Method for enhancing yield of unsaturated fatty acid in chlorella through fungi

Abstract

The invention provides a method for enhancing the yield of unsaturated fatty acid in chlorella by fungi, which comprises the following steps: adding maltose and yeast extract into a basic BG11 culture medium, and adjusting the pH value to 5 to obtain a domesticated BG11 culture medium, the content of the yeast extract is 5.0-7.5 g / L, the content of NaNO3 is 1.5-2.25 g / L, and the content of K2HPO4 is 0.04-0.06 g / L; the method comprises the following steps: selecting chlorella and at least one fungus as culture species, and inoculating the fungus and chlorella into a domesticated BG11 culture medium according to a ratio of 1: 10 to 1: 50; continuously culturing under set culture conditions to enable the fungi and the chlorella to form a phycomycete symbiotic system; and collecting and centrifuging the phycomycete symbiotic system to obtain the phycomycete symbiont. By optimizing the phycomycete symbiotic system, the yield and variety of the unsaturated fatty acid of the chlorella are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture technology, specifically relating to a method for enhancing the production of unsaturated fatty acids from Chlorella using fungi. Background Technology

[0002] Microalgae are among the most photosynthetically efficient primitive organisms in nature, capable of fully utilizing sunlight and CO2 for photosynthesis to synthesize organic matter and release O2. Microalgal cells are rich in lipids, proteins, polysaccharides, and pigments, thus finding wide applications in food, feed, medicine, and chemical industries. In particular, their unsaturated fatty acids, such as ω-3 and ω-6 series fatty acids, possess important physiological functions and nutritional value. In recent years, microalgae have gained increasing attention as a source of renewable energy and high-value nutrients. However, traditional microalgae cultivation techniques suffer from low yields and high costs, limiting their large-scale application. The development of new technologies to improve microalgae cultivation efficiency and target product yields is urgently needed. Summary of the Invention

[0003] Based on the above background, the purpose of this invention is to provide a method for enhancing the production of unsaturated fatty acids in Chlorella through fungi, thereby significantly increasing the production and variety of unsaturated fatty acids in Chlorella by optimizing the algae-fungus symbiotic system.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] A method for enhancing the production of unsaturated fatty acids from Chlorella using fungi, the method comprising the following steps:

[0006] Maltose and yeast extract were added to the basic BG11 medium and the pH was adjusted to 5 to obtain the domesticated BG11 medium. The contents of yeast extract, NaNO3 and K2HPO4 in the domesticated BG11 medium were 5.0-7.5 g / L, 1.5-2.25 g / L, and 0.04-0.06 g / L, respectively.

[0007] Chlorella and at least one fungus were selected as culture species, and the fungus and Chlorella were inoculated into the domesticated BG11 medium at a fungus-to-algae ratio of 1:10 to 1:50.

[0008] Under the set culture conditions, the fungi and Chlorella were cultured for 10-15 days to form a symbiotic system.

[0009] The algae-bacterial symbiotic system was collected and centrifuged to obtain the algae-bacterial symbiotic.

[0010] The Plackett-Burman (PB) and Box-Behnken (BBD) experiments determined the three key components of yeast extract, NaNO3, and K2HPO4 in the BG11 culture medium and their optimal concentration range. These three components have a significant impact on the biomass, total lipid content, and total protein content in the algae-bacteria symbiotic system. Reasonable adjustment of their concentration can maximize the synthesis of unsaturated fatty acids.

[0011] Preferably, the fungus is selected from one or more of Ganoderma lucidum, Cordyceps militaris, and Aspergillus niger.

[0012] All three fungi exhibit excellent symbiotic relationships with microalgae. *Ganoderma lucidum* is an important medicinal fungus with various biological activities; *Cordyceps militaris* possesses strong biosynthetic capabilities; and *Aspergillus niger* is a commonly used industrial microorganism. When these fungi are co-cultured with *Chlorella vulgaris*, they can form a stable algae-fungus symbiotic system, significantly enhancing the synthesis of unsaturated fatty acids.

[0013] Preferably, fungi and Chlorella are inoculated into the domesticated BG11 culture medium at a fungus-to-algae ratio of 1:20.

