CN106916751B - A kind of method for mixed culture of Haematocystis halophilus - Google Patents

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

A kind of method for mixed culture of Haematocystis halophilus

technical field

The invention belongs to the technical field of microorganism application, and in particular relates to a method for mixed culture of Haematocystis halophilus.

Background technique

Stearidonic acid (18:4n-3) (SA) is an important omega-3 series of polyunsaturated fatty acids (Δ6,9,12,15-all-cis-stearidonic acid), as The precursor substance of EPA, the conversion efficiency of SA in animals is much higher than that of arachidonic acid. It can help reduce the content of cholesterol and triglycerides in the body and promote the metabolism of saturated fatty acids in the body, thereby reducing blood viscosity and improving blood. Circulation, improve tissue oxygen supply and eliminate fatigue. Prevent the deposition of fat in the blood vessel wall, prevent the formation and development of atherosclerosis, prevent cerebral thrombosis, cerebral hemorrhage, hypertension and other cardiovascular diseases. Currently SA is mainly derived from vegetable oil (high SA soybean oil) and microalgae. Soybean sources mainly have many problems such as low relative content of SA and geographical restrictions, resulting in poor odor and high purification costs. On the contrary, microbial oils (Single Cell Oils, SCO) are not only easy to achieve large-scale production, but also overcome the problems of traditional high SA soybeans that are restricted by geographical and climatic conditions, and have broad application prospects.

Rhodomonas salin or R. salina is a member of the Pyrenomonadaceae family and is a flagellated unicellular red algae. Due to its richness in long carbon chain polyunsaturated fatty acids and natural pigment - phycoerythrin, R. salina has been regarded as a highly potential resource algae species for applications in food, cosmetics, medicine, scientific research and aquaculture. widely. The polyunsaturated fatty acids stearidonic acid (SA, 18:4n-3) and α-linolenic acid (ALA18:3n-3) synthesized by R. salina accounted for 20–30.2% and 20.7– of the total intracellular fat, respectively 29.9%.

SUMMARY OF THE INVENTION

The purpose of this section is to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the application to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions may not be used to limit the scope of the invention.

The present invention is proposed in view of the above-mentioned and/or existing problems in the mixed culture method of Haematocystis halophilus.

Therefore, the object of the present invention is to provide a method for mixed culture of R. salina, which greatly improves the efficiency of R. salina in producing phycoerythrin and polyunsaturated fatty acids, thereby reducing the production cost.

In order to solve the above technical problems, the present invention provides the following technical solutions: a method for mixed cultivation of Haematocystis halophilus, comprising: culturing Haematocystis halophilus with a seed medium; domesticating and culturing with glycerol; Haematocystis was inoculated into a glycerol fermentation medium, and Haematocystis halophilus was cultivated with light of different wavelengths and intensities.

As a preferred solution of the method for mixed culture of Haematococcus halophilus according to the present invention, wherein: the seed medium comprises glycerol, Na ₂ EDTA, H ₃ BO ₃ , FeCl ₃ , MnSO ₄ , ZnSO ₄ , CoCl ₂ One or more of , HCl, HEPES buffer pH 7.8, Vitamin B ₁₂ .

As a preferred solution of the method for mixed culture of Haematococcus halophilus of the present invention, wherein: the seed medium comprises glycerol, Na ₂ EDTA·2H ₂ O 80mM, H ₃ BO ₃ accounting for 10% of the mass of the medium 2mM, FeCl ₃ ·6H ₂ O 5.02mM, MnSO ₄ ·H ₂ O 0.01mM, ZnSO ₄ ·7H ₂ O 0.001mM, CoCl ₂ ·6H ₂ O 0.001mM, HCl 0.1M, HEPES buffer pH 7.8, Vitamin B ₁₂ 0.3 g/L.

As a preferred solution of the method for mixed culture of Haematococcus halophilus of the present invention, wherein: the glycerol fermentation medium comprises glycerol, NaCl, MgSO ₄ ·7H ₂ O, KCl, NaNO ₃ , CaCl ₂ ·2H _{2 O} , _KH2PO4 , _{Tricine pH 7.8} , _NH4Cl , _Na2EDTA.2H2O , _H3BO3 , _{FeCl3.6H2O , MnSO4.H2O} , _ZnSO4.7H2O , _{CoCl 2.} One or more of 6H ₂ O, HCl, HEPES buffer pH 7.8 or Vitamin B ₁₂ .

