Method for producing tetraacetyl phytosphingosine through semi-continuous fermentation

Abstract

The invention discloses a method for producing tetraacetyl phytosphingosine through semi-continuous fermentation. The method comprises the following steps: inoculating a fermentation seed solution of a tetraacetyl phytosphingosine production strain into a fermentation tank containing a fermentation culture medium; fermenting and culturing until the glucose concentration of the fermentation liquor is reduced to 10g / L, and adding a fed-batch culture medium in a continuous fed-batch manner; continuously carrying out fermentation culture, continuously feeding the fed-batch culture medium until the volume of the fermentation liquid reaches 70-80% of the volume of the fermentation tank, and discharging part of the fermentation liquid in the fermentation tank; the last step is repeated for the remaining fermentation liquor in the fermentation tank after discharging; and mixing the discharged fermentation liquor with the fermentation liquor in the fermentation tank to obtain the fermentation liquor containing tetraacetyl phytosphingosine. According to the method disclosed by the invention, the high-speed synthesis time of the tetraacetyl phytosphingosine is prolonged through circulating intermittent discharging and continuous feeding, the utilization rate of fermentation equipment and the strain production capacity are improved, the single-tank productivity is greatly improved, and the method is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fermentation engineering, and particularly relates to a method for producing tetraacetyl phytosphingosine through semi-continuous fermentation. BACKGROUND

[0002] Tetraacetyl phytosphingosine is a natural high-efficiency multifunctional cosmetic raw material with multiple functions such as anti-wrinkle, moisturizing, anti-inflammatory and anti-aging, and is suitable for anti-aging, repair and eye cream products. Phytosphingosine is generated after tetraacetyl phytosphingosine is completely deacetylated, is a signal lipid molecule participating in various cell processes, plays many important functions in living cells, and is also an active ingredient widely used in cosmetics. Phytosphingosine series and ceramide series cosmetic raw materials can be synthesized by taking phytosphingosine as a starting raw material, and are applied to high-end cosmetics and skin care products. At present, more than ten phytosphingosine series raw materials such as phytosphingosine, acetyl phytosphingosine, lactyl phytosphingosine and salicyl phytosphingosine are recorded in the International Cosmetic Ingredient Dictionary and Handbook (16th Edition) compiled by the American Personal Care Products Council, and relevant ceramide series raw materials such as ceramide AP, ceramide NP and ceramide EOP are also recorded. Therefore, the application potential and market prospect of tetraacetyl phytosphingosine in the field of cosmetics are very broad.

[0003] Since Wickerham and Stodol discovered tetracosanoyl phytosphingosine from Wickerhamiella sp. in 1956, the research on the biosynthesis mechanism of tetracosanoyl phytosphingosine, high-yield strain breeding, fermentation process optimization, etc. has never stopped in academia and industry. In recent years, significant progress has been made. Not only has a commercially viable tetracosanoyl phytosphingosine high-yield strain been obtained through classical mutagenesis breeding, but also multiple strains with improved substrate utilization and reduced byproduct content have been constructed through synthetic biology technology. The current highest level of tetracosanoyl phytosphingosine produced by Wickerhamiella sp. has broken through 20 g / L, making it the most potential host microorganism for synthesizing tetracosanoyl phytosphingosine. The synthesis of tetracosanoyl phytosphingosine using Wickerhamiella sp. has the advantages of easy access to raw materials, short fermentation period, low production cost, and mild production conditions, which conforms to the trend of green and low-carbon development. The fermentation production methods of tetracosanoyl phytosphingosine are disclosed in US patents US5910425, US5627056, US5618706, US5598742 and Chinese patents CN115323012A, CN115820440A, CN114736815A, CN101098968B, etc. However, all of them use traditional batch fermentation or fed-batch fermentation, i.e. after single-tank fermentation is completed, all the fermentation broth is simultaneously sent to the downstream for extraction and purification. The batch fermentation and fed-batch fermentation require operations such as dosing, sterilization, inoculation, feeding, tank release, and cleaning, which are complicated and result in low production efficiency. The utilization rate of fermentation tank equipment is low, which cannot fully utilize the strain production capacity, and the single-tank capacity is low. SUMMARY

