An avian eggshell membrane macromolecular collagen and a preparation method thereof

Abstract

This application discloses a method for preparing macromolecular collagen from poultry eggshell membranes. The method includes: pulverizing the poultry eggshell membrane, mixing it with a reducing agent and urea, and intermittently sonicating it under gentle stirring to dissolve and remove keratin; extracting the precipitate with citric acid and pepsin, adjusting the pH of the supernatant to neutral, adding ammonium sulfate for precipitation, dialysis after dissolving the precipitate, and freeze-drying to obtain macromolecular collagen from the poultry eggshell membrane. This application selectively removes keratin under non-thermal, neutral conditions, preserving the natural conformation of the collagen. The obtained collagen exhibits a strong absorption peak at 235 nm and a shoulder peak at 280 nm in its ultraviolet spectrum, with a thermal denaturation temperature of 75 °C, and possesses a complete triple helix structure. This method is mild and yields high results, filling a technological gap in the extraction of macromolecular collagen from poultry eggshell membranes. The obtained product can be used in fields such as biological scaffold materials, tissue engineering, and drug carriers.

Description

Technical Field

[0001] This application relates to the field of high-value utilization technology of agricultural by-products, specifically to a method for separating and extracting conformationally intact macromolecular collagen from poultry eggshell membranes. Background Technology

[0002] my country is the world's largest producer and consumer of poultry eggs, with an estimated output of 34.98 million tons in 2025. However, the current primary processing rate of poultry eggs is only about 10%, leaving a large amount of byproducts, such as eggshell membranes, unutilized and resulting in resource waste. Eggshell membranes are rich in collagen, a major structural protein of the extracellular matrix, possessing excellent biocompatibility and biomimetic properties, making them an ideal raw material for preparing bioactive materials. Therefore, developing efficient technologies for separating and extracting macromolecular collagen from eggshell membranes is of great significance for achieving high-value utilization of byproducts and promoting the development of the biomaterials industry.

[0003] However, extracting macromolecular collagen from eggshell membranes has long been hampered by technical bottlenecks. Within the shell membrane, collagen and disulfide-rich keratin interact strongly to form a highly organized and stable structure. Conventional acid, alkali, or enzymatic methods are ineffective in disrupting this structure, resulting in extremely low collagen dissolution rates. Studies have shown that removing or dissociating keratin is a crucial prerequisite for efficient release of shell membrane collagen, but keratin, due to the stability of its intramolecular disulfide bonds, is poorly soluble in common solvents.

[0004] Existing technologies for processing shell membrane keratin have the following main drawbacks:

[0005] On the one hand, the lack of a mild, non-thermal, and non-alkaline pretreatment process for keratin removal makes it difficult to protect the structural integrity of collagen. For example, the earliest published "Enzymatic Extraction Process of Eggshell Membrane Collagen" (Food Science, 2012, 33(16):96-99) directly enzymatically hydrolyzes the shell membrane without any pretreatment to remove keratin, resulting in a collagen yield of only 0.91% (the total collagen content in the shell membrane is about 10%). Patent CN118813744A (A Method for Ultrasonic-Assisted Enzymatic Extraction of Eggshell Membrane Protein Based on Response Surface Methodology) also lacks a keratin removal step, and the acetic acid it uses is not the optimal choice for dissolving collagen in a weak acid. The NaCl used during salting out has a far less effective precipitation effect on large-molecule collagen than the conventional (NH4)2SO4. More importantly, although patent CN121022964A (an eggshell membrane peptide for enhancing bone density and its preparation method and application) uses keratinase, its processing conditions are harsh (pH 10.5–11.5, temperature 50–60 °C). Collagen is an acid-soluble and heat-sensitive protein, which will rapidly denature and degrade into gelatin or small molecule peptides under strong alkaline and heating conditions, completely failing to retain the natural conformation of large-molecule collagen. None of these methods can achieve the goal of protecting the integrity of collagen structure while removing keratin.

