A method for producing a magnetosome

Abstract

The application discloses a method for producing magnetosomes, comprising the following steps: inoculating M. gryphiswaldense MSR-1 into culture medium I for culture; inoculating the seed liquid obtained through the culture into culture medium II for fermentation to produce magnetosomes; and collecting magnetotactic bacteria and extracting magnetosomes. The application optimizes the culture conditions and culture medium of the magnetotactic bacteria, significantly improves the growth of the strain, and greatly shortens the culture period of the strain. The application optimizes the extraction and purification method of the magnetosomes, and the method does not involve expensive equipment, is simple to operate, and has high extraction efficiency. Compared with the chemically synthesized magnetic nanoparticles, the magnetosomes prepared by the application have the advantages of uniform particle size distribution, good biocompatibility, easy surface functionalization and modification, and can be applied to the fields of in vitro detection, disease diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a method for producing magnetosomes. Background Technology

[0002] Magnetotactic bacteria are Gram-negative bacteria capable of directional movement under the influence of an external magnetic field, generally either microaerobic or anaerobic. A rich variety of magnetotactic bacteria exist in nature, including bacilli, cocci, spirilla, vibrio, and oomycetes, and they hold broad application prospects in disease diagnosis and treatment, and pollution control. However, the culture conditions for magnetotactic bacteria are quite demanding; currently, only a small percentage of magnetotactic bacteria have been successfully cultured in the laboratory, and their growth rate is slow. Therefore, it is necessary to establish a mature method for the simple and rapid cultivation of magnetotactic bacteria in the laboratory.

[0003] Magnetic nanoparticles have great application potential in drug delivery, tumor magnetothermal therapy, and magnetic resonance imaging. However, chemically synthesized magnetic nanoparticles have disadvantages such as difficulty in controlling morphology, difficulty in controlling surface modification, and poor biocompatibility, which limit their application. Magnetosomes formed by the biomineralization of magnetotactic bacteria are natural magnetic nanoparticles. Compared with chemically synthesized magnetic nanoparticles, magnetosomes have many superior properties: (1) uniform particle size and stable crystal form; (2) good biocompatibility; (3) a large number of amino and hydroxyl groups on the outer membrane of magnetosomes, which are easy to modify for surface functionalization. Extracting magnetosomes from cultured magnetotactic bacteria is an effective means to make up for the lack of artificial magnetic nanoparticles. At present, the culture of magnetotactic bacteria and the production of magnetosomes face the following two obstacles: (1) due to the harsh requirements of magnetotactic bacteria for nutrition and environmental conditions, the growth rate of the strain is slow and the culture cycle is long, resulting in low magnetosome yield; (2) in the process of magnetosome extraction, cell lysis requires special equipment (such as ultrasonic homogenizer or high-pressure homogenizer), which has problems such as limited sample size, complicated operation or expensive equipment. Therefore, it is necessary to establish a simple and rapid method for producing magnetosomes under laboratory conditions, including optimizing the components of the magnetotactic bacterial culture medium, culture conditions, and magnetosome extraction method, in order to improve the growth rate of the strain, increase the yield of magnetosomes, and simplify the magnetosome extraction method. Summary of the Invention

[0004] In view of the above-mentioned technical difficulties, this invention provides a simple and rapid method for producing magnetosomes. This method, by optimizing the culture medium composition and culture conditions of magnetotactic bacteria, increases the growth rate of magnetotactic bacteria and the yield of magnetosomes by 1.2 and 1.6 times, respectively. This invention also optimizes the magnetosome extraction method, eliminating the need for special equipment to disrupt cells and achieving excellent extraction results.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a method for producing magnetic particles, comprising the following steps:

[0007] (1) Magnetotactic bacteria were inoculated into culture medium I and cultured.

[0008] (2) The seed culture obtained from the culture was inoculated into culture medium II for fermentation to produce magnetosomes;

[0009] (3) Collect magnetotactic bacterial cells and extract magnetosomes;

[0010] The magnetotactic bacteria was *M. gryphiswaldense* MSR-1.

