Method for improving grease titer by regulating schizochytrium limacinum fermentation process through plant hormones and application

Abstract

The invention belongs to the technical field of biology, and discloses a method and application for regulating and controlling the fermentation process of schizochytrium limacinum through plant hormones to improve the oil titer, and the method is characterized in that in the fermentation process of schizochytrium limacinum, one or more plant hormones are added into a culture medium to promote accumulation of oil. Plant hormones are added into a fermentation culture medium of schizochytrium limacinum, the plant hormones comprise gibberellin (GB), 6-benayl aminopurine (6-BAP), forchlorfenuron (CPPU) and brassinolide (BR), and the plant hormones promote grease accumulation by adjusting activity of enzymes related to fatty acid synthesis, so that the plant hormones can adjust metabolic pathways of the schizochytrium limacinum and promote grease accumulation. The synthesis efficiency and the yield of the grease are obviously improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to microbial fermentation and oil production, in particular to a method and application of utilizing plant hormones to regulate the fermentation process of schizochytrium to increase the oil titer. Background Art

[0002] Schizochytrium can efficiently synthesize and accumulate oils through the fermentation process. These oils are mainly composed of long-chain polyunsaturated fatty acids (PUFAs), especially DHA (docosahexaenoic acid, C22:6, ω-3) and EPA (eicosapentaenoic acid, C20:5, ω-3). The oils produced by Schizochytrium have great market potential due to their efficient DHA and EPA production, pollution-free, pure and safe characteristics, and their wide application prospects in food, medicine, feed, skin care products and biofuels. With the continuous growth of green environmental protection and health needs, Schizochytrium oils will occupy an important position in the global functional oil market.

[0003] However, the current Schizochytrium fermentation production process still faces several technical bottlenecks. First, small-scale fermentation under laboratory conditions is easy to control, but in large-scale industrial fermentation, the problem of unstable production process is often encountered. Secondly, in current fermentation processes, oil production is usually increased by optimizing the composition of the culture medium and regulating fermentation parameters (such as temperature, pH and oxygen supply), but these methods have limited effects and poor sustainability.

[0004] In recent years, plant hormones, as a class of naturally occurring compounds, have shown great potential in regulating plant metabolic activities, promoting growth and responding to environmental stress. Studies have found that plant hormones can not only regulate oil synthesis in plants, but also activate the oil metabolism pathways of certain microorganisms through exogenous addition. For example, plant hormones such as 2-chlorolongic acid (2-chloroethylphosphonic acid), salicylic acid (SA), and abscisic acid (ABA) have been shown to regulate the activity of fatty acid synthesis-related enzymes in different fermentation systems, increasing oil accumulation by 10.00-18.78%.

[0005] So far, research on the application of plant hormones in the fermentation process of Schizochytrium is relatively limited. There is no effective method to systematically use plant hormones to regulate the metabolic pathways of Schizochytrium, thereby significantly increasing the oil titer. Therefore, it is urgent to develop a new technology to regulate Schizochytrium fermentation through plant hormones to achieve efficient oil production and provide new solutions for industrial applications. 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 regulating the fermentation process of Schizochytrium by using plant hormones to increase the oil titer.

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

[0008] A method for increasing the oil titer by regulating the fermentation process of Schizochytrium using plant hormones. The method comprises adding one or more plant hormones to a culture medium during the fermentation process of Schizochytrium to promote the accumulation of oil.

[0009] Furthermore, the plant hormone is selected from the following categories: Gibberellin (GB), 6-Benzylaminopurine (6-BAP), Forchlorfenuron (CPPU), and 24-Epibrassinolide (BR).

[0010] Furthermore, the added amount of the plant hormone is 0.1 μM to 200 μM, preferably 10 μM to 100 μM.

[0011] Further, the plant hormone is added during 24 to 72 hours of fermentation.

[0012] Furthermore, during the fermentation process, the fermentation temperature is 25-29° C., the rotation speed is 150-200 rpm, the time is 72-120 h, and the pH value is maintained between 5.5 and 7.0.

