Method for preparing surfactin-rich bacillus subtilis preparation by liquid fermentation-adsorption method

Abstract

This invention relates to the field of bioengineering technology, and in particular to a method for preparing Bacillus subtilis preparations rich in surfactantin using a liquid fermentation-adsorption method. The method involves preparing and selecting a fermentation substrate liquid, inoculating with a seed liquid followed by liquid fermentation, and using a specific Bacillus subtilis (Bacillus subtilis) S21. This liquid fermentation method avoids the technical drawbacks of solid fermentation, which is prone to contamination by other microorganisms and results in low surfactantin yield. Furthermore, the method combines fermentation sludge and extrusion residue with mixing, adsorption, and airflow drying, avoiding the high costs associated with traditional liquid fermentation and spray drying. This improves both surfactantin content and spore count in the Bacillus subtilis preparation, thereby enhancing the quality of Bacillus subtilis preparations for feed additives.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, in particular to a method for preparing a surfactin-rich Bacillus subtilis preparation by liquid fermentation-adsorption method. BACKGROUND

[0002] At present, in order to improve the utilization rate of feed and promote the growth of livestock and poultry, growth-promoting antibiotics are usually added to the feed. However, with the use of growth-promoting antibiotics, antibiotic residues, environmental pollution, and increased drug resistance and drug tolerance have caused livestock and poultry to be in a sub-healthy state for a long time, resulting in decreased immunity and high breeding costs. Moreover, antibiotic residues greatly affect the health of consumers. Therefore, the search for new feed additives to replace antibiotics has attracted the attention of technical personnel in the field of antibiotic replacement.

[0003] Surfactin is a cyclic lipopeptide with surface activity produced by Bacillus subtilis through a non-ribosomal pathway. It is composed of a cyclic peptide of 7 amino acid residues and a β-hydroxy fatty acid ester with a chain length of 13-15 carbon atoms. Surfactin has been widely used in livestock and poultry feed additives and can effectively improve the production performance, immunity, and feed conversion rate of livestock and poultry.

[0004] Based on this, our research team has also conducted research on the preparation of microecological preparations using Bacillus subtilis S21 as a seed in the early stage. For example, patent No. 201810608662.5 discloses the preparation of a Vitex negundo microecological preparation by semi-solid fermentation. However, the surfactin content in this microecological preparation is very low, and the cost of Vitex negundo medicinal materials is high, resulting in high use cost of the Vitex negundo microecological preparation. For another example, patent application No. 20211001334.X discloses the production of a functional microbial feed additive containing surfactin by solid-state fermentation using soybean meal as the main raw material component. However, this method uses solid-state fermentation, which is easily infected with miscellaneous bacteria, resulting in low surfactin content and spore content in the functional microbial feed additive produced by fermentation.

[0005] In view of this, our research team has explored the preparation of surfactin-rich feed preparations using Bacillus subtilis S21 based on previous research and practical experience, providing a new process for surfactin feed preparations. SUMMARY

[0006] To solve the above technical problems in the prior art, the present application provides a method for preparing a surfactin-rich Bacillus subtilis preparation by liquid fermentation-adsorption method.

[0007] Specifically, the technical solutions are achieved by the following technical solutions.

[0008] The method for preparing the surfactin-rich Bacillus subtilis preparation by liquid fermentation-adsorption method comprises the following steps:

[0009] (1) mixing fresh corncob and rice husk, ball milling with water, sieving, and centrifugal separation to obtain a separation liquid and a separation slurry;

[0010] (2) adding auxiliary materials to the separation liquid to prepare a fermentation substrate liquid, and obtaining an extrusion liquid and an extrusion residue by extruding the liquid in the separation slurry;

[0011] (3) culturing Bacillus subtilis S21 by using an LB culture medium to obtain a seed liquid;

[0012] (4) inoculating the seed liquid in step (3) into the fermentation substrate liquid in step (2), uniformly stirring, and introducing sterile air to perform fermentation treatment at 25-37°C for 2-4 days, and then performing centrifugal separation to obtain a fermentation liquid and a fermentation mud;

[0013] (5) uniformly mixing the fermentation mud in step (4) with the extrusion residue in step (2), performing air flow drying, crushing, and packaging to obtain the product.

