A fermentation method for a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine, comprising the following steps: step (1), glycerol tube preservation; step (2), primary shake flask culture: taking a strain preservation glycerol tube, inoculating 1% (v / v) seed volume into a seed culture medium of a primary seed bottle, and culturing in a box shaker at 28°C and 240rpm for 72h; step (3), secondary shake flask culture: taking a primary seed bottle, inoculating 1% (v / v) inoculation volume into a secondary seed bottle, and culturing in a box shaker at 28°C and 240rpm for 48h; step (4), seed tank culture; step (5), transfer to a fermentation tank; step (6), fermentation. The invention adopts Wickham syringa to simultaneously ferment and produce TAPS and TriASa in the same fermentation system, and can obtain mixtures of TAPS and TriASa in different proportions by regulating dissolved oxygen, thereby greatly improving production efficiency and saving production costs.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to a fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine. Background Art

[0002] Sphingolipids refer to a group of lipid substances derived from sphingosine bases. They are a general term for ceramides or ceramide derivatives in the human body with sphingosine, phytosphingosine and dihydrosphingosine as the basic skeleton. In human skin, ceramide, cholesterol, cholesterol sulfate and free fatty acids, as the main components of the stratum corneum, form a specific layered quasi-crystalline structure, forming a permeation barrier that blocks moisture and protects the skin to prevent moisture loss and microbial invasion. Therefore, ceramide has a high application potential in many application fields such as antimicrobial drugs, active pharmaceutical ingredients, cosmetics, and skin care products.

[0003] Currently, there are three main methods for obtaining ceramide: 1. Natural extraction. Extracting ceramide from plants (such as konjac) is affected by weather, season, pests and diseases, and the yield is low and unstable; 2. Chemical synthesis. There are currently ways to chemically synthesize ceramide, but there are two stereoisomer centers and three functional groups, so chemical synthesis of ceramide is difficult and costly; 3. Microbial fermentation. There are mainly two forms, direct fermentation to obtain ceramide and fermentation to obtain ceramide precursors and then chemical synthesis to obtain ceramide; currently, the yield of ceramide obtained by direct fermentation is low, so fermenting ceramide precursors and then chemically synthesizing ceramide has become the first choice.

[0004] Tetraacetyl Phytosphingosine (TAPS) and Triacetyl Sphinganine (TriASa) are important precursors of ceramide and are natural metabolites of Wickerhamomyces ciferrii. In addition, TAPS and TriASa are also excellent natural skin care ingredients with moisturizing, anti-oxidation, whitening and freckle removal, and promoting skin barrier self-repair. Acetylated phytosphingosine and dihydrosphingosine are extremely water-soluble and can be mixed with a variety of cosmetic raw materials, and have been widely used.

[0005] At present, there are many papers and patents related to the fermentation of tetraacetyl phytosphingosine by microorganisms, but there are few reports on the fermentation production of triacetyl dihydrosphingosine, and even fewer reports on the simultaneous production and fermentation of the two. Therefore, triacetyl dihydrosphingosine and tetraacetyl phytosphingosine are mostly fermented and cultured separately, with low production efficiency and high cost, which affects production. In this regard, the present invention provides a new fermentation method. Summary of the invention

[0006] The present invention aims to provide a method for fermenting a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine, so as to solve the problem that there are few methods for simultaneously fermenting triacetyl dihydrosphingosine and tetraacetyl phytosphingosine in the prior art, resulting in low production efficiency and high cost.

