Method and application for improving squalene production by a small molecule additive during the fermentation process of Schizochytrium

Abstract

The present invention belongs to the technical field of microbial fermentation, and discloses a method and application of a small molecule additive for increasing the squalene yield during the fermentation process of Schizochytrium, comprising the following steps: activation; transferring the obtained activated bacteria to a seed culture medium to obtain first-generation seeds; transferring to the seed culture medium for further cultivation to obtain second-generation seeds; transferring to the seed culture medium for further cultivation to obtain third-generation seeds; transferring to a fermentation culture medium containing terbinafine for fermentation culture to produce squalene. The present invention increases the squalene yield of Schizochytrium by adding small molecule substances to the Schizochytrium fermentation system. A small amount of terbinafine in a specific proportion can effectively inhibit the oxidation of squalene into sterol substances during the fermentation process of Schizochytrium, thereby reducing the consumption of squalene. Compared with the case without adding terbinafine, when adding an appropriate concentration of terbinafine, the squalene content is 2.63 times that of the control group, and the ratio of squalene to biomass is 2 times that of the control group.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and in particular to a method and application for improving the squalene yield during the fermentation of Schizochytrium sp by a small molecule additive. Background Art

[0002] Squalene is an unsaponifiable lipophilic terpene compound and can serve as a precursor for the biosynthesis of all steroids in animals and plants. As an acyclic triterpene, squalene is a type of functionally derived lipid. Squalene can be isolated from animals and plants such as shark liver oil, olives, and amaranth seeds. However, due to the cumbersome processes of extraction and purification, such production methods have great limitations. Therefore, using microorganisms as hosts to continuously, efficiently, and safely synthesize squalene is a current research hotspot. Currently, two pathways for producing squalene have been discovered. One is the mevalonate pathway (MVA), and the other is the non-mevalonate pathway (MEP). Which pathway an organism adopts to synthesize squalene mainly depends on the characteristics of the organism itself. For example, prokaryotes such as bacteria and cyanobacteria synthesize squalene through the MEP pathway, while eukaryotes such as yeast, higher fungi, plants, animals, and humans synthesize squalene through the MVA pathway.

[0003] Schizochytrium sp is a marine fungus of the family Thraustochytriaceae, rich in oil, with an oil content of up to more than 50%, and a simple fatty acid composition. Schizochytrium sp is considered to be a highly potential industrial fermentation strain for squalene due to its short fermentation cycle, fast growth, and complete MVA pathway.

[0004] So far, the method of improving squalene by adding exogenous compounds has been widely applied in the fermentation production process of Schizochytrium sp, mainly manifested in the following aspects: Chinese Patent CN117821530A enhances the activity of terpene synthase in the terpene synthesis pathway by adding Mn 2+ to the culture medium, thereby disturbing the metabolic network and increasing the carbon flow to the biosynthesis of squalene, and can effectively promote the fermentation production of squalene by Schizochytrium sp. Chinese Patent CN115820749A controls the content of squalene during the fermentation of Schizochytrium sp by adding an exogenous compound - sesamol. Compared with the control group without sesamol, the content of squalene in Schizochytrium sp in the experimental groups with different concentrations of sesamol added can be increased. Chinese Patent CN105803005A adds a certain concentration of an exogenous regulatory factor - alcohol to the fermentation medium before or during fermentation to increase the content of squalene in Schizochytrium sp, with obvious effects and can be applied to large-scale production.

[0005] Currently, there is no report on regulating the content of squalene during the fermentation of Schizochytrium sp by adding exogenous terbinafine. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies in the prior art and provide a method and application for improving the squalene yield during the fermentation of Schizochytrium sp. by using a small molecule additive.

[0007] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0008] An application of a small molecule additive in improving the squalene yield in Schizochytrium sp.

[0009] Further, the small molecule additive is terbinafine.

[0010] Further, the exogenous substance terbinafine is added to Schizochytrium sp. during the fermentation process;

[0011] Or, adding the exogenous inhibitor terbinafine to Schizochytrium sp. during the fermentation process can significantly improve the squalene yield.

