A method for promoting the synthesis of metabolites of dunaliella salina

Abstract

This invention belongs to the field of bacterial and algal cultivation, specifically relating to a method for promoting the synthesis of metabolites from Dunaliella salina. This invention employs a bacterial-algal co-culture system and utilizes algae isolated from saline-alkali soil. Bacillus safensis The method involves the co-cultivation of bacterial strains, including strain isolation, identification, and co-cultivation. This invention significantly improves the growth rate and carotenoid synthesis and accumulation efficiency of Dunaliella salina. The strain exhibits strong salt tolerance and is well-suited to the high-salt culture environment of Dunaliella salina, providing a low-cost and efficient new process for the large-scale production of carotenoids from Dunaliella salina.

Description

Technical Field

[0001] This invention belongs to the field of bacterial and algal culture, and specifically relates to a method for promoting the synthesis of metabolites from Dunaliella salina. Background Technology

[0002] Carotenoids are a class of widely used, physiologically active natural fat-soluble pigments. Among them, β-carotene, as a precursor to vitamin A, possesses strong antioxidant, immunomodulatory, and anti-tumor effects, and is widely used in functional foods, pharmaceuticals, cosmetics, and feed additives, with continuously growing market demand. Dunaliella salina (… Dunaliella salina It is an extreme halophilic single-celled green algae and the most β-carotene-accumulating photosynthetic microorganism known in nature. Its intracellular β-carotene content can reach up to 14% of the cell dry weight, far exceeding that of other microalgae species. It can also synthesize high-value-added metabolites such as glycerol, unsaturated fatty acids, and polysaccharides, making it a core algae species for the industrial production of natural carotenoids.

[0003] Currently, in the large-scale production of Dunaliella salina, the induced synthesis of carotenoids mainly relies on abiotic stress methods such as high salt, high light, and nitrogen and phosphorus starvation. While this method can induce product accumulation, it significantly inhibits algal cell growth and proliferation, resulting in a trade-off between biomass and product accumulation. This leads to industry pain points such as long production cycles, low product yields, and high overall costs. To address these issues, existing technologies mainly employ methods such as culture medium optimization, the addition of exogenous chemical inducers, and genetic engineering modification. However, the improvement achieved through culture medium optimization is limited and struggles to overcome metabolic bottlenecks. Chemical inducers pose food safety risks and residue issues, restricting the application scenarios of the products. Genetically engineered strains present biosafety risks and industrial compliance barriers, hindering large-scale promotion and application.

[0004] Algal-microbe co-culture technology, based on the mutualistic symbiotic relationship between microorganisms, provides growth factors, CO2, and trace elements to microalgae through bacterial metabolism, while simultaneously consuming inhibitory substances produced by microalgal metabolism. This achieves a synergistic enhancement of algal cell growth and product synthesis, offering advantages such as environmental friendliness, low cost, and no exogenous chemical residues, making it a research hotspot in the field of microalgae cultivation. However, current research on algal-microbe co-culture of Dunaliella salina still has significant shortcomings: First, existing co-culture strains have poor salt tolerance and cannot adapt to the high-salt culture environment of Dunaliella salina, rapidly inactivating in high-salt systems and failing to form a stable symbiotic system; second, existing strains can only increase the biomass of Dunaliella salina to a limited extent, with poor promotion of the synthesis of core metabolites such as carotenoids, and may even inhibit product synthesis, failing to balance biomass and product yield; third, strains have not been screened in situ for the saline-alkali growth environment of Dunaliella salina, resulting in poor symbiotic adaptability and hindering industrial application.

