Method for improving production of palm oleic acid by schizochytrium through fermentation regulation and application

Abstract

The application belongs to the technical field of microbial fermentation, and discloses a method for improving the production of palm oleic acid in Schizochytrium sp. by fermentation regulation and application. In the fermentation process of Schizochytrium sp., one or more combined regulation strategies of controlling the fermentation temperature, optimizing the carbon-nitrogen ratio of the fermentation medium and adjusting the pH value of the fermentation liquid are adopted to change the metabolic flow direction, so as to improve the accumulation of palm oleic acid C16:1 in oil. Compared with genetic engineering modification, the method does not need complex genetic operation and has high safety. Through verification of a 5L fermentation tank, the proportion of palm oleic acid in oil can be significantly increased from less than 1% in conventional fermentation to 16.12%, and the oil yield is stable, so that the method has good industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular to a method and application for increasing palmitoleic acid production by Schizochytrium. Background Technology

[0002] Palmitoleic acid (C16:1 ω-7) is a monounsaturated fatty acid with important physiological functions. Studies have shown that it has significant effects in anti-inflammation, improving the body's sensitivity to insulin, improving lipid metabolism, and inhibiting hepatic steatosis, and has broad market prospects in the pharmaceutical, health food, and high-end cosmetic fields. Currently, palmitoleic acid is mainly obtained from plant and animal extracts, such as sea buckthorn fruit oil, macadamia nut oil, or deep-sea fish oil. However, plant sources are limited by season, climate, and arable land, resulting in unstable yields and high extraction costs; animal sources face the risks of resource depletion and marine pollutant accumulation. Developing a microbial synthetic route with relatively simple product components and industrial scale-up potential has become key to overcoming the bottleneck in obtaining high-purity palmitoleic acid.

[0003] Schizochytrium ( Schizochytrium As a marine heterotrophic fungus, docosahexaenoic acid (DHA) has industrial advantages such as fast growth rate, high fermentation density, and strong oil accumulation capacity (up to 50% or more of dry weight), and has become the main engineered strain for industrial production of docosahexaenoic acid (DHA).

[0004] However, under conventional fermentation conditions (25-28°C, neutral pH), the metabolic flux within *Schizochytrium* is significantly directed towards the synthesis of long-chain polyunsaturated fatty acids (especially DHA). The accumulation of palmitoleic acid in wild-type *Schizochytrium* oil is extremely low, typically less than 1% of total fatty acids (e.g., only 0.46%-0.68% in the strain used in this invention), greatly limiting its potential application as a palmitoleic acid-producing strain.

[0005] Therefore, how to efficiently produce palmitic acid using Schizochytrium, a mature industrial chassis cell, has become a research hotspot in the fields of synthetic biology and fermentation engineering.

[0006] Specifically, the existing technology has the following drawbacks:

[0007] 1. The accumulation of palmitic acid can be achieved through metabolic engineering techniques (such as knocking out fatty acid elongase genes or overexpressing Δ9 desaturase genes), but the application of genetically modified microorganisms (GMOs) in the food and cosmetic fields faces challenges such as strict regulatory oversight, ethical review, and low public acceptance.

[0008] 2. Methods to increase DHA production by optimizing fermentation conditions are relatively mature, but research on increasing palmitic acid production is limited.

[0009] Therefore, developing a process that can significantly increase the proportion of palmitoleic acid in wild-type Schizochytrium without genetic modification, simply by regulating fermentation process parameters (temperature, pH, nutrient source), has significant industrial value and is the core objective of this invention. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for increasing palmitoleic acid production by Schizochytrium through fermentation regulation, involving the utilization of wild-type Schizochytrium (… Schizochytrium (sp.) A method to increase the proportion of palmitoleic acid (C16:1) in oils by controlling fermentation temperature, fermentation broth pH and optimizing the carbon-nitrogen ratio (C / N) of the culture medium.

[0011] The technical solution adopted by this invention to solve its technical problem is:

[0012] A method for increasing palmitoleic acid production in Schizochytrium by fermentation regulation, wherein the method employs one or more combined regulatory strategies, including controlling fermentation temperature, optimizing the carbon-nitrogen ratio of the fermentation medium, and adjusting the pH of the fermentation broth, to change the metabolic flow of the microorganisms and thereby increase the accumulation of palmitoleic acid C16:1 in the oil.

[0013] Furthermore, the fermentation temperature is controlled by maintaining a constant value throughout the fermentation process, between 18°C ​​and 26°C.

[0014] Alternatively, the optimized fermentation medium carbon-nitrogen ratio is to control the carbon-nitrogen ratio between 5 and 30;

[0015] Alternatively, adjusting the pH of the fermentation broth means maintaining the pH of the fermentation broth at 7.0-9.0;

[0016] Alternatively, the Schizochytrium fungus is Schizochytrium sp. HX-308, its accession number is CCTCC No.M209059.

