Raw material pretreatment method for improving ACE (angiotensin converting enzyme) inhibition ratio of oyster peptide
By fermenting and pretreating oyster meat and enzymatically decomposed using complex enzymes, the problem of low ACE inhibitory activity of oyster peptides in the prior art was solved, significantly improving the ACE inhibitory rate, and providing a more efficient method for preparing oyster peptides.
Patent Information
- Application Number
- CN202411632388.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-02-07
AI Technical Summary
In the prior art, the ACE inhibitory activity of oyster peptides is relatively low, and a preparation method that can improve the ACE inhibitory rate of oyster peptides is urgently needed.
After fermenting oyster meat, it is performed by using complex enzymes for enzymatic decomposition. The specific steps include mixing oyster meat with soy flour, beating, sterilizing, and sterilizing Lactobacillus rhamnosus and natto fermenting agent for fermentation. After completion, enzymatic lysis is performed, and complex enzyme I or complex enzyme II is used for enzymatic lysis.
The ACE inhibitory activity of oyster peptides was significantly improved, with the ACE inhibitory rate of ≥95%, laying the foundation for the preparation of oyster peptides and related blood pressure-lowering agents and foods in the subsequent high ACE inhibitory activity.
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Figure CN119391801A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to a raw material pretreatment method for improving ACE inhibition rate of oyster peptides. Background Art
[0002] The protein content of oysters accounts for 45-57% of the dry weight, which is currently a raw material with high utilization of marine protein sources. Oysters have the functions of increasing male serum testosterone levels, regulating blood lipids, inhibiting platelet aggregation, improving hyperglycemia symptoms, improving human immunity, promoting metabolism, etc., and also have certain effects in fighting cancer and preventing the spread of cancer cells. Oyster peptides are small molecule peptides hydrolyzed by biological enzymes, which have the functions of protecting the liver and sobering up.
[0003] Existing studies have shown that some oyster peptides have angiotensin converting enzyme (ACE) inhibitory activity and can effectively regulate blood pressure. However, the ACE inhibitory activity of existing oyster peptides is relatively low, and a method for preparing oyster peptides with a high ACE inhibition rate is urgently needed.
[0004] Jiang Naiyi et al. used chitin / graphene oxide / cellulose acetate composite membrane and ultrafiltration ultrafrequency vibration separation technology to separate and purify oysters after enzymatic hydrolysis, and improved the ACE inhibitory activity and antioxidant activity of oyster peptides by improving the separation efficiency and effect. Mou Haijin et al. homogenized oyster meat with water and obtained oyster peptide solution after enzymatic hydrolysis, and then mixed the oyster peptide solution with soy protein peptide, maltose, sodium chloride and water to prepare a fermentation medium, inoculated thrombolytic metabolism-enhanced recombinant bacteria for fermentation, and thus prepared a fermentation product; the fermentation product has strong ACE inhibitory activity.
[0005] As can be seen from the above, in the prior art, the activity of oyster peptides is mostly improved by further processing the oyster peptides after enzymatic hydrolysis. However, there are few methods for improving the ACE inhibitory activity of oyster peptides by improving the enzymatic hydrolysis efficiency. Summary of the invention
[0006] In view of the above problems, the main purpose of the present invention is to provide a raw material pretreatment method for improving the ACE inhibition rate of oyster peptides. The method of the present invention significantly improves the ACE inhibition activity of oyster peptides by fermenting and pretreating oyster meat and then using a composite enzyme for enzymolysis. The present invention lays a foundation for the subsequent preparation of oyster peptides with higher ACE inhibition activity and related antihypertensive agents and foods.
[0007] To achieve the above object, the present invention adopts the following technical solutions:
[0008] The invention provides a raw material pretreatment method for improving the ACE inhibition rate of oyster peptides. The method comprises the following steps: mixing oyster meat with soybean powder, beating, sterilizing, inoculating rhamnosus lactobacillus and natto fermentation bacteria agent for fermentation pretreatment, and performing enzymolysis after the fermentation is completed to obtain the raw material.
[0009] Furthermore, the soybean powder accounts for 0.05% to 0.1% of the dry matter weight of the oyster meat.
[0010] Furthermore, the water content of the oyster meat is 85% to 90%.
[0011] Furthermore, the inoculation amounts of Lactobacillus rhamnosus and natto fermentation agent were 10 7 ~10 10 cfu / mL.
[0012] Further, the fermentation is allowed to stand at 30-37° C. for 10-20 hours, maintaining the pH at 5.5-6.0.