[0005] Therefore, developing a co-culture method for Dunaliella salina that is suitable for the high-salt culture environment of Dunaliella salina, can simultaneously improve the growth rate of algal cells and the efficiency of carotenoid synthesis and accumulation, and is stable and easy to industrialize has important theoretical significance and application value. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for promoting the synthesis of Dunaliella salina metabolites. The method employs a co-culture system of bacteria and algae, and the selected bacterial strain is... Bacillus safensis Specifically, it includes the following steps: (I) Isolation and treatment of microbial strains in saline-alkali soil (I-1) After mixing the saline-alkali soil sample with water, a preliminary separation was performed to obtain the bacterial solution; (I-2) Spread the bacterial solution onto a culture medium for incubation, thus completing the isolation of the bacterial strain; (II) Isolation and identification of bacterial strains The isolated bacterial strain was expanded into a large-scale culture, and then sequenced and compared to confirm the isolated strain as [missing information]. Bacillus safensis ; (III) Co-culture with Dunaliella salina (III-1) Bacillus safensis Co-culture with Dunaliella salina yielded a co-culture system; (III-2) The salt tolerance of the bacterial strain, the growth rate of Dunaliella salina, and the efficiency of carotenoid synthesis and accumulation were measured in the co-culture system. After completion, the results were determined. Bacillus safensis Co-culturing with Dunaliella salina can promote the synthesis of Dunaliella salina metabolites.

[0007] Preferably, in step (I-1), the initial separation is performed by filtration, including the following steps: (i) Mix the saline-alkali soil sample with water and centrifuge at 25-28 ℃ and 2000-2500×g for 3-5 min and discard the supernatant; (ii) After resuspending, the solution was filtered through 0.45 µm and 0.22 µm filters in sequence, and the filtrate was collected to obtain bacterial solution.

[0008] Preferably, in step (I-1), the initial separation method is vortex separation, which includes the following steps: (i) Mix the saline-alkali soil sample with water and centrifuge at 25-28 ℃ and 2000-2500×g for 3-5 min and discard the supernatant; (ii) After resuspending, filter through a 0.45 µm filter screen and collect the filtrate; (iii) Centrifuge the filtrate at 25-28 ℃ and 1000-1200×g for 8-10 min, and take the supernatant.

[0009] Preferably, in step (I-1), the initial separation is performed by grinding, which includes the following steps: (i) Mix the saline-alkali soil sample with water and centrifuge at 25-28 ℃ and 2000-2500×g for 3-5 min and discard the supernatant; (ii) After resuspending, filter through a 0.45 µm filter screen and collect the filtrate; (iii) Mix the filtrate with mortar and grind for 15-20 min, then centrifuge at 25-28 ℃ and 1000-1200×g for 8-10 min, and take the supernatant.

[0010] Preferably, in step (I-2), the bacterial solution is spread onto the culture medium and cultured at 28-30 °C for 48-52 h.

[0011] Preferably, in step (II), the isolated bacterial strain is cultured at 28-30 ℃ and 120-140 rpm for 48-52 h.

[0012] The beneficial effects of this invention are as follows: 1. Strong strain adaptability and excellent salt tolerance: The strain selected in this invention... Bacillus safensis Isolated from the in-situ environment of saline-alkali soil, it is highly matched with the natural growth environment of Dunaliella salina. It can grow normally at a high salt concentration of 10%, and shows the best growth performance at a salinity of 7.5%, which is close to the actual culture conditions of Dunaliella salina. It can survive stably for a long time in the high salt culture system of Dunaliella salina, which solves the industry pain point of poor salt tolerance and easy instability of existing co-culture strains.

[0013] 2. Significantly promotes the growth and proliferation of Dunaliella salina, and greatly increases biomass: The invention constructs... Bacillus safensis - The co-culture system of Dunaliella salina can significantly accelerate the growth rate of Dunaliella salina, with the algal cell density reaching 4.093 × 10⁻⁶ after 48 hours of culture. 6 The number of cells / mL was increased by 31.84% compared with the blank control group, achieving efficient accumulation of Dunaliella salina biomass and significantly shortening the production cycle.

[0014] 3. Simultaneously and significantly improves the efficiency of carotenoid synthesis and accumulation, balancing yield and quality: The co-culture system of this invention can simultaneously achieve a dual increase in the total carotenoid yield and cell yield of Dunaliella salina. After 48 hours of culture, the carotenoid concentration in the culture medium can reach 430.68 mg / L, an increase of 80.52% compared with the blank control group; the carotenoid cell yield can reach 282.23 pg / cell, an increase of 85.28% compared with the blank control group. This breaks through the technical bottleneck of "the inability to balance biomass improvement and product accumulation" in the existing technology, and significantly improves the production efficiency and economic value of Dunaliella salina.

