Extraction method and application of oenococcus oeni extracellular vesicles
By performing multi-step centrifugation and filtration treatment on the fermentation broth of fermented fermentation broth, combined with acid or ethanol stress culture, the purity and yield of the extracellular vesicles of fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented ferment
Patent Information
- Application Number
- CN202510191197.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-05-16
AI Technical Summary
The prior art lacks effective methods for extracting and purifying extracellular vesicles of ycocci, and fails to fully utilize these vesicles to improve the tolerance of the bacteria to acid and ethanol stress.
The fermentation broth of jujubecaca was centrifuged twice, followed by filtration of impurities using tangential flow ultrafiltration membrane pack and filter membrane, and then purified by ultracentrifugation to obtain high-purity extracellular vesicles of jujubecacacaca. At the same time, the extraction yield of extracellular vesicles is increased by acid or ethanol stress culture.
The efficient extraction and purification of extracellular vesicles of jujubecocci was achieved, and the tolerance of bacteria to acid and ethanol stress was improved. The method was simple, efficient and low-cost, and it would not destroy the structure of extracellular vesicles.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological separation technology, and in particular to an extraction method and application of extracellular vesicles of Oenococcus oeni. Background Art
[0002] In the initial stage of winemaking, the sugars in the grapes are fermented into alcohol by one or more yeast strains (especially Saccharomyces cerevisiae). After the alcoholic fermentation is completed, malolactic fermentation (MLF) occurs, although this process sometimes occurs earlier. Malic acid is one of the main organic acids in grapes, with a content of about 3-5g / L. The presence of malic acid in wine will significantly affect the acidity, pH and taste of the wine. At the same time, malic acid is also one of the nutrient sources for several spoilage bacteria. Malolactic fermentation is a key process to improve the sensory quality of wine. This process is a process in which winemaking lactic acid bacteria (mostly Oenococcus oeni) use malic acid-lactic enzymes to convert L-malic acid, which has a strong sour and astringent feeling in the wine, into L-lactic acid with a softer taste through decarboxylation reaction, while releasing CO2. This process can not only improve the flavor of the wine, including taste and aroma, but also prevent some spoilage yeasts from using L-malic acid to reproduce and grow, thereby improving the stability of the wine. In addition, MLF also contributes to the production of other metabolites, such as ethyl lactate, 2,3-butanediol, acrolein, diacetyl, etc., enriching terpenes, alcohols, polyphenols and other substances in wine.
[0003] Oenococcus oeni (O.oeni) is the most commonly used malic acid-lactic acid fermentation bacteria and plays an important role in the wine production process. First, Oenococcus oeni can produce bacteriocins to inhibit the growth of foreign bacteria and improve the microbial stability of the wine. Second, Oenococcus oeni can improve the acidity of wine. The bacteria decompose the dibasic acid malic acid to produce the monobasic acid lactic acid to increase the pH of the wine. Wine environmental stress factors such as ethanol, low pH, SO2 and low temperature are the main factors affecting the MLF of Oenococcus oeni. At present, many studies have focused on the stress response mechanism of Oenococcus oeni, such as bacterial integrity, intracellular pH stability, nucleic acid and protein repair, stress proteins, and exopolysaccharide synthesis (EPS). Other studies have shown that when Oenococcus oeni and planktonic Oenococcus oeni with biofilm characteristics were inoculated in fermented grape juice, the results showed that the biofilm could significantly improve the tolerance of the bacteria to wine stress and the rate of malic acid-lactic acid fermentation. Therefore, exploring the production mechanism of Oenococcus oeni biofilm is helpful to the study of the stress tolerance mechanism of Oenococcus oeni.
[0004] Extracellular vesicles (EVs) are nanoscale vesicles with a lipid bilayer structure that bacteria secrete from the cell membrane to the extracellular space during their growth. EVs are a secretion and delivery system of bacteria and are indispensable for bacterial survival. EVs can enhance bacterial defense and resistance through quorum sensing and the removal of harmful substances; EVs can serve as nucleation sites for extracellular DNA or promote biofilm formation by providing beneficial proteins; EVs can also serve as an effective platform to facilitate the interaction between bacteria, proteins, nucleic acids and extracellular polysaccharides. In general, extracellular vesicles are complex and functionally rich nanoparticles that play a key role in many fields such as intercellular communication, immune regulation, inflammatory response, cell migration and tumor biology, and are closely related to the function and state of their source cells. They can affect the function of target cells, deliver bioactive molecules, and regulate intercellular signal transduction. At present, there is a lack of methods for obtaining EVs from brewing lactic acid bacteria, and there are no reports on the use of stress regulation treatment to increase the production of EVs in malic acid-lactic acid fermentation bacteria.