[0013] Furthermore, a composite enzyme I is selected for enzymolysis, and the composite enzyme I includes the following components and amounts based on the dry matter of oyster meat:
[0014] Alkaline protease 50-100U / g, neutral protease 50-150U / g, flavor protease 10-40U / g, aminopeptidase 2-15U / g and weak alkaline protease 5-20U / g.
[0015] Furthermore, complex enzyme II is selected for enzymolysis. Calculated on the dry matter of oyster meat, the complex enzyme II includes the following components and dosages: alkaline protease 50-100U / g, neutral protease 50-150U / g, papain 50-150U / g, trypsin 2-8U / g and weak alkaline protease 5-20U / g.
[0016] The present invention obtains compound enzyme I and compound enzyme II through a large number of experimental screenings. The oyster peptide obtained by enzymolysis of oysters pretreated by fermentation using the compound enzyme I or compound enzyme II has high ACE inhibitory activity.
[0017] Furthermore, during enzymatic hydrolysis, the pH value is adjusted to 7.0-7.5, and the enzymatic hydrolysis is carried out at 40-55° C. for 1.5 h to 3 h.
[0018] The present invention also provides a method for preparing oyster peptide, which comprises the following steps: pretreating the raw material by the above method, separating and purifying the pretreated raw material by a membrane integrated system, collecting a filtrate with a molecular weight of ≤3000Da, and concentrating and drying the obtained filtrate.
[0019] The present invention also provides oyster peptides prepared by the above method, wherein small peptides with a molecular weight distribution of 300-600 Da account for more than 80% of the oyster peptides.
[0020] Compared with the prior art, the present invention has the following technical advantages:
[0021] The method of the present invention significantly improves the ACE inhibition rate of oyster peptides by fermenting and pretreating oyster meat and then performing enzymolysis with a composite enzyme, and the ACE inhibition rate is ≥95%. The method of the present invention improves the ACE inhibition activity of the oyster peptides obtained in the enzymolysis stage, thereby laying a foundation for the subsequent purification and separation to prepare oyster peptide products with higher ACE inhibition activity.
[0022] The oyster peptide prepared by the method of the present invention has a molecular weight distribution of small peptides in the range of 300-600 Da accounting for more than 80%, and can be well used in food processing, development of health products for assisting blood pressure regulation and other fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a chromatogram of the molecular weight distribution of oyster peptides obtained in Example 1 of the present invention. DETAILED DESCRIPTION
[0024] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.
[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.
[0026] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.
[0027] The rhamnosus lactobacillus and natto fermentation bacteria used in the embodiments of the present invention are all commercial products. The alkaline protease, flavor protease, trypsin, aminopeptidase and weak alkaline protease used are all purchased from finished enzyme preparations on the market, including: alkaline protease, enzyme activity 300000U / ml; flavor protease, enzyme activity 100000U / g; trypsin, enzyme activity 4000U / g; aminopeptidase, enzyme activity 5000000U / g; weak alkaline protease, enzyme activity 480000U / g; neutral protease, enzyme activity 50000U / g; papain, enzyme activity 1000000U / g.
[0028] Other related reagents and detection methods are prior art unless otherwise specified.
[0029] Example 1
[0030] A raw material pretreatment method for improving the ACE inhibition rate of oyster peptides comprises the following steps: using oyster meat as raw material, adding soybean powder accounting for 0.05% of the dry matter of the oyster meat, beating the oyster meat and the soybean powder together, and sterilizing at 121°C for 30 minutes.
[0031] According to the inoculation volume 10 7 cfu / mL were inoculated with freeze-dried Lactobacillus rhamnosus powder and natto fermentation agent, respectively, and cultured at 37℃ for 18h, maintaining pH 5.5.
[0032] After fermentation, the pH was adjusted to 7.0, complex enzyme I was added, the temperature was raised to 55°C for enzymatic hydrolysis for 1.5 hours, the temperature was raised to 100°C for enzyme inactivation for 20 minutes, the mixture was cooled and centrifuged, the supernatant was filtered using a conventional membrane integrated system, the filtrate with a molecular weight of ≤3000Da was collected, and the filtrate was concentrated and dried to obtain the oyster peptide product.
[0033] Calculated on the basis of oyster meat dry matter, the complex enzyme I is composed of the following components and amounts: 50 U / g alkaline protease, 80 U / g neutral protease, 10 U / g flavor protease, 5 U / g aminopeptidase and 10 U / g weak alkaline protease.
