Method for generating and extracting microalgae metabolite
By applying adversarial treatment to microalgae cultures and using emulsifiers and hydrolytic enzymes to decompose cell walls, the problems of low yield and low extraction efficiency in microalgae metabolite generation and extraction are solved, and efficient and economical metabolite extraction is achieved.
Patent Information
- Application Number
- CN202410137473.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-31
- Publication Date
- 2025-08-08
AI Technical Summary
In the prior art, the generation and extraction of microalgae metabolites have problems with low yield and anti-degradation characteristics of cell walls, resulting in low extraction efficiency and high cost.
By applying adversity treatment to the microalgae culture, such as salt adversity, temperature adversity, acid-base adversity, etc., and using it in combination with an emulsifier, then adding hydrolytic enzymes or fungal cultures to decompose the cell walls, and finally performing isolation and purification.
Significantly improve metabolites yield and improve extraction efficiency. It is suitable for different microalgae and culture stages, reduces costs, and is suitable for large-scale applications.
Smart Images
Figure CN120442720A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microalgae processing, and in particular to a method for generating and extracting microalgae metabolites. Background Art
[0002] Microalgae have attracted widespread attention as a sustainable bioresource. They can not only efficiently utilize solar energy for photosynthesis, converting carbon dioxide and water into organic matter, but also produce a variety of valuable metabolites, such as growth factors, fats, proteins, vitamins, and antioxidants. However, significant technical challenges currently exist in the production and extraction of microalgae metabolites.
[0003] First, the metabolite yields of microalgae in a normal growth state are relatively low, which necessitates extensive cultivation to obtain sufficient metabolites (target products). Furthermore, the cell walls of microalgae are robust and resistant to degradation, making efficient extraction of metabolites from within the cells of microalgae a major challenge.
[0004] The anti-degradation properties of microalgae cell walls refer to their complex composition of polysaccharides, proteins, lipids, and other biopolymers. For example, the cell walls of Chlorella contain large amounts of cellulose, while Spirulina contains a complex of polysaccharides and polypeptides. Furthermore, the polymer structures in microalgae cell walls are cross-linked, further enhancing their anti-degradation properties.
[0005] Currently, common methods for disrupting microalgae cell walls include mechanical methods (such as high-pressure homogenization), physical methods (such as ultrasonication), and chemical or biochemical methods (such as the use of specific enzymes). However, each of these methods has limitations. Mechanical methods may damage the intracellular fluid components of the algae; physical methods are ineffective in treating algae cells; and chemical solvent methods are subject to high costs and may negatively impact the quality of microalgae metabolites.
[0006] In summary, the development of efficient and economical methods for the production and extraction of microalgae metabolites is a pressing task. Such technologies should be able to effectively destroy the cell wall without damaging the components of the algal intracellular fluid, and should be low-cost and easy to operate to facilitate large-scale application. Summary of the Invention
[0007] The main purpose of the present invention is to provide a method for producing microalgae metabolites.
[0008] The present invention provides a method for producing microalgae metabolites, comprising:
[0009] Step 1, subjecting a microalgae culture to stress treatment to increase the yield of metabolites secreted by the microalgae in the microalgae culture; and
[0010] Step 2: Stop the adversity processing.
[0011] The adversity treatment is at least one of salt adversity, temperature adversity, pressure adversity, acid-base adversity and nutritional adversity.
[0012] When the stress treatment includes at least salt stress, before performing step 1, the method further includes the following steps: adding an emulsifier to the microalgae culture to coat the microalgae with the emulsifier to enhance the effect of the salt stress.
[0013] The emulsifier has biocompatibility characteristics and includes, but is not limited to, oils, fatty acid esters, nonionic surfactants, colloids, and waxes. Furthermore, the emulsifier is added to the microalgae culture at a final concentration of 0.1 to 0.3 weight percent (wt%).
[0014] The salts used in the salt stress include but are not limited to urea, sodium chloride, magnesium sulfate, potassium nitrate, potassium chloride, and calcium chloride. Furthermore, the salts are added to the microalgae culture at a final concentration of 1 to 3 weight percent (wt%).
