Production process of freeze-dried algae species of compound chlamydomonas

Abstract

The invention relates to the technical field of production of aquaculture microalgae preparations, in particular to a production process of freeze-dried algae species of compound chlamydomonas. Comprising the following steps: obtaining algae species, expanding culture, storing and using. The algae species acquisition comprises purchase approach optimization and self-separation technology upgrading, and purchase approach optimization: establishing a long-term stable cooperation relationship with a national algae species library. The method has the beneficial effects that the method is convenient to preserve: the chlamydomonas is prepared into the freeze-dried algae species through a vacuum freeze-drying technology, so that the preservation time of the chlamydomonas is greatly prolonged, the transportation and the storage are convenient, and the preservation cost of the algae species is reduced. A proper freeze-drying protective agent and a carrier are added in the freeze-drying process, and a proper freeze-drying curve is used, so that the cell integrity rate and the good dormancy state of the chlamydomonas in the freeze-drying and preservation process are effectively improved, and the chlamydomonas can be quickly resurgent in use. After reviving, a culture medium can be added for expanding culture propagation, and stable passage capacity is achieved.

Description

Technical Field

[0001] This invention relates to the field of microalgae preparation production technology for aquaculture, specifically to a process for producing a compound Chlamydomonas freeze-dried algae strain. Background Technology

[0002] In aquaculture, high-quality algae strains are of great significance for improving water quality and providing natural feed for farmed animals. However, traditional methods of preserving and using algae strains have many problems. For example, fresh algae strains are difficult to preserve and transport for a long time, and liquid algae solutions are prone to deterioration and have unstable content of effective ingredients. Chlamydomonas, as an algae with high efficiency in purifying water quality and rich in nutrients, would have a positive impact on the aquaculture industry if an effective method for its preservation and application could be developed.

[0003] Currently, there is a lack of a production process on the market that can effectively solve the problem of preserving and facilitating the use of Chlamydomonas. Existing technologies have technical defects in the expansion, concentration, and freeze-drying preservation of Chlamydomonas, and cannot meet the demand of aquaculture for high-quality and stable algae strains. Summary of the Invention

[0004] To address the problems mentioned in the background art, the present invention aims to provide a process for producing composite Chlamydomonas freeze-dried algal strains.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a process for producing compound Chlamydomonas freeze-dried algal strains, comprising the following steps: algal strain acquisition, expansion and cultivation, concentration, compounding and blending, filling and freeze-drying, storage and use;

[0006] Algae Acquisition

[0007] Optimize procurement channels: Establish a long-term and stable cooperative relationship with the National Algal Seed Bank to ensure that the purchased fresh Chlamydomonas seeds have detailed genetic background information and physiological characteristic data. At the same time, introduce third-party testing institutions to conduct purity and activity tests on the purchased algal seeds to ensure that the quality of the algal seeds meets production requirements.

[0008] Upgraded self-separation technology: Flow cytometry is combined with fluorescent labeling to efficiently separate Chlamydomonas from collected natural water samples. Fluorescent labeling specifically identifies Chlamydomonas cell surface markers, and flow cytometry is used to precisely screen Chlamydomonas cells, greatly improving separation efficiency and purity.

[0009] Expansion

[0010] Sealed mixed storage:

[0011] Culture medium improvement: On the basis of traditional culture medium, a compound additive rich in various vitamins and trace elements, such as vitamin B12, iron ions, zinc ions, etc., is added to meet the diverse nutritional needs of Chlamydomonas during polyculture and further improve its growth rate.

[0012] Refined gas control: Using intelligent gas mixing equipment, the ratio of carbon dioxide and organic carbon source introduced is monitored and controlled in real time, and the gas ratio is dynamically adjusted according to different growth stages of Chlamydomonas.

[0013] Open-mouth self-sufficiency:

[0014] Intelligent light control system: Install intelligent light equipment, monitor the ambient light intensity and spectral distribution in real time through sensors, and automatically adjust the light intensity and light duration according to the light demand curve of Chlamydomonas growth to achieve precise light control;

[0015] Dynamic nutrient replenishment: An online nutrient monitoring system is established to detect the concentration of inorganic nutrients in the culture medium in real time. Based on the growth and consumption of Chlamydomonas, an automated replenishment device is used to accurately replenish inorganic nutrients such as sodium nitrate, magnesium sulfate, and calcium chloride to maintain a stable nutrient concentration and provide continuous nutritional support for the growth of Chlamydomonas.

