Rhynchophylla culture method for improving accumulation amount of fucoxanthine, oleic acid and arachidonic acid

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for culturing fucoxanthine, oleic acid and arachidonic acid to increase the accumulation amount of fucoxanthine, oleic acid and arachidonic acid. According to the method, the microcystis is added into the palmitotheca for mixed culture, and meanwhile, continuous inflation is performed, so that the microcystis is swallowed by the palmitotheca. The mode of swallowing the microcystis by utilizing the palmitothecium is utilized to realize higher fucoxanthine accumulation in the palmitothecium. The experiment proves that the palmitotheca has high removal efficiency on non-toxic microcystis, and can effectively ingest toxic microcystis, so that a new way is provided for biomanufacturing of fucoxanthine with higher accumulation amount.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a brown flagellate culture method for improving accumulation of fucoxanthin, oleic acid and arachidonic acid. BACKGROUND

[0002] Fucoxanthin is a precious marine carotenoid with unique chemical structure (containing propylene bond and epoxy group) and wide biological activities such as antioxidant, anti-inflammatory, anti-obesity and anti-tumor, etc., and has great application prospects in the fields of medicine, health products and cosmetics. At present, fucoxanthin is mainly extracted from large brown algae, but there are bottlenecks such as low yield, complex extraction process, and resource limitation by season and region.

[0003] At the same time, brown flagellate, as a member of Chrysophyta, is an important producer of fucoxanthin in freshwater ecosystems. It is known that brown flagellate is a typical mixotrophic protist that can not only autotroph through photosynthesis, but also ingest particulate food (such as bacteria and other algae) from the outside through phagotrophy. This flexible nutritional strategy makes it have strong ecological adaptability and competitive advantage in the variable nutritional environment. SUMMARY

[0004] In view of the above status, the present application provides a brown flagellate culture method for improving accumulation of fucoxanthin, oleic acid and arachidonic acid, which also provides an integrated solution for resource utilization of microalgae and a new way to obtain higher accumulation of fucoxanthin.

[0005] In order to achieve the above purpose, the technical scheme of the present application is as follows: The brown flagellate culture method for improving accumulation of fucoxanthin, oleic acid and arachidonic acid adds microcystis for mixed culture in brown flagellate, and the microcystis is phagocytosed by brown flagellate during the mixed culture process. The brown flagellate is an algal strain separated from microcystis culture, and is a species of Ochromonas; the microcystis is non-toxic Microcystis aeruginosa, and the algal species number is FACHB1005.

[0006] Further, the mixed culture is carried out on BG11 liquid medium, and the culture conditions are as follows: light intensity is 35 μmol·m -2 ·s -1 , light / dark ratio (L / D) is 16 h:8 h, temperature is 20-24 ℃, aeration culture, and culture time is 5-9 d.

[0007] Furthermore, the initial inoculation density of the non-toxic Microcystis aeruginosa is 6 × 10⁻⁶. 6 The initial inoculation density of the *Phaeocystis jirovecii* was 5 × 10⁶ cells / mL. 5 cells / mL.

[0008] Furthermore, after the culture is completed, the product of the culture medium is collected, centrifuged at 8000 rpm for 6-10 min and the supernatant is discarded. The precipitated algal mud sample is freeze-dried and then extracted with anhydrous ethanol to obtain crude fucoxanthin.

[0009] Furthermore, after the culture is completed, the product of the culture medium is collected, centrifuged and the supernatant is discarded. The precipitated algal mud sample is freeze-dried and lipids are extracted from the freeze-dried sample using a mixed solvent of chloroform, methanol and water in a volume ratio of 2:1:1.

[0010] Furthermore, when the non-toxic Microcystis aeruginosa (FACHB1005) is co-cultured with Pterocarya chinensis in this invention, the accumulation of phycoxanthin, oleic acid (C18:1n9), and arachidonic acid (C20:4n6) in Pterocarya chinensis is higher than that in the toxic Microcystis aeruginosa (PCC7806).

[0011] Compared with the prior art, the present invention has the following advantages: This invention incorporates Microcystis aeruginosa into the culture of *Phaeocystis chinensis*. *Phaeocystis chinensis* exhibits higher clearance efficiency against non-toxic Microcystis aeruginosa, meaning it demonstrates a stronger phagocytic and clearance capacity for non-toxic Microcystis aeruginosa. Furthermore, when *Phaeocystis chinensis* phagocytoses Microcystis aeruginosa, compared to autotrophically cultured *Phaeocystis chinensis*, it accumulates more of the products fucoxanthin, oleic acid (C18:1n9), and arachidonic acid (C20:4n6).

