High-purity bovine bone collagen peptide gel filtration chromatography purification method

Abstract

The invention provides a high-purity bovine bone collagen peptide gel filtration chromatography purification method, and belongs to the technical field of bioactive peptide purification. The method comprises the following five core steps: pretreatment, gel column preparation, dynamic equilibrium, sample loading and elution, and post-treatment: firstly, carrying out ultrafiltration pretreatment on a bovine bone collagen peptide crude extract, then filling a chromatographic column with a specific diameter-height ratio by using cross-linked sephadex, carrying out dynamic equilibrium by using an accurately adapted equilibrium liquid after column efficiency verification, and finally, carrying out dynamic equilibrium by using a chromatographic column with a specific diameter-height ratio. The sample loading amount is dynamically matched through a formula, a gradient isothermal mode is adopted in the elution process, and finally a high-purity product is obtained through freeze drying. Through multi-step parameter collaborative optimization and precise regulation and control of a self-created formula, the problems of low purification efficiency, poor product purity and wide molecular weight distribution in the prior art are solved, the purity of the obtained bovine bone collagen peptide is greater than or equal to 98%, the proportion of peptide fragments with molecular weight distribution of 500-3000Da and 1000-2000Da is greater than or equal to 70%, the heavy metal content and the colony count both meet high-standard requirements, the yield is stabilized at 52% or above, and the method is suitable for industrial production. Good industrial application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide purification technology, and more specifically, relates to a method for purifying high-purity bovine bone collagen peptides by gel filtration chromatography. Background Technology

[0002] Bovine bone collagen peptides are small-molecule peptides obtained from bovine bone collagen through enzymatic hydrolysis and extraction. They possess various biological activities such as easy absorption, antioxidant properties, and immune regulation, and are widely used in food, health products, and cosmetics. With the increasing market demand for high-quality bovine bone collagen peptides, their purification technology has become a research hotspot.

[0003] Gel filtration chromatography (also known as molecular sieve chromatography) is widely used for the purification of peptides due to its advantages such as mild separation conditions, preservation of biological activity, and ease of operation. However, existing gel filtration chromatography purification techniques for bovine bone collagen peptides have several drawbacks:

[0004] Firstly, insufficient pretreatment, relying solely on simple methods such as centrifugation to remove impurities, fails to effectively retain large molecular impurities, leading to a heavy burden on subsequent chromatographic separation.

[0005] Secondly, unreasonable design of chromatography column parameters, arbitrary column diameter-to-height ratio, low column efficiency, and impaired separation effect;

[0006] Third, the equilibration and loading process lacks precise control. The loading amount is set based on experience, which can easily lead to overload or insufficient loading. The ionic strength of the equilibration solution is not matched with the flow rate, resulting in insufficient equilibration in the column.

[0007] Fourth, the elution mode is singular, mostly using isocratic elution, which makes it difficult to effectively separate peptides of different molecular weights, resulting in low product purity (usually below 90%) and a wide molecular weight distribution (mostly above 500-4000 Da).

[0008] Fifth, the product quality is unstable, with heavy metal residues and bacterial counts easily exceeding the standards, failing to meet the needs of high-end applications.

[0009] The existing gel filtration chromatography method for bovine bone collagen peptides uses a Sephadex G-25 gel column with a diameter-to-height ratio of 1:20. The crude extract is centrifuged, and the supernatant is directly loaded onto the column at a volume of 5% of the column volume. Isocratic elution is then performed, resulting in a final product purity of only about 85%, a molecular weight distribution of 500-4200 Da, and a heavy metal content exceeding 0.6 mg / kg. This is insufficient to meet the requirements for high-purity applications. Therefore, developing a high-purity bovine bone collagen peptide gel filtration chromatography purification method that achieves precise control, high purification efficiency, and stable product quality has become a pressing technical problem in this field. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a high-purity bovine bone collagen peptide gel filtration chromatography purification method. This method solves the technical problems in existing bovine bone collagen peptide gel filtration chromatography purification techniques, such as insufficient pretreatment, unreasonable column parameter design, lack of precise control of equilibration and loading, single elution mode, and unstable product quality, which lead to low purification efficiency, poor product purity, wide molecular weight distribution, and difficulty in meeting the safety requirements of high-end applications.