[0015] When the stress treatment includes at least the temperature stress, and the microalgae are Chlorella and / or Spirulina, the temperature stress is 27 to 35 degrees Celsius, and the duration of the temperature stress is less than 48 hours.
[0016] Wherein, when the stress treatment includes at least the acid-base stress, and the microalgae are Chlorella and / or Spirulina, the acid-base stress is a pH value lower than 5.0 or higher than 11.0.
[0017] In accordance with the above objectives, the present invention provides a method for extracting microalgae metabolites, comprising:
[0018] performing the method for producing microalgae metabolites as described above;
[0019] Step 3, adding at least one of a hydrolytic enzyme and a fungal culture to the microalgae culture to decompose the cell walls of the microalgae; and
[0020] Step 4: Isolate the metabolites released from the cell wall pores of the microalgae.
[0021] The fungal culture continues to grow in the culture medium of the microalgae culture and continuously releases the hydrolytic enzyme.
[0022] The duration of adversity treatment is less than 48 hours, in particular any time point between 24 and 48 hours, such as 30, 36, or 42 hours.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] (1) Increase the production of metabolites: By applying stress treatment (such as salt stress, temperature stress, pressure stress, acid-base stress and nutritional stress) to microalgae cultures, the amount of metabolites secreted by microalgae can be significantly increased, including growth factors, algal oil, antioxidants, protective proteins, fatty acids, etc.
[0025] (2) Adaptive culture: The selection and application of stress treatment (such as temperature and salt concentration adjustment) can be optimized according to the characteristics of microalgae to ensure that the microalgae are activated to produce more metabolites without causing their death.
[0026] (3) Increased metabolite extraction efficiency: By adding hydrolytic enzymes or fungal cultures to decompose the cell walls of microalgae, intracellular metabolites can be released more effectively, thereby improving extraction efficiency. When using fungal cultures, fungal metabolites such as polysaccharides, hydrolytic enzymes, and organic acids can also be extracted.
[0027] (4) Applicable to different microalgae and cultivation stages: This technology is applicable to different types of microalgae (especially Chlorella and Spirulina) and takes into account different stages of microalgae cultivation (such as the stationary phase) to achieve optimal growth and metabolite production.
[0028] (5) Application of emulsifiers: Adding emulsifiers (such as lecithin) during stress treatment can promote the more uniform distribution of substances added under stress conditions (such as salts) in the microalgae culture, thereby improving the effect of stress treatment and the survival rate of microalgae.
[0029] The following text provides a detailed description of the specific embodiments in conjunction with the accompanying drawings, which should make it easier to understand the purpose, technical content, characteristics and effects achieved by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 A flow chart of a method for producing microalgae metabolites according to an embodiment of the present invention; and
[0031] Figure 2 This is a flow chart of the method for extracting microalgae metabolites according to an embodiment of the present invention.
[0032] Description of the accompanying figures: S1, S2, S3, S4: steps. DETAILED DESCRIPTION
[0033] The embodiments of the present invention are further explained below with reference to the accompanying drawings. Whenever possible, identical reference numerals are used throughout the drawings and the specification to represent identical or similar components. It should be understood that elements not specifically shown in the drawings or described in the specification are well known to those skilled in the art. Those skilled in the art may make various changes and modifications based on the teachings of the present invention.
[0034] Please see first Figure 1 , Figure 1 This is a flow chart of the method for producing microalgae metabolites according to an embodiment of the present invention.
[0035] like Figure 1 As shown, according to one embodiment, the method for producing microalgae metabolites includes steps S1 and S2.
[0036] In step S1 , the microalgae culture is subjected to stress treatment to increase the yield of metabolites secreted by the microalgae in the microalgae culture.
[0037] In step S2, the adverse processing is stopped.
[0038] In this embodiment, steps S1 to S2 can be repeated as needed, for example, to apply a milder degree of stress to the microalgae culture (e.g. Figure 1 , step S1 applies salts close to the optimal growth concentration), and re-applies the stress treatment after a period of time, which is different from applying extreme stress at one time (such as Figure 1 , step S1 applies salts close to the maximum tolerance concentration), multiple mild adversities will help the microalgae survive.
