Fermentation method for improving yield of D-biotin

Abstract

The invention belongs to the technical field of fermentation engineering, and particularly relates to an improved method of a liquid fermentation method for producing D-biotin. According to the production method, pseudomonas variabilis is taken as a fermentation strain, glycerol and glucose are taken as a composite carbon source, a three-stage temperature control method is adopted in the fermentation process, and dissolved oxygen in fermentation liquor in the whole fermentation process is further controlled, so that the yield of D-biotin is increased. The method for controlling the dissolved oxygen comprises the following steps: maintaining relatively high DO in the early stage to enable thalli to rapidly start growth, and then properly reducing the DO level of a fermentation system to enable the thalli to be massively produced and accumulate D-biotin, or maintaining relatively low DO level in the whole process, so that the method disclosed by the invention is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fermentation engineering, and particularly relates to an improved method for a liquid fermentation method for producing D-biotin. BACKGROUND

[0002] Biotin, also known as vitamin B7, vitamin H or coenzyme R, mainly exists in animals and plants in nature, is a vitamin indispensable to maintain normal physiological activities of the human body, and is a necessary substance for synthesizing vitamin C. It is a bicyclic compound containing 3 asymmetric carbon atoms, and there are 8 isomers, but only D-biotin with a full cis structure has physiological activity.

[0003] D-biotin was first discovered in the research of yeast, and is involved in nitrogen metabolism in yeast, so that a small amount of D-biotin added in brandy can improve the flavor of the wine. With in-depth research, D-biotin can be gradually applied to many fields such as animal husbandry, biotechnology and fermentation industry. It is reported that D-biotin can promote rapid decomposition of fat and convert it into energy needed for human movement, which lays the foundation for the application of D-biotin in weight loss sports health care products.

[0004] Biotin synthesis can be divided into biosynthesis and chemical synthesis. Chemical synthesis is the main method for synthesizing biotin in industry. The total synthesis method was first reported by chemists Sternbach and Goldberg of Hoffmann-La-Roche in Switzerland in the 1940s. The main disadvantages of chemical synthesis are high cost, complex process and heavy environmental burden. In addition, a small amount of biotin is prepared by microbial fermentation (relatively low cost, safer, simpler process and environmentally friendly), but due to the low yield of D-biotin produced by the existing fermentation method, it is still in the research and development stage, and the domestic fermentation level is far behind the international leading level, which cannot meet the needs of industrial production. Therefore, it is necessary to develop an improved microbial fermentation method to improve the yield of D-biotin, which is of great significance to break through the technical barriers of existing chemical synthesis and improve the existing fermentation process. On the other hand, it can also meet the needs of industrialization.

[0005] The applicant finds through theoretical analysis and experimental research that if the bacterial growth is too fast, the carbon source is rapidly consumed for cell growth, thereby being not conducive to metabolic flow to the D-biotin synthesis direction, and too high DO (dissolved oxygen, DO for short) will lead to vigorous respiration of the bacterial cells, which is not conducive to the synthesis of D-biotin. Moreover, D-biotin is a secondary metabolite, and a short growth period is not conducive to the accumulation of D-biotin. In order to realize the accumulation of a large amount of D-biotin, it is necessary to appropriately switch the growth stage of the bacterial cells and the accumulation stage of D-biotin, maintain a relatively high DO in the early stage to enable the bacterial cells to start growing rapidly, and then appropriately reduce the DO level of the fermentation system, so that the bacterial cells can produce and accumulate a large amount of D-biotin, or maintain a low DO level throughout the process to realize the present application.

[0006] The applicant of the present application has previously optimized the flow process and temperature control in the D-biotin fermentation process (Chinese invention patent application: CN114480525A), and the present application further optimizes the DO control in the fermentation process on the basis of the above-mentioned process, so that the yield of D-biotin is further improved. SUMMARY

[0007] In order to achieve the above-mentioned purpose, the present application discloses a production method for improving the yield of D-biotin, which uses Pseudomonas variabilis as a fermentation strain, uses glycerol and glucose as a composite carbon source, uses a three-stage temperature control method in the fermentation process, and further controls the dissolved oxygen in the fermentation broth throughout the fermentation process, thereby improving the yield of D-biotin.

[0008] The present application achieves the above-mentioned purpose by using the following technical route:

[0009] One of the technical solutions provided by the present application is a method for producing D-biotin by controlling low DO, which is as follows: the fermentation strain seed liquid is inoculated into the fermentation medium at an inoculation amount of 1-5%, the DO content is controlled to be lower than 2% throughout the fermentation, when the glycerol content is reduced to 1 g / L, glycerol is added to maintain the glycerol content in the system at 1 g / L, the pH in the system is maintained at 6.8-7.4 during the fermentation, and a three-stage temperature control method is used: the fermentation temperature is controlled to be 30-32℃ from the beginning of fermentation to when the glucose content in the fermentation broth is 0; the fermentation temperature is controlled to be 28-29℃ from when the glucose content is 0 to when the glycerol content in the fermentation broth naturally reduces to 1 g / L; and the fermentation temperature is controlled to be 25-27℃ from when the glycerol content in the fermentation broth naturally reduces to 1 g / L to the end of fermentation.