[0007] To achieve the above object, the present invention provides the following technical solution: a fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine, comprising the following steps:

[0008] Step (1), glycerol tube storage:

[0009] The fermented strain was added to a glycerol tube and stored at -80°C with a 60% (v / v) glycerol concentration;

[0010] Step (2), primary shake flask culture:

[0011] Take the glycerol tube for bacterial preservation and inoculate it into the seed medium of the first-level seed bottle at a seed volume of 1% (v / v), and culture it in a box shaker at 28°C and 240 rpm for 72 hours;

[0012] Step (3), secondary shake flask culture:

[0013] Take the first-level seed bottle and inoculate it into the second-level seed bottle at a 1% (v / v) inoculation amount, and culture it in a box shaker at 28°C and 240 rpm for 48 hours;

[0014] Step (4), seed tank cultivation:

[0015] Transfer the expansion culture solution from the secondary seed bottle into the seed tank and continue culturing;

[0016] Step (5), transfer to fermentation tank:

[0017] The seed liquid in the seed tank is transferred to the fermentation tank; when transferring, it is preferred to transfer to the mid-exponential growth stage and OD 600 =20-25 seed solution, the transfer amount should be controlled at 1%-15% (v / v);

[0018] Step (6), fermentation:

[0019] The seed liquid in the fermentation tank is subjected to batch feeding fermentation, and the entire fermentation process is a four-stage, five-stage or six-stage fermentation;

[0020] The fermentation conditions are as follows:

[0021] Temperature: 22℃-36℃;

[0022] pH: 3-7;

[0023] Dissolved oxygen: The dissolved oxygen in the TAPS metabolic stage is controlled at more than 30%, and the dissolved oxygen in the TriASa metabolic stage is controlled at 0% to 20%;

[0024] Ventilation volume: controlled between 1VVM-2VVM according to dissolved oxygen;

[0025] Stirring speed: controlled at 50rpm-1000rpm according to dissolved oxygen;

[0026] Feeding: When the central control detects that the glucose is lower than 20g / L, glucose is added in the form of feeding, and the carbon source concentration in the fermentation broth is maintained at 10g / L-20g / L;

[0027] Fermentation period: 120h.

[0028] Preferably, the fermentation temperature is 27-29° C., and the fermentation pH is 5-6.

[0029] Preferably, the composition and content (w / v, %) of the seed culture medium of the primary seed bottle and the secondary seed bottle are as follows: 3-5% glucose, 0.2-0.4% yeast extract powder, 0.1-0.5% potassium dihydrogen phosphate, 0.5-1% dipotassium hydrogen phosphate, 0.1-0.5% ammonium sulfate, 0.2-0.5% calcium chloride, and 0.1-0.6% defoaming agent.

[0030] Preferably, the transfer amount in step (5) is 8%-12%.

[0031] Preferably, the components and contents (w / v, %) of the fermentation medium are as follows: 5-8 glucose, 0.2-0.8 yeast extract powder, 0.5-1 potassium dihydrogen phosphate, 0.1-0.5 ammonium sulfate, 0.2-0.5 calcium chloride, 0.2-0.8 magnesium sulfate heptahydrate, 0.001-0.01 citric acid monohydrate, 0.001-0.005 nicotinic acid, 0.002-0.005 ferrous sulfate heptahydrate, 0.002-0.005 thiamine hydrochloride, and 0.1-0.6 defoaming agent.

[0032] Preferably, the steps for detecting the content of TAPS and TriASa in the fermentation broth in step (6) are as follows:

[0033] S1, preprocessing:

[0034] Take the fermentation broth and add 4 times volume of methanol, ultrasonically treat for 30 min, and centrifuge to obtain the supernatant;

[0035] S2, Liquid Chromatography:

[0036] The S1 supernatant was subjected to liquid chromatography detection under the following conditions:

[0037] Detector: UV detector;

[0038] Chromatographic column model: Sepax HP-C18 4.6×250mm 5μm;

[0039] Mobile phase A: ultrapure water;

[0040] Mobile phase B: acetonitrile;

[0041] Flow rate: 1.0ml / min;

[0042] Column temperature: 40°C;

[0043] Detection wavelength: 195nm;

[0044] Injection volume: 10uL.

