CN117947119A - Fermentation method of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine mixture - Google Patents

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a fermentation method of a tetraacetyl phytosphingosine and triacetyl dihydrosphingosine mixture, which comprises the following steps: step (1), the glycerol pipe is stored; step (2), primary shake flask culture: inoculating seed-preserving glycerol pipe to seed culture medium of first-stage seed bottle at 1% (v/v), and culturing at 28deg.C in box-type shaking table at 240rpm for 72 hr; step (3), secondary shake flask culture: taking a first-level seed bottle, inoculating the first-level seed bottle with an inoculum size of 1% (v/v) to a second-level seed bottle, and culturing for 48 hours at 28 ℃ and 240rpm in a box-type shaking table; step (4), culturing in a seed tank; step (5), transferring the seed fermentation tank; and (6) fermenting. The invention adopts Wick ham sevoflurane to ferment and produce TAPS and TriASa simultaneously under the same fermentation system, and can obtain the mixture of TAPS and TriASa with different proportions by regulating and controlling dissolved oxygen, thereby greatly improving the production efficiency and saving the production cost.

Description

Fermentation method of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine mixture

Technical Field

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

Background

Sphingolipids are a group of lipid substances derived from sphingosine base, and are a generic term for ceramides or ceramide derivatives having sphingosine, phytosphingosine and dihydrosphingosine as basic skeletons in human body, and in human skin, ceramides, cholesterol sulfate and free fatty acids constitute a specific lamellar quasicrystal structure as main components of the stratum corneum, forming a permeation barrier for blocking moisture and protecting skin from water diversion and microbial attack. Therefore, the ceramide has high application potential in various application fields such as antibacterial drugs, active pharmaceutical ingredients, cosmetics, skin care products and the like.

There are three main methods for obtaining ceramide: 1. natural extraction, the ceramide (such as konjak) extracted from plants is affected by weather, season and plant diseases and insect pests, and the yield is low and unstable; 2. chemical synthesis, which is a way for chemically synthesizing ceramide at present, but has two stereogenic centers and three functional groups, so that the chemical synthesis of ceramide is difficult and high in cost; 3. microbial fermentation, mainly comprising two forms, namely directly fermenting to obtain ceramide and preparing a ceramide precursor substance by fermenting first and then synthesizing chemically to obtain ceramide; at present, the direct fermentation to obtain ceramide has low yield, so that the ceramide is obtained by fermenting ceramide precursor substances and then chemically synthesizing the ceramide is the first choice.

Tetraacetylphytosphingosine (TETRAACETYL PHYTOSPHINGOSINE, TAPS) and triacetyldihydrosphingosine (TRIACETYL SPHINGANINE, TRIASA) are important precursors for ceramides, and are natural metabolites of Welch Hance (Wickerhamomyces ciferrii); in addition, TAPS and TriASa are excellent natural skin care raw materials, and have the effects of moisturizing, resisting oxidation, whitening, removing freckles, promoting skin barrier self-repair and the like; and acetylated phytosphingosine and dihydrosphingosine have excellent water solubility, can be mixed with various cosmetic raw materials, and have been widely used.

At present, related papers and patents of microbial fermentation of tetraacetyl phytosphingosine are not few, but reports of production of triacetyl dihydrosphingosine by fermentation are rare, and production of triacetyl dihydrosphingosine and tetraacetyl phytosphingosine are extremely rare, so that most of triacetyl dihydrosphingosine and tetraacetyl phytosphingosine are fermented and cultured independently, the production efficiency is low, the cost is high, and the production is influenced.

Disclosure of Invention

The invention aims to provide a fermentation method of a tetraacetyl phytosphingosine and triacetyl dihydrosphingosine mixture, which aims to solve the problems of low production efficiency and high cost caused by few methods for simultaneously producing triacetyl dihydrosphingosine and tetraacetyl phytosphingosine by fermentation in the prior art.