[0012] A method for improving the squalene yield during the fermentation of Schizochytrium sp. by using a small molecule additive, comprising the following steps:

[0013] (1) Activation: Inoculating the Schizochytrium sp. strain stored at low temperature into a seed medium for activation;

[0014] (2) Transferring the obtained activated Schizochytrium sp. to a seed medium to obtain first-generation seeds;

[0015] (3) Transferring the first-generation seeds obtained in step (2) to a seed medium for further cultivation to obtain second-generation seeds;

[0016] (4) Transferring the second-generation seeds obtained in step (3) to a seed medium for further cultivation to obtain third-generation seeds;

[0017] (5) Transferring the third-generation seeds in step (4) to a fermentation medium containing terbinafine for fermentation cultivation, and obtaining squalene after the fermentation ends.

[0018] Further, the concentration of terbinafine added to the fermentation medium is 1 mg / L - 100 mg / L.

[0019] Further, the seed medium includes a carbon source, a nitrogen source, inorganic salt ions, and trace elements. The pH of the seed medium is 6.5 - 7.5, and the seed medium formula is: glucose 40 g / L, sodium sulfate 15 g / L, yeast powder 8 g / L, magnesium sulfate heptahydrate 3 g / L, dipotassium hydrogen phosphate 3 g / L, potassium chloride 0.5 g / L, glycerol 2 ml / L, 0.2% vitamin B 1 、0.2% vitamin B 6 、0.2% vitamin B 12, the solvent is water; the above percentages are mass percentages.

[0020] Furthermore, the fermentation medium comprises a carbon source, a nitrogen source, inorganic salt ions and terbinafine. The pH of the fermentation medium is 6.5 - 7.5, and the formula of the fermentation medium is: glucose 80 g / L, sodium sulfate 10 g / L, yeast powder 8 g / L, magnesium sulfate heptahydrate 3 g / L, dipotassium hydrogen phosphate 3 g / L, potassium chloride 0.5 g / L, calcium chloride 0.2 g / L, glycerol 2 ml / L, 0.2% vitamin B1, 0.2% vitamin B 6 , 0.2% vitamin B 12 , the solvent is water; the above percentages are mass percentages.

[0021] Furthermore, the conditions for the seed culture are all 30 °C and 180 rpm for 24 h;

[0022] The conditions for the fermentation culture are 30 °C and 180 rpm for 120 h.

[0023] Furthermore, the specific steps are as follows:

[0024] (1) Inoculate the Schizochytrium strain stored at low temperature into the seed medium for activation. After microscopic examination shows no contamination, transfer the obtained activated Schizochytrium to the seed medium. The inoculation amount of the activated bacteria is 1% - 2% (volume percentage, v / v), and culture at 28 - 30 °C and 170 - 180 rpm for 24 h to obtain the first-generation seeds;

[0025] (2) After microscopic examination shows no contamination of the first-generation seeds obtained in step (1), inoculate them into the seed medium for further culture. The inoculation amount of the first-generation seeds is 1% - 2% (volume percentage, v / v), and culture at 28 - 30 °C and 170 - 180 rpm for 24 h to obtain the second-generation seeds;

[0026] (3) After microscopic examination shows no contamination of the second-generation seeds obtained in step (2), inoculate them into the seed medium for further culture. The inoculation amount of the second-generation seeds is 1% - 2% (v / v), and culture at 28 - 30 °C and 170 - 180 rpm for 24 h to obtain the third-generation seeds;

[0027] (4) After microscopic examination shows no contamination of the third-generation seeds in step (3), inoculate the third-generation seeds into the fermentation medium. The inoculation amount is 8% - 10% (v / v, volume ratio), and culture at a temperature of 28 - 30 °C and a rotation speed of 170 - 180 rpm for 120 h. During this period, at 36 h of fermentation, add terbinafine with a concentration of 100 mg / L to the fermentation medium. After fermentation ends, squalene is obtained.

[0028] Further, the inoculation amount of the activated bacteria in step (1) is 1% (volume percentage, v / v), and it is cultured at 30°C and 180 rpm for 24 h to obtain the first-generation seeds;

[0029] In step (2), the inoculation amount of the first-generation seeds is 2% (volume percentage, v / v), and it is cultured at 30°C and 180 rpm for 24 h to obtain the second-generation seeds;

[0030] In step (3), the inoculation amount of the second-generation seeds is 2% (volume percentage, v / v), and it is cultured at 30°C and 180 rpm for 24 h to obtain the third-generation seeds;

[0031] In step (4), the third-generation seeds are inoculated into the fermentation medium, and the inoculation amount is 8% (v / v, volume ratio), and it is cultured at a temperature of 30°C and a rotation speed of 180 rpm for 120 h.

