BACILLUS NATTO FOR PRODUCING MENAQUINONE-7 AND USE THEREOF

Abstract

The present invention provides Bacillus natto for producing menaquinone-7. Bacillus natto is deposited at the China General Microbiological Culture Collection Center, the accession number being CGMCC No. 21799. The strain has few requirements for nutrients, has a high yield, has mild metabolism, is easy to regulate and control, and is suitable for industrial scale-up.

Claims

1-12. (canceled)

13. A Bacillus natto strain HDCC00023, is preserved in the China General Microbiological Culture Collection Center on Feb. 1, 2021, with an accession number of CGMCC NO. 21799.

14. Use of the Bacillus natto strain according to claim 13 in preparing menaquinone-7; or preparing one or more of a medicament, a healthcare product, a food, a beverage, a cosmetic product, an additive, and a feed comprising menaquinone-7.

15. A fermentation broth comprising the Bacillus natto strain according to claim 13.

16. Use of the fermentation broth according to claim 15 in preparing menaquinone-7; or preparing one or more of a medicament, a healthcare product, a food, a beverage, a cosmetic product, an additive, and a feed comprising menaquinone-7.

17. A fermentation method for producing menaquinone-7 by using the Bacillus natto strain according to claim 13.

18. The fermentation method according to claim 17, comprising performing a fermentation in a fermentation medium comprising an assimilable carbon source and/or nitrogen source.

19. The fermentation method according to claim 18, wherein the assimilable carbon source is selected from one of or a combination of any of starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, glycerol, soybean oil, sorbitol, and mannitol.

20. The fermentation method according to claim 19, wherein the assimilable carbon source is selected from one of or a combination of glycerol and starch.

21. The fermentation method according to claim 18, wherein the assimilable nitrogen source is selected from one of or a combination of any of yeast extract powder, yeast powder, yeast cream, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea, and ammonium salt.

22. The fermentation method according to claim 21, wherein the assimilable nitrogen source is soybean peptone.

23. The fermentation method according to claim 18, wherein the fermentation medium further comprises an inorganic salt, wherein the inorganic salt is selected from one of or a combination of any of trisodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, and manganese sulfate.

24. The fermentation method according to claim 23, wherein the inorganic salt is selected from one of or a combination of any of potassium dihydrogen phosphate, magnesium sulfate, potassium chloride, and calcium chloride.

25. The fermentation method according to claim 17, wherein the seed solution is fermented in the fermentation medium, with an amount of dissolved oxygen of a fermentation broth controlled to be not less than 5% or to be 5%-15%.

26. The fermentation method according to claim 17, wherein the seed solution is fermented in the fermentation medium, with a total residual sugar concentration of a fermentation broth maintained to be not less than 1.0% or to be 1.0%-1.5%.

27. The fermentation method according to claim 17, wherein the seed solution is fermented in the fermentation medium, and the fermentation is performed at a temperature of not lower than 10? C. or not lower than 20? C., or a temperature of 20-60? C., or 20-50? C., or 20-40? C., or 30-50? ? C., or 30-40? C., or 35-40? C., or 37? C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows a comparison of fermentation yield, substrate consumption, and by-product formation (viscosity characterization) of different strains in 50 L fermenters.

[0036] FIG. 2 shows a comparison of the parameters of the fermentation process of different strains in 50 L fermenters.

DETAILED DESCRIPTION

[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0038] Unless otherwise specified, the materials, reagents, and the like used in the following examples are all ordinary and commercially available products that can be purchased in the market.

[0039] The present invention will be further described by way of the examples below, and these descriptions are not intended to further limit the content of the present invention. It should be appreciated by those skilled in the art that equivalent substitutions or corresponding modifications made to the content of the present invention still fall within the scope of the present invention. Unless otherwise specified, in the culture media used in the following examples, the units of measurement for the percentages (%) used for the proportion of materials are all mass percentages.

Example 1: Mutagenesis Screening of Strains

[0040] A pure strain (HDCC00001) producing MK-7 was isolated from the feces of an adult who ate natto throughout the year. The strain was identified as Bacillus natto by 16SRNA sequencing (the sequence is set forth in SEQ ID NO: 1 in detail). Using adult feces as a source of isolation, the strain obtained has higher safety and is more suitable for the production of healthcare products. 0.1 mL of a thawed liquid of the original strain (HDCC00001) in a glycerol tube obtained after isolation was taken, inoculated into 50 mL of an LB liquid medium, and then cultured overnight at 37? C. The seed solution was mutagenized for 30 s using lithium chloride with a final concentration of 0.2%, serially diluted to 10-2-10-5 with sterile normal saline, then separated in an LB solid plate medium, and cultured for 24 h at a constant temperature of 37? ? C. in the dark. After colonies grew out, the colonies were transferred to a fresh LB solid medium using a toothpick and then cultured overnight at 37? ? C. The seed was then inoculated into a test tube (15 mL) containing 3 mL of a liquid primary screening fermentation medium using an inoculating needle, and then the tube was shaken for 96 h at 37? C. and 250 rpm, followed by addition of an equal volume of analytical pure isopropanol for soaking. After the mixture was centrifuged, the supernatant was taken for potency determination, and finally, an MK19283 strain was obtained, which had a fermentation level of 112 ?g/mL for the primary screening in the test tube.

