Bacillus subtilis GH1 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a bacillus subtilis (Bacillus subtilis) GH1 and application thereof. Bacillus subtilis The strain provided by the application has high safety, can improve the content of various active substances when used for fermenting coix seed, can effectively protect the stability of active substances such as plasmin and tetramethylpyrazine in the freeze-drying process in combination with the protection of vitamin C, and can effectively regulate the taste characteristics of the product, reduce the bitter aftertaste and astringent aftertaste.

Description

[0001] Technical field preparation This invention belongs to the field of microbial technology, specifically a Bacillus subtilis (… Bacillus subtilis GH1 and its applications. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are among the leading threats to human health and life worldwide. The "China Cardiovascular Health and Disease Report 2024" shows that the number of people suffering from cardiovascular diseases in my country has reached 330 million, including 245 million with hypertension, and over ten million each with coronary heart disease and stroke. Cardiovascular diseases account for nearly 50% of all deaths, ranking first among all diseases. Faced with such a severe situation, actively exploring safe and effective functional foods and active ingredients has become an important direction in the prevention and treatment strategies for cardiovascular and cerebrovascular diseases.

[0003] Ligustrazine, as a bioactive alkaloid, has the effects of dilating blood vessels, inhibiting platelet aggregation, and improving microcirculation. Meanwhile, fibrinolytic enzymes are proteases that can directly degrade fibrin, effectively dissolving thrombi and playing a crucial role in the prevention and treatment of cardiovascular and cerebrovascular diseases. The two have complementary mechanisms of action in thrombolysis and antithrombosis: ligustrazine focuses on improving blood circulation and inhibiting platelet aggregation, while fibrinolytic enzymes directly act on fibrin to degrade thrombi. Their synergy allows for a more comprehensive protective effect on cardiovascular and cerebrovascular health. (Bacillus spp.) Bacillus Microorganisms (MICs) are a common and dominant group of microorganisms in fermented foods, possessing good food safety and the ability to metabolize active substances. Previous research by our group has found that... B. subtilis BJ3-2 Fermented Job's tears can accumulate ligustrazine and fibrinolytic enzymes. Therefore, B. subtili *S. s* is an excellent microbial resource for the production of tetramethylpyrazine and fibrinolytic enzyme. Currently, reports of bifunctional Bacillus strains capable of simultaneously producing high levels of both tetramethylpyrazine and fibrinolytic enzyme are still relatively scarce, especially systematic studies on the synergistic accumulation of the two active ingredients by the same strain in the same fermentation system are rare.

[0004] In recent years, fermented foods and their functional active ingredients have received widespread attention, and the application of health foods in the prevention and treatment of chronic diseases is becoming a cutting-edge hot topic in food science research. Therefore, the development of functional fermented foods rich in both tetramethylpyrazine and fibrinolytic enzymes has significant theoretical value and broad application prospects.

[0005] Furthermore, thrombosis is also closely related to gut health. Studies have shown that an imbalance in the gut microbiota can lead to an increased Firmicutes / Bacteroidetes ratio and an increase in pro-inflammatory bacteria, resulting in increased local inflammation and permeability in the gut. This triggers a low-grade systemic inflammatory response, which in turn promotes thrombosis. However, current health supplements or functional foods for preventing thrombosis mainly target single cardiovascular targets, neglecting the link between gut microbiota and thrombosis risk. Summary of the Invention

[0006] To address the lack of systematic studies on the synergistic accumulation of tetramethylpyrazine and fibrinolytic enzymes by the same bacterial strain in the same fermentation system, and the absence of "intestinal thrombosis"-related health-promoting natural foods, this invention screens and obtains a Bacillus strain with high production of both tetramethylpyrazine and fibrinolytic enzymes. This strain possesses both high fibrinolytic enzyme activity and high tetramethylpyrazine production capacity. Using this strain to ferment Job's tears, a standardized method for synergistic high production of tetramethylpyrazine and fibrinolytic enzymes in fermented Job's tears and a method for producing solid beverages were constructed.

[0007] Specifically, this is achieved through the following technical solutions: In a first aspect, the present invention provides a Bacillus subtilis ( Bacillus subtilis GH1, the Bacillus subtilis GH1, was deposited at the China General Microbiological Culture Collection Center on May 8, 2026, with accession number: CGMCC NO.38585.

[0008] The Bacillus subtilis GH1 was isolated from fermented soybeans.

[0009] The Bacillus subtilis GH1 was grown on LB agar medium at 37°C. The colonies were round, slightly yellow, dry, opaque, irregular in shape, and had irregular edges. The Gram staining test was positive (+), and the bacteria were rod-shaped under a microscope. The hydrogen sulfide test was positive, while the indole test and nitrate reduction test were negative.

[0010] The second invention provides the application of Bacillus subtilis GH1 in increasing the content of active ingredients in coix seed fermentation products, wherein the active ingredients are at least one of tetramethylpyrazine, fibrinolytic enzyme, γ-aminobutyric acid, coixol, polyphenols, flavonoids, triterpenes and coixol ester.

[0011] And the application of Bacillus subtilis GH1 in the enrichment of tetramethylpyrazine and fibrinolytic enzyme.

[0012] Thirdly, the present invention provides a method for preparing fermented Job's tears products, comprising the following steps: (1) Activation of strain: Bacillus subtilis GH1 with preservation number CGMCC No. 38585 was activated, and after passing the purity test, it was cultured on slant for later use; (2) Seed culture preparation: Bacillus subtilis GH1 was inoculated into LB broth medium and cultured in shake flasks to obtain a bacterial suspension. The suspension was then separated by centrifugation, and the bacterial cells were precipitated and washed with physiological saline until the bacterial concentration reached 1.0 × 10⁻⁶. 8 CFU / mL ~ 1.8×10 8 CFU / mL, which yields the seed solution; The conditions for the shake flask culture were: a culture temperature of 37 ℃, a shake speed of 180 r / min, and a culture time of 24 h; the conditions for the centrifugation were: a speed of 10000 rpm and a time of 5 min. (3) Solid-state fermentation: After soaking the washed Job's tears overnight, add ultrapure water for sterilization. Then, according to the inoculation amount (v / m) being 5%-9% of the Job's tears mass, inoculate the seed liquid obtained in step (2) into the sterilized Job's tears, mix well, and then ferment in a solid state at 34℃-40℃ for 72-96 h to obtain the fermentation product. The sterilization conditions are as follows: time is 15 to 35 minutes, preferably 30 minutes; material-to-water ratio (m / v) is 1:(1.4 to 1.8), preferably 1:1.6; Preferably, the solid-state fermentation conditions are: inoculum size (v / m) of 7%, fermentation temperature of 37°C, and fermentation time of 87 hours. (4) Preparation of freeze-dried powder: Add ascorbic acid to the fermentation product and mix evenly. Then freeze-dry, pulverize and pass through a 40-mesh sieve to obtain fermented Job's tears freeze-dried powder, and seal and refrigerate at 4°C for later use.

