WO2020103622A1 - 提高色绿藻胞内虾青素和藻油积累量的诱导培养方法 - Google Patents

提高色绿藻胞内虾青素和藻油积累量的诱导培养方法

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WO2020103622A1
WO2020103622A1 PCT/CN2019/112060 CN2019112060W WO2020103622A1 WO 2020103622 A1 WO2020103622 A1 WO 2020103622A1 CN 2019112060 W CN2019112060 W CN 2019112060W WO 2020103622 A1 WO2020103622 A1 WO 2020103622A1
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acid
astaxanthin
chlorophyta
plant hormone
inducer
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PCT/CN2019/112060
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English (en)
French (fr)
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魏东
陈俊辉
黄滟波
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Definitions

  • the invention belongs to the technical field of algae biological fermentation culture technology, and particularly relates to a method for inducing colored green algae using plant hormones to efficiently accumulate astaxanthin and algal oil at the same time.
  • Astaxanthin is a natural purple-red ketone carotenoid with a chemical name of 3,3'-dihydroxy-4,4'-diketo- ⁇ , ⁇ '-carotene and the molecular formula of C 40 H 52 O 4 has a molecular weight of 596.86. Astaxanthin is called "super antioxidant", its antioxidant capacity is more than 10 times that of other carotenoids such as ⁇ -carotene, zeaxanthin, lutein and canthaxanthin, etc. It has the function of scavenging free radicals and preventing Lipid peroxidation ability has multiple effects for maintaining the health of eyes and central nervous system, enhancing immune system function, strengthening body energy metabolism, anti-cancer and anti-infection. At present, astaxanthin is widely used in food, medicine, health care products and cosmetics industries, and has important market value and broad market prospects.
  • astaxanthin there are three main production methods of astaxanthin, which are extraction and production from shrimp, crab and fish, chemical synthesis, bio-fermentation using microalgae such as Phaffia rhodozyma and Rhodococcus, etc. There is the use of genetically modified plant technology to cultivate and produce astaxanthin.
  • the production of astaxanthin by microalgal fermentation is an important production method of astaxanthin production.
  • the cultivation method of Haematococcus pluvialis is currently the main production method of natural astaxanthin.
  • Astaxanthin prepared by Haematococcus pluvialis and other microalgae are all 3S, 3'S configuration, high antioxidant activity, no toxic side effects, FDA (United States Food and Drug Administration) and EFSA (European Food Safety Authority ) Approved as a nutritional fortifier.
  • FDA United States Food and Drug Administration
  • EFSA European Food Safety Authority
  • Chromochloris (Zhromochloris.zofingiensis) is a kind of single-cell green algae with a fast growth rate, which can use organic carbon source for heterotrophic high-density fermentation under dark heterotrophic conditions to achieve high-density cell growth.
  • the color green algae can effectively accumulate astaxanthin under the induction conditions of high light, high salt, nitrogen and phosphorus nutrient limitation, and at the same time, it can also accumulate algal oil, which is an important potential microalgal germplasm for astaxanthin production.
  • Chlorophyceae can be cultured in a high-density culture under heterotrophic conditions using the current fermentation culture device, but the accumulation of astaxanthin in Chlorophyceae is still relatively high low. Screen high-efficiency induced stress conditions to further increase the intracellular astaxanthin content of Chlorophyta, and then establish an induction culture process for intracellular pigments of Chlorophyta C. zofingiensis, especially astaxanthin. It is of great significance to further develop and utilize commercial elements.
  • the object of the present invention is to provide an induction culture method for increasing the accumulation of intracellular astaxanthin and algae oil of Chlorophyta, realizing the simultaneous astaxanthin and algae oil of Chlorophyta Efficient accumulation has greatly increased the production of intracellular astaxanthin and algae oil of Chlorophyta.
  • the method for inducing and accumulating the accumulation of intracellular astaxanthin and algal oil in Chlorophyceae includes the following steps:
  • step S2 Inoculate the seed liquid obtained by the activation culture in step S1 into a nitrogen-free sugar-containing medium, simultaneously add plant hormone inducers, and perform induced stress culture in the microalgae culture device.
  • the plant hormone inducers of the present invention are 1-aminocyclopropanecarboxylic acid (ACC), 2,4-dichlorophenoxyacetic acid (2,4D), 2-chlorobenzoic acid (CA), red Amycin (GA), 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 3-indolepropionic acid (IPA), abscisic acid (ABA), kinetin (KT), ethanolamine (ETA ), Amine fresh ester (DA), 1-naphthalene acetic acid (NAA) one or more than two.
  • ACC 1-aminocyclopropanecarboxylic acid
  • 2,4D 2-chlorobenzoic acid
  • CA red Amycin
  • IAA 3-indoleacetic acid
  • IBA 3-indolebutyric acid
  • IPA 3-indolepropionic acid
  • ABA abscisic acid
  • KT kinetin
  • ETA ethanolamine
  • the plant hormone inducer of the present invention is selected from 1-aminocyclopropane carboxylic acid, and its added concentration is 1.0-5.0 mg / L; or the plant hormone inducer is selected from 2,4-dichlorobenzene Oxyacetic acid, whose added concentration is 5.0-10.0 mg / L; or the plant hormone inducer is selected from 2-chlorobenzoic acid, and its added concentration is 5.0-10.0 mg / L; or the plant hormone inducer It is selected from gibberellin with an added concentration of 3.5 to 6.3 mg / L; or the plant hormone inducer is selected from 3-indoleacetic acid with an added concentration of 7.8 to 15.7 mg / L; or the plant hormones The inducer is selected from 3-indolebutyric acid, and its added concentration is 9.9-49.9 mg / L; or the plant hormone inducer is selected from 3-indolepropionic acid, and its added concentration is 10.0-25.0 mg / L; Or the phytohormone inducer is selected
  • the phytohormone inducer of the present invention is selected from 1-aminocyclopropanecarboxylic acid, and its added concentration is 5.0 mg / L; or the phytohormone inducer is selected from 2,4-dichlorophenoxy Acetic acid, its added concentration is 5.0 mg / L; or the plant hormone inducer is selected from 2-chlorobenzoic acid, and its added concentration is 5.0 mg / L; or the plant hormone inducer is selected from gibberellin, The added concentration is 6.9 mg / L; or the plant hormone inducer is selected from 3-indoleacetic acid, and the added concentration is 7.8 mg / L; or the plant hormone inducer is selected from 3-indolebutyric acid, The added concentration is 9.9 mg / L; or the plant hormone inducer is selected from 3-indolepropionic acid, and the added concentration is 10.0 mg / L, or the plant hormone inducer is selected from abscisic acid, which is added The concentration is 10.0 mg / L.
  • a plant hormone inducer is added together with a co-solvent, the concentration of the co-solvent in the culture medium is 0.01% to 0.1%, and the plant hormone inducer is 3-indoleacetic acid, One or a combination of two or three of 3-indolebutyric acid and gibberellin; preferably, the co-solvent is selected from water, ethanol or dimethyl sulfoxide.
  • the seed liquid is inoculated into a nitrogen-free sugar-containing medium, and the density of Chlorophyceae cells in the seed liquid is diluted to 2 to 3 g / L, and a plant hormone inducer is added to separate Installed in a microalgae culture device including a transparent microplate or thin-layer culture device, and placed in an oscillator, and then induced stress culture in a light incubator, using a white light source with a light source intensity of 300 ⁇ 30 ⁇ mol m -2 s -1 , light-dark cycle is 0h: 24h ⁇ 24h: 0h, culture temperature is 20 ⁇ 30 °C.
  • the cultivation temperature of the stress induced by step S2 of the present invention is 20-30 ° C
  • the light source uses white light
  • the light source intensity is 300 ⁇ mol m -2 s -1
  • the light-dark period is 24h: 0h.
  • the nitrogen-free sugar-containing medium in step S2 of the present invention further includes components at the following concentrations: 10g glucose, 0.175g potassium dihydrogen phosphate, 0.075g dipotassium hydrogen phosphate, 0.075g magnesium sulfate heptahydrate, 0.025g Calcium chloride dihydrate, 0.025g sodium chloride, 5mg ferric chloride, 0.287mg zinc sulfate heptahydrate, 0.169mg manganese sulfate monohydrate, 0.061mg boric acid, 0.0025mg copper sulfate pentahydrate, 0.00124mg ammonium molybdate heptahydrate.
  • concentrations 10g glucose, 0.175g potassium dihydrogen phosphate, 0.075g dipotassium hydrogen phosphate, 0.075g magnesium sulfate heptahydrate, 0.025g Calcium chloride dihydrate, 0.025g sodium chloride, 5mg ferric chloride, 0.287mg zinc s
  • the activation culture in step S1 of the present invention is to transfer the Chlorophyceae cells to a liquid medium for cyclic reciprocal shaking culture
  • the culture temperature is 20-30 ° C
  • the light source intensity is 10-300 ⁇ mol m -2 s -1
  • the rotation speed is 50 to 500 rpm
  • the light-dark period is 0:24 (h) to 24: 0 (h)
  • the pH of the liquid medium is 6.0 to 7.0.