[0014] Preferably, the fresh corncob is crushed and ground to pass through a 100-mesh sieve, the rice husk is crushed to pass through a 100-mesh sieve, and then the fresh corncob and the rice husk are uniformly mixed according to a mass ratio of 1:0.4-0.6.

[0015] Preferably, the ball milling with water is performed by adjusting a liquid-solid volume ratio to 1:0.1-0.3, and the sieving is performed by passing through a 300-mesh sieve.

[0016] Preferably, the auxiliary materials include, in terms of mass parts, 4-5 parts of glucose, 0.2-0.3 parts of potassium dihydrogen phosphate, 0.1 part of magnesium sulfate, and 20-30 parts of protein powder; and the auxiliary materials are added in the separation liquid in an amount of 10-30% of the mass of the separation liquid.

[0017] Preferably, the seed liquid accounts for 10% of the mass of the fermentation substrate liquid.

[0018] Preferably, the mass ratio of the fermentation mud to the extrusion residue is 1:2-3 on a dry basis.

[0019] Preferably, the air flow drying is performed by using hot air at a temperature of 50-70°C to dry the mixture to a water content of less than 8%.

[0020] Preferably, the extrusion liquid is returned and uniformly mixed with the separation liquid.

[0021] Preferably, the fermentation liquid is returned, mixed with the seed liquid, and then added to the separation liquid.

[0022] Preferably, the fermentation liquor is first adsorbed by the adsorption carrier, and then separated by liquid-solid separation to obtain tail liquor and adsorption residue; the tail liquor is returned and mixed with the seed liquor before being added to the separation liquor; the adsorption residue is mixed with the fermentation mud, and then dried by airflow to a water content of less than 8%; and the adsorption carrier is obtained by drying the extrusion residue to a water content of less than 8%.

[0023] Preferably, the drying of the extrusion residue is carried out at 50°C to a water content of less than 8%. The adsorption carrier is mixed with the fermentation liquor at a liquid-solid volume ratio of 1:0.2-0.3. The adsorption residue is mixed with the fermentation mud at an equal mass ratio.

[0024] Compared with the prior art, the technical effects of the present invention are embodied in:

[0025] The present invention selects fresh corn cobs and rice husks to prepare extrusion residue and separation liquor, so that nutrients enter the separation liquor, and then the fermentation substrate liquid for liquid culture is prepared by combining with excipients, so as to meet the nutritional needs of Bacillus subtilis culture. In addition, liquid fermentation is introduced to avoid the defects of semi-solid or solid fermentation, such as easy bacterial infection and low surfactin yield. Moreover, the dispersibility of the extrusion residue is improved, the drying energy consumption is reduced during reuse of the extrusion residue, the surfactin content and spore content are guaranteed, and the cost of feed additive preparation is reduced.

[0026] The present invention uses extrusion residue drying treatment, which not only improves the adsorbability of the extrusion residue, but also makes the adsorption of surfactin and spores in the fermentation liquor more significant, which helps to improve the content of surfactin in the preparation and improve the quality of the preparation. Moreover, the dispersibility of the extrusion residue is improved, so that the overall adhesion is reduced after mixing with the fermentation mud, thereby reducing the drying difficulty and energy consumption. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The process flow chart of the present invention is shown.

[0028] Figure 2 The process flow chart of another embodiment of the present invention is shown. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention are further limited by combining the drawings and specific embodiments, but the scope of protection is not limited to the description.

[0030] As shown in Figure 1 some embodiments, the liquid fermentation-adsorption method for preparing Bacillus subtilis preparation rich in surfactin includes the following steps:

[0031] (1) Mix fresh corn cobs with rice husks, add water and ball mill, sieve, centrifuge to separate, and obtain separation liquid and separation slurry; using fresh corn cobs can make full use of the sugar in the corn cobs and ensure that the viscosity after mixing with rice husks is high, which promotes the formation of slurry during the ball milling process.