[0010] Further, the method for controlling DO includes but is not limited to adjusting the rotation speed and / or adjusting the ventilation; the rotation speed ranges from 200 rpm to 1000 rpm, and the ventilation ranges from 2 L / min to 5 L / min.

[0011] Further, the carbon source in the fermentation medium is a composite carbon source, which comprises glucose and glycerol; the concentration ratio of the glucose and glycerol is 1-2:1-2;

[0012] Further, the fermentation medium is (g / L): glucose 15-30, glycerol 15-30, yeast extract 10-39, (NH4)2SO41-4, Na2HPO4·12H2O 10-20, KH2PO43-10, MgSO4·7H2O 0.2-2, FeSO4·7H2O 0.08-0.1; pimelic acid 0.1-1.0, pH 7.0-7.3.

[0013] As an alternative to the technical solution one, the application regulates DO in stages to further improve the yield of D-biotin.

[0014] The technical solution two provided by the application is a D-biotin production method for regulating DO in stages, and the method is specifically as follows:

[0015] The fermentation strain seed liquid is inoculated into the fermentation medium at an inoculation amount of 1-5%, glycerol is added to maintain the glycerol content in the system at 1g / L when the glycerol content is reduced to 1g / L, the pH of the system is maintained at 6.8-7.4 during the fermentation process, the fermentation temperature is controlled at 30-32℃ from the beginning of the fermentation to when the glucose content in the fermentation liquid is 0 in the first stage, the fermentation temperature is controlled at 28-29℃ from when the glucose content is 0 to when the glycerol content in the fermentation liquid naturally reduces to 1g / L in the second stage, the fermentation temperature is controlled at 25-27℃ when the glycerol content in the fermentation liquid naturally reduces to 1g / L to the end of the fermentation in the third stage, and the DO content is controlled in stages throughout the fermentation process.

[0016] Further, the method for controlling DO includes but is not limited to adjusting the rotation speed and / or adjusting the ventilation; the rotation speed ranges from 200 to 1000 rpm, and the ventilation ranges from 2 to 5 L / min.

[0017] Further, the carbon source in the fermentation medium is a composite carbon source, which comprises glucose and glycerol; the concentration ratio of the glucose and glycerol is 1-2:1-2;

[0018] Further, the fermentation medium is (g / L): glucose 15-30, glycerol 15-30, yeast extract 10-39, (NH4)2SO41-4, Na2HPO4·12H2O 10-20, KH2PO43-10, MgSO4·7H2O 0.2-2, FeSO4·7H2O 0.08-0.1; pimelic acid 0.1-1.0, pH 7.0-7.3.

[0019] Further, the method for controlling DO content in stages is: the DO content in the first stage is higher than 20%, the DO content in the second and third stages is lower than 2%;

[0020] Further, the method for controlling DO content in stages is: the DO content in the first stage is higher than 20%, the DO content in the second and third stages is lower than 2%;

[0021] Further, the method for controlling DO content in stages is: the DO content in the first, second stages is higher than 20%, the DO content in the third stage is lower than 2%;

[0022] Further, the method for controlling DO content in stages is: the DO content in the first, second stages is higher than 20%, the DO content in the third stage is lower than 2%.

[0023] Beneficial effects:

[0024] On the basis of the prior art of producing D-biotin by using glycerol and glucose as a compound carbon source and by using a three-stage temperature control method in a fermentation process, the present application further controls the dissolved oxygen in the fermentation broth in the whole fermentation process, thereby improving the yield of D-biotin. The method for controlling the dissolved oxygen includes:

[0025] (1) The whole process is controlled at a low DO, and the DO content is maintained to be lower than 2%, which can make the yield of D-biotin increase to 773.8 mg / L, which is increased by 191.1% compared with 265.8 mg / L in the whole process of constant temperature and constant DO, increased by 30.5% compared with 592.8 mg / L in the three-stage control, and increased by 117.2% compared with 356.2 mg / L in the low-DO control only.

