collagen. The proline hydroxylation degree of the sturgeon cartilage type ii collagen prepared in example 3 was calculated to be 43.8%.
Ultraviolet-visible absorption spectrum analysis of non-denatured II type collagen of sturgeon cartilage
The UV absorption of collagen is characterized by a peak maximum at 220-240nm, which is associated with C ═ O, -COOH and-CO-NH in the polypeptide chain2Groups are related and are primarily due to n → pi transitions of C ═ O. As shown in FIG. 4, the maximum absorption wavelength of CII is near 234nm, which is consistent with the UV absorption properties of collagen. Typically, aromatic amino acids (primarily tryptophan and tyrosine) will have absorption maxima at 280nm and 257 nm. CII showed no significant absorption in these regions, indicating a low tryptophan and tyrosine content. This is consistent with the results of the amino acid analysis. The extraction process of the invention can also be reflected to achieve good impurity removal effect, and the obtained type II collagen sample has higher purity.
Three, Fourier transform Infrared Spectroscopy (FTIR)
Table 5: characteristic absorption chart of functional group
Figure BDA0003261763170000121
FIG. 5 and Table 5 show the IR spectra and major peaks of sturgeon cartilage type II collagen and their assignment, the FTIR spectra of CII produced toThe following main absorption peaks: amide A band: 3316cm-1Amide B band: 2941cm-1Amide I band: 1654cm-1Amide II band: 1558cm-1 and amide III band: 1240cm-1. The secondary structure of collagen is associated with amide I, II and III regions; the amide A band is associated with N-H stretching vibrations. At 3400--1Free N-H stretching vibrations occur in the range and when the N-H groups in the peptide chain participate in hydrogen bonding, the position will shift to lower frequencies. The band B of the amide represents-CH2The absorption peak position of the asymmetric stretching vibration of (1) is 2930-2944cm-1. The generation of the amide I band results from the stretching vibration of the protein polypeptide backbone C ═ O and the coupling of hydrogen bonds to COO-.
Analysis of circular dichroism spectrum of non-denatured II type collagen in sturgeon cartilage
Circular dichroism spectroscopy is the most widely used method for determining the secondary structure of collagen, and can determine the difference in absorption coefficient of an optically active substance with respect to left and right circularly polarized light constituting plane polarized light. The absolute value of the ratio of the positive and negative absorption peak intensities (RPN) is commonly used to indicate the helicity of the three helical structures in the collagen solution. The natural collagen is an optically active protein, and the characteristic CD spectrum of the natural collagen presents a positive peak at about 220nm and a negative peak at 195-197nm[14]The RPN value was 0.15. When the triple helix is changed, the positive absorption peak disappears and a red shift of the negative absorption peak is found.
As shown in FIG. 6, the CD spectrum of CII shows a maximum rotation (positive peak) at 221nm, a minimum (negative peak) at 195nm, a crossing point (zero rotation) around 215nm, and an RPN value of 0.12. These all indicate that CII has the typical features of collagen triple helix structure.
X-ray diffraction analysis of sturgeon cartilage non-denatured type II collagen
The x-ray spectrum of the collagen sample is shown in fig. 7. There are two diffraction peaks at diffraction angles (2 θ) of about 7.47 ° and 21.73 °, and the first is sharp and the second is broad, which fit the characteristic diffraction peaks of collagen. Diffraction peaks indicate that the spatial conformation of the CII molecules is not an irregular coil, but an ordered arrangement. According to the Bragg equation
Figure BDA0003261763170000131
Figure BDA0003261763170000132
Calculating the minimum value (d) of the repetition interval, d value of the sharp peak being
Figure BDA0003261763170000133
Figure BDA0003261763170000134
Broad peak d value of
Figure BDA0003261763170000135
This is related to the diameter of the three chains of the supercoiled molecule and the structure of the single-chain left-handed helix in collagen; indicating that the extracted CII sample has a triple helix structure.
Sixthly, peptide quality fingerprint analysis of sturgeon cartilage non-denatured type II collagen
Data processing is carried out by MaxQuant software, and the generated result is detected by contrasting a collagen database in UNIPROT. The results (see table 6) show that the confidence level is higher than 95%, the peptide fragments are relatively high in content, the protein sources of the peptide fragments are type II collagen, the peptide fragments are concentrated among 12-24 amino acids, the peptide fragments mainly form Gly-X-Y, most of X and Y are proline and hydroxyproline respectively, and the peptide fragments accord with the characteristics of the primary structure of collagen.
Table 6: sturgeon CII trypsin hydrolyzed peptide fragment and protein source table
Figure BDA0003261763170000141
Figure BDA0003261763170000151
The optimal extraction process conditions are obtained through a response surface experiment: liquid-material ratio of 15:1, acetic acid concentration: 0.5mol/L, 0.4 percent of enzyme addition amount and 75.80 +/-1.97 percent of CII extraction rate. Amino acid composition results showAs a result, glycine accounted for 32.8% of the total amino acid residues, and the imino acid content was 20.1%, and no tryptophan residue was found. The maximum ultraviolet absorption wavelength is near 234nm, and no visible absorption peak exists in other regions, which shows that the purity of the extracted collagen is higher, and the method also embodies that the method of the invention achieves good effects of separating collagen and removing impurities. The result of FTIR analysis shows that the extracted CII conforms to the infrared spectrum characteristics of typical collagen. Results of circular dichroism analysis showed that the RPN value of CII was 0.12, corresponding to the native triple-helical conformation of collagen; the results of X-ray diffraction analysis showed that the d1 values of CII were respectively
Figure BDA0003261763170000152
Indicating the distance between the collagen molecular chains; d2 value of
Figure BDA0003261763170000153
The diameters of the individual left-handed helices of the collagen indicate that the extracted CII is not denatured. The analysis of peptide fingerprint spectrum shows that amino acid composition of polypeptide generated by hydrolyzing CII by trypsin is mainly Gly-X-Y, which accords with the characteristics of primary structure of collagen, and the source is type II collagen.