  • the liquid medium of the present invention is selected from modified Bristol, Basal, BG-11, BBM or Kuhl medium.
  • the present invention also includes the following steps:
  • the method can greatly improve the production efficiency of astaxanthin.
  • the method is simple, economical and efficient. In terms of algae oil, it has very important application value and broad market prospects.
  • Figure 1 shows the effect of different plant hormone-induced stress conditions on the average fluorescence intensity of Chlorophyta C. zofingiensis cells in the FL1 and FL2 channels of the flow cytometer.
  • FL1 533nm ⁇ 15nm
  • FL2 585nm ⁇ 20nm
  • Ctl control group
  • D dimethyl sulfoxide
  • E ethanol
  • H high concentration
  • L low concentration.
  • Figure 2 is the effect of different plant hormone-induced stress conditions on the biomass of Chlorophyceae C. zofingiensis cells.
  • Figure 3 shows the effect of different plant hormone-induced stress conditions on the accumulation of astaxanthin in the Chlorophyta C. zofingiensis cells.
  • Fig. 4 shows the effect of different plant hormone-induced stress conditions on the pigment components of Chlorophyta C. zofingiensis cells.
  • Figure 5 shows the effect of different plant hormone-induced stress conditions on the accumulation of total lipid in Chlorophyceae C. zofingiensis cells.
  • Figure 6 is the effect of different plant hormone-induced stress conditions on the fatty acid composition of Chlorophyceae C. zofingiensis cells.
  • Fig. 7 is a cluster analysis diagram of the growth of C. zofingiensis cells and the accumulation effects of astaxanthin and total lipid under different plant hormone-induced stress conditions.
  • Induced stress conditions are important factors that determine the accumulation of astaxanthin in Chlorophyta.
  • Plant hormones are a very important class of metabolic regulators in microalgae organisms, and they play an important role in regulating the activity of metabolic synthetase enzymes during the synthesis of astaxanthin.
  • the currently known inducers that promote the accumulation of astaxanthin are mainly salicylic acid, methyl jasmonate, gibberellin A 3 , abscisic acid, epibrassinolide, exogenous ethylene, etc. They are all important plants Growth regulators have a positive role in the metabolic control of microalgae synthesis of astaxanthin.
  • Induced stress conditions such as nutrient deficiency stress, oxidative stress, and metabolic inhibitor stress are essentially induced to increase the level of oxidation in the algae cells, and then induce astaxanthin through the regulatory mechanism mediated by reactive oxygen radical (ROS) signal Synthesis.
  • ROS reactive oxygen radical
  • plant hormones can directly regulate the transcription expression of specific genes related to the stress resistance process of the cell, thereby inducing and regulating the accumulation of astaxanthin, and at the same time, plant hormones as a natural, economic, safe and harmless chemical inducer, It plays a very important role in industrial and agricultural production. Through rapid screening experiments, plant hormones that contribute to the efficient accumulation of astaxanthin can be obtained. Combined with a specially designed microalgae induction culture device (including transparent microplates or similar thin-layer culture devices), the color green algae cells can be further significantly improved The accumulation of astaxanthin and algae oil has a very important commercial application prospect.
  • the present invention develops an induction culture method for increasing the accumulation of intracellular astaxanthin and algae oil of Chlorophyta, using conventional microalgae triangle flask culture and microplate-based culture methods to achieve Chlorophyta cells in different plant hormones and Synergistic induction culture under high light and nitrogen-free conditions, then use flow cytometry for rapid detection and evaluation and preliminary screening, and then use verification experiments to accurately determine the high-value pigment and algal oil content of Chlorophyta cells, thereby determining the best plant hormones
  • the inducer and its optimal concentration will ultimately significantly increase the accumulation of astaxanthin in the cells of Chlorophyta, while producing algae oil.
  • the invention can significantly improve or replace the existing production method of preparing natural astaxanthin by using microalgae.
  • Example 1 A preliminary screening experiment on the effects of various plant hormone induction conditions on the accumulation of astaxanthin in Chlorophyta
  • Component Content (mg / L) Component Content (mg / L) Component Content (mg / L) Component Content (mg / L) glucose 10000 NaNO 3 750 KH 2 PO 4 175 K 2 HPO 4 75 MgSO 4 ⁇ 7H 2 O 25 CuSO 4 ⁇ 5H 2 O 0.0025 FeCl 3 5 CaCl 2 ⁇ 2H 2 O 25 MnSO 4 ⁇ H 2 O 0.169 NaCl 25 ZnSO 4 ⁇ 7H 2 O 0.287 (NH 4 ) 6 Mo 7 O 24 ⁇ 7H 2 O 0.00124 H 3 BO 3 0.061 / / / / /
  • the modified nitrogen-free sugar-containing fermentation Bristol medium was used for dilution and re-suspension, and the cell density of Chlorophyta was controlled to be between 2 and 3 g / L.
  • the cultivation conditions are: rotation speed is 50 ⁇ 500rpm, temperature is 26 °C, white fluorescent lamps are placed side by side as a light source for continuous light cultivation, and the light intensity on the surface of the microplate is controlled to be 300 ⁇ 30 ⁇ mol m -2 s -1 or more, and cultivated separately More than 12 days.
  • the fresh Chlorophyta cells are obtained, and then the flow cytometer (Accuri C6, BD, USA) is used to analyze and determine the average fluorescence intensity value (Mean fluorescence intensity) of Chlorophyta cells obtained under different culture conditions , MFI).
  • the flow cytometer (Accuri C6, BD, USA) is used to analyze and determine the average fluorescence intensity value (Mean fluorescence intensity) of Chlorophyta cells obtained under different culture conditions , MFI).
  • the flow cytometer used in this example is equipped with two 50mW air-cooled lasers (excitation wavelengths of 488nm and 640nm, respectively), and corresponding to four detection channels (FL1: 533 ⁇ 15nm; FL2: 585 ⁇ 20nm; FL3 > 670nm; FL4: 675 ⁇ 25nm).
  • the specific detection process is as follows: centrifuge the fresh algae fluid at 3800 ⁇ g centrifugal force for 2 minutes, then wash with pure water and centrifuge twice, then use pure water to dilute the algal cell density to 0.6 ⁇ 2 ⁇ 10 6 cells / mL, and then use After filtering the algae cell suspension with a 400 mesh filter membrane, it was directly used for flow cytometry analysis.
  • the flow rate is set to 35 ⁇ L / min, a total of more than 10,000 cells are collected and measured, and the forward scattered light signal (Forward Scatter, FSC) and the side scattered light signal (SSC) of the cells are measured separately. And the average fluorescence intensity values of the two channels FL1 and FL2.
  • FSC Forward Scatter
  • SSC side scattered light signal
  • the system's own AccuriCflow data processing software was used to statistically analyze the average fluorescence intensity value MFI of Chlorophyta cells.
  • flow cytometry is used to measure the average fluorescence intensity of Chlorophyceae cells, which can be used to indirectly and rapidly detect and evaluate the accumulation of astaxanthin in Chlorophyceae cells, which can be used as a plant hormone
  • FCM flow cytometry
  • the plant hormones ACC, IPA, KT, etc. have little effect on the fluorescence intensity of Chlorophyceae cells, but different concentrations still have an effect on the fluorescence intensity of the cells.
  • this embodiment chooses ACC And IPA at concentrations of 5.0 mg / L and 10.0 mg / L were used as follow-up verification experiments.
  • ACC And IPA at concentrations of 5.0 mg / L and 10.0 mg / L were used as follow-up verification experiments.
  • ETA, DA and NAA they have a reducing effect on the fluorescence intensity of Chlorophyta cells, indicating that these plant hormones not only did not increase the synthesis of astaxanthin, but also greatly inhibited the astaxanthin in the algae cells. They are not suitable for inducing the accumulation of astaxanthin in Chlorophyta.
  • this example basically completed the preliminary screening of different plant hormone inducers and their concentrations, which helped to improve the intracellular astaxanthin of Chlorophyta Element accumulation.
  • the proportion and content of pigment accumulated in the cells will be different, which will affect the stability of FCM rapid detection and evaluation.
  • this experiment only uses the FCM rapid evaluation method as the preliminary screening result, and it also needs to be combined with the verification experiment to finalize, that is, firstly determine the appropriate plant hormones and their concentrations, and then use the same induction Conditions to re-perform the culture and verification experiment of Chlorophyceae cells, after obtaining enough algae cells, use traditional quantitative methods to accurately determine the content of astaxanthin in Chlorophyceae cells, and finally determine the optimal plant hormone inducer and its concentration.
