Method for extracting extracellular polymeric substance from cladophora
By controlling the enzyme-thermal synergistic extraction process with temperature gradient and using a specific microalgae culture medium, the problem of low extraction efficiency of extracellular polymers from Cladosporium was solved, achieving efficient extraction of EPS with high polysaccharide content and ensuring biological activity.
Patent Information
- Application Number
- CN202511285567.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-10
- Publication Date
- 2025-12-23
AI Technical Summary
Existing technologies are insufficient for efficiently extracting extracellular polymers from Cladophora, especially tightly bound EPS. Furthermore, traditional methods are prone to introducing impurities or damaging the active structure, and there is a lack of targeted extraction solutions.
A temperature gradient controlled enzyme-thermal synergistic extraction process was adopted, which combines inorganic salts, organic nutrients and trace elements in microalgae culture medium. By precisely controlling the temperature and time, dissolved, loosely attached and tightly bound EPS were extracted respectively.
This method increases the extraction yield of extracellular polymers and polysaccharide content, ensuring the bioactivity of EPS and avoiding cell rupture and impurity introduction caused by excessive enzymatic hydrolysis.
Smart Images
Figure CN121181718A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology and natural product extraction, and particularly relates to a method for extracting extracellular polymeric substance from Cladophora. BACKGROUND
[0002] Cladophora is a filamentous macroalgae belonging to the order Cladophorales of the division Chlorophyta, which is widely distributed in freshwater, brackish water and marine environments. The cell wall of Cladophora is composed of cellulose, pectin and sulfated polysaccharides, and has high mechanical strength and chemical stability.
[0003] Extracellular polymeric substance (EPS) secreted by Cladophora during growth has significant biological activity and industrial application value. Studies have shown that EPS of Cladophora is mainly composed of polysaccharides, proteins and a small amount of lipids, and the structure of sulfated polysaccharides endows it with biological activities such as antioxidant, anti-inflammatory and antiviral, making it have broad application prospects in the fields of medicine, cosmetics and environmental protection. For example, active groups such as carboxyl and sulfate groups in EPS can effectively adsorb heavy metal ions, making it a potential water treatment material; its gelation and biocompatibility are also suitable for the development of drug release carriers.
[0004] At present, the extraction methods of EPS from microorganisms mainly include physical methods such as centrifugation and ultrasonic, chemical methods such as acid-base treatment, chelating agents and enzymatic hydrolysis. However, these methods have obvious limitations when applied to the extraction of EPS from Cladophora: physical methods are difficult to effectively destroy the dense cell wall of Cladophora, resulting in low extraction rate of tightly bound EPS (TB-EPS); chemical methods can improve the extraction efficiency, but are easy to introduce impurities and destroy the active structure of EPS; and enzymatic hydrolysis requires optimization of enzyme combination and reaction conditions due to the complex composition of Cladophora cell wall, which is high in cost and lacks process stability. In addition, existing technologies are mostly developed for bacteria or unicellular microalgae such as Chlorella, and lack specific extraction schemes for filamentous algae such as Cladophora. SUMMARY
[0005] The present application aims to provide a method for extracting extracellular polymeric substance from Cladophora, which has high EPS extraction amount, high polysaccharide content in EPS, high biological activity and good extraction effect.
[0006] The technical scheme adopted by the present application to achieve the above-mentioned purpose is as follows: The application discloses a method for extracting extracellular polymers from Cladophora sp., and comprises the following steps: culturing initial Cladophora sp. by using a microalgae culture medium to obtain Cladophora sp. algae, and then performing an extraction process on the Cladophora sp. algae to obtain extracellular polymers; the extraction process comprises a tightly bound EPS extraction process, the tightly bound EPS extraction process adopts temperature gradient controlled enzyme-heat synergistic extraction, the extraction temperature is 20-40 DEG C, and the enzyme is cellulase.
