Method for increasing squalene yield in schizochytrium limacinum fermentation process by using small-molecule additive and application of squalene yield in schizochytrium limacinum fermentation process

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses a method for increasing the squalene yield in the schizochytrium limacinum fermentation process through a small molecule additive and application of the squalene yield in the schizochytrium limacinum fermentation process. Transferring the obtained activated bacteria to a seed culture medium to obtain a first generation of seeds; transferring into a seed culture medium and culturing again to obtain second-generation seeds; transferring into a seed culture medium, and culturing again to obtain third-generation seeds; and transferring into a fermentation culture medium containing terbinafine for fermentation culture so as to generate squalene. According to the method, the yield of squalene produced by the schizochytrium limacinum is increased by adding small molecular substances into a schizochytrium limacinum fermentation system. A small amount of specific proportion of terbinafine can effectively inhibit squalene from being oxidized into sterol substances in the fermentation process of schizochytrium limacinum, so that the consumption of squalene is reduced. Compared with the method without adding the terbinafine, the method has the advantages that the content of squalene is 2.63 times of that of a control group by adding the terbinafine with proper concentration, and the ratio of the squalene to the biomass is 2 times of that of the control group.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular to a method and application of using a small molecule additive to increase the yield of squalene in a schizochytrium fermentation process. Background Art

[0002] Squalene is an unsaponifiable lipophilic terpenoid compound that can be used as a precursor for the biosynthesis of all steroids in plants and animals. As an acyclic triterpenoid, squalene is a class of functionally derived lipids. Squalene can be isolated from plants and animals such as shark liver oil, olives, and amaranth seeds, but due to the cumbersome extraction and purification process, this type of production method has great limitations. Therefore, using microorganisms as hosts to synthesize squalene in a sustainable, efficient, and safe manner is currently a hot topic of research. Currently, two pathways for the production of squalene have been discovered, one is the mevalonate pathway (MVA) and the other is the non-mevalonate pathway (MEP). The pathway that an organism takes to synthesize squalene depends mainly on the characteristics of the organism itself. For example, prokaryotes such as bacteria and cyanobacteria synthesize squalene through the MEP pathway; while eukaryotic organisms such as yeast, advanced fungi, plants, animals, and humans synthesize squalene through the MVA pathway.

[0003] Schizochytrium sp is a marine fungus of the Thraustochytriaceae family. It is rich in oil, with an oil content of up to 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 increasing squalene by adding exogenous compounds has been widely used in the fermentation production process of Schizochytrium, mainly in the following aspects: Chinese patent CN117821530A adds Mn 2+ To enhance the activity of terpene synthase in the terpene synthesis pathway, thereby disturbing the metabolic network to increase the carbon flow to the biosynthesis of squalene, which can effectively promote the fermentation and production of squalene by Schizochytrium. Chinese patent CN115820749A controls the content of squalene in the fermentation process of Schizochytrium by adding an exogenous compound-sesamol. Compared with the control group without sesamol, the squalene content of Schizochytrium in the experimental group with different concentrations of sesamol can be increased. Chinese patent CN105803005A increases the squalene content in Schizochytrium by adding a certain concentration of exogenous regulatory factor-alcohol to the fermentation medium before or during fermentation. The effect is obvious and can be applied to large-scale production.

[0005] At present, there are no reports on regulating the squalene content in the Schizochytrium fermentation process by adding exogenous terbinafine. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method and application of a small molecule additive to increase the yield of squalene in the fermentation process of Schizochytrium.

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

[0008] Application of a small molecule additive in improving the production of squalene in Schizochytrium.

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

[0010] Furthermore, the Schizochytrium adds exogenous substance terbinafine during the fermentation process;

[0011] Alternatively, the addition of exogenous inhibitor terbinafine during the fermentation process of Schizochytrium can significantly increase the production of squalene.

[0012] A method for increasing the yield of squalene in a Schizochytrium fermentation process by using a small molecule additive, comprising the following steps:

[0013] (1) Activation: The low-temperature frozen Schizochytrium strain is inoculated into the seed culture medium for activation;

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

[0015] (3) transferring the first-generation seeds obtained in step (2) to a seed culture medium to culture again to obtain second-generation seeds;

[0016] (4) transferring the second-generation seeds obtained in step (3) to a seed culture medium and culturing them again to obtain third-generation seeds;

[0017] (5) The third generation seeds of step (4) are transferred to a fermentation medium containing terbinafine for fermentation culture, and squalene is obtained after the fermentation is completed.

