Method for enhancing biomass and astaxanthin accumulated by haematococcus pluvialis based on gradient salt stress

Abstract

The invention discloses a method for enhancing biomass and astaxanthin accumulated by haematococcus pluvialis based on gradient salt stress, and belongs to the technical field of bioengineering. According to the method, low-salt pre-stress is firstly carried out in the haematococcus pluvialis logarithmic culture stage to enhance the stress resistance of algae cells, and then high-salt is applied to induce the haematococcus pluvialis to efficiently accumulate biomass and astaxanthin in the stress stage. According to the method, the biomass of haematococcus pluvialis and the yield and stress resistance of astaxanthin can be effectively improved, the process is simple, operation is easy, dependence on the environment is small, the environment cannot be harmed in the culture process, and the method has a strong application prospect in large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for enhancing the accumulation of biomass and astaxanthin of Haematococcus pluvialis based on gradient salt stress, belonging to the field of bioengineering technology. BACKGROUND

[0002] As a highly valuable ketone carotenoid, astaxanthin exhibits super strong antioxidant capacity in free radical scavenging. Its antioxidant activity is 550 times that of vitamin E and 10 times that of beta-carotene, and it is called "super antioxidant". It has been widely used in many fields. Haematococcus pluvialis is currently the best biological source of natural astaxanthin in nature, and Haematococcus pluvialis has become the first choice for industrial production of natural astaxanthin. However, there is a core problem in the industrial cultivation of Haematococcus pluvialis. Algal cells have weak stress resistance in the vegetative growth stage, and in the high salinity and strong light stress environment required for astaxanthin synthesis, algal cells are easily damaged, metabolically disordered, or even die, which limits the improvement of astaxanthin synthesis efficiency and final yield.

[0003] Currently, the traditional cultivation technology of Haematococcus pluvialis mainly relies on single stress treatment to induce the synthesis of astaxanthin. Although this method can promote the accumulation of astaxanthin to a certain extent, the biomass is significantly reduced due to the inability of algal cells to adapt to severe environmental changes. In addition, the activity of algal cells under long-term high-intensity stress conditions is inhibited, thereby limiting the overall yield of astaxanthin. In addition, the existing cultivation method has strict requirements for environmental conditions, which is difficult to realize large-scale stable production, and the complex process increases the operation difficulty and pollution risk. Therefore, developing a cultivation method that can both enhance the stress resistance of algal cells and achieve high-efficiency accumulation of biomass and astaxanthin is the key to breaking the bottleneck of Haematococcus pluvialis industrialization, and it is of great significance to promote the sustainable development of astaxanthin industry. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides a method for enhancing the accumulation of biomass and astaxanthin of Haematococcus pluvialis based on gradient salt stress, and the specific cultivation method is as follows: (1) First stage cultivation: cultivate Haematococcus pluvialis to the logarithmic growth phase, and inoculate the algal cells obtained in the logarithmic growth phase into a culture medium containing a low concentration of NaCl for cultivation.

[0005] (2) Second stage cultivation: inoculate the Haematococcus pluvialis subjected to low-salt pre-stress in step (1) into a culture medium containing a high concentration of NaCl for cultivation.

[0006] Preferably, the culture medium for cultivating Haematococcus pluvialis in step (1) and step (2) is BG-11 culture medium.

[0007] Preferably, the culture conditions in step (1) are: ambient temperature of 24-26°C, and sterile air ventilation volume of 1 vvm.

[0008] Preferably, the culture conditions in step (2) are: ambient temperature of 27-29°C, and sterile air ventilation volume of 1 vvm.

[0009] Preferably, the light intensity in step (1) is 2900~3100LUX.

[0010] Preferably, the light intensity in step (2) is 22000 LUX.

[0011] Preferably, the concentration of NaCl in the culture medium in step (1) is 25 mg L -1 ~100mg L -1 .

[0012] Further preferably, the concentration of NaCl in the culture medium in step (1) is 25 mg L -1 ~50mg L -1 .

