Preparation method and application of fermented soybean containing bioactive peptide

Abstract

According to the fermented soybean containing the bioactive peptide and the preparation method of the fermented soybean, the preparation process of the fermented soybean is optimized by setting different conditions such as fermentation temperature, cooking time and fermentation humidity, and the polypeptide with remarkable neuroprotective activity is identified for the first time. Experiments of the polypeptides in a PC12 cell model show that the polypeptides have good antioxidant and neuroprotective effects, and show the potential of being developed into functional foods or drugs for treating neurodegenerative diseases.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a preparation method of light fermented black beans containing bioactive peptides and application thereof. Background Art

[0002] With the aging of the population, the incidence of neurodegenerative diseases continues to rise, and the incidence of neurological diseases (such as Alzheimer's disease, Parkinson's disease) and neuropsychiatric diseases (such as depression, anxiety) has also increased dramatically worldwide. These diseases have seriously affected the quality of human life and brought a huge burden to the social economy. Most of the existing treatments can only relieve symptoms and cannot cure them fundamentally. At the same time, existing drugs are usually accompanied by major adverse reactions, and the safety of long-term use by patients is questioned.

[0003] Traditional Chinese medicine and its active ingredients have shown great potential in the field of neuroprotection in recent years due to their relatively safe characteristics. However, the low content of active ingredients in some traditional Chinese medicines and the complex and high cost of extraction processes have limited their widespread clinical application.

[0004] The present invention aims to provide a simple preparation process, new active ingredient discovery and structural description, and biological activity verification in view of the limitation that there is a lack of safe and effective neuroprotective ingredients in the market. Summary of the invention

[0005] The present invention aims to solve one of the technical problems of the existing lack of bioactive molecules to at least a certain extent. To this end, the present invention provides a preparation method of light fermented black beans containing bioactive peptides and application thereof.

[0006] According to one aspect of the present invention, a method for identifying active ingredients from light black beans is provided, comprising: analyzing the light black beans by liquid chromatography-mass spectrometry to obtain a data file to be analyzed; establishing a database of cyclic dipeptides; opening the data file to be analyzed in Agilent MassHunter Qualitative Analysis software based on an FBF (Find by Formula) algorithm and importing the data file into the database of cyclic dipeptides; screening for potential active ingredients; and confirming the structure of the potential active ingredients by analyzing the MS2 spectrum.

[0007] Preferably, the active ingredient is at least one of cyclo(Pro-Val), cyclo(Pro-Ile), cyclo(Pro-Thr), cyclo(Pro-Leu), cyclo(Pro-Pro), cyclo(Pro-Phe) and cyclo(Pro-His).

[0008] Preferably, the structure of the active ingredient is confirmed by analyzing the MS2 spectrum, including: determining the MS2 characteristic fragment ions of the cyclic dipeptide; determining the imine ions of the cyclic dipeptide; determining the retention time information of the cyclic dipeptide under RP-LC (reverse phase liquid chromatography) conditions, and confirming the structure of the active ingredient according to the MS2 characteristic fragment ions, imine ions, and retention time.

[0009] According to another aspect of the present invention, a method for preparing fermented soybean containing bioactive peptides is provided, including taking mulberry leaves and artemisia annua, adding water, soaking and decocting, and filtering out the decoction; mixing black beans into the decoction, steaming for 1.5 - 3 h after the decoction is completely absorbed; covering the black beans with the decocted artemisia annua and mulberry leaf residues, and then fermenting at a fermentation temperature of 25 - 35 °C and a relative humidity of 50 - 80% until the yellow coat covers all; taking out, washing off the yellow coat, and then placing it in an incubator at a temperature of 50 - 60 °C for anaerobic fermentation; turning it out and stirring during the anaerobic fermentation to ensure sufficient fermentation.

[0010] Preferably, steam for 1.5 h, ferment at a temperature of 25 °C - 35 °C, and a relative humidity of 80%.

[0011] According to another aspect of the present invention, the use of the fermented soybean containing bioactive peptides obtained by the above preparation method in the preparation of a drug for preventing or improving neurodegenerative diseases is provided.

[0012] According to another aspect of the present invention, the use of active ingredients I - VII in the preparation of a drug for preventing or improving neurodegenerative diseases is provided.