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

[0019] Furthermore, 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 seed culture medium formula is: 40g / L glucose, 15g / L sodium sulfate, 8g / L yeast powder, 3g / L magnesium sulfate heptahydrate, 3g / L dipotassium hydrogen phosphate, 0.5g / L potassium chloride, 2ml / 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.

[0020] Furthermore, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt ion and terbinafine, the pH of the fermentation medium is 6.5-7.5, and the fermentation medium formula is: 80g / L glucose, 10g / L sodium sulfate, 8g / L yeast powder, 3g / L magnesium sulfate heptahydrate, 3g / L dipotassium hydrogen phosphate, 0.5g / L potassium chloride, 0.2g / L calcium chloride, 2ml / 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.

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

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

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

[0024] (1) The low-temperature frozen Schizochytrium strain was inoculated into the seed culture medium for activation. After microscopic examination showed that there was no bacterial contamination, the activated Schizochytrium was transferred into the seed culture medium. The inoculation amount of the activated bacteria was 1%-2% (volume percentage, v / v), and the culture was carried out at 28-30°C and 170-180 rpm for 24 hours to obtain the first generation of seeds;

[0025] (2) After the first-generation seeds obtained in step (1) are free of bacteria under microscopic examination, they are inoculated into a seed culture medium for further cultivation. The inoculation amount of the first-generation seeds is 1%-2% (volume percentage, v / v), and the seeds are cultured at 28-30° C. and 170-180 rpm for 24 h to obtain second-generation seeds;

[0026] (3) After the second-generation seeds obtained in step (2) are free of bacteria under microscopic examination, they are inoculated into a seed culture medium and cultured again. The inoculation amount of the second-generation seeds is 1%-2% (v / v), and the seeds are cultured at 28-30°C and 170-180 rpm for 24 hours to obtain third-generation seeds;

[0027] (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 inoculum amount of 8%-10% (v / v, volume ratio), and cultured 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, 100 mg / L of terbinafine is added to the fermentation medium, and squalene is obtained after the fermentation is completed.

[0028] Furthermore, in step (1), the inoculation amount of the activated bacteria is 1% (volume percentage, v / v), and the first generation of seeds is obtained by culturing at 30° C. and 180 rpm for 24 hours;

[0029] In step (2), the inoculation amount of the first generation seeds is 2% (volume percentage, v / v), and the second generation seeds are obtained by culturing at 30° C. and 180 rpm for 24 h;

[0030] In step (3), the inoculation amount of the second generation seeds is 2% (volume percentage, v / v), and the third generation seeds are obtained by culturing at 30° C. and 180 rpm for 24 h;

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

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

[0033] The advantages and positive effects achieved by the present invention are:

[0034] 1. The present invention increases the yield of squalene produced by Schizochytrium by adding a small molecule substance, terbinafine, to the Schizochytrium fermentation system. A small amount of terbinafine in a specific proportion can effectively inhibit the oxidation of squalene into sterols during the fermentation of Schizochytrium, thereby reducing the consumption of squalene. Compared with the case where no terbinafine was added, the squalene content of the control group was 2.63 times that of the control group when terbinafine was added at an appropriate concentration, and the ratio of squalene to biomass was 2 times that of the control group.

[0035] 2. The present invention provides a method for increasing the production of squalene by exogenously adding metabolites during the fermentation production process. Specifically, a small amount of terbinafine is additionally added during the production of squalene by Schizochytrium by fermentation. The present invention can significantly increase the squalene content in Schizochytrium through simple fermentation regulation. Meanwhile, terbinafine is inexpensive and easy to obtain, so that the production cost of the method is relatively reduced, and the method has great economic benefits.

[0036] 3. The method of the present invention achieves a final squalene production of 3.65 g / L by adding exogenous substances in a simple, convenient, economical and efficient manner. However, the current production of squalene by wild-type strains of Schizochytrium is generally 1-2 g / L, 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 tested to be free of terbinafine, which can ensure the purity and safety of the oil and avoid the problem of drug residues. These advantages make the Schizochytrium oil free of terbinafine have a wider application prospect in the fields of food, health products and cosmetics.