[0013] The amount of ascorbic acid added is 0.1% to 1.0% of the mass of the fermentation product.

[0014] The freeze-drying refers to first pre-freezing the fermentation product mixed with ascorbic acid at -80 ℃ for 4 to 8 hours, and then drying it for 36 to 60 hours under the conditions of cold trap temperature of -50 ℃ to -40 ℃ and vacuum degree of 2 to 5 Pa.

[0015] Preferably, the freeze-drying refers to dispensing the fermentation product mixed with ascorbic acid into a petri dish with a thickness of 9 mm, pre-freezing it at -80°C for 6 hours, and then drying it at a cold trap temperature of -50°C and a vacuum degree of 3 Pa for 48 hours.

[0016] Fourthly, the present invention provides a fermented Job's tears solid beverage, which is obtained by mixing fermented Job's tears freeze-dried powder and probiotic powder, adding a binder, and extruding and granulating the mixture; wherein the fermented Job's tears freeze-dried powder is obtained by solid-state fermentation of Job's tears by Bacillus subtilis GH1 with preservation number CGMCC No. 38585, followed by mixing with ascorbic acid and freeze-drying.

[0017] The binder is maltodextrin, and the addition of maltodextrin accounts for 15% to 20% of the total mass of the fermented Job's tears solid beverage.

[0018] The mass ratio of the fermented Job's tears powder to the probiotic powder is (3~7):(7~3), preferably 7:3.

[0019] Beneficial effects 1. This invention provides a strain of Bacillus subtilis GH1 (CGMCC NO.38585), which is negative in hydrogen sulfide, indole, and nitrate reduction tests, indicating high safety and suitability for functional food fermentation. This strain possesses the dual functions of high fibrinolytic enzyme activity and high tetramethylpyrazine production. When fermenting Job's tears, the fibrinolytic enzyme activity reaches 1437.195 U / g, the acetoin yield reaches 10.433 mg / g, and the tetramethylpyrazine yield reaches 1.32 mg / g, significantly superior to the previously reported Bacillus subtilis BJ3-2.

[0020] 2. This invention optimized the process parameters of Bacillus subtilis GH1 fermentation of Job's tears using single-factor and response surface methodology. The optimal conditions were: inoculum size 7%, material-to-water ratio 1:1.6, fermentation temperature 37℃, and fermentation time 87 h. Under these conditions, the fibrinolytic enzyme activity in fermented Job's tears reached 2088.28 U / g, and the yield of tetramethylpyrazine reached 4.10 mg / g DW, achieving synergistic high yield of two active ingredients by the same strain in the same fermentation system.

[0021] 3. The fermented Job's tears product of this invention exhibits a significantly increased content of active substances. Specifically, the γ-aminobutyric acid (GABA) content is 6.21 mg / g, which is 310.5 times that of unfermented Job's tears. The coixol content is 7.54 μg / g, which is 4.31 times that of unfermented Job's tears. The polyphenol and flavonoid contents are 2.37 mg GAE / g and 3.32 mg RE / g, respectively, which are 8.78 and 6.64 times that of unfermented Job's tears. Furthermore, the contents of triterpenes and coixol esters are also significantly increased. This is attributed to the favorable fermentation characteristics of Bacillus subtilis GH1, and also to the addition of ascorbic acid (VC) as a protective agent during freeze-drying, which significantly improves the stability of active substances, especially fibrinolytic enzymes and tetramethylpyrazine. The fibrinolytic enzyme activity in freeze-dried coix seed powder with added ascorbic acid ranged from 6781.59 to 8254.33 U / g DW, an increase of 23% to 40% compared to the unadded group; its tetramethylpyrazine content ranged from 3.27 to 3.74 mg / g DW, an increase of 0.22% to 16.9% compared to the unadded group. After storage at -20℃ for 4 weeks, the enzyme activity retention rate remained as high as 93%, effectively solving the problem of easy inactivation of active ingredients during processing and storage.

[0022] 4. This invention combines fermented Job's tears freeze-dried powder with probiotic powder and adds 15%~20% maltodextrin to prepare a solid beverage. This beverage exhibits extremely strong fibrin degradation ability in in vitro thrombolysis experiments. The 3:7 (probiotic powder: fermented Job's tears freeze-dried powder) ratio group showed a thrombus residual rate of only 4.08% after 9 hours, comparable to the urokinase-positive control group. Simultaneously, this ratio group showed an 83.42% inhibition rate against COX-2, demonstrating significant anti-inflammatory activity; a DPPH free radical scavenging rate of 94.81% and an ABTS scavenging rate of 99.47%, also demonstrating good antioxidant capacity.

[0023] 5. The solid beverage prepared by this invention exhibits excellent tetramethylpyrazine release and fibrinolytic enzyme gastrointestinal stability in in vitro simulated digestion. In the 3:7 ratio group, the tetramethylpyrazine content still reached 1.50 mg / g DW at the end of intestinal digestion, and the fibrinolytic enzyme activity remained at 1304.22 U / g, still possessing significant thrombolytic efficacy. The product is rich in basic nutrients, and the addition of Lactobacillus reuteri freeze-dried powder effectively modulates the product's taste characteristics, reducing bitter and astringent aftertastes. Attached Figure Description

