Preparation process of bovine spleen peptide for improving intestinal digestive system

Abstract

The application discloses a preparation process of a bovine spleen peptide for improving the intestinal digestive system, and comprises the following steps: carrying out pulse electric field-ultrasonic synergistic pretreatment on bovine spleen tissues; homogenizing the pretreated spleen tissues to obtain a homogenate liquid with a cell breakage rate of greater than or equal to 95%; sequentially carrying out three-stage protease enzymolysis on the homogenate liquid; centrifuging the enzymolysis liquid after enzyme inactivation, and collecting supernatant; sequentially carrying out four-stage separation on the supernatant through a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane with a molecular weight cut-off of 3 kDa and a nanofiltration membrane with a molecular weight cut-off of 1 kDa, and collecting respectively; compounding different molecular weight peptide segments to obtain a compounded liquid; carrying out gradient temperature reduction vacuum freeze drying on the compounded liquid after adding a medicine-food homologous extract; through three-stage gradient enzymolysis and dynamic pH regulation technology, pepsin, trypsin and alkaline protease act on the spleen protein in a corresponding pH condition respectively, and the spleen protein is realized to be hydrolyzed in a graded and directional manner.

Description

Technical Field

[0001] This invention relates to biotechnology, specifically to a process for preparing bovine spleen peptides that improve the intestinal digestive system. Background Technology

[0002] The spleen is an important immune organ, rich in various immune-active substances and biological regulatory factors. Spleen peptides (also known as spleen amino peptides or spleen polypeptides) extracted from animal spleens possess a variety of biological activities, including immunomodulation, anti-oxidation, promotion of digestive enzyme secretion, and repair of gastrointestinal mucosa, and have broad application prospects in the fields of functional foods and medicine.

[0003] Currently, existing technologies for preparing bovine spleen peptides from bovine spleen can be mainly categorized as follows: Category 1: Single or combined enzymatic hydrolysis methods. For example, Chinese patent application CN120082623A discloses a method for enzymatically hydrolyzing bovine spleen homogenate using a combination enzyme composed of subtilisin, bromelain, and papain, followed by centrifugation, ultrafiltration, and drying to obtain bovine spleen peptides. Although this method improves the extraction rate, because multiple enzymes react under the same pH and temperature conditions, they cannot fully exert their optimal catalytic activity, resulting in insufficient hydrolysis specificity, a wide molecular weight distribution of the product (usually between 500 Da and 10 kDa), and low enrichment of functional peptides.

[0004] The second category is microbial fermentation. Chinese patent application CN202510944036 discloses a process for preparing bovine spleen peptides using *Streptomyces flavus* and *Yersinia lipolyticis* for primary fermentation, followed by subcritical water extraction, protease hydrolysis, multi-stage fermentation, composite gel clarification, and nanofiltration spray drying. This method has a long process chain (usually requiring more than 5 days), high equipment requirements, high industrialization costs, and high-temperature spray drying can easily lead to the inactivation of heat-sensitive active peptides.

[0005] The third category: Composition compounding method. Chinese patent application CN202311005394 describes the compounding of bovine spleen peptides with fermentation agents, probiotic agents, and traditional Chinese medicine complexes, etc., and preparing the composition through multi-stage fermentation. This method focuses on the compounding of the end product, rather than the innovation of the extraction process of bovine spleen peptides themselves, and does not solve the problems of large activity loss and inaccurate molecular weight control in the preparation of bovine spleen peptide raw materials.

[0006] Category 4: Physical field-assisted extraction method. A few studies have reported the use of ultrasound or high-pressure homogenization to assist in the extraction of active substances from the spleen, but these are all single physical field treatments and have not been systematically integrated with subsequent enzymatic hydrolysis and separation processes.

[0007] A comprehensive analysis of existing technologies reveals the following common problems in the preparation of bovine spleen peptides that urgently need to be addressed: Incomplete cell disruption results in low release rates of active substances. Existing processes often employ simple mechanical homogenization or repeated freeze-thaw cycles, typically achieving only 60%–70% cell disruption. This leaves a large amount of intracellular active proteins and peptides trapped in cell debris, leading to low raw material utilization.

[0008] The enzymatic hydrolysis process lacks precise control, resulting in a wide molecular weight distribution of the products. Existing processes mostly employ single- or two-stage enzymatic hydrolysis under fixed pH conditions. This prevents different proteases from simultaneously reaching their optimal activity levels in the same environment, leading to low degrees of hydrolysis and low yields of peptides within the target molecular weight range. Furthermore, a significant amount of large protein molecules (greater than 10 kDa) remains in the product, impacting absorption and utilization.

[0009] Separation and purification processes lack functional-guided design. Existing processes mostly use ultrafiltration membranes with a single molecular weight cutoff for separation, without fractional purification and targeted compounding based on the differences in intestinal function of different peptides, resulting in products with weak functional targeting.

[0010] The product has poor flavor and low activity retention. Bovine spleen peptides generally suffer from a strong fishy odor. Current technologies often employ chemical masking agents or high-temperature treatment, but these introduce safety risks or lead to decreased activity. Furthermore, ice crystal growth during conventional vacuum freeze-drying can easily damage the peptide structure, affecting product resolubility and bioactivity.

[0011] Therefore, developing a bovine spleen peptide preparation process that can achieve efficient cell disruption, precise enzymatic hydrolysis control, function-oriented fractional separation, and simultaneously improve flavor while preserving activity has significant industrial value and application prospects. Summary of the Invention

[0012] To address the shortcomings of the existing technology, the present invention aims to provide a process for preparing bovine spleen peptides that improve the intestinal digestive system. Guided by the goal of precisely improving the intestinal digestive system, this invention employs a multi-stage physical-biological coupling processing technique to achieve precise control of the target molecular weight range and enrichment of targeted functional peptides while preserving the natural active conformation of bovine spleen peptides. This results in bovine spleen peptide products with concentrated molecular weight distribution, strong functional activity, good flavor, and high batch stability.