[0038] 5. The method of the present invention uses Schizochytrium as a production strain and removes the addition of trace elements in the culture medium to increase the yield of squalene. The present invention increases the yield of squalene produced by Schizochytrium by adding exogenous compounds to the Schizochytrium fermentation system. Terbinafine can effectively inhibit the oxidation of squalene to sterols during the fermentation of Schizochytrium, thereby reducing the consumption of squalene. Compared with the case where no terbinafine was added, the squalene content was increased when 100 mg / L of terbinafine was added at 36 hours of fermentation. Terbinafine can inhibit squalene epoxidase and increase the yield of squalene during the fermentation of Schizochytrium. The present invention applies terbinafine to the regulation of terpenoid production by Schizochytrium, provides a new idea for increasing the content of squalene in Schizochytrium, and has great potential in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] Figure 2 This is a graph showing the results of terbinafine fermentation regulating the production of squalene by Schizochytrium in the present invention. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0042] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0043] Application of a small molecule additive in improving the production of squalene in Schizochytrium.

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

[0045] Alternatively, the addition of exogenous inhibitor terbinafine during the fermentation process of Schizochytrium can significantly increase the production of squalene.

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

[0047] (1) Activation: The low-temperature frozen Schizochytrium strain is inoculated into the seed culture medium for activation;

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

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

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

[0051] (5) The third generation seeds of step (4) are transferred to a 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 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;

[0054] The above percentages are mass percentages.

[0055] Preferably, the fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt ion and terbinafine, the pH of the fermentation medium is 6.5-7.5, and the fermentation medium formula is: glucose 80g / L, sodium sulfate 10g / L, yeast powder 8g / L, magnesium sulfate heptahydrate 3g / L, dipotassium hydrogen phosphate 3g / L, potassium chloride 0.5g / L, calcium chloride 0.2g / L, glycerol 2ml / L, 0.2% vitamin B1, 0.2% vitamin B6, 0.2% vitamin B 12 , the solvent is water;

[0056] The above percentages are mass percentages.

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

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

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

[0060] (1) The frozen Schizochytrium strain was inoculated into the seed culture medium for activation. After microscopic examination showed that there was no bacterial contamination, the activated bacteria were transferred into the seed culture medium. The inoculation amount of the activated bacteria was 1%-2% (volume percentage, v / v), and the culture was carried out at 28-30°C and 170-180 rpm for 24 hours to obtain the first generation of seeds;

[0061] (2) After the first-generation seeds obtained in step (1) are free of bacteria under microscopic examination, they are inoculated into a seed culture medium for further cultivation. The inoculation amount of the first-generation seeds is 1%-2% (volume percentage, v / v), and the seeds are cultured at 28-30° C. and 170-180 rpm for 24 h to obtain second-generation seeds;

[0062] (3) After the second-generation seeds obtained in step (2) are free of bacteria under microscopic examination, they are inoculated into a seed culture medium and cultured again. The inoculation amount of the second-generation seeds is 1%-2% (v / v), and the seeds are cultured at 28-30°C and 170-180 rpm for 24 hours to obtain third-generation seeds;

[0063] (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 inoculum amount of 8%-10% (v / v, volume ratio), and cultured 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, 100 mg / L of terbinafine is added to the fermentation medium, and squalene is obtained after the fermentation is completed.

[0064] Preferably, the inoculation amount of the activated bacteria in step (1) is 1% (volume percentage, v / v), and the first generation of seeds is obtained by culturing at 30°C and 180 rpm for 24 hours;

[0065] In step (2), the inoculation amount of the first generation seeds is 2% (volume percentage, v / v), and the second generation seeds are obtained by culturing at 30° C. and 180 rpm for 24 h;

[0066] In step (3), the inoculation amount of the second generation seeds is 2% (volume percentage, v / v), and the third generation seeds are obtained by culturing at 30° C. and 180 rpm for 24 h;

[0067] In step (4), the third generation seeds are inoculated into the fermentation medium at an inoculation amount of 8% (v / v, volume ratio), and cultured 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 relevant 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 seed culture medium formula is: glucose 40g / L, sodium sulfate 15g / L, yeast powder 8g / L, magnesium sulfate heptahydrate 3g / L, dipotassium hydrogen phosphate 3g / L, potassium chloride 0.5g / L, glycerol 2ml / L, 0.2% vitamin B1, 0.2% vitamin B6, 0.2% vitamin B 12 , the solvent is water; the above percentages are mass percentages.

[0072] The fermentation medium includes a carbon source, a nitrogen source, an inorganic salt ion and terbinafine. The pH of the fermentation medium is 6.5-7.5, and the formula of the fermentation medium is: 80 g / L glucose, 10 g / L sodium sulfate, 8 g / L yeast powder, 3 g / L magnesium sulfate heptahydrate, 3 g / L potassium 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.