[0016] Seated heteroculture in fermentation tanks:

[0017] Intelligent monitoring of the fermenter environment: Multi-parameter sensors are installed in the fermenter to monitor parameters such as temperature, pH value, dissolved oxygen, and oxidation-reduction potential in real time. The monitoring data is transmitted to the central processor through the data acquisition and control system. Using the preset control algorithm, the operating parameters such as stirring speed, aeration rate, and feed rate of the fermenter are automatically adjusted to ensure that the environment inside the fermenter is always in the most suitable state for Chlamydomonas growth.

[0018] Application of novel carbon and nitrogen sources: Explore the use of novel organic carbon sources, such as fructooligosaccharides and xylose, as well as novel nitrogen sources, such as amino acid chelated nitrogen, to replace some traditional carbon and nitrogen sources;

[0019] concentrate

[0020] Centrifugation equipment optimization: Select centrifuges with variable frequency speed regulation and automatic slag discharge functions. According to the concentration and volume of algal liquid, the speed and centrifugation time of the centrifuge are precisely adjusted by the PLC control system. During the centrifugation process, the centrifugation effect is monitored in real time. When the set water content of algal sludge is reached, the slag discharge program is automatically started to discharge the concentrated algal sludge, thereby improving centrifugation efficiency and automation.

[0021] Concentration-assisted technology: Before centrifugation, add an appropriate amount of flocculant, such as polyacrylamide, to the algal solution. Through flocculation, Chlamydomonas cells aggregate to form larger floc particles, reducing the viscosity of the algal solution and improving the centrifugation separation effect.

[0022] Compound blending

[0023] Screening and optimization of freeze-drying protectants: Large-scale freeze-drying protectant screening experiments were conducted. In addition to traditional freeze-drying protectants such as trehalose and mannitol, novel freeze-drying protectants such as chitosan derivatives and cyclodextrin were introduced. The effects of different freeze-drying protectants and their combinations on the survival rate of Chlamydomonas were studied through experiments. A freeze-drying protectant screening model was established, and the formulation of freeze-drying protectants was optimized using response surface methodology to determine the optimal combination and concentration of freeze-drying protectants, so as to maximize the survival rate of Chlamydomonas during freeze-drying and preservation.

[0024] Carrier optimization and innovation: In addition to Chlorella liquid, Haematococcus pluvialis liquid or photosynthetic bacteria liquid as carriers, the feasibility of using nanomaterials as carriers is studied. For example, nano-silica or nano-cellulose carriers are prepared, and Chlamydomonas cells are loaded on the surface or inside of nanomaterials. The high specific surface area and good dispersibility of nanomaterials are used to improve the stability and activity of Chlamydomonas.

[0025] Filling freeze-dried

[0026] Optimization of freeze-drying process curve: Thermal analysis technology is used to analyze the thermal characteristics of Chlamydomonas algal mud and composite concentrated algal liquid to obtain key thermal parameters such as phase change temperature and moisture content change. Based on these thermal parameters and combined with the heat and mass transfer principle in the freeze-drying process, a mathematical model of the freeze-drying process is established. By simulating different freeze-drying process conditions, the freeze-drying process curve is optimized to determine the optimal pre-freezing temperature, pre-freezing time, sublimation drying temperature, sublimation drying time, desorption drying temperature, and desorption drying time, so as to improve freeze-drying efficiency and product quality while ensuring the activity of Chlamydomonas.

[0027] Improved vial packaging technology: Before packaging, the vials are pretreated, such as by high-temperature sterilization and siliconization, to improve their cleanliness and surface properties and reduce damage to Chlamydomonas cells. During the packaging process, nitrogen protection packaging technology is used to reduce the impact of oxygen and moisture on Chlamydomonas. At the same time, the sealing method of the vials is optimized by using new sealing materials and processes to ensure the airtightness of the vials during storage, preventing the entry of external air and moisture and ensuring the quality stability of the freeze-dried Chlamydomonas strain.

