Method for producing fermented extracellular vesicles comprising sterilization step

Abstract

The present invention relates to a method for producing fermented extracellular vesicles, the method comprising a sterilization step. The method for producing fermented extracellular vesicles, according to the present invention, comprises: a fermentation step of obtaining a fermented product using microorganisms; and a sterilization step of separating a first material containing extracellular vesicles and impurities from a biological solution obtained by sterilizing the fermented product to thereby obtain the first material. The present invention can provide a method for producing fermented extracellular vesicles, which can efficiently and economically separate, from a biological solution obtained by sterilizing a microbial fermentation product, impurities such as microorganisms, bacteria, cell debris, wastes, proteins, and large particles, extracellular vesicles, and / or exosomes.

Description

Method for producing fermented extracellular vesicles including a sterilization process

[0001] The present invention relates to a method for producing fermented extracellular vesicles including a sterilization process.

[0002] Extracellular vesicles (EVs) are nano-sized vesicles enclosed by a lipid bilayer that all cells secrete into the external environment. EVs possess a wide range of biological functions and are involved in various physiological and pathological processes. EVs are formed and secreted by cells, and they function to transmit signals and regulate cells within and between neighboring cells.

[0003] Extracellular vesicles (EVs) are a general term for vesicles secreted outside of cells, not only from plants but also from animals, including bacteria. Depending on their origin, secretion mechanism, and size, EVs have been called by various names, including exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, shedding vesicles, nanovesicles, and outer membrane vesicles.

[0004] Exosomes are a type of extracellular vesicle (EV) and are nanomaterials measuring 50 to 200 nm in size. With the discovery of various types of vesicles secreted from cells, exosomes can be considered part of the broader concept of extracellular vesicles (EVs).

[0005] Exosomes were first discovered in 1983. Initially, their biological functions were unknown, and they were initially considered "garbage bags" released by cells. However, exosomes have been found to contain a variety of ligands, cell-derived proteins, growth factors, and nucleic acids, and play a crucial role in intercellular communication. Because they contain a wealth of biological information released by cells, they are being used in disease diagnosis and the discovery of new biomarkers. Furthermore, they are being developed as therapeutics utilizing effective substances that influence cellular signaling, and as drug delivery systems utilizing their double-lipid membrane structure for drug detection.

[0006] All living organisms, composed of cells, constantly exchange signals with one another. These intercellular signals are facilitated by exosomes, which act as signaling mediators, transmitting long-distance intercellular signals through nearby cells or through intracellular circulation. Exosomes exist not only in eukaryotic animals and plants, but also in prokaryotes such as microorganisms and algae. Exosomes are intercellular communication molecules secreted from all cells, from microorganisms to humans.

[0007] Exosomes are composed of a phospholipid bilayer. By enclosing their contents with a phospholipid bilayer, exosomes can protect their contents from external degradants and transport hydrophobic substances. One example is the RNA transport mechanism, where RNA within exosomes is protected from degradants and can be smoothly delivered to target cells. Another example is membrane protein transport. Hydrophobic membrane proteins within the phospholipid bilayer have difficulty being transported between cells through the bloodstream. Exosomes, composed of a phospholipid bilayer, can transport membrane proteins.

[0008] There are many different methods for isolating exosomes or extracellular vesicles, including precipitation using polyethylene glycol (PEG), differential ultracentrifugation (UC), ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography (SEC), gradient UC or density gradient ultracentrifugation, affinity isolation, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, total exosome isolation kit, and polymer-based precipitation.

[0009] Korean Patent Publication No. 10-2015-0145720 relates to a method for isolating adipose-derived stem cells and exosomes from adipose tissue. The method comprises adding collagenase to the adipose tissue layer and centrifuging the process. Specifically, the adipose tissue layer is a connective tissue containing a significant number of blood vessels. However, adipose-derived stem cells and exosomes are distributed around blood vessels, so the connecting portions between the adipose cells and blood vessels must be disrupted to effectively harvest adipose-derived stem cells. The tissue at the connecting portion is not as rigid as fat, but is primarily composed of collagen type II, a type II collagenase secreted in large quantities by cartilage cells. This type II collagenase is fluid and easily reassembled or separated, making it possible to increase the yield of stem cells. Therefore, by disrupting the perivascular tissue, where a large number of adipose-derived stem cells are distributed, collagenase type II, a collagen type II-degrading enzyme, can be used to increase the yield of stem cells.

