Preparation method of bovine placenta peptide, product and application thereof

Abstract

The application discloses a preparation method of bovine placenta peptide and application thereof, and belongs to the technical field of biological products. The bovine placenta peptide is prepared from the placenta of a Galla Sahiwal cow, and the specific preparation steps comprise raw material pretreatment, complex enzymolysis, enzyme inactivation and separation, desalting and decoloration, concentration and drying. The complex enzymolysis adopts trypsin and neutral protease, and the adding ratio of the trypsin to the neutral protease is 3:2. More than 90% of the peptide segments in the obtained bovine placenta peptide have molecular weights distributed in the range of 500-1500 Da, the protein content is greater than 90%, and the free amino acid content is less than 3%. The bovine placenta peptide has a narrow peptide molecular weight distribution range, strong antioxidant and immunoregulatory activities, and health care functions of preventing and assisting in treating lung nodules and premature ovarian failure.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a method for preparing bovine placental peptides, as well as the products and applications thereof. Background Technology

[0002] Bovine placenta is rich in protein, amino acids, vitamins, minerals, and various bioactive components. Among them, placental peptides are a class of small molecule peptides with multiple biological functions, including immune regulation, anti-oxidation, anti-fatigue, and promotion of cell repair and regeneration. They have broad application prospects in the fields of medicine, health food, and cosmetics.

[0003] Currently, traditional methods for extracting bovine placental peptides mostly employ single enzymatic hydrolysis or acid hydrolysis, which have problems such as wide peptide molecular weight distribution, low activity, complex processes, high costs, and easy environmental pollution.

[0004] Based on this, this application creates a new method for preparing bovine placental peptides, as well as their products and applications, by screening different varieties of placental raw materials and improving the extraction method. This method enables the more efficient acquisition of high-purity bovine placental peptides with a narrow molecular weight distribution, stronger activity, and wider applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing bovine placental peptides, which enables the simple and convenient acquisition of high-purity bovine placental peptides with a narrow molecular weight distribution range, stronger activity, and wider application, thereby overcoming the shortcomings of existing placental peptides.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for preparing bovine placental peptides, wherein the bovine placental peptides are prepared from the placenta of Simmental cattle of the Canadian breed, and the specific preparation method is as follows:

[0007] (1) Raw material pretreatment: Select fresh placenta within 2 hours after calving from female Simmental cattle of the Canadian breed, remove fetal membranes, blood vessels and connective tissue, take placental leaf, remove residual blood, dust and other dirt, wash with 0.85% physiological saline until there is no blood, chop and repeatedly freeze and thaw to obtain placental homogenate;

[0008] (2) Compound enzymatic hydrolysis: Add deionized water to placental homogenate, adjust pH to 7.8-8.2 with NaOH solution, add trypsin and neutral protease, and enzymatically hydrolyze in a constant temperature water bath at 50℃ for 3-5 hours to obtain enzymatic hydrolysate; wherein, the ratio of trypsin to neutral protease is 3:2.

[0009] (3) Enzyme inactivation and separation: The enzyme hydrolysate was inactivated in a 90℃ water bath for 15 min, cooled to room temperature, and then centrifuged at 8000 r / min at 4℃ for 20 min. The supernatant was then collected.

[0010] (4) Desalting and decolorizing: The supernatant was separated by an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was desalted by a 732 type cation exchange resin and then decolorized by a D101 macroporous adsorption resin. The purified solution was collected.

[0011] (5) Concentration and drying: The purified solution is concentrated under vacuum to a solid content of 25%, sterilized by UHT instantaneous sterilization, and spray dried to obtain the bovine placental peptide powder.

[0012] Furthermore, fresh placentas were obtained from Canadian Simmental female cattle, with breed source numbers including 22115033, 22117059, 22118035, 22119039, 22121191, and 22120137.

[0013] Furthermore, in step (1), the freeze-thaw process is repeated 3 times, and the conditions for each freeze-thaw process are: freezing at -20℃ for 6 hours and thawing in an ice-water bath for 3 hours.

[0014] Furthermore, the specific conditions for the compound enzymatic hydrolysis in step (2) are as follows: add 5 times the amount of deionized water to the placental homogenate, adjust the pH to 7.8~8.2 with 1mol / L NaOH, add trypsin and neutral protease, and enzymatically hydrolyze in a constant temperature water bath at 50℃ for 4h to obtain the enzymatic hydrolysate; wherein, the amount of trypsin added is 1.2% w / w and the amount of neutral protease added is 0.8% w / w.