[0045] The present invention has at least the following beneficial effects:

[0046] The present invention provides a fermentation method for a mixture of tetraacetylphytosphingosine and triacetyldihydrosphingosine, which adopts Wickham syringa to simultaneously ferment and produce TAPS and TriASa in the same fermentation system, and can obtain mixtures of TAPS and TriASa in different proportions by regulating dissolved oxygen, thereby greatly improving production efficiency and saving production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The fermentation process of the present invention;

[0048] Figure 2 It is a typical microscopic examination diagram of the present invention;

[0049] Figure 3 It is the TAPS:TriASa≈8:1HPLC-UV spectrum;

[0050] Figure 4 It is the TAPS:TriASa≈4:1HPLC-UV spectrum;

[0051] Figure 5 This is the HPLC-UV spectrum of TAPS:TriASa≈2:1. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The tetraacetyl phytosphingosine and triacetyl dihydrosphingosine of the present invention are obtained by using Wickham syringa yeast to carry out deep liquid fermentation for 120 hours in an aerated stirring fermenter with automatic control of pH, temperature and feed addition. The fermentation process is divided into two stages, namely the TAPS metabolism stage and the TriASa metabolism stage.

[0054] like Figure 1 As shown, the fermentation strain is connected to the seed bottle by the glycerol preservation tube for expansion culture in the shaking bottle stage, and then the expansion culture solution in the seed bottle is transferred to the seed tank, and finally the seed solution in the seed tank is transferred to the fermentation tank for batch feeding fermentation. The entire fermentation process can be four-stage, five-stage or six-stage fermentation, and the specific number of stages depends on the fermentation scale, but five-stage fermentation is preferred, and four-stage fermentation is more preferred.

[0055] The details are as follows:

[0056] A fermentation method for a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine comprises the following steps:

[0057] Step (1), glycerol tube storage:

[0058] The fermentation strain (Saccharomyces cerevisiae Wickhamii) was added to a glycerol tube and stored at -80°C with a 60% (v / v) glycerol concentration;

[0059] Step (2), primary shake flask culture:

[0060] Take the glycerol tube for bacterial preservation and inoculate it into the seed medium of the first-level seed bottle at a seed volume of 1% (v / v), and culture it in a box shaker at 28°C and 240 rpm for 72 hours;

[0061] Step (3), secondary shake flask culture:

[0062] Take the first-level seed bottle and inoculate it into the second-level seed bottle at a 1% (v / v) inoculation amount, and culture it in a box shaker at 28°C and 240 rpm for 48 hours;

[0063] Step (4), seed tank cultivation:

[0064] Transfer the secondary seed bottle expansion culture solution into the seed tank and continue to culture; during the seed tank culture process, add seed tank separation sugar;

[0065] Seed culture medium for primary and secondary seed bottles (w / v, %): 3-5% glucose, 0.2-0.4% yeast extract powder, 0.1-0.5% potassium dihydrogen phosphate, 0.5-1% potassium hydrogen phosphate, 0.1-0.5% ammonium sulfate, 0.2-0.5% calcium chloride, and 0.1-0.6% defoaming agent;

[0066] Step (5), transfer to fermentation tank:

[0067] The seed liquid in the seed tank is transferred to the fermentation tank; during the transfer to the fermentation tank, glucose and phosphate are added to the fermentation tank;

[0068] The size of the seed liquid biomass and the activity of the strain seriously affect the fermentation process and results. When transferring the seeds, it is preferred to select the mid-exponential growth stage and OD 600 =20-25, the transfer rate should be controlled at 1%-15% (v / v), preferably 8%-12%;

[0069] Step (6), fermentation:

[0070] The seed liquid in the fermentation tank is subjected to batch feeding fermentation, and the entire fermentation process is a four-stage, five-stage or six-stage fermentation. During the fermentation process, ammonia water and defoaming agent are fed, and glucose is supplemented through the feeding tank.