[0007] Preferably, the microalgae culture medium comprises inorganic salts, organic nutrients and trace elements.
[0008] More preferably, the inorganic salts comprise at least one of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate and sodium carbonate.
[0009] More preferably, the organic nutrients comprise at least one of citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid.
[0010] More preferably, the trace elements comprise at least one of boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate and cobalt nitrate hexahydrate.
[0011] Preferably, the microalgae culture medium comprises inorganic salts, organic nutrients, trace elements and water.
[0012] Preferably, the inorganic salts comprise sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate and sodium carbonate.
[0013] Preferably, the organic nutrients comprise citric acid, ferric ammonium citrate and disodium ethylenediaminetetraacetate.
[0014] Preferably, the organic nutrients further include L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid. The content of L-(-)-diacetyl tartaric acid in the microalgae culture medium is 0.1-1wt%, and the content of 3,4-dihydroxyphenylacetic acid is 0.08-0.6wt%.
[0015] Preferably, the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate; the content of boric acid in the microalgae culture medium is 1-5mg / L, the content of manganese chloride tetrahydrate is 0.5-3mg / L, the content of zinc sulfate heptahydrate is 0.1-0.4mg / L, the content of sodium molybdate dihydrate is 0.2-0.8mg / L, the content of copper sulfate pentahydrate is 0.04-0.16mg / L, and the content of cobalt nitrate hexahydrate is 0.02-0.5mg / L.
[0016] Preferably, the extraction process further includes an extraction process of dissolved EPS and an extraction process of loosely attached EPS.
[0017] More preferably, in the extraction process of dissolved EPS, the extraction liquid is a PBS buffer.
[0018] More preferably, in the extraction process of loosely attached EPS, the extraction liquid is a Tris-HCl buffer containing EDTA.
[0019] Preferably, in the extraction process of tightly bound EPS, the extraction liquid is a NaCl buffer containing cellulase.
[0020] More preferably, the content of cellulase in the NaCl buffer is 0.05-0.1wt%.
[0021] Preferably, in the cultivation of microalgae, the initial Cladophora sp. is added to the microalgae culture medium, and the Cladophora sp. is cultured at 20-40℃ for 7-10d, and then centrifuged at 0-5℃ to obtain the Cladophora sp. algal body.
[0022] More preferably, in the cultivation of microalgae, the addition amount of the initial Cladophora sp. is 30-40wt% of the microalgae culture medium.
[0023] Preferably, in the extraction of dissolved EPS, the Cladophora sp. algal body is suspended in a PBS buffer, oscillated at 50-100rpm for 15-20min, and then centrifuged at 6000-8000rpm for 15-20min, and the supernatant is taken, filtered by a filter membrane, to obtain the dissolved EPS. By precisely controlling the pH value and ionic strength of the buffer system, and optimizing the mechanical action parameters, the release of dissolved EPS from the surface of the algal body is effectively promoted, and the cell rupture caused by excessive mechanical action is avoided, thereby ensuring the integrity and extraction efficiency of the EPS.
[0024] More preferably, in the extraction of the dissolved EPS, the pH of the PBS buffer is 7.4-7.8, and the amount of the Cladophora algae used is 30-40wt% of the PBS buffer. The filter membrane pore size is 0.22 μm.
[0025] Preferably, in the extraction of the loosely attached EPS, the Cladophora algae after the extraction of the dissolved EPS is resuspended in the Tris-HCl buffer containing EDTA, ultrasonically treated in an ice water bath for 5-7 min, centrifuged at 0-10℃ and 60-80 rpm for 20-30 min, and the supernatant is collected to obtain the loosely attached EPS. The EDTA in a specific concentration range can effectively chelate divalent metal ions, and the optimized pH condition can maintain the optimal chelation activity of EDTA.