[0024] Figure 1 The activity of fibrinolytic enzyme and the content of acetoin in coix seed fermented by different strains were investigated. Figure 2 The images show the colony morphology and microscopic examination of the bacterial strain. Figure 3 This is a growth curve of the strain; Figure 4 Phylogenetic tree of strain GH1; Figure 5 For the inoculum amount (A) and feed-to-water ratio (B) B. subtilis Effects of GH1 fermentation on the yield of ligustrazine and the activity of fibrinolytic enzyme; Figure 6 For the effect of fermentation temperature (A) and time (B) on B. subtilis Effects of GH1 fermentation on the yield of ligustrazine and the activity of fibrinolytic enzyme; Figure 7 The changes in cellulase levels during the storage of fermented Job's tears powder; Figure 8 The content of probiotic live bacteria count, fibrinolytic enzyme activity, and ligustrazine in fermented coix seed solid beverage; Figure 9 The content of active ingredients in fermented Job's tears solid beverage; Figure 10 The in vitro thrombolytic effect of fermented Job's tears probiotic solid beverage. Detailed Implementation

[0025] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0026] Example 1: Screening of superior strains with high enzyme activity in fermented black soybeans (1) Separation and purification: Weigh 10 g of fermented soybeans and rice straw samples (pre-ground and mixed), add 90 mL of sterile physiological saline, and shake at 200 r / min for 30 min in a constant temperature shaker at 37 ℃ to fully elute the microorganisms in the sample. After standing, take 1 mL of the supernatant and prepare 10 μL of the solution using a 10-fold serial dilution method. -1 ~ 10 -6 Series of diluents. Take 10 μL of each... -4 10 -5 10 -6 100 μL of each dilution solution was spread onto LB agar plates and incubated upside down in a 37 ℃ incubator for 24–48 h. Colony growth was observed, and single colonies with significant morphological differences were selected for streak isolation and purification. The purified strains were then transferred to slant agar for storage and future use.

[0027] (2) Safety Performance Evaluation: During the natural fermentation of soy products, harmful substances such as H2S, indole, and nitrite are produced by microorganisms. These substances not only affect the aroma and color of the product but also reduce its palatability and safety, posing a safety hazard to fermented soy products. Therefore, H2S, indole, and nitrite are used as important indicators for evaluating the safety of fermented soy products. Thus, the strains initially screened in the above steps were subjected to safety tests, including hydrogen sulfide, indole, and nitrate reduction tests (results are shown in Table 1).

[0028] ① Hydrogen sulfide test: Sterilize and dry filter paper strips are immersed in a sterile, saturated lead acetate solution. After ensuring the filter paper strips fully absorb the solution, they are removed and placed in a sterile Petri dish for further drying. The strain to be tested is then inoculated into nutrient broth medium and cultured under suitable conditions. After the strain has fully cultured, the pre-prepared lead acetate filter paper strips are removed and suspended in a test tube containing culture medium, and allowed to stand for 1-2 days in a suitable incubation environment. If the filter paper strips turn black or brownish-black during the test, the result is considered positive, indicating that the strain produces hydrogen sulfide.

[0029] ② Indole test: Inoculate the bacterial strain into peptone water medium and incubate at 37 ℃ for 24 h. After incubation, add 1-2 mL of diethyl ether to the culture medium, followed by indole reagent, and gently shake the test tube. If the liquid in the test tube turns red, the result is considered positive, indicating that the strain produces indole.

[0030] ③ Nitrate reduction test: Inoculate the strain into a test tube containing nitrate medium and incubate at 37 °C for 24 h. Then add one drop of nitrate reduction reagent solution A and solution B to each test tube. When the culture solution turns red or brown, the test result is positive, indicating that the strain has the ability to reduce nitrate.

[0031] According to Table 1, GH1 and GD-5 are strains that are expected to be relatively safe in the experiment.

[0032] Table 1 Safety evaluation of different strains

[0033] Note: "-" indicates negative, and "+" indicates positive.

[0034] (4) Screening of fibrinolytic enzyme and tetramethylpyrazine-producing strains The isolated strain and laboratory-preserved bacteria were fermented separately in Job's tears, and the fibrinolytic enzyme activity was determined using the fibrin plate method. 10 μL of the sample was spotted onto a fibrin plate and reacted at 37 ℃ for 18 h. The two diameters of the dissolution zone were measured perpendicularly, and the area of ​​the dissolution zone was calculated. The fibrinolytic enzyme activity was calculated based on the standard curve equation. 1 g of sample was weighed, and 14 mL of water was added for ultrasonic extraction for 20 min. After centrifugation, the volume was adjusted to 15 mL. 100 μL of the extract was taken, and 2.4 mL of water, 0.5 mL of 0.5% creatine solution, 0.5 mL of 5% 1-naphthol solution, and 0.5 mL of 10% NaOH solution were added. The mixture was stirred and incubated at 30 ℃ for 1 h. The absorbance was measured at 520 nm. A series of acetoin standard solutions of different concentrations were used for the same method, and a concentration-absorbance standard curve was plotted to calculate the acetoin content in the sample. Figure 1As shown, all 10 Bacillus strains produced acetoin and also possessed fibrinolytic activity. Among them, strain GH1 showed the best effect, with fibrinolytic activity of 1437.195 U / g, acetoin content of 10.433 mg / g, and tetramethylpyrazine content of 1.32 mg / g in fermented Job's tears. Under the same fermentation conditions, both fibrinolytic activity and acetoin content were higher than those of the previously reported Bacillus subtilis BJ3-2. Figure 1 Except for BJ3-2, all of them were isolated from fermented soybeans.

[0035] Example 2: Identification of superior strains with high enzyme activity in fermented black soybeans (1) The high fibrinolytic enzyme activity of the excellent strains screened in Example 1 were identified in the order of morphological identification → growth curve plotting → molecular biological identification.

[0036] (2) Morphological identification: The colony morphology of the selected strains after growing on agar plates for 24 h was observed and recorded. Gram staining and methylene blue staining were performed on the strains to observe their morphology, arrangement, special structures and staining reactions under a microscope.

[0037] (3) Growth curve plotting: The growth curve was determined using photoelectric turbidimetry. Bacteria were sampled every 2 hours, and their OD values ​​were measured. 600 A growth curve was plotted using absorbance values ​​in nm, with incubation time on the x-axis and OD values ​​on the y-axis.

[0038] (4) Molecular biological identification: The purified bacterial strain was sequenced for 16S rDNA and ITS region. The sequencing results were compared with the sequence in the ribosome database. The homology of the sequence was compared with the sequences of related strains in NCBI. A phylogenetic tree was constructed using MAGA software.