[0012] Therefore, the mixed culture method of Phaeocystis chinensis and Microcystis thunbergii proposed in this invention provides an integrated solution for the biological management of cyanobacterial blooms and the utilization of microalgal resources. At the same time, it provides a new way to obtain higher accumulations of fucoxanthin, oleic acid (C18:1n9), and arachidonic acid (C20:4n6) in organisms. Attached Figure Description

[0013] Figure 1 These are actual observation images of *Microcystis aeruginosa* feeding on non-toxic and toxic *Microcystis aeruginosa* in Examples 1 and 2.

[0014] Figure 2 The figures show the growth status of *Phaeocystis aeruginosa* and *Microcystis aeruginosa* in Examples 1 and 2, where a is the cell density of *Microcystis aeruginosa*, b is the cell density of *Phaeocystis aeruginosa*, and c is the feeding rate of *Phaeocystis aeruginosa*.

[0015] Figure 3 The effects of non-toxic Microcystis aeruginosa (Example 1) and toxic Microcystis aeruginosa (Example 2) on fucoxanthin accumulation in Pterocarya spp., where a represents the change in fucoxanthin concentration and b represents dry weight, fucoxanthin content, and daily yield.

[0016] Figure 4 The effects of non-toxic Microcystis aeruginosa (Example 1) and toxic Microcystis aeruginosa (Example 2) on the fatty acid composition and content of Pterygodium japonicum are shown. In this example, a represents the change in the proportion of each fatty acid to the total fat, and b represents the change in the proportion of each fatty acid to the dry weight content. Detailed Implementation

[0017] The present invention will be further illustrated below through specific embodiments. The following embodiments are specific implementations of the present invention, but the implementation of the present invention is not limited to the following embodiments.

[0018] I. The laboratory culture methods for the *Phaeocystis chinensis* and *Microcystis aeruginosa* involved in this invention are as follows: *Ochromonas sp. strain-01* was isolated from a contaminated culture of *Microcystis aeruginosa*. After 18S rDNA sequencing and morphological observation under an optical microscope, *Ochromonas sp. strain-01* was identified as a species of the genus *Ochromonas*, recorded in CN114376118A, with the name *Ochromonas sp.*. The deposit date was January 25, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No. M 2022112. The culture conditions for this *Ochromonas sp.* were as follows: inoculation into sterile BG11 medium with aeration (formula shown in Table 1), and incubation in a constant temperature and light incubator (ZGX-380A-LED, Ningbo Kesheng) at 23℃ and a light intensity of 35 μmol·m². -2 ·s -1 The light / dark ratio (L / D) was 16 h:8 h. BG11 medium needed to be adjusted to pH 7.1 (1 M HCl or NaOH) before sterilization.

[0019] Both the toxin-producing *Microcystis aeruginosa* (species number PCC7806) and the non-toxic *Microcystis aeruginosa* (species number FACHB1005) were obtained from the Freshwater Algae Collection Center of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The toxin-producing *Microcystis aeruginosa* PCC7806 can produce at least three types of microcystin toxins (MC-LR, MC-RR, and MC-YR). The culture conditions for both algal strains were as follows: inoculation into sterile BG11 culture medium and cultured in a constant temperature and light incubator at 23℃ and 25 μmol·m² light intensity. -2 ·s -1 The photoperiod ratio (L / D) was 16h:8h, and the cells were cultured until the logarithmic growth phase.

[0020] Table 1. BG11 Culture Medium Formulation II. Methods for measuring and calculating experimentally relevant indicators in the following embodiments of the present invention 1. Biomass determination Cell counting of *Phaeodinella pulmonale* and *Microcystis aeruginosa* was performed under a Nikon microscope using a cell counting chamber. *Phaeodinella pulmonale* was fixed with 1% Luger's reagent. Each sample was counted six times, and the average value was taken. *Phaeodinella pulmonale* was counted from day 1 to day 12, and *Microcystis aeruginosa* was counted on days 0, 6, 12, 24, and 36. Finally, on day 9, 10 mL of culture was collected for dry weight (DW) determination. A 0.22 μm filter membrane (GF / C, Waterman, USA) was prepared in advance and dried in an oven at 85°C for 6 hours until constant weight. The culture was then transferred to the dried filter membrane using a vacuum filtration flask and dried again at 85°C for 6 hours until constant weight. The mass of the membrane before and after filtration was measured using an analytical balance, and the results were recorded. The dry weight was then calculated using the following formula. Where M1 and M2 are the membrane masses before and after filtration, respectively, and V is the filtration volume.