[0032] Further, the Schizochytrium is Schizochytrium sp. HX-308.

[0033] The advantages and positive effects obtained by the present invention are as follows:

[0034] 1. In the present invention, terbinafine, a small molecule substance, is added to the Schizochytrium fermentation system to increase the yield of squalene produced by Schizochytrium. A small amount of terbinafine in a specific proportion can effectively inhibit the oxidation of squalene into sterol substances during the fermentation process of Schizochytrium, thereby reducing the consumption of squalene. Compared with the case without adding terbinafine, when adding an appropriate concentration of terbinafine, the squalene content is 2.63 times that of the control group, and the ratio of squalene to biomass is 2 times that of the control group.

[0035] 2. The method of the present invention for increasing the squalene yield by exogenously adding metabolites during the fermentation production process specifically uses a small amount of terbinafine to be additionally added during the process of producing squalene by Schizochytrium fermentation to increase the squalene yield of Schizochytrium. Through simple fermentation regulation, the present invention can significantly increase the squalene content in Schizochytrium. At the same time, terbinafine is inexpensive and easy to obtain, making the production cost of this method relatively reduced and having great economic benefits.

[0036] 3. The method of the present invention enables the final squalene yield to reach 3.65 g / L through a simple, convenient, economical and efficient way of adding exogenous substances. However, the squalene yield of wild-type strains of the genus Schizochytrium is generally 1 - 2 g / L at present, indicating that the method provided by the present invention has extremely high application potential.

[0037] 4. The oil extracted after fermentation by the method of the present invention is detected to contain no terbinafine, which can ensure the purity and safety of the oil and avoid the problem of drug residues. These advantages make the Schizochytrium oil without terbinafine have a broader application prospect in the fields of food, health products, cosmetics, etc.

[0038] 5. The method of the present invention uses Schizochytrium as the production strain and removes the addition of trace elements in the culture medium to increase the yield of squalene. The present invention adds exogenous compounds to the Schizochytrium fermentation system to increase the yield of squalene produced by Schizochytrium. Terbinafine can effectively inhibit the oxidation of squalene into sterols during the fermentation process of Schizochytrium, thereby reducing the consumption of squalene. Compared with the case without adding terbinafine, when 100 mg / L of terbinafine is added at 36 h of fermentation, the squalene content is increased. Terbinafine can inhibit squalene epoxidase and increase the yield of squalene during the fermentation process of Schizochytrium. The present invention applies terbinafine to the regulation of terpene production by Schizochytrium, providing a new idea for increasing the content of squalene in Schizochytrium and having great potential in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the gas chromatography of squalene in the present invention;

[0040] Figure 2 It is a result diagram of the regulation of Schizochytrium production of squalene content by terbinafine fermentation in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The following further illustrates the present invention in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0042] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For the parts not specifically noted in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the filing date of the present invention application for implementation.

[0043] Application of a small molecule additive in increasing the yield of squalene in Schizochytrium.

[0044] Preferably, the small molecule additive is terbinafine, and an exogenous substance terbinafine is added during the fermentation process of Schizochytrium;

[0045] Alternatively, adding an exogenous inhibitor terbinafine during the fermentation process of Schizochytrium can significantly increase the yield of squalene.

[0046] A method for increasing the yield of squalene during the fermentation process of Schizochytrium by a small molecule additive, comprising the following steps:

[0047] (1) Activation: Inoculate the Schizochytrium sp. strain cryopreserved at low temperature into the seed medium for activation;

[0048] (2) Transfer the activated bacteria obtained to the seed medium to obtain first-generation seeds;

[0049] (3) Transfer the first-generation seeds obtained in step (2) to the seed medium and culture again to obtain second-generation seeds;

[0050] (4) Transfer the second-generation seeds obtained in step (3) to the seed medium and culture again to obtain third-generation seeds;

[0051] (5) Transfer the third-generation seeds in step (4) to the fermentation medium containing terbinafine for fermentation culture to produce squalene.