[0010] Korean Patent Publication No. 10-2016-0116802 relates to a method for separating extracellular vesicles using an aqueous two-phase system, and is characterized by the fact that the separation of extracellular vesicles is accomplished in a short time of 10 to 20 minutes without the need for ultracentrifugation by using an aqueous two-phase system. According to this document, ultracentrifugation is the most widely used method for separating extracellular vesicles, and is the most reliable separation method due to its simple principle, but it has the disadvantages of low yield, long separation time, and expensive equipment. However, since it can effectively separate different types of particles in a short time, it is said that the aqueous two-phase system, which has been widely used for particle separation, can be applied to the separation of extracellular vesicles, enabling high yield, selectivity, and rapid separation of extracellular vesicles in a short time.

[0011] Korean Patent Publication No. 10-2125567 relates to a method for mass production of plant exosomes, and is characterized by a method for mass production of plant exosomes using ultracentrifugation and a circular filtration method, replacing the conventional density gradient centrifugation method, which is limited to small-scale production of plant exosomes, to extract high-purity plant exosomes.

[0012] Generally, proteins denature at about 60 to 70°C, and denatured proteins exhibit coagulation and precipitation. The inventor of the present invention confirmed that extracellular vesicles are not denatured even under sterile conditions, and thought that if extracellular vesicles have excellent thermal stability even under sterile conditions, it would be possible to efficiently and economically separate extracellular vesicles from impurities such as microorganisms, bacteria, cell debris, waste products, proteins, and large particles in a biological solution obtained by sterilizing a microbial fermentation product, and completed the present invention by confirming this through experiments.

[0013] The present invention aims to provide a method for producing fermented extracellular vesicles by sterilizing a fermented product obtained through fermentation.

[0014] The present invention seeks to provide a composition comprising fermented extracellular vesicles.

[0015] The present invention seeks to provide a pharmaceutical composition comprising fermented extracellular vesicles.

[0016] The present invention aims to provide a cosmetic composition comprising fermented extracellular vesicles.

[0017] The present invention seeks to provide a food composition comprising fermented extracellular vesicles.

[0018] In addition, the present invention aims to provide a method for producing fermented exosomes by sterilizing a fermented product obtained through fermentation.

[0019] The present invention seeks to provide a composition comprising fermented exosomes.

[0020] The present invention seeks to provide a pharmaceutical composition comprising fermented exosomes.

[0021] The present invention seeks to provide a cosmetic composition comprising fermented exosomes.

[0022] The present invention seeks to provide a food composition comprising fermented exosomes.

[0023]

[0024] The tasks of the invention are not limited to the technical tasks mentioned above, and other technical tasks not mentioned will be clearly understood by those skilled in the art from the description below.

[0025] The method for producing fermented extracellular vesicles according to the present invention includes a sterilization process for sterilizing a fermented product obtained through fermentation. The method for producing fermented extracellular vesicles according to the present invention includes a fermentation process for obtaining a fermented product using a microorganism, and a sterilization process for separating a first substance containing extracellular vesicles and impurities from a biological solution obtained by sterilizing the fermented product, and obtaining the first substance. In one example, the extracellular vesicles may be exosomes.

[0026] The manufacturing method according to the present invention may further include a centrifugation process. In this case, the centrifugation process is added after the sterilization process. The centrifugation process uses centrifugal force to separate the second material containing extracellular vesicles and impurities from the first material, thereby obtaining the second material.

[0027] The manufacturing method according to the present invention may further include an ultrafiltration process. In this case, the ultrafiltration process is added after the centrifugation process. The ultrafiltration process uses an ultrafiltration membrane to separate a third substance containing extracellular vesicles and impurities from the second substance, thereby obtaining the third substance.