[0013] Furthermore, the OD value of the fermentation bacteria is 8-10; and the inoculation amount of the fermentation bacteria is 0.8-1.5% of the volume of the fermentation medium.

[0014] Furthermore, the Schizochytrium is Schizochytrium sp. HX-308.

[0015] Furthermore, the specific steps are as follows:

[0016] The Schizochytrium is inoculated into a seed culture medium, and cultured at a temperature of 28°C for 24 hours to obtain first-level seeds; the first-level seeds are inoculated into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and cultured again at a temperature of 28°C for 24 hours to obtain second-level seeds; the second-level seeds are inoculated into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and cultured again at a temperature of 28°C for 24 hours, and after the above activation process, a strain for fermentation is obtained;

[0017] A fermentation strain with an OD600 value of 8-10 is inoculated into a fermentation medium at an inoculation rate of 1%, and the fermentation culture is carried out for 120 hours at a temperature of 25°C and a rotation speed of 170 rpm; wherein, at the 24th hour of the fermentation process, a plant hormone is added to the fermentation tank, and the plant hormone is one of gibberellin with a final concentration of 50 μM + chlorfenapyr with a final concentration of 75 μM, gibberellin with a final concentration of 50 μM + 6-benzylaminopurine with a final concentration of 50 μM, gibberellin with a final concentration of 50 μM + 50 μM brassinolide, chlorfenapyr with a final concentration of 75 μM + 6-benzylaminopurine with a final concentration of 50 μM, and chlorfenapyr with a final concentration of 75 μM + 50 μM brassinolide.

[0018] Further, the composition of the seed culture medium is: glucose 45g / L, yeast powder 2g / L, sodium glutamate 15g / L, MgCl2·7H2O 4g / L, Na2SO4 15g / L, KCl 1g / L, NaCl 1g / L, MgSO4·7H2O 5g / L, KH2PO4 3g / L, and the solvent is water;

[0019] The fermentation medium consisted of 90 g / L glucose, 4 g / L yeast powder, 15 g / L sodium glutamate, 4 g / L MgCl2·7H2O, 15 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, 5 g / L MgSO4·7H2O, 3 g / L KH2PO4 and (NH 44 )2SO44 g / L, the solvent is water.

[0020] Application of the method as described above in improving the oil titer during Schizochytrium fermentation.

[0021] The advantages and positive effects achieved by the present invention are:

[0022] 1. The present invention adds plant hormones to the fermentation medium of Schizochytrium, and the plant hormones include gibberellins (GB), 6-benzylaminopurine (6-BAP), chlorpyrifos (CPPU), and brassinolide (BR). The plant hormones promote the accumulation of oil by regulating the activity of enzymes related to fatty acid synthesis. Therefore, the plant hormones can regulate the metabolic pathway of Schizochytrium and significantly improve the synthesis efficiency and yield of oil.

[0023] 2. The present invention uses exogenous addition of plant hormones to regulate the fatty acid synthesis pathway of Schizochytrium. The present invention adds plant hormones to the fermentation medium of wild-type Schizochytrium, which can regulate the activity of enzymes related to fatty acid synthesis and increase the oil titer by 15-30%.

[0024] 3. The method of the present invention optimizes the metabolic pathway of the strain by adding specific plant hormones to the fermentation medium, thereby significantly increasing the oil titer. The present invention has the characteristics of simple operation, high efficiency, environmental protection, and low cost, and can effectively increase the oil titer of Schizochytrium fermentation, and the titer is increased by 15% to 30% compared with the wild-type Schizochytrium.

[0025] 4. The present invention aims to solve the problem of low titer in the current production of oil by Schizochytrium fermentation, especially the insufficient production of polyunsaturated fatty acids (PUFAs). The present invention introduces plant hormones into the fermentation process to regulate the metabolic pathway of Schizochytrium, thereby significantly improving the synthesis efficiency and yield of oil. For example, the addition of 50 μM gibberellin increased the oil yield by 25.01% and the DHA content by 7.93%.