[0013]

[0014]

[0015] The present invention provides a fermented soybean containing bioactive peptides and a preparation method thereof. By optimizing the preparation process of fermented soybean, polypeptides with significant neuroprotective activity are identified for the first time. Experiments in the PC12 cell model show that these polypeptides have good antioxidant and neuroprotective effects, indicating the potential for development into functional foods or drugs for treating neurodegenerative diseases.

[0016] By the optimized preparation process of the present invention, the content of bioactive peptides in fermented soybean is increased, and its neuroprotective effect is enhanced. Through LC-MS analysis, the present invention identifies multiple bioactive peptides with neuroprotective effects from fermented soybean for the first time. Experimental results show that these polypeptides show significant neuroprotective effects in the in vitro PC12 cell model, indicating their application prospects in the prevention and treatment of neurodegenerative diseases. Description of the Drawings

[0017] The accompanying drawings described herein are used to provide a further understanding of the present invention and form a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0018] Figure 1 Shows the differential analysis of 7 representative cyclic dipeptides provided according to the present invention under different fermentation conditions.

[0019] Figure 2 Shows the peak areas of 7 representative cyclic dipeptides provided according to the present invention under different fermentation conditions.

[0020] Figure 3 Shows the effect of representative CDPs provided according to the embodiments of the present invention on the survival rate of H 2 O 2 induced PC12 cell damage ([ ## P < 0.01 vs blank group; *P < 0.05, **P < 0.01 vs model group). Detailed implementation manners

[0021] The following listed embodiments are to enable those skilled in the art to understand the present invention more clearly. It should be noted that the following embodiments do not constitute a limitation to the protection scope required by the present invention and are only illustrative embodiments. The raw materials, reagents or devices mentioned in the following embodiments can be obtained from commercial channels or obtained by known existing methods without special instructions.

[0022] Example 1

[0023] Preparation process of fermented soybean

[0024] A: Take 9 g of mulberry leaves and 10 g of Artemisia annua, add 18 times the amount of water, soak for 3 h, decoct 3 times, 1 h each time, and filter out the decoction; mix 100 g of washed black beans into the decoction, soak for 5 h, and after the decoction is completely absorbed, steam for 1.5 h, take out, cool slightly, and then place in a container; cover with the residue of Artemisia annua and mulberry leaves after decoction; then carry out fermentation, the fermentation temperature is 35 °C, the relative humidity is 80%, and ferment for 6 - 8 d until the yellow mold covers the surface (the fermentation time is controlled according to the degree of the yellow mold covering the surface. The yellow mold covering the surface is the sign of the end of the pre-fermentation of fermented soybean, and the yellow mold covering the surface means that the surface presents a uniform light yellow mold layer); take out, wash off the yellow mold, place in a container, and place in an incubator at a temperature of 50 - 60 °C and stuffy for 15 d (mainly for anaerobic fermentation to prevent the interference of foreign miscellaneous bacteria); during the stuffing period, pour out every 3 d, turn over, dry slightly, and repeat 4 - 5 times until fully fermented and the aroma overflows, then take out, and finally steam slightly and dry.

[0025] Example 2

[0026] Identification process of polypeptides in fermented soybean

[0027] Preparation of the test solution: The self-made fermented soybean powder was pulverized thoroughly and passed through a 50-mesh sieve. 150 mg of the sample was accurately weighed and successively extracted with 1 mL of MeOH, 1 mL of 50% MeOH, and 1 mL of water at room temperature for 1 h each. After centrifugation at 4 °C and 13,500 rpm for 10 min, the supernatant solutions extracted with the 3 different solvents were combined for LC-MS analysis.