[0015] Furthermore, in step (4), when the permeate is desalted using a 732 type cation exchange resin column, the flow rate of the permeate onto the column is 1.5 BV / h, and when it is decolorized using a D101 macroporous adsorption resin column, the flow rate of the permeate onto the column is 2 BV / h; in step (5), the purified solution is concentrated under vacuum conditions of 60℃ and -0.08 MPa; during spray drying, the inlet air temperature is 130℃ and the outlet air temperature is 80℃.

[0016] As a further improvement of the present invention, the present invention also provides a bovine placental peptide, which is prepared by the above-described method for preparing bovine placental peptide.

[0017] Furthermore, in the bovine placental peptides, more than 90% of the peptides have a molecular weight distribution between 500 and 1500 Da, and the protein content is > 90%, while the free amino acid content is < 3%.

[0018] Furthermore, the bovine placental peptides prepared by this invention have health benefits in preventing and assisting in the treatment of pulmonary nodules.

[0019] Furthermore, the bovine placental peptides prepared by this invention have health benefits in preventing and assisting in the treatment of premature ovarian failure.

[0020] Furthermore, the bovine placental peptides prepared by this invention can be applied to more health foods with antioxidant and immunomodulatory activities.

[0021] With this design, the present invention has at least the following advantages:

[0022] 1. Raw material specificity: Fresh placenta from Canadian Simmental cattle is selected, which has a protein content that is nearly 30% higher than that of local yellow cattle. This high protein content lays the foundation for the preparation of highly active peptides.

[0023] 2. Process optimization: By screening the number of repeated freeze-thaw cycles and studying the enzyme combination, enzyme ratio, hydrolysis time, and enzyme dosage of compound enzymatic hydrolysis, a preparation method that can significantly improve the degree of protein hydrolysis and peptide yield was derived. Then, by using ultrafiltration and resin in combination to achieve an integrated desalting and decolorization process, the resulting bovine placental peptide product has high purity, protein content greater than 90%, free amino acid content less than 3%, concentrated peptide molecular weight, and significantly improved activity.

[0024] 3. Outstanding activity: The obtained bovine placental peptide products have peptide molecular weights concentrated in the range of 500~1500 Da, which are easily absorbed. Their antioxidant and immunomodulatory activities are superior to existing products, and they have health care functions of preventing and adjuvant treatment of pulmonary nodules and premature ovarian failure. Attached Figure Description

[0025] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Figure 1 This is a schematic diagram showing the relationship between the number of freeze-thaw cycles and the degree of protein hydrolysis in the embodiments of the bovine placental peptide preparation method of the present invention.

[0027] Figure 2 This is a schematic diagram illustrating the influence of different combinations of complex enzymes on the degree of protein hydrolysis and peptide yield in the embodiments of the bovine placental peptide preparation method of the present invention.

[0028] Figure 3 This is a schematic diagram showing the effect of different ratios of compound enzymes on the degree of protein hydrolysis and peptide yield in the embodiments of the bovine placental peptide preparation method of the present invention.

[0029] Figure 4 This is a schematic diagram illustrating the effect of different amounts of the compound enzyme added on the degree of protein hydrolysis and peptide yield in the embodiments of the bovine placental peptide preparation method of the present invention.

[0030] Figure 5 This is a schematic diagram illustrating the effect of different enzymatic hydrolysis times on the degree of protein hydrolysis and peptide yield in the embodiments of the bovine placental peptide preparation method of the present invention.

[0031] Figure 6This is a schematic diagram of the molecular weight distribution of small molecule peptides in the detection of the physicochemical properties of bovine placental peptides in this invention.

[0032] Figure 7 This is a schematic diagram showing the results of DPPH free radical scavenging rate in the antioxidant activity test of bovine placental peptides in this invention.

[0033] Figure 8 This is a schematic diagram of the ABTS+ free radical scavenging rate results in the test of the antioxidant activity of bovine placental peptides in this invention. Detailed Implementation

[0034] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that a more thorough understanding of the invention will be achieved and that the full scope of the invention will be conveyed to those skilled in the art.