[0017] Furthermore, the optimization of the carbon-nitrogen ratio of the fermentation medium is achieved by adjusting the content of yeast powder or monosodium glutamate in the fermentation medium components; the adjustment of the pH value of the fermentation broth is achieved by adjusting sodium hydroxide.

[0018] Furthermore, the yeast content in the fermentation medium is from 3 g / L to 52 g / L;

[0019] Alternatively, the sodium glutamate content in the fermentation medium is from 2 g / L to 72 g / L.

[0020] Furthermore, the fermentation temperature is 24°C;

[0021] Alternatively, the fermentation medium has a carbon-to-nitrogen ratio of 15;

[0022] Alternatively, the pH of the fermentation broth may be maintained at 8.5;

[0023] Alternatively, a combined control strategy can be adopted, which involves simultaneously setting the fermentation temperature to 24°C, adjusting the carbon-to-nitrogen ratio of the fermentation medium to 15, and controlling the pH of the fermentation broth to 8.5.

[0024] Furthermore, the fermentation process includes the following steps:

[0025] After activating the Schizochytrium fungus by inoculating it into a seed culture medium, a fermentation strain is obtained. The fermentation strain is then inoculated into a fermentation culture medium for fermentation. After fermentation, the cells are collected to extract the oil.

[0026] Furthermore, the OD600 value of the fermentation strain is 8-10; the inoculation amount of the fermentation strain is 0.8%-1.5% of the fermentation medium volume.

[0027] Furthermore, the specific steps are as follows:

[0028] Strain activation and seed culture preparation: Wild-type Schizochytrium ( Schizochytrium sp HX-308 was inoculated into seed culture medium and cultured at 28℃ for 24 hours to obtain primary seed; the primary seed was transferred to fresh seed culture medium at an inoculation rate of 10% and cultured at the same temperature for 24 hours to obtain secondary seed; the secondary seed was then transferred again at an inoculation rate of 10% and cultured at the same temperature for 24 hours to obtain fermentation strain.

[0029] Fermentation culture: Select the above-mentioned fermentation strains with an OD600 value of 8-10 and inoculate them into the fermentation medium at an inoculation rate of 1%. Fermentate for 120 h at a temperature of 18℃ to 26℃ and a rotation speed of 170 rpm. Adjust the initial pH of the fermentation medium to 8.5 by adding 1M NaOH. During the fermentation process, the pH is kept constant at 8.5 by adding NaOH solution continuously.

[0030] Furthermore, the seed culture medium comprises: 45 g / L glucose, 2 g / L yeast extract, 15 g / L monosodium glutamate, 4 g / L MgCl2·7H2O, 15 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, 5 g / L MgSO4·7H2O, and 3 g / L KH2PO4, with water as the solvent;

[0031] The fermentation medium consisted of: 90 g / L glucose, 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 4 g / L (NH4)2SO4, along with yeast extract and sodium glutamate adjusted according to the required carbon-to-nitrogen ratio. The solvent was a 0.1 M phosphate buffer system. The initial pH of the fermentation medium was 8.5, and the initial carbon-to-nitrogen ratio was 15.

[0032] The method described above is applied to increase palmitoleic acid production in Schizochytrium.

[0033] The advantages and positive effects of this invention are as follows:

[0034] 1. This invention increases the content of the target product C16:1 from less than 0.5% to about 16% through the synergistic regulation of environmental factors.

[0035] 2. Compared with genetic engineering, this invention does not require complex gene manipulation; the goal can be achieved simply by changing conventional fermentation parameters such as temperature and pH. This method is simple to operate, low in cost, and suitable for large-scale industrial production.

[0036] 3. This invention optimizes the proportion of palmitoleic acid in oils without significantly negatively impacting the growth performance and fermentation efficiency of Schizochytrium. The strain maintains high DHA yield and fermentation stability, ensuring the feasibility of industrial production.

[0037] 4. This invention breaks with the conventional understanding that Schizochytrium mainly produces DHA. Through precise regulation of environmental factors, it successfully enriches palmitic acid, significantly improving the application value of Schizochytrium fermentation products and giving them greater market competitiveness.

[0038] 5. The method of the present invention exhibits good stability and repeatability under different fermentation batches and production conditions.

[0039] 6. This invention directly uses wild-type strains, without the need for complex gene editing, complies with food safety regulations, and the strains have good genetic stability.

[0040] 7. The successful verification of this invention on a 5L fermenter shows that the process has good industrial scale-up potential, overcomes the defect of extremely low palmitoleic acid yield of wild-type Schizochytrium in the prior art, and significantly increases the proportion of palmitoleic acid in oil by changing the metabolic flow of the cells through multi-dimensional fermentation regulation.