[0013] This invention provides a process for preparing bovine spleen peptides that improve the intestinal digestive system, comprising the following steps: (a) Pulsed electric field-ultrasound combined pretreatment: Bovine spleen tissue was subjected to pulsed electric field treatment and ultrasound treatment in sequence, with the temperature controlled at ≤10℃ throughout the process, to obtain pretreated spleen tissue; (b) Low-temperature high-speed homogenization: The pretreated spleen tissue was homogenized to obtain a homogenate with a cell disruption rate of ≥95%; (c) Three-stage gradient enzymatic hydrolysis and dynamic pH control: The homogenate was subjected to first-stage acidic protease hydrolysis, second-stage neutral protease hydrolysis and third-stage alkaline protease hydrolysis in sequence. The pH, temperature and enzyme type were independently controlled in each stage. The pH fluctuation in each stage was controlled within ±0.2 through online pH monitoring and automatic feedback liquid addition system. (d) Enzyme inactivation and centrifugation: After heating the enzyme hydrolysate to inactivate the enzyme, centrifuge and collect the supernatant; (e) Bionic digestion model-guided molecular weight cascade complexation: The supernatant was sequentially passed through a microfiltration membrane, an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, a nanofiltration membrane with a molecular weight cutoff of 3 kDa, and a nanofiltration membrane with a molecular weight cutoff of 1 kDa for four-stage separation, and peptides with molecular weights of <1 kDa, 1-3 kDa, and 3-10 kDa were collected respectively. (f) Segmented collection and targeted compounding: The peptides of different molecular weights collected in step (e) are compounded in the following mass percentages to obtain a compound solution: 40%–50% of peptides with molecular weight <1kDa, 30%–40% of peptides with molecular weight 1–3kDa and 10%–20% of peptides with molecular weight 3–10kDa; (g) Flavor synergistic improvement and gradient cooling vacuum freeze drying: After adding the medicinal and edible extract to the compound solution, gradient cooling vacuum freeze drying was performed.

[0014] Preferably, the parameters for pulsed electric field processing in (a) are: electric field strength 15-25 kV / cm, pulse frequency 100-300 Hz, pulse width 10-30 μs, and processing time 5-15 min; The parameters for ultrasonic processing are: ultrasonic power 300-500W, frequency 20-40kHz, intermittent processing mode, working for 5s / pause for 10s, and total effective processing time of 10-20min.

[0015] Preferably, the specific parameters for the three-stage gradient enzymatic hydrolysis in (c) are as follows: First stage: pH 3.5-4.5, temperature 35-40℃, add pepsin, the amount of pepsin added is 1.5%-2.5% of the substrate protein mass, enzymatic hydrolysis for 1.5-2.5 hours; Second stage: pH 6.5-7.5, temperature 38-42℃, add trypsin, the amount of trypsin added is 1.0%-2.0% of the substrate protein mass, enzymatic hydrolysis for 2.0-3.0h; Third stage: pH 8.0-9.0, temperature 48-52℃, add alkaline protease, the amount of alkaline protease added is 0.8%-1.5% of the substrate protein mass, and enzymatic hydrolysis for 1.0-2.0h.

[0016] Preferably, the microfiltration membrane in (e) has a pore size of 0.45 μm, and both the ultrafiltration membrane and the nanofiltration membrane are hydrophilic polyethersulfone membranes or regenerated cellulose membranes. The operating pressure of the membrane separation process is 0.2–0.8 MPa, and the operating temperature is 15–25 °C.

[0017] Preferably, the (g) traditional Chinese medicine and food homology extract includes hawthorn extract, tangerine peel extract, licorice extract and monk fruit extract. Based on 100 parts of the weight of bovine spleen peptide solids in the compound solution, the amount of hawthorn extract added is 3-5 parts, the amount of tangerine peel extract added is 2-4 parts, the amount of licorice extract added is 1-3 parts, and the amount of monk fruit extract added is 0.5-2 parts.

[0018] Preferably, the gradient cooling vacuum freeze-drying in (g) includes: Pre-freezing stage: Cool to -10℃ at a rate of 0.5℃ / min and hold for 1 hour; then cool to -30℃ at a rate of 0.3℃ / min and hold for 2 hours; finally cool to -50℃ at a rate of 0.5℃ / min and hold for 4 hours. One-time drying: plate temperature -20℃~-10℃, vacuum degree 10~30Pa, duration 12~18h; Secondary drying: The temperature of the plate is gradually increased to 25℃~30℃, the vacuum degree is 5~10Pa, and it is maintained for 6~10h.

[0019] Preferably, the biomimetic digestion model in (f) is constructed by the following method: placing bovine spleen peptide samples in simulated gastric juice and simulated intestinal juice for digestion in sequence, measuring the changes in molecular weight distribution and residual activity at different time points, and establishing a quantitative relationship between the digestive stability and absorption characteristics of peptides with different molecular weights.

[0020] On the other hand, the present invention also provides a bovine spleen peptide obtained according to the above-described preparation process, wherein the molecular weight distribution of the bovine spleen peptide includes: Peptides with a molecular weight <1kDa account for 40%–50%, peptides with a molecular weight of 1–3kDa account for 30%–40%, peptides with a molecular weight of 3–10kDa account for 10%–20%, and components with a molecular weight >10kDa account for 0%–5%.

[0021] Preferably, the content of dipeptides to eicoseptides in the bovine spleen peptide accounts for more than 80% of the total peptide mass.

[0022] Thirdly, the present invention also provides an application of the above-mentioned bovine spleen peptide in the preparation of products that improve the function of the intestinal digestive system; the improvement of the function of the intestinal digestive system includes at least one of the following: promoting the secretion and activity of digestive enzymes, regulating the balance of intestinal flora, repairing damage to the gastrointestinal mucosa, and promoting small intestinal peristalsis to improve digestive and absorptive functions.

[0023] The beneficial effects of this invention are: This invention utilizes a synergistic pretreatment process involving pulsed electric field and ultrasound. The pulsed electric field induces irreversible electroporation of the cell membrane, while the cavitation effect of ultrasound further disrupts the cytoskeleton and organelle membranes. The two processes work synergistically under low-temperature conditions, resulting in a cell disruption rate of over 95% and a protein release rate that is more than 40% higher than that of the traditional homogenization method, significantly improving the utilization rate of raw materials.

[0024] This invention utilizes a three-stage gradient enzymatic hydrolysis and dynamic pH control technology, where pepsin, trypsin, and alkaline protease act sequentially under their respective pH conditions, achieving graded and targeted hydrolysis of spleen proteins. Compared to traditional single-stage enzymatic hydrolysis, the proportion of peptides with a molecular weight <3kDa in the product increases from 40%–55% to over 70%, and the enrichment of functional peptides is improved by 35%–50%.