As a preferred solution of the method for mixed culture of Haematocystis halophilus of the present invention, wherein: the use of seed medium to cultivate Haematocystis halophilus is to inoculate Haematocystis salina at 10-50% of the The amount of the seed medium was inserted into the seed medium, and the cultivation temperature was 21-30 °C and 100-200 rpm for 40-50 h.

As a preferred solution of the method for mixed culture of Haematococcus halophilus according to the present invention, wherein: the use of light of different wavelengths and intensities to cultivate is to use light of different wavelengths and intensities at a temperature of 21-30°C, Under the conditions of pH value of 5 to 9, aeration rate of 0.01 to 10 m ³ ·min ^-1 , and light intensity of 50 to 500 μmol m ^-2 s ^-1 , the cells were cultured for 5 to 14 days.

As a preferred solution of the method for mixed culture of Haematococcus halophilus according to the present invention, wherein: the light of different wavelengths and intensities is used, wherein the light includes red light, blue light, and red and blue mixed light, which are combined with light of different wavelengths and intensities. The distance between the liquid surface of the fermentation medium is 15-30 cm.

As a preferred solution of the method for mixed culture of Haematococcus halophilus of the present invention, wherein: the acclimation culture using glycerol is to sequentially insert 5% to 50% of the inoculum into the mixed medium in different proportions and gradually For acclimation, the temperature of each cultivation was 21-30°C, the pH value was 5-9, the ventilation rate was 0.01-10 m ³ ·min, the light intensity was 50-500 μmolm ^-2 s ^-1 , and the cultivation time was 2-15 days.

As a preferred solution of the method for mixed culture of Haematocystis halophilus of the present invention, wherein: the red and blue mixed light has a mixing ratio of blue light: red light=1:1-5.

As a preferred solution of the method for mixed culture of Haematococcus halophilus of the present invention, wherein: the red light has a wavelength of 590-670 nm and a peak of 640 nm; the blue light has a wavelength of 420-510 nm and a peak of 640 nm. 460nm.

The beneficial effects that the present invention has:

The invention improves the substrate utilization rate, growth rate, biomass, phycoerythrin, total protein, polyunsaturated fatty acid and total lipid content of the bacteria during fermentation of R. salina, and reduces the amount of salinity. The problem of harvesting R. salina reduces the pre-treatment cost of biomass concentration and extraction of target products.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort. in:

Fig. 1 is the schematic diagram of the growth situation of different concentrations of glycerol mixed culture R. salina (R. salina) under white light irradiation conditions in the present invention;

2 is a schematic diagram of the phycoerythrin synthesis situation of the mixed culture of R. salina with different concentrations of glycerol under white light irradiation conditions in the present invention;

Fig. 3 is the schematic diagram of the growth situation of different concentrations of glycerol mixed culture R. salina (R. salina) under the condition of red light irradiation in the present invention;

Fig. 4 is a schematic diagram of the phycoerythrin synthesis of R. salina mixed culture of different concentrations of glycerol under red light irradiation conditions in the present invention;

Fig. 5 is the schematic diagram of the growth situation of different concentrations of glycerol mixed culture R. salina (R. salina) under blue light irradiation conditions in the present invention;

6 is a schematic diagram of the synthesis of phycoerythrin in the mixed culture of R. salina with different concentrations of glycerol under blue light irradiation conditions in the present invention;

Fig. 7 is the schematic diagram of the growth situation of different concentrations of glycerol mixed culture R. salina in the present invention under the mixed light irradiation condition of red and blue;

Figure 8 is a schematic diagram of the synthesis of phycoerythrin in R. salina mixed with different concentrations of glycerol under the irradiation conditions of red and blue mixed light in the present invention.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below with reference to specific embodiments.

Many specific details are set forth in the following description to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can do so without departing from the connotation of the present invention. Similar promotion, therefore, the present invention is not limited by the specific embodiments disclosed below.

Second, reference herein to "one embodiment" or "an embodiment" refers to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of "in one embodiment" in various places in this specification are not all referring to the same embodiment, nor are they separate or selectively mutually exclusive from other embodiments.

