Production of high-purity iron oxide nanoparticles in cells

Abstract

To provide a method for producing high-purity iron oxide nanoparticles by using nanoparticle producing cells.SOLUTION: A method includes the followings: a) a preliminary growth step including amplifying nanoparticle producing cells in preliminary growth medium and / or fed-batch medium; and b) a growth step including amplifying the nanoparticle producing cells generated from the preliminary growth step in the growth medium and / or the fed-batch medium, in which the preliminary growth medium and / or the growth medium and / or the fed-batch medium contains the followings per kilogram or per liter of the preliminary growth medium and / or the growth medium and / or the fed-batch medium: i) 0.005 gram or less of yeast extract and ii) 0.001 gram or less of CMR agent selected from boric acid and nitrilotriacetic acid, in which the fed-batch medium is medium supplementing preliminary growth and / or growth medium. Further, a larger number of nanoparticles is created in the growth step than the preliminary growth step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The field of this invention relates to the biological production of nanoparticles with low impurity content. [Background technology]

[0002] Bacteria that produce nanoparticles, such as magnetotactic bacteria, are known to accumulate impurities in their crystalline structure. For example, when magnetotactic bacteria grow in the presence of cobalt, magnetosomes containing iron oxide and cobalt are produced (see non-patent reference 1). For medical applications, it is desirable that nanoparticles have a low content of toxic impurities such as cobalt. References

[0003] [Non-Patent Document 1] S. Stanil et al., Nature nanotechnology, Vol. 3, p. 158 (2008) [Overview of the project] [Problems that the invention aims to solve]

[0004] [Means for solving the problem]

[0005] The present invention relates to a method for producing high-purity iron oxide nanoparticles using nanoparticle-producing cells, and includes the following: a) A preliminary growth step comprising preferentially growing nanoparticle-producing cells in a pre-growth and / or fed-batch medium such that the nanoparticle-producing cells do not essentially produce nanoparticles, and b) A growth step comprising preferentially growing nanoparticle-producing cells derived from a growth and / or preliminary growth step in a fed-batch medium so that nanoparticle-producing cells produce nanoparticle-producing cells, Here, the pre-growth or growth or fed-batch medium contains, in less than the following amounts, per kilogram or liter of pre-growth or growth or fed-batch medium: i) 5.10 5 , 5.10 3, 50, 5, 0.5, 0.005, 0.0005, 0.00005 or 5.10 -10 grams of yeast extract, and / or ii) 10 5 , 10 3 , 10, 1, 0.1, 0.001, 10 -5 or 10 -10 grams of a CMR agent preferentially selected from the group consisting of boric acid and nitrilotriacetic acid, wherein, when a fed-batch medium is present, it is preferentially a medium that supplements the pre-growth and / or growth medium, wherein at least 0, 0.1, 0.5, 2, 5, 10, 10 3 , 10 5 or 10 10 of the coefficient, more nanoparticles are produced preferentially in the growth phase than in the pre-growth phase, and when this coefficient is Q2 / Q1, Q1 and Q2 are respectively the amount of nanoparticles produced in the pre-growth and growth step or sub-step. And / or, preferentially, the pre-growth and / or growth and / or fed-batch medium comprises at least one other compound preferentially involved in bacterial growth and / or magnetosome production or synthesis, and the other compound is preferentially a source of carbon, nitrogen, calcium, vitamins, oxygen, iron, phosphate, phosphorus, and / or magnesium. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] [Figure 1]TGA-DSC analysis of magnetosomes extracted from magnetotactic bacteria according to the conditions for whole magnetotactic bacteria and lysis. (a) Change in weight percent as a function of temperature when a sample containing 3 mg of lyophilized magnetotactic bacteria is heated at a rate of 6°C / min between 20°C and 600°C, and the derivative of this change as a function of temperature. (b) Heat flow rate in mW as a function of temperature when a sample containing 3 mg of lyophilized magnetotactic bacteria is heated at a rate of 6°C / min between 20°C and 600°C. (c) Variation in weight percentage as a function of temperature when a sample containing 3 mg of lyophilized magnetosomes is heated at a rate of 6°C / min between 20°C and 600°C under Condition 2, and the derivative of this variation as a function of temperature. (d) Heat flow rate in mW as a function of temperature when a sample containing 3 mg of lyophilized magnetosomes is heated at a rate of 6°C / min between 20°C and 600°C under Condition 2. For Figures 1(a) and 1(c), the y-axis can be replaced with percentage of mass and the plots will be the same. [Figure 2] and TGA-DSC analysis of chemically synthesized SIGMA nanoparticles. (a) Variation in weight percentage as a function of temperature of a sample containing 3 mg of lyophilized magnetosomes extracted from magnetotactic bacteria according to condition 3, and the derivative of this variation as a function of temperature. (b) Heat flow rate in mW as a function of temperature of a sample containing 3 mg of lyophilized magnetosomes extracted from magnetotactic bacteria according to condition 3. (c) Variation in weight percentage as a function of temperature of a sample containing 3 mg of lyophilized SIGMA nanoparticles, and the derivative of this variation as a function of temperature. (d) Heat flow rate in mW as a function of temperature of a sample containing 3 mg of lyophilized SIGMA nanoparticles. For Figures 2(a) and 2(c), the same plots are obtained by replacing the y-axis with percentage of mass. [Figure 3]This is an exemplary example of the use of the method according to the present invention after a series of preliminary growth steps 1, i, and i+1, where the preliminary growth step is initiated by inserting nanoparticle-producing cells into a cell bank (usually 107 cells) in a volume of preliminary growth step VPGS1 (usually ~50 mL). The nanoparticle-producing cells are grown in this volume for typically ~7 days. The nanoparticle-producing cells are then transferred from VPGS1 to VPGSi (VPGSi typically ~500 mL), where they are grown in this volume for typically ~3 days. The nanoparticle-producing cells are then transferred from VPGSi to VPGSi+1 (VPGS i+1 typically ~5 L), where they are grown in this for typically 3 days to typically reach an OD (optical density) of 1. They are then transferred to VGS0 (usually 45 L), where VGS0 is supplemented with fed-batch medium (usually 5-10 L) during a ~5-day growth phase in a growth medium with oxygen bubbles, allowing bacterial growth to typically reach an OD of 5 to 40, and the production of 5 to 500 mg of magnetosomes per liter of growth medium. Here, the pre-growth medium contains a limited concentration of iron or an iron source (preferably <2 μM) to prevent nanoparticle formation, while the growth medium contains iron or an iron source (preferably >2 μM) to promote nanoparticle formation. [Figure 4] Concentrations of carbon, nitrogen, and iron sources C1 and C2 at the start and end of the preliminary growth stage. [Figure 5] and [Figure 6] Concentrations of carbon source, nitrogen source, and iron source C1 and C2 at the start, 20 hours after the start, 40 hours after the start, and for more than 40 hours after the start of the growth step. Detailed description of the invention

[0007] In one embodiment of the present invention, the nanoparticle-producing cell is preferably a cell having the ability to produce or synthesize nanoparticles, preferably a eukaryote or prokaryote, and when inserted into or grown in a medium containing at least one compound, it produces 10 nanoparticles. -6 , 1 or 10 6 It is present at concentrations exceeding μM.

[0008] In one embodiment of the present invention, nanoparticle-producing cells are characterized in that these cells undergo preliminary growth or growth or 10 per liter of fed-batch medium. 50 , 10 10 , 10 5 , 10 3 , 10, 1, 10 -1 , 10 -3 or 10 -5 When producing nanoparticles of a mg or less, or when the pre-growth or growth or fed-batch medium produces nanoparticle-producing cells (s) 10 per liter of pre-growth or growth or fed-batch medium 50 , 10 10 , 10 5 , 10 3 , 10, 1, 10 -1 , 10 -3 or 10 -5 If it contains nanoparticles in mg or less, it does not inherently produce nanoparticles.

[0009] In one embodiment of the present invention, nanoparticle-producing cells are characterized by these cells having a preliminary growth or growth or a production of 0.10 per liter of fed-batch medium. -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1, 10, 10 3 or 10 5 If producing more than mg, or if the amount is 0.10 per liter of pre-growth, growth, or fed-batch medium. -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1, 10, 10 3 or 10 5 If the amount of nanoparticles exceeds a certain mg, nanoparticles will be produced.

[0010] In one embodiment of the present invention, the amount of nanoparticles produced by the nanoparticle cell is equal to Q2-Q1, where Q1 and Q2 are the amounts of nanoparticles produced at times t1 and t2 of the preliminary growth and / or growth step, where t2 is greater than t1, and preferably t2 / t1 is 1, 2, 5, 10, or 10 3Larger and preferred, t2 is the end of preliminary growth and / or, and t1 is the start of preliminary growth and / or.

[0011] In one embodiment of the present invention, the amount of nanoparticles generated by the nanoparticle cell in the growth step is equal to Q'2 - Q'1, where Q'1 and Q'2 are the amounts of nanoparticles generated at times t'1 and t'2. The growth step t'2 is greater than t'1, and t'2 / t'1 is 1, 2, 5, 10, or 10 3 Larger, t'2 is the end of the growth step, and t'1 is the beginning of the growth step. Preferentially, Q'2-Q'1 is at least 0, 10⁻¹, 10⁻¹, 1, 5, 10, 10³, or 10⁵ times larger than Q2-Q1. Prioritizing the total amount of nanoparticles produced by nanoparticle-producing cells, Q total This is equal to = Q'2-Q'1 + Q2-Q1.

[0012] In some cases, Qtotal is preferably greater than 10⁻⁵, 10⁻¹, 10⁻⁵, 10⁻³, 10⁻¹, 0, 1, 5, 10, or 100 mg of iron per liter of pre-growth medium and / or growth medium, preferably in nanoparticles, preferably in the nanoparticles.

[0013] In some other cases, Q total This preferentially contains 10% iron in nanoparticles per liter of pre-growth and / or growth medium / culture medium. 50 , 10 10 , 10 5 , 10 3 The dosage may be 100, 50, 10, or less than 100 mg.

[0014] In one embodiment of the present invention, yeast extract is i) all yeast extract, ii) a culture medium containing 1, 10, 50, or 90% or more of compounds from the whole yeast extract, and iii) one element of a chemical equivalent of yeast extract.

[0015] In one embodiment of the present invention, peptone is an element selected from the group consisting of i) total peptone, ii) a medium containing more than 1, 10, 50, or 90% of compounds derived from total peptone, and iii) a chemical equivalent of peptone.

[0016] The present invention relates to a method for producing high-purity iron oxide nanoparticles using nanoparticle-producing cells, comprising a preliminary growth step which includes preferentially growing nanoparticle-producing cells without essentially generating nanoparticles, followed by a growth step which includes growing the nanoparticle-producing cells resulting from the preliminary growth step in a growth medium while generating nanoparticles, wherein the growth step differs from the preliminary growth step by at least one feature selected from the group consisting of: i) CFeGS / CFePGS ratio of 0, 10 -5 , 10 -3 , 1, 10, 10 3 , or 10 5 Greater than, where CFeGS and CFePGS are the concentrations of iron or iron source in the growth medium and pre-growth medium, respectively. ii) CCGS / CCPGS ratio of 0, 10 -5 , 10 -3 , 1, 10, 10 3 , or 10 5 Larger values, where CCGS and CCPGS are the concentrations of carbon or carbon source in the growth medium and pre-growth medium, respectively. iii) CNGS / CNPGS ratio of 0, 10 -5 , 10 -3 , 1, 10, 10 3 , or 10 5 Larger values, where CNGS and CNPGS are the concentrations of nitrogen or nitrogen source in the growth medium and pre-growth medium, respectively. iv) D pHGS / DpH PGS The ratio is 0, 10 10 , 10 5 , 10 3 , 10 2 , 1, 0.5 or less, or 0.1 or less, where DpHGS and DpH PGS These represent the pH changes of the growth medium and the preliminary growth medium, respectively. v) QGGS / QGPGS ratio of 0, 10 10 , 10 5 , 10 3 , 10 2 , 0.5 or less, where QGGS and QGPGS are the amounts of gas, oxygen, or air introduced or bubbled into the growth medium and pre-growth medium, respectively. vi) Ratio of NSSGS / NSSPGS is 0, 10 -5 , 10 -3 , 1, 10, 10 3 , or 10 5 Larger than . Here, NSSGS and NSSPGS are the number of substeps in the growth step and the number of substeps in the preliminary growth step, respectively, and the two substeps are separated from each other by transferring the nanoparticle-producing cells from the first substep to the second substep, and vii) The growth medium is supplemented with fed-batch medium, but the reserve growth medium is not supplemented with such medium. Here, preliminary growth, growth, or sulfurized medium may or may not include: I. At least one compound or aggregate of compounds contained in or derived from a yeast extract selected from the following groups: I1) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium with more than gram / liter of protein, I2) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium for nucleic acids in grams / liter or more, I3) 0, 10 -10 , 10 -5 , 10 -1 , 1, 103 , or 10 5 a pre-growth, growth or fed-batch medium containing not less than grams per liter of glutathione, I4) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 a pre-growth, growth or fed-batch medium containing not less than grams per liter of at least one compound selected from the group consisting of dextran, mannan, trehalose, flavor nucleotides, B vitamins, biotin, and volatile aromatic compounds, I5) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 a pre-growth, growth or fed-batch medium containing not less than grams per liter of calcium, I6) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 a pre-growth, growth or fed-batch medium containing not less than grams per liter of phosphorus, I7) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 a pre-growth, growth or fed-batch medium containing not less than grams per liter of zinc, I8) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 a pre-growth, growth or fed-batch medium containing not less than grams per liter of chromium, I9) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 a pre-growth, growth or fed-batch medium containing not less than grams per liter of potassium, I10) 0, 10 -10 , 10 -5, 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium with cobalt in grams / liter or more, I11) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing manganese in grams / liter or more, I12) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or feeding-batch medium of strontium in grams / liter or more, I13) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or feeding-batch medium with magnesium in grams / liter or more, I14) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium of yeast extract in grams / liter or more, and I15) Ingredients derived from 0, 1, 5, 10, 15, 20 or 50 or more types of yeast extracts, II. At least one compound or set of compounds contained in or derived from peptone selected from the following groups: II 1) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium of ash in grams / liter or more, II 2) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 105 Pre-growth or growth or fed-batch medium of protein in grams / liter or more, II 3) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium with sucrose in grams / liter or more, II 4) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium with raffinose in grams / liter or more, II 5) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium of neutral detergent fibers at a concentration of grams / liter or more, II 6) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium of ether extracts in grams / liter or more, II 7) 0, 10 -10 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium with peptone in grams / liter or more, and II 8) Components derived from 0, 1, 5, 10, 15, 20 or 50 or more types of peptones III. At least one compound or aggregate of compounds contained in or derived from a wolf mineral or mineral elixir, selected from the following groups: III 1) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5Pre-growth, growth, or fed-batch medium containing more than one mole of nitrilotriacetic acid per liter, III 2) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than 1 mole of magnesium sulfate per liter, III 3) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of sodium chloride per liter, III 4) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than 1 mole of manganese sulfate per liter, III 5) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of ferrous sulfate per liter, III 6) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of cobalt nitrate per liter, III 7) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of calcium chloride per liter, III 8) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 105 Pre-growth or growth or fed-batch medium containing more than one mole of zinc sulfate per liter, III 9) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of copper sulfate per liter, III 10) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole per liter of Wolf's mineral or mineral elixir-derived potassium aluminum sulfate, III 11) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of boric acid per liter, III 12) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than 1 mole of sodium molybdate per liter, III 13) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than 1 mole of sodium selenite per liter, III 14) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of sodium tungstate per liter, III 15) 0, 10 -20 , 10 -5 , 10-1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of nickel chloride per liter, III 16) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Wolf's Minerals or Mineral Elixir 22 in pre-growth, growth, or fed-batch medium in amounts of more than 1 molar per liter. III 17) Various components of Wolf's Minerals or Mineral Elixirs, 0, 10 or more IV. 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one molar of EDTA per liter, and / or V. Select from the following groups: at least one compound or group of compounds contained in or derived from Wolf's vitamin. i) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or feeding-batch medium containing more than one mole of biotin per liter, ii) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of calcium pantothenate per liter, iii) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or feeding-batch medium containing more than one mole of folic acid per liter, iv) 0, 10 -20 , 10 -5 , 10-1 , 1, 10 3 , or 10 5 Pre-growth, growth, or fed-batch medium containing more than one mole of inositol per liter, v) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of nicotinic acid per liter, vi) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of p-aminobenzoic acid per liter, vii) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth, growth, or fed-batch medium containing more than one molar of pyridoxine HCl per liter, viii) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of riboflavin per liter, ix) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of thiamine HCl per liter, x) 0, 10 -20 , 10 -5 , 10 -1 , 1, 10 3 , or 10 5 Pre-growth or growth or fed-batch medium containing more than one mole of thioctic acid per liter, xi) 0, 10 -20 , 10 -5 , 10 -1 , 1, 103 , or 10 5 At least one component of wolf vitamins in amounts greater than or equal to one mole per liter, and xii) 0, 1, 5, 10, 15, 20 or more types of wolf vitamins, And, or, here, the pre-growth, growth, or fed-batch medium, when measured, contains or does not contain the following per mg, per g, per milliliter, or per liter of pre-growth, growth, or fed-batch medium: 1) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of protein derived from yeast extract. 2) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of nucleic acid, derived from yeast extract. 3) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of glutathione, derived from yeast extract. 4) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 or at least one compound derived from yeast extract selected from the group of dextran, mannan, trehalose, flavor nucleotides, B vitamins, biotin, and volatile aromatic compounds, 5) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of calcium, derived from yeast extract. 6) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of phosphorus, derived from yeast extract. 7) 0, 10 -20 , 10 -10 , 10-5 , 10 -3 Or 1 gram of zinc, derived from yeast extract. 8) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of chromium, derived from yeast extract. 9) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of potassium, derived from yeast extract. 10) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of cobalt, derived from yeast extract. 11) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of manganese, derived from yeast extract. 12) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of strontium, derived from yeast extract. 13) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of magnesium, derived from yeast extract. 14) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of yeast extract, 15) 0, 1, 5, 10, 15, 20 or more ingredients derived from yeast extract, 16) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of ash, derived from peptone. 17) 0, 10-20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of protein derived from peptone, 18) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of sucrose, derived from peptone. 19) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of raffinose, derived from peptone. 20) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of neutral detergent fiber derived from peptone. 21) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of ether extract, derived from peptone. 22) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of raffinose, derived from peptone. 23) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of peptone, 24) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of nitrilotriacetic acid derived from Wolf's mineral or mineral elixir. 25) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of magnesium sulfate derived from Wolf's mineral or mineral elixir. 26) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of sodium chloride derived from Wolf's mineral or mineral elixir. 27) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of manganese sulfate derived from Wolf's mineral or mineral elixir. 28) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of ferrous sulfate derived from Wolf's mineral or mineral elixir. 29) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of cobalt nitrate derived from Wolf's mineral or mineral elixir. 30) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of calcium chloride derived from Wolf's mineral or mineral elixir. 31) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of zinc sulfate derived from Wolf's mineral or mineral elixir. 32) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of copper sulfate derived from Wolf's mineral or mineral elixir. 33) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of aluminum potassium derived from Wolf's mineral or mineral elixir. 34) 0, 10 -20 , 10-10 , 10 -5 , 10 -3 Or 1 gram of boric acid derived from Wolf's mineral or mineral elixir. 35) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of sodium molybdate derived from Wolf's mineral or mineral elixir. 36) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of sodium selenite derived from Wolf's mineral or mineral elixir. 37) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of sodium tungstate derived from Wolf's mineral or mineral elixir. 38) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of nickel chloride derived from Wolf's mineral or mineral elixir. 39) 0, 10 -20 , 10 -10 , 10 -5 , 10 -3 Or 1 gram of Wolf's mineral or mineral elixir, 40) Wolf's Minerals or Mineral Elixir with 0, 1, 5, 10, or 20 ingredients, 41) 0, 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1 mole of EDTA, 42) 0, 10 derived from wolf vitamins -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or 1 mole of biotin, 43) 0, 10 -50, 10 -30 , 10 -9 , 10 -3 , 10 -1 Or calcium derived from 1 mole of wolf vitamins. 44) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or folic acid derived from 1 mole of wolf vitamins, 45) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or inositol derived from 1 mole of wolf vitamins, 46) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or nicotinic acid derived from 1 mole of wolf vitamin, 47) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or p-aminobenzoic acid derived from 1 mole of wolf vitamin, 48) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or pyridoxine HCl derived from 1 mole of wolf vitamin, 49) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or riboflavin derived from 1 mole of wolf vitamin, 50) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or thiamine HCl derived from 1 mole of wolf vitamins, 51) 0, 10-50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or thioctic acid derived from 1 mole of wolf vitamin, 52) 0, 10 -50 , 10 -30 , 10 -9 , 10 -3 , 10 -1 Or one component of 1 mole of wolf vitamins, 53) 0, 1, 5, 10 or 20 types of wolf vitamins, 54) 0, 1, 2, 3, 6, 10 or 100 kinds of vitamins, 55) 0, 10 -50 , 10 -20 , 10 -9 , 10 -8 , 10 -7 , 10 -5 , 10 -3 , 10 -1 Or at least one vitamin in 1 mole, 56) 0, 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -4 , 10 -2 , 10 -1 , 1, 10 or 10 3 g of yeast extract, 57) 0, 10 -50 , 10 -20 , 10 -9 , 10 -3 , 10 -1 , 1, 5 or 10 moles of yeast extract, at least one component 58) 0, 1, 2, 5, 10 or 100 types of yeast extract components, 59) 0, 10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 10 or 10 3 g of peptone, 60) 0, 10 -50 , 10 -9 , 10 -5, 10 -3 , 10 -1 , 1 or 10 moles of peptone component, 61) 0, 1, 2, 5, 10, or 100 types of peptone components, 62) 0, 1, 2, 5, 10 or 100 types of CMR catalysts, 63) 0, 10 -50 , 10 -9 , 10 -5 , 0.05, 10 -1 , 1, 10, 10 3 or 10 6 mg of at least one CMR catalyst, 64) 0, 1, 2, 5, 10 or 100 types of chelating agents, 65) 0, 10 -50 , 10 -20 , 10 -9 , 10 -8 , 10 -3 , 10 -1 , 1, 5, 10 or 10 3 Moles of at least one chelating agent, 66) 0, 1, 2, 5, 10 or 100 types of amino acids, 67) 0, 10 -50 , 10 -10 , 10 -5 , 10 -3 , 1, 10, 10 3 , 10 5 or 10 10 mg of at least one amino acid, 68) 0, 1, 2, 5, 10, or 100 types of toxic or cytotoxic compounds, 69) 0, 10 -50 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 mg of at least one toxic or cytotoxic compound, 70) 0, 1, 3 or 7 types of heavy metals other than iron, 71) 0, 10 -50 , 10 -10 , 10-5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 mg of heavy metals other than iron, 72) A metal or chemical element selected from cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, and copper, in the order of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. 73) 1 mg of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, copper, 74) 0, 10 -50 , 10 -10 , 0.5, 1, 5, 10, 10 3 or 10 6 mL or 10 -50 , 10 -30 , 10 -10 , 10 -8 , 10 -5 , 10 -3 , 1, 10, 10 3 or 10 6 Mole Wolf Vitamins 75) 10 -50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 Moles have at least one wolf vitamin, 76) 0, 1, 2, 5, 10 or 100 types of wolf vitamin components, 77) 0, 10 -50 , 10 -10 , 0.5, 1, 5, 10, 10 3 or 10 6 mL or 10 -50 , 10 -30 , 10 -10 , 10 -8 , 10 -5 , 10 -3 , 1, 10, 10 3 or 10 6 Mole Wolf Minerals 78) 10-50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 Mole's at least one wolf mineral component, 79) 0, 1, 2, 5, 7, 10, 15 or 100 types of wolf mineral components, 80) 10 -50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 Mole mineral elixir, 81) 10 -50 , 10 -20 , 10 -9 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 or 10 5 At least one component of the mole mineral elixir, and / or 82) 0, 1, 2, 5, 10, 14, or 100 types of mineral elixirs.