[0026] (2) The DO is controlled in stages according to a three-stage temperature control method, and different DO contents are controlled in different stages, specifically:

[0027] The DO content in the first stage is higher than 20%, the DO content in the second and third stages is lower than 2%, and the yield of D-biotin reaches 807.5 mg / L;

[0028] The DO content in the first stage is 2%-20%, the DO content in the second and third stages is lower than 2%, and the yield of D-biotin reaches 783.4 mg / L;

[0029] The DO content in the first and second stages is higher than 20%, the DO content in the third stage is lower than 2%, and the yield of D-biotin reaches 790.4 mg / L;

[0030] The DO content in the first and second stages is 2%-20%, the DO content in the third stage is lower than 2%, and the yield of D-biotin reaches 762.4 mg / L. DETAILED DESCRIPTION

[0031] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and techniques related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunology, and laboratory procedures used herein are those commonly used in the corresponding fields and are well known and commonly practiced by those skilled in the art. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below. It should also be understood that the terms used herein are only for the purpose of describing the specific embodiments and are not intended to be limiting.

[0032] The articles "a" and "the" are used herein to refer to one or more than one, independent of any other quantifier.

[0033] The use of the alternative (for example, "or") should be understood to mean one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean one or both of the alternatives. As used herein and unless otherwise indicated, the term "about" or "approximately" means that a measurable value such as an amount, a time period, and the like, is within 10% of the stated value ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1%, as long as this variation is appropriate for the method being performed.

[0034] For example, the biomass OD 600 To about 20 means that its range is 15-25, preferably 18-22, more preferably 19.5-20.5.

[0035] The genetic engineering terms or gene names used in the present application have the usual meaning in the art, and can also refer to the prior application document CN114480525A cited in the present application.

[0036] Biomass OD 600 Means the absorbance value of a certain solution at a wavelength of 600 nm, OD 600 Is a standard method for tracking microbial growth in liquid culture; the culture solution without bacteria is used as a blank, and then the bacterial-containing culture solution after culture is quantified. OD 600 Commercialized instruments can be used for detection.

[0037] The synthesis method of the present application will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above content of the present application is covered within the scope intended to be protected by the present application. Unless otherwise specified, the raw materials and reagents used in the following examples are all commonly commercially available goods, or can be prepared by known methods.

[0038] In the embodiments, the culture medium components are as follows:

[0039] LB medium (g / L): Tryptone 10.0, yeast powder 5.0, and NaCl 10.0.

[0040] SM seed medium (g / L): Glucose 10.0, yeast extract powder 10.0, tryptone 20.0, Na2HPO4·12H2O 0.5, MgSO4·7H2O 0.8, (NH4)2SO4 2.0, pH 7.0.

[0041] IFM fermentation medium (g / L): Glycerol 40.0, yeast tryptone 20.0, (NH4)2SO4 2.0, Na2HPO4·12H2O 18.0, KH2PO4 4.05, MgSO4·7H2O 2.0, FeSO4·7H2O 0.1, pimelic acid 0.5, pH 7.0.

[0042] D-biotin concentration determination: The D-biotin standard was purchased from MERCK (https: / / www.merck.com / ). 1.0 mL of fermentation broth was centrifuged at 10,000 rpm for 10 min to remove the bacterial cells, and the supernatant was diluted by an appropriate multiple and filtered through a 0.22 μm filter membrane. The biotin concentration was determined by LC-MS. The instrument used was Shimadzu triple quadrupole LCMS8045, the chromatographic column was ultimate LP-C18 4.0*250mm 5μm, the mobile phase flow composition, the proportion change, the flow rate and the chromatographic column temperature were set according to the method of the previous application file.

[0043] Strain: The fermentation strain used in the patent and embodiments is Pseudomonas mutabilis engineering strain PM / WIABFD, which is derived from the Chinese invention patent with the publication number CN114480525B and the name “A production method for improving the yield of D-biotin”. The specific construction method is shown in Example 1 of the patent (the engineering strain PM / WIABFD is obtained by using Pseudomonas mutabilis ATCC31014 as the starting strain and performing genetic engineering construction. The strain is derived from the Chinese invention patent application with the application number CN201910591872.2 and the name “Method for promoting biotin synthesis, recombinant cell for promoting biotin synthesis, and genetically engineered bacteria”. The specific construction method is derived from the description of Examples 1 and 2).

[0044] The application is further explained and described by specific embodiments.

[0045] Example 1 (comparative example 1: initial process)

[0046] Seed medium is the same as SM medium above, and fermentation medium is the same as IFM medium above.

[0047] The P. variabilis engineering strain PM / WIABFD preserved in glycerol was inoculated into seed medium and cultured in a shaker at 28°C and 200 rpm. When the OD 600 reached 2.0, seed liquid was prepared.

[0048] The prepared seed liquid was inoculated into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio), and the initial pH was 7.0. During the fermentation process, the temperature was controlled at 28°C, the dissolved oxygen was controlled at more than 2% throughout the process, the rotation speed was 200-1000 rpm, the aeration amount was 2-5L / min, NH3·H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2; when the glycerol concentration in the fermentation liquid decreased to 1.0g / L, 800g / L glycerol was added to control the final glycerol concentration in the fermentation liquid at about 1.0g / L.