[0026] 5. The present invention optimizes the concentration of plant hormones and the combined use of plant hormones. Under the premise of keeping costs controllable, the present invention increases the oil yield and DHA content of Schizochytrium, reduces production costs, and facilitates industrial application.

[0027] 6. The present invention uses natural plant hormones to regulate the fermentation process, avoiding the use of chemical additives, and helps to achieve a green and environmentally friendly biological fermentation process, which meets the needs of modern green industry.

[0028] 7. The present invention is not only applicable to the fermentation production of Schizochytrium, but also provides a reference for the optimization of other microbial fermentation systems. It is simple to operate and has application potential and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the effect of adding plant hormones on oil yield and DHA content in Example 2 of the present invention;

[0030] Figure 2 This is a graph showing the effects of different concentrations of gibberellins on oil yield and DHA content in Example 3 of the present invention;

[0031] Figure 3 This is a graph showing the effects of different concentrations of chlorfenapyr on oil yield and DHA content in Example 4 of the present invention;

[0032] Figure 4 This is a graph showing the effect of the combined use of different plant hormones on oil yield and DHA content in Example 5 of the present invention;

[0033] Figure 5 This is a gas chromatogram without adding plant hormone regulation in the present invention;

[0034] Figure 6This is a gas chromatogram after adding 50 μM gibberellin + 75 μM chlorfenapyr for fermentation regulation in the present invention;

[0035] Figure 7 The diagram is a comparison photo of the oil yield without adding plant hormones and with adding 50 μM gibberellin + 75 μM chlorpyrifos; wherein, the left side is a photo of the oil yield without adding plant hormones, and the right side is a photo of the oil yield with adding 50 μM gibberellin + 75 μM chlorpyrifos.

[0036] Figure 8 This is a diagram showing the optimization results of the effects of different addition times of plant hormones on oil yield and DHA content in Example 6 of the present invention. DETAILED DESCRIPTION

[0037] 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.

[0038] 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.

[0039] A method for increasing the oil titer by regulating the fermentation process of Schizochytrium using plant hormones. The method comprises adding one or more plant hormones to a culture medium during the fermentation process of Schizochytrium to promote the accumulation of oil.

[0040] Preferably, the plant hormone is selected from the following categories: Gibberellin (GB), 6-Benzylaminopurine (6-BAP), Forchlorfenuron (CPPU), and 24-Epibrassinolide (BR).

[0041] Preferably, the added amount of the plant hormone is 0.1 μM to 200 μM, preferably 10 μM to 100 μM.

[0042] Preferably, the phytohormone is added during the 24 to 72 hours of fermentation.

[0043] Preferably, the fermentation temperature during the fermentation process is 25-29°C, the rotation speed is 150-200 rpm, the time is 72-120 h, and the pH value is maintained between 5.5 and 7.0.

[0044] Preferably, the OD value of the fermentation bacteria is 8-10; and the inoculation amount of the fermentation bacteria is 0.8-1.5% of the volume of the fermentation medium.

[0045] Preferably, the Schizochytrium is Schizochytrium sp. HX-308.

[0046] Preferably, the specific steps are as follows:

[0047] The Schizochytrium is inoculated into a seed culture medium, and cultured at a temperature of 28°C for 24 hours to obtain first-level seeds; the first-level seeds are inoculated into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and cultured again at a temperature of 28°C for 24 hours to obtain second-level seeds; the second-level seeds are inoculated into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and cultured again at a temperature of 28°C for 24 hours, and after the above activation process, a strain for fermentation is obtained;

[0048] A fermentation strain with an OD600 value of 8-10 is inoculated into a fermentation medium at an inoculation rate of 1%, and the fermentation culture is carried out for 120 hours at a temperature of 25°C and a rotation speed of 170 rpm; wherein, at the 24th hour of the fermentation process, a plant hormone is added to the fermentation tank, and the plant hormone is one of gibberellin with a final concentration of 50 μM + chlorfenapyr with a final concentration of 75 μM, gibberellin with a final concentration of 50 μM + 6-benzylaminopurine with a final concentration of 50 μM, gibberellin with a final concentration of 50 μM + 50 μM brassinolide, chlorfenapyr with a final concentration of 75 μM + 6-benzylaminopurine with a final concentration of 50 μM, and chlorfenapyr with a final concentration of 75 μM + 50 μM brassinolide.