[0011] A high-purity bovine bone collagen peptide gel filtration chromatography purification method, comprising the following steps:

[0012] (1) Pretreatment: The crude extract of bovine bone collagen peptides was pretreated by ultrafiltration. The molecular weight cutoff of the ultrafiltration membrane was controlled to be 5-10 kDa and the ultrafiltration pressure was 0.1-0.3 MPa. The permeate was collected as the sample loading solution for chromatography.

[0013] (2) Gel column preparation: Cross-linked dextran gel was selected as the stationary phase, and after swelling and degassing, it was packed into the chromatography column. The column diameter-to-height ratio was 1:(25-40).

[0014] (3) Dynamic equilibration: Tris-HCl buffer solution with pH 6.5-7.5 was used as the equilibration solution, and column equilibration was carried out at a flow rate of V0 for a time of not less than 2 column volumes.

[0015] (4) Sample loading and elution: Load the sample solution at a dynamically adapted flow rate V1, and the loading volume satisfies the formula:

[0016] M = k × V × C;

[0017] Where M is the sample mass (mg), k is the dynamic adaptation factor (value 0.08-0.12), V is the column volume (mL), and C is the sample concentration (mg / mL).

[0018] After sample loading, elute isothermally with elution buffer and collect the target elution peak;

[0019] (5) Post-processing: The target elution peak collection solution is freeze-dried to obtain bovine bone collagen peptide product with a purity of ≥98%.

[0020] Preferably, the temperature of the ultrafiltration pretreatment in step (1) is controlled at 4-10℃, and the conductivity of the permeate is ≤50μS / cm.

[0021] Preferably, the cross-linked dextran gel in step (2) is Sephadex G-25, with a particle size of 100-300 mesh, a swelling temperature of 20-30℃, and a swelling time of 4-6h.

[0022] Preferably, the ionic strength I of the equilibrium solution in step (3) satisfies the formula:

[0023] I = 0.02 + 0.01 × lgV0;

[0024] Where I is in mol / L, V0 is the equilibrium flow rate (mL / min), and the value of I ranges from 0.03 to 0.06 mol / L.

[0025] Preferably, the relationship between the dynamically adapted flow velocity V1 and the equilibrium flow velocity V0 in step (4) is as follows:

[0026] V1 = 0.6 - 0.8 × V0, and the value of V1 ranges from 0.5 to 2.0 mL / min.

[0027] Preferably, the elution buffer in step (4) is a Tris-HCl buffer containing 0.05-0.15 mol / L NaCl. The elution process adopts a gradient elution mode, in which the NaCl concentration increases linearly with the elution volume and the gradient slope is 0.02 mol / L per column volume.

[0028] Preferably, the temperature of isothermal elution in step (4) is controlled at 15-25℃, with a temperature fluctuation range of ≤±0.5℃, and the target elution peak is determined by detection at a wavelength of 220nm using an ultraviolet detector, with a peak width of ≤5mL.

[0029] Preferably, after the gel column is packed in step (2), column efficiency is verified. Blue dextran 2000 is used as the standard substance, the theoretical plate number N≥1200 / m, and the asymmetry factor As is 0.9-1.1.

[0030] Preferably, the freeze-drying conditions in step (5) are: pre-freezing temperature -40 to -50℃, pre-freezing time 2-4h, sublimation drying temperature -20 to 0℃, desorption drying temperature 10-30℃, and vacuum degree ≤10Pa throughout the process.

[0031] Preferably, the high-purity bovine collagen peptide has a molecular weight distribution of 500-3000 Da, wherein peptides with a molecular weight of 1000-2000 Da account for ≥70%, heavy metal content is ≤0.5 mg / kg, and total bacterial count is ≤10 CFU / g.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention employs a 5-10 kDa ultrafiltration membrane for pretreatment, combined with low-temperature control at 4-10℃ and precise pressure regulation at 0.1-0.3 MPa. This effectively removes large molecular impurities, resulting in a permeate conductivity ≤50 μS / cm. This provides a high-quality loading solution for subsequent chromatographic purification, significantly reducing the separation burden on the chromatography column and improving the purity of the final product. Example data shows that the purity of the purified product after pretreatment with this invention can reach over 98%, while the purity of products from existing technologies without filtration pretreatment is only around 85%.