[0025] Based on research results from a biomimetic digestion model, this invention combines peptides of different molecular weight ranges in a specific ratio to form a spatiotemporal synergistic effect chain of "rapid absorption for energy (<1kDa) — promotion of digestive enzyme secretion (1-3kDa) — regulation of intestinal immunity (3-10kDa)". Animal experiments show that the bovine spleen peptides prepared in this invention have a comprehensive effect of more than 50% higher than that of ordinary bovine spleen peptides without graded compounding in promoting digestive enzyme secretion, regulating intestinal flora, and repairing intestinal mucosal damage.

[0026] This invention utilizes extracts of hawthorn, dried tangerine peel, licorice, and monk fruit—all traditional Chinese medicine and food ingredients—to synergistically improve flavor. Through hydrogen bonding and hydrophobic interactions, it effectively masks fishy odors. Simultaneously, these components themselves possess spleen-strengthening and stomach-nourishing effects, which synergize with bovine spleen peptides. The gradient cooling vacuum freeze-drying process avoids mechanical damage to the peptide structure from ice crystals, improving product resolubility by 30% and increasing the retention rate of active peptides by more than 20% compared to conventional freeze-drying.

[0027] The entire process of this invention does not use organic solvents and leaves no harmful residues; the membrane separation technology enables continuous operation and is easy to scale up; the medicinal and edible homologous ingredients are safe and non-toxic, which is in line with the development direction of the big health industry. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments. The described embodiments are some, but not all, of the embodiments of the present invention.

[0029] This invention provides a preparation process for bovine spleen peptides that improve the intestinal digestive system, including the following steps: raw material pretreatment; take fresh spleens from healthy cattle or frozen spleens transported by cold chain, remove fat, fascia and connective tissue, cut into small pieces of 1-2 cm³, rinse repeatedly with 4°C physiological saline until no blood is present, and drain for later use.

[0030] (a) Pulsed electric field-ultrasound co-processing The spleen mass was placed in a pulsed electric field treatment chamber and subjected to pulsed electric field treatment at a low temperature of 4℃. The treatment parameters were: electric field strength 15-25kV / cm, pulse frequency 100-300Hz, pulse width 10-30μs, and treatment time 5-15min.

[0031] After pulsed electric field treatment, the spleen tissue block was transferred to an ultrasonic treatment container, and 1.0–2.0 times the spleen mass of 4°C physiological saline was added for ultrasound-assisted treatment. The treatment parameters were: ultrasonic power 300–500W, frequency 20–40kHz, intermittent treatment mode (5s working / 10s rest), total effective treatment time 10–20 min, and temperature ≤10°C throughout the process.

[0032] (b) Low-temperature high-speed homogenization The spleen tissue after co-processing in step (a) was homogenized at high speed at 4°C for 3-5 minutes at a speed of 10,000-15,000 rpm to obtain a homogenate with a cell disruption rate of ≥95%.

[0033] (c) Three-stage gradient enzymatic hydrolysis and dynamic pH regulation Add protease to the homogenate obtained in step (b) and employ a three-stage gradient enzymatic hydrolysis process, with pH, ​​temperature, and enzyme type independently controlled in each stage, as detailed below: First stage: Acidic protease hydrolysis is performed to adjust the pH of the homogenate to 3.5-4.5 (preferably pH 4.0±0.2), and pepsin is added. The amount of pepsin added is 1.5%-2.5% of the substrate protein in the homogenate. Enzymatic hydrolysis is carried out at a temperature of 35-40℃ (preferably 37℃) for 1.5-2.5 hours.

[0034] Second stage: Neutral protease hydrolysis After the first stage of hydrolysis is completed, adjust the pH of the system to 6.5-7.5 (preferably pH 7.0±0.2) with an alkaline solution (such as 1M NaOH), add trypsin, and add trypsin at a rate of 1.0%-2.0% of the substrate protein mass. Hydrolyze for 2.0-3.0 h at a temperature of 38-42℃ (preferably 40℃).

[0035] Third stage: Alkaline protease hydrolysis After the second stage of hydrolysis is completed, adjust the pH of the system to 8.0-9.0 (preferably pH 8.5±0.2), add alkaline protease, the amount of alkaline protease added is 0.8%-1.5% of the substrate protein mass, and hydrolyze for 1.0-2.0 h at a temperature of 48-52℃ (preferably 50℃).

[0036] During the three-stage enzymatic hydrolysis process described above, an online pH monitoring and automatic feedback liquid addition system was used to control the pH fluctuations at each stage within ±0.2.

[0037] (d) Enzyme inactivation and centrifugation After the three-stage enzymatic hydrolysis is completed, the hydrolysate is heated to 90-95℃ and held for 10-15 minutes to inactivate the enzyme. After enzyme inactivation, it is cooled to room temperature and centrifuged at 3000-5000 rpm for 10-20 minutes, and the supernatant is collected.

[0038] (e) Bionic digestion model-guided molecular weight cascade compounding The supernatant obtained in step (d) is subjected to four-stage membrane separation, as follows: First stage: Microfiltration, using a microfiltration membrane with a pore size of 0.45μm to remove cell debris and insoluble macromolecular impurities to obtain a clear liquid.

[0039] The second stage is ultrafiltration, which uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to separate the microfiltration clarified liquid and collect the permeate; the retentate (molecular weight > 10 kDa) can be recycled for the next batch of enzymatic hydrolysis substrate.

[0040] The third stage is nanofiltration, which uses a nanofiltration membrane with a molecular weight cutoff of 3 kDa to separate the 10 kDa permeate. The cutoff liquid (molecular weight 3-10 kDa) and the permeate (molecular weight <3 kDa) are collected separately.

[0041] Fourth stage: Nanofiltration, using a nanofiltration membrane with a molecular weight cutoff of 1 kDa to separate the 3 kDa permeate, and collect the cutoff liquid (molecular weight 1-3 kDa) and the permeate liquid (molecular weight <1 kDa) separately.

[0042] The operating pressure of the above membrane separation process is 0.2–0.8 MPa, and the operating temperature is 15–25 °C.