[0071] Fermentation conditions:

[0072] Temperature: 22°C-36°C, preferably 27-29°C;

[0073] pH: 3-7, preferably 5-6;

[0074] Dissolved oxygen: The dissolved oxygen in the TAPS metabolic stage is controlled at more than 30%, and the dissolved oxygen in the TriASa metabolic stage is controlled at 0% to 20%;

[0075] Ventilation volume: controlled at 1VVM-2VVM according to dissolved oxygen;

[0076] Stirring speed: controlled at 50rpm-1000rpm according to dissolved oxygen;

[0077] Feeding: When the central control detects that the glucose is lower than 20g / L, glucose is added in the form of feeding, and the carbon source concentration in the fermentation broth is maintained at 10g / L-20g / L;

[0078] Fermentation period: 120h.

[0079] Fermentation medium (w / v, %): glucose 5-8, yeast extract powder 0.2-0.8, potassium dihydrogen phosphate 0.5-1, ammonium sulfate 0.1-0.5, calcium chloride 0.2-0.5, magnesium sulfate heptahydrate 0.2-0.8, citric acid monohydrate 0.001-0.01, niacin 0.001-0.005, ferrous sulfate heptahydrate 0.002-0.005, thiamine hydrochloride 0.002-0.005, defoaming agent 0.1-0.6.

[0080] Based on the above method, the present invention provides the following partial embodiments:

[0081] Example 1

[0082] 1. Seed propagation

[0083] 1.1 First-level seed bottle

[0084] Take the glycerol tube containing the bacteria and inoculate it into the seed culture medium at a 1% (v / v) seed volume, and culture it in a box shaker at 28°C and 240 rpm for 72 h.

[0085] 1.2 Secondary Seed Bottle

[0086] Take the first-level seed bottle and inoculate it into the second-level seed bottle at a 1% (v / v) inoculation rate, and culture it in a box shaker at 28°C and 240 rpm for 48 hours.

[0087] 1.3 Transformation indicators

[0088] OD600=20~25; pH=3.0-3.6, typical microscopic examination pictures are as attached Figure 2 shown.

[0089] 2. Fermentation

[0090] Fermentation tank scale: 50L aerated stirred fermentation tank;

[0091] Seed amount: 10% (v / v);

[0092] Fermentation temperature: 28℃±0.2℃;

[0093] Fermentation pH: automatically controlled at 5.5±0.02 with ammonia water throughout the process;

[0094] Tank pressure: 0.05Mpa;

[0095] Dissolved oxygen: controlled at more than 30% in the TAPS metabolic stage and 10% to 15% in the TriASa metabolic stage;

[0096] Ventilation volume: controlled at 1VVM-2VVM depending on dissolved oxygen;

[0097] Stirring speed: controlled at 200rpm-800rpm depending on dissolved oxygen;

[0098] Feeding: When the central control detects that the glucose is lower than 20g / L, glucose is added in the form of feeding, and the glucose concentration in the fermentation broth is maintained at 10g / L-20g / L;

[0099] Fermentation cycle: Fermentation ends in 120 hours.

[0100] 4. Fermentation results

[0101] At the end of 120h of fermentation, the TAPS fermentation unit was 15.97g / L, the TriASa fermentation unit was 2.04g / L, TAPS:TriASa≈8:1, and the HPLC-UV spectrum was as follows: Figure 3 .

[0102] Example 2

[0103] The fermentation process (except for dissolved oxygen control) was the same as in Example 1, with dissolved oxygen being controlled at more than 30% in the TAPS metabolic stage and 5% to 10% in the TriASa metabolic stage.

[0104] Fermentation results

[0105] At the end of 120h of fermentation, the TAPS fermentation unit was 14.54g / L, the TriASa fermentation unit was 3.67g / L, TAPS:TriASa≈4:1, and the HPLC-UV spectrum was as follows: Figure 4 .

[0106] Example 3

[0107] The fermentation process (except for dissolved oxygen control) was as in Example 1, with dissolved oxygen: controlled at more than 30% in the TAPS metabolic stage and at 0% to 5% in the TriASa metabolic stage.

[0108] Fermentation results

[0109] At the end of 120h of fermentation, the TAPS fermentation unit was 10.78g / L, the TriASa fermentation unit was 5.24g / L, TAPS:TriASa≈2:1, and the HPLC-UV spectrum was as follows: Figure 5 .