[0017] The present invention also relates to a fed-batch medium comprising at least one compound selected from the group consisting of iron, iron source, carbon, carbon source, nitrogen, nitrogen source, and combinations thereof, and wherein the concentration of at least one compound in the fed-batch medium is 10 -6 μM, 1 μM, or 10 6 The present invention relates to a method with a magnitude greater than μM.

[0018] In one embodiment of the present invention, at least one vitamin, a component of Wolf's vitamin, or a vitamin solution is selected from the following group: folic acid, folate, pyridoxine, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, nicotinic acid, pantothenic acid, vitamin B 12This product contains alternative provitamin A functional carotenoids including aminobenzoic acid, thionic acid, all-trans retinol, retinal, all-trans beta-carotene, niacin, niacinamide, nicotinamide, riboside, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ergocalciferol, tocopherol, tocotrienol, phylloquinone, menaquinone, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, Vitamin V i (V is any letter from A to Z, i is any integer from 1 to 100), and their derivatives.

[0019] In another embodiment of the present invention, at least one component of Wolf's mineral is selected from the following group: nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate heptahydrate, copper sulfate hydrate, aluminum potassium sulfate decahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate dihydrate, nickel chloride, and derivatives thereof.

[0020] In another embodiment of the present invention, at least one component of the yeast extract is selected from the following groups: i) at least one protein, ii) at least one nucleic acid, iii) at least one functional peptide, iv) glutathione, v) dextran, vi) mannan, vii) trehalose, viiii) flavor nucleotide, ix) vitamin B, x) biotin, x) at least one volatile aromatic compound, xi) calcium, xii) phosphorus, xiii) zinc, xiv) iron, xv) chromium, xvi) potassium, xvii) cobalt, xviii) manganese, xix) strontium, xx) magnesium, and xxi) derivatives thereof.

[0021] In another embodiment of the present invention, at least one component of the mineral elixir is selected from the following group: nitrilotriacetic acid, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3, and derivatives thereof.

[0022] In one embodiment of the present invention, the statement that a pre-growth, growth, or fed-batch medium contains or does not contain a certain amount or concentration or number of at least one compound is equivalent to the statement that a pre-growth, growth, or fed-batch medium contains no more than or equal to a certain amount or concentration or number of at least one compound.

[0023] In one embodiment of the present invention, nanoparticle-producing cells that are grown or expanded in a group of pre-growth and / or growth and / or fed-batch media are preferably grown or expanded in the pre-growth and / or growth media and not in the fed-batch media.

[0024] In one embodiment of the present invention, the growth and / or pre-growth medium comprises either i) a pre-growth medium and / or growth medium before and / or in which a fed-batch medium is inserted into the growth medium, or ii) a growth medium and / or pre-growth medium in which a fed-batch medium is present and after it has been inserted.

[0025] In one embodiment of the present invention, the fed-batch medium is the medium before it is inserted into the fed-batch medium or growth and / or pre-growth medium.

[0026] In another embodiment of the present invention, the fed-batch medium is a portion of the pre-growth and / or growth medium after being inserted into the growth and / or pre-growth medium.

[0027] In one embodiment of the present invention, the parameters CFEGS, CFePGS, CCGS, CCPGS, CNGS, CNPGS, and ΔpH are used. GS , and / or ΔpH PGSIt is present in or measured in the preliminary growth, growth, and / or fed-batch medium.

[0028] In one embodiment of the present invention, parameter Q GGS Q GPGS , N SSGS , and N SSPGS Furthermore, the situation in which feed-batch medium is added to the growth medium occurs during, at the start of, or at the end of the pre-growth and / or growth stage, although such medium is not added to the pre-growth medium.

[0029] In one embodiment of the present invention, the derivative is a derivative of at least one first compound selected from the following groups: i) a second compound which differs from at least one first compound by at least one different atom or functional group, selectively derived from at least one first compound after conversion of at least one first compound, and selectively sharing at least one atom or functional group common to at least one first compound; ii) an unhydrated form of at least one first compound; iii) a hydrated form of at least one first compound; iv) a reduced form of at least one first compound; v) an oxidized form of at least one first compound; vi) an acidic form of at least one first compound; vii) a basic form of at least one first compound; viiii) a crystalline or solid form of at least one first compound; ix) a soluble or solubilized form of at least one first compound; and x) a salt of at least one first compound.

[0030] The present invention relates to a method according to the present invention, wherein the growth step differs from the preliminary growth step by at least one characteristic selected from the group consisting of: I C FeGS / C FePGS The ratio is 10 -5 , 10 -3 , 1, 10, 10 3 , or 10 5 It is larger than C FeGS and C FePGS These represent the concentrations of iron or iron source in the growth medium and pre-growth medium, respectively. ii) CCGS / C CPGS The ratio is 10 -5 , 10 -3 , 1, 10, 10 3 , or 10 5 It is larger than C CGS and C CPGS These represent the concentrations of carbon or carbon source in the growth medium and pre-growth medium, respectively. iii) C NGS / C NPGS The ratio is 10 -5 , 10 -3 , 1, 10, 10 3 , or 10 5 It is larger than C NGS and C NPGS These represent the concentrations of nitrogen or nitrogen source in the growth medium and pre-growth medium, respectively. iv) DpH GS / DpH PGS The ratio is 10 10 , 10 5 , 10 3 , 10 2 It is 1, 0.5, or 0.1 or less. Here, D pHGS and DpH PGS These represent the pH changes of the growth medium and the preliminary growth medium, respectively. v)Q GGS / Q GPGS 10 in proportion 10 , 10 5 , 10 3 , 10 2 Q GGS and Q GPGS These represent the amounts of gas, oxygen, or air introduced or bubbled into the growth medium and pre-growth medium, respectively. vi)N SSGS / N SSPGS 10 in proportion -5 , 10 -3 , 1, 10, 10 3 , or 10 5 Something larger than N. SSGS and N SSPGSThese represent the number of substeps in the growth step and the number of substeps in the preliminary growth step, where the two substeps are separated from each other by the movement of nanoparticle-producing cells from the first substep to the second substep, which is preferentially related to the growth of nanoparticle-producing cells in the first quantity, and from the second substep, which is preferentially related to the growth of nanoparticle-producing cells in the second quantity. vii) The growth medium is supplemented with fed-batch medium, but the reserve growth medium is not supplemented with such medium.

[0031] The present invention relates to a method according to the present invention, wherein the pre-growth and / or growth and / or fed-batch medium comprises no more than the following amounts per kilogram or liter of pre-growth and / or growth and / or fed-batch medium: i) 103% by mass or volume, or 1% by mass or volume, or 10-2% by mass or volume, or 5.10-3% by mass or volume, or 103 grams, or 10 2 grams, or 10 grams, or 10 10 mL, or 10 5 mL, or 10 3 mL, or 10 mL, or 5 mL, or 1 mL, or 0.5 mL, or 10 -5 mL, or 10 3 mol, or 10 mol, or 1 mol, or 10 -5 mol, or 10 -8 mol, or 10 -9 mol, or 10 -10A mole of Wolf's vitamins or vitamin solutions or chemical compounds selected from the following groups: folic acid, pyridoxine, pyridoxine hydrochloride, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, thiamine HCl, nicotinic acid, pantothenic acid, calcium pantothenate, inositol, p-aminobenzoic acid, aminobenzoic acid, thionic acid, all-trans-retinol, retinal, all-trans-β-carotene, niacin, niacinamide, nicotinamide, riboside, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ercocalciferone, tocopherol, tocopherol, tocopherol, tocopherol, tocopherol, tocopherol, tocopherol, tocopherol Vitamin A, Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B7, Vitamin B9, Vitamin B 12 Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, Vitamin V i Any letter from A to Z, where i is any integer from 1 to 100, and its derivatives. ii) iii) Wolf's vitamins or vitamin solutions selected from the following groups of 1, 5, 6, 10, or 20: folic acid, folate, pyridoxine, pyridoxine HCl, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, thiamine HCl, nicotinic acid, pantothenic acid, calcium pantothenate, vitamin B12, aminobenzoic acid, inositol, p-aminobenzoic acid, pyridoxine HCl, thionic acid, all-trans retinol, retinal, all-trans beta-carotene, niacin, niacinamide, nicotinamide, riboside, cyanocobalamin, hydroxocobalamin, methylcobalamin, adenosylcobalamin, ascorbic acid, cholecalciferol, turquoise, ergocalcopherol, ergocalcopherol, ergocalcopherol, ergocalco phylloquinone, menaquinone, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 Vitamin C, Vitamin D, Vitamin D2, Vitamin D3, Vitamin E, Vitamin K, V i Here, V is any letter from A to Z, i is an integer from 1 to 100, and their derivatives, iv) 1 mL or 10 mL selected from the following groups -7 Moles of Wolf's minerals or mineral elixirs or trace elements: nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, copper sulfate hydrate, aluminum potassium sulfate, aluminum potassium sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, and their derivatives. v) 103% of mass or volume, or 10% of mass or volume, or 1% of mass or volume, or 10-2% of mass or volume, or 10% of mass or volume -5 %, or 10 3 grams, or 10 grams, or 1 gram, or 10 5 mL, or 103 mL, or 10 mL, or 1 mL, or 10 -3 mL, or 10 3 mol, or 10 mol, or 1 mol, or 10 -3 mol, or 10 - 7 moles, or 10 -8 moles, or 10 -10 Molar minerals, preferably Wolf's minerals or mineral elixirs, or chemical components selected from the following group: nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, copper sulfate hydrate, aluminum potassium sulfate, aluminum potassium sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3, and their derivatives. vi) Minerals, preferably 1, 3, 7, or 10 components of Wolf's Minerals or Mineral Elixir, or chemical components selected from the following group: nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, copper sulfate hydrate, aluminum potassium sulfate, aluminum potassium sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, MgSO4, MnSO4, NaCl, FeSO4, CoSO4, CaCl2, ZnSO4, CuSO4, KAl(SO4)2, H3BO3, Na2MoO4, NiCl2, Na2SeO3, and their derivatives. vii)10 -50 gram, 10 -10 gram, 10 -5 grams, 0.005 grams, 10 -1gram, 1 gram, 10 grams or 10 5 grams or 10 -50 M, 10 -8 M, 10 -3 M, 1M, 10 3 At least one component of M's yeast extract or at least one component derived from the yeast extract, selected from the following group of compounds: at least one protein, at least one nucleic acid, at least one functional peptide, glutathione, dextran, mannan, trehalose, flavor nucleotide, vitamin B, biotin, at least one volatile aromatic compound, calcium, phosphorus, zinc, iron, chromium or chromium, potassium, cobalt, manganese, strontium, magnesium, and their derivatives. viii) 1, 2, 3, 5, 10, 15, 20, or 50 components of yeast extract or components derived from yeast extract selected from the following groups: at least one protein, at least one nucleic acid, at least one functional peptide, glutathione, dextran, mannan, trehalose, flavor nucleotides, vitamin B, biotin, at least one volatile aromatic compound, calcium, phosphorus, zinc, iron, chromium or chromium, potassium, cobalt, manganese, strontium, magnesium, and their derivatives. ix)10 -50 gram, 10 -10 gram, 10 -3 gram, 0.01 gram, 1 gram, 5 grams, 10 grams or 10 5 grams, or 10 -50 M, 10 -20 M, 10 -8 M, 10 -3 M, 10 -1 M, 1M, 10M, or 10 3 At least one component of peptone M, or at least one compound derived from peptone selected from the following group: ash, protein, sucrose, stachyose, raffinose, neutral detergent fiber, ether extract, and derivatives thereof. x) 1, 3, 5, 10, 20, or 50 types of peptone components, or compounds derived from peptone selected from the following groups: ash, protein, sucrose, stachyose, raffinose, neutral detergent fibers, ether extracts, and their derivatives. xi)10 -50 , 10 -10 , 10 -5 , 0.001, 10 -1 , 1, 10, 10 3 or 10 5 Grams of EDTA, xii)10 -50 , 10 -10 , 10 -5 , 0.001, 10 -1 , 1, 10, 10 3 or 10 5 Grams of at least one amino acid, xiii) 1, 3, 5, 10, 20, or 50 types of amino acids, xiv) 1, 5, 7, 12, 15, 20 or 50 CMRs, toxic or cytotoxic compounds selected from the following groups: nitrilotriacetic acid, manganese sulfate, cobalt nitrate, zinc sulfate, copper sulfate, aluminum potassium sulfate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, nickel chloride, and their derivatives. xv) Elements or heavy metals selected from the following groups: 1, 2, 5, 10, 50, or 100: cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, and copper. xvi)10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 10, 10 3 , or 10 5 Gram's toxic or cytotoxic compounds selected from the following groups: nitrilotriacetic acid, manganese sulfate, cobalt nitrate, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, nickel chloride, and / or their derivatives, and / or xvii)10 -50 , 10 -10 , 10 -5 , 0.01, 10-1, 1, 10, 10 3 or 10 5 Grams of peptone.

[0032] The present invention also relates to a method according to the present invention, wherein, preferentially, the concentration of at least one compound in the pre-growth and / or growth medium is concentration C2 or concentration C total = C1 + C2, and here, - C1 is the concentration of at least one compound in the pre-growth and / or growth medium that is not consumed by the nanoparticle-producing cells. - C2 is the concentration of at least one compound in the pre-growth and / or growth medium consumed by the nanoparticle-producing cells. - C1 and / or C2 are preferentially measured, separated, or distinguished using a method capable of separating at least one compound consumed by nanoparticle-producing cells from at least one compound not consumed by nanoparticle-producing cells, such as centrifugation or tangential filtration, and further, - C1 and / or C2 are preferentially measured or considered at the start, during, or end of the preliminary growth and / or growth step.

[0033] In one embodiment of the present invention, one compound of the pre-growth and / or growth medium consumed by the nanoparticle-producing cells is one compound contained in the nanoparticle cells if the nanoparticle-producing cells consume such a compound.

[0034] In another embodiment of the present invention, one compound of the pre-growth and / or growth medium that is not consumed by nanoparticle-producing cells is, preferentially, one compound that is contained outside the nanoparticle-producing cells, if such a compound is not consumed by the nanoparticle-producing cells.

[0035] In one embodiment, a method is used to separate consumable compounds from non-consumable compounds. This method preferentially separates the entire bacteria from the pre-growth and / or growth medium that does not contain such bacteria. This method is capable of separating the entire bacteria from a liquid medium by centrifugation or filtration, preferably tangential filtration, or by separating the low-size and / or low-weight elements contained in the pre-growth and / or growth medium from the large-size and / or high-weight elements in the total bacteria.

[0036] The present invention also relates to a method according to the present invention, wherein the concentration of at least one compound contained in a fed-batch medium, preferably iron, an iron source, carbon, a carbon source, nitrogen, and / or a nitrogen source, is 10 -6 μM, 1 μM, and / or 10 6 Larger than μM

[0037] The present invention also relates to a method according to the present invention, wherein the pre-growth, growth, and / or fed-batch medium does not contain at least one component selected from the following group in a concentration that preferentially affects the growth of nanoparticle-producing cells / or the production of nanoparticles, and / or the pre-growth, growth, and / or fed-batch medium does not contain at least one component selected from the following group: 1) A medium containing more than half of the total number of components of Wolf's Vitamin or various components of Wolf's Vitamin, 2) One component of Wolf's Vitamin, 3) Folic acid, 4) Pyridoxine, 5) Riboflavin, 6) Biotin, 7) Thiamine, 8) Nicotinic acid, 9) Pantothenic acid, 10) Vitamin B 1211) Aminobenzoic acid, 12) Thioic acid, 13) Medium containing Wolf's Mineral or more than half of the total number of different components of Wolf's Mineral, 14) Nitrilotriacetic acid, 15) Magnesium sulfate, 16) Sodium chloride, 17) Manganese sulfate, 18) Ferrous sulfate heptahydrate, 19) Cobalt nitrate, 20) Calcium chloride, 21) Zinc sulfate heptahydrate, 22) Copper sulfate hydrate, 23) Aluminum sulfate dodecahydrate, 24) Boric acid, 25) Sodium molybdate, 26) Sodium selenite, 27) Sodium tungstate dihydrate, 28) Culture medium containing more than half the total number of different components of yeast extract, 29) Culture medium containing more than half the total number of different components of yeast extract or yeast extract equivalent, 30) 1, 2, or 5 proteins derived from or contained in yeast extract, 31) 1, 2, or 5 nucleic acids derived from or contained in yeast extract, 32) 1, 2, or 5 peptides or functional peptides derived from or contained in yeast extract, 33) Glutathione, 34 ) Dextran, 35) Mannan, 36) Trehalose, 37) Flavor nucleotides derived from or contained in yeast extract, 38) Vitamin B, 39) Biotin, 40) Volatile aromatic compounds 1, 2 or 5 derived from or contained in yeast extract, 41) Chromium, 42) Cobalt, 43) Strontium, 44) Nickel chloride, 45) or a medium containing more than half of the total number of different components of a mineral elixir, 46) MnSO4, 47) NaCl, 48) FeSO4, 49) CoSO4, 50) CaCl2, 51) ZnSO4, 52) CuSO4, 53) KAl(SO4)2, 54) H3BO3, 55) Na2MoO4, 56) NiCl2, 57) Na2SeO3, 58) a medium containing more than half the total number of peptone or various components of peptone, 59) one component of peptone, 60) one, two or five proteins derived from or contained in peptone, 61) sugars derived from or contained in peptone, 62) one amino acid derived from or contained in peptone, 63) ash derived from or contained in peptone, 64) one fiber derived from or contained in peptone, 65) one CMR agent,66) Boric acid, 67) One amino acid, 68) Alanine, 69) Arginine, 70) Asparagine, 71) Aspartic acid, 72) Cysteine, 73) Glutamine, 74) Glutamic acid, 75) Glycine, 76) Histidine, 77) Isoleucine, 78) Leucine, 79) Lysine, 80) Methionine, 81) Phenylalanine, 82) Proline, 83) Serine, 84) Threonine, 85) Tryptophan, 86) Tyrosine, 87) Valine, 88) One cytotoxic or toxic compound, 89) Manganese sulfate, 90) Copper sulfate, 91) Aluminum potassium Sulfates, 92) Boric acid, 93) Sodium tungstate, 94) Heavy metals other than iron, 95) Titanium, 96) Vanadium, 97) Manganese, 98) Nickel, 99) Copper, 100) Zinc, 101) Gallium, 102) Germanium, 103) Arsenic, 104) Zirconium, 105) Niobium, 106) Molybdenum, 107) Technetium, 108) Ruthenium, 109) Rhodium, 110) Palladium, 111) Silver, 112) Cadmium, 113 ) Indium, 114) Tin, 115) Tellurium, 116) Lutetium, 117) Hafnium, 118) Tantalum, 119) Tungsten, 120) Rhenium, 121) Osmium, 122) Iridium, 123) Platinum, 125) Gold, 126) Mercury, 127) Thallium, 128) Lead, 129) Bismuth, 130) Polonium, 131) Astatine, 132) Lanthanum, 133) Cerium, 134) Praseodymium, 135) Neodymium, 136) Promethium, 137) Samarium, 138) Europium, 139) Gadolinium, 140) Terbium, 141) Dysprosium, 142) Holmium, 143) Erbium, 144) Thulium, 145) Ytterbium, 146) Actinium, 147) Thorium, 148) Protactinium, 149) Uranium, 150) Neptunium, 151) Plutonium, 152) Americium, 153) Curium, 154) Berkerium, 155) Californium, 156) Einsteinium, 157) Fermium, 158) Novelium, 159)Radium, 160) Lawrencium, 161) Rutherfordium, 162) Dubnium, 163) Seaborgium, 164) Bohrium, 165) Hassium, 166) Meitnerium, 167) Darmstadium, 168) Roentgenium, 169) Copernicium, 170) Elements 113-118, 171) Helium, 172) Lithium, 173) Beryllium, 174) Bore, 175) Fluorine, 176) Aluminum, 177) Silicon, 178) Argon, 179) Scandium, 180) Chromium, 181) Nickel, 182) Copper, 183) Selenium, 184) Brom, 185 )Krypton, 186)Rubidium, 187)Yttrium, 188)Sn, 189)Antimony, 190)Iodine, 91)Xenon, 192)Cesium, 193)Barium, 194)Lutesium, 195)Asthete, 196)Radon, 197)Francium, 198)Mendelevium, 199)Mount, 200)Ununbium, 201)Ununtrium, 202)Ununquadium, 203)Ununpentium, 204)Ununhexium, 205)Ununseptium, 206)Ununoctium, 207)Compounds of these, salts of 1) to 206), and 208)Derivatives of these.

[0038] The present invention also relates to a method according to the present invention in which the concentration of a compound that affects the growth and / or production of nanoparticle-producing cells is the following concentration in a pre-growth, growth, and / or fed-batch medium: i) 1 picomole, 1 micromol, 1 millimol, 10 -50 M, 10 -10 or 10 -5 ii) Larger than M, or ii) Pre-growth, growth and / or 10 per liter of fed-batch medium -50 , 10 - 10, 10 -5 or 10 -3 It's larger than a gram.

[0039] In one embodiment of the present invention, the concentration of a compound that affects the growth and / or production of nanoparticle-producing cells is the following concentration in the pre-growth, growth, and / or fed-batch medium: i) 10 per liter of pre-growth, growth, and / or fed-batch medium50 , 1, 10 -5 , 10 -6 , or 10 -9 M or less, or ii) 10 10 , 1, 10 -10 , or 10 -20 Less than a gram.

[0040] The present invention is a method in which a growth medium and / or a fed-batch medium is supplemented with fed-batch medium and: i) The pH of the fed-batch medium shall be at least 10 degrees higher than the pH of the pre-growth and / or pre-growth medium. -5 , 0.1, 0.5, 1, 2, 3 or 5 pH units lower, and or ii) The concentration of at least one chemical element preferentially selected from the group consisting of a) phosphorus or phosphate source, b) potassium source, c) magnesium source, d) iron source, e) vitamin source, f) calcium source, g) KH2PO4, h) MgSO4, i) FeCl3, j) thiamine, k) CaCl2, and l) derivatives thereof, is preferred to be at least 10 in the fed-batch medium than in the pre-growth and / or growth medium. -50 , 10 -10 , 0, 1.1, 5, 10 or 10 3 Twice as big.