[0014] An excessively high bacteria-to-algae ratio can lead to rapid algal death due to insufficient space and nutrients caused by excessively rapid reproduction; conversely, an excessively low ratio results in a low concentration of algae and bacteria, making it difficult to form stable algal-bacterial balls. Experiments have shown that when the bacteria-to-algae ratio is 1:20, algal-bacterial ball formation is optimal, with a large number of balls, a high ball formation rate, and the best results in terms of biomass, total lipid content, and total protein content.

[0015] Preferably, the contents of yeast extract, NaNO3, and K2HPO4 in the BG11 acclimatization medium are as follows: yeast extract 5.688 g / L, NaNO3 1.703 g / L, and K2HPO4 0.047 g / L.

[0016] This ratio was optimized using Box-Behnken experiments and is particularly suitable for increasing the total lipid content of algae-bacterial symbiotic systems. Experiments show that algae-bacterial symbionts cultured under this ratio have a significantly higher total lipid content than those with other ratios, and are also richer in unsaturated fatty acids.

[0017] Preferably, the contents of yeast extract, NaNO3, and K2HPO4 in the BG11 acclimatization medium are: yeast extract 6.239 g / L, NaNO3 1.977 g / L, and K2HPO4 0.050 g / L, respectively.

[0018] This set of proportions represents the optimal culture medium formulation for the biomass of the algae-bacterial symbiotic system, optimized using response surface methodology. This formulation maximizes the biomass of the algae-bacterial symbiotic system, thus improving overall yield.

[0019] Preferably, the contents of yeast extract, NaNO3, and K2HPO4 in the BG11 acclimatization medium are: yeast extract 6.748 g / L, NaNO3 1.931 g / L, and K2HPO4 0.052 g / L, respectively.

[0020] This ratio is specifically designed to increase the total protein content of algae-bacteria symbiotic systems and is calculated based on a multi-factor response surface optimization model. Algae-bacteria symbionts cultured under this ratio not only have high unsaturated fatty acid content but also achieve optimal protein levels, which is beneficial for improving the overall nutritional value of the product.

[0021] Preferably, the culture conditions are as follows: cultured at a temperature of 25-26℃, a light intensity of 2000 Lux, and a cycle of 12 hours of light / 12 hours of darkness, and the culture is shaken for 2 hours daily at a speed of 160 r / min.

[0022] Temperature control at 25-26℃ is conducive to the co-growth of microalgae and fungi; light intensity of 2000 Lux and 12-hour light / dark cycle can meet the needs of microalgae for photosynthesis; shaking at 160r / min for 2 hours a day on a constant temperature shaker can ensure sufficient contact and material exchange between algae and fungi without damaging the already formed algal-fungus ball structure.

[0023] Preferably, after obtaining the algal-bacterial symbiosis, the method further includes the following steps:

[0024] The algal-bacterial symbiont was dried to constant weight, and a mixed solvent of anhydrous diethyl ether and petroleum ether in a volume ratio of 1:2 was added. The mixture was then shaken for 10 minutes under ultrasonic conditions in a water bath and extracted at 20°C for 4 hours.

[0025] After extraction, 10% NaOH solution was added to precipitate the cells, and the supernatant was collected after centrifugation.

[0026] The resulting clear liquid was evaporated to constant weight in a 60°C water bath, and then an equal volume of saturated NaOH-methanol solution was added for saponification.

[0027] Adjust the pH to 1-2 with hydrochloric acid, add deionized water and centrifuge to separate the oil layer. Take the upper oil layer and evaporate it to constant weight in an oven at 40°C to obtain a fatty acid mixture.

[0028] This series of post-processing steps aims to efficiently extract unsaturated fatty acids from algal symbionts. A mixed solvent of anhydrous diethyl ether and petroleum ether provides good solubility for lipids; ultrasonic oscillation in a water bath accelerates cell disruption and lipid release; saponification with NaOH-methanol solution converts fatty acid glycerides into fatty acid salts; and acidification and separation steps yield a high-purity mixture of fatty acids. This extraction method maximizes the preservation of the activity of unsaturated fatty acids and minimizes oxidative losses.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] This invention discloses a method for enhancing the production of unsaturated fatty acids in Chlorella using fungi. The production of unsaturated fatty acids is 30%-50% higher than that of Chlorella cultured alone. Experimental data show that when the fungus-to-algae ratio is 1:20, the total lipid content of the algae-fungus symbiotic system can reach 0.2248 g / g, while the total lipid content of Chlorella cultured alone under the same conditions is only 0.1256 g / g. The algae-fungus symbiotic system cultured by this invention can produce a variety of high-value unsaturated fatty acids, including 9-Hexadecenoic acid, 6,9-Octadecadienoic acid, Docosanoic acid, 9,12-Octadecadienoic acid (Z,Z), and 1-Octadecene, etc., with both the types and contents significantly higher than those of microalgae cultured alone. Attached Figure Description

