CN112812986A

CN112812986A - Bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield and application thereof

Info

Publication number: CN112812986A
Application number: CN202011521142.4A
Authority: CN
Inventors: 郭仁妹; 韩峰; 黄洁
Original assignee: Suzhou Weike Life Technology Co ltd
Current assignee: Suzhou Weike Life Technology Co ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-05-18

Abstract

The invention discloses bacillus subtilis XTK1001 with high yield of nattokinase and vitamin K2 and application thereof. The preservation number of the Bacillus subtilis is as follows: GDMCC No.61351, with the collection address: the preservation unit of No. 59 building 5 of No. 100 college of the Pieli Zhou city is as follows: the preservation time of the Guangdong province microorganism strain preservation center is as follows: 12/7/2020. The bacillus subtilis XTK1001 disclosed by the invention has the characteristics of high yield of nattokinase and high yield of vitamin K2, and is expected to solve the problems that the liquid state fermentation of the high yield of nattokinase and vitamin K2 in China lacks excellent strains and the production cost of products is high.

Description

Bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and in particular relates to bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield and application thereof.

Background

Natto is a traditional food in japan, and nattokinase is originally extracted from natto. Nattokinase (EC 3.4.21.62) is serine protease, and is composed of 275 amino acid residues and has a molecular weight of 27.7 kDa. Nattokinase has strong fibrinolysis effect, can digest fibrin and can also crack plasmin substrates [ A pilot on the serum pharmaceuticals of nattokinase in humans down a single, oral, daily dose. alternative therapeutics in Health and Medicine,2013,19(3):16-19], and is helpful for reducing cardiovascular diseases and improving blood circulation. In the last 80 years, the thrombolysis property of nattokinase was verified, and the anticoagulation property of nattokinase in vitro and in vivo systems is well proved. Nattokinase has other functions besides thrombolytic effect, including blood pressure reduction, blood fat reduction, atherosclerosis resistance, nerve protection, inhibition of formation of collagen-activated platelet thromboxane B2 and the like [ Nattokinase activity and stability research progress, Chinese seasoning 2019,44(07): 160-. In addition, nattokinase has been used in other diseases such as chronic fatigue syndrome, hysteromyoma, hypertension, vitreoretinal disease and Alzheimer's disease [ Effect of Tb (III) on activity and stability of nattokinase. journal of Rare Earth, 2017,35(05): 510-. However, the activity of the current domestic nattokinase products is generally lower than that of foreign products, and the screening of the strains with high nattokinase production capacity has important significance.

Vitamin K2 is a fat-soluble vitamin, a derivative of naphthoquinone group having the biological activity of phylloquinone, and is one of important vitamins indispensable to the human body. Studies have shown that Vitamin K2 can improve bone density, reduce vascular calcification, reduce cardiovascular risk, improve mitochondrial function, relieve insulin resistance, relieve inflammation and oxidative stress, and treat cancer [ Vitamin K2 in multiple sclerosis Patients. Wien Klin Wochenschr,2018,130(9/10):307-313 ]. Vitamin K2 is named in the form of MK-n, such as M-4, MK-5, MK-6 and MK-7, etc., according to the length of the isoprene side chain carried by the main ring. Vitamin K2 was originally isolated from natto, mostly MK-7, accounting for about 98%. MK-7 is vitamin K2 with the broadest function and strongest activity, and the longest half-life in blood and safest. Vitamin K2 is mainly synthesized by chemical synthesis, however, the isoprene side chain of chemically synthesized vitamin K2 is often cis-structure and has low activity, while natural vitamin K2(MK-7) with higher activity can only be obtained by fermentation. At present, the patent CN 104328064A reports that the NK-7 type vitamin K2 is prepared by a fermentation method of bacillus natto.

In view of the important role of nattokinase and vitamin K2(MK-7) on human health, and a method for co-producing vitamin K2 and nattokinase by utilizing bacillus natto is reported in patent CN 106701719A, which shows that the feasibility and the important application prospect of simultaneously obtaining high nattokinase and vitamin K2 by utilizing microbial fermentation are realized, so that the important significance of developing and obtaining strains capable of simultaneously producing nattokinase and vitamin K2 at high yield is realized, and the method is beneficial to developing foods with higher health care value.