In order to achieve the above purpose, the present invention provides the following technical solutions: a process for fermenting a tetraacetylphytosphingosine and triacetyldihydrosphingosine mixture comprising the steps of:

Step (1), glycerol tube preservation:

adding the fermentation strain into glycerol tube, and storing at-80deg.C with 60% (v/v) glycerol concentration;

Step (2), primary shake flask culture:

Inoculating seed-preserving glycerol pipe to seed culture medium of first-stage seed bottle at 1% (v/v), and culturing at 28deg.C in box-type shaking table at 240rpm for 72 hr;

Step (3), secondary shake flask culture:

Taking a first-level seed bottle, inoculating the first-level seed bottle with an inoculum size of 1% (v/v) to a second-level seed bottle, and culturing for 48 hours at 28 ℃ and 240rpm in a box-type shaking table;

Step (4), seed tank culture:

transferring the secondary seed bottle expanding culture solution into a seed tank, and continuing culturing;

step (5), transferring the seeds into a fermentation tank:

Transferring the seed liquid in the seed tank to a fermentation tank; during seed transfer, seed liquid with an exponential growth medium phase and an OD ₆₀₀ =20-25 is preferred, and the seed transfer amount is controlled to be 1% -15% (v/v);

step (6), fermentation:

The seed liquid in the fermentation tank is fed-batch fermented, and the whole fermentation process can be four-stage, five-stage or six-stage fermentation.

Preferably, the fermentation conditions are as follows:

temperature: 22-36 ℃;

pH：3-7；

Dissolved oxygen: the dissolved oxygen in the TAPS metabolic stage is controlled to be more than 30 percent, and the dissolved oxygen in the TriASa metabolic stage is controlled to be 0 to 20 percent;

ventilation volume: controlling the temperature between 1VVM and 2VVM according to dissolved oxygen;

Stirring rotation speed: controlling the speed at 50rpm-1000rpm according to dissolved oxygen;

And (3) material supplementing: when the central control detects that the glucose is lower than 20g/L, glucose is supplemented in a fed-batch mode, and the concentration of a carbon source in the fermentation liquor is maintained at 10g/L-20g/L;

Fermentation period: 120h.

Preferably, the fermentation temperature is preferably 27-29 ℃, and the fermentation pH is preferably 5-6.

Preferably, 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 to 5 portions of glucose, 0.2 to 0.4 portion of yeast extract powder, 0.1 to 0.5 portion of monopotassium phosphate, 0.5 to 1 portion of dipotassium phosphate, 0.1 to 0.5 portion of ammonium sulfate, 0.2 to 0.5 portion of calcium chloride and 0.1 to 0.6 portion of defoaming agent.

Preferably, the amount of the transferred seeds in the step (5) is preferably 8% -12%.

Preferably, the components and contents (w/v,%) of the fermentation medium are as follows: glucose 5-8, yeast extract powder 0.2-0.8, monopotassium 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, nicotinic acid 0.001-0.005, ferrous sulfate heptahydrate 0.002-0.005, thiamine hydrochloride 0.002-0.005, and defoamer 0.1-0.6.

Preferably, the step (6) of detecting the content of TAPS and TriASa in the fermentation broth comprises the following steps:

S1, pretreatment:

Adding 4 times of methanol into the fermentation broth, performing ultrasonic treatment for 30min, and centrifuging to obtain supernatant;

S2, liquid chromatography:

And (3) carrying out liquid chromatography detection on the S1 supernatant, wherein the detection conditions are as follows:

A detector: a UV detector;

chromatographic column model: sepax HP-C184.6X105 mm 5 μm;

Mobile phase a: ultrapure water;

Mobile phase B: acetonitrile;

Flow rate: 1.0ml/min;

Column temperature: 40 ℃;

Detection wavelength: 195nm;

the sample injection amount was 10uL.

