Brown flagellum culture method for improving accumulation of fucoxanthin, oleic acid and arachidonic acid

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a method for improving accumulation of fucoxanthin, oleic acid and arachidonic acid in brown flagellate culture. The method is to add microcystis to brown flagellate for mixed culture, and continuously aerate, and the microcystis is phagocytosed by the brown flagellate. The method of phagocytosis of microcystis by brown flagellate is used to realize higher accumulation of fucoxanthin in brown flagellate. Through the experiment, it is proved that the brown flagellate has higher removal efficiency for non-toxic microcystis, and can also effectively ingest toxic microcystis, and a new way for obtaining higher accumulation of fucoxanthin in biological manufacturing is provided.

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, 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:

[0006] The brown flagellate culture method for improving accumulation of fucoxanthin, oleic acid and arachidonic acid adds microcystis to the brown flagellate for mixed culture, and continuously aerates during the culture process, and the microcystis is phagocytosed by the brown flagellate during the mixed culture process.

[0007] The brown flagellate is an algal strain separated from the microcystis culture, and is a species of Ochromonas; the microcystis is non-toxic Microcystis aeruginosa, and the algal strain number is FACHB1005.

[0008] Further, the mixed culture is carried out on the 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.

[0009] Further, the initial inoculation density of the non-toxic Microcystis aeruginosa is 6x10 6 cells / mL, and the initial inoculation density of the brown flagellum is 5x10 5 cells / mL.

[0010] Further, after the culture is completed, the product of the culture medium is collected, centrifuged at a speed of 8000 rpm for 6-10 min, and the supernatant is discarded, and the frozen and dried algal mud sample is extracted with anhydrous ethanol to obtain a fucoxanthin crude product.

[0011] Further, after the culture is completed, the product of the culture medium is collected, centrifuged at a speed of 8000 rpm for 6-10 min, and the supernatant is discarded, and the frozen and dried algal mud sample is extracted with anhydrous ethanol to obtain a fucoxanthin crude product.

[0012] Further, in the present application, when the non-toxic Microcystis aeruginosa FACHB1005 is mixed and cultured with the brown flagellum, the accumulation amount of fucoxanthin, oleic acid (C18:1n9) and arachidonic acid (C20:4n6) of the brown flagellum is higher than that of the toxic Microcystis aeruginosa PCC7806.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] In the present application, the brown flagellum is added with Microcystis aeruginosa, and the brown flagellum has a higher removal efficiency for the non-toxic Microcystis aeruginosa, that is, the brown flagellum has a strong phagocytosis and removal capacity for the non-toxic Microcystis aeruginosa; at the same time, the brown flagellum has a higher accumulation amount of products, i.e., fucoxanthin, oleic acid (C18:1n9) and arachidonic acid (C20:4n6), when phagocytizing the Microcystis aeruginosa, compared with the brown flagellum cultured in an autotrophic manner.

[0015] Therefore, the mixed culture method of the brown flagellum and the Microcystis aeruginosa provided in the present application provides an integrated solution for the biological treatment of cyanobacterial blooms and the resource utilization of microalgae, and provides a new way for obtaining a higher accumulation amount of fucoxanthin, oleic acid (C18:1n9) and arachidonic acid (C20:4n6) in organisms. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The figure is a real observation figure of the brown flagellum feeding the non-toxic and toxic Microcystis aeruginosa in Example 1 and Example 2.

[0017] Figure 2The growth conditions of brown flagellate and Microcystis aeruginosa in Example 1 and Example 2, wherein a is the cell density of Microcystis aeruginosa, b is the cell density of brown flagellate, and c is the feeding rate of brown flagellate.

[0018] Figure 3 The effects of non-toxic Microcystis aeruginosa in Example 1 and toxic Microcystis aeruginosa in Example 2 on the accumulation of fucoxanthin in brown flagellate, wherein a is the change in fucoxanthin concentration, and b is the dry weight, fucoxanthin content and daily production rate.

[0019] Figure 4 The effects of non-toxic Microcystis aeruginosa in Example 1 and toxic Microcystis aeruginosa in Example 2 on the fatty acid composition and content of brown flagellate, wherein a is the change in the proportion of each fatty acid in total fat, and b is the change in the dry weight content of each fatty acid. DETAILED DESCRIPTION

[0020] The present application will be further described below by means of specific embodiments, and the following examples are specific embodiments of the present application, but the embodiments of the present application are not limited by the following examples.