[0006] On the other hand, existing technologies generally focus on extracting shell membrane active peptides or mixed proteins, with very little attention paid to the extraction of conformationally intact macromolecular collagen. For example, patents CN120025425A (A method for preparing and applying X-type collagen peptides to improve bone growth), CN112553280B (A method for preparing eggshell membrane peptide calcium), and CN102517363A (A method for producing and applying egg membrane collagen polypeptides) all use shell membrane powder as a direct raw material, preparing collagen peptides or mixed polypeptide products through single or combined enzymatic hydrolysis, microbial fermentation, etc. While these technologies achieve the utilization of shell membrane proteins, their target products are small molecular weight peptides with disrupted conformation, rather than macromolecular collagen with a complete triple helix structure. Furthermore, due to the lack of selective keratin removal pretreatment, the products obtained by these methods are actually mixtures of collagen, keratin, and their peptides, with low purity.

[0007] In summary, existing technologies for processing poultry eggshell membranes either employ harsh conditions such as high temperature, high pressure, and strong alkali, leading to collagen denaturation and degradation, or skip the keratin removal step altogether, resulting in extremely low collagen yields and impure products. Furthermore, these technologies primarily target small molecule peptides rather than large molecule collagen. Currently, there is a lack of a process that can selectively remove keratin under mild, non-thermal, and non-alkali conditions, and then efficiently separate and extract conformationally intact, high-purity large molecule collagen from the eggshell membrane.

[0008] Therefore, developing a method for separating and extracting macromolecular collagen from poultry eggshell membranes that meets the above requirements is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0009] This application aims to overcome the shortcomings of existing technologies in the extraction of macromolecular collagen from poultry eggshell membranes, such as the lack of a mild keratin removal pretreatment process and the fact that existing methods often lead to collagen denaturation and degradation or only yield small peptides. It provides a method for efficiently separating and extracting conformationally intact macromolecular collagen. This method first involves selectively reducing disulfide bonds in shell membrane keratin under non-thermal, neutral conditions using a chemical reducing agent-assisted ultrasonic treatment, causing it to dissolve in the aqueous phase while the target collagen component remains stably retained in the solid phase, simultaneously loosening the shell membrane structure. Subsequently, a process involving citric acid combined with pepsin extraction, ammonium sulfate precipitation, dialysis, and freeze-drying is employed to obtain high-purity, conformationally intact macromolecular collagen from poultry eggshell membranes. This method effectively removes keratin barriers under mild conditions, avoiding thermal denaturation or alkaline degradation of collagen, significantly improving the extraction yield and structural integrity of macromolecular collagen, thus providing an efficient, mild, and controllable technical solution for the high-value utilization of poultry eggshell membrane byproducts.

[0010] To achieve the above objectives, this application provides the following technical solution:

[0011] In a first aspect, this application provides a method for preparing macromolecular collagen from poultry eggshell membranes, comprising the following steps:

[0012] (1) The eggshell membrane of poultry eggs is crushed;

[0013] (2) The crushed poultry eggshell membrane is mixed with a solution containing reducing agent and urea, and gently stirred. During the stirring process, ultrasonic treatment is performed intermittently. Then, solid-liquid separation is performed, the precipitate is collected, and poultry eggshell membrane with keratin removed is obtained.

[0014] (3) The keratin-free eggshell membrane obtained in step (2) is mixed with an extract containing citric acid and pepsin for extraction, and then solid-liquid separation is performed to collect the supernatant.

[0015] (4) Adjust the pH of the supernatant obtained in step (3) to neutral, add ammonium sulfate for salting out, and collect the precipitate after solid-liquid separation;

[0016] (5) After dissolving the precipitate obtained in step (4), it is placed in a dialysis bag for dialysis desalting, and then freeze-dried to obtain poultry eggshell membrane macromolecular collagen.

[0017] In some embodiments, in step (1), the eggshell membrane is cut into 1-3 cm pieces before crushing. 3 After being flaked, it is crushed and then passed through a 45-mesh sieve.

[0018] In some embodiments, in step (2), the concentration of urea in the solution containing reducing agent and urea is 2-4 mol / L, and the reducing agent is sodium bisulfite with a mass concentration of 1%-3%; the ratio of the crushed eggshell membrane to the solution is 1:100-1:300; the gentle stirring time is 18-24 h, and the stirring is stopped intermittently 4-8 times during the stirring process; the conditions for the ultrasonic treatment are: frequency 30-40 kHz, power 400-600 W, each ultrasonic treatment is 20 min, and the total ultrasonic time is 80-160 min.

[0019] In some embodiments, in step (2), the solid-liquid separation conditions are: centrifugation at 10 °C and 3000~6000 r / min for 20 min, taking the precipitate and washing it with running water and air drying it.