[0031] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 This is an experimental diagram of algae-bacterial symbiosis culture, showing the formation of algae-bacterial spheres during the culture process;

[0033] Figure 2 This is an experimental diagram of algal-bacterial symbiotic biomass, showing the biomass extraction process and results;

[0034] Figure 3 This is an experimental diagram of total lipids from an algal-bacterial symbiotic organism, showing the fatty acid extraction process and results.

[0035] Figure 4 These are the GC-MS spectra of the experimental group, showing the analysis results of fatty acids in the algae-bacterial symbiosis under different bacterial-algae ratios;

[0036] Figure 5This is the GC-MS spectrum of the control group, showing the analysis results of fatty acids when Chlorella was cultured alone;

[0037] Figure 6 This is the GC-MS main time peak spectrum of the experimental group, showing the characteristic peaks of the main unsaturated fatty acids in the algal-bacterial symbiosis;

[0038] Figure 7 This is the GC-MS elution spectrum of the control group, showing the characteristic peaks of the main unsaturated fatty acids in Chlorella cultured alone. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below through specific embodiments and in conjunction with the accompanying drawings. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0040] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the components or equipment in the following embodiments are general standard parts or components known to those skilled in the art, and their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods.

[0041] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings. In this detailed description, numerous specific details are set forth to facilitate explanation and provide a thorough understanding of the embodiments of the present invention. However, one or more embodiments may be practiced by those skilled in the art without these specific details.

[0042] This invention discloses a method for enhancing the production of unsaturated fatty acids from Chlorella using fungi. Through the construction and optimization of an algae-fungus symbiotic system, the method significantly improves the production and diversity of ω-3 and ω-6 unsaturated fatty acids. The method includes the following steps:

[0043] Maltose and yeast extract were added to the basic BG11 medium and the pH was adjusted to 5 to obtain the domesticated BG11 medium. The contents of yeast extract, NaNO3 and K2HPO4 in the domesticated BG11 medium were 5.0-7.5 g / L, 1.5-2.25 g / L, and 0.04-0.06 g / L, respectively.

[0044] Chlorella and at least one fungus were selected as culture species, and the fungus and Chlorella were inoculated into domesticated BG11 medium at a fungus-to-algae ratio of 1:10 to 1:50.

[0045] Under the set culture conditions, the fungi and Chlorella were cultured for 10-15 days to form a symbiotic system.

[0046] The algae-bacterial symbiotic system was collected and centrifuged to obtain the algae-bacterial symbiotic body.

[0047] To efficiently extract unsaturated fatty acids from algal-bacterial symbionts, the method further includes the following steps after obtaining the algal-bacterial symbionts:

[0048] The algae-bacterial symbiont was dried to constant weight, and a mixed solvent of anhydrous diethyl ether and petroleum ether in a volume ratio of 1:2 was added. The mixture was then shaken for 10 minutes under ultrasonic conditions in a water bath and extracted at 20°C for 4 hours.

[0049] After extraction, 10% NaOH solution was added to precipitate the cells, and the supernatant was collected after centrifugation.

[0050] The resulting clear liquid was evaporated to constant weight in a 60°C water bath, and then an equal volume of saturated NaOH-methanol solution was added for saponification.

[0051] Adjust the pH to 1-2 with hydrochloric acid, add deionized water and centrifuge to separate the oil layer. Take the upper oil layer and evaporate it to constant weight in an oven at 40°C to obtain a fatty acid mixture.

[0052] The method will be described in detail below with reference to the accompanying drawings.

[0053] The formulation of the basic BG11 medium is shown in Table 1, which contains the following components: NaNO3 (not added), K2HPO4 (not added), MgSO4·7H2O (10 ml / L, stock solution concentration 0.75 g / 100 ml dH2O), CaCl2·2 H2O (10 ml / L, stock solution concentration 0.36 g / 100 ml dH2O), citric acid (1 ml / L, stock solution concentration 0.6 g / 100 ml dH2O), ferric citrate (1 ml / L, stock solution concentration 0.6 g / 100 ml dH2O), EDTANa2 (1 ml / L, stock solution concentration 0.1 g / 100 ml dH2O), Na2CO3 (1 ml / L, stock solution concentration 2.0 g / 100 ml dH2O), and A5 trace element solution (1 ml / L).