[0043] (f) Segmented collection and targeted compounding Based on the intestinal functional characteristics of peptides in different molecular weight ranges determined by the biomimetic digestion model, the different molecular weight peptides collected in step (e) were compounded according to the following mass percentages: Peptides with a molecular weight <1kDa: 40%–50%; Peptides with a molecular weight of 1–3 kDa: 30%–40%; Peptides with molecular weights of 3–10 kDa: 10%–20%.

[0044] The biomimetic digestion model was constructed by the following method: bovine spleen peptide samples were digested sequentially in simulated gastric juice (pH 2.0, containing pepsin) and simulated intestinal juice (pH 7.5, containing pancreatic enzymes and bile salts), and the changes in molecular weight distribution and residual activity were measured at different time points to establish a quantitative relationship between the digestive stability and absorption characteristics of peptides with different molecular weights.

[0045] (g) Flavor synergistic improvement and gradient cooling vacuum freeze drying Add the medicinal and edible extracts to the compound solution obtained in step (f), based on 100 parts by weight of bovine spleen peptide solids in the compound solution: 3-5 parts hawthorn extract, 2-4 parts tangerine peel extract, 1-3 parts licorice extract, and 0.5-2 parts monk fruit extract. After mixing evenly, perform gradient cooling vacuum freeze-drying. Pre-freezing stage: The mixture is cooled to -10℃ at a rate of 0.5℃ / min and kept at this temperature for 1 hour; then cooled to -30℃ at a rate of 0.3℃ / min and kept at this temperature for 2 hours; finally cooled to -50℃ at a rate of 0.5℃ / min and kept at this temperature for 4 hours.

[0046] Primary drying (sublimation drying): The plate temperature is controlled at -20℃ to -10℃, the vacuum degree is controlled at 10 to 30 Pa, and the drying time is 12 to 18 hours.

[0047] Secondary drying (analytical drying): The temperature of the plate is gradually increased to 25℃~30℃, and the vacuum degree is controlled at 5~10Pa for 6~10h.

[0048] After drying, lyophilized bovine spleen peptide powder is obtained.

[0049] Quality Inspection and Packaging: Quality testing is performed on the lyophilized powder, including molecular weight distribution (HPLC-SEC), active peptide content (Lowry method or BCA method), moisture content (Karl Fischer method), and microbial limits. Qualified products are sealed in packaging under nitrogen protection and stored in a dark, low-temperature environment.

[0050] Preferred technical solution: Preferably, in step (a), the electric field strength of the pulsed electric field treatment is 20 kV / cm, the pulse frequency is 200 Hz, the pulse width is 20 μs, and the treatment time is 10 min; the ultrasonic power of the ultrasonic treatment is 400 W, the frequency is 30 kHz, and the total effective treatment time is 15 min.

[0051] Preferably, in step (c), the amount of pepsin added in the first stage is 2.0% of the substrate protein mass, and the enzymatic hydrolysis time is 2.0 h; the amount of trypsin added in the second stage is 1.5% of the substrate protein mass, and the enzymatic hydrolysis time is 2.5 h; and the amount of alkaline protease added in the third stage is 1.0% of the substrate protein mass, and the enzymatic hydrolysis time is 1.5 h.

[0052] Preferably, the microfiltration, ultrafiltration, and nanofiltration membranes mentioned in step (e) are all hydrophilic polyethersulfone membranes or regenerated cellulose membranes.

[0053] Preferably, the mass ratio of the peptides with a molecular weight of <1kDa, 1-3kDa, and 3-10kDa in step (f) is 45:35:20.

[0054] Preferably, the hawthorn extract, tangerine peel extract, licorice extract, and monk fruit extract mentioned in step (g) are all water-extracted freeze-dried powders with a particle size ≥200 mesh.

[0055] Embodiments of the present invention also provide bovine spleen peptides obtained by the preparation process described in any of the above technical solutions.

[0056] Preferably, the molecular weight distribution of the bovine spleen peptide is as follows: peptides with a molecular weight <1kDa account for 40% to 50%, peptides with a molecular weight of 1 to 3kDa account for 30% to 40%, peptides with a molecular weight of 3 to 10kDa account for 10% to 20%, and the proportion of components with a molecular weight >10kDa is less than 5%.

[0057] Preferably, the content of dipeptides to eicoseptides in the bovine spleen peptide accounts for more than 80% of the total peptide mass.

[0058] The present invention also provides the application of the above-mentioned bovine spleen peptide in the preparation of products that improve the function of the intestinal digestive system.

[0059] The improvement of intestinal digestive system function includes at least one of the following: 1. Promotes the secretion and activity of digestive enzymes (such as pepsin, trypsin, and lipase); 2. Regulates the balance of intestinal flora, promotes the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus, and inhibits the growth of pathogenic bacteria; 3. Repairs gastrointestinal mucosal damage and enhances the intestinal mucosal immune barrier function; 4. Promotes intestinal peristalsis and improves digestive and absorptive functions.

[0060] The products include functional foods, health foods, special medical foods, or medicines.

[0061] Example 1 Raw material pretreatment: Take 10 kg of fresh spleen from a healthy cow within 2 hours of slaughter. Remove the fat, fascia, and connective tissue from the spleen surface. Cut the spleen into 1.5 cm³ pieces using surgical scissors. Place the chopped spleen pieces in a stainless steel container and add 4°C pre-cooled physiological saline (0.9% NaCl solution). Rinse repeatedly 3 times, adding 20 L of physiological saline each time. Gently stir and let stand for 1 minute. Discard the supernatant and blood water until the rinsing solution is basically clear and no visible blood streaks remain. After rinsing, place the spleen pieces on a stainless steel sieve to drain the surface water and set aside.

[0062] Pulsed electric field-ultrasound synergistic preprocessing: (1) Pulse electric field processing The drained spleen pieces were evenly placed in a PTFE tray within the pulsed electric field treatment chamber, with a thickness not exceeding 2 cm. The electrode spacing within the treatment chamber was 2 cm. The pulsed electric field generator parameters were set as follows: electric field strength 20 kV / cm, pulse frequency 200 Hz, pulse width 20 μs, and treatment time 10 min. The pulsed electric field treatment was initiated, and the chamber temperature was maintained at 4 ± 1 °C during the process using a circulating cooling system. The pulsed electric field treatment caused irreversible electroporation of the spleen cell membranes, resulting in the initial release of intracellular substances.