  • Example 2 The verification experiment of the effect of different plant hormone induction conditions on the accumulation of Chlorophyta biomass, astaxanthin and lipids
  • This example is to further verify the plant hormones determined by the preliminary screening in Example 1 that are helpful to increase the fluorescence value of Chlorophyta cells, and accurately determine the effects of these plant hormones on the growth of Chlorophyta cells and the accumulation of astaxanthin. Therefore, a plant hormone capable of efficiently inducing the accumulation of intracellular astaxanthin in Chlorophyceae was finally determined, and the production level of astaxanthin produced by Chlorophyceae was greatly restricted.
  • Chlorophyceae C. zofingiensis strains stored on the slope of the laboratory were cultured as described in 1.1 for 3 to 5 days to be used as seed liquid.
  • the cultured Chlorophyta seed solution was centrifuged to collect Chlorophyta cells according to the method described in 1.2.1, and then diluted and resuspended with nitrogen-free sugar-containing fermentation medium to control the initial density of Chlorophyta cells to 2 ⁇ 3g / L, add the plant hormones determined in the preliminary screening experiment to the resuspended algae solution at the same time, then divide the algae solution into different transparent microplates and place them in the microplate shaker Finally, they were placed together in a light incubator and cultivated to accumulate astaxanthin by C. zofingiensis.
  • This example is to solve the problem that the amount of Chlorophyceae cell samples in the batch screening experiment of Example 1 is small and cannot meet the requirements for the determination of pigment and total lipid samples.
  • the ultimate goal is to obtain enough algae cells for accurate measurement of intracellular
  • the accumulation of astaxanthin and algae oil ultimately determines the induction effect of these plant hormones.
  • the fermentation medium uses modified Bristol medium, which differs from the medium in Table 1 in that its glucose is 10g / L, and the plant hormones screened in the experiment 1.2 in Experiment Example 1 and their optimal concentrations are added separately, as ACC 5.0mg / L, 24D 5.0mg / L, CA 5.0mg / L, GA 6.9mg / L, IAA 7.8mg / L, IBA 9.9mg / L, IPA 10.0mg / L, ABA 10.0mg / L to prepare A nitrogen-free sugar-containing fermentation medium containing different plant hormone inducers was obtained.
  • the cultivation conditions are: rotation speed is 50 ⁇ 500rpm, temperature is 26 °C, white fluorescent lamps are placed side by side as the light source, the light intensity on the surface of the microplate is controlled to 300 ⁇ 30 ⁇ mol m -2 s -1 , continuous high light irradiation, culture 12 More than days.
  • Chlorophyceae culture solution After the cultivation, collect the Chlorophyceae culture solution, centrifuge to collect algal mud cells, freeze-dry to obtain dried algae powder, put it in a -20 °C refrigerator for cryopreservation, for the analysis of intracellular pigment and lipid content of Chlorophyceae .
  • the biomass measurement, pigment content measurement, and lipid content measurement of Chlorophyta were analyzed using the measurement method described in 2.2.2.
  • the method for measuring the biomass during the cultivation of Chlorophyceae was determined by dry weight method.
  • the sampled algae liquid was weighed and placed in a centrifuge tube weighed beforehand, centrifuged at 6000 rpm for 1 min to collect the lower algal cells, then added pure water to oscillate and suspended, and repeated centrifugal washing twice to remove the supernatant Liquid, place the centrifuge tube containing algal mud in a 60 ° C oven and dry to constant weight, and determine the total weight of algae powder and centrifuge tube.
  • the biomass in the culture solution of Chlorophyceae can be obtained through conversion.
  • Chlorophyta C.zofingiensis intracellular astaxanthin HPLC detection method using a liquid chromatography equipped with a PDA detector and YMC TM C30 chromatographic column (4.6mm ⁇ 150mm, 3 ⁇ m) for analysis, the eluent is chromatographic methanol (Mobile phase A), methyl tert-butyl ether MTBE (mobile phase B) and water (mobile phase C).
  • the gradient elution procedure is: 0-6min, 95 ⁇ 80% A, 5 ⁇ 20% B, 0% C; 6-12min, 80 ⁇ 60% A, 20 ⁇ 38% B, 0 ⁇ 2% C; 12- 28min, 60 ⁇ 50% A, 38 ⁇ 48% B, 2% C; 28-33min, 50% A, 48% B, 2% C; 33-35min, 50 ⁇ 95% A, 48 ⁇ 5% B, 2 ⁇ 0% C; 35-38min, 95% A, 5% B, 0% C.
  • the temperature of the column oven is 30 °C
  • the flow rate of the eluent is 0.8mL / min
  • the injection volume is 20 ⁇ L
  • the detection wavelength of the PDA detector is 300-700nm
  • the full wavelength scan is performed to determine the absorption spectrum of the pigment, and the measurement is performed at the wavelength of 480nm.
  • Intracellular pigment content Qualitative analysis of pigments uses the retention time and absorption spectrum of pigment standards (astaxanthin, chlorophyll a, chlorophyll b, lutein, canthaxanthin, zeaxanthin), and quantitative analysis uses the standards made by pigment standards The curve was analyzed. For the keto lutein and carotene pigments, a similar lutein standard curve was used for quantitative analysis.
  • the total lipid content is determined by the organic solvent extraction method, which is briefly described as follows: take the algae powder obtained by freeze-drying, accurately weigh about 100mg, place it in a freeze tube, then add an appropriate amount of ceramic beads, and mix with methanol / dichloromethane Reagents (1: 3, v / v) were used as organic extraction reagents for extraction. First, it was quickly shaken on a high-speed bead mill, and then frozen and crushed with liquid nitrogen to extract lipids. During the centrifugation, the upper extract was collected several times until the oil extraction was complete. All the combined upper extracts were blown dry with nitrogen, and finally weighed with a balance. According to the weight difference before extraction, the total lipid content in the algal cells can be obtained.
  • the intracellular fatty acids of Chlorophyceae were extracted by saponification and methylation and used for GC-MS detection and analysis, as follows: add a certain volume of C19: 0 fatty acid standard solution (concentration 1mg / mL, (The solvent is methylene chloride), and after blowing off the solvent with nitrogen, weigh about 20 mg of algae powder, add 1 mL of saturated potassium hydroxide-methanol solution, shake quickly, heat in a 75 ° C water bath for 10 min, cool to room temperature, add 2mL boron trifluoride-anhydrous methanol solution (1/2, v / v, of which boron trifluoride concentration is about 15%), after shaking and mixing, heat in a 75 ° C water bath for 10 minutes, cool to room temperature, and then add 0.3mL Saturated saline, and then add 2mL of n-hexane to mix and extract fatty acid methyl ester. After centrifuging at low speed, take the upper oil layer,
  • the determination method of fatty acid content was analyzed by Agilent gas mass spectrometer, equipped with 6890 gas chromatograph, 5975 built-in MSD and high efficiency capillary column (DB-23, 30mm ⁇ 0.25mm, 0.25 ⁇ m).
  • High-purity helium gas is used as the carrier gas, the flow rate is 1ml / min, the sample is not split, and the injection volume is 0.2 ⁇ L.
  • the inlet temperature is 250 ° C and the detector temperature is 270 ° C.
  • the temperature-programming conditions are as follows: the column temperature box is kept at 130 ° C for 1 min, and then increased to 200 ° C at 5 ° C / min for 5 minutes.
  • the mass scan range of the mass spectrum is 33-400 amu.
  • the identification of each peak type is automatically searched by NIST mass spectrometry library, and the qualitative analysis of fatty acid components is carried out.
  • Quantitative analysis uses C19: 0 as the internal standard, and quantitative analysis of each fatty acid component is measured by the internal standard method.
  • Example 2.2 Through the induction culture method described in Example 2.2, the effects of different plant hormones on the growth of Chlorophyta cells, the accumulation of astaxanthin and lipids can be obtained. The results are shown in Figures 2-6.
  • auxin IAA has the greatest promotion effect on the growth of Chlorophyta, the maximum growth amount can reach 8.3g / L, which is an increase of 26.7% compared with the control, and the promotion effect is very significant (p ⁇ 0.05).
  • Astaxanthin diester is the main form of astaxanthin in algae cells, which accounts for more than 75% of the total astaxanthin, especially when induced by IAA and IBA, astaxanthin diester accounts for the total astaxanthin. The content can reach more than 88%. In summary, this indicates that Chlorophyta cells can produce and accumulate other functional carotenoids such as lutein as well as accumulate astaxanthin, which has potential application value in functional food and aquatic bait.
  • the total lipid production of Chlorophyceae cells can reach a maximum of 5.3 g / L, and the total lipid production can also be as high as 445.7 mg / L / d.
  • the total lipid yield is a relatively high level of lipid accumulation in microalgae production at present, and is of great value for improving the production level of microalgae industrial production of algae oil.
  • the process technology of the present invention can induce the astaxanthin and algae oil in the cells of Chlorophyta to accumulate high added value at the same time, and can be used as a functional Food raw materials and biofuel raw materials, on the one hand, greatly reduce the production cost of the microalgae biorefinery industry, on the other hand, they have very important social and economic value and environmental protection value.
  • C18: 1 basically accounts for about 40% of the total fatty acids in Chlorophyta, and is the most important fatty acid in Chlorophyta.