[0005] In view of this, the present invention is proposed. Summary of the invention
[0006] The first objective of the present invention is to provide a method for extracting extracellular vesicles of Oenococcus oeni to solve the above-mentioned technical problems.
[0007] The second object of the present invention is to provide an application of the extracellular vesicles of Oenococcus oeni extracted by the above extraction method in improving the acid and ethanol stress resistance of Oenococcus oeni.
[0008] In order to achieve the above objectives, the following technical solutions are adopted:
[0009] In a first aspect, the present invention provides a method for extracting extracellular vesicles of Oenococcus oeni, comprising the following steps:
[0010] a. The fermentation broth of Oenococcus oeni is centrifuged for the first time at a centrifugal force of 1000-2000×g to remove most of the bacteria, collect the first supernatant, and then the supernatant is centrifuged for the second time at a centrifugal force of 10000-20000×g to remove the remaining bacteria and bacterial fragments, and collect the second supernatant;
[0011] b. Concentrating the second supernatant obtained in step a by a tangential flow ultrafiltration membrane package, and then diluting it with a buffer solution and concentrating it again by a tangential flow ultrafiltration membrane package to obtain a concentrate; the molecular weight cutoff of the tangential flow ultrafiltration membrane package is smaller than the minimum diameter of the extracellular vesicles of Oenococcus oeni;
[0012] c. filtering the concentrated solution obtained in step b through a filter membrane, and collecting the filtrate; the molecular weight cutoff of the filter membrane is greater than the maximum diameter of the extracellular vesicles of Oenococcus oeni;
[0013] d. The filtrate collected in step c is centrifuged for the first time at a centrifugal force of 100,000 to 150,000 × g to obtain preliminarily purified extracellular vesicles, and then the preliminarily purified extracellular vesicles are resuspended in a buffer and centrifuged for a second time at a centrifugal force of 100,000 to 150,000 × g to obtain purified extracellular vesicles.
[0014] As a further technical solution, in step a, the first centrifugation time is 5 to 10 minutes, and the second centrifugation time is 5 to 10 minutes.
[0015] As a further technical solution, the molecular weight cutoff of the tangential flow ultrafiltration membrane package is 100 kDa.
[0016] As a further technical solution, in step b, the dilution with buffer is dilution with a buffer having an equal volume to the concentrated solution of the first tangential flow ultrafiltration membrane package;
[0017] In step b, the buffer comprises PBS buffer;
[0018] In step b, the first tangential flow ultrafiltration membrane package is concentrated 20 times, and the second tangential flow ultrafiltration membrane package is concentrated until the volume remains unchanged.
[0019] As a further technical solution, in step c, the pore size of the filter membrane is 0.45 μm.
[0020] As a further technical solution, in step d, the first centrifugation time is 1.5 to 2.5 hours, and the second centrifugation time is 1.5 to 2.5 hours;
[0021] In step d, the buffer comprises PBS buffer.
[0022] As a further technical solution, the extraction temperature is 0-4°C.
[0023] As a further technical solution, the fermentation broth of Oenococcus oeni is a liquid obtained by culturing Oenococcus oeni to a stable phase.
[0024] As a further technical solution, Oenococcus oeni is cultured in an Oenococcus oeni culture medium containing ethanol to obtain a fermentation broth of Oenococcus oeni; the concentration of the ethanol is within 8% (v / v);
[0025] Alternatively, a culture medium of Oenococcus oeni with a pH of 3.5 to 4.8 is used for culturing to obtain a fermentation broth of Oenococcus oeni.
[0026] In a second aspect, the present invention provides the use of extracellular vesicles of Oenococcus oeni extracted by the above extraction method in improving the acid and ethanol stress resistance of Oenococcus oeni.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] The invention provides a method for extracting extracellular vesicles of O. oeni. The method comprises the following steps: firstly, the fermentation broth of O. oeni is centrifuged twice under a specific centrifugal force to remove the bacteria and its fragments, so as to avoid the reduction of the yield of extracellular vesicles due to the adhesion of the bacteria; then, the broth is concentrated by two tangential flow ultrafiltration membrane packs and filtered by a filter membrane to fully remove impurities with a diameter larger than or smaller than the extracellular vesicles; and then, after two ultracentrifugal separation and purification treatments, the extracellular vesicles of O. oeni are obtained. The extraction and purification method is simple, efficient, and low in cost, and the structure of the extracellular vesicles will not be destroyed. The extracellular vesicles of O. oeni extracted are of high purity.