[0028] The above sterilization process can sterilize the fermented product using an autoclave.

[0029] The above sterilization process may have a temperature of 121°C or higher.

[0030] The above sterilization process may be performed at a pressure of 1 bar or more.

[0031] The above sterilization process may sterilize the fermented product for 10 minutes or more.

[0032] In one example, the sterilization process may be performed at a temperature of 121°C or higher and a pressure of 1 bar or higher. In another example, the sterilization process may be performed by sterilizing the fermented product at a temperature of 121°C or higher and a pressure of 1 bar or higher for 10 minutes or longer.

[0033] The centrifugal separation process may have a rotation speed of 6,000 to 20,000 rpm. In one example, the centrifugal separation process may have a bowl rotation speed of 6,000 to 20,000 rpm.

[0034] The above ultrafiltration process may use an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 10,000 Daltons (Da). In one example, the ultrafiltration process may pass the second material through an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 10,000 Daltons (Da).

[0035] In the above fermentation process, the microorganism may be one or more selected from the group consisting of yeast, Bacillus subtilis, lactic acid bacteria, and Aspergillus oryzae. In other words, the fermented product may be obtained by fermentation of one or more microorganisms selected from the group consisting of yeast, Bacillus subtilis, lactic acid bacteria, and Aspergillus oryzae.

[0036] The yeast may be one or more selected from the group consisting of, but is not limited to, Aureobasidium pullulans, Galactomyces Candidum, Pichia Cifferrii, Rhodosporidium Toruloides, Saccharomyces boulardii, Saccharomyces Cerevisiae, and Schizosaccharomyces Ellipsoids.

[0037] The lactic acid bacteria include Bifidobacterium Bifidum, Bifidobacterium Breve, Bifidobacterium Lactis, Bifidobacterium Longum, Enterococcus Faecium, Enterococcus Faecalis, Lactobacillus Acidophilus, Lactobacillus Brevis, Lactobacillus Bulgaricus, Lactobacillus Casei, Lactobacillus Fermentum, Lactobacillus Gacceri, Lactobacillus Helceticus, Lactobacillus Kunkeei, Lactobacillus ParacaseiLactobacillus Plantarum, Lactobacillus Reuteri, Lactobacillus Rhamnosus, Lactobacillus Salivarius, Lactococcus Lactis, Leuconostoc Citreum, Leuconostoc Mesenteroides, Pediococcus acidilactici, Pediococcus Pentosaceus and Streptococcus Can be one or more selected from the group consisting of Thermophilus However, it is not limited to these.

[0038] Examples of the above-mentioned bacteria include, but are not limited to, Bacillus subtilis.

[0039] The above yeast may be one or more selected from the group consisting of Aspergillus Oryzae, Rhizopus Javanicus, and Monascus Anka, but is not limited thereto.

[0040] The composition according to the present invention comprises the fermented extracellular vesicles. In one example, the composition according to the present invention may comprise the fermented extracellular vesicles as an active ingredient.

[0041] The above-mentioned content may be a pharmaceutical composition comprising the fermented extracellular vesicles. In one example, the pharmaceutical composition may comprise the fermented extracellular vesicles as an active ingredient.

[0042] The above-mentioned content may be a cosmetic composition comprising the fermented extracellular vesicles. In one example, the cosmetic composition may comprise the fermented extracellular vesicles as an active ingredient. The cosmetic composition may have any one formulation selected from the group consisting of skin products, lotions, essences, creams, packs, foundations, and makeup bases.

[0043] The above-mentioned content may be a food composition comprising the fermented extracellular vesicles. In one example, the food composition may be a health functional food composition comprising the fermented extracellular vesicles as an active ingredient.

[0044] The present invention can provide a method for producing fermented extracellular vesicles, which can efficiently and economically separate impurities such as microorganisms, bacteria, cell debris, waste, proteins, and large particles, and extracellular vesicles and / or exosomes from a biological solution obtained by sterilizing a fermented microbial product.

[0045] The present invention can provide a composition comprising fermented extracellular vesicles and / or exosomes.