[0004] The purpose of the present application is to provide a method for semi-continuous fermentation production of tetracosanoyl phytosphingosine. Through optimization of full-nutrient feeding medium, intermittent feeding and continuous feeding, semi-continuous fermentation is formed, and after several cycles, fermentation is completed, thereby improving the utilization rate of fermentation tank equipment, strain production capacity, and single-tank capacity.

[0005] The application provides a method for producing tetraacetyl phytosphingosine through semi-continuous fermentation.

[0006] The application aims to achieve the above-mentioned technical problems through the following technical scheme.

[0007] The application provides a method for producing tetraacetyl phytosphingosine through semi-continuous fermentation, which comprises the following steps.

[0008] S1, inoculating a fermentation seed liquid of a tetraacetyl phytosphingosine producing strain into a fermenter containing a fermentation medium;

[0009] Preferably, the tetraacetyl phytosphingosine producing strain is Xiphophorus hellerii.

[0010] Preferably, the preparation method of the fermentation seed liquid comprises the following steps: inoculating the tetraacetyl phytosphingosine producing strain into a first-stage seed culture medium to culture, so as to obtain a first-stage seed; inoculating the first-stage seed into a second-stage seed culture medium to culture, so as to obtain a second-stage seed, i.e., the fermentation seed liquid.

[0011] Preferably, the first-stage seed culture medium and the second-stage seed culture medium have the same composition and comprise the following components: glucose 40-60 g / L, yeast extract powder 4-6 g / L, ammonium sulfate 2-4 g / L, potassium dihydrogen phosphate 15-20 g / L, magnesium sulfate heptahydrate 3-5 g / L, calcium chloride dihydrate 0.5-1 g / L, trace element mother liquor 10-15 ml / L, vitamin mother liquor 10-15 ml / L, and pH 5.5-6.0.

[0012] Preferably, the culture conditions of the first-stage seed comprise the following: culture temperature 26-28 DEG C, shaking bed rotating speed 200-250 rpm, culture time 40-60 h, and culture until OD600 reaches 10-15.

[0013] Preferably, the culture conditions of the secondary seed include: culture temperature 26-28℃, stirring speed 300-400rpm, aeration amount 0.5-1.0VVM, culture time 70-90h, and culture to OD600 of 15-22; the inoculation amount of the secondary seed is 0.5-1.0%;

[0014] Preferably, the trace element mother liquor includes the following components: ferrous sulfate heptahydrate 30-40g / L, zinc sulfate heptahydrate 8-12g / L, copper sulfate pentahydrate 2-4g / L, manganese sulfate monohydrate 1-2g / L, sodium molybdate monohydrate 1-2g / L, boric acid 1-2g / L, potassium iodide 0.2-0.4g / L, cobalt chloride hexahydrate 0.2-0.4g / L, concentrated sulfuric acid 5-10g / L;

[0015] Preferably, the vitamin mother liquor includes the following components: D-calcium pantothenate 2-4g / L, nicotinic acid 2-4g / L, thiamine hydrochloride 1-3g / L, pyridoxine 0.2-0.4g / L, D-biotin 0.1-0.2g / L, concentrated sulfuric acid 5-10g / L;

[0016] Preferably, the inoculation amount of the fermentation seed liquid is 10-12%;

[0017] Preferably, the fermentation medium includes the following components: glucose 60-80g / L, yeast extract powder 6-8g / L, ammonium sulfate 3-5g / L, potassium dihydrogen phosphate 20-25g / L, magnesium sulfate heptahydrate 4-6g / L, calcium chloride dihydrate 1.0-1.5g / L, trace element mother liquor 10-15ml / L, vitamin mother liquor 10-15ml / L, and pH 5.5-6.0. The culture conditions of the fermentation include: culture temperature 24-26℃, stirring speed 200-600rpm, aeration amount 0.5-1.5VVM, tank pressure 0.04-0.06Pa, dissolved oxygen DO value 20-30%, and ammonia water is added in a continuous flow manner to control the pH of the fermentation broth to 5.5-6.0, and the concentration of the added ammonia water is preferably 25-28%.