[0029] In addition, the inventors have found that subjecting Oenococcus oeni to acid or ethanol stress during the cultivation process can further increase the extraction yield of extracellular vesicles. Therefore, using acid or ethanol stressed culture medium for the extraction of extracellular vesicles can help further increase the yield of extracellular vesicles of Oenococcus oeni. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0031] Figure 1 This is a transmission electron micrograph of the extracellular vesicles of Oenococcus oeni;
[0032] Figure 2 This is the particle size diagram of extracellular vesicles of Oenococcus oeni;
[0033] Figure 3 Figure 2. Stress treatment increases extracellular vesicle production in Oenococcus oeni;
[0034] Figure 4 Figure 2 shows that stress treatment increases the content of extracellular vesicle proteins in Oenococcus oeni;
[0035] Figure 5 This is a quantitative diagram of extracellular vesicle protein identification in normal culture and stress conditions of Oenococcus oeni;
[0036] Figure 6 The extracellular vesicles of Oenococcus oeni promote its growth under multiple stresses. DETAILED DESCRIPTION
[0037] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0038] In a first aspect, the present invention provides a method for extracting extracellular vesicles of Oenococcus oeni, comprising the following steps:
[0039] a. The fermentation broth of Oenococcus oeni is centrifuged for the first time at a centrifugal force of 1000-2000×g to remove most of the bacteria, collect the first supernatant, and then the supernatant is centrifuged for the second time at a centrifugal force of 10000-20000×g to remove the remaining bacteria and bacterial fragments, and collect the second supernatant;
[0040] b. Concentrating the second supernatant obtained in step a by a tangential flow ultrafiltration membrane package, and then diluting it with a buffer solution and concentrating it again by a tangential flow ultrafiltration membrane package to obtain a concentrate; the molecular weight cutoff of the tangential flow ultrafiltration membrane package is smaller than the minimum diameter of the extracellular vesicles of Oenococcus oeni;
[0041] c. filtering the concentrated solution obtained in step b through a filter membrane, and collecting the filtrate; the molecular weight cutoff of the filter membrane is greater than the maximum diameter of the extracellular vesicles of Oenococcus oeni;
[0042] d. The filtrate collected in step c is centrifuged for the first time at a centrifugal force of 100,000 to 150,000 × g to obtain preliminarily purified extracellular vesicles, and then the preliminarily purified extracellular vesicles are resuspended in a buffer and centrifuged for a second time at a centrifugal force of 100,000 to 150,000 × g to obtain purified extracellular vesicles.
[0043] The invention provides a method for extracting extracellular vesicles of O. oeni. The method comprises the following steps: firstly, the fermentation broth of O. oeni is centrifuged twice under a specific centrifugal force to remove the bacteria and its fragments, so as to avoid the reduction of the yield of extracellular vesicles due to the adhesion of the bacteria; then, the broth is concentrated by two tangential flow ultrafiltration membrane packs and filtered by a filter membrane to fully remove impurities with a diameter larger than or smaller than the extracellular vesicles; and then, after two ultracentrifugal separation and purification treatments, the extracellular vesicles of O. oeni are obtained. The extraction method is simple, efficient, and low in cost, and the structure of the extracellular vesicles will not be destroyed. The extracted extracellular vesicles of O. oeni are of high purity.
[0044] In some optional embodiments, in step a, the time of the first centrifugation may be, for example, but not limited to, 5 min, 8 min or 10 min, and the time of the second centrifugation may be, for example, but not limited to, 5 min, 8 min or 10 min.
[0045] The centrifugation was performed twice, with the first centrifugation being at a low speed to ensure that the bacteria were precipitated in large quantities and to prevent the extracellular vesicles in the supernatant from being precipitated with the bacteria due to bacterial adhesion; the second centrifugation was at a high speed to ensure that the bacteria and debris were completely precipitated.
[0046] Usually the diameter of bacterial extracellular vesicles is in the range of 20 to 400 nm, therefore, tangential flow ultrafiltration membrane packages with pore sizes less than 20 nm and filter membranes with pore sizes greater than 400 nm are preferred.
[0047] In some optional embodiments, the molecular weight cutoff of the tangential flow ultrafiltration membrane package is 100 kDa.
[0048] In some optional embodiments, in step b, the dilution with buffer is dilution with a buffer having an equal volume to the concentrated solution of the first tangential flow ultrafiltration membrane package;
[0049] In step b, the buffer comprises PBS buffer;
[0050] In step b, the first tangential flow ultrafiltration membrane package is concentrated 20 times, and the second tangential flow ultrafiltration membrane package is concentrated until the volume remains unchanged.
[0051] Conventional ultrafiltration methods have problems such as clogging due to dead-end filtration, which affects the extraction efficiency. However, tangential flow ultrafiltration membrane packages can greatly improve ultrafiltration efficiency and avoid clogging problems. The inventors have found that diluting the concentrate of the first tangential flow ultrafiltration with a buffer solution and then performing a second tangential flow ultrafiltration until the concentrated volume remains unchanged can effectively filter out some macromolecular substances in the fermentation broth, which helps to obtain purer extracellular vesicles.