[0054] Table 1. Formulation of basic BG11 medium

[0055]

[0056] The formulation of the A5 trace element solution is shown in Table 2, which includes: H3BO3 (2.86 g / L dH2O), MnCl2·H2O (1.86 g / L dH2O), ZnSO4·7 H2O (0.22 g / L dH2O), Na2MoO4·2 H2O (0.39 g / L dH2O), CuSO4·5 H2O (0.08 g / L dH2O) and Co(NO3)2·6 H2O (0.05 g / L dH2O).

[0057] Table 2 Formula for A5 Trace Element Solution

[0058]

[0059] To construct the most suitable culture environment for algae-bacterial symbiosis, the basic BG11 medium needs to be optimized. Therefore, this invention identified yeast extract, NaNO3, and K2HPO4 as the three key components affecting the production of unsaturated fatty acids in the algae-bacterial symbiotic system through PB experiments. In the PB experiments, yeast extract, as an organic nitrogen source and growth factor source, had the most significant impact on the algae-bacterial symbiotic system. In the total lipid optimization model, the contribution of yeast extract reached 19.56%; in the total protein optimization model, its contribution was as high as 38.17%. NaNO3, as an inorganic nitrogen source, is mainly used by Chlorella, and its contribution reached 23.14% in the total lipid optimization model. K2HPO4 provides the essential phosphorus element and also has a buffering effect; in the biomass optimization model, its contribution reached 20.64%; in the total protein optimization model, its contribution was 10.83%.

[0060] Based on the results of the PB experiment, this invention further optimizes the concentration of these three key components using the Box-Behnken response surface methodology. By designing multiple groups with different ratios and comparing the biomass, total lipid content, and total protein content of the algal-bacterial symbiont under different concentration combinations, three optimization models were established, and finally, the optimal ratios of the three key components under different optimization objectives were determined.

[0061] Therefore, based on different optimization objectives, these three key components can be accurately weighed and added to the basic BG11 medium components that do not contain NaNO3 and K2HPO4, while the remaining components are added according to the formulations in Tables 1 and 2, thus obtaining three types of acclimatized BG11 mediums.

[0062] The optimization objective for the first acclimatization of BG11 medium was to increase the total lipid content, with the following three key components:

[0063] Yeast extract: 5.688 g / L;

[0064] NaNO3: 1.703 g / L;

[0065] K2HPO4: 0.047 g / L.

[0066] The optimization objective for the second type of BG11 culture medium was to increase biomass, with the following content for the three key components:

[0067] Yeast extract: 6.239 g / L;

[0068] NaNO3: 1.977 g / L;

[0069] K2HPO4: 0.050 g / L.

[0070] The second optimization goal for the BG11 culture medium was to increase the total protein content, with the following three key components:

[0071] Yeast extract: 6.748 g / L;

[0072] NaNO3: 1.931 g / L;

[0073] K2HPO4: 0.052 g / L.

[0074] Based on the above adjustments, 20 g / L maltose was added to each acclimatization BG11 medium as an additional carbon source to promote fungal growth. The pH of the acclimatization BG11 medium was adjusted to 5.0 using HCl or NaOH solution. The prepared acclimatization BG11 medium was dispensed into appropriate containers and autoclaved at 121°C for 20 minutes before use.

[0075] The following experiment was conducted using the first type of acclimatized BG11 culture medium.

[0076] This experiment used *Chlorella vulgaris* (FACHB-8) from the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences, as the microalgae material. The activation process was as follows: the preserved *Chlorella vulgaris* solution was shaken well and transferred to a sterile Erlenmeyer flask (20-50 ml) under aseptic conditions; it was then cultured in a light incubator under the following conditions: temperature 25℃, light intensity 2000 Lux, and light / dark cycle of 12 hours / 12 hours; 5-10 ml of the algal solution was added to 10-20 ml of fresh culture medium for activation, and cultured for about 15 days until the algal strain showed good growth; scale-up culture was then carried out at a transfer ratio of 1:5 (algae solution: culture medium), and the Erlenmeyer flask was shaken twice daily to promote growth; the morphology and viability of the algal cells were examined under a microscope to ensure the use of healthy algal strains.

