CN106916751B - Method for mixed culture of haematococcus salina - Google Patents

Method for mixed culture of haematococcus salina Download PDF

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CN106916751B
CN106916751B CN201710143729.8A CN201710143729A CN106916751B CN 106916751 B CN106916751 B CN 106916751B CN 201710143729 A CN201710143729 A CN 201710143729A CN 106916751 B CN106916751 B CN 106916751B
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常明
刘睿杰
王兴国
金青哲
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Jiangnan University
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Abstract

The invention provides a method for mixed culture of erythrozoon salina, which comprises the steps of culturing the erythrozoon salina by utilizing a seed culture medium; domesticating and culturing by using glycerol; inoculating the domesticated erythrozoon salina to a glycerol fermentation culture medium, and culturing the erythrozoon salina by using light with different wavelengths and intensities. The invention improves the substrate utilization rate, the growth speed, the biomass, the phycoerythrin, the total protein, the polyunsaturated fatty acid and the total lipid content of the thallus during the fermentation of the erythromonas salina (R.salina), reduces the problem of harvesting the erythromonas salina (R.salina), and reduces the pretreatment cost of biomass concentration and target product extraction.

Description

Method for mixed culture of haematococcus salina
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to a mixed culture method of haematococcus salina.
Background
Octadecatetraenoic acid (18:4n-3) (SA) is an important omega-3 series polyunsaturated fatty acid ( delta 6,9,12, 15-all-cis-octadecatetraenoic acid), is used as a precursor substance of EPA, has higher conversion efficiency in animal bodies than arachidonic acid, has the effects of assisting in reducing the content of cholesterol and triglyceride in the bodies and promoting the metabolism of saturated fatty acid in the bodies, thereby reducing the blood viscosity, promoting the blood circulation, improving the oxygen supply of tissues and eliminating fatigue. Preventing fat deposition on blood vessel wall, preventing atherosclerosis formation and development, and preventing cardiovascular diseases such as cerebral thrombosis, cerebral hemorrhage, hypertension, etc. Currently SA is mainly derived from vegetable oils (high SA soybean oil) and microalgae. The soybean source mainly has the problems of low relative content of SA, geographical limitation and the like, so that the odor is bad, the purification cost is high and the like. On the contrary, the microbial oil (SCO) not only is easy to realize large-scale production, but also overcomes the problem that the traditional high-SA soybean is restricted by regions and climatic conditions, and has wide application prospect.
Rhodosporium salina (Rhodomonas salina or R.salina) belongs to Pyrenomonadaceae family member, and is unicellular rhodosporium flagellatum. Because of being rich in long-carbon-chain polyunsaturated fatty acid and natural pigment phycoerythrin, R.salina is regarded as a source phycophyta with great potential, and is widely applied to the aspects of food, cosmetics, medicine, scientific research, aquaculture and the like. The polyunsaturated fatty acids stearidonic acid (SA,18:4n-3) and alpha-linolenic acid (ALA18:3n-3) synthesized by salana account for 20-30.2% and 20.7-29.9% of the total fat in the cell, respectively.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or other problems occurring in the conventional mixed culture method of H.salina.
Therefore, the invention aims to provide a method for mixed culture of the erythrozoon salina, which greatly improves the efficiency of producing phycoerythrin and polyunsaturated fatty acid by the erythrozoon salina (R.salina), thereby reducing the production cost.
In order to solve the technical problems, the invention provides the following technical scheme: a method for mixed culture of Rhodosporidium salina comprises culturing Rhodosporidium salina with seed culture medium; domesticating and culturing by using glycerol; inoculating the domesticated erythrozoon salina to a glycerol fermentation culture medium, and culturing the erythrozoon salina by using light with different wavelengths and intensities.
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the seed culture medium comprises glycerol and Na2EDTA、H3BO3、FeCl3、MnSO4、ZnSO4、CoCl2、HCl、HEPES bufferpH 7.8、Vitamin B12One or more of them.
