Preparation method and functional component loading method of microalgae exosome-like nanovesicles

Abstract

The application relates to a preparation method and a functional component loading method of microalgae exosome-like nanovesicles, and a multistage purification system of gradient differential centrifugation combined with multistage filtration and ultrafiltration enrichment is constructed to realize step-by-step removal of different scale fragments, aggregates and impurities in a microalgae culture system, high-efficiency preparation of the microalgae exosome-like nanovesicles is realized while damage of high centrifugal force to the structure of the nanovesicles is avoided, and the functional component loading is realized by using ultrasonic action to induce transient reversible nanopores of a lipid bilayer membrane or using phase transition stress caused by ice crystal formation and melting in a freeze-thaw cycle process to make the nanovesicle membrane have transient structure reconstruction and form a controllable channel structure.

Description

Technical Field

[0001] This invention belongs to the field of natural nanodelivery systems and food functional factor encapsulation technology, specifically involving a method for preparing microalgae-derived exosome-like nanovesicles and a method for differentially loading food functional factors of different polarities. Background Technology

[0002] Curcumin, carotenoids, anthocyanins, and other food functional components possess antioxidant, anti-inflammatory, and metabolic regulatory bioactivities, but they often suffer from poor solubility, low stability, and weak intestinal absorption. Static encapsulation of these functional components using nanodelivery systems is an important approach to promote their targeted delivery and the full expression of their bioactivities.

[0003] As a novel delivery system, food-derived exosome-like nanovesicles are attracting increasing attention. Compared to synthetic nanodelivery systems such as nanocomposites, nanoemulsions, and liposomes, exosome-like nanovesicles exhibit better safety and stability, and possess the ability to overcome the intestinal absorption barrier. Furthermore, the endogenous miRNAs, lipids, and small-molecule active metabolites within exosome-like nanovesicles can synergistically enhance health effects with the loaded exogenous molecules. Current research focuses primarily on exosome-like nanovesicles derived from plants, microorganisms, and milk, while the development and application of exosome-like nanovesicles derived from microalgae are relatively limited. Microalgae, with their rapid growth, short cultivation cycle, and scalability, are suitable as a raw material for the preparation of natural nanovesicles.

[0004] At present, the application of microalgae-derived exosome-like nanovesicles in the food industry is still very limited. The main bottlenecks are reflected in three aspects: (1) Protein, polysaccharide and pigment impurities in the microalgae system cause serious interference, making separation and purification difficult; (2) Functional component loading technology is lacking, resulting in low encapsulation efficiency; (3) Microalgae-derived nanovesicles are prone to structural damage and non-uniform particle size during separation, preparation and nutrient encapsulation, affecting delivery efficiency. Summary of the Invention

[0005] To address the aforementioned existing problems and shortcomings, this invention provides a method for preparing microalgal exosome-like nanovesicles and a method for loading functional components.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: a method for preparing microalgal exosome-like nanovesicles, comprising the following steps: S1, Microalgae culture: Spirulina, Chlamydomonas reinhardtii, Chlorella vulgaris and Euglena were selected for culture. The culture conditions were: temperature 28±5℃, relative humidity 70±5%RH, light intensity 3000±500Lux, culture for 2-3 weeks. S2, the mixture obtained from step S1 was subjected to multi-stage differential centrifugation at 4±1℃: centrifugation at 1000g for 10 min to remove intact cells and large particles; centrifugation at 2000g for 20 min to remove cell wall fragments and aggregates; centrifugation at 3500g for 30 min to remove proteoglycan aggregates; and finally centrifugation at 7000g for 30 min to remove small-scale membrane fragments and colloidal impurity particles. S3, the supernatant obtained in step S2 is filtered sequentially through filter paper and filter membrane to remove micron-sized particles; S4. The filtrate obtained in step S3 is concentrated by ultrafiltration to remove free proteins and small molecule impurities while retaining the membrane structure nanovesicles. S5. The concentrate obtained in step S4 is centrifuged at 150,000g for 1-3 hours, and the precipitate is collected and resuspended by pipetting with PBS buffer. S6, placed below -75 o Store under C conditions.

[0007] Furthermore, the supernatant described in step S3 is filtered through filter paper, and then sequentially filtered through a 0.45-micron filter membrane and a 0.22-micron filter membrane.

[0008] Furthermore, the ultrafiltration concentration in step S4 uses a filter membrane with a molecular weight of 5 kDa, and the volume of the concentrated liquid after concentration is 1 / 20 to 1 / 40 of that before concentration.

[0009] Furthermore, steps S2-S5 are repeated multiple times before step S6 to ensure the hydrodynamic diameter D of the obtained microalgal exosome-like nanovesicles. Z Between 100-200nm, PDI is less than 0.3.