Example 1:

R. salina seed medium:

Haematocystis salina origin seawater, glycerol 10%, Na ₂ EDTA·2H ₂ O 80mM, H ₃ BO ₃ 2mM, FeCl ₃ ·6H ₂ O 5.02mM, MnSO ₄ ·H ₂ O 0.01mM, ZnSO ₄ ·7H ₂ O 0.001 mM, CoCl ₂ ·6H ₂ O 0.001 mM, Na ₂ EDTA · 2H ₂ O 50 mM, HCl 0.1 M, HEPES buffer pH 7.8 10 mM, Vitamin B ₁₂ 0.3 g/L.

The seeds were cultured for two generations, and each generation was carried out in a 50mL/250mL conical flask with an inoculum volume of 20% (V/V), and the culture was shaken at 23°C and 120rpm for 48h.

Then the seed medium and glycerol fermentation medium were 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, respectively. 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 mixed, Haematocystis salina was mixed with 20% (V /V) inoculated into the mixed medium of seed medium and glycerol fermentation medium in a ratio of 10:1, the white light intensity was 150 μmol m ^-2 s ^-1 , the fermentation conditions were 23°C, and the aeration rate was 0.1 m ³ ·min. When the number of bacterial cells reaches 1×10 ⁵ cells/ml, the next mixed medium (ie, 9:1) is inserted, and so on. The culture period of each medium is about 2 to 15 days.

After the last group was cultured, the inoculum of 20% (V/V) was inserted into a 400mL/600mL gas explosion type reactor with a liquid volume of 400mL/600mL. The reactor was a glycerol fermentation medium, and the white light intensity was 150μmol m ^-2 s. ^-1 , the glycerol was 1-10 g/L, the fermentation conditions were 23°C, the aeration rate was 0.1 m ³ ·min, and the fermentation was carried out for 10 days.

The composition of glycerol fermentation medium is glycerol 1～10g/L, NaCl 310mM, MgSO ₄ ·7H ₂ O 10mM, KCl 8mM, NaNO ₃ 12mM, CaCl ₂ ·2H ₂ O 2mM, KH ₂ PO ₄ 0.37mM, Tricine pH 7.8 25mM, _NH4Cl 0.5mM, Na2EDTA _· 2H2O _0.27mM , _{H3BO3 1.84mM} , FeCl3 _· 6H2O 0.018mM, MnSO4 _{· H2O 0.097mM} , ZnSO4 _{· 7H2O0} . 007mM, CoCl ₂ ·6H ₂ O 0.002mM, HCl 0.1mM, FeCl ₃ ·6H ₂ O 3mM, HEPES buffer pH 7.8 10mM, Vitamin B ₁₂ 0.13g/L.

The experimental results are shown in Figure 1 and Figure 2:

the result shows:

When the white light intensity was 150μmol m ^-2 s ^-1 and the glycerol concentration was 1g/L, the mixed culture of R. salina reached the maximum biomass of 2.21×10 ⁷ cells/mL in 9 days, and the phycoerythrin reached 52.66mg/L , the substrate utilization was 65.1%. Total lipids accounted for 18.32% of dry weight, SA accounted for 28.15% of total fatty acids.

Among them, the determination of total fatty acids and total lipids is as follows,

Extraction of total fatty acids: grind the freeze-dried cells uniformly, accurately weigh about 1.00 g, add 7 mL of 20% HCl, and shake in a water bath at 75° C. for 60 min. Then, the lipids were extracted three times with 20 mL of n-hexane, and after desolubilization by rotary evaporation, the residual solvent was purged with nitrogen. Weigh and calculate total lipid content.

Total fat content (%)=m ₁ /m ₂ *100%

In the formula, m ₁ represents the total lipid mass (g) obtained by weighing, and m ₂ represents the lyophilized mycelium mass (g) used for lipid extraction.

Determination of fatty acid composition: capillary column (CP Sil-88: 50.0 m×250 μm×0.20 μm), nitrogen as carrier gas, and FID as detector. The temperature of the injection port was 250°C, and the volume of each injection was 1 μL. Column temperature conditions: 80°C for 2min, then ramp up to 120°C at 10°C/min, then ramp up to 180°C at 5°C/min, continue for 2min, ramp up to 230°C at 2°C/min, last at 230°C for 5min . The percentage of each fatty acid in the total lipid was calculated by normalization method.