[0011] The culture medium I and / or culture medium II contain: 20–40 mM sodium pyruvate, 1–5 g / L peptone, 10–40 mM sodium gluconate, 5–20 mM ferric nitrate, 80–120 μM ferric acetylacetone, 0.1–0.5 g / L yeast extract, 0.1–0.5 g / L magnesium sulfate heptahydrate, 0.3–0.7 g / L dipotassium hydrogen phosphate, 0.02–0.07 g / L sodium mercaptoglycolate, 0.1–0.5 g / L humic acid, and 3–7 mL of a mineral element mixture; the mineral element mixture comprises 12 ~18g / L aminotriacetic acid, 25-35g / L magnesium sulfate, 8-13g / L sodium chloride, 0.5-1.5g / L ferrous sulfate, 1-2g / L cobalt sulfate, 0.5-1.5g / L calcium chloride, 1-2g / L zinc sulfate, 0.05-0.15g / L copper sulfate, 0.15-0.25g / L potassium aluminum sulfate, 0.05-0.15g / L boric acid, 0.05-0.15g / L sodium molybdate, 0.2-0.3g / L nickel chloride, 2-5mg / L sodium selenite, and 3-6g / L manganese sulfate.

[0012] In a preferred embodiment, in step (1), the rotation speed of the culture is 100-200 rpm; and the culture temperature is 28-32°C.

[0013] In a preferred embodiment, step (1) includes primary culture and secondary culture; the secondary culture involves transferring the seed liquid obtained from the primary culture to a fresh culture medium I for further culture to obtain a secondary seed liquid; the time for the primary and secondary cultures is 18 to 36 hours; in the technical solution of the present invention, a certain number of metabolically vigorous seeds can be obtained by progressively scaling up the culture through two cultures.

[0014] In some specific embodiments, in step (1), the culture is carried out in a serum bottle with an inoculum of 10-25% (v / v) and a liquid volume of 20-40% (v / v).

[0015] In a preferred embodiment, in step (2), the fermentation temperature is 28-32°C, the rotation speed is 100-200 rpm, and the time is 24-48 h.

[0016] In some specific embodiments, in step (2), the fermentation is carried out in a serum bottle with a seed culture inoculation amount of 10-25% (v / v) and a liquid volume of 60-90% (v / v).

[0017] In a preferred embodiment, step (3) includes the following specific steps:

[0018] S1. Centrifuge the fermented broth to remove the supernatant;

[0019] S2. Add alkaline solution and heat treatment;

[0020] S3. Centrifuge to remove alkaline solution, and resuspend the magnetic borosiforms in PBS solution;

[0021] S4. After magnetic suction, discard the supernatant and ultrasonically clean; repeat 4 to 8 times.

[0022] Preferably, the alkaline solution contains an alkali, polyethylene glycol, and water; the alkali is selected from at least one of NaOH and KOH; the concentration of the alkali is 1–5 M; the molecular weight of the polyethylene glycol is 4000–20000; and the concentration of the polyethylene glycol is 1%–50% (w / v, g / 100 mL).

[0023] In some specific embodiments, the amount of alkali solution used is 3-7 mL / g of bacterial cells, that is, 3-7 mL of alkali solution is added to each gram of wet bacterial cells.

[0024] Preferably, the temperature of the heat treatment is 50-60°C, and the time of the heat treatment is 1.5-3 hours.

[0025] Preferably, the cleaning solution for ultrasonic cleaning is selected from at least one of PBS solution and distilled water.

[0026] Preferably, the repetition is repeated until the protein concentration in the supernatant is ≤0.01 mg / mL.

[0027] In some specific implementations, a drying post-processing is also included.