Disclosure of Invention

The invention aims to solve the technical problem of providing the bacillus subtilis capable of producing the nattokinase and the vitamin K2 at high yield so as to solve the problems that the liquid state fermentation of the high-yield nattokinase and the vitamin K2 in China lacks excellent strains and the production cost of products is high.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, the invention provides Bacillus subtilis XTK1001 with high nattokinase and vitamin K2 yield, wherein the Bacillus subtilis (Bacillus subtilis) has a preservation number of: GDMCC No.61351, with the collection address: the preservation unit of No. 59 building 5 of No. 100 college of the Pieli Zhou city is as follows: the preservation time of the Guangdong province microorganism strain preservation center is as follows: 12/7/2020.

The preparation method of the bacillus subtilis XTK1001 with high yield of nattokinase and vitamin K2 comprises the following steps:

1. putting 1-10 g of natto sample into a sterilization test tube, adding 1-10 mL of sterilization normal saline for washing, subpackaging in a centrifuge tube, centrifuging for 3-10 min at a rotating speed of 3000-8000 r/min, carrying out water bath at 65-95 ℃ for 5min to kill most of nutrients and mixed bacteria, respectively absorbing 0.01-0.5 mL of bacterial liquid to coat a casein plate (5.0-15.0 g/L of casein, 2.0-10.0 g/L of glucose, 0.1-2.0 g/L of yeast extract, and K₂HPO₄·3H₂O 0.5～2.0g/L，KH₂PO₄ 0.1～3g/L，MgSO₄0.05-0.5 g/L and 1.0-2.0 g/L of agar powder, adding water for dissolution, carrying out pH 7.2-6.4 and autoclaving at 110-125 ℃ for 10-30 min), carrying out inverted culture at 35-40 ℃ for 12-48 h, observing single colonies on a casein plate, selecting a casein plate with a large dissolution circle, using a sterile toothpick for spotting, carrying out culture at 35-40 ℃ for 12-48 h, and screening strains with the colony ratio of less than 0.5 to obtain prescreened strains.

2. Inoculating the primary screened strain to 20-150 mL of seed culture medium (glucose 2.0-10.0 g/L, tryptone 5.0-20.0 g/L, beef extract 2.0-10.0 g/L, sodium chloride 2.0-10.0 g/L, 110-125 ℃ autoclaving for 10-30 min), culturing at 35-40 ℃ at 150-250 r/min for 12-48 h to obtain bacterial suspension, and inoculating to 20-150 mL of fermentation culture medium (glucose 5.0-20.0 g/L, tryptone 20.0g/L, K) according to 1-5% inoculation amount₂HPO₄·3H₂O 0.5～5.0g/L，KH₂PO₄0.5-2.0 g/L, MgSO40.1-1.0 g/L, CaCl 20.05-0.5 g/L, adding water to dissolve, carrying out high-pressure sterilization at the temperature of 110-125 ℃ for 10-30 min at the pH of 7.2-6.4), fermenting for 12-48 h at the temperature of 35-40 ℃, centrifuging for 3-10 min at the temperature of 0-10 ℃ at 3000-8000 r/min, collecting supernatant to obtain fermentation liquor, measuring the nattokinase activity of the fermentation liquor, and screening a strain with the fibrinolytic activity of more than or equal to 1000U/mL, namely a re-screened strain.