[0041] 8. This invention targets wild-type Schizochytrium ( SchizochytriumThis invention addresses the low palmitoleic acid accumulation under conventional fermentation conditions by employing a multi-dimensional control strategy. This strategy involves controlling fermentation temperature, optimizing the carbon-to-nitrogen ratio (C / N) of the fermentation broth, and adjusting the pH of the fermentation broth. By altering the metabolic flow of the microorganisms, the invention significantly increases the proportion of palmitoleic acid in oils through multi-dimensional fermentation parameter control. Compared to genetic engineering, this method requires no complex gene manipulation and is highly safe. Validated in a 5L fermenter, this process significantly increases the proportion of palmitoleic acid in oils from less than 1% in conventional fermentation to 16.12%, with stable oil yield, demonstrating excellent potential for industrial application. Attached Figure Description

[0042] Figure 1 This is a graph showing the effect of different fermentation temperatures on the proportion of palmitoleic acid in the oil of Schizochytrium in Example 1 of this invention;

[0043] Figure 2 This is a graph showing the effect of different carbon-nitrogen ratios on the proportion of palmitoleic acid in the oil of Schizochytrium in Example 2 of this invention;

[0044] Figure 3 This is a graph showing the effect of different fermentation broth pH environments on the proportion of palmitoleic acid in the oil of Schizochytrium in Example 3 of this invention;

[0045] Figure 4 This is a comparison chart of the proportion of palmitic acid in Example 4 of this invention under conventional fermentation and the optimized process of this invention (joint regulation and 5L tank scale-up);

[0046] Figure 5 This is a gas chromatogram under the conventional fermentation conditions of this invention (usually 28°C, pH around 6.5);

[0047] Figure 6 This is a gas chromatogram of the 5L fermenter under optimized conditions in Example 4 of this invention.

[0048] Schizochytrium in this invention Schizochytrium sp. The HX-308 strain is a strain in the prior art. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M209059, as disclosed in Chinese patent publication CN116478835A. Detailed Implementation

[0049] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0050] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0051] A method for increasing the proportion of palmitoleic acid in the oil of Schizochytrium can be proposed by regulating fermentation. During the fermentation process of Schizochytrium, the metabolic flow of the cells is changed by controlling the fermentation temperature, optimizing the carbon-nitrogen ratio of the fermentation broth, and adjusting the pH value of the fermentation broth, thereby increasing the accumulation of palmitoleic acid.

[0052] Specifically, the control method includes a combination of one or more of the following strategies:

[0053] Furthermore, a low-temperature fermentation strategy is adopted. This low-temperature fermentation strategy refers to maintaining a constant fermentation temperature throughout the entire fermentation process, controlling it between 18°C ​​and 26°C, for 120 hours.

[0054] Preferably, when the fermentation temperature is controlled at 24°C, the method can significantly increase the proportion of palmitoleic acid in the oil of Schizochytrium. The proportion of palmitoleic acid is significantly increased from 0.68% in conventional fermentation at 28°C to 4.06%.

[0055] Furthermore, at the preferred fermentation temperature, the carbon-nitrogen ratio of the fermentation broth system is adjusted by regulating the content of yeast powder or monosodium glutamate in the fermentation medium.

[0056] Furthermore, the yeast content in the fermentation medium ranges from 3 g / L to 52 g / L.

[0057] Alternatively, the sodium glutamate content in the fermentation medium can be between 2 g / L and 72 g / L.

[0058] Furthermore, the carbon-nitrogen ratio is adjusted to be between 5 and 30 to promote the synthesis and accumulation of palmitoleic acid.

[0059] Preferably, by changing the sodium glutamate content to make the carbon-nitrogen ratio of the fermentation medium 15, the method can significantly increase the proportion of palmitoleic acid in the oil of Schizochytrium, increasing the proportion of palmitoleic acid in the oil to 8.27% compared with conventional fermentation.

[0060] Furthermore, under the preferred fermentation temperature and carbon-to-nitrogen ratio conditions, the pH of the fermentation broth was maintained at 7.0-9.0 by adding sodium hydroxide.

[0061] Preferably, the pH of the fermentation broth is maintained at around 8.5. Under this alkaline environment, the proportion of palmitoleic acid increases significantly, reaching 12.63%.

[0062] Furthermore, the above-mentioned preferred conditions were simultaneously adopted: fermentation temperature 24℃, carbon-nitrogen ratio adjusted to 15, and fermentation broth pH controlled at 8.5. The process was scaled up to a 5L fermenter, and temperature, pH, and feeding were precisely controlled at the fermenter level. The proportion of palmitoleic acid in the oil was ultimately increased to 16.12%, with no significant impact on oil yield (65g / L), and palmitoleic acid yield reached 10.4g / L.

[0063] Furthermore, the fermentation medium used during fermentation contains glucose, ammonium sulfate, and phosphate.