[0052] Preferably, the concentration of terbinafine added to the fermentation medium is 1 mg / L - 100 mg / L.

[0053] Preferably, the seed medium includes a carbon source, a nitrogen source, inorganic salt ions, and trace elements. The pH of the seed medium is 6.5 - 7.5, and the formula of the seed medium is: glucose 40 g / L, sodium sulfate 15 g / L, yeast powder 8 g / L, magnesium sulfate heptahydrate 3 g / L, dipotassium hydrogen phosphate 3 g / L, potassium chloride 0.5 g / L, glycerol 2 ml / L, 0.2% vitamin B 1 , 0.2% vitamin B 6 , 0.2% vitamin B 12 , and the solvent is water;

[0054] The above percentages are mass percentages.

[0055] Preferably, the fermentation medium includes a carbon source, a nitrogen source, inorganic salt ions, and terbinafine. The pH of the fermentation medium is 6.5 - 7.5, and the formula of the fermentation medium is: glucose 80 g / L, sodium sulfate 10 g / L, yeast powder 8 g / L, magnesium sulfate heptahydrate 3 g / L, dipotassium hydrogen phosphate 3 g / L, potassium chloride 0.5 g / L, calcium chloride 0.2 g / L, glycerol 2 ml / L, 0.2% vitamin B1, 0.2% vitamin B 6 , 0.2% vitamin B 12 , and the solvent is water;

[0056] The above percentages are mass percentages.

[0057] Preferably, the conditions for seed culture are all 30 °C, 180 rpm for 24 h;

[0058] The conditions for fermentation culture are 30 °C, 180 rpm for 120 h.

[0059] Preferably, the specific steps are as follows:

[0060] (1) Inoculate the Schizochytrium strain cryopreserved at low temperature into the seed medium for activation. After examining under the microscope and finding no contamination, transfer the obtained activated bacteria to the seed medium. The inoculation amount of the activated bacteria is 1%-2% (volume percentage, v / v), and culture at 28-30 °C and 170-180 rpm for 24 h to obtain the first-generation seeds;

[0061] (2) After examining the first-generation seeds obtained in step (1) under the microscope and finding no contamination, inoculate them into the seed medium for further culture. The inoculation amount of the first-generation seeds is 1%-2% (volume percentage, v / v), and culture at 28-30 °C and 170-180 rpm for 24 h to obtain the second-generation seeds;

[0062] (3) After examining the second-generation seeds obtained in step (2) under the microscope and finding no contamination, inoculate them into the seed medium for further culture. The inoculation amount of the second-generation seeds is 1%-2% (v / v), and culture at 28-30 °C and 170-180 rpm for 24 h to obtain the third-generation seeds;

[0063] (4) After examining the third-generation seeds in step (3) under the microscope and finding no contamination, inoculate the third-generation seeds into the fermentation medium. The inoculation amount is 8%-10% (v / v, volume ratio), and culture at a temperature of 28-30 °C and a rotation speed of 170-180 rpm for 120 h. During this period, at 36 h of fermentation, add terbinafine with a concentration of 100 mg / L to the fermentation medium. After the fermentation is completed, squalene is obtained.

[0064] Preferably, in step (1), the inoculation amount of the activated bacteria is 1% (volume percentage, v / v), and culture at 30 °C and 180 rpm for 24 h to obtain the first-generation seeds;

[0065] In step (2), the inoculation amount of the first-generation seeds is 2% (volume percentage, v / v), and culture at 30 °C and 180 rpm for 24 h to obtain the second-generation seeds;

[0066] In step (3), the inoculation amount of the second-generation seeds is 2% (volume percentage, v / v), and culture at 30 °C and 180 rpm for 24 h to obtain the third-generation seeds;

[0067] In step (4), inoculate the third-generation seeds into the fermentation medium. The inoculation amount is 8% (v / v, volume ratio), and culture at a temperature of 30 °C and a rotation speed of 180 rpm for 120 h.

[0068] Preferably, the Schizochytrium is Schizochytrium sp. HX-308.