[0020] In some embodiments, in step (3), the ratio of the keratin-free eggshell membrane to the extract is 1:100 to 1:300; the concentration of citric acid in the extract is 0.3 to 0.9 mol / L, the amount of pepsin is 2% to 6% of the dry weight of the shell membrane, and the enzyme activity of the pepsin is 1:3000; the extraction conditions are: gentle stirring at 20 to 25 °C for 12 to 24 h.

[0021] In some embodiments, in step (3), the solid-liquid separation conditions are: centrifugation at 10 °C and 6000~10000 r / min for 10 min, and then taking the supernatant.

[0022] In some embodiments, in step (4), NaOH is used to adjust the pH to 7.0~7.5; ammonium sulfate is added to make its final concentration 1.5 mol / L; after adding ammonium sulfate, it is allowed to stand for 12 h, and then solid-liquid separation is performed; the conditions for solid-liquid separation are: centrifugation at 10℃, 6000~10000 r / min for 10~30 min, and the precipitate is taken.

[0023] In some embodiments, in step (5), the precipitate is dissolved by adding 10 to 20 times the volume of 0.4 to 0.8 mol / L citric acid solution; the molecular weight cutoff of the dialysis bag is 7000 D to 14000 D; the dialysis desalting time is 3 to 5 days, of which the first 1 to 2 days are dialysis with 0.04 M sodium dihydrogen phosphate solution and the solution is changed every 6 hours, and the next 2 to 3 days are dialysis with deionized water and the solution is changed every 6 hours, and the dialysis endpoint is the conductivity value of the dialysate is <10 μS / cm; the freeze-drying conditions are: freezing at -80 ℃ overnight, followed by freeze-drying at -80 ℃ and 0.03 to 0.05 MPa for 48 hours.

[0024] In some embodiments, in step (2), the supernatant obtained after solid-liquid separation is tested and found to be free of hydroxyproline, indicating that the removed protein is keratin rather than collagen.

[0025] Secondly, this application provides a macromolecular collagen for poultry eggshell membranes, which is prepared using the preparation method described in any one of the first aspects.

[0026] The aforementioned poultry eggshell membrane macromolecular collagen has the following characteristics:

[0027] (a) The ultraviolet spectrum has a strong absorption peak at 235 nm and a shoulder peak at 280 nm; (b) The thermal denaturation temperature measured by differential scanning calorimetry is 75 ± 2 °C.

[0028] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0029] 1. This application proposes for the first time a combined treatment process of "reducing agent (sodium bisulfite) + urea + intermittent ultrasound," which selectively reduces the disulfide bonds and breaks the hydrogen bonds of shell membrane keratin under non-thermal, neutral conditions, promoting the dissolution of keratin in the aqueous phase while the target collagen component is stably retained in the solid phase. This method avoids the thermal denaturation or alkaline degradation of collagen under harsh conditions such as high temperature, high pressure, and strong alkali, effectively protecting the natural conformation of macromolecular collagen.

[0030] 2. This application significantly improves the collagen dissolution rate and recovery rate by pretreatment before keratin removal, combined with citric acid and ammonium sulfate precipitation, overcoming the defect of extremely low yield in existing technologies.

[0031] 3. The shell-membrane collagen prepared in this application, after freeze-drying, exhibits a sponge-like appearance, similar in morphology to the type I collagen standard. Ultraviolet spectroscopy analysis shows that the shell-membrane collagen has a strong absorption peak at 235 nm for the π→π* transition of the carbonyl C=O double bond, and characteristic shoulder peaks of the benzene rings of tyrosine and phenylalanine side chains around 280 nm. This spectrum is highly consistent with the type I collagen standard, but significantly different from the spectrum of gelatin (a collagen degradation product), indicating that the extracted collagen molecules have an intact conformation and have not undergone significant degradation. The thermal denaturation temperature (Tm) of the shell-membrane macromolecular collagen obtained in this application is 75 ℃, close to that of the type I collagen standard. This proves that the collagen prepared in this application maintains a good natural conformation. Furthermore, through dialysis desalting and freeze-drying processes, the final product purity can reach over 90%, with extremely low keratin residue, meeting the stringent requirements for raw material purity in biomedical materials.