[0049] Preferably, the composition of the seed culture medium is: glucose 45g / L, yeast powder 2g / L, sodium glutamate 15g / L, MgCl2·7H2O 4g / L, Na2SO4 15g / L, KCl 1g / L, NaCl 1g / L, MgSO4·7H2O 5g / L, KH2PO4 3g / L, and the solvent is water;

[0050] The components of the fermentation medium are: glucose 90 g / L, yeast powder 4 g / L, sodium glutamate 15 g / L, MgCl2·7H2O4 g / L, Na2SO4 15 g / L, KCl 1 g / L, NaCl 1 g / L, MgSO4·7H2O 5 g / L, KH2PO4 3 g / L and (NH4)2SO4 4 g / L, and the solvent is water.

[0051] Application of the method as described above in improving the oil titer during Schizochytrium fermentation.

[0052] Specifically, the relevant preparation and detection are as follows:

[0053] Example 1

[0054] The culture medium components used in the following examples are as follows:

[0055] The components of the seed culture medium are: glucose 45g / L, yeast powder 2g / L, sodium glutamate 15g / L, MgCl2·7H2O4g / L, Na2SO4 15g / L, KCl 1g / L, NaCl 1g / L, MgSO4·7H2O 5g / L, KH2PO4 3g / L, and the solvent is water.

[0056] The components of the fermentation medium are: glucose 90 g / L, yeast powder 4 g / L, sodium glutamate 15 g / L, MgCl2·7H2O4 g / L, Na2SO4 15 g / L, KCl 1 g / L, NaCl 1 g / L, MgSO4·7H2O 5 g / L, KH2PO4 3 g / L and (NH4)2SO4 4 g / L, and the solvent is water.

[0057] The cultivation method is as follows:

[0058] Inoculate Schizochytrium sp. HX-308 into a seed culture medium, and culture it at a temperature of 28°C for 24 hours to obtain first-level seeds; take the first-level seeds and inoculate them into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and culture it again at a temperature of 28°C for 24 hours to obtain second-level seeds; take the second-level seeds and inoculate them into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and culture it again at a temperature of 28°C for 24 hours, and after the above activation process, obtain a strain for fermentation;

[0059] Take 0.5 L of the fermentation bacteria with an OD600 value of 8-10 and inoculate it into 50 L of fermentation medium. Fermentation culture is carried out for 120 hours at a temperature of 25° C. and a rotation speed of 170 rpm.

[0060] The biomass was determined as follows:

[0061] Take 1 mL of the fermentation liquid that has just been fermented, centrifuge it at 4000 r / min for 5 min, discard the supernatant, add 1 mL of 2% NaCl solution to wash once, then wash once with 1 mL of distilled water, dry it at 105°C to constant weight, and finally weigh and calculate.

[0062] The gas chromatography analysis method is as follows:

[0063] Take the fermentation broth after the fermentation and culture, adjust the pH to 10 with NaOH, add 0.3% of the fermentation broth mass of lytic enzyme, hydrolyze at 55℃ for 1h, add n-hexane for extraction, let stand and wait for stratification, take the upper yellow organic phase liquid, repeat the extraction until the upper color becomes colorless, combine the organic phase liquid, evaporate the solvent, and get the total oil; add 2ml of 15% mass concentration NaOH solution and 5.5ml of 70% methanol content aqueous solution to the total oil. React in a water bath at 65℃ for 1.5h; cool to room temperature, add 2ml of n-hexane (chromatographic grade) for extraction, collect the upper organic phase; remove impurities from the upper organic phase through micro-membrane pores, and then analyze by gas chromatography.