[0032] (2) Add auxiliary materials to the separation liquid to prepare a fermentation substrate liquid, and squeeze the liquid in the separation slurry to obtain extruded liquid and extruded residue; by adding auxiliary materials, the influence composition required for the growth of strains can be adjusted to meet the requirements of subsequent fermentation to prepare surfactantin and increase the yield of surfactantin.

[0033] (3) Bacillus subtilis S21 was cultured in LB medium to obtain seed culture. According to previous studies, this strain has a better surfactant production effect. For example, Bacillus subtilis S21, which was described in the patent application No. 201810608662.5 filed by our research team, was deposited at the China General Microbiological Culture Collection Center on April 2, 2018, with the strain preservation number: CGMCCNo: 15544.

[0034] (4) Inoculate the seed liquid from step (3) into the fermentation substrate liquid from step (2), stir evenly, introduce sterile air, ferment at 25-37℃ for 2-4 days, centrifuge to separate, and obtain fermentation liquid and fermentation mud; realize liquid fermentation, avoid the technical defects of traditional solid fermentation that is easily infected by miscellaneous bacteria, and improve the efficiency of fermentation production of surfactantin.

[0035] (5) Mix the fermented mud from step (4) with the extruded residue from step (2) evenly, dry it with airflow, crush and package it to obtain the final product. The introduction of solid mixing and airflow drying process avoids the high cost caused by spray drying of the liquid produced by fermentation.

[0036] This invention creates a method for preparing and selecting a fermentation substrate liquid. It utilizes liquid fermentation after seed liquid inoculation and selects a specific Bacillus subtilis S21. The combination of liquid fermentation and solid fermentation avoids the technical defects of easy contamination by other bacteria, which leads to low surfactant yield. Furthermore, the combination of mixing, adsorption, and airflow drying of fermentation sludge and extrusion residue avoids the technical defects of high cost caused by traditional liquid fermentation and spray drying. As a result, the surfactant content and spore count in Bacillus subtilis preparations are improved, thus enhancing the quality of Bacillus subtilis preparations for feed additives.

[0037] In some embodiments, the mixing involves crushing and grinding fresh corn cobs and passing them through a 100-mesh sieve, and crushing rice husks and passing them through a 100-mesh sieve. The fresh corn cobs and rice husks are then mixed evenly at a mass ratio of 1:0.4-0.6, for example: 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6, etc. The water-added ball milling adjusts the liquid-to-solid volume ratio to 1:0.1-0.3, for example: 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, etc., and the sieving is done through a 300-mesh sieve. This ensures the formation of the slurry and maximizes the utilization of nutrients in the corn cobs and rice husks. The auxiliary materials include, by mass, 4-5 parts glucose, 0.2-0.3 parts potassium dihydrogen phosphate, 0.1 parts magnesium sulfate, and 20-30 parts protein powder; and the amount of these auxiliary materials added to the separation liquid is 10-30% of the separation liquid's mass. This ensures that the separated liquid meets the fermentation requirements of Bacillus subtilis S21, thereby increasing the yield of surfactantin produced during fermentation. The seed liquid accounts for 10% of the mass of the fermentation substrate liquid.

[0038] In some embodiments, the mass ratio of the fermented sludge to the extruded residue is 1:2-3 on a dry basis. This improves airflow drying efficiency and reduces drying difficulty. The airflow drying uses hot air at a temperature of 50-70°C to dry to a moisture content of <8%.

[0039] In some embodiments, the extruded liquid is returned to the separated liquid and mixed thoroughly.

[0040] In some embodiments, the fermentation broth is returned to be mixed with the seed broth and then added to the separation broth.

[0041] like Figure 2 As shown, in some embodiments, the fermentation broth is first adsorbed using an adsorption carrier, then separated by liquid-solid separation to obtain tailings and adsorption residue; the tailings are returned to be mixed with the seed broth and then added back to the separated broth; the adsorption residue is mixed with the fermentation sludge and then dried by airflow to a moisture content of <8%; the adsorption carrier is obtained by drying the extrusion residue to a moisture content of <8%. This improves the adsorption efficiency of nutrients in the fermentation broth while ensuring that a large number of spores in the fermentation sludge can be collected, thereby improving the surfactantin content in the Bacillus subtilis preparation and resulting in a higher spore count.