[0069] Specifically, the related preparation and detection are as follows:

[0070] Example 1

[0071] The seed culture medium includes a carbon source, a nitrogen source, inorganic salt ions and trace elements. The pH of the seed culture medium is 6.5 - 7.5, and the formula of the seed culture medium is: glucose 40 g / L, sodium sulfate 15 g / L, yeast powder 8 g / L, magnesium sulfate heptahydrate 3 g / L, dipotassium hydrogen phosphate 3 g / L, potassium chloride 0.5 g / L, glycerol 2 ml / L, 0.2% vitamin B 1 、0.2% vitamin B 6 、0.2% vitamin B 12 , and the solvent is water; the above percentages are mass percentages.

[0072] The fermentation culture medium includes a carbon source, a nitrogen source, inorganic salt ions and terbinafine. The pH of the fermentation culture medium is 6.5 - 7.5, and the formula of the fermentation culture medium is: glucose 80 g / L, sodium sulfate 10 g / L, yeast powder 8 g / L, magnesium sulfate heptahydrate 3 g / L, dipotassium hydrogen phosphate 3 g / L, potassium chloride 0.5 g / L, calcium chloride 0.2 g / L, glycerol 2 ml / L, 0.2% vitamin B1, 0.2% vitamin B 6 、0.2% vitamin B 12 , and the solvent is water; the above percentages are mass percentages.

[0073] The added concentration of terbinafine is: 1 mg / L, 10 mg / L, 100 mg / L. During the experiment, the components and addition amounts of the culture medium remain unchanged. Only the above different concentrations of terbinafine need to be added to different fermentation culture media, and a control experimental group, namely the group with an added amount of exogenous terbinafine of 0 mg / L, should be set up.

[0074] The strain used in the present invention is the wild-type Schizochytrium sp. HX-308, which is stored at -80 °C in a glycerol tube.

[0075] A method for the content of squalene during the fermentation of Schizochytrium sp. by adding a small molecule additive, the specific steps are as follows:

[0076] 1. Inoculate Schizochytrium sp. HX-308 stored in a glycerol tube into 40 mL of the seed culture medium, with an inoculation amount of 1% (v / v, volume ratio), and culture at a temperature of 30 °C and a rotation speed of 180 rpm for 24 h to obtain first-generation seeds;

[0077] 2. Inoculate the above first-generation seeds into 40 mL of the seed culture medium, with an inoculation amount of 2% (v / v, volume ratio), and culture at a temperature of 30 °C and a rotation speed of 180 rpm for 24 h to obtain second-generation seeds;

[0078] 3. Inoculate the above-mentioned second-generation seeds into 40 mL of seed medium at an inoculation amount of 2% (v / v, volume ratio), and culture at a temperature of 30 °C and a rotation speed of 180 rpm for 24 h to obtain third-generation seeds;

[0079] 4. Inoculate the third-generation seeds into 100 mL of fermentation medium at an inoculation amount of 8% (v / v, volume ratio), and culture at a temperature of 30 °C and a rotation speed of 180 rpm for 120 h. Squalene is obtained at the end of fermentation. During this period, at 0 h, 36 h, and 72 h of fermentation, terbinafine with different concentrations are added to the fermentation medium, and the concentrations are 1 mg / L, 10 mg / L, and 100 mg / L in sequence. Judge the effects of adding terbinafine with different concentrations on biomass, oil yield, and squalene yield respectively.

[0080] 5. After 120 h of fermentation, the extraction of substances such as oil is carried out according to the following steps:

[0081] (1) Accurately pipette a quantitative fermentation broth into a stoppered flask, adjust the pH to 10 - 11 with 2 mol / L sodium hydroxide aqueous solution, mix well, and then add 0.2% of the weight of the fermentation broth of cell wall-lysing enzyme, and enzymolyze at 50 °C for 3 h until the cells are completely digested;

[0082] (2) Add anhydrous ethanol in a ratio of 1:1 by volume to the fermentation broth to precipitate the protein;

[0083] (3) Then add n-hexane in a ratio of 1:1 by volume to the fermentation broth, shake well, let it stand for stratification, take the upper organic phase and place it in a pre-weighed and dried round-bottom conical flask, and wash it about 3 times with n-hexane until the upper organic phase is colorless and transparent;

[0084] (4) Perform rotary evaporation extraction on the obtained upper liquid. Weigh and record the rotary evaporation flask before rotary evaporation (g1), and perform rotary evaporation at 40 °C until no more condensed water droplets drip;