[0052] In some optional embodiments, in step c, the pore size of the filter membrane is 0.45 μm.
[0053] In some optional embodiments, in step d, the time of the first centrifugation may be, for example, but not limited to 1.5 h, 2 h or 2.5 h, and the time of the second centrifugation may be, for example, but not limited to 1.5 h, 2 h or 2.5 h;
[0054] In step d, the buffer includes but is not limited to PBS buffer, or other buffers well known to those skilled in the art.
[0055] The inventors have found that the combination of two ultracentrifugations can help further improve the purity of extracellular vesicles.
[0056] In some optional embodiments, the extraction temperature may be, for example, but not limited to, 0°C, 2°C or 4°C.
[0057] In some optional embodiments, the fermentation broth of Oenococcus oeni is a liquid obtained by growing Oenococcus oeni to a stable phase.
[0058] The bacterial content in the fermentation broth during this period reaches its maximum value, which helps to increase the production of extracellular vesicles of Oenococcus oeni.
[0059] In some optional embodiments, Oenococcus oeni is cultured in an Oenococcus oeni culture medium containing ethanol to obtain a fermentation broth of Oenococcus oeni; the concentration of the ethanol is within 8% (v / v);
[0060] It should be noted that the Oenococcus oeni culture medium in the present invention refers to a culture medium that can be used for culturing Oenococcus oeni, which contains nutrients required for the growth of Oenococcus oeni.
[0061] In some optional embodiments, a culture medium of Oenococcus oeni with a pH of 3.5 to 4.8 is used for culturing to obtain a fermentation broth of Oenococcus oeni.
[0062] The inventors have found that subjecting Oenococcus oeni to acid or ethanol stress during its cultivation can further increase the extraction yield of extracellular vesicles. Therefore, it is preferred to cultivate Oenococcus oeni under acid or ethanol stress.
[0063] In a second aspect, the present invention provides the use of extracellular vesicles of Oenococcus oeni extracted by the above extraction method in improving the acid and ethanol stress resistance of Oenococcus oeni.
[0064] For example, when O. oeni is subjected to acid or ethanol stress, adding extracellular vesicles of O. oeni to the culture medium can help improve the stress tolerance of O. oeni and promote the growth of O. oeni.
[0065] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.
[0066] In the following examples or comparative examples, unless otherwise specified, the preparation method of ATB extracellular vesicle culture medium is as follows:
[0067] Peptone (10 g / L, w / v), glucose (10 g / L, w / v), yeast extract (5 g / L, w / v), MgSO4·7H2O (0.2 g / L, w / v), MnSO4·H2O (0.038 g / L, w / v) and tomato juice (25%, v / v) were mixed, distilled water was added to 1 L, and the pH was adjusted to 4.8 with hydrochloric acid or sodium hydroxide to obtain a liquid culture medium. The liquid culture medium was then filtered through a vacuum filtration device with a 0.22 μm filter membrane to remove insoluble particles, and ultrafiltration was performed using a 100 kDa tangential filter to remove macromolecular substances and possible extracellular vesicles in the tomato juice, and the filtrate was sterilized at 121°C for 15 min.
[0068] Before inoculating the culture medium, 0.5 mL of 1.0% (w / v) cysteine hydrochloride solution boiled at normal pressure for 15 min was aseptically added to every 10 mL of culture medium.
[0069] Example 1
[0070] A method for extracting extracellular vesicles of Oenococcus oeni, comprising the following steps:
[0071] 1. Culture: After Oenococcus oeni was activated twice in ATB extracellular vesicle culture medium, the culture medium was 1×10 8 The inoculum of CFU was inoculated in ATB extracellular vesicle medium (1 L) and cultured for 6 days.
[0072] 2. Extraction of extracellular vesicles: The fermentation broth was centrifuged at 4°C and 1000g for 10 min, and the supernatant was taken to remove the intact bacteria. The obtained supernatant was centrifuged at 4°C and 10000g for 10 min, and the bacteria and bacterial fragments were removed to obtain the supernatant containing extracellular vesicles. The supernatant was filtered with a 100kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to 100mL. 100mL PBS buffer (NaCl, 137mmol / L; KCl, 2.7mmol / L; Na2HP04, 10mmol / L; KH2P04, 2mmol / L; pH7.4, 0.22μm filter membrane) was added to the concentrate and filtered again with a 100kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to a constant volume.