[0028] Compared with existing methods, the present invention has the following advantages:

[0029] This invention provides a simple and rapid method for producing magnetosomes. The method utilizes a specific culture medium, and compared to other methods, the magnetotactic bacteria in this invention are stabilized after cultivation and subculturing. The growth rate of the magnetotactic bacteria and the magnetosome yield are increased by 1.2 and 1.6 times, respectively, and the cell culture cycle is significantly shortened. Furthermore, this invention optimizes the extraction and purification methods for magnetosomes, resulting in a simple, efficient, and effective extraction process without the need for expensive equipment. Compared to chemically synthesized magnetic nanoparticles, the magnetosomes prepared by this invention have advantages such as uniform particle size distribution, good biocompatibility, and ease of surface functionalization modification. With further modification, the application scope of magnetotactic bacterial magnetosomes can be expanded in in vitro detection, disease diagnosis, and treatment. Attached Figure Description

[0030] Figure 1 This shows the growth of the strains before and after the culture medium optimization in Example 1 and Comparative Example 1 of the present invention.

[0031] Figure 2 This describes the production status of magnetosomes before and after culture medium optimization in Example 2 and Comparative Example 2 of this invention.

[0032] Figure 3 This is a microscopic (100X) observation of the cell lysate in Example 2 of the present invention.

[0033] Figure 4 This is a TEM image of the magnetic particles produced in Embodiment 2 of the present invention. Detailed Implementation

[0034] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0035] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0036] Example 1:

[0037] In this embodiment, the magnetotactic bacteria M. gryphiswaldense MSR-1 was cultured according to the culture medium formulation shown in Table 1 to screen for the optimal culture medium.

[0038] Table 1. Bacterial growth and magnetosome production under different culture medium components.

[0039] 1-1 1-2 1-3 1-4 1-5 1-6 1-7 Sodium pyruvate / mM 20 20 20 25 25 30 30 <![CDATA[Peptone / g·L -1 > 0.5 0.5 0.5 1 1 2 2 Sodium gluconate / mM 20 20 20 20 20 20 20 Ferric nitrate / mM 5 10 15 10 15 5 15 Iron acetylacetone / μM 80 120 100 120 80 100 120 <![CDATA[Yeast extract / g·L -1 > 0.1 0.1 0.1 0.1 0.1 0.1 0.1 <![CDATA[magnesium sulfate heptahydrate / g·L -1 > 0.1 0.1 0.1 0.1 0.1 0.1 0.1 dipotassium hydrogen phosphate 0.5 0.5 0.5 0.5 0.5 0.5 0.5 <![CDATA[Sodium thioglycolate / g·L -1 > 0.05 0.05 0.05 0.05 0.05 0.05 0.05 <![CDATA[Humic acid / g·L -1 > 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Mineral element mixture / mL 5 5 5 5 5 5 5 <![CDATA[OD 600 ]]> 0.9 0.6 0.6 0.6 0.6 1.1 0.6 <![CDATA[Magnetosome yield / mg·L -1 > 0.8 2.5 4.2 4.2 5.0 9.7 5.0

[0040] Table 1 shows that the mineral element mixture contains: 15 g / L nitroglycerin, 30 g / L magnesium sulfate, 10 g / L sodium chloride, 1 g / L ferrous sulfate, 1.8 g / L cobalt sulfate, 1 g / L calcium chloride, 1.8 g / L zinc sulfate, 0.1 g / L copper sulfate, 0.2 g / L potassium aluminum sulfate, 0.1 g / L boric acid, 0.1 g / L sodium molybdate, 0.25 g / L nickel chloride, 3 mg / L sodium selenite, and 5 g / L manganese sulfate.

[0041] The specific culture process is as follows: a single colony of M. gryphiswaldense MSR-1 was picked and inoculated into a 100mL serum bottle containing 20mL of the above-mentioned culture medium and cultured for 36h to obtain the primary seed culture. Then, 10mL of the primary seed culture was transferred to a 500mL serum bottle containing 100mL of the above-mentioned fresh liquid culture medium and cultured for 24h to obtain the secondary seed culture. The culture conditions were as follows: the liquid volume was 20% (v / v), the culture temperature was 30℃, the rotation speed was 100rpm, and the inoculum size was 10% (v / v).