[0015] 4. Simple process, low cost, environmentally friendly, and easy to industrialize: The strain isolation and screening method of this invention is simple and controllable. The co-culture system does not require the addition of additional chemical inducers or complex equipment modifications. The efficient synthesis of metabolites can be achieved simply by co-culturing the symbiotic strains screened in situ with Dunaliella salina. There are no food safety risks or biosafety hazards. The production cost is low and the environment is environmentally friendly. It can be directly adapted to the existing large-scale cultivation process of Dunaliella salina and has a very strong prospect for industrial application.

[0016] 5. Diverse and widely applicable microbial isolation methods: This invention optimizes three initial microbial isolation methods—filtration, vortexing, and grinding—for saline-alkali soil samples. These methods can be flexibly selected according to sample characteristics and can efficiently isolate the target microbial species. The methods are highly stable and widely applicable, providing a reliable technical solution for the isolation and screening of functional microorganisms in saline-alkali environments. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 yes Bacillus safensis A diagram showing the phylogenetic classification results of the bacterial strain.

[0019] Figure 2 yes Bacillus safensis OD at different salinity culture media 600 Values ​​(left) and actual image of the culture medium (right).

[0020] Figure 3 Bacillus safensis Comparison of growth curves of Dunaliella salina in the co-culture group and the blank group.

[0021] Figure 4 yes Bacillus safensisComparison of carotenoid concentrations in the culture medium of the co-culture group and the blank group.

[0022] Figure 5 yes Bacillus safensis Comparison of carotenoid production per cell in Dunaliella salina between the co-culture group and the control group. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Example

[0024] This embodiment provides a method for promoting the synthesis of Dunaliella salina metabolites. The method employs a co-culture system of bacteria and algae, and the selected bacterial strain is... Bacillus safensis Specifically, it includes the following steps: (I) Isolation and treatment of microbial strains in saline-alkali soil (I-1) Knead the saline-alkali soil sample thoroughly, weigh 0.50 g of soil sample, add 1.00 mL of Milli-Q Water, and transfer 1.00 mL of the dissolved saline-alkali soil sample to a 1.50 mL microcentrifuge tube. Centrifuge at 25 ℃, 2000×g, for 3 min, remove the supernatant, and resuspend the sample in 1.00 mL of sterile Milli-Q Water. Filter the sample under aseptic conditions through a sterile 0.45 µm aqueous mixed cellulose MCE filter to remove extramicrobial impurities. Rinse the filter repeatedly with 100 µL of sterile Milli-Q Water, and repeat the rinsing solution three times. Filter the collected filtrate under aseptic conditions through a 0.22 µm aqueous mixed cellulose MCE filter, rinsing the filter repeatedly with 100 µL of sterile Milli-Q Water. Collect the filtrate as a bacterial culture for later use.

[0025] (I-2) Prepare TSB solid culture medium and sterilize it. After sterilization, pour the solid culture medium into disposable petri dishes in an aseptic environment and let it cool and solidify before use.

[0026] (I-3) Under aseptic conditions, take 20 µL of the bacterial solution, add 80 µL of Milli-Q Water to dilute it, and then spread 100 µL of the bacterial solution onto a solid TSB medium plate.

[0027] (I-4) Incubate at 28 °C for 48 h in a constant temperature incubator.

[0028] (II) Isolation and identification of microbial strains isolated from saline-alkali soil (II-1) Prepare TSB liquid culture medium and sterilize it. After sterilization, dispense the liquid culture medium into 15.00 mL centrifuge tubes under aseptic conditions for later use.

[0029] (II-2) Under aseptic conditions, pick single colonies of different morphologies from the plates cultured in step (I) and transfer them to 15.00 mL centrifuge tubes containing TSB liquid medium for expansion culture.

[0030] (II-3) Incubate in a constant temperature shaker at 28 ℃ and 120 rpm for 48 h.

[0031] (II-4) Design 16S primers. Primers were designed using Primer Premier V6.0 software. The specific primer sequences are shown in Table 1.

[0032] Table 1. 16S Primers for Strain Identification

[0033] (II-5) Perform 16S PCR on the expanded bacterial culture, as shown in Table 2. The PCR procedure follows the instructions for the 2×Hieff® Robust PCR Master Mix (With Dye).