[0026] More preferably, in the extraction of the loosely attached EPS, the pH of the Tris-HCl buffer is 8.0-8.5, the content of EDTA in the Tris-HCl buffer is 0.1-0.3wt%, and the amount of the Cladophora algae after the extraction of the dissolved EPS is 30-40wt% of the Tris-HCl buffer.
[0027] Preferably, in the extraction of the tightly bound EPS, the Cladophora algae after the extraction of the loosely attached EPS is suspended in the NaCl buffer containing cellulase, enzymatically treated at 20-37℃ for 10-60 min, and then treated at 38-40℃ for 10-30 min, and then the reaction is terminated in an ice water bath, centrifuged at 10000-12000 rpm for 20-25 min, and the supernatant is collected to obtain the tightly bound EPS.
[0028] More preferably, in the extraction of the tightly bound EPS, the pH of the NaCl buffer is 6.0-6.8, the content of NaCl in the NaCl buffer is 50-60mM, and the content of cellulase in the NaCl buffer is 0.05-0.1wt%.
[0029] Preferably, in the preparation of the EPS, the dissolved EPS, the loosely attached EPS and the tightly bound EPS are mixed to obtain a combined EPS extract, and then anhydrous ethanol is added, and the mixture is placed at 0-10℃ for 6-24h, centrifuged at 3000-5000 rpm for 10-15 min, the precipitate is collected, and freeze-dried to obtain the EPS.
[0030] More preferably, in the preparation of the EPS, the dissolved EPS, the loosely attached EPS and the tightly bound EPS are extracted from the same initial Cladophora, and the amount of the anhydrous ethanol used is 600-1800wt% of the dissolved EPS.
[0031] Preferably, methyl p-formylcinnamate can also be added to the organic nutrients. The content of methyl p-formylcinnamate in the microalgae culture medium is 0.03-0.3 wt%. In this invention, after using citric acid, ferric ammonium citrate, disodium EDTA, L-(-)-diacetyltartaric acid, and 3,4-dihydroxyphenylacetic acid, the further addition of methyl p-formylcinnamate can increase the yield of extracellular polymers in Cladosporium and increase the polysaccharide content, thereby enhancing the bioactivity of the extracellular polymers.
[0032] This invention employs different extraction steps for dissolved EPS, loosely attached EPS, and tightly bound EPS to extract as much extracellular polymeric substance (EPS) as possible. The amount of EPS extracted is related to the production of Cladophora algae. In this invention, Cladophora algae are cultured in a microalgae culture medium containing inorganic salts, organic nutrients, and trace elements before extraction. The different amounts of EPS obtained indicate that the components in the microalgae culture medium can increase the amount of EPS produced by Cladophora algae. The organic nutrients in this invention include at least one of citric acid, ferric ammonium citrate, disodium EDTA, L-(-)-diacetyl tartaric acid, and 3,4-dihydroxyphenylacetic acid, thus having the following beneficial effects: high EPS extraction yield and high polysaccharide content in EPS. Therefore, this invention is a method for extracting extracellular polymeric substances from Cladophora algae with high EPS extraction yield and high polysaccharide content. Attached Figure Description
[0033] Figure 1 This is an AFM image of extracellular polymeric substances from Cladophora.
[0034] Figure 2 This is a graph showing the amount of EPS extracted.
[0035] Figure 3 This is a graph showing the polysaccharide content. Detailed Implementation
[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0037] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0038] Example 1: A method for extracting extracellular polymers from Cladophora. Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, and disodium ethylenediaminetetraacetate; and the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0039] Microalgae cultivation: Initial Cladophora was added to the microalgae culture medium and cultured at 30℃ for 10 days. The Cladophora cells were obtained by centrifugation at 4℃. The initial Cladophora addition amount was 40 wt% of the microalgae culture medium.
