Method for increasing fucoxanthine accumulation amount by using microcystis subjected to depolymerization treatment to feed palmarthria formosana

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for increasing the fucoxanthine accumulation amount by using microcystis subjected to depolymerization treatment to feed palmarthria formosana. According to the method, microcystis subjected to depolymerization treatment are added in the culture process of the palmitothecium for heterotrophic culture, meanwhile, continuous inflation is conducted, the microcystis subjected to depolymerization treatment are swallowed by the palmitothecium, and the higher fucoxanthine accumulation amount in the palmitothecium is obtained by means of the culture mode. The experiment proves that after the microcystis is subjected to pretreatment and depolymerization, the removal efficiency of the microcystis is higher by the brown algae, the limitation of treatment of harmful cyanobacteria and ingestion of protozoa is made up, and meanwhile, an integrated solution is provided for biological treatment of cyanobacterial bloom and resource utilization of the microalgae; meanwhile, a new way is provided for obtaining fucoxanthine with higher accumulation amount.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of algal culture, and particularly relates to a method for improving accumulation of fucoxanthin by feeding brown flagellate with microcystis subjected to depolymerization treatment. BACKGROUND

[0002] Brown algae is a typical mixotrophic protist that can both photosynthesize and phagotrophically ingest particulate food (such as bacteria, other algae). At the same time, as a member of the Chrysophyta, brown algae is an important producer of fucoxanthin in marine and freshwater ecosystems. Fucoxanthin is a valuable marine carotenoid with a unique chemical structure (containing a propylene bond and an epoxy group) and a wide range of biological activities, such as antioxidant, anti-inflammatory, anti-obesity, and anti-tumor, etc. It has great application prospects in the fields of medicine, health products, and cosmetics. Currently, fucoxanthin is mainly extracted from large brown algae, but there are bottlenecks such as low yield, complex extraction process, and resource limitations by season and region. Currently, the fermentation production of fucoxanthin by microalgae (such as diatoms and chrysophytes) is considered a more promising alternative approach. The examples disclosed in "CN114376118A-A method for preparing high-quality aquatic feed" disclose the use of algal feed to feed chrysophytes to produce fucoxanthin. However, when the diameter of the microcystis used as chrysophyte feed is not more than 8-10 μm, it is beneficial to the phagocytosis of chrysophytes. Therefore, the microcystis used as chrysophyte feed currently needs to be artificially cultured into single-cell algae to be used as feed. However, the microcystis captured in natural water bodies often exists in a gelatinous colony form with a size greater than 10 μm, a tough cell wall, a spherical or nearly spherical cell shape, a blue-green color, and algae floating on the surface layer of water. The widely distributed common species often grow in still water bodies, such as sub-tropical or warm-temperate lakes, ponds, fish farming ponds, etc. They grow vigorously in spring and summer, forming "water blooms". The size and strength of the toxins produced by the copper green microcystis vary significantly depending on the population, and even the same toxic algae strain has a large difference in toxicity in different water environments, or is completely non-toxic (i.e., does not produce toxins). Some species also contain toxins, such as the copper green microcystis strain PCC7806, which can synthesize a strong hepatotoxicity - classic biological manipulation (such as the use of large filter-feeding zooplankton) is often limited when facing toxic blue-green algae, because microcystis toxins have a strong inhibitory or even lethal effect on many predators. Microcystis toxins are stable in nature and difficult to degrade, and can be transmitted and enriched through the food chain, disrupting the stability of the structure and function of the ecosystem. Microcystis aggregates in a gelatinous colony in natural water bodies, forming a significant water environmental problem. One of the most significant ecological consequences is the frequent outbreak of harmful algal blooms. Traditional physical and chemical algae removal methods are often costly, easy to cause secondary pollution, and only treat the symptoms rather than the root cause. Therefore, exploring environmentally friendly, efficient, and economic strategies for blue-green algae bloom control has become a research hotspot in the field of water environmental science and ecology. SUMMARY

[0003] In view of the above situation, the present application provides a method for improving the accumulation of fucoxanthin in brown flagellate by feeding microcystis through depolymerization treatment, which makes up for the limitations of harmful blue-green algae control and protozoan feeding, and provides an integrated solution for the biological control of blue-green algae blooms and the resource utilization of microalgae, and provides a new way to obtain organisms with higher accumulation of fucoxanthin.

