Haematococcus pluvialis rapid culture and astaxanthin high-yield coupling method
By constructing a symbiosis system of Rhodococcus irrigation and using functional strains such as Bacillus subtilis and Lactobacillus plantarum, the problem of low biomass and astaxanthin yields was solved, and rapid culture and high yields were achieved.
Patent Information
- Application Number
- CN202510438509.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-06-20
AI Technical Summary
In the prior art, the biomass of Radix Chronicus is relatively low, astaxanthin yield is low, the process flow is complex, and the culture cycle is long, resulting in high production efficiency and cost.
By constructing a symbiosis system of Rhodococcus rabidacea-functional bacteria, using Bacillus subtilis and/or Lactobacillus plantarum as functional strains, the culture conditions such as light intensity, temperature and algae ratio are optimized, and the culture and induction time are shortened.
It significantly improves the biomass and astaxanthin production of Radix Chronicus, shortens the culture time, reduces production costs, and improves production efficiency.
Smart Images
Figure CN120173746A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology. Specifically, it relates to a method for coupling rapid cultivation of Haematococcus pluvialis and high-yield production of astaxanthin. Background Art
[0002] Haematococcus pluvialis ( Haematococcus pluvialis ) is a freshwater single-celled alga. When the algal cells are under stress, a large amount of astaxanthin will accumulate in the cells. The astaxanthin content can account for 1.5% - 4.0% of the cell dry weight, and all of it is in the highly bioactive 3S, 3S' form. It is currently recognized as the best natural source of astaxanthin production in nature. However, there are still many bottleneck problems in the large-scale cultivation and commercial production of Haematococcus pluvialis, such as low biomass, low accumulation rate of target products, complex technological process, long cultivation cycle, etc. Currently, for the problems of low biomass and astaxanthin production, mainly by adding carbon sources to increase biomass accumulation. Adding exogenous carbon sources can increase biomass, but also increases production costs. Organic carbon sources such as sodium acetate and glucose are relatively expensive, which limits large-scale promotion and application; and during the use process, it is necessary to strictly control the addition amount and timing to avoid adverse effects on the growth of algal cells and astaxanthin accumulation. In addition, the growth cycle of Haematococcus pluvialis is relatively long, and it usually takes several weeks to complete the transformation from motile cells to immotile spores. The long cultivation cycle not only reduces production efficiency but also increases production costs, such as equipment depreciation, energy consumption, etc. During the astaxanthin accumulation stage, it is necessary to induce stress on the algal cells, such as increasing light intensity, restricting nutrients, etc. The induction process usually takes a long time to enable the algal cells to accumulate sufficient astaxanthin, thus further prolonging the entire production cycle. Therefore, exploring a low-cost and high-efficiency cultivation method is the technical key to promoting the large-scale production and application of Haematococcus pluvialis.
[0003] The algal-bacterial symbiotic system has significant advantages in promoting algal growth and shortening the cultivation cycle. Constructing a synthetic microbial symbiotic system of Haematococcus pluvialis - growth-promoting bacteria has important significance. Summary of the Invention
[0004] The purpose of the present invention is to solve at least one of the above-mentioned deficiencies existing in the prior art. For example, one of the purposes of the present invention is to promote the growth of Haematococcus pluvialis, the second purpose is to increase the astaxanthin yield of Haematococcus pluvialis, and the third purpose is to shorten the cultivation and induction time.
[0005] To achieve the above purpose, the present invention provides a method for coupling rapid cultivation of Haematococcus pluvialis and high-yield production of astaxanthin.
[0006] The method is realized by constructing a symbiotic system of Haematococcus pluvialis - functional bacteria, and the functional strains include Bacillus subtilis and / or Lactobacillus plantarum.
[0007] Optionally, the Haematococcus pluvialis - functional bacteria symbiotic system is constructed by adding the functional bacteria Bacillus subtilis and Lactobacillus plantarum to the Haematococcus pluvialis culture system.
[0008] Optionally, the initial inoculation amount of the Haematococcus pluvialis is 3.0 - 4.0×10 4 cells / mL.
[0009] Optionally, the inoculation ratio of the functional bacteria to the algae is 1:1.5 - 2.5.
[0010] For example, when the functional bacteria is Bacillus subtilis, the inoculation ratio of the algae to the bacteria is 1:1.5 - 2.5; when the functional bacteria is Lactobacillus plantarum, the inoculation ratios of the algae to the bacteria are respectively 1:1.5 - 2.5.