[0028] Storage and Use

[0029] Optimization of storage conditions: Establish a dedicated room temperature and cool storage room, install an automatic temperature and humidity control system, monitor and adjust the temperature and humidity in the storage room in real time, place the vacuum freeze-dried Chlamydomonas sylvestris strain on storage racks with moisture-proof and light-proof functions, regularly conduct quality tests on the stored freeze-dried Chlamydomonas sylvestris strain, including tests on indicators such as survival rate, activity, and nutrient content, and adjust the storage conditions according to the test results to ensure the quality stability of the freeze-dried Chlamydomonas sylvestris strain during storage;

[0030] Usage guidance and technical services: We provide users with detailed user manuals and technical guidance manuals, including key information such as the revival method of freeze-dried Chlamydomonas sylvestris, propagation conditions, dosage and timing of release. We also develop personalized usage plans for different aquaculture species and environments to ensure that freeze-dried Chlamydomonas sylvestris can fully play its role in improving water quality and providing live algae feed in aquaculture water.

[0031] The beneficial effects of this invention are:

[0032] Convenient storage: By using vacuum freeze-drying technology, Chlamydomonas can be made into freeze-dried algal strains, which greatly extends the storage time of Chlamydomonas, facilitates transportation and storage, and reduces the storage cost of algal strains.

[0033] High revival rate: By adding appropriate freeze-drying protectants and carriers and using appropriate freeze-drying curves during the freeze-drying process, the cell integrity rate and good dormancy state of Chlamydomonas are effectively improved, ensuring rapid revival when used.

[0034] It has stable genetic characteristics: after resurrection, it can be expanded and propagated by adding culture medium, and has a stable passage ability;

[0035] Wide range of applications: The compound Chlamydomonas freeze-dried algae strain can not only improve water quality, but also provide a rich source of live algae feed for aquatic animals, meeting the needs of different aquatic species and showing good application prospects.

[0036] High flexibility: Diverse propagation methods are available, allowing for the selection of appropriate propagation methods based on different production conditions and needs; the freeze-drying process curve, freeze-drying protectant, and finished product compounding can be adjusted according to actual conditions, demonstrating strong flexibility and adaptability.

[0037] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0038] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0039] The present invention is illustrated below with specific embodiments, which are not intended to limit the scope of the invention.

[0040] The process includes the following steps: algal strain acquisition, propagation, concentration, compounding, filling and freeze-drying, storage and use;