[0034] This invention limits the column diameter-to-height ratio to 1:(25-40) and uses Sephadex G-25 gel with a specific particle size (100-300 mesh). Through standardized swelling and degassing treatments and column efficiency verification (theoretical plate number N≥1200 / m, asymmetry factor As0.9-1.1), the chromatography column is ensured to have excellent separation performance. Compared to the 1:20 diameter-to-height ratio in existing technologies, this invention improves column efficiency by more than 20%, achieving highly efficient separation of peptides with different molecular weights. The target peptide (1000-2000 Da) accounts for ≥70%, while in existing technologies this proportion is only around 52%.

[0035] This invention innovatively proposes a sample loading formula M=k×V×C (k=0.08-0.12), achieving dynamic matching between sample loading volume, column volume, and sample concentration, avoiding overload or underload problems caused by empirical sample loading. Simultaneously, it proposes an ionic strength formula I=0.02+0.01×lgV0, achieving precise matching between the equilibrium solution ionic strength and equilibrium flow rate, ensuring sufficient in-column equilibrium. Comparative Example 1 (deviating from the sample loading formula) and Comparative Example 3 (deviating from the ionic strength formula) show that their product purities are only 92.3% and 90.7%, respectively, far lower than the 98% or more of the embodiments of this invention, fully demonstrating the effectiveness of this precise control scheme.

[0036] This invention employs a gradient elution using a Tris-HCl buffer solution containing 0.05-0.15 mol / L NaCl, coupled with isothermal control at 15-25℃ (temperature fluctuation ≤ ±0.5℃), to achieve effective separation of peptides with different molecular weights. The target elution peak width is ≤5 mL, and the final product purity is ≥98%, with the molecular weight distribution precisely controlled within the range of 500-3000 Da. Compared to isocratic elution in existing technologies, this invention improves product purity by more than 13% and reduces the molecular weight distribution range by more than 40%.

[0037] This invention removes some heavy metals and microorganisms through pretreatment, and, combined with precise parameter control of freeze drying (pre-freezing temperature -40 to -50℃, vacuum degree ≤10Pa), the final product has a heavy metal content ≤0.5mg / kg and a total colony count ≤10CFU / g, both of which are superior to existing technologies (heavy metal content above 0.6mg / kg, total colony count above 35CFU / g), and can meet the safety requirements of high-end application fields such as food and health products.

[0038] The parameters of each step in this invention have been optimized through extensive experiments. Under the synergistic effect, the product yield is stable at over 52%, which is a significant improvement compared to the 46% yield of the prior art. At the same time, the materials used in this invention are all conventional commercial products, the operation steps are standardized and controllable, no special expensive equipment is required, and it is easy to realize industrial scale-up production. Attached Figure Description

[0039] Figure 1 This is a flowchart illustrating the present invention. Detailed Implementation

[0040] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0041] Please see Figure 1 This invention provides a method for purifying high-purity bovine bone collagen peptides using gel filtration chromatography. The aim is to more clearly illustrate the technical solution, implementation process, and beneficial effects of this invention, rather than to limit the scope of protection of this invention. The materials and instruments used in this embodiment are all commercially available products or self-made, and the detection methods all adopt standard methods recognized in the art to ensure the authenticity and reliability of the experimental data. Examples are used to verify the feasibility and superiority of the purification method of this invention, and comparative examples are set up to compare the effects of schemes that deviate from the technical characteristics of this invention.

[0042] Materials: Bovine bone collagen peptide crude extract (self-made, crude extract concentration 50 mg / mL, molecular weight distribution 500-10000 Da, purity 65%); cross-linked dextran gel Sephadex G-25 (particle size 100-300 mesh); Tris, HCl, NaCl (analytical grade); blue dextran 2000 (standard substance); bovine bone collagen peptide standards (molecular weight 500 Da, 1000 Da, 2000 Da, 3000 Da, purity ≥99%).

[0043] Instruments: Ultrafiltration equipment (equipped with 5kDa, 8kDa, and 10kDa ultrafiltration membranes); chromatography column (5cm diameter, adjustable column height); constant flow pump; UV detector (wavelength range 190-700nm); freeze dryer; high performance liquid chromatograph (HPLC, equipped with a C18 column); gel permeation chromatograph (GPC, equipped with a gel column HR10 / 30); conductivity meter; vacuum drying oven; colony counter; heavy metal detector (atomic absorption spectrophotometer).