3. Extracting vitamin K2 from the fermentation liquor of the re-screened strain by using an organic solvent (the volume ratio of normal hexane to isopropanol is 1-3: 1), extracting according to the volume ratio of a water phase to an organic phase being 1: 1-3, placing the mixture on a vortex oscillator for oscillation for 3-10 min during extraction, centrifuging the mixture for 5-15 min at 2000-5000 r/min, and absorbing the upper layer liquid for concentration. Concentrating the organic phase for 2-10 min by using a rotary evaporator, wrapping an evaporation bottle with tinfoil paper, concentrating to obtain yellow oily liquid, adding 1-5 mL of isopropanol to dissolve, and placing the yellow oily liquid in a centrifuge tube. The method comprises the steps of measuring vitamin K2 by adopting a High Performance Liquid Chromatography (HPLC) method, using an Agilent C18 as a chromatographic column, using methanol and dichloromethane (5-10: 1) as mobile phases, using a flow rate of 0.5-2.0 mL/min, using a column temperature of 30-50 ℃, using a sample injection amount of 10-50 mu L and using an ultraviolet detection wavelength of 230-260 nm, filtering an extracting solution by a microporous filtration membrane (0.22-0.45 mu m), using a sample injection method, and synthesizing nattokinase activity and vitamin K2 yield to obtain the strain with highest activity and yield.

4. The strain with the highest nattokinase activity and vitamin K2 yield is subjected to colony morphology identification and 16s rDNA determination (primers are 27F and 1492R), and the strain is identified as the bacillus subtilis.

Through verification, the strain is used for producing the nattokinase and the vitamin K2 by fermentation, the activity of the nattokinase in fermentation liquor reaches 1423.72U/mL, and the yield of the vitamin K2 reaches 22.4 mg/L.

In a second aspect, the invention provides application of the bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield in preparation of pharmaceutical compositions and fermented foods of natto kinase and vitamin K2.

In the invention, the pharmaceutical composition is prepared from fermentation liquor obtained after fermentation of the bacillus subtilis, and the dosage forms of the pharmaceutical composition include, but are not limited to, granules, capsules, tablets, pills and liquid preparations.

Further, the preparation process of the fermentation liquor comprises the following steps:

(a) inoculating the bacillus subtilis strain of claim 1 into 20-150 mL of seed culture medium, and culturing at 35-40 ℃ and 150-250 r/min for 12-48 h to obtain a bacterial suspension;

(b) inoculating the activated bacterial suspension into 20-150 mL of fermentation medium according to the inoculation amount of 1-5%, fermenting at 35-40 ℃ for 12-48 h, centrifuging at 0-10 ℃ and 3000-8000 r/min for 3-10 min, and collecting supernatant to obtain fermentation liquor.

Further, in step (a), the seed culture medium comprises: 2.0-10.0 g/L of glucose, 5.0-20.0 g/L of tryptone, 2.0-10.0 g/L of beef extract and 2.0-10.0 g/L of sodium chloride, and the sterilization condition is that autoclaving is carried out for 10-30 min at 110-125 ℃.

Further, in step (b), the fermentation medium comprises: 5.0-20.0 g/L of glucose, 20.0g/L of tryptone and K₂HPO₄·3H₂O 0.5～5.0g/L，KH₂PO₄ 0.5～2.0g/L，MgSO₄ 0.1～1.0g/L，CaCl₂0.05-0.5 g/L, pH 7.2-6.4, and sterilization conditions of 110-125 ℃ high-pressure sterilization for 10-30 min.

In the present invention, the fermented food includes, but is not limited to, fermented natto, fermented soybean, and fermented soybean paste.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, bacillus subtilis XTK1001 with high yield of nattokinase and vitamin K2 is screened from natto food, the bacillus subtilis XTK1001 has the characteristics of high yield of nattokinase and vitamin K2, the practical problems that excellent strains are lacked in liquid state fermentation of high yield of nattokinase and vitamin K2 in China, the production cost of products is high and the like are solved, and the application of the bacillus subtilis XTK1001 can promote market development of functional nutritional health care products.

Drawings

FIG. 1 is a standard curve for urokinase;

fig. 2 is a standard curve of vitamin K2.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available without otherwise specified.

Example 1: bacterial strain primary screen for high yield of nattokinase

1. Preparation of prescreening solid culture medium (Casein plate)

Weighing casein 10.0g/L, glucose 5.0g/L, yeast extract 1.0g/L, and K₂HPO₄·3H₂O 1.31g/L，KH₂PO₄ 0.5g/L，MgSO₄0.1g/L of agar powder and 1.0ml/L of agar powder, adding water for dissolution, and carrying out autoclaving at the temperature of 115 ℃ for 20min, wherein the pH value is 7.2-6.4.

