Natto oligopeptide for relieving formation of artery plaque as well as preparation method and application of natto oligopeptide

Abstract

The invention discloses natto oligopeptide capable of relieving formation of arterial plaques and a preparation method and application thereof, belongs to the field of active peptide preparation, aims to solve the problem of obtaining natto or oligopeptide thereof capable of relieving formation of atherosclerotic plaques, and is characterized in that soybeans are soaked, sterilized, inoculated with compound bacteria, fermented at constant temperature and subjected to after-ripening to obtain natto, the volume ratio of the bacillus natto to the lactobacillus plantarum to the bifidobacterium bifidum in the compound bacteria is 1: (0.3-0.5): (0.1-0.3); compound protease is added into the aqueous solution of the freeze-dried natto powder for hydrolysis, centrifugation and filtration, components below 30000 Da are intercepted to obtain the natto oligopeptide, the ratio of chymotrypsin to papain in the compound protease is (1-3): 1, and the natto oligopeptide has the effects that the content of nattokinase in natto is higher, the protein level is also improved, and the natto oligopeptide can be used for preparing the natto oligopeptide. The nattokinase activity is well reserved in hydrolysis, the effect of promoting macrophage autophagy is achieved, and atherosclerotic plaque formation can be relieved.

Description

Technical Field

[0001] The invention belongs to the field of active peptide preparation, and specifically relates to a natto oligopeptide and its application in influencing plasmin activation and alleviating arterial plaque formation. Background Art

[0002] With the change of lifestyle, the incidence of cardiovascular and cerebrovascular diseases in China has been increasing year by year. Carotid atherosclerosis (intima-media thickening and plaque formation) is a clinical manifestation of systemic atherosclerosis and is closely related to cardiovascular and cerebrovascular diseases. At present, the research on its pathogenesis mainly focuses on the local vascular lesions. Endothelial injury and inflammatory response are considered to be the initiating factors of atherosclerotic plaque formation. When the endothelium is damaged, lipids in the blood (such as low-density lipoprotein, LDL) are more likely to enter the subintima of the blood vessels. At the same time, endothelial cell dysfunction reduces its ability to metabolize and clear lipids, providing a material basis for the formation of the lipid core of atherosclerotic plaques. Inflammatory cells (such as monocytes, lymphocytes, etc.) will gather at the site of injury. These cells release a variety of inflammatory mediators, such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), etc. These inflammatory mediators not only stimulate the proliferation and migration of intimal smooth muscle cells, leading to intimal hyperplasia, but also promote lipid oxidation and the uptake of oxidized low-density lipoprotein (ox-LDL) by inflammatory cells, thereby accelerating the formation of atherosclerotic plaques. In this process, intimal hyperplasia and plaque formation are mutually reinforcing.

[0003] A large number of studies have shown that natto has medical and health benefits such as preventing osteoporosis, lowering blood pressure, and improving blood sugar, especially its effects on cardiovascular health. Most of the research on the active ingredients of natto focuses on nattokinase (NK) and vitamin K2. Related studies have shown that nattokinase has multiple activities closely related to cardiovascular health, such as anti-platelet aggregation, direct thrombolysis, increasing the level of tissue plasminogen activator, and regulating blood lipids. The improvement of natto fermentation process to increase the content of nattokinase or K2 and the development of the functional activity of nattokinase are the main contents of natto-related research at present. Natto protein, as the main nutritional component of natto, has not received enough attention. For example, the influence of fermentation process on the composition of active peptides contained in natto, the identification and analysis of natto active peptide components, etc. are still in a blank state. If natto active peptide products with good anti-inflammatory and blood lipid regulation effects can be developed, it will help to improve the application value of natto in alleviating the formation of atherosclerotic plaques. Summary of the invention

[0004] In order to solve the above problems, in a first aspect, according to some embodiments of the present application, the natto preparation method comprises soaking and sterilizing soybeans, inoculating composite bacteria, fermenting at a constant temperature, and post-ripening the natto; wherein the inoculation amount of the composite bacteria is 2-5% of the volume of the soybeans;

[0005] Among them, the constant temperature fermentation temperature is 35-40°C, and the fermentation time is 18-36h;

[0006] Wherein, the volume ratio of Bacillus natto, Lactobacillus plantarum and Bifidobacterium bifidum in the composite bacteria is 1:(0.3-0.5):(0.1-0.3).