[0018] S2, fermentation culture to the glucose concentration of the fermentation broth reduced to 10g / L, and a feed medium is added in a continuous flow manner;

[0019] Preferably, the feed medium comprises the following components: glucose 500-600 g / L, yeast extract 3-4 g / L, serine 10-15 g / L, ammonium sulfate 2-3 g / L, potassium dihydrogen phosphate 10-15 g / L, magnesium sulfate heptahydrate 2-3 g / L, calcium chloride dihydrate 1.0-1.5 g / L, pH natural, after sterilization, add sterile filtered low alcohol to a final concentration of 50-60 ml / L, wherein the low alcohol is preferably methanol or ethanol; the feed medium is a full nutrient feed medium, wherein serine as a direct precursor of tetraacetyl phytosphingosine can promote the production of tetraacetyl phytosphingosine, and low alcohol can play a role in the fermentation process of tetraacetyl phytosphingosine producing strain by increasing the expression level of genes related to tetraacetyl phytosphingosine biosynthesis, which has a significant effect on the fermentation level of tetraacetyl phytosphingosine, and yeast extract and inorganic salts promote the growth of bacteria and maintain the metabolic activity of bacteria.

[0020] S3, continue to ferment and continuously add the feed medium to the fermentation broth until the volume of the fermentation broth reaches 70-80% of the volume of the fermentation tank, and then discharge part of the fermentation broth in the fermentation tank; preferably, 10-15% of the total volume of the fermentation broth in the fermentation tank; the above two numerical ranges can effectively avoid the phenomenon of fermentation tank liquid escape during the fermentation process, and ensure the fermentation tank loading coefficient and fermentation capacity.

[0021] S4, repeating the treatment of step S3 on the remaining fermentation broth in the fermentation tank after discharging;

[0022] The semi-continuous fermentation process provided by the application is carried out in the stage where the conventional fed-batch fermentation has the fastest growth rate of fermentation units, and through recycling, intermittent discharging and continuous addition of the full nutrient feed medium containing glucose, low alcohol, yeast extract, serine and inorganic salts are carried out, so that the glucose concentration in the fermentation broth is maintained at 5-10 g / L, and other nutrient components are also maintained at the optimum concentration, so that the metabolic activity of the bacteria in the fermentation broth and the product synthesis capacity are maintained at a high level for a long time. The recycling number of the semi-continuous fermentation provided by the application is 5-6 times, the more the recycling number, the higher the equipment utilization rate, but as the recycling number increases, the proportion of aging bacteria in the fermentation broth will also be higher, and the concentration of cell metabolic waste accumulated in the fermentation tank will also be higher, and overall the growth rate of fermentation units shows a downward trend. When the recycling number is 5-6 times and the total volume of the intermittent discharging during the fermentation process is 50-70% of the total volume of the fermentation tank, the semi-continuous fermentation process has obvious advantages over the traditional fed-batch fermentation.

[0023] S5, combining the discharged fermentation broth with the fermentation broth in the fermentation tank to obtain a fermentation broth containing tetraacetyl phytosphingosine;

[0024] The fermentation liquor of the present application is stored in a tank at low temperature, and after the semi-continuous fermentation is completed, all the fermentation liquor is discharged at one time, and then enters the downstream for further extraction and purification to obtain tetraacetyl phytosphingosine.