[0041] The MK19283 strain in a glycerol tube was taken, serially diluted to 10-3-10-6 with sterile normal saline, then separated in an LB solid plate medium, and cultured for 24 h at a constant temperature of 37? ? C. After colonies grew out, 40 colonies were randomly selected, then each inoculated in one loop into a seed flask containing 20 mL of a liquid LB medium using an inoculating needle, and then cultured overnight at 37? C. and 250 rpm. The seed was then inoculated into a 250 ml triangular flask containing 30 mL of a liquid re-screening fermentation medium at a ratio of 4% (v/v), and then the flask was shaken for 96 h at 37? C. and 250 rpm. 1 mL of the fermentation broth was taken, followed by addition of an equal volume of analytical pure isopropanol for soaking. After the mixture was centrifuged, the supernatant was taken for potency determination. Highly productive strains were selected, and natural isolation and purification were continuously performed using the method described above until the fermentation level of each colony was stable. After three consecutive rounds of purification, a strain (HDCC00003) was obtained, which had a maximum shake-flask fermentation level of 228 ?g/mL, and the distribution range of shake-flask fermentation levels for 40 single colonies was 200?10 ?g/mL.

[0042] 0.1 mL of a thawed liquid of the HDCC00003 strain in a glycerol tube was taken, inoculated into 50 mL of an LB liquid medium, and cultured overnight at 37? C. After the seed solution was centrifuged, the bacteria were collected and resuspended in normal saline. The bacterial suspension was mutagenized for 10 s under a 15 W ultraviolet lamp and then mutagenized for another 10s using lithium chloride with a final concentration of 0.2%. By referring to the isolation and purification method for the obtained strain (HDCC00003) described above, a strain (HDCC00023) was obtained, which had a maximum shake-flask fermentation level of 584 ?g/mL, and the distribution range of shake-flask fermentation levels for 40 single colonies was 564?20 ?g/mL. The strain has been preserved in the China General Microbiological Culture Collection Center on Feb. 1, 2021, with an accession number of CGMCC NO. 21799.

[0043] Primary screening fermentation medium: 1% of glycerol, 0.5% of soybean peptone, pH 7.0, sterilized at 121? C. for 15 min, 3 mL/15 mL.

[0044] Re-screening fermentation medium: 4% of glycerol, 1.5% of soybean peptone, pH 7.0, sterilized at 121? ? C. for 20 min, 30 mL/250 mL.

Example 2: Fermentation Test of Primary Strain (HDCC00001) in 50 L Fermenter

1. Strain Activation

[0045] The original strain (HDCC00001) in a glycerol tube was taken and thawed at room temperature. 0.1 mL of the bacterial suspension was pipetted, inoculated onto an LB solid plate, plated uniformly, and then cultured in an incubator at 37? ? C. for 16 h to obtain activated single colonies.

2. Preparation of the Primary Seed

[0046] The activated single colonies were taken, and a loop of the colonies was scraped and then inoculated into a 500 mL triangular flask containing 100 mL of an LB liquid medium using an inoculating loop. The triangular flask was then wrapped well and placed on a shaker at 37? C. and 250 rpm for 8 h of shaking culture, with the OD value of the seed solution controlled to be ?4.0. The pH of the LB liquid medium was adjusted to 7.0 before sterilization, wherein the conditions for the sterilization were 121-123? C. and 30 min.

3. Preparation of the Secondary Seed

[0047] Qualified primary seed was taken and inoculated into a seed tank containing 10 L of an LB liquid medium at a ratio of 0.05% (V/W), the temperature was set at 37? C., the tank pressure was set at 0.05 MPa, and the air flow rate was set at 1vvm. Initial stirring was performed at 100 rpm, and the amount of dissolved oxygen was controlled to be ?40% with a stirring linkage. The culture period was 7 h, with the OD value of the seed solution controlled to be ?6.0.

[0048] The pH of the LB liquid medium was adjusted to 7.0 before sterilization, wherein the conditions for the sterilization were 121-123? C. and pressure-holding for 30 min.