[0041] In one embodiment, conditions i) and ii) above are verified by considering the pH and concentration of at least one compound as follows: i) Before the fed-batch medium is inserted into the preliminary growth and / or growth medium as a fed-batch medium, and / or ii) Before, during, and / or after the feed-in medium is inserted into the pre-growth and / or growth medium.

[0042] The present invention relates to a method according to the present invention, wherein: - Nanoparticle-producing cells are magnetotactic bacteria, and / or -Nanoparticles are magnetosomes.

[0043] In another embodiment of the present invention, the nanoparticles are high-purity nanoparticle-producing cells.

[0044] In another embodiment of the present invention, the nanoparticles are high-purity nanoparticles, preferably high-purity iron oxide-based nanoparticles, wherein the high-purity nanoparticles preferably contain 100, 99, 90, 50, 20, 10, 1, or 0.1% or less by mass number of an element selected from the following group: cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, copper, and derivatives, where the high-purity iron oxide-based nanoparticles contain 1, 50, 90, 93, or 99% or more of iron and / or oxygen and / or iron oxide by atomic number or mass.

[0045] The present invention also relates to a method according to the present invention, wherein the pre-growth and / or growth medium comprises a source of calcium, a source of carbon, a source of nitrogen, a source of phosphate or phosphorus, a source of sulfur, a source of iron, a source of vitamins, and a source of calcium, and: - The concentration of the carbon source in the pre-growth and / or growth medium is at least 0, 0.5, 1.1, 2, 4, 10 or 100 times greater than the concentration of at least one component selected from the group consisting of the phosphate or phosphorus source, sulfur source, vitamin source, and calcium source in the pre-growth and / or growth medium, and / or - The concentration of the nitrogen source in the pre-growth and / or growth medium is at least 0, 0.5, 1.1, 2, 4, 10, or 100 times greater than the concentration of at least one compound selected from the group consisting of phosphates or phosphorus sources, sulfur sources, vitamin sources, and calcium sources in the pre-growth and / or growth medium.

[0046] The present invention also relates to a method according to the present invention, further comprising the step of storing, growing, preparing or inserting a bank of nanoparticle-producing cells into a preliminary growth and / or growth and / or fed-batch medium, wherein such a bank preferentially contains at least 10 of the same components in the preliminary growth and / or growth and / or fed-batch medium in terms of the number of components. -50 , 10 -10 , 10 -1Preferentially stored, grown, and prepared in bank media containing 0, 1, 5, 10, 50, 70, 90, or 99% of the following: At least one compound such as vitamins, minerals, chelating agents, sucrose, and / or cryoprotectants, which is preferentially different from those in preliminary growth and / or growth and / or fed-batch media: i) Pre-growth and / or during growth and / or, or not present in the fed-batch medium, ii) Lower concentrations in bank media for preliminary growth and / or growth and / or feeding medium.

[0047] In one embodiment of the present invention, the nanoparticle-producing cells are a master, working, or research cell bank of nanoparticle-producing cells. In some cases, such a bank may preferentially contain 1, 10, or 10 nanoparticles per milliliter or liter of pre-growth and / or growth and / or feeding medium. 3 , 10 10 or 10 20 Contains more than 10 nanoparticle-producing cells. In some other cases, such a bank contains 10 100 , 10 50 , 10 20 , 10 10 , or 10 3 Contains nanoparticle-producing cells of a certain size or less.

[0048] In another embodiment of the present invention, a cell bank is an aggregate of at least one cell used to initiate the growth of nanoparticle-producing cells in pre-growth and / or growth and / or fed-batch medium.

[0049] In one embodiment of the present invention, the cell bank is prepared under the same or similar conditions as those for the preliminary growth and / or growth steps.

[0050] In another embodiment of the present invention, the cell bank is prepared by bubbling or inserting a gas into a bank medium that preferably contains a low oxygen concentration of 50, 10, or 1% or less relative to the volume of the bank medium.

[0051] The present invention further relates to a method according to the present invention, which starts with nanoparticles isolated from nanoparticle-producing cells obtained at the end of a growth step and includes a purification step for obtaining high-purity iron oxide-based nanoparticles. The purification step preferentially includes removing at least one impurity from the nanoparticles produced in the growth step, preferably using at least one heating step which increases the temperature of the nanoparticles according to the present invention produced in the growth step. The temperature of the nanoparticles in that heating step rises to a temperature Ti and is maintained at Ti for a heating time thi which is included between 1 second and 20 years, where Ti is included between 50°C and 700°C.

[0052] In one embodiment of the present invention, T i is -273, -100, -50, 0, 1, 10, 20, 50, 100, 200, 500, 700 or 10 3 It's higher than ℃.

[0053] In one embodiment of the present invention, T i is 10 20 , 10 10 , 10 5 , 10 3 , 100, 50, 10, 0, -10 or below -50°C.

[0054] In yet another embodiment of the present invention, T i is 10~10 5 , 50~10 4 , 100~10 3 The temperature ranges from 150 to 700 degrees Celsius, or from 200 to 500 degrees Celsius.

[0055] In one embodiment of the present invention, th i is 10 -30 , 10 -10 , 10 -8 , 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 It's longer than a second.

[0056] In another embodiment of the present invention, thithi is 10 100, 10 50 , 10 10 , 10 5 , 10 3 Shorter than 10, 10, 5, 2, or 1 second.

[0057] In yet another embodiment of the present invention, thi is 10 -5 It is between a second and a year, a second and 20 years, a second and a year, a second and a month, a second and a week, a second and a day, or a second and an hour.

[0058] In one embodiment of the present invention, when nanoparticles are inserted into a device used for heating, the time taken to raise the temperature from an initial temperature that preferentially corresponds to the temperature of the nanoparticles to Ti is preferentially 1.1, 5, 10, or 10 than thi. 3 It is short by coefficient.

[0059] In another embodiment of the present invention, the time to raise the temperature to Ti is at least 1.1, 5, 10, or 10 times longer than thi. 3 Twice as long.

[0060] In one embodiment of the present invention, the iron oxide-based nanoparticles are nanoparticles that preferentially contain more than 1%, 50%, 70%, 90%, or 99% by mass of iron oxide, without considering coating or excipient materials in this proportion.

[0061] The present invention also relates to high-purity nanoparticle-producing cells that can be preferentially obtained by the method according to the present invention, the high-purity nanoparticle-producing cells include: i)M FeC / M MC Based on the ratio, iron is 10 -10 Contains more than 1, 5, 10, 50, 75, 80, 90, 95, 99, or 99.9%. Here, M FeC M is the mass of iron in cells that produce high-purity nanoparticles. MC This is the mass of iron and other metals or metalloids within nanoparticle-producing cells that produce high-purity cells. ii) Based on the M1 / M2 ratio, at least one non-iron metal selected from the following group, where M1 is the mass of at least one metal selected from the following group in the high-purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high-purity iron oxide nanoparticles: sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungstates, and iii) At least one nonmetal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the M3 / M4 ratio, where M3 is the mass of at least one other nonmetal selected from the above group, and M4 is the mass of all chemical elements contained in the high-purity iron oxide nanoparticles.

[0062] The present invention also relates to 1,10 heavy metals selected from the following group. -3 , 10 -6 or 10 -9 Regarding high-purity nanoparticle-producing cells or high-purity iron oxide-based nanoparticles obtained by growing or culturing nanoparticle-producing cells in a pure culture medium containing less than %: Cobalt, manganese, zinc, nickel, silver, aluminum, arsenic, barium, cadmium, chromium, copper, molybdenum, lead, antimony, selenium, silica, titan, thallium, mercury, vanadium, gold, iridium, osmium, rhodium, ruthenium, platinum, lithium, antimony, tin, tungsten, and their derivatives. Here, this percentage is C FeM / C MM It is based on C FeM This is the concentration of iron in the pure culture medium, and CMM This refers to the concentration of iron and other metals or metalloids in the pure culture medium.

[0063] The present invention also relates to high-purity iron oxide-based nanoparticles preferentially obtained by the method according to the present invention, wherein the high-purity nanoparticle-producing cells include: i) M FeC / M MC Based on the ratio, iron is 10 -10 Contains more than 1, 5, 10, 50, 75, 80, 90, 95, 99, or 99.9%. Here, M FeC M is the mass of iron in cells that produce high-purity nanoparticles. MC This is the mass of iron and other metals or metalloids within nanoparticle-producing cells that produce high-purity cells. ii) Based on the M1 / M2 ratio, iron and at least one non-iron metal selected from the following groups, where M1 is the mass of iron and at least one metal selected from the following groups in the high-purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high-purity iron oxide nanoparticles: sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungstates, and iii) Iron and at least one nonmetal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, based on the M3 / M4 ratio, where M3 is the mass of iron and at least one other nonmetal selected from the above group, and M4 is the mass of all nonmetallic chemical elements contained in high-purity iron oxide nanoparticles.

[0064] The present invention also relates to high-purity nanoparticle-producing cells and / or high-purity iron oxide-based nanoparticles according to the present invention, wherein the metal or metalloid other than iron in the high-purity iron oxide nanoparticles is selected from the following group: cobalt, manganese, zinc, nickel, silver, aluminum, arsenic, barium, cadmium, chromium, copper, molybdate, lead, antimony, selenium, silica, titan, thallium, mercury, vanadium, gold, iridium, osmium, rhodium, ruthenium, platinum, lithium, antimony, tin, tungsten, and their derivatives.

[0065] The present invention also relates to high-purity iron oxide-based nanoparticles in which the high-purity iron oxide-based nanoparticles are magnetosomes.

[0066] The present invention also relates to a composition comprising high-purity iron oxide-based nanoparticles.

[0067] The present invention also relates to high-purity nanoparticle-producing cells, also called high-purity nanoparticle-producing cells according to the present invention, wherein the high-purity nanoparticle-producing cells are magnetotactic bacteria.

[0068] The present invention also relates to a composition comprising high-purity nanoparticle-producing cells.

[0069] The present invention also relates to compositions comprising high-purity nanoparticle-producing cells and high-purity iron oxide nanoparticles preferentially obtained by the method according to the present invention, wherein:

[0070] High-purity nanoparticle-producing cells contain 0, 1, 10, 50, 70, 90, 95, or 99% or more of the following compositions: i) Iron based on the MFeC / MMFe ratio, where MFeC is the mass of iron in high-purity nanoparticle-producing cells, and MMC is the mass of iron and other metals or metalloids in high-purity nanoparticle-producing cells. ii) Iron and at least one non-iron metal selected from the following groups based on the M1 / M2 ratio: sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungstates, where M1 is the mass of iron and at least one non-iron metal in the high-purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high-purity iron oxide nanoparticles, and / or iii) Iron based on the M3 / M4 ratio, and at least one nonmetal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, where M3 is the mass of iron and at least one other nonmetal selected from the above group in the high-purity iron oxide nanoparticles, and M4 is the mass of all nonmetals contained in the high-purity iron oxide nanoparticles.

[0071] The present invention also relates to a composition comprising high-purity nanoparticle-producing cells and / or high-purity iron oxide nanoparticles. High-purity nanoparticle-producing cells contain the following at 0%, 10⁻⁵, 10⁻¹, 10⁻⁵, 10⁻², 1%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, and 99%: i) Iron based on the MFeC / MMC ratio, where MFeC is the mass of iron in high-purity nanoparticle-producing cells, and MMC is the mass of iron and nonmetallic or metalloidal elements in high-purity nanoparticle-producing cells. ii) Iron based on the M1 / M2 ratio, and other metals selected from the following groups: sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungstates, where M1 is the mass of iron and at least one other metal selected from the above groups in the high-purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high-purity iron oxide nanoparticles, and / or iii) Iron and at least one nonmetal selected from the group consisting of the following, based on the M3 / M4 ratio: hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, oxygen, where M3 is the mass of iron and at least one other nonmetal selected from the above group in the high-purity iron oxide nanoparticles, and M4 is the mass of all nonmetal elements contained in the high-purity iron oxide nanoparticles.

[0072] High-purity iron oxide-based nanoparticles contain the following in proportion to 0, 10⁻⁵, 10⁻¹, 10⁻⁵, 10⁻³, 10⁻¹, 0, 1, 5, 10, 25, 50, 75, 93, 95, 99, or 99.9% or more: i) Iron based on the MFeN / MMN ratio, where MFeN is the mass of iron in high-purity iron oxide nanoparticles and MMN is the mass of iron and other metals or metalloids in high-purity iron oxide nanoparticles. ii) Iron and at least one non-iron metal selected from the following groups based on the ratio of M1 / M2: sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, chromium, manganese, zinc, gallium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, indium, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, lutetium, hafnium, rhenium, and tungstates, where M1 is the mass of iron and at least one non-iron metal selected from the above groups in the high-purity iron oxide nanoparticles, and M2 is the mass of all metals contained in the high-purity iron oxide nanoparticles. and or iii) Iron based on the M3 / M4 ratio, and at least one nonmetal selected from the group consisting of hydrogen, carbon, nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, iodine, helium, neon, argon, krypton, xenon, radon, and oxygen, where M3 is the mass of iron and at least one nonmetal selected from the above group in the high-purity iron oxide nanoparticles, and M4 is the mass of all nonmetals contained in the high-purity iron oxide nanoparticles.

[0073] Here, nanoparticle-producing cells of high-purity and / or high-purity iron oxide-based nanoparticles are substantially free of at least one metal or nonmetal, or contain at least one metal or nonmetal in amounts of less than 10 M, or 1 M, or 10⁻¹ M, or 10⁻³ M, or 1 nanomolar, wherein the at least one metal or nonmetal is selected from the following groups. They are preferentially obtained by culturing nanoparticle-producing cells in growth medium: 1) Cadmium, 2) Lead, 3) Arsenic, 4) Mercury, 5) Cobalt, 6) Vanadium, 7) Nickel, 8) Lithium, 9) Antimony, 10) Copper, 11) Barium, 12) Molybdate, 13) Selenium, 14) Barium, 15) Chromium, 16) Strontium, 17) Radiochemical elements, 18) Beryllium, 19) Rubidium, 20) Ruthenium, 21) Rhodium, 22) Palladium, 23) Promethium, 24) Ytterbium, 25) Tantalum, 26) Osmium, 27) Iridium, 28 ) Bismuth, 29) Polonium, 30) Francium, 31) Radium, 32) Actinium, 33) Thorium, 34) Protactinium, 35) Uranium, 36) Neptunium, 37) Plutonium, 38) Americium, 39) Curium, 40) Berkelium, 41) Californium, Einsteinium, 42) Fermium, 43) Mendelevium, 44) Nobelium, 45) Laurencium, 46) Rutherfordium, 47) Dabunium, 48) Seaborgium, 49) Bohrium, 50) Hassium, 51) Meitnerium, 52) Darmstadium 53) Roentgen, 54) Copernicium, 55) Nihonium, 56) Flerovium, 57) Moscovium, 58) Livermorium, 59) Astatine, 60) Tennessine, 61) Oganesson, and 62) its derivatives.

[0074] In one embodiment of the present invention, high-purity nanoparticle-producing cells and / or high-purity iron oxide-based nanoparticles are obtained by culturing and / or growing nanoparticle-producing cells in the following: a) Pre-growth and / or growth medium, and or b) Culture media that are free of or free in concentrations that affect the growth of nanoparticle-producing cells at least one metal or metalloid other than iron, preferably selected from the following groups: cobalt, manganese, zinc, nickel, silver, aluminum, arsenic, barium, cadmium, chromium, copper, molybdenum, lead, antimony, selenium, silica, titanium, thallium, mercury, vanadium, gold, iridium, osmium, rhodium, ruthenium, platinum, lithium, antimony, tin, tungsten, and their derivatives.

[0075] The present invention also relates to high-purity nanoparticle-producing cells according to the present invention, and / or high-purity iron oxide-based nanoparticles preferentially obtained from high-purity nanoparticle-producing cells according to the present invention, wherein: - High-purity nanoparticle-producing cells are magnetotactic bacteria / bacteria, and / or - High-purity iron oxide nanoparticles are magnetosomes.

[0076] The present invention also relates to compositions, medical devices, drugs, formulations, suspensions, cosmetic compositions, plant compositions, biological compositions, mineral compositions, and / or nanoparticle compositions comprising nanoparticle-producing cells according to the present invention, and / or high-purity iron oxide nanoparticles according to the present invention.

[0077] This invention also, M FeC / M MC Regarding high-purity nanoparticle-producing cells containing more than 1, 25, 50, 75, 90, or 99% iron based on the ratio, where M FeC M is the mass of iron in nanoparticle-producing cells. MC M is the mass of iron and other metals or metalloids in the high-purity nanoparticle-producing cell, and / or high-purity iron oxide-based nanoparticles. FeN / M MN Based on the ratio, it contains more than 1, 10, 50, 75, 93 or 99% iron, where M FeN M is the mass of iron in high-purity iron oxide nanoparticles. MN is the mass of iron and non-ferrous metals or metalloids in high-purity iron oxide-based nanoparticles, and high-purity nanoparticle-producing cells and / or high-purity iron oxide-based nanoparticles are obtained by culturing nanoparticle-producing cells in a growth medium that is substantially free of metals selected from the following groups: cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, and copper.

[0078] In this specification, the term "substantially absent" refers to a substance that is not intentionally added to the culture medium but may be present as an impurity.