[0040] Extraction of soluble EPS: The algae of *Cladophora* were suspended in PBS buffer and shaken at 100 rpm for 20 min, then centrifuged at 8000 rpm for 20 min. The supernatant was collected and filtered through a filter membrane to obtain soluble EPS. The pH of the PBS buffer was 7.4, and the amount of *Cladophora* algae used was 40 wt% of the PBS buffer. The filter membrane had a pore size of 0.22 μm.
[0041] Extraction of loosely attached EPS: After extraction of the dissolved EPS, the Cladophora algae were resuspended in a Tris-HCl buffer containing EDTA, sonicated in an ice-water bath for 5 min, and centrifuged at 4℃ and 80 rpm for 30 min. The supernatant was collected to obtain the loosely attached EPS. The pH of the Tris-HCl buffer was 8.0, the EDTA content in the Tris-HCl buffer was 0.3 wt%, and the amount of Cladophora algae used after extraction of the dissolved EPS was 40 wt% of the Tris-HCl buffer.
[0042] Extraction of tightly bound EPS: After extraction of loosely attached EPS, the Cladophora algae were suspended in NaCl buffer containing cellulase and enzymatically digested at 37°C for 20 min, followed by treatment at 40°C for 20 min. The reaction was then terminated by ice bath, and the mixture was centrifuged at 12000 rpm for 20 min. The supernatant was collected to obtain tightly bound EPS. The pH of the NaCl buffer was 6.8, the NaCl concentration was 50 mM, and the cellulase concentration was 0.1 wt%.
[0043] Preparation of EPS: Dissolved EPS, loosely attached EPS, and tightly bound EPS were mixed to obtain a combined EPS extract. Anhydrous ethanol was then added, and the mixture was allowed to stand at 5°C for 12 hours. After centrifugation at 5000 rpm for 15 minutes, the precipitate was collected and freeze-dried to obtain EPS. The dissolved EPS, loosely attached EPS, and tightly bound EPS were extracted from the same initial Cladophora species. The amount of anhydrous ethanol used was 1200 wt% of the dissolved EPS.
[0044] Example 2: A method for extracting extracellular polymers from Cladophora. The difference between this embodiment and Example 1 lies in the microalgae culture medium.
[0045] Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, L-(-)-diacetyltartaric acid, and 3,4-dihydroxyphenylacetic acid; and the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, L-(-)-diacetyl tartaric acid 0.8 mg / L, 3,4-dihydroxyphenylacetic acid 0.5 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0046] Example 3: A method for extracting extracellular polymers from Cladophora. The difference between this embodiment and Example 2 lies in the microalgae culture medium.
[0047] Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, L-(-)-diacetyltartaric acid, and 3,4-dihydroxyphenylacetic acid; and the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, L-(-)-diacetyl tartaric acid 0.3 mg / L, 3,4-dihydroxyphenylacetic acid 0.12 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0048] Example 4: A method for extracting extracellular polymers from Cladophora. The difference between this embodiment and Example 2 lies in the microalgae culture medium.
[0049] Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, disodium EDTA, L-(-)-diacetyl tartaric acid, 3,4-dihydroxyphenylacetic acid, and methyl p-formylcinnamate; the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, L-(-)-diacetyl tartaric acid 0.8 mg / L, 3,4-dihydroxyphenylacetic acid 0.5 mg / L, methyl p-formylcinnamate 0.2 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0050] Example 5: A method for extracting extracellular polymers from Cladophora. The difference between this embodiment and Example 2 lies in the microalgae culture medium.
[0051] Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, disodium EDTA, L-(-)-diacetyl tartaric acid, 3,4-dihydroxyphenylacetic acid, and methyl p-formylcinnamate; the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, L-(-)-diacetyl tartaric acid 0.8 mg / L, 3,4-dihydroxyphenylacetic acid 0.5 mg / L, methyl p-formylcinnamate 0.07 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0052] Comparative Example 1: A method for extracting extracellular polymers from Cladophora. The difference between this comparative example and Example 1 lies in the microalgae culture medium.