[0021] Firstly, the culture method of brown flagellate and Microcystis aeruginosa involved in the present application in the laboratory is as follows:

[0022] The brown flagellate (Ochromonas sp. strain-01) was isolated from a contaminated Microcystis aeruginosa culture. After 18S rDNA gene sequencing and basic morphological observation under an optical microscope, it was identified as a species of Ochromonas sp. strain-01, recorded in CN114376118A, named Ochromonas sp., with a preservation date of January 25, 2022, a preservation unit of China Center for Type Culture Collection, a preservation unit address of Wuhan University, Wuhan, China, and a preservation number of CCTCC No. M2022112. The culture conditions of the brown flagellate are as follows: inoculation into sterilized BG11 medium for aeration culture (formula see Table 1), placement in a constant temperature and light incubator (ZGX-380A-LED, Ningbo Kesheng), culture temperature 23℃, light intensity 35 μmol·m -2 ·s -1 , light / dark ratio (L / D) 16 h:8 h. The BG11 medium needs to be adjusted to pH 7.1 (1 M HCl or NaOH) before sterilization.

[0023] Microcystis aeruginosa (strain PCC7806) and Microcystis aeruginosa (strain FACHB1005) were obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Microcystis aeruginosa PCC7806 can produce at least three types of microcystins (MC-LR, MC-RR and MC-YR). The culture conditions of the two strains were as follows: inoculated into sterilized BG11 medium and placed in a constant temperature and light incubator, the culture temperature was controlled at 23℃, the light intensity was 25 μmol·m -2 ·s -1 , the light period ratio (L / D) was 16h:8h, and the culture was carried out to the logarithmic growth phase.

[0024] Table 1 BG11 medium formula

[0025]

[0026] II. Determination and calculation methods of experimental related indexes in the following examples of the present application

[0027] 1. Biomass determination

[0028] Cell counting of brown flagellate and Microcystis aeruginosa was carried out under a microscope (Nikon), and 1% Lugol's reagent was used to fix the brown flagellate, and each sample was counted 6 times, and the average value was taken. The brown flagellate was counted on the 1st day to the 12th day of the experiment, and the Microcystis aeruginosa was counted on the 0th day, the 6th day, the 12th day, the 24th day and the 36th day. Finally, 10 mL of culture was taken on the 9th day of the experiment for determination of dry weight (DW). 0.22 μm filter membrane (GF / C, Whatman, USA) was prepared in advance and dried in an oven at 85℃ for 6 h to constant weight, and the culture was filtered into the dried filter membrane using a suction filter bottle, and then placed in an oven at 85℃ for 6 h to constant weight. The mass of the filter membrane before and after use was weighed using an analytical balance, and the results were recorded and calculated according to the following formula. , wherein M1 and M2 are the masses of the filter membrane before and after filtration, respectively, and V is the filtration volume.

[0029] Ingestion rate (cells predator -1 d -1 ) of brown flagellate: , wherein M t0 and M t represent the densities (cells / mL) of Microcystis aeruginosa in the treatment group at the beginning of the experiment and at t, respectively, and N0 and N tDensity of B. subsala (cells / mL) at the beginning of the experiment and at t, respectively, t denoting the duration of the experiment.

[0030] 2. Fucoxanthin assay

[0031] The culture collected for fucoxanthin assay was centrifuged at 8000 rpm for 6 min and the supernatant was discarded. The pellet of algal slurry was stored in an ultra-low temperature freezer at -80°C for later detection. The sample was dried in a freeze dryer beforehand. The biomass was transferred into a homogenizer (IKA, Germany) with steel beads for extraction for 10 min with anhydrous ethanol. The supernatant was concentrated with a protein concentrator and then diluted to 2 mL with chromatographic grade ethanol. The sample was filtered with a 0.22 pm organic filter head and then detected with a Waters 1525 HPLC-PDA (Waters, USA). The detection conditions were as follows: the mobile phase was 85% acetonitrile, the flow rate was 1 mL / min, the column temperature was 35°C, the injection volume was 10 pL, the detection wavelength was set to 300 nm to 600 nm, and the single wavelength was 450 nm for the detection of fucoxanthin.

[0032] 3. Fatty acid assay

[0033] For the detection of fatty acids, accurate determination was performed by the internal standard method. The culture collected for fatty acid assay was centrifuged at 8000 rpm for 6 min and the supernatant was discarded. The pellet of algal slurry was pre-frozen in an ultra-low temperature freezer at -80°C for 12 h and then dried in a freeze dryer. Lipids were extracted from the dried biomass with a mixed solvent of chloroform, methanol and water (2:1:1, v / v / v), and an internal standard heptadecanoic acid was added to the lipid sample. The solvent brought in by the internal standard solution was first volatilized with a nitrogen blower, and then 2 mL of 2% sulfuric acid methanol solution was added for methyl esterification in an oven at 85°C for 2.5 h, with shaking every 30 min. After methyl esterification, 1 mL of saturated sodium chloride and 1 mL of n-hexane were added, shaken for 2 min, and the upper solution was collected by centrifugation at 4000 rpm for 6 min. The above steps were repeated by adding 1 mL of n-hexane. Finally, the collected upper solutions were combined and blown dry with a nitrogen blower, and diluted to 1 mL with chromatographic grade n-heptane. Then, detection was performed with an Agilent 5890 GC-FID (Agilent, Japan). The detection conditions were as follows: the carrier gas was high-purity N2, the injection port temperature was 250°C, the programmed temperature was 80°C for 2 min, then increased to 260°C at 4°C / min, and maintained for 10 min, the injection amount was 1.0 min.