[0034] Table 2 16S PCR reaction system

[0035] (II-6) Take 10 µL of the 16S PCR stock solution for electrophoresis to verify the band. After confirming the existence of a single band, send the remaining PCR stock solution to Qingke Biotechnology Co., Ltd. for sequencing.

[0036] (II-7) The 16S sequence of the isolated strain was compared with the NCBI database, and phylogenetic analysis was performed using Mega 11.0 to confirm that the isolated strain was... Bacillus safensis .

[0037] (III) Co-culture with Dunaliella salina (III-1) Dunaliella salina culture After activating Dunaliella salina to the logarithmic growth phase, 30.00 mL of the algal strain was taken out and inoculated into sterilized D Medium (as shown in Table 3). The medium was then placed in a light-controlled culture room for later use. The culture conditions were 2000 Lux white light, alternating 16 h light and 8 h dark cycles, and a temperature of 28 ℃.

[0038] Table 3. Components of D Medium

[0039] Note: Except for sodium chloride (NaCl), all other components of D Medium are prepared as stock solutions and added when preparing the medium.

[0040] (III-2) Co-culture of bacterial strains with Dunaliella salina (1) Dunaliella salina and Bacillus safensis The bacterial strains were inoculated into their respective culture media for later use.

[0041] (2) The optical density (OD) of the algal solution at 680 nm was measured using a spectrophotometer. 680 ) and OD at 600 nm of bacterial culture 600 Determine the inoculation ratio to ensure that the initial optical density of the algae and bacteria solutions is equal at the corresponding wavelengths during co-culture of bacteria and bacteria.

[0042] (3) Bacillus safensis The bacterial strain and Dunaliella salina were co-cultured. The bacterial and algal strains were inoculated into sterilized D Medium medium according to the inoculation ratio. A blank control group was set up, that is, Dunaliella salina was cultured alone without the addition of bacterial strain. Three biological replicates were set up in the same group.

[0043] (4) Place the co-cultured bacteria and algae and blank algae samples into a light culture room for culture. The culture conditions are the same as those for Dunaliella salina culture. After the culture reaches the appropriate stage, conduct subsequent experiments.

[0044] (III-3) Adaptability screening of co-culture strains (1) Take 1.00 mL of the above-mentioned bacterial-algae co-culture solution and place it in a 1.50 mL microcentrifuge tube. Then filter it according to the above method.

[0045] (2) Prepare TSB solid culture medium and sterilize it. After sterilization, pour the solid culture medium into disposable petri dishes in an aseptic environment and let it cool and solidify before use.

[0046] (3) In an aseptic environment, take 20 µL of the isolated bacterial solution, add 80 µL of Milli-Q Water to dilute it, and then spread 100 µL of the bacterial solution onto a solid TSB medium plate.

[0047] (4) Incubate at 28 °C for 48 h in a constant temperature incubator.

[0048] (5) Pick colonies with different morphologies from solid TSB medium plates for 16S PCR. The reaction system and reaction procedure are as described in Tables 1 and 2 above, and then sequence them.

[0049] (6) Compare sequencing results to screen whether the strain can survive in the Dunaliella salina co-culture environment.

[0050] (III-4) Salt tolerance test of bacterial strains (1) Prepare Tryptone Soya Broth (Casein soya bean digestbroth) culture media with different NaCl content ratios, namely NaCl%=0.05 (original culture medium), 2.50, 5.00, 7.50, 10.00, respectively. After sterilization, dispense into 15.00 mL sterile centrifuge tubes under aseptic conditions for later use.

[0051] (2) Bacillus safensis The bacterial strains were inoculated into culture media with different NaCl% concentrations and cultured in a constant temperature shaker at 28℃, 120 rpm for 16 h.

[0052] (3) Use a spectrophotometer to detect the OD of the bacterial solution. 600 To compare the growth of bacteria.

