A method and application of optimizing the Schizochytrium fermentation process by chemical regulators to increase the DHA / DPA ratio in oils and fats

Abstract

The present invention belongs to the field of microbial fermentation technology and discloses a method and application for optimizing the Schizochytrium fermentation process using chemical regulators to increase the DHA / DPA ratio in oils and fats. During the Schizochytrium fermentation process, one or more chemical regulators selected from one or more of melatonin, ethanolamine, betaine, and inositol are added to the fermentation broth to increase the DHA / DPA ratio in the oil. By adding specific chemical regulators, the present invention can increase DHA content or reduce DPA accumulation, thereby achieving specific optimization of the DHA / DPA ratio. Compared with genetic engineering, the present invention does not require complex genetic manipulation; the goal is achieved by simply adding chemical regulators during the fermentation process. This method is simple to operate, low-cost, and suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, relates to microbial fermentation and oil production, and in particular to a method and application of optimizing the Schizochytrium fermentation process by using a chemical regulator to increase the DHA / DPA ratio in oil. Background Art

[0002] Schizochytrium is an important industrial microorganism that efficiently synthesizes polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA, C22:6 ω-3) and docosapentaenoic acid (DPA, C22:5 ω-3). These two ω-3 fatty acids have widespread application in food, health supplements, and pharmaceuticals. DHA, due to its important role in nervous system development, retinal function, and cardiovascular health, has a significantly higher market demand than DPA.

[0003] The Schizochytrium sp. HX-308 strain used in the present invention has a strong capacity for both DHA and DPA synthesis, resulting in a high DHA content. However, DPA accumulation is also relatively high, resulting in a suboptimal DHA / DPA ratio (there is no specific optimal value for the DHA / DPA ratio, but based on the current situation of this strain, a higher DHA / DPA ratio is preferred, typically around 2.6 in this strain). This ratio limits the potential application of the fermentation product in high-value-added applications, such as infant formula and high-purity DHA preparations. Therefore, further optimizing the DHA / DPA ratio without significantly reducing DHA production is a core objective of the present invention.

[0004] Specifically, the prior art has the following defects:

[0005] 1. The DHA and DPA synthesis process of Schizochytrium involves multiple enzymatic reactions and complex metabolic pathways, making it difficult to accurately regulate the activity of key enzymes in the metabolic pathways.

[0006] 2. There are few exogenous regulatory factors used to regulate the ratio of DHA and DPA, and the effects of these factors are often not significant or stable.

[0007] 3. The method of increasing DHA production by optimizing fermentation conditions is relatively mature, but its effect in adjusting the ratio of DHA and DPA is limited. Summary of the Invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method and application of optimizing the Schizochytrium fermentation process by chemical regulators to increase the DHA / DPA ratio in oils and fats.

[0009] The technical solution adopted by the present invention to solve its technical problem is:

[0010] A method for optimizing the Schizochytrium fermentation process by using chemical regulators to increase the DHA / DPA ratio in oils and fats. During the Schizochytrium fermentation process, one or more chemical regulators are added to the fermentation broth. The chemical regulators are selected from one or more of melatonin (MT), ethanolamine (ETA), betaine (Bet), and inositol (Inositol) to increase the DHA / DPA ratio in the oils and fats.

[0011] Furthermore, the added amount of the chemical regulator is 1 mM to 200 mM, preferably 1 mM to 100 mM.

[0012] Alternatively, the chemical regulator is added to the fermentation medium at 0 to 36 hours of fermentation production;

[0013] Alternatively, the fermentation temperature is 25-29° C., the rotation speed is 150-200 rpm, and the total fermentation time is 72-120 h.

[0014] Furthermore, the chemical regulator is inositol, which is added to the fermentation broth at a concentration of 50 mM;

[0015] Alternatively, the chemical regulators are inositol and betaine, the concentration of inositol added to the fermentation broth is 50 mM, and the concentration of betaine added to the fermentation broth is 75 mM.

[0016] Furthermore, the fermentation medium used during the fermentation contains glucose, ammonium sulfate, and phosphate.