[0039] Colony morphology and microscopic images of the strain are shown below. Figure 2 As shown in the figure, the growth curve is as follows: Figure 3 As shown, molecular biological identification confirmed that this strain is Bacillus subtilis (B. subtilis). Bacillus subtilis ), named Bacillus subtilis GH1 (e.g. Figure 4 (As shown), its nucleotide sequence is as follows:

[0040] Example 3: Optimization of Fermented Job's Tears Process Conditions (1) Activation of strain: Bacillus subtilis GH1 was activated, purified and cultured on slant after passing the purity test, and stored for later use.

[0041] (2) Seed culture preparation: Select fully activated strains and culture them in LB broth medium with shaking at 37 ℃ for 24 h to obtain the expanded seed culture for later use.

[0042] (3) Optimization of fermentation conditions for Job's tears: Single-factor experiments were conducted to optimize the material-to-water ratio (m / v), inoculum size (v / m), fermentation time, and fermentation temperature. Results are shown below. Figure 5 and Figure 6 At a material-to-water ratio of 1:1.6 (m / v), an inoculum size of 7% (v / m), a temperature of 37℃, and a fermentation time of 84 h, both the content of tetramethylpyrazine and the activity of fibrinolytic enzyme were relatively high. Based on the single-factor experiments, four factors were selected: material-to-water ratio (v / m), inoculum size (v / m), fermentation temperature, and fermentation time. A three-level response surface methodology was designed using the Box-Behnken principle. Design Expert software was used for experimental design and data analysis. The optimal fermentation conditions were determined through model fitting and optimization, with tetramethylpyrazine content and fibrinolytic enzyme activity as the response values. The factors and levels of the response surface methodology are shown in Table 2. The results of the response surface methodology experiment are shown in Table 3. Based on the response surface model predictions, B. subtilis The theoretically optimal process parameters for high-yield tetramethylpyrazine production from GH1 fermented coix seed are: material-to-water ratio (m / v) 1:1.6, inoculum size (v / m) 7%, fermentation temperature 40 ℃, and fermentation time 91 h. Under these conditions, the predicted yield of tetramethylpyrazine is 4.533 mg / g DW. B. subtilis The theoretically optimal process parameters for high-yield ligustrazine and thiolytic enzyme production from GH1 fermented Job's tears are: a material-to-water ratio (m / v) of 1:1.6, an inoculum size (v / m) of 7%, a fermentation temperature of 37 ℃, and a fermentation time of 87 h. Under these conditions, the predicted thiolytic enzyme activity is 2128.54 U / g. Since the thiolytic enzyme activity decreases sharply at 40 ℃ and 96 h, and TMP still maintains a relatively high yield at 37 ℃ and 87 h of fermentation, the final fermentation conditions were selected as a material-to-water ratio (m / v) of 1:1.6, an inoculum size (v / m) of 7%, a fermentation temperature of 37 ℃, and a fermentation time of 87 h, in order to achieve the best overall production of ligustrazine and thiolytic enzyme.

[0043] Table 2 B. subtilis Fermentation factor level table of fermented Job's tears

[0044] Table 3 B. subtilis Box-Behnken optimization results of GH1 fermentation process for producing TMP and fibrinolytic enzyme from Job's tears.

[0045] Example 4: Preparation of Fermented Job's Tears Powder (1) Activation of strain: Bacillus subtilis GH1 was activated, purified and cultured on slant after passing the purity test, and stored for later use.

[0046] (2) Seed culture preparation: Fully activated bacterial strains were picked and cultured in LB broth medium at 180 r / min and 37 ℃ in a shaker until the logarithmic growth phase to obtain a bacterial suspension. The suspension was then centrifuged at 10000 rpm for 5 min to precipitate the bacterial cells, which were then washed with physiological saline until the bacterial concentration was 1.6 × 10⁻⁶. 8 CFU / mL was used to obtain the seed culture for later use.

[0047] (3) Coix seed fermentation: Soak the washed coix seeds overnight, add ultrapure water at a ratio of 1:1.6, sterilize for 30 minutes, and then inoculate the seed liquid obtained in step (2) into the sterilized coix seeds at a ratio of 7% of the mass of coix seeds (v / m). After mixing, ferment at a fermentation temperature of 37 ℃ for 87 h to obtain the fermented coix seed sample, which is denoted as GA-87.

[0048] Example 5: Preparation of Fermented Job's Tears Freeze-Dried Powder Ascorbic acid (VC) was mixed with fermented Job's tears sample GA-87 at a concentration of 0.5%, and dispensed into petri dishes to a thickness of 9 mm. The mixture was pre-frozen at -80 °C for 6 h, then freeze-dried in a freeze dryer at a cold trap temperature of -50 °C and a vacuum of 3 Pa for 48 h. After drying, the freeze-dried sample was ground and sieved through a 40-mesh sieve, then placed in a sealed container and refrigerated at 4 °C for later use. The resulting freeze-dried Job's tears powder sample was designated GA-VC.

[0049] Example 6: Preparation of Fermented Job's Tears Solid Beverage Bacillus subtilis-fermented Job's tears powder (GA-VC) was mixed with 18% maltodextrin as a binder and then extruded and granulated to obtain a fermented Job's tears solid beverage, denoted as GAP.

[0050] Comparative Example 1: Coix seed fermented with Bacillus subtilis GH1 (1) Activation of strain: Bacillus subtilis GH1 was activated, purified and cultured on slant after passing the purity test, and stored for later use.

[0051] (2) Seed culture preparation: Fully activated bacterial strains were picked and cultured in LB broth medium at 180 r / min and 37 ℃ for 24 h to obtain a bacterial suspension. The suspension was then centrifuged at 10000 rpm for 5 min to precipitate the bacterial cells, which were then washed with physiological saline until the bacterial concentration was 1.6 × 10⁻⁶. 8CFU / mL was used to obtain the seed culture for later use.

[0052] (3) Coix seed fermentation: Soak the washed coix seed overnight, add ultrapure water at a material-to-water ratio (m / v) of 1:1.6, sterilize for 30 minutes, and then inoculate the seed liquid obtained in step (2) into the sterilized coix seed at an inoculation amount (v / m) of 7%, mix well, and ferment for 0 hours to obtain an unfermented coix seed sample, which is denoted as GA-0.