[0039] According to another embodiment, the microalgae culture is pre-cultured in liquid or semi-solid conditions to allow the microalgae culture to reach a target concentration. Methods for measuring the microalgae in the microalgae culture include cell count (cells per milliliter, cells / mL), dry weight (dry weight, g / L or mg / L), wet weight (wet weight, g / L or mg / L), optical density (OD), chlorophyll concentration, and biomass volume concentration. Based on the four stages of algae cultivation: lag phase, log phase, stationary phase, and death phase, under the same culture environment, the target concentration recommends selecting a microalgae culture in the stationary phase to obtain the maximum amount of microalgae with the best growth status.
[0040] Please see Table 1, which shows the test results of the concentrations of Chlorella and Spirulina during the stable period.
[0041] Table 1: Concentration of algae during the stable period
[0042] Cell number (cells / mL) Dry weight (g / L) Optical density (OD) Chlorella <![CDATA[10 6 ~10 9 ]]> 2 to 5 grams 0.6 to 1.5 Spirulina <![CDATA[10 6 ~10 9 *]]> 2 to 5 grams 0.6 to 1.5
[0043] *The cell size of Spirulina is smaller than that of Coccophyllum, so the actual cell value will be slightly lower.
[0044] According to another embodiment, the stress treatment is at least one of saline stress, thermal stress, pressure stress, pH stress, and nutritional stress. Testing has shown that each stress condition will cause some physiological changes in the microalgae, as briefly described below:
[0045] During salt stress, adding high concentrations of salts (such as urea, sodium chloride, or magnesium sulfate) to the algae culture medium causes changes in extracellular osmotic pressure, forcing the algae to adjust their internal balance. Salt stress can stimulate algae to produce antioxidants, protective proteins, fatty acids, and other metabolites to resist stress.
[0046] Please refer to Table 2, which shows the test results of the suitable salt concentration and maximum tolerable concentration for the growth of Chlorella and Spirulina.
[0047] Table 2: Analysis of algae's adaptability to salt concentration
[0048] Optimal growth concentration (wt%) Maximum tolerable concentration (wt%) Chlorella Less than 0.1 1 Spirulina 0.2 to 0.5 3
[0049] As shown in Table 2, when the microalgae in the microalgae culture is Chlorella, salt stress can adjust the salt concentration of the microalgae culture to 0.1-1 wt%; when the microalgae in the microalgae culture is Spirulina, salt stress can adjust the salt concentration of the microalgae culture to 0.5-3 wt%. Short-term salt stress treatment (24-48 hours) at or near the maximum tolerated concentration (wt%) can help maintain microalgae survival and increase metabolite production.
[0050] Temperature stress is when algae are exposed to suboptimal growth temperatures, either above or below their normal growth range. Temperature stress activates algae to increase the production of heat shock proteins and related metabolites.
[0051] Please refer to Table 3, which shows the test results of the suitable temperature and extreme tolerance temperature for the growth of Chlorella and Spirulina.
[0052] Table 3: Analysis of algae's adaptability to temperature
[0053] Optimum growth temperature (℃) Minimum tolerance temperature (℃) Maximum temperature tolerance (℃) Chlorella 20～28 4 35 Spirulina 25～35 20 40
[0054] As shown in Table 3, when the microalgae in the microalgae culture is Chlorella, the culture temperature can be adjusted to 4-20°C or 30-35°C by applying temperature stress; when the microalgae in the microalgae culture is Spirulina, the culture temperature can be adjusted to 20-25°C or 35-40°C by applying salt stress. Furthermore, a short-term (24-48 hours) temperature stress treatment close to or equal to the maximum or minimum tolerance temperature (°C) can help maintain microalgae survival and increase metabolite production.
[0055] Stress refers to the application of pressure higher than the optimal growth pressure of algae. Stress may affect the cell membrane and protein function of algae, forcing algae to make adaptive changes.