[0034] Example 2
[0035] A raw material pretreatment method for improving the ACE inhibition rate of oyster peptides, the method comprising the following steps:
[0036] Oyster meat was used as a raw material, and soybean powder accounting for 0.1% of the dry matter of the oyster meat was added. The oyster meat and the soybean powder were beaten together and sterilized at 121° C. for 30 minutes.
[0037] According to the inoculation volume 10 7 cfu / mL were inoculated with freeze-dried powder of Lactobacillus prunus and natto fermentation agent, respectively, and cultured at 37℃ for 18h, maintaining pH 6.0.
[0038] After fermentation, the pH was adjusted to 7.5, complex enzyme I was added, the temperature was raised to 50°C for enzymatic hydrolysis for 2 hours, the temperature was raised to 100°C for enzyme inactivation for 20 minutes, the mixture was cooled and centrifuged, the supernatant was filtered using a conventional membrane integrated system, the filtrate with a molecular weight ≤3000Da was collected, and the filtrate was concentrated and dried to obtain the oyster peptide product.
[0039] Calculated on the basis of oyster meat dry matter, the complex enzyme I is composed of the following components and amounts: 100 U / g alkaline protease, 50 U / g neutral protease, 30 U / g flavor protease, 15 U / g aminopeptidase and 5 U / g weak alkaline protease.
[0040] Example 3
[0041] A raw material pretreatment method for improving the ACE inhibition rate of oyster peptides, the method comprising the following steps:
[0042] Oyster meat was used as a raw material, and soybean powder accounting for 0.08% of the dry matter of the oyster meat was added. The oyster meat and the soybean powder were beaten together and sterilized at 121° C. for 30 minutes.
[0043] According to the inoculation volume 10 7 cfu / mL were inoculated with freeze-dried powder of Lactobacillus prunus and natto fermentation agent, respectively, and cultured at 37℃ for 18h, maintaining pH 6.0.
[0044] After fermentation, the pH was adjusted to 7.5, complex enzyme II was added, the temperature was raised to 40°C for enzymatic hydrolysis for 3 hours, the temperature was raised to 100°C for enzyme inactivation for 20 minutes, the mixture was cooled and centrifuged, the supernatant was filtered using a conventional membrane integrated system, the filtrate with a molecular weight of ≤3000Da was collected, and the filtrate was concentrated and dried to obtain the oyster peptide product.
[0045] Calculated on the basis of oyster meat dry matter, the complex enzyme II is composed of the following components and amounts: 50 U / g alkaline protease, 100 U / g neutral protease, 50 U / g papain, 8 U / g trypsin and 20 U / g weak alkaline protease.
[0046] Example 4
[0047] A raw material pretreatment method for improving the ACE inhibition rate of oyster peptides, the method comprising the following steps:
[0048] Oyster meat was used as a raw material, and soybean powder accounting for 0.06% of the dry matter of the oyster meat was added. The oyster meat and the soybean powder were beaten together and sterilized at 121° C. for 30 minutes.
[0049] According to the inoculation volume 10 7 cfu / mL were inoculated with freeze-dried Lactobacillus rhamnosus powder and natto fermentation agent, respectively, and cultured at 37°C for 18 h, maintaining pH 5.8.
[0050] After the fermentation, the pH was adjusted to 7.0, complex enzyme II was added, the temperature was raised to 50°C for enzymatic hydrolysis for 2 hours, the temperature was raised to 100°C for enzyme inactivation for 20 minutes, the mixture was cooled and centrifuged, the supernatant was filtered using a conventional membrane integrated system, the filtrate with a molecular weight of ≤3000Da was collected, and the filtrate was concentrated and dried to obtain the oyster peptide product.
[0051] Calculated on the basis of oyster meat dry matter, the complex enzyme II is composed of the following components and amounts: 100 U / g alkaline protease, 80 U / g neutral protease, 150 U / g papain, 2 U / g trypsin and 5 U / g weak alkaline protease.
[0052] Comparative Example 1
[0053] The difference from Example 1 is that soybean powder is not added, and the rest is the same as Example 1.
[0054] Comparative Example 2
[0055] The difference from Example 1 is that only the freeze-dried powder of Lactobacillus rhamnosus is added, and the rest is the same as Example 1.
[0056] Comparative Example 3
[0057] The difference from Example 1 is that only the natto fermentation agent is introduced, and the rest is the same as Example 1.
[0058] Comparative Example 4
[0059] The difference from Example 1 is that the following composite protease is added: 500 U / g alkaline protease, 200 U / g neutral protease, 100 U / g flavor protease, 30 U / g aminopeptidase and 50 U / g weak alkaline protease, and the rest is the same as Example 1.