2. Bacterial strain preliminary screening

Putting 5g of natto sample into a sterilization test tube, adding 5mL of sterilization normal saline, washing, subpackaging into centrifuge tubes, centrifuging at a rotation speed of 5000r/min for 5min, and carrying out water bath at 85 ℃ for 5min to kill most of nutriments and mixed bacteria; then respectively sucking 0.1mL of bacterial liquid to coat a casein plate, carrying out inverted culture at 37 ℃ for 24h, observing a single colony on the casein plate, selecting a casein plate with a large dissolving ring and using a sterile toothpick to spot the casein plate, carrying out culture at 37 ℃ for 24h, observing the forming condition of a transparent ring, recording the ratio of the diameter of the colony to the diameter of the transparent ring, and carrying out three parallel averaging on each strain.

TABLE 1 Primary screen results (farmhouse fermented soya beans)

As shown in Table 1, 92 strains of fermented soybeans were separated from the fermented soybeans, and the colony ratio of 30 strains is less than 0.5, which shows that the fermented soybeans have higher proteolytic activity and can be used for next fibrin plate rescreening. The symbol "-" means no hydrolysis ring or too small a hydrolysis ring.

Example 2: bacterial strain re-screening and fibrinolysis capacity determination of high-yield nattokinase

1. Preparation of seed culture medium

Weighing the components according to the concentration ratio of 5.0g/L glucose, 10.0g/L tryptone, 5.0g/L beef extract and 6.0g/L sodium chloride, adding water to dissolve the components, and sterilizing at 115 ℃ for 20min to obtain the seed culture medium.

2. Preparation of fermentation medium

According to glucose 10.0g/L, tryptone 20.0g/L, K₂HPO₄·3H₂O 2.62g/L，KH₂PO₄ 1.0g/L，MgSO₄ 0.5g/L，CaCl₂Weighing the components according to the concentration ratio of 0.15g/L, adding water to dissolve the components, adjusting the pH value to 7.2-6.4, and carrying out autoclaving at 115 ℃ for 20min to obtain the fermentation medium.

3. Preparation of fibrin solid culture medium

A proper amount of fibrinogen was added with PBS buffer to prepare a fibrinogen solution of 1.5 mg/mL. (PBS buffer needs to be added slowly to prevent air bubbles, ice added to dissolve ultrasonically).

Taking 39mL of fibrinogen solution in a 100mL beaker, heating in a water bath at 50 ℃ for 5min, adding 39mL of agarose solution and 3mL of thrombin while stirring, immediately mixing, quickly pouring into a culture dish, and horizontally placing at room temperature for 1 h. (bacteriostatic ring preparation plus enzyme wells).

4. Preparation of fermentation broth

Inoculating the screened 30 strains of bacteria into 50mL of seed culture medium, and culturing for 24h at 37 ℃ under the condition of 180r/min to obtain a bacterial suspension; inoculating the bacterial suspension into 50mL (250mL triangular flask) fermentation medium according to the inoculation amount of 2%, fermenting at 37 ℃ for 48h, centrifuging at 4 ℃ under 6000r/min for 5min, and collecting supernatant to obtain fermentation liquor.

5. Measurement of urokinase Standard Curve

TABLE 2 preparation of urokinase Standard series solutions

Urokinase control series solution solubility (IU/ml)	Urokinase control solution sample size (μ L)	PBS buffer sample size (μ L)
			1000	100	0
800	80	20
			600	60	40
500	50	50
			400	40	60
200	20	80
			100	10	90
50	10	190
			25	10	390

A urokinase standard series solution was prepared as shown in Table 2, 10. mu.L of urokinase was dropped on a fibrin plate using a micro syringe, incubated at 37 ℃ for 18 hours, the diameter of the lysis ring was measured, the area of the lysis ring was calculated, and a urokinase standard curve was prepared as shown in FIG. 1.

6. Dripping 10 μ L fermentation liquid on fibrin plate with micro injector, incubating at 37 deg.C for 18h, measuring diameter of lysis ring, calculating area of lysis ring, and calculating enzyme activity according to standard curve.