The invention has at least the following beneficial effects:

According to the fermentation method of the tetraacetyl phytosphingosine and triacetyl dihydrosphingosine mixture, disclosed by the invention, wick ham sevoflurane is adopted to simultaneously ferment and produce TAPS and TriASa under the same fermentation system, and the mixture of the TAPS and the TriASa in different proportions can be obtained by regulating and controlling dissolved oxygen, so that the production efficiency is greatly improved, and the production cost is saved.

Drawings

FIG. 1 is a fermentation process flow of the present invention;

FIG. 2 is a representative microscopic view of the present invention;

FIG. 3 is a TAPS: triASa.apprxeq.8:1 HPLC-UV spectrum;

FIG. 4 is a TAPS: triASa ≡4:1HPLC-UV spectrum;

FIG. 5 is a TAPS: triASa.apprxeq.2:1 HPLC-UV spectrum.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The tetraacetyl phytosphingosine and triacetyl dihydrosphingosine are obtained by deep liquid fermentation for 120 hours in an aeration stirring type fermentation tank with self-control pH, temperature and feed supplement by adopting Wick ham schiff, and the fermentation process is divided into two stages, namely a TAPS metabolic stage and a TriASa metabolic stage.

As shown in figure 1, the fermentation strain is inoculated into a seed bottle by a glycerol preservation tube for expansion culture in a shaking period, then the expansion culture solution of the seed bottle is transferred into a seed tank, and finally the seed solution in the seed tank is transferred into a fermentation tank for fed-batch fermentation. The whole fermentation process can be four-stage, five-stage or six-stage fermentation, the specific stage number depends on the fermentation scale, but five-stage fermentation is preferred, and four-stage fermentation is more preferred.

The method comprises the following steps:

A process for fermenting a tetraacetylphytosphingosine and triacetyldihydrosphingosine mixture comprising the steps of:

Step (1), glycerol tube preservation:

Adding the fermentation strain (Wick ham Schiff yeast) into glycerol tube, and preserving at-80deg.C at 60% (v/v) glycerol concentration;

Step (2), primary shake flask culture:

Inoculating seed-preserving glycerol pipe to seed culture medium of first-stage seed bottle at 1% (v/v), and culturing at 28deg.C in box-type shaking table at 240rpm for 72 hr;

Step (3), secondary shake flask culture:

Taking a first-level seed bottle, inoculating the first-level seed bottle with an inoculum size of 1% (v/v) to a second-level seed bottle, and culturing for 48 hours at 28 ℃ and 240rpm in a box-type shaking table;

Step (4), seed tank culture:

transferring the secondary seed bottle expanding culture solution into a seed tank, and continuing culturing; adding seed tanks for sub-desugaring in the seed tank culture process;

Seed culture medium (w/v,%) of primary seed bottle and secondary seed bottle: 3 to 5 portions of glucose, 0.2 to 0.4 portion of yeast extract powder, 0.1 to 0.5 portion of monopotassium phosphate, 0.5 to 1 portion of dipotassium phosphate, 0.1 to 0.5 portion of ammonium sulfate, 0.2 to 0.5 portion of calcium chloride and 0.1 to 0.6 portion of defoaming agent;

step (5), transferring the seeds into a fermentation tank:

Transferring the seed liquid in the seed tank to a fermentation tank; during the process of transferring the seed fermentation tank, glucose digestion and phosphate digestion are added into the fermentation tank;

The biomass size of the seed liquid and the activity of the strain seriously influence the fermentation process and the fermentation result, the exponential growth medium period and the OD ₆₀₀ = 20-25 are preferable during the transfer, and the transfer amount is controlled to be 1% -15% (v/v), preferably 8% -12%;

step (6), fermentation:

the seed liquid in the fermentation tank is fed-batch fermented, and the whole fermentation process can be four-stage, five-stage or six-stage fermentation. And ammonia water and a defoaming agent are fed in the fermentation process, and glucose is fed through a feeding tank.