[0042] In some embodiments, the extruded residue is dried at 50°C until the moisture content is <8%. The amount of adsorbent added to the fermentation broth is such that the liquid-to-solid volume ratio is 1:0.2-0.3, for example: 1:0.20, 1:0.22, 1:0.25, 1:0.28, 1:0.30, etc. The adsorbent residue and the fermentation sludge are mixed in an equal mass ratio.

[0043] In order to verify the technical effects of the invention, the researchers prepared samples of the Bacillus subtilis preparation created in this invention in the laboratory and conducted research on the surfactant and spore count of the obtained samples.

[0044] 1. Sample preparation

[0045] Example 1

[0046] Fresh corn cobs are crushed and ground through a 100-mesh sieve, and rice husks are crushed and ground through a 100-mesh sieve. The sieved fresh corn cobs and sieved rice husks are then mixed evenly at a mass ratio of 1:0.4. The mixture is then placed in a ball mill and ball-milled into a slurry. Water is added during the ball milling process to adjust the liquid-to-solid volume ratio to 1:0.1. The mixture is then passed through a 300-mesh sieve and centrifuged at 1000 r / min to separate the liquid and slurry.

[0047] According to the mass parts (kg), take 4 parts of glucose, 0.2 parts of potassium dihydrogen phosphate, 0.1 parts of magnesium sulfate, and 20 parts of egg white protein powder, mix them evenly to prepare the excipients; take the separation liquid, add 10% of the excipients according to the mass of the separation liquid to the separation liquid, stir evenly at 1000 r / min to obtain the fermentation substrate liquid; boil the fermentation substrate liquid in a water bath for 30 min at room temperature and pressure, and set aside for use.

[0048] Bacillus subtilis S21 was cultured in LB medium to obtain seed culture. The seed culture was inoculated into the fermentation substrate at 10% of the mass of the fermentation substrate, stirred evenly, and sterile air was introduced. The mixture was fermented at 25-37℃ for 2 days. The fermentation liquid and fermentation sludge were obtained by centrifugation at 1000 r / min.

[0049] The separated slurry is filtered by a plate and frame press to obtain extruded liquid and extruded residue. The extruded residue is mixed with fermented mud at a dry weight ratio of 2:1, and then dried with hot air at 50°C until the moisture content is <8%. The mixture is then crushed and packaged to obtain the final product.

[0050] Example 2

[0051] Fresh corn cobs are crushed and ground through a 100-mesh sieve, and rice husks are crushed and ground through a 100-mesh sieve. The sieved fresh corn cobs and sieved rice husks are then mixed evenly at a mass ratio of 1:0.6. The mixture is then placed in a ball mill and ball-milled into a slurry. Water is added during the ball milling process to adjust the liquid-to-solid volume ratio to 1:0.3. The mixture is then passed through a 300-mesh sieve and centrifuged at 1000 r / min to separate the liquid and slurry.

[0052] According to the mass parts (kg), take 5 parts of glucose, 0.3 parts of potassium dihydrogen phosphate, 0.1 parts of magnesium sulfate, and 30 parts of egg white protein powder, mix them evenly to prepare the excipients; take the separation liquid, add the excipients accounting for 30% of the mass of the separation liquid to the separation liquid, stir evenly at 1000 r / min to obtain the fermentation substrate liquid; boil the fermentation substrate liquid in a water bath at room temperature and pressure for 30 min, and set aside for use.

[0053] Bacillus subtilis S21 was cultured in LB medium to obtain seed culture. The seed culture was inoculated into the fermentation substrate at 10% of the mass of the fermentation substrate, stirred evenly, and sterile air was introduced. The mixture was fermented at 25-37℃ for 4 days. The fermentation liquid and fermentation sludge were obtained by centrifugation at 1000 r / min.