[0064] Furthermore, the Schizochytrium fungus is Schizochytrium sp.HX-308, its accession number is CCTCCNo.M209059.

[0065] Further, after activating the Schizochytrium fungus by inoculating it into a seed culture medium, a fermentation strain is obtained; the fermentation strain is then inoculated into a fermentation culture medium for fermentation culture, and after fermentation, the cells are collected to extract the oil.

[0066] Furthermore, the OD value of the fermentation strain is 8-10; the inoculation amount of the fermentation strain is 0.8-1.5% of the fermentation medium volume.

[0067] Furthermore, the specific steps are as follows:

[0068] Strain activation and seed culture preparation: Wild-type Schizochytrium ( Schizochytrium sp. HX-308 was inoculated into seed culture medium and cultured at 28℃ for 24h to obtain primary seed; the primary seed was transferred to fresh seed culture medium at an inoculation rate of 10% and cultured at the same temperature for 24h to obtain secondary seed; the secondary seed was then transferred again at an inoculation rate of 10% and cultured at the same temperature for 24h to obtain the fermentation strain.

[0069] Fermentation culture: Select the above-mentioned fermentation strains with an OD600 value of 8-10 and inoculate them into the fermentation medium at a 1% inoculum rate. Fermentation conditions are controlled as follows: rotation speed 170 rpm, culture period 120 h, and temperature set as needed. Process samples are collected at 48 h, 72 h, 96 h, and 120 h of culture. The remaining glucose level in each bottle is measured after each sampling. After the first three measurements, any missing glucose is replenished to maintain a stable fermentation environment.

[0070] Furthermore, the seed culture medium comprises: 45 g / L glucose, 2 g / L yeast extract, 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, and 3 g / L KH2PO4, with water as the solvent.

[0071] Furthermore, the fermentation medium is composed of, for example, sodium glutamate adjusted to a C / N ratio of 15; the fermentation medium consists of: glucose 90 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, yeast extract 4 g / L, sodium glutamate 13.9 g / L, and a 0.1 M phosphate buffer system as the solvent; the initial pH of the fermentation medium is 8.5, and the initial carbon-nitrogen ratio (C / N) of the fermentation medium is 15.

[0072] The fermentation medium for conventional fermentation consists of: 4 g / L yeast extract, 20 g / L monosodium glutamate, and water as the solvent. The conventional fermentation medium also consists of: 90 g / L glucose, 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 4 g / L (NH₄)₂SO₄, 4 g / L yeast extract, 20 g / L monosodium glutamate, and water as the solvent. The initial pH of the fermentation medium is 6.5, and the initial carbon-to-nitrogen ratio (C / N) is 13.

[0073] Specifically, the relevant preparation and testing methods are as follows:

[0074] Specific fermentation method:

[0075] Schizochytrium was inoculated into a seed culture medium and cultured at 28°C for 24 hours to obtain primary seeds. Primary seeds were then inoculated into the seed culture medium at an inoculation rate of 10% of the seed culture medium volume and cultured again at 28°C for 24 hours to obtain secondary seeds. Secondary seeds were then inoculated into the seed culture medium at an inoculation rate of 10% of the seed culture medium volume and cultured again at 28°C for 24 hours. Through this activation process, the fermentation strain was obtained.

[0076] Take 0.5L of fermentation strain with an OD600 value of 8-10 and inoculate it into 50L of fermentation medium. Ferment and culture for 120h at a temperature of 25℃ and a rotation speed of 170rpm.

[0077] Lipid content determination: To determine lipid content, 100 mL of fermentation broth was taken at specific time points, and the pH was adjusted to 11-13 with 3M sodium hydroxide aqueous solution. Then, *Schizochytrium* cell-wall-breaking enzyme was added at a mass ratio of 0.3%, and the mixture was shaken at 50℃ and 100 rpm for 5 hours. After confirming complete cell disruption by microscopic examination, an equal volume of anhydrous ethanol and n-hexane were added sequentially, mixed thoroughly, and allowed to stand for separation. The upper n-hexane extract was collected, and the extraction was repeated 2-3 times with an equal volume of n-hexane. All n-hexane phases were combined. The obtained extract was evaporated to remove the solvent using a vacuum rotary evaporator to obtain crude oil. The rotary evaporator flask was dried in a 65℃ oven to constant weight, cooled, and weighed to calculate the oil content.

[0078] Methyl esterification of oils: First, weigh approximately 10 mg of oil or 50 mg of wet bacterial cells into a 2 mL EP tube, then add 0.5 mL of 1 M sodium hydroxide-methanol solution. Initiate methyl esterification by shaking at 1200 rpm for 5 hours at room temperature. After the reaction is complete, add 40 μL of concentrated sulfuric acid to terminate the reaction. Next, add 1 mL of n-hexane and extract by shaking at 1200 rpm for 25-30 minutes. Then, centrifuge at 12000 rpm for 5 minutes, and finally use the upper n-hexane phase for gas chromatography analysis.