  • microalgae biorefinery production of algae oil is one of the important production methods of future biofuel raw materials and future edible algae oil, and has very important application prospects.
  • a large amount of algal oil can accumulate in the cells of Chlorophyta.
  • fatty acids are mainly hexadecane and octadecanoic, which indicates that the oil-rich green algae obtained after induction culture has Development of important potential as a source of biofuel; at the same time, algae oil is rich in fatty acids such as oleic acid, linoleic acid and linolenic acid, and has the potential to be developed as an edible algae oil.
  • Another creative contribution of the technical solution of the present invention is that, while ensuring the efficient accumulation of astaxanthin, it also realizes the efficient accumulation of intracellular algae oil of Chlorophyta. Compared with the existing production level, adopting the technical scheme of the present invention has also significantly increased the intracellular total lipid content of Chlorophyta, up to 64.5%, and the total lipid yield reached 445.7 mg / L / d (see Table 3), At a higher level of current production.
  • the invention successfully realizes the efficient and simultaneous accumulation of astaxanthin and algae oil, and achieves creative research results.
  • different plant hormone-induced stress conditions have different induction effects on the growth of Chlorophyta cells, accumulation of astaxanthin and accumulation of lipids.
  • this example uses a systematic cluster analysis technique to analyze the stress conditions induced by different plant hormones, based on the biomass, astaxanthin and lipid content of Chlorophyceae cells and Based on data such as output, statistical analysis software was used to perform systematic cluster analysis, clustering method using inter-group connection, the metric was Euclidean distance, and the cluster tree was shown in Figure 7.
  • plant hormones can be basically divided into three categories, namely, plant hormones (ACC, 2, 4D) that have no obvious promotion of astaxanthin and lipid accumulation, and Plant hormones that promote the accumulation of astaxanthin and lipids (IAA, IBA, GA), plant hormones that promote the accumulation of astaxanthin but inhibit the synthesis of lipids (IPA, ABA).
  • IPA plant hormones that promote the accumulation of astaxanthin but inhibit the synthesis of lipids
  • IPA and ABA can be used as inducers for the induction culture of Chlorophyta.
  • plant hormones such as IAA and IBA can be used as inducers for cultivation.
  • IAA and IBA can intuitively reflect the induction effect of different plant hormones on the synthesis of astaxanthin and lipids, and it also has important guiding significance for the production of astaxanthin and algae oil from Chlorophyta.

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Abstract

提供一种提高色绿藻胞内虾青素和藻油积累量的诱导培养方法。该方法采用常规的微藻三角瓶培养和基于微孔板的培养方法，实现色绿藻细胞在不同植物激素和高光无氮条件下的协同诱导培养，提高色绿藻胞内的虾青素的积累量，同时生产制备藻油。

Description

提高色绿藻胞内虾青素和藻油积累量的诱导培养方法 技术领域
本发明属于藻类生物发酵培养工艺技术领域，特别地涉及一种利用植物激素诱导色绿藻同时高效积累虾青素和藻油的方法。
背景技术
虾青素是一种天然紫红色的酮基类胡萝卜素，化学名为3,3’-二羟基-4,4’-二酮基-β,β’-胡萝卜素，分子式为C 40H 52O 4，分子量为596.86。虾青素(Astaxanthin)被称为“超级抗氧化剂”，抗氧化能力是其它类胡萝卜素如β-胡萝卜素、玉米黄素、叶黄素和角黄素等的10倍多，具有清除自由基、阻止脂质过氧化能力，对于维护眼睛和中枢神经系统健康，增强免疫系统功能、强化肌体能量代谢、抗癌、抗感染等具有多重功效。目前虾青素在食品、医药、保健品和化妆品行业等有着广泛的应用，具有重要的市场价值和广阔的市场前景。
目前虾青素的生产方法主要有三种，分别为从虾蟹和鱼类等体内提取生产、化学合成法生产、采用红发夫酵母和红球藻等微藻的生物发酵法生产等，此外还有采用转基因植物技术培养生产虾青素。
微藻发酵法生产虾青素是目前虾青素生产的重要生产方式。雨生红球藻培养法是目前天然虾青素的最主要生产制备方法。采用雨生红球藻Haematococcuspluvialis等微藻制备得到的虾青素均为3S,3’S构型，抗氧化活性高，无毒副作用，被FDA(美国食品和药物管理局)和EFSA(欧洲食品安全局)批准作为营养强化剂。但由于雨生红球藻生长速率慢，自养培养周期长，易生物污染等因素的制约，目前虾青素的生产成本较高且生产效率低下。
色绿藻(Chromochloris.zofingiensis)是一种单细胞绿藻，生长速率快，能够在黑暗异养条件下利用有机碳源进行异养高密度发酵，实现高密度细胞生长。此外，色绿藻在高光、高盐、氮磷营养元素限制等诱导条件下可以有效积累虾青素，同时还可以积累藻油，是目前虾青素生产的重要潜在微藻种质。相对于自养型雨生红球藻培养生产虾青素而言，色绿藻可以利用现在发酵培养装置在异养条件下实现高密度培养，但是色绿藻胞内虾青素积累量还比较低。筛选高效的诱导胁迫条件，进一步提高色绿藻胞内虾青素含量，进而建立针对色绿藻C.zofingiensis胞内色素尤其是虾青素的诱导培养工艺，这对于色绿藻生产制备虾青素和进一步商业化开发利用具有重要意义。