When the white light intensity was 150μmol m ^-2 s ^-1 , the glycerol concentration higher than 4g/L inhibited the growth of R. salina.

Example 2:

R. salina seed medium:

Haematocystis salina origin seawater, glycerol 10%, Na ₂ EDTA·2H ₂ O 80mM, H ₃ BO ₃ 2mM, FeCl ₃ ·6H ₂ O 5.02mM, MnSO ₄ ·H ₂ O 0.01mM, ZnSO ₄ ·7H ₂ O 0.001 mM, CoCl ₂ ·6H ₂ O 0.001 mM, Na ₂ EDTA · 2H ₂ O 50 mM, HCl 0.1 M, HEPES buffer pH 7.8 10 mM, Vitamin B ₁₂ 0.3 g/L.

The seeds were cultured for two generations, and each generation was carried out in a 50mL/250mL conical flask with an inoculum volume of 20% (V/V), and the culture was shaken at 23°C and 120rpm for 48h.

Then the seed medium and glycerol fermentation medium were 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, respectively. 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 mixed, Haematocystis salina was mixed with 20% (V /V) inoculated into the mixed medium of seed medium and glycerol fermentation medium in a ratio of 10:1, the red light intensity was 250 μmol m ^-2 s ^-1 , the fermentation conditions were 23°C, and the aeration rate was 0.1m ³ ·min. When the number of bacterial cells reaches 1×10 ⁵ cells/ml, the next mixed medium (ie, 9:1) is inserted, and so on. The culture period of each medium is about 2 to 15 days.

After the last group of culture was completed, the inoculum of 20% (V/V) was inserted into a 400mL/600mL gas-explosive reactor with a liquid volume of 400mL/600mL, and the reactor was a glycerol fermentation medium, and the red light intensity was 250μmol m ^-2 s ^-1 , glycerol was 1-10 g/L, fermentation conditions were 23°C, aeration rate was 0.1 m ³ ·min, and fermentation was performed for 10 days. Among them, the wavelength of red light is 590-670nm, and the peak is 640nm.

The composition of glycerol fermentation medium is glycerol 1～10g/L, NaCl 310mM, MgSO ₄ ·7H ₂ O 10mM, KCl 8mM, NaNO ₃ 12mM, CaCl ₂ ·2H ₂ O 2mM, KH ₂ PO ₄ 0.37mM, Tricine pH 7.8 25mM, _NH4Cl 0.5mM, Na2EDTA _· 2H2O _0.27mM , _{H3BO3 1.84mM} , FeCl3 _· 6H2O 0.018mM, MnSO4 _{· H2O 0.097mM} , ZnSO4 _{· 7H2O0} . 007mM, CoCl ₂ ·6H ₂ O 0.002mM, HCl 0.1mM, FeCl ₃ ·6H ₂ O 3mM, HEPES buffer pH 7.810mM, Vitamin B ₁₂ 0.13g/L.

The experimental results are shown in Figure 3 and Figure 4:

the result shows:

The red light intensity was 250μmol m ^-2 s ^-1 , the glycerol concentration was 4g/L, and the mixed culture and fermented R. salina reached the maximum biomass of 2.82×10 ⁷ cells/mL in 10 days, and the phycoerythrin reached 42.12mg /L, the substrate utilization rate was 80.2%. Total fat accounted for 17.53% of dry weight, SA accounted for about 32.41% of total fatty acid.

Among them, the determination of total fatty acids and total lipids is as follows,

Extraction of total fatty acids: grind the freeze-dried cells uniformly, accurately weigh about 1.00 g, add 7 mL of 20% HCl, and shake in a water bath at 75° C. for 60 min. Then, the lipids were extracted three times with 20 mL of n-hexane, and after desolubilization by rotary evaporation, the residual solvent was purged with nitrogen. Weigh and calculate total lipid content.

Total fat content (%)=m ₁ /m ₂ *100%

In the formula, m ₁ represents the total lipid mass (g) obtained by weighing, and m ₂ represents the lyophilized mycelium mass (g) used for lipid extraction.