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the seed culture medium comprises glycerol and Na accounting for 10% of the mass of the culture medium2EDTA·2H2O 80mM、H3BO32mM、FeCl3·6H2O 5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、HCl 0.1M、HEPES bufferpH 7.8、Vitamin B120.3g/L。
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the glycerol fermentation medium comprises glycerol, NaCl and MgSO4·7H2O、KCl、NaNO3、CaCl2·2H2O、KH2PO4、Tricine pH 7.8、NH4Cl、Na2EDTA·2H2O、H3BO3、FeCl3·6H2O、MnSO4·H2O、ZnSO4·7H2O、CoCl2·6H2O, HCl, HEPES buffer pH 7.8 or Vitamin B12One or more of them.
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the method for culturing the erythrozoon salina by utilizing the seed culture medium comprises the step of inoculating the erythrozoon salina into the seed culture medium by 10-50% of inoculation amount, and culturing for 40-50 hours at the culture temperature of 21-30 ℃ and at the rotation speed of 100-200 rpm.
As a preferred method for the mixed culture of the haematococcus salinaA table, wherein: the light culture with different wavelengths and intensities is carried out by using light with different wavelengths and intensities at the temperature of 21-30 ℃, the pH value of 5-9 and the aeration rate of 0.01-10 m3·min-1The illumination intensity is 50 to 500 [ mu ] mol m-2s-1Culturing for 5-14 days.
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the method comprises the steps of utilizing light with different wavelengths and intensities, wherein the light comprises red light, blue light and red-blue mixed light, and the distance between the light and the liquid level of a fermentation medium is 15-30 cm.
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the domestication culture by using the glycerol comprises the steps of sequentially inoculating 5-50% of inoculum size into mixed culture media with different proportions for gradual domestication, wherein the temperature of each culture is 21-30 ℃, the pH value is 5-9, and the aeration rate is 0.01-10 m3Min, illumination intensity of 50 to 500. mu. mol-2s-1The culture time is 2-15 days.
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the red and blue mixed light is mixed in a ratio of blue light: the red light is 1: 1-5.
As a preferable scheme of the method for mixed culture of the haematococcus salina, the method comprises the following steps: the wavelength of the red light is 590-670 nm, and the peak is 640 nm; the wavelength of the blue light ranges from 420 nm to 510nm, and the peak is 460 nm.
The invention has the following beneficial effects:
the invention improves the substrate utilization rate, the growth speed, the biomass, the phycoerythrin, the total protein, the polyunsaturated fatty acid and the total lipid content of the thallus during the fermentation of the erythromonas salina (R.salina), reduces the problem of harvesting the erythromonas salina (R.salina), and reduces the pretreatment cost of biomass concentration and target product extraction.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a schematic diagram showing the growth of Haematococcus salina (R.salina) cultured in a mixture of glycerol at different concentrations under white light irradiation in the present invention;
FIG. 2 is a schematic diagram showing the synthesis of phycoerythrin from mixed culture of Rhodophyta salina (R.salina) with different concentrations of glycerol under white light irradiation in the present invention;
FIG. 3 is a schematic diagram showing the growth of Haematococcus salina (R.salina) cultured in mixed glycerol at different concentrations under red light irradiation in the present invention;
FIG. 4 is a schematic diagram showing the synthesis of phycoerythrin from Haematococcus salina (R.salina) cultured in mixed glycerol with different concentrations under the condition of red light irradiation in the present invention;
FIG. 5 is a schematic diagram showing the growth of Haematococcus salina (R.salina) cultured in mixed glycerol at different concentrations under blue light irradiation in the present invention;
FIG. 6 is a schematic diagram showing the synthesis of phycoerythrin from Haematococcus salina (R.salina) cultured in mixed glycerol with different concentrations under blue light irradiation in the present invention;
FIG. 7 is a schematic diagram showing the growth of Haematococcus salina (R.salina) cultured in mixed glycerol at different concentrations under the irradiation of red and blue mixed light according to the present invention;
fig. 8 is a schematic diagram of the synthesis of phycoerythrin from mixed culture of red and blue mixed glycerol with different concentrations in the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
sargassum salina (r. salina) seed medium:
the marine water from Haematococcus salina, glycerol 10%, and Na2EDTA·2H2O 80mM、H3BO32mM、FeCl3·6H2O5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B120.3g/L。
The seeds were cultured for two generations, each generation was carried out in a flask containing 50mL/250mL of liquid, and the inoculum size was 20% (V/V), and the shaking culture was carried out at 23 ℃ and 120rpm for 48 hours.