[0079] In some cases, M FeN / M MN and or M FeN / M MN The percentage may be 100, 99, 95, 90, 50, 25, 10, 5, or less than 1%. The present invention also relates to a method according to the present invention for producing high-purity iron oxide nanoparticles using nanoparticle-producing cells grown in a preliminary growth stage in a subsequent growth step, here: a) The preliminary growth step is characterized by at least one of the following properties: a1) comprising at least one substep i, during which the nanoparticle-producing cells are in a volume V containing a preliminary growth medium. PGSi It is propagated by a2)V PGSi The value does not change by more than 1, 10, 50, 80, 90, or 99% between the start and end of substep i. a3)i is primarily composed of 1-5, 1-10, or 1-1000. a4) VPGSi + 1 / V PGSi It consists of 1.001 to 1000 or 1.1 to 50. a5) Each substep i lasts for a period of time from 1 second to 1 year, 1 minute to 1 month, 10 minutes to 1 week, or 60 minutes to 3 days. a6) At least one substep i lasts for 1 second or 1 hour or longer, a7) The temperature of the preliminary growth medium in at least one substep consists of 10–60°C, 20–50°C, or 30–40°C. a8) The oxygen, air, or compressed air concentration in the pre-growth medium decreases as follows: —From each of the following values: a8i) At the start of at least one substep i, the partial pressure of oxygen, air, or compressed air is greater than 50 mbar and equal to at least 210 mbar, or a8ii) The ratio of the volume of oxygen, air, or compressed air to the maximum volume of saturated pre-growth medium occupied by oxygen, air, or compressed air is equal to 25-100% at the start of at least one substep i, or a8iii) At the start of at least one substep i, the volume of oxygen, air, or compressed air is 5-25% of the volume of the pre-growth medium. - The following values ​​are reduced: iv) The partial pressure of oxygen, air, or compressed air is lower than 100 millibars and equal to at least 0 millibars at the end of at least one substep i, or a8v) The ratio of oxygen, air, or compressed air to the maximum volume of oxygen, air, or compressed air in the saturated pre-growth medium is equal to 0-50% at the end of at least one substep i, or a8vi) The volume of oxygen, air, or compressed air to the volume of pre-growth medium is equal to 0-10% at the end of at least one substep i. a9) Volume V in at least one substep i of the preliminary growth step PGSi The amount of oxygen, air, or compressed air delivered is preferably less than 100 liters, 1, 5, 10, or 50 percent of the total duration of at least one substep i, at least 100 liters, 1,000 mL, or 100 mL of oxygen, air, or compressed air per minute. a10) During all or part of substep i, stir the pre-growth medium at a speed of 0 to 100 revolutions per minute or 0 to 103 meters per minute. This speed is such that the pre-growth medium is at least one compound in the state of liquid, gas, or solid. a11) By not adding fed-batch medium or another medium other than the pre-growth medium to the pre-growth medium containing an iron source, the pH of the pre-growth medium is not maintained at a fixed pH. a12) The pH of the preliminary growth medium should range from a minimum value less than 7 to a maximum value greater than 7 between the start and end of at least one substep i. -5, 10 -1 The pH changes by 0.5 or 1 unit or more. a13) Add 10 to the preliminary growth medium 5 , 10 3 , 10 2 By adding a fed-batch medium containing 10 or 2 μM or less of iron or an iron source, the pH of the preliminary growth medium is maintained between 3 and 11 or 6 and 8. a14) The preliminary growth medium comprises iron, an iron source, carbon, a carbon source, nitrogen, and at least one of the nitrogen source, and it changes to 100, 50, 20, 10, 5 or 1% or less between the start and end of at least one substep i. a15) The preliminary growth medium is 10 per liter of preliminary growth medium. 5 mM, 10 3 10 mM, 10 mM, 2 mM or less iron or iron source, or 10 5 , 10 3 , 10 2 , containing total concentrations of iron or iron sources of 50, 10, 5, 2, 1, or 0.5 g or less, a16) Preliminary growth medium: 10 per liter of preliminary growth medium -50 Contains iron or iron sources at a total concentration exceeding M or 1 pM, or 0.4 ng of iron or iron sources. a17) The preliminary growth medium contains carbon or a carbon source at a total concentration less than 2 M or 260 g of carbon or carbon source per liter of preliminary growth medium. a18) The preliminary growth medium contains carbon or a carbon source at a total concentration greater than 0.1 nM or 0.1 ng per liter of preliminary growth medium. a19) The preliminary growth medium contains nitrogen or a nitrogen source at a total concentration of 740 mM or 40 g of nitrogen or nitrogen source per liter of preliminary growth medium. a20) The preliminary growth medium contains nitrogen or a nitrogen source at a total concentration of more than 0.1 nM or 0.1 ng of nitrogen or nitrogen source per liter of preliminary growth medium. a21) The amount, concentration, or percentage of carbon or carbon source consumed by the nanoparticle-producing cell between the start and end of at least one substep i is greater than the following value: - per liter of preliminary growth medium, 10 -50 , 0.01, 1 or 10 g of carbon or a carbon source, or 1 mM of carbon or a carbon source, and / or -10- 10 , 1, 50, or 75%, wherein this ratio is equal to (Q C f - Q Ci ) / Q Ci , wherein here Q Cf and Q Ci are respectively the amount of carbon contained in the preliminary growth medium at the end and the start of at least one sub-step i of growth, a22) the amount, concentration or proportion of nitrogen or a nitrogen source consumed by the nanoparticle-producing cells between the start and end of at least one sub-step i is greater than: - per liter of preliminary growth medium, 10 -50 , 0.001, 1 or 10 g of nitrogen or a nitrogen source, or 0.1 mM of nitrogen or a nitrogen source, and / or -10 -10 , 1, 50, or 75%. wherein this ratio is equal to (Q Nf - Q Ni ) / Q Ni , and Q Nf and Q Ni are respectively the amount of nitrogen contained in the preliminary growth medium at the end and the start of at least one sub-step i of growth. a23) the amount, concentration or proportion of iron or an iron source consumed by the nanoparticle-producing cells between the start and end of at least one sub-step i is greater than: - per liter of preliminary growth medium, 10 -10 , 0.0001, 1, 10 or 10 10 mg of iron or an iron source, or 0.5 μM of iron or an iron source, and / or -10 -10 , 1, 20, 50 or 75%, wherein this percentage value is preferentially based on the ratio of (Q Fef - Q Fei ) / Q Fei , and Q Fef and Q Feieach represents the amount of iron contained in the medium for the first preliminary growth and at the end of at least one sub-step i, respectively. a24) the amount of carbon, carbon source, nitrogen, nitrogen source, iron, and / or iron source consumed by the nanoparticle-producing cells between the start and end of at least one sub-step i is less than the total concentration of carbon, carbon source, nitrogen, nitrogen source, iron, and / or iron source in the preliminary growth medium, a25) the total concentration of carbon, carbon source, nitrogen, nitrogen source, iron, and / or iron source in the preliminary growth medium does not change by more than 1, 10, 20, 50, 80, or 99% between the start and end of sub-step i. a26) the concentration of carbon, carbon source, nitrogen, nitrogen source, iron, and / or iron source consumed by the nanoparticle-producing cells increases by 1, 10, 20, 50, 80, 90 or 99% or more between the start and end of sub-step i, wherein such conditions preferentially result in nanoparticle-producing cells having at least one of the following properties: a27) the nanoparticle-producing cells essentially do not produce nanoparticles, or the nanoparticle-producing cells produce 10 10 , 10 5 , 1, 10 -3 or 10 -6 mg or less of nanoparticles per liter of preliminary growth medium, wherein this amount is the difference between the amount of nanoparticles produced at the end of at least one sub-step i, or the amount of nanoparticles produced at the end of at least one sub-step i, and the amount of nanoparticles produced at the start of at least one sub-step i. a28) the nanoparticle-producing cells, without concentrating at the end of at least one sub-step i, preferentially provide an optical density measured from 0 to 10 5 nm, most preferably at 565 nm, and is characterized by at least one of the following properties: a28i) during at least one sub-step i of the preliminary growth step, it varies within an optical density range of 10 -20 to 10 20 or 0.0001 to 40. a28ii) Between the start and end of at least one substep i, 0, 0.5, 1, 1.1, 5, 10, 10 3 , or 10 5 It increases by a coefficient exceeding . This coefficient is preferentially the ratio between the optical density measured at the end of substep i and the optical density measured at the beginning of substep i. a28iii) Between the start and end of at least one substep i, 10 10 , 10 5 , 2000, 10 3 , 10 2 It increases by a coefficient of 10, 5, 2, or less than or equal to 1. a28iv) At the end of at least one substep i, 10 5 , 10 3 , or there is a maximum value of 100 or less. a28v) At the start of at least one substep i, 0, 10 -50 , 10 -10 , 0.0001, 10 -3 or 10 -1 There is a larger minimum value. a29) Nanoparticle-producing cells are characterized by a doubling time or a period multiplied by a factor of 2 during at least one substep i of the preliminary growth step, which is: a29i)10 -50 , 10 -5 , 1, 10, 10 2 or 10 3 Longer than a minute, a29ii)10 3 , 10, 1 or 0.1 months or less; and or a29iii) Between 1 second and 1 month, or between 1 minute and 1 month. b) A growth step comprising growing nanoparticle-producing cells arising from and / or generated during the preliminary growth step, in at least one growth step, preferably only one growth step, most preferably a number of growth steps less than the number of substep i of the preliminary growth step. Herein, at least one growth step is characterized by properties selected from the following group: b1) Nanoparticle-producing cells are in volume V containing growth medium. GS It multiplies there. b2)V GS is at least 0, 1, 1.1, 5, 10, or 10 times the volume of at least one substep i of the preliminary growth step. 3 It doubles in size. b3)V GS V GS0 + V FB It is equal to V. GS0 This is the volume containing the growth medium at the start of the growth step, V FB This is the volume of fed-batch medium added to the growth medium during the growth step. b4)V GS This is preferably done by adding fed-batch medium to the growth medium during the growth step, between the start and end of at least one growth step. -3 , 10 -1 It changes by more than 1, 5, 10, 25, 50, or 75%. b5) Volume V containing growth medium at the start of at least one growth step GS0 This is the volume V of the fed-batch medium added to the growth medium during at least one growth step. FB Preferably 0, 1, 1.1, 1.5, 2, 5, 10, or 10 3 The coefficient is large. b6) At least one growth step should consist of a duration ranging from one minute to one month, preferably from 40 hours to 15 days. b7) The duration of at least one growth step is preferred over the duration of at least one substep i of the preliminary growth step by at least 0, 1, 1.1, 2, 5, 10, or 10. 3 It's twice as long. b8) The temperature of the growth medium consists of 10-60°C, 20-50°C, or 30-40°C. b9) The concentration of oxygen, air, or compressed air in the growth medium decreases: — from b9i) at the start of at least one growth step, the partial pressure of oxygen, air, or compressed air is preferably greater than 1 or 10 mbar, most preferably equal to 210 mbar, or b9ii) at the start of at least one growth step, the ratio of the volume of compressed air to the maximum volume occupied by oxygen, air, or compressed air in the saturated growth medium is preferably equal to a ratio of 10–100%. or 9iii) at the start of at least one growth step, the volume of oxygen, air, or compressed air to the volume of the growth medium is preferably between 1 and 25%. — b1iv) At the end of at least one growth step, the partial pressure of oxygen, air, or compressed air is preferably equal to 50 or 500 mbar or less, most preferably equal to 0 mbar, or b9v) At the end of at least one growth step, the volume of oxygen or air or compressed air relative to the maximum volume of saturated growth medium is preferably equal to 0-25%, or b9vi) At the end of at least one growth step, the volume of oxygen or air or compressed air relative to the volume of pre-growth medium is preferably between 0-5%. b10) During 1% of the entire growth step, volume V is generated during the entire growth step or in part of the entire growth step. GS The amount of oxygen, air, or compressed air delivered is greater than 1, 10, or 200 mL of oxygen, air, or compressed air per minute. b11) Volume V during the growth step GS The amount of oxygen, air, or compressed air delivered is preferentially at least 0, 1, 1.1, 5, 10, or 10³ times greater at the end of the growth step than at the beginning. b12) In the entire growth step or in part of the entire growth step, volume V GS The amount of oxygen, air, or compressed air delivered is 10 per minute. -10 , 10 -5 , 1, 10, or 10 5 More than mL of oxygen, air, or compressed air. b13) During all or part of the growth stage, the growth medium is turned at a rate faster than the growth medium, preferably at a rate greater than 1, 10 or 100 revolutions per minute, or 0 to 10 revolutions per minute. 3 The mixture is stirred between meters, and this rate is the rate of at least one component of the growth medium, regardless of whether the growth medium is in a liquid, gaseous, or solid state. b14) By preferentially adding fed-batch medium or a different medium to the growth medium, the pH of the growth medium changes less than the pH of the pre-growth medium, is maintained at a fixed pH of 1–14 or 6.5–7.5, or does not change by more than 0.1, 0.5, or 10 pH units. b15) Between the start and end of the growth step, the pH of the growth medium changes from a minimum value of 10, 0.5, or 0.1 pH units, preferably less than 7.5, to a maximum value preferably greater than 6.5. b16) The growth medium has a total concentration of carbon, carbon source, nitrogen, nitrogen source, iron, and / or iron source between the start and end of at least one growth step, and is 0 to 10 -5 It changes by 1 or 50% or more. b17) The growth medium contains more than 10⁻⁵ mM, 0.1 mM, 10⁻⁵ g, or 0.01 g of carbon or carbon source in total concentration per liter of growth medium. b18) The growth medium should have a total concentration of 10 per liter of growth medium. 3 M or 2 M or 10 3 Contains 180 g or less of carbon or a carbon source. b19) The growth medium should have a total concentration of 10 per liter of growth medium. -50 mM or 0.01 mM or 5.10 -10 Contains more than 1 g or 0.00005 g of nitrogen or nitrogen source. b20) The growth medium should have a total concentration of 10 per liter of growth medium. 10 mM or 111 mM or 10 5 Contains less than 6g of nitrogen or a nitrogen source. b21) The growth medium should have a total concentration of 10 per liter of growth medium. -5 nM or 1 nM or 10 -10 g or 3.10-7 g of iron or an iron source. b22) the growth medium contains, in total concentration, 10 5 or 1 mM or 10 5 g or less than 0.3 g of iron or an iron source. b23) the amount, concentration or proportion of carbon or a carbon source consumed by the nanoparticle-producing cells between the start and end of at least one growth step is greater than: - 10 g of carbon or a carbon source per liter of growth medium, -10 , 10 -5 , 0.1, 1, 10 or 10 3 g of carbon or a carbon source, or 1 mM of carbon or a carbon source, and / or - 10 -50 , 10 -10 , 10 -5 , 10-1, 1, 5, 10, 50, or 75%. This percentage is (Q Cf - Q Ci ) / Q Ci is equal to, wherein Q Cf and Q Ci are respectively the amount of carbon contained in the growth medium at the end and at the start of the at least one growth step. b24) the amount, concentration or proportion of nitrogen or a nitrogen source consumed by the nanoparticle-producing cells between the start and end of at least one growth step is greater than: - 10 -10 , 10 -5 , 0.01, 1 or 10 g of nitrogen or a nitrogen source, or 0.6 mM of nitrogen or a nitrogen source, and / or - 10 -50 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 50, or 75%. This proportion is preferentially equal to (Q Nf - Q Ni ) / Q Ni , wherein Q Nf and Q Ni are nitrogen respectively contained in the growth medium at the end and at the start of the at least one growth step. b25) The amount, concentration, or proportion of iron or iron source consumed by nanoparticle-producing cells between the start and end of at least one growth step is greater than the following: - 10 per liter of growth medium -10 , 10 -5 , 0.01 or 1 mg of iron or iron source, or 0.04 μM of iron or iron source, and or -10 -50 , 10 -10 , 10 -5 , 10 -1 1, 5, 10, 50, or 75%. This percentage is preferred over (Q Fef - Q fei ) / Q Fe Equal to i, Q Fef and Q Fei These represent the amount of iron present in the growth medium at the end and beginning of at least one growth step. b26) Between the start and end of at least one growth step, the amount of iron or iron source delivered to the growth medium shall preferentially be 10 per liter of growth medium. -10 mg or 0.3 mg of iron or iron source, or 10 -3 More than μM or 1 μM of iron or iron source. b27) The amount of carbon or carbon source preferentially supplied to the growth medium by the fed-batch medium between the start and end of at least one growth step shall be 10 per liter of growth medium. -50 , 10 -10 , 10 -5 Or 0.07 g of carbon or carbon source, or 10 -10 , 10 -5 , 10 -3 , 0.8, 1 or 10 3 Carbon exceeding a certain mM, or larger than a carbon source. b28) The amount of nitrogen or nitrogen source delivered to the growth medium between the start and end of at least one growth step shall be, preferentially, 10 per liter of growth medium. -10 , or more than 0.006 or 1 g of nitrogen or nitrogen source or 0.4 mM of nitrogen or nitrogen source. b29) The amount of carbon, carbon source, nitrogen, nitrogen source, iron, and / or iron source consumed by the nanoparticle-producing cell between the start and end of at least one growth step is less in total concentration than the carbon, carbon source, nitrogen, nitrogen source, iron, and iron source of the growth source. Here, such conditions result in nanoparticle-producing cells having at least one of the following characteristics: b30) Nanoparticle-producing cells produce nanoparticles, or nanoparticle-producing cells produce 10 per liter of growth medium -50 , 10 -10 The system produces more than 0.01 or 1 mg of nanoparticles, where this amount is the amount of nanoparticles produced at the end of the growth step, or the difference between the amount of nanoparticles produced at the end of the growth step and the amount of nanoparticles produced at the start of the growth step. b31) Nanoparticle-producing cells yield an optical density measured from 0 to 104 nm, preferably at 565 nm, without being enriched preferentially at the end of at least one growth step, and are characterized by at least one of the following properties: b31i) The optical density at the end of the growth step is at least 0, 0.5, 1, 1.1, 1.5, 2, 5, 10, or 10 times higher than the optical density at the end of at least one preliminary growth step. 3 Twice as big. b31ii) The optical density of the growth step varies within the range of optical densities between 0.001 and 300. b31iii) The optical density is 0, 0.5, 1, 1.1, 5, 10, or 10 between the start and end of at least one growth step. 3 It increases by a coefficient exceeding , where this coefficient is the ratio between the optical density measured at the end of at least one growth step and the optical density measured at the beginning of at least one growth step. b31iv) The optical density is 10 between the start and end of at least one growth step. 10 , 10 4 Alternatively, it increases by a coefficient of 10 or less. b31v) The optical density is 10 at the end of at least one growth step. 10 , 10 5It has a maximum value less than 300 or 10. b31vi) The optical density is 10 at the start of at least one growth step. -50 , 10 -10 , having a minimum value greater than 0.001 or 0.01. And / or b32) Nanoparticle-producing cells are characterized by a doubling time or period for doubling, which is between the entire growth step or a portion of the entire growth step, and furthermore, it is the next period: b32i) Longer than 1 minute b32ii) Less than 1 month b32iii) It is included between 1 minute and 1 month. and or b32iv) Preferably shorter than the doubling time of at least one substep i of the preliminary growth step by at least 1.1 times. Herein, preferentially, pre-growth, growth, and / or fed-batch media shall preferentially not contain, by measurement per liter of pre-growth, growth, and / or fed-batch media: i) 1, 2, 3, or 6 types of vitamins, ii) 10 -9 3) 10 -4 g yeast extract, iv) 10 -9 v) at least one component of mol of yeast extract, vi) 1, 2, 5, or 10 components of yeast extract -5 g of peptone, vii) 1 or 2 different CMR agents, viiii) 0.05 mg of at least one CMR agent, ix) 1, 2, or 5 chelating agents, x) 10 -810 mol of at least one chelating agent, xi) 1, 2 or 5 different amino acids, xii) 1 mg of at least one amino acid, xiii) 1, 2 or 5 different toxic or cytotoxic compounds, xiv) 1 mg of at least one toxic or cytotoxic compound, xv) 1, 3 or 7 different heavy metals and iron, xvi) 1 mg of at least one different heavy metal and iron, xvii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 metals or chemical elements selected from cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, and copper, xviii) 1 mg of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, and / or copper, xix) 0.5 mL or 10 -8 mol of wolf vitamins, xx)10 -9 xx) at least one component of wolf vitamins in mol, xxi) 1, 2, 5 or 10 components of wolf vitamins, xxii) 5 mL or 10 -8 Mole Wolf Minerals, xxiii)10 -8 At least one component of the wolf mineral of moles, xxiv) 1, 2, 5, 7, 10 or 15 components of the wolf mineral of moles, xxv) 10 -9 Mole mineral elixir, xxvi) 10 -9 9 moles of at least one component of the mineral elixir, and / or 1, 2, 5, 10, or 14 different components of the mineral elixir.

[0080] The present invention also relates to a method according to the present invention, wherein the preliminary growth and / or completion of the growth step is characterized by at least one of the following characteristics: i) The optical density of the pre-growth / growth medium is saturated or increases by less than 1 OD unit per hour during the pre-growth and / or growth step. ii) The biomass of the pre-growth and / or growth medium is saturated or the number of nanoparticle-producing cells increases to 1 g or less per hour during the pre-growth and / or growth step. iii) Nanoparticle-producing cells cease nanoparticle production or produce no more than 0.01 mg of nanoparticles per hour during the pre-growth and / or growth phase. iv) No further fed-batch medium is added to the pre-growth and / or growth medium. v) No further oxygen is introduced into the preliminary growth and / or growth medium.

[0081] The present invention also relates to a method according to the present invention, wherein the initiation of the preliminary growth and / or growth step is characterized by at least one of the following characteristics: i) The optical density of the pre-growth and / or growth medium begins to increase by at least 0.1 OD units per day during the pre-growth and / or growth stage. ii) The biomass of the pre-growth and / or growth medium begins to increase by 0.1 g or more of nanoparticle-producing cells per day during the pre-growth and / or growth stage. iii) Nanoparticle-producing cells initiate nanoparticle production, preferentially producing more than 0.01 mg of nanoparticles per day during the pre-growth and / or growth phases. iv) Start adding the fed-batch medium to the preliminary growth and / or growth medium. v) Oxygen begins to introduce bubbles into the pre-growth and / or growth medium.

[0082] The present invention also relates to a method according to the present invention in which a pre-growth, growth, and / or fed-batch medium composition is defined as follows: i) Preferentially the pre-growth medium is at least 1, 2, 3, 4, 5, 6, or 7 of the following chemical elements or derivatives, which derivatives preferentially contain carbon, nitrogen, phosphate, manganese, potassium, calcium, vitamins, and / or chlorine or their sources. i) Sodium lactate or carbon source, preferably 10 per liter of preliminary growth medium -20 A sodium lactate or carbon source at a molar or gram concentration between 0.0023 and 0.23 or 10 per liter of preliminary growth medium. 5Molars or grams of sodium lactate or carbon source, most preferably at a concentration of 0.023 ± 0.01 moles of sodium lactate or carbon source per liter of preliminary growth medium. ii) Ammonium chloride or nitrogen source, preferably 10 per liter of preliminary growth medium -20 and 7.4 10 -4 Between moles or grams of ammonium chloride or nitrogen source and 7.4 10 per liter of pre-growth medium -2 or 10 5 The concentration of ammonium chloride or nitrogen source contained between moles, most preferably (7.5 ± 1) per liter of preliminary growth medium. -3 Molars of ammonium chloride or nitrogen source, iii) KH2PO4 or potassium or phosphorus source, preferably 10 per liter of preliminary growth medium -20 or 1.55·10 -5 A concentration of KH2PO4 or potassium or phosphorus source, 1.55·10 per liter of preliminary growth medium. -3 or 10 5 Molar concentration of KH2PO4 or potassium or phosphorus source, most preferably (1.5±1) 10 per liter of preliminary growth medium. -4 Molar concentration of KH2PO4 or potassium source or phosphorus source, iv) MgSO4 or magnesium source, preferably 10 per liter of preliminary growth medium -10 or 4.1·10 -5 MgSO4 or magnesium source in molar or gram concentrations, and 4.1·10 per liter of preliminary growth medium. -3 or 10 5 MgSO4 or magnesium source in molar or gram concentration, with the highest preference being (4±1)·10 per liter of preliminary growth medium. -4 Molar concentration of MgSO4 or magnesium source medium, v) Iron source or FeCl3, preferably 10 per liter of pre-growth medium -20 and 2.10 -7 A source of iron or FeCl3 at a concentration between molars or grams, and 10 per liter of pre-growth medium.-5 or 10 5 A source of FeCl3 or iron in molar or gram concentration, with the highest preference being (2±1)10 per liter of preliminary growth medium. -6 Sources of FeCl3 or iron in molar or gram concentrations, vi) Thiamine or vitamin, preferably 10 per liter of preliminary growth medium -20 and 8.10 -9 Vitamin or thiamine in molar or gram concentrations between 8.10-7 or 10 per liter of preliminary growth medium. 5 Vitamins or thiamines in molar or gram concentrations, with the highest preference being (8±2)10 per liter of preliminary growth medium. -8 Molar concentration of vitamin or thiamine, vii) CaCl2 or calcium or chlorine source, preferably 10 per liter of preliminary growth medium -20 or 10 -5 CaCl2 or calcium or chlorine source in molar or gram concentrations between 10 and 10 per liter of pre-growth medium. -3 or 10 5 Molar or gram concentration of CaCl2 or calcium or chlorine source, most preferably (1 ± 0.8)·10 per liter of preliminary growth medium. -4 A molar concentration of CaCl2 or a source of calcium or chlorine.

[0083] The growth medium preferentially contains at least 1, 2, 3, 4, 5, 6, or 7 of the following chemical elements or derivatives before preferentially adding the fed-batch medium to the growth medium, wherein the derivative is or contains carbon, nitrogen, phosphate, manganese, potassium, calcium, vitamins, and / or chlorine. i) Sodium lactate or carbon source, preferably 10 per liter of growth medium -20 Alternatively, sodium lactate or carbon source at a concentration of molar or gram between 0.0014 and 0.14 or 10 per liter of growth medium. 5Sodium lactate or carbon source in molar or gram concentrations, most preferably (0.014 ± 0.01) molar concentrations per liter of growth medium. ii) Ammonium chloride or nitrogen source, preferably 10 per liter of growth medium -20 and 4.1·10 -4 Between moles or grams of ammonium chloride or nitrogen source and 4.1-10 per liter of growth medium -2 or 10 5 The concentration of ammonium chloride or nitrogen source contained between moles, most preferably (4.1 ± 1) per liter of growth medium. -3 Molars of ammonium chloride or nitrogen source, iii) KH2PO4 or potassium or phosphorus source, preferably 10 per liter of growth medium -20 or 1.55·10 -5 A concentration of KH2PO4 or potassium or phosphorus source, 1.55·10 per liter of growth medium. -3 or 10 5 Molar concentration of KH2PO4 or potassium or phosphorus source, most preferably (1.5±1) 10 per liter of growth medium. -4 Molar concentration of KH2PO4 or potassium source or phosphorus source, iv) MgSO4 or magnesium source, preferably 10 per liter of growth medium -20 or 4.1·10 -5 MgSO4 or magnesium source in molar or gram concentrations, and 4.1·10 per liter of growth medium. -3 or 10 5 MgSO4 or magnesium source in molar or gram concentration, most preferably (4±1)·10 per liter of growth medium. -4 Molar concentration of MgSO4 or magnesium source medium, v) Iron source or FeCl3, preferably 10 per liter of growth medium -20 and 10 -7 A source of iron or FeCl3 at a concentration between molars or grams, and 10 per liter of growth medium. -5 or 10 5A source of FeCl3 or iron in molar or gram concentration, with the highest preference being (2±1)10 per liter of growth medium. -6 Sources of FeCl3 or iron in molar or gram concentrations, vi) Thiamine or vitamin, preferably 10 per liter of growth medium -20 and 8.10 -9 Vitamins or thiamine in molar or gram concentrations between 8 and 10 per liter of growth medium. -7 or 10 5 Vitamins or thiamine in molar or gram concentrations, with the highest preference being (8±2)10 per liter of growth medium. -8 Molar concentration of vitamin or thiamine, vii) CaCl2 or calcium or chlorine source, preferably 10 per liter of growth medium -20 and 10 -5 CaCl2 or calcium or chlorine source in molar or gram concentrations between 10 and 10 per liter of growth medium. -3 or 10 5 CaCl2 or calcium or chlorine source in molar or gram concentrations, most preferably (1 ± 0.8)·10 per liter of growth medium. -4 A molar concentration of CaCl2 or a source of calcium or chlorine.

