TWI901003B - Method for the generation and extraction of metabolites from microalgae - Google Patents

Abstract

This invention provides a method for the generation and extraction of metabolites from microalgae. By subjecting microalgal cultures to stress treatments, the microalgae are stimulated to increase the secretion of metabolites. Additionally, this invention utilizes hydrolytic enzymes or fungal cultures to break down the cell walls of the microalgae, thereby releasing the microalgal metabolites more effectively. Finally, methods such as filtration, centrifugation, and ultrafiltration are employed to separate and purify the released metabolites from the microalgae. This technique enhances the yield of microalgal metabolites and is of significant value for applications in biotechnology, food, and pharmaceutical industries involving microalgae.

Description

Production and extraction methods of microalgae metabolites

The present invention relates to the field of microalgae processing technology, and in particular to a method for producing and extracting microalgae metabolites.

Microalgae have garnered widespread attention as a sustainable bioresource. They 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.

First, the production of metabolites from microalgae in a normal growth state is relatively low, necessitating large-scale cultivation to obtain sufficient metabolites (target products). Furthermore, the cell walls of microalgae are strong and resistant to degradation, making efficient extraction of metabolites from these cells a significant challenge.

The anti-degradation properties of microalgae cell walls refer to the fact that microalgae cell walls are typically composed of complex polysaccharides, proteins, lipids, and other biopolymers. For example, the cell walls of Chlorella contain a large amount of cellulose, while Spirulina contains a complex of polysaccharides and polypeptides. Furthermore, the polymer structures in microalgae cell walls form interlinked structures, further enhancing their anti-degradation properties.

Currently, common methods for disrupting microalgae cell walls include mechanical methods (such as high-pressure homogenization), physical methods (such as ultrasonic treatment), and chemical or biochemical methods (such as the use of specific enzymes). However, each of these methods has limitations. Mechanical methods may cause damage to the components of the algae's intracellular fluid; physical methods are ineffective in treating algae cells; and chemical solvent methods face the problems of high costs and the potential for negative effects on the metabolites of the microalgae.

In summary, the development of efficient and economical methods for the production and extraction of microalgae metabolites is a pressing need. Such a technology should be able to effectively destroy the cell walls without damaging the algal cellular fluid components, and should also be low-cost and simple to operate, facilitating large-scale application.

The primary objective of the present invention is to provide a method for producing microalgae metabolites, comprising: step 1, subjecting a microalgae culture to stress treatment to increase the production of multiple microalgae-secreted metabolites in the microalgae culture; and step 2, ceasing the stress treatment.

The stress treatment is at least one of salt stress, temperature stress, pressure stress, acid-base stress, and nutritional stress.

When the stress treatment includes at least salt stress, the following step is further included before step 1: adding an emulsifier to the microalgae culture to coat the microalgae with the emulsifier to enhance the effect of the salt stress.

The emulsifier has biocompatibility properties and includes, but is not limited to, oils, fatty acid esters, non-ionic surfactants, colloids, and waxes. Furthermore, the final concentration of the emulsifier added to the microalgae culture is 0.1 to 0.3 weight percent (wt%).

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%).

When the stress treatment includes at least temperature stress, and the microalgae are Chlorella and/or Spirulina, the temperature stress is between 27 and 35 degrees Celsius, and the duration of the temperature stress is less than 48 hours.

When the stress treatment includes at least the acid-alkaline stress and the microalgae are Chlorella and/or Spirulina, the acid-alkaline stress has a pH value lower than 5.0 or higher than 11.0.

In accordance with the above-mentioned objectives, the present invention provides a method for extracting microalgae metabolites, comprising: performing the aforementioned method for producing microalgae metabolites; 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 step 4, isolating the metabolites released from the pores in the cell walls of the microalgae.

The fungal culture continues to grow in the culture medium of the microalgae culture and continuously releases the hydrolytic enzyme.

Among them, the time required to cope with adversity is less than 48 hours. In particular, it is any time point between 24 and 48 hours, such as 30, 36, or 42 hours.

Compared with the prior art, the claimed benefits of this invention include:

(1) Increase the production of metabolites: By applying stress treatments (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, algae oil, antioxidants, protective proteins, fatty acids, etc.

(2) Adaptive cultivation: The selection and application of stress treatment (such as temperature and salt concentration adjustment) can be optimized according to the characteristics of the microalgae to ensure that the microalgae are activated to produce more metabolites while not causing their death.