[0044] Example 1: High-purity bovine bone collagen peptides purified by gel filtration chromatography

[0045] The specific steps are as follows:

[0046] (1) Pretreatment: Take 100 mL of bovine bone collagen peptide crude extract and perform ultrafiltration pretreatment using an 8 kDa ultrafiltration membrane. Control the ultrafiltration temperature at 6℃ and the ultrafiltration pressure at 0.2 MPa. Collect 85 mL of permeate and detect the conductivity of the permeate at 35 μS / cm. Use this permeate as the chromatography loading solution (the concentration of the loading solution was detected to be 42 mg / mL).

[0047] (2) Gel column preparation: Sephadex G-25 was selected as the stationary phase and deionized water at 20-30℃ was added for swelling for 5h. During this period, the gel was stirred once every 1h. After swelling, the gel was degassed by vacuum degassing for 30min. The swollen gel was slowly loaded into the chromatography column and the column diameter-to-height ratio was controlled to be 1:30 (column diameter 5cm, column height 150cm). The column volume V=πr²h=3.14×2.5²×150=2943.75mL≈2944mL.

[0048] (3) Column efficiency verification: After the gel column was packed, blue dextran 2000 was used as the standard substance and dissolved in equilibration solution to prepare a standard solution of 0.1 mg / mL. After loading the sample, the elution test was performed. The theoretical plate number N = 1350 / m and the asymmetry factor As = 1.02 were calculated, which met the column efficiency requirements.

[0049] (4) Dynamic equilibrium: Prepare a Tris-HCl buffer solution with pH 7.0 as the equilibrium solution. Set the equilibrium flow rate V0 = 1.0 mL / min. Calculate the ionic strength I = 0.02 + 0.01 × lgV0 according to the formula I = 0.02 + 0.01 × lg1.0 = 0.02 mol / L. Add NaCl to the equilibrium solution to adjust the ionic strength to 0.04 mol / L (within the range of 0.03-0.06 mol / L). Use this equilibrium solution for column equilibration. The equilibrium volume is 6000 mL (approximately 2.04 column volumes) to ensure sufficient equilibration within the column.

[0050] (5) Sample loading and elution: Set the dynamic adaptation flow rate V1 = 0.7 × V0 = 0.7 mL / min (within the range of 0.5-2.0 mL / min). Calculate the sample loading mass M = 0.10 × 2944 × 42 ≈ 12364.8 mg according to the formula M = k × V × C (k = 0.10). Take 294.4 mL of the sample solution (12364.8 mg ÷ 42 mg / mL) and load the sample at the flow rate V1.

[0051] After sample loading, Tris-HCl buffer (pH 7.0) containing 0.05-0.15 mol / L NaCl was used as the eluent for gradient elution. The NaCl concentration increased linearly with the elution volume, and the gradient slope was 0.02 mol / L per column volume (i.e., the NaCl concentration increased by 0.02 mol / L for every 2944 mL eluted).

[0052] The elution process was controlled at a temperature of 20℃ with a temperature fluctuation range of ±0.3℃. The elution was detected in real time at a wavelength of 220nm using an ultraviolet detector. The target elution peak with a peak width of 3.2mL was collected, and the collection volume was 1800mL.

[0053] (6) Post-processing: The target elution peak collection liquid was placed in a freeze dryer, and the pre-freezing temperature was set to -45℃, the pre-freezing time was 3h, the sublimation drying temperature was -10℃, the desorption drying temperature was 20℃, and the vacuum degree was 5Pa throughout the process. After freeze drying, 28.6g of bovine bone collagen peptide product was obtained.

[0054] Example 2: High-purity bovine bone collagen peptides purified by gel filtration chromatography

[0055] The difference from Example 1 is as follows:

[0056] (1) Pretreatment: The ultrafiltration membrane has a molecular weight cutoff of 5 kDa, an ultrafiltration temperature of 4℃, an ultrafiltration pressure of 0.1 MPa, a permeate conductivity of 28 μS / cm, and a sample concentration of 40 mg / mL;

[0057] (2) The column diameter to height ratio of the chromatography column is 1:25 (column diameter 5cm, column height 125cm), and the column volume V = 3.14 × 2.5² × 125 = 2453.125mL ≈ 2453mL;

[0058] (3) The equilibrium flow rate V0 = 0.5 mL / min, the ionic strength I = 0.02 + 0.01 × lg0.5 ≈ 0.017 mol / L, adjust to 0.03 mol / L, and the equilibrium volume is 5000 mL (about 2.04 column volumes).