Then mixing the seed culture medium and the glycerol fermentation culture medium according to the proportion of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 respectively, inoculating the haematococcus halophilus into the mixed culture medium with the proportion of the seed culture medium and the glycerol fermentation culture medium being 10:1 in sequence according to the inoculation amount of 20% (V/V), wherein the white light intensity is 150 mu mol m-2s-1The fermentation condition is 23 ℃, and the aeration rate is 0.1m3Min. when the number of cells reached 1 × 105After one/ml, the next mixed culture medium (9: 1) is inoculated, and so on, the culture period of each culture medium is about 2-15 days.
After the last group of culture is finished, inoculating the mixture into an aeration reactor with the liquid loading capacity of 400mL/600mL by the inoculation amount of 20% (V/V), wherein the reactor is a glycerol fermentation culture medium, and the white light intensity is 150 mu mol m-2s-1Glycerol is 1About 10g/L, fermentation conditions 23 ℃ and aeration rate 0.1m3Min, fermentation for 10 days.
The glycerol fermentation medium comprises 1-10 g/L, NaCl 310mM of glycerol and MgSO 14·7H2O 10mM、KCl 8mM、NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.8 10mM、Vitamin B120.13g/L。
The experimental results are shown in fig. 1 and 2:
the results show that:
white light intensity 150 mu mol m-2s-1Mixed culture of Haematococcus salina (R.salina) with glycerol concentration of 1g/L for 9 days to reach maximum biomass of 2.21 × 107The yield of phycoerythrin per mL reaches 52.66mg/L, and the substrate utilization rate is 65.1%. Total lipid accounted for 18.32% of dry weight and SA accounted for 28.15% of total fatty acids.
Wherein the total fatty acids and total lipids are determined as follows,
extraction of total fatty acids: the freeze-dried cells were ground uniformly, and about 1.00g of the ground cells was accurately weighed, added to 7mL of 20% HCl, and shaken in a water bath at 75 ℃ for 60 min. The lipids were then extracted three times with 20mL of n-hexane, desolventized by rotary evaporation, and the residual solvent was purged with nitrogen. Weigh and calculate the total lipid content.
Total lipid content (%) ═ m1/m2*100％
In the formula, m1Represents the total lipid mass (g), m2Represents the mass (g) of the lyophilized mycelium used for lipid extraction.
Determination of fatty acid composition: capillary column (CP Sil-88: 50.0. mu. m.times.250. mu. m.times.0.20. mu.m), nitrogen as carrier gas, FID as detector. The temperature of the injection port was 250 ℃ and the volume of injection per time was 1. mu.L. Column temperature conditions: the temperature is kept at 80 ℃ for 2min, then the temperature is increased to 120 ℃ at the speed of 10 ℃/min, then the temperature is increased to 180 ℃ at the speed of 5 ℃/min for 2min, the temperature is increased to 230 ℃ at the speed of 2 ℃/min, and finally the temperature is kept at 230 ℃ for 5 min. The percentage of each fatty acid in the total lipid was calculated by normalization.
White light intensity 150 mu mol m-2s-1When the concentration of glycerol is higher than 4g/L, the growth of the salt-producing red blood cell algae (R.salina) is inhibited.
Example 2:
sargassum salina (r. salina) seed medium:
the marine water from Haematococcus salina, glycerol 10%, and Na2EDTA·2H2O 80mM、H3BO32mM、FeCl3·6H2O5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B120.3g/L。
The seeds were cultured for two generations, each generation was carried out in a flask containing 50mL/250mL of liquid, and the inoculum size was 20% (V/V), and the shaking culture was carried out at 23 ℃ and 120rpm for 48 hours.