[0046] The present invention can provide a pharmaceutical composition comprising fermented extracellular vesicles and / or exosomes. The present invention can provide a medicine comprising the pharmaceutical composition.

[0047] The present invention can provide a cosmetic composition comprising fermented extracellular vesicles and / or exosomes. The present invention can provide a cosmetic comprising the cosmetic composition.

[0048] The present invention can provide a food composition comprising fermented extracellular vesicles and / or exosomes. The present invention can provide a food or health functional food comprising the food composition.

[0049]

[0050] The effects according to the invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0051] Figure 1 shows the results of NTA (Nanoparticle Tracking Analysis) analysis of the Lactobacillus fermented exosome obtained in Example 1.

[0052] Figure 2 is a TEM (Transmission Electron Microscope) photograph of the Lactobacillus fermented exosome obtained in Example 1.

[0053] Figure 3 shows the results of NTA (Nanoparticle Tracking Analysis) analysis of the Galactomyces fermented exosome obtained in Example 2.

[0054] Figure 4 is a TEM (Transmission Electron Microscope) photograph of the Galactomyces fermented exosome obtained in Example 2.

[0055] The invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and the embodiments are provided only to ensure that the disclosure of the invention is complete and to fully inform a person skilled in the art of the invention of the scope of the invention, and the invention is defined only by the scope of the claims.

[0056] Although terms like "first" and "second" are used to describe various components, these components are not limited by these terms. These terms are merely used to distinguish one component from another. Therefore, it should be understood that a "first" component referred to below may also be a "second" component within the technical scope of the present invention.

[0057] In this specification, A to B are defined as being greater than or equal to A and less than or equal to B.

[0058] In this specification, A~B are defined as A to B.

[0059] In this specification, “A and / or B”, “at least one of A and B” are all concepts including A or B or a combination of A and B.

[0060]

[0061] The fermented extracellular vesicles and / or exosomes according to the present invention are efficiently and economically obtained by sterilizing the fermented product. Proteins generally denature at about 60 to 70°C, and the denatured proteins exhibit coagulation and precipitation. According to the inventors of the present invention, in a biological solution obtained by sterilizing a fermented microbial product, impurities such as microorganisms, bacteria, cell debris, waste products, proteins, and large particles denature and coagulate and / or precipitate, whereas the extracellular vesicles and / or exosomes have excellent thermal stability that does not denature even under sterilization conditions, and thus the first substance including the extracellular vesicles and / or exosomes was separated from the impurities and the like in the supernatant. In other words, the first substance including the extracellular vesicles and / or exosomes was separated from the denatured protein impurities and the like that coagulated and precipitated in the supernatant.

[0062] Extracellular vesicles (EVs) have a phospholipid membrane structure similar to the cell membrane, allowing them to pass through biological tissues and walls, enabling the active ingredients contained within them to be delivered to any tissue within the body. Furthermore, EVEs can be loaded with large proteins and various types of RNA using cell engineering and genetic engineering methods. Smaller chemicals can also be loaded into EVEs to deliver drugs to specific tissues and sites. EVEs themselves are derived from cells and have low toxicity.

[0063] Exosomes are recognized as a new concept of therapeutics as cell-free therapeutics that overcome stability issues of cell therapy, such as immune rejection, carcinogenesis, and vascular occlusion, while maintaining the functional role of existing cell therapy, and are advantageous for storage, transportation, and management because they are not living cells.

[0064] There are currently no commercially available extracellular vesicle and / or exosome therapeutics available domestically or internationally, due to technical limitations in quality control and mass production. Taking exosomes as an example, developing highly heterogeneous exosomes as therapeutics presents significant challenges. Therapeutics require consistency and comparability across batches, and maintaining consistency and comparability in highly heterogeneous exosomes is a significant challenge for therapeutic development.