[0025] Compared with the prior art, the present application has the following advantages:

[0026] The present application provides a method for producing tetraacetyl phytosphingosine by semi-continuous fermentation, which significantly prolongs the high-speed synthesis period of tetraacetyl phytosphingosine produced by fermentation of Wickerhamiella sp., thereby improving the utilization rate of the fermentation tank, the production capacity of the strain and the yield per tank, and reducing the production cost. According to the average cycle of 5 times, the fermentation index can be increased by 30-40%, the equipment utilization rate and the tank batch yield are significantly improved, and it is suitable for industrial production. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application. The Wickerhamiella sp. used in the following embodiments is a tetraacetyl phytosphingosine industrial production strain, which is obtained through commercial channels. Unless otherwise specified, the materials, reagents, instruments and methods used in the embodiments are conventional materials, reagents, instruments and methods in the art, which can be obtained through commercial channels.

[0028] The culture medium composition involved in the following embodiments is as follows:

[0029] Seed flask culture medium: glucose 50g / L, yeast extract powder 5g / L, ammonium sulfate 3g / L, potassium dihydrogen phosphate 18g / L, magnesium sulfate heptahydrate 4g / L, calcium chloride dihydrate 0.8g / L, trace element mother liquor 12ml / L, vitamin mother liquor 12ml / L, adjust pH to 5.8 with alkali before sterilization, and the sterilization condition is 121℃, 25min.

[0030] Seed tank culture medium: the seed tank culture medium formula is the same as the seed flask culture medium, and 0.05% Si-O-C type polyether modified organosilicon defoaming agent is added, the pH is adjusted to 5.8 with alkali before sterilization, and the sterilization condition is 121℃, 25min, wherein the glucose is sterilized separately and then mixed with other components.

[0031] Fermentation tank medium: glucose 70 g / L, yeast extract 7 g / L, ammonium sulfate 4 g / L, potassium dihydrogen phosphate 22 g / L, magnesium sulfate heptahydrate 5 g / L, calcium chloride dihydrate 1.2 g / L, trace element mother liquor 10 ml / L, vitamin mother liquor 10 ml / L, before sterilization, adjust pH to 5.8 with alkali, sterilization conditions are 121°C, 25 min, wherein the glucose is sterilized separately and then mixed with other components.

[0032] Full nutrition medium: glucose 550 g / L, yeast extract 3.5 g / L, serine 12 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 12 g / L, magnesium sulfate heptahydrate 2.5 g / L, calcium chloride dihydrate 1.2 g / L, natural pH, sterilization conditions are 121°C, 25 min, wherein the glucose is sterilized separately and then mixed with other components, after mixing, add filtered sterilized ethanol with a concentration of 95% to a final concentration of 55 ml / L.

[0033] Trace element mother liquor: ferrous sulfate heptahydrate 25 g / L, zinc sulfate heptahydrate 10 g / L, copper sulfate pentahydrate 2.5 g / L, manganese sulfate monohydrate 1.5 g / L, sodium molybdate monohydrate 1.5 g / L, boric acid 1.5 g / L, potassium iodide 0.25 g / L, cobalt chloride hexahydrate 0.25 g / L, concentrated sulfuric acid 10 g / L.

[0034] Vitamin mother liquor: D-calcium pantothenate 2.5 g / L, nicotinic acid 2.5 g / L, thiamine hydrochloride 2 g / L, pyridoxine 0.25 g / L, D-biotin 0.15 g / L, concentrated sulfuric acid 10 g / L.

[0035] Example 1 Preparation of seed liquid for tetraacetyl phytosphingosine fermentation

[0036] A small amount of Hamsi Fermenting Yeast strain was picked from a well-grown slant and inoculated into 500 ml of shake flask seed medium in a flask for shake flask seed culture, the flask was filled with 80 ml of medium, the culture temperature was 26°C, the rotation speed of the shaker was 250 rpm, and the culture period was 60 h, and the OD600 value of the well-cultured shake flask seed liquid was 11.2.