[0079] Gas chromatography analysis: This experiment used a DB-23 column (60m × 0.25mm × 0.25μm) and a flame ionization detector (FID). The injection port and detector temperatures were set to 250℃ and 280℃, respectively. 1μL of sample was injected under the conditions of high-purity nitrogen as carrier gas and a split ratio of 30:1. The temperature program was as follows: initial column temperature 100℃, increased to 196℃ at 25℃ / min, then increased to 220℃ at 2℃ / min and held for 12 minutes. Gas flow rates were set as follows: column flow rate 0 mL / min, make-up gas 30 mL / min, hydrogen 40 mL / min, and air 400 mL / min.

[0080] Example 1: Effect of isothermal fermentation at different temperatures on the proportion of palmitoleic acid in oils

[0081] This embodiment aims to investigate the effects of a low-temperature fermentation strategy on Schizochytrium (… Schizochytrium The effect of sp. HX-308 on palmitoleic acid production was investigated to screen for the optimal fermentation temperature.

[0082] This invention aims to investigate the effect of temperature on increasing the growth of Schizochytrium (…). SchizochytriumThe effect of palmitoleic acid content during the fermentation of sp. HX-308 was investigated using a single-factor experiment. Following the specific fermentation method described above, the prepared fermentation strain was inoculated at a 1% inoculum into the basal fermentation medium (the formulation of which was: yeast extract 4 g / L, monosodium glutamate 20 g / L, C / N ratio approximately 13. The basal fermentation medium is the conventional fermentation medium, with the following components: glucose 90 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, (NH4)2SO4 4 g / L, yeast extract 4 g / L, monosodium glutamate 20 g / L, and water as the solvent; the initial pH of the fermentation medium was 6.5, and the initial C / N ratio was 13). The fermentation speed was 170 rpm, and the fermentation cycle was 120 h. In this embodiment, the initial pH of fermentation is 6.5, and no artificial pH adjustment is performed during the fermentation process, allowing it to change naturally with the metabolism of the cells.

[0083] Different constant fermentation temperatures were set, preferably 18℃, 20℃, 22℃, 24℃, and 26℃. A conventional fermentation temperature of 28℃ was set as a control group.

[0084] Fermentation ended at 120 hours, and samples were taken using the above method. The contents of DHA, DPA, and palmitoleic acid (C16:1) in the oil were then detected by gas chromatography.

[0085] During the screening process, the proportion of palmitoleic acid in the fermented oil of Schizochytrium was tested at fermentation temperatures of 18℃, 20℃, 22℃, 24℃, 26℃, and 28℃. The results are shown in Table 1 below.

[0086] Table 1

[0087]

[0088] Figure 1 This study demonstrated the effect of different fermentation temperatures (20℃, 22℃, 24℃, 26℃, and 28℃) on the palmitoleic acid (C16:1) ratio in oils fermented by *Schizochytrium*. The results showed that, compared with the control group fermented at conventional 28℃, suitable low-temperature fermentation conditions significantly increased the palmitoleic acid ratio. The optimal temperature was 24℃, increasing the palmitoleic acid ratio from 0.687% in the control group (28℃) to 4.061%. Furthermore, under fermentation conditions of 20℃ and 22℃, the palmitoleic acid ratio also increased to 1.845% and 2.954%, respectively. This indicates that temperature regulation can effectively induce the synthesis of monounsaturated fatty acids by altering the metabolic flow of *Schizochytrium*, thereby significantly optimizing the palmitoleic acid ratio in the oils.

[0089] Example 2: Effect of Optimized Carbon-to-Nitrogen Ratio on the Proportion of Palmitoleic Acid in Oils

[0090] To further increase the proportion of palmitic acid in the fermented oil of Schizochytrium, this example further investigates the effect of the carbon-nitrogen ratio on the accumulation of C16:1 at the preferred temperature (24°C) determined in Example 1.

[0091] The specific fermentation method is basically the same as in Example 1, except that: the glucose concentration in the fermentation medium is kept constant, and the initial carbon-to-nitrogen ratio (C / N) is set by adjusting the content of yeast extract or monosodium glutamate in the fermentation medium, preferably 5, 10, 15, or 25. The fermentation temperature is kept constant at 24℃. For example, using monosodium glutamate to adjust the C / N ratio to 15: the composition of the fermentation medium is: glucose 90 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, yeast extract 4 g / L, monosodium glutamate 13.9 g / L, and water as the solvent; the initial pH of the fermentation medium is 6.5, and the initial carbon-to-nitrogen ratio (C / N) of the fermentation medium is 15.