[0053] (III-5) Determination of the growth rate of Dunaliella salina in the bacterial-algal co-culture system Related studies have shown that when Dunaliella salina is cultured to a cell count of approximately 2.5 × 10⁻⁶, the cell count is... 6 cell / mL (approximately OD of the algal strain of this invention) 680 Carotenoids begin to accumulate when the growth rate reaches 0.3%. To better compare the various physiological indicators of Dunaliella salina in the co-culture system, this growth stage was also used as the starting point for the growth rate.

[0054] (1) Wait for the blank bacterial strain to grow to the OD level in the pure culture group. 680 The initial value was set at 0.3, and the number of Dunaliella salina cells in the control group and each bacterial-algae co-culture system was measured. Measurements were taken at four growth time points: 0 h, 12 h, 24 h, and 48 h.

[0055] (2) In an aseptic environment, take 3.00 mL of culture medium from the three biological replicates of each group and place it in a 5.00 mL sterile centrifuge tube. Then use a pipette to draw 100 µL of culture medium and put it into an opaque 96-well plate. Add 12 wells for each biological replicate sample.

[0056] (3) The fluorescence signal of the culture medium was detected using a full-function continuous wavelength microplate reader (which can eliminate the influence of bacterial cells on algal cell counting). The excitation wavelength was set to 450 nm and the emission wavelength to 685 nm, and the fluorescence intensity of the sample was measured (MFI450 / 685 nm).

[0057] (4) Determine the functional relationship between MFI 450 / 685 and the number of algal cells, and calculate the number of algal cells in the culture medium (the functional relationship between MFI 450 / 685 and the number of algal cells is y=0.02087x, where x is the value of MFI 450 / 685, and the unit of y is 10). 5 (cells / mL).

[0058] (III-6) Determination of the efficiency of Dunaliella salina carotenoid synthesis and accumulation in a bacterial-algae co-culture system The total amount of carotenoids produced by Dunaliella salina is affected by algal biomass. Therefore, it is necessary to provide feedback on the influence of bacterial strains on the efficiency of carotenoid synthesis and accumulation in Dunaliella salina. This invention compares various co-culture systems from two aspects: total carotenoid production and algal cell yield.

[0059] (1) Wait for the blank bacterial strain to grow to the OD level in the pure culture group. 680 The initial value was set at 0.3, and the number of Dunaliella salina cells in the control group and each bacterial-algae co-culture system was measured. Measurements were taken at four growth time points: 0 h, 12 h, 24 h, and 48 h.

[0060] (2) In an aseptic environment, take 30.00 mL of culture medium from three biological replicates of each group and place it in a 50.00 mL sterile centrifuge tube. Centrifuge at 4 ℃, 7000×g, for 5 min and discard the supernatant.

[0061] (3) Resuspend the precipitate in 1.00 mL of sterile Milli-Q Water, place it in a 5.00 mL sterile centrifuge tube, centrifuge at 4 ℃, 7000×g for 5 min and discard the supernatant.

[0062] (4) Add 3.00 mL of 80% acetone solution to a 5.00 mL sterile centrifuge tube containing precipitate, and extract on ice for 45-120 min (depending on the culture time and the amount of precipitate in the culture medium).

[0063] (5) Prepare a new 5.00 mL sterile centrifuge tube and add 2.70 mL of 80% acetone. After extraction, centrifuge at 4 °C, 11100×g for 3 min and take 300 µL of the supernatant after extraction. Add it to the new 5.00 mL sterile centrifuge tube containing 2.70 mL of 80% acetone and mix well.

[0064] (6) Use a spectrophotometer to detect the OD of the sample solution. 470 OD 647 OD 663 .

[0065] (7) Calculate the total amount of carotenoids in each sample according to the calculation formula, which is shown in Table 4.

[0066] Table 4. Formula for calculating carotenoid content in Dunaliella salina acetone extract

[0067] Note: After conversion based on the volume ratio of the experimental design, the value calculated by Car is exactly equal to the carotenoid concentration in the Dunaliella salina culture medium.

[0068] Based on the algal cell count of Dunaliella salina measured in each time period and system in step (III-5), the carotenoid cell yield of Dunaliella salina in each system was calculated (unit: pg / cell).