[0085] (5) After rotary evaporation, place it in an oven to dry the excess water to a constant weight, weigh and record (g2), then the oil weight is g2 - g1;

[0086] 6. After extracting the oil, the methylation of the oil is carried out according to the following steps (for preparation of gas chromatography):

[0087] ① Add 0.6 mL of 1 M potassium hydroxide methanol solution to a 2 mL centrifuge tube, and add 20 μL of the obtained oil to it and mix well;

[0088] ② Under room temperature conditions, shake at 1500 rpm for 7 h. Add 50 μL of concentrated sulfuric acid to terminate the reaction;

[0089] ③ Add 1 mL of n-hexane for extraction and shake at 1500 rpm for 1 h;

[0090] ④ Then centrifuge at 12,000 rpm for 5 min, take the upper n - hexane phase, filter it through an organic filter membrane to remove impurities, add it to a gas - phase vial, and then it can be detected by gas chromatography. Gas chromatography test method: Gas analysis conditions: Use a DB - 23 (60 m * 0.25 mm * 0.25 μm) chromatographic column; select FID as the detector; use high - purity nitrogen as the carrier gas; the split ratio is 30 / 1; the inlet temperature is set at 250 °C; the detector temperature is set at 280 °C; the injection volume is 1 μL; temperature - rising program: At the beginning of the program, the column temperature of the gas chromatographic column is set at 100 °C. First, the program rises to 196 °C at a rate of 25 °C / min. Subsequently, the program rises to 220 °C at a rate of 2 °C / min and holds at 220 °C for 12 min. The flow rate of the gas chromatographic column is set at 3.0 mL / min; the tail - blow flow rate is set at 30 mL / min; the hydrogen flow rate is 40 mL / min and the air flow rate is 400 mL / min. The gas chromatogram of the prepared squalene is as Figure 1 shown.

[0091] 7. Determination of biomass: Take 1 mL of the fermentation broth fermented for 120 h, centrifuge at 4000 rpm for 5 min, discard the supernatant, add 1 mL of 2% (mass fraction) NaCl solution for washing once, and then wash once with 1 mL of 1 mol / L sorbitol solution. Dry it at 110 °C to a constant weight, and finally weigh and calculate.

[0092] 8. Prepare a squalene standard sample, obtain the standard curve equation, and then calculate the squalene yield according to the analysis results of the gas chromatogram.

[0093] 9. When the fermentation time is 120 h, after the fermentation ends, measure the biomass, oil yield, and squalene content after adding terbinafine at different concentrations. The measurement methods for oil extraction and purification are as described above. Detect the biomass, oil yield, and squalene content. The data is shown in Table 1. Draw a graph based on the squalene content results in Table 1 Figure 2 .

[0094]

[0095] As shown in Table 1, after adding different concentrations of terbinafine, the biomass, oil content, and squalene content of Schizochytrium sp. all increased. When 100 mg / L of terbinafine was added at 36 h of fermentation, the highest biomass reached 19.5 g / L, and at this time, the oil content of Schizochytrium sp. produced by the strain was up to 40.9 g / L, and the squalene content reached 3.36 g / L. Compared with the control group, that is, the group with an exogenous terbinafine addition amount of 0 mg / L, the biomass increased by 23%; the oil content increased by 17%; the squalene content increased by 140%. Through the simple, convenient, economical, and efficient method of adding exogenous substances, the present invention can effectively increase the squalene yield.

[0096] At the same time, it can also be seen from Table 1 that there is a synergistic effect between 36 h of fermentation and 100 mg / L of terbinafine in the method of the present invention, which can synergistically increase the biomass, oil content, and squalene content obtained.

[0097] Example 2

[0098] Fed-batch fermentation culture of Schizochytrium sp. strain HX-308 in a 5 L fermenter:

[0099] The preserved seed liquid of Schizochytrium sp. HX-308 (the third-generation seed liquid in Example 1) was inoculated into a 5 L fermenter containing 3 L of sterilized fermentation medium at an inoculation amount of 8% (v / v, volume ratio). When the fermentation reached 36 h, 100 mg / L of terbinafine was added to the medium (other preparation methods, formulas, etc. were the same as in Example 1). The fermentation was carried out at 30 °C and a stirring speed of 180 rpm for 120 h, and the fermentation broth was extracted to measure the content of each index. The results are shown in Table 2.