[0007] According to the natto preparation method in some embodiments of the present application, the volume ratio of Bacillus natto, Lactobacillus plantarum and Bifidobacterium bifidum in the composite bacteria is 1:0.4:0.2.

[0008] According to the natto preparation method in some embodiments of the present application, the composite bacteria are inoculated after cooling to room temperature after sterilization.

[0009] According to the natto preparation method in some embodiments of the present application, wherein the sterilization is performed by high-pressure steaming at high temperature for 20 to 30 minutes;

[0010] According to the natto preparation method in some embodiments of the present application, the high temperature is 110-120° C.;

[0011] According to the natto preparation method in some embodiments of the present application, the high temperature is 115° C.;

[0012] According to the natto preparation method in some embodiments of the present application, wherein the constant temperature fermentation temperature is 37°C

[0013] According to the natto preparation method in some embodiments of the present application, the post-ripening temperature is 2 to 6°C;

[0014] According to the natto preparation method in some embodiments of the present application, the post-ripening temperature is 4°C;

[0015] According to the natto preparation method in some embodiments of the present application, wherein the natto is post-ripened in a refrigerator at 4°C;

[0016] According to the natto preparation method in some embodiments of the present application, the natto Bacillus natto was purchased from the China Microbial Strain Query Network, strain number: bio-132038;

[0017] According to the natto preparation method in some embodiments of the present application, the Lactobacillus Plantarum was purchased from the China Microbial Strain Query Network, strain number: bio-67374;

[0018] According to the natto preparation method in some embodiments of the present application, the Bifidobacterium Bifidum was purchased from the China Microbial Strain Query Network, strain number: bio-80361.

[0019] In a second aspect, natto prepared by any method according to the first aspect.

[0020] On the third aspect, the natto oligopeptide prepared from the natto described in the second aspect.

[0021] According to some embodiments of the present application, the method for preparing natto oligopeptide comprises adding a composite protease to the aqueous solution of the lyophilized powder of natto in the second aspect for hydrolysis, centrifugation, filtration, and intercepting components below 30,000 Da to obtain the natto oligopeptide;

[0022] Wherein, the mass of the composite protease is 1 to 3% of the mass of the lyophilized powder;

[0023] The enzymatic hydrolysis temperature is 40-50°C, the enzymatic hydrolysis pH is 7.0-8.0, and the enzymatic hydrolysis time is 3-5h;

[0024] Among them, the ratio of chymosin to papain in the composite protease is (1-3):1.

[0025] According to the preparation method of natto oligopeptide in some embodiments of the present application, the volume ratio of water to natto freeze-dried powder in the aqueous solution of natto freeze-dried powder is (5-10):1;

[0026] According to the preparation method of natto oligopeptide in some embodiments of the present application, the ratio of chymosin to papain in the composite protease is 2:1;

[0027] According to the preparation method of natto oligopeptide in some embodiments of the present application, the hydrolysis time is 4h;

[0028] According to the preparation method of natto oligopeptide in some embodiments of the present application, the centrifugal speed is 8000r / min;

[0029] According to the preparation method of natto oligopeptide in some embodiments of the present application, the centrifugation time is 10 minutes;

[0030] According to the preparation method of natto oligopeptide in some embodiments of the present application, the filtration is membrane separation filtration.

[0031] In a fourth aspect, the natto oligopeptide is prepared by the method described in any one of the third aspects.