[0063] (2) Ultrasonic treatment After pulsed electric field treatment, the spleen masses were transferred to an ultrasonic treatment container (20L volume, equipped with a focused ultrasonic probe, adjustable frequency), and 1.5 times the weight of the spleen masses in 4℃ physiological saline (i.e., 15L) was added. The ultrasonic generator parameters were set as follows: ultrasonic power 400W, frequency 30kHz, intermittent treatment mode (5s operation, 10s pause), with a total effective treatment time of 15min (i.e., actual ultrasonic radiation time 5min, total treatment cycle 15min). During ultrasonic treatment, the container was placed in an ice-water bath, and the temperature of the solution was monitored in real time using a temperature probe to ensure that the temperature remained ≤10℃. The cavitation effect of ultrasound further disrupted the cytoskeleton and organelle membranes, producing a synergistic cell disruption effect with the pulsed electric field.

[0064] Low-temperature high-speed homogenization: The co-processed spleen tissue and fluid were transferred to a low-temperature high-speed homogenizer (equipped with a stainless steel homogenizer blade and a jacketed cooling system), with 4°C cooling water circulated through the jacket. The homogenization speed was set to 12000 rpm, and homogenization was performed for 4 minutes at 4°C. After homogenization, samples were taken, and cell disruption was counted under a microscope using a hemocytometer. The results showed a cell disruption rate of 96.5%. A bovine spleen homogenate with a total volume of 22 L was obtained.

[0065] Three-stage gradient enzymatic hydrolysis and dynamic pH regulation: (1) Preparation before enzymatic hydrolysis The above homogenate was taken and its total protein content was determined (using the Kjeldahl method). The total protein content was found to be 32 g / L, which translates to a total substrate protein content of 704 g. The homogenate was then transferred to a jacketed enzymatic hydrolysis vessel (50 L capacity, equipped with an online pH meter, automatic acid / alkali pump, temperature control, and stirring device), and the stirring was started at a speed of 100 rpm.

[0066] (2) First stage: Acidic protease hydrolysis Adjust the pH of the homogenate to 4.0 ± 0.1 using 1M HCl solution. After the pH stabilizes, add pepsin (enzyme activity ≥ 3000 U / mg), at a rate of 2.0% of the substrate protein content in the homogenate, i.e., 14.1 g of pepsin. Pour constant-temperature water into the jacket of the enzymatic hydrolysis tank to raise the temperature of the solution to 37℃ ± 0.5℃, and maintain this temperature with stirring for 2.0 h. During the enzymatic hydrolysis process, monitor the pH value in real time using an online pH meter. When the pH deviates from 4.0 ± 0.2, the automatic feedback pump adds 1M HCl or 1M NaOH dropwise to adjust the pH, ensuring it remains within the range of 4.0 ± 0.2. Maintain the stirring speed at 100 rpm.

[0067] (3) Second stage: neutral protease digestion After the first stage of enzymatic hydrolysis, the pH of the system was adjusted to 7.0 ± 0.1 using 1M NaOH solution. Once the pH stabilized, trypsin (enzyme activity ≥ 2500 U / mg) was added at a rate of 1.5% of the substrate protein mass, i.e., 10.6 g of trypsin. The jacket water temperature was adjusted to 40℃ ± 0.5℃, and the mixture was kept at this temperature with stirring for 2.5 hours. An online pH feedback control system was used to maintain pH fluctuations within the range of 7.0 ± 0.2.

[0068] (4) Third stage: alkaline protease hydrolysis After the second stage of enzymatic hydrolysis, the pH of the system was adjusted to 8.5 ± 0.1 using 1M NaOH solution. Once the pH stabilized, alkaline protease (enzyme activity ≥ 2000 U / mg) was added at a rate of 1.0% of the substrate protein mass, i.e., 7.0 g of alkaline protease. The jacket water temperature was adjusted to 50℃ ± 0.5℃, and the mixture was kept at this temperature with stirring for 1.5 hours. Online pH feedback control kept pH fluctuations within the range of 8.5 ± 0.2.

[0069] Enzyme inactivation and centrifugation: After the three-stage enzymatic hydrolysis is completed, the automatic pH control system is turned off, and the jacket of the hydrolysis tank is switched to steam heating, rapidly raising the temperature to 90°C (heating rate 3°C / min). Once the feed solution temperature reaches 90°C, it is maintained for 10 minutes for enzyme inactivation. After enzyme inactivation, cooling water is introduced into the jacket to cool the feed solution to room temperature (below 25°C).

[0070] The cooled enzymatic hydrolysate was transferred to a tube centrifuge and centrifuged at 4000 rpm for 15 minutes. The supernatant was collected after centrifugation, and the bottom precipitate was discarded. The total volume of the supernatant was 19 L, and it was a pale yellow, clear liquid.

[0071] Bionic digestion model-guided molecular weight cascade compounding: A four-stage membrane separation system was used to separate the supernatant. All membrane modules were hydrophilic polyethersulfone membranes. The operating temperature was controlled at 20±2℃, and the operating pressure was set according to the membrane type.

[0072] (1) First stage: microfiltration The supernatant was passed through a hollow fiber microfiltration membrane module with a pore size of 0.45 μm at an operating pressure of 0.2 MPa. After the permeate pressure differential stabilized, the microfiltration permeate was collected. The microfiltration retentate (containing cell debris and insoluble macromolecular impurities) was discarded. The volume of the microfiltration permeate was 17 L.

[0073] (2) Second stage: Ultrafiltration (10kDa) The microfiltration permeate is passed through a spiral-wound ultrafiltration membrane module with a molecular weight cutoff of 10 kDa at an operating pressure of 0.5 MPa, and the permeate is collected. The retentate (large proteins with a molecular weight >10 kDa and incompletely hydrolyzed peptides) can be collected and mixed with the next batch of enzymatic hydrolysis substrate for reuse. The volume of the ultrafiltration permeate is 14 L.

[0074] (3) Third stage: Nanofiltration (3kDa) The 10 kDa ultrafiltration permeate was passed through a nanofiltration membrane module with a molecular weight cutoff of 3 kDa at an operating pressure of 0.6 MPa. The retentate (molecular weight 3–10 kDa) and the permeate (molecular weight <3 kDa) were collected separately. The volume of the retentate was 3 L and the volume of the permeate was 11 L.

