Method for improving biotin fermentation yield and stability by controlling temperature and humidity

Abstract

The invention discloses a method for improving biotin fermentation yield and stability by controlling temperature and humidity. The method comprises the following steps: inoculating a strain capable of synthesizing biotin into a solid culture medium, and culturing at constant temperature and humidity within the temperature range of 34-38 DEG C and the humidity range of 10-60% until the size of a bacterial colony or the thickness of a bacterial lawn is qualified; directly transferring the bacterial colony to a fermentation culture medium for culturing; or firstly transferring to a liquid culture medium and then transferring to a fermentation culture medium for fed-batch culture to finally obtain biotin. The method provided by the invention can effectively improve the titer of biotin produced by a fermentation method.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a method for improving the fermentation yield and stability of biotin by controlling temperature and humidity. Background Art

[0002] Biotin, a type of B vitamin, is a water-soluble vitamin that is ubiquitous in nature. It is also known as vitamin H, vitamin B7 or coenzyme R. It is abundant in the liver, kidney, spleen and other organs of animals, egg yolk and yeast. The chemical name of biotin is (3aS, 4S, 6aR)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid. Its structure contains an imidazole ring, a tetrahydrothiophene ring and a pentanoic acid side chain. Its structure determines that it has multiple isomers, and only D-biotin has biological activity. Biotin is not only a coenzyme of carboxylase, but also a coenzyme of protein, sugar and fat intermediate metabolism. Biotin is also closely related to the metabolism of various nutrients such as sugar, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, etc. Biotin is widely used in medicine, food and cosmetics, biotechnology, feed additives, etc.

[0003] Biotin has a complex structure and is technically difficult to produce on an industrial scale. In industry, biotin is usually obtained by chemical synthesis, such as the Scheme process route. The Sternbach route is the basis of industrial production so far, and the current industrial production routes are all improved on this basis. However, chemical synthesis has the disadvantages of complex routes, high toxicity, low yield, and high danger that are difficult to overcome. Biotin can also be obtained by extraction. The extraction method can obtain biotin from a variety of substances such as animal tissues, Antarctic krill, corn pulp, beet molasses, and young betel nut fruits. However, the extraction method has high costs and low yields, making it difficult to achieve industrialization.

[0004] Fermentation is an ideal method for obtaining biotin. The reaction conditions are mild, the raw materials are non-toxic or low-toxic, and the reaction process is controllable or adjustable. However, the current potency of the fermentation method is relatively low, and compared with chemical synthesis, it is not cost-effective. Therefore, in order to achieve industrialization of biotin production by fermentation, it is necessary to continuously optimize the fermentation process of biotin. Zhejiang Shengda Biopharmaceutical Co., Ltd. has continuously optimized the fermentation process, and the potency has increased by 21%-170% compared with the original process output, and the cycle has been shortened by 13%-28%. The strain obtained by Zhejiang University through genetic engineering has the ability to efficiently synthesize biotin. When fed-batch fermentation is carried out in a culture medium with pimelic acid as a substrate, the biotin yield can reach 417 mg / L, which is 200 times that of the original Escherichia coli, and the yield reaches 10.4 mg / (L·h). These process optimizations have made a big step forward in the realization of industrial fermentation synthesis of biotin.

[0005] The process conditions that have a significant impact on fermentation include temperature and dissolved oxygen. In the laboratory, dissolved oxygen is generally controlled by adjusting the rotation speed. In production, in addition to adjusting the rotation speed, it can also be combined with air flow control. In addition to affecting the growth rate of the bacteria, the culture temperature also affects the activity of various enzymes in the bacteria, thereby affecting the production capacity of the bacteria. However, there is currently no specific research on the impact of humidity on fermentation. Summary of the invention

[0006] The purpose of the present invention is to improve the current status of biotin fermentation preparation, and to propose a method for increasing the fermentation yield and stability of biotin by controlling temperature and humidity. Air humidity can affect the evaporation of water in the culture medium, further affect the concentration of nutrients in the culture medium, the osmotic pressure of the culture medium and the water activity of the culture medium, and affect the aggregation of miscellaneous bacteria in the environment, thereby further comprehensively affecting the growth of the strains and production capacity. The method of the present invention can effectively improve the titer of biotin produced by fermentation.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] A method for increasing the yield and stability of biotin fermentation by controlling temperature and humidity, comprising the following steps:

[0009] (1) inoculating a strain capable of synthesizing biotin into a solid culture medium, maintaining a constant temperature and humidity of 34-38° C. and a humidity of 10-60%, and culturing until the colony size or the thickness of the bacterial moss meets the requirements;

[0010] (2) directly transferring the colony to a fermentation medium for fermentation culture; or first transferring the colony to a liquid culture medium and then transferring the colony to a fermentation medium for fed-batch fermentation culture, wherein the fermentation temperature is 28-38° C. and the humidity is the same as in step (1), and finally biotin is obtained.

[0011] Preferably, the bacterial species capable of synthesizing biotin is genetically engineered Escherichia coli.

[0012] Preferably, the solid culture medium comprises: 5-15 g / L of tryptone, 1-10 g / L of yeast extract powder, 1-10 g / L of sodium chloride, 0.5-2 g / L of glucose, and 0.5-2 g / L of beef extract.

[0013] Preferably, the liquid culture medium comprises: 5-15 g / L of a composite carbon source, 5-15 g / L of a composite nitrogen source, 10-20 g / L of an inorganic salt, and 1-5 ml / L of a trace element mixture.

[0014] Preferably, the dosage of the fermentation medium is: 15-30 g / L of a composite carbon source, 10-20 g / L of a composite nitrogen source, 10-20 g / L of an inorganic salt, 1-10 ml / L of a trace element mixture, and 0.5-2 ml / L of a defoaming agent.

[0015] More preferably, the composite carbon source is at least one of glucose, sucrose, and maltose; the composite nitrogen source is at least one of peptone, yeast extract, yeast powder, beef extract, corn steep liquor, corn steep liquor powder, and urea; the inorganic salt is at least one of sodium chloride, sodium sulfate, sodium glutamate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, calcium carbonate, and calcium chloride; the component dosages of the trace element mixture are: 0.1-5g / L zinc sulfate, 0.1-3g / L manganese sulfate, 0.1-10g / L ferrous sulfate, 0.1-2g / L cobalt chloride, 0.1-1g / L betaine, 0.1-2g / L choline chloride, 0.1-1g / L nicotinic acid, 0.1-1g / L folic acid, and 0.1-1g / L thiamine.

[0016] Preferably, in step (2), the colonies are directly transferred to a fermentation medium with a liquid volume of (20-50) ml / 250 ml, a rotation speed of 220-280 rpm, and a temperature of 28-38°C for 40-48 hours; the colonies are first transferred to a liquid culture medium with a liquid volume of (20-50) ml / 250 ml, a rotation speed of 220-280 rpm, and a temperature of 34-38°C for 4-8 hours, and then transferred to a fermentation medium with a liquid volume of (20-50) ml / 250 ml, a rotation speed of 220-280 rpm, and a temperature of 28-38°C for 40-48 hours.

[0017] Preferably, the feed is at least one of glucose, MOPS and ammonia water.

[0018] The beneficial effects of the present invention are:

[0019] The present invention can reduce the fluctuation of biotin fermentation potency from the original 30% to 10% by strictly controlling the temperature and humidity of the biotin fermentation process, and improve the potency by 20% as a whole. DETAILED DESCRIPTION

[0020] To facilitate a thorough understanding of the present invention, the present invention will be described in more detail below. It should be noted that the present invention can be implemented in many different ways and is not limited to the embodiments described herein.

[0021] In a specific embodiment of the present invention, the air humidity is controlled to 10%-60% by using equipment, and the genetically engineered Escherichia coli capable of synthesizing biotin is inoculated into a solid culture medium and cultured at 34-38°C in a constant humidity until the colony size or the thickness of the bacterial moss is qualified, and then the fermentation culture medium is transferred, and the liquid volume is (20-50) ml / 250 ml, the rotation speed is 220-280 rpm, the temperature is 28-38°C, and the titer is detected for 40-48 hours. Alternatively, the liquid culture medium is directly transferred, and the liquid volume is (20-50) ml / 250 ml, the rotation speed is 220-280 rpm, the temperature is 34-38°C, and the fermentation culture medium is transferred, and the rotation speed is 220-280 rpm, and the temperature is 28-38°C. Culture for 40-48 hours to detect the titer.