[0010] Another objective of this invention is to provide a method for preparing curcumin-loaded microalgal exosome-like nanovesicles. The method involves mixing a prepared curcumin organic solution with the microalgal exosome-like nanovesicles obtained by the above preparation method. The resulting mixture is then subjected to ultrasonic treatment to generate transient nanopores in the lipid bilayer. Curcumin enters the membrane phase through these transient nanopores. Subsequently, the mixture is incubated at 37±2℃ to rearrange the membrane structure and seal the pores, thus completing the stable encapsulation.

[0011] Furthermore, the mixture was subjected to the following ultrasonic treatment conditions: ultrasonic treatment with a 650W ultrasonic instrument for 1 min, using 15s / 15s intermittent pulse ultrasonic treatment with an amplitude of 25%; followed by incubation in an ice bath for 2 min, and the ultrasonic and incubation process was repeated at least twice.

[0012] Another objective of this invention is to provide a method for preparing microalgal exosome-like nanovesicles loaded with anthocyanins. The method is characterized in that anthocyanins are mixed with microalgal exosome-like nanovesicles obtained by the above preparation method, and then subjected to freeze-thaw cycle treatment to induce the formation of ice crystals to create a transient channel in the membrane. Anthocyanins enter the internal chamber of the vesicle through the transient channel. After thawing, the membrane is reconstructed and the channel is sealed to complete the stable encapsulation.

[0013] Furthermore, the anthocyanin is an anthocyanin extract from black rice, blueberries, purple sweet potatoes, or other anthocyanin-rich plant materials, with a purity between 10% and 95%, preferably 30% to 90%.

[0014] Furthermore, the freeze-thaw cycle process is as follows: the mixed system is frozen at -80°C for 2 minutes, and then completely thawed at 25°C to form one freeze-thaw cycle; the freeze-thaw cycle is repeated 3 times.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) This invention uses gradient differential centrifugation combined with multi-stage filtration and ultrafiltration enrichment to remove fragments, aggregates, impurities of different sizes in the microalgae culture system step by step, avoiding the damage of vesicle structure to high centrifugal force, achieving the structure preservation and efficient separation of target nanovesicles, overcoming the problems of many impurities in the microalgae system and the difficulty of vesicle separation and purification, thereby improving the purity, structural integrity and particle size uniformity of microalgae exosome-like nanovesicles.

[0016] (2) This invention induces the formation of transient nanopores in the lipid bilayer by ultrasound, allowing hydrophobic functional factors to enter the membrane phase and complete the structural closure after rewarming. At the same time, it induces the formation of transient channel structures by ice crystals during freeze-thaw, allowing hydrophilic functional factors to enter the internal chamber of the vesicle and achieve membrane reconstruction and closure after thawing. This enables differentiated and efficient loading of functional factors with different polarities and improves the encapsulation stability, while protecting the vesicle structure to the greatest extent. Attached Figure Description

[0017] Figure 1 This is a flowchart of the preparation method and functional component loading method of the microalgal exosome-like nanovesicles described in this invention.

[0018] Figure 2 The figures represent the yield and purity of different algal exosome-like nanovesicles in the embodiments of the present invention. A is Spirulina platensis exosome-like nanovesicles, B is Chlamydomonas reinhardtii exosome-like nanovesicles, C is Euglena scoparia exosome-like nanovesicles, and D is Chlorella proteoglycans exosome-like nanovesicles.

[0019] Figure 3These are TEM images of different algal exosome-like nanovesicles in the embodiments of the present invention. A is an exosome-like nanovesicle of Spirulina platensis, B is an exosome-like nanovesicle of Chlamydomonas reinhardtii, C is an exosome-like nanovesicle of Euglena scoparia, and D is an exosome-like nanovesicle of Chlorella proteoglycans.

[0020] Figure 4 This is a DLS particle size distribution diagram of different algal exosome-like nanovesicles in the embodiments of the present invention. A is Spirulina platensis exosome-like nanovesicles, B is Chlamydomonas reinhardtii exosome-like nanovesicles, C is Euglena exosome-like nanovesicles, and D is Chlorella proteoglycans exosome-like nanovesicles.

[0021] Figure 5 This is an NTA particle size distribution diagram of different algal exosome-like nanovesicles in the embodiments of the present invention. A is Spirulina platensis exosome-like nanovesicles, B is Chlamydomonas reinhardtii exosome-like nanovesicles, C is Euglena spp. exosome-like nanovesicles, and D is Chlorella proteoglycans exosome-like nanovesicles.