[0049] After the fermentation was completed, 1mL of fermentation liquid was taken and the D-biotin content was determined according to the above method. The D-biotin yield was 265.8mg / L, and the fermentation period was 168h.

[0050] In this application, the OD 600 of each batch of fermentation liquid gradually decreased, and the pH gradually increased, which was considered as the end of fermentation. The following examples were all based on this as the standard for the end of fermentation.

[0051] Example 2 (combined carbon source + three-stage temperature control):

[0052] The seed medium was the same as in Example 1, and the fermentation medium was changed from 40g / L glycerol in Example 1 to 20g / L glycerol and 20g / L glucose, with the rest unchanged.

[0053] The preparation process of the seed liquid was the same as in Example 1.

[0054] The prepared seed liquid was inoculated into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio) and initial pH of 7.0. Temperature control was divided into three stages: in the first stage, the fermentation temperature was controlled at 30.0°C from the beginning of fermentation to the time when the glucose content in the fermentation liquid was 0, so as to facilitate rapid proliferation of the bacteria; in the second stage, the fermentation temperature was controlled at 28.0°C when the glucose content in the fermentation liquid was 0 to the time when the glycerol content in the fermentation liquid naturally decreased to 1.0g / L; in the third stage, the fermentation temperature was controlled at 26.0°C when the glycerol content in the fermentation liquid naturally decreased to 1.0g / L to the end of fermentation; during the fermentation process, when the glycerol content in the fermentation liquid naturally decreased to 1.0g / L, glycerol was automatically added to control the glycerol content in the fermentation liquid at about 1.0g / L until the end of fermentation, so as to facilitate product generation; during the fermentation process, the rotation speed was 200-1000rpm, the aeration amount was 2-5L / min, the DO was controlled above 2%, NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2.

[0055] After the fermentation was completed, 1mL of the fermentation liquid was taken and the D-biotin content was determined according to the above method; the D-biotin yield was detected to be 592.8mg / L. Compared with the yield of Example 1, the yield was increased by 123.1%, and the fermentation period was 120h, which was shortened by 28.6% compared with the process period of Example 1.

[0056] Example 3 (whole process low DO)

[0057] The seed medium and the fermentation medium were the same as in Example 1.

[0058] The seed liquid preparation process was the same as in Example 1.

[0059] The prepared seed liquid was inoculated into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio) and initial pH of 7.0. During the fermentation process, the temperature was controlled at 28°C, the whole process DO was controlled within 2%, the rotation speed and DO were linked to maintain the rotation speed at 200-400rpm, the aeration amount was 2L / min, NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2; when the glycerol content in the fermentation liquid decreased to 1.0g / L, 800g / L glycerol was added to control the final glycerol content in the fermentation liquid at about 1.0g / L.

[0060] After the fermentation was completed, 1mL of the fermentation liquid was taken and the D-biotin content was determined according to the above method; the D-biotin yield was detected to be 356.2mg / L. Compared with the yield of Example 1, the yield was increased by 34.0%, and the fermentation period was 144h, which was shortened by 14.3% compared with the process period of Example 1.

[0061] Example 4 (composite carbon source + three-stage temperature control + whole process low DO):

[0062] The seed culture medium and fermentation culture medium are the same as in Example 2.

[0063] The seed liquid preparation process is the same as in Example 1.

[0064] The cultured seed culture was transferred at an inoculation rate of 1% (volume ratio) into a 5L mechanically stirred fermenter containing 2L of fermentation medium, with an initial pH of 7.0. Temperature control was implemented in three stages: Stage 1, from the start of fermentation until the glucose content in the fermentation broth reached 0, the fermentation temperature was kept constant at 30.0℃ to facilitate rapid cell proliferation; Stage 2, from when the glucose content in the fermentation broth reached 0 until the glycerol content naturally decreased to 1.0 g / L, the fermentation temperature was kept at 28.0℃; Stage 3, from when the glycerol concentration naturally decreased to 1.0 g / L until the end of fermentation, the fermentation temperature was kept at 26.0℃. During fermentation, when the glycerol concentration naturally decreased to 1.0 g / L, glycerol was automatically added to maintain the glycerol concentration at approximately 1.0 g / L until the end of fermentation, facilitating product formation. During fermentation, the rotation speed was controlled between 200-400 rpm, the aeration rate was 2 L / min, the dissolved oxygen (DO) was controlled below 2%, and NH3·H2O and H3PO4 were used to maintain the pH of the fermentation broth at approximately 7.2.