(3) Increase metabolite extraction efficiency: By adding hydrolytic enzymes or fungal cultures to decompose the cell walls of microalgae, metabolites within the cells 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.

(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 stabilization period) to achieve optimal growth and metabolite production.

(5) Application of emulsifiers: Adding emulsifiers (such as lecithin) during stress treatment can promote the more even distribution of substances (such as salts) added under stress conditions in the microalgae culture, thereby improving the effect of stress treatment and the survival rate of microalgae.

The following text provides detailed descriptions of specific embodiments with accompanying drawings, which will make it easier to understand the purpose, technical content, features, and effects achieved by the present invention.

S1, S2, S3, S4: Steps

Figure 1 is a flow chart of a method for producing microalgae metabolites according to an embodiment of the present invention; and Figure 2 is a flow chart of a method for extracting microalgae metabolites according to an embodiment of the present invention.

The embodiments of this invention are further explained below with reference to the relevant figures. Whenever possible, identical reference numerals in the figures and the specification represent identical or similar components. It should be understood that components not specifically shown in the figures or described in the specification are of a form known to those skilled in the art. Those skilled in the art may make various changes and modifications based on the contents of this invention.

Please refer to Figure 1, which is a flow chart of the method for producing microalgae metabolites according to an embodiment of the present invention.

As shown in Figure 1, according to one embodiment, the method for producing microalgae metabolites includes steps S1 and S2.

As in step S1, the microalgae culture is subjected to stress treatment to increase the production of microalgae-secreted metabolites in the microalgae culture.

As in step S2, stop the adversity processing.

In this embodiment, steps S1 and S2 can be repeated as needed. For example, the microalgae culture can be subjected to a mild stress (e.g., in Figure 1, step S1 applies a salt concentration close to the optimal growth concentration) and then reapplied to the stress treatment at intervals. This is different from applying extreme stress all at once (e.g., in Figure 1, step S1 applies a salt concentration close to the maximum tolerance concentration). Multiple mild stress treatments will help the microalgae survive.

According to another embodiment, the microalgae culture is pre-cultured in liquid or semi-solid medium to achieve a target concentration. Methods for measuring the microalgae in the microalgae culture include cell count (cells per milliliter, cells/mL), dry weight (g/L or mg/L), 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 cultivation environment, the target concentration recommends selecting microalgae cultures in the stationary phase to obtain the maximum amount of microalgae with optimal growth.

Please see Table 1, which shows the test results of Chlorella and Spirulina at stable concentrations.

According to another embodiment, the stress treatment is at least one of saline stress, temperature stress, pressure stress, pH stress, and nutritional stress. Testing has shown that each stress condition causes physiological changes in microalgae. A brief explanation is as follows: In saline 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 combat stress.

Please see Table 2, which shows the test results of the suitable salt concentrations and maximum tolerable concentrations for the growth of Chlorella and Spirulina.

As shown in Table 2, when the microalgae culture is Chlorella, the salt concentration can be adjusted to 0.1-1 wt% during salt stress. When the microalgae culture is Spirulina, the salt concentration can be adjusted to 0.5-3 wt%. Short-term salt stress treatment (24-48 hours) at or near the maximum tolerable concentration (wt%) can help maintain microalgae survival and increase metabolite production.

Temperature stress involves exposing algae to suboptimal growth temperatures, either above or below their normal growth range. Temperature stress activates algae and increases the production of heat shock proteins and related metabolites.

Please see Table 3, which shows the test results of the suitable growth temperature and maximum tolerance temperature of Chlorella and Spirulina.

As shown in Table 3, when the microalgae culture is Chlorella, the culture temperature can be adjusted to 4-20°C or 30-35°C. When the microalgae culture is Spirulina, the culture temperature can be adjusted to 20-25°C or 35-40°C. A short-term (24-48 hours) temperature stress treatment at or near the maximum or minimum tolerance temperature (°C) can help maintain microalgae survival and increase metabolite production.

Stress refers to conditions that are higher than the algae's optimal growth pressure. Stress can affect the algae's cell membranes and protein functions, forcing the algae to undergo adaptive changes.

In acid-base stress, by changing the pH of the algae culture medium, exposing the algae to acidic or alkaline conditions, the stress can affect the algae's metabolic pathways. pH changes affect the enzyme activity and metabolite stability of the algae, and therefore can be used to induce the production of specific metabolites.

Please see Table 4, which shows the test results of the suitable pH values and maximum tolerance pH values for the growth of Chlorella and Spirulina.