[0028] LC-MS analysis conditions: An Agilent 1290 Infinity UHPLC system was used, combined with a dual-jet electrospray 6550 UHD accurate mass Q-TOF mass spectrometer as the ion source to analyze the fermented soybean samples. A Waters BEH C18 chromatographic column (2.1×100 mm, 1.7 μm) was selected for chromatographic separation. The column temperature was 35 °C and the injection volume was 2 μL. The mobile phase was 0.1% formic acid solution (solvent A) and acetonitrile solution containing 0.1% formic acid (solvent B), with gradient elution (c) 0 - 6 min, 5% B; 6 - 14 min, 5% - 10% B; 14 - 21 min, 10% - 25% B; 21 - 23 min, 25% - 47% B; 23 - 26.5 min, 47% - 85% B; 26.5 - 27.5 min, 85% - 95% B; 27.5 - 28 min, 95% - 5% B), and the flow rate was set at 0.3 mL / min. The ESI source of the mass spectrometer was operated as follows: the drying gas flow rate was 10 L / min, the temperature was 325 °C, the nebulizer pressure was 35 psi, the sheath gas flow rate was 10 L / min, the temperature was 350 °C, the capillary voltage was 5000 V, and the nozzle voltage was 350 V. Non-targeted MS / MS mode was used for the acquisition of the second-stage mass spectrum, and the collision energy was set according to the formula [(slope)×(m / z) / 100 + Offset]: when the slope was 0.5, the initial collision energy was set at 12 eV; when the slope was 1, the initial collision energy was set at 20 eV; when the slope was 1.3, the initial collision energy was set at 27 eV.

[0029] The FBF algorithm (built into the Agilent MassHunter qualitative analysis software) was used to characterize the CDPs in fermented soybeans. This method is a targeted feature search algorithm using an imported database, which can achieve a more sensitive detection level and higher accuracy. It can search for compounds by extracting ions calculated according to the molecular formula from the compound database. A special *.csv file was required to be established as the chemical composition database. Therefore, a database of CDPs (cyclic dipeptides) was established. Any two of the 20 common amino acids were selected for combination, and a total of 210 cyclic dipeptides were obtained. In most cases, CDPs with the same molecular formula are isomers of each other. The database should include the molecular formula and the compound name.

[0030] The content of the *.csv file is as follows:

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037] Open the raw data file in the Agilent MassHunter qualitative analysis software and import the CDPs.csv database file in the method editor. Then, set important filtering parameters (such as the mass error of the compound being less than 5 ppm and the matching score being greater than 70%), and automatically extract the extracted ion chromatogram (EIC).

[0038] Click the Run button in the toolbar of the Agilent MassHunter qualitative analysis software to obtain all candidates that meet the set conditions in the compound list. Finally, 117 candidate compounds that meet the set conditions are obtained in the compound list. The results show that the FBF method can achieve rapid and effective analysis of CDPs in Semen Sojae Preparatum. All possible CDP molecular formulas can be listed in the csv file. Once the database file is established, it will be a major breakthrough in avoiding matrix interference. Compared with traditional manual inspection and fragmentation analysis-based methods, the FBF method makes the analysis of raw data faster and more accurate by reducing the potential interference of matrix ions.

[0039] All candidate compounds were considered potential CDPs and confirmed by analyzing their MS2 (second-stage mass spectrometry). First, the MS2 characteristics of CDPs were revealed using standards, including cyclo(Pro-Val), cyclo(Pro-Ile), cyclo(Pro-Thr), cyclo(Pro-Leu), cyclo(Pro-Pro), cyclo(Pro-Phe), and cyclo(Pro-His). Ions with m / z of 70.06, 72.08, 74.06, 120.08, 86.09, and 110.07 were observed and attributed to the imine ions of Pro (proline), Val (valine), Thr (threonine), Phe (phenylalanine), Leu / Ile (leucine / isoleucine), and His (histidine), respectively. The imine ions were used as indicators to judge the presence of specific amino acid residues in the peptide chain sequence. Table 1 lists the m / z of the imine ions of 20 common amino acids. In addition, [M+H-28] was also observed. + 、[M+H-45] + and [M+H-73] + These characteristic ions and were attributed to the neutral loss of one molecule of CO, one molecule of CO and NH 3 、and one molecule of NH 3 and two molecules of CO.

[0040] Finally, for CDPs containing the amino acid isomers Leu (leucine) and Ile (isoleucine), their liquid-phase elution information can partially support the differentiation between them. The present invention found that under the conditions of RP-LC (reversed-phase liquid chromatography), the elution time of cyclo(Pro-Leu) was later than that of cyclo(Pro-Ile). In addition, when analyzing the MS2 data, the Leu and Ile residues can also be distinguished: CDPs containing Leu showed two characteristic product ions with m / z of 86.09 and 72.04, while CDPs containing Ile showed two characteristic product ions with m / z of 86.09 and 69.07.