[0075] (4) Fourth stage: Nanofiltration (1kDa) The 3 kDa nanofiltration permeate was passed through a nanofiltration membrane module with a molecular weight cutoff of 1 kDa, operating at a pressure of 0.6 MPa. The retentate (molecular weight 1–3 kDa) and the permeate (molecular weight < 1 kDa) were collected separately. The volume of the retentate was 4 L, and the volume of the permeate was 7 L.

[0076] Segmented collection and targeted blending: The peptide solutions collected by the above membrane separation were sampled and the peptide concentration was determined (using the Lowry method), and the solid content was calculated.

[0077] The peptides are formulated with a mass percentage of 45% for peptides <1kDa, 35% for peptides between 1 and 3kDa, and 20% for peptides between 3 and 10kDa.

[0078] Synergistic flavor improvement and gradient cooling vacuum freeze-drying: (1) Add medicinal and edible extracts Based on 100 parts of the bovine spleen peptide solids in the compound solution (57.4g of solids in this batch), weigh the following water-extracted freeze-dried powder (particle size ≥200 mesh): Hawthorn extract: 4 parts, tangerine peel extract: 3 parts, licorice extract: 2 parts, and monk fruit extract: 1 part.

[0079] After dispersing the above extract powder evenly with a small amount of purified water (100 mL), add it to the compound solution and stir for 15 min to fully dissolve it and form an intermolecular complex with bovine spleen peptide.

[0080] (2) Gradient cooling vacuum freeze drying Dispense the above mixture into stainless steel freeze-drying trays, each tray filled to a thickness of 1.5 cm, for a total of 6 trays. Place the freeze-drying trays on the plates of a vacuum freeze dryer and freeze-dry according to the following procedure: Pre-freezing stage: The plate temperature was lowered from room temperature to -10℃ at a rate of 0.5℃ / min, and then held at -10℃ for 1 hour. The plate temperature was then lowered from -10℃ to -30℃ at a rate of 0.3℃ / min, and held at -30℃ for 2 hours. Finally, the plate temperature was lowered from -30℃ to -50℃ at a rate of 0.5℃ / min, and then held at -50℃ for 4 hours.

[0081] First drying (sublimation drying): Raise the plate temperature to -20℃ to -10℃ (program set to -15℃), turn on the vacuum pump, and evacuate the drying chamber to a vacuum level of 20Pa. Continue drying under these conditions for 15 hours. Determine the end point of the first drying by the pressure rise method (pressure rise rate <5Pa / min after closing the vacuum valve).

[0082] Secondary drying (analytical drying): After the primary drying, the plate temperature is gradually increased to 25℃ at a rate of 0.2℃ / min, while the vacuum degree is reduced to 8Pa, and drying continues for 8 hours. During the secondary drying process, samples are taken periodically to determine the moisture content until the moisture content is below 3%.

[0083] After freeze-drying, the vacuum is turned off, and dry nitrogen gas filtered through a 0.22μm filter membrane is introduced until atmospheric pressure is reached. The freeze-dried bovine spleen peptide powder is then removed from the chamber. The product is a white to light yellow loose block that crumbles into powder with a gentle tap.

[0084] Example 2 The difference from Example 1 is that the electric field strength in the pulsed electric field-ultrasound co-processing is 15kV / cm and the ultrasonic power is 300W.

[0085] In the three-stage gradient enzymatic hydrolysis and dynamic pH regulation, the first stage of enzymatic hydrolysis takes 1.5 h, the second stage takes 2.0 h, and the third stage takes 1.0 h.

[0086] In the segmented collection and targeted compounding, the peptides were compounded according to the following mass percentages: <1kDa peptides 40%, 1-3kDa peptides 40%, and 3-10kDa peptides 20%.

[0087] In the process of flavor synergistic improvement and gradient cooling vacuum freeze drying, hawthorn extract: 3 parts, tangerine peel extract: 2 parts, licorice extract: 1 part and monk fruit extract: 0.5 parts were weighed.

[0088] Example 3 The difference from Example 1 is that the electric field strength in the pulsed electric field-ultrasound co-processing is 25kV / cm and the ultrasonic power is 500W.

[0089] In the three-stage gradient enzymatic hydrolysis and dynamic pH regulation, the first stage of enzymatic hydrolysis takes 2.5 hours, the second stage takes 3.0 hours, and the third stage takes 2.0 hours.

[0090] In the segmented collection and targeted compounding process, the peptides were compounded according to the following mass percentages: <1kDa peptides 50%, 1-3kDa peptides 30%, and 3-10kDa peptides 20%.

[0091] In the process of flavor synergistic improvement and gradient cooling vacuum freeze drying, hawthorn extract: 5 parts, tangerine peel extract: 4 parts, licorice extract: 3 parts and monk fruit extract: 2 parts were weighed.

[0092] Example 4 The difference from Example 1 is that the homogenization speed is 10,000 rpm and the homogenization time is 5 min; the membrane separation operation pressure is finely adjusted as follows: microfiltration is 0.25 MPa, 10 kDa ultrafiltration is 0.55 MPa, 3 kDa nanofiltration is 0.65 MPa and 1 kDa nanofiltration is 0.55 MPa.

[0093] Example 5 The difference from Example 1 is that the homogenization speed is 15000 rpm and the homogenization time is 3 min; the membrane separation operation pressure is finely adjusted as follows: microfiltration is 0.18 MPa, 10 kDa ultrafiltration is 0.45 MPa, 3 kDa nanofiltration is 0.50 MPa and 1 kDa nanofiltration is 0.55 MPa.

[0094] Example 6 The difference from Example 1 is that the pH of the three-stage gradient enzymatic hydrolysis is set to the minimum boundary value: the pH of the first stage is 3.5, the pH of the second stage is 6.5, and the pH of the third stage is 8.0.

[0095] Example 7 The difference from Example 1 is that the pH of the three-stage gradient enzymatic hydrolysis is set to the minimum value of the boundary: the pH of the first stage is 4.5, the pH of the second stage is 7.5, and the pH of the third stage is 9.0.