[0084] The fed-batch medium preferentially contains at least 1, 2, 3, 4, 5, 6, or 7 of the following chemical elements or derivatives before being preferentially added to the growth medium, wherein the derivatives are or contain carbon, nitrogen, phosphate, manganese, potassium, calcium, vitamins, and / or chlorine. i) Sodium lactate or carbon source, preferably 10 per liter of fed-batch medium -20 or 10 -1 Sodium lactate or carbon source in molar or gram concentrations between 10 or 10 per liter of fed-batch medium, and 10 or 10 5 Sodium lactate or carbon source in molar or gram concentrations, most preferably (1 ± 0.5) molar concentrations per liter of fed-batch medium. ii) Ammonium or nitrogen source, preferably 10 per liter of fed-batch medium -20 and 2.8·10 -2 Between moles or grams of ammonium or nitrogen source and 2.8 or 10 per liter of fed-batch medium 5 The concentration of ammonium or nitrogen source contained between moles, most preferably (2.8 ± 1) per liter of fed-batch medium. -1 Molar ammonium or nitrogen source, iii) KH2PO4 or potassium or phosphorus source, preferably 10 per liter of fed-batch medium -20 or 1.7·10 -3 A concentration of KH2PO4 or potassium or phosphorus source, 1.7-10 per liter of fed-batch medium. -1 or 10 5 Molar concentration of KH2PO4 or potassium or phosphorus source, most preferably (1.7±1) 10 per liter of fed-batch medium. -2 Molar concentration of KH2PO4 or potassium source or phosphorus source, iv) MgSO4 or magnesium source, preferably 10 per liter of fed-batch medium -20 or 2.10 -4 A molar or gram concentration of MgSO4 or magnesium source, and 2.10 per liter of fed-batch medium. -2 or 10 5 MgSO4 or magnesium source in molar or gram concentration, most preferably (2±1)·10 per liter of fed-batch medium. -3 Molar concentration of MgSO4 or magnesium source medium, v) Iron source or FeCl3, preferably 10 per liter of fed-batch medium -20 and 10 -4 A source of iron or FeCl3 at a concentration between molars or grams, and 10 per liter of fed-batch medium. -1 or 10 5 A source of FeCl3 or iron in molar or gram concentration, with the highest preference being (7±4)10 per liter of fed-batch medium. -3 Sources of FeCl3 or iron in molar or gram concentrations, vi) Thiamine or vitamin, preferably 10 per liter of fed-batch medium -20 and 10 -8 Vitamins or thiamine in molar or gram concentrations between 10 per liter of fed-batch medium. -4 or 10 5 Vitamins or thiamines in molar or gram concentrations, most preferably (2 ± 1.5) 10 per liter of fed-batch medium. -6 Molar concentration of vitamin or thiamine, vii) CaCl2 or calcium or chlorine source, preferably 10 per liter of fed-batch medium -20 and 10 -5 CaCl2 or calcium or chlorine source in molar or gram concentrations between 10 per liter of fed-batch medium, and 10 -2 or 10 5 Molar or gram concentration of CaCl2 or calcium or chlorine source, most preferably (1 ± 0.8)·10 per liter of fed-batch medium. -3 A molar concentration of CaCl2 or a source of calcium or chlorine.

[0085] The present invention also relates to a method by which the preliminary growth, growth, and / or fed-batch medium comprises at least one source selected from the following group. i) A carbon source selected from the following group at a concentration of 1 nM to 2 Mol / L, namely, at least one compound containing at least one carbon atom, i.e., lactic acid, sodium lactate, lactic acid, acetic acid, glycolic acid, glucose, pyruvic acid, succinic acid, carbon dioxide, glycerol, and combinations thereof. ii) A source of iron with a concentration of 1 nM to 2.10-3 Mol / L, at least one compound containing at least one iron atom, namely iron citrate, iron quinate, iron chloride, iron sulfate, FeCl3, and combinations thereof. iii) A source of nitrogen of a concentration of 1 nM to 4 mol / L, preferably a compound containing at least one nitrogen atom, i.e., nitrates, nitrogen gas, ammonium, ammonia, ammonium salts, urea, amino acids, ammonia gas, and combinations thereof. iv) An oxygen source selected from the following group, at least one compound containing at least one oxygen atom, i.e., oxygen or air or compressed air, the oxygen source preferably in gaseous form, and optionally foamed or introduced into the growth medium at a gas rate between 5 mL of gas per minute and 50,000 mL of gas per minute. v) A phosphate source comprising at least one compound containing at least one phosphorus atom, preferably at a concentration between 1 nM and 2.10-1 Mol / L. vi) A potassium source comprising at least one compound containing at least one potassium atom, preferably at a concentration between 1 nM and 2.10-1 Mol / L. vii) A source of sulfur or sulfate consisting of at least one compound containing at least one atom of sulfur or sulfate, preferably in concentrations between 1 nM and 4.10-1 Mol / L, viii) A manganese source consisting of at least one compound containing at least one manganese atom at a concentration preferentially between 1 nM and 4.10-1 Mol / L, ix) A vitamin source selected from the group consisting of at least one vitamin, biotin, calcium, pantothenic acid, folic acid, inositol, nicotinic acid, p-aminobenzoic acid, pyridoxine HCl, riboflavin, thiamine, thiamine HCl, and its derivatives and combinations thereof, preferably containing concentrations between 1 nM and 10⁻⁴ mol / L. x) A calcium source comprising at least one compound containing at least one calcium atom at a concentration preferentially between 1 nM and 10⁻¹ Mol / L.

[0086] In one embodiment of the present invention, a source of carbon, nitrogen, potassium, phosphorus, magnesium, calcium, vitamins, iron, oxygen, and / or chlorine preferentially contains at least 1, 2, 5, 10, or 103 carbon, nitrogen, potassium, phosphorus, magnesium, calcium, vitamins, iron, oxygen, and / or chlorine atoms within the chemical formula, molecule, or component of these sources.

[0087] In yet another embodiment of the present invention, if a compound, quantity, element, or property P1 is higher, longer, or greater than a compound, quantity, element, or property P2, then it means that P1 = a·P2, where a is preferably a number or integer greater than 1, or P1 = a + P2, where a is preferably a number or integer greater than 0.

[0088] In yet another embodiment of the present invention, if a compound, quantity, element, or property P1 is lower, shorter, or smaller than a compound, quantity, element, or property P2, then P1 = P2 / a, where a is preferably a number or integer greater than 1, or P1 = P2-a, where a is preferably a number or integer greater than 0.

[0089] In one embodiment of the present invention, the CMR compound is a carcinogenic, mutagenic, and / or reproductive toxic compound. In some cases, a carcinogenic compound is a compound that preferentially induces cancer, produces or is suspected of producing cancer, in living organisms or humans. In some cases, a mutagenic compound is a compound that produces or induces, or is suspected of producing or inducing, mutation, modification, change in number or size of at least one gene, DNA, RNA, DNA strand, RNA strand, and / or nucleic acid in an organism or human. In some cases, a reproductive toxic compound is a compound that produces or induces, or is suspected of producing or inducing, toxicity, mutation, modification, or change in the reproductive organs, embryos, or fetuses in living organisms or humans.

[0090] In one embodiment of the present invention, a toxic or cytotoxic compound is a compound that produces or induces, or is suspected to produce or induce, toxicity, death, weight loss, organ damage, behavioral changes, changes in food or water consumption, necrosis, apoptosis, intracellular integration, changes in the number, shape and / or arrangement of cells in an individual or organism.

[0091] In some cases, the compound is 10 -6 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 6 CMR, cytotoxicity, or toxicity are observed at concentrations above μM. In other cases, the compound is 10 20 , 10 6 , 10 3 , 10, 1, 10 -1 , 10 -3 or 10 -6 At concentrations of μM or less, it is CMR, cytotoxic, or toxic.

[0092] The present invention relates to a method for producing high-purity iron oxide nanoparticles using nanoparticle-generating cells, and includes the following: i) A preliminary growth step consisting of growing nanoparticle-producing cells in a preliminary growth medium so that the nanoparticle-producing cells do not inherently produce nanoparticles, and ii) A growth step comprising growing nanoparticle-producing cells that have been generated from the pre-growth stage in a growth medium so that the nanoparticle-producing cells produce nanoparticles.

[0093] The present invention relates to a method for producing high-purity iron oxide nanoparticles using nanoparticle-generating cells, and includes the following: i) A preliminary growth step consisting of growing nanoparticle-producing cells in a preliminary growth medium so that the nanoparticle-producing cells do not inherently produce nanoparticles, or ii) A growth step comprising growing nanoparticle-producing cells that have been generated from the pre-growth stage in a growth medium so that the nanoparticle-producing cells produce nanoparticles.

[0094] In one embodiment of the present invention, fed-batch medium is added to the growth medium.

[0095] In one embodiment of the present invention, the growth medium supplemented by the fed-batch medium is the growth medium.

[0096] In one embodiment of the present invention, the growth medium shares at least one property with the pre-growth medium and / or fed-batch medium.

[0097] One embodiment of the present invention includes a growth medium and / or a pre-growth medium, comprising at least one source selected from the following: i) Carbon or sodium or lactic acid, preferably sodium lactate, ii) Ammonium source, preferentially ammonium chloride, iii) Magnesium source, preferably magnesium sulfate, iv) Potassium source, preferentially potassium phosphate, v) Vitamin sources, preferentially thiamine, vi) Calcium source, preferentially calcium chloride, and vii) Iron source, iron chloride preferred. Each source is 10 -6 ~10 3 , 10 -3 It is preferentially included in growth media and / or pre-growth media at concentrations of ~100 mM, 0.01–10 mM, or 0.1–10 mM.

[0098] In one embodiment of the present invention, in the pre-growth medium and / or growth medium, the concentrations of vitamins and / or calcium sources and / or iron sources are at least 0, 0.5, 1, 1.1, 1.2, 1.5, 5, 10, 10 3 or 10 5 The coefficient is smaller than that, and the concentration is higher than that of the sodium source and / or ammonium source and / or magnesium source and / or potassium source.

[0099] In one embodiment of the present invention, the fed-batch medium comprises at least one source selected from the following groups: i) a carbon source, preferably lactic acid; ii) ammonia; iii) a potassium source, preferably potassium; iv) a magnesium source, preferably magnesium sulfate; v) an iron source, preferably iron chloride; vi) a vitamin source, preferably thiamine; vii) a calcium source, preferably calcium chloride; and viiii) an iron source, preferably iron chloride. Each source is preferentially included in the growth and / or pre-growth medium at concentrations between 0.001 and 100 mM, between 0.01 and 10 mM, or between 0.1 and 10 mM.

[0100] In one embodiment of the present invention, in a fed-batch medium, the concentrations of vitamins and / or calcium sources are at least 0, 0.5, 1, 1.1, 1.2, 1.5, 5, 10, 10 3 or 10 5 The coefficient is twice as large, and the concentrations are smaller than those of the sodium source and / or ammonium source and / or magnesium source and / or potassium source and / or iron source.

[0101] In one embodiment of the present invention, the nanoparticles according to the present invention are 1, 2, 5, 10, 10 nanoparticles per liter of growth medium or per nanoparticle-producing cell. 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 It is an aggregate of nanoparticles exceeding 1, 10, 10. In some cases, iron oxide is 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 iron atoms and / or 1,10,10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100It represents or is an aggregate of oxygen atoms. In some other cases, the chemical elements and / or impurities contained in nanoparticles are 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 These are impurities contained in chemical substances and / or nanoparticles exceeding a certain level.

[0102] In another embodiment of the present invention, the nanoparticles according to the present invention are nanoparticles per liter of growth medium or nanoparticles per nanoparticle-producing cell, 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 100, 50, 10, 5 or aggregates of 2 or fewer nanoparticles, or containing them. In some cases, iron oxide is 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 Iron atoms and / or 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 The following are aggregates of oxygen atoms or represent them. Furthermore, in some other cases, the chemical elements and / or impurities contained in the nanoparticles are 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 100, 50, 10, 5 or 2 or less chemical elements, and / or impurities contained in nanoparticles, or representing them.

[0103] In one embodiment of the present invention, at least one impurity is contained in the nanoparticles.

[0104] In one embodiment of the present invention, the high-purity iron oxide nanoparticles contain small amounts of impurities, for example, to enable the production of nanoparticles containing small amounts of impurities by this method. In some cases, the nanoparticles contain at least one impurity, or more than 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10, 5, 2, 5, 1, 10 -2 , 10 -10 , 10 -20 or 10 -50 The following impurities are not present, per gram of nanoparticles, or per gram of impurities. In some other cases, the proportion of impurities contained inside or on the surface of the nanoparticles is preferably less than or equal to 100, 90, 80, 70, 60, 50, 30, 20, 10, 105, 1, 0.1, or 0.001% by mass. According to the present invention, this proportion of impurities can, in some cases, be defined as the ratio of the number, amount, mass, or volume of atoms of impurities contained in the nanoparticles divided by the total number, amount, mass, or volume of atoms of all chemical elements contained in the nanoparticles. In some cases, all chemical elements contained in the nanoparticles are the sum of iron oxides, doping materials, and impurities contained in the nanoparticles. In yet another few cases, the concentration of impurities contained inside or on the surface of the nanoparticles is less than or equal to 10% of impurities per gram of nanoparticles. 50 , 10 30 , 10 10 , 10 5 , 10 3 , 500, 100, 50, 10, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 or 10 -50 It is lower than μg. Furthermore, in several other cases, the concentration of impurities contained inside or on the surface of nanoparticles is than 10 per gram of nanoparticles. -100 , 10 -50 , 10 -20 , 10 -10 , 10-5 , 10 -3 , 10 -2 , 10 -1 , 1, 10, 50, 100, 10 3 , 10 5 or 10 10 Higher than μg.

[0105] In some cases, impurities may be the same impurities, that is, impurities that preferentially contain the same chemical elements.

[0106] In some other cases, the impurities may be a variety of impurities, i.e., impurities containing at least one different chemical element.

[0107] In one embodiment of the present invention, the chemical element is selected from the following group: Actinides, actinium, aluminum, americium, antimony, argon, arsenic, astatine, barium, beryllium, beryllium, bismuth, boric acid, boron, bromine, cesium, calcium, californium, carbon, cerium, chlorine, chromium, cobalt, copernicum, cadmium, copper, curium, darmustatium, dubnium, dysprosium, eusteinium, erbium, europium, fermium, phlevium, fluorine, francium, gadolinium, gallium, germanium, gold, hafnium, helium, hesium, holmium, hydrogen, indium, iodine, iridium, iron, krypton, lanthanides, lanthanum, lawrencium, lead, lithium, hepatomolium, lutetium, magnesium, manganese, magnesium, mendelevium, mercury Molybdenum, neodymium, neon, neptunium, nickel, niobium, nitrogen, novelium, osmium, oxygen, palladium, phosphorus, platinum, plutonium, polonium, potassium, praseodymium, proctinium, promethium, radium, radon, rhenium, rhodium, lenthenium, rubidium, ruthenium, razafordium, samarium, selenium, silicon, silver, sodium, strontium, sulfur, scandium, sieborgium, tellurium, terbium, thorium, thulium, tin, tantalum, technetium, thallium, titanium, tungsten, ununoctium, ununpentium, ununseptium, ununthorium, uranium, vanadium, xenon, ytterbium, yttrium, zinc, zirconium, and some combinations of these chemical elements.

[0108] The present invention also relates to a method according to the present invention in which the impurity is at least one chemical element different from iron, oxygen, and / or iron oxide.

[0109] The present invention also relates to a method according to the present invention in which the impurities are preferentially carbon or carbonaceous material.

[0110] In yet another embodiment of the present invention, the carbon or carbonaceous material is derived from, produced by, or originates from nanoparticle-producing cells.

[0111] The present invention also relates to a method by which nanoparticles obtained by the method according to the present invention contain iron oxide, and the iron oxide has at least one of the following properties: i) containing at least one iron atom and one oxygen atom, ii) To form a crystalline or mineral structure. iii) The chemical formulas may be FeO, FeO2, Fe3O4, Fe4O5, Fe5O6, Fe5O7, Fe25O32, Fe13O19, α-Fe2O3, β-Fe2O3, γ-Fe2O3, and ε-Fe2O3. iv) It may consist of wustite, iron dioxide, magnetite, hematite, and maghemite. v) It can be in the epsilon phase, alpha phase, beta phase, or gamma phase. vi) It could be oxidation at various levels. vii) The chemical formula FeaObDg is given by a molecule where a, b, and / or g are coefficients, preferably stoichiometric coefficients. In some cases, a, b, and / or g are equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or 20. In some other cases, a, b, and / or g are greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or 20. In some other cases, a, b, and / or g are lower than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or 20. In some other cases, D is the doping material for the nanoparticles. In some cases, doping materials can be selected from a group consisting of aluminum, antimony, barium, chromium, copper, gold, manganese, silver, tin, titanium, and zinc.

[0112] In one embodiment of the present invention, iron oxide contained in nanoparticles is the primary chemical element of the nanoparticles. In some cases, high-purity iron oxide nanoparticles may contain a large amount of iron oxide. In some cases, the proportion of iron oxide contained in nanoparticles is preferably greater by mass than 10⁻⁴⁰, 10⁻²⁰, 10⁻⁰, 10⁻⁵, 10⁻², 10⁻¹, 1, 5, 10, 25, 50, 75, 80, 90, 99, or 99.9%. According to the present invention, this proportion of iron oxide can, in some cases, be defined as the ratio of the number, amount, mass, or volume of iron oxide atoms in the nanoparticles to the total number, amount, mass, or volume of all chemical elements contained in the nanoparticles. In several other cases, the concentration of iron oxide contained in nanoparticles is 10⁻⁶ in terms of iron oxide per gram of nanoparticles. -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 10, 50, 100, 10 3 , 10 5 or 10 10 Higher than μg.

[0113] In one embodiment of the present invention, high-purity iron oxide nanoparticles contain a small amount of iron oxide, for example, when the nanoparticles are processed and / or partially or completely destroyed and / or administered to a living organism, or when the method makes it impossible to incorporate a large amount of iron oxide. In some cases, the proportion of iron oxide contained inside or on the surface of the nanoparticles is preferentially 100, 90, 80, 70, 50, 30, 10, 5, 1, 0.1 or 0.001% or less by mass. In other cases, the concentration of iron oxide contained in the nanoparticles is 10% or less in terms of iron oxide per gram of nanoparticles. 50 , 10 30 , 10 10 , 10 5 , 10 3 , 500, 100, 50, 10, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 , or 10 -50 It is less than or equal to μg.

[0114] In another embodiment of the present invention, the percentage, concentration, number of atoms, amount, mass, or volume of iron oxide contained in the nanoparticles is preferably 1.00001, 1.001, 1.1, 2, 5, 10, 50, 10 2 , 10 3 , 10 5 , 10 10 , 10 20 or 10 50 The coefficient is greater than the percentage, concentration, number of atoms, amount, mass, or volume of impurities contained in the nanoparticles.

[0115] In one embodiment of the present invention, iron oxide and / or impurities are present or inserted in the following locations: i) inside the nanoparticles, ii) on the surface of the nanoparticles, iii) outside the nanoparticles, iv) in the crystalline or amorphous structure of the nanoparticles, v) in defects of the nanoparticles, and / or vi) in vacancies of the nanoparticles.

[0116] In one embodiment of the present invention, iron oxide and / or impurities are in interaction with nanoparticles, such as electrostatic, strong, weak, nuclear, metallic, van der Waals, Debye, London, or hydrogen interactions.

[0117] In one embodiment of the present invention, iron oxide and / or impurities are preferably separated from nanoparticles at a distance of 1050 or less from the center or surface of the nanoparticles. 50 , 10 20 , 10 10 , 10 5 , 10 3 It is located within 100, 10, 5, or 1 nm. In some cases, the center of a nanoparticle is the region, volume, location, or aggregate of chemical elements, which is the center of the maximum, minimum location, and / or average dimension of the nanoparticle, such as half the diameter of a spherical nanoparticle or half the maximum, minimum, and / or average location of the nanoparticle. In some other cases, the surface of a nanoparticle is the region, location, or aggregate of chemical elements that remains within the nanoparticle while being at the maximum distance from the center of the nanoparticle.

[0118] In yet another embodiment of the present invention, iron oxide and / or impurities are derived from nanoparticles, preferably from the center or surface of the nanoparticles, in amounts of 0.001, 0.01, 0.1, 1, 10, 100, 10 3 , 10 5 , 10 10 , 10 20 or 10 50 It is located at a distance greater than nm. II 1) In another embodiment of the present invention, the nanoparticles according to the present invention consist of a core and / or coating preferentially surrounding the core of the nanoparticle. II 2) In one embodiment of the present invention, the core and / or coating of the nanoparticles has at least one property common to the nanoparticles, such as the concentration of iron oxide and / or impurities.

[0119] In one embodiment of the present invention, the nanoparticles, the nanoparticle core and / or coating have at least one of the following properties: (a) Magnetic, diamagnetic, superparamagnetic, ferromagnetic, ferrimagnetic, and / or paramagnetic behavior or properties are preferred. -50 , 10 -40 , 10 -20 , 10 -10 , 10 -5 , 10 -2 or 10 -1 With a strength of T or higher, 10 10 , 10 5 , 10 3 , 10 2 These properties are preferentially observed at temperatures below 10°C or 1 K. In some cases, the core may have different magnetic properties from the coating; for example, the core may be ferromagnetic or superparamagnetic, while the coating may be diamagnetic or paramagnetic. b) at least 1, 2, 5, 10, 50, 100, 10 3 , 10 5 , 10 7 , 10 9 , 10 20 or 10 50It includes crystalline portions or structures containing crystal planes or crystalline ordered structures that can be preferentially observed or measured with an electron microscope. In some cases, the core may have a different crystalline structure from the coating. For example, the core may have 1, 5, 10, 10 3 or 10 5 The coating may contain crystal planes or crystal order structures exceeding 10 5 , 10 3 It may contain 10, 5 or 2 or fewer crystal planes or an ordered crystalline structure. (c) A composition comprising a metal or metal oxide, preferably iron oxide, most preferably maghemite and / or magnetite. In some cases, the core may contain a different composition from the coating. For example, the core may contain more than 1, 5, 10, 25, 50, 75, 90, 95 or 99 percent or mass percent of iron oxide, and the coating may contain 99, 95, 90, 75, 50, 10, 5 or 1 percent or mass percent of iron oxide. This ratio is the ratio obtained by dividing the amount, volume, number of atoms, and mass of iron oxide contained in the core and / or coating by the total amount, total volume, total number of atoms, and total mass of all chemical elements contained in the core and / or coating. (d) Single domain, or magnetically single domain, (e) Magnetic microstructures characterized by the presence of magnetic field lines. These can be oriented in a preferred direction, such as the easy magnetization axis or crystallographic direction of the core of nanoparticles like

[0111] . Such magnetic microstructures may be observable under certain conditions, particularly by electron holography. (f) Dimensions of 1 nm and 10 5 mm, 1 nm and 10 3mm, 1 nm and 100 mm, 1 nm and 10 mm, 1 nm and 1 μm, 5 nm and 1 mm, 5 and 500 nm, 5 and 250 nm, 5 and 100 nm, 5 and 80 nm, 5 and 60 nm, 10 nm and 1 mm, 10 and 500 nm, 10 and 250 nm, 10 and 100 nm, 10 and 80 nm, 10 and 60 nm, 15 nm and 1 mm, 15 and 500 nm, 15 and 250 nm, 15 and 100 nm, 15 and 80 nm, 15 and 60 nm, 20 nm and 1 mm, 20 and 500 nm, 20 and 250 nm, 20 and 100 nm, 20 and 80 nm, or between It is between 20 and 60 nm. (g) In some cases, sizes larger than 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35, or 40 nm. (h) In other cases than 10 10 , 10 5 , 10 4 Dimensions of 2000, 1000, 500, 400, 300, 200, 150, 120, 100, 95, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or less than 5 nm. (i) Preferably at pH levels of 0-14, 1-13, 2-12, 3-11, 4-10, 5-9, or 6-8, 10 10 mV and 10 10 mV, -10 5 mV and 10 5 mV, -10 4 mV and 10 4 mV, -10 3 mV, -10 2 mV and 10 2 Zeta potential, charge, or surface charge between mV, -10, and 10 mV. (j) Zeta potential, charge, or surface charge, which may in some cases preferentially at pH greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 than -10 50 -10 20 -10 10 -10 5 -10 3 Higher than -10, -5, -1, 0, 5, 10, 20, 50, or 100 mV. (k) Zeta potential, charge, or surface charge, which in other cases preferentially at pH values ​​of 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less than -10 50 -10 20 -10 10 -10 5 -10 3 , -10, -5, -1, 0, 5, 10, 20, 50, or lower than 100 mV. (l) Zeta potential, charge, or surface charge, which in other cases preferentially at pH greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, than 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 5, 1, 0, -5, -10, -20, -50, or higher than -100 mV. (m) Zeta potential, charge, or surface charge. In other cases, 1050, 1020, 1010, 105, 103, 10, 5, 1, 0, -5, -10, -20, -50, or -100 mV, preferably low at pH values ​​of 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 or less. (n) Isoelectric points that fall between 0 and 14, 1 and 13, 2 and 12, 3 and 11, 4 and 10, 5 and 9, or 6 and 8. (o) In some cases, an isoelectric point greater than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. (p) In other cases, an isoelectric point less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.