[0032] 4. The technology system of this application integrates the entire process of "pulverization → dekeratinization → acid enzyme extraction → salting out → dialysis → freeze drying". The process is mild, controllable and scalable, filling the technological gap of direct extraction of macromolecular collagen and expanding the application prospects of poultry eggshell membrane in the fields of biomaterials, tissue engineering and drug carriers. Attached Figure Description

[0033] Figure 1 This is a flowchart of the extraction process for macromolecular collagen from the shell membrane in this application.

[0034] Figure 2 This study compares the effectiveness of different pretreatment methods in removing keratin from poultry eggshell membranes.

[0035] Figure 3 Curve showing the effect of different concentrations of citric acid extraction on the hydroxyproline content in the shell membrane supernatant.

[0036] Figure 4 The images show the changes in the microstructure of the eggshell membrane during the treatment process. A represents the fresh eggshell membrane, B represents the membrane after treatment with reducing agent and ultrasound, and C represents the membrane after collagen extraction with citric acid.

[0037] Figure 5 These are comparative photographs of the physical appearance of poultry eggshell membrane macromolecular collagen, poultry eggshell membrane raw material, and type I collagen standard, where A is poultry eggshell membrane raw material, B is poultry eggshell membrane macromolecular collagen extracted in this application, and C is type I collagen standard.

[0038] Figure 6 Comparison of the ultraviolet spectra of macromolecular collagen in poultry eggshell membranes, type I collagen standards, and gelatin.

[0039] Figure 7 Comparison of differential scanning calorimetry (DSC) of macromolecular collagen in eggshell membranes with type I collagen standards and gelatin. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0041] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0042] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0043] Unless otherwise specified, in this application, "%" represents mass fraction when indicating concentration.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before providing a detailed description of this application, the following terms and definitions are provided to better understand this application:

[0045] 1. Macromolecular collagen: The “macromolecular collagen” mentioned in this application refers to a collagen molecule with a right-handed superhelical structure (also known as a triple helix structure) formed by three left-handed polyproline α-chains intertwined with each other. Its molecular weight is about 300 kDa, which is different from collagen peptides (usually with a molecular weight <10 kDa) and gelatin (partially hydrolyzed collagen products, where the triple helix structure has been destroyed and the peptide chains have dissociated).

[0046] 2. Conformational Integrity: "Conformational integrity" as described in this application refers to collagen molecules maintaining their natural triple helix structure, which can be characterized by one or a combination of the following features: (i) a characteristic absorption peak for the π→π* transition of the carbonyl C=O double bond near 235 nm in the ultraviolet spectrum, and characteristic shoulder peaks for the tyrosine and phenylalanine side chains near 280 nm; (ii) a thermal denaturation temperature (Tm) determined by differential scanning calorimetry (DSC) not lower than 70 °C; (iii) a positive absorption peak near 220 nm and a negative absorption peak near 195 nm in the circular dichroism (CD) spectrum; (iv) polyacrylamide gel electrophoresis (SDS-PAGE) bands showing that the α chain, β chain, and γ chain are near 110 kDa, 220 kDa, and 330 kDa, respectively.

[0047] 3. Keratin Removal: "Keratin removal" as described in this application refers to the process of using a chemical reducing agent to break the disulfide bonds within keratin molecules, causing them to selectively dissolve in the aqueous phase from the shell membrane tissue, while the collagen components remain in the solid phase. The term "selective" means that the removed protein is primarily keratin, with minimal loss of collagen components; that is, no or only trace amounts of hydroxyproline (a characteristic amino acid of collagen) are detected in the removal solution.

[0048] 4. Hydroxyproline: This is an amino acid unique to collagen, produced by the modification of proline with hydroxylase, and is almost absent in keratin and most other proteins. Therefore, in this application, hydroxyproline can serve as a specific marker for collagen: the detection of hydroxyproline in a sample indicates the presence of collagen; its absence indicates the absence of collagen. This application utilizes this property to verify the composition of the supernatant during the keratin removal step (i.e., to prove that pure keratin is removed without collagen loss).

[0049] 5. Thermal denaturation temperature (Tm): This refers to the characteristic temperature at which collagen undergoes conformational collapse and protein denaturation and dissociation due to heat, and is usually determined using differential scanning calorimetry (DSC). Conformally intact natural collagen has a higher Tm value than its degradation products such as gelatin (e.g., the type I collagen standard in this application has a Tm value of 83 °C). The shell-membrane macromolecular collagen prepared in this application has a Tm value of 75 ± 2 °C.