[0096] Example 8 The difference from Example 1 is that the ratio of segmented collection and targeted compounding is: 40% for <1kDa peptides, 40% for 1-3kDa peptides and 20% for 3-10kDa peptides.

[0097] Example 9 The difference from Example 1 is that the ratio of segmented collection and targeted compounding is: 50% for <1kDa peptides, 30% for 1-3kDa peptides and 20% for 3-10kDa peptides.

[0098] Example 10 The difference from Example 1 is that the minimum values ​​of the amounts of the traditional Chinese medicine and food homology extracts added to improve the flavor synergistically (based on 100 parts of bovine spleen peptide solids) are: hawthorn extract is changed from 4 parts to 3 parts, tangerine peel extract is changed from 3 parts to 2 parts, licorice extract is changed from 2 parts to 1 part, and monk fruit extract is changed from 1 part to 0.5 parts.

[0099] Comparative Example 1 After cutting and homogenizing the beef spleen, a combination of subtilisin, bromelain, and papain (1.5% each) was added. The mixture was subjected to single-stage enzymatic hydrolysis for 6 hours at pH 7.0 and 50°C. After enzyme inactivation and centrifugation, the mixture was filtered through a 10 kDa ultrafiltration membrane, and the permeate was collected and freeze-dried using conventional methods.

[0100] Comparative Example 2 The difference from Example 1 is that the pulsed electric field-ultrasound co-processing pretreatment is omitted, and only low temperature high speed homogenization (4℃, 12000rpm, 5min) is used.

[0101] Comparative Example 3 The difference from Example 1 is that, instead of segmented recombining after cascade separation, the 10kDa ultrafiltration permeate is directly freeze-dried.

[0102] To verify the effect of the bovine spleen peptide of the present invention on improving the intestinal digestive system, the following unified method was used to test the samples of Examples 1-10 and Comparative Examples 1-3.

[0103] 3.1 Laboratory Animals and Grouping Animals: SPF-grade Kunming mice, male, weighing 18-22g, a total of 140 mice (10 example groups × 10 mice + 3 comparative groups × 10 mice + blank control group × 10 mice).

[0104] Blank control group: Gavaged with an equal volume of physiological saline.

[0105] Each treatment group was administered the corresponding bovine spleen peptide sample by gavage at a dose of 200 mg / kg body weight (based on peptide powder) once daily for 14 consecutive days.

[0106] 3.2 Detection Indicators and Methods (1) Pepsin activity Mice were fasted for 24 hours after the last administration, then sacrificed. Gastric mucosa homogenate was collected, and pepsin activity (unit: U / mg protein) was determined using the Anson method.

[0107] (2) Trypsin activity Pancreatic tissue homogenate was collected, and trypsin activity (unit: U / mg protein) was determined using the BAEE method.

[0108] (3) Intestinal flora analysis Cecal contents were collected and selectively cultured for counting: Bifidobacteria (BL medium), Lactobacillus (LBS medium), and Escherichia coli (EMB medium). Results are expressed as log10 (CFU / g).

[0109] (4) Small intestinal villus height / crypt depth ratio Duodenal tissue was harvested, stained with hematoxylin and eosin (HE), and the villus height and crypt depth were measured. The ratio (V / C) was calculated. This value reflects the intestinal mucosal absorption area and repair status.

[0110] The obtained data is shown in Table 1 below: Table 1

[0111] Table 1 shows that the pepsin activity in the blank control group was 32.4 U / mg, and the trypsin activity was 45.2 U / mg. In Examples 1-10, the pepsin activity increased to 64.3-68.7 U / mg (approximately 2.0-2.1 times that of the blank), and the trypsin activity increased to 85.2-89.5 U / mg (approximately 1.9-2.0 times that of the blank). Comparative Example 1 (traditional single-enzyme digestion method) only increased to 40.5 and 56.3 U / mg, respectively, significantly lower than the examples (p<0.01). Comparative Example 2 (no pulsed electric field-ultrasound pretreatment) showed 51.2 and 68.4 U / mg, and Comparative Example 3 (no biomimetic compound) showed 55.6 and 72.1 U / mg, all lower than the examples. This demonstrates that the three-stage gradient enzymatic digestion, physical field pretreatment, and targeted compounding of the present invention have a synergistic effect in improving digestive enzyme activity.

[0112] In Example 1, the levels of Bifidobacteria and Lactobacillus reached 9.45 and 9.82 logCFU / g, respectively, significantly higher than the blank control group (7.82, 8.05), while the level of harmful Escherichia coli decreased from 8.44 to 6.71 logCFU / g. In Comparative Example 1, the increase in beneficial bacteria was not significant (8.15, 8.42), and Escherichia coli only slightly decreased to 8.15. The effects of Comparative Examples 2 and 3 were between those of Comparative Example 1 and Example 1. This indicates that the product of the present invention can more effectively promote the proliferation of beneficial bacteria, inhibit pathogenic bacteria, and improve the intestinal microecology.

[0113] The villus volume (V / C) ratio of the small intestine reflects the intestinal absorptive surface area and the health status of the mucosa. The control group had a V / C ratio of 1.52, indicating some mucosal damage (normal mice have a V / C ratio of approximately 2.0–2.5). The ratio in Example 1 increased to 2.68, significantly higher than the control group and Comparative Example 1 (1.78), Comparative Example 2 was 2.05, and Comparative Example 3 was 2.18. This indicates that the product of this invention can effectively repair intestinal mucosal damage, which may be related to the rapid absorption and energy supply of the <1kDa peptide segment and the immunomodulatory effect of the 3–10kDa peptide segment.

[0114] Example 1 (optimal parameter combination) is slightly better than other examples in all indicators, but there is no statistically significant difference between examples (p>0.05), indicating that the parameter adjustments within the scope of the claims of this invention can achieve significantly better results than the prior art and have a robust process window.

[0115] Comparative Example 1 (traditional process) had the worst performance across all indicators, only slightly better than the blank control, indicating that traditional single-stage enzymatic hydrolysis and simple ultrafiltration cannot effectively release active peptides.

[0116] Comparative Example 2 (without physical field pretreatment) showed significantly better results than Comparative Example 1 but worse results than the Example 1, demonstrating that pulsed electric field-ultrasound synergistic pretreatment can greatly improve the release rate of active substances.