[0035] Example 1: Screening of bovine placenta raw materials

[0036] Ten fresh placentas from local yellow cattle and ten from Canadian Simmental cattle (both within 2 hours postpartum) were selected and processed separately: fetal membranes, blood vessels, and connective tissue were removed; the placentas were washed with 0.85% physiological saline until no blood was found; they were steamed for 40 minutes, dried at 60℃, pulverized, passed through an 80-mesh sieve, and weighed. The protein content of the two types of placentas was then determined using the Kjeldahl method. The results are shown in Table 1 below.

[0037] Table 1 Comparison of weight and protein content of the two types of placentas

[0038]

[0039] Table 1 shows that the average weight of a single Simmental cattle placenta is nearly 30% higher than that of a native yellow cattle placenta. While the protein content of placentas from native yellow cattle and Simmental cattle is relatively similar, the significant difference in placental weight results in a substantially higher protein content per placenta from Simmental cattle compared to native yellow cattle, increasing by nearly 30%. Therefore, this application uses Simmental cattle placentas to extract bovine placental peptides.

[0040] Example 2: Screening of bovine placental peptide extraction methods

[0041] (a) Number of freeze-thaw cycles of bovine placenta

[0042] Select fresh Simmental cattle placentas within 2 hours postpartum, remove the fetal membranes, blood vessels and connective tissue, take the cotyledons of the placenta, remove residual blood, dust and other dirt, wash with 0.85% saline until no blood is left, chop and divide into 5 portions for later use.

[0043] Five samples were treated under different freeze-thaw conditions: freezing at -20℃ for 6 hours and thawing in an ice-water bath for 3 hours. The samples were treated with no freeze-thaw, freeze-thaw cycles of 1, 2, 3, and 4 times to obtain placental homogenates under different treatment conditions.

[0044] Deionized water (material-to-liquid ratio 1:5, w / v) was added to the placental homogenate. The pH was adjusted to 7.8-8.2 with 1 mol / L NaOH. Trypsin (1.2%, w / w) and neutral protease (0.8%, w / w) were added, and the mixture was enzymatically hydrolyzed in a 50℃ water bath for 4 h. Then, the enzyme was inactivated in a 90℃ water bath for 15 min. After cooling to room temperature, the mixture was centrifuged at 8000 r / min for 20 min at 4℃. The supernatant was collected, and the degree of protein hydrolysis was determined.

[0045] The degree of protein hydrolysis was determined using the OPA (ortho-phthalaldehyde) method with serine as a standard. The degree of protein hydrolysis (DH) was calculated using the following formula (1):

[0046] (1)

[0047] Where h is the number of millimoles of free amine groups in the sample (mmol / g protein), h tot This is the total number of peptide bonds in the raw protein (usually taken as 8 mmol / g protein, based on an average amino acid molecular weight of 125 g / mol).

[0048] The results are attached. Figure 1 Appendix Figure 1 The effects of different freeze-thaw cycles on the hydrolysis of placental proteins in Canadian Simmental cattle are shown.

[0049] from Figure 1 It can be seen that the number of freeze-thaw cycles has a significant impact on protein hydrolysis in bovine placenta, indicating that repeated freeze-thaw cycles are beneficial for protein hydrolysis. However, at 3 and 4 freeze-thaw cycles, the degree of protein hydrolysis was 84.9% and 83.2%, respectively, with the effects becoming similar. The results show that the degree of protein hydrolysis reached its maximum after 3 freeze-thaw cycles of bovine placenta. Therefore, 3 freeze-thaw cycles were chosen as the optimal number of freeze-thaw cycles for bovine placenta peptide extraction.

[0050] (II) Enzyme screening

[0051] Fresh placentas from Canadian Simmental cattle within 2 hours postpartum were selected. The fetal membranes, blood vessels, and connective tissue were removed. The cotyledons of the placenta were removed, and residual blood, dust, and other contaminants were removed. The placenta was washed with 0.85% saline solution until no blood was found. After being chopped, the placenta was subjected to repeated freeze-thaw cycles (freezing at -20℃ for 6 hours and thawing in an ice-water bath for 3 hours) three times to obtain a placental homogenate, which was then divided into 6 portions for later use.