[0013] Preferably, the concentration of NaCl in the culture medium in step (2) is 1 g L -1 .

[0014] Preferably, the inoculation amount of algal cells during low-salt pre-stress culture in step (1) is 1.5×10 5 cells mL -1 .

[0015] Preferably, in step (1), the number of days for low-salt pre-stress culture of Haematococcus pluvialis during its logarithmic growth phase is 4 days.

[0016] Preferably, the number of days for culturing Haematococcus pluvialis in step (2) is 13 days.

[0017] Beneficial effects of the present invention: (1) The results of the present invention show that gradient salt stress enhances the accumulation of biomass and astaxanthin in Haematococcus pluvialis. This culture method significantly improves the stress resistance and astaxanthin production potential of Haematococcus pluvialis, and increases the biomass and astaxanthin accumulation of Haematococcus pluvialis. The highest biomass of Haematococcus pluvialis reached 2.13 g L -1 , which was significantly increased by 14.24% compared with that without gradient salt stress; the astaxanthin yield reached 4.37 mg L -1 d -1 , which was significantly increased by 34.95% compared with that without gradient salt stress; the highest cell density of Haematococcus pluvialis was 4.55×10 5 cells mL -1Compared with no gradient salt stress, it is significantly improved by 24.52%.

[0018] (2) The present application creates the best environment for Haematococcus pluvialis biomass and astaxanthin accumulation by controlling the culture conditions (such as temperature, light intensity, ventilation, etc.), which helps to improve the astaxanthin yield of algae.

[0019] (3) The present application helps to promote Haematococcus pluvialis to improve the production efficiency of astaxanthin, which is expected to reduce the dependence on natural resources and the impact on the environment, thereby promoting environmental sustainability.

[0020] (4) The present application provides an efficient culture strategy for large-scale production of Haematococcus pluvialis astaxanthin, which has high commercial application value.

[0021] (5) The present application has simple process, easy operation and high efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a flowchart of the method described in the present application.

[0023] Figure 2 is an influence diagram of Haematococcus pluvialis biomass of Examples 1~3 and Comparative Examples 1~2.

[0024] Figure 3 is an influence diagram of Haematococcus pluvialis astaxanthin yield of Examples 1~3 and Comparative Examples 1~2.

[0025] Figure 4 is an influence diagram of Haematococcus pluvialis cell density of Examples 1~3 and Comparative Examples 1~2. DETAILED DESCRIPTION

[0026] The technical solutions of the present application will be further described below in conjunction with the drawings and through specific embodiments. However, the following examples are only simple examples of the present application, and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0027] Example 1 A method for enhancing Haematococcus pluvialis biomass and astaxanthin accumulation based on gradient salt stress, in the first culture stage, the logarithmic growth period is pre-stressed with low salt NaCl concentration of 25mg / L -1 The specific culture method is as follows: (1) The first stage culture: in 3L round bottom flask, add 2.5L BG-11 medium to culture Haematococcus pluvialis, the environmental temperature of culture is 26℃, the light intensity is 2900LUX, the sterile air ventilation volume is 1vvm, and the Haematococcus pluvialis is grown to logarithmic growth phase; 400mL BG-11 medium is used as the basic medium, and NaCl is added in the medium to make the concentration of NaCl in the medium 25mg / L -1 The logarithmic growth phase algae cells obtained by culture are inoculated into the medium for low salt pre-stress to enhance the stress resistance of the algae cells, and the inoculation amount of Haematococcus pluvialis is 1.5×10 5 cells / mL -1 , the environmental temperature of culture is 26℃, the light intensity is 2900LUX, the sterile air ventilation volume is 1vvm, and the Haematococcus pluvialis is grown to logarithmic growth phase; 400mL BG-11 medium is used as the basic medium, and NaCl is added in the medium to make the concentration of NaCl in the medium 25mg / L