[0073] 3. Purification of extracellular vesicles: Take 100 mL of the concentrate obtained in step 2, first filter it with a 0.45 μm filter, and then centrifuge the filtered supernatant at 120,000 g for 2 h at 4 ° C to obtain a crude extracellular vesicle precipitate. Resuspend the precipitate in PBS buffer (NaCl, 137 mmol / L; KCl, 2.7 mmol / L; Na2HP04, 10 mmol / L; KH2P04, 2 mmol / L; pH 7.4, 0.22 μm filter) and centrifuge it again at 4 ° C for 120,000 g for 2 h to obtain a purified extracellular vesicle precipitate. Finally, add 200 μL PBS buffer to the extracellular vesicle precipitate obtained, resuspend it, and store it at -80 ° C for use.
[0074] Example 2
[0075] A method for extracting extracellular vesicles of Oenococcus oeni, comprising the following steps:
[0076] 1. Culture: After Oenococcus oeni was activated twice in ATB extracellular vesicle culture medium, the culture medium was 1×10 8 The inoculum of CFU was inoculated in ATB extracellular vesicle medium (1 L) and cultured for 6 days.
[0077] 2. Extraction of extracellular vesicles: The fermentation broth was centrifuged at 4°C and 2000g for 5 minutes, and the supernatant was taken to remove the intact bacteria. The obtained supernatant was centrifuged at 4°C and 20000g for 5 minutes, and the bacteria and bacterial fragments were removed to obtain the supernatant containing extracellular vesicles. The supernatant was filtered with a 100kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to 100mL. 100mL PBS buffer (NaCl, 137mmol / L; KCl, 2.7mmol / L; Na2HP04, 10mmol / L; KH2P04, 2mmol / L; pH7.4, 0.22μm filter membrane) was added to the concentrate and filtered again with a 100kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to a constant volume.
[0078] 3. Purification of extracellular vesicles: Take 100 mL of the concentrate obtained in step 2, first filter it with a 0.45 μm filter, and then centrifuge the filtered supernatant at 150,000 g for 1.5 h at 4 ° C to obtain a crude extracellular vesicle precipitate. Resuspend the precipitate in PBS buffer (NaCl, 137 mmol / L; KCl, 2.7 mmol / L; Na2HP04, 10 mmol / L; KH2P04, 2 mmol / L; pH 7.4, 0.22 μm filter) and centrifuge it again at 4 ° C for 150,000 g for 1.5 h to obtain a purified extracellular vesicle precipitate. Finally, add 200 μL PBS buffer to the extracellular vesicle precipitate obtained, resuspend it, and store it at -80 ° C for use.
[0079] Comparative Example 1
[0080] A method for extracting extracellular vesicles of Oenococcus oeni, comprising the following steps:
[0081] 1. Culture: After Oenococcus oeni was activated twice in ATB extracellular vesicle culture medium, the culture medium was 1×10 8 The inoculum of CFU was inoculated in ATB extracellular vesicle medium (1 L) and cultured for 6 days.
[0082] 2. Extraction of extracellular vesicles: The fermentation broth was centrifuged at 4°C and 10,000 g for 10 min, and the supernatant was taken to remove the bacteria and bacterial fragments to obtain the supernatant containing extracellular vesicles. The supernatant was filtered with a 100 kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to 100 mL. 100 mL of PBS buffer (NaCl, 137 mmol / L; KCl, 2.7 mmol / L; Na2HP04, 10 mmol / L; KH2P04, 2 mmol / L; pH 7.4, 0.22 μm membrane filtration) was added to the concentrate and filtered again with a 100 kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to a constant volume.
[0083] 3. Purification of extracellular vesicles: Take 100 mL of the concentrate obtained in step 2, first filter it with a 0.45 μm filter, and then centrifuge the filtered supernatant at 120,000 g for 2 h at 4 ° C to obtain a crude extracellular vesicle precipitate. Resuspend the precipitate in PBS buffer (NaCl, 137 mmol / L; KCl, 2.7 mmol / L; Na2HP04, 10 mmol / L; KH2P04, 2 mmol / L; pH 7.4, 0.22 μm filter) and centrifuge it again at 4 ° C for 120,000 g for 2 h to obtain a purified extracellular vesicle precipitate. Finally, add 200 μL PBS buffer to the extracellular vesicle precipitate obtained, resuspend it, and store it at -80 ° C for use.
[0084] Comparative Example 2
[0085] A method for extracting extracellular vesicles of Oenococcus oeni, comprising the following steps:
[0086] 1. Culture: After Oenococcus oeni was activated twice in ATB extracellular vesicle culture medium, the culture medium was 1×10 8 The inoculum of CFU was inoculated in ATB extracellular vesicle medium (1 L) and cultured for 6 days.
[0087] 2. Extraction of extracellular vesicles: The fermentation broth was centrifuged at 4°C and 1000g for 10 min, and the supernatant was taken to remove the intact bacteria. The obtained supernatant was centrifuged at 4°C and 10000g for 10 min, and the bacteria and bacterial fragments were removed to obtain the supernatant containing extracellular vesicles. The supernatant was filtered with a 100kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to a constant volume.