[0017] Furthermore, by regulating the addition time and concentration of chemical regulators, the DHA / DPA ratio in the oil of Schizochytrium can be significantly increased, with the increase in the ratio ranging from 40% to 95%.

[0018] Furthermore, the Schizochytrium sp. is Schizochytrium sp. HX-308.

[0019] Furthermore, the Schizochytrium is inoculated into a seed culture medium for activation to obtain a fermentation strain; the fermentation strain is inoculated into a fermentation culture medium for fermentation culture, and after the fermentation is completed, the bacteria are collected to extract oil.

[0020] 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.

[0021] Furthermore, the specific steps are as follows:

[0022] Schizochytrium sp. HX-308 was inoculated into a seed culture medium and cultured at 28°C for 24 hours to obtain first-level seeds; the first-level seeds were inoculated into a seed culture medium at an inoculation rate of 10% of the volume of the seed culture medium and cultured again at 28°C for 24 hours to obtain second-level seeds; the second-level seeds were inoculated into a seed culture medium at an inoculation rate of 10% of the volume of the seed culture medium and cultured again at 28°C for 24 hours. After the above activation process, a fermentation strain was obtained; a fermentation strain with an OD600 value of 8-10 was inoculated into a fermentation medium at an inoculation rate of 1%, and fermented and cultured at a temperature of 25°C and a rotation speed of 170 rpm for 120 hours.

[0023] Furthermore, the components of the seed culture medium are: 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;

[0024] 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.

[0025] The application of the above method in improving the DHA / DPA ratio in Schizochytrium fermented oil.

[0026] The advantages and positive effects achieved by the present invention are:

[0027] 1. The present invention increases DHA content or reduces DPA accumulation by adding specific chemical regulators, thereby achieving specific optimization of the DHA / DPA ratio. Compared with genetic engineering, the present invention does not require complex genetic manipulation and only requires the addition of chemical regulators during the fermentation process to achieve the desired results. This method is simple to operate, low-cost, and suitable for large-scale industrial production.

[0028] 2. While optimizing the DHA / DPA ratio, the present invention does not significantly negatively impact the growth performance and fermentation efficiency of Schizochytrium. The strain still maintains high DHA production and fermentation stability, ensuring the feasibility of industrial production.

[0029] 3. The method of the present invention shows good stability and repeatability under different fermentation batches and production conditions.

[0030] 4. The chemical regulators used in the present invention, melatonin (MT), betaine (Bet), and inositol (Inositol), are biocompatible and environmentally friendly substances that will not pollute the environment.

[0031] 5. The present invention optimizes the fermentation process of Schizochytrium sp. HX-308 by adding one or more chemical regulators to the culture medium. By adding these regulators, the DHA / DPA ratio is increased. This invention specifically targets Schizochytrium sp. HX-308 and precisely modulates its metabolic pathways, significantly increasing the DHA content or reducing DPA accumulation. Chemical regulators, including melatonin (MT), ethanolamine (ETA), betaine (Bet), and inositol, are added to the Schizochytrium fermentation medium. The addition of 50 mM inositol increased the DHA / DPA ratio by 51.15%. The combined use of these chemical regulators yielded even more significant results. For example, the addition of 50 mM inositol and 75 mM betaine increased the DHA / DPA ratio by 93.92%. The present invention improves the DHA / DPA ratio and has the advantages of simple operation, low cost, suitability for industrial production, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the effect of adding a chemical regulator on the DHA / DPA ratio in oils and fats in Example 1 of the present invention;

[0033] Figure 2 This is a graph showing the effect of different inositol concentrations on the DHA / DPA ratio in oils and fats in Example 2 of the present invention;

[0034] Figure 3 This is a graph showing the effect of different concentrations of betaine on the DHA / DPA ratio in oils and fats in Example 3 of the present invention;

[0035] Figure 4 This is a graph showing the effect of the combined use of different chemical regulators on the DHA / DPA ratio in oils and fats in Example 4 of the present invention;

[0036] Figure 5 This is a gas chromatogram obtained without adding any chemical regulator in the present invention;

[0037] Figure 6 This is a gas chromatogram after fermentation regulation with the addition of 50 mM inositol + 75 mM betaine in the present invention. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following embodiments.