[0004] In order to achieve the above-mentioned purpose, the technical scheme of the present application is: A method for improving the accumulation of fucoxanthin in brown flagellate by feeding microcystis through depolymerization treatment, comprising adding microcystis treated by depolymerization to feed brown flagellate during the cultivation of brown flagellate, and continuously aerating during the cultivation process. The brown flagellate is an algal strain isolated from microcystis culture, and is a species of Ochromonas; The microcystis is wild Microcystis aeruginosa, and the wild Microcystis aeruginosa includes toxic Microcystis aeruginosa and non-toxic Microcystis aeruginosa; The method for depolymerization treatment includes high-temperature sterilization treatment, microwave heating treatment or ultrasonic treatment.

[0005] Further, the cultivation of the brown flagellate is carried out on BG11 liquid medium, and the cultivation conditions are: light intensity of 35 μmol·m -2 ·s -1 , light-dark ratio (L / D) of 16 h:8 h, temperature of 20~24 ℃, aeration culture, and cultivation time of 5~9d.

[0006] Further, the single-cell particle size range of the wild Microcystis aeruginosa after depolymerization treatment is 2~10 μm.

[0007] Further, the initial feeding amount of the wild Microcystis aeruginosa treated by depolymerization is 6×10 6 cells / mL, and the initial inoculation density of the brown flagellate is 5×10 5 cells / mL.

[0008] Further, after the cultivation of the brown flagellate is completed, the product of the culture medium is collected, the supernatant is discarded after centrifugation, and the algal mud sample is freeze-dried and then extracted with anhydrous ethanol to obtain a crude product of fucoxanthin.

[0009] The high-temperature sterilization treatment includes heating at 115℃ for 30min.

[0010] The microwave heating treatment includes microwave oven power of 560W and heating for 3min.

[0011] The ultrasonic treatment includes 20kHz, 100W, and treatment for 2min.

[0012] Compared with the prior art, the present application has the following beneficial effects: The present application adds wild Microcystis aeruginosa treated by depolymerization to the culture of brown flagellate, and the brown flagellate has higher removal efficiency for the wild Microcystis aeruginosa treated by depolymerization, that is, the brown flagellate has stronger phagocytosis and removal capacity for the wild Microcystis aeruginosa treated by depolymerization in natural lakes; meanwhile, compared with the brown flagellate cultured in an autotrophic way, the brown flagellate has higher accumulation of fucoxanthin and fatty acid when phagocytizing Microcystis aeruginosa.

[0013] Therefore, the heterotrophic culture method of brown flagellate and depolymerized Microcystis aeruginosa provided by the present application makes up for the limitations of harmful blue-green algae treatment and protozoan feeding, so that the wild Microcystis aeruginosa can be directly captured as bait for industrial culture of brown flagellate, the harmful Microcystis aeruginosa bloom can be treated by brown flagellate, and high-value fucoxanthin can be efficiently co-produced. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a micrograph of wild Microcystis aeruginosa collected in the wild.

[0015] Figure 2 It is a micrograph of brown flagellate feeding Microcystis aeruginosa, wherein a is brown flagellate feeding laboratory-cultured non-toxic Microcystis aeruginosa, and b is brown flagellate feeding wild Microcystis aeruginosa.

[0016] Figure 3 It is the growth of brown flagellate after feeding wild Microcystis aeruginosa treated by different depolymerization methods, wherein a is the cell density of brown flagellate, b is the cell density of Microcystis aeruginosa, c is the growth rate of brown flagellate, and d is the feeding rate of brown flagellate.

[0017] Figure 4 It is the influence of different depolymerization treatment methods of wild Microcystis aeruginosa on the accumulation of fucoxanthin of brown flagellate, wherein a is the change of the concentration of fucoxanthin, b is the content of fucoxanthin per cell, and c is the dry weight, the content of fucoxanthin and the daily yield.