[0077] This experiment used Ganoderma lucidum as the fungal material. Of course, Cordyceps or Aspergillus niger can also be used in other examples. The activation process of the fungus is as follows: Ganoderma lucidum is inoculated into BG11 acclimatization medium; it is cultured at 25°C for 7 days to allow it to fully adapt to the culture environment; the mycelial growth is observed under a microscope to ensure good fungal activity.

[0078] After confirming that the algae are healthy and the bacteria are active, such as Figure 1 As shown, algae and bacteria were co-cultured.

[0079] The concentrated Chlorella (7.53×10) 8 spores / L) and Ganoderma lucidum (1×10 5 Mix the bacteria and algae (spores / L) in different ratios. Inoculate the resulting bacterial-algae mixture into 100 mL of acclimatization BG11 medium and gently shake to ensure uniform distribution. The bacterial-algae ratio is 1:1, consisting of 1 mL of bacterial solution and 14 µL of algae solution.

[0080] The inoculated culture was placed in a light incubator under the following conditions: temperature 25℃, light intensity 2000 Lux, and light / dark cycle of 12 hours / 12 hours.

[0081] The culture was placed in a constant-temperature shaker at 160 rpm and 26°C for 2 hours each day, and then returned to a light incubator for further cultivation. This periodic shaking promotes contact and material exchange between algae and bacteria, which is beneficial for the formation of algal-bacterial balls.

[0082] Continue culturing for 10-15 days, during which the formation of algal balls is observed regularly under a microscope. Ideal algal balls exhibit a uniform spherical structure and are dark green in color.

[0083] During cultivation, fungal hyphae gradually form a stable symbiotic relationship with Chlorella. The fungal hyphal network provides an attachment substrate for Chlorella, while simultaneously secreting CO2, vitamins, and other growth factors to promote Chlorella growth; in turn, Chlorella produces oxygen and organic matter through photosynthesis, providing nutrients for the fungi. This mutually beneficial relationship significantly enhances the synthesis of unsaturated fatty acids.

[0084] After cultivation is completed, such as Figure 2 As shown, the algal-bacterial symbiotic organism was collected and its biomass was measured.

[0085] Add the cultured algae-bacteria symbiotic fluid directly into a 50mL centrifuge tube, centrifuge at 4000r / min for 8-10 minutes, and discard the supernatant.

[0086] Wash the precipitate with distilled water, centrifuge again, and repeat the washing process twice to remove residual culture medium.

[0087] The washed algae-bacterial symbiont was placed in an 80℃ drying oven and dried to constant weight.

[0088] Weigh the dry weight three times and take the average value, which is recorded as the total biomass of the algae-bacteria symbiosis.

[0089] After that, as Figure 3 As shown, fatty acids were extracted from the algal-bacterial symbiosis and analyzed:

[0090] Grind 0.1g of algal symbiotic powder and add a mixture of anhydrous diethyl ether and petroleum ether (volume ratio 1:2).

[0091] The sample was oscillated for 10 minutes under ultrasonic conditions in a water bath, and then extracted at 20°C for 4 hours, with appropriate oscillation and mixing during the process.

[0092] After extraction, add 10% NaOH solution to precipitate the cells, centrifuge at 4000 r / min for 15 minutes, and collect the supernatant.

[0093] The supernatant was evaporated to constant weight in a 60°C water bath to obtain an extract containing lipids.

[0094] Add an equal volume of saturated NaOH-methanol solution (0.4 mol / L) for saponification, and let the reaction proceed overnight in a 4°C refrigerator.

[0095] Adjust the pH to 1-2 with a dilute HCl solution (concentrated hydrochloric acid and deionized water diluted at a volume ratio of 9:1), add a small amount of deionized water, centrifuge, and take the upper oil layer.

[0096] The oil layer was evaporated to constant weight in a 40°C oven to obtain the total lipids of the algae-bacterial symbiotic organism.