[0039] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0040] A method for optimizing the Schizochytrium fermentation process by using chemical regulators to increase the DHA / DPA ratio in oils and fats. During the Schizochytrium fermentation process, one or more chemical regulators are added to the fermentation broth. The chemical regulators are selected from one or more of melatonin (MT), ethanolamine (ETA), betaine (Bet), and inositol (Inositol) to increase the DHA / DPA ratio in the oils and fats.

[0041] Preferably, the amount of the chemical regulator added is 1 mM to 200 mM, preferably 1 mM to 100 mM.

[0042] Alternatively, the chemical regulator is added to the fermentation medium at 0 to 36 hours of fermentation production;

[0043] Alternatively, the fermentation temperature is 25-29° C., the rotation speed is 150-200 rpm, and the total fermentation time is 72-120 h.

[0044] Preferably, the chemical regulator is inositol, which is added to the fermentation broth at a concentration of 50 mM;

[0045] Alternatively, the chemical regulators are inositol and betaine, the concentration of inositol added to the fermentation broth is 50 mM, and the concentration of betaine added to the fermentation broth is 75 mM.

[0046] Preferably, the fermentation medium used during fermentation contains glucose, ammonium sulfate and phosphate.

[0047] Preferably, by regulating the addition time and concentration of the chemical regulator, the DHA / DPA ratio in the oil of Schizochytrium can be significantly increased, with the ratio increase ranging from 40% to 95%.

[0048] Preferably, the Schizochytrium sp. is Schizochytrium sp. HX-308.

[0049] Preferably, the Schizochytrium is inoculated into a seed culture medium for activation to obtain a fermentation strain; the fermentation strain is inoculated into a fermentation culture medium for fermentation culture, and after the fermentation is completed, the bacteria are collected to extract oil.

[0050] 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.

[0051] Preferably, the specific steps are as follows:

[0052] Schizochytrium sp. HX-308 was inoculated into a seed culture medium and cultured at 28°C for 24 hours to obtain first-level seeds; the first-level seeds were inoculated into a seed culture medium at an inoculation rate of 10% of the volume of the seed culture medium and cultured again at 28°C for 24 hours to obtain second-level seeds; the second-level seeds were inoculated into a seed culture medium at an inoculation rate of 10% of the volume of the seed culture medium and cultured again at 28°C for 24 hours. After the above activation process, a fermentation strain was obtained; a fermentation strain with an OD600 value of 8-10 was inoculated into a fermentation medium at an inoculation rate of 1%, and fermented and cultured at a temperature of 25°C and a rotation speed of 170 rpm for 120 hours.

[0053] 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;

[0054] 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.

[0055] Specifically, the relevant preparation and detection are as follows:

[0056] The culture medium components used in each embodiment are as follows:

[0057] 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.

[0058] 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.

[0059] Specific fermentation method:

[0060] The Schizochytrium fungus 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 inoculum 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 inoculum amount of 10% of the volume of the seed culture medium and cultured again at a temperature of 28° C. for 24 hours. After the above activation process, a fermentation strain is obtained;

[0061] 0.5 L of fermentation bacteria with an OD600 value of 8-10 was inoculated into 50 L of fermentation medium, and fermented for 120 h at a temperature of 25° C. and a rotation speed of 170 rpm.

[0062] Gas chromatography analysis: After fermentation, adjust the pH of the fermentation broth to 10 with NaOH. Add 0.3% of the fermentation broth's mass of lytic enzyme. Hydrolyze at 55°C for 1 hour, then extract with n-hexane. Allow to stand for separation. Collect the yellow upper organic phase and repeat the extraction until the upper layer becomes colorless. Combine the organic phases and evaporate the solvent to obtain the total oil. Add 2ml of 15% NaOH solution and 5.5ml of 70% methanol in water to the total oil. Incubate in a 65°C water bath for 1.5 hours. Cool to room temperature, extract with 2ml of n-hexane (chromatographic grade), collect the upper organic phase, pass it through a micro-membrane to remove impurities, and analyze it by gas chromatography.