[0041] The acquisition of algal strains includes optimizing procurement channels and upgrading self-separation technology. Optimizing procurement channels involves establishing a long-term and stable cooperative relationship with the National Algal Strain Bank to ensure that the purchased fresh *Chlamydomonas* strains have detailed genetic background information and physiological characteristic data. Simultaneously, third-party testing institutions are introduced to conduct purity and activity tests on the purchased algal strains, guaranteeing that the quality meets production requirements. Upgrading self-separation technology involves using flow cytometry combined with fluorescent labeling to efficiently separate *Chlamydomonas* from collected natural water samples. Fluorescent labels specifically identify *Chlamydomonas* cell surface markers, and flow cytometry is used to precisely screen *Chlamydomonas* cells, greatly improving separation efficiency and purity. Propagation includes culture medium improvement, refined gas control, an intelligent light control system, and dynamic nutrient supplementation. Culture medium improvement: Based on traditional culture media, a compound additive rich in various vitamins and trace elements, such as vitamin B12, iron ions, and zinc ions, is added to meet the diverse nutrient requirements of *Chlamydomonas* during polyculture, further improving its growth rate; Refined gas control: Intelligent gas mixing equipment is used to monitor and control the ratio of carbon dioxide and organic carbon sources in real time, dynamically adjusting the gas ratio according to different growth stages of *Chlamydomonas*; Intelligent light control system: Intelligent light equipment is installed, using sensors to monitor ambient light intensity and spectral distribution in real time. Based on the light requirement curve of *Chlamydomonas* growth, the light intensity and duration are automatically adjusted to achieve precise light control; Dynamic nutrient supplementation: An online nutrient monitoring system is established to detect the culture medium in real time. The concentration of inorganic nutrients in the culture medium is precisely replenished using an automated replenishment device based on the consumption of Chlamydomonas aeruginosa to maintain a stable nutrient concentration and provide continuous nutritional support for its growth. The fermenter is sealed and heterotrophically cultured with intelligent environmental monitoring: multi-parameter sensors are installed inside the fermenter to monitor parameters such as temperature, pH, dissolved oxygen, and redox potential in real time. The data acquisition and control system transmits the monitored data to a central processor, which automatically adjusts operating parameters such as stirring speed, aeration rate, and feed rate using a preset control algorithm to ensure the fermenter environment is always optimal for Chlamydomonas aeruginosa growth. The application of novel carbon and nitrogen sources is explored, such as fructooligosaccharides. Xylose, as well as novel nitrogen sources such as amino acid chelated nitrogen, are used to replace some traditional carbon and nitrogen sources. Concentration includes centrifugation equipment optimization and concentration auxiliary technology. Centrifugation equipment optimization: Centrifuges with variable frequency speed control and automatic sludge discharge are selected. According to the concentration and volume of the algal solution, the speed and centrifugation time of the centrifuge are precisely adjusted by the PLC control system. During the centrifugation process, the centrifugation effect is monitored in real time. When the set water content of the algal sludge is reached, the sludge discharge program is automatically started to discharge the concentrated algal sludge, thereby improving centrifugation efficiency and automation. Concentration auxiliary technology: Before centrifugation, an appropriate amount of flocculant, such as polyacrylamide, is added to the algal solution. Through flocculation, Chlamydomonas cells aggregate to form larger floc particles, reducing the viscosity of the algal solution and improving the centrifugation separation effect.The compound formulation includes the screening and optimization of freeze-drying protectants and the optimization and innovation of carriers. The screening and optimization of freeze-drying protectants involves conducting large-scale screening experiments, introducing novel freeze-drying protectants such as chitosan derivatives and cyclodextrins in addition to traditional ones like trehalose and mannitol. Experiments are conducted to study the effects of different freeze-drying protectants and their combinations on the survival rate of *Chlamydomonas*, establishing a freeze-drying protectant screening model, and using response surface methodology to optimize the freeze-drying protectant formulation, determining the optimal combination and concentration of freeze-drying protectants to maximize the survival rate of *Chlamydomonas* during freeze-drying and preservation. The optimization and innovation of carriers involves studying the feasibility of using nanomaterials as carriers, in addition to *Chlorella vulgaris* liquid, *Haematococcus pluvialis* liquid, or photosynthetic bacteria liquid. For example, the preparation of nano-dioxanone... Silicon carbide or nanocellulose carriers are used to load Chlamydomonas cells onto the surface or interior of nanomaterials. The high specific surface area and good dispersibility of nanomaterials enhance the stability and activity of Chlamydomonas. The filling and freeze-drying process includes optimization of the freeze-drying process curve and improvement of vial packaging technology. Optimization of the freeze-drying process curve involves using thermal analysis techniques to analyze the thermal characteristics of Chlamydomonas algal sludge and composite concentrated algal solution, obtaining key thermal parameters such as phase transition temperature and moisture content changes. Based on these thermal parameters and the principles of heat and mass transfer during freeze-drying, a mathematical model of the freeze-drying process is established. By simulating different freeze-drying process conditions, the freeze-drying process curve is optimized to determine the optimal pre-freezing temperature, pre-freezing time, sublimation drying temperature, sublimation drying time, desorption drying temperature, and desorption drying time, ensuring optimal performance. To improve freeze-drying efficiency and product quality while ensuring the viability of *Chlamydomonas*, improvements were made to the vial packaging technology: Before filling, the vials underwent pretreatment, such as high-temperature sterilization and siliconization, to improve their cleanliness and surface properties, reducing damage to *Chlamydomonas* cells. During packaging, nitrogen-protected packaging technology was used to reduce the impact of oxygen and moisture on *Chlamydomonas*. Simultaneously, the sealing method of the vials was optimized, employing new sealing materials and processes to ensure the vials remained airtight during storage, preventing the entry of external air and moisture and guaranteeing the quality stability of the freeze-dried *Chlamydomonas* strain. Storage and use included optimized storage conditions, usage guidance, and technical services. Optimized storage conditions included establishing a dedicated cool, room-temperature storage room and installing an automatic temperature and humidity control system. The system monitors and regulates the temperature and humidity within the preservation chamber in real time. Vacuum-dried Chlamydomonas aeruginosa is placed on moisture-proof and light-proof storage racks. Regular quality testing is conducted on the preserved strains, including measurements of survival rate, activity, and nutrient content. Preservation conditions are adjusted based on test results to ensure stable quality during preservation. User guidance and technical services include detailed instruction manuals and technical support, covering key information such as revival methods, propagation conditions, dosage, and timing of the freeze-dried Chlamydomonas aeruginosa. Personalized usage plans are developed for different aquaculture species and environments to ensure the freeze-dried Chlamydomonas aeruginosa fully performs its function of improving water quality and providing live algae feed in aquaculture water.