[0022] The detection method refers to the article entitled “Determination of biotin content by high performance liquid chromatography” published by Pan Yibin et al. in the journal “China Pharmaceutical Industry”, which is as follows: chromatographic column: C18 column (150mm×4.6mm, 5μm), mobile phase: 0.05% trifluoroacetic acid solution / acetonitrile (75:25), detection wavelength: 210nm, column temperature: 30°C, flow rate: 1mL / min, injection volume: 20μL.

[0023] The present invention does not limit the strains that produce biotin, and the strains that produce biotin can be any strain that has the ability to synthesize biotin. The present invention does not limit the solid, liquid culture medium and fermentation medium components used to cultivate the strains, including all culture medium compositions known in the art that are suitable for producing biotin strains. The present invention does not limit the method of strain transfer fermentation, and can determine the method of solid direct transfer fermentation, solid transfer liquid strain culture medium and then transfer fermentation according to needs. The present invention does not limit the scope and method of controlling humidity, that is, it includes all humidity control conditions such as the internal humidity of the equipment for controlling strain culture, the environmental humidity, and humidifying and dehumidifying the environment by any means.

[0024] In some embodiments, the humidity control range is 10%-60%. In an optional specific embodiment, the humidity control value is: 15%, 25%, 35%, 40%, 45%, 55%.

[0025] In some embodiments, the bacterial culture temperature is controlled in the range of 34° C. to 38° C. In an optional specific embodiment, the temperature control value is: 34° C., 36° C., 37° C., 38° C.

[0026] In some embodiments, the fermentation culture temperature is controlled in the range of 28° C.-38° C. In an optional specific embodiment, the temperature control values ​​are: 30° C., 32° C., 34° C., 36° C. throughout the culture process; and 36° C. in the early stage and 32° C. in the middle and late stages.

[0027] In some embodiments, the control range of the bacterial strain and fermentation culture speed is 220rpm-280rpm. In an optional specific embodiment, the control value of the speed is: 220rpm, 240rpm, 260rpm, 280rpm.

[0028] In an optional specific embodiment, the composition of the solid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 1 g / L glucose, and 1 g / L beef extract.

[0029] In an optional specific embodiment, the liquid seed culture medium has a liquid volume of 25 ml / 250 ml and is composed of: 10 g / L glucose, 5 g / L corn syrup powder, 7 g / L yeast extract powder, 3 g / L ammonium sulfate, 3 g / L magnesium sulfate, 4 g / L sodium chloride, and 2 ml / L trace element mixture.

[0030] In an optional specific embodiment, the liquid volume of the fermentation medium is 50ml / 250ml, and the composition is: 20g / L glucose, 15g / L corn steep liquor, 5g / L beef extract, 2g / L sodium sulfate, 2g / L sodium glutamate, 3g / L dipotassium hydrogen phosphate, 4g / L magnesium sulfate, 2g / L ammonium sulfate, 2g / L calcium carbonate, 5ml / L trace element solution, and 1ml / L defoaming agent.

[0031] In an optional specific embodiment, the trace element solution formula is as follows:

[0032]

[0033]

[0034] In an optional specific embodiment, the feeding method is to feed 2 ml of a glucose solution with a mass volume ratio of 50% and 1 ml of a MOPS solution with a mass volume ratio of 40% after 20-24 hours of fermentation.

[0035] In order to make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. When the reagents and instruments used in the examples are not specifically described, they are conventionally selected in the art. The experimental methods for which specific conditions are not specified in the examples are performed with reference to the conventional conditions in the literature, books and other materials. However, the following examples are only descriptive and not restrictive, and the scope of protection of the present invention cannot be limited by them.