As shown in Table 4, when the microalgae used in the microalgae culture is Chlorella, the pH of the culture can be adjusted to 5-6 or 7-9 during acid-alkaline stress. When the microalgae used in the microalgae culture is Spirulina, the pH of the culture can be adjusted to 8-8.5. Short-term acid-alkaline stress treatment (24-48 hours) at or near the minimum or maximum tolerable pH can help maintain microalgae survival and increase metabolite production.

Nutritional stress is achieved by limiting the availability of 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 activate a low-consumption mode and reallocate resources, which affects the production of certain metabolites.

According to another embodiment, when the stress treatment includes at least salt stress, before proceeding to step 1, the following step is further included: 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, non-ionic surfactants, colloids, and waxes.

Furthermore, the emulsifier is added to the microalgae culture to a final concentration of 0.1 to 0.3 weight percent (wt%).

Furthermore, the advantage of adding an emulsifier to the microalgae culture lies in the fact that the microalgae may contain some lipophilic molecules (such as algal oil) secreted by the microalgae. These lipophilic molecules can cause uneven distribution of salts applied during salt stress. Therefore, adding an emulsifier beforehand will help distribute the salts applied during salt stress more evenly within the microalgae culture, ensuring that each microalgae species experiences the same level of salt stress. On the other hand, according to the inventors' tests, when two microalgae cultures with the same cell number were treated with the same salt concentration, the survival rate of the microalgae culture group that had been pre-treated with an emulsifier was higher than that of the microalgae culture group that had not been treated with an emulsifier. This may be because the microalgae culture group without an emulsifier had a higher local salt concentration, causing the microalgae to dehydrate and die.

Example: Qualitative testing of algae metabolites under salt stress with the addition of emulsifier.

This example tested microalgae cultures of Chlorella and Spirulina at ^1x10 cells/mL. Urea was used as the salt stress agent (added to the microalgae culture at final concentrations of 1% and 3%), and lecithin was used as the 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 the 24th and 48th hours.

Qualitative tests include: microscopic observation (to observe chlorophyll production), enzyme-linked immunosorbent assay (to detect growth factors), Western blot (to detect proteins secreted by microalgae), and centrifugation or oil-water separation to determine metabolite volume (to detect algal oil in the supernatant).

In this embodiment, qualitative tests were performed on sampled microalgae cultures (a salt stress-treated group and a normal culture group). This embodiment used at least one of the aforementioned qualitative tests to compare Chlorella and Spirulina in the salt stress-treated group and the normal culture group, respectively. If the metabolite production in the salt stress-treated group was statistically significantly higher than that in the normal culture group, a △ was assigned; if there was no difference, a ◇ was assigned; if there was a significant decrease, a ▽ was assigned. If the production of various metabolites showed both a significant increase and a significant decrease, the result was assigned a △, ◇, or ▽ based on the production of the target metabolite of interest. For example, if Chlorella Growth Factor (CGF) is used as the target metabolite, as long as the production of CGF increases after stress treatment, it is judged as △.

Please see Tables 5 and 6, which show the qualitative test results of the growth factor production of Chlorella and Spirulina.

As shown in Tables 5-6, qualitative testing results were identical for microalgae cultures sampled at either 24 or 48 hours. Qualitative testing revealed that microalgae cultures cultured with an emulsifier and subjected to saline stress exhibited higher yields of green algae growth factor than those cultured normally.

Because chlorella and spirulina salts need to increase their ability to survive in adverse environments, this example demonstrated a significant increase in the production of chlorella growth factor after exposure. 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.

Example: Qualitative testing of algal metabolites under nutritional stress.

In this example, microalgae cultures of Chlorella and Spirulina were grown to a density of ¹⁰ cells/mL at a nitrogen:phosphorus:potassium ratio of 110:35:10. The phosphorus ratio can be adjusted to 30-35, and the potassium ratio can 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, involve removing all nitrogen from the culture medium for 48 hours. Sampling was performed at the 24th and 48th hours.

Qualitative tests include: microscopic observation (to observe chlorophyll production), enzyme-linked immunosorbent assay (to detect growth factors), Western blot (to detect proteins secreted by microalgae), and centrifugation or oil-water separation to determine metabolite volume (to detect algal oil in the supernatant).

In this example, qualitative testing was performed on sampled microalgae cultures (both those subjected to nutritional stress and those maintained under normal conditions). If the metabolite production in the nutritional stress group showed a statistically significant increase compared to the normal culture group, it was marked with a triangle; if there was no difference, it was marked with a circle; if there was a significant decrease, it was marked with a square. If the production of various metabolites showed both a significant increase and a significant decrease, the result was assigned a triangle, a circle, or a square, depending on the production of the target metabolite of interest. For example, if algal oil was the target metabolite, an increase in the production of chlorophyll growth factor after stress treatment would be assigned a triangle.