[0022] Figure 6 This is a lipid composition diagram of different algal exosome-like nanovesicles in the embodiments of the present invention. Detailed Implementation

[0023] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims. Example

[0024] 1. Preparation of microalgal exosome-like nanovesicles Spirulina, Chlamydomonas reinhardtii, Chlorella vulgaris, and Euglena were cultured separately under the following conditions: temperature 28℃, relative humidity 70% RH, light intensity 3000 Lux, for 2-3 weeks. The culture systems of Spirulina, Chlamydomonas reinhardtii, Euglena vulgaris, or Chlorella vulgaris were then mixed thoroughly and the mixture was incubated at 4... o Differential centrifugation was performed under C conditions: centrifugation at 1000g for 10 min; centrifugation at 2000g for 20 min; centrifugation at 3500g for 30 min; centrifugation at 7000g for 30 min; the supernatant was filtered sequentially through paper, a 0.45-micron filter membrane, and a 0.22-micron filter membrane; the filtrate was then subjected to ultrafiltration concentration with a filter membrane molecular weight of 5 kDa, reducing the volume to 1 / 30 of the initial volume; The concentrate was placed in 1500,000g, 4 o Centrifuge at C for 1.5 h, collect the precipitate, resuspend it in PBS buffer (pH 7.4, 10 mM) by pipetting, and repeat the centrifugation process twice. After resuspending, incubate at -80°C. oStorage at C conditions resulted in the production of Spirulina exosome-like nanovesicles, Chlamydomonas reinhardtii exosome-like nanovesicles, Microcystis exosome-like nanovesicles, and Euglena exosome-like nanovesicles, respectively. Example 2: Preparation of microalgal exosome-like nanovesicles encapsulating curcumin Curcumin ethanol solution was mixed with Chlamydomonas reinhardtii exosome-like nanovesicles to achieve a curcumin-to-vesicle protein ratio of 1:1. The sample was then sonicated in an ice bath for 1 min (15 s / 15 s, 25% amplitude, rated maximum power 650 W), followed by incubation in an ice bath for 2 min. This process was repeated 3 times. The sample was then incubated at 37°C. o Incubate in a constant temperature incubator at C for 1 hour to promote membrane recovery. Centrifuge the above system at 5000g for 10 minutes, then transfer the supernatant to an ultrafiltration centrifuge tube for ultrafiltration to remove unencapsulated curcumin. Finally, centrifuge at 150,000g (440°C) for 1 hour. o C) Isolate the vesicles encapsulating curcumin, resuspend them, and then -80°C. o Store at C. The collected system was lysed using 1% Triton X-100 to extract curcumin, which was then quantitatively analyzed to calculate the encapsulation efficiency.

[0025] Example 3: Preparation of microalgal exosome-like nanovesicles encapsulated with anthocyanins Chlamydomonas reinhardtii exosome-like nanovesicles were resuspended in acetate buffer (pH 5.0, 10 mM), and anthocyanins were dissolved in the same buffer. The anthocyanin solution and exosome-like nanovesicles were mixed at a 1:1 mass ratio and incubated at 25°C in the dark for 20 min. The mixture was then frozen at -80°C for 2 min, followed by complete thawing at 25°C, forming one freeze-thaw cycle. This freeze-thaw cycle was repeated three times. After the freeze-thaw cycle, the mixture was allowed to stand at 25°C for 30 min to promote membrane structure recovery. Aggregates were removed by centrifugation at 5000g for 10 min. Free anthocyanins were removed by centrifugation at 12000g using a 5 kDa ultrafiltration tube, followed by adding an equal volume of acetate buffer to make up the volume. Ultrafiltration washing was repeated three times. The encapsulated product was collected by centrifugation at 150,000g at 4°C for 60 min, resuspended in the same buffer, and then frozen at -80°C. o Store at C. The collected system was lysed using 1% Triton X-100 to extract anthocyanins, which were then quantitatively analyzed to calculate the encapsulation efficiency.

[0026] The core innovation of this invention lies in: 1. This invention constructs a graded purification system that combines gradient differential centrifugation with multi-stage filtration and ultrafiltration enrichment to achieve the stepwise removal of fragments, aggregates, and impurities of different sizes in the microalgae culture system. This achieves efficient preparation of microalgae exosome-like nanovesicles while avoiding damage to the nanovesicle structure caused by high centrifugal force.

[0027] 2. This invention utilizes ultrasound to induce transiently reversible nanopores in a lipid bilayer membrane, which temporarily enhances the local permeability of the membrane. This allows hydrophobic functional factors to enter the lipid bilayer under the drive of hydrophobic interactions, and the pores are closed through spontaneous rearrangement of the membrane lipids during the rewarming process, thereby achieving efficient and stable encapsulation of hydrophobic functional factors.