[0110] The above shows and describes the basic principles, main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0111] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fermentation method for a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine, characterized in that: The following steps are involved: Step (1), glycerol tube storage: The fermented strain was added to a glycerol tube and stored at -80°C with a 60% (v / v) glycerol concentration; Step (2), primary shake flask culture: Take the glycerol tube for bacterial preservation and inoculate it into the seed medium of the first-level seed bottle at a seed volume of 1% (v / v), and culture it in a box shaker at 28°C and 240 rpm for 72 hours; Step (3), secondary shake flask culture: Take the first-level seed bottle and inoculate it into the second-level seed bottle at a 1% (v / v) inoculation rate, and culture it in a box shaker at 28°C and 240 rpm for 48 hours; Step (4), seed tank cultivation: Transfer the secondary seed bottle expansion culture solution into the seed tank and continue to culture; Step (5), transfer to fermentation tank: The seed liquid in the seed tank is transferred to the fermentation tank; when transferring, it is preferred to transfer to the mid-exponential growth stage and OD 600 =20-25 seed solution, the transfer amount should be controlled at 1%-15% (v / v); Step (6), fermentation: The seed liquid in the fermentation tank is subjected to batch feeding fermentation, and the entire fermentation process is a four-stage, five-stage or six-stage fermentation; The fermentation conditions are as follows: Temperature: 22℃-36℃; pH: 3-7; Dissolved oxygen: The dissolved oxygen in the TAPS metabolic stage is controlled at more than 30%, and the dissolved oxygen in the TriASa metabolic stage is controlled at 0% to 20%; Ventilation volume: controlled between 1VVM-2VVM according to dissolved oxygen; Stirring speed: controlled at 50rpm-1000rpm according to dissolved oxygen; Feeding: When the central control detects that the glucose is lower than 20g / L, glucose is added in the form of feeding, and the carbon source concentration in the fermentation broth is maintained at 10g / L-20g / L; Fermentation period: 120h.

2. The fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, characterized in that: The fermentation temperature is 27-29° C., and the fermentation pH is 5-6.

3. The fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, characterized in that: The components and contents (w / v, %) of the seed culture medium of the primary seed bottle and the secondary seed bottle are as follows: 3-5% glucose, 0.2-0.4% yeast extract powder, 0.1-0.5% potassium dihydrogen phosphate, 0.5-1% dipotassium hydrogen phosphate, 0.1-0.5% ammonium sulfate, 0.2-0.5% calcium chloride, and 0.1-0.6% defoaming agent.

4. The fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, characterized in that: The transfer amount in step (5) is 8%-12%.

5. The fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, characterized in that: The components and contents (w / v, %) of the fermentation medium are as follows: 5-8 glucose, 0.2-0.8 yeast extract powder, 0.5-1 potassium dihydrogen phosphate, 0.1-0.5 ammonium sulfate, 0.2-0.5 calcium chloride, 0.2-0.8 magnesium sulfate heptahydrate, 0.001-0.01 citric acid monohydrate, 0.001-0.005 nicotinic acid, 0.002-0.005 ferrous sulfate heptahydrate, 0.002-0.005 thiamine hydrochloride, and 0.1-0.6 defoaming agent.

6. The fermentation method of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, characterized in that: The steps for detecting the content of TAPS and TriASa in the fermentation broth in step (6) are as follows: S1, preprocessing: Take the fermentation broth and add 4 times volume of methanol, ultrasonically treat for 30 min, and centrifuge to obtain the supernatant; S2, Liquid Chromatography: The S1 supernatant was subjected to liquid chromatography detection under the following conditions: Detector: UV detector; Chromatographic column model: Sepax HP-C18 4.6×250mm 5μm; Mobile phase A: ultrapure water; Mobile phase B: acetonitrile; Flow rate: 1.0ml / min; Column temperature: 40°C; Detection wavelength: 195nm; Injection volume: 10uL.

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