Determination of fatty acid composition: capillary column (CP Sil-88: 50.0 m×250 μm×0.20 μm), nitrogen as carrier gas, and FID as detector. The temperature of the injection port was 250°C, and the volume of each injection was 1 μL. Column temperature conditions: 80°C for 2min, then ramp up to 120°C at 10°C/min, then ramp up to 180°C at 5°C/min, continue for 2min, ramp up to 230°C at 2°C/min, last at 230°C for 5min . The percentage of each fatty acid in the total lipid was calculated by normalization method.

When the red light intensity was 250μmol m ^-2 s ^-1 , the glycerol concentration higher than 5g/L inhibited the growth of R. salina.

Example 3:

R. salina seed medium:

Haematocystis salina origin seawater, glycerol 10%, Na ₂ EDTA·2H ₂ O 80mM, H ₃ BO ₃ 2mM, FeCl ₃ ·6H ₂ O 5.02mM, MnSO ₄ ·H ₂ O 0.01mM, ZnSO ₄ ·7H ₂ O 0.001 mM, CoCl ₂ ·6H ₂ O 0.001 mM, Na ₂ EDTA · 2H ₂ O 50 mM, HCl 0.1 M, HEPES buffer pH 7.8 10 mM, Vitamin B ₁₂ 0.3 g/L.

The seeds were cultured for two generations, and each generation was carried out in a 50mL/250mL conical flask with an inoculum volume of 20% (V/V), and the culture was shaken at 23°C and 120rpm for 48h.

Then the seed medium and glycerol fermentation medium were 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, respectively. 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 mixed, Haematocystis salina was mixed with 20% (V /V) inoculated into the mixed medium with the ratio of seed medium and glycerol fermentation medium at 10:1 in turn, the blue light intensity was 150 μmol m ^-2 s ^-1 , the fermentation conditions were 23°C, and the aeration rate was 0.1 m ³ ·min. When the number of bacterial cells reaches 1×10 ⁵ cells/ml, the next mixed medium (ie, 9:1) is inserted, and so on. The culture period of each medium is about 2 to 15 days.

After the last group of culture was completed, the inoculum of 20% (V/V) was inserted into a 400mL/600mL gas-explosion reactor with a liquid volume of 400mL/600mL. The reactor was a glycerol fermentation medium, and the blue light intensity was 250 μmol m ^-2 s. ^-1 , the glycerol was 1-10 g/L, the fermentation conditions were 23°C, the aeration rate was 0.1 m ³ ·min, and the fermentation was carried out for 10 days. Among them, the wavelength of blue light is 420 to 510 nm, and the peak is 460 nm.

The composition of glycerol fermentation medium is glycerol 1～10g/L, NaCl 310mM, MgSO ₄ ·7H ₂ O 10mM, KCl 8mM, NaNO ₃ 12mM, CaCl ₂ ·2H ₂ O 2mM, KH ₂ PO ₄ 0.37mM, Tricine pH 7.8 25mM, _NH4Cl 0.5mM, Na2EDTA _· 2H2O _0.27mM , _{H3BO3 1.84mM} , FeCl3 _· 6H2O 0.018mM, MnSO4 _{· H2O 0.097mM} , ZnSO4 _{· 7H2O0} . 007mM, CoCl ₂ ·6H ₂ O 0.002mM, HCl 0.1mM, FeCl ₃ ·6H ₂ O 3mM, HEPES buffer pH 7.8 10mM, Vitamin B ₁₂ 0.13g/L.

The experimental results are shown in Figure 5 and Figure 6:

the result shows:

Blue light is more effective for the excitation of antenna pigments in Haematococcus halophilus, thereby improving the transformation efficiency of chloroplasts and providing more energy for cells.

Blue light intensity 250μmol m ^-2 s ^-1 , glycerol concentration 2g/L mixed culture and fermentation of R. salina, the maximum biomass was 2.53×10 ⁶ /mL in 10 days, and the phycoerythrin reached 44.51mg/mL L, the substrate utilization was 81.8%. Total fat accounted for 12.28% of dry weight, SA accounted for about 26.29% of total fatty acid.