TABLE 3 rescreening results

5 strains with high fibrinolytic activity (more than or equal to 1000U/mL) are obtained by re-screening on a fibrin plate, and are respectively No. 42 (1121.02U/mL), No. 56 (1341.97U/mL), No. 59 (1423.72U/mL), No. 77 (1136.78U/mL) and No. 79 (1120.36U/mL).

Example 3: production assay for the selected Strain vitamin K2

1. Screening strains 42, 56, 59, 77, 79 fermentation broths were prepared as in example 2.

2. Extraction of vitamin K2

Extracting vitamin K2 from the fermentation liquor by adopting an organic solvent extraction mode, selecting normal hexane and isopropanol (the volume ratio is 2: 1) as extraction liquid, and changing the proportion of an aqueous phase (fermentation liquor) and an organic phase (extraction liquid) according to the following steps: extracting with organic phase at a volume ratio of 1: 2. During extraction, the mixture is placed on a vortex oscillator to oscillate for 5min, then the mixture is centrifuged for 10min at 3000r/min, and the upper liquid is sucked up to be concentrated. Concentrating the organic phase for 5min by using a rotary evaporator, wrapping an evaporation flask with tinfoil paper for operation, concentrating to obtain yellow oily liquid, adding 2mL of isopropanol for dissolving, placing in a 1.5mL centrifuge tube, wrapping with tinfoil paper for dark storage.

3. Determination of vitamin K2

Vitamin K2 was measured by High Performance Liquid Chromatography (HPLC). The chromatographic column is Agilent C18, the mobile phase is methanol: dichloromethane (9: 1), the flow rate is 1.0mL/min, the column temperature is 40 ℃, the sample injection amount is 20 muL, and the ultraviolet detection wavelength is 248 nm. The extract was filtered through a microfiltration membrane (0.22 μm) and injected, all manipulations were performed in the dark.

Preparing a standard substance: 10mg of vitamin K2(MK7) standard substance is accurately weighed into a 50mL brown volumetric flask, 30mL of isopropanol is added to fully dissolve and fix the volume, and the mixture is shaken for 1 hour in a constant temperature water bath kettle. Then, the samples were diluted in a gradient to give standard solutions having concentrations of 0, 5, 10, 15, 20, 25, 30 and 35. mu.g/mL, filtered through a 0.22 μm microporous membrane, subjected to liquid chromatography, and a standard curve was prepared using the peak area and the standard concentration as shown in FIG. 2.

According to a standard curve, the yield of the screened strain vitamin K2 (shown in table 4) is determined, wherein the yield of the No. 56 strain is up to 22.4mg/L, and the No. 56 strain is the most preferable strain by integrating the enzyme activity of the nattokinase, so that the No. 56 strain of the strain with high yield of the nattokinase and the vitamin K2 is obtained.

TABLE 4 screening of the Strain vitamin K2(MK-7) production

Numbering	Vitamin K2(MK-7) yield (mg/L)
		42	9.4
56	22.4
		59	11.4
77	12.5
		79	7.4

Example 4: identification of strains with high yield of nattokinase and vitamin K2

1. Preparation of seed culture medium and agar culture medium

The seed medium was prepared as in example 2, and the agar medium was prepared by adding 2% agar to the seed medium to prepare a plate.

2. After morphological observation, physiological and biochemical identification and 16s rDNA strain identification analysis are carried out on the bacillus subtilis strain, the No. 56 strain is identified to be the bacillus subtilis strain. The specific identification results are as follows:

and (3) morphological observation: the bacterial colony is coated on an agar culture medium plate and cultured for 24h, the morphology of the bacterial colony is observed to be grey white, nearly circular, dry in surface, opaque, wrinkled, matt, unsmooth in edge and leaf-like, and the central color is darker than the edge part.

16s rDNA strain identification: the whole genome of strain No. 56 was extracted by a genome kit, 16s rDNA amplification was performed using universal primers (27F and 1492R), and the sequencing results were compared at NCBI.

TABLE 5 primer information

The sequencing result of 16s rDNA is shown in SEQ ID NO. 1.