[0065] After fermentation, 1 mL of the fermentation broth was taken and the D-biotin content was determined according to the above method. The D-biotin yield was found to be 773.8 mg / L. This represents a 191.1% increase in yield compared to Example 1, and the fermentation cycle was 136 h, a 19.1% reduction in the process cycle compared to Example 1.

[0066] Example 5 (Composite carbon source + three-stage temperature control + continuous DO):

[0067] The seed culture medium and fermentation culture medium are the same as in Example 2.

[0068] The seed liquid preparation process is the same as in Example 1.

[0069] The cultivated seed liquid was transferred into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio), and the initial pH was 7.0. Temperature control was divided into three stages: in the first stage, the fermentation temperature was controlled at 30.0°C from the beginning of fermentation to the time when the glucose content in the fermentation liquid was 0, so as to facilitate rapid proliferation of the bacteria; in the second stage, the fermentation temperature was controlled at 28.0°C when the glucose content in the fermentation liquid was 0 to the time when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L; in the third stage, the fermentation temperature was controlled at 26.0°C when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L to the end of fermentation; during the fermentation process, when the glycerol content in the fermentation liquid dropped to 1.0g / L, glycerol was automatically added to control the glycerol content in the fermentation liquid at about 1.0g / L until the end of fermentation, so as to facilitate product generation; during the fermentation process, the rotation speed was adjusted to be within 200-600rpm, the aeration amount was 2-3L / min, the DO was controlled to be within 2-20%, NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2.

[0070] After the fermentation was completed, 1mL of the fermentation liquid was taken and the D-biotin content was determined according to the above method; the detected D-biotin yield was 705.4mg / L. Compared with the yield of Example 1, the yield was increased by 165.4%, and the fermentation period was 130h, which was shortened by 22.6% compared with the process period of Example 1.

[0071] Example 6 (composite carbon source + three-stage temperature control + high DO throughout the process):

[0072] The seed culture medium was the same as that of Example 2.

[0073] The seed liquid preparation process was the same as that of Example 1.

[0074] The cultivated seed liquid was transferred into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio), and the initial pH was 7.0. Temperature control was divided into three stages: in the first stage, the fermentation temperature was controlled at 30.0°C from the beginning of fermentation to the time when the glucose content in the fermentation liquid was 0, so as to facilitate rapid proliferation of the bacteria; in the second stage, the fermentation temperature was controlled at 28.0°C when the glucose content in the fermentation liquid was 0 to the time when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L; in the third stage, the fermentation temperature was controlled at 26.0°C when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L to the end of fermentation; during the fermentation process, when the glycerol content in the fermentation liquid dropped to 1.0g / L, glycerol was automatically added to control the glycerol content in the fermentation liquid at about 1.0g / L until the end of fermentation, so as to facilitate product generation; during the fermentation process, the rotating speed was controlled at 400-1000rpm by adjusting the rotating speed, and the aeration amount was controlled at 2-5L / min, so as to control the DO at more than 20%, and NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2.

[0075] After the fermentation was completed, 1mL of the fermentation liquid was taken, and the D-biotin content was determined according to the above method; the detected D-biotin yield was 644.4mg / L. Compared with the yield of Example 1, the yield was increased by 142.3%, and the fermentation period was 124h, which was shortened by 26.2% compared with the process period of Example 1.

[0076] Example 7 (composite carbon source + three-stage temperature control + one-stage high DO, two-stage and three-stage low DO):

[0077] The seed culture medium was the same as that of Example 2.

[0078] The seed liquid preparation process was the same as that of Example 1.

[0079] The cultivated seed liquid was transferred into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio), and the initial pH was 7.0. The temperature was controlled in three stages: in the first stage, the fermentation temperature was controlled at 30.0℃ from the beginning of fermentation to the time when the glucose content in the fermentation liquid was 0, the rotating speed was controlled at 400-1000rpm, and the aeration amount was controlled at 2-5L / min, so that the DO was controlled above 20% to facilitate the rapid proliferation of the bacteria; in the second stage, the fermentation temperature was controlled at 28.0℃ from the time when the glucose content in the fermentation liquid was 0 to the time when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L, the rotating speed was controlled at 200-400rpm, and the aeration amount was 2L / min, so that the DO was controlled within 2%; in the third stage, the fermentation temperature was controlled at 26.0℃ from the time when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L to the end of fermentation, the rotating speed was controlled at 200-400rpm, and the aeration amount was 2L / min, so that the DO was controlled within 2%; during the fermentation process, when the glycerol content in the fermentation liquid dropped to 1.0g / L, glycerol was automatically added to control the glycerol content in the fermentation liquid at about 1.0g / L until the end of fermentation to facilitate the production of the product, and NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2.