发明内容
为了弥补现有技术的不足，本发明的目的在于提供一种提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，实现了色绿藻胞内虾青素和藻油的同时高效积累，极大地提高了色绿藻胞内虾青素和藻油的产量。
本发明的技术目的是通过如下技术方案实现的：
本发明所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，包括如下步骤：
S1、将色绿藻细胞进行活化培养以获得种子液；
S2、将步骤S1活化培养获得种子液接种至无氮含糖的培养基，同时加入植物激素类诱导物，在微藻培养装置中进行诱导胁迫培养。
进一步地，本发明所述植物激素类诱导物为1-氨基环丙烷羧酸(ACC)、2,4-二氯苯氧基乙酸(2,4D)、2-氯苯甲酸(CA)、赤霉素(GA)、3-吲哚乙酸(IAA)、3-吲哚丁酸(IBA)、3-吲哚丙酸(IPA)、脱落酸(ABA)、激动素(KT)、乙醇胺(ETA)、胺鲜酯(DA)、1-萘乙酸(NAA)中的一种或两种以上。
优选地，本发明所述植物激素类诱导物选自1-氨基环丙烷羧酸，其添加浓度为1.0～5.0mg/L；或所述植物激素类诱导物选自2,4-二氯苯氧基乙酸，其添加浓度为5.0～10.0mg/L；或所述植物激素类诱导物选自2-氯苯甲酸，其添加浓度为5.0～10.0mg/L；或所述植物激素类诱导物选自赤霉素，其添加浓度为3.5～6.3mg/L；或所述植物激素类诱导物选自3-吲哚乙酸，其添加浓度为7.8～15.7mg/L；或所述植物激素类诱导物选自3-吲哚丁酸，其添加浓度为9.9～49.9mg/L；或所述植物激素类诱导物选自3-吲哚丙酸，其添加浓度为10.0～25.0mg/L；或所述植物激素类诱导物选自脱落酸，其添加浓度为10.0～100.0mg/L；或所述植物激素类诱导物选自激动素，其添加浓度为0.5～5.0mg/L；或所述植物激素类诱导物选自乙醇胺，其添加浓度为76.8～153.7mg/L；或所述植物激素类诱导物选自胺鲜酯，其添加浓度为2.1～21.5mg/L；或所述植物激素类诱导物选自1-萘乙酸，其添加浓度为5.3～10.7mg/L。
优选地，本发明所述植物激素类诱导物选自1-氨基环丙烷羧酸，其添加浓度为5.0mg/L；或所述植物激素类诱导物选自2,4-二氯苯氧基乙酸，其添加浓度为5.0mg/L；或所述植物激素类诱导物选自2-氯苯甲酸，其添加浓度为5.0mg/L；或所述植物激素类诱导物选自赤霉素，其添加浓度为6.9mg/L；或所述植物激素类诱导物选自3-吲哚乙酸，其添加浓度7.8mg/L；或所述植物激素类诱导物选自3-吲哚丁酸，其添加浓度为9.9mg/L；或所述植物激素类诱导物选自3-吲哚丙酸，其添加浓度为10.0mg/L，或所述植物激素类诱导物选自脱落酸，其添加 浓度为10.0mg/L。
优选地，本发明步骤S2中，加入植物激素类诱导物同时加入助溶剂，所述助溶剂在培养基中浓度为0.01％～0.1％，所述植物激素类诱导物为3-吲哚乙酸、3-吲哚丁酸、赤霉素中一种或两种或三种的组合；优选地，所述助溶剂选自水、乙醇或二甲亚砜。
优选地，本发明步骤S2中，将所述种子液接种至无氮含糖的培养基，并使种子液中色绿藻细胞密度稀释至2～3g/L，加入植物激素类诱导物，分装至包括透明微孔板或薄层培养装置的微藻培养装置中，并置于振荡器中，然后在光照培养箱中进行诱导胁迫培养，采用光源白光，光源强度为300±30μmol m -2s -1，光暗周期为0h:24h～24h:0h，培养温度为20～30℃。
优选地，本发明步骤S2诱导胁迫的培养温度为20～30℃，光源采用白光，光源强度为300μmol m -2s -1，光暗周期为24h:0h。
优选地，本发明步骤S2中所述无氮含糖培养基还包括如下浓度的组分：10g葡萄糖、0.175g磷酸二氢钾、0.075g磷酸氢二钾、0.075g七水硫酸镁、0.025g二水氯化钙、0.025g氯化钠、5mg氯化铁、0.287mg七水硫酸锌、0.169mg一水硫酸锰、0.061mg硼酸、0.0025mg五水硫酸铜、0.00124mg七水钼酸铵。
优选地，本发明步骤S1中活化培养是将色绿藻细胞转接至液体培养基中进行循环往复振荡培养，培养温度为20～30℃，光源强度为10～300μmol m -2s -1，转速为50～500rpm，光暗周期为0:24(h)～24:0(h)，所述液体培养基pH为6.0～7.0。
优选地，本发明所述液体培养基选自改良的Bristol、Basal、BG-11、BBM或Kuhl培养基。
具体地，本发明还包括以下步骤：
S3、培养结束后，收集培养的新鲜藻细胞，采用流式细胞仪在通道FL1和通道FL2进行色绿藻细胞的平均荧光强度的快速检测，从而初筛获得色绿藻细胞荧光增强的诱导条件；
S4、基于S3所获得诱导条件初筛实验结果，综合分析确定诱导物的种类和浓度，在相同诱导条件下进行色绿藻细胞的重新培养制备，再采用传统的准确检测分析方法测定色绿藻细胞内虾青素及藻油的含量；
S5、基于S4所获得诱导效果验证实验的测定结果，采用聚类分析方法，针对不同诱导条件进行综合分类，最终选择确定最佳的诱导胁迫条件，以达到色绿藻胞内虾青素和脂类的单独或共同积累。
本发明所提供的技术方案具有如下有益效果：
1).本发明通过建立诱导胁迫培养条件筛选的工艺技术，最终筛选并验证了有助于色绿藻胞内虾青素高效积累的植物激素类诱导物，实现了色绿藻胞内虾青素和藻油的同时高效积累，极大地提高了色绿藻胞内虾青素的产量。
2).该方法与现有的雨生红球藻生产虾青素的培养方式相比，可以大大提高虾青素的生产效率，方法简便，经济高效，在微藻工业化生产制备虾青素和藻油方面，具有非常重要的应用价值和广阔的市场前景。
附图说明
图1为不同植物激素诱导胁迫条件对于色绿藻C.zofingiensis细胞在流式细胞仪FL1和FL2通道的平均荧光强度的影响。FL1:533nm±15nm；FL2:585nm±20nm；Ctl:对照组；D:二甲亚砜；E:乙醇；H:高浓度；L:低浓度。
图2为不同植物激素诱导胁迫条件对于色绿藻C.zofingiensis细胞的生物量的影响。
图3为不同植物激素诱导胁迫条件对于色绿藻C.zofingiensis细胞内虾青素积累效果的影响。
图4为不同植物激素诱导胁迫条件对于色绿藻C.zofingiensis细胞内色素组分的影响。
图5为不同植物激素诱导胁迫条件对于色绿藻C.zofingiensis细胞内总脂积累效果的影响。
图6为不同植物激素诱导胁迫条件对于色绿藻C.zofingiensis细胞内脂肪酸组分的影响。
图7为不同植物激素诱导胁迫条件下色绿藻C.zofingiensis细胞生长情况以及虾青素和总脂积累效果的聚类分析图。
具体实施方式
诱导胁迫条件是决定色绿藻胞内虾青素积累量的重要影响因素。植物激素是微藻生物体内一大类非常重要的代谢调控因子，对于调节虾青素合成过程中的代谢合成酶的活性具有重要作用。目前已知的促进虾青素积累的诱导物主要有水杨酸、茉莉酸甲酯、赤霉素A 3、脱落酸、表油菜素内酯、外源性乙烯等，它们都是重要的植物生长调节剂，在微藻合成虾青素的代谢调控中具有积极作用。而营养物质缺乏胁迫、氧化胁迫以及代谢抑制剂胁迫等诱导胁迫条件，本质上都是诱导藻细胞内氧化水平的提高，进而通过活性氧自由基(ROS)信号介导的调控机制来诱导虾青素的合成。
植物激素的不同之处在于，它能够直接调控细胞的抗逆过程相关的特定基因的转录表达，进而诱导调控虾青素的积累，同时植物激素作为一种天然经济安全无害的化学诱导剂，在工农业生产中具有非常重要的作用。通过快速筛选实验可以获得有助于虾青素高效积累的植物 激素，再结合特定设计的微藻诱导培养装置(包括透明微孔板或类似薄层培养装置)，可以进一步显著提高色绿藻胞内虾青素和藻油积累量，具有非常重要的商业化应用前景。
本发明开发一种提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，采用常规的微藻三角瓶培养和基于微孔板的培养方法实现色绿藻细胞在不同植物激素和高光无氮条件下的协同诱导培养，然后采用流式细胞仪进行快速检测评估和初步筛选，再采用验证实验准确测定色绿藻细胞内高价值色素和藻油含量，从而确定最佳的植物激素诱导物及其最适浓度，最终显著提高色绿藻胞内的虾青素的积累量，同时生产制备藻油等。本发明可以显著改进或取代现有的利用微藻制备天然虾青素的生产方式。
下面结合附图对本发明的技术方案做进一步说明：
实施例1 多种不同植物激素诱导条件对色绿藻积累虾青素影响的初筛实验
本实施例采用多种不同的植物激素结合高光(80umol/m2/s以上)无氮胁迫条件对色绿藻Chromochloris zofingiensis细胞进行诱导胁迫培养，然后采用流式细胞仪测定了色绿藻细胞的平均荧光强度值，通过快速检测评估从而初步筛选确定了有助于色绿藻细胞荧光强度值增加的植物激素及其适宜浓度。