[0053] Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, and 3,4-dihydroxyphenylacetic acid; and the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, 3,4-dihydroxyphenylacetic acid 0.5 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0054] Comparative Example 2: A method for extracting extracellular polymers from Cladophora. The difference between this comparative example and Example 1 lies in the microalgae culture medium.
[0055] Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, and L-(-)-diacetyl tartaric acid; and the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, L-(-)-diacetyl tartaric acid 0.8 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0056] Comparative Example 3: A method for extracting extracellular polymers from Cladophora. The difference between this comparative example and Example 1 lies in the microalgae culture medium.
[0057] Microalgae culture medium consists of inorganic salts, organic nutrients, trace elements, and water. The inorganic salts in the microalgae culture medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate; the organic nutrients include citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, L-(-)-diacetyltartaric acid, and 3,4-dihydroxyphenylacetic acid; and the trace elements include boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate. The microalgae culture medium contained the following components: sodium nitrate 1.5 g / L, dipotassium hydrogen phosphate 40 mg / L, magnesium sulfate heptahydrate 75 mg / L, calcium chloride dihydrate 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium ethylenediaminetetraacetate 1 mg / L, L-(-)-diacetyl tartaric acid 0.08 mg / L, 3,4-dihydroxyphenylacetic acid 0.05 mg / L, sodium carbonate 20 mg / L, boric acid 2.86 mg / L, manganese chloride tetrahydrate 1.86 mg / L, zinc sulfate heptahydrate 0.22 mg / L, sodium molybdate dihydrate 0.39 mg / L, copper sulfate pentahydrate 0.08 mg / L, and cobalt nitrate hexahydrate 0.05 mg / L.
[0058] Experimental example: The extracellular polymeric material of *Cladophora* prepared in Example 1 was characterized using AFM, and the results are as follows: Figure 1As shown, the surface of the extracellular polymer of Cladosporium exhibits obvious nanoscale undulations and heterogeneous structure. The color difference reflects the force deviation when different regions come into contact with the probe, indirectly indicating the difference in local mechanical properties. The bright areas may correspond to polysaccharide aggregation areas with relatively high stiffness or protrusions, while the dark areas may represent softer or less aggregated areas. This indicates that the surface of the extracellular polymer of Cladosporium presents a complex micro-nano structure, providing a potential physical basis for its adhesion function.
[0059] 1. Extracellular polymeric material extraction amount The results of preparing extracellular polymers in the examples and comparative examples of this invention are as follows: Figure 2 As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. The extracellular polymers include soluble EPS, loosely attached EPS, and tightly bound EPS. This invention employs different extraction processes for soluble EPS, loosely attached EPS, and tightly bound EPS to extract as much extracellular polymer as possible. The amount of extracellular polymer extracted is related to the amount of Cladophora produced. In this invention, Cladophora is cultured in a microalgae culture medium containing inorganic salts, organic nutrients, and trace elements. The different amounts of extracellular polymers obtained indicate that the components in the microalgae culture medium can increase the amount of extracellular polymers produced by Cladophora. The organic nutrients in this invention include citric acid, ferric ammonium citrate, and disodium ethylenediaminetetraacetate. With the use of citric acid, ferric ammonium citrate, and disodium ethylenediaminetetraacetate, L-(-)- Using diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid as organic nutrients can increase the yield of extracellular polymers in the resulting microalgae culture medium. The combined use of L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid can also increase the yield of extracellular polymers, but the amount of L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid used needs to be within a certain range. If the amount of L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid used is too small, the yield of extracellular polymers cannot be effectively increased. Similarly, using only L-(-)-diacetyl tartaric acid or 3,4-dihydroxyphenylacetic acid will also fail to increase the yield of extracellular polymers. In this invention, when using citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, L-(-)-diacetyl tartaric acid, and 3,4-dihydroxyphenylacetic acid as organic nutrients, methyl p-formylcinnamate can also be added. The use of methyl p-formylcinnamate can further increase the yield of extracellular polymers.