Among them, the determination of total fatty acids and total lipids is as follows,

Extraction of total fatty acids: grind the freeze-dried cells uniformly, accurately weigh about 1.00 g, add 7 mL of 20% HCl, and shake in a water bath at 75° C. for 60 min. Then, the lipids were extracted three times with 20 mL of n-hexane, and after desolubilization by rotary evaporation, the residual solvent was purged with nitrogen. Weigh and calculate total lipid content.

Total fat content (%)=m ₁ /m ₂ *100%

In the formula, m ₁ represents the total lipid mass (g) obtained by weighing, and m ₂ represents the lyophilized mycelium mass (g) used for lipid extraction.

Determination of fatty acid composition: capillary column (CP Sil-88: 50.0 m×250 μm×0.20 μm), nitrogen as carrier gas, and FID as detector. The temperature of the injection port was 250°C, and the volume of each injection was 1 μL. Column temperature conditions: 80°C for 2min, then ramp up to 120°C at 10°C/min, then ramp up to 180°C at 5°C/min, continue for 2min, ramp up to 230°C at 2°C/min, last at 230°C for 5min . The percentage of each fatty acid in the total lipid was calculated by normalization method.

When the blue light intensity was 250μmol m ^-2 s ^-1 , the glycerol concentration higher than 3g/L inhibited the growth of R. salina.

Example 4:

R. salina seed medium:

Haematocystis salina origin seawater, glycerol 10%, Na ₂ EDTA·2H ₂ O 80mM, H ₃ BO ₃ 2mM, FeCl ₃ ·6H ₂ O 5.02mM, MnSO ₄ ·H ₂ O 0.01mM, ZnSO ₄ ·7H ₂ O 0.001 mM, CoCl ₂ ·6H ₂ O 0.001 mM, Na ₂ EDTA · 2H ₂ O 50 mM, HCl 0.1 M, HEPES buffer pH 7.8 10 mM, Vitamin B ₁₂ 0.3 g/L.

The seeds were cultured for two generations, and each generation was carried out in a 50mL/250mL conical flask with an inoculum volume of 20% (V/V), and the culture was shaken at 23°C and 120rpm for 48h.

Then the seed medium and glycerol fermentation medium were 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, respectively. 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 mixed, Haematocystis salina was mixed with 20% (V /V) inoculated into the mixed medium with the ratio of seed medium and glycerol fermentation medium at 10:1 in turn, the mixed light intensity of red and blue is 150 μmol m ^-2 s ^-1 , and the fermentation conditions are 23° C., aeration The rate was 0.1 m ³ ·min. When the number of bacterial cells reaches 1×10 ⁵ cells/ml, the next mixed medium (ie, 9:1) is inserted, and so on. The culture period of each medium is about 2 to 15 days.

After the last group was cultured, the inoculum of 20% (V/V) was inserted into a 400mL/600mL gas-explosion reactor with a liquid volume of 400mL/600mL. The reactor was a glycerol fermentation medium, and the red and blue mixed light intensity was 250 μmol m. ^-2 s ^-1 , glycerol was 1-10 g/L, fermentation conditions were 23°C, aeration rate was 0.1 m ³ ·min, and fermentation was performed for 10 days. Among them, the wavelength of blue light is 420 to 510 nm, and the peak is 460 nm. Among them, the wavelength of red light is 590-670nm, and the peak is 640nm.

The composition of glycerol fermentation medium is glycerol 1～10g/L, NaCl 310mM, MgSO ₄ ·7H ₂ O 10mM, KCl 8mM, NaNO ₃ 12mM, CaCl ₂ ·2H ₂ O 2mM, KH ₂ PO ₄ 0.37mM, Tricine pH 7.8 25mM, _NH4Cl 0.5mM, Na2EDTA _· 2H2O _0.27mM , _{H3BO3 1.84mM} , FeCl3 _· 6H2O 0.018mM, MnSO4 _{· H2O 0.097mM} , ZnSO4 _{· 7H2O0} . 007mM, CoCl ₂ ·6H ₂ O 0.002mM, HCl 0.1mM, FeCl ₃ ·6H ₂ O 3mM, HEPES buffer pH 7.810mM, Vitamin B ₁₂ 0.13g/L.