[0037] The well-cultured shake flask seed liquid was inoculated into a seed tank containing seed tank medium for expansion culture, the inoculation amount was 0.5%, the culture temperature was 26°C, the ventilation ratio was 0.5 VVM, the tank pressure was 0.04 Mpa, the correlation 300 rpm stirring controlled DO at 40-50%, and the pH was natural. With the growth of the strain, the pH of the seed liquid showed a gradual downward trend, the culture period was 80 h, and the OD600 value of the well-cultured seed liquid was 21.8, and the pH was 2.76.

[0038] Example 2 Tetraacetyl phytosphingosine semi-continuous fermentation

[0039] The seed tank seed liquid is inoculated into the fermentation tank medium at about 10% (5L) of the inoculation amount. The fermentation tank culture process is as follows: the culture temperature is 24-26°C, the stirring speed is 200-600 rpm, the ventilation ratio is 0.5-1.5 VVM, the tank pressure is 0.04-0.06 MPa, when the dissolved oxygen is lower than 20%, the stirring speed, ventilation ratio and tank pressure are alternately increased to increase the dissolved oxygen to the range of 20-30%, the pH of the fermentation broth is controlled in the range of 5.5-6.0 by continuously adding 25-28% ammonia water. After 20-24 hours of fermentation, the residual glucose in the fermentation broth is reduced to 10 g / L, and a full-nutrient feed medium containing glucose, ethanol, yeast extract, serine and inorganic salts is continuously added. The glucose content and OD600 value in the fermentation broth are determined every 2 hours, and the flow rate of the feed medium is adjusted according to the glucose content to maintain the glucose content in the fermentation broth at 5-10 g / L. The fermentation unit of tetraacetyl phytosphingosine is detected every 8 hours during the fermentation process.

[0040] After 65 hours of fermentation, the volume of the fermentation broth in the fermentation tank is increased to 75% of the volume of the fermentation tank. The flow rate of the full-nutrient feed medium is 1.2 L / h, and the fermentation unit of tetraacetyl phytosphingosine is about 15.3 g / L. The fermentation broth equivalent to 12% (about 9L) of the liquid volume of the fermentation tank is discharged into a storage tank for low-temperature static preservation. The remaining culture solution in the fermentation tank continues to be fermented by continuously adding the full-nutrient feed medium. After 72 hours of fermentation, the volume is increased to 75% of the volume of the fermentation tank again. The flow rate of the full-nutrient feed medium is 1.4 L / h, and the fermentation unit of tetraacetyl phytosphingosine is about 17.5 g / L. Again, the fermentation broth equivalent to 12% (about 10L) of the liquid volume of the fermentation tank is discharged into a storage tank for low-temperature static preservation. This cycle of intermittent discharge, continuous fermentation and continuous feeding is repeated 6 times. At the end of the 6th cycle, the fermentation unit of tetraacetyl phytosphingosine has a certain downward trend due to the influence of cell aging and metabolic waste. No more discharge is performed, and the fermentation continues for 6 hours to 102 hours. The total fermentation broth in the fermentation tank is discharged at once, with a volume of 75L and a unit of 20.5 g / L. The final measurement results show that the total discharge volume is about 135L, the final discharge of tetraacetyl phytosphingosine is about 1530g, the continuous discharge of tetraacetyl phytosphingosine is 1330g, and the total tetraacetyl phytosphingosine production is 2860g. The cumulative glucose consumption is about 38 kg, the ammonia water consumption is 7.5L, the fermentation index (the amount of fermentation product produced per unit of fermentation tank volume per unit of time) is 0.28 kg / h / m 3 , and the sugar alcohol conversion rate is 13.3 g / g. In this example, the starting period of each cycle, OD600 value, feed flow rate, fermentation unit and discharge volume are shown in Table 1:

[0041] Table 1 Start of each cycle period, OD600 value, feed flow rate, fermentation units, and bleed volume statistics

[0042] fermentation cycle 65h 72h 78h 84h 90h 96h 102h feed number 1 2 3 4 5 6 — fermentation broth volume in tank 75L 75L 75L 75L 75L 75L 75L OD600 value 207 236 255 271 284 296 291 feed flow rate 1.2 L / h 1.4 L / h 1.5 L / h 1.5 L / h 1.5 L / h 1.5 L / h 1.5 L / h fermentation unit 15.3 g / L 17.5 g / L 19.0 g / L 20.4 g / L 21.3 g / L 20.8 g / L 20.5 g / L feed volume ≈9L ≈10L ≈10L ≈10L ≈10L ≈10L ≈75L

[0043] Comparative Example 1 Tetraacetyl phytosphingosine fed-batch fermentation

[0044] A 100 L fermenter was used, and the fermenter medium volume was 45 L. The seed tank seed liquid was inoculated into the fermenter at about 10% of the inoculation volume (5 L). The fermenter culture process was as follows: culture temperature 24-26 °C, stirring speed 200-600 rpm, ventilation ratio 0.5-1.5 VVM, tank pressure 0.04-0.06 Mpa. When the dissolved oxygen was less than 20%, the stirring speed, ventilation ratio, and tank pressure were alternately increased to increase the dissolved oxygen to the range of 20-30%. The pH of the fermentation broth was controlled in the range of 5.5-6.0 by adding 25-28% ammonia water throughout the process. When the residual glucose in the fermentation broth was reduced to 10 g / L after 20-24 h of fermentation, a full-nutrient feed medium containing glucose, ethanol, yeast extract, serine, and inorganic salts was continuously fed. The glucose content and OD600 value in the fermentation broth were measured every 2 h, and the feeding rate of the feed medium was adjusted according to the glucose content to maintain the glucose content in the fermentation broth at 5-10 g / L. The fermentation units of tetraacetyl phytosphingosine were detected every 8 h during the fermentation process.

[0045] The fermentation broth volume in the fermenter increased to 75% of the fermenter volume after 68 h of fermentation, and the feed medium feeding rate was 1.3 L / h. The tetraacetyl phytosphingosine fermentation units were 16.7 g / L. The fermentation broth volume in the fermenter increased to 85% of the fermenter volume after 75 h of fermentation, and the tetraacetyl phytosphingosine fermentation units were 18.5 g / L. The fermentation started to leak, and a one-time tank discharge treatment was performed. The final calculation showed that the single batch tank discharge volume was about 85 L, the tetraacetyl phytosphingosine yield was about 1570 g, the cumulative glucose consumption was about 20 kg, and the tank discharge fermentation index (the amount of fermentation product produced per unit of fermenter volume per unit of time) was 0.21 kg / h / m 3 , and the sugar alcohol conversion rate was 12.7 g / g.

[0046] Comparing the results of Example 2 and Comparative Example 1, it can be seen that the fermentation period of Comparative Example 1 was 75 h, the tetraacetyl phytosphingosine was obtained with a purity of 1570 g, the glucose consumption was about 20 kg, the tank discharge fermentation index was 0.21 kg / h / m 3 , and the sugar alcohol conversion rate was 12.7 g / g. The fermentation period of Example 2 was 105 h, a total of 2860 g of tetraacetyl phytosphingosine was obtained with a purity of 2860 g, the glucose consumption was about 38 kg, and the fermentation index was 0.28 kg / h / m 3, the sugar alcohol conversion rate was 13.3 g / g. Therefore, compared with the fed-batch fermentation process, the fermentation index of the semi-continuous fermentation process of the present application was increased by 33.3%, the same weight of tetraacetylphytosphingosine was produced, the sugar alcohol conversion rate was similar, but the fermentation time was shorter, and the production efficiency was higher.