[0032] In the fifth aspect, according to the use of the composite protease in the embodiment of the present application for retaining the activity of nattokinase in natto hydrolysis, wherein the ratio of chymosin to papain in the composite protease is (1-3):1.

[0033] According to the use of the composite protease in the embodiment of the present application for retaining the activity of nattokinase in natto hydrolysis, wherein the ratio of chymosin to papain in the composite protease is 2:1.

[0034] According to the use of the composite protease in the embodiment of the present application for promoting macrophage autophagy in natto hydrolysis, wherein the ratio of chymosin to papain in the composite protease is (1-3):1.

[0035] According to the use of the composite protease in the examples of the present application for promoting macrophage autophagy in natto hydrolysis, the ratio of chymosin to papain in the composite protease is 2:1.

[0036] In the sixth aspect, use of the natto described in the second aspect or the natto oligopeptide described in the third aspect or the fourth aspect in preparing food.

[0037] Use of the natto described in the second aspect or the natto oligopeptide described in the third aspect or the fourth aspect in preparing functional foods.

[0038] The use of the natto described in the second aspect or the natto oligopeptide described in the third aspect or the fourth aspect in retaining the activity of nattokinase.

[0039] Use of the natto described in the second aspect or the natto oligopeptide described in the third aspect or the fourth aspect in promoting macrophage autophagy.

[0040] Use of the natto described in the second aspect or the natto oligopeptide described in the third aspect or the fourth aspect in the preparation of a medicament for preventing or treating arterial plaque or its formation.

[0041] Beneficial effects:

[0042] In the first aspect, the present invention uses a composite bacteria composed of Bacillus natto, Lactobacillus plantarum, and Bifidobacterium bifidum to ferment natto under the fermentation conditions. Compared with fermentation using Bacillus natto alone, the nattokinase content in the natto is higher, the protein level is also improved, and the protein and polypeptide composition is also changed, which is helpful for the subsequent enzymatic hydrolysis step to obtain natto oligopeptides with high nattokinase content that can alleviate arterial plaque formation.

[0043] On the second aspect, nattokinase, as a protein component, is easily hydrolyzed and destroyed by the added protease preparation during the hydrolysis of natto. The present invention found in the process of developing the natto enzymatic hydrolysis process that the use of a composite protease composed of chymotrypsin and papain to directly hydrolyze natto can well retain the activity of nattokinase while obtaining an active peptide component that promotes macrophage autophagy. As the main cellular component in the development of arterial plaques, macrophages can promote cholesterol outflow through the autophagy-lysosome system, thereby inhibiting the formation of foam cells, while inhibiting the activation of inflammasomes and reducing IL-1β secretion, thereby alleviating the inflammatory response of plaques. The natto oligopeptides obtained by the present invention reduce the deposition of lipids and platelets in the vascular wall through the blood lipid regulation and antiplatelet aggregation effects of nattokinase, improve blood flow, and reduce the inflammatory infiltration of the vascular wall through the active peptide component that promotes macrophage autophagy, reduce the formation and expansion of lipid cores in plaques, thereby alleviating the formation and development of arterial plaques. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Nattokinase content.

[0045] Figure 2 Macrophage LC3Ⅱ / LC3Ⅰ value.

[0046] Figure 3 The percentage of carotid artery plaque area in rabbits. DETAILED DESCRIPTION

[0047] The present invention is further described below by way of examples. It should be noted that the examples do not limit the scope of protection claimed for the present invention.

[0048] The present invention provides a natto oligopeptide and its application in influencing plasmin activation and alleviating arterial plaque formation, wherein the preparation of the natto oligopeptide comprises the following steps:

[0049] S100. Wash and soak the soybeans, drain and sterilize under high pressure at 110-120° C. for 20-30 min, cool naturally to room temperature, inoculate with composite bacteria at 2-5% of the volume of the soybeans, ferment at a constant temperature of 35-40° C. for 18-36 h, and post-ripen in a refrigerator at 2-6° C. to obtain fresh natto.