[0050] Based on the inventive concept of "selectively removing shell membrane keratin under non-thermal, neutral conditions to extract conformationally intact macromolecular collagen," the inventors of this application have proposed a complete process for the separation and extraction of macromolecular collagen from poultry eggshell membranes. For example... Figure 1As shown, the core process of this method includes: first, using a reducing agent (sodium bisulfite) in combination with urea and intermittent ultrasonic treatment to selectively reduce keratin disulfide bonds and disrupt hydrogen bonds, allowing keratin to dissolve in the aqueous phase while collagen remains in the solid phase, simultaneously loosening the shell membrane structure; then, through citric acid-pepsin combined extraction, ammonium sulfate precipitation, dialysis desalting, and freeze-drying, high-purity, conformationally intact poultry eggshell membrane macromolecular collagen is finally obtained. Experiments have shown that this method can effectively remove keratin (hydroxyproline was not detected in the eluent), the UV spectral characteristics of the obtained collagen are consistent with those of type I collagen standards, the thermal denaturation temperature reaches 75℃, the triple helix structure remains intact, and the process is mild and highly controllable.

[0051] The following are specific examples:

[0052] Example 1: Removal of eggshell keratin using a combination of reduction and ultrasound

[0053] This embodiment provides a method for selectively removing keratin from avian eggshell membranes under non-thermal, neutral conditions, specifically including the following steps:

[0054] (1) Crushing the shell membrane: Take fresh poultry eggs, manually peel off the shell membrane, and cut the eggshell membrane into pieces of 1-3 cm. 3 The flakes are quickly pulverized into powder using a pulverizer, passed through a 45-mesh sieve, and the sieved powder is collected for later use.

[0055] (2) Combined reduction method and ultrasound to remove keratin

[0056] Weigh 25 g of the shell membrane powder obtained in step (1) and place it in a glass container. Add a mixed solution of 3 mol / L urea and 2% (mass concentration) sodium bisulfite (i.e., add 5000 mL of the mixed solution) according to the material-liquid ratio of 1 g: 200 mL.

[0057] The mixture was gently stirred with a stirring paddle for 21 h, with six intermittent stops during the stirring process. During stirring, the mixture was sonicated at 35 kHz and 500 W for 20 min each time, for a total sonication time of 120 min (i.e., six sonication sessions of 20 min each). After sonication, the mixed sample was centrifuged at 10 ℃ and 4500 r / min for 20 min. The precipitate was collected, washed with running water, and air-dried to obtain the keratin-free eggshell membrane.

[0058] In the above process, urea mainly disrupts hydrogen bonds, while sodium bisulfite reduces keratin disulfide bonds. The synergistic effect of urea and sodium bisulfite on the shell membrane promotes keratin dissolution while simultaneously breaking the hydrogen bonds between keratin and collagen, thus facilitating their dissociation. The cavitation effect of ultrasound is utilized to allow the reagent to penetrate the dense structure of the shell membrane, thereby improving the contact efficiency between the reagent and keratin.

[0059] (3) Verification of keratin removal effect

[0060] Collect the supernatant after centrifugation (i.e., shell membrane eluent), centrifuge at 10 ℃ and 7000 r / min for 10 min, take 20 mL of supernatant and divide it into two equal parts.

[0061] The protein concentration in the supernatant was determined using the BCA method in the first part, and the calculated protein concentration was 40.36 mg / mL.

[0062] Part II: Hydroxyproline concentration was determined according to GB / T 9695.23-2008: 6 M HCl was added, and the mixture was hydrolyzed at 110℃ for 6 h. After adjusting the pH to 7.0 with 2 M NaOH solution, the concentration was measured. The result was: Hydroxyproline was not detected (the content was below the detection limit of this method).

[0063] Calculate the keratin removal rate using the following formula:

[0064] Keratin removal rate (%) = C × V / m

[0065] Where C is the protein concentration of the supernatant (mg / mL), V is the volume of the supernatant (20 mL in this example), and m is the dry weight of the shell membrane (25 g).

[0066] The keratin removal rate of this embodiment was calculated to be 3.229%.