4. Fermentation Culture

[0049] Qualified secondary seed was taken and inoculated into a fermenter containing 30 L of a liquid fermentation medium at a ratio of 4% (V/W), the temperature was set at 37? C., the tank pressure was set at 0.05 MPa, and the air flow rate was set at 0.7 vvm. Initial stirring was performed at 150 rpm, and when the dissolved oxygen dropped below 15%, the stirring linkage was turned on to control the amount of dissolved oxygen to be 5%-15%. The total residual sugar concentration was maintained to be 1.0%-1.5%, and the fermentation period was 7 days.

[0050] The formulation of the liquid fermentation medium was as follows: 2% of glycerol, 5% of soluble starch, and 2% of soybean peptone, with the pH adjusted to 8.0 before sterilization, wherein the conditions for the sterilization were 121-123? C. and pressure-holding for 30 min.

5. Index Monitoring During Fermentation

[0051] Since the beginning of fermentation, samples were taken every 12 h to determine the total sugar, amino nitrogen, and potency, and the statistical recording of the feeding amount was completed. According to the fermentation process and results of the strain and referring to FIG. 1 and FIG. 2, the strain (HDCC00001) had a maximum potency of 20.3 ?g/mL after 3 days of fermentation, and then the amount of dissolved oxygen rebounded rapidly, the CER (which refers to the amount of carbon dioxide released from the cultures in the biological fermentation process) dropped completely to zero, a large number of spores were formed as determined by microscopic examination, and the potency no longer increased. After a large amount of feeding, the CER appeared to rebound and the amount of dissolved oxygen dropped, indicating that the activity of the bacteria was recovered, but the potency still could not be effectively increased, and even fell backward. Meanwhile, by-products were continuously accumulated, so that the viscosity of the feeding liquid was continuously increased along with the extension of the fermentation culture period, resulting in a poorer mass transfer effect of the fermentation broth.

Example 3: Fermentation Test of Mutagenized Strain (HDCC00003) in 50 L Fermenter

[0052] The procedures in this example were the same as those in Example 2 except that the strain was a mutagenized strain (HDCC00003).

[0053] According to the fermentation process and results of the strain and referring to FIG. 1 and FIG. 2, the fermentation period of the strain (HDCC00003) could be extended to 4 days, after which the maximum potency of 124 ?g/mL was achieved. In the mid-prophase of fermentation, the carbon and nitrogen sources were rapidly consumed, the amount of dissolved oxygen dropped rapidly to zero, the stirring was carried out at the maximum rotation speed, and the feeding amount was increased sharply. The results of microscopic examination showed that the increase in the feeding amount was effective in avoiding the formation of spores, but was still unable to maintain the rate of increase in potency. In addition, the curves of stirring, dissolved oxygen, and CER in the fermentation process showed that the fermentation parameters fluctuated repeatedly after 84 h, indicating that although the potency of the strain obtained after mutagenesis with lithium chloride was improved, the fermentation process of the strain was poor in stability, resulting in the difficulty in scale-up control.

Example 4: Fermentation Test of Mutagenized Strain (HDCC00023) in 50 L Fermenter

[0054] The procedures in this example were the same as those in Example 2 except that the strain was a mutagenized strain (HDCC00023).

[0055] According to the fermentation process and results of the strain and referring to FIG. 1 and FIG. 2, the effective fermentation period of the strain (HDCC00023) could last for 7 days, and the fermentation potency could be continuously increased, reaching a maximum of 526 ?g/mL, thus successfully demonstrating the production capacity of the strain exhibited in the shake-flask fermentation. A comparison between the potency and cumulative feeding revealed that the strain (HDCC00023) consumed the least nutrients and produced the most MK-7 compared with the strain (HDCC00003) and the strain (HDCC00001). However, the viscosity of the feeding liquid was maintained at the level of about 100 cps, indicating that the formation of by-products was effectively controlled. That is, the strain (HDCC00023) produced more MK-7 with a significantly improved effective conversion rate under the condition of consuming very few nutrients.

[0056] The difficulty of the entry of oxygen into the fermentation broth was reduced due to the reduction of the viscosity of the feeding liquid, making it easier to control the dissolved oxygen through stirring, thus eliminating the need to frequently increase the stirring speed to maintain dissolved oxygen. The required amount of dissolved oxygen could be achieved by maintaining the stirring speed at about 400 rpm, and the dissolved oxygen in the entire fermentation process was controllable. In addition, because it was not necessary to carry out at a high rotation speed frequently, the machine loss and the energy consumption were correspondingly reduced. Furthermore, the reduced viscosity of the fermentation broth made the difficulty of subsequent purification correspondingly reduced.