Then mixing the seed culture medium and the glycerol fermentation culture medium according to the ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 respectively, inoculating the halorhodosporidium salina into the mixed culture medium with the ratio of the seed culture medium and the glycerol fermentation culture medium of 10:1 in sequence according to the inoculation amount of 20% (V/V), wherein the red light intensity is 250 mu mol m-2s-1The fermentation condition is 23 ℃, and the aeration rate is 0.1m3Min. when the number of cells reached 1 × 105After one/ml, the next mixed culture medium (9: 1) is inoculated, and so on, the culture period of each culture medium is about 2-15 days.
After the last group of culture is finished, inoculating the mixture into an aeration reactor with the liquid loading capacity of 400mL/600mL by the inoculation amount of 20% (V/V), wherein the reactor is a glycerol fermentation culture medium, and the red light intensity is 250 mu mol m-2s-11-10 g/L of glycerol, 23 ℃ of fermentation condition and 0.1m of aeration rate3Min, fermentation for 10 days. WhereinThe wavelength of the red light is 590-670 nm, and the wave peak is 640 nm.
The glycerol fermentation medium comprises 1-10 g/L, NaCl 310mM of glycerol and MgSO 14·7H2O 10mM、KCl 8mM、NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.810mM、Vitamin B120.13g/L。
The experimental results are shown in fig. 3 and 4:
the results show that:
the red light intensity is 250 mu mol m-2s-1Mixed culture of fermented Haematococcus salina (R.salina) with glycerol concentration of 4g/L for 10 days to reach maximum biomass of 2.82 × 107The yield of phycoerythrin per mL reaches 42.12mg/L, and the utilization rate of a substrate is 80.2%. Total lipid accounted for 17.53% of dry weight and SA accounted for about 32.41% of total fatty acids.
Wherein the total fatty acids and total lipids are determined as follows,
extraction of total fatty acids: the freeze-dried cells were ground uniformly, and about 1.00g of the ground cells was accurately weighed, added to 7mL of 20% HCl, and shaken in a water bath at 75 ℃ for 60 min. The lipids were then extracted three times with 20mL of n-hexane, desolventized by rotary evaporation, and the residual solvent was purged with nitrogen. Weigh and calculate the total lipid content.
Total lipid content (%) ═ m1/m2*100％
In the formula, m1Represents the total lipid mass (g), m2Represents the mass (g) of the lyophilized mycelium used for lipid extraction.
Determination of fatty acid composition: capillary column (CP Sil-88: 50.0. mu. m.times.250. mu. m.times.0.20. mu.m), nitrogen as carrier gas, FID as detector. The temperature of the injection port was 250 ℃ and the volume of injection per time was 1. mu.L. Column temperature conditions: the temperature is kept at 80 ℃ for 2min, then the temperature is increased to 120 ℃ at the speed of 10 ℃/min, then the temperature is increased to 180 ℃ at the speed of 5 ℃/min for 2min, the temperature is increased to 230 ℃ at the speed of 2 ℃/min, and finally the temperature is kept at 230 ℃ for 5 min. The percentage of each fatty acid in the total lipid was calculated by normalization.
The red light intensity is 250 mu mol m-2s-1When the concentration of glycerol is higher than 5g/L, the growth of the salt-producing red blood cell algae (R.salina) is inhibited.
Example 3:
sargassum salina (r. salina) seed medium:
the marine water from Haematococcus salina, glycerol 10%, and Na2EDTA·2H2O 80mM、H3BO32mM、FeCl3·6H2O5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B120.3g/L。
The seeds were cultured for two generations, each generation was carried out in a flask containing 50mL/250mL of liquid, and the inoculum size was 20% (V/V), and the shaking culture was carried out at 23 ℃ and 120rpm for 48 hours.