[0069] Results and Analysis (I) Isolation and Identification of Microbial Species in Saline-Alkali Soils Microbial strains were isolated and identified from samples collected from saline-alkali soil. A total of [number missing] strains were successfully isolated and identified. Bacillus safensis The 16S sequence of the bacterial strain (B3) was compared with the NCBI database to construct a phylogenetic tree and perform phylogenetic analysis. Bacillus safensis The phylogenetic classification results of the strains are as follows Figure 1 As shown.

[0070] (ii) Salt tolerance of the strain This invention describes the isolation, identification, and co-culture adaptability screening of substances obtained from saline-alkali soil. Bacillus safensis Salt tolerance experiments were conducted on the bacterial strains. Considering the high osmotic pressure (salinity 8.769%) of the growth environment and culture medium of Dunaliella salina, different salinity gradients (NaCl% = 0.05, 2.50, 5.00, 7.50, 10.00) were set up to evaluate the growth ability of each strain under this environment. The results are as follows: Figure 2 As shown.

[0071] Experimental results show that Bacillus safensis The bacterial strain can grow at a salinity of 10%, indicating that it possesses the basic ability to survive in the Dunaliella salina culture medium environment. Among other things, Bacillus safensis The strain exhibited high biomass at a salinity of 7.5%, which is close to the actual culture conditions of Dunaliella salina (8.769%). Bacillus safensis It also maintained a certain growth level at this salinity, indicating that it may be more suitable for survival in the growth environment of Dunaliella salina.

[0072] (III) Growth rate of Dunaliella salina in the bacterial-algal co-culture system Will Bacillus safensis The bacterial strain was co-cultured with Dunaliella salina. The growth-promoting effect of the bacterial strain on Dunaliella salina was evaluated by comparing the cell density of Dunaliella salina at each growth stage (48 h). The results are as follows:Figure 3 As shown.

[0073] Experimental results show that Bacillus safensis The bacterial strain showed a significant overall growth-promoting effect on Dunaliella salina, with the cell density reaching 4.093 × 10⁻⁶ cells / day during the 48-hour growth phase. 6 The number of cells / mL increased by 31.84% compared to the blank control group. The Dunaliella salina co-cultured with this strain maintained a relatively obvious growth trend, which is conducive to the construction of a long-term stable co-culture system.

[0074] (iv) Efficiency of carotenoid synthesis and accumulation of Dunaliella salina in the bacterial-algae co-culture system Dunaliella salina, an important model microalga, relies heavily on the synthesis and accumulation of carotenoids, particularly β-carotene, within its cells for its economic value and physiological functions. Carotenoids are not only key photoprotective molecules for algal cells to resist abiotic stresses such as high light and high salinity, but also core indicators for measuring algal metabolic activity and product quality. Therefore, when screening strains suitable for co-culturing with Dunaliella salina, using cell density alone is insufficient; further investigation is needed to examine the impact of the co-culturing system on the efficiency of carotenoid synthesis and accumulation in algal cells.

[0075] The efficiency of carotenoid synthesis and accumulation is an important physiological function of Dunaliella salina. Ideally, co-cultured microorganisms should maintain or enhance the carotenoid synthesis capacity of Dunaliella salina within the system. If the co-cultured microorganisms only promote algal cell division and proliferation but inhibit carotenoid accumulation, it may lead to decreased algal stress resistance and reduced product value, which is detrimental to practical applications. Therefore, carotenoid synthesis and accumulation efficiency should be considered one of the important physiological indicators for evaluating the quality of co-culture systems, forming a multi-dimensional screening system along with indicators such as growth rate.

[0076] Because the total amount of carotenoids in Dunaliella salina culture medium is affected by biomass, comparing the carotenoid concentrations in the culture medium of each co-culture system cannot directly reflect the carotenoid synthesis capacity of Dunaliella salina. In addition to comparing the carotenoid concentrations in the culture medium of each co-culture system, this invention will also combine the results of the growth rate of Dunaliella salina in the bacterial-algae co-culture system to evaluate the influence of each bacterial species on the efficiency of carotenoid synthesis and accumulation in Dunaliella salina.

[0077] Will Bacillus safensis The bacterial strains were initially co-cultured with Dunaliella salina, with pure cultures of Dunaliella salina serving as a blank control. The concentration of carotenoids in the culture medium and the amount of carotenoid synthesis in algal cells were compared at each growth stage (48-hour culture cycle) in different systems. The results are as follows: Figure 4 , Figure 5 As shown.