Claims (8)

1. A preparation method of sturgeon cartilage II type non-denatured collagen is characterized by comprising the following steps: treating sturgeon cartilage with alkali liquor, homogenizing, washing with Tris-HCl buffer solution containing guanidine hydrochloride, performing enzymolysis with pepsin in acid liquor, centrifuging the enzymolysis solution, and collecting supernatant; salting out the supernatant; and centrifuging and collecting a precipitate product; and redissolving the precipitated product by using an acid solution, dialyzing the solution, and freeze-drying the dialyzate to obtain the sturgeon cartilage II type non-denatured collagen.
2. The method of claim 1, wherein the alkali solution is 0.02-0.1mol/L NaOH solution.
3. The method according to claim 1, wherein the Tris-HCl buffer to which guanidine hydrochloride is added is 0.05mol/L Tris-HCl buffer to which 1mol/L guanidine hydrochloride is added.
4. The preparation method of claim 1, wherein the enzymolysis is carried out in a liquid-material ratio of 1:10-1:20, the concentration of acetic acid is 0.5mol/L, the amount of enzyme is 0.3-0.5% of the mass of the substrate, and the enzymolysis time is 48 h.
5. The process according to claim 4, wherein the enzymolysis is carried out in a liquid-to-liquid ratio of 15:1, an acetic acid concentration of 0.5mol/L and an enzyme addition of 0.4%.
6. The method according to claim 1, wherein the salting-out is carried out by adding NaCl to a final concentration of 0.9 to 1.2 mol/L.
7. The method of claim 1, wherein the centrifugation is performed at 4 ℃ and 9000g for 20-30 min.
8. Sturgeon cartilage type ii non-denatured collagen, prepared using the method of any one of claims 1 to 7.
CN202111074561.2A 2021-09-14 2021-09-14 Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof Pending CN113735965A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111074561.2A CN113735965A (en) 2021-09-14 2021-09-14 Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111074561.2A CN113735965A (en) 2021-09-14 2021-09-14 Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN113735965A true CN113735965A (en) 2021-12-03

Family

ID=78738652

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111074561.2A Pending CN113735965A (en) 2021-09-14 2021-09-14 Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN113735965A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114921511A (en) * 2022-06-29 2022-08-19 晶昌明科技贸易(上海)有限公司 Preparation method of fish cartilage hydrolysate rich in small-molecule chondroitin sulfate

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102154425A (en) * 2011-03-18 2011-08-17 北京华达杰瑞生物技术有限公司 Production method of non-denatured II-type collagen
JP2012176926A (en) * 2011-02-25 2012-09-13 Shigemi Sawada Method for producing unmodified type ii collagen
CN105112481A (en) * 2015-07-30 2015-12-02 上海市水产研究所 Method for extracting undenatured type II collagen from squid cartilage
CN105331662A (en) * 2015-11-30 2016-02-17 四川大学 Non-denatured II type collagen of animal cartilage source and preparation method for non-denatured II type collagen
CN106319015A (en) * 2016-11-18 2017-01-11 上海海洋大学 Preparation method of II-type collagen with biological activity in shark cartilage
CN107385005A (en) * 2017-09-12 2017-11-24 海力生集团有限公司 A kind of preparation method of sturgeon bone II Collagen Type VI peptides
KR20180018055A (en) * 2016-08-12 2018-02-21 한국국제대학교 산학협력단 Anti-wrinkle Cosmetic Composition Comprising Functional Components from Sturgeon, Method for Obtaining the Functional Components and Method for Producing Anti-wrinkle Cosmetic Using the Functional Components
CN111662375A (en) * 2020-06-11 2020-09-15 湖北和格复合骨胶原生物科技有限公司 Preparation method for extracting non-denatured type II collagen from animal cartilage
CN111748030A (en) * 2020-06-24 2020-10-09 华南理工大学 Soluble non-denatured II type collagen and preparation method thereof
CN112079913A (en) * 2020-09-25 2020-12-15 康洛信(广东)生物科技有限公司 Process for extracting non-denatured type II collagen from sturgeon cartilage