[0080] After the fermentation was completed, 1ml of the fermentation liquid was taken and the D-biotin content was determined according to the above method, and the D-biotin yield was detected to be 807.5mg / L. The yield was increased by 203.8% compared to that of Example 1, and the fermentation period was 130h, which was shortened by 22.6% compared to the process period of Example 1.

[0081] Example 8 (composite carbon source + three-stage temperature control + one-stage medium DO, low DO in the second and third stages):

[0082] The seed culture medium was the same as that of Example 2.

[0083] The seed liquid preparation process was the same as that of Example 1.

[0084] The cultivated seed liquid was transferred into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio), and the initial pH was 7.0. The temperature was controlled in three stages: in the first stage, the fermentation temperature was controlled at 30.0°C from the beginning of fermentation to the time when the glucose content in the fermentation liquid was 0, the rotation speed was controlled within 200-600rpm, the aeration amount was 2-3L / min, and the DO was controlled within 2-20% to facilitate the rapid proliferation of the bacteria; in the second stage, the fermentation temperature was controlled at 28.0°C from the time when the glucose content in the fermentation liquid was 0 to the time when the glycerol content in the fermentation liquid naturally decreased to 1.0g / L, the rotation speed was controlled at 200-400rpm, the aeration amount was 2L / min, and the DO was controlled within 2%; in the third stage, the fermentation temperature was controlled at 26.0°C from the time when the glycerol content in the fermentation liquid naturally decreased to 1.0g / L to the end of fermentation, the rotation speed was controlled at 200-400rpm, the aeration amount was 2L / min, and the DO was controlled within 2%; during the fermentation process, when the glycerol content in the fermentation liquid decreased to 1.0g / L, glycerol was automatically added to control the glycerol content in the fermentation liquid at about 1.0g / L until the end of fermentation to facilitate the production of the product, and NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2.

[0085] After the fermentation was completed, 1mL of the fermentation liquid was taken and the D-biotin content was determined according to the above method, and the D-biotin yield was detected to be 783.4mg / L. The yield was increased by 194.7% compared to that of Example 1, and the fermentation period was 136h, which was shortened by 19.1% compared to the process period of Example 1.

[0086] Example 9 (composite carbon source + three-stage temperature control + high DO in the first and second stages and low DO in the third stage):

[0087] The seed culture medium was the same as that of Example 2.

[0088] The seed liquid preparation process was the same as that of Example 1.

[0089] The cultivated seed liquid was transferred into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio), and the initial pH was 7.0. The temperature was controlled in three stages: in the first stage, the fermentation temperature was controlled at 30.0°C from the beginning of fermentation to the time when the glucose content in the fermentation liquid was 0; in the second stage, the fermentation temperature was controlled at 28.0°C from the time when the glucose content in the fermentation liquid was 0 to the time when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L; in the first and second stages, the rotation speed was controlled at 400-1000rpm, the aeration amount was controlled at 2-5L / min, and the DO was controlled above 20% to facilitate the multiplication of the bacteria; in the third stage, the fermentation temperature was controlled at 26.0°C from the time when the glycerol content in the fermentation liquid dropped to 1.0g / L to the end of fermentation, the rotation speed was controlled at 200-400rpm, the aeration amount was 2L / min, and the DO was controlled within 2%; during the fermentation, glycerol was automatically added when the glycerol content in the fermentation liquid dropped to 1.0g / L to control the glycerol content in the fermentation liquid at about 1.0g / L until the end of fermentation to facilitate the production of the product, and NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2.

[0090] After the fermentation, 1ml of the fermentation liquid was taken and the D-biotin content was determined according to the above method. The D-biotin yield was 790.4mg / L. Compared with the yield in Example 1, the yield was increased by 197.4%, and the fermentation period was 130h, which was shortened by 22.6% compared with the process period in Example 1.

[0091] Example 10 (complex carbon source + three-stage temperature control + DO in the first and second stages, low DO in the third stage):

[0092] The seed culture medium was the same as in Example 2.

[0093] The seed liquid preparation process was the same as in Example 1.

[0094] The cultivated seed liquid was transferred into a 5L mechanical stirring fermenter containing 2L fermentation medium at an inoculation amount of 1% (volume ratio), and the initial pH was 7.0. The temperature was controlled in three stages: in the first stage, the fermentation temperature was controlled at 30.0°C from the beginning of fermentation to the time when the glucose content in the fermentation liquid was 0; in the second stage, the fermentation temperature was controlled at 28.0°C when the glucose content in the fermentation liquid was 0 to the time when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L; in the first and second stages, the rotation speed was controlled within 200-600rpm, the aeration amount was 2-3L / min, and the DO was controlled between 2-20% to facilitate the propagation of the bacteria; in the third stage, the fermentation temperature was controlled at 26.0°C when the glycerol content in the fermentation liquid naturally dropped to 1.0g / L to the end of fermentation, the rotation speed was controlled at 200-400rpm, the aeration amount was 2L / min, and the DO was controlled within 2% to facilitate the generation of the product; during the fermentation, glycerol was automatically added when the glycerol content in the fermentation liquid dropped to 1.0g / L to control the glycerol content in the fermentation liquid at about 1.0g / L until the end of fermentation to facilitate the generation of the product, and NH3-H2O and H3PO4 were used to maintain the pH of the fermentation liquid at about 7.2.

