High-yield nattokinase bacillus subtilis with biological preservation function and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses bacillus subtilis with a high nattokinase yield and a biological preservation function and application of the bacillus subtilis. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.34222. The serine protease gene of the strain carries specific mutations (SEQ ID NO: 1) which are mutated from G to C at the 217th site and the 230th site, so that the yield and the activity of nattokinase are remarkably improved. In the fermentation process, through an induction stage (38 DEG C heat preservation for 8 hours), a main fermentation stage (50 DEG C heat preservation for 7 hours) and a cooling after-ripening stage (4 DEG C heat preservation for 24 hours), the enzyme activity of the prepared natto product is larger than or equal to 57028 IU / mL, and the natto product is light in flavor, excellent in wiredrawing effect and appropriate in taste. In addition, the strain can inhibit the growth of Candida albicans (the inhibition zone is greater than or equal to 25mm), and the shelf life of natto is effectively prolonged. The problems that an existing strain is poor in stability, products are prone to pollution, and the flavor is poor are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Bacillus subtilis CGMCC No. 34222 strain with high nattokinase production and its application in natto fermentation, food preservation and functional food for preventing thrombotic diseases. Background Art

[0002] In modern society, thrombotic diseases have become a major threat to human health due to their high morbidity and mortality risk. Although traditional thrombolytic drugs (such as urokinase and streptokinase) are widely used, their short half-life, high bleeding risk, and high cost limit their clinical application. Therefore, natural thrombolytic active substances have attracted much attention, among which nattokinase has become a research hotspot due to its high thrombolytic efficiency, high safety, and food-derived properties. Nattokinase is a serine protease produced by fermentation of Bacillus subtilis. It can not only directly degrade fibrin but also activate the human plasminogen system. This dual mechanism of action gives it significant advantages in preventing thrombosis and improving blood circulation, and it is widely used in the development of functional foods and drugs.

[0003] However, there are many technical bottlenecks in the current natto production strains: First, some strains have poor genetic stability, and the production and activity of nattokinase decrease significantly after passage, resulting in batch quality fluctuations in industrial production; second, natto products produced by traditional fermentation processes have a strong ammonia smell, high viscosity or a rough taste, which makes it difficult to meet consumers' diverse demands for flavor and texture, hindering market promotion; third, most strains lack antibacterial function, and natto is easily contaminated by fungi (such as Candida albicans) during storage, leading to corruption and deterioration, shortening the product shelf life and increasing economic losses to enterprises. These problems not only restrict the large-scale development of the natto industry, but also limit the in-depth application of highly active nattokinase in the health field.

[0004] Therefore, the development of a Bacillus subtilis strain with high enzyme activity, strong genetic stability, significant antibacterial function, and the ability to improve product flavor has become an urgent need in the industry. Based on this background, the present invention aims to break through the limitations of existing technologies by screening natural mutant strains and analyzing their functional genes, combined with process innovation, and provide technical support for the efficient production of nattokinase and the development of functional foods. Summary of the Invention

[0005] The present invention aims to provide a Bacillus subtilis strain with high nattokinase production and biological preservation function, thereby solving the problems of poor stability of existing strains, easy product contamination, and poor flavor, while improving nattokinase production and product quality.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A high-yield nattokinase-producing Bacillus subtilis strain with biological preservation function is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration with the deposit number CGMCC No. 34222 and the deposit date of April 16, 2025. The strain is isolated from natto food. The colonies on TSA plate culture medium are milky white, round, and opaque. The surface has wrinkles, micro-protrusions, and irregular edges. It is Gram-positive and the bacteria are rod-shaped. The spores are mesogenous, oval, and do not expand significantly. Under a microscope, the bacteria are long rod-shaped and Gram-positive, with spores located in the middle or at both ends of the cell.