[0036] Example 1

[0037] Take a frozen strain and evenly spread 0.1 ml on the slant of the test tube. The culture medium formula is: tryptone 10g / L, yeast extract powder 5g / L, sodium chloride 5g / L, glucose 1g / L, beef extract 1g / L. Culture overnight at 34℃ and humidity controlled at 40%.

[0038] Scrape a ring of the cultured bacterial moss and transfer it into the liquid culture medium. The culture medium formula is: 10g / L glucose, 5g / L corn steep liquor powder, 7g / L yeast extract powder, 3g / L ammonium sulfate, 3g / L magnesium sulfate, 4g / L sodium chloride, 2ml / L trace element mixture, liquid volume 25ml / 250ml, 260rpm, 34℃, humidity controlled at 40% and culture until the logarithmic phase.

[0039] The cultured seed liquid is inoculated into the fermentation medium at a 1% inoculation rate. The medium formula is: 20g / L glucose, 15g / L corn steep liquor, 5g / L beef extract, 2g / L sodium sulfate, 2g / L sodium glutamate, 3g / L dipotassium hydrogen phosphate, 4g / L magnesium sulfate, 2g / L ammonium sulfate, 2g / L calcium carbonate, 5ml / L trace element solution, 1ml / L defoamer. The liquid volume is 50ml / 250ml, the rotation speed is 240rpm, the humidity is controlled at 40%, the culture is 36℃ in the early stage, and 32℃ in the middle and late stages. After fermentation for 20-24h, add 2ml of 50% glucose solution by mass volume and 1ml of 40% MOPS solution by mass volume.

[0040] After 48h of fermentation, the titer was tested by high performance liquid chromatography. The results are shown in Table 1.

[0041] Example 2

[0042] The strains and fermentation culture process used in this embodiment are substantially the same as those in embodiment 1. The difference is that the humidity is controlled at 15%, the strain culture temperature is 36°C, the rotation speed is 240rpm, the fermentation temperature is controlled at 34°C throughout the fermentation process, and the fermentation speed is 260rpm.

[0043] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0044] Example 3

[0045] The strains and fermentation culture process used in this embodiment are substantially the same as those in embodiment 1. The difference is that the humidity is controlled at 25%, the strain culture temperature is 37°C, the rotation speed is 240 rpm, and the fermentation temperature is controlled at 36°C throughout the whole process.

[0046] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0047] Example 4

[0048] The strains and fermentation culture process used in this embodiment are substantially the same as those in embodiment 1. The differences are as follows: the humidity is controlled at 35%, the strain culture temperature is 34°C, the fermentation temperature is controlled at 32°C throughout the fermentation process, and the rotation speed is 260 rpm.

[0049] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0050] Example 5

[0051] The strains and fermentation culture process used in this embodiment are substantially the same as those in embodiment 1. The difference is that the humidity is controlled at 45%, the temperature is controlled at 30°C throughout the fermentation process, and the fermentation speed is 280 rpm.

[0052] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0053] Example 6

[0054] The strains and fermentation culture process used in this embodiment are substantially the same as those in embodiment 1. The differences are as follows: the humidity is controlled at 55%, the strain culture temperature is 36°C, and the rotation speed is 240 rpm; the fermentation temperature is controlled at 32°C throughout the fermentation process, and the rotation speed is 260 rpm.

[0055] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0056] Comparative Example 1

[0057] The strains and fermentation culture process used in this comparative example are substantially the same as those in Example 1. The difference is that the humidity is not controlled.

[0058] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0059] Comparative Example 2

[0060] The strains and fermentation culture process used in this comparative example are substantially the same as those in Example 2. The difference is that the humidity is not controlled.

[0061] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0062] Comparative Example 3

[0063] The strains and fermentation culture process used in this comparative example are substantially the same as those in Example 3. The difference is that the humidity is not controlled.

[0064] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0065] Comparative Example 4

[0066] The strains and fermentation culture process used in this comparative example are substantially the same as those in Example 1. The difference is that the humidity is not controlled.

[0067] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0068] Comparative Example 5

[0069] The strains and fermentation culture process used in this comparative example are substantially the same as those in Example 5. The difference is that the humidity is not controlled.