[0052] Deionized water (solid-to-liquid ratio 1:5, w / v) was added to each of the six placental homogenates. The pH was adjusted to 7.8–8.2 with 1 mol / L NaOH. Then, 2.0% of different enzyme preparations (papain, bromelain, trypsin, animal protein hydrolysis complex enzyme, neutral protease, and alkaline protease) were added to each. The mixture was incubated in a 50°C water bath for 4 h. The enzymes in the hydrolysate were then inactivated in a 90°C water bath for 15 min. After cooling to room temperature, the mixture was centrifuged at 8000 rpm for 20 min at 4°C, and the supernatant was collected. The degree of protein hydrolysis in each supernatant was determined using the OPA (o-phthalaldehyde) method.

[0053] Meanwhile, the obtained supernatant was separated by an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was desalted using a 732 type cation exchange resin (flow rate 1.5 BV / h) and then decolorized using a D101 macroporous adsorption resin (flow rate 2 BV / h). The purified solution was collected. The nitrogen content in the purified solution was measured using the Kjeldahl nitrogen analyzer. The peptide yield PY was calculated according to the following formula (2):

[0054] (2)

[0055] Where N is the total nitrogen content of the purified solution and W is the sample weight.

[0056] The results are shown in Table 2 below.

[0057] Table 2. Results of protein hydrolysis degree and peptide yield obtained from different enzyme preparations.

[0058]

[0059] As shown in Table 2, neutral protease yielded the highest degree of protein hydrolysis and peptide yield when used as the enzyme preparation. Trypsin had a slightly lower degree of hydrolysis, but its peptide yield was second only to neutral protease. Papain had the lowest degree of hydrolysis and peptide yield. Bromelain had both low degree of hydrolysis and peptide yield, while alkaline protease had a higher degree of hydrolysis but a lower peptide yield. It should be noted that although the animal protein hydrolysis complex enzyme had a high degree of hydrolysis and peptide yield, its hydrolysate had a strong fishy odor. Therefore, papain, bromelain, and the animal protein hydrolysis complex enzyme were excluded, and the complex enzyme combination was selected from trypsin, neutral protease, and alkaline protease.

[0060] (III) Screening of compound enzyme preparations

[0061] Repeat the enzyme screening steps in (II) above, except that a complex enzyme is selected, such as a complex enzyme composed of trypsin + neutral protease, an alkaline protease + neutral protease, or a trypsin + alkaline protease. The degree of protein hydrolysis and peptide yield obtained from different complex enzyme preparations are also measured. All other conditions and detection methods are the same to determine the effect of the complex enzyme preparation on the extraction method. The results are shown in the appendix. Figure 2 .

[0062] Appendix Figure 2 The effects of different complex enzyme preparations on the degree of protein hydrolysis and peptide yield are shown. From Figure 2 The results showed that the complex enzyme combination of trypsin and neutral protease yielded the highest peptide yield, reaching 13.3%, with a degree of hydrolysis of 38.4%. The complex enzyme combination of alkaline protease and neutral protease had the highest degree of hydrolysis (39.5%), but the lowest peptide yield. The complex enzyme combination of trypsin and alkaline protease exhibited both low degree of hydrolysis and low peptide yield. These results indicate that the complex enzyme combination of trypsin and neutral protease is the optimal enzyme combination and can be used in the extraction of bovine placental peptides.

[0063] (iv) Enzyme ratio screening

[0064] Repeat the enzyme screening steps in (II) above, except that a complex enzyme combination of trypsin and neutral protease is selected, and the proportions of trypsin and neutral protease added are shown in Table 3 below. All other conditions and detection methods are the same to determine the effect of the ratio of the two enzymes in the complex enzyme preparation on the extraction method. The results are attached. Figure 3 .

[0065] Table 3. Proportion of trypsin and neutral protease added to the complex enzyme

[0066]

[0067] Appendix Figure 3 The effects of different ratios of compound enzyme preparations on protein hydrolysis degree and peptide yield are shown. From Figure 3 It can be seen that different ratios of trypsin and neutral protease have a significant impact on the degree of protein hydrolysis and peptide yield. For example, when the ratio of trypsin to neutral protease is 1.2% and the ratio is 0.8%, i.e., the ratio of trypsin to neutral protease is 3:2, the degree of protein hydrolysis and peptide yield are the highest, which is most suitable for the extraction of placental peptides from Simmental cattle.