[0056] In acid-base stress, by changing the pH of the algae culture medium, exposing the algae to acidic or alkaline conditions, the acid-base stress can affect the metabolic pathways of the algae. The pH change can affect the enzyme activity and metabolite stability of the algae, and can therefore be used to induce the production of specific metabolites.
[0057] Please refer to Table 4, which shows the test results of the suitable pH value and the extreme tolerance pH value for the growth of Chlorella and Spirulina.
[0058] Table 4: Analysis of algae adaptability to pH
[0059] Optimal growth pH Minimum tolerable pH Maximum tolerable pH value Chlorella 6.0～7.0 5.0 9.0 Spirulina 8.5～11 8 11
[0060] As shown in Table 4, when the microalgae used in the microalgae culture is Chlorella, acid-base stress can adjust the culture pH to 5-6 or 7-9; when the microalgae used in the microalgae culture is Spirulina, acid-base stress can adjust the culture pH to 8-8.5. A short-term (24-48 hours) acid-base stress treatment at or near the minimum or maximum tolerable pH can help maintain microalgae survival and increase metabolite production.
[0061] Nutritional stress is achieved by limiting key nutrients in the culture medium, such as nitrogen (nitrates, urea), phosphorus (phosphates), potassium (potassium salts), sulfur or other trace elements. When algae experience nutrient limitation, they will activate a low-consumption mode and reallocate resources, which will affect the production of some specific metabolites.
[0062] According to another embodiment, when the stress treatment includes at least salt stress, prior to step 1, the process further includes adding an emulsifier to the microalgae culture to coat the algae and enhance the effect of the salt stress. Furthermore, the emulsifier is biocompatible and includes, but is not limited to, oils, fatty acid esters, nonionic surfactants, colloids, and waxes.
[0063] Furthermore, the final concentration of the emulsifier added to the microalgae culture is 0.1 to 0.3 weight percent (wt %).
[0064] Furthermore, the advantage of adding an emulsifier to microalgae cultures is that, since microalgae cultures may contain some lipophilic molecules (such as algal oil) secreted by the microalgae, these molecules can cause uneven distribution of salts applied by salt stress. Therefore, adding an emulsifier beforehand helps distribute the salts applied by salt stress more evenly within the microalgae culture, ensuring that each microalgae experiences the same degree of salt stress. Otherwise, according to the inventors' tests, when the same salt concentration was applied to two microalgae cultures with the same cell number, the survival rate of the microalgae culture group with the emulsifier added beforehand was higher than that of the microalgae culture group without the emulsifier. This may be because the microalgae culture group without the emulsifier had a higher local salt concentration, which caused dehydration of the microalgae and led to their death.
[0065] Example 1: Qualitative testing of algal metabolites under salt stress with the addition of emulsifier.
[0066] In this example, 1×10 8 cells / mL of Chlorella and Spirulina were tested. Urea was used as a salt stress agent (added to the microalgae culture at final concentrations of 1% and 3%). Lecithin was used as an emulsifier (added to the microalgae culture at final concentrations of 0.1% and 0.3%). The salt and / or emulsifier exposure time was 48 hours. Samples were collected at 24 and 48 hours.
[0067] Qualitative tests: microscopic observation (observation of chlorophyll production), enzyme-linked immunosorbent assay (detection of growth factors), Western Blotting (detection of proteins secreted by microalgae), and centrifugation or oil-water separation to determine the volume of metabolites (detection of algal oil in the supernatant), etc.
[0068] In this embodiment, the sampled microalgae cultures (salt stress treatment group and normal culture group) are subjected to qualitative testing. In this embodiment, the Chlorella and Spirulina of the salt stress treatment group and the normal culture group are compared respectively through at least one of the above-mentioned qualitative tests. If the output of the metabolites in the salt stress treatment group is statistically significantly increased compared with the normal culture group, it is marked as △; if there is no difference, it is marked as ◇; if it is significantly decreased, it is marked as ▽. If the results of significant increases and decreases in the output of various metabolites appear at the same time, it is determined as △, ◇ or ▽ according to the output of the target metabolite of interest. For example, taking Chlorella Growth Factor (CGF) as the target metabolite, as long as the output of Chlorella Growth Factor increases after adversity treatment, it is determined as △.