[0067] To verify the effectiveness of the combined treatment of reducing agent, urea, and ultrasound in removing keratin in this embodiment, the following four treatment groups were set up for comparative experiments:

[0068] Group A (blank control group): 25 g of shell membrane powder obtained in step (1) was placed in a glass container, and deionized water was added at a material-to-liquid ratio of 1:200 (without adding reducing agent or urea). The mixture was gently stirred with a stirrer for 21 h, with 6 intermittent stops during the process. No ultrasonic treatment was performed. After stirring, the mixed sample was centrifuged at 10 ℃ and 4500 r / min for 20 min, and the supernatant (elution) was collected.

[0069] Group B (2% sodium bisulfite treatment group): Take 25 g of the shell membrane powder obtained in step (1), place it in a glass container, add 2% sodium bisulfite solution (without adding urea) at a material-to-liquid ratio of 1:200, and gently stir with a stirrer for 21 h, stopping the stirring intermittently 6 times during the process, without ultrasonic treatment. After stirring, centrifuge the mixed sample at 10 ℃ and 4500 r / min for 20 min, and collect the supernatant (elution).

[0070] Group C (2% sodium bisulfite + 3M urea treatment group): 25 g of shell membrane powder obtained in step (1) was placed in a glass container, and a mixed solution containing 2% sodium bisulfite and 3M urea was added at a material-to-liquid ratio of 1:200. The mixture was gently stirred with a stirrer for 21 h, with 6 intermittent stops during the process. No ultrasonic treatment was performed. After stirring, the mixed sample was centrifuged at 10℃ and 4500 r / min for 20 min, and the supernatant (elution) was collected.

[0071] Group D (2% sodium bisulfite + 3M urea treatment group + cumulative ultrasound for 2 hours treatment group): that is, the treatment method in Example 1 above.

[0072] Take the supernatant from groups A, B, C, and D after centrifugation, and centrifuge at 10 ℃ and 7000 r / min for 10 min. Take 20 mL of the supernatant and divide it into two equal parts. Calculate the removal rate of keratin from the eggshell membrane using the method described above.

[0073] The test results for each group are as follows Figure 2 As shown, the keratin removal rate was 0.003% in group A, 0.129% in group B, 1.229% in group C, and 3.229% in group D. These results indicate that the keratin removal rate in group D was significantly higher than that in groups A, B, and C. Furthermore, the removal rates in groups B and C were higher than those in group A, and group C was higher than that in group B. This suggests that sodium bisulfite and urea have a synergistic effect, and ultrasonic treatment can further improve the keratin removal efficiency. Meanwhile, hydroxyproline was not detected in the supernatant of groups A, B, C, and D, indicating that there was no significant loss of collagen components in any of the treatment groups.

[0074] Example 2: Extraction, purification, and drying of macromolecular collagen from poultry eggshell membranes

[0075] This embodiment provides a method for extracting, purifying, and drying macromolecular collagen from the keratin-free eggshell membrane. The keratin-free shell membrane used in this embodiment was prepared using the method in Example 1. The specific steps are as follows:

[0076] (1) Optimization of citric acid concentration

[0077] Weigh 10 g portions of the keratinized shell membrane prepared according to Example 1 and place them in multiple glass containers. Prepare citric acid solutions of different concentrations: 0.3 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, and 0.9 mol / L. Add the above-mentioned citric acid solutions at a material-to-liquid ratio of 1 g:200 mL, and add 4% pepsin (enzyme activity 1:3000) to each solution. Stir gently at 22 °C for 18 h. Centrifuge each mixed sample at 10 °C and 8000 r / min for 10 min, and collect the supernatant.

[0078] The hydroxyproline content in each supernatant was determined according to GB / T 9695.23-2008: Take the supernatant, add 6 M HCl, hydrolyze at 110 ℃ for 6 h, adjust the pH to 7.0 with 2 M NaOH solution and then perform the determination.

[0079] The measurement results are as follows Figure 3 As shown, as the citric acid concentration increased from 0.3 mol / L to 0.9 mol / L, the hydroxyproline content in the supernatant showed an increasing trend, reaching a relatively high level in the range of 0.6–0.7 mol / L. Considering both extraction efficiency and reagent cost, 0.6 mol / L was selected as the optimal extraction concentration for subsequent processes. [Basis:] Figure 3 [and its explanation]

[0080] (2) Acid-enzyme extraction

[0081] Weigh 15 g of the keratinized shell membrane prepared according to the method in Example 1 and place it in a glass container.