[0060] Comparative Example 5
[0061] The difference from Example 1 is that the following composite protease is added: alkaline protease 50 U / g, neutral protease 80 U / g, flavor protease 10 U / g, aminopeptidase 5 U / g, and the rest is the same as Example 1.
[0062] Comparative Example 6
[0063] The difference from Example 1 is that the following composite protease is added: 50 U / g alkaline protease, 80 U / g neutral protease, 10 U / g flavor protease, and 10 U / g weak alkaline protease. The rest is the same as Example 1.
[0064] Comparative Example 7
[0065] The difference from Example 1 is that the following composite protease is added: alkaline protease 50 U / g, neutral protease 80 U / g, flavor protease 10 U / g, trypsin 5 U / g, weak alkaline protease 10 U / g, and the rest is the same as Example 1.
[0066] Test example
[0067] Figure 1 The middle peak corresponds to oyster peptide samples with a molecular weight of 300-600Da. Figure 1 It can be seen that, in the oyster peptides prepared by the method of the present invention, small peptides with a molecular weight distribution of 300-600 Da account for more than 80%.
[0068] The oyster peptides prepared by the methods described in Examples 1 to 4, the oyster peptides prepared by the methods described in Comparative Examples 1 to 7, and the commercially available oyster peptide products 1, 2, and 3 were respectively prepared into solutions with the same protein concentration, and the ACE inhibition rate was tested. The results are shown in Table 1 below.
[0069] Table 1 ACE inhibition rate of different active peptide products
[0070] Oyster peptide products ACE inhibition rate% Oyster peptide products ACE inhibition rate% Example 1 95.32 Comparative Example 1 62.90 Example 2 94.47 Comparative Example 2 73.49 Example 3 95.49 Comparative Example 3 76.78 Example 4 95.17 Comparative Example 4 61.12 Commercially available products 1 40.31 Comparative Example 5 54.36 Commercially available product 2 32.21 Comparative Example 6 52.52 Commercially available product 3 30.17 Comparative Example 7 60.07
[0071] As can be seen from Table 1, the ACE inhibition rate of the oyster peptide prepared in the embodiment of the present invention is significantly higher than that of the market product and the product described in the comparative example.
[0072] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A raw material pretreatment method for improving the ACE inhibition rate of oyster peptides, characterized in that: The following steps are involved: The oyster meat is mixed with soybean powder, beaten, sterilized, and then inoculated with Lactobacillus rhamnosus and natto fermentation bacteria for fermentation pretreatment, and then enzymatic hydrolysis is performed after the fermentation is completed.
2. The method according to claim 1, characterized in that: The soybean powder accounts for 0.05% to 0.1% of the dry matter weight of the oyster meat.
3. The method according to claim 1, characterized in that: The inoculation amount of Lactobacillus rhamnosus and natto fermentation agent was 10 7 ~10 10 cfu / mL.
4. The method according to claim 1, characterized in that: Ferment at 30-37℃ for 10-20h, maintaining pH 5.5-6.
0.
5. The method according to claim 1, characterized in that: A composite enzyme is selected for enzymolysis, and the composite enzyme includes the following components and dosages based on the dry matter of oyster meat: Alkaline protease 50-100U / g, neutral protease 50-150U / g, flavor protease 10-40U / g, aminopeptidase 2-15U / g and weak alkaline protease 5-20U / g.
6. The method according to claim 1, characterized in that: A composite enzyme is selected for enzymolysis, and the composite enzyme includes the following components and dosages based on the dry matter of oyster meat: Alkaline protease 50-100U / g, neutral protease 50-150U / g, papain 50-150U / g, trypsin 2-8U / g and weak alkaline protease 5-20U / g.
7. The method according to claim 1, characterized in that: During enzymatic hydrolysis, the pH value was adjusted to 7.0-7.5, and the enzymatic hydrolysis was carried out at 40-55°C for 1.5h-3h.
8. The method according to claim 1, characterized in that: The water content of the oyster meat is 85% to 90%.
9. A method for preparing oyster peptide, characterized in that: The following steps are involved: The raw material is pretreated by the method according to any one of claims 1 to 7, the pretreated raw material is separated and purified by a membrane integrated system, a filtrate with a molecular weight of ≤3000Da is collected, and the obtained filtrate is concentrated and dried to obtain the product.
10. The oyster peptide prepared by the method of claim 9, characterized in that: The oyster peptides have a molecular weight distribution of 300-600 Da and small peptides account for more than 80%.