[0034] Example 1

[0035] 300 mL of BG11 liquid medium was added into a 500 mL flask, and inoculated with 5 x 10 5 cells / mL of B. brownianum and 6 x 10 6 cells / mL of M. aeruginosa (FACHB1005) at 23℃, light intensity of 35 μmol·m -2 ·s -1 , light-dark ratio (L / D) of 16 h:8 h, and aerated for 9 days of mixed culture.

[0036] The blank group was only inoculated with 5 x 10 5 cells / mL of B. brownianum in BG11 liquid medium, and placed in a constant temperature and light incubator for culture, with the culture temperature controlled at 23℃, light intensity of 35 μmol·m -2 ·s -1 , and light-dark ratio (L / D) of 16 h:8 h.

[0037] The control group was only inoculated with 6 x 10 6 cells / mL of M. aeruginosa in BG11 liquid medium, and placed in a constant temperature and light incubator for culture, with the culture temperature controlled at 23℃, light intensity of 25 μmol·m -2 ·s -1 , and light-dark ratio (L / D) of 16 h:8 h.

[0038] Example 2

[0039] 300 mL of BG11 liquid medium was added into a 500 mL flask, and inoculated with 5 x 10 5 cells / mL of B. brownianum and 6 x 10 6 cells / mL of toxic M. aeruginosa (PCC7806) at 23℃, light intensity of 35 μmol·m -2 ·s -1 , light-dark ratio (L / D) of 16 h:8 h, and aerated for 9 days of mixed culture.

[0040] The blank group was the blank group of Example 1.

[0041] The control group was only inoculated with 6 x 10 6 cells / mL of toxic M. aeruginosa in BG11 liquid medium, and placed in a constant temperature and light incubator for culture, with the culture temperature controlled at 23℃, light intensity of 25 μmol·m -2 ·s -1, photoperiod ratio (L / D) was 16 h:8 h.

[0042] The following is the determination and calculation method of relevant indicators during the experiment process to observe the feeding growth and related product accumulation of brown flagellate.

[0043] On the 0th, 1st, 3rd, 5th and 9th days of the experiment of Example 1 and Example 2, the actual observation and photography were carried out, and the results are shown in Figure 1 .

[0044] As shown in Figure 2 , the cell density of brown flagellate in the mixed nutrition mode of phagocytizing Microcystis aeruginosa increased significantly faster than that in the autotrophic nutrition (blank group of Example 1 and Example 2); and the feeding rate of brown flagellate on non-toxic Microcystis aeruginosa was higher, which was more conducive to the biomass growth of brown flagellate.

[0045] The culture collected for the determination of fucoxanthin in Example 1 and Example 2 was detected according to the above method. As shown in Figure 3 , the mixed nutrition mode of brown flagellate feeding on Microcystis aeruginosa promoted the synthesis and accumulation of fucoxanthin. The concentration of fucoxanthin was closely related to the cell density, and the growth rate of brown flagellate feeding on non-toxic Microcystis aeruginosa was higher, which promoted the accumulation of fucoxanthin. In summary, by the 9th day of the end of the experiment, the daily production rate of fucoxanthin and the dry weight accumulation of fucoxanthin in the non-toxic Microcystis aeruginosa feeding group were higher.

[0046] The culture collected for the determination of fatty acid in Example 1 and Example 2 was detected according to the above method. As shown in Figure 4 , the fatty acid composition of brown flagellate was mainly composed of palmitic acid (C16:0) and linolenic acid (C18:3n3), and the SFA (saturated fatty acid) and PUFA (polyunsaturated fatty acid) accounted for more than 80% of the total fatty acid. Compared with the autotrophic nutrition of brown flagellate (blank group of Example 1 or blank group of Example 2), the content of high economic value fatty acid (C18:1n9 and C20:4n6) in the non-toxic Microcystis aeruginosa feeding group was significantly increased, which revealed that the mixed nutrition mode of brown flagellate and Microcystis aeruginosa existed specific regulation on the metabolic products.

[0047] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the review principles of the present application shall be included in the protection scope of the present application.

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 Pleuronectiformis for mixed culture, and aeration was continuously provided during the culture process. Microcystis was consumed by Pleuronectiformis during the mixed culture process. The brown dinoflagellate was *Ochromonas* sp. strain-01, deposited at CCTCC, accession number: M2022112; the microcystis was the non-toxic *Microcystis aeruginosa* FACHB1005. The mixed culture was carried out on BG11 liquid medium under the following conditions: light intensity of 35 μmol / m². -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. 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.

2. 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.

3. 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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