[0088] 3. Purification of extracellular vesicles: Take 100 mL of the concentrate obtained in step 2, first filter it with a 0.45 μm filter, and then centrifuge the filtered supernatant at 120,000 g for 2 h at 4 ° C to obtain a crude extracellular vesicle precipitate. Resuspend the precipitate in PBS buffer (NaCl, 137 mmol / L; KCl, 2.7 mmol / L; Na2HP04, 10 mmol / L; KH2P04, 2 mmol / L; pH 7.4, 0.22 μm filter) and centrifuge it again at 4 ° C for 120,000 g for 2 h to obtain a purified extracellular vesicle precipitate. Finally, add 200 μL PBS buffer to the extracellular vesicle precipitate obtained, resuspend it, and store it at -80 ° C for use.
[0089] Comparative Example 3
[0090] A method for extracting extracellular vesicles of Oenococcus oeni, comprising the following steps:
[0091] 1. Culture: After Oenococcus oeni was activated twice in ATB extracellular vesicle culture medium, the culture medium was 1×10 8 The inoculum of CFU was inoculated in ATB extracellular vesicle medium (1 L) and cultured for 6 days.
[0092] 2. Extraction of extracellular vesicles: The fermentation broth was centrifuged at 4°C and 1000g for 10 minutes, and the supernatant was taken to remove the intact bacteria. The obtained supernatant was centrifuged at 4°C and 10000g for 10 minutes, and the bacteria and bacterial fragments were removed to obtain the supernatant containing extracellular vesicles. The supernatant was filtered with a 100kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to 100mL. 100mL PBS buffer (NaCl, 137mmol / L; KCl, 2.7mmol / L; Na2HP04, 10mmol / L; KH2P04, 2mmol / L; pH7.4, 0.22μm filter membrane) was added to the concentrate and filtered again with a 100kDa tangential filter (the supernatant containing extracellular vesicles was placed in an ice-water mixture for ice bath during the whole process) and concentrated to a constant volume.
[0093] 3. Purification of extracellular vesicles: Take 100 mL of the concentrate obtained in step 2, first filter it with a 0.45 μm filter, then centrifuge the filtered supernatant at 120,000 g for 2 h at 4°C to obtain a crude extracellular vesicle precipitate. Finally, add 200 μL of PBS buffer to the extracellular vesicle precipitate, resuspend it, and store it at -80°C for later use.
[0094] Test Example 1
[0095] The extracellular vesicles of Examples 1-2 and Comparative Examples 1-3 were characterized.
[0096] 1. Quantification of extracellular vesicles: The particle count and size distribution of extracellular vesicles isolated from different sample types were measured by a nanoparticle tracking analyzer (Nanosight NS 3000, Malvern). Take 10 μL of the extracellular vesicle sample and dilute it 100 times with PBS buffer. Use Nanosight software for analysis. Finally, the concentration and particle size distribution of the extracellular vesicles are calibrated by NTA by inputting the sample volume, the volume of the extracellular vesicle resuspension, and the dilution factor. The particle size diagram of the extracellular vesicles of Oenococcus oeni in Example 1 is shown in Figure 1. Figure 2 shown.
[0097] 2. Structural characteristics of extracellular vesicles: Fix 50 μL of extracellular vesicle sample with an equal amount of 4% paraformaldehyde. Take 5 μL of extracellular vesicles and drop them on a 200-mesh copper grid with a support membrane, dry and incubate at room temperature for 5 minutes. Use filter paper to absorb excess liquid from the edge of the copper grid. Stain the sample with 1% uranyl acetate stain (1 minute). Rinse the copper grid twice with distilled water, and then remove excess liquid with filter paper. After drying, observe with a HT7700 transmission electron microscope (accelerating voltage 100 kV). The transmission electron microscopy image of the extracellular vesicles of Oenococcus oeni in Example 1 is as follows Figure 1 shown.
[0098] 3. Protein concentration of extracellular vesicles: Use SDT lysis buffer (4% SDS, 100mM DTT, 100mM Tris-HCl pH 8.0) to extract protein from the isolated and purified extracellular vesicle sample, boil the sample for 3 minutes, and then further perform ultrasonic treatment. Centrifuge at 16000g for 15 minutes to remove undissolved sample fragments. Collect the supernatant and use the BCA protein detection kit to detect the protein concentration of the extracellular vesicle lysate as follows: Follow the instructions of the kit. Use BSA as the protein standard to prepare a standard curve. Dilute the extracellular vesicle lysate appropriately and add 20μL to the sample well. Add 200μL BCA working solution and place at 37℃ for 20-30min. Use an enzyme reader to measure the absorbance at a wavelength of 562nm, and calculate the protein content in the extracellular vesicle sample according to the standard curve.