[0008] In one possible embodiment, the serine protease gene of the strain has two non-synonymous mutation sites, located at positions 217 and 230 of the gene sequence, respectively, from G to C, and its nucleic acid sequence is shown in SEQ ID NO: 1. The screening process is as follows:

[0009] A strain with a significant clearing zone for nattokinase hydrolysis was identified using the agarose-fibrinogen plate method. The genome sequence of this strain was then obtained using whole-genome sequencing. This sequence was then compared with the Bacillus subtilis genome in a public database to precisely locate differential sites within the strains. These differential sites play a key role in regulating gene function, resulting in unique physiological characteristics and functional differences between strains by influencing gene expression levels, protein structure, or enzyme activity. Functional annotation of the strains revealed the genetic location of differential sites within serine protease genes. Serine protease genes play an important role in the strain's metabolic processes, protein degradation, and regulation of nattokinase synthesis and activity. These differential sites may directly influence the strain's nattokinase hydrolysis capacity by altering the enzyme's catalytic efficiency, substrate specificity, or stability, providing important genetic insights for in-depth analysis of the strain's functional properties and optimization of nattokinase production.

[0010] In one possible embodiment, the strain adopts the following process conditions during the fermentation process:

[0011] Induction stage: product temperature 38℃, keep warm for 8 hours;

[0012] Main fermentation stage: product temperature 50℃, keep warm for 7 hours;

[0013] Cooling stage: the product temperature is reduced to 4°C and kept warm for 24 hours.

[0014] In one possible embodiment, the strain can inhibit the growth of Candida albicans (C.albicans), and the diameter of the inhibition zone is ≥25 mm.

[0015] The present invention also provides a use of a high-yield nattokinase Bacillus subtilis CGMCC No. 34222 having a biological preservation function in preparing a natto product, comprising the following steps:

[0016] S1. Soaking soybeans and then sterilizing them, inoculating the seed liquid of Bacillus subtilis CGMCC No.34222 at an inoculation rate of 2%;

[0017] S2, sequentially carrying out the induction stage, the main fermentation stage and the cooling stage, and finally obtaining the natto product.

[0018] Furthermore, the nattokinase activity of the natto product is ≥57028 IU / mL, and in sensory evaluation, the product has a light ammonia taste, an excellent stringy effect, and a suitable taste.

[0019] The present invention also provides a high-yield nattokinase Bacillus subtilis with biological preservation function

[0020] The application of CGMCC No.34222 in food preservation can extend the shelf life of natto products by inhibiting the growth of Candida albicans, a contaminating bacteria in food.

[0021] The strain provided by the present invention has the ability to secrete highly active nattokinase. The strain is fermented with beans as raw materials to obtain natto fermented products, and the application steps are as follows:

[0022] Soak soybeans overnight, drain water, sterilize under high pressure, cool to room temperature, and then inoculate with Bacillus subtilis seed liquid at a rate of 2%. After the induction stage, germination and reproduction stage, main fermentation stage and low-temperature ripening stage, natto product is obtained.

[0023] The strain provided by the present invention has a significant inhibitory effect on Candida albicans, a common contaminant in food. The main steps are: inoculating Bacillus subtilis on an MEA plate evenly coated with Candida albicans, culturing in a 37°C constant temperature incubator for 18-24 hours, and observing the antibacterial transparent zone.

[0024] The present invention also provides a method for screening high-yield nattokinase Bacillus subtilis CGMCC No. 34222 with biological preservation function, which is characterized by comprising the following steps:

[0025] S1. Screen strains with clear zones using the agarose-fibrinogen plate method;

[0026] S2. Determine the mutation site of the serine protease gene of the strain through whole genome sequencing and comparison analysis.

[0027] Furthermore, the formula of the agarose-fibrinogen plate method is: 5 mL of 1.5% agarose solution, 5 mL of 0.15% fibrinogen solution, and 100 μL of thrombin (10 U / mL).

[0028] The present invention also provides a use of high-yield nattokinase Bacillus subtilis CGMCC No. 34222 with biological preservation function in preparing functional food for preventing or treating thrombotic diseases.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. High-yield nattokinase: The nattokinase produced by fermentation of Bacillus subtilis CGMCC No.34222 has significantly higher activity than existing strains, with an enzyme activity of ≥57028IU / mL. This can meet the market demand for high-activity nattokinase products and provide high-quality raw materials for the development of functional foods and drugs for the prevention and treatment of thrombotic diseases.