[0042] Comparative Example 1:

[0043] The magnetotactic bacterium M. gryphiswaldense MSR-1 used in this comparative example was cultured in the conventional OFM medium reported in the literature (Applied Microbiology and Biotechnology, 2008, 79:389-397), and the culture process of the strain was the same as in Example 1.

[0044] The fermentation results of culture media 1-6 and Comparative Example 1 in Example 1 are as follows: Figure 1 As shown, the culture medium provided by this invention can significantly improve the growth rate of the strain and the OD of the secondary seed culture. 600 The highest value can reach 1.1, while the OD of the secondary seed solution in Comparative Example 1 is... 600 With a concentration of only 0.5, its growth rate is only 45.5% of that in Example 1. That is, the culture medium provided by the present invention can increase the growth rate of the strain by up to 1.2 times and shorten the culture cycle of the strain by about half.

[0045] Example 2:

[0046] This embodiment utilizes the magnetotactic bacterium Magnetospirillum mgryphiswaldense MSR-1, cultured in media 1-6 of Example 1, to produce magnetosomes via fermentation. The specific process is as follows:

[0047] (1) Take 60 mL of secondary seed liquid and inoculate it into a 1 L serum bottle containing 600 mL of the culture medium shown in Table 1 for fermentation to produce magnetosomes. The fermentation conditions are: liquid volume of 60% (v / v), culture temperature of 30℃, rotation speed of 100 rpm, inoculation amount of 10% (v / v), and fermentation time of 30 h.

[0048] (2) Centrifuge the cultured magnetotactic bacteria solution at 4000 rpm for 15 min, remove the supernatant and obtain a wet weight of 1.5 g of bacterial cells;

[0049] (3) Add 7.5 mL of 1 M NaOH solution (containing polyethylene glycol-4000, with a polyethylene glycol concentration of 20% (w / v, g / 100 mL) and suspend the bacterial cells, then heat at 55 °C for 1.5 h.

[0050] (4) Centrifuge the cell lysate obtained after heating (12000 rpm, 30 min) to remove NaOH solution, and resuspend the magnetic bodies in PBS buffer solution;

[0051] (5) The magnetic particles were adsorbed overnight using neodymium iron boron magnets;

[0052] (6) Discard the supernatant, use PBS solution to ultrasonically clean the magnetosomes, and then use a neodymium iron boron magnet to adsorb the magnetosomes for 2 hours; repeat step (6) 4 times until the protein concentration of the supernatant is ≤0.01mg / mL;

[0053] (7) The magnetic bobbin obtained in step (6) is dried at 70°C for 6 hours and then weighed.

[0054] In this embodiment, the production of magnetic particles is as follows: Figure 2 As shown, the magnetosome yield was 9.7 mg / L (meaning that each 1 L of fermentation broth contained 9.7 mg of magnetosomes). Compared with Comparative Example 2, the magnetosome yield in this example was increased by about 1.6 times.

[0055] In this embodiment, the remaining culture media from Example 1 were used to produce magnetosomes using the same method, and the yields are shown in Table 1.

[0056] like Figure 3 As shown, in this embodiment, after using NaOH solution to disrupt the cells, the cell disruption effect is good (no intact cells are visible under the microscope, only cell fragments), and no special cell disruption instruments (such as ultrasonic disruptors and high-pressure homogenizers) are required. The operation is simple and efficient, and it is suitable for large and small-scale magnetosome extraction.

[0057] The TEM of the magnetosome prepared in this embodiment is as follows: Figure 4 As shown, the magnetic borosiforms have a regular cubic octahedral shape and a size between 20-60 nm, which is consistent with the basic characteristics of magnetic borosiforms.