[0050] Preferably, the composite bacteria is Bacillus natto. The volume ratio of Lactobacillus plantarum to Bifidobacterium bifidum is 1: 0.3-0.5: 0.1-0.3.

[0051] More preferably, the composite bacteria is Bacillus natto. The volume ratio of Lactobacillus plantarum to Bifidobacterium bifidum is 1:0.4:0.2.

[0052] Preferably, the Bacillus natto is purchased from the China Microbial Strain Query Network (https: / / www.biobw.org / ), strain number: bio-132038.

[0053] Preferably, the Lactobacillus plantarum is purchased from the China Microbial Strain Query Network, strain number: bio-67374.

[0054] Preferably, the Bifidobacterium bifidum is purchased from the China Microbial Strain Query Network, strain number: bio-80361.

[0055] S200. Grind the fresh natto in S100 into slurry and freeze-dry it to obtain natto freeze-dried powder.

[0056] S300. Take natto freeze-dried powder and add 5 to 10 times the volume of pure water to mix evenly, add 1 to 3% of the mass of natto composite protease, enzymatically hydrolyze for 3 to 5 hours at 40 to 50° C. and pH 7.0 to 8.0, centrifuge at 8000 r / min for 10 minutes, remove the residue, separate and filter by membrane, intercept and collect components below 30,000 Da, and freeze-dry to obtain natto oligopeptide powder.

[0057] Preferably, the composite protease is chymosin:papain=1-3:1.

[0058] More preferably, the composite protease is chymotrypsin:papain=2:1.

[0059] Based on the above, the present invention relates to the application of natto oligopeptides in influencing the activation of plasminogen lysate and alleviating the formation of arterial plaques.

[0060] The preparation method of the natto oligopeptide of the present invention first ferments soybeans with Bacillus natto, Lactobacillus plantarum and Bifidobacterium bifidum to obtain natto with a high nattokinase content, thereby changing the protein composition in the natto. Then, a composite enzyme combination that does not destroy the activity of nattokinase is selected to enzymolyze the natto to obtain a natto oligopeptide with a high nattokinase content that promotes macrophage autophagy.

[0061] While retaining the lipid-regulating and anti-platelet aggregation activity of nattokinase, natto oligopeptides reduce foam cells containing a large amount of ox-LDL by promoting macrophage autophagy and reduce the release of pro-inflammatory factors, thereby alleviating plaque formation. It has broad application prospects in improving cardiovascular health.

[0062] Example NP1: A method for preparing natto oligopeptides, comprising the following steps:

[0063] S1. Wash and soak 1000 g soybeans.

[0064] S2. After draining, sterilize under high pressure at 115℃ for 30min and cool naturally to room temperature.

[0065] S3. Inoculate 4% of the volume of soybean with composite bacteria, and ferment at 37°C for 36 hours. The composite bacteria composition is Bacillus natto, Lactobacillus plantarum and Bifidobacterium bifidum = 1:0.4:0.2.

[0066] S4. Post-ripen the natto in a 4°C refrigerator to obtain fresh natto.

[0067] S5. Grind the fresh natto in S4 into a slurry and freeze-dry it to obtain natto freeze-dried powder N1.

[0068] S6. Take 100 g of freeze-dried natto powder and add 10 times the volume of pure water and mix well.

[0069] S7. Add 3% of the mass of natto with a composite protease composition of chymosin:papain = 2:1, and perform enzymolysis at 50°C and pH 7.5 for 4 hours.

[0070] The mixture was centrifuged at S8.8000 r / min for 10 minutes to remove the residue, and the mixture was separated and filtered by a membrane to intercept and collect the components below 30000 Da, and then freeze-dried to obtain natto oligopeptide powder NP1.

[0071] Example NP2: The preparation steps are the same as those of Example 1, except that in step S3, 4% of the soybean volume is inoculated with composite bacteria, Lactobacillus plantarum and Bifidobacterium bifidum are removed, and 2.5% of the soybean volume is inoculated with Bacillus natto, finally obtaining natto oligopeptide powder NP2.