[0097] Fatty acid composition was analyzed by gas chromatography-mass spectrometry (GC-MS). The chromatographic conditions were: 100 m × 0.25 mm × 0.20 μm capillary column, helium as carrier gas, and a flow rate of 0.5 mL / min. -1 The pressure was 280 kPa, the nozzle and detector temperatures were both 260 °C, the split ratio was 30:1, and the injection volume was 1 μL. The temperature program was as follows: initial column temperature 140 °C, held for 5 min, then increased at 4 °C / min. -1 Raise the temperature to 240°C and maintain for 20 minutes.

[0098] The inoculation ratios of Chlorella vulgaris and Ganoderma lucidum in the experimental and control groups, as well as the experimental results, are shown in Tables 3 and 4.

[0099] Table 3. Experimental results of different bacterial-algae ratios

[0100]

[0101] As shown in Table 3, an excessively high proportion of algae in the culture medium can prevent the growth of Ganoderma lucidum. Furthermore, longer cultivation times can lead to rapid algal death due to insufficient space and nutrients caused by excessive reproduction. A bacterial-to-algae ratio of less than 1:10, or a low algae-bacteria concentration, makes it difficult for the two to combine, or the algae and bacteria may combine loosely and fail to form clusters.

[0102] Table 4. Experimental results of the proportion of single microalgae

[0103]

[0104] Table 4 shows that, except for a slightly larger biomass of single microalgae compared to the algae-bacterial symbiotic system, the total lipid and total protein contents were lower in the algae-bacterial symbiotic system. At an algae-bacterial ratio of 1:20, the biomass of the algae-bacterial symbiotic system was 431.50 mg / L, slightly lower than that of Chlorella cultured alone (642.00 mg / L), but the overall yield was still considerable. The total lipid content of the algae-bacterial symbiotic system was 0.2248 g / g, significantly higher than that of Chlorella cultured alone (0.1256 g / g), an increase of approximately 79%. Meanwhile, the total protein content of the algae-bacterial symbiotic system was 492.50 OD / g. -1 The concentration was higher than that of Chlorella cultured alone (356.77 OD / g). -1 This increased by approximately 38%. Therefore, a bacterial-to-algae ratio of 1:20 showed the best overall performance in terms of biomass, total lipid content, and total protein content.

[0105] Reference Figure 4-7 Analysis revealed that the experimental group exhibited distinct characteristic peaks, all of which were relatively large. In terms of the number of peaks, the number began to decrease when the algae-to-bacteria ratio reached 1:50 or higher, with more peaks observed at ratios of 1:1, 1:10, and 1:20. Regarding peak size, the peak size decreased when the algae-to-bacteria ratio reached 1:50 or higher, while the peaks were larger at ratios of 1:1, 1:10, and 1:20. The GC-MS spectra of the experimental group are shown below. Figure 4 As shown, from left to right, they are experimental groups 1-6.

[0106] The control group had fewer peaks than the experimental group, and the peak sizes were relatively smaller. Therefore, the algae-bacterial symbiotic system has a significant advantage in terms of fatty acid content and variety. The GC-MS spectrum results of the control group are shown below. Figure 5 As shown, from left to right, these are control groups 1-5.

[0107] The main elution times of the experimental group were 15 min, 19 min, and 45 min, and the main fatty acids in these three time periods were 9−Hexadecenoic acid and (Z)−(C 16 H 30 O2); 6,9-Octadecadienoic acid (C18 H 32 O2); Docosanoic acid (C 22 H 44 O2);9,12-Octadecadienoic(Z,Z)-(C 18 H 32 O2). These four fatty acids reappeared in every algal-bacterial symbiotic sample, along with another fatty acid, 1-Octadecene (C). 18 H 36 It was detected at bacterial-to-algae ratios of 1:10 and 1:20. Representative spectra at 15 min, 19 min, and 45 min are shown below. Figure 6 As shown.

[0108] The main elution times of the control group were 15 min and 45 min, and the main fatty acids in these two time periods were 6,9-Octadecadienoic acid (C6O4). 18 H 32 O2);9,12-Octadecadienoic(Z,Z)-(C 18 H 32 O2). Representative spectra for the 15-minute and 45-minute time intervals are as follows: Figure 7 As shown.