[0069] Please refer to Tables 5 and 6, which are the qualitative test results of the green algae growth factor production of Chlorella and Spirulina.
[0070] Table 5: Qualitative results of green algae growth factor in samples collected over 24 hours
[0071]
[0072] Table 6: Qualitative results of green algae growth factor in samples collected at 48 hours
[0073]
[0074] As shown in Tables 5 and 6, qualitative analysis results were similar for microalgae cultures sampled 24 or 48 hours apart. Qualitative analysis revealed that microalgae cultures grown under conditions of salt stress and emulsifier production of Chlorella Growth Factor (CGF) were higher than those grown under normal conditions.
[0075] Because Chlorella and Spirulina salts need to increase their ability to survive in adverse environments, this example demonstrated a significant increase in Chlorella Growth Factor production after adverse treatment. Chlorella Growth Factor is primarily composed of nucleic acids, amino acids, vitamins, minerals, and other bioactive substances. These active substances also promote human health and strengthen the immune system.
[0076] Example 2: Qualitative testing of algal metabolites in response to added nutritional stress.
[0077] In this example, the number of microalgae cultures was accumulated to 10 using a nitrogen:phosphorus:potassium ratio of 110:35:10. 8cells / mL of Chlorella and Spirulina. The phosphorus ratio could be adjusted to 30-35, and the potassium ratio could be adjusted to less than 10 but not equal to 0, while still maintaining normal algal growth. Nutritional stress conditions, defined as a nitrogen:phosphorus:potassium ratio of 0:35:10, meant removing all nitrogen from the culture medium for 48 hours. Samples were taken at the 24th and 48th hours, respectively.
[0078] Qualitative tests: microscopic observation (observation of chlorophyll production), enzyme-linked immunosorbent assay (detection of growth factors), Western Blotting (detection of proteins secreted by microalgae), and centrifugation or oil-water separation to determine the volume of metabolites (detection of algal oil in the supernatant), etc.
[0079] In this example, qualitative testing was performed on sampled microalgae cultures (a group treated with nutritional stress and a group cultured normally). If the metabolite production in the group treated with nutritional stress showed a statistically significant increase compared to the group cultured normally, it was marked as △; if there was no difference, it was marked as ◇; if it decreased significantly, it was marked as ▽. If the production of various metabolites increased and decreased significantly at the same time, it was determined as △, ◇, or ▽ based on the production of the target metabolite of interest. For example, if algae oil is the target metabolite, as long as the production of green algae growth factor increases after stress treatment, it is determined as △.
[0080] Please refer to Table 7, which shows the qualitative test results of the yields of Chlorella and Spirulina algae oil.
[0081] Table 7: Qualitative results of algae oil from sampled samples
[0082]
[0083] As shown in Table 7, the qualitative test results were similar for microalgae cultures sampled at either 24 or 48 hours. In the qualitative test results, the algal oil yields were higher in microalgae cultures after nitrogen source removal than in microalgae cultures cultured normally.
[0084] This embodiment utilizes the lack of nitrogen sources in the nutritionally adverse environment of Chlorella and Spirulina to reduce protein synthesis and redistribute energy metabolism, thereby increasing the yield of algal oil in Chlorella and Spirulina.
[0085] The above embodiment is not limited to open or closed culture. However, given that prolonged stress exposure may cause algae death, it is more appropriate to subject the algae to stress exposure for a limited time, such as 48 hours. Specifically, within 24 to 48 hours, this allows the algae to survive the stress environment, produce sufficient metabolites, and be less likely to die.
[0086] Please see first Figure 2 , Figure 2 This is a flow chart of the method for extracting microalgae metabolites according to an embodiment of the present invention.
[0087] like Figure 2 As shown, according to one embodiment, the method for extracting microalgae metabolites is to first perform Figure 1 After step S1 to step S2, step S3 to step S4 are performed.
[0088] In step S3, at least one of a hydrolytic enzyme and a fungal culture is added to the microalgae culture to decompose the cell walls of the microalgae.