[0011] Optionally, the initial culture pH of the Haematococcus pluvialis - functional bacteria symbiotic system is 7.0 - 7.5, the rotation speed during the culture process is 120 - 160 rpm, the temperature is 22 - 26°C, the light intensity is 2500 - 3500 Lux, with a light - dark cycle, and the co - culture lasts for 9 - 12 days.
[0012] Optionally, the construction of the Haematococcus pluvialis - functional bacteria symbiotic system is carried out under oscillating light culture conditions.
[0013] Optionally, before constructing the Haematococcus pluvialis - functional bacteria symbiotic system, the method further includes: culturing the Haematococcus pluvialis seed liquid.
[0014] Optionally, after the green - phase culture is completed, high - light stress is induced to synthesize astaxanthin.
[0015] Optionally, the high - light stress induction lasts for 6 - 9 days.
[0016] Optionally, Bacillus subtilis and Lactobacillus plantarum are inoculated into Haematococcus pluvialis, and green - phase culture is carried out for 9 - 12 days, and then high - light continuous light stress is used to induce Haematococcus pluvialis to accumulate astaxanthin.
[0017] Optionally, the conditions for inducing the algal cells of Haematococcus pluvialis to accumulate astaxanthin include: temperature 28 - 32°C, light intensity 6500 - 7500 Lux.
[0018] Optionally, the Haematococcus pluvialis - functional bacteria symbiotic system can be cultured by an oscillating light method.
[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention has identified strains that significantly promote the growth of Haematococcus pluvialis, and established an algal-bacterial symbiotic system. Under this system, the green growth period of Haematococcus pluvialis can be significantly shortened, the growth rate, biomass, and astaxanthin yield of Haematococcus pluvialis can be increased, and the culture and induction time can be shortened. Among them, the biomass of Haematococcus pluvialis can be increased to 1.35 g / L, the astaxanthin yield can be increased to 45 mg / L, and the culture time can be significantly shortened, for example, by 30%. The culture method of the present invention has the advantages of simplicity, high efficiency, low cost, etc., and has broad commercial application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Through the following description in conjunction with the drawings, the above and other objects and / or features of the present invention will become clearer, wherein: Figure 1 Shows the influence of different algal / bacterial inoculation ratios on the biomass of Haematococcus pluvialis; Figure 2 Shows the SEM image of the algal-bacterial symbiotic result in the algal-bacterial symbiotic system; Figure 3 Shows the influence of different algal / bacterial inoculation ratios on the astaxanthin yield and content of Haematococcus pluvialis. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0021] Hereinafter, the present invention will be described in detail in conjunction with exemplary embodiments, but the embodiments do not limit the present invention in any form.
[0022] To promote the growth of Haematococcus pluvialis and the accumulation of astaxanthin, and at the same time effectively improve the application effect and safety, the present invention creatively applies the functional strains Bacillus subtilis and Lactobacillus plantarum in the cultivation of Haematococcus pluvialis, and establishes a symbiotic system of Haematococcus pluvialis-functional bacteria. Specifically: First, the present invention inoculates the algal species of Haematococcus pluvialis into the culture medium (such as BG11 medium), and then inoculates Bacillus subtilis and Lactobacillus plantarum in proportion to detect the influence of these two functional strains on Haematococcus pluvialis and astaxanthin production. The results show that adding functional strains in a certain algal-bacterial ratio can significantly promote the growth of Haematococcus pluvialis. After high-light stress, the astaxanthin content and yield of Haematococcus pluvialis are measured. The results show that, compared with the treatment without adding functional bacteria, the astaxanthin content and yield are significantly increased; the culture and induction time are further shortened, which is beneficial to reducing the astaxanthin production cycle and improving production efficiency.
[0023] Exemplary Embodiment 1 This exemplary embodiment provides a method for coupling the rapid culture of Haematococcus pluvialis and high-yield production of astaxanthin.
[0024] The method includes: adding functional bacteria to the Haematococcus pluvialis culture system to construct an algal-bacterial symbiotic system. This method can improve the biomass, astaxanthin content, and astaxanthin yield of Haematococcus pluvialis, shorten the culture cycle and the astaxanthin accumulation time, and achieve efficient production of astaxanthin.
[0025] Specifically, the method may include: culturing the Haematococcus pluvialis seed liquid; inoculating Haematococcus pluvialis and functional strains in a certain proportion to construct an algal-bacterial symbiotic system, and performing it under the conditions of oscillating light culture; after the green period culture ends, performing high-light stress induction for astaxanthin synthesis.