[0018] Figure 5 It is the influence of non-toxic Microcystis aeruginosa of Comparative Example 1 and toxic Microcystis aeruginosa of Comparative Example 2 on the accumulation of fucoxanthin of brown flagellate, wherein a is the change of the concentration of fucoxanthin, b is the content of fucoxanthin per cell, and c is the dry weight, the content of fucoxanthin and the daily yield. DETAILED DESCRIPTION

[0019] The present application will be further described by specific embodiments below, 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.

[0020] Firstly, the culture method of brown flagellate and single-cell Microcystis aeruginosa in the laboratory is as follows: Ochromonas sp. strain-01 was isolated from a contaminated Microcystis aeruginosa culture. It was identified as a species of Ochromonas based on 18S rDNA gene sequencing and basic morphological observation under an optical microscope. It was recorded in CN114376118A as 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. M 2022112. The culture conditions of the Ochromonas sp. are as follows: inoculation into sterilized BG11 medium for aeration culture (see Table 1 for the formula), placement in a constant-temperature light incubator (ZGX-380A-LED, Ningbo Kesheng), culture temperature of 23℃, light intensity of 35 μmol·m -2 ·s -1 , light-dark ratio (L / D) of 16 h:8 h. The BG11 medium needs to be adjusted to pH 7.1 (1 M HCl or NaOH) before sterilization.

[0021] Microcystis aeruginosa (strain number PCC7806) and non-toxic Microcystis aeruginosa (strain number FACHB1005) were obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The toxic Microcystis aeruginosa PCC7806 can produce at least three types of microcystin (MC-LR, MC-RR and MC-YR). The culture conditions of the two strains are as follows: inoculation into sterilized BG11 medium and placement in a constant-temperature light incubator for culture, culture temperature control at 23℃, light intensity of 25 μmol·m -2 ·s -1 , light period ratio (L / D) of 16 h:8 h, and culture to the logarithmic growth phase.

[0022] Table 1 BG11 medium formula

[0023] II. Determination and calculation methods of experimental related indicators in the following examples of the present application 1. Biomass determination Cell counting of B. subsala and M. aeruginosa was performed under microscope (Nikon) using cell counting chamber, where B. subsala was fixed with 1% Lugol's reagent, and each sample was counted 6 times, and the results were averaged. B. subsala was counted from day 1 to day 12, and M. aeruginosa was counted on day 0, 6, 12, 24, and 36. Finally, 10 mL of culture was taken on day 9 for dry weight (DW) determination. 0.22 μm filter membrane (GF / C, Whatman, USA) was prepared in advance by drying in an oven at 85°C for 6 h to constant weight, and the culture was filtered using a suction filter bottle, and then placed in an oven at 85°C for 6 h to constant weight. The mass of the filter membrane before and after was weighed using an analytical balance, and the results were recorded and calculated according to the following formula. Where M1 and M2 are the mass of the filter membrane before and after filtration, respectively, and V is the filtration volume.

[0024] Ingestion rate (cells predator -1 d -1 ) of B. subsala: Where M t0 and M t represent the density of M. aeruginosa (cells / mL) in the treatment group at the beginning of the experiment and at time t, respectively, and N0 and N t represent the density of B. subsala (cells / mL) at the beginning of the experiment and at time t, respectively, and t represents the duration of the experiment.

[0025] 2. Fucoxanthin determination The culture collected for fucoxanthin determination was centrifuged at 8000 rpm for 6 min and the supernatant was discarded, and the algal slurry sample was stored in a ultra-low temperature freezer at -80°C for subsequent detection. The sample was dried in advance in a freeze dryer. The biomass was transferred to a homogenizer (IKA, Germany) with steel beads for 10 min of grinding extraction, and centrifuged at 8000 rpm for 6 min, and the supernatant was concentrated with a protein concentrator, and then diluted to 2 ml with chromatographic grade ethanol, and the test solution was filtered with a 0.22 μm organic filter head, and then detected with a Waters 1525 HPLC-PDA (Waters, USA). Detection conditions: mobile phase is 85% acetonitrile, flow rate is 1 mL / min, column temperature is 35°C, sample injection volume is 10 μL, detection wavelength is set to 300 nm to 600 nm, single wavelength is 450 nm, and is used for detection of fucoxanthin.