[0072] Example NP3: The preparation steps are the same as those of Example 1, except that the composite protease (chymotrypsin and papain) is replaced by alkaline protease in step S7, and finally natto oligopeptide powder NP3 is obtained.

[0073] Example NP4: The preparation steps are the same as those in Example 1, except that the composite protease (chymotrypsin and papain) is replaced by a neutral protease in step S7, and finally natto oligopeptide powder NP4 is obtained.

[0074] Example NP5: The preparation steps are the same as those in Example 1, except that the composite protease (chymotrypsin and papain) is replaced by trypsin in step S7, and finally natto oligopeptide powder NP5 is obtained.

[0075] Example NP6: The preparation steps are the same as those in Example 1, except that the composite protease (chymotrypsin and papain) is replaced with chymotrypsin in step S7, and finally natto oligopeptide powder NP6 is obtained.

[0076] Example NP7: The preparation steps are the same as those in Example 1, except that the composite protease (chymotrypsin and papain) is replaced with papain in step S7, and finally natto oligopeptide powder NP7 is obtained.

[0077] Experimental description:

[0078] (1) Determination of nattokinase content: The nattokinase content in the natto oligopeptide powder obtained in each example was determined by ultraviolet spectrophotometry according to the nattokinase determination method described in Appendix A of DBS44 / 013-2019 "Local Food Safety Standard Natto Powder".

[0079] (2) Protein content determination: The protein content in the natto oligopeptide in each example was determined using a BCA protein concentration kit.

[0080] (3) Effect of natto oligopeptide on the expression of autophagy in RAW264.7 cells in vitro: RAW264.7 macrophages were cultured in a constant temperature incubator at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody. The macrophages were divided into 6 groups, namely control group, N1 group, NP1 group, NP2 group, NP6 group, and NP7 group. The experimental groups were treated with 10ug / L natto freeze-dried powder or natto oligopeptide aqueous solution for 12h. After 12h, the cell survival rate was determined by cell counting kit-8 reagent, and the cell survival rate of each group was greater than 97%. Total cell protein was extracted, and the levels of autophagy markers LC3Ⅰ and LC3Ⅱ were determined by immunoblotting. LC3 initially existed in the cytoplasm in the form of LC3Ⅰ. After its C-terminus was processed, it combined with phosphatidylethanolamine to form LC3Ⅱ. LC3Ⅱ can be specifically localized to the autophagosome membrane and is an important marker for the formation of autophagosomes.

[0081] (4) Effect of natto oligopeptide on carotid atherosclerotic plaque formation in rabbits: New Zealand white rabbits were used as materials and induced to form atherosclerotic plaque models by feeding them with high-fat diet (containing 1% cholesterol, 7.5% egg yolk powder, 8% lard, 0.5% sodium cholate, 0.1% propylthiouracil and 81% ordinary rabbit feed) for 4 weeks. After the model was successfully established, each medication group was continuously gavaged once a day for 8 consecutive weeks. 24 New Zealand white rabbits were divided into 3 groups, 8 in each group, including a blank group (ordinary diet + normal saline), a model group (high-fat diet modeling + normal saline), and experimental groups such as N1 group, NP1 group, NP2 group, NP3 group, NP4 group, NP5 group, NP6 group, and NP7 group (high-fat diet modeling + 1g / kg / day dose of natto freeze-dried powder / natto oligopeptide powder). After 8 weeks, the carotid arteries were cut, embedded in paraffin, sliced, and stained with HE. The sections were sealed and observed morphologically under a microscope. The plaque area was calculated using ImagePro Plus image processing software. Plaque area ratio (%) = plaque area / aortic inner wall cross-sectional area × 100%.