[0117] Comparative Example 3 (without biomimetic compound) showed better results than Comparative Example 2 but worse results than the Example 1, demonstrating that function-oriented compounding can produce synergistic effects.

[0118] In summary, the embodiments of the present invention are significantly superior to the comparative examples in promoting digestive enzyme secretion, regulating intestinal flora, and repairing intestinal mucosal damage, and the effects are stable, demonstrating the inventiveness, practicality, and industrial value of the present invention.

[0119] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A preparation process for bovine spleen peptides that improve the intestinal digestive system, characterized in that, Includes the following steps: (a) Pulsed electric field-ultrasound combined pretreatment: Bovine spleen tissue was subjected to pulsed electric field treatment and ultrasound treatment in sequence to obtain pretreated spleen tissue; (b) Low-temperature high-speed homogenization: The pretreated spleen tissue was homogenized to obtain a homogenate with a cell disruption rate of ≥95%; (c) Three-stage gradient enzymatic hydrolysis and dynamic pH control: The homogenate was subjected to first-stage acidic protease hydrolysis, second-stage neutral protease hydrolysis and third-stage alkaline protease hydrolysis in sequence, and the pH fluctuation in each stage was controlled within ±0.

2. (d) Enzyme inactivation and centrifugation: After heating the enzyme hydrolysate to inactivate the enzyme, centrifuge and collect the supernatant; (e) Bionic digestion model-guided molecular weight cascade complexation: The supernatant was sequentially passed through a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane with a molecular weight cutoff of 3 kDa, and a nanofiltration membrane with a molecular weight cutoff of 1 kDa for four-stage separation, and peptides of different molecular weights were collected respectively. (f) Segmented collection and targeted compounding: The peptides of different molecular weights collected in step (e) are compounded to obtain a compound solution; (g) Flavor synergistic improvement and gradient cooling vacuum freeze drying: After adding the medicinal and edible extract to the compound solution, gradient cooling vacuum freeze drying was performed.

2. The preparation process of bovine spleen peptide for improving the intestinal digestive system according to claim 1, characterized in that, The parameters for pulsed electric field processing in (a) are: electric field strength 15-25 kV / cm, pulse frequency 100-300 Hz, pulse width 10-30 μs, and processing time 5-15 min. The parameters for ultrasonic processing are: ultrasonic power 300-500W, frequency 20-40kHz, intermittent processing mode, working for 5s / pause for 10s, and total effective processing time of 10-20min.

3. The preparation process of bovine spleen peptide for improving the intestinal digestive system according to claim 1, characterized in that, The specific parameters for the three-stage gradient enzymatic hydrolysis in (c) are as follows: First stage: pH 3.5-4.5, temperature 35-40℃, add pepsin, the amount of pepsin added is 1.5%-2.5% of the substrate protein mass, enzymatic hydrolysis for 1.5-2.5 hours; Second stage: pH 6.5-7.5, temperature 38-42℃, add trypsin, the amount of trypsin added is 1.0%-2.0% of the substrate protein mass, enzymatic hydrolysis for 2.0-3.0h; Third stage: pH 8.0-9.0, temperature 48-52℃, add alkaline protease, the amount of alkaline protease added is 0.8%-1.5% of the substrate protein mass, and enzymatic hydrolysis for 1.0-2.0h.

4. The preparation process of bovine spleen peptide for improving the intestinal digestive system according to claim 1, characterized in that, The microfiltration membrane in (e) has a pore size of 0.45 μm, and both the ultrafiltration membrane and nanofiltration membrane are hydrophilic polyethersulfone membranes or regenerated cellulose membranes. The operating pressure of the membrane separation process is 0.2–0.8 MPa, and the operating temperature is 15–25 °C.

5. The preparation process of bovine spleen peptide for improving the intestinal digestive system according to claim 1, characterized in that, The (g) Chinese medicinal and edible extracts include hawthorn extract, tangerine peel extract, licorice extract and monk fruit extract. Based on 100 parts of the weight of bovine spleen peptide solids in the compound solution, the amount of hawthorn extract added is 3-5 parts, the amount of tangerine peel extract added is 2-4 parts, the amount of licorice extract added is 1-3 parts, and the amount of monk fruit extract added is 0.5-2 parts.

6. The preparation process of bovine spleen peptide for improving the intestinal digestive system according to claim 5, characterized in that, The gradient cooling vacuum freeze-drying in (g) includes: Pre-freezing stage: Cool to -10℃ at a rate of 0.5℃ / min and hold for 1 hour; then cool to -30℃ at a rate of 0.3℃ / min and hold for 2 hours; finally cool to -50℃ at a rate of 0.5℃ / min and hold for 4 hours. One-time drying: plate temperature -20℃~-10℃, vacuum degree 10~30Pa, duration 12~18h; Secondary drying: The temperature of the plate is gradually increased to 25℃~30℃, the vacuum degree is 5~10Pa, and it is maintained for 6~10h.

7. The preparation process of bovine spleen peptide for improving the intestinal digestive system according to claim 5, characterized in that, The biomimetic digestion model in (f) is constructed by the following method: bovine spleen peptide samples are digested in simulated gastric juice and simulated intestinal juice in sequence, the changes in molecular weight distribution and residual activity at different time points are measured, and a quantitative relationship between the digestive stability and absorption characteristics of peptides with different molecular weights is established.

8. A bovine spleen peptide obtained by the preparation process according to any one of claims 1-7, characterized in that, The molecular weight distribution of the bovine spleen peptide includes: Peptides with a molecular weight <1kDa account for 40%–50%, peptides with a molecular weight of 1–3kDa account for 30%–40%, peptides with a molecular weight of 3–10kDa account for 10%–20%, and components with a molecular weight >10kDa account for 0%–5%.

9. The bovine spleen peptide according to claim 8, characterized in that, The bovine spleen peptide contains dipeptides to eicoseptides accounting for more than 80% of the total peptide mass.

10. An application of bovine spleen peptide according to any one of claims 8 or 9, characterized in that, The application of the bovine spleen peptide in the preparation of products that improve the function of the intestinal digestive system; the improvement of the function of the intestinal digestive system includes at least one of the following: promoting the secretion and activity of digestive enzymes, regulating the balance of intestinal flora, repairing damage to the gastrointestinal mucosa, and promoting small intestinal peristalsis to improve digestive and absorptive functions.

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