[0028] 3. This invention utilizes the phase transformation stress caused by ice crystal formation and melting during freeze-thaw cycles to induce instantaneous structural reconstruction of the nanovesicle membrane and form a controllable channel structure. This allows hydrophilic functional factors to enter the aqueous chamber inside the vesicle, and the channels are closed through membrane structure rearrangement after thawing, thereby achieving stable encapsulation of hydrophilic functional factors.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing microalgal exosome-like nanovesicles, characterized in that, Includes the following steps: S1, Microalgae culture: Spirulina, Chlamydomonas reinhardtii, Chlorella vulgaris and Euglena were selected for culture. The culture conditions were: temperature 28±5℃, relative humidity 70±5%RH, light intensity 3000±500Lux, culture for 2-3 weeks. S2, the mixture obtained from step S1 was subjected to multi-stage differential centrifugation at 4±1℃: centrifugation at 1000g for 10 min to remove intact cells and large particles; centrifugation at 2000g for 20 min to remove cell wall fragments and aggregates; centrifugation at 3500g for 30 min to remove proteoglycan aggregates; and finally centrifugation at 7000g for 30 min to remove small-scale membrane fragments and colloidal impurity particles. S3, the supernatant obtained in step S2 is filtered sequentially through filter paper and filter membrane to remove micron-sized particles; S4. The filtrate obtained in step S3 is concentrated by ultrafiltration to remove free proteins and small molecule impurities while retaining the membrane structure nanovesicles. S5. The concentrate obtained in step S4 is centrifuged at 150,000g for 1-3 hours, and the precipitate is collected and resuspended by pipetting with PBS buffer. S6, placed below -75 o Store under C conditions.

2. The method for preparing microalgal exosome-like nanovesicles according to claim 1, characterized in that, The supernatant obtained in step S3 is filtered through filter paper, and then sequentially filtered through a 0.45-micron filter membrane and a 0.22-micron filter membrane.

3. The method for preparing microalgal exosome-like nanovesicles according to claim 1, characterized in that, The ultrafiltration concentration in step S4 uses a filter membrane with a molecular weight of 5 kDa, and the volume of the concentrated liquid after concentration is 1 / 20 to 1 / 40 of that before concentration.

4. The method for preparing microalgal exosome-like nanovesicles according to claim 1, characterized in that, Repeat steps S2-S5 multiple times before step S6 to ensure the hydrodynamic diameter D of the obtained microalgal exosome-like nanovesicles. Z Between 100-200nm, PDI is less than 0.

3.

5. A method for preparing microalgal exosome-like nanovesicles loaded with curcumin, characterized in that, The prepared curcumin organic solution was mixed with microalgal exosome-like nanovesicles obtained by any of the preparation methods in claims 1-4. The resulting mixture was ultrasonically treated to generate transient nanopores in the lipid bilayer. Curcumin entered the membrane phase through the transient nanopores. Subsequently, the membrane structure was rearranged and the pores were sealed by incubation at 37±2℃, thus completing the stable encapsulation.

6. The method for preparing curcumin-loaded microalgal exosome-like nanovesicles according to claim 5, characterized in that, The mixture was subjected to the following ultrasonic treatment conditions: ultrasonic treatment with a 650W ultrasonic instrument for 1 min, using 15s / 15s intermittent pulse ultrasonic treatment with an amplitude of 25%; followed by incubation in an ice bath for 2 min, and the ultrasonic and incubation process was repeated at least twice.

7. A method for preparing anthocyanin-loaded microalgal exosome-like nanovesicles, characterized in that, After mixing anthocyanins with microalgal exosome-like nanovesicles obtained by any of the preparation methods in claims 1-4, the mixture is subjected to freeze-thaw cycles to induce the formation of ice crystals and create transient channels in the membrane. Anthocyanins then enter the internal chamber of the vesicle through these transient channels. After thawing, the membrane is reconstructed and the channels are sealed, thus completing the stable encapsulation.

8. The method for preparing curcumin-loaded microalgal exosome-like nanovesicles according to claim 7, characterized in that, The anthocyanins are anthocyanin extracts from black rice, blueberries, purple sweet potatoes, or other anthocyanin-rich plant materials, with a purity between 10% and 95%, preferably between 30% and 90%.

9. The method for preparing curcumin-loaded microalgal exosome-like nanovesicles according to claim 7, characterized in that, The freeze-thaw cycle process is as follows: the mixture is frozen at -80°C for 2 minutes, then completely thawed at 25°C, forming one freeze-thaw cycle; the freeze-thaw cycle is repeated 3 times.

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