[0054] The separated slurry is filtered by a plate and frame extrusion filter to obtain extruded liquid and extruded residue. The extruded residue is mixed with fermented mud at a dry weight ratio of 3:1, and then dried with hot air at 70℃ until the moisture content is <8%. The mixture is then crushed and packaged to obtain the final product.

[0055] Example 3

[0056] Fresh corn cobs are crushed and ground through a 100-mesh sieve, and rice husks are crushed and ground through a 100-mesh sieve. The sieved fresh corn cobs and sieved rice husks are then mixed evenly at a mass ratio of 1:0.5. The mixture is then placed in a ball mill and ball-milled into a slurry. Water is added during the ball milling process to adjust the liquid-to-solid volume ratio to 1:0.2. The mixture is then passed through a 300-mesh sieve and centrifuged at 1000 r / min to separate the liquid and slurry.

[0057] Take 5 parts glucose, 0.2 parts potassium dihydrogen phosphate, 0.1 parts magnesium sulfate, and 20 parts egg white protein powder by weight (kg), mix them evenly to prepare the excipients; take the separation liquid, add 20% of the excipients by weight of the separation liquid to the separation liquid, stir evenly at 1000 r / min to obtain the fermentation substrate liquid; boil the fermentation substrate liquid in a water bath at room temperature and pressure for 30 min, and set aside for use.

[0058] Bacillus subtilis S21 was cultured in LB medium to obtain seed culture. The seed culture was inoculated into the fermentation substrate at 10% of the mass of the fermentation substrate, stirred evenly, and sterile air was introduced. The mixture was fermented at 25-37℃ for 3 days. The fermentation liquid and fermentation sludge were obtained by centrifugation at 1000 r / min.

[0059] The separated slurry is filtered by a plate and frame press to obtain extruded liquid and extruded residue. The extruded residue is mixed with fermented mud at a dry weight ratio of 2:1 and then dried with hot air at 60°C until the moisture content is <8%. The mixture is then crushed and packaged to obtain the final product.

[0060] Example 4

[0061] Based on Example 3, the extrusion residue was dried at a constant temperature of 50°C until the moisture content was <8% and then used as an adsorption carrier. The adsorption carrier was added to the fermentation broth at a liquid-to-solid volume ratio of 1:0.25 and stirred until uniform. After liquid-solid separation, tail liquid and adsorption residue were obtained. The tail liquid was returned to be mixed with the seed liquid. The adsorption residue and fermentation mud were then mixed uniformly at an equal mass ratio and dried with hot air at 60°C until the moisture content was <8%. The mixture was then pulverized and packaged. All other steps were the same as in Example 3.

[0062] Example 5

[0063] Based on Example 3, the extrusion residue was dried at a constant temperature of 50°C until the moisture content was <8% and then used as an adsorption carrier. The adsorption carrier was added to the fermentation broth at a liquid-to-solid volume ratio of 1:0.2 and stirred until uniform. After liquid-solid separation, tail liquid and adsorption residue were obtained. The tail liquid was returned to be mixed with the seed liquid. The adsorption residue and fermentation mud were then mixed uniformly at an equal mass ratio and dried with hot air at 70°C until the moisture content was <8%. The mixture was then pulverized and packaged. All other steps were the same as in Example 3.

[0064] Example 6

[0065] Based on Example 3, the extrusion residue was dried at a constant temperature of 50°C until the moisture content was <8% and then used as an adsorption carrier. The adsorption carrier was added to the fermentation broth at a liquid-to-solid volume ratio of 1:0.3 and stirred until uniform. After liquid-solid separation, tail liquid and adsorption residue were obtained. The tail liquid was returned to be mixed with the seed broth. The adsorption residue and fermentation mud were then mixed uniformly at an equal mass ratio and dried with hot air at 60°C until the moisture content was <8%. The mixture was then pulverized and packaged. All other steps were the same as in Example 3.