[0120] In one embodiment of the present invention, the core and / or coating are synthesized by nanoparticle-producing cells.

[0121] In another embodiment of the present invention, the core and / or coating are not synthesized by nanoparticle-producing cells.

[0122] In one embodiment of the present invention, the nanoparticle-producing cells, also called nanoparticle-producing cells, are eukaryotic or prokaryotic cells. In some cases, they are cells produced, contained within, or grown within a pre-growth and / or growth medium.

[0123] In one embodiment of the present invention, the nanoparticle-producing cells are 100, 80, 70, 50, 10, 20, 10, 5, 10, 2, 10, 0.1 or 10 -10 Less than % contains or produces at least one nanoparticle. In some cases, this percentage is the number of cells in the pre-growth and / or growth medium that contain or produce at least one nanoparticle divided by the total number of cells in the pre-growth and / or growth medium / media.

[0124] In another embodiment of the present invention, nanoparticle-producing cells exceeding 100, 80, 70, 50, 10, 20, 10, 5, 2, 1, 0.1, or 10-10% contain or produce at least one nanoparticle.

[0125] In one embodiment of the present invention, the nanoparticle-producing cells are whole cells.

[0126] In yet another embodiment of the present invention, the nanoparticle-producing cell is a part of a cell such as a cell membrane, vesicle, enzyme, protein, lipid, DNA, RNA, organelle, compartment, cytoplasm, or virus, which is contained in, originates from, replicates within, or is produced therein.

[0127] In one embodiment of the present invention, the synthetic cells are cells that preferentially grow and / or are contained in a fed-batch medium and preferentially synthesize nanoparticles when they grow or divide, but do not synthesize nanoparticles when they grow, divide or are composed in a preliminary growth medium.

[0128] In one embodiment of the present invention, nanoparticles synthesized by cells are called cell-synthesized nanoparticles.

[0129] In one embodiment of the present invention, a nanoparticle-producing cell synthesizes nanoparticles within its cell. Preferentially, nanoparticles are synthesized within the cell as follows when they are synthesized, assembled, crystallized, partially or completely synthesized: i) organelles, Golgi vesicles or apparatus, endosomes, exosomes, ribosomes, endoplasmic reticulum, actin filaments, nuclei, peroxisomes, microtubules, lysosomes, mitochondria, filaments, centrosomes, flagella, or cell membranes, etc., on the side of the cell, in or near or inside the cell ii) In a region within a cell, or iii) From a portion of the cells 10 5 , 10 3 In regions at distances of 100, 10, or 1 nm or less, That is the case.

[0130] In another embodiment of the present invention, nanoparticle-producing cells synthesize nanoparticles extracellularly. Preferably, nanoparticles are synthesized extracellularly, either partially or completely, when they are synthesized, assembled, and crystallized, as follows: i) Extracellular regions, or ii) From a portion of the cell, 1, 10, 100, 10 3 , or 10 5 A region located at a distance greater than nm.

[0131] In some cases, cells preferentially grow in quantities of 1, 10, or 10 per liter of growth medium. 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10100 It is an aggregate of more than one cell. In some other cases, cells preferentially grow at a rate of 10 per liter of growth medium. 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 It is an aggregate of 100, 50, 10, 5, or 2 or fewer cells.

[0132] In one embodiment of the present invention, the nanoparticle-producing cells are eukaryotic cells, preferably belonging to humans, animals, plants, trees, wheat flour, branches, mushrooms, fungi, archaea, birds, fish, pigeons, trout, mammals, ants, bees, or insects.

[0133] In one embodiment of the present invention, the nanoparticle-producing cell is a prokaryotic cell or a bacterium.

[0134] In some cases, nanoparticle-producing cells may be Mycobacterium, preferably Mycobacterium paratuberculosis, Shewanella, preferably Shewanella oneidensi, and Diospirix, preferably Diospirix fermentans. These bacteria preferentially synthesize nanoparticles extracellularly.

[0135] In some other cases, nanoparticle-producing cells are magnetotactic bacteria such as Magnetospirillum magneticum strain AMB-1, magnetotactic cocci strain MC-1, three Vibrio strains (facultative anaerobic Vibrio strains MV-1, MV-2, and MV-4), magnetotactic strain MS-1, Magnetospirillum glutiswaldens strain MSR-1, facultative aerobic magnetotactic Spirillum, Magnetospirillum magneticum strain MGT-1, and obligate anaerobic bacteria, Desulfovibrio megneticus anaerobic respiratory bacterium RS-1. These bacteria synthesize nanoparticles within their cells.

[0136] In one embodiment of the present invention, nanoparticle-producing cells are cultured in or using a pre-growth medium for and between a pre-growth step, and / or in or using a growth medium for and between a growth step. A fed-batch medium is used or used during the growth step. In some cases, the pre-growth medium and the growth medium are the media in which the nanoparticle-producing cells are grown. In some cases, the fed-batch medium is a medium that is preferentially added to the growth medium during the growth step.

[0137] In one embodiment of the present invention, all pre-growth media and growth media may include a chemical element, water, and at least one source of nanoparticle-producing cells. In some other cases, the partial pre-growth and / or growth media may include water and at least one source of chemical elements that do not include nanoparticle-producing cells. In yet another few cases, the pre-growth and / or growth media may include only nanoparticle-producing cells.

[0138] In one embodiment of the present invention, the pre-growth and / or growth and / or fed-batch medium comprises at least one source, preferably one source of chemical elements, or preferably at least one chemical element in a liquid, gaseous, and / or solid state. In some cases, the pre-growth and / or growth and / or fed-batch medium is in a liquid, gaseous, and / or solid state.

[0139] In one embodiment of the present invention, the concentration of a chemical element such as iron in the pre-growth and / or growth medium is the concentration of the chemical element in: i) the entire pre-growth and growth medium, ii) a portion of the pre-growth and growth medium, or iii) nanoparticle-producing cells.

[0140] In one embodiment of the present invention, a specific amount or volume of cells may be a specific amount or volume of growth medium containing those cells. In some other cases, a specific amount or volume of cells may be a specific amount or volume of cells without water or without an aqueous environment for the cells, or after water or an aqueous environment for the cells has been removed, for example by freeze-drying.

[0141] In one embodiment of the present invention, it becomes possible to prevent genetic modification of nanoparticle-producing cells by the growth conditions of the cells during the preliminary growth and / or growth step. In some cases, the genetic modification of nanoparticle-producing cells is a modification of the gene, a portion of the gene, a portion of the DNA, or at least 10 nucleotides. -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 The modifications are 1, 5, 10, 50, 75, 90, or 95%, where the percentage is the ratio of the number or amount of genes, gene portions, DNA portions, or nucleotides modified in the nanoparticle-producing cells to the total number or amount of all genes, gene portions, DNA portions, or nucleotides belonging to the nanoparticle-producing cells.

[0142] In yet another embodiment of the present invention, the preliminary growth medium and / or growth medium is preferably purified water, deionized water, or ultrapure water, and preferably 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10, 50, 75, 80, 90, 99, 99.99 or 99.99999 percent or more of water or mass percent of water. This percentage is the ratio of the amount, mass, volume, or number of atoms of water in the pre-growth medium and / or growth medium, divided by the total amount, mass, volume, or number of atoms of all chemicals present in the pre-growth medium and / or growth medium.

[0143] In one embodiment of the present invention, the pre-growth medium and / or growth medium includes one source of at least one chemical element or chemical component. In some cases, the concentration of a chemical component, such as iron, in the pre-growth medium and / or growth medium is the concentration of this chemical component at any point in the pre-growth and / or growth stage. In some cases, this concentration can be measured by dividing the number of moles, mass, or volume of this chemical component or its occupancy by the number of moles, total mass, or total volume of all chemical components or their occupancy in the pre-growth and / or growth medium.

[0144] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one carbon source. The carbon source may include chemical elements of the periodic table C. The carbon source may also include acetates, glycolates, glucose, lactates, pyruvates, succinates, carbon dioxide, glycerol, and derivatives or combinations thereof.

[0145] In one embodiment of the present invention, the growth and / or pre-growth medium comprises at least one nitrogen source. Optionally, the nitrogen source comprises a chemical element of the periodic table N. Optionally, the nitrogen source can be selected from the group consisting of ammonium salts, nitrates, urea, amino acids, ammonia, nitrogen gas, and derivatives or combinations thereof.

[0146] In one embodiment of the present invention, the pre-growth and / or growth medium includes at least one source of sulfur or sulfate. The sulfur or sulfate source may include chemical elements of the periodic table S. The sulfur or sulfate source may also be sulfate or hydrogen sulfide.

[0147] In one embodiment of the present invention, the pre-growth and / or growth medium includes at least one source of phosphorus or phosphate. In some cases, the source of phosphorus or phosphate includes a chemical element of the periodic table P. In some cases, the source of phosphorus or phosphate may be a phosphate.

[0148] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one calcium source. In some cases, the calcium source comprises a chemical element of the periodic table Ca. In some cases, the calcium source may be a calcium salt.

[0149] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one potassium source. In some cases, the potassium source comprises a chemical element of the periodic table K. In some cases, the potassium source is a potassium salt.

[0150] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one magnesium source. In some cases, the magnesium source comprises the chemical element Mg of the periodic table. In some cases, the magnesium source is a magnesium salt.

[0151] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one oxygen source. Optionally, the oxygen source comprises a chemical element of the periodic table O. Optionally, the oxygen source is an organic compound, carbon dioxide, or dioxygen.

[0152] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one hydrogen source. Optionally, the hydrogen source comprises a chemical element of the periodic table H. Optionally, the hydrogen source is an organic compound or a dihydrogen.

[0153] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one iron source. In some cases, the iron source comprises a chemical element of the periodic table Fe. In some cases, the iron source is iron, consists of iron, or contains iron. In some cases, the iron source is iron citrate, iron quinate, iron chloride, or iron sulfate.

[0154] In one embodiment of the present invention, the pre-growth and / or growth medium comprises at least one sulfur source. Optionally, the sulfur source comprises a chemical element of the periodic table S. Optionally, the sulfur source comprises at least one vitamin.

[0155] In one embodiment of the present invention, the source of carbon, nitrogen, sulfur, phosphorus sulfate, phosphate, calcium, potassium, magnesium, oxygen, hydrogen, or iron is 10 -100 , 10 -50 , 10 -20, 10 -10 , 10 -5 , 10 -3 , 10 -1 It contains 1, 5, 10, 25, 50, 75, 80, 90 or 95 percent by mass of carbon, nitrogen, sulfur, sulfates, phosphorus, phosphates, calcium, potassium, magnesium, oxygen, hydrogen, or iron. In some cases, they are in gaseous, liquid, or solid state. In other cases, they can be used to prepare preliminary growth and / or growth media. In some cases, preliminary growth and / or growth media may be 2, 3, 4, 5, 10, 50, 100, 10 3 , 10 5 , 10 10 or 10 50 Contains sources of different carbon, nitrogen, and sulfur, sulfates, phosphorus, phosphates, calcium, potassium, magnesium, oxygen, hydrogen, and / or iron. In some other cases, the pre-growth and / or growth medium may be 2, 3, 4, 5, 10, 50, 100, or 10 3 , 10 5 , 10 10 or 10 50 Contains carbon, nitrogen, sulfur sulfates, phosphorus, phosphates, calcium, potassium, magnesium, oxygen, hydrogen, and / or iron from one or fewer different sources.

[0156] In one embodiment of the present invention, at least one source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen, and / or iron in the preliminary growth medium is the same as that of the growth medium.

[0157] In another embodiment of the present invention, at least one source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen, and / or iron in the pre-growth medium is different from those in the growth medium.

[0158] In another embodiment of the present invention, the pre-growth and / or growth medium comprises a source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron. -100 , 10-50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 It contains concentrations greater than mM.

[0159] In another embodiment of the present invention, the pre-growth and / or growth medium comprises a source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen and / or iron. 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10, 1, 10 -1 , 10 -3 , 10 -6 , 10 -9 , 10 -20 , 10 -50 or 10 -100 It contains at a concentration of mM.

[0160] In another embodiment of the present invention, the pre-growth and / or growth medium is prepared using pharmaceutical-grade or ultra-high-purity chemicals or elements.

[0161] In yet another embodiment of the present invention, the culture medium impurities are impurities contained in the pre-growth and / or growth medium and / or fed-batch medium.

[0162] In one embodiment of the present invention, the pre-growth and / or growth medium contains small amounts of medium impurities. In some cases, the proportion of impurities in the medium is 100, 10 20 , 10 10 , 10 5 , 10 2 , 10, 5, 1, 0.1, or 0.001% or less. Preferentially, the pre-growth and / or growth mediums shall have a quantity or concentration of medium impurities of at least 1.00001, 1.1, 1.5, 2, 5, 10, 103 , 10 10 or 10 20 The coefficient is lower than the amount or concentration of at least one source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen, and / or iron. According to the present invention, the proportion of impurities in the medium can, in some cases, be defined as the ratio of the number, amount, mass, or volume of atoms of impurities in the medium contained in the pre-growth and / or growth medium divided by the total number, amount, mass, or volume of atoms of all chemical elements contained in the pre-growth and / or growth medium. In some other cases, the concentration of medium impurities contained in the pre-growth and / or growth medium is 10 per mL of medium impurities. 50 , 10 30 , 10 10 , 10 5 , 10 3 , 500, 100, 50, 10, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 or 10 -50 It is lower than μg.

[0163] In one embodiment of the present invention, the pre-growth and / or growth medium contains a considerable amount of medium impurities. In some cases, the mass percentage of impurities in the medium is 0, 10 -40 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , greater than 1, 5, 10, 25, 50, 75, 80, or 90%. In some other cases, the concentration of media impurities in the pre-growth and / or growth medium is greater than 10% of the pre-growth and / or growth medium per 1 mL of medium impurities. -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 10, 50, 100, 10 3 , 10 5 , or 1010 Larger than μg

[0164] In some cases, nanoparticles generated, obtained, or produced from pre-growth and / or growth media may be magnetosomes.

[0165] In one embodiment of the present invention, the method according to the present invention comprises a preliminary growth step in which nanoparticle-producing cells are grown in a preliminary growth medium to produce nanoparticle-producing cells, wherein the nanoparticle-producing cells essentially do not produce nanoparticles.

[0166] In one embodiment of the present invention, the nanoparticle-producing cell used to initiate the preliminary growth step is a cell having at least one of the following characteristics: i) These are 0.001, 0.1, 1, 5, 10, 10 of the start of the preliminary growth step. 3 , 10 5 or 10 10 These are cells from over an hour ago. ii) These are included in cell banks such as master cell banks, working cell banks, or research cell banks. iii) 1, 5, 10, 10 per cell 3 , 10 5 or 10 10 It is composed of one or more nanoparticles. iv) They preferentially contain the majority of water and are preferably liquids or media that are identical or similar in composition to the pre-growth and / or growth media. v) They are 100, 10, 1, 10 -1 , 10 -2 , 10 -3 , 10 -5 or 10 -10 It is contained in culture media with impurity concentrations of μM or less. vi) They are contained in or held in a culture medium that allows them to maintain or have no more than 100, 10, 1, 0.1, or 0.01 grams of impurities per gram of nanoparticles. vii) They are 10 -100 and 10 100, 10 -50 and 10 50 , 10 -30 and 10 30 , 10 -20 and 10 20 , 10 -10 and 10 10 , 10 -6 and 10 5 , 10 -6 and 10 4 , 10 -6 and 10 2 , or 10 -6 It consists of volumes between and 1 liter. viii) They consist of a volume at least 10 times lower than the volume of the initial preliminary growth step. ix) They preferentially use 1 and 10 cells per liter of pre-growth and / or growth medium. 100 , 2, and 10 50 , 3 and 10 20 , or between 10 and 10 10 cells, vii), they have an optical density comprised between 10 -50 and 10 50 , 10 -20 and 10 20 , 10 -10 and 10, 10 -5 and 10 5 , 10 -5 and 10 3 , 10 -5 and 10 2 , 10 -5 and 1, 10 -5 and 10 -1 , 10 -5 and 10 -2 , or between 10 -5 and 10 -3 It is either the optical density contained between or represents. x) Preferably 1, 10, 10 per hour or 1 liter of pre-growth and / or growth medium per hour. 3 , 105 , 10 10 , 10 20 , 10 50 or 10 100 It has the following number of cell divisions. Store or keep at temperatures below 100, 50, 25, or 0°C, preferably at 77 K or -20°C.

[0167] In one embodiment of the present invention, the nanoparticle-producing cells used to initiate the preliminary growth step have at least one of the following characteristics: i) They have a culture medium impurity concentration of 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 It is contained in a culture medium with a concentration of 1 or 10 μM or higher. ii) Impurities per gram of nanoparticles are 10 -40 , 10 -20 or 10 -10 It is contained in or maintained in a culture medium that allows it to maintain or hold more than a certain amount (in grams). vii) Preferentially, cell divisions per hour or per hour of preliminary growth and / or per liter of growth medium are 1, 10, 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 There are more. viii) 1, 5, 10, 10 per cell 3 , 10 5 or 10 10 This includes the following nanoparticles, which are maintained from or originate from a medium containing a sufficiently low iron concentration to prevent the formation of nanoparticles.

[0168] In one embodiment of the present invention, at least one of the properties of the nanoparticle-producing cells used to initiate a preliminary growth step allows for the prevention of death, destruction, disappearance, denaturation, or inactivation of the nanoparticle-producing cells.

[0169] In one embodiment of the present invention, the optical density of cells is measured when the cells are contained in pre-growth and / or growth medium, solution, or water, preferably when the growth medium is removed and the cells are resuspended in water. In some cases, the optical density of cells is 1, 2, 5, 10, 50, 100, 200, 300, 400, 450, 500, 550, 600, 900, 10 3 , 10 5 or 10 7 It is measured at wavelengths of 10 nm or greater. 7 , 10 5 , 10 3 , 600, 550, 500, 450, 400, 300, 200, 100, 50, 10, 5, 2 or shorter wavelengths than 1 nm. In some other cases, the optical density of cells is measured at wavelengths of 1–107 nm, 50–105 nm, 100–103 nm, 200–900 nm, or 400–800 nm.

[0170] In one embodiment of the present invention, the number of cell proliferations between two time points t0 and t1 of the preliminary growth and / or growth step is equal to or proportional to: i) the number of cells n(t1) measured at t1 and / or ii) the ratio between the number of cells n(t1) measured at t1 and the number of cells n(t0) measured at t0.

[0171] In one embodiment of the present invention, the rate or speed of cell division is [n(t1)-n(t0)] / (t1-t0) That is the case.

[0172] In another embodiment, the rate or speed of cell division is [n(t1)- n(t0)] / [(t1-t0)·V] V is the volume of the preliminary growth and / or growth medium in which the cells are cultured or grown.

[0173] In one embodiment of the present invention, the preliminary growth step is initiated by thawing or heating to start the preliminary growth step from a temperature of 100, 50, 25, 10, or 0°C or lower to a temperature of 0, 10, 25, 50, or 100°C or higher. The nanoparticle-producing cells are then inserted into or added to the preliminary growth medium. In some cases, this initial stage of the preliminary growth step is 10 -50 and 10 50 , 10 -50 and 10 10 , 10 -30 and 10 5 , 10 -20 and 10 3 , 10 -10 and 10 2 , or between 10 -5 And it takes place during the time elapsed between 10 and 10.

[0174] In another embodiment of the present invention, the preliminary growth step involves increasing different, preferentially increased volumes V0, V1, ..., V i … V j The proliferation is divided into substeps 0, 1, ..., i, ..., j, where i is an integer (0) that specifies the number of proliferations on different volumes. 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 When initiating the preliminary growth stage with small amounts of cells, such as 10, 5, 3, or 2 or fewer, the number of cells in different volumes of cell proliferation or cell proliferation may be important, for example, 10 cells preferentially contained in 1 liter, 1 milliliter, or 1 microliter of preliminary growth medium or aqueous solution. 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10​3 , 10 2 This is when the preliminary growth step begins with 10, 5, 3, or 2 or fewer cells. In these cases, i and / or j are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 10, 10 3 , 10 5 or 10 10 It can be made larger. In some other cases, different amounts or numbers of cell proliferations can be used, for example, if the preliminary growth step is large, it can be preferentially contained in 1 liter, 1 milliliter, or 1 microliter of preliminary growth medium or aqueous solution, preferably 2, 3, 5, 10, or 10. 2 , 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 If starting with more cells, the value may be lower. In these cases, i and / or j are 10. 10 , 10 5 , 10 3 , 10 2 , 10, 5, 4, 3, 2, or 1 or less.

[0175] In one embodiment of the present invention, ratio V i / V i-1 The largest is 10, with priority given to 10. -50 , 10 -30 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 1.00001, 1.0001, 1.001, 1.01, 1.1, 1.2, 1.5, 2, 3, 4, 5, 7, 10, 10 2 , 10 3 , 10 5 or 10 10 Larger. In some cases, the number of cell divisions between substep i-1 and substep i of the preliminary growth step is 1, 5, 10, or 10 per hour, or per liter of preliminary growth medium per hour. 3 , 10 10 or 1020 More, or when the optical density of cells is 1.00001, 1.1, 2, 5, 10, 10 per hour 3 , 10 5 or 10 7 If it increases beyond V i / V i-1 It's big.

[0176] In another embodiment of the present invention, ratio V i / V i-1 It is low, and 10 is preferred. 100 , 10 50 , 10 10 , 10 5 , 10 3 , 10 2 , 10, 5, 3, 2, 1.01, 1.001, 1.000001, 1, 10 -5 , 10 -10 or 10 -50 In some cases, the number of cell divisions per hour, or per liter of pre-growth medium per hour, is preferred to be 10. 50 , 10 10 , 10 3 , 10 2 If the values ​​are less than 10, 5 or 1, or if the optical density of the cell is 1.00001, 1.1, 2, 5, 10, 10 per hour 3 , 10 5 or 10 7 If it increases by the following coefficient, V i / V i-1 It is low.