[0082] Experimental Example 1. Determination of Nattokinase Content, the results are as follows Figure 1 As shown, compared with the natto freeze-dried powder N1 obtained by compound bacteria fermentation, the nattokinase content in the natto oligopeptide powder NP6, NP7, and NP1 obtained after hydrolysis by chymotrypsin alone or papain alone, and the combination of the two under specific conditions did not decrease significantly (P>0.05). After being hydrolyzed by alkaline protease, neutral protease, and trypsin, the nattokinase in the natto oligopeptide powder NP3, NP4, and NP5 was damaged to varying degrees, and the content decreased significantly (P<0.05), indicating that nattokinase will be affected by a variety of commonly used protease preparations, and the active site is damaged, and the composite protease combination provided by the present invention better retains the nattokinase activity in natto during the enzymolysis process. At the same time, the nattokinase content of NP1 fermented by compound bacteria is increased by about 50% compared with NP2 fermented by Bacillus natto alone, indicating that the natto fermentation carried out by the composite bacteria combination helps to improve the nattokinase content in the product.

[0083] Experimental Example 2. Determination of protein content. The protein content in NP1 was 41.25±0.21%, significantly higher than 34.08±0.15% of NP2 (P<0.05). The composite bacteria fermentation effectively increased the protein content in natto.

[0084] Experimental Example 3. Effect of Natto Oligopeptide on the Expression of Autophagy in RAW264.7 Cells in Vitro. The results of LC3Ⅱ / LC3Ⅰ in each group are as follows: Figure 2As shown in the results, compared with the control group, there was no significant difference in the LC3Ⅱ level in macrophages treated with natto freeze-dried powder N1, NP4, NP5, and NP7 after being hydrolyzed by neutral protease, trypsin, and papain (P>0.05). The LC3Ⅱ levels of NP3 and NP6 groups hydrolyzed by alkaline protease and chymotrypsin were significantly increased (P<0.05), and the effect of NP6 group was better than that of NP3 group (P<0.05). The LC3Ⅱ level of NP1 group obtained by hydrolyzing with chymotrypsin and papain was further increased compared with that of NP6 group (P<0.05). The above results indicate that active peptide components with enhanced macrophage autophagy ability were obtained after chymotrypsin-papain composite hydrolysis. At the same time, the LC3Ⅱ level of NP1 group was also significantly higher than that of NP2 obtained by fermentation of Bacillus natto alone. The differences in protein components caused by composite fermentation affected the preparation of active peptides that promote macrophage autophagy.

[0085] Experimental Example 4. Effect of Natto Oligopeptide on the Formation of Carotid Artery Atherosclerotic Plaque in Rabbits. Figure 3 As shown in the figure, after the model group was fed with a high-fat diet, the area of ​​carotid artery plaques rapidly expanded, and the model was successfully established. After oral gavage intervention, the plaque area of ​​each experimental group was reduced to varying degrees compared with the model group (P < 0.05). Among them, the NP1 group had a better effect on alleviating plaque formation than the non-hydrolyzed natto freeze-dried powder N1 group (P < 0.05), and was also better than the NP6 group that was only hydrolyzed by chymosin (P < 0.05). The NP2 group fermented by Bacillus natto alone had a lower content of nattokinase and a weaker effect on promoting macrophage autophagy, and its effect on alleviating plaque formation was significantly lower than that of the NP1 group (P < 0.05), and was slightly better than the NP3 group, NP4 group, and NP5 group in which nattokinase was severely damaged and had no obvious effect on promoting macrophage autophagy (P < 0.05).

[0086] As described above, the preparation method of the natto oligopeptide of the present invention first ferments soybeans with Bacillus natto, Lactobacillus plantarum, and Bifidobacterium bifidum to obtain natto with a high nattokinase content, thereby changing the protein composition in the natto. Then, a composite enzyme combination that does not destroy the activity of nattokinase is screened to enzymolyze the natto to obtain a natto oligopeptide with a high nattokinase content that promotes macrophage autophagy. While retaining the blood lipid and anti-platelet aggregation activities of nattokinase, the natto oligopeptide reduces foam cells containing a large amount of ox-LDL by promoting macrophage autophagy, reduces the release of proinflammatory factors, and achieves the effect of alleviating plaque formation, and has broad application prospects in improving cardiovascular health.