[0026] In this embodiment, the algal-bacterial symbiotic system may include Haematococcus pluvialis and functional strains, and the functional strains are Bacillus subtilis and Lactobacillus plantarum.
[0027] In this embodiment, the initial number of Haematococcus pluvialis cells inoculated can be 3.0 - 4.0×10 4 cells / mL, such as 3.1×10 4 、3.3×10 4 、3.6×10 4 、3.9×10 4 cells / mL. Preferably, the number of Haematococcus pluvialis cells inoculated can be 3.5×10 4 cells / mL.
[0028] In this embodiment, Bacillus subtilis and Lactobacillus plantarum can be inoculated into the Haematococcus pluvialis culture system on the same day when inoculating the algae.
[0029] Preferably, when the functional bacterial species is Bacillus subtilis, Bacillus subtilis can be inoculated according to an algal-bacterial ratio of 1:1.5 - 2.5, such as 1:1.6, 1:1.8, 1:2.0, 1:2.1, 1:2.4, etc.
[0030] Preferably, when the functional bacterial species is Lactobacillus plantarum, Lactobacillus plantarum can be inoculated according to an algal-bacterial ratio of 1:1.5 - 2.5, such as 1:1.6, 1:1.8, 1:2.0, 1:2.1, 1:2.4, etc.
[0031] In addition, Bacillus subtilis and Lactobacillus plantarum can be inoculated simultaneously as functional bacterial species, and the algal-bacterial ratio is 1:1.5 - 2.5.
[0032] In this embodiment, after the access of functional bacteria, the culture can be carried out in a constant temperature oscillation mode. Among them, the oscillation intensity of the oscillation can be 120-160 rpm, such as 130, 140, 150, 158 rpm, etc., the initial culture pH can be 7.0-7.5, such as 7.1, 7.2, 7.3, 7.4, the temperature can be 22-26 °C, such as 23, 24, 25, 25.5 °C, etc., the light intensity can be 2500-3500 Lux, such as 2600, 2700, 2800, 3000, 3100, 3200, 3300, 3400 Lux, etc., and the light-dark cycle is 12h / 12h.
[0033] Preferably, the oscillation intensity of the oscillation is 140 rpm, the initial culture pH is 7.2, the temperature is 24 °C, and the light intensity is 3000 Lux.
[0034] In this embodiment, after Bacillus subtilis and Lactobacillus plantarum are inoculated into Haematococcus pluvialis, the algal cells can be induced to accumulate astaxanthin by high-light continuous illumination for 9-12 days.
[0035] In this embodiment, the conditions for inducing astaxanthin accumulation in Haematococcus pluvialis can be: temperature 28-32 °C, such as 29, 30, 31 °C, light intensity 6500-7500 Lux, such as 6600, 6800, 7000, 7200, 7400 Lux.
[0036] Preferably, the conditions for inducing astaxanthin accumulation in Haematococcus pluvialis are temperature 30 °C and light intensity 7500 Lux.
[0037] In this embodiment, the stress of astaxanthin accumulation in Haematococcus pluvialis can be 6-9 days. Preferably, the stress of astaxanthin accumulation in Haematococcus pluvialis is 7 days.
[0038] Exemplary Embodiment 2 This exemplary embodiment provides an algal-bacterial symbiotic system.
[0039] The algal-bacterial symbiotic system can be the algal-bacterial symbiotic system in Exemplary Embodiment 1. For example, it can include Haematococcus pluvialis and functional strains. The functional strains are Bacillus subtilis and / or Lactobacillus plantarum. The algal-bacterial symbiotic system can be constructed by adding functional bacteria to the Haematococcus pluvialis culture system.
[0040] To better understand the above exemplary embodiments, further description will be given below with specific examples.
[0041] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available. For example: The BG11 medium was cultured as follows: NaNO3 was 1500 mg / L; K2HPO4 was 40 mg / L; MgSO4 was 36.6 mg / L; CaCl2·2H2O was 27.2 mg / L; Na2CO3 was 20 mg / L, citric acid was 6 mg / L; ammonium ferric citrate was 6 mg / L; disodium EDTA was 1 mg / L; H3BO3 was 2.86 mg / L; MnCl2·4H2O was 1.81 mg / L; ZnSO4 was 0.22 mg / L; Na2MoO4·2H2O was 0.39 mg / L; CuSO4·5H2O was 0.08 mg / L; CoCl2·6H2O was 0.0409 mg / L. The above components were dissolved in distilled water, the pH was adjusted, and autoclaved at 121 °C for 15 minutes.