Fermentation conditions:

Temperature: 22-36 ℃, preferably 27-29 ℃;

pH:3-7, preferably 5-6;

Dissolved oxygen: the dissolved oxygen in the TAPS metabolic stage is controlled to be more than 30 percent, and the dissolved oxygen in the TriASa metabolic stage is controlled to be 0 to 20 percent;

ventilation volume: controlling the temperature to be 1VVM-2VVM according to dissolved oxygen;

Stirring rotation speed: controlling the speed at 50rpm-1000rpm according to dissolved oxygen;

And (3) material supplementing: when the central control detects that the glucose is lower than 20g/L, glucose is supplemented in a fed-batch mode, and the concentration of a carbon source in the fermentation liquor is maintained at 10g/L-20g/L;

Fermentation period: 120h.

Fermentation medium (w/v,%): glucose 5-8, yeast extract powder 0.2-0.8, monopotassium 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, nicotinic acid 0.001-0.005, ferrous sulfate heptahydrate 0.002-0.005, thiamine hydrochloride 0.002-0.005, and defoamer 0.1-0.6.

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

Example 1

1. Seed expansion culture

1.1 First-level seed bottle

Inoculating seed culture medium with seed oil pipe at 1% (v/v), and culturing at 28deg.C and 240rpm in box type shaking table for 72 hr.

1.2 Second grade seed bottle

The primary seed flask was inoculated to the secondary seed flask at an inoculum size of 1% (v/v), and cultured in a box shaker at 28℃and 240rpm for 48 hours.

1.3 Seed conversion index

Od600=20 to 25; ph=3.0-3.6, and a typical microscopic image is shown in fig. 2.

2. Fermentation culture

Fermenter scale: a 50L aeration-agitation type fermenter;

The amount is as follows: 10% (v/v);

fermentation temperature: 28 ℃ +/-0.2 ℃;

Fermentation pH: ammonia water is used for automatically controlling the temperature to be 5.5+/-0.02 in the whole process;

tank pressure: 0.05Mpa;

dissolved oxygen: the TAPS metabolic stage is controlled to be more than 30 percent, and the TriASa metabolic stage is controlled to be 10 to 15 percent;

ventilation volume: controlling the oxygen dissolution to be 1VVM-2VVM;

Stirring rotation speed: controlling the dissolved oxygen at 200rpm-800rpm;

And (3) material supplementing: when the central control detects that the glucose is lower than 20g/L, glucose is supplemented in a fed-batch mode, and the glucose concentration in the fermentation liquor is maintained at 10g/L-20g/L;

Fermentation period: and (5) ending the fermentation after 120 hours.

4. Fermentation results

At the end of 120h fermentation, TAPS fermentation unit 15.97g/L, triASa fermentation unit 2.04g/L, TAPS: triASa ≡8:1, HPLC-UV spectrum as shown in FIG. 3.

Example 2

Fermentation process (dissolved oxygen removal control) as in example 1, dissolved oxygen: the TAPS metabolic stage is controlled to be more than 30 percent, and the TriASa metabolic stage is controlled to be 5 to 10 percent.

Fermentation results

At the end of 120h fermentation, TAPS fermentation unit 14.54g/L, triASa fermentation unit 3.67g/L, TAPS: triASa. Apprxeq.4:1, HPLC-UV spectrum as shown in FIG. 4.

Example 3

Fermentation process (dissolved oxygen removal control) as in example 1, dissolved oxygen: the TAPS metabolic stage is controlled to be more than 30%, and the TriASa metabolic stage is controlled to be 0% -5%.

Fermentation results

At the end of 120h fermentation, TAPS fermentation unit 10.78g/L, triASa fermentation unit 5.24g/L, TAPS: triASa. Apprxeq.2:1, HPLC-UV spectrum as shown in FIG. 5.