[0063] Example 1 Screening of chemical regulators

[0064] To improve oil yield and EPA content during Schizochytrium sp. HX-308 fermentation, this study screened the most suitable chemical regulator from four sources: melatonin (MT), ethanolamine (ETA), betaine (Bet), and inositol (MI). By comparing the effects of each regulator, the one with the best fermentation performance was identified.

[0065] According to the specific fermentation method described above, melatonin, ethanolamine, betaine, and inositol were added to the fermenter at a concentration of 50 mM at 24 hours of the fermentation process.

[0066] The fermentation was completed at 120 h, and the oil was extracted by the above method and the DHA content and DPA content in the oil were detected by gas chromatography, thereby calculating the DHA / DPA ratio.

[0067] During the screening process, the effects of melatonin, ethanolamine, betaine, and inositol on the DHA / DPA ratio in Schizochytrium fermented oil were tested. The screening results are shown in Table 1 below.

[0068] Table 1

[0069]

[0070] Figure 1 The effects of different chemical modulators (melatonin, ethanolamine, betaine, and inositol) at a concentration of 50 mM on the DHA / DPA ratio in oil fermented by Schizochytrium sp. were demonstrated. The results showed that all four chemical modulators significantly increased the DHA / DPA ratio compared to the control group without chemical modulators. Inositol exhibited the greatest effect, increasing the DHA / DPA ratio from 2.62 in the control group to 3.93, a 50.00% increase. Melatonin, ethanolamine, and betaine also increased the DHA / DPA ratio by 40.83%, 41.22%, and 47.33%, respectively. This suggests that chemical modulators can optimize the DHA / DPA ratio by manipulating the metabolic pathways of Schizochytrium sp., either reducing DPA accumulation or increasing DHA content.

[0071] Example 2 Optimization of inositol concentration

[0072] In order to further increase the DHA / DPA ratio in the oil fermented by Schizochytrium, this example optimizes the concentration of inositol, which has the best effect in Example 1, and determines the most suitable inositol concentration to increase the DHA / DPA ratio.

[0073] The specific fermentation method was essentially the same as in Example 1, except that, in this example, different concentrations of inositol were added to the fermentation tanks at the 24th hour of fermentation, ranging from 1 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM. At the 120th hour of fermentation, the fermentation broth was collected, and the oil was extracted and assayed for DHA and DPA content. The results are shown in Table 2.

[0074] Table 2

[0075]

[0076] Figure 2The effects of different inositol concentrations (1 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM) on the DHA / DPA ratio were analyzed. Results showed that 50 mM inositol had the most significant effect on increasing the DHA / DPA ratio, reaching a ratio of 3.93, a 51.15% increase over the control group. When the inositol concentration exceeded 50 mM, the DHA / DPA ratio began to decrease, indicating that excessive inositol concentrations may inhibit metabolic regulation. This further confirms that 50 mM is the optimal concentration for inositol, maximizing the DHA / DPA ratio.

[0077] Example 3 Optimization of betaine concentration

[0078] In Example 1, the chemical regulator most effective in increasing the DHA / DPA ratio was inositol. Betaine also significantly increased the DHA / DPA ratio. Therefore, the present invention investigated the optimal concentration of betaine to further increase the DHA / DPA ratio in Schizochytrium fermented oils.

[0079] The specific fermentation method was essentially the same as in Example 1, except that, in this example, different concentrations of betaine were added to the fermentation tanks at the 24th hour of fermentation, ranging from 1 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM. At the 120th hour of fermentation, the fermentation broth was collected, the oil was extracted, and the DHA and DPA contents in the oil were measured. The results are shown in Table 3.

[0080] Table 3

[0081]

[0082] Figure 3 The study demonstrated the effects of different betaine concentrations (1 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM) on the DHA / DPA ratio. The results showed that betaine at a concentration of 75 mM had the greatest effect on increasing the DHA / DPA ratio, reaching a ratio of 3.92, a 49.05% increase over the control group. However, when the betaine concentration exceeded 75 mM, the DHA / DPA ratio decreased slightly, indicating that betaine's metabolic regulatory effect is optimal at 75 mM, and that higher concentrations may increase metabolic burden.