[0099] The extracellular vesicles of Example 1 and Example 2 were characterized and it was found that there was no significant difference in the yield, protein concentration and purity of the extracellular vesicles obtained by the two embodiments. The difference between Comparative Example 1 and Example 1 is that 10000×g is directly used for centrifugation. After testing and comparison, it was found that the yield and protein concentration of the extracellular vesicles obtained by the two centrifugation methods of Example 1 were 20% higher than those in Comparative Example 1. The difference between Comparative Example 2 and Example 1 is that no buffer dilution and a second tangential flow ultrafiltration were performed. After testing and comparison, it was found that the extracellular vesicles obtained in Example 1 had a clearer transmission electron microscopy background than the extracellular vesicles in Comparative Example 2, fewer impurities, and no large particle agglomerates. The difference between Comparative Example 3 and Example 1 is that only one ultracentrifugation was performed. After testing and comparison, it was found that the two ultracentrifugations in Example 1 increased the yield of extracellular vesicles by more than 15% compared with Comparative Example 3, and the transmission electron microscopy background was clearer.
[0100] Test Example 2 Selection of Oenococcus oeni culture method
[0101] Control medium (NS): ATB extracellular vesicle medium;
[0102] Acid stress medium (AS): ATB extracellular vesicle medium was adjusted to pH 4.0 with hydrochloric acid;
[0103] Ethanol stress medium (ES): After the ATB extracellular vesicle medium was prepared, 5% anhydrous ethanol (v / v) was added under sterile conditions.
[0104] The same inoculum size was used to inoculate Oenococcus oeni into a control medium, an acid stress medium and an ethanol stress medium, respectively. The culture was carried out at 28°C for 6 days to reach a stable period. The fermentation broth was harvested, and then extracellular vesicles were extracted from the fermentation broth according to the extraction method of Example 1. The yield of extracellular vesicles was detected by a nanoparticle tracking analyzer and a BCA protein detection kit, and the structure of the extracellular vesicles was observed using a transmission electron microscope.
[0105] The results showed that the production of extracellular vesicles of O. oeni cultured in acid stress medium (NS) and ethanol stress medium (ES) increased to 166% and 153% respectively compared with that in conventional medium (NS). Figure 3 ), protein concentration detection and scanning electron microscopy observation found that the extracellular vesicle protein content obtained in acid stress medium (AS) and ethanol stress medium (ES) increased significantly, and the vesicle capacity increased significantly ( Figure 4 ).
[0106] Experimental Example 3: Enzymatic hydrolysis of extracellular vesicle proteins from different culture methods of Oenococcus oeni and DIA mass spectrometry data analysis
[0107] Proteins were extracted from the isolated and purified extracellular vesicle samples using SDT lysis buffer (4% SDS, 100mM DTT, 100mM Tris-HCl pH 8.0). The samples were boiled for 3 minutes and then further sonicated. The undissolved sample fragments were removed by centrifugation at 16000g for 15 minutes. DTT was added to the protein sample to a final concentration of 100mM, and the sample was placed in a boiling water bath for 5 minutes. The protein sample cooled to room temperature was mixed with 8M urea (pH8.0), ultrafiltration centrifuged (12000g, 15min), and then 8M urea was added again for ultrafiltration centrifugation. The filtrate was removed, 50mM IAA was added, and the sample was gently shaken and incubated in the dark for 30min. Centrifuged at 12000g for 10min. 8M urea was added again and centrifuged twice. NH4HCO3 was added to the sample and centrifuged twice. Finally, trypsin was added and incubated at 37℃ with shaking for 16-18h. The peptides were collected by centrifugation and vacuum freeze-dried. Chromatographic separation was performed using a Vanquish Neo UHPLC system, operated Neo UHPLC system (Thermo Scientific). 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile aqueous solution were selected as buffers. All mass spectrometry data were merged using the software DIA-NN to complete database retrieval of DIA mass spectrometry data and protein DIA quantitative analysis, using Q-value ≤ 0.01 as the screening parameter.
[0108] After testing, it was found that the number of proteins in the extracellular vesicles of O. oeni cultured in acid stress medium (AS) and ethanol stress medium (ES) was not significantly different from that in the extracellular vesicles of normal medium (NS) ( Figure 5 ).