[0087] Finally, it should be noted that the above examples are only some specific embodiments of the present invention, and all derivatives that can be directly derived or associated with the contents disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for preparing natto, characterized in that: The method comprises soaking and sterilizing soybeans, inoculating composite bacteria, fermenting at a constant temperature, and post-ripening to obtain the natto; wherein the inoculation amount of the composite bacteria is 2-5% of the volume of the soybeans; Among them, the constant temperature fermentation temperature is 35-40°C, and the fermentation time is 18-36h; Wherein, the volume ratio of Bacillus natto, Lactobacillus plantarum and Bifidobacterium bifidum in the composite bacteria is 1:(0.3-0.5):(0.1-0.3).

2. The method for preparing natto according to claim 1, characterized in that: in, The volume ratio of Bacillus natto, Lactobacillus plantarum and Bifidobacterium bifidum in the composite bacteria is 1:0.4:0.

2.

3. The method for preparing natto according to any one of claims 1 to 2, characterized in that: Preferably, the composite bacteria are inoculated after being cooled to room temperature after sterilization. Preferably, the sterilization is performed by high pressure cooking at high temperature for 20 to 30 minutes; More preferably, the high temperature is 110-120°C; More preferably, the elevated temperature is 115°C; Preferably, the constant temperature fermentation temperature is 37°C Preferably, the ripening temperature is 2 to 6°C; Preferably, the ripening temperature is 4°C; Preferably, the mixture is post-ripened in a refrigerator at 4°C; Preferably, the Bacillus Natto is purchased from the China Microbial Strain Query Network, strain number: bio-132038; Preferably, the Lactobacillus Plantarum is purchased from the China Microbial Strain Query Network, strain number: bio-67374; Preferably, the Bifidobacterium Bifidum is purchased from the China Microbial Strain Query Network, strain number: bio-80361.

4. Natto prepared by the method according to any one of claims 1 to 3.

5. The natto oligopeptide prepared from natto according to claim 4.

6. A method for preparing natto oligopeptide, characterized in that: The method comprises adding a composite protease into the aqueous solution of the freeze-dried powder of natto according to claim 4 to perform hydrolysis, centrifugation, filtration, and intercepting components below 30,000 Da to obtain the natto oligopeptide; Wherein, the mass of the composite protease is 1 to 3% of the mass of the lyophilized powder; The enzymatic hydrolysis temperature is 40-50°C, the enzymatic hydrolysis pH is 7.0-8.0, and the enzymatic hydrolysis time is 3-5h; Among them, the ratio of chymosin to papain in the composite protease is (1-3):

1.

7. The method for preparing natto oligopeptide according to claim 6, characterized in that: Preferably, the volume ratio of water to natto freeze-dried powder in the aqueous solution of natto freeze-dried powder is (5-10):1; Preferably, in the composite protease, chymotrypsin:papain=2:1; Preferably, the hydrolysis time is 4h; Preferably, the centrifugal speed is 8000r / min; Preferably, the centrifugation time is 10 min; Preferably, the filtration is membrane separation filtration.

8. Natto oligopeptide prepared by the method according to any one of claims 6 to 7.

9. Use of a composite protease for retaining the activity of nattokinase in natto hydrolysis or for promoting macrophage autophagy, wherein: In the composite protease, chymotrypsin:papain=(1-3):1; Preferably, the ratio of chymosin to papain in the composite protease is 2:

1.

10. Use of the natto according to claim 4 or the natto oligopeptide according to claim 5 or 8 in preparing food, or in preparing functional food, or in retaining the activity of nattokinase, or in promoting macrophage autophagy, or in preparing a drug for preventing or treating arterial plaque or its formation.

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