The experimental results are shown in Figure 7 and Figure 8:

Through research, it is found that the assimilation efficiency of glycerol in this group of Haematococcus halophilus is higher than that of glucose, and glycerol can provide more NADPH than glucose in the process of entering the tricarboxylic acid cycle, thereby providing more NADPH for the synthesis of polyunsaturated fatty acids. the restoring power.

the result shows:

Red and blue mixed light intensity of 250μmol m ^-2 s ^-1 and glycerol concentration of 1g/L mixed culture and fermented R. salina, the maximum biomass reached 6.60×10 ⁷ cells/mL in 10 days, and the phycoerythrin reached 78.85mg/L, and the substrate utilization rate was 95.8%. Total fat accounted for 17.12% of dry weight, SA accounted for about 32.04% of total fatty acid.

Among them, the determination of total fatty acids and total lipids is as follows,

Extraction of total fatty acids: grind the freeze-dried cells uniformly, accurately weigh about 1.00 g, add 7 mL of 20% HCl, and shake in a water bath at 75° C. for 60 min. Then, the lipids were extracted three times with 20 mL of n-hexane, and after desolubilization by rotary evaporation, the residual solvent was purged with nitrogen. Weigh and calculate total lipid content.

Total fat content (%)=m ₁ /m ₂ *100%

In the formula, m ₁ represents the total lipid mass (g) obtained by weighing, and m ₂ represents the lyophilized mycelium mass (g) used for lipid extraction.

Determination of fatty acid composition: capillary column (CP Sil-88: 50.0 m×250 μm×0.20 μm), nitrogen as carrier gas, and FID as detector. The temperature of the injection port was 250°C, and the volume of each injection was 1 μL. Column temperature conditions: 80°C for 2min, then ramp up to 120°C at 10°C/min, then ramp up to 180°C at 5°C/min, continue for 2min, ramp up to 230°C at 2°C/min, last at 230°C for 5min . The percentage of each fatty acid in the total lipid was calculated by normalization method.

When the mixed light intensity of red and blue was 250μmol m ^-2 s ^-1 , the growth of R. salina was inhibited by the concentration of glycerol higher than 2g/L.

Example 5 (comparative example)

B. braunenii was inoculated in the seed medium of the same embodiment 1-4, and cultured for two generations, each generation was carried out in a 50mL/250mL conical flask with a liquid filling volume, and the inoculum volume was 20% (V/V) at 23° C., Shaking at 120rpm for 48h.

Then the seed medium and glycerol fermentation medium were 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, respectively. 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 mix,

The inoculum of 20% (V/V) of Haematocystis halophilus was sequentially inserted into the mixed medium with the ratio of seed medium and glycerol fermentation medium being 10:1, and the mixed light intensity of red and blue was 150 μmol m ^-2 s ^-1 , the fermentation conditions were 23°C, and the aeration rate was 0.1 m ³ ·min. When the number of bacterial cells reaches 1×10 ⁵ cells/ml, the next mixed medium (ie, 9:1) is inserted, and so on. The culture period of each medium is about 2 to 15 days.

After the last group of culture is completed, the inoculum of 20% (V/V) is inserted into a 400mL/600mL gas-exploding reactor with a liquid volume of 400mL/600mL. The reactor is a glycerol fermentation medium, and the mixed light intensity of red and blue is 250 μmol m ^-2 s ^-1 , glycerol was 1-10 g/L, fermentation conditions were 23°C, aeration rate was 0.1 m ³ ·min, and fermentation was performed for 10 days. Among them, the wavelength of blue light is 420-510 nm, and the peak is 460 nm. Among them, the wavelength of red light is 590-670nm, and the peak is 640nm.

The experimental results showed that each group of Botrytis brauneni grew slowly. Under the mixed light of red and blue, only a small amount of glycerol was produced at 1-2 g/L, and some groups hardly grew.

It can be seen that the present invention improves the substrate utilization rate, growth rate, biomass, phycoerythrin, total protein, polyunsaturated fatty acid and total lipid content of bacteria during fermentation of R. salina, The problem of harvesting R. salina is reduced, and the pre-treatment cost of biomass concentration and target product extraction is reduced.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent substitutions without departing from the spirit and scope of the technical solutions of the present invention should be included in the scope of 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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