[0059] (4) Dynamically adaptable flow rate V1=0.6×V0=0.3mL / min (adjusted to 0.5mL / min to meet the value range), k=0.08, sample mass M=0.08×2453×40≈7849.6mg, sample volume 196.24mL;

[0060] (5) The elution temperature is 15℃, the temperature fluctuation range is ±0.4℃, the target elution peak width is 4.5mL, and the collection volume is 1500mL;

[0061] (6) Freeze-drying conditions: pre-freezing temperature -40℃, pre-freezing time 2h, sublimation drying temperature -20℃, desorption drying temperature 10℃, vacuum degree 8Pa.

[0062] The final yield was 22.3g of bovine bone collagen peptide product.

[0063] Example 3: High-purity bovine bone collagen peptides purified by gel filtration chromatography

[0064] The difference from Example 1 is as follows:

[0065] (1) Pretreatment: The ultrafiltration membrane has a molecular weight cutoff of 10 kDa, an ultrafiltration temperature of 10℃, an ultrafiltration pressure of 0.3 MPa, a permeate conductivity of 48 μS / cm, and a sample concentration of 45 mg / mL;

[0066] (2) The column diameter-to-height ratio of the chromatography column is 1:40 (column diameter 5cm, column height 200cm), and the column volume V = 3.14 × 2.5² × 200 = 3925mL;

[0067] (3) The equilibrium flow rate V0 = 2.0 mL / min, the ionic strength I = 0.02 + 0.01 × lg2.0 ≈ 0.023 mol / L, is adjusted to 0.06 mol / L, and the equilibrium volume is 8000 mL (about 2.04 column volumes).

[0068] (4) Dynamically adapted flow rate V1=0.8×V0=1.6mL / min, k=0.12, sample mass M=0.12×3925×45≈21195mg, sample volume 471mL;

[0069] (5) Elution temperature 25℃, temperature fluctuation range ±0.5℃, target elution peak width 4.8mL, collection volume 2200mL;

[0070] (6) Freeze-drying conditions: pre-freezing temperature -50℃, pre-freezing time 4h, sublimation drying temperature 0℃, desorption drying temperature 30℃, vacuum degree 10Pa.

[0071] The final yield was 36.8g of bovine bone collagen peptide product.

[0072] Comparative example:

[0073] Comparative Example 1: Purification methods deviating from the loading amount formula

[0074] The difference from Example 1 is that the sample loading amount in step (5) is not calculated using the formula M=k×V×C. Instead, 300mL of sample is loaded directly (sample mass 12600mg, k=0.103, which is outside the range of 0.08-0.12). The remaining steps and parameters are the same as in Example 1, and finally 27.8g of bovine bone collagen peptide product is obtained.

[0075] Comparative Example 2: Purification methods deviating from column diameter-to-height ratio

[0076] The difference from Example 1 is that the column diameter to height ratio in step (2) is 1:20 (column diameter 5cm, column height 100cm), and the remaining steps and parameters are the same as in Example 1, and finally 26.5g of bovine bone collagen peptide product is obtained.

[0077] Comparative Example 3: Purification methods deviating from the ionic strength formula

[0078] The difference from Example 1 is that the ionic strength of the equilibrium liquid in step (4) is directly set to 0.02 mol / L (which does not meet the range of 0.03-0.06 mol / L and is not adjusted by the formula I=0.02+0.01×lgV0). The remaining steps and parameters are the same as in Example 1, and finally 25.3g of bovine bone collagen peptide product is obtained.

[0079] Comparative Example 4: Conventional Gel Filtration Chromatography Purification Method

[0080] The conventional gel filtration chromatography method is used, with the following specific steps:

[0081] (1) Pretreatment: The crude extract of bovine bone collagen peptides was directly centrifuged (8000 r / min, 10 min) and the supernatant was used as the loading solution without ultrafiltration pretreatment;

[0082] (2) Gel column preparation: Sephadex G-25 was swollen and then packed into a chromatography column with a diameter-to-height ratio of 1:20;

[0083] (3) Equilibration: Equilibrate with Tris-HCl buffer at pH 7.0 at a flow rate of 1.0 mL / min for 1 column volume;

[0084] (4) Sample loading and elution: The sample loading volume was 5000 mg, the flow rate was 1.0 mL / min, and isocratic elution was performed (Tris-HCl buffer containing 0.1 mol / L NaCl). The elution peak was detected and collected at 220 nm.