[0047] Effect of low molecular alcohol on semi-continuous fermentation of tetraacetylphytosphingosine

[0048] The fermentation equipment, medium loading, inoculum, culture process, method of controlling pH by adding ammonia water, and method of controlling glucose concentration in the fermentation broth by adding the feed medium were the same as those in Example 2, the only difference was that no low molecular alcohol was added in the feed medium during the fermentation (the remaining components were added), and the fermentation was stopped at 100 h, and the final tetraacetylphytosphingosine fermentation unit was 16.7 g / L.

[0049] Effect of serine on semi-continuous fermentation of tetraacetylphytosphingosine

[0050] The fermentation equipment, medium loading, inoculum, culture process, method of controlling pH by adding ammonia water, and method of controlling glucose concentration in the fermentation broth by adding the feed medium were the same as those in Example 2, the only difference was that no serine was added in the feed medium during the fermentation (the remaining components were added), and the fermentation was stopped at 104 h, and the final tetraacetylphytosphingosine fermentation unit was 15.1 g / L.

[0051] Effect of yeast extract powder on semi-continuous fermentation of tetraacetylphytosphingosine

[0052] The fermentation equipment, medium loading, inoculum, culture process, method of controlling pH by adding ammonia water, and method of controlling glucose concentration in the fermentation broth by adding the feed medium were the same as those in Example 2, the only difference was that no yeast extract powder was added in the feed medium during the fermentation (the remaining components were added), and the fermentation was stopped at 98 h, and the final tetraacetylphytosphingosine fermentation unit was 14.3 g / L.

[0053] Effect of inorganic salt on semi-continuous fermentation of tetraacetylphytosphingosine

[0054] The fermentation equipment, medium loading, inoculum, culture process, method of controlling pH by adding ammonia water, and method of controlling glucose concentration in the fermentation broth by adding the feed medium were the same as those in Example 2, the only difference was that no inorganic salt (ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and calcium chloride dihydrate) was added in the feed medium during the fermentation (the remaining components were added), and the fermentation was stopped at 105 h, and the final tetraacetylphytosphingosine fermentation unit was 13.8 g / L.

[0055] It can be seen from comparative example 2 and comparative examples 2-5 that the comparative examples 2-5 change the composition of the fed-batch medium based on example 2, and the final tetraacetyl phytosphingosine fermentation units of example 2 are 20.5 g / L, and those of comparative examples 2-5 are 16.7 g / L, 15.1 g / L, 14.3 g / L and 13.8 g / L respectively, the fermentation units of example 2 are obviously improved compared with those of each comparative example, which indicates that the present application further improves the fermentation yield by optimizing the composition of the medium on the basis of semi-continuous fermentation.

[0056] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A method for the semi-continuous fermentation production of tetraacetyl phytosphingosine, characterized in that, The method comprises: S1, inoculating the fermentation seed liquid of the tetraacetyl phytosphingosine production strain into a fermenter containing a fermentation medium; S2, fermenting to a glucose concentration of 10 g / L in the fermentation liquid, and adding a feed medium in a continuous flow manner; S3, continuing the fermentation and continuously adding the feed medium until the volume of the fermentation liquid reaches 70-80% of the volume of the fermenter, and discharging part of the fermentation liquid in the fermenter; S4, repeating the step S3 for the remaining fermentation liquid in the fermenter after discharging; and S5, combining the discharged fermentation liquid with the fermentation liquid in the fermenter to obtain a fermentation liquid containing tetraacetyl phytosphingosine.

2. The method of claim 1, wherein, The step S4 is repeated 5-6 times.

3. The method of claim 1, wherein, The part of the fermentation liquid accounts for 10-15% of the total volume of the fermentation liquid in the fermenter.