[0177] In some cases, V i / V i-1 By reducing V, the number of preliminary growth steps at different volumes can be increased. In other cases, V i / V i-1 By increasing this, the number of preliminary growth steps at different volumes can be reduced.

[0178] In one embodiment of the present invention, the preliminary growth step and at least one of its substeps is a volume V iOptical density of bacterial suspension inside (1 i) 10 -50 , 10 -30 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 5, 10, 15, 50, 10 2 , 10 3 or reaches a value of 10 or more. and or ii) Preferably 1.000001, 1.0001, 1.01, 1.1, 1.5, 2, 5, 10, 10 at the end of the preliminary growth step rather than at the start of the preliminary growth step, and / or at the end of at least one substep of the preliminary growth step rather than at the start of the substep. 2 , 10 3 , 10 5 , 10 10 or 10 20 A coefficient exceeding this value indicates a larger value.

[0179] In one embodiment of the present invention, in the preliminary growth step and / or at least one of its substeps, volume V i Optical density of bacterial suspension inside (1 i) 10 -50 , 10 -30 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 5, 10, 15, 50, 10 2 , 10 3 or 10 5 Reaching the following values, and / or ii) Prioritize 1.000001, 1.0001, 1.01, 1.1, 1.5, 2, 5, 10, 10 2 , 10 3 , 10 5 , 10 10 or 10 20 A coefficient greater than , which is lower at the end of the preliminary growth step than at the start of the preliminary growth step, and / or lower at the end of at least one substep of the preliminary growth step than at the start of the preliminary growth step.

[0180] ​​ In one embodiment of the present invention, the start of volume V0 proliferation is time t PG0b The process is carried out at time t, and the end of volume V0 proliferation is at time t PG0e This is done at volume V i The start of proliferation is at time t PGib It was carried out at V i The end of proliferation is time t PGie , performed in volume V j The start of proliferation is at time t PGjb It is done in and / or volume V j The end of proliferation is time t PGje It will be held at [location].

[0181] In one embodiment of the present invention, i) time t PG0b The start of the preliminary growth step at time t PGje The length of time separating the end of the preliminary growth step is t PGje -t PG0b and or ii) the start of substep i PGib , and the end of substep i t PGie The length of time for separating them is t PGie -t PGib It is equal to 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 2, 5, 10, 24, 100, 10 3 , 10 5 or 10 7 It is more than an hour. In some cases, tPGje-tPG0b and / or t PGie -t PGib This refers to a condition where cell division is difficult or slow, with 10 cells per hour. -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 It grows larger when there are one cell division or one cell division per hour per liter of preliminary growth medium.

[0182] In yet another embodiment of the present invention, t PGje -t PG0b and or t PGie -t PGib is 10 40 , 10 30 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , 10, 5, 2, 1, 10 -1 , 10 -3 , 10 -5 or 10 -10 The following applies. In some cases, t PGje -t PG0b and or t PGie -t PGib The cells divide easily or rapidly, preferably 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 This is considered short if it is the number of cell divisions per hour or the number of cell divisions per hour per liter of preliminary growth medium.

[0183] In another embodiment of the present invention, nanoparticle-producing cells are t PGib or t PG0b By introducing it, or during the preliminary growth step or at least one of its substeps, the iron concentration of the preliminary growth medium is increased to 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 or 10 -1 Maintaining a concentration of above μM promotes proliferation during the preliminary growth step.

[0184] In another embodiment of the present invention, nanoparticle-producing cells are grown during the pre-growth step or at least one of its substeps by consuming oxygen. In some cases, the percentage of oxygen in the pre-growth medium decreases from the following values: i)t PGib or t PG0b in 10 -50 , 10 -10 , 10 -5 , 10 -3 , 1, 5, 10, 20, 50, 75, 90, 95, 99 or values ​​above 99.9%, preferentially from values ​​between 21% and 10-30%, t PGie or t PGje In this case, 99.9, 95, 90, 80, 75, 50, 20, 5, 2, 1, 10 -3 , 10 -5 , 10 -10 or 10 -50 The oxygen content is reduced preferentially to 0%, or to a value between 0 and 10%, and or ii) the oxygen content in the medium for preliminary growth is t PG0b and t PGje between and or t PGib and t PGie Between 1.0001, 1.001, 1.1, 1.2, 1.5, 2, 5, 10, 50, 10 2 , 10 3 , 10 5 or 10 10 The oxygen content decreases by the coefficients described above. In some cases, oxygen is not added to the pre-growth medium in the pre-growth step or at least one of its substeps, and oxygen is consumed by bacteria, resulting in a decrease in the oxygen content of the pre-growth medium. In some other cases, oxygen is added to the pre-growth medium in the pre-growth step or at least one of its substeps, resulting in fluctuations in the oxygen content of the pre-growth medium due to oxygen consumption by bacteria and the addition of oxygen to the pre-growth medium.

[0185] In one embodiment of the present invention, the proportion of oxygen, preferably O2 in the pre-growth medium and / or growth medium, is the proportion of dissolved oxygen in the pre-growth and / or growth medium, preferably O2. In some cases, a proportion of 100% corresponds to the maximum amount of O2 solubilized in the pre-growth and / or growth medium, and 10 per liter of pre-growth and / or growth medium. -5 from 10 20 It consists of mg, preferably 1 to 10 mg of dissolved O2.

[0186] In one embodiment of the present invention, the preliminary growth step consists of cell proliferation, which relates to, corresponds to, and is the following: i) The rate or number of cell divisions is preferably than 1.000001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 10 per unit volume, such as 1 liter of preliminary growth medium. 2 , 10 3 , 10 5 , 10 10 , 10 20 or 10 50 The coefficient is more than double the starting point of the preliminary growth step or its substep, or greater than tPGib or tPG0b than tPGie or tPGje. ii) The rate or number of cell divisions per unit volume, such as 1 liter of pre-growth medium, at the start of one of the pre-growth steps or substeps, or t PGib or t PG0b So, 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 From a number of cells of 10, 5, or 2 or fewer per hour, at the end of one of the preliminary growth steps or substeps or t PGie or t PGje So, 2, 5, 10, 10 2 , 10 3 , 10 5 , 1010 , 10 20 , 10 50 or 10 100 Up to the above number of cells or cells per hour, iii) The optical density preferentially measured for cells contained in a fixed preliminary growth volume such as 1 liter is measured at the start of the preliminary growth step or any of its substeps or t PGib or t PG0b In this case, the optical density is 10, 1, 10 -1 , 10 -2 or 10 -3 From a point equal to or lower than, at the end of one of the preliminary growth steps or its substeps or t PGie or t PGje The optical density is 10 -10 , 10 -2 , 10 -1 up to 1 or 10, such as 1.00001, 1.1, 1.2, 1.5, 2, 5, 10, 10 3 , 10 5 , 10 10 , 10 20 or 10 50 It increases with a larger coefficient.

[0187] In one embodiment of the present invention, cells that do not inherently produce nanoparticles during the preliminary growth step, or during, at the start, or at the end of at least one of its substeps, are characterized by at least one of the following properties: i) The number of nanoparticles contained in the cell is 10 3 ; 10 2 , 50, 20, 10, 5, 2 or less than 1, preferentially 10 or 5, or 0-10 3 The numbers are preferably between 0 and 10 or between 0 and 5. ii) The percentage of cells containing at least one nanoparticle is 100, 99, 90, 80, 50, 20, 10, 1, 0.1%, preferably 10 or 1%, or between 0 and 99%, 0 and 50%, 0 and 10%, preferably between 0 and 5%, where this percentage is preferably the ratio of the number of cells containing at least one nanoparticle to the total number of cells in the preliminary growth medium. iii) Optical density is 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 0.1, 0.2, 0.5, 1, 5, 10, or 100 or more. iv) Number of cells: 1, 5, 10, 10 2 , 10 3 , 10 5 , 10 10 , 10 20 , 10 50 or 10 100 That's all. v) They are 0.0001, 0.001, 0.1, 1, 10, 50, 10 2 , 10 3 , 10 5 or 10 10 It is contained in a volume of more than one liter. vi) Number of cells generated: 1 and 10 10 , 1 and 10 3 During this period, it will be preferentially composed of 50 to 300, vii) End of preliminary growth step OD PGE Optical density measured and the start of the preliminary growth step OD PGB The ratio of optical density measured by ODPGE / ODPGB is 1, 2, 5, 10, 15, 25, 50, 100, 10 3 , 10 5 , 10 10 , 10 50 or 10 100 , greater than; and or viii) Optical density OD measured at the end of substep i of the preliminary growth step PGiE And the optical density O measured at the beginning of substep i of the preliminary growth stepDPGiB The ratio, ODPGiE / ODPGiB, is 1, 2, 5, 10, 15, 25, 50, 100, 10 3 , 10 5 , 10 10 , 10 50 or 10 100 It is equal to or greater than that.

[0188] In one embodiment of the present invention, cells that do not inherently produce nanoparticles are non-magnetic cells.

[0189] In one embodiment of the present invention, the proportion of non-magnetic cells is preferably 10 during the preliminary growth step or at least one of its substeps, at the start or end. -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 Values ​​greater than 1, 5, 10, 50, or 75% can be obtained. In some cases, the percentage of non-magnetic cells is equal to nNMC / (nMC + nNMC), where nMC and nNMC are the number of magnetic and non-magnetic cells, respectively.

[0190] In another embodiment of the present invention, non-magnetic cells do not exhibit a magnetic response. Here, the magnetic response may be the movement of at least one cell at a velocity parallel to the magnetic field or proportional to the strength of the magnetic field, where the strength of the magnetic field is 10 -9 , 10 -3 , 10 -1 , 1, 10 3 or 10 6 The magnetic field may be greater than mT and / or preferentially applied to the cells.

[0191] In one embodiment of the present invention, the method includes a growth step comprising growing nanoparticle-producing cells arising from a preliminary growth step in a growth medium, thereby causing the nanoparticle-producing cells to produce nanoparticles. In some cases, the growth step is carried out in a fermenter or apparatus that allows control of temperature, pH, iron concentration, and / or oxygen concentration of the growth medium.

[0192] In another embodiment of the present invention, the growth step begins by inserting cells obtained from the preliminary growth step into a growth medium. In some cases, at least one of the growth step or its substeps is 10 -50 and 10 50 , 10 -50 and 10 10 , 10 -30 and 10 5 , 10 -20 and 10 3 , 10 -10 and 10 2 , or between 10 -5 and is carried out during the process which is included within 24 hours. In some other cases, the growth step or at least one of its substeps is 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , 10, 5, 2, 1, 10 -1 , 10 -2 , 10 -3 , 10 -5 , 10 -10 or 10 -20 It takes place within an elapsed time of less than 10 hours. Furthermore, in some other cases, the growth step or at least one of its substeps is 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 10 -1 , 1, 2, 5, 10, 10 2 , 10 3 , 10 5 , 10 10 or 10 20 This takes place during the above elapsed time. In some other cases, i and / or i are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 10, 10 3 , 10 5 or 10 10 The following applies:

[0193] In one embodiment of the present invention, each subset i is to bubble or bring different amounts of oxygen into or into the growth medium, and / or bring different amounts of iron into or into the growth medium, preferably with the help of a fed-batch medium.

[0194] In one embodiment of the present invention, the growth step is to preferentially use a gas such as compressed air or a gas containing more than 1% O2 from 1 to 10 10 , 5 to 10 5 , 10 to 10 4 , 100 to 10 3 , or including at least one of the following substeps, which involves introducing into the growth medium under stirring conditions of 100 to 300 revolutions per minute: i) 10 -3 ~10 3 During the first substep, which typically lasts 2-16 hours, the gas is 0-10 units per liter of growth medium. 10 The flow rate is preferentially composed of 0.001 to 40 mL / min, resulting in a cell optical density of 10 -10 ~10 3 Preferably, the value between the start of the first substep (between 0.08 and 0.12) and the end of the first substep is 1.0000001, 1.1, 1.5, 2, 5, 10, or 10. 3 It increases with a larger coefficient, 10 -9 and 10 4 During this period, the value will preferentially be between 0.2 and 1. ii) In the second substep lasting 10⁻³ to 10³ hours, or preferentially 2 to 120 hours, the gas shortage is 1.000000¹, 1.1, 1.5, 2, 5, 10, or 10 3 The coefficient increases by , compared to the first substep, or from 0 to 10 per liter of growth medium. 10 The flow rate is preferentially composed of 1 to 50 mL / min, resulting in an increase in the optical density of cells from the second starting value, equal to or equal to the substep obtained at the end of the first stub step. -9 from 10 4Between these values, preferably between 0.2 and 1, and at the end of the second substep a coefficient greater than the value at the start of the second substep by 1.0000001, 1.1, 1.5, 2, 5, 10, or 103, or 10 -9 from 10 4 During this period, the preferred values ​​are between 0.5 and 4. iii) 10 -3 ~10 3 In the third substep, which lasts for a certain number of hours, or preferentially 2 to 120 hours, the gas shortage is 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 3 It increases significantly more than in the second substep, or 0-10 per liter of growth medium. 10 The flow rate is predominantly 50-120 mL / min, and the optical density of the cells increases from the value at the start of the third substep, equal to the value obtained at the end of the second substep, or 10. -9 from 10 4 Between 0.5 and 4, preferably at the end of the third substep, 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 3 With the above coefficients, the value increases from the value at the start of the third substep, and preferentially increases at the end of the third substep or 10 -9 from 10 4 During this period, the values ​​will preferentially be between 1 and 8. iv) 10 -3 ~10 3 In the fourth substep, which preferentially lasts 2 to 120 hours, the gas shortage is 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 3 It increases significantly more than in the third substep, or 0-10 per liter of growth medium. 10 The flow rate is predominantly 200-300 mL / min, and the optical density of the cells increases from the value at the start of the fourth substep, equal to the value obtained at the end of the third substep, or 10. -9 from 10 4During this time, the values ​​between 1 and 8 are preferred, with the end of the 4th substep preferentially being 1, 1.0000001, 1.1, 1.5, 2, 5, 10 3 It increases by a coefficient of 10 -9 from 10 4 During this time, the preferred value will be between 2 and 16 at the end of the fourth substep. v) 10 -3 from 10 3 In the fifth substep, which preferentially lasts from 2 to 120 hours, the gas shortage is 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 times longer than in the fourth substep. 3 The coefficients above increase, and the growth medium is 0-10 per liter. 10 The flow rate is preferentially composed of 300-500 mL / min, resulting in an increase in the optical density of cells from the value at the start of the fifth substep to the value obtained at the end of the fourth substep, 10 -9 from 10 4 Between 2 and 16, preferably, at the end of the 5th substep, the value should be 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 greater than the value at the start of the 5th substep. 3 It increases by a coefficient of , or 10 -9 from 10 4 The value between these two values ​​will be prioritized, with the fifth substep being the last value between 4 and 32.

[0195] In one embodiment of the present invention, during substep i, preferably substeps 2 to 5, i) The oxygen percentage is maintained between 0.01% or 0.1 mBar and 0.9% or 9 mBar due to oxygen consumption by cells and lack of air. ii) Gas deficiency of 0 to 10 per liter of growth medium 10 , 1 to 10 5 , 5 to 10 4 , 10 to 10 3 It is composed of values ​​between mL / min. iii) The growth medium is 1 to 10 5 , 10 to 10 4, 50 to 10 3 It is being stirred at a speed consisting of 100 to 500 revolutions per minute. iv) Gas shortage can be reduced by increasing the stirring speed of the medium. v) A gas deficiency can be increased by reducing the stirring speed of the culture medium, and / or vi) The optical density of the cells is preferentially 10 at the start of substep i. -50 from 10 3 From the values ​​included in between, preferentially select 10 at the end of substep i. -20 from 10 5 The value increases to the value contained within that range.

[0196] In one embodiment of the present invention, the growth step comprises at least one substep during which the pH of the medium is fixed or determined or selected between 0 and 14, 2 and 13, i.e., between 4 and 11, between 5 and 10, preferably between 5 and 8, and most preferably between 6.9. It is preferably rotated at 1 to 10 per minute. 10 This is preferentially done by adding an acidic source of iron contained in the fed-batch medium under stirring conditions of 100-300. In some cases, the growth medium contains the iron concentration at the start of the growth step or its substep, and it i), 10 10 , 10 5 or 10 2 Less than μM, preferably 10 or 2 μM or less, and / or ii)10 -10 ~10 10 , 10 -5 ~10 5 , 10 -3 ~10 3 It consists of μM, preferably 0.2 to 20 μM. In some other cases, during the growth step or one of its substeps, the iron concentration in the growth medium increases preferentially with the addition of fed-batch medium to the growth medium, reaching the following value at the end. i) 10 -10 , 10 -5 , 10 -1 or greater than 1 μM, preferably greater than 2 μM, and or ii)10 -10 from 10 10 The concentrations are preferably between 2 μM and 5 mM, or between 2 μM and 0.5 mM.

[0197] In one embodiment of the present invention, the growth step includes at least one of the following substeps, in which: 10 -3 from 10 3 Preferably, during the first substep lasting 2 to 16 hours, the feed-in medium is inserted into the growth medium by means of 10 -10 from 10 10 Between μM, the iron concentration in the growth medium is preferentially 2–20 μM. Bacterial iron consumption may or may not be considered. This results in 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 from the start to the end of the first substep. 3 It increases by a coefficient exceeding . Preferably, 10 nanoparticles per liter of growth medium at the start of the first substep. -10 from 10 10 Between preferably 0.001 and 0.1 mg, at the end of the first substep, 10 nanoparticles per liter of growth medium. -10 and 10 10 Nanoparticles are produced with a value between 1 and 10 mg, preferably between 1 and 10 mg. 10 -3 from 10 3 Preferably, during the second substep lasting 2 to 120 hours, the feed-in medium is inserted into the growth medium by means of 10 -10 from 10 10 This results in an iron concentration in the growth medium that is between 20 and 40 μM. Bacterial iron consumption may or may not be taken into account. As a result, from the start to the end of the second substep, the nanoparticles are 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 3 It increases by a coefficient exceeding 10 at the start of the second substep, or 10 -10 and 10 10From a value between 1 and 10 mg, preferably a value between 1 and 10 mg, at the end of the second substep, 10 nanoparticles per liter of growth medium. -10 ~10 10 During this period, values ​​preferably ranging from 2 to 20 mg are preferentially produced. 10 -3 from 10 3 Preferably, during the third substep lasting 2 to 120 hours, the feed-in medium is inserted into the growth medium by means of 10 -10 from 10 10 This results in iron concentrations in the growth medium being between μM, preferably between 40 and 150 μM. Bacterial iron consumption may or may not be taken into account. As a result, from the start to the end of the third substep, the nanoparticles are 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 3 It increases by a coefficient exceeding 10 at the start of the third substep. -10 and 10 10 From a value between 2 and 20 mg, at the end of the third substep, 10 nanoparticles per liter of growth medium. -10 ~10 10 Between these values, preferably values ​​between 4 and 40 mg are preferentially produced. 10 -3 from 10 3 Preferably, during the fourth substep lasting 2 to 120 hours, by inserting fed-batch medium into the growth medium, 10 -10 from 10 10 The resulting iron concentration in the growth medium is between μM, preferably between 150 and 500 μM. Bacterial consumption of iron may or may not be taken into account. As a result, from the start to the end of the fourth substep, the nanoparticles are 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 3 It increases by a coefficient exceeding 10 at the start of the fourth substep. -10 and 10 10 From a value between 4 and 40 mg, preferably a value between 4 and 40 mg, at the end of the fourth substep, 10 nanoparticles per liter of growth medium -10 ~10 10Between these values, preferably values ​​between 8 and 80 are preferentially generated. 10 -3 from 10 3 Preferably, during the fifth substep which lasts from 2 to 120 hours, the feeding medium is inserted into the growth medium by means of 10 -10 from 10 10 The resulting iron concentration in the growth medium is between μM, preferably between 500 and 1000 μM. Bacterial consumption of iron may or may not be taken into account. As a result, from the start to the end of the fifth substep, the nanoparticles are 1.0000001, 1.1, 1.5, 2, 5, 10, or 10 3 It increases by a coefficient exceeding 10 at the start of the fifth substep. -10 and 10 10 From a value between 8 and 80 mg, preferably a value between 8 and 80 mg, at the end of the fifth substep, 10 nanoparticles per liter of growth medium -10 ~10 10 During this period, values ​​preferably between 16 and 160 mg are preferentially produced.

[0198] In one embodiment of the present invention, the growth step or substep i of the growth step, preferably substeps 1 to 5, is: i) Meanwhile, the iron concentration in the growth medium is 10 -10 from 10 10 The concentration increases to over μM, preferably more than 2 μM. Preferably, iron-containing fed-batch medium is added to the growth medium, and 10 times the amount is used for iron consumption by the cells. 10 Add 5 mM or less, preferentially. ii) The total amount of iron per liter of growth medium is 10 per liter of growth medium. -6 From 15, preferentially 2·10⁻⁴ to 1.5g of iron. and or iii) The amount of nanoparticles should be 0 to 500 mg per liter of growth medium at the start of a growth step or substep, preferably 0 to 80 mg, and 1 to 10 mg per liter of growth medium at the end of one of the growth steps or substeps. 5The values ​​are between mg, with a preference for growing to nanoparticles between 10 and 200 mg.

[0199] In one embodiment of the present invention, the growth step consists of cell proliferation, and such proliferation is related to or corresponds to the rate or number or optical density of cell division. The amount is preferentially 1.000001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 10 in the growth step or at least one of its substeps over the preliminary growth step or at least one of its substeps. 2 , 10 3 , 10 5 , 10 10 , 10 20 or 10 50 It grows in multiples of the above.

[0200] In one embodiment of the present invention, nanoparticle-producing cells preferentially have or are characterized by having at least one of the following properties during the growth step or during, at the start of, or at the end of at least one of its substeps: i) The number of nanoparticles in the cell is 1, 2, 5, 10, 50, 10 2 or 10 3 Greater than 0, preferentially 0, 1 or 2 or 0 and 10 3 It is greater than the range between 0 and 100, preferably between 0 and 100, or between 0 and 10. ii) The percentage of cells having at least one nanoparticle is greater than 10⁻⁴, 10⁻², 10⁻¹, 1, 5, 10, 50, 75, or 95%, preferably greater than 10 or 50%, or between 0–99%, 10–75%, 5–90%, preferably between 20–100%. iii) Optical density is 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -2 , 0.1, 0.2, 0.5, 1, 5, 10 or greater than 100, iv) The number of cells is 1, 5, 10, 10 2 , 10 3 , 105 , 10 10 , 10 20 , 10 50 or 10 100 Larger. v) Cell volume is 0.0001, 0.001, 0.1, 1, 10, 50, 10 2 , 10 3 , 10 5 or 10 10 It's larger than a liter. vi) Number of cells generated: 1 to 10 10 , 1 to 10 3 They are primarily included in the range of 50 to 300. vii) Optical density OD measured at the end of the growth step GE , and optical density OD measured at the start of the growth step GB The ratio, OD GE / OD GB is than 1, 2, 5, 10, 15, 25, 50, 100, 10 3 , 10 5 , 10 10 , 10 50 or 10 100 Larger. Or viii) Optical density OD measured at the end of substep i of the growth step GiE And the optical density OD measured at the beginning of substep i of the growth step GiB The ratio between OD GiE / OD GiB The numbers are 1, 2, 5, 10, 15, 25, 50, 100, and 10. 3 , 10 5 , 10 10 , 10 50 or 10 100 Larger.