[0028] (2) The second stage culture: the Haematococcus pluvialis subjected to low salt pre-stress in step (1) is inoculated into the BG-11 medium added with NaCl for stress culture, the concentration of NaCl in the medium is 1g / L -1 , the environmental temperature of culture is 27℃, the light intensity is 22000LUX, the sterile air ventilation volume is 1vvm, and the Haematococcus pluvialis is grown to logarithmic growth phase; 400mL BG-11 medium is used as the basic medium, and NaCl is added in the medium to make the concentration of NaCl in the medium 25mg / L

[0029] Example 2 A method for enhancing the accumulation of biomass and astaxanthin of Haematococcus pluvialis based on gradient salt stress, in the first culture stage, the logarithmic growth phase is subjected to low salt pre-stress, and the concentration of NaCl is 50mg / L -1 , and the specific culture method is as follows: (1) The first stage culture: in 3L round bottom flask, add 2.5L BG-11 medium to culture Haematococcus pluvialis, the environmental temperature of culture is 24℃, the light intensity is 3100LUX, the sterile air ventilation volume is 1vvm, and the Haematococcus pluvialis is grown to logarithmic growth phase; 400mL BG-11 medium is used as the basic medium, and NaCl is added in the medium to make the concentration of NaCl in the medium 50mg / L -1 The logarithmic growth phase algae cells obtained by culture are inoculated into the medium for low salt pre-stress to enhance the stress resistance of the algae cells, and the inoculation amount of Haematococcus pluvialis is 1.5×10 5 cells / mL -1 , the environmental temperature of culture is 24℃, the light intensity is 3100LUX, the sterile air ventilation volume is 1vvm, and the Haematococcus pluvialis is grown to logarithmic growth phase; 400mL BG-11 medium is used as the basic medium, and NaCl is added in the medium to make the concentration of NaCl in the medium 25mg / L

[0030] (2) Second stage of cultivation: 1 g L -1 NaCl stress was used, the culture environment temperature was 29°C, the light intensity was 22000LUX, the sterile air ventilation volume was 1vvm, and the culture was continued for 13 days to obtain Haematococcus pluvialis containing astaxanthin.

[0031] Example 3 A method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis based on gradient salt stress, wherein the NaCl concentration for low salt pre-stress during the logarithmic growth phase in the first culture stage is 100 mg L -1 The specific culture method is as follows: (1) The first stage of culture: 2.5 L of BG-11 medium was added to a 3 L round-bottom flask to culture Haematococcus pluvialis. The culture environment temperature was 25 °C, the light intensity was 3000 LUX, and the ventilation volume of sterile air was 1 vvm. The Haematococcus pluvialis grew to the logarithmic growth phase. 400 mL of BG-11 medium was used as the base medium. NaCl was added to the medium to make the concentration in the medium 100 mg L -1 The algal cells in the logarithmic growth phase were inoculated into the culture medium for low-salt pre-stress to enhance the stress resistance of the algal cells. The inoculation amount of Haematococcus pluvialis was ensured to be 1.5×10 5 cells mL -1 The culture environment temperature was 25°C, the light intensity was 3000LUX, the sterile air ventilation volume was 1vvm, and the culture was continued for 4 days to obtain Haematococcus pluvialis that had undergone low salt pre-stress.

[0032] (2) Second stage of training: For the Haematococcus pluvialis that was pre-stressed with low salt in step (1), 1 g L -1 NaCl stress was used, the culture environment temperature was 28°C, the light intensity was 22000LUX, the sterile air ventilation volume was 1vvm, and the culture was continued for 13 days to obtain Haematococcus pluvialis containing astaxanthin.