[0065] Another challenge is the ability to mass-produce exosomes in sufficient quantities to be used in many patients. Various isolation methods are being used to obtain highly pure exosomes. However, despite their high purity, the small amount of exosomes obtained hinders their entry into preclinical and clinical trials. Ultracentrifugation, the most widely used method for exosome isolation, has low yields, is time-consuming and labor-intensive, requires expensive equipment, and can damage exosomes during the isolation process. Ultrafiltration, used in conjunction with ultracentrifugation, can increase exosome purity, but exosomes adhere to the filter, resulting in low yields after isolation. Furthermore, immunoaffinity isolation, which separates exosomes by attaching antibodies to them, offers the advantage of high specificity. However, it requires antibody preparation and post-isolation removal, is expensive, and is not suitable for scale-up.

[0066] Recently, various exosome isolation kits such as exosome precipitation, total exosome isolation kit, or polymer-based precipitation have been commercially sold as a method for isolating exosomes. However, although they are easy to use, the reagents are expensive, so although they can be used to isolate exosomes at the laboratory level, they are not suitable for isolating and purifying exosomes in large quantities.

[0067] Above all, the isolation process for extracellular vesicles and / or exosomes presents challenges due to the presence of impurities, such as proteins, leading to low yields. Furthermore, the isolation and purification of extracellular vesicles and / or exosomes is time-consuming, cumbersome, and expensive. Furthermore, existing separation methods developed to increase purity present challenges in scale-up and are not suitable for Good Manufacturing Practice (GMP).

[0068] The manufacturing method according to the present invention is a technology that can economically and efficiently isolate and purify extracellular vesicles and / or exosomes, while also being scale-up-capable and suitable for Good Manufacturing Practice (GMP). In other words, the manufacturing method according to the present invention is a novel technology that economically and efficiently isolates and purifies extracellular vesicles and / or exosomes with high purity and a uniform particle size distribution.

[0069] Specifically, the method for producing fermented extracellular vesicles according to the present invention includes a sterilization process for sterilizing the fermented product obtained through fermentation. Fermentation refers to the process of decomposing organic matter using enzymes secreted by microorganisms. Furthermore, fermentation also involves the production of useful substances by microorganisms during the process of obtaining energy.

[0070] The manufacturing method according to the present invention comprises a fermentation process for obtaining a fermented product using microorganisms, and a sterilization process for separating a first substance containing extracellular vesicles and impurities from a biological solution obtained by sterilizing the fermented product, and obtaining the first substance. In one example, the extracellular vesicles may be exosomes.

[0071] The manufacturing method according to the present invention may further include a centrifugation process. In this case, the centrifugation process is added after the sterilization process. The centrifugation process uses centrifugal force to separate the second material containing extracellular vesicles and impurities from the first material, thereby obtaining the second material.

[0072] The manufacturing method according to the present invention may further include an ultrafiltration process. In this case, the ultrafiltration process is added after the centrifugation process. The ultrafiltration process uses an ultrafiltration membrane to separate a third substance containing extracellular vesicles and impurities from the second substance, thereby obtaining the third substance.

[0073] The above sterilization process can sterilize the fermented product using an autoclave.

[0074] The above sterilization process may have a temperature of 121°C or higher.

[0075] The above sterilization process may be performed at a pressure of 1 bar or more.

[0076] The above sterilization process may sterilize the fermented product for 10 minutes or more.

[0077] In one example, the sterilization process may be performed at a temperature of 121°C or higher and a pressure of 1 bar or higher. In another example, the sterilization process may be performed by sterilizing the fermented product at a temperature of 121°C or higher and a pressure of 1 bar or higher for 10 minutes or longer.

[0078] The centrifugal separation process may have a rotation speed of 6,000 to 20,000 rpm. In one example, the centrifugal separation process may have a bowl rotation speed of 6,000 to 20,000 rpm.

[0079] The above ultrafiltration process may use an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 10,000 Daltons (Da). In one example, the ultrafiltration process may pass the second material through an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 10,000 Daltons (Da).

[0080] In the above fermentation process, the microorganism may be one or more selected from the group consisting of yeast, Bacillus subtilis, lactic acid bacteria, and Aspergillus oryzae. In other words, the fermented product may be obtained by fermentation of one or more microorganisms selected from the group consisting of yeast, Bacillus subtilis, lactic acid bacteria, and Aspergillus oryzae.