[0031] 2. Antibacterial effect ensures product quality: The diameter of the inhibition zone for Candida albicans is ≥25mm. The antibacterial properties of this strain against fungal contaminants can effectively inhibit the growth of harmful microorganisms during natto production and storage, reduce microbial contamination, improve the safety and stability of natto products, and extend the shelf life of the product.

[0032] 3. Improve the quality of natto products: Natto has a light ammonia smell, excellent stringiness and a suitable taste. The natto products prepared using this strain have excellent flavor and taste, are more easily accepted by consumers, and are conducive to promoting the market promotion of natto products and promoting the development of the natto industry.

[0033] 4. Strong genetic stability: There is no significant decrease in enzyme activity and yield after subculture, which is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This figure shows the effect of the Bacillus subtilis strains described in the present invention forming a clearing zone on a fibrinogen plate during the initial screening process for nattokinase. The figure shows that among the screened strains, including CGMCC No. 34222, CICC 10460, CICC 21076, CICC 23916, and CICC 20642, the clearing zone diameters all exceeded 20 mm, indicating that they have strong fibrinolytic ability.

[0035] Figure 2Figure 2 shows the antibacterial effects of different screened strains against Candida albicans (C. albicans). The results are based on MEA plate assays. CGMCC No. 34222 formed a significant zone of inhibition with an average diameter of 25 mm, significantly outperforming the control strain, demonstrating its potential for anti-contamination applications in food preservation.

[0036] Figure 3 This graph shows the stringiness of natto products fermented with the strain CGMCC No. 34222 described in this invention. The graph shows the long, stringy strands formed when picking up the fermented natto with chopsticks, demonstrating that this strain can improve the stickiness and sensory quality of natto products.

[0037] Figure 4 This is a standard curve plotting the activity-absorbance of a lumbrokinase standard used for nattokinase activity determination. The absorbance of the standard at different concentrations was measured using the fibrin plate method, and a fitting curve was established to provide a reference standard for the quantitative determination of nattokinase activity in fermented natto. DETAILED DESCRIPTION

[0038] To further illustrate the strains of the present invention and their application effects, the technical solutions of the present invention are described in detail below with reference to the accompanying drawings. However, it should be understood that the present invention is not limited to the following embodiments, and any equivalent substitutions or modifications made within the spirit and technical principles of the present invention should be considered within the scope of protection of the present invention.

[0039] Example 1: Screening of high-yield nattokinase-producing strains

[0040] In order to screen functional Bacillus subtilis with the ability to efficiently secrete nattokinase, this example collected samples from traditional natto products and related fermentation companies' applied strains to obtain multiple candidate Bacillus subtilis strains for subsequent activity screening and evaluation.

[0041] Initial strain screening uses the fibrinogen enzymatic clearing zone assay, also known as the agarose-fibrinogen plate method, to determine activity. This method uses clearing zone formation as a visual indicator of serine protease (including nattokinase) activity and can quickly evaluate the strain's enzyme secretion capacity.

[0042] The specific steps are as follows:

[0043] First, prepare a fibrin plate for screening. Mix 1.5% (mass-to-volume ratio) agarose solution and 0.15% fibrinogen solution at a 1:1 volume ratio. Add 100 μL of 10 U / mL thrombin solution. Immediately after mixing, pour the mixture into sterile culture dishes, with a volume of approximately 10 mL per dish. Let it stand at room temperature for 1 hour to fully solidify and form a uniform, transparent fibrin gel layer.

[0044] After the plate solidifies, use a sterile 3mm diameter hole puncher to punch a hole in the center of the plate. Add 10 μL of Bacillus subtilis fermentation broth (the resulting supernatant) that has been pre-fermented for 18 hours to each test well to complete the spot inoculation of all strain samples.

[0045] Place the inoculated plates in a 37°C incubator for 18 hours to facilitate the diffusion of enzyme activity and visualize the fibrin degradation reaction. After the incubation period, observe the formation of hydrolysis clearing zones around each sample well. Use a vernier caliper or image analysis software to accurately measure the diameter of each clearing zone to evaluate and screen Bacillus subtilis strains, focusing on strains with larger hydrolysis clearing zones.