[0064] Example 2 Plant hormone screening

[0065] In order to screen out the plant hormone with the best effect on regulating Schizochytrium fermentation, four plant hormones, namely gibberellin (GB), 6-Benzylaminopurine (6-BAP), Forchlorfenuron (CPPU) and 24-Epibrassinolide (BR), were added during the fermentation process, and the oil yield was compared to screen out the plant hormone with the best effect.

[0066] The culture was carried out according to the specific implementation method of Example 1, and at the 24th hour of the fermentation process, gibberellin, 6-benzylaminopurine, chlorfenapyr, and brassinolide were added to the fermenter to a final concentration of 10-100 μM.

[0067] The fermentation was completed at 120 h, and the oil content was extracted and detected by the method described in Example 1. The results are shown in Table 1 and Figure 1 shown.

[0068]

[0069] The results showed that under the action of gibberellin (50μM), 6-benzylaminopurine (50μM), chlorfenapyr (50μM), and brassinolide (50μM), the biomass increased to 120.65g / L, 117.60 g / L, 119.63 g / L, and 119.37 g / L, respectively, which was significantly higher than the control group of 113.36 g / L. The oil yields were 75.03g / L, 69.24g / L, 73.37g / L, and 71.45g / L, respectively, and the DHA contents were 46.98%, 45.12%, 46.49%, and 46.23%, respectively. Compared with the control group without the addition of plant hormones, the oil yields increased by 25.01%, 15.36%, 22.24%, and 19.04%, respectively, and the DHA contents increased by 7.93%, 3.65%, 6.80%, and 6.20%, respectively. Among them, gibberellin and chlorfenapyr showed more significant effects at a concentration of 50 μM.

[0070] Example 3 Optimization of gibberellin concentration

[0071] In Example 2, the gibberellin was screened out to have the best promoting effect on regulating the fermentation production of oil by Schizochytrium. In order to further optimize the use concentration of gibberellin, in this example, different concentration gradients were set to explore the effect of gibberellin on the oil production and DHA content of Schizochytrium to determine the optimal addition concentration.

[0072] The specific fermentation method is basically the same as that in Example 2, except for the added plant hormones and the concentration of the plant hormones. In this example, at the 24th hour of fermentation, different concentrations of gibberellins were added to the fermentation tank group, respectively, and the concentration range was set to 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM. At the 120th hour of fermentation, the fermentation liquid was collected and the oil and DHA content was tested. The results are shown in Table 2 and Figure 2 shown.

[0073]

[0074] The results showed that when 10μM, 25μM, 50μM, 75μM, and 100μM gibberellins were added, the biomass increased to 115.32g / L, 118.21g / L, 120.45g / L, 117.03g / L, and 116.24g / L, respectively, the oil yield increased by 7.79%, 15.37%, 24.56%, 19.73%, and 16.13%, respectively, and the DHA content increased by 3.86%, 4.96%, 7.54%, 7.11%, and 6.38%, respectively. Among them, when the added concentration was 50μM, the increase in oil yield and DHA content was the highest. Therefore, the optimal concentration of gibberellins in regulating Schizochytrium fermentation is 50μM. In addition, when higher concentrations (75 μM and 100 μM) of gibberellins were added, the oil production and DHA content decreased compared to when 50 μM gibberellins were added, but it still promoted the increase in oil production and DHA content, indicating that excessively high concentrations of gibberellins may have an inhibitory effect on the oil synthesis of Schizochytrium.

[0075] Example 4 Optimization of the concentration of chlorfenapyr

[0076] In Example 2, chlorfenapyr also showed a good promoting effect on regulating the fermentation of Schizochytrium. In order to further optimize the concentration of chlorfenapyr, different concentration gradients were also designed in this example to determine the optimal concentration.

[0077] The specific fermentation method is basically the same as that in Example 2, except for the added plant hormones and the concentration of the plant hormones. In this example, at the 24th hour of fermentation, different concentrations of chlorfenapyr were added to the fermentation tank group, and the concentration range was set to 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM. At the 120th hour of fermentation, the fermentation liquid was collected for the detection of oil and DHA content. The results are shown in Tables 3 and Figure 3 shown.