[0081] The yeast may be one or more selected from the group consisting of, but is not limited to, Aureobasidium pullulans, Galactomyces Candidum, Pichia Cifferrii, Rhodosporidium Toruloides, Saccharomyces boulardii, Saccharomyces Cerevisiae, and Schizosaccharomyces Ellipsoids.

[0082] The lactic acid bacteria include Bifidobacterium Bifidum, Bifidobacterium Breve, Bifidobacterium Lactis, Bifidobacterium Longum, Enterococcus Faecium, Enterococcus Faecalis, Lactobacillus Acidophilus, Lactobacillus Brevis, Lactobacillus Bulgaricus, Lactobacillus Casei, Lactobacillus Fermentum, Lactobacillus Gacceri, Lactobacillus Helceticus, Lactobacillus Kunkeei, Lactobacillus ParacaseiLactobacillus Plantarum, Lactobacillus Reuteri, Lactobacillus Rhamnosus, Lactobacillus Salivarius, Lactococcus Lactis, Leuconostoc Citreum, Leuconostoc Mesenteroides, Pediococcus acidilactici, Pediococcus Pentosaceus and Streptococcus Can be one or more selected from the group consisting of Thermophilus However, it is not limited to these.

[0083] Examples of the above-mentioned bacteria include, but are not limited to, Bacillus subtilis.

[0084] The above yeast may be one or more selected from the group consisting of Aspergillus Oryzae, Rhizopus Javanicus, and Monascus Anka, but is not limited thereto.

[0085] The composition according to the present invention comprises fermented extracellular vesicles and / or exosomes. In one example, the composition according to the present invention may comprise the fermented extracellular vesicles and / or exosomes, and may be, as non-limiting examples, a pharmaceutical composition, a food composition, a cosmetic composition, etc. In one example, the composition according to the present invention may comprise the fermented extracellular vesicles and / or exosomes as an active ingredient, and may be, as non-limiting examples, a pharmaceutical composition, a food composition, a cosmetic composition, etc.

[0086] When the above-mentioned content is developed into a pharmaceutical composition, the present invention can provide a pharmaceutical product; when the above-mentioned content is developed into a food composition, the present invention can provide a food or health functional food; and when the above-mentioned content is developed into a cosmetic composition, the present invention can provide a cosmetic product.

[0087] In the case of the above cosmetic composition, it may have any one formulation selected from the group consisting of skin products, lotions, essences, creams, packs, foundations, and makeup bases.

[0088]

[0089] The present invention is described in more detail in the following examples, but the present invention is not limited thereto.

[0090]

[0091] Example 1: Preparation and Evaluation of Lactobacillus Fermented Exosomes

[0092] After inoculating the culture medium with Lactobacillus, the fermentation was performed in a fermenter at 35 to 40°C for 24 to 48 hours to produce a fermented product. To complete the fermentation, the product was sterilized by heating using an autoclave at 121°C and 1 bar of pressure for 20 minutes, and the supernatant was obtained. The supernatant was centrifuged at 6,000 to 20,000 rpm for 20 to 30 minutes, and the obtained supernatant was purified using an ultrafiltration membrane with a molecular weight cutoff of 100,000 to 10,000 Daltons (Da), to produce fermented exosomes.

[0093]

[0094] Example 2: Preparation and Evaluation of Galactomyces Fermented Exosomes

[0095] After inoculating the culture medium with Galactomyces, the fermentation was performed in a fermenter at 28–32°C for 24–48 hours to produce a fermented product. To complete the fermentation, the fermentation was sterilized by heating using an autoclave at 121°C and 1 bar of pressure for 20 minutes, and the supernatant was obtained. The supernatant was centrifuged at 6,000–20,000 rpm for 20–30 minutes, and the obtained supernatant was purified using an ultrafiltration membrane with a molecular weight cutoff of 100,000–10,000 Daltons (Da), to produce fermented exosomes.