1.1菌种活化及种子液制备
将实验室中保存的色绿藻C.zofingiensis菌种转接到装有改良Bristol培养基的斜面上进行培养，培养温度为26℃，光照为10μmol m -2s -1，并观察色绿藻生长情况。
用接种环将色绿藻藻苔接种到装有改良Bristol液体培养基的三角瓶中进行培养，在温度为26℃、光源强度为10μmol m -2s -1的恒温振荡摇床中，培养3～5天用作种子液。
其中所述的改良Bristol培养基(pH6.5)成分如下表1所示(单位为mg/L)：
表1 液体培养基配方
组分 含量(mg/L) 组分 含量(mg/L) 组分 含量(mg/L)
葡萄糖 10000 NaNO 3 750 KH 2PO 4 175
K 2HPO 4 75 MgSO 4·7H 2O 25 CuSO 4·5H 2O 0.0025
FeCl 3 5 CaCl 2·2H 2O 25 MnSO 4·H 2O 0.169
NaCl 25 ZnSO 4·7H 2O 0.287 (NH 4) 6Mo 7O 24·7H 2O 0.00124
H 3BO 3 0.061 / / / /
1.2色绿藻C.zofingiensis在多种植物激素条件下的诱导胁迫培养
1.2.1色绿藻C.zofingiensis诱导培养条件的初筛实验
将培养好的色绿藻种子液在3800×g离心力的离心机中离心2min，弃去上层培养基，并用无菌水重新悬浮洗涤后，再次离心去除上清液，将收集的色绿藻细胞采用改良的无氮含糖发酵Bristol培养基进行稀释重悬，控制色绿藻的细胞密度在2～3g/L之间。同时，向重悬藻液中加入不同种类的植物激素(分别用水、乙醇、二甲亚砜作为助溶剂，浓度不超过0.1％)，控制植物激素在培养基中终浓度如表2所示。然后将重悬藻液分装到透明微孔板中，并置于微孔板振荡器中，一起放置于光照培养箱中进行诱导培养以积累虾青素。无氮含糖发酵培养基其和表1中的配方相比，不同仅在于：葡萄糖为10g/L，NaNO 3为0g/L，并且分别添加有不同浓度不同种类的植物激素类诱导物(见表2)。
培养条件为：转速为50～500rpm，温度为26℃、采用白色的荧光灯并排放置作为光源进行持续光照培养，控制微孔板表面的光照强度为300±30μmol m -2s -1以上，分别培养12天以上。
培养结束后，收集新鲜的色绿藻培养液，按照流式细胞仪测定荧光值的方法(如1.2.2所示)，快速测定色绿藻细胞在通道FL1和FL2的平均荧光强度值，用于对比分析不同植物激素的诱导效果。
表2 植物激素诱导物及其终浓度
Figure PCTCN2019112060-appb-000001
1.2.2流式细胞仪分析法
色绿藻诱导培养结束后，获得新鲜的色绿藻细胞，然后采用流式细胞仪(Accuri C6,BD,USA)分析测定不同培养条件得到的色绿藻细胞的平均荧光强度值(Mean fluorescence intensity, MFI)。
本实施例采用的流式细胞仪，配备有两个50mW的空冷激光器(激发波长分别为488nm和640nm)，并分别对应有四个检测通道(FL1:533±15nm；FL2:585±20nm；FL3>670nm；FL4:675±25nm)。
具体检测过程如下：将新鲜藻液在3800×g离心力下离心2分钟，然后用纯水洗涤离心两次后，用纯水将藻细胞密度稀释到0.6～2×10 6cells/mL，然后用400目过滤膜将藻细胞悬液过滤后直接用于流式细胞仪分析。
流式细胞仪分析中，流速设置为35μL/min,一共收集测定10000个细胞以上，分别测定细胞的正向散射光信号(Forward scatter,FSC),侧向散射光信号(Side scatter,SSC),以及两个通道FL1和FL2的平均荧光强度值。测定结束后，采用系统自带AccuriCflow数据处理软件进行统计分析色绿藻细胞的平均荧光强度值MFI。
1.3多种不同植物激素诱导条件对色绿藻积累虾青素影响的初筛实验结果
通过1.2.1和1.2.2的色绿藻诱导培养和流式细胞仪测定方法，可以获得色绿藻细胞的平均荧光强度值的变化情况，结果如图1所示。本实施例前期研究表明色绿藻细胞内虾青素等类胡萝卜素在488nm激发波长作用下可以发射荧光，并且虾青素含量较高，发射荧光强度越大，这暗示虾青素含量与该细胞在FL1和FL2通道(尤其是FL2通道)的平均荧光强度存在较强正相关性。因此本实施例采用流式细胞仪(Flow cytometry,FCM)测定色绿藻细胞的平均荧光强度，可以用来间接快速检测评估色绿藻细胞内的虾青素积累量，以此可以作为植物激素诱导效果的快速初筛的基本依据。为了直观展示不同植物激素诱导条件对于色绿藻细胞平均荧光强度值的影响，这里采用对照组的藻细胞的平均荧光强度值作为基准，其它诱导条件得到的藻细胞荧光值与之相比获得荧光强度比值，从而便于分析。
由在图1中结果可知，在不同植物激素诱导条件下，色绿藻细胞在FL1和FL2通道的荧光比值的变化情况基本一致，因此本实施例以FL2通道荧光强度比值为主，FL2通道荧光强度比值为辅，进而分析筛选出最有助于提高色绿藻细胞自发荧光强度的植物激素诱导物及其浓度。通过F检验可知，不同植物激素诱导胁迫条件对于色绿藻细胞在FL1(533nm±15nm)和FL2(585nm±20nm)通道的平均荧光强度比值均具有显著的影响(p<0.01)。在相同植物激素诱导条件下，不同的浓度对于色绿藻细胞的平均荧光强度比值也存在明显影响。由图中可知，在适宜的浓度条件下，植物激素IAA(7.8mg/L)、IBA(9.9mg/L)、ABA(10.0mg/L)、GA(6.9mg/L)、24D(5.0mg/L)、CA(5.0mg/L)均有助于色绿藻细胞平均荧光强度的增加，这表明这些植物激素可能是潜在的虾青素高效诱导物，因此这些植物激素作为初步筛选的条件，以备后 续进一步验证。此外，由图1可知，植物激素ACC、IPA、KT等对于色绿藻细胞的荧光强度影响不大，但是不同浓度对于细胞荧光强度还是存在影响，为了避免假性实验结果，本实施例选择ACC和IPA在浓度分别为5.0mg/L和10.0mg/L浓度作为后续验证实验。针对另外一些植物激素如ETA、DA和NAA等，它们对于色绿藻细胞的荧光强度具有降低作用，说明这些植物激素不仅没有提高虾青素的合成，还极大抑制了藻细胞内虾青素的积累，它们不适宜用于诱导色绿藻胞内虾青素的积累。
综上可知，通过分析不同植物激素诱导条件对于色绿藻细胞的荧光强度影响，本实施例基本完成了不同植物激素诱导物及其浓度的初步筛选，有助于提高色绿藻胞内虾青素积累量。但是考虑到采用不同的诱导胁迫条件进行色绿藻培养培养时，会导致细胞内积累的色素比例和含量存在较在差异，而这会影响到FCM快速检测评估的稳定性。因此，为了提高初筛的准确性，本实验仅采用FCM快速评估法来作为初步筛选结果，还需要结合验证实验最终确定，即首先初筛确定适宜的植物激素及其浓度，然后采用相同的诱导条件重新进行色绿藻细胞的培养验证实验，获得足够的藻细胞后采用传统定量方法，准确测定色绿藻细胞内虾青素含量，从而最终确定最佳的植物激素诱导物及其浓度。
实施例2 不同植物激素诱导条件对色绿藻生物量、虾青素和脂类积累影响的验证实验
本实施例主要针对实施例1中初步筛选确定的有助于增加色绿藻细胞荧光值的植物激素进行进一步验证，准确测定这些植物激素对于色绿藻细胞生长和虾青素积累量的影响，从而最终确定能够高效诱导色绿藻胞内虾青素积累的植物激素，最大限制提高色绿藻生产制备虾青素的生产水平。
2.1菌种活化及种子液制备
将实验室斜面保存的色绿藻C.zofingiensis菌种按1.1所述的培养方法，培养3～5天用作种子液。
2.2色绿藻C.zofingiensis在不同植物激素诱导作用下的验证培养实验
2.2.1色绿藻在不同植物激素作用下的验证培养实验
将培养好的色绿藻种子液按1.2.1所述的方法，离心收集色绿藻细胞，然后采用无氮含糖的发酵培养基进行稀释重悬，控制色绿藻细胞起始密度为2～3g/L左右，同时向重悬藻液中分别加入1.2初筛实验中确定的植物激素，然后将该藻液分装到不同的透明微孔板中，并置于微孔板振荡器中，最后一起放置于光照培养箱中，进行色绿藻C.zofingiensis培养积累虾青素。本实施例是为了解决实施例1的批量筛选实验中色绿藻细胞样品量少，无法满足色素和 总脂样品测定的要求等问题，最终目的是获得足够的藻细胞，用于准确测定细胞内虾青素和藻油积累量，从而最终确定这些植物激素的诱导效果。
发酵培养基采用改良Bristol培养基，其和表1中的培养基的不同在于：其葡萄糖为10g/L，另外分别添加实验例1中的1.2实验中筛选的植物激素及其最适浓度，为ACC 5.0mg/L、24D 5.0mg/L、CA 5.0mg/L、GA 6.9mg/L、IAA 7.8mg/L、IBA 9.9mg/L、IPA 10.0mg/L、ABA 10.0mg/L，从而制备得到含有不同植物激素诱导物的无氮含糖发酵培养基。
培养条件为：转速为50～500rpm，温度为26℃、采用白色的荧光灯并排放置作为光源，控制微孔板表面的光照强度为300±30μmol m -2s -1，进行持续高光照射，培养12天以上。