[0053] Comparative Example 2: Bacillus subtilis BJ3-2 fermented Job's tears (1) Activation of strain: Bacillus subtilis BJ3-2 was activated, purified and cultured on slant after passing the purity test, and stored for later use.

[0054] (2) Seed culture preparation: Fully activated bacterial strains were picked and cultured in LB broth medium at 180 r / min and 37 ℃ for 24 h to obtain a bacterial suspension. The suspension was then centrifuged at 10000 rpm for 5 min to precipitate the bacterial cells, which were then washed with physiological saline until the bacterial concentration was 1.6 × 10⁻⁶. 8 CFU / mL was used to obtain the seed culture for later use.

[0055] (3) Coix seed fermentation: Washed coix seed overnight, add ultrapure water at a material-to-water ratio of 1:1.6, sterilize for 30 min, then inoculate the seed liquid obtained in step (2) into the sterilized coix seed at an inoculation amount (v / m) of 7%, mix well, and ferment at a fermentation temperature of 37 ℃ for 87 h to obtain the fermented coix seed sample, which is denoted as BA-87.

[0056] Comparative Example 3: Preparation of freeze-dried coix seed fermented with Bacillus subtilis GH1 Fermented Job's tears sample GA-87 was aliquoted into petri dishes, approximately 9 mm thick, and pre-frozen at -80 ℃ for 6 h. Then, it was freeze-dried for 48 h in a freeze dryer at a cold trap temperature of -50 ℃ and a vacuum of 3 Pa. After drying, the freeze-dried sample was ground and sieved through a 40-mesh sieve, then placed in a sealed container and refrigerated at 4 ℃ for later use. The resulting freeze-dried Job's tears powder sample was denoted as GA.

[0057] Comparative Example 4: Preparation of a solid beverage made from fermented Job's tears using Bacillus subtilis GH1 Probiotic powder (LAP) and Bacillus subtilis fermented coix seed powder (GA-VC) are mixed in different proportions (LAP:GAP=3:7, 4:6, 5:5, 6:4, 7:3), and 18% maltodextrin is added as a binder. The mixture is then extruded and granulated to obtain a fermented coix seed solid beverage.

[0058] The specific embodiments are compared with the comparative examples below. Example 4 was compared with Comparative Example 1. (1) Analysis of basic physicochemical indicators Moisture content is a key factor in the fermentation process of Job's tears, directly affecting the growth and metabolism of microorganisms and the activity of related enzymes. Proteins, fats, and carbohydrates are not only flavor precursors in food but can also be transformed into unique flavors under the action of microorganisms. Table 4 shows that with prolonged fermentation, the moisture content of the Job's tears samples increased, while the fat and carbohydrate content decreased. The increase in moisture content is mainly due to the aerobic respiration of Bacillus subtilis GH1 during its growth and metabolism, which decomposes organic matter such as carbohydrates and produces water. Simultaneously, during fermentation, extracellular enzymes secreted by Bacillus subtilis GH1 continuously hydrolyze large-molecule nutrients, generating smaller molecules such as sugars, peptides, and amino acids. These smaller molecules have stronger hydrophilicity, enhancing the water-holding capacity of the material and leading to a decrease in carbohydrate content. The moisture content of GA-87 sample was 77.09%, and the contents of protein, ash, fat and carbohydrates were 5.29 g / 100 g, 2.19 g / 100 g, 2.39 g / 100 g and 13.06 g / 100 g, respectively.

[0059] Table 4. Analysis of basic physicochemical properties of different samples

[0060] (2) Analysis of active ingredients The changes in the active components of Job's tears fermented by Bacillus subtilis GH1 are shown in Table 5. Compared with GA-0, the contents of GABA, polyphenols, flavonoids, triterpenes, coixol, and coixol esters in the GA-87 sample were significantly increased, but the polysaccharide content decreased. Triterpenoids have been widely reported to have various biological activities such as anti-inflammatory, hepatoprotective, immunomodulatory, and antitumor effects. Polyphenols in Job's tears mainly exist in bound form. B. subtilis During the growth and metabolism of Job's tears substrate, degradative enzymes such as cellulase are secreted, hydrolyzing structural polysaccharides such as cellulose and hemicellulose in Job's tears, disrupting the dense structure of plant cell walls, and promoting the release of bound phenols / flavonoids. GABA, as a neurotransmitter regulator, has physiological functions such as sedation, lowering blood pressure, and improving sleep; the fermentation process can significantly increase the GABA content in grains. Microbial transformation is the main source of GABA. In summary, triterpenes, polysaccharides, and polyphenols are the three most abundant active ingredients in fermented Job's tears, constituting the main material basis for its efficacy.

[0061] Table 5. Analysis of active ingredients in different samples

[0062] (3) Analysis of volatile flavor compounds The characteristic flavors formed during the fermentation of Job's tears mainly originate from the synergistic effect of volatile compounds. Table 6 shows that the composition and relative content of various flavor substances differ at different fermentation times, exhibiting a dynamic change pattern of weakening raw material aroma and gradual formation and intensification of fermentation-specific aromas. This indicates that fermentation time is a key factor in regulating the flavor quality of Job's tears. Table 4 shows that alcohols, aldehydes, ketones, and carboxylic acids are the three most abundant volatile compounds in fermented Job's tears, forming the core framework of its basic flavor. Pyrazines make a significant contribution to flavor, accumulating substantially during fermentation and serving as hallmark components for the characteristic soy sauce and nutty aromas of fermented Job's tears.

[0063] Table 6. Analysis of volatile flavor compounds in different samples (%)

[0064] (4) Analysis of the content of fibrinolytic enzyme, acetoin and tetramethylpyrazine Fibrinolytic enzymes are key proteolytic enzymes that can directly or indirectly degrade fibrin, thereby dissolving thrombi. Ligustrazine acts as a novel calcium ion antagonist and free radical scavenger, preventing calcium ion influx and reducing free radical production. Simultaneously, ligustrazine also acts as a thromboxane A2 synthase inhibitor, effectively inhibiting platelet aggregation, dilating arterioles, and improving microcirculation and cerebral circulation. Furthermore, ligustrazine can regulate the fibrinolytic system, enhance plasmin activity, and inhibit excessive activation of coagulation factors, thus exerting a dual antithrombotic effect. Table 7 shows that Bacillus subtilis GH1-fermented Job's tears yielded extremely high plasmin activity and ligustrazine content after 87 h of fermentation, exceeding those of the known Bacillus subtilis BJ3-2-fermented Job's tears, indicating that Bacillus subtilis GH1-fermented Job's tears have extremely high thrombolytic and antithrombotic effects.