[0021] Ingestion rate of brown dinoflagellates (cell predator) -1 d -1 ): M t0 and M t The numbers N0 and N2 represent the densities (cells / mL) of Microcystis aeruginosa in the treatment groups at the beginning of the experiment and at time t, respectively. t t represents the density (cells / mL) of *Phaeocystis chinensis* at the start of the experiment and at time t, respectively, where t represents the duration of the experiment.

[0022] 2. Fucoxanthin Determination The culture collected for fucoxanthin determination was centrifuged at 8000 rpm for 6 min and the supernatant was discarded. The precipitated algal sludge sample was stored in an ultra-low temperature freezer at -80℃ for subsequent analysis. The sample was pre-dried in a freeze dryer. The biomass was transferred to a homogenizer (IKA, Germany) with anhydrous ethanol and extracted by grinding with steel balls for 10 min. After centrifugation at 8000 rpm for 6 min, the supernatant was concentrated using a protein concentrator and then diluted to 2 ml with chromatographic grade ethanol. The test solution was filtered through a 0.22 μm organic filter and then analyzed using a Waters 1525 HPLC-PDA (Waters, USA). Detection conditions: mobile phase: 85% acetonitrile; flow rate: 1 mL / min; column temperature: 35℃; injection volume: 10 μL; detection wavelength: 300 nm to 600 nm, single wavelength: 450 nm, for fucoxanthin detection.

[0023] 3. Fatty acid determination For fatty acid detection, accurate determination was performed using the internal standard method. Cultures used for fatty acid determination were collected, centrifuged at 8000 rpm for 6 min, and the supernatant was discarded. The precipitated algal sludge was pre-frozen at -80℃ for 12 h, and then dried in a freeze dryer. Lipids were extracted from the dried biomass using a mixed solvent of chloroform, methanol, and water (2:1:1, v / v / v). Heptadecanoic acid, an internal standard, was added to the lipid sample. The solvent introduced by the internal standard solution was first evaporated using a nitrogen evaporator. Then, 2 mL of 2% sulfuric acid methanol solution was added, and the sample was methylated in an oven at 85℃ for 2.5 h, with shaking every 30 min. After methylation, 1 mL of saturated sodium chloride and 1 mL of n-hexane were added to the sample, shaken for 2 min, and centrifuged at 4000 rpm for 6 min to collect the supernatant. Another 1 mL of n-hexane was added, and the above steps were repeated. Finally, the collected supernatant solutions were combined, dried using a nitrogen evaporator, and brought to a final volume of 1 mL with chromatographic-grade n-heptane. The solution was then analyzed using an Agilent 5890 GC-FID (Agilent, Japan). Detection conditions: high-purity N2 carrier gas, injection port temperature 250℃, temperature program: initial temperature 80℃, hold for 2 min, increase to 260℃ at 4℃ / min, hold for 10 min, injection volume 1.0 min.

[0024] Example 1 Add 300 mL of BG11 liquid culture medium to a 500 mL Erlenmeyer flask and inoculate with 5 × 10⁶ cells / day. 5 Brown dinoflagellates cells / mL and 6×10 6Non-toxic Microcystis aeruginosa (FACHB1005) cells / mL was obtained at 23℃ under a light intensity of 35 μmol·m⁻². -2 ·s -1 The light-to-dark ratio (L / D) was 16 h:8 h, and the mixture was cultured under aeration for 9 days.

[0025] The control group consisted of only 5 × 10⁵ cells inoculated into BG11 liquid medium. 5 Brown dinoflagellates with a concentration of cells / mL were cultured in a constant-temperature, light-controlled incubator at a temperature of 23℃ and a light intensity of 35 μmol·m⁻¹. -2 ·s -1 The photoperiod ratio (L / D) is 16h:8h.

[0026] The control group consisted of only 6 × 10⁶ inoculated in BG11 liquid medium. 6 Non-toxic Microcystis aeruginosa cells / mL were cultured in a constant temperature and light incubator at 23℃ and 25 μmol·m⁻². -2 ·s -1 The photoperiod ratio (L / D) is 16 h: 8 h.

[0027] Example 2 Add 300 mL of BG11 liquid culture medium to a 500 mL Erlenmeyer flask and inoculate with 5 × 10⁶ cells / day. 5 Brown dinoflagellates cells / mL and 6×10 6 Toxic-producing Microcystis aeruginosa (PCC7806) cells / mL, at a light intensity of 35 μmol·m⁻¹ -2 ·s -1 Under conditions of a light-to-dark ratio (L / D) of 16 h:8 h and a temperature of 23℃, the mixture was cultured under aeration for 9 days.