[0092] At 120 hours of fermentation, the fermentation broth was collected, and the proportion of fatty acids in the oil was determined by the internal standard method. The results are shown in Table 2.

[0093] Table 2

[0094]

[0095] Figure 2 This study demonstrates the effects of different initial C / N ratios (5, 10, 15, 25) and different nitrogen source adjustments (monosodium glutamate, yeast extract) on the proportion of palmitoleic acid (C16:1) in the fermented oil of *Schizochytrium* at an optimal temperature (24°C). The results show that adjusting the C / N ratio with monosodium glutamate significantly promotes palmitoleic acid accumulation more effectively than adjusting with yeast extract. Specifically, when the C / N ratio was adjusted to 15 with monosodium glutamate, the palmitoleic acid proportion reached a peak of 8.271%, a significant increase compared to 1.386% at a lower C / N ratio (C / N=5). However, when the C / N ratio was further increased to 25, the palmitoleic acid proportion decreased to 3.852%. This indicates that excessively high or low C / N ratios are detrimental to the targeted enrichment of C16:1, and the optimal fermentation condition is a C / N ratio of 15 to enhance the C16:1 proportion.

[0096] Comparative Example 1

[0097] The specific fermentation method is basically the same as in Example 1, except that: the glucose concentration in the fermentation medium is kept constant, and the initial carbon-to-nitrogen ratio (C / N) is set to 15 by adjusting the content of yeast powder or monosodium glutamate in the fermentation medium. The fermentation temperature is 28℃. After 120 hours of fermentation, the fermentation broth is collected, and the proportion of fatty acids in the oil is determined by the internal standard method. The fermentation medium consists of: glucose 90 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, with water as the solvent.

[0098] The results are shown in Table 3.

[0099] Table 3

[0100]

[0101] As shown in Table 3, at the conventional fermentation temperature (28°C), even with the C / N ratio optimized to 15 (adjusted by monosodium glutamate), the proportion of palmitoleic acid (C16:1) in the *Schizochytrium* oil was only 1.054%, while the DHA content remained at 51.252%. Compared to the results in Example 2 with the same C / N conditions but at 24°C (C16:1 was 8.271%), the proportion of palmitoleic acid decreased by 7.2%. This strongly demonstrates that low temperature (24°C) is a prerequisite for C16:1 accumulation; simply optimizing the nutrient source while ignoring temperature control cannot change the main metabolic shift of the microorganism towards long-chain fatty acid (DHA) synthesis.

[0102] Meanwhile, by comparing Example 2 and Comparative Example 1, it can be seen that the fermentation temperature of 24°C and the initial carbon-nitrogen ratio (C / N) of the fermentation medium in the method of the present invention have a synergistic effect, which can synergistically increase the C16:1 ratio in the prepared fermentation broth.

[0103] Example 3: Effect of fermentation broth pH on palmitoleic acid ratio

[0104] In Example 1, the optimal fermentation temperature for increasing the proportion of palmitic acid (C16:1) is 24°C; in Example 2, the optimal sodium glutamate-adjusted carbon-nitrogen ratio of the fermentation medium for increasing the proportion of palmitic acid is 15.

[0105] Based on the preferred temperature (24°C) and preferred carbon-nitrogen ratio (C / N=15), this embodiment further explores the effect of pH value on the proportion of palmitoleic acid in the oil of Schizochytrium.

[0106] The specific fermentation method is basically the same as in Example 1, except that in the preparation of the fermentation medium, this example abandons the traditional deionized water and uses a 0.1M phosphate buffer system instead, and adjusts the initial pH to the target value by adding 1M NaOH or HCl. This improvement aims to enhance the system's resistance to acid and alkali shocks through a chemical buffering mechanism, maintaining the optimal physiological pH environment for microbial growth. The preferred pH values ​​for the fermentation broth are 7.0, 8.0, 8.5, and 9.0. A pH of 6.5 is used as a control. The results are shown in Table 4.

[0107] For example, the C / N ratio was adjusted to 15 using monosodium glutamate: The fermentation medium consisted of: glucose 90 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, yeast extract 4 g / L, monosodium glutamate 13.9 g / L, and a 0.1 M phosphate buffer system as the solvent; the initial pH of the fermentation medium was 6.5, and the initial C / N ratio was 15.

[0108] Table 4

[0109]

[0110] Figure 3 This study demonstrates that, under optimal temperature (24°C) and optimal carbon-to-nitrogen ratio (C / N=15), the proportion of palmitoleic acid in the oil rapidly increases with pH from 6.5 to 8.5. At pH 6.5, the C16:1 ratio is 7.491%; however, when the pH is adjusted to 8.5, the C16:1 ratio reaches a peak of 12.631%, an increase of approximately 68.6% compared to the control group. Furthermore, when the pH is increased to 9.0, the C16:1 ratio decreases to 9.855%, indicating that an excessively alkaline environment may inhibit cell metabolism. Simultaneously, DHA decreases from 43.151% (control group) to 39.942% (pH 8.5). This indicates that pH adjustment of the fermentation broth can significantly increase the proportion of palmitoleic acid in the oil by altering the metabolic flow of Schizochytrium.