[0078] The experimental results showed that, compared with the blank control group, Bacillus safensis The bacterial strain, in a co-culture system with Dunaliella salina, exhibited a promoting effect on the total synthesis of carotenoids. For example... Figure 4 As shown, the carotenoid concentration in the co-culture system was significantly higher than that in the control group during the 48-h growth phase. The carotenoid concentration in the co-culture system reached 430.68 mg / L, which was 80.52% higher than that in the blank control group.

[0079] Figure 5 show, Bacillus safensis It exhibited a highly significant promoting effect. In its co-culture system, the maximum cellular synthesis of carotenoids in Dunaliella salina reached 282.23 pg / cell, an increase of 85.28% compared to the blank control group. It is worth noting that... Bacillus safensis It maintained a significant promoting effect on cell synthesis efficiency throughout the entire culture period.

[0080] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for promoting the synthesis of Dunaliella salina metabolites, characterized in that, The method employs a bacterial-algae co-culture system, and the selected bacterial strains are... Bacillus safensis Specifically, it includes the following steps: (I) Isolation and treatment of microbial strains in saline-alkali soil (I-1) After mixing the saline-alkali soil sample with water, a preliminary separation was performed to obtain the bacterial solution; (I-2) Spread the bacterial solution onto a culture medium for incubation, thus completing the isolation of the bacterial strain; (II) Isolation and identification of bacterial strains The isolated bacterial strain was expanded into a large-scale culture, and then sequenced and compared to confirm the isolated strain as [missing information]. Bacillus safensis ; (III) Co-culture with Dunaliella salina (III-1) Bacillus safensis Co-culture with Dunaliella salina yielded a co-culture system; (III-2) The salt tolerance of the bacterial strain, the growth rate of Dunaliella salina, and the efficiency of carotenoid synthesis and accumulation were measured in the co-culture system. After completion, the results were determined. Bacillus safensis Co-culturing with Dunaliella salina can promote the synthesis of Dunaliella salina metabolites.

2. The method for promoting the synthesis of Dunaliella salina metabolites according to claim 1, characterized in that, In step (I-1), the initial separation method is filtration separation, which includes the following steps: (i) Mix the saline-alkali soil sample with water and centrifuge at 25-28 ℃ and 2000-2500×g for 3-5 min and discard the supernatant; (ii) After resuspending, the solution was filtered through 0.45 µm and 0.22 µm filters in sequence, and the filtrate was collected to obtain bacterial solution.

3. The method for promoting the synthesis of Dunaliella salina metabolites according to claim 1, characterized in that, In step (I-1), the initial separation method is vortex separation, which includes the following steps: (i) Mix the saline-alkali soil sample with water and centrifuge at 25-28 ℃ and 2000-2500×g for 3-5 min and discard the supernatant; (ii) After resuspending, filter through a 0.45 µm filter screen and collect the filtrate; (iii) Centrifuge the filtrate at 25-28 ℃ and 1000-1200×g for 8-10 min, and take the supernatant.

4. The method for promoting the synthesis of Dunaliella salina metabolites according to claim 1, characterized in that, In step (I-1), the initial separation method is grinding separation, which includes the following steps: (i) Mix the saline-alkali soil sample with water and centrifuge at 25-28 ℃ and 2000-2500×g for 3-5 min and discard the supernatant; (ii) After resuspending, filter through a 0.45 µm filter screen and collect the filtrate; (iii) Mix the filtrate with mortar and grind for 15-20 min, then centrifuge at 25-28 ℃ and 1000-1200×g for 8-10 min, and take the supernatant.

5. The method for promoting the synthesis of Dunaliella salina metabolites according to claim 1, characterized in that, In step (I-2), the bacterial solution is spread onto the culture medium and cultured at 28-30 °C for 48-52 h.

6. The method for promoting the synthesis of Dunaliella salina metabolites according to claim 1, characterized in that, In step (II), the isolated bacterial strains are cultured at 28-30 ℃ and 120-140 rpm for 48-52 h.

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