[0089] In step S4, the metabolites released from the cell wall pores of the microalgae are separated.
[0090] According to another embodiment, the fungal culture continues to grow in the culture medium of the microalgae culture and continuously releases the hydrolytic enzyme.
[0091] The hydrolytic enzymes include, but are not limited to, cellulase, lysozyme, pectinase, mannanase, hemicellulase, and protease.
[0092] The fungal culture includes, but is not limited to, Pleurotus ostreatus, Agaricus bisporus, Ganoderma lucidum, and Hericium erinaceus. Furthermore, fungi classified under the genera Aspergillus, Penicillium, and Trichoderma also have the ability to secrete at least one hydrolytic enzyme selected from the group consisting of cellulase, lyase, pectinase, mannanase, hemicellulase, and protease. Therefore, as in step S3, if no exogenous hydrolytic enzyme is added and the microalgae culture and the fungal culture are placed in the same culture environment, the hydrolytic enzyme produced by the fungal culture can also be used to decompose the cell walls of the microalgae.
[0093] According to another embodiment, as in step S4, the methods for isolating and purifying the metabolites include, but are not limited to, filtration and centrifugation, ultrafiltration, solvent extraction, chromatography (high performance liquid chromatography (HPLC) or gel permeation chromatography (GPC)), rotary evaporation, and lyophilization.
[0094] The above description is only used to illustrate the preferred embodiment of the present invention and is not intended to limit the scope of implementation. Any simple replacement and equivalent changes made in accordance with the claims and description of the present invention are within the scope of protection of the patent of the present invention.
Claims
1. A method for producing microalgae metabolites, characterized in that: include: Step 1: subjecting a microalgae culture to stress treatment to increase the yield of metabolites secreted by the microalgae in the microalgae culture; as well as Step 2: Stop the adversity processing.
2. The method for producing microalgae metabolites according to claim 1, wherein: The adversity treatment is at least one of salt adversity, temperature adversity, pressure adversity, acid-base adversity and nutritional adversity.
3. The method for producing microalgae metabolites according to claim 2, wherein: When the stress treatment includes at least the salt stress, the method further comprises the following steps before performing step 1: An emulsifier is added to the microalgae culture to coat the microalgae and enhance the effect of salt stress.
4. The method for producing microalgae metabolites according to claim 3, wherein: The emulsifier has the characteristic of biocompatibility and is selected from the group consisting of oil, fatty acid ester, nonionic surfactant, colloid and wax.
5. The method for producing microalgae metabolites according to claim 4, wherein: The emulsifier is added to the microalgae culture to a final concentration of 0.1 to 0.3 weight percent (wt %).
6. The method for producing microalgae metabolites according to claim 2, wherein: The salt used in the salt environment is selected from the group consisting of urea, sodium chloride, magnesium sulfate, potassium nitrate, potassium chloride and calcium chloride.
7. The method for producing microalgae metabolites according to claim 6, wherein: The salts are added to the microalgae culture to a final concentration of 1 to 3 weight percent (wt %).
8. The method for producing microalgae metabolites according to claim 2, wherein: When the stress treatment includes at least the nutritional stress, and the microalgae are Chlorella and / or Spirulina, the nutritional stress is a lack of nitrogen source.
9. The method for producing microalgae metabolites according to claim 2, wherein: When the stress treatment includes at least the acid-base stress, and the microalgae are Chlorella and / or Spirulina, the acid-base stress has a pH value lower than 5.0 or higher than 11.
0.
10. The method for producing microalgae metabolites according to any one of claims 1 to 9, wherein: The duration of the adverse condition treatment is less than 48 hours.
11. A method for extracting microalgae metabolites, characterized by: include: Carrying out the method for producing microalgae metabolites according to any one of claims 1 to 10; adding at least one of a hydrolytic enzyme and a fungal culture to the microalgae culture to decompose the cell walls of the microalgae; and Isolate metabolites released from the cell wall pores of these microalgae.
12. The method for extracting microalgae metabolites according to claim 11, wherein: The fungal culture continues to grow in the culture medium of the microalgae culture and continuously releases the hydrolytic enzyme.