[0078]

[0079] The results showed that when 10μM, 25μM, 50μM, 75μM, and 100μM of chlorfenapyr were added, the biomass increased to 115.32g / L, 117.58g / L, 118.96g / L, 119.75g / L, and 116.84g / L, respectively, the oil yield increased by 5.56%, 15.43%, 22.27%, 24.68%, and 19.29%, respectively, and the DHA content increased by 2.58%, 4.95%, 6.83%, 8.42%, and 5.87%, respectively. Among them, when the added concentration was 75μM, the increase in oil yield and DHA content was the highest. Therefore, the optimal concentration of chlorfenapyr in regulating Schizochytrium fermentation is 75μM. In addition, when chlorfenapyr was added at a concentration of 100 μM, the oil yield and DHA content decreased slightly compared to when 75 μM chlorfenapyr was added, but it still promoted the increase in oil yield and DHA content, indicating that the promoting effect of chlorfenapyr on oil accumulation was weakened at higher concentrations.

[0080] Example 5 Optimization of fermentation conditions combined with plant hormones

[0081] In the above examples, the use of plant hormones such as gibberellin (GB) and chlorfenapyr (CPPU) alone has a positive effect on the accumulation of oil in Schizochytrium fermentation. In order to further increase the oil yield and DHA content, the effect of the combined use of different plant hormones was explored in this example, and the application conditions in Schizochytrium fermentation were optimized. The results are shown in Table 4.

[0082] The specific fermentation method is basically the same as that in Example 2, the only difference being the added plant hormones and the concentrations of the plant hormones. In this example, at the 24th hour of fermentation, gibberellin (50 μM) + chlorfenapyr (75 μM), gibberellin (50 μM) + 6-benzylaminopurine (50 μM), gibberellin (50 μM) + brassinolide (50 μM), chlorfenapyr (75 μM) + 6-benzylaminopurine (50 μM), chlorfenapyr (75 μM) + brassinolide (50 μM) were added to the fermentation tank group, respectively. When the fermentation reached the 120th hour, the fermentation broth was collected for the detection of oil and DHA content. The results are shown in Tables 4 and Figure 4 shown.

[0083]

[0084] The results showed that with the addition of gibberellin (50μM) + chlorfenapyr (75μM), gibberellin (50μM) + 6-benzylaminopurine (50μM), gibberellin (50μM) + brassinolide (50μM), chlorfenapyr (75μM) + 6-benzylaminopurine (50μM), and chlorfenapyr (75μM) + brassinolide (50μM), the biomass increased to 125.40g / L, 124.25g / L, 123.17g / L, 123.64g / L, and 124.86g / L, respectively, the oil yield increased by 33.84%, 30.07%, 31.71%, 28.78%, and 32.14%, respectively, and the DHA content increased by 10.07%, 9.11%, 8.37%, 7.66%, and 8.30%, respectively. Among them, the combined use of gibberellin (50 μM) and chlorfenuron (75 μM) has the best effect. It can also be seen that the combined use of gibberellin and chlorfenuron in the method of the present invention can play a synergistic role and can synergistically improve the oil yield and DHA content of the prepared oil, especially the combined use of gibberellin with a final concentration of 50 μM and chlorfenuron with a final concentration of 75 μM can synergistically improve the oil yield and DHA content of the prepared oil.

[0085] In addition, if Figure 5 and Figure 6 As shown in the figure, by comparing the gas chromatogram without adding plant hormone regulation and the gas chromatogram after adding 50μM gibberellin + 75μM chlorfenapyr for fermentation regulation, the following differences can be intuitively seen through the gas chromatogram:

[0086] 1) Significant increase in DHA peak value: In the gas phase graph without the addition of plant hormones, the peak value of DHA is low, while in the gas phase graph with the addition of a combination of gibberellins and chlorfenapyr, the peak value of DHA increases significantly, proving that the combination of plant hormones promotes the synthesis of DHA.