[0057] The CER was relatively stable and a large number of spores are not formed throughout the microscopic examination, indicating that the strain (HDCC00023) had stronger activity and could realize continuous fermentation production. In addition, the fluctuation amplitude of pH can reflect the stability of the strains in metabolizing nutrients. The strain (HDCC00023) had a more moderate consumption of nutrients as reflected by the small fluctuation amplitude of pH, so the fermentation process of the strain (HDCC00023) was easier to control compared with other strains.

Example 5: Fermentation Test of Mutagenized Strain (HDCC00023) in 30-Ton (T) Fermenter

1. Strain Activation

[0058] The production strain (HDCC00023) in a working glycerol tube was taken and thawed at room temperature. 0.1 mL of the bacterial suspension was pipetted, inoculated onto an LB solid plate, plated uniformly, and then cultured in an incubator at 37? C. for 16 h to obtain activated single colonies.

2. Preparation of the Primary Seed

[0059] The activated single colonies were taken, and a loop of the colonies was scraped and then inoculated into a 2800 mL triangular flask containing 500 mL of an LB liquid medium using an inoculating loop. The triangular flask was then wrapped well and placed on a shaker at 37? C. and 220 rpm for 8 h of shaking culture, with the OD value of the seed solution controlled to be ?4.0. The pH of the LB liquid medium was adjusted to 7.0 before sterilization, wherein the conditions for the sterilization were 121-123? C. and 30 min.

3. Preparation of the Secondary Seed

[0060] Qualified primary seed was taken and inoculated into a seed tank containing 2 tons of an LB liquid medium at a ratio of 0.05% (V/W), the temperature was set at 37? C., the tank pressure was set at 0.05 MPa, and the air flow rate was set at 1vvm. Initial stirring was performed at 100 rpm, and the amount of dissolved oxygen was controlled to be ?40% with a stirring linkage. The culture period was 7 h, with the OD value of the seed solution controlled to be ?6.0.

[0061] The pH of the LB liquid medium was adjusted to 7.0 before sterilization, wherein the conditions for the sterilization were 121-123? C. and pressure-holding for 30 min.

4. Fermentation Culture

[0062] Qualified secondary seed was taken and inoculated into a fermenter containing 20 tons of a liquid fermentation medium at a ratio of 4% (V/W), the temperature was set at 37? C., the tank pressure was set at 0.05 MPa, and the air flow rate was set at 0.7 vvm. Initial stirring was performed at 80 rpm, and when the amount of dissolved oxygen dropped below 15%, the stirring linkage was turned on to control the dissolved oxygen to be 5%-15%. The total sugar concentration was controlled to be 1.0%-1.5%, and the fermentation period was 7 days.

[0063] The formulation of the liquid fermentation medium was as follows: 2% of glycerol, 5% of soluble starch, and 2% of soybean peptone, with the pH adjusted to 8.0 before sterilization, wherein the conditions for the sterilization were 121-123? C. and pressure-holding for 30 min.

[0064] The results of the fermentation culture showed that, after the strain (HDCC00023) was scaled up to 30 T scale, the process was stable and controllable with good reproducibility, and the effective fermentation period could last for more than 7 days with a fermentation potency up to 384 ?g/mL.

[0065] In conclusion, the strain (HDCC00023) had a much higher potency than that of the strain (HDCC00003) and the strain (HDCC00001), and its fermentation potency could be continuously increased; the formation of by-products was effectively controlled, the growth and metabolism were mild, the dissolved oxygen in the entire fermentation process was controllable, the control of nutrient feeding was easier, and the process stability was good, so that the strain (HDCC00023) is suitable for industrial scale-up. The present invention fundamentally solves the key challenges of difficulty in scale-up (poor repeatability of process regulation), low effective conversion rate of substrate, and high production cost in the industrialization process of MK-7, and reduces the difficulty of purification of fermentation broth.

[0066] There are a variety of fermentation methods for producing MK-7 using Bacillus natto in the prior art, and the above examples are used only for exemplary illustration of the fermentation method employed for the Bacillus natto strain provided by the present invention. It can be understood that, after learning the Bacillus natto strain provided by the present invention, those skilled in the art can utilize the Bacillus natto strain provided herein for the fermentation production of MK-7 and other products (e.g., the seed solution, fermentation broths, fermentation products, and bacterial residues), and utilize the Bacillus natto strain provided herein or the MK-7 or other products produced therefrom for the production of other products, such as the manufacture of medicaments, healthcare products, foods, beverages, cosmetic products, additives, and feeds.