[0026] Example 1 Considering the time and cost spent on culturing non-toxic or toxic Microcystis aeruginosa in the laboratory, the wild Microcystis aeruginosa collected in the field is subjected to disaggregation treatment and then used for feeding culture of brown flagellate.

[0027] The treatment method of the wild Microcystis aeruginosa collected in the field includes the following: (way 1) high-temperature sterilization: heating at 115°C for 30 min, so that the wild Microcystis aeruginosa population is dispersed into single small cells; (way 2) microwave heating: microwave oven 560W, heating for 3 min, so that the wild Microcystis aeruginosa population is dispersed into single small cells; (way 3) ultrasonic wave: 20 kHz, 100W (500W 20%), treatment for 2 min, so that the wild Microcystis aeruginosa population is dispersed into single small cells.

[0028] In this embodiment, the wild Microcystis aeruginosa population collected in the field is dispersed into single cells by "disaggregation" treatment, and the particle size of the single cells is controlled to be 2-10 μm, which is convenient for brown flagellate feeding.

[0029] The wild Microcystis aeruginosa includes toxic Microcystis aeruginosa and non-toxic Microcystis aeruginosa.

[0030] 300 mL of BG11 liquid medium is added to a 500 mL flask, and 5×10 5 cells / mL of brown flagellate is inoculated to obtain four groups of cultured brown flagellate, and then three groups of cultured brown flagellate are fed with Microcystis aeruginosa treated by one way, and the feeding amount is 6×10 6 cells / mL, as the experimental group. Three groups of brown flagellate are aerated and cultured at a temperature of 24°C for 9 days of heterotrophic culture, 35 μmol·m -2 ·s -1 , light-dark ratio (L / D) is 16 h:8 h. Another group of cultured brown flagellate is not fed with Microcystis aeruginosa, as a blank group, and is aerated and cultured at a temperature of 24°C for 9 days of autotrophic culture, 35 μmol·m -2 ·s -1 , light-dark ratio (L / D) is 16 h:8 h.

[0031] Comparative Example 1 300 mL of BG11 liquid medium is added to a 500 mL flask, and 6×10 6 cells / mL of brown flagellate and 5×10 5 cells / mL of non-toxic Microcystis aeruginosa (Microcystis aeruginosa, FACHB1005) are inoculated, and aerated and cultured at a temperature of 23°C for 9 days of autotrophic culture, 35 μmol·m-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.

[0032] The control group consisted of only 6 × 10⁶ cells inoculated into BG11 liquid medium. 6 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.

[0033] Comparative Example 2 Add 300 mL of BG11 liquid culture medium to a 500 mL Erlenmeyer flask and inoculate with 6 × 10⁶ cells / year. 6 Brown dinoflagellates cells / mL and 5×10 5 The toxin-producing Microcystis aeruginosa (PCC7806) at a temperature of 23℃ and a light intensity of 35 μmol·m⁻¹ was detected. -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.

[0034] The blank group is the same as that of comparative example 1.

[0035] like Figure 1 As shown, wild Microcystis aeruginosa collected in the field mainly exists in a colony form, dispersing into single cells after heating and deaggregation, with an average cell diameter of 8.2 μm. Figure 2 As shown, *Phaeocystis chinensis* can consume up to 4-5 wild *Microcystis* cells, with a diameter reaching 20.5 μm, which is more than twice the size of the unfed cells.

[0036] like Figure 3 As shown, the cell size of *Microcystis aeruginosa* is the main determinant of the feeding characteristics of the protozoan *Dinoflagellate*. Pretreatment of wild-type *Microcystis aeruginosa* with depolymerization compensates for the limitations of the treatment method and protozoan feeding. During days 1-5, the cell density of *Dinoflagellate* did not increase significantly, suggesting that metabolites produced by wild-type *Microcystis aeruginosa* may have inhibited its reproduction. Figure 3 As shown in Figure b, high temperature, microwave and ultrasonic treatment methods all improved the removal efficiency of wild Microcystis aeruginosa by Phaeodactylum chinense, with Phaeodactylum chinense exhibiting a higher feeding rate on wild Microcystis aeruginosa treated with high temperature.