[0042] The Haematococcus pluvialis (FACHB-712) used in the example was from the Freshwater Algae Collection of the Institute of Hydrobiology, Chinese Academy of Sciences. Bacillus subtilis ( Bacillus subtilis ), Lactobacillus plantarum ( Lactiplantibacillus plantarum ) were all purchased from Beijing BioWin Biotechnology Co., Ltd.
[0043] Bacillus subtilis seed culture medium: It was cultured in a 100 mL conical flask containing sterilized LB medium and incubated at 28 °C with constant shaking for 48 hours.
[0044] Lactobacillus plantarum seed culture medium: It was cultured in a 100 ml conical flask containing sterilized MRS medium and incubated at 24 °C with constant shaking for 48 hours.
[0045] Figure 1 And Figure 3 The CK in Figure 2 represents the control group, and B4:1, B2:1, B1:1, B1:2, B1:4 represent the treatment groups inoculated with Bacillus subtilis at different algal-bacterial ratios, and L4:1, L2:1, L1:1, L1:2, L1:4 represent the treatment groups inoculated with Lactobacillus plantarum at different algal-bacterial ratios.
[0046] In the following examples, the incubator used for culturing has lamps surrounding it on three sides. The light intensity is stronger near the lamps and weaker farther away from the lamps. Therefore, the light intensity corresponds to a corresponding parameter range.
[0047] Example 1 S1. Preparation of high-activity algal seeds: The Haematococcus pluvialis cells were inoculated into the BG11 medium, and the inoculated cell density was 5×10 4cells / mL, the culture temperature is 24 °C, the light intensity is 1200 - 2000 lux, the light exposure time is 12 hours per day, and the green motile flagellated highly active cells account for 90%, with the cell density reaching 1×10 6 cells / mL, which is used as the algal strain.
[0048] S2. Transfer the above algal strain to a culture reactor. The number of Haematococcus pluvialis cells is 3.5×10 4 cells / mL, which is the initial inoculation amount.
[0049] S3. Add the functional strains Bacillus subtilis and Lactobacillus plantarum to the Haematococcus pluvialis culture system respectively. Among them, each functional strain is inoculated according to the algal - bacterium cell ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 respectively.
[0050] S4. Algal - bacterium symbiotic system culture: Transfer the two constructed algal - bacterium symbiotic systems to an illuminated shaking incubator for culture. The rotation speed is 140 rpm, the temperature is controlled at 24 °C, the light intensity is 2500 - 3500 Lux, and the light - dark cycle is 12 h / 12 h. Culture for 9 days.
[0051] S5. Determination of the biomass of Haematococcus pluvialis. Collect 50 mL of Haematococcus pluvialis in a centrifuge tube, transfer it to an oven after centrifugation, dry it at 60 °C for 12 h, and weigh it to calculate the biomass of Haematococcus pluvialis.
[0052] The results are as Figure 1 shown. Bacillus subtilis and Lactobacillus plantarum can increase the biomass of Haematococcus pluvialis. The biomass reaches the highest at the ratio of 1:2 for both, which are 1.32 g / L and 1.23 g / L respectively, being 2.58 times and 2.41 times that of the control group (0.51 g / L).
[0053] Example 2 Step 1. Construct an algal - bacterium symbiotic system according to a method basically the same as that in Example 1. Specifically, S1, S2, and S4 are the same as in Example 1, and in S3, both Bacillus subtilis and Lactobacillus plantarum are inoculated according to the algal - bacterium cell ratio of 1:2.
[0054] Step 2. Conduct scanning electron microscope observations on the 0th and 9th days of the culture of each algal - bacterium symbiotic system. The results are as Figure 2 shown. Figure 2 The three columns from left to right respectively represent the scanning electron microscope observation results of the control group, the Bacillus subtilis treatment group, and the Lactobacillus plantarum treatment group.
[0055] Figure 2The SEM results showed that the algal cells in the control group were plump on the 0th day (Figure a) and the 9th day (Figure A), and there were no obvious changes on the surface. While in the algal-bacterial symbiotic treatment group on the 9th day, both types of bacteria were attached to the surface of the algal cells in large numbers, but did not change the morphology of the algal cells to cause phenomena such as collapse and shrinkage.
[0056] Example 3 Step 1: Construct an algal-bacterial symbiotic system according to the method of Example 1 (that is, according to Steps S1-S4 in Example 1).