[0095] After the fermentation was completed, 1ml of the fermentation liquid was taken and the D-biotin content was determined according to the above method, and the D-biotin yield was detected to be 762.4mg / L. The yield was increased by 186.8% compared to that of Example 1, and the fermentation period was 132h, which was shortened by 21.4% compared to the process period of Example 1.

[0096] Example 11 (complex carbon source + one-stage high DO, two-stage and three-stage low DO):

[0097] The seed culture medium was the same as that of Example 2.

[0098] The seed liquid preparation process was the same as that of Example 1.

[0099] The cultivated seed liquid was transferred into a 5L mechanically stirred fermenter containing 2L fermentation medium at an inoculation amount of 1% (by volume), with an initial pH of 7.0 and a constant temperature of 28℃. The DO was controlled in three stages: in the first stage, from the beginning of fermentation to when the glucose content in the fermentation broth was 0, the rotation speed was controlled at 400-1000rpm and the aeration rate was controlled at 2-5L / min to control the DO above 20% to facilitate rapid proliferation of the bacteria; in the second stage, when the glucose content in the fermentation broth was 0 to when the glycerol content in the fermentation broth naturally decreased to 1.0g / L, the rotation speed was controlled at 200-400rpm and the aeration rate was controlled at 2L / min to control the DO below 2%; in the third stage, when the glycerol concentration in the fermentation broth naturally decreased to 1.0g / L to the end of fermentation, the rotation speed was controlled at 200-400rpm and the aeration rate was controlled at 2L / min to maintain the DO below 2%; during the fermentation process, when the glycerol concentration in the fermentation broth decreased to 1.0g / L, glycerol was automatically added to control the glycerol concentration in the fermentation broth at about 1.0g / L until the end of fermentation to facilitate product formation. NH3·H2O and H3PO4 were used to maintain the pH of the fermentation broth at about 7.2.

[0100] After the fermentation was completed, 1mL of the fermentation broth was taken and the D-biotin content was determined according to the above method. The D-biotin yield was detected to be 495.8mg / L. Compared with the yield of Example 1, the yield was increased by 86.5%, and the fermentation period was 136h, which was shortened by 19.0% compared with the process period of Example 1.

[0101] By comparing Example 1 with Example 3, it was proved that DO control during the fermentation of Pseudomonas alcaliphila can increase the yield of D-biotin. By comparing Example 1 with Examples 2-4, it was unexpectedly found that the DO control process and the process of patent CN114480525B (composite carbon source + three-stage temperature control) had a significant synergistic effect on the production of D-biotin.

[0102] For optimization of the DO control process, by comparing Example 4 with Examples 5 and 6, it was found that low DO throughout the process was more conducive to the accumulation of D-biotin than medium / high DO throughout the process. By integrating the stage-wise DO control with the three-stage temperature control process, by comparing Example 4 with Examples 7-10, it was proved that providing sufficient DO during the rapid growth period of the bacteria and reducing DO during the production and accumulation of D-biotin can further increase the yield of the product. The yield of biotin obtained by the process of Example 7 was the highest, and the comparison of Examples 2, 7 and 11 further illustrated that the combination of three-stage temperature control and DO control had a synergistic effect on the increase of biotin yield.

[0103] Example 12 A method for preparing D-biotin by fermentation

[0104] Seed medium: glucose 10.0, yeast extract 10.0, tryptone 20.0, Na2HPO4·12H2O 0.5, MgSO4·7H2O 0.8, (NH4)2SO4 2.0, pH 7.0.

[0105] Fermentation medium: glucose 15, glycerol 30, yeast extract 30, (NH4)2SO4 4, Na2HPO4·12H2O 20, KH2PO4 10, MgSO4·7H2O 2, FeSO4·7H2O 0.1; pimelic acid 1.0, pH 7.0-7.3.

[0106] Seed medium preparation process is same as example 1.