[0109] It can be seen that the main fatty acid detected in the algal-bacterial symbiotic organism of the experimental group is 9-Hexadecenoic acid,(Z)-(C 16 H 30 O2), 6,9-Octadecadienoic acid(C 18 H 32 O2), Docosanoic acid (C 22 H 44 O2)、9,12-Octadecadienoic(Z,Z)-(C 18 H 32 O2) and 1-Octadecene (C 18 H 36 Five types of unsaturated fatty acids were detected, including 6,9-Octadecadienoic acid and 9,12-Octadecadienoic (Z,Z)-, while only two fatty acids, 6,9-Octadecadienoic acid and 9,12-Octadecadienoic (Z,Z)-, were detected in the control group of individually cultured Chlorella. This indicates that the algae-bacteria symbiotic system can significantly increase the diversity of unsaturated fatty acids.

[0110] In summary, this invention significantly improves the yield and variety of unsaturated fatty acids by optimizing the culture conditions and culture medium composition of the algae-bacteria symbiotic system.

[0111] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for enhancing the production of unsaturated fatty acids from Chlorella using fungi, characterized in that: The method includes the following steps: Maltose and yeast extract were added to the basic BG11 medium and the pH was adjusted to 5 to obtain the domesticated BG11 medium. The contents of yeast extract, NaNO3 and K2HPO4 in the domesticated BG11 medium were 5.0-7.5 g / L, 1.5-2.25 g / L, and 0.04-0.06 g / L, respectively. Chlorella and at least one fungus were selected as culture species, and the fungus and Chlorella were inoculated into the domesticated BG11 medium at a fungus-to-algae ratio of 1:10 to 1:

50. Under the set culture conditions, the fungi and Chlorella were cultured for 10-15 days to form a symbiotic system. The algae-bacterial symbiotic system was collected and centrifuged to obtain the algae-bacterial symbiotic.

2. The method for enhancing the yield of unsaturated fatty acids from Chlorella using fungi according to claim 1, characterized in that: The fungus is selected from one or more of Ganoderma lucidum, Cordyceps militaris, and Aspergillus niger.

3. The method for enhancing the yield of unsaturated fatty acids from Chlorella using fungi according to claim 1, characterized in that: Fungi and Chlorella were inoculated into the domesticated BG11 medium at a fungus-to-algae ratio of 1:

20.

4. The method for enhancing the yield of unsaturated fatty acids from Chlorella using fungi according to claim 1, characterized in that: The contents of yeast extract, NaNO3, and K2HPO4 in the BG11 acclimatization medium were as follows: yeast extract 5.688 g / L, NaNO3 1.703 g / L, and K2HPO4 0.047 g / L.

5. The method for enhancing the yield of unsaturated fatty acids from Chlorella using fungi according to claim 1, characterized in that: The contents of yeast extract, NaNO3, and K2HPO4 in the BG11 acclimatization medium were as follows: yeast extract 6.239 g / L, NaNO3 1.977 g / L, and K2HPO4 0.050 g / L.

6. The method for enhancing the yield of unsaturated fatty acids from Chlorella using fungi according to claim 1, characterized in that: The contents of yeast extract, NaNO3, and K2HPO4 in the BG11 acclimatization medium were as follows: yeast extract 6.748 g / L, NaNO3 1.931 g / L, and K2HPO4 0.052 g / L.

7. The method for enhancing the yield of unsaturated fatty acids from Chlorella using fungi according to claim 1, characterized in that: The culture conditions were as follows: the culture was carried out at a temperature of 25-26℃, a light intensity of 2000 Lux, and a cycle of 12 hours of light / 12 hours of darkness, and the culture was shaken for 2 hours at a speed of 160 r / min every day.

8. The method for enhancing the yield of unsaturated fatty acids from Chlorella using fungi according to claim 1, characterized in that: After obtaining the algal-bacterial symbiotic relationship, the method further includes the following steps: The algal-bacterial symbiont was dried to constant weight, and a mixed solvent of anhydrous diethyl ether and petroleum ether in a volume ratio of 1:2 was added. The mixture was then shaken for 10 minutes under ultrasonic conditions in a water bath and extracted at 20°C for 4 hours. After extraction, 10% NaOH solution was added to precipitate the cells, and the supernatant was collected after centrifugation. The resulting clear liquid was evaporated to constant weight in a 60°C water bath, and then an equal volume of saturated NaOH-methanol solution was added for saponification. Adjust the pH to 1-2 with hydrochloric acid, add deionized water and centrifuge to separate the oil layer. Take the upper oil layer and evaporate it to constant weight in an oven at 40°C to obtain a mixture of fatty acids.

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