Then mixing the seed culture medium and the glycerol fermentation culture medium according to the ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 respectively, inoculating the halorhodosporidium salina into the mixed culture medium with the ratio of the seed culture medium and the glycerol fermentation culture medium of 10:1 in sequence according to the inoculation amount of 20% (V/V), and enabling the blue light intensity to be 150 mu mol m-2s-1The fermentation condition is 23 ℃, and the aeration rate is 0.1m3Min. when the number of cells reached 1 × 105After one/ml, the next mixed culture medium (9: 1) is inoculated, and so on, the culture period of each culture medium is about 2-15 days.
After the last group of culture is finished, inoculating the mixture into an aeration reactor with the liquid loading capacity of 400mL/600mL by the inoculation amount of 20% (V/V), wherein the reactor is a glycerol fermentation culture medium, and the blue light intensity is 250 mu mol m-2s-11-10 g/L of glycerol, 23 ℃ of fermentation condition and 0.1m of aeration rate3Min, fermentation for 10 days. Wherein the blue light has a wavelength of 420510nm with a peak at 460 nm.
The glycerol fermentation medium comprises 1-10 g/L, NaCl 310mM of glycerol and MgSO 14·7H2O 10mM、KCl 8mM、NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.8 10mM、Vitamin B120.13g/L。
The experimental results are shown in fig. 5 and 6:
the results show that:
the blue light can excite the antenna pigment in the halorhodophyta more effectively, so that the transformation efficiency of chloroplast is improved, and more energy is provided for cells.
Blue light intensity 250 μmol m-2s-1Mixed culture of fermented Haematococcus salina (R.salina) with glycerol concentration of 2g/L for 10 days to reach maximum biomass of 2.53 × 106The yield of phycoerythrin per mL reaches 44.51mg/L, and the substrate utilization rate is 81.8%. Total lipid accounted for 12.28% of dry weight and SA accounted for around 26.29% of total fatty acids.
Wherein the total fatty acids and total lipids are determined as follows,
extraction of total fatty acids: the freeze-dried cells were ground uniformly, and about 1.00g of the ground cells was accurately weighed, added to 7mL of 20% HCl, and shaken in a water bath at 75 ℃ for 60 min. The lipids were then extracted three times with 20mL of n-hexane, desolventized by rotary evaporation, and the residual solvent was purged with nitrogen. Weigh and calculate the total lipid content.
Total lipid content (%) ═ m1/m2*100％
In the formula, m1Represents the total lipid mass (g), m2Represents the mass (g) of the lyophilized mycelium used for lipid extraction.
Determination of fatty acid composition: capillary column (CP Sil-88: 50.0. mu. m.times.250. mu. m.times.0.20. mu.m), nitrogen as carrier gas, FID as detector. The temperature of the injection port was 250 ℃ and the volume of injection per time was 1. mu.L. Column temperature conditions: the temperature is kept at 80 ℃ for 2min, then the temperature is increased to 120 ℃ at the speed of 10 ℃/min, then the temperature is increased to 180 ℃ at the speed of 5 ℃/min for 2min, the temperature is increased to 230 ℃ at the speed of 2 ℃/min, and finally the temperature is kept at 230 ℃ for 5 min. The percentage of each fatty acid in the total lipid was calculated by normalization.
Blue light intensity 250 μmol m-2s-1When the concentration of glycerol is higher than 3g/L, the growth of the salt-producing red blood cell algae (R.salina) is inhibited.
Example 4:
sargassum salina (r. salina) seed medium:
the marine water from Haematococcus salina, glycerol 10%, and Na2EDTA·2H2O 80mM、H3BO32mM、FeCl3·6H2O5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B120.3g/L。
The seeds were cultured for two generations, each generation was carried out in a flask containing 50mL/250mL of liquid, and the inoculum size was 20% (V/V), and the shaking culture was carried out at 23 ℃ and 120rpm for 48 hours.