[0065] Table 7. Analysis of the contents of fibrinolytic enzyme, acetoin, and tetramethylpyrazine.

[0066] In summary, the fermentation of Job's tears using the Bacillus subtilis GH1 strain of this invention not only enhances the activity of active substances but also exhibits extremely high thrombolytic and antithrombotic effects. As a safe fermentation strain, this strain has extremely strong application prospects.

[0067] Comparative analysis of Example 5 and Comparative Example 3 (1) Analysis of the content of fibrinolytic enzyme and ligustrazine in freeze-dried coix seed fermentation powder Fibrinolytic enzyme is a protease that is easily inactivated by oxidative stress during freeze-drying. Vitamin C, as a strong antioxidant, can scavenge free radicals and inhibit carbonyl reactions even at low concentrations, thereby stabilizing the enzyme conformation. As shown in Table 8, the fibrinolytic enzyme activity in the freeze-dried powder sample with added 0.5% VC was 8254.33 U / g DW, which increased the retention rate by 40% compared to the unadded group; its tetramethylpyrazine content was 3.74 mg / g DW, which increased the retention rate by 16.9% compared to the unadded group.

[0068] Table 8. Analysis of fibrinolytic enzyme and tetramethylpyrazine content

[0069] (2) Stability analysis of fibrinolytic enzyme activity in fermented coix seed powder during storage Depend on Figure 7 It was found that the enzyme activity of all treatment groups decreased to varying degrees with prolonged storage time. However, the enzyme activity decrease of the samples with added VC was significantly lower than that of the GA group without the protective agent at all temperature conditions, indicating that 0.5% VC as a lyophilization protectant has a significant protective effect on plasmin. In particular, at week 4, the residual enzyme activity of the GA-VC group remained above 93% at -20℃, while that of the G group was only 82.85% under the same conditions, further verifying the stabilizing effect of VC during long-term storage.

[0070] (3) Analysis of active substances in fermented Job's tears powder Table 9 shows that, compared with the GA group, the contents of polyphenols, polysaccharides, and flavonoids in the GA-VC sample increased after the addition of the VC protectant. The polysaccharide content in GA-VC was 67.37 mg / g, with a retention rate increased by 18.26% compared to the GA group; the total polyphenol content was 16.97 mg / g, with a retention rate increased by 26.17% compared to the GA group; and the flavonoid content was 5.06 mg / g, with a retention rate increased by 11.45% compared to the GA group. Conversely, the contents of triterpenes and coixol decreased significantly: triterpenes decreased from 23.53 mg / g to 14.80 mg / g; and coixol decreased from 0.03 mg / g to 0.02 mg / g. The GABA content remained basically unchanged, while the coixol content increased from 0.03 mg / g to 0.04 mg / g.

[0071] Table 9 Active substances in fermented Job's tears powder

[0072] Comparative analysis of Example 6 and Comparative Example 4 (1) Basic nutrients in fermented Job's tears solid beverage Table 10 shows that the basic nutrient content ranges of the blended products (3:7 to 7:3) are as follows: moisture content 7.01%–13.65%, fat content 8.82–10.91 g / 100g, protein content 12.09–15.68 g / 100g, ash content 8.27–9.18 g / 100g, and carbohydrate content 53.01–63.92 g / 100g. Overall, as the proportion of Lactobacillus reuteri freeze-dried powder (LAP) increases, the moisture content of the product gradually decreases, while the protein, ash, and carbohydrate contents all show an upward trend.

[0073] Table 10 Basic Nutritional Contents of Fermented Job's Tears Solid Beverage

[0074] Note: Different letters in the same column indicate significant differences. p<0.05 ) (2) Analysis of fibrinolytic enzyme activity and ligustrazine content in fermented coix seed compound solid beverage Depend on Figure 8 It can be seen that as the proportion of LAP in the compound system increases, the contents of tetramethylpyrazine and fibrinolytic enzyme show a continuous decreasing trend. Conversely, the number of viable probiotics increases significantly with the increase of LAP proportion, indicating that Lactobacillus reuteri can survive effectively in the mixed system and its number increases with the increase of its proportion. The content of tetramethylpyrazine decreases monotonically with the increase of LAP proportion and is completely absent in pure LAP. Although the activity of fibrinolytic enzyme also decreases with the increase of LAP proportion, the loss is smaller at low LAP proportions.

[0075] (3) Analysis of active ingredients in fermented coix seed compound solid beverage The content of active substances was analyzed using different ratios of Lactobacillus reuteri and Bacillus subtilis fermented freeze-dried powder (LAP:GAP = 0:10, 3:7, 4:6, 5:5, 6:4, 7:3, 10:0). The results are shown in the table below. Figure 9The GABA content in the GAP group was 2.71 mg / g. It initially increased slightly with increasing LAP ratio, reaching a maximum of 3.01 mg / g at a 5:5 ratio, and then decreased. It was undetectable in the LAP group, indicating that GABA is mainly synthesized by Bacillus subtilis GH1 fermentation. The polyphenol content was highest in the LAP group (13.55 mg / g), followed by the GAP group (11.27 mg / g), with slightly lower levels in intermediate ratios. The polysaccharide content in the GAP group was 63.71 mg / g, significantly increasing to 75.73 mg / g in the 3:7 ratio group, and then decreased with increasing LAP ratio, reaching only 16.93 mg / g in the LAP group. The flavonoid content continuously decreased with increasing LAP ratio, reaching 4.31 mg / g in the GAP group and 0.25 mg / g in the LAP group. The triterpenoid content was significantly higher in the 3:7 ratio group (15.44 mg / g) and the 7:3 ratio group (14.55 mg / g) than in other groups. The coixol content gradually decreased with increasing LAP ratio, reaching 17.28 μg / g in the GAP group and not detected in the LAP group. Coixol esters showed the opposite trend, increasing overall with increasing LAP ratio, reaching a maximum of 37.58 mg / g in the 7:3 ratio group and only 23.31 mg / g in the LAP group.