Please see Table 7, which shows the qualitative test results of the yield of Chlorella and Spirulina algae oil.

As shown in Table 7, qualitative testing results were similar for microalgae cultures sampled at either 24 or 48 hours. Qualitative testing revealed that algal oil production was higher in microalgae cultures after nitrogen removal compared to cultures maintained normally.

This embodiment utilizes the lack of nitrogen sources in the nutritionally stressful environment of Chlorella and Spirulina, resulting in reduced protein synthesis and redistribution of energy metabolism, thereby increasing the yield of algal oil from Chlorella and Spirulina.

The above embodiments are not limited to open or closed culture. However, given that prolonged stress exposure may cause algae death, it is more appropriate to conduct stress exposure for a limited time, such as within 48 hours. Specifically, a duration between 24 and 48 hours allows the algae to survive the stress environment, produce sufficient metabolites, and be less likely to die.

Please refer to Figure 2, which is a flow chart of the microalgae metabolite extraction method according to an embodiment of the present invention.

As shown in Figure 2, according to one embodiment, the method for extracting microalgae metabolites first performs steps S1 to S2 of Figure 1, and then proceeds to steps S3 to S4.

As 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.

As in step S4, the metabolite released from the cell wall pores of the microalgae is isolated.

According to another embodiment, the fungal culture continues to grow in the culture medium of the microalgae culture and continuously releases the hydrolytic enzyme.

The hydrolytic enzymes include but are not limited to cellulase, lysozyme, pectinase, mannanase, hemicellulase, and protease.

The fungal cultures include, but are not limited to, Pleurotus ostreatus, Agaricus bisporus, Ganoderma lucidum, and Hericium erinaceus. Furthermore, fungi belonging to the genera Aspergillus, Penicillium, and Trichoderma also have the ability to secrete hydrolytic enzymes such as at least one of cellulase, lyase, pectinase, mannanase, hemicellulase, and protease. Therefore, as in step S3, if no exogenous hydrolytic enzymes are added and the microalgae culture and the fungal culture are co-cultured in the same environment, the hydrolytic enzymes produced by the fungal culture can also be used to decompose the cell walls of the microalgae.

According to another embodiment, as in step S4, specific methods for separating and purifying 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.

The above is merely an example of the preferred embodiment of this invention and is not intended to limit the scope of implementation. Any simple substitutions and equivalent changes made within the scope of the patent application and the content of the patent specification of this invention are within the scope of the patent application of this invention.

S1, S2: Steps

Claims (5)

A method for producing and extracting microalgae metabolites comprises: adding an emulsifier to a microalgae culture, coating a plurality of microalgae with the emulsifier, wherein the microalgae are Chlorella and/or Spirulina, and the final concentration of the emulsifier added to the microalgae culture is 0.1 to 0.3 weight percent (wt%), wherein the emulsifier is lecithin; subjecting the microalgae culture to an adverse environment, wherein the adverse environment treatment comprises at least a salt adverse environment, and in the salt adverse environment, The final concentration of a salt added to the microalgae culture is 1 to 3 weight percent (wt%); the stress treatment is terminated after 24 to 48 hours; at least one fungal culture is added to the microalgae culture to decompose a cell wall of the microalgae; and the metabolites released from the pores of the cell wall of the microalgae are separated, wherein the fungal culture continues to grow in the culture medium of the microalgae culture and continuously releases the hydrolytic enzyme. The method for producing and extracting microalgae metabolites as described in claim 1, wherein the salt used in the salt stress is selected from the group consisting of urea, sodium chloride, magnesium sulfate, potassium nitrate, potassium chloride, and calcium chloride. The method for producing and extracting microalgae metabolites as described in claim 1, wherein the stress treatment further includes nutritional stress, and the nutritional stress condition is a nitrogen:phosphorus:potassium ratio of 0:35:10. The method for producing and extracting microalgae metabolites as described in claim 1, wherein the fungal culture is selected from the group consisting of Agaricus edulis, Pleurotus ostreatus, Ganoderma lucidum, and Hericium erinaceus. The method for producing and extracting microalgae metabolites as described in claim 1, wherein the fungal culture is selected from the group consisting of Aspergillus, Penicillium, and Trichoderma.