4. The method of claim 1, wherein, The feed medium comprises the following components: glucose 500-600 g / L, yeast extract powder 3-4 g / L, serine 10-15 g / L, ammonium sulfate 2-3 g / L, potassium dihydrogen phosphate 10-15 g / L, magnesium sulfate heptahydrate 2-3 g / L, calcium chloride dihydrate 1.0-1.5 g / L, pH is natural, after sterilization, add sterile filtered low molecular alcohol to a final concentration of 50-60 ml / L.

5. The method of claim 4, wherein, The low molecular alcohol is methanol or ethanol.

6. The method of claim 1, wherein, The fermentation medium comprises the following components: glucose 60-80 g / L, yeast extract powder 6-8 g / L, ammonium sulfate 3-5 g / L, potassium dihydrogen phosphate 20-25 g / L, magnesium sulfate heptahydrate 4-6 g / L, calcium chloride dihydrate 1.0-1.5 g / L, trace element mother liquor 10-15 ml / L, vitamin mother liquor 10-15 ml / L, pH is 5.5-6.

0.

7. The method of claim 1, wherein, The fermentation conditions comprise: culture temperature 24-26℃, stirring speed 200-600 rpm, aeration amount 0.5-1.5 VVM, tank pressure 0.04-0.06 Pa, dissolved oxygen DO value 20-30%, and adding ammonia water in a continuous flow manner to control the pH of the fermentation liquid to 5.5-6.

0.

8. The method of claim 1, wherein, The preparation method of the fermentation seed liquid comprises: Inoculating the tetraacetyl phytosphingosine production strain into a primary seed medium for culture to obtain a primary seed; inoculating the primary seed into a secondary seed medium for culture to obtain a secondary seed, which is the fermentation seed liquid; Preferably, the primary seed medium comprises the following components: glucose 40-60 g / L, yeast extract powder 4-6 g / L, ammonium sulfate 2-4 g / L, potassium dihydrogen phosphate 15-20 g / L, magnesium sulfate heptahydrate 3-5 g / L, calcium chloride dihydrate 0.5-1 g / L, trace element mother liquor 10-15 ml / L, vitamin mother liquor 10-15 ml / L, pH is 5.5-6.0; Preferably, the culture conditions of the primary seed comprise: culture temperature 26-28℃, shaking speed 200-250 rpm, culture time 40-60 h, and culturing to OD600 of 10-15. Preferably, the secondary seed culture medium comprises the following components: glucose 40-60 g / L, yeast extract powder 4-6 g / L, ammonium sulfate 2-4 g / L, potassium dihydrogen phosphate 15-20 g / L, magnesium sulfate heptahydrate 3-5 g / L, calcium chloride dihydrate 0.5-1 g / L, trace element mother liquor 10-15 ml / L, vitamin mother liquor 10-15 ml / L, and pH 5.5-6.

0. Preferably, the culture conditions of the secondary seed culture comprise: culture temperature 26-28℃, stirring speed 300-400 rpm, aeration amount 0.5-1.0 VVM, culture time 70-90 h, and culture to OD600 of 15-22.

9. The method according to claim 6 or 8, characterized in that, The trace element mother liquor comprises the following components: ferrous sulfate heptahydrate 30-40 g / L, zinc sulfate heptahydrate 8-12 g / L, copper sulfate pentahydrate 2-4 g / L, manganese sulfate monohydrate 1-2 g / L, sodium molybdate monohydrate 1-2 g / L, boric acid 1-2 g / L, potassium iodide 0.2-0.4 g / L, cobalt chloride hexahydrate 0.2-0.4 g / L, and concentrated sulfuric acid 5-10 g / L.

10. The method of claim 6 or 8, wherein, The vitamin mother liquor comprises the following components: D-calcium pantothenate 2-4 g / L, nicotinic acid 2-4 g / L, thiamine hydrochloride 1-3 g / L, pyridoxine 0.2-0.4 g / L, D-biotin 0.1-0.2 g / L, and concentrated sulfuric acid 5-10 g / L.