[0068] (v) Screening of enzyme addition amount

[0069] The enzyme screening steps in section (II) were repeated, except that a 3:2 ratio of trypsin and neutral protease was selected as the enzyme, and the amounts of the enzyme added were 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% by weight, respectively. All other conditions and detection methods remained the same to determine the effect of enzyme dosage on the extraction method. The results are attached. Figure 4 .

[0070] Appendix Figure 4 The effect of different addition amounts of the compound enzyme preparation on the degree of protein hydrolysis and peptide yield is shown. From Figure 4 It can be seen that when the amount of compound enzyme preparation added is 2.0%, the degree of protein hydrolysis and peptide yield are the highest.

[0071] (vi) Screening of enzymatic hydrolysis time

[0072] The enzyme screening steps in section (II) were repeated, except that a 3:2 complex enzyme combination of trypsin and neutral protease was selected. Experiments were conducted with hydrolysis times of 1, 2, 3, 4, and 5 hours. All other conditions and detection methods remained the same to determine the effect of hydrolysis time on the extraction method. Results are attached. Figure 5 .

[0073] Appendix Figure 5 The effect of the compound enzyme preparation on the degree of protein hydrolysis and peptide yield under different enzymatic hydrolysis time conditions is shown. From Figure 5 It can be seen that the degree of protein hydrolysis and peptide yield are highest when the enzymatic hydrolysis time of the compound enzyme preparation is 4 hours.

[0074] Based on the experimental results, the optimal enzyme preparation for extracting placental peptides from Simmental cattle is a complex enzyme combination of trypsin and neutral protease, with a trypsin to neutral protease ratio of 3:2, an added amount of 2.0%, an enzymatic hydrolysis temperature of 50℃, and an enzymatic hydrolysis time of 4 hours.

[0075] Example 3: Detection of the physicochemical properties of bovine placental peptides

[0076] Preparation of bovine placental peptide powder

[0077] Fresh placentas from Canadian Simmental cattle within 2 hours postpartum were selected. The breed numbers included, but were not limited to, 22115033, 22117059, 22118035, 22119039, 22121191, and 22120137. The fetal membranes, blood vessels, and connective tissue were removed, and the cotyledons of the placenta were taken. Residual blood, dust, and other contaminants were removed, and the placenta was washed with 0.85% saline until no blood was found. After being chopped, the placenta was subjected to repeated freeze-thaw treatment (freezing at -20℃ for 6 hours and thawing in an ice-water bath for 3 hours) three times to obtain a placental homogenate.

[0078] Add deionized water (material-to-liquid ratio 1:5, w / v) to placental homogenate, adjust pH to 7.8-8.2 with 1 mol / L NaOH, add trypsin (1.2% w / w) and neutral protease (0.8% w / w) sequentially, and enzymatically hydrolyze in a 50℃ constant temperature water bath for 4 h; then inactivate the enzyme in a 90℃ water bath for 15 min, cool to room temperature, centrifuge at 8000 r / min at 4℃ for 20 min, and collect the supernatant.

[0079] The obtained supernatant was separated by an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was desalted using a 732 type cation exchange resin (flow rate 1.5 BV / h) and then decolorized using a D101 macroporous adsorption resin (flow rate 2 BV / h). The purified solution was collected.

[0080] The purified solution was concentrated under vacuum (temperature 60℃, vacuum degree -0.08MPa) to a solid content of 25%, then sterilized by UHT and spray-dried (inlet air temperature 130℃, outlet air temperature 80℃) to obtain bovine placental peptide powder.

[0081] Physicochemical property detection of bovine placental peptides

[0082] The molecular weight distribution and composition of the bovine placental peptides obtained above were detected.

[0083] Molecular weight distribution determination: High-performance gel filtration chromatography (HPLC) was used. The chromatographic column was a Thermo Scientific BioBasic SEC 120 (7.8 mm × 300 mm, 5 μm). The mobile phase was acetonitrile / water / trifluoroacetic acid (40 / 60 / 0.05, v / v / v), the flow rate was 0.5 mL / min, and the detector wavelength was 220 nm. The standards were bacitracin and acetaminophen-acetaminophen-tyrosine-arginine, with molecular weights of 1422.7 Da and 451.5 Da, respectively. Results are attached. Figure 6 .

[0084] from Figure 6 It can be concluded that more than 90% of the peptides obtained by this extraction method have molecular weights between 500 and 1500 Da, and the molecular weight distribution is narrow.