[0033] Comparative Example 1 For comparison, compared with Example 2, the difference of this experiment is that no exogenous NaCl was applied for low-salt pre-stress treatment during the logarithmic growth phase of the first culture stage. The other steps are the same as those in Example 2, specifically: (1) The first stage culture: in 3L round bottom flask, add 2.5L BG-11 medium to culture Haematococcus pluvialis, the environmental temperature of culture is 24℃, the light intensity is 3100LUX, the sterile air ventilation volume is 1vvm, when Haematococcus pluvialis grows to logarithmic growth phase, take 400mL BG-11 medium as the basic medium, inoculate the logarithmic growth phase algal cells obtained by culture into the medium, ensure that the inoculation amount of Haematococcus pluvialis is 1.5×10 5 cells mL -1 , the environmental temperature of culture is 24℃, the light intensity is 3100LUX, the sterile air ventilation volume is 1vvm, continuously culture for 4 days.

[0034] (2) The second stage culture: to Haematococcus pluvialis in step (1), add 1g L -1 NaCl again to stress, the environmental temperature of culture is 29℃, the light intensity is 22000LUX, the sterile air ventilation volume is 1vvm, continuously culture for 13 days, obtain Haematococcus pluvialis containing astaxanthin.

[0035] Comparative example 2 A method for enhancing the accumulation of biomass and astaxanthin of Haematococcus pluvialis based on gradient salt stress, in the first culture stage, the low salt pre-stress NaCl concentration in the logarithmic growth phase is 12.5mg L -1 , the specific culture method is as follows: (1) The first stage culture: in 3L round bottom flask, add 2.5L BG-11 medium to culture Haematococcus pluvialis, the environmental temperature of culture is 26℃, the light intensity is 2900LUX, the sterile air ventilation volume is 1vvm, when Haematococcus pluvialis grows to logarithmic growth phase; take 400mL BG-11 medium as the basic medium, add NaCl in the medium to make its concentration in the medium 12.5mg L -1 , inoculate the logarithmic growth phase algal cells obtained by culture into the medium to perform low salt pre-stress, so as to enhance the stress resistance of algal cells, ensure that the inoculation amount of Haematococcus pluvialis is 1.5×10 5 cells mL -1 , the environmental temperature of culture is 26℃, the light intensity is 2900LUX, the sterile air ventilation volume is 1vvm, continuously culture for 4 days, obtain Haematococcus pluvialis subjected to low salt pre-stress.

[0036] (2) The second stage culture: To Haematococcus pluvialis subjected to low salt pre-stress in step (1), add 1g L -1The Haematococcus pluvialis is cultured under stress of NaCl, at an ambient temperature of 27℃, an illumination intensity of 22000LUX, and a sterile air ventilation of 1vvm, for 13 days to obtain the Haematococcus pluvialis containing astaxanthin.

[0037] The extraction and determination methods of astaxanthin in all examples and comparative examples are as follows: 5mL of the culture solution obtained in the second stage culture is transferred into a centrifuge tube in a sterile clean bench, centrifuged at 3500r / min for 5min, and the supernatant is discarded, and the algal cells are collected, 5ml of dimethyl sulfoxide and glacial acetic acid mixture (volume ratio of dimethyl sulfoxide: glacial acetic acid is 100:1) is added, 65℃ water bath for 15min, then 3000rpm centrifugation for 5min, collect the supernatant into a new centrifuge tube. Add 5ml of dimethyl sulfoxide and glacial acetic acid mixture to the precipitate, repeat the above water bath and centrifugation operation until the algal body is white. The supernatant is combined, and the absorbance value is measured at 530nm using a spectrophotometer, and the astaxanthin concentration of Haematococcus pluvialis is calculated by substituting the standard curve.

[0038] The biomass of Haematococcus pluvialis is as shown in Figure 2 From the figure, it can be seen that: when the stress treatment is 13 days, the biomass of Haematococcus pluvialis in Example 1 is 2.13g / L -1 , which is significantly increased by 14.24% compared with Comparative Example 1, and increased by 9.4% compared with Comparative Example 2; the biomass of Haematococcus pluvialis in Example 2 is 2.13g / L -1 , which is significantly increased by 14.24% compared with Comparative Example 1, and increased by 9.4% compared with Comparative Example 2; the biomass of Haematococcus pluvialis in Example 3 is 2.10g / L -1 , which is significantly increased by 12.6% compared with Comparative Example 1, and increased by 8.5% compared with Comparative Example 2.