[0073] The concentrations of added terbinafine are: 1 mg / L, 10 mg / L, and 100 mg / L. During the experiment, the composition and amount of the culture medium remain unchanged, and only the above-mentioned different concentrations of terbinafine need to be added to different fermentation media, and a control experimental group, i.e., a group in which the exogenous terbinafine addition amount is 0 mg / L, is prepared.

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

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

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

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

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

[0079] 4. The third generation seeds were inoculated into 100 mL of fermentation medium with an inoculation amount of 8% (v / v, volume ratio), and cultured at a temperature of 30°C and a rotation speed of 180 rpm for 120 hours. Squalene was obtained after the fermentation. During this period, different concentrations of terbinafine were added to the fermentation medium at 0 hours, 36 hours and 72 hours of fermentation, respectively, with the concentrations of 1 mg / L, 10 mg / L and 100 mg / L. The effects of adding different concentrations of terbinafine on biomass, oil yield and squalene yield were determined respectively.

[0080] 5. After 120 hours of fermentation, the extraction of oil and other substances follows the following steps:

[0081] (1) Accurately pipette a certain amount of fermentation liquid into a stopper bottle, adjust the pH to 10-11 with a 2 mol / L sodium hydroxide aqueous solution, mix well, then add 0.2% of the weight of the fermentation liquid cell wall-breaking enzyme, and perform enzymolysis at 50°C for 3 h until the bacteria are completely digested;

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

[0083] (3) Add n-hexane in a ratio of 1:1 to the volume of the fermentation broth, shake well, let stand to separate the layers, take the upper organic phase and place it in a round-bottom conical flask that has been constant weight and dried, and wash it with n-hexane for about 3 times until the upper organic phase is colorless and transparent;

[0084] (4) The upper layer of liquid is subjected to rotary evaporation extraction. Before rotary evaporation, the rotary evaporation bottle is weighed and recorded (g1). The rotary evaporation is performed at 40°C until no condensed water drips;

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

[0086] 6. After extracting the oil, methylate the oil according to the following steps (to prepare for 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 and mix well;

[0088] ② Under room temperature, 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 12000 rpm for 5 min, take the upper n-hexane phase and filter it through an organic filter membrane to remove impurities, add it to a gas phase vial and then perform gas chromatography detection. Gas chromatography test method: Gas phase analysis conditions: Use DB-23 (60 m*0.25 mm*0.25μm) chromatographic column; select FID as the detector; use high-purity nitrogen as the carrier gas; split ratio is 30 / 1; the injection port temperature is set to 250°C; the detector temperature is set to 280°C; the injection volume is 1 μL; temperature program: At the beginning of the program, the gas chromatography column temperature is set to 100°C, and the program first increases to 196°C at a rate of 25°C / min. Subsequently, the program increases to 220°C at a rate of 2°C / min and maintains at 220°C for 12 min. The gas phase column flow rate is set to 3.0 mL / min; the tail blow flow rate is set to 30 mL / min; the hydrogen flow rate is 40mL / min and the air flow rate is set to 400 mL / min. The gas chromatogram of the prepared squalene is as follows: Figure 1 shown.

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

[0092] 8. Prepare a squalene standard sample, obtain the standard curve equation, and then calculate the squalene production based on the gas chromatographic analysis results.

[0093] 9. The fermentation time was 120 hours. After the fermentation was completed, the biomass, oil yield and squalene content of the plants with different concentrations of terbinafine were measured. The oil extraction and purification methods were as described above. The biomass, oil yield and squalene content were measured, and the data are shown in Table 1. The squalene content results in Table 1 were used to draw Figure 2 .

[0094]

[0095] As shown in Table 1, after adding different concentrations of terbinafine, the biomass, oil content, and squalene content of Schizochytrium were all increased. When 100 mg / L of terbinafine was added at 36 hours of fermentation, the biomass reached a maximum of 19.5 g / L, and the oil content of Schizochytrium produced by the strain reached a maximum of 40.9 g / L, and the squalene content reached 3.36 g / L. Compared with the control group, i.e., the group with 0 mg / L of exogenous terbinafine added, the biomass increased by 23%; the oil content increased by 17%; and the squalene content increased by 140%. The present invention can effectively increase the production of squalene by adding exogenous substances in a simple, convenient, economical and efficient manner.

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

[0097] Example 2

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

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

[0100]

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

[0102] At present, there is a study on the application of terbinafine in increasing the production of squalene by Rhodosporidium toroculans. According to the results, the maximum production of squalene can reach 863.06 mg / L, which is much lower than the content of squalene produced by Schizochytrium in the present invention, which is 3.9 g / L. Moreover, the oil extracted after fermentation by the method of the present invention is tested to be free of terbinafine, which can ensure the purity and safety of the oil, avoid the problem of drug residues, ensure the biological activity and nutritional value of the oil, and meet regulatory requirements and market demand. These advantages make Schizochytrium oil without terbinafine have a wider application prospect in the fields of food, health products and cosmetics.