[0085] Composition analysis: The protein content of bovine placental peptide powder, determined by the Kjeldahl method, was 92.3%, the free amino acid content was 2.1%, and the heavy metal (Pb, As, Hg) content was all <0.1 mg / kg.

[0086] Example 4: Antioxidant Activity Test

[0087] Following the preparation method of bovine placental peptide powder in Example 3, placental peptide powder from Canadian Simmental cattle and placental peptide powder from native yellow cattle were prepared. The obtained Canadian Simmental cattle placental peptide powder and native yellow cattle placental peptide powder were respectively prepared into solutions with concentrations of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, and 6 mg / mL, which were used as test solutions for DPPH free radical scavenging rate and ABTS+ free radical scavenging ability detection.

[0088] DPPH free radical scavenging rate: Take the above test solution and mix it with 1 mL of distilled water and 3 mL of anhydrous ethanol solution containing 0.1 mmol / L DPPH. React at 25°C in the dark for 30 min. After centrifugation at 4000 r / min for 20 min, measure the absorbance value As at 517 nm. At the same time, use anhydrous ethanol instead of DPPH solution to conduct parallel experiments and measure the absorbance value Ax. Then use distilled water instead of bovine placental peptide solution to conduct parallel experiments and measure the absorbance value Ac. Calculate the DPPH scavenging rate (%) according to the following formula (3).

[0089] (3)

[0090] The results are attached. Figure 7 As shown. From Figure 7 The results showed that the DPPH free radical scavenging rate of placental peptides from Canadian Simmental cattle increased the fastest in the concentration range of 2-5 mg / mL. When the concentration was 5 mg / mL, the scavenging rate was as high as 85.4%. In the concentration range of 5-6 mg / mL, the DPPH free radical scavenging rate was stable in the range of 85.4-85.8%, with no significant difference (p>0.05). The DPPH scavenging rate of placental peptides from native yellow cattle was significantly lower than that of placental peptides from Canadian Simmental cattle at the same concentration.

[0091] The DPPH scavenging capacity IC50 of the placental peptide from Simmental bovine lineage was calculated. 50 =2.53 mg / mL, significantly higher than the DPPH scavenging capacity IC50 of native bovine placental peptides. 50 =3.44 mg / mL.

[0092] ABTS + Free radical scavenging ability: The ABTS colorimetric method was used. The test solution was taken and mixed with ABTS free radical working solution (ABTS solution (7 mmol / L) and potassium persulfate solution (2.45 mmol / L), mixed in a 1:1 ratio). After adding the solutions, the microplate was placed in the dark for 10 minutes to ensure complete reaction. Then, the microplate was transferred to a microplate reader, and the absorbance was measured at 734 nm. The free radical scavenging rate was calculated. The results are attached. Figure 8 As shown.

[0093] from Figure 8 The results show that the clearance rate of placental peptides from Canadian Simmental cattle was 90.7% at a concentration of 5 mg / mL, indicating that it has strong antioxidant activity; the clearance rate of ABTS+ from placental peptides from native yellow cattle was significantly lower than that from Canadian Simmental cattle at the same concentration.

[0094] In summary, placental peptides from Canadian Simmental cattle exhibit stronger antioxidant activity than placental peptides from native yellow cattle.

[0095] Example 5: Immunomodulatory Activity Test

[0096] Following the preparation method of bovine placental peptide powder in Example 3, placental peptide powder from Canadian Simmental cattle and placental peptide powder from native yellow cattle were prepared. The obtained Canadian Simmental cattle placental peptide powder and native yellow cattle placental peptide powder were respectively prepared into solutions of 50 μg / mL, 100 μg / mL, and 200 μg / mL, which were used as test solutions for mouse spleen lymphocyte proliferation experiments and macrophage phagocytic capacity determination.

[0097] Mouse spleen lymphocyte proliferation experiment: The above test solution was used to co-culture ConA-induced mouse spleen lymphocytes for 48 h. The proliferation rate was detected by MTT assay. The results are shown in Table 4 below.

[0098] Table 4. Effects of Simmental bovine placental peptides and native yellow bovine placental peptides on T cell proliferation rate.

[0099]

[0100] As shown in Table 4, both local yellow cattle and Canadian Simmental cattle placental peptides have a promoting effect on T cell proliferation, but the proliferation effect of Canadian Simmental cattle placental peptides is significantly better than that of local yellow cattle placental peptides.