[0111] Meanwhile, by comparing Examples 2 and 3, it can be seen that the initial carbon-nitrogen ratio (C / N) of the fermentation medium of the present invention being 15 and the initial pH of the fermentation medium being 8.5 have a synergistic effect, which can synergistically increase the C16:1 ratio in the prepared fermentation broth.

[0112] Comparative Example 2

[0113] The specific fermentation method is basically the same as in Example 1, except that in the preparation of the fermentation medium, this example abandons the traditional deionized water and uses a 0.1M phosphate buffer system instead, and adjusts the initial pH to the target value by adding 1M NaOH or HCl. This improvement aims to enhance the system's resistance to acid and alkali shocks through a chemical buffering mechanism, maintaining the optimal physiological pH environment for microbial growth. The initial pH of the fermentation medium was set at 8.5. The fermentation temperature was kept constant at 28℃. After 120 hours of fermentation, the fermentation broth was collected, and the proportion of fatty acids in the oil was determined using the internal standard method. For example, the C / N ratio was adjusted to 15 using monosodium glutamate: The fermentation medium consisted of: 90 g / L glucose, 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 4 g / L (NH4)2SO4, 4 g / L yeast extract, and 13.9 g / L monosodium glutamate, with a 0.1 M phosphate buffer system as the solvent; the initial pH of the fermentation medium was 8.5, and the initial C / N ratio was 15.

[0114] The results are shown in Table 5.

[0115] Table 5

[0116]

[0117] As shown in Table 5, at the conventional fermentation temperature (28℃), adjusting the pH of the fermentation broth to 8.5 only resulted in a limited increase in the proportion of palmitoleic acid (C16:1) in the oil, reaching only 2.125%. In contrast, the combined regulation strategy of "24℃ + pH 8.5" used in Example 3 of this invention achieved a C16:1 proportion as high as 12.631%, which is 5.9 times that of the comparative example. This significant difference indicates that the effect of alkaline stress (pH 8.5) on the interception of metabolic flux is temperature-dependent. Only by applying alkaline stress under low-temperature conditions (24℃) can a synergistic effect of "1+1>2" be produced, leading to an explosive accumulation of palmitoleic acid.

[0118] Meanwhile, by comparing Example 3 and Comparative Example 2, it can be seen that the fermentation temperature of 24°C and the initial pH of the fermentation medium of 8.5 in the method of the present invention have a synergistic effect, which can synergistically increase the C16:1 ratio in the prepared fermentation broth.

[0119] A comparison of the palmitoleic acid ratio in oils fermented by Schizochytrium under optimized fermentation conditions in a 5L fermenter and under conventional fermentation conditions.

[0120] The process of Example 3 was scaled up to a 5L fermenter to verify its industrialization potential.

[0121] The specific fermentation method is basically the same as in Example 1, except that in this example, the prepared seed culture is inoculated into the fermenter at a rate of 10%. The temperature is precisely controlled at 24±0.5℃; the pH is kept constant at 8.5 by automatically adding NaOH solution; and the dissolved oxygen (DO) is maintained above 20% by a cascaded control of the rotation speed. Based on the monitoring results, glucose feed solution is automatically added, and an appropriate amount of nitrogen source is added according to a C / N ratio of 15 to maintain a stable fermentation environment.

[0122] The specific components of the fermentation medium are as follows:

[0123] Control group: The fermentation medium consisted of: glucose 90 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, yeast extract 4 g / L, monosodium glutamate 20 g / L, and water as the solvent; the initial pH of the fermentation medium was 6.5, and the initial carbon-nitrogen ratio (C / N) of the fermentation medium was 20.

[0124] Preferred conditions: The fermentation medium consisted of: glucose 90 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, yeast extract 4 g / L, monosodium glutamate 13.9 g / L, and a 0.1 M phosphate buffer system as the solvent; the initial pH of the fermentation medium was 8.5, and the initial carbon-nitrogen ratio (C / N) of the fermentation medium was 15.

[0125] The results are shown in Table 6.

[0126] Conventional fermentation conditions: The prepared seed culture is inoculated into the fermenter at a rate of 10%. A suitable dissolved oxygen level is maintained by adjusting the stirring speed or aeration rate. More preferred fermentation parameters are 28°C, 170 rpm, and 120 h of incubation.