[0042] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0043] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

[0044] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for producing a compound Chlamydomonas freeze-dried algal strain, characterized in that, Includes the following steps: Algal strain acquisition, propagation, concentration, compounding, filling, freeze drying, storage and use; The acquisition of algal strains includes using flow cytometry to separate Chlamydomonas strains from natural water samples, and using a third-party testing agency to test the purity and activity of the purchased or self-separated algal strains. The expansion culture includes open autotrophy, utilizing an intelligent light control system to automatically adjust light intensity and time, and employing an automated liquid replenishment device to accurately replenish inorganic nutrients based on data from an online nutrient monitoring system. The concentration process includes using a centrifuge with variable frequency speed control and automatic slag discharge function, adjusting the speed and centrifugation time precisely according to the algal solution concentration and volume through a PLC control system, adding flocculant to the algal solution before centrifugation and using ultrasonic-assisted centrifugation technology. The compound formulation includes conducting large-scale lyophilization protectant screening experiments, using response surface methodology to establish a screening model and optimize the formulation, and studying the use of nanomaterials as carriers and surface modification of the carriers; the filling lyophilization includes establishing a mathematical model of the lyophilization process to optimize the lyophilization process curve, performing high-temperature sterilization and siliconization pretreatment on the vials before filling, and using nitrogen protection packaging technology and optimizing the sealing method. The preservation and use include establishing a cool, room-temperature storage facility with an automatic temperature and humidity control system, regularly conducting quality tests on the preserved freeze-dried Chlamydomonas sylvestris, establishing a technical service team, and developing personalized usage plans for different aquaculture conditions.

2. The process for producing a composite Chlamydomonas freeze-dried algal strain according to claim 1, characterized in that, In the process of obtaining algal strains, when separating Chlamydomonas strains by hand, specific inducing factors are added to the separation culture medium to promote the growth and differentiation of Chlamydomonas cells.

3. The process for producing a compound Chlamydomonas freeze-dried algal strain according to claim 1, characterized in that, The modified sealed mixed culture medium contains a compound additive rich in vitamin B12, iron ions, zinc ions, and other vitamins and trace elements.

4. The process for producing a compound Chlamydomonas freeze-dried algal strain according to claim 1, characterized in that, The open-type self-sustaining intelligent lighting control system uses a combination of LED light sources of different wavelengths to simulate the natural light spectrum.

5. The process for producing a compound Chlamydomonas freeze-dried algal strain according to claim 1, characterized in that, The novel organic carbon sources explored in the sealed heterotrophic fermentation tank include fructooligosaccharides, and the novel nitrogen sources include amino acid chelated nitrogen.

6. The process for producing a compound Chlamydomonas freeze-dried algal strain according to claim 1, characterized in that, The novel freeze-drying protectants introduced in the compound formulation include chitosan derivatives and cyclodextrin.

7. The process for producing a compound Chlamydomonas freeze-dried algal strain according to claim 1, characterized in that, The nanomaterials used in the optimization and innovation of the carrier are nano-silica or nano-cellulose.