[0076] (4) Analysis of the in vitro antioxidant capacity of fermented coix seed compound solid beverage Table 11 shows that the LAP group exhibited the strongest DPPH and hydroxyl radical scavenging abilities, particularly in hydroxyl radical scavenging, which was significantly superior to other groups (P<0.05), indicating that the LAP fermentation product is rich in specific antioxidant active substances. In contrast, the GAP group showed better ABTS radical scavenging performance, comparable to the LAP group, but weaker DPPH and hydroxyl radical scavenging abilities.

[0077] In the mixed-ratio samples, the 7:3 ratio group showed better DPPH scavenging rate than the GAP group and other mixed groups, and its hydroxyl radical scavenging rate was second only to the LAP group, indicating that appropriately increasing the LAP ratio (≥60%) can retain the antioxidant advantages of the LAP group to some extent. With further increases in the GAP ratio (4:6, 3:7), the hydroxyl radical scavenging rate continued to decline. Overall, *Lactobacillus reuteri* fermentation alone showed significant advantages in scavenging hydroxyl and DPPH radicals, while the mixed ratios did not surpass the LAP group in most indicators, but the 7:3 ratio group showed some application potential.

[0078] Table 11. In vitro antioxidant capacity of fermented Job's tears probiotic solid beverage

[0079] Note: Different letters in the same column indicate significant differences. p<0.05 ) (5) Analysis of the in vitro thrombolytic effect of fermented coix seed compound solid beverage Results of in vitro thrombolysis experiment as follows Figure 10 As shown in the figure, during the 9-hour experimental period, the percentage of residual thrombus weight in the blank control group (CK) remained stable between 97.22% and 100.00%, while the residual thrombus in the LAP group showed no significant change from 0 to 9 hours. In contrast, the GAP group exhibited extremely strong thrombolytic ability. The in vitro residual thrombus rate rapidly decreased to 47.10% at 3 hours, further decreasing to 19.12% at 6 hours, and reaching only 1.12% at the 9-hour endpoint. The thrombolytic effect of the GAP group at the 9-hour endpoint was statistically at the same extremely low level as that of the urokinase-positive control group (4.57% residual thrombus), demonstrating that the fibrinolytic enzyme produced by Bacillus subtilis fermentation of Job's tears has the ability to efficiently degrade fibrin and has great potential as a source for the development of novel thrombolytic agents. In different ratio systems of the LAP and GAP groups, the thrombolytic efficiency showed a gradient change pattern positively correlated with the proportion of GAP, with the 3:7 (LAP:GAP) ratio group showing consistent in vitro thrombolytic effect with the GAP group. The residual thrombus rates in the 3:7 group at 3 h, 6 h and 9 h were 56.40%, 24.55% and 4.08%, respectively. The residual thrombus rate at each time point was significantly lower than that at the previous time point. Moreover, the residual thrombus rate at the 9 h endpoint (4.08%) was much lower than that in the 4:6 ratio group (12.11%) and the 5:5 ratio group (22.57%).

[0080] (6) Analysis of the in vitro anti-inflammatory and lipid-lowering effects of fermented coix seed solid beverage COX-2 and 5-LOX are key rate-limiting enzymes in the arachidonic acid metabolic pathway. Their overexpression can catalyze the production of large amounts of prostaglandins and leukotrienes, which are core drivers of the inflammatory response. Table 12 shows that the inhibition rates of 5-LOX by the samples ranged from 74.81% to 80.10%, with no significant difference between groups. However, the inhibitory activity on COX-2 showed significant differences among different ratios. The pure GAP group had the lowest inhibition rate, while all samples containing LAP components showed significantly higher inhibition rates than the pure GAP group, with the 3:7 ratio reaching the highest value of 83.42%. The residual cholesterol levels after treatment ranged from 86.31 to 89.69 μg / mL. This indicates that neither the fermentation products from a single strain nor the compound products with different ratios exhibited significant cholesterol binding or scavenging ability under in vitro conditions.

[0081] In summary, different formulation ratios significantly regulate the anti-inflammatory activity of the product. Lactobacillus reuteri fermented Job's tears powder is the main contributor to COX-2 inhibition activity, while Bacillus subtilis fermented Job's tears powder shows weaker activity in this regard. Considering both thrombolytic and anti-inflammatory activities, a 3:7 ratio maintains high thrombolytic capacity while exhibiting optimal COX-2 inhibition, making it the better choice for balancing functional activities.

[0082] Table 12 COX-2 and 5-LOX enzyme inhibition rates and residual cholesterol levels Table 5-6 Inhibition rates of COX-2 and 5-LOX enzymes and residualcholesterol content

[0083] Note: Different letters in the same row indicate significant differences. p<0.05 ) (7) Analysis of ligustrazine in in vitro digestion of fermented coix seed compound solid beverage To investigate the release pattern and stability of tetramethylpyrazine in solid beverages of fermented coix seed probiotics with different ratios under gastrointestinal conditions, an in vitro simulated digestion model was used. The mixture was digested in three stages: oral cavity, stomach, and intestine. The content of tetramethylpyrazine in the supernatant of each stage of digestion was measured. The results are shown in Table 13.

[0084] The GAP group had the highest tetramethylpyrazine content (2.61 mg / g DW) during the oral digestion stage (K), and the content decreased gradually with the increase of the LAP ratio. Tetramethylpyrazine was not detected in the pure LAP group. After entering the gastric digestion stage, the content in the GAP group dropped to 1.61 mg / g DW at W0. With the extension of gastric digestion time, the tetramethylpyrazine content gradually rebounded from W1 to W3, reaching 3.16 mg / g DW at W3. The 3:7 and 4:6 ratio groups had relatively stable contents during the gastric digestion stage, while the 5:5, 6:4, and 7:3 ratio groups showed slight fluctuations or a decreasing trend. After entering the intestinal digestion stage, the tetramethylpyrazine content in all ratio groups decreased to varying degrees compared with the end of gastric digestion. In the pure GAP group, the tetramethylpyrazine content decreased to 1.24 mg / g DW at C0, which was only 39.2% of the W3 value; it remained between 0.91 and 1.24 mg / g DW during C1 to C3. The 3:7 ratio group maintained the highest tetramethylpyrazine content throughout the entire intestinal digestion stage (1.50 mg / g DW at C3), which was significantly higher than other ratios.