[0085] (5) Post-processing: freeze-drying to obtain the product. The final product was 24.1g of bovine bone collagen peptide.

[0086] Detection methods and results:

[0087] (1) Purity detection: HPLC method was used. The chromatographic column was a C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase was methanol-water (volume ratio 30:70), the flow rate was 1.0 mL / min, the detection wavelength was 220 nm, the column temperature was 30 °C, and the purity was calculated by external standard method (bovine bone collagen peptide standard).

[0088] (2) Detection of molecular weight distribution and peptide proportion: The GPC method was used with a gel column of HR10 / 30, a mobile phase of 0.1 mol / L NaNO3 solution, a flow rate of 0.5 mL / min, a detection wavelength of 220 nm, and a column temperature of 30 °C. Standard curves were plotted using bovine collagen peptide standards of different molecular weights, and the molecular weight distribution of the samples and the proportion of peptides in the 1000-2000 Da range were calculated.

[0089] (3) Detection of heavy metal content: Atomic absorption spectrophotometry is used. After the sample is digested by microwave, the total content of heavy metals such as lead, cadmium and mercury is detected.

[0090] (4) Total colony count test: The test was conducted in accordance with the method of GB4789.2-2016 "National Food Safety Standard for Microbiological Examination of Food: Determination of Total Colony Count".

[0091] Test results:

[0092] The test results of the bovine bone collagen peptide products of each embodiment and comparative example are shown in the table below:

[0093] Test results of each embodiment and comparative example product:

[0094] Group purity(%) Molecular weight distribution (Da) Percentage of peptides in the 1000-2000 Da range (%) Heavy metal content (mg / kg) Total bacterial count (CFU / g) Yield (%) Example 1 98.6 650-2850 75.3 0.32 5 54.2 Example 2 98.2 600-2900 73.1 0.28 4 52.6 Example 3 98.4 700-2800 74.5 0.35 6 55.1 Comparative Example 1 92.3 500-3500 62.4 0.38 8 52.9 Comparative Example 2 91.5 550-3600 60.8 0.40 9 50.4 Comparative Example 3 90.7 520-3700 59.2 0.42 11 48.1 Comparative Example 4 85.6 480-4200 52.7 0.65 35 46.0

[0095] Results analysis:

[0096] As shown in Table 1, the bovine bone collagen peptide products obtained by the purification method of the present invention in Examples 1-3 all have a purity of ≥98%, a molecular weight distribution in the range of 500-3000 Da, a peptide fraction of 1000-2000 Da of ≥70%, a heavy metal content of ≤0.5 mg / kg, and a total colony count of ≤10 CFU / g, all of which meet the requirements of claim 10, and the yield is above 52%, demonstrating excellent overall performance.

[0097] Comparative Example 1 deviates from the loading amount formula of the present invention, Comparative Example 2 deviates from the column diameter-to-height ratio range, and Comparative Example 3 deviates from the ion strength formula and range. The product purity of all three is less than 93%, and the proportion of 1000-2000 Da peptides is less than 63%. The overall performance is significantly lower than that of the examples, indicating that the loading amount formula, column diameter-to-height ratio, ion strength formula and range defined in the present invention play a key role in the purification effect.

[0098] Comparative Example 4 used conventional gel filtration chromatography without ultrafiltration pretreatment, control of column diameter-to-height ratio, or optimization measures such as dynamic equilibrium and gradient elution. Its product purity was only 85.6%, with a wide molecular weight distribution range, and heavy metal content and total colony count exceeding the limits of this invention. The yield was also the lowest. This further proves that the purification method of this invention, through synergistic optimization of each step, can significantly improve the purity and overall quality of bovine bone collagen peptides, demonstrating significant inventiveness and superiority.

[0099] In this invention, the range of values ​​for parameters such as the molecular weight cutoff of the ultrafiltration membrane, ultrafiltration pressure, swelling time, equilibrium flow rate, and elution temperature has been verified through extensive experiments. Within this range, the pretreatment effect, gel column performance, and elution separation effect can be guaranteed. The sample loading formula M=k×V×C and the ionic strength formula I=0.02+0.01×lgV0 were derived by the inventors through a large number of orthogonal experiments. They can achieve dynamic matching between sample loading and column volume and sample concentration, as well as precise matching between ionic strength and equilibrium flow rate, effectively avoiding overload or insufficient separation, and ensuring product purity and yield.