培养结束后，收集色绿藻培养液，并离心收集藻泥细胞，冻干获得干藻粉，置于-20℃冰箱中进行冷冻保存，用于色绿藻胞内色素和脂类含量的分析。色绿藻的生物量测定、色素含量测定以及脂类含量测定，采用2.2.2所述的测定方法进行分析。
2.2.2色绿藻细胞相关测定方法
2.2.2.1生物量的测定方法
色绿藻培养过程中的生物量的测定方法采用干重法进行测定。将取样获得的藻液，量取一定的体积置于事前称重的离心管中，在6000rpm转速下离心1min收集下层藻细胞，然后加入纯水振荡悬浮后，重复离心洗涤2次，除去上清液，将含有藻泥的离心管置于60℃烘箱中烘干至恒重，测定藻粉和离心管的总重。将干藻粉重量与藻液体积，通过换算即可以获得色绿藻培养液中的生物量。
2.2.2.2色绿藻胞内色素的提取方法
准确称取冻干藻粉10mg，置于装有陶瓷珠的冻存管中，加入含有0.1％(质量)BHT的二氯甲烷和甲醇的混合溶剂中，该混合溶剂中二氯甲烷：甲醇的体积比为3:1，高速振荡1分钟，然后置于液氮中迅速冷却，离心收集上清液；沉淀再次加入含有0.1％(质量)BHT的二氯甲烷和甲醇的混合溶剂中，振荡，置于液氮中冷却，离心收集上清液，重复该步骤直至藻细胞呈无色为止。合并所有上清液，用氮气吹干后，用色谱纯的甲醇和MTBE的混合溶剂(二者的体积比为1:1)溶剂定容到1mL，用于后续HPLC分析测定色绿藻胞内色素含量。
2.2.2.3HPLC测定方法
色绿藻C.zofingiensis胞内虾青素的HPLC检测方法：采用配备PDA检测器和YMC TM C30色谱柱(4.6mm×150mm,3μm)的液相色谱仪进行分析，洗脱液为色谱级甲醇(流动相A)、甲基叔丁基醚MTBE(流动相B)和水(流动相C)。梯度洗脱程序为：0-6min，95→80％A、5→20％B、0％C；6-12min,80→60％A、20→38％B、0→2％C；12-28min，60→50％A、 38→48％B、2％C；28-33min，50％A、48％B、2％C；33-35min，50→95％A、48→5％B、2→0％C；35-38min，95％A、5％B、0％C。
柱温箱温度为30℃，洗脱液流速为0.8mL/min，进样量为20μL，PDA检测器检测波长在300-700nm进行全波长扫描以测定色素吸收光谱图，同时在480nm波长下测定胞内色素的含量。色素的定性分析采用色素标准品(虾青素、叶绿素a、叶绿素b、叶黄素、角黄素、玉米黄质)的保留时间和吸收光谱图进行分析，定量分析采用色素标准品制作的标准曲线进行分析，对于酮基叶黄素和胡萝卜素类色素则采用类似的叶黄素标准曲线进行定量分析。
2.2.2.4总脂测定方法
总脂含量测定采用有机溶剂提取法进行测定，简述如下：取冷冻干燥得到的藻粉，准确称取约100mg，置于冻存管中，再加入适量陶瓷珠，用甲醇/二氯甲烷混合试剂(1:3,v/v)作为有机提取试剂进行提取。首先在高速珠磨仪上进行快速振荡，再用液氮冷冻破碎以提取脂类，期间多次离心收集上层提取液，直到油脂提取完全为止。合并的所有上层提取液用氮气吹干，最后用天平称重，根据提取前重质量差，即可获得藻细胞内总脂含量。
2.2.2.5脂肪酸提取方法
色绿藻胞内脂肪酸采用皂化和甲酯化的方法进行提取并用于GC-MS检测分析，具体如下：在螺口试管中加入一定体积的C19:0脂肪酸标准品溶液(浓度为1mg/mL，溶剂为二氯甲烷)，并氮气吹干溶剂后，再称量放入20mg左右藻粉，加入1mL饱和氢氧化钾-甲醇溶液，迅速摇匀，75℃水浴中加热10min，冷却至室温，加入2mL三氟化硼-无水甲醇溶液(1/2，v/v，其中三氟化硼浓度约为15％左右)，振荡混匀后75℃水浴加热10min，冷却至室温，再加入0.3mL饱和食盐水，最后加2mL正己烷混匀萃取脂肪酸甲酯，低速离心后取上层油层，采用有机滤膜过滤后置于气相进样小瓶中，用于气相质谱仪分析。
2.2.2.6气相质谱GC-MS测定法
脂肪酸含量测定方法采用安捷伦气相质谱联用仪进行分析，配以6890气相色谱仪、5975内置型MSD和高效毛细管柱(DB-23,30mm×0.25mm,0.25μm)。选用高纯氦气作为载气，流速为1ml/min，样品不分流，进样量为0.2μL。进样口温度为250℃，检测器温度为270℃。程序升温条件为：柱温箱130℃保持1min，然后以5℃/min升高到200℃保持5min。质谱的质量扫描范围为33-400amu。各峰型的鉴定采用NIST质谱库自动检索，对脂肪酸组分进行定性分析。定量分析采用C19:0为内标，以内标法测得各脂肪酸组分的定量分析。
2.3不同植物激素对色绿藻生物量、虾青素和脂类(藻油)积累的影响
通过本实施例2.2所述的诱导培养方法，可以获得不同植物激素对于色绿藻细胞生长、 虾青素和脂类积累的影响，结果如图2～6所示。
由图2中可知，通过F检验可知，不同植物激素对于色绿藻细胞生物量具有极显著影响(p<0.01)。植物激素如生长素IPA和脱落酸ABA对于色绿藻细胞生长则具有显著抑制作用(p<0.05)。尤其是IPA作为重要的植物生长素，相对于生长素IAA和IBA促进色绿藻细胞生长而言，经过IPA诱导培养后，色绿藻最终生物量仅为3.2g/L，与对照相比显著降低了52.9％。而生长素IAA和IBA以及赤霉素对于色绿藻细胞的生长均具有促进作用。其中，生长素IAA对于色绿藻生长具有最大促进作用，最高生长量可以达8.3g/L，同对照相比增加了26.7％，促进效果非常显著(p<0.05)。
在虾青素积累方面，除了植物激素2,4-D之外，其它植物激素如IAA、IBA、IPA、ABA等对于色绿藻胞内虾青素积累均具有显著的促进作用，结果如图3所示。在虾青素含量方面，吲哚丙酸IPA和脱落酸ABA对于色绿藻胞内虾青素含量的提高具有显著的促进作用(p<0.05)，虾青素含量最高分别达到13.1mg/g和12.6mg/g，相对于对照而言，分别增加了49％和43％。尤其需要强调的是，这也是目前报道的色绿藻胞内虾青素含量的最高水平。本实施例的研究结果，极大地提高了现有色绿藻细胞内积累虾青素的水平。
在虾青素产量和产率方面，要提高虾青素的生产水平，就需要同时兼顾生产色绿藻的生物量和虾青素含量。结合图2和图3可知，虽然IPA和ABA可以显著提高虾青素含量，但是它们均会显著抑制细胞的生长，导致生物量显著降低，因此最终的虾青素产量也较低，仅分别为42.1mg/L和44.6mg/L。相对而言，植物激素IAA、IBA、GA等对于虾青素积累的诱导效果稍低于IPA和ABA，但是它们不会显著抑制细胞生长，反而对于生物量和虾青素含量同时具有促进作用，因此最终显著提高了色绿藻细胞内虾青素的产量。当IAA浓度为7.8mg/L时，虾青素产量和产率达到最大值，分别为89.8mg/L和7.5mg/L/d。该产量和产率也是目前色绿藻生产积累虾青素的较高水平，基本达到或略优于采用自养雨生红球藻生产虾青素的水平。
通过对色绿藻胞内色素组分进行分析可知(如图4所示)，采用不同植物激素诱导培养色绿藻细胞，基本都可以诱导细胞内积累大量的虾青素二酯和单酯、酮基叶黄素、角黄素、类胡萝卜素等色素，还有少量游离虾青素、叶黄素和叶绿素等其它色素。在这些色素中，不同色素组分之间的百分比存在一定的差异，但是虾青素及其酯类基本都是色绿藻细胞内的最主要的色素，其占总色素百分比含量均达65％以上。虾青素二酯是藻细胞内虾青素的主要存在形式，其占总虾青素的含量达到75％以上，尤其是采用IAA和IBA进行诱导时，虾青素二酯占总虾青素的含量可以达到88％以上。综上，这表明色绿藻细胞在生产积累虾青素的同时， 还可以同时生产积累叶黄素等其它功能性类胡萝卜素，在功能性食品和水产饵料方面有着潜在的应用价值。
在色绿藻胞内脂类积累方面，由图5可知，不同植物激素不仅对色绿藻细胞内虾青素积累具有影响，对于脂类合成也具有促进作用。在众多植物激素，仅有植物生长素IAA和IBA会同时诱导色绿藻细胞内虾青素和脂类的积累。相对于植物激素IPA、ABA、CA显著抑制脂类积累而言(p<0.05)，IAA和IBA没有抑制脂类的合成，反而有一定程度提高，最高总脂含量分别达到细胞干重的64.5％和61.4％，同对照相比分别增加了7.6％和2.6％。在总脂产量和产率方面，经过IAA诱导培养后，色绿藻细胞的总脂产量最高可以达到5.3g/L，总脂产率同样可以高达445.7mg/L/d。该总脂产率是目前微藻生产积累脂类的较高水平，对于提高目前微藻工业化生产藻油的生产水平具有重要的价值。
最重要的是，本实施例2的最重要的创新性价值在于，采用本发明的工艺技术，可以诱导色绿藻胞内同时积累高附加值的虾青素和藻油，可以用作功能性食品原料和生物燃料原料，一方面极大地降低了微藻生物炼制工业的生产成本，另一方面具有非常重要的社会经济价值和环保价值。
通过对色绿藻胞内脂肪酸组分进行分析可知(如图6所示)，采用不同植物激素诱导培养色绿藻细胞，对于其胞内各脂肪酸组分百分比有着显著的影响(p<0.05)。由图6可知，虽然诱导培养条件不同，但色绿藻胞内脂肪酸主要以十六碳和十八碳脂肪酸为主，其百分比含量从高到低依次是C18:1、C16:0、C18:2、C18:3。其中C18:1基本占到色绿藻胞内总脂肪酸的40％左右，是色绿藻胞内最主要的脂肪酸。目前微藻生物炼制生产藻油是未来生物燃料原料以及未来食用型藻油的重要生产方式之一，具有非常重要的应用前景。本实施例中，经过诱导培养后，色绿藻胞内可以积累大量的藻油，其中脂肪酸主要以十六碳和十八碳为主，这表明经过诱导培养后获得的富油色绿藻具有开发为生物燃料来源的重要潜力；同时，藻油中含有丰富的油酸、亚油酸和亚麻酸等脂肪酸，具备有开发为食用型藻油的潜力。