Then mixing the seed culture medium and the glycerol fermentation culture medium according to the ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 respectively, inoculating the halorhodosporidium salina with the inoculation amount of 20% (V/V) into the mixed culture medium with the ratio of the seed culture medium and the glycerol fermentation culture medium of 10:1 in sequence, wherein the intensity of the red and blue mixed light is 150 mu mol m-2s-1The fermentation condition is 23 ℃, and the aeration rate is 0.1m3Min. when the number of cells reached 1 × 105After one/ml, the next mixed culture medium (9: 1) is inoculated, and so on, the culture period of each culture medium is about 2-15 days.
After the last group of culture is finished, inoculating the mixture into an aeration reactor with the liquid loading capacity of 400mL/600mL by the inoculation amount of 20% (V/V), wherein the reactor is filled with a glycerol fermentation culture medium and the intensity of red and blue mixed light250μmol m-2s-11-10 g/L of glycerol, 23 ℃ of fermentation condition and 0.1m of aeration rate3Min, fermentation for 10 days. Wherein the wavelength of the blue light is 420-510 nm, and the peak is 460 nm. Wherein the wavelength of the red light is 590-670 nm, and the wave peak is 640 nm.
The glycerol fermentation medium comprises 1-10 g/L, NaCl 310mM of glycerol and MgSO 14·7H2O 10mM、KCl 8mM、NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.810mM、Vitamin B120.13g/L。
The experimental results are shown in fig. 7 and 8:
through research, the anabolic rhodomonas salina is higher in assimilation efficiency of glycerol than glucose, and the glycerol can provide more NADPH than the glucose in the process of entering a tricarboxylic acid cycle, so that more reducing power is provided for the synthesis of polyunsaturated fatty acid.
The results show that:
the mixed light intensity of red and blue is 250 mu mol m-2s-1Mixed culture of fermented Haematococcus salina (R.salina) with 1g/L glycerol concentration for 10 days to reach maximum biomass of 6.60 × 107The yield of phycoerythrin per mL reaches 78.85mg/L, and the substrate utilization rate is 95.8%. Total lipid accounted for 17.12% of dry weight and SA accounted for about 32.04% of total fatty acids.
Wherein the total fatty acids and total lipids are determined as follows,
extraction of total fatty acids: the freeze-dried cells were ground uniformly, and about 1.00g of the ground cells was accurately weighed, added to 7mL of 20% HCl, and shaken in a water bath at 75 ℃ for 60 min. The lipids were then extracted three times with 20mL of n-hexane, desolventized by rotary evaporation, and the residual solvent was purged with nitrogen. Weigh and calculate the total lipid content.
Total lipid content (%) ═ m1/m2*100％
In the formula, m1Represents the total lipid mass (g), m2Represents the mass (g) of the lyophilized mycelium used for lipid extraction.
Determination of fatty acid composition: capillary column (CP Sil-88: 50.0. mu. m.times.250. mu. m.times.0.20. mu.m), nitrogen as carrier gas, FID as detector. The temperature of the injection port was 250 ℃ and the volume of injection per time was 1. mu.L. Column temperature conditions: the temperature is kept at 80 ℃ for 2min, then the temperature is increased to 120 ℃ at the speed of 10 ℃/min, then the temperature is increased to 180 ℃ at the speed of 5 ℃/min for 2min, the temperature is increased to 230 ℃ at the speed of 2 ℃/min, and finally the temperature is kept at 230 ℃ for 5 min. The percentage of each fatty acid in the total lipid was calculated by normalization.
The mixed light intensity of red and blue is 250 mu mol m-2s-1When the concentration of glycerol is higher than 2g/L, the growth of the salt-producing red blood cell algae (R.salina) is inhibited.
Example 5 (comparative example)
The Botryococcus braunii was inoculated into the seed culture medium of examples 1 to 4, and cultured for two generations, each generation being carried out in a flask containing 50mL/250mL of liquid in an amount of 20% (V/V) and cultured with shaking at 23 ℃ and 120rpm for 48 hours.