[0058] Comparative Example 2

[0059] This comparative example used magnetotactic bacteria M. gryphiswaldense MSR-1 to produce magnetosomes via fermentation. The culture medium was the conventional OFM medium reported in the literature (Applied Microbiology and Biotechnology, 2008, 79:389-397). The fermentation conditions and magnetosome extraction and purification processes were consistent with those in Examples 1 and 2. The fermentation results are as follows: Figure 2 As shown, the magnetosome yield of the strain was only 3.8 mg / L, which was only 39.2% of that in Example 2.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing magnetic microparticles, characterized in that, Includes the following steps: (1) Magnetotactic bacteria were inoculated into culture medium I and cultured. (2) The seed culture obtained from the culture was inoculated into culture medium II for fermentation to produce magnetosomes; (3) Collect magnetotactic bacterial cells and extract magnetosomes; The magnetotactic bacteria was *M. gryphiswaldense* MSR-1. The culture medium I and / or culture medium II contain: 20–40 mM sodium pyruvate, 1–5 g / L peptone, 10–40 mM sodium gluconate, 5–20 mM ferric nitrate, 80–120 μM ferric acetylacetone, 0.1–0.5 g / L yeast extract, 0.1–0.5 g / L magnesium sulfate heptahydrate, 0.3–0.7 g / L dipotassium hydrogen phosphate, 0.02–0.07 g / L sodium mercaptoglycolate, 0.1–0.5 g / L humic acid, and 3–7 mL of a mineral element mixture; the mineral element mixture comprises 12 ~18g / L aminotriacetic acid, 25-35g / L magnesium sulfate, 8-13g / L sodium chloride, 0.5-1.5g / L ferrous sulfate, 1-2g / L cobalt sulfate, 0.5-1.5g / L calcium chloride, 1-2g / L zinc sulfate, 0.05-0.15g / L copper sulfate, 0.15-0.25g / L potassium aluminum sulfate, 0.05-0.15g / L boric acid, 0.05-0.15g / L sodium molybdate, 0.2-0.3g / L nickel chloride, 2-5mg / L sodium selenite, and 3-6g / L manganese sulfate.

2. The method according to claim 1, characterized in that, In step (1), the rotation speed of the culture is 100-200 rpm; the culture temperature is 28-32℃.

3. The method according to claim 1, characterized in that, In step (1), the culture includes primary culture and secondary culture; the secondary culture is to transfer the seed culture obtained from the primary culture to fresh culture medium I and continue to culture to obtain the secondary seed culture; the time for the primary culture and the secondary culture is 18 to 36 hours.

4. The method according to claim 1, characterized in that, In step (1), the culture is carried out in a serum bottle with an inoculation amount of 10-25% (v / v) and a liquid volume of 20-40% (v / v).

5. The method according to claim 1, characterized in that, In step (2), the fermentation temperature is 28-32℃, the rotation speed is 100-200rpm, and the time is 24-48h.

6. The method according to claim 1, characterized in that, In step (2), the fermentation is carried out in a serum bottle with a seed culture inoculation amount of 10-25% (v / v) and a liquid volume of 60-90% (v / v).

7. The method according to claim 1, characterized in that, The specific steps of step (3) include: S1. Centrifuge the fermented broth to remove the supernatant; S2. Add alkaline solution and heat treatment; S3. Centrifuge to remove alkaline solution, and resuspend the magnetic borosiforms in PBS solution; S4. After magnetic suction, discard the supernatant and ultrasonically clean; repeat 4 to 8 times.

8. The method according to claim 7, characterized in that, The alkaline solution contains alkali, polyethylene glycol, and water; the alkali is selected from at least one of NaOH and KOH; the concentration of the alkali is 1–5 M; the molecular weight of the polyethylene glycol is 4000–20000; and the concentration of the polyethylene glycol is 1%–50% (w / v, g / 100 mL).

9. The method according to claim 7, characterized in that, The amount of alkaline solution used is 3-7 mL / g of bacterial cells.

10. The method according to claim 7, characterized in that, The temperature of the heat treatment is 50–60°C; the time of the heat treatment is 1.5–3 hours. The cleaning solution for ultrasonic cleaning is selected from at least one of PBS solution and distilled water; The repetition is repeated until the protein concentration in the supernatant is ≤0.01 mg / mL.

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