[0041] Next, the above-mentioned second-stage fragment characteristics were used to confirm 117 CDPs in fermented soybean. The present invention first revealed the existence of CDPs in fermented soybean.

[0042] Example 3

[0043] Based on the identified polypeptides, different preparation processes (B-F) of fermented soybean were set up.

[0044] B: a) Take 9 g of mulberry leaves and 10 g of sweet wormwood, add 18 times the amount of water, soak for 3 h, decoct 3 times, 1 h each time, and filter out the decoction; b) Mix 100 g of washed black beans into the decoction, soak for 5 h, after the decoction is completely absorbed, steam for 1 h, take out, let it cool slightly, and then place it in a container; c) Cover with the residue of the decocted sweet wormwood and mulberry leaves; d) Then carry out fermentation, the fermentation temperature is 35 °C, the relative humidity is 80%, and ferment for 6 - 8 d until the yellow mold covers all (the fermentation time is controlled according to the degree of the yellow mold covering all, and the yellow mold covering all is the sign of the end of the pre-fermentation of fermented soybean); e) Take out, wash off the yellow mold, place it in a container, and then place it in an incubator at 55 °C and keep it sealed for 15 d (mainly for anaerobic fermentation to prevent the interference of foreign miscellaneous bacteria); f) During the sealing period, take it out every 3 d, turn it over, dry it slightly, repeat 4 - 5 times, until it is fully fermented and the aroma overflows, then take it out, and finally steam it slightly and dry it.

[0045] C: a) Take 9 g of mulberry leaves and 10 g of sweet wormwood, add 18 times the amount of water, soak for 3 h, decoct 3 times, 1 h each time, and filter out the decoction; b) Mix 100 g of washed black beans into the decoction, soak for 5 h, after the decoction is completely absorbed, steam for 2 h, take out, let it cool slightly, and then place it in a container; c) Cover with the residue of the decocted sweet wormwood and mulberry leaves; d) Then carry out fermentation, the fermentation temperature is 35 °C, the relative humidity is 80%, and ferment for 6 - 8 d until the yellow mold covers all (the fermentation time is controlled according to the degree of the yellow mold covering all, and the yellow mold covering all is the sign of the end of the pre-fermentation of fermented soybean); e) Take out, wash off the yellow mold, place it in a container, and then place it in an incubator at 55 °C and keep it sealed for 15 d (mainly for anaerobic fermentation to prevent the interference of foreign miscellaneous bacteria); f) During the sealing period, take it out every 3 d, turn it over, dry it slightly, repeat 4 - 5 times, until it is fully fermented and the aroma overflows, then take it out, and finally steam it slightly and dry it.

[0046] D: a) Take 9 g of mulberry leaves and 10 g of sweet wormwood, add 18 times the amount of water, soak for 3 h, decoct 3 times, 1 h each time, and filter out the decoction; b) Mix 100 g of washed black beans into the decoction, soak for 5 h, after the decoction is completely absorbed, steam for 3 h, take out, let it cool slightly, and then place it in a container; c) Cover with the residue of the decocted sweet wormwood and mulberry leaves; d) Then carry out fermentation, the fermentation temperature is 35 °C, the relative humidity is 80%, and ferment for 6 - 8 d until the yellow mold covers all (the fermentation time is controlled according to the degree of the yellow mold covering all, and the yellow mold covering all is the sign of the end of the pre-fermentation of fermented soybean); e) Take out, wash off the yellow mold, place it in a container, and then place it in an incubator at 55 °C and keep it sealed for 15 d (mainly for anaerobic fermentation to prevent the interference of foreign miscellaneous bacteria); f) During the sealing period, take it out every 3 d, turn it over, dry it slightly, repeat 4 - 5 times, until it is fully fermented and the aroma overflows, then take it out, and finally steam it slightly and dry it.