[0096]

[0097] [Experimental Example 1] Evaluation of Lactobacillus-fermented exosomes

[0098] The obtained Lactobacillus fermentation exosomes were evaluated using Nano Tracking Analyzer (NTA) and Transmission electron microscopy (TEM). Figure 1 shows the results of NTA (Nanoparticle Tracking Analysis) analysis of the Lactobacillus fermentation exosomes obtained in Example 1, and Figure 2 shows a TEM (Transmission Electron Microscope) image of the Lactobacillus fermentation exosomes obtained in Example 1. The Lactobacillus fermentation exosomes maintained their spherical shape even after sterilization, and their sizes were measured to be around 200 nm on average.

[0099]

[0100] [Experimental Example 2] Evaluation of Galactomyces-fermented exosomes

[0101] The obtained Galactomyces fermentation exosomes were evaluated using Nano Tracking Analyzer (NTA) and Transmission electron microscopy (TEM). Figure 3 shows the results of NTA (Nanoparticle Tracking Analysis) analysis of the Galactomyces fermentation exosomes obtained in Example 2, and Figure 4 shows a TEM (Transmission Electron Microscope) image of the Galactomyces fermentation exosomes obtained in Example 2. The Galactomyces fermentation exosomes maintained their spherical shape even after sterilization, and their sizes were measured to be around 150 nm on average.

[0102]

[0103] Although embodiments of the present invention have been described with reference to the attached drawings, the present invention is not limited to the above embodiments, but can be manufactured in various different forms. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

[0104] The mode for carrying out the invention has been described together with the best mode for carrying out the invention above.

[0105] The present invention has industrial applicability because it can efficiently and economically separate impurities such as microorganisms, bacteria, cell debris, waste, proteins, and large particles, as well as extracellular vesicles and / or exosomes from a biological solution obtained by sterilizing a microbial fermentation product.

Claims

1. A fermentation process for obtaining a fermented product using at least one microorganism among yeast and lactic acid bacteria; A sterilization process for separating the supernatant containing exosomes and impurities from the biological solution obtained by sterilizing the above fermented product and obtaining the supernatant; A centrifugation process for separating the supernatant containing exosomes and impurities from the supernatant using centrifugal force and obtaining the supernatant; and A method for producing fermented exosomes, comprising an ultrafiltration process for purifying the supernatant using an ultrafiltration membrane and obtaining fermented exosomes.

2. In paragraph 1, A method for producing fermented exosomes, characterized in that the above sterilization process is performed at a temperature of 121°C or higher, a pressure of 1 bar or higher, and for sterilization for 10 minutes or longer.

3. In paragraph 1, A method for producing fermented exosomes, characterized in that the centrifugation process above has a rotation speed of the bowl of 6,000 to 20,000 rpm.

4. In paragraph 1, A method for producing fermented exosomes, characterized in that the above ultrafiltration process passes the supernatant through an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 10,000 daltons (Da).

5. In paragraph 1, A method for producing a fermented exosome, characterized in that the yeast is at least one selected from the group consisting of Aureobasidium pullulans, Galactomyces Candidum, Pichia Cifferrii, Rhodosporidium Toruloides, Saccharomyces boulardii, Saccharomyces Cerevisiae, and Schizosaccharomyces Ellipsoids.

6. In paragraph 1, A method for producing fermented exosomes, characterized in that the above lactic acid bacteria are one or more selected from the group consisting of Bifidobacterium Bifidum, Bifidobacterium Breve, Bifidobacterium Lactis, Bifidobacterium Longum, Enterococcus Faecium, Enterococcus Faecalis, Lactobacillus Acidophilus, Lactobacillus Brevis, Lactobacillus Bulgaricus, Lactobacillus Casei, Lactobacillus Fermentum, Lactobacillus Gacceri, Lactobacillus Helceticus, Lactobacillus Kunkeei, Lactobacillus Paracasei, Lactobacillus Plantarum, Lactobacillus Reuteri, Lactobacillus Rhamnosus, Lactobacillus Salivarius, Lactococcus Lactis, Leuconostoc Citreum, Leuconostoc Mesenteroides, Pediococcus acidilactici, Pediococcus Pentosaceus, and Streptococcus Thermophilus.

Images