[0037] The cultures collected from the experimental and blank groups in Comparative Examples 1 and 2 for fucoxanthin determination were analyzed according to the above method. The results are as follows: Figure 5As shown, the mixed culture mode of feeding Microcystis aeruginosa by B. brownii promoted the synthesis and accumulation of fucoxanthin. The concentration of fucoxanthin was closely related to the cell density, and the growth rate of B. brownii feeding non-toxic Microcystis aeruginosa was higher, and the cell growth promoted the accumulation of fucoxanthin.

[0038] The culture collected for the determination of fucoxanthin in each experimental group in Example 1 was detected according to the above method. The results are shown in Table 1. Figure 4 As shown, the microwave and ultrasonic treatment groups accumulated more biomass, and the yield and daily yield of fucoxanthin were higher. Compared with feeding the laboratory cultured non-toxic or toxic Microcystis aeruginosa in Comparative Example 1 and Comparative Example 2, the yield of fucoxanthin of B. brownii feeding the depolymerized wild Microcystis aeruginosa was increased by 1.4 times, and the daily yield was increased by 2.1 times.

[0039] The above only describes the preferred embodiments of the present application and is not intended 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. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for increasing the fucoxanthin accumulation of *Ochromonas* by feeding it with depolymerized *Microcystis*, comprising adding depolymerized *Microcystis* to the culture of *Ochromonas* during the culture process, and continuously aerating the culture; wherein the *Ochromonas* is an algal strain isolated from the *Microcystis* culture, and is a species of the genus *Ochromonas*; characterized in that... The microcystis species is wild Microcystis aeruginosa. The depolymerization process includes high-temperature sterilization, microwave heating, or ultrasonic treatment.

2. The method for increasing the fucoxanthin accumulation of *Microcystis aeruginosa* by feeding it with depolymerized *Microcystis* according to claim 1, characterized in that... The *Phaeodinella* species were cultured 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 for increasing the fucoxanthin accumulation of *Microcystis aeruginosa* by feeding it with depolymerized *Microcystis* according to claim 1, characterized in that... After depolymerization treatment, the single-cell particle size of the wild Microcystis aeruginosa ranges from 2 to 10 μm.

4. The method for increasing the fucoxanthin accumulation of *Microcystis aeruginosa* by feeding it with depolymerized *Microcystis* according to claim 3, characterized in that... The initial feeding amount of the depolymerized wild Microcystis aeruginosa was 6 × 10⁻⁶. 6 The initial inoculation density of the *Phaeocystis jirovecii* was 5 × 10⁶ cells / mL. 5 cells / mL.

5. The method for increasing the fucoxanthin accumulation of *Microcystis aeruginosa* by feeding it with depolymerized *Microcystis* according to claim 1, characterized in that... After the culturing of *Phaeodinella pulveratum* is completed, the product of the culture medium is collected, the supernatant is discarded by centrifugation, and the precipitated algal mud sample is freeze-dried and then extracted with anhydrous ethanol to obtain crude fucoxanthin.

6. The method for increasing the fucoxanthin accumulation of *Microcystis aeruginosa* by feeding it with depolymerized *Microcystis* according to claim 1, characterized in that... The high-temperature sterilization process includes heating at 115°C for 30 minutes.

7. The method for increasing the fucoxanthin accumulation of *Microcystis aeruginosa* by feeding it with depolymerized *Microcystis* according to claim 1, characterized in that... The microwave heating process includes: a microwave oven with a power of 560W, heating for 3 minutes.

8. The method for increasing the fucoxanthin accumulation of *Microcystis aeruginosa* by feeding it with depolymerized *Microcystis* according to claim 1, characterized in that... The ultrasonic treatment includes: 20kHz, 100W, treatment for 2 minutes.

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