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012176926A (en) * 2011-02-25 2012-09-13 Shigemi Sawada Method for producing unmodified type ii collagen
CN102154425A (en) * 2011-03-18 2011-08-17 北京华达杰瑞生物技术有限公司 Production method of non-denatured II-type collagen
CN105112481A (en) * 2015-07-30 2015-12-02 上海市水产研究所 Method for extracting undenatured type II collagen from squid cartilage
CN105331662A (en) * 2015-11-30 2016-02-17 四川大学 Non-denatured II type collagen of animal cartilage source and preparation method for non-denatured II type collagen
KR20180018055A (en) * 2016-08-12 2018-02-21 한국국제대학교 산학협력단 Anti-wrinkle Cosmetic Composition Comprising Functional Components from Sturgeon, Method for Obtaining the Functional Components and Method for Producing Anti-wrinkle Cosmetic Using the Functional Components
CN106319015A (en) * 2016-11-18 2017-01-11 上海海洋大学 Preparation method of II-type collagen with biological activity in shark cartilage
CN107385005A (en) * 2017-09-12 2017-11-24 海力生集团有限公司 A kind of preparation method of sturgeon bone II Collagen Type VI peptides
CN111662375A (en) * 2020-06-11 2020-09-15 湖北和格复合骨胶原生物科技有限公司 Preparation method for extracting non-denatured type II collagen from animal cartilage
CN111748030A (en) * 2020-06-24 2020-10-09 华南理工大学 Soluble non-denatured II type collagen and preparation method thereof
CN112079913A (en) * 2020-09-25 2020-12-15 康洛信(广东)生物科技有限公司 Process for extracting non-denatured type II collagen from sturgeon cartilage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
胡艺: "鲟鱼软骨中II 型胶原蛋白的提取及其结构鉴定", 《食品安全质量检测学报》, vol. 12, no. 6, pages 2433 - 2438 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114921511A (en) * 2022-06-29 2022-08-19 晶昌明科技贸易(上海)有限公司 Preparation method of fish cartilage hydrolysate rich in small-molecule chondroitin sulfate
CN114921511B (en) * 2022-06-29 2023-12-22 晶昌明科技贸易(上海)有限公司 Preparation method of fish cartilage hydrolysate rich in small molecular chondroitin sulfate

Similar Documents

Publication Publication Date Title
CN104710525A (en) Tuna fishbone collagen sourced zinc chelated collagen peptide, preparation method and application thereof
CN113402599A (en) Optimization method for extracting and purifying type I collagen from human placenta
CN104710511A (en) Iron chelating peptide derived from hairtail fish protein and preparation method and application thereof
WO2023040084A1 (en) Method for extracting collagen tripeptide and prepared collagen tripeptide
CN108893515B (en) High F value oligopeptide and preparation method thereof
AU2021105562A4 (en) Preparation Method of Tilapia Protein Peptide with Antioxidant and Antifatigue Activities
CN113735965A (en) Sturgeon cartilage II type non-denatured collagen and preparation method and application thereof
Bisht et al. Uncovering the potential of aqueous solutions of deep eutectic solvents on the extraction and purification of collagen type I from Atlantic codfish (Gadus morhua)
CN111269290B (en) Preparation method of sturgeon anti-inflammatory peptide
CN1803836A (en) Jellyfish collagen and method for preparing the same
CN112410392A (en) Extraction method and application of type I collagen
Dong et al. Steam explosion-assisted extraction of protein from fish backbones and effect of enzymatic hydrolysis on the extracts
CN110564802A (en) Extraction method of yak achilles tendon bone collagen
Hu et al. Effects of ultrasound on the structural and functional properties of sheep bone collagen
CN112501229B (en) Production process of bovine bone collagen peptide
CN113789361A (en) Cod peptide and preparation method and application thereof
CN109111515B (en) Method for preparing main egg yolk protein from body cavity fluid of sea cucumber
CN114573682A (en) Elastin peptide and preparation method thereof
CN112877390A (en) Preparation method of functional alcohol-soluble sturgeon cartilage preparation
CN111574612B (en) Preparation method of hepatocyte growth-promoting factor
CN112280816A (en) Large-scale preparation method of marine source type I collagen subunit
CN114933648B (en) Method for producing collagen dipeptide from fish scales
CN110484586A (en) A method of flavor peptides are prepared from Tilapia bone albumen
CN115651066B (en) Sea cucumber polypeptide, preparation process and application thereof
CN114106148A (en) Fish skin-derived medical-grade non-denatured collagen and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20211203

WD01 Invention patent application deemed withdrawn after publication