[0046] The results showed that some candidate strains, such as CGMCC No. 34222, CICC 10460, CICC 23916, CICC 20642, and CICC 21076, formed distinct clear zones on the fibrin plates. Among them, the average clear zone diameter of strain CGMCC No. 34222 was greater than 20 mm, significantly superior to the other strains, preliminarily indicating that the nattokinase activity secreted by it was significantly enhanced.

[0047] like Figure 1 As shown, CGMCC No. 34222 formed a clear, broad, and clear zone on the fibrinogen plate, demonstrating excellent hydrolysis capacity and potential industrial application. This result laid the experimental foundation for subsequent screening of highly active enzyme-producing strains.

[0048] The primary screening strain CGMCC No. 34222 in this example was isolated by the applicant's team and has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 34222.

[0049] The CICC 10460, CICC 23916, CICC 21076 and CICC 20642 primary screening strains were isolated by the applicant's team and deposited in the China Industrial Microbiology Culture Collection Center.

[0050] Example 2: Analysis of Bacillus subtilis serine protease genes

[0051] In order to further reveal the key functional differences at the molecular level of the high-yield nattokinase Bacillus subtilis strains provided by the present invention, representative strains with excellent enzyme production ability and significant antibacterial activity, including CGMCC No. 34222, CICC 10460 and CICC 21076, were specially selected for whole genome sequencing and systematic bioinformatics analysis to clarify the genetic basis of their enzyme production mechanism and provide molecular guidance for subsequent strain improvement and industrial application.

[0052] Whole-genome sequencing of each of the above strains was performed using the Illumina high-throughput sequencing platform to obtain complete chromosomal sequences and annotation information. Subsequently, strain-specific sites were identified through genome sequence alignment analysis using the NCBI public database (https: / / www.ncbi.nlm.nih.gov / ). Cluster of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were performed for the specific strain CGMCC No. 34222. Ultimately, two non-synonymous mutations in the serine protease gene of strain CGMCC No. 34222 were identified. The differential sites, at positions 217 and 230, respectively, were G to C changes. The nucleic acid sequence and differential site information are shown in SEQ ID NO: 1.

[0053] Table 1: Functional properties analysis of Bacillus subtilis CGMCC No.34222

[0054]

[0055] Example 3: Bacillus subtilis fungus inhibition experiment

[0056] To verify the natural inhibitory ability of the screened Bacillus subtilis strains against common food contaminating fungi such as Candida albicans (C. albicans), this example designed and conducted an inhibition zone assay based on the agar diffusion method to evaluate its practical application potential in food safety control.

[0057] First, prepare a culture plate for the target contaminant, Candida albicans. Activate the C. albicans slant by inoculating it onto malt extract agar (MEA) medium: 3% malt extract, 0.3% peptone, 1.5% agar, and distilled water to 1000 mL. Adjust the pH to 7.0 ± 0.2. Autoclave the prepared medium at 121°C for 15 minutes, then pour it into a sterile plate. Incubate in a 28°C incubator for 24–48 hours until the C. albicans grows and forms a uniform biofilm. Set aside.

[0058] Meanwhile, the Bacillus subtilis strains to be tested (including CICC 21076, CGMCC No. 34222, CICC 10460, CICC 23916, and CICC 20642) were inoculated into TSB liquid seed medium containing 1.7% trypticase, 0.3% soy peptone, 0.25% glucose, 0.5% NaCl, and 0.25% dipotassium phosphate. The pH was adjusted to 7.3 ± 0.2, and the medium was autoclaved at 121°C for 15 minutes and then cooled. The strains were incubated in a 37°C incubator at 200 rpm for 18 hours to obtain a highly active fermentation supernatant, which served as an antibacterial source.

[0059] The antibacterial test was conducted using the agar diffusion method. The specific steps were as follows: A sterile hole punch was used to create standard 6mm diameter holes on a pre-prepared Candida albicans culture plate. 10μL of the fermentation broth of the corresponding strain was added to each well, keeping the liquid level flush with the edge of the well to prevent overflow. After spotting, the plate was placed in a 37°C incubator for 18–24 hours, and the formation of inhibition zones for each strain was observed.