Then mixing the seed culture medium and the glycerol fermentation culture medium according to the ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 respectively,
inoculating the Haematococcus salina into mixed culture medium of seed culture medium and glycerol fermentation culture medium at a ratio of 10:1 in sequence at an inoculation amount of 20% (V/V), wherein the intensity of red and blue mixed light is 150 μmol m-2s-1The fermentation condition is 23 ℃, and the aeration rate is 0.1m3Min. when the number of cells reached 1 × 105After one/ml, the next mixed culture medium (9: 1) is inoculated, and so on, the culture period of each culture medium is about 2-15 days.
After the last group of culture is finished, inoculating the mixture into an aeration reactor with the liquid loading capacity of 400mL/600mL by the inoculation amount of 20% (V/V), wherein the reactor is a glycerol fermentation culture medium, and the red and blue mixed light intensity is 250 mu mol m-2s-11-10 g/L of glycerinThe fermentation conditions were 23 ℃ and the aeration rate was 0.1m3Min, fermentation for 10 days. Wherein the wavelength of the blue light is 420-510 nm, and the peak is 460 nm. Wherein the wavelength of the red light is 590-670 nm, and the wave peak is 640 nm.
The experimental result shows that all groups of botryococcus braunii grow slowly, only a small amount of products exist in 1-2 g/L of glycerin under red and blue mixed light, and the individual groups hardly grow.
Therefore, the method improves the substrate utilization rate, the growth speed, the biomass, the phycoerythrin, the total protein, the polyunsaturated fatty acid and the total lipid content of the thallus during the fermentation of the erythromonas salina (R.salina), reduces the problem of harvesting of the erythromonas salina (R.salina), and reduces the pretreatment cost of biomass concentration and target product extraction.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (3)

1. A method for mixed culture of erythrozoon salina is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
culturing the erythrozoon salina by using a seed culture medium, inoculating 10-50% of the erythrozoon salina into the seed culture medium, and culturing at 21-30 ℃ and 100-200 rpm for 40-50 h;
domesticating and culturing by using glycerol, sequentially inoculating 5-50% of inoculum size into mixed culture medium with different proportion for gradual domestication, wherein the temperature of each culture is 21-30 ℃, the pH value is 5-9, and the aeration rate is 0.01-10 m3Min, illumination intensity of 50 to 500. mu. mol m-2s-1The culture time is 2-15 days;
inoculating domesticated Haematococcus salina to a glycerol fermentation culture medium, wherein the glycerol fermentation culture medium is prepared from glycerol, NaCl and MgSO4·7H2O、KCl、NaNO3、CaCl2·2H2O、KH2PO4、Tricine pH 7.8、NH4Cl、Na2EDTA·2H2O、H3BO3、FeCl3·6H2O、MnSO4·H2O、ZnSO4·7H2O、CoCl2·6H2O, HCl, HEPES buffer pH 7.8 and Vitamin B12, wherein glycerol is 1 g/L;
culturing the erythrozoon salina by using light with different wavelengths and intensities, wherein the temperature is 21-30 ℃, the pH value is 5-9, and the aeration rate is 0.01-10 m3·min-1The illumination intensity is 50 to 500 [ mu ] mol m-2s-1Culturing for 5-14 days, wherein the light is red-blue mixed light, the distance between the red-blue mixed light and the liquid level of the fermentation medium is 15-30 cm, and the mixed proportion of the red-blue mixed light is blue light: 1: 1-5 of red light, wherein the wavelength of the red light is 590-670 nm, and the peak is 640 nm; the wavelength of the blue light ranges from 420 nm to 510nm, and the peak is 460 nm.
2. The method for mixed culture of H.salina according to claim 1, wherein: the seed culture medium is composed of glycerol and Na2EDTA、H3BO3、FeCl3、MnSO4、ZnSO4、CoCl2HCl, HEPES buffer pH 7.8, Vitamin B12.
3. The method for mixed culture of H.halophila as claimed in claim 1 or 2, wherein: the seed culture medium comprises glycerol and Na accounting for 10% of the mass of the culture medium2EDTA·2H2O 80mM、H3BO32mM、FeCl3·6H2O5.02mM、 MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O0.001mM、HCl 0.1M、HEPESbuffer pH 7.8、Vitamin B12 0.3g/L。
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