[0103] Patent CN118240858A mainly uses genetic engineering to transform Rhodosporidium toruloides to obtain recombinant bacteria to increase the production of squalene. The present invention increases the production of squalene by Schizochytrium through fermentation regulation.

[0104] Patent CN116970656A increases the yield of squalene produced by Rhodosporidium toruloides through fermentation regulation, which is different from the strain used in the present invention. The present invention applies terbinafine to Schizochytrium for the first time to increase the yield of squalene, providing a new idea and method for Schizochytrium to produce squalene.

[0105] Patent CN1927181A focuses on introducing a drug and a method for preparing the drug. The present invention mainly studies the increase of squalene production by microbial fermentation regulation. The focus is inconsistent.

[0106] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate 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 contents disclosed in the embodiments.

Claims

1. Application of a small molecule additive in increasing the yield of squalene in Schizochytrium, Features: The small molecule additive is terbinafine, and the exogenous substance terbinafine is added to Schizochytrium during the fermentation process.

2. A method for increasing the production of squalene in the fermentation process of Schizochytrium using a small molecule additive, Features: The following steps are involved: (1) Activation: The low-temperature frozen Schizochytrium strain is inoculated into the seed culture medium for activation; (2) transferring the activated Schizochytrium obtained to a seed culture medium to obtain first-generation seeds; (3) transferring the first-generation seeds obtained in step (2) to a seed culture medium to culture again to obtain second-generation seeds; (4) transferring the second-generation seeds obtained in step (3) to a seed culture medium and culturing them again to obtain third-generation seeds; (5) The third generation seeds of step (4) are transferred to a fermentation medium containing terbinafine for fermentation culture to produce squalene.

3. The method according to claim 2, Features: The concentration of terbinafine added to the fermentation medium is 1 mg / L-100 mg / L.

4. The method according to claim 2, Features: 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 seed culture medium formula is: 40g / L glucose, 15g / L sodium sulfate, 8g / L yeast powder, 3g / L magnesium sulfate heptahydrate, 3g / L dipotassium hydrogen phosphate, 0.5g / L potassium chloride, 2ml / L glycerol, 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.

5. The method according to claim 2, Features: 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 potassium hydrogen phosphate, 0.5 g / L potassium chloride, 0.2 g / L calcium chloride, 2 ml / L glycerol, 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.

6. The method according to claim 2, Features: The seed culture conditions were 30°C, 180 rpm, and 24 h; The fermentation culture conditions are 30° C. and 180 rpm for 120 h.

7. The method according to claim 2, Features: The specific steps are as follows: 1) The low-temperature frozen Schizochytrium strain is inoculated into the seed culture medium for activation. After microscopic examination shows that there is no bacterial contamination, the activated Schizochytrium is transferred to the seed culture medium. The inoculation amount of the activated bacteria is 1%-2% by volume, and the culture is carried out at 28-30°C and 170-180rpm for 24h 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 1%-2% by volume, and the seeds are cultured at 28-30° C. and 170-180 rpm for 24 hours to obtain second generation seeds; 3) The second generation seeds obtained in step 2) are inoculated in a seed culture medium and cultured again after being examined under a microscope to be free of bacteria. The inoculation amount of the second generation seeds is 1%-2% by volume, and cultured at 28-30°C and 170-180 rpm for 24 hours to obtain 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%-10% by volume, and cultured at a temperature of 28-30° C. and a rotation speed of 170-180 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.

8. The method according to claim 7, Features: In the step 1), the inoculation amount of the activated Schizochytrium is 1% by volume, and the first generation of seeds is obtained by culturing at 30° C. and 180 rpm for 24 hours; In step 2), the inoculation amount of the first generation seeds is 2% by volume, and the second generation seeds are obtained by culturing at 30° C. and 180 rpm for 24 h; In step 3), the inoculation amount of the second generation seeds is 2% by volume, and the third generation seeds are obtained by culturing at 30° C. and 180 rpm for 24 h; In step 4), the third generation seeds are inoculated into the fermentation medium at an inoculation volume of 8% and cultured at a temperature of 30° C. and a rotation speed of 180 rpm for 120 h.

9. The method according to any one of claims 2 to 8, Features: The schizochytrium is Schizochytrium sp. HX-308.

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