[0101] Macrophage phagocytic capacity assay: Mouse peritoneal macrophages were treated with the above-mentioned test solutions, and the macrophage phagocytic capacity of native yellow cattle placental peptides and Canadian Simmental cattle placental peptides was evaluated by a neutral red phagocytic assay. The results are shown in Table 5 below.

[0102] Table 5. Phagocytic index results of placental peptides from Canadian Simmental cattle and native yellow cattle.

[0103]

[0104] Table 5 shows that both native yellow cattle placental peptides and Canadian Simmental cattle placental peptides promoted the phagocytic activity of macrophages, but the promoting effect of Canadian Simmental cattle placental peptides on macrophage phagocytosis was significantly better than that of native yellow cattle placental peptides. For example, at 200 μg / mL, the phagocytic index of Canadian Simmental cattle placental peptides was 66% higher than that of the negative control group and 24% higher than that of native yellow cattle placental peptides.

[0105] In summary, placental peptides from Canadian Simmental cattle exhibit stronger immunomodulatory activity than placental peptides from native yellow cattle.

[0106] Example 6: The preventive and adjunctive therapeutic effects of Simmental bovine placental peptides on pulmonary nodules

[0107] This embodiment uses the placental peptide from Simmental bovine strain prepared in Example 3 as the sample group to conduct experiments on a rat model of pulmonary nodules induced by Mycobacterium tuberculosis. The specific method is as follows:

[0108] Rats were randomly divided into 5 groups (n=10 per group): normal control group, model control group, low-dose placental peptide group, medium-dose placental peptide group, and high-dose placental peptide group. The model control group, low-dose placental peptide group, medium-dose placental peptide group, and high-dose placental peptide group all used rats with pulmonary nodules induced by Mycobacterium tuberculosis. Rats in each placental peptide dosage group were administered Simmental bovine placental peptide by gavage at low, medium, and high doses (200 mg / kg, 400 mg / kg, and 600 mg / kg), respectively. The normal control group and model control group received an equal volume of physiological saline. Administration was once daily for 4 weeks. The rats' mental state, activity level, food and water intake, weight changes, and mortality were observed daily. After the last administration, the rats were fasted for 12 hours but allowed free access to water. After weighing, the rats were anesthetized, and their lungs were quickly removed and weighed.

[0109] The lung index (lung wet weight / body weight × 100%) was calculated based on the rat's body weight and lung tissue weight. The results are shown in Table 6 below.

[0110] Table 6. Effects of different doses of bovine placental peptide on lung index.

[0111]

[0112] As shown in Table 6, the lung index of the rats in the model control group was significantly different from that in the normal control group (P < 0.05), indicating that the rat model of pulmonary nodules was successfully established. The lung indices of the rats in the low-, medium-, and high-dose placental peptide groups were all significantly different from those in the model control group (P < 0.05), indicating that the low-, medium-, and high-dose placental peptide groups all had an adjuvant therapeutic effect on pulmonary nodules, and this effect was dose-dependent. In particular, the lung index of the high-dose placental peptide group was close to that of the normal control group.

[0113] The results showed that the placental peptide from the Canadian Simmental bovine strain prepared in Example 3 had an adjunctive therapeutic effect on pulmonary nodules. This may be because pulmonary sarcoidosis is a systemic disease characterized by non-caseating necrotizing epithelioid cell granulomas, and improving the body's own immunity can effectively prevent and treat this type of disease.

[0114] Example 7: The preventive and adjunctive therapeutic effects of Simmental bovine placental peptides on premature ovarian failure.

[0115] This embodiment uses the Simmental bovine placental peptide prepared in Example 3 as the sample group to conduct experiments on a rat premature ovarian failure model constructed using cyclophosphamide. The specific method is as follows:

[0116] Establishment of a rat model of premature ovarian failure: Rats were given an initial loading dose of cyclophosphamide 200 mg / kg via intraperitoneal injection, followed by continuous intraperitoneal injections of 8 mg / kg once daily for 14 days, for a total of 15 days. The remaining rats were injected intraperitoneally with an equal volume of physiological saline as a normal control group.

[0117] Rats were randomly divided into two groups of 10 each: a model control group and a bovine placental peptide group. Rats in the bovine placental peptide group were administered 600 mg / kg of Cilic acid Simmental bovine placental peptide by gavage, while the normal control group and the model control group were administered an equal volume of physiological saline. The administration was once daily for 21 consecutive days.