Example 5: the bacterial strain of the invention is used for preparing the drug combination of nattokinase and vitamin K2

The seed medium and the fermentation medium were prepared as in example 2.

The bacterial strain is inoculated into 50mL of seed culture medium and cultured for 24h under the conditions of 37 ℃ and 180r/min to prepare bacterial suspension. Then, the mixture was inoculated into 250mL (1L Erlenmeyer flask) of a fermentation medium at an inoculum size of 2%, and cultured at 37 ℃ for 24 hours at 180r/min to prepare a bacterial suspension. Inoculating the bacterial suspension into a 10L fermentation tank according to the proportion of 2%, fermenting for 48h at 180r/min, centrifuging for 5min at 4 ℃ and 6000r/min, collecting supernatant to obtain fermentation liquor, concentrating the fermentation liquor, and freeze-drying to prepare the drug dry powder rich in nattokinase and vitamin K2.

Example 6: natto product prepared by using the strain of the invention

The seed medium was prepared as in example 2.

Preparing a solid fermentation medium: cleaning soybeans, adding deionized water with the volume being 3 times that of the soybeans, soaking for 14-18 h at normal temperature, draining, adding 3% of salt and 3% of cane sugar, and sterilizing for 20min at 121 ℃.

The natto product is prepared by the following steps:

inoculating the strain of the invention into a liquid seed culture medium, culturing for 18h under the conditions of 37 ℃ and 180r/min, then inoculating the strain onto a sterilized solid fermentation culture medium according to the inoculation amount of 4%, culturing for 36h under the condition of 37 ℃, and stirring once every 12h to obtain the natto fermented food with high yield of natto kinase and vitamin K2.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

SEQUENCE LISTING

<110> Suzhou microgram Life technology Co., Ltd

<120> bacillus subtilis XTK1001 with high yield of nattokinase and vitamin K2 and application thereof

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1398

<212> DNA

<213> (Artificial sequence)

<400> 1

tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60

acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120

atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc acttacagat 180

ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240

ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300

ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360

gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac cgttcgaata 420

gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480

ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540

tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600

aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660

tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720

gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780

agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840

gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900

catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960

tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020

gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080

ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140

tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200

caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260

gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320

cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380

cccgaagtcg gtgaggta 1398

Claims

1. A Bacillus subtilis XTK1001 with high nattokinase and vitamin K2 yield is characterized in that the Bacillus subtilis collection number is as follows: GDMCC No.61351, with the collection address: the preservation unit of No. 59 building 5 of No. 100 college of the Pieli Zhou city is as follows: the preservation time of the Guangdong province microorganism strain preservation center is as follows: 12/7/2020.

2. The application of the bacillus subtilis XTK1001 with high nattokinase and vitamin K2 yield in claim 1 in preparing pharmaceutical compositions and fermented foods of nattokinase and vitamin K2.

3. The use of claim 2, wherein the pharmaceutical composition is prepared from a fermentation broth obtained after fermentation of bacillus subtilis XTK1001 of claim 1.

4. The use according to claim 3, wherein the pharmaceutical composition is in the form of granules, capsules, tablets, pills and liquids.

5. The use according to claim 3, wherein the fermentation broth is prepared by:

6. The use according to claim 5, wherein in step (a), the seed medium comprises: 2.0-10.0 g/L of glucose, 5.0-20.0 g/L of tryptone, 2.0-10.0 g/L of beef extract and 2.0-10.0 g/L of sodium chloride, and the sterilization condition is that autoclaving is carried out for 10-30 min at 110-125 ℃.

7. The use according to claim 5, wherein in step (b) the fermentation medium comprises: 5.0-20.0 g/L of glucose, 20.0g/L of tryptone and K₂HPO₄·3H₂O 0.5～5.0g/L，KH₂PO₄ 0.5～2.0g/L，MgSO₄ 0.1～1.0g/L，CaCl₂0.05-0.5 g/L, pH 7.2-6.4, and sterilization conditions of 110-125 ℃ high-pressure sterilization for 10-30 min.

8. The use according to claim 2, wherein the fermented food products include fermented natto, fermented soybean and fermented soybean paste.