[0060] The experimental results showed that different strains had significant differences in their inhibitory effects on Candida albicans. Among them, CGMCC No. 34222 formed a significant inhibition zone with a diameter of about 25 mm on the surface of the culture medium, showing stronger antifungal activity than other strains (such as Figure 2 This antibacterial effect indicates that CGMCC No. 34222 can inhibit the growth of Candida albicans through its metabolites and synthesize proteins or small molecules with antibacterial or antifungal activities.

[0061] In summary, strain CGMCC No.34222 not only has a high nattokinase secretion ability, but also exhibits excellent natural inhibitory ability against Candida albicans. It can effectively inhibit the growth of common contaminating fungi in the production, storage and transportation of fermented products such as natto, thereby improving the hygiene safety and shelf life of the products, and has important practical application value.

[0062] Example 4: Production of Natto by Bacillus subtilis Fermentation and Evaluation

[0063] To verify the applicability and fermentation performance of the high-yield nattokinase Bacillus subtilis CGMCC No. 34222 provided by the present invention in the actual natto fermentation process, a natto production experiment using soybeans as the base material was carried out, and the enzyme activity and sensory quality of the produced natto products were measured.

[0064] 1. Seed Solution Preparation

[0065] Fresh, well-grown strain CGMCC No. 34222 was selected from the preserved slant and inoculated into TSB liquid seed medium. The medium formulation was consistent with that in Example 2, comprising: 1.7% trypticase peptone, 0.3% soy peptone, 0.25% glucose, 0.5% NaCl, and 0.25% dipotassium hydrogen phosphate, with the pH adjusted to 7.3 ± 0.2. After inoculation, the medium was incubated at 37°C and 200 rpm in a shaker for 18 hours to obtain a highly active Bacillus subtilis seed solution, which served as the inoculum for subsequent fermentation.

[0066] 2. Raw material processing

[0067] Select high-quality soybeans that are intact, plump, and free of mold. Wash and remove impurities, then soak in room temperature water for 18 hours to allow them to fully absorb and swell. Drain thoroughly, place in an autoclave, and autoclave at 120°C for 30 minutes to kill any bacteria. Cool to room temperature before use.

[0068] 3. Fermentation process control

[0069] Place the soybeans on a plate and add the seed solution prepared above at a ratio of 2% of the soybean weight. Mix well and then ferment under controlled temperature. The entire fermentation process is divided into three stages for temperature program control:

[0070] Induction stage: The inoculated sample was kept at 38°C for 8 hours to promote rapid adaptation of the strain to the environment and early enzyme expression;

[0071] Main fermentation stage: The temperature is then raised to 50°C and kept warm for 7 hours to enhance the synthesis and accumulation of nattokinase;

[0072] Cooling stage: Rapidly lower the temperature to 4°C and keep it at low temperature for 24 hours to facilitate the formation of product flavor and the stabilization of microbial metabolism.

[0073] 4. Post-ripening treatment

[0074] After the fermentation is completed, the fermented sample is moved to a 4°C refrigerator for post-ripening for 24 to 48 hours to promote the formation of good surface stickiness and complete flavor structure of natto, and finally the finished natto (such as Figure 3 shown).

[0075] 5. Nattokinase Activity Assay

[0076] Weigh 5g of sample from mature natto, add 20mL of sterile distilled water, and shake at 37℃ and 200rpm for 2 hours. Centrifuge the extract at 12000rpm for 10 minutes, take the supernatant as the crude enzyme solution, and use the fibrin plate method to determine the nattokinase activity. The enzyme activity is calculated by the size of the transparent zone combined with the standard curve drawn by the lumbrokinase standard (such as Figure 4The results showed that the activity of nattokinase obtained by fermentation of strain CGMCC No.34222 was as high as 57028IU / mL, which was much higher than that of common commercial strains and had a significant production efficiency advantage.