[0070] Similarly, the titer was detected by HPLC after 48h of fermentation. The results are shown in Table 1.

[0071] Comparative Example 6

[0072] The strains and fermentation culture process used in this comparative example are substantially the same as those in Example 6. The difference is that the humidity is not controlled.

[0073] The test conditions and results of Examples 1-6 and Comparative Examples 1-6 are summarized in Table 1:

[0074]

[0075] Wherein, biotin content (%) is based on the content of Comparative Example 1, and the ratios of other examples / comparative examples to it.

[0076] From the results in Table 1, it can be seen that the biotin titer is significantly improved by stably controlling the humidity within 10-60% and the temperature is suitable for bacterial growth and fermentation.

[0077] The process parameters of the above-mentioned embodiments can be combined arbitrarily. To simplify the description, not all combinations of the invention are described. However, as long as there is no contradiction between the process parameters, they should be considered to be within the scope of this specification.

Claims

1. A method for increasing the yield and stability of biotin fermentation by controlling temperature and humidity, characterized in that: The steps include: (1) inoculating a strain capable of synthesizing biotin into a solid culture medium, maintaining a constant temperature and humidity of 34-38° C. and a humidity of 10-60%, and culturing until the colony size or thickness of the bacterial moss meets the requirements; (2) directly transferring the colony to a fermentation medium for fermentation culture; or first transferring the colony to a liquid culture medium and then transferring the colony to a fermentation medium for fed-batch fermentation culture, wherein the fermentation temperature is 28-38° C. and the humidity is the same as in step (1), and finally biotin is obtained.

2. The method according to claim 1, characterized in that: The bacterial species capable of synthesizing biotin is preferably genetically engineered Escherichia coli.

3. The method according to claim 1, characterized in that: The solid culture medium comprises: 5-15 g / L of tryptone, 1-10 g / L of yeast extract powder, 1-10 g / L of sodium chloride, 0.5-2 g / L of glucose and 0.5-2 g / L of beef extract.

4. The method according to claim 1, characterized in that: The composition and dosage of the liquid culture medium are: 5-15 g / L of a composite carbon source, 5-15 g / L of a composite nitrogen source, 10-20 g / L of an inorganic salt, and 1-5 ml / L of a trace element mixed solution.

5. The method according to claim 1, characterized in that The dosage of the fermentation medium is: 15-30 g / L of a composite carbon source, 10-20 g / L of a composite nitrogen source, 10-20 g / L of an inorganic salt, 1-10 ml / L of a trace element mixture, and 0.5-2 ml / L of a defoaming agent.

6. The method according to claim 4 or 5, characterized in that: The composite carbon source is at least one of glucose, sucrose and maltose; the composite nitrogen source is at least one of peptone, yeast extract powder, yeast powder, beef extract, corn steep liquor, corn steep liquor dry powder and urea; the inorganic salt is at least one of sodium chloride, sodium sulfate, sodium glutamate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, calcium carbonate and calcium chloride; the component dosages of the trace element mixed solution are: 0.1-5g / L zinc sulfate, 0.1-3g / L manganese sulfate, 0.1-10g / L ferrous sulfate, 0.1-2g / L cobalt chloride, 0.1-1g / L betaine, 0.1-2g / L choline chloride, 0.1-1g / L nicotinic acid, 0.1-1g / L folic acid and 0.1-1g / L thiamine.

7. The method according to claim 1, characterized in that In the step (2), the colony is directly transferred to a fermentation medium with a liquid volume of (20-50) ml / 250 ml, a rotation speed of 220-280 rpm, and a temperature of 28-38° C. for 40-48 hours; the colony is first transferred to a liquid culture medium with a liquid volume of (20-50) ml / 250 ml, a rotation speed of 220-280 rpm, and a temperature of 34-38° C. for 4-8 hours, and then transferred to a fermentation medium with a liquid volume of (20-50) ml / 250 ml, a rotation speed of 220-280 rpm, and a temperature of 28-38° C. for 40-48 hours.

8. The method according to claim 1, characterized in that: The feed is at least one of glucose, MOPS and ammonia water.

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