While the fundamental principles, principal features, and advantages of the present invention have been shown and described, it will be apparent to those skilled in the art that the present invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (7)

1. A process for fermenting a tetraacetyl phytosphingosine and triacetyl dihydrosphingosine mixture, comprising the steps of:

Step (1), glycerol tube preservation:

adding the fermentation strain into glycerol tube, and storing at-80deg.C with 60% (v/v) glycerol concentration;

Step (2), primary shake flask culture:

Inoculating seed-preserving glycerol pipe to seed culture medium of first-stage seed bottle at 1% (v/v), and culturing at 28deg.C in box-type shaking table at 240rpm for 72 hr;

Step (3), secondary shake flask culture:

Taking a first-level seed bottle, inoculating the first-level seed bottle with an inoculum size of 1% (v/v) to a second-level seed bottle, and culturing for 48 hours at 28 ℃ and 240rpm in a box-type shaking table;

Step (4), seed tank culture:

transferring the secondary seed bottle expanding culture solution into a seed tank, and continuing culturing;

step (5), transferring the seeds into a fermentation tank:

Transferring the seed liquid in the seed tank to a fermentation tank; during seed transfer, seed liquid with an exponential growth medium phase and an OD ₆₀₀ =20-25 is preferred, and the seed transfer amount is controlled to be 1% -15% (v/v);

step (6), fermentation:

The seed liquid in the fermentation tank is fed-batch fermented, and the whole fermentation process can be four-stage, five-stage or six-stage fermentation.

2. The process for the fermentation of a mixture of tetraacetyl phytosphingosine and triacetdihydrosphingosine according to claim 1, characterized in that the fermentation conditions are as follows:

temperature: 22-36 ℃;

pH：3-7；

Dissolved oxygen: the dissolved oxygen in the TAPS metabolic stage is controlled to be more than 30 percent, and the dissolved oxygen in the TriASa metabolic stage is controlled to be 0 to 20 percent;

ventilation volume: controlling the temperature between 1VVM and 2VVM according to dissolved oxygen;

Stirring rotation speed: controlling the speed at 50rpm-1000rpm according to dissolved oxygen;

And (3) material supplementing: when the central control detects that the glucose is lower than 20g/L, glucose is supplemented in a fed-batch mode, and the concentration of a carbon source in the fermentation liquor is maintained at 10g/L-20g/L;

Fermentation period: 120h.

3. A process for the fermentation of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 2, characterized in that the fermentation temperature is preferably between 27 and 29 ℃, and the fermentation pH is preferably between 5 and 6.

4. The method for fermenting a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, wherein 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 to 5 portions of glucose, 0.2 to 0.4 portion of yeast extract powder, 0.1 to 0.5 portion of monopotassium phosphate, 0.5 to 1 portion of dipotassium phosphate, 0.1 to 0.5 portion of ammonium sulfate, 0.2 to 0.5 portion of calcium chloride and 0.1 to 0.6 portion of defoaming agent.

5. The process for the fermentation of a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, wherein the amount of trans-seed in step (5) is preferably 8% to 12%.

6. The method for fermenting a tetraacetyl phytosphingosine and triacetyl dihydrosphingosine mixture according to claim 1, wherein the components and contents (w/v,%) of the fermentation medium are as follows: glucose 5-8, yeast extract powder 0.2-0.8, monopotassium 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, nicotinic acid 0.001-0.005, ferrous sulfate heptahydrate 0.002-0.005, thiamine hydrochloride 0.002-0.005, and defoamer 0.1-0.6.

7. The method for fermenting a mixture of tetraacetyl phytosphingosine and triacetyl dihydrosphingosine according to claim 1, wherein the step (6) of detecting the TAPS and TriASa contents in the fermentation broth comprises the steps of:

S1, pretreatment:

Adding 4 times of methanol into the fermentation broth, performing ultrasonic treatment for 30min, and centrifuging to obtain supernatant;

S2, liquid chromatography:

And (3) carrying out liquid chromatography detection on the S1 supernatant, wherein the detection conditions are as follows:

A detector: a UV detector;

chromatographic column model: sepax HP-C184.6X105 mm 5 μm;

Mobile phase a: ultrapure water;

Mobile phase B: acetonitrile;

Flow rate: 1.0ml/min;

Column temperature: 40 ℃;

Detection wavelength: 195nm;

the sample injection amount was 10uL.