[0087] 2) Changes in the overall peak: The other fatty acid peaks in the gas phase diagram with the addition of gibberellins and chlorfenapyr were also enhanced compared to the gas phase diagram without the addition of plant hormones, indicating that the addition of plant hormones promoted the oil production of Schizochytrium, which not only increased the DHA content, but also promoted the synthesis of other oil components.

[0088] pass Figure 7 It can be seen that the combination of gibberellins and chlorfenapyr has a significant effect on improving the oil production of Schizochytrium. This result is consistent with the previous gas chromatography analysis, further supporting the positive effect of the plant hormone combination on oil synthesis and providing a practical technical path for optimizing the fermentation production of Schizochytrium.

[0089] Example 6 Optimization of the time of adding plant hormones

[0090] In the above examples, the plant hormones were added at the 24th hour of fermentation. In order to explore the effects of different plant hormone addition times on oil production and DHA content during Schizochytrium fermentation, this example adds a plant hormone combination of gibberellin (50 μM) + chlorfenapyr (75 μM) at different times to determine the optimal plant hormone addition time, thereby further improving the fermentation effect. The specific fermentation method is basically the same as that of Example 2, except that the plant hormone combination of gibberellin (50 μM) + chlorfenapyr (75 μM) is added at the 24th, 12th, 36th, and 48th hours of fermentation. The results are shown in Tables 5 and Figure 8 shown.

[0091]

[0092] The results showed that the addition of the plant hormone combination of gibberellin (50μM) + chlorfenapyr (75μM) at the 12th, 24th, 36th and 48th hours increased the oil yield by 22.90%, 33.46%, 25.98% and 20.85%, respectively, and the DHA content by 6.23%, 9.97%, 7.70% and 6.32%, respectively. Among them, the plant hormone combination of gibberellin (50μM) + chlorfenapyr (75μM) added at the 24th hour of fermentation had the highest increase in oil yield and DHA content. Therefore, the best time to add plant hormones is the 24th hour of fermentation.

[0093] At the same time, by comparing Examples 5 to 6, it can be seen that the addition time of gibberellin (50 μM) + chlorfenapyr (75 μM) and the plant hormone in the present invention is the 24th hour of fermentation. There is a synergistic effect between the two, which can synergistically improve the oil yield and DHA content of the prepared oil, especially the combined use of gibberellin with a final concentration of 50 μM and chlorfenapyr with a final concentration of 75 μM can synergistically improve the oil yield and DHA content of the prepared oil.

[0094] The results showed that when a single plant hormone was added, each plant hormone could increase the oil yield and DHA content of Schizochytrium sp. HX-308 fermentation. Compared with Schizochytrium sp. HX-308 without the addition of plant hormones, the addition of a final concentration of 50 μM single plant hormones gibberellin, 6-benzylaminopurine, chlorfenapyr, and brassinolide increased the oil yield by 25.01%, 15.36%, 22.24%, and 19.04%, respectively, and the DHA content by 7.93%, 3.65%, 6.80%, and 6.20%, respectively. The combined use of plant hormones with final concentrations of 50μM gibberellin + 75μM chlorfenapyr, 50μM gibberellin + 50μM 6-benzylaminopurine, 50μM gibberellin + 50μM brassinolide, 75μM chlorfenapyr + 50μM 6-benzylaminopurine, and 75μM chlorfenapyr + 50μM brassinolide resulted in oil yields of 80.65g / L, 78.38g / L, 79.37g / L, 77.60g / L, and 79.63g / L, respectively, and DHA contents of 47.89%, 47.44%, 47.12%, 46.81%, and 47.09%, respectively. Therefore, compared with the addition of a single plant hormone, the combined use of plant hormones to regulate Schizochytrium fermentation can play a synergistic role and more significantly increase the oil production and DHA content of Schizochytrium.

[0095] The present invention can significantly increase the oil yield of Schizochytrium by regulating the addition time, concentration, type and combination of plant hormones, and the oil titer is increased by 15% to 30%. The present invention increases the oil yield by adding plant hormones to the fermentation medium of Schizochytrium, providing a reference for subsequent metabolic engineering transformation.