[0047] E: a) Take 9 g of mulberry leaves and 10 g of Artemisia annua, add 18 times the amount of water, soak for 3 h, decoct 3 times, 1 h each time, and filter out the decoction; b) Mix 100 g of washed black beans into the decoction, soak for 5 h, and after the decoction is completely absorbed, steam for 1.5 h, take out, let it cool slightly, and then place it in a container; c) Cover with the residue of Artemisia annua and mulberry leaves after decoction; d) Then carry out fermentation, the fermentation temperature is 25 °C, the relative humidity is 80%, and ferment for 6 - 8 d until the yellow coating covers all (the fermentation time is controlled according to the degree of the yellow coating covering all, and the yellow coating covering all is the sign of the end of the pre-fermentation of fermented soya beans); e) Take out, wash off the yellow coating, place it in a container, and place it in an incubator at 55 °C and keep it sealed for another 15 d (mainly for anaerobic fermentation to prevent the interference of foreign miscellaneous bacteria); f) During the sealing period, take it out every 3 d, turn it over, let it dry slightly, repeat 4 - 5 times, until it is fully fermented and the aroma overflows, then take out, and finally steam slightly and dry it.

[0048] F: a) Take 9 g of mulberry leaves and 10 g of Artemisia annua, add 18 times the amount of water, soak for 3 h, decoct 3 times, 1 h each time, and filter out the decoction; b) Mix 100 g of washed black beans into the decoction, soak for 5 h, and after the decoction is completely absorbed, steam for 1.5 h, take out, let it cool slightly, and then place it in a container; c) Cover with the residue of Artemisia annua and mulberry leaves after decoction; d) Then carry out fermentation, the fermentation temperature is 35 °C, the relative humidity is 50%, and ferment for 6 - 8 d until the yellow coating covers all (the fermentation time is controlled according to the degree of the yellow coating covering all, and the yellow coating covering all is the sign of the end of the pre-fermentation of fermented soya beans); e) Take out, wash off the yellow coating, place it in a container, and place it in an incubator at 55 °C and keep it sealed for another 15 d (mainly for anaerobic fermentation to prevent the interference of foreign miscellaneous bacteria); f) During the sealing period, take it out every 3 d, turn it over, let it dry slightly, repeat 4 - 5 times, until it is fully fermented and the aroma overflows, then take out, and finally steam slightly and dry it.

[0049] The results show that ( Figure 1 and Figure 2 ), when fermenting fermented soya beans, it is more appropriate to control the steaming time within 1.5 h - 2 h, the optimal steaming time is 1.5 h, cyclic dipeptides can be produced at a fermentation humidity of 50% - 80%, the optimal fermentation humidity is 80%, and there is no significant difference in temperature between 25 °C and 35 °C (the LC-MS analysis conditions are the same as those described above, and the peak areas are calculated).

[0050] Table 1 Characteristic imine ions of 20 kinds of amino acids

[0051]

[0052] Example 4 Activity evaluation of polypeptides in fermented soya beans

[0053] Cell line: Rat adrenal pheochromocytoma cells (PC12 cells)

[0054] Preparation of Test Samples: Analytes: Accurately weigh 1.00 mg of 7 representative polypeptides (CDPs including cyclo(Pro-Ile), cyclo(Pro-Leu), cyclo(Pro-Phe), cyclo(Pro-Val), cyclo(Pro-Thr), cyclo(Pro-His) and cyclo(Pro-Pro) were synthesized by Synpeptide Co., Ltd. (Shanghai, China, purity > 95%)), dissolve them in DMSO to prepare a 10 mM stock solution, and dilute it to 20, 10, 5 μM with DMEM medium containing 10% FBS, 5% HS and 1% penicillin / streptomycin, so that the final DMSO concentration entering the cells is less than 0.05% to avoid toxicity to the cells. H 2 O 2 : Initial concentration 10 M, serially dilute it to 6600 μM (i.e., 11 times the final concentration) with blank DMEM medium.

[0055] Activity Test: Take PC12 cells in the logarithmic growth phase, after digestion with trypsin solution and centrifugation, resuspend the cells with DMEM medium containing 10% FBS, 5% HS and 1% penicillin / streptomycin, and pipette them evenly. Seed them at 1.0×10 4 cell / well in a 96-well cell culture plate, add 100 μL to each well, incubate at 37 °C and 5% CO 2 for 24 h. After that, discard the original culture medium, and divide the PC12 cells into blank, normal control group, H 2 O 2 model group, positive control group (edaravone) and experimental group. The blank group, normal control group and model group do not add drugs. Discard the original medium and add 100 μL of new medium. The experimental group and positive control group add the test drugs (20, 10, 5 μM) and pre-administer for 4 h. After 4 h, add H 2 O 2 solution (so that the final concentration of H 2 O 2 is 600 μM, the median lethal dose) 10 μL, and culture at 37 °C and 5% CO 2 for 24 h. Then add 10 μL of CCK-8 to each well, and culture at 37 °C and 5% CO 2 for 1 h. Measure the OD value at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader, and set 3 parallel replicates for each concentration. The experiment is repeated 3 times.