[0118] During the experiment, the estrous cycle of rats was observed daily via vaginal smears. After the intervention, the rats were fasted for 12 hours but allowed free access to water. Blood was then collected from the abdominal aorta, allowed to coagulate naturally at room temperature for 20 minutes, and centrifuged at 3500 r / min for 15 minutes. The supernatant was collected, and estradiol levels were measured. The results are shown in Table 7 below.

[0119] Table 7. Effects of bovine placental peptides on premature ovarian failure.

[0120]

[0121] Table 7 shows that the model control group rats exhibited absent estrus, prolonged interestrus, or estrous cycle disorders, as well as significantly reduced estradiol levels, indicating successful model establishment. Compared with the normal control group, the estrous cycle disorders in the capri-masked Simmental bovine placental peptide group were improved, with many rats experiencing a return to normal estrous cycles, and estradiol levels were also significantly increased. This indicates that the capri-masked Simmental bovine placental peptide has a protective effect on the ovarian structure and function of rats damaged by cyclophosphamide, and can enhance the body's immunity and alleviate premature ovarian failure. The reason may be that premature ovarian failure (POF) refers to amenorrhea before the age of 40 due to ovarian dysfunction, characterized by primary or secondary amenorrhea accompanied by elevated gonadotropin levels and decreased estrogen levels, along with varying degrees of hypoestrogenic symptoms such as hot flashes, night sweats, facial flushing, and decreased libido. Premature ovarian failure is related to autoimmunity; improving the body's own immunity can effectively prevent and treat this type of disease.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A method for preparing bovine placental peptides, characterized in that, The bovine placental peptides were prepared from the placenta of Simmental cattle of the Canadian breed, and the specific preparation method is as follows: (1) Raw material pretreatment: Fresh placentas from female Simmental cattle within 2 hours after calving were selected, and the fetal membranes, blood vessels and connective tissue were removed. Placental plates were taken, and residual bloodstains, dust and other dirt were removed. They were washed with 0.85% physiological saline until no blood was left. After being chopped, they were subjected to repeated freeze-thaw treatment to obtain placental homogenate. The number of freeze-thaw treatments was 3 times, and the conditions for each freeze-thaw treatment were: freezing at -20℃ for 6 hours and thawing in an ice water bath for 3 hours. (2) Compound enzymatic hydrolysis: Add 5 times the amount of deionized water to the placental homogenate, adjust the pH to 7.8~8.2 with 1mol / L NaOH solution, add trypsin and neutral protease, and enzymatically hydrolyze in a constant temperature water bath at 50℃ for 4h to obtain the enzymatic hydrolysate; wherein, the ratio of trypsin to neutral protease is 3:2, the amount of trypsin added is 1.2% w / w, and the amount of neutral protease added is 0.8% w / w. (3) Enzyme inactivation and separation: The enzyme hydrolysate was inactivated in a 90℃ water bath for 15 min, cooled to room temperature, and then centrifuged at 8000 r / min at 4℃ for 20 min. The supernatant was then collected. (4) Desalting and decolorizing: The supernatant was separated by an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was desalted by a 732 type cation exchange resin and then decolorized by a D101 macroporous adsorption resin. The purified solution was collected. When the permeate was desalted by a 732 type cation exchange resin column, the flow rate of the permeate on the column was 1.5 BV / h. When the permeate was decolorized by a D101 macroporous adsorption resin column, the flow rate of the permeate on the column was 2 BV / h. (5) Concentration and drying: The purified solution is concentrated under vacuum to a solid content of 25%, sterilized by UHT instantaneous sterilization, and spray dried to obtain the bovine placental peptide powder; wherein, the purified solution is concentrated under vacuum conditions of 60°C and -0.08Mpa; the inlet air temperature is 130°C and the outlet air temperature is 80°C during spray drying.

2. A bovine placental peptide, characterized in that, It is prepared by the method for preparing bovine placental peptides according to claim 1.

3. The bovine placental peptide according to claim 2, characterized in that, The bovine placental peptides contain over 90% peptides with a molecular weight distribution between 500 and 1500 Da, and have a protein content > 90% and a free amino acid content < 3%.

4. The application of the bovine placental peptide according to claim 3 in the preparation of health food with antioxidant and immunomodulatory activities.

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