[0057] Step 2: Subject the algal-bacterial symbiotic system completed in the green cultivation period to high-light induction for astaxanthin accumulation. The environmental conditions for high-light induction are a temperature of 30 °C, a light intensity of 6500-7500 lux, and a high-light induction time of 7 days.
[0058] Step 3: After the induction is completed, measure the biomass and astaxanthin content of Haematococcus pluvialis in each group. The astaxanthin concentration is determined by spectrophotometry. Specifically: Treat the cells with 5% (w / v) KOH and 30% (w / v) methanol, centrifuge at 10000 rpm for 5 min, and remove the cell supernatant to eliminate the interference of total chlorophyll. Subsequently, add 1 mL of dimethyl sulfoxide containing 1% acetic acid, incubate the mixture in a water bath (70 °C) and shake constantly for 10 min. Collect the supernatant, and repeat the extraction procedure until the cell debris is almost colorless. Measure the absorbance of the combined extract at 490 nm, and calculate the astaxanthin concentration (C) per unit volume according to Equation (1): C (mg L -1 )=A490×4.5×Vb / Va Equation (1) In the formula, Va is the volume of the culture sample, L; Vb is the volume of the extract, L; A490 is the absorbance of the extract at 490 nm.
[0059] The results are as Figure 3 shown. When Bacillus subtilis and Lactobacillus plantarum were inoculated according to the algal-bacterial ratio of 1:2, the astaxanthin yields of Haematococcus pluvialis were 42.84 mg / L and 40.39 mg / L, far higher than 7.85 mg / L of the control group. At the same time, the astaxanthin contents (32.50 mg / g and 32.97 mg / g) were also higher than that of the control group (15.35 mg / g).
[0060] Although the present invention has been described above in conjunction with exemplary embodiments and the accompanying drawings, those of ordinary skill in the art should understand that various modifications can be made to the above embodiments without departing from the spirit and scope of the claims.
Claims
1. A method for coupling rapid cultivation of Haematococcus pluvialis and high production of astaxanthin, characterized in that: The method is achieved by constructing a symbiotic system of Haematococcus pluvialis and functional bacteria, wherein the functional strains include Bacillus subtilis and / or Lactobacillus plantarum.
2. The method for coupling rapid cultivation of Haematococcus pluvialis and high yield of astaxanthin according to claim 1, characterized in that: The Haematococcus pluvialis-functional bacteria symbiotic system is constructed by adding the functional strain into the Haematococcus pluvialis culture system.
3. The method for coupling rapid cultivation of Haematococcus pluvialis and high yield of astaxanthin according to claim 2, characterized in that: The initial inoculation amount of the Haematococcus pluvialis is 3.0-4.0×10 4 cells / mL.
4. The method for coupling rapid cultivation of Haematococcus pluvialis and high yield of astaxanthin according to claim 1, characterized in that: The algae-bacteria ratio of the functional strain inoculation is 1:1.5-2.
5.
5. The method for coupling rapid cultivation of Haematococcus pluvialis and high yield of astaxanthin according to claim 1, characterized in that: The initial culture pH of the Haematococcus pluvialis-functional bacteria symbiotic system is 7.0-7.5, the rotation speed during the culture process is 120-160 rpm, the temperature is 22-26° C., the light intensity is 2500-3500 Lux, the light-dark cycle is adopted, and the culture is carried out for 9-12 days.
6. The method for coupling rapid cultivation of Haematococcus pluvialis and high yield of astaxanthin according to claim 1, characterized in that: Before constructing the Haematococcus pluvialis-functional bacteria symbiotic system, the method further comprises: culturing Haematococcus pluvialis seed liquid.
7. The method for coupling rapid cultivation of Haematococcus pluvialis and high yield of astaxanthin according to claim 1, characterized in that: After the green stage cultivation, high light stress was applied to induce astaxanthin synthesis.
8. The method for coupling rapid cultivation of Haematococcus pluvialis and high production of astaxanthin according to claim 7, characterized in that: The high light stress induction lasts for 6 to 9 days.
9. The method for coupling rapid cultivation of Haematococcus pluvialis and high yield of astaxanthin according to claim 1, characterized in that: The conditions for inducing the algal cells Haematococcus pluvialis to accumulate astaxanthin include: temperature 28~32℃, light intensity 6500~7500 Lux.
10. The method for coupling rapid cultivation of Haematococcus pluvialis and high production of astaxanthin according to claim 1, characterized in that: The Haematococcus pluvialis-functional bacteria symbiotic system is cultured in an oscillating illumination manner.