[0066] 2. Determination of spore count and surfactantin content

[0067] The spore count determination method and surfactantin determination method in this invention were performed in accordance with the methods described in the literature with patent application number 202110013347.X filed by the researcher in a previous study. The determination results are shown in Table 1 below.

[0068] Table 1 Results of spore number and surfactantin content determination

[0069] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Surfactin content (mg / kg) 719.36 722.43 718.54 803.12 789.65 790.31 Spore count (cfu / g) x 10 11 ]] 9.16 9.57 10.03 10.16 10.21 10.09

[0070] Note: The data in Table 1 is the average of three tests.

[0071] As shown in Table 1, the surfantin content in the Bacillus subtilis preparation obtained in this invention reached over 0.7 mg / g, and the spore count reached 9.16 × 10⁻⁶.11 In addition, this method also utilizes liquid fermentation to treat Bacillus subtilis S21, replacing the shortcomings of previous semi-solid or solid-state fermentation methods, thereby increasing surfantin yield and avoiding the high cost associated with spray drying required for liquid fermentation.

[0072] For any matters not covered in this invention, reference can be made to existing technologies or common knowledge known to those skilled in the art, and conventional technical means can be used for implementation. For example, the determination of spore count and surfantin content in this invention refers to the technical content disclosed in patent application number 202110013347.X. The Bacillus subtilis S21 used in this invention was deposited at the China General Microbiological Culture Collection Center on April 2, 2018, with the culture collection number: CGMCC No: 15544.

[0073] The step of mixing the extruded residue with the fermented mud in this invention has the technical effect of reducing the viscosity of the fermented mud, promoting better dispersibility of the fermented mud, and also adsorbing the liquid components in the fermented mud, thereby improving the effect of subsequent drying and dispersion, and reducing the energy consumption and cost of crushing and drying.

[0074] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing Bacillus subtilis preparations rich in surfactantin via liquid fermentation-adsorption, characterized in that, Includes the following steps: (1) Crush and grind fresh corn cobs and pass them through a 100-mesh sieve, and crush rice husks and pass them through a 100-mesh sieve. Mix fresh corn cobs and rice husks at a mass ratio of 1:0.

5. Add water to adjust the liquid-solid volume ratio to 1:0.2 and ball mill. Pass the mixture through a 300-mesh sieve and centrifuge at a speed of 1000 r / min to obtain the separation liquid and separation slurry. (2) Add 20% of the mass of the excipient to the separation liquid, the excipient being composed of 5 parts glucose, 0.2 parts potassium dihydrogen phosphate, 0.1 parts magnesium sulfate and 20 parts protein powder, to prepare a fermentation substrate liquid; extrude the liquid in the separation slurry to obtain extruded liquid and extruded residue; dry the extruded residue at a constant temperature of 50°C until the moisture content is <8% and then use it as an adsorption carrier. (3) Bacillus subtilis S21 with preservation number CGMCC No:15544 was cultured in LB medium to obtain seed culture; (4) Inoculate the seed liquid from step (3) into the fermentation substrate liquid from step (2) at 10% of the mass of the fermentation substrate liquid, stir evenly, introduce sterile air, ferment at 25-37℃ for 2-4 days, centrifuge to separate, and obtain fermentation liquid and fermentation mud. The adsorption carrier was added to the fermentation broth at a liquid-to-solid volume ratio of 1:0.2 and stirred until homogeneous. Then, the mixture was separated into liquid and solid to obtain the tail liquid and adsorption residue. The tail liquid was returned to be mixed with the seed liquid and then added back to the separated liquid. (5) Mix the adsorption residue and fermentation mud in equal mass ratio, dry them with hot air at 70°C until the moisture content is <8%, crush and package them to obtain the preparation.

2. The method for preparing Bacillus subtilis preparations rich in surfactantin using the liquid fermentation-adsorption method as described in claim 1, characterized in that, The extruded liquid is returned to the separated liquid and mixed thoroughly.

3. The method for preparing Bacillus subtilis preparations rich in surfactantin using the liquid fermentation-adsorption method as described in claim 1, characterized in that, The fermentation broth is returned to be mixed with the seed broth and then added to the separation broth.

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