[0060] 2. Determination of polysaccharide content in extracellular polymers Extracellular polymers mainly consist of micromolecules such as polysaccharides, proteins, and small amounts of lipids. Polysaccharides possess high biological activity and ecological functions. This invention uses the anthrone-sulfuric acid method to detect the polysaccharide content in extracellular polymers, and the results are as follows: Figure 3 As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. The *Cladophora* species in this invention were cultured in a microalgae culture medium containing inorganic salts, organic nutrients, and trace elements. The resulting extracellular polymers showed varying polysaccharide content, indicating that the components in the microalgae culture medium can increase the amount of polysaccharides produced by *Cladophora* in its extracellular polymers. The organic nutrients in this invention include citric acid, ferric ammonium citrate, and disodium EDTA. Using L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid as organic nutrients in the microalgae culture medium resulted in increased polysaccharide production in the extracellular polymers. The combined use of L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid can increase the yield of polysaccharides in extracellular polymers. However, the amount of L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid used needs to be within a certain range. If the amount of L-(-)-diacetyl tartaric acid and 3,4-dihydroxyphenylacetic acid used is too small, the yield of polysaccharides in extracellular polymers cannot be effectively increased. Similarly, if only L-(-)-diacetyl tartaric acid or 3,4-dihydroxyphenylacetic acid is used, the yield of polysaccharides in extracellular polymers cannot be increased. In this invention, when using organic nutrients such as citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, L-(-)-diacetyl tartaric acid, and 3,4-dihydroxyphenylacetic acid, methyl p-formylcinnamate can also be added. The use of methyl p-formylcinnamate can further increase the yield of polysaccharides in extracellular polymers.
[0061] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.
[0062] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.
Claims
1. A method for extracting extracellular polymers from Cladophora, comprising: Initial Cladophora was cultured in a microalgae culture medium to obtain Cladophora algae thallus. Then, the Cladophora algae thallus was extracted to obtain extracellular polymers. The extraction process included the extraction of tightly bound EPS. The extraction of tightly bound EPS adopted temperature gradient controlled enzyme-thermal synergistic extraction. The extraction temperature was 20-40℃, and the enzyme was cellulase.
2. The method for extracting extracellular polymers from Cladophora according to claim 1, characterized in that, The microalgae culture medium includes inorganic salts, organic nutrients, and trace elements.
3. The method for extracting extracellular polymers from Cladophora according to claim 2, characterized in that, The inorganic salt includes at least one of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, and sodium carbonate.
4. The method for extracting extracellular polymers from Cladophora according to claim 2, characterized in that, The organic nutrients include at least one of citric acid, ferric ammonium citrate, disodium EDTA, L-(-)-diacetyl tartaric acid, and 3,4-dihydroxyphenylacetic acid.
5. The method for extracting extracellular polymers from Cladophora according to claim 2, characterized in that, The trace elements include at least one of boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate, and cobalt nitrate hexahydrate.
6. The method for extracting extracellular polymers from Cladophora according to claim 1, characterized in that, The extraction process also includes the extraction process of soluble EPS and the extraction process of loosely attached EPS.
7. The method for extracting extracellular polymers from Cladophora according to claim 6, characterized in that, In the extraction process of the dissolved EPS, the extraction solution is PBS buffer.
8. The method for extracting extracellular polymers from Cladophora according to claim 1, characterized in that, In the extraction process of the loosely attached EPS, the extraction solution is a Tris-HCl buffer solution containing EDTA.
9. A method for extracting extracellular polymers from Cladophora according to claim 1, characterized in that, In the extraction process of the tightly bound EPS, the extraction solution is a NaCl buffer solution containing cellulase.
10. A method for extracting extracellular polymers from Cladosporium according to claim 9, characterized in that, The NaCl buffer solution contains 0.05-0.1 wt% cellulase.