[0056] Data processing: Cell viability was expressed as a percentage, with the cell viability of the control group regarded as 100%. Result calculation: Cell viability = (OD value of the experimental group - OD value of the blank group) / (OD value of the normal control group - OD value of the blank group) * 100%. GraphPad software was used for data processing, and the statistical results were analyzed. The data were analyzed by one-way ANOVA and expressed as mean ± standard deviation (mean ± SD). LSD was used for comparison between groups. P < 0.01 indicated significant difference, and P < 0.001 indicated extremely significant difference. See Figure 1 .

[0057] Experimental results: Compared with the normal control group, the cell viability of the model group was significantly decreased (P < 0.001). After polypeptide intervention, the cell viability increased in a concentration-dependent manner at the concentration of 5 - 20 μM, and the cell viability was significantly increased under the treatment of 20 μM. The above experimental results showed that H 2 O 2 could oxidatively damage nerve cells, causing a large number of PC12 cell apoptosis and decreased cell viability. After polypeptide intervention, the apoptosis of PC12 cells could be effectively improved, indicating that these compounds had significant neuroprotective effects.

[0058] In summary, the polypeptides in Semen Sojae Preparatum provided by the present invention could resist oxidative stress, inhibit nerve cell apoptosis, had significant neuroprotective effects, and could be used as potential therapeutic drugs for preventing and treating neurodegenerative diseases, providing a drug option for clinical use.

[0059] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for identifying active ingredients from light fermented black beans, characterized in that: include: Liquid chromatography-mass spectrometry was used to analyze the light black beans to obtain the data file to be analyzed; Establish a database of cyclic dipeptides; Based on the FBF (Find by Formula) algorithm, potential active ingredients are screened according to the data file to be analyzed and the database of the cyclic dipeptide; The structure of the active ingredient was confirmed by MS2 analysis.

2. The method according to claim 1, characterized in that The active ingredient is at least one of cyclo(Pro-Val), cyclo(Pro-Ile), cyclo(Pro-Thr), cyclo(Pro-Leu), cyclo(Pro-Pro), cyclo(Pro-Phe) and cyclo(Pro-His).

3. The method according to claim 2, characterized in that The structure of the active ingredients was confirmed by MS2 analysis, including: Determine the MS2 characteristic fragment ions of cyclic dipeptides; Determine the iminium ion of cyclic dipeptides; Determine the retention time information of cyclic dipeptides under RP-LC (reverse phase liquid chromatography) conditions; The structure of the active ingredient is confirmed based on the MS2 characteristic fragment ions, imide ions, and retention time information.

4. A method for preparing light fermented black beans containing bioactive peptides, characterized in that: include, Take mulberry leaves and Artemisia annua, add water, soak and boil, and filter out the decoction; Add black beans to the decoction until the decoction is completely absorbed, then steam for 1.5 to 3 hours; After covering the black beans with boiled Artemisia annua and mulberry leaf residue, fermenting at a temperature of 25 to 35° C. and a relative humidity of 50 to 80% until the black beans are covered with yellow skin; After taking out and washing off the yellow coat, place it in an incubator at a temperature of 50-60°C and then keep it in a ventilated state for anaerobic fermentation; Pour out and stir during the period of steaming to allow for full fermentation.

5. The preparation method according to claim 4, characterized in that: Steam for 1.5 hours, fermentation temperature is 25℃~35℃, relative humidity is 80%.

6. Use of the light fermented black beans containing bioactive peptides obtained by the preparation method according to claim 4 or 5 in preparing a drug for preventing or improving neurodegenerative diseases.

7. Use of active ingredients I to VII in the preparation of drugs for preventing or improving neurodegenerative diseases.

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