[0107] Fermentation process: the fermentation seed seed liquid is inoculated into 5L mechanical stirring fermentation tank containing 2L fermentation medium with 5% inoculation amount, the initial pH is 7.0, the rotation speed is controlled to be 200-400rpm, the aeration amount is 2L / min, the DO content is controlled to be lower than 2% in the whole fermentation process, when the glycerol content is reduced to 1g / L, the glycerol is added to maintain the glycerol content in the system to be 1g / L, NH3·H2O and H3PO4 are used to maintain the pH of the fermentation liquid to be about 7.2 in the fermentation process, three-stage temperature control method is adopted in the fermentation process: the fermentation temperature is controlled to be 32℃ from the initial fermentation to the glucose content in the fermentation liquid being 0; the fermentation temperature is controlled to be 29℃ from the glucose content being 0 to the glycerol content in the fermentation liquid naturally reducing to 1g / L; the fermentation temperature is controlled to be 27℃ when the glycerol content in the fermentation liquid naturally reduces to 1g / L to the end of the fermentation.

[0108] After the fermentation is completed, 1mL of the fermentation liquid is taken, and the D-biotin content is determined according to the above method, and the D-biotin yield is 710.7mg / L. The fermentation period is 130h.

[0109] The above examples only express several embodiments of the present application, which are described in detail, but cannot be understood as the limitation of the patent scope. It should be pointed out that for ordinary skilled persons in the art, the above embodiments can be deformed, combined and improved without departing from the concept of the present patent, and these all belong to the protection scope of the present patent. Therefore, the protection scope of the present patent should be subject to the claims.

Claims

1. A method for producing D-biotin, characterized by, The method is as follows: the fermentation strain seed liquid is connected to the fermentation medium, the DO content is controlled to be lower than 2% in the whole fermentation process, when the glycerol content is reduced to 1 g / L, the glycerol is added to maintain the glycerol content in the system to be 1 g / L, and the three-stage temperature control method is adopted in the fermentation process: the fermentation temperature is controlled to be 30-32 ℃ from the initial fermentation to the glucose content in the fermentation liquid being 0; the fermentation temperature is controlled to be 28-29 ℃ from the glucose content being 0 to the glycerol content in the fermentation liquid being naturally reduced to 1 g / L; and the fermentation temperature is controlled to be 25-27 ℃ when the glycerol content in the fermentation liquid is naturally reduced to 1 g / L to the end of the fermentation.

2. A method of producing D-biotin according to claim 1, wherein, The carbon source in the fermentation medium is a composite carbon source, and the composite carbon source comprises glucose and glycerol; the concentration ratio of the glucose and the glycerol is 1-2:1-2.

3. A method of producing D-biotin according to claim 2, wherein, The fermentation medium is (g / L): glucose 15-30, glycerol 15-30, yeast protein peptone 10-39, (NH4)2SO4 1-4, Na2HPO4·12H2O 10-20, KH2PO4 3-10, MgSO4·7H2O 0.2-2, FeSO4·7H2O 0.08-0.1; pimelic acid 0.1-1.0, and the pH is adjusted to 7.0-7.

3.

4. The method of producing D-biotin according to claim 1, wherein, The DO is controlled in stages; the method for controlling the DO content in stages is that the DO content is controlled to be higher than 20% in the first stage, and the DO content is controlled to be lower than 2% in the second and third stages.

5. The method of producing D-biotin according to claim 1, wherein, The DO is controlled in stages; the method for controlling the DO content in stages is that the DO content is controlled to be in the range of 2%-20% in the first and second stages, and the DO content is controlled to be lower than 2% in the third stage.

6. The method of producing D-biotin according to claim 1, wherein, The DO is controlled in stages; the method for controlling the DO content in stages is that the DO content is controlled to be higher than 20% in the first and second stages, and the DO content is controlled to be lower than 2% in the third stage.

7. The method of producing D-biotin according to claim 1, wherein, The DO is controlled in stages; the method for controlling the DO content in stages is that the DO content is controlled to be in the range of 2%-20% in the first and second stages, and the DO content is controlled to be lower than 2% in the third stage.

8. The method of producing D-biotin according to any one of claims 1 to 7, wherein The method for controlling the DO includes but is not limited to adjusting the rotating speed and / or adjusting the ventilation; the rotating speed ranges from 200 rpm to 1000 rpm, and the ventilation ranges from 2 L / min to 5 L / min.

9. The method of producing D-biotin according to any one of claims 1 to 7, wherein The rotating speed is 200-400 rpm, the ventilation is 2 L / min, the DO content is controlled to be lower than 2%; the rotating speed is 200-600 rpm, the ventilation is 2-3 L / min, the DO content is controlled to be in the range of 2%-20%; and the rotating speed is 400-1000 rpm, the ventilation is 2-5 L / min, and the DO content is controlled to be higher than 20%.

10. The method of producing D-biotin according to any one of claims 1 to 7, wherein The fermentation strain is Pseudomonas mutabilis engineering strain PM / WIABFD.