[0100]

[0101] In the final 5 L fermenter, when 100 mg / L of terbinafine was added at 36 h of fermentation, compared with the original strain, the biomass, oil content, and squalene content of Schizochytrium sp. were all significantly increased. The biomass increased by 16.2%, the oil content increased by 35%, and the squalene content increased by 116%.

[0102] At present, there are studies on applying terbinafine to improve the squalene production of Rhodosporidium toruloides. According to the results, the highest squalene production can reach 863.06 mg / L, which is far lower than the squalene content of 3.9 g / L produced by Schizochytrium sp. in the present invention. Moreover, it is detected that the extracted oil after fermentation by the method of the present invention does not contain terbinafine, which can ensure the purity and safety of the oil, avoid the problem of drug residues, guarantee the biological activity and nutritional value of the oil, and meet the regulatory requirements and market demands. These advantages enable the Schizochytrium sp. oil without terbinafine to have a broader application prospect in the fields of food, health products, cosmetics, etc.

[0103] Patent CN118240858A mainly uses genetic engineering means to transform Rhodosporidium toruloides to obtain a recombinant bacterium to increase the squalene production. The present invention improves the squalene production of Schizochytrium sp. through fermentation regulation.

[0104] Patent CN116970656A improves the squalene production of Rhodosporidium toruloides through fermentation regulation. It is different from the strain used in the present invention. The present invention first applies terbinafine to Schizochytrium sp. to increase the squalene production, providing new ideas and methods for the production of squalene by Schizochytrium sp.

[0105] Patent CN1927181A mainly introduces a drug and its preparation method. The present invention mainly focuses on improving the squalene production through microbial fermentation regulation. The focuses are inconsistent.

[0106] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A method for increasing the yield of squalene in a Schizochytrium fermentation process by using a small molecule additive, characterized in that: The following steps are involved: 1) The low-temperature frozen Schizochytrium strain was inoculated into the seed culture medium for activation. After microscopic examination showed no bacterial contamination, the activated Schizochytrium was transferred into the seed culture medium with an inoculation amount of 1% by volume, and cultured at 30°C and 180 rpm for 24 h to obtain the first generation of seeds; 2) The first generation seeds obtained in step 1) are inoculated in a seed culture medium for further cultivation after being examined under a microscope to be free of bacteria. The inoculation amount of the first generation seeds is 2% by volume, and the seeds are cultured at 30° C. and 180 rpm for 24 h to obtain the second generation seeds; 3) The second generation seeds obtained in step 2) are inoculated in a seed culture medium for further cultivation after being examined under a microscope to be free of bacteria. The inoculation amount of the second generation seeds is 2% by volume, and the seeds are cultured at 30° C. and 180 rpm for 24 h to obtain the third generation seeds; 4) After the third-generation seeds of step 3) are examined under a microscope and found to be free of bacteria, the third-generation seeds are inoculated into a fermentation medium at an inoculation amount of 8% by volume, and cultured at a temperature of 30° C. and a rotation speed of 180 rpm for 120 hours. During this period, at 36 hours of fermentation, 100 mg / L of terbinafine is added to the fermentation medium, and squalene is obtained after the fermentation is completed; The Schizochytrium Schizochytrium sp. HX-308; The seed culture medium comprises a carbon source, a nitrogen source, inorganic salt ions and trace elements, the pH of the seed culture medium is 6.5-7.5, and the seed culture medium formula is: 40 g / L glucose, 15 g / L sodium sulfate, 8 g / L yeast powder, 3 g / L magnesium sulfate heptahydrate, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium chloride, 2 ml / L glycerol, 0.2% vitamin B1, 0.2% vitamin B6, 0.2% vitamin B 12 , the solvent is water; The above percentages are mass percentages; The fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt ion, trace elements and terbinafine, the pH of the fermentation medium is 6.5-7.5, and the fermentation medium formula is: 80 g / L glucose, 10 g / L sodium sulfate, 8 g / L yeast powder, 3 g / L magnesium sulfate heptahydrate, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium chloride, 0.2 g / L calcium chloride, 2 ml / L glycerol, 0.2% vitamin B1, 0.2% vitamin B6, 0.2% vitamin B 12 , the solvent is water; The above percentages are mass percentages.

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