[0082] Add 0.6 mol / L citric acid solution at a material-to-liquid ratio of 1 g:200 mL, then add 4% (mass fraction) pepsin (enzyme activity 1:3000, purchased from Shanghai Yuanye Biotechnology Co., Ltd., derived from pig stomach, product number: S36152-100g, ≥400 NF.U / mg), and gently stir at 22 ℃ for 18 h. Centrifuge the mixed sample at 10 ℃ and 8000 r / min for 10 min, and collect the supernatant.

[0083] (3) Salting out

[0084] Take the supernatant and adjust the pH to 7.2 with NaOH. Add ammonium sulfate solid to the neutral solution in small amounts several times until the final concentration is 1.5 mol / L, and let it stand for 12 h. After standing, centrifuge at 10 ℃ and 8000 r / min for 20 min, and collect the precipitate.

[0085] (4) Dissolving, dialysis and freeze drying

[0086] The precipitate was dissolved by adding 15 times its volume of 0.6 mol / L citric acid solution. The solution was then placed into a dialysis bag with a molecular weight cutoff of 10,000 D and dialyzed for desalting for a total of 4 days: the first 2 days were dialysis with 0.04 M sodium dihydrogen phosphate solution, with the solution changed every 6 hours; the last 2 days were dialysis with deionized water, with the solution changed every 6 hours. The conductivity of the dialysate was measured to be 8 μS / cm (<10 μS / cm), indicating that the dialysis endpoint had been reached.

[0087] The liquid in the dialysis bag was transferred to a plastic petri dish, frozen at -80°C overnight, and then sent to a freeze dryer for freeze drying at -80°C and 0.04 MPa for 48 h to obtain poultry eggshell membrane macromolecular collagen.

[0088] Figure 4 The images show scanning electron microscopy (SEM) images of the changes in the microstructure of the eggshell membrane during the treatment process. A represents the fresh shell membrane, B represents the shell membrane after treatment with a reducing agent and ultrasound, and C represents the shell membrane after collagen extraction with citric acid. The images show that the fibrous network of the fresh shell membrane (before treatment) is dense. After treatment with a reducing agent and ultrasound as described in Example 1, the shell membrane fibers show a tendency to dissolve and break, resulting in a significantly looser structure, which creates favorable conditions for subsequent acid-enzyme extraction of collagen. After collagen extraction with citric acid, the shell membrane fibers become looser, indicating successful collagen dissolution.

[0089] The extracted macromolecular collagen from the eggshell membrane was compared with the original eggshell membrane and the physical images of type I collagen standards. Figure 5 As shown in the figure, the shell membrane collagen prepared in this embodiment of the application, compared with the shell membrane and collagen standard, has a similar appearance to the commercially available type I collagen standard, both being spongy in shape.

[0090] The poultry eggshell membrane macromolecular collagen, type I collagen standard (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number C390609, source: bovine Achilles tendon), and gelatin (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number V32300) prepared in this example were dissolved in 0.5 mol / L citric acid solution to prepare sample solutions with a concentration of 0.5 mg / mL. A UV-Vis spectrophotometer was used to scan in the wavelength range of 190–400 nm at a scanning speed of 200 nm / min, and the spectra of each sample were recorded. Figure 6 As shown, the shell-membrane collagen prepared in this embodiment exhibits a strong absorption peak at 235 nm and a shoulder peak near 280 nm. This spectral characteristic is basically consistent with that of the type I collagen standard. The shoulder peak at around 280 nm is attributed to the π→π* transition of the carbonyl C=O double bond, while the shoulder peak at around 280 nm is attributed to the UV absorption of the benzene rings of the tyrosine and phenylalanine side chains, indicating that the extracted shell-membrane macromolecular collagen has a complete conformation.

[0091] Approximately 5 mg each of the poultry eggshell membrane macromolecular collagen, type I collagen standard, and gelatin prepared in this embodiment were placed in an aluminum crucible and sealed. Differential scanning calorimetry (DSC) was used for measurement, with an empty crucible as a reference. The heating rate was 5 °C / min, and the scanning temperature range was 20–120 °C under nitrogen protection. The endothermic curve of the sample was recorded, and the peak temperature of the endothermic peak was recorded as the thermal denaturation temperature (Tm). Figure 7 As shown, the Tm value of the poultry eggshell membrane macromolecular collagen prepared in this embodiment is 75 °C.