[0039] The astaxanthin yield is as shown in Figure 3 From the figure, it can be seen that: the astaxanthin yield in Example 1 is 3.83mg / L -1 d -1 , which is significantly increased by 18.36% compared with Comparative Example 1, and significantly increased by 13.46% compared with Comparative Example 2; the astaxanthin yield in Example 2 is 4.37mg / L -1 d -1 , which is significantly increased by 34.95% compared with Comparative Example 1, and increased by 29.37% compared with Comparative Example 2; the astaxanthin yield in Example 3 is 3.48mg / L -1 d -1 , which is significantly increased by 7.39% compared with Comparative Example 1, and significantly increased by 2.93% compared with Comparative Example 2.

[0040] The cell density is as shown in Figure 4As shown in the figure, at the second stage of culture for 13 days, the cell density in Example 1 was 4.55 x 10 5 cells mL -1 , which was significantly improved by 24.52% compared with Comparative Example 1 and by 11.13% compared with Comparative Example 2; the cell density in Example 2 was 4.46 x 10 5 cells mL -1 , which was significantly improved by 22.04% compared with Comparative Example 1 and by 8.92% compared with Comparative Example 2.

[0041] In summary, based on the method for enhancing the accumulation of biomass and astaxanthin of Haematococcus pluvialis under gradient salt stress, by applying 25 mg / L -1 ~50mg L -1 of NaCl in the low-salt pre-stress in the logarithmic culture period of the first stage and then applying 1 g / L -1 of NaCl in the stress stage, biomass and astaxanthin can be efficiently accumulated.

Claims

1. A method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis based on gradient salt stress, characterized in that: The specific culture method is as follows: (1) The first stage of culture: Cultivate Haematococcus pluvialis to the logarithmic growth phase, and inoculate the algal cells obtained in the logarithmic growth phase into a medium containing low concentration of NaCl for low salt pre-stress culture; (2) Second stage of cultivation: All the Haematococcus pluvialis that had been pre-stressed with low salt in step (1) were inoculated into a culture medium containing a high concentration of NaCl for cultivation.

2. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis under gradient salt stress according to claim 1, wherein: The culture medium used for culturing Haematococcus pluvialis in step (1) and step (2) is BG-11 culture medium.

3. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis under gradient salt stress according to claim 1, characterized in that: The culture conditions in step (1) are: ambient temperature of 24-26°C, and sterile air ventilation volume of 1 vvm.

4. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis under gradient salt stress according to claim 1, characterized in that: The culture conditions in step (2) are: ambient temperature of 27-29°C, and sterile air ventilation volume of 1 vvm.

5. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis based on gradient salt stress according to claim 1, characterized in that: The light intensity in step (1) is 2900~3100LUX; the light intensity in step (2) is 22000LUX.

6. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis under gradient salt stress according to claim 1, characterized in that: Step (1) The concentration of NaCl in the culture medium is 25 mg L -1 ~100mg L -1 .

7. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis under gradient salt stress according to claim 6, characterized in that: The concentration of NaCl in the culture medium was 25 mg L -1 ~50mg L -1 .

8. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis under gradient salt stress according to claim 1, characterized in that: Step (2) The concentration of NaCl in the culture medium is 1 g L -1 .

9. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis under gradient salt stress according to claim 1, characterized in that: The inoculation amount of algal cells during low-salt pre-stress culture in step (1) was 1.5×10 5 cells mL -1 .

10. The method for enhancing the accumulation of biomass and astaxanthin in Haematococcus pluvialis based on gradient salt stress according to claim 1, characterized in that: The low-salt pre-stress culture period in step (1) is 4 days; the Haematococcus pluvialis culture period in step (2) is 13 days.

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