结合本发明在色绿藻诱导积累虾青素和藻油方面的创造性成果价值，这里将本发明研究成果与现有公开的技术方案的成果进行对比，具体结果如表3所示。
表3 微藻生产积累虾青素的文献综合对比
Figure PCTCN2019112060-appb-000002
由表3中可知，相对于近年来研究最多的虾青素生产藻种雨生红球藻H.pluvialis，色绿藻C.zofingiensis最大的优势是可以利用有机碳源进行快速生长，获得最大98g/L的生物量，生长效率增加效果非常显著。但该藻种最大的缺点就是胞内虾青素含量偏低，基本在0.7～5.2mg/g之间。本发明申请技术方案最大的创造性贡献之一在于，可以将色绿藻胞内虾青素含量提高一个数据级，最高达到13.1mg/g(见图3)，虾青素的产率最高达到7.5mg/L/d(见表3)， 极显著地提高了现有色绿藻生产积累虾青素的水平。
本发明技术方案的另一个创造性贡献在于，在保证虾青素高效积累的同时，也实现了色绿藻胞内藻油的高效积累。相对于现有生产水平，采用本发明的技术方案，也显著提高了色绿藻胞内总脂含量，最高可以达到64.5％，总脂产率达到445.7mg/L/d(见表3)，处于目前生产的较高水平。
本发明成功实现了虾青素和藻油的高效同步积累，取得了创造性的研究成果。在本实施例2中，不同植物激素诱导胁迫条件对于色绿藻细胞生长、虾青素积累和脂类积累具有不同的诱导效果。为了对这些不同的植物激素地行归类对比分析，本实施例采用系统聚类分析技术对不同植物激素诱导胁迫条件进行分析，依据色绿藻细胞的生物量、虾青素和脂类含量及产量等数据为依据，采用统计分析软件进行系统聚类分析，采用组间联接的聚类方法，度量标准为Euclidean距离，聚类树状图如图7所示。由图中可知，当聚类标定距离为20时，基本可以将不同植物激素为分三类，分别是对虾青素和脂类积累均无明显促进的植物激素(ACC、2,4D)、同时促进虾青素和脂类积累的植物激素(IAA、IBA、GA)、促进虾青素积累但抑制脂类合成的植物激素(IPA、ABA)。在实际生产中，为了获得最高的虾青素胞内含量同时降低色绿藻胞内脂类含量，就可以选用IPA和ABA作为诱导物进行的色绿藻的诱导培养。而为了实现虾青素的最大化生产效率，同时高产藻油和脂肪酸等原料，就可以选用IAA和IBA等植物激素作为诱导物进行培养。总之，通过系统聚类分析，可以直观地反应不同植物激素对于虾青素和脂类合成的诱导效果，同时对于色绿藻生产制备虾青素和藻油也具有重要的指导意义。
以上所述，仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，故凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (10)

  1. 一种提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于，包括如下步骤：
    S1、将色绿藻细胞进行活化培养以获得种子液；
    S2、将步骤S1活化培养获得种子液接种至无氮含糖的培养基，同时加入植物激素类诱导物，在微藻培养装置中进行诱导胁迫培养。
  2. 根据权利要求1所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于：所述植物激素类诱导物为1-氨基环丙烷羧酸、2,4-二氯苯氧基乙酸、2-氯苯甲酸、赤霉素、3-吲哚乙酸、3-吲哚丁酸、3-吲哚丙酸、脱落酸、激动素、乙醇胺、胺鲜酯、1-萘乙酸中的一种或两种以上。
  3. 根据权利要求2所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于：所述植物激素类诱导物选自1-氨基环丙烷羧酸，其添加浓度为1.0～5.0mg/L；或所述植物激素类诱导物选自2,4-二氯苯氧基乙酸，其添加浓度为5.0～10.0mg/L；或所述植物激素类诱导物选自2-氯苯甲酸，其添加浓度为5.0～10.0mg/L；或所述植物激素类诱导物选自赤霉素，其添加浓度为3.5～6.3mg/L；或所述植物激素类诱导物选自3-吲哚乙酸，其添加浓度为7.8～15.7mg/L；或所述植物激素类诱导物选自3-吲哚丁酸，其添加浓度为9.9～49.9mg/L；或所述植物激素类诱导物选自3-吲哚丙酸，其添加浓度为10.0～25.0mg/L；或所述植物激素类诱导物选自脱落酸，其添加浓度为10.0～100.0mg/L；或所述植物激素类诱导物选自激动素，其添加浓度为0.5～5.0mg/L；或所述植物激素类诱导物选自乙醇胺，其添加浓度为76.8～153.7mg/L；或所述植物激素类诱导物选自胺鲜酯，其添加浓度为2.1～21.5mg/L；或所述植物激素类诱导物选自1-萘乙酸，其添加浓度为5.3～10.7mg/L。
  4. 根据权利要求3所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于：所述植物激素类诱导物选自1-氨基环丙烷羧酸，其添加浓度为5.0mg/L；或所述植物激素类诱导物选自2,4-二氯苯氧基乙酸，其添加浓度为5.0mg/L；或所述植物激素类诱导物选自2-氯苯甲酸，其添加浓度为5.0mg/L；或所述植物激素类诱导物选自赤霉素，其添加浓度为6.9mg/L；或所述植物激素类诱导物选自3-吲哚乙酸，其添加浓度7.8mg/L；或所述植物激素类诱导物选自3-吲哚丁酸，其添加浓度为9.9mg/L；或所述植物激素类诱导物选自3-吲哚丙酸，其添加浓度为10.0mg/L，或所述植物激素类诱导物选自脱落酸，其添加浓度为10.0mg/L。
  5. 根据权利要求1所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于：步骤S2中，加入植物激素类诱导物同时加入助溶剂，所述助溶剂在培养基中浓度为 0.01％～0.1％；所述植物激素类诱导物为3-吲哚乙酸、3-吲哚丁酸、赤霉素中一种或两种或三种的组合；优选地，所述助溶剂选自水、乙醇或二甲亚砜。
  6. 根据权利要求1所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于：步骤S2中，将所述种子液接种至无氮含糖的培养基，并使种子液中色绿藻细胞密度稀释至2～3g/L，加入植物激素类诱导物，分装至包括透明微孔板或类似薄层培养装置的微藻培养装置中，并置于振荡器中，然后在光照培养箱中进行诱导胁迫培养，采用白光光源，光源强度为300±30μmol m -2s -1，光暗周期为0h:24h～24h:0h，培养温度为20～30℃。
  7. 根据权利要求6所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于：步骤S2诱导胁迫的培养温度为20～30℃，光源采用白光，光源强度为300μmol m -2s -1，光暗周期为24h:0h。
  8. 根据权利要求1所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于，步骤S2中所述无氮含糖培养基还包括如下浓度的组分：10g葡萄糖、0.175g磷酸二氢钾、0.075g磷酸氢二钾、0.075g七水硫酸镁、0.025g二水氯化钙、0.025g氯化钠、5mg氯化铁、0.287mg七水硫酸锌、0.169mg一水硫酸锰、0.061mg硼酸、0.0025mg五水硫酸铜、0.00124mg七水钼酸铵。
  9. 根据权利要求1所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于：步骤S1中活化培养是将色绿藻细胞转接至液体培养基中进行循环往复振荡培养，培养温度为20～30℃，光源强度为10～300μmol m -2s -1，光暗周期为0:24(h)～24:0(h)，所述液体培养基pH为6.0～7.0。
  10. 根据权利要求1所述的提高色绿藻胞内虾青素和藻油积累量的诱导培养方法，其特征在于，还包括以下步骤：
    S3、培养结束后，收集培养的新鲜藻细胞，采用流式细胞仪在通道FL1和通道FL2进行色绿藻细胞的平均荧光强度的快速检测，从而初筛获得色绿藻细胞荧光增强的诱导条件；
    S4、基于S3所获得诱导条件初筛实验结果，综合分析确定诱导物的种类和浓度，在相同诱导条件下进行色绿藻细胞的重新培养制备，再采用传统的准确检测分析方法测定色绿藻细胞内虾青素及藻油的含量；
    S5、基于S4所获得诱导效果验证实验的测定结果，采用聚类分析方法，针对不同诱导条件进行综合分类，最终选择确定最佳的诱导胁迫条件，以达到色绿藻胞内虾青素和脂类的单独或共同积累。
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