[0092] In this embodiment, the optimal extraction concentration (0.6 mol / L) was determined through citric acid concentration optimization experiments, and macromolecular collagen was successfully extracted from the keratinized shell using this concentration. The product was spongy, and its UV spectral characteristics were consistent with those of type I collagen standards. The thermal denaturation temperature was 75 °C, indicating that conformationally intact macromolecular collagen was obtained.

[0093] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for preparing macromolecular collagen from poultry eggshell membranes, comprising the following steps: (1) The eggshell membrane of poultry eggs is crushed; (2) The crushed poultry eggshell membrane is mixed with a solution containing reducing agent and urea, and gently stirred. During the stirring process, ultrasonic treatment is performed intermittently. Then, solid-liquid separation is performed, the precipitate is collected, and poultry eggshell membrane with keratin removed is obtained. (3) The keratin-free eggshell membrane obtained in step (2) is mixed with an extract containing citric acid and pepsin for extraction, and then solid-liquid separation is performed to collect the supernatant. (4) Adjust the pH of the supernatant obtained in step (3) to neutral, add ammonium sulfate for salting out, and collect the precipitate after solid-liquid separation; (5) After dissolving the precipitate obtained in step (4), it is placed in a dialysis bag for dialysis desalting, and then freeze-dried to obtain poultry eggshell membrane macromolecular collagen.

2. According to the preparation method of claim 1, in step (1), before crushing, the eggshell membrane is cut into pieces of 1-3 cm. 3 After being flaked, it is crushed and then passed through a 45-mesh sieve.

3. According to the preparation method of claim 1, in step (2), the concentration of urea in the solution containing reducing agent and urea is 2~4 mol / L, the reducing agent is sodium bisulfite, and its mass concentration is 1%~3%; the ratio of the crushed poultry eggshell membrane to the solution is 1:100~1:300; the time for gentle stirring is 18~24 h, and the stirring is stopped intermittently 4~8 times during the stirring; the conditions for ultrasonic treatment are: frequency 30~40 kHz, power 400~600 W, ultrasonic treatment for 20 min each time, and the total ultrasonic time is 80~160 min.

4. According to the preparation method of claim 1, in step (2), the solid-liquid separation conditions are: centrifugation at 10 ℃ and 3000~6000 r / min for 20 min, taking the precipitate and washing it with running water and air drying it.

5. According to the preparation method of claim 1, in step (3), the ratio of the keratin-free eggshell membrane to the extract is 1:100 to 1:300; the concentration of citric acid in the extract is 0.3 to 0.9 mol / L, the amount of pepsin is 2% to 6% of the dry weight of the shell membrane, and the enzyme activity of the pepsin is 1:3000; the extraction conditions are: gentle stirring at 20 to 25 °C for 12 to 24 h.

6. According to the preparation method of claim 1, in step (3), the solid-liquid separation conditions are: centrifugation at 10 °C and 6000~10000 r / min for 10 min, and taking the supernatant.

7. According to the preparation method of claim 1, in step (4), NaOH is used to adjust the pH to 7.0~7.5; ammonium sulfate is added to make its final concentration 1.5 mol / L; after adding ammonium sulfate, it is allowed to stand for 12 h, and then solid-liquid separation is performed; the solid-liquid separation conditions are: centrifugation at 10 ℃ and 6000~10000 r / min for 10~30 min, and the precipitate is taken.

8. According to the preparation method of claim 1, in step (5), the precipitate is dissolved by adding 10 to 20 times the volume of 0.4 to 0.8 mol / L citric acid solution; the molecular weight cutoff of the dialysis bag is 7000 D to 14000 D; the dialysis desalting time is 3 to 5 days, wherein the first 1 to 2 days are dialysis with 0.04 M sodium dihydrogen phosphate solution and the solution is changed every 6 h, and the next 2 to 3 days are dialysis with deionized water and the solution is changed every 6 h, and the dialysis endpoint is the conductivity value of the dialysate <10 μS / cm; the freeze-drying conditions are: freezing at -80 ℃ overnight, and then freeze-drying at -80 ℃ and 0.03 to 0.05 MPa for 48 h.

9. According to the preparation method of claim 1, in step (2), the supernatant obtained after solid-liquid separation is found to be free of hydroxyproline, indicating that the removed protein is keratin rather than collagen.

10. A macromolecular collagen for poultry eggshell membrane, prepared by the method described in any one of claims 1-9.

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