[0085] (8) Analysis of ligustrazine in in vitro digestion of fermented coix seed compound solid beverage As shown in Table 14, the GAP group exhibited the highest fibrinolytic enzyme activity during the oral digestive stage (K), reaching 6785.87 U / g DW. With increasing L ratio, the K values ​​in each formulation group showed a gradient decreasing trend. During the gastric digestion phase, the activity of fibrinolytic enzymes exhibited different patterns depending on the ratio of LAP to GAP. Fibrinolytic enzyme activity was detectable throughout the entire gastric digestion process (W0-W3) in both the GAP and 3:7 groups, but showed a continuous decreasing trend, with a total retention rate of approximately 27.3%. When the LAP ratio reached 40% or higher (in the 4:6, 5:5, 6:4, and 7:3 ratio groups), fibrinolytic enzyme activity was completely lost by W1 (1 hour of gastric digestion). The LAP:GAP ratio is a key factor affecting the gastric stability of fibrinolytic enzymes; when the LAP ratio is ≤30%, fibrinolytic enzymes can maintain a certain level of activity, but above this threshold, they are rapidly inactivated.

[0086] After entering the intestinal digestion stage, the fibrinolytic enzyme activity in both the GAP group and the 3:7 ratio group further decreased. In the GAP group, the activity dropped to 2449.37 U / g at C0, representing 84.4% of the W3 value, and then slightly rebounded during C1-C3, reaching 2732.57 U / g at C3. The 3:7 ratio group showed a similar trend during the intestinal digestion stage, decreasing to 1042.28 U / g at C0 and rebounding to 1304.22 U / g at C3, approximately 21.9% of the initial value during oral digestion.

[0087] Considering the entire in vitro digestion process, the gastrointestinal stability of fibrinolytic enzymes is a key bottleneck restricting their oral efficacy. Although the GAP group had the highest initial enzyme activity, the retention rate dropped to 42.7% at the end of gastric digestion and further decreased after intestinal digestion. While the 3:7 ratio group had lower initial enzyme activity and absolute retention values ​​than the pure GAP group, its enzyme activity remained detectable after gastrointestinal digestion. Combined with the aforementioned in vitro thrombolysis results, this enzyme activity level was still sufficient to exert a significant thrombolytic effect in a relatively short time. Therefore, from the perspective of fibrinolytic enzyme gastrointestinal stability, the 3:7 ratio is a critical ratio that can balance probiotic function and thrombolytic activity, and the fibrinolytic enzyme retains a certain level of activity in the intestinal phase, which can provide a reference for subsequent in vivo efficacy evaluation.

[0088] Table 13 Changes in tetramethylpyrazine content in samples with different ratios during in vitro simulated digestion.

[0089] Note: Different letters in the same column indicate significant differences between different ratios at the same digestion stage. p <0.05). K represents the oral digestion stage, W0-W3 represents the gastric digestion stage (0-3 hours), and C0-C3 represents the intestinal digestion stage (0-3 hours).

[0090] Table 14 Changes in fibrinolytic enzyme activity during in vitro simulated digestion of samples with different ratios

[0091] Note: Different letters in the same column indicate significant differences between different ratios at the same digestion stage. p <0.05). Enzyme activity units are U / g DW. K represents the oral digestion stage, W0-W3 represents the gastric digestion stage (0-3 h), and C0-C3 represents the intestinal digestion stage.

Claims

1. A type of Bacillus subtilis ( Bacillus subtilis GH1, characterized in that, The Bacillus subtilis GH1 has the accession number CGMCC No. 38585, the accession date is May 8, 2026, and the accession address is China General Microbiological Culture Collection Center.

2. The application of Bacillus subtilis GH1 as described in claim 1 in increasing the content of active ingredients in coix seed fermentation products, wherein the active ingredients are at least one selected from tetramethylpyrazine, fibrinolytic enzyme, γ-aminobutyric acid, coixol, polyphenols, flavonoids, triterpenes and coixol esters.

3. The application of Bacillus subtilis GH1 as described in claim 1 in the enrichment of tetramethylpyrazine and fibrinolytic enzyme.

4. A method for preparing a fermented Job's tears product, characterized in that, Includes the following steps: Using Job's tears as a fermentation substrate, Bacillus subtilis GH1 seed liquid was inoculated for solid-state fermentation to obtain fermentation products. Ascorbic acid was then added to the fermentation products for mixing, and after freeze-drying, fermented Job's tears freeze-dried powder was obtained. The preservation number of Bacillus subtilis GH1 is CGMCC No. 38585.

5. The method for preparing a fermented Job's tears product as described in claim 4, characterized in that, The concentration of the Bacillus subtilis GH1 seed solution is 1.0 × 10⁻⁶. 8 CFU / mL ~1.8×10 8 CFU / mL.

6. The method for preparing a fermented Job's tears product as described in claim 4, characterized in that, The solid-state fermentation refers to inoculating Bacillus subtilis GH1 seed liquid into sterilized Job's tears and fermenting it at 34℃~40℃ for 72~96 hours; the inoculation amount of Bacillus subtilis GH1 seed liquid is 5%~9%.

7. The method for preparing a fermented Job's tears product as described in claim 4, characterized in that, The amount of ascorbic acid added is 0.1% to 1.0% of the mass of the fermentation product.

8. The method for preparing a fermented Job's tears product as described in claim 4, characterized in that, The freeze-drying process involves pre-freezing the fermentation product mixed with ascorbic acid at -80°C for 4 to 8 hours, and then drying it for 36 to 60 hours at a cold trap temperature of -50°C to -40°C and a vacuum of 2 to 5 Pa.

9. A fermented Job's tears solid beverage, characterized in that, The fermented Job's tears solid beverage is obtained by mixing fermented Job's tears freeze-dried powder and probiotic powder, adding a binder, and then extruding and granulating. The fermented Job's tears freeze-dried powder is obtained by solid-state fermentation of Job's tears by Bacillus subtilis GH1 with preservation number CGMCCNo.38585, followed by mixing with ascorbic acid and freeze-drying.

10. A fermented Job's tears solid beverage as described in claim 9, characterized in that, The binder is maltodextrin, and the amount of maltodextrin added accounts for 15% to 20% of the total mass of the fermented Job's tears solid beverage.

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