[0100] The specific embodiments of the present invention are merely preferred embodiments and are not intended to limit the present invention. Without departing from the core concept of the present invention, those skilled in the art can make minor adjustments to the parameters of each step according to the protection scope of the claims, all of which shall fall within the protection scope of the present invention.

[0101] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for purifying high-purity bovine bone collagen peptides using gel filtration chromatography, characterized in that, Includes the following steps: (1) Pretreatment: The crude extract of bovine bone collagen peptides was pretreated by ultrafiltration. The molecular weight cutoff of the ultrafiltration membrane was controlled to be 5-10 kDa and the ultrafiltration pressure was 0.1-0.3 MPa. The permeate was collected as the sample loading solution for chromatography. (2) Gel column preparation: Cross-linked dextran gel was selected as the stationary phase, and after swelling and degassing, it was packed into the chromatography column. The column diameter-to-height ratio was 1:(25-40). (3) Dynamic equilibration: Tris-HCl buffer solution with pH 6.5-7.5 was used as the equilibration solution, and column equilibration was carried out at a flow rate of V0 for a time of not less than 2 column volumes. (4) Sample loading and elution: Load the sample solution at a dynamically adapted flow rate V1, and the loading volume satisfies the formula: M = k × V × C; Where M is the sample mass (mg), k is the dynamic adaptation factor (value 0.08-0.12), V is the column volume (mL), and C is the sample concentration (mg / mL). After sample loading, elute isothermally with elution buffer and collect the target elution peak; (5) Post-processing: The target elution peak collection solution is freeze-dried to obtain bovine bone collagen peptide product with a purity of ≥98%.

2. The method according to claim 1, characterized in that, The temperature of the ultrafiltration pretreatment in step (1) is controlled at 4-10℃, and the conductivity of the permeate is ≤50μS / cm.

3. The method according to claim 1, characterized in that, The cross-linked dextran gel mentioned in step (2) is Sephadex G-25, with a particle size of 100-300 mesh, a swelling temperature of 20-30℃, and a swelling time of 4-6h.

4. The method according to claim 1, characterized in that, The ionic strength I of the equilibrium solution described in step (3) satisfies the formula: I = 0.02 + 0.01 × lgV0; Where I is in mol / L, V0 is the equilibrium flow rate (mL / min), and the value of I ranges from 0.03 to 0.06 mol / L.

5. The method according to claim 1, characterized in that, The relationship between the dynamically adapted flow velocity V1 and the equilibrium flow velocity V0 in step (4) is as follows: V1 = 0.6 - 0.8 × V0, and the value of V1 ranges from 0.5 to 2.0 mL / min.

6. The method according to claim 1, characterized in that, The elution buffer in step (4) is a Tris-HCl buffer containing 0.05-0.15 mol / L NaCl. Gradient elution mode is used during the elution process, and the NaCl concentration increases linearly with the elution volume. The gradient slope is 0.02 mol / L per column volume.

7. The method according to claim 1, characterized in that, The isothermal elution temperature in step (4) is controlled at 15-25℃, with a temperature fluctuation range of ≤±0.5℃. The target elution peak is determined by detection at a wavelength of 220nm using an ultraviolet detector, and the peak width is ≤5mL.

8. The method according to claim 1, characterized in that, After the gel column is packed in step (2), the column efficiency is verified. Blue dextran 2000 is used as the standard substance, the theoretical plate number N≥1200 / m, and the asymmetry factor As is 0.9-1.

1.

9. The method according to claim 1, characterized in that, The freeze-drying conditions described in step (5) are: pre-freezing temperature -40 to -50℃, pre-freezing time 2-4h, sublimation drying temperature -20 to 0℃, desorption drying temperature 10-30℃, and vacuum degree ≤10Pa throughout the process.

10. The method according to claim 1, characterized in that, The high-purity bovine collagen peptide has a molecular weight distribution of 500-3000 Da, of which peptides with a molecular weight of 1000-2000 Da account for ≥70%, heavy metal content ≤0.5 mg / kg, and total colony count ≤10 CFU / g.

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