[0096] At present, in the prior art:

[0097] 1. A method for producing DHA by fermentation of Schizochytrium (Patent Publication No.: CN116622788A), providing a method for producing DHA by fermentation of Schizochytrium, the obtained product biomass is 100g / L, the oil yield is 45.01g / L, and the DHA content is 37.78%. Taking the addition of gibberellin (50μM) as an example, compared with this, the oil yield of the present invention is increased by 67.3%, and the DHA content is increased by 24.51%.

[0098] 2. A method for increasing the DHA content and yield of Schizochytrium by synergistically inducing two synthetic plant growth regulators (patent publication number: CN116179619A). The DHA yield of this invention is 5.17 g / L. Taking the addition of gibberellin (50 μM) as an example, the DHA content of the invention reaches 35.43 g / L.

[0099] It can be seen from this that the method of the present invention has significant advantages and can utilize plant hormones to regulate the fermentation process of Schizochytrium to increase the oil titer.

[0100] 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. A method for increasing oil titer by regulating the fermentation process of Schizochytrium using plant hormones, characterized in that: The method comprises adding a plant hormone during the fermentation process of Schizochytrium, wherein the plant hormone is one of gibberellin with a final concentration of 50 μM and chlorfenapyr with a final concentration of 75 μM, gibberellin with a final concentration of 50 μM and 6-benzylaminopurine with a final concentration of 50 μM, gibberellin with a final concentration of 50 μM and 50 μM brassinolide with a final concentration, chlorfenapyr with a final concentration of 75 μM and 6-benzylaminopurine with a final concentration of 50 μM, and chlorfenapyr with a final concentration of 75 μM and 50 μM brassinolide with a final concentration; The schizochytrium is Schizochytrium sp. HX-308.

2. The method according to claim 1, characterized in that: The phytohormones are added during the 24 to 72 hours of fermentation.

3. The method according to claim 1, characterized in that: During the fermentation process, the fermentation temperature is 25-29° C., the rotation speed is 150-200 rpm, the time is 72-120 h, and the pH value is maintained between 5.5 and 7.

0.

4. The method according to claim 1, characterized in that: The OD value of the fermentation bacteria is 8-10; the inoculation amount of the fermentation bacteria is 0.8-1.5% of the volume of the fermentation medium.

5. The method according to any one of claims 1 to 4, characterized in that: The specific steps are as follows: The Schizochytrium is inoculated into a seed culture medium, and cultured at a temperature of 28°C for 24 hours to obtain first-level seeds; the first-level seeds are inoculated into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and cultured again at a temperature of 28°C for 24 hours to obtain second-level seeds; the second-level seeds are inoculated into a seed culture medium at an inoculation amount of 10% of the volume of the seed culture medium, and cultured again at a temperature of 28°C for 24 hours, and after the above activation process, a strain for fermentation is obtained; A fermentation strain with an OD600 value of 8-10 was inoculated into a fermentation medium at a rate of 1%, and the fermentation culture was carried out for 120 hours at a temperature of 25°C and a rotation speed of 170 rpm; wherein, plant hormones were added to the fermentation tank at the 24th hour of the fermentation process.

6. The method according to claim 5, characterized in that: The composition of the seed culture medium is: glucose 45g / L, yeast powder 2g / L, sodium glutamate 15g / L, MgCl2·7H2O 4g / L, Na2SO4 15g / L, KCl 1g / L, NaCl 1g / L, MgSO4·7H2O 5g / L, KH2PO4 3g / L, the solvent is water; The components of the fermentation medium were: glucose 90 g / L, yeast powder 4 g / L, sodium glutamate 15 g / L, MgCl2·7H2O 4 g / L, Na2SO4 15 g / L, KCl 1 g / L, NaCl 1 g / L, MgSO4·7H2O 5 g / L, KH2PO4 3 g / L and (NH4)2SO4 4 g / L, and the solvent was water.

7. Use of the method according to any one of claims 1 to 6 in increasing the oil titer during Schizochytrium fermentation.

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