[0127] Table 6

[0128]

[0129] like Figure 4 , Figure 5 and Figure 6As shown, this study compares the effects of optimized fermentation conditions (24°C, C / N=15, pH 8.5) and conventional fermentation conditions on the proportion of palmitoleic acid (C16:1) in *Schizochytrium* oil during scale-up validation in a 5L fermenter. The palmitoleic acid proportion in the 5L fermenter control group (1.427%) was higher than that in the shake flask control group in Example 1 (0.687%). This is mainly due to the fact that the fermenter system's control precision for pH and dissolved oxygen is far superior to that of the shake flask, enabling it to maintain an optimal alkaline stress environment during large-scale, high-density fermentation and improve the basal metabolic state of the cells. The experimental results show that the optimized conditions significantly increased the proportion of palmitoleic acid in the oil. Under conventional fermentation conditions (control group), the C16:1 proportion was only 1.427%; while under the combined regulation of the optimized conditions, the C16:1 proportion reached as high as 16.154%, approximately 11.3 times that of the control group. Simultaneously, DHA was adjusted from 52.355% (control group) to 37.525% (optimized group). This indicates that the combined regulation strategy still exhibits excellent metabolic regulation effects at the 5L fermenter level, successfully achieving high-yield industrial production of palmitoleic acid by altering the metabolic flow. This demonstrates the high industrial application value of the proposed "low temperature-alkaline environment-carbon-nitrogen ratio" combined regulation strategy.

[0130] This invention improves the proportion of palmitoleic acid (C16:1) in oils by precisely controlling fermentation parameters such as temperature, pH, and carbon-nitrogen ratio during the fermentation process of Schizochytrium, providing a reference for subsequent metabolic engineering modifications.

[0131] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for increasing palmitoleic acid production from Schizochytrium fungi through fermentation regulation, characterized in that: The method employs a combination of regulatory strategies, including controlling fermentation temperature, optimizing the carbon-nitrogen ratio of the fermentation medium, and adjusting the pH of the fermentation broth, during the fermentation process of Schizochytrium, to alter the metabolic flow of the cells and thereby increase the accumulation of palmitoleic acid C16:1 in the oil. The Schizochytrium fungus is Schizochytrium sp. HX-308, its accession number is CCTCC No. M209059; A combined control strategy was adopted, which simultaneously set the fermentation temperature to 24℃, adjusted the carbon-nitrogen ratio of the fermentation medium to 15, and controlled the pH of the fermentation broth to 8.

5. During the fermentation process, 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; after fermentation, the cells are collected to extract oil. The seed culture medium consists of: 45 g / L glucose, 2 g / L yeast extract, 15 g / L monosodium glutamate, 4 g / L MgCl2·7H2O, 15 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, 5 g / L MgSO4·7H2O, and 3 g / L KH2PO4, with water as the solvent. The fermentation medium consisted of: 90 g / L glucose, 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 4 g / L (NH4)2SO4, along with yeast extract and sodium glutamate adjusted according to the required carbon-to-nitrogen ratio. The solvent was a 0.1 M phosphate buffer system. The initial pH of the fermentation medium was 8.5, and the initial carbon-to-nitrogen ratio was 15.

2. The method according to claim 1, characterized in that: The optimization of the carbon-nitrogen ratio of the fermentation medium is achieved by adjusting the content of yeast powder or monosodium glutamate in the fermentation medium components; the adjustment of the pH value of the fermentation broth is achieved by adjusting sodium hydroxide.

3. The method according to claim 2, characterized in that: The yeast powder content in the fermentation medium is from 3 g / L to 52 g / L; The sodium glutamate content in the fermentation medium is from 2 g / L to 72 g / L.

4. The method according to claim 1, characterized in that: The OD600 value of the fermentation strain is 8-10; the inoculum amount of the fermentation strain is 0.8%-1.5% of the fermentation medium volume.

5. The method according to any one of claims 1 to 4, characterized in that: The specific steps are as follows: Strain activation and seed culture preparation: Wild-type Schizochytrium fungi Schizochytrium sp HX-308 was inoculated into seed culture medium and cultured at 28℃ for 24 hours to obtain primary seed; the primary seed was transferred to fresh seed culture medium at an inoculation rate of 10% and cultured at the same temperature for 24 hours to obtain secondary seed; the secondary seed was then transferred again at an inoculation rate of 10% and cultured at the same temperature for another 24 hours to obtain the fermentation strain. Fermentation culture: Select the above-mentioned fermentation strains with an OD600 value of 8-10 and inoculate them into the fermentation medium at an inoculation rate of 1%. Fermentate for 120 h at a temperature of 24℃ and a rotation speed of 170 rpm. Adjust the initial pH of the fermentation medium to 8.5 by adding 1M NaOH. During the fermentation process, the pH is kept constant at 8.5 by adding NaOH solution continuously.

6. The application of the method according to any one of claims 1 to 5 in increasing palmitoleic acid production from Schizochytrium.

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