[0109] Experimental Example 4 Extracellular vesicles of Oenococcus oeni improve bacterial stress resistance
[0110] Preparation of multi-stress ATB medium: Peptone (10 g / L, w / v), glucose (10 g / L, w / v), yeast extract (5 g / L, w / v), MgSO4·7H2O (0.2 g / L, w / v), MnSO4·H2O (0.038 g / L, w / v) and tomato juice (25%, v / v) were mixed, and distilled water was added to 1 L. The pH was adjusted to 3.5 with hydrochloric acid or sodium hydroxide to obtain a liquid medium. The liquid medium was then filtered through a vacuum filtration device with a 0.22 μm filter membrane to remove insoluble particles, and the filtrate was sterilized at 121°C for 15 min. Before inoculation of the medium, 8% anhydrous ethanol (v / v) was added under aseptic conditions, and 0.5 mL of 1.0% (w / v) cysteine hydrochloride solution boiled at normal pressure for 15 min was added aseptically for every 10 mL of the medium.
[0111] Extracellular vesicles of Oenococcus oeni obtained under normal culture conditions were added to the multi-stress ATB medium to prepare low-concentration groups (L-EVs) and high-concentration groups (H-EVs) with final concentrations of 5 μg / mL and 20 μg / mL, respectively. The control group without the addition of extracellular vesicles (No-EVs) was used, and the OD was measured every 24 h. 600 , preparation of Oenococcus oeni growth curve.
[0112] After testing, it was found that the addition of low concentration of extracellular vesicles did not have a good effect on the growth of Oenococcus oeni under adversity, but the addition of high concentration of extracellular vesicles had a significant promoting effect on the growth of Oenococcus oeni under adversity, indicating that extracellular vesicles help improve the stress resistance of Oenococcus oeni.
[0113] ( Figure 6 ).
[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for extracting extracellular vesicles of Oenococcus oeni, characterized in that: The steps include: a. The fermentation broth of Oenococcus oeni is centrifuged for the first time at a centrifugal force of 1000-2000×g to remove most of the bacteria, collect the first supernatant, and then the supernatant is centrifuged for the second time at a centrifugal force of 10000-20000×g to remove the remaining bacteria and bacterial fragments, and collect the second supernatant; b. Concentrating the second supernatant obtained in step a by a tangential flow ultrafiltration membrane package, and then diluting it with a buffer solution and concentrating it again by a tangential flow ultrafiltration membrane package to obtain a concentrate; the molecular weight cutoff of the tangential flow ultrafiltration membrane package is smaller than the minimum diameter of the extracellular vesicles of Oenococcus oeni; c. filtering the concentrated solution obtained in step b through a filter membrane, and collecting the filtrate; the molecular weight cutoff of the filter membrane is greater than the maximum diameter of the extracellular vesicles of Oenococcus oeni; d. The filtrate collected in step c is centrifuged for the first time at a centrifugal force of 100,000 to 150,000 × g to obtain preliminarily purified extracellular vesicles, and then the preliminarily purified extracellular vesicles are resuspended in a buffer and centrifuged for a second time at a centrifugal force of 100,000 to 150,000 × g to obtain purified extracellular vesicles.
2. The extraction method according to claim 1, characterized in that In step a, the first centrifugation time is 5 to 10 minutes, and the second centrifugation time is 5 to 10 minutes.
3. The extraction method according to claim 1, characterized in that The molecular weight cut-off of the tangential flow ultrafiltration membrane package is 100 kDa.
4. The extraction method according to claim 1, characterized in that In step b, the dilution with buffer is dilution with a volume of buffer equal to that of the concentrated solution of the first tangential flow ultrafiltration membrane package; In step b, the buffer comprises PBS buffer; In step b, the first tangential flow ultrafiltration membrane package is concentrated 20 times, and the second tangential flow ultrafiltration membrane package is concentrated until the volume remains unchanged.
5. The extraction method according to claim 1, characterized in that In step c, the pore size of the filter membrane is 0.45 μm.
6. The extraction method according to claim 1, characterized in that In step d, the first centrifugation time is 1.5 to 2.5 hours, and the second centrifugation time is 1.5 to 2.5 hours; In step d, the buffer comprises PBS buffer.
7. The extraction method according to claim 1, characterized in that The extraction temperature is 0-4°C.
8. The extraction method according to claim 1, characterized in that The fermentation liquid of Oenococcus oeni is a liquid obtained by culturing Oenococcus oeni to a stable phase.
9. The extraction method according to claim 1, characterized in that Cultivating Oenococcus oeni in an Oenococcus oeni culture medium containing ethanol to obtain a fermentation liquid of Oenococcus oeni; the concentration of the ethanol is within 8% (v / v); Alternatively, a culture medium of Oenococcus oeni with a pH of 3.5 to 4.8 is used for culturing to obtain a fermentation broth of Oenococcus oeni.
10. Use of the extracellular vesicles of Oenococcus oeni extracted by the extraction method according to any one of claims 1 to 9 in improving the acid and ethanol stress resistance of Oenococcus oeni.