[0077] 6. Sensory quality evaluation

[0078] The products were placed in a standard sensory evaluation environment, and a five-person panel evaluated them based on factors such as odor, string length, surface mucilage, and mouthfeel. The results showed that natto produced with CGMCC No. 34222 had a mild ammonia smell, excellent stringiness (>6cm), a distinct and uniform surface mucilage, a moderately dry but not astringent taste, and a pleasant chewiness, resulting in a high overall acceptance rate, meeting modern consumers' demand for foods that combine health and flavor.

[0079] In summary, Example 4 fully verified the high enzyme production capacity, excellent sensory properties and stable fermentation adaptability of the strain CGMCC No. 34222 of the present invention in the actual fermentation production of natto, and is suitable for the development of industrial functional natto products, with good application prospects and commercial promotion value.

[0080] The present invention systematically demonstrates a series of process flows and technical parameters of the high-yield nattokinase Bacillus subtilis CGMCC No.34222 strain in activity screening, gene identification, antibacterial verification, and natto product fermentation process through specific embodiments. The above embodiments not only verify that the strain has significant advantages in secreting nattokinase, but also further confirm its comprehensive performance in food safety control, flavor improvement, and product stability through functional annotation and application experiments at the molecular level. In particular, in the large-scale production of functional natto products, the strain exhibits good fermentation adaptability and process compatibility, and has a technology promotion value that is significantly superior to existing strains. Based on the relevant technical solutions and implementation methods of the present invention, a more efficient, safe, and stable biological solution can be provided for the development of nattokinase functional foods, which has broad application prospects in the fields of modern health foods, biological health products, and the food industry.

[0081] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A high-yield nattokinase Bacillus subtilis with biological preservation function, characterized by: Deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.34222 and the deposit date April 16, 2025.

2. The high-yield nattokinase Bacillus subtilis CGMCC No. 34222 according to claim 1, wherein: The serine protease gene of the strain has two non-synonymous mutation sites, located at positions 217 and 230 of the gene sequence, respectively, mutating from G to C. The nucleic acid sequence is shown in SEQ ID NO:

1.

3. The high-yield nattokinase Bacillus subtilis CGMCC No. 34222 according to claim 1, wherein: The strain was fermented under the following process conditions: Induction stage: product temperature 38℃, keep warm for 8 hours; Main fermentation stage: product temperature 50℃, keep warm for 7 hours; Cooling stage: the product temperature is reduced to 4°C and kept warm for 24 hours.

4. The high-yield nattokinase Bacillus subtilis CGMCC No. 34222 according to claim 1, wherein: The strain can inhibit the growth of Candida albicans, and the diameter of the inhibition zone is ≥25 mm.

5. The use of the high-yield nattokinase Bacillus subtilis CGMCC No. 34222 according to claim 1 in preparing natto products, characterized in that: The following steps are involved: S1. Soaking soybeans and then sterilizing them, inoculating the seed liquid of Bacillus subtilis CGMCC No.34222 at an inoculation rate of 2%; S2, sequentially carrying out the induction stage, the main fermentation stage and the cooling stage, and finally obtaining the natto product.

6. The use according to claim 5, characterized in that: The nattokinase activity of the natto product is ≥57028 IU / mL, and in sensory evaluation, the ammonia taste is light, the stringy effect is excellent, and the taste is suitable.

7. The use of the high-yield nattokinase Bacillus subtilis CGMCC No. 34222 in food preservation according to claim 1, characterized in that: By inhibiting the growth of Candida albicans, a contaminating bacteria in food, the shelf life of natto products can be extended.

8. The screening method of the high-yield nattokinase Bacillus subtilis CGMCC No. 34222 according to claim 1, wherein: The following steps are involved: S1. Screen strains with clear zones using the agarose-fibrinogen plate method; S2. Determine the mutation site of the serine protease gene of the strain through whole genome sequencing and comparison analysis.

9. The screening method according to claim 8, wherein: The formula of the agarose-fibrinogen plate method is: 5 mL of 1.5% agarose solution, 5 mL of 0.15% fibrinogen solution, and 100 μL of thrombin (10 U / mL).

10. Use of the high-yield nattokinase Bacillus subtilis CGMCC No. 34222 according to claim 1 in preparing functional foods for preventing or treating thrombotic diseases.

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