[0083] Example 4 Combined use of chemical regulators

[0084] As shown in the previous examples, the use of chemical regulators significantly increases the DHA / DPA ratio in Schizochytrium fermented oils, with betaine and inositol being the most effective. Therefore, in this example, we investigated whether the combined use of multiple chemical regulators could achieve even better results. The results are shown in Table 4.

[0085] Table 4

[0086]

[0087] Figures 4 to 6 The study demonstrated the effects of no chemical modulators and the combined use of different chemical modulators on the DHA / DPA ratio in Schizochytrium fermented oil. The results showed that the combined use of inositol (50 mM) and betaine (75 mM) exhibited a significant synergistic effect compared to the unmodified control (DHA / DPA ratio of 2.63), increasing the DHA / DPA ratio to 5.10, a 93.92% increase. In contrast, the DHA / DPA ratios of inositol (50 mM) or betaine (75 mM) alone were 3.93 (a 49.43% increase) and 3.92 (a 49.05% increase), respectively. Furthermore, the combined use of inositol and melatonin (50 mM) resulted in a DHA / DPA ratio of 4.37 (a 66.16% increase), and the combined use of inositol and ethanolamine (50 mM) resulted in a DHA / DPA ratio of 4.09 (a 55.51% increase). These results indicate that the combination strategy of chemical regulators can work synergistically to significantly enhance the optimization effect of the DHA / DPA ratio.

[0088] Comparative Example Comparison of DHA / DPA ratio in oils fermented with Schizochytrium using chemical regulators and without adding chemical regulators

[0089] The DHA / DPA ratios in oils fermented with Schizochytrium using chemical regulators and those fermented without chemical regulators were compared. As described in Example 1, the DHA / DPA ratio in the control oil, which did not use chemical regulators to optimize Schizochytrium fermentation, was 2.60. However, the DHA / DPA ratios in the oils fermented with chemical regulators increased to varying degrees. The DHA / DPA ratios in the oils fermented with Schizochytrium using melatonin (50 mM), ethanolamine (50 mM), betaine (75 mM), and inositol (50 mM) were 3.69, 3.70, 3.93, and 3.92, respectively, representing increases of 41.92%, 42.31%, 51.15%, and 50.77%, respectively.

[0090] The present invention increases the DHA / DPA ratio in oil by adding a chemical regulator to the fermentation medium of Schizochytrium, thereby providing a reference for subsequent metabolic engineering modification.

[0091] Although the embodiments of the present invention have been 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 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 optimizing the Schizochytrium fermentation process by using chemical regulators to increase the DHA / DPA ratio in oils and fats, characterized in that: During the fermentation process of Schizochytrium, chemical regulators are added to the fermentation broth, wherein the chemical regulators are inositol and betaine, the concentration of inositol added to the fermentation broth is 50 mM, and the concentration of betaine added to the fermentation broth is 75 mM; At 24 hours of fermentation production, adding the chemical regulator to the fermentation medium; The fermentation temperature is 25-29°C, the rotation speed is 150-200 rpm, and the total fermentation time is 72-120 hours; The fermentation medium used during fermentation contains glucose, ammonium sulfate, and phosphate; The schizochytrium is Schizochytrium sp. HX-308; The Schizochytrium is inoculated into a seed culture medium for activation to obtain a fermentation strain; the fermentation strain is inoculated into a fermentation culture medium for fermentation and culture; after the fermentation is completed, the bacteria are collected to extract oil; 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; The specific steps are as follows: Schizochytrium sp. HX-308 was inoculated into a seed culture medium and cultured at 28°C for 24 hours to obtain first-level seeds; the first-level seeds were inoculated into a seed culture medium at an inoculum amount of 10% of the volume of the seed culture medium and cultured again at 28°C for 24 hours to obtain second-level seeds; the second-level seeds were inoculated into a seed culture medium at an inoculum amount of 10% of the volume of the seed culture medium and cultured again at 28°C for 24 hours, and after the above activation process, a fermentation strain was obtained; a fermentation strain with an OD600 value of 8-10 was inoculated into a fermentation medium at an inoculum amount of 1%, and fermented at 25°C and a rotation speed of 170 rpm for 120 hours; 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; 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.

2. Use of the method according to claim 1 in increasing the DHA / DPA ratio in oils fermented by Schizochytrium.

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