[0028] The blank group is the blank group of Example 1.

[0029] The control group consisted of only 6 × 10⁶ inoculated in BG11 liquid medium. 6 Toxin-producing Microcystis aeruginosa cells / mL were cultured in a constant temperature and light incubator at 23℃ and 25 μmol·m⁻². -2 ·s -1 The photoperiod ratio (L / D) is 16 h: 8 h.

[0030] The following describes how, in Examples 1 and 2, relevant indicators were measured and calculated to observe the feeding growth and accumulation of related products of *Phaeodinium gloeosum* during the experiment.

[0031] On days 0, 1, 3, 5, and 9 of the experiments in Examples 1 and 2, physical observations and photographs were taken, and the results are as follows. Figure 1 As shown.

[0032] like Figure 2 As shown, under the mixed nutrient mode of phagocytosis of Microcystis aeruginosa, the cell density of Pterygodium monnieri increased significantly faster than that under autotrophic nutrient mode (the blank groups of Examples 1 and 2); and Pterygodium monnieri had a higher feeding rate on non-toxic Microcystis aeruginosa, which was more conducive to the biomass growth of Pterygodium monnieri.

[0033] The cultures collected for fucoxanthin determination in Examples 1 and 2 were analyzed according to the above method. The results are as follows: Figure 3 As shown, the polyculture model of *Phaeodinium chinense* feeding on *Microcystis aeruginosa* promoted the synthesis and accumulation of fucoxanthin. Fucoxanthin concentration was closely related to cell density; *Phaeodinium chinense* feeding on non-toxic *Microcystis aeruginosa* showed a higher growth rate, and cell growth promoted fucoxanthin accumulation. Overall, by day 9 of the experiment, the group feeding on non-toxic *Microcystis aeruginosa* had a higher daily fucoxanthin production rate and a higher fucoxanthin dry weight accumulation.

[0034] The cultures collected for fatty acid determination in Examples 1 and 2 were analyzed according to the methods described above. The results are as follows: Figure 4 As shown, the fatty acid composition of *Phaeodinella pulcherrima* is dominated by palmitic acid (C16:0) and linolenic acid (C18:3n3), with SFAs (saturated fatty acids) and PUFAs (polyunsaturated fatty acids) accounting for more than 80% of the total fatty acids. Compared with the autotrophic nutrition of *Phaeodinella pulcherrima* (the blank group in Example 1 or the blank group in Example 2), the content of high-economic-value fatty acids (C18:1n9 and C20:4n6) in the non-toxic *Microcystis aeruginosa* feeding group was significantly increased, which reveals that the mixed nutrition mode of *Phaeodinella pulcherrima* and *Microcystis aeruginosa* has a specific regulatory effect on its metabolites.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the scope of the review principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for cultivating *Phaeodinium oryzae* to increase the accumulation of fucoxanthin, oleic acid, and arachidonic acid, characterized in that, Microcystis was added to Pyrodinium for mixed culture, and aeration was continuously carried out during the culture process. Microcystis was consumed by Pyrodinium during the mixed culture process. The *Ochromonas* strain is an algal strain isolated from a *Microcystis* culture and is a species of the genus *Ochromonas*; the *Microcystis* strain is the non-toxic *Microcystis aeruginosa* FACHB1005.

2. The method according to claim 1, characterized in that, The mixed culture was carried out on BG11 liquid medium under the following conditions: light intensity of 35 μmol·m⁻¹. -2 ·s -1 The light-dark ratio (L / D) was 16 h:8 h, the temperature was 20~24 ℃, and the culture was carried out under aeration for 5~9 days.

3. The method according to claim 1, characterized in that, The initial inoculation density of the non-toxic Microcystis aeruginosa was 6 × 10⁻⁶. 6 The initial inoculation density of the *Phaeocystis jirovecii* was 5 × 10⁶ cells / mL. 5 cells / mL.

4. The method according to claim 1, characterized in that, After the culture is completed, the product of the culture medium is collected, centrifuged and the supernatant is discarded. The precipitated algal mud sample is freeze-dried and then extracted with anhydrous ethanol to obtain crude fucoxanthin.

5. The method according to claim 1, characterized in that, After the culture is completed, the product of the culture medium is collected, centrifuged and the supernatant is discarded. The precipitated algal mud sample is freeze-dried and lipids are extracted from the freeze-dried sample using a mixed solvent of chloroform, methanol and water in a volume ratio of 2:1:1.

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