[0201] In one embodiment of the present invention, cells that do not inherently produce nanoparticles are non-magnetic cells.

[0202] In one embodiment of the present invention, during the growth step or at least one of its substeps, at the start or end, the proportion of magnetocytes is 10 -50 , 10 -20 , 10-10 , 10 -5 , 10 -3 , 10 -1 1, 5, 10, 50, or 75% can be obtained. In some cases, the percentage of magnetocytes is n MC / (n MC + n NMC ) is equal to n MC and n NMC These represent the number of magnetic cells and non-magnetic cells, respectively.

[0203] In some cases, the preliminary growth step, the growth step, or at least one of its substeps is carried out at -250, -200, -150, -100, -50, -20, -10, -5, -2, -1, 0, 1, 2, 5, 10, 20, 50, 75, 100, 103, 105, 107°C, and also at temperatures of 10⁻⁵, 10⁻³, 10⁻², 0.1, 1, 5, 10, 50, 100 or 150°C or higher. In some other cases, the preliminary growth step, the growth step, or at least one of its substeps is carried out at 10 7 , 10 5 , 10 3 , 100, 75, 50, 40, 30, 20, 10, 5, 2, 1, 0, -1, -2, -5, -10, -20, -50, -100, -150, -200 or -250 °C or temperatures lower than -250 °C or temperature fluctuations, 10 5 , 10 3 , 10 2 The tests are conducted at temperatures of 50, 20, 10, 5, 2, 1, or below 0.1 °C.

[0204] In some cases, the preliminary growth step, the growth step, or at least one of its substeps is at a pH greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or the pH variation is 10 -10 , 10 -7 , 10 -5 , 10 -4 , 10 -3 , 10 -1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 pH units or higher. In some other cases, the preliminary growth step, the growth step, or at least one of its substeps is performed at a pH lower than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 -10 , 10 -7 , 10 -5 , 10 -4 , 10 -3 , 10 -1 It is performed at pH units of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or less.

[0205] In some cases, temperature, temperature fluctuations, pH, or pH fluctuations may be at least 1, 5, 10, or 10 per hour. 3 , 10 5 , 10 10 or 10 50 It is large enough to allow for cell division or cell division per hour.

[0206] In other cases, temperature, temperature fluctuations, pH, or pH fluctuations are sufficiently low, and 1, 5, 10, 10 3 , 10 5 , 10 10 or 10 50 Prevents the destruction, disappearance, or degeneration of more than a certain number of cells or cells per hour.

[0207] The present invention also relates to a method by which a preliminary growth medium is free of iron or at least one iron source.

[0208] The present invention relates to a method according to the present invention, wherein the pre-growth medium comprises iron or at least one iron source, and the properties and / or quantity of the iron or iron source preferentially do not enable the production of nanoparticles by the cells, while enabling cell growth.

[0209] In one embodiment of the present invention, the properties of the iron source are its composition, chemical formula, type of iron source, or the iron source itself. In some cases, the iron source is ferrous or a source of ferrous iron. In some cases, it may be, contain, consist of, or have the following chemical formula: Cl3Fe, C 10 H 12 FeN2NaO8, Fe2O 12 S3, C6H8FeNO7, C6H5FeO7, FeH 18 N3O 18 , C 30 H 21 FeN3O 15 -3 FeO4P, C6H7FeO8, Fe2H2O 13 S3, Fe2H 12 O 18 S3, C 10 H 12 FeN2NaO8, C 10 H 13 FeN2O8, FeH 28 NO 20 S2, C 10 H 15 FeN2NaO8, C 10 H 14 FeN2NaO8 +4 , C 14 H 21 FeN3O 10 , C 18 Fe7N 18 Fe4H2O 22 S5, Fe4O 21 P6, F3Fe, C6H 11 FeNO7 +3 , C6H 11 FeNO7, C 18 H 15 FeO9, C 12 H 29 Fe5Na2O 23 , C 12 H 22 Fe2O 14 , C 15 H 21 FeO6, C 15 H 24 FeO6, C6H5FeO7, C 10 H 16 FeN3O8, C4H10 FeO5, C 54 H 105 FeO6, AsFeH 13 O9 + AsFeO4, Fe +3 C6H 12 FeN3O 12 C6H 18 As3FeO6, FeH2O5P, C 21 H 21 FeO9S3, C6H 11 FeNaO7 +3 C 14 H 22 FeN3NaO 10 FeNaO7P2, C3H9As3Fe3O9, C 18 H 24 Fe4O 42 P6, C6H 11 FeO 10 C9H 18 FeN3S6, Cl3FeO 12 C6H9FeNO7 + Cr3Fe2O 12 C6H 10 FeNO8, FeH3O3, C 15 H 30 FeN3S6、C 30 H 27 FeN3O 15 C3FeN3S3, C6H 12 FeKO6 +4 FeH3O3, FeN3O9, C3H3FeO6, C6H8FeO7, C 24 H 45 FeO6, FeO6P3, Fe2H 14 O 19 S3、C 18 H 33 FeO 21 C6H9FeO9, C 18 H 27 FeO 24 、C6FeN6 -3 C 10 H 12 FeN2O8 - C 22 H 36 N4O 13 C3FeN3, C6H 12 FeN3O 12、C6H9FeO6、C 15 H 27 FeO6、FeH4O6P、C 21 H 15 FeO9、FeH8O8P、C6H6FeNO6、C4FeKO8、C 12 H 12 Fe2O 18 、C 33 H 35 FeN4O4、Cl3FeH4O2、C 24 H 45 FeO6、C 10 H 15 FeN2O7、FeH4NO8S2、C 32 H 31 FeN4O5、Fe2H6O3、AlF6Fe、C4H4FeNO8、C 81 H 84 FeN3O 33 、Fe2S3、Cl3FeH 14 O7、C 18 H6FeN9O 21 、Cl3FeO9、FeI3、C6H 14 FeO 10 、C6H 10 FeO8、C 55 H 80 FeN 17 O 21 S3、 C 10 H 16 FeN5O 13 P3、C 18 H 34 FeO 16 +3 、C 12 H 12 Fe2O 15 、C6FeNa3O 12 、C 10 H 12 FeKN2O8、C 21 H 24 FeN3O9、 C6H6Fe2O 12 、C6Fe2O 12 、AsFe、C 35 H 33 FeN5O 11 -3 、Cl3FeH2O、C 18 H 30 Fe2N6O 12 、FeI3O9、C10 H 18 FeN2NaO 11 、Cl3FeH 18 O9、Cr2FeH4NO8、C9H 21 Fe2O 18 P3、C 18 H 34 FeO2、C 30 H 27 FeO6、C 30 H 24 FeN3O 15 、C 54 H 102 FeO6、Fe4H 18 O 30 P6、Fe2Se3、C 54 H 99 FeO6、C 15 H 21 FeO6、C 10 H 18 FeN2O7 +2 、C 10 H 18 FeN2O7 +2 、C 10 H 19 FeN3O8、C 22 H 14 FeO4、C 39 H 63 FeN6O 15 +3 、C 10 H 19 FeN3O8、C4FeNaO8、FeO4V、C6H 15 FeN3O 12 、C6Fe2O 12 、C 18 H 24 Fe2O 24 -6 、C 18 H 19 FeN2NaO6、C 18 H 19 FeN2NaO6、C 12 H 18 Fe2O 12 、C6FeK3N6、C 24 H 47 FeO 25 - 、 C 18 H 38 FeO 19 、C 15H 21 FeO6, C 18 H 39 FeO 24 C6H 11 FeNO7, C6H 12 FeO6, C 12 H 28 FeO 14 FeHO2, C 45 H 36 FeN3O6, Fe3H2O4, Fe2O3, C 36 H 72 FeO6, C 12 H 18 Fe2O 15 C9H 18 FeO9, FeH6O3, C 54 H 102 FeO6, C 42 H 84 FeO6, C 16 H 31 FeO2 +2 C 36 H 69 FeO6, Fe3H8O4, C8H 15 Fe2O2 +4 C 12 H 48 Fe2N 12 O 12 S3、C 48 H 96 FeO6, C9H 15 FeO9, C 35 H 39 N5O 11 C 42 H 81 FeO6, C 48 H 93 FeO6, C 10 H 24 O2, Fe2H 18 O 21 S3、FeH 12 N3O 15 C 24 H 23 FeN 10 O6S2、C 18 H 14 Cl3FeN 10 S2、C 21 H 15 FeO6, Fe2H 10 O 17 S3、C10 H 19 FeN3O8、C 18 H 20 FeN2NaO6、C3F9FeO9S3、C5H 14 FeO4、C6H 19 FeNO 11 、C 18 H 16 FeN2NaO6、C 32 H 36 N4O9、C 15 H 30 FeO6、C 15 H 24 FeO6、C 15 H 15 F9FeO6、C 21 H 21 Cl3FeIS、C6H 12 Fe2O 18 、C6H 18 FeO 12 、C6H 15 FeO 12 、C6H 18 Fe2O 18 、C6H8FeO7、C6H 13 FeO 11 、C6H4Fe2N7、FeH2O4S、C 42 H 60 N 12 O 16 、C6Fe2N6、C3Fe2O9、C 162 H 297 FeO 27 -6 、C 21 H 27 Cl4FeN2O、C6H4FeNaO7、C 27 H 50 FeN6O 10 、C 25 H 48 FeN6O8、C 27 H 48 FeN6O9、C6H7FeO6 +2 、Fe2H2O4、C 14 H 26 FeN5O 10 、Cl4FeH4N、Cl3FeH 12 O 18 、C6H 17 FeN2NaO7 +3 、C10 H 11 FeNO6、C 15 H 15 F9FeO6、C6Fe2N6Na、C9H 21 Fe2O 18 P3、C 21 H 27 ClN2 O、C2H3FeO、C 10 H 12 Fe2N2O8、FeH3O3P、C7H5FeO2、C7H5FeO2、FeI3O 12 、C3H4FeNO2S、C2H2FeNO2、C 12 H 12 Fe2O 12 、C8H7FeNO3、C2HFe、C6H7FeO2S4、C6H 11 FeO6、C 14 H 19 FeO 12 、BFeH3O3、C 21 H 18 FeO 15 、C 35 H 56 FeN6O 13 、C 12 H 30 FeO3、CHFe、C 47 H 48 FeNO 14 、Fe2H6O3、Fe2O9Sn3、C 18 H 18 FeO3、Fe2O9Se3、Fe2O9Si3、Fe2O9S3、Br3FeO9、FeN3O6、C 24 H 54 FeO3、C 66 H 129 FeO6、FeP、C6H 18 FeO 24 P6 +3 、C 33 H 72 FeO3、C 40 H 75 FeO4、C2H3FeS、C3FeN3、C 21 H 39 FeO6、FeSi、C 30 H 29 FeN3O 16 、C 22 H 36 FeN4O13 、C 30 H 57 FeO6、C 60 H 117 FeO6、C 18 H 12 FeN3O6、C 18 H 31 FeO2 +2 、FeS2、C6H 11 FeN4O2、C6H5FeO7、C6H5FeS、C 10 H 13 FeN2O 10 - 、C8H 13 FeOS2、C 27 H 51 FeO6、C 24 H 44 FeO 25 - ,C6H 15 FeN3O6、C6H 12 FeO9、Cl3FeO9S3、CFeNS、Fe4H 12 O 12 Si3、C3H6FeO 12 、C4H3FeO4S2、C4H4FeO6、C6H3FeN3O6、C5H5FeO2、C 10 H 24 FeN4O9、C 14 H 19 FeN3NaO 10 、C 10 H 14 FeN2Na2O8、C 36 H 44 FeN4、C6FeNa3O 12 、Fe2H3OS3、C 16 H 27 FeO4、C6H8Fe2O 13 、C6H7FeO3、C4H4FeO6S2、C2H5FeN2、C5H7FeOS2、C 18 H 18 FeNa6O 21 、C3H9FeO9S3、C 24 H 54 FeO 12 P3、C 36 H 55 FeN6O 11 、Fe2H2O 10Si3、C2H4FeNO2、C4H 11 FeN2O4、AsFeH2O5、C 12 H 13 FeO 13 、C 36 H 67 FeO6、C 12 H 13 FeO 13 、C3H6FeN3O6、C 18 H 15 FeO9S3、C 36 H 75 FeO 12 S3、Fe2H4O5、C 28 H 24 FeN4 +3 、F3Fe、C 30 H 30 FeO6、BFe、C2H8N2O4、C8H5FeN2O5、Fe2H4O 11 Se3、C6H7FeO6S4、C4H 10 FeN3、C6H 12 Fe2O 15 、C 15 H 23 FeO5、C8H 12 FeNO 12 、C 49 H 56 ClFeN4O6、FeH4NO8S2、C 36 H 75 FeO9S3、B3F 12 Fe、FeP、Fe2H 20 O 22 S3、Cl3FeH 12 O 15 、C 18 H9FeN6、Fe2H 12 O 15 Se3、C 56 H 51 FeN4、Fe2H8O 13 Se3、C 44 H 27 FeN4、C 33 H 30 FeN4O6 -2 、CrFeO3、C 18 H 12 FeN3O 15 S3、Cl3FeH 18 O 21 、C6H5FeNa3O13 、C 18 H 14 FeN 13 O9S2、C 15 H 24 FeO6、C 24 H 27 FeO9S3、C 27 H 54 FeN3S6、Cl3FeH 12 O6、C 16 H 36 Pb、C8H 18 Fe2O 12 P2、Cl3FeH 24 O 12 、C 24 H 30 FeO9S3、C 21 H 24 FeO9S3、C 18 H 15 FeO 12 S3、Cl3FeH 20 O 10 、C 28 H 24 FeN6O6 + ,C 66 H 121 Fe2NaO 65 、Cr3FeH3O 12 、C 12 H 28 Fe2O 14 、C3H8FeNS2Zn - ,F3FeH6O3、C 30 H 51 FeO6、C 30 H 48 Fe4N6O 24 、C 30 H 18 FeN3O6、C 20 H 36 FeO4、C6H6FeK3O 15 、C 15 H6F 18 FeO6、C 10 H 13 FeN2O8、C6FeN6、C 15 H3F 18 FeO6、C 15 H 12 FeN3O3S3、C 21 H 23 FeO 10S3, FeH2O +3 C 24 H 44 FeNaO 28 Cr3FeO6, Fe2H2O +6 C6H 12 FeN9, FeH5NO4S, C2K2O4, C 18 H 13 FeN6, C 30 H 27 FeO6, C 34 H 38 N4O4, Cl3FeH 15 O 18 C6H 18 FeO6P3S6, C6H 11 ClFeNO 10 S2, C5H4F3FeO2 +2 C6H6Cr2O 12 C4H3CrKO8, C2MgO4, C 12 H 25 FeO 14 C2H2MgO4 +2 C2CrO4 + C2HNaO4, C2HKO4, C6Cr2O 12 、C2H2FeO4、C2H4MgO6、C6AlO 12 -3 C6Al2O 12 C2Li2O4, C2MgO4, C 44 H 30 N4O 12 S4、C 10 H 19 FeN2NaO 10 C5H4CuFeN6O3, C 10 H 14 FeN2NaO9, C 30 H 15 FeN3Na3O 15 S3、C 27 H 15 FeN 12 O6, C9H 18 FeN3S6、C 30 H 30 FeN3O 15 +3 C9H 18 FeN3S6, C6FeN6, C 18 Fe7N 18 C18 H 18 FeN2NaO6、C 30 H 21 FeN 12 O6、C 44 H 30 FeN4 +3 、C 14 H 18 FeK2N3O 10 、C 10 H 16 FeN2NaO8、C 33 H 29 FeNO 11 + 、C 25 H 18 FeN4O6S + ,C 35 H 24 FeN6O2S + ,C 32 H 32 ClFeN4O6、C 30 H 12 F9FeN 12 O6、C 30 H 18 Cl3FeN 12 O9、C 60 H 72 FeN9O9 +3 、C 60 H 66 FeN9O9 +3 、C 15 H 24 FeO6、C 22 H 25 Cl2FeN3O9 + ,C 18 H 23 Cl3FeN3O 12 、C 11 H 24 FeNO 11 、C 49 H 54 FeN4O9 + ,C 42 H 54 Cl8Fe2N4O2、C 44 H 26 Cl4 FeN4 +3 、C 34 H 32 FeN4O4 + ,C 44 H38 FeN8 +7 C9H 11 Cl2FeN4O2S, C 18 H 32 FeN4O8 +3 , C 34 H 32 ClFeN4O6, or C 19 H 25 FeN4O6. In some other cases, the iron source has the following chemical formula, contains it, is made of it, or can have it: Fe +2 FeH 14 O 11 S, FeH8N2O8S2, FeO4S, Cl2Fe, FeS, C4H2FeO4, C 12 H 26 FeO 16 , C4H5FeNO4, C 12 H 10 Fe3O 14 , C 16 H 30 FeO4, FeH2O5S, C 10 H 12 FeN2Na2O8, As2Fe3O8, CFeO3, C6H 12 FeO6, FeH 12 N2O 12 , C 12 H 10 Fe3O 14 , C6H5FeNaO7, C 34 H 32 FeN4O4, C 12 H 22 FeO 14 , C 12 H 14 FeO 12 , C6H 10 FeO6, C4H8FeN2O4, C 12 H 28 FeO 16 FeI2, FeH4N2O6S2, C 34 H 32 FeN4O4 -2 , C 34 H 32 FeN4O4, F2Fe, C6H 18 FeO9, C6H5FeO7 -、C2FeO4、C4H4FeO4、Cl2FeO8、Fe3O8P2、FeO、B2F8Fe、FeH8O8S、C4H6FeO4、C4H4FeO4、C 12 H 10 FeNa4O 14 、C 22 H 14 FeO4、C2H4FeO6、C 12 H 24 FeO 14 、C 14 H 20 FeN3O 10 - 、Cl2FeH8O4、C 12 H8FeN2O4、C4H8FeO4、C5H7FeNO4、C8H 12 FeN2O8、C 12 H 10 Fe3O 14 、C6H 16 FeO9、C 19 H 19 FeN7O 10 S、C 10 H 16 FeN2O8、C 12 H 10 Ca2FeO 14 、C2H6FeO6、C 36 H 70 FeO4、C6H6FeO7、C4H2FeO4、C 36 H 21 Cl2FeN9O 14 、C 32 H 62 FeO4、FeH2O2、C4H6FeO6、C6H8CaFeO7 +4 、C4H 10 Cl2FeN2O4、C 36 H 24 Cl2FeN6O8、C6H 14 FeO7、C 12 H 16 FeO 12 、BFe、C 32 H 16 FeN8、C 12 H 26 FeO 15 、C 12 H 10 Fe3O 14 、FeH8I2O4、C4H10 FeN2O8S、C 30 H 24 Cl2FeN6O8, C 39 H 30 Cl2FeN6O8, C 12 H 14 FeO 12 C 30 H 24 FeN6 +2 C4H2FeO4 -2 C4H4FeO4, C 10 H 16 FeO4, C 36 H 24 FeN6O4S、C2H4FeO6、C2H2FeO6、C8H 15 Fe2O2 +4 C 32 H 16 FeN8, C 12 H 16 Fe3O 14 C 12 H 24 FeO 14 C2FeN2S2, C 12 H 16 FeN6O4, C 14 H 20 FeN3O 10 C 12 H7FeN3O6S、C 20 H 12 FeN4, C 12 H 16 Ca2FeO 14 C 46 H 54 FeO9, C6H5FeO7, FeH4O6S, C 10 H 15 FeN2NaO7, C 10 H6FeN4O8、Fe2P、C4H4FeO6、C 14 H 26 FeO 16 Cl2FeH 12 O 14 C4H8Cl2FeN2O4, C6Fe3N6, C4H 12 As2FeO8, C 10 H 16 FeO4, FeH 20 N2O 14 S2、C 16 H30 FeO4、C 40 H 40 FeN8O4 + 、Fe2Na8O 21 P6、C 14 H8FeO 10 、C 14 H8FeO4、C 12 H 20 FeO4、C8H8FeS、C5H4FeO、C2H3FeNO2、C 10 H 14 FeN2O8、C6H2FeN3O7 + 、C2H2Fe、C 10 H6FeN2、C6H 15 FeN3O7、C 72 H 124 FeO8 -2 、FeH 22 N2O 15 S2、C 40 H 78 FeO4、FeH2N2O6 +2 、C 44 H 86 FeO4、C 10 H 20 FeN2O8S2、C 20 H 38 FeO4、C 36 H 66 FeO4、C 24 H 46 FeO4、C 29 H 26 FeP + 、C 36 H 64 FeO6、C 14 H 26 FeO4、C 26 H 28 FeNP、C 28 H 54 FeO4、C 36 H 32 FeN4O4、C 36 H 36 FeN4O8、C6H9FeNO7 + 、C5H6FeO2、C4H 11 BFeO4、C8H 19 BFeO4、C4H4FeO4S2、C6H6FeO7、C 18 H 34 FeO4、C12 H 20 FeO 13 、C4H4FeO6、C5H7FeNO3、Fe3H8O4、C2FeN2S2、FeH2O2、Fe3H2O4、C 44 H 28 FeN4, C2H6FeO5, Fe2H6O 11 S2, C3H4FeN2O3, Fe3H2O9P2, C6H 14 Fe3N3O7 -3 C4H 10 FeN2O6、Cl2FeH2O、FeO4W、C6H5FeO3P、C6H8FeO7、FeTe、C4H2FeO4、C 20 H 20 Cl2FeN8, C 14 H 12 FeO6, C3H3FeO7P, C4H7FeNO4, FeO3Si, Cl2FeH 12 O6, Cl2FeH2O9, FeH 10 O9S、FeH 12 O 10 S、C8H 17 FeO3P, C4H 14 FeO8, Fe3H 16 O 16 P2, F6FeSi, C 72 H 42 FeN6Na6O 22 S7, FeH4O5S, C 39 H 30 FeN6O4S、C 40 H 50 O4, C4H 10 FeN2O4、C2H4FeN2O4S、Br2FeH2O、C 98 H 200 FeN 10 C 36 H 21 FeN9O 10 S、C 10 H 10 Fe, C2H6FeN2, F6FeH 12 O6Si、C 48 H 48 FeN6O4S, FeO4S, C2H 10 FeN2O8S2, C 44 H 27 FeN5O、C 30 H24 FeN6O4S、C6H8O6、C6H7NaO6、FeH4O2 +2 、FeH2O +2 、C3H7FeNO7S、C 30 H 18 FeN3NaO6、C2H 18 FeN2O 12 S2、C4H4FeO4、C7H7FeN4O + 、Br2Fe、C 18 H 22 Cl2FeN2、C 32 H 28 FeN6O6S2、C 12 H 14 MgO 12 、C2H5FeNO6S、C 45 H 60 FeN2O8、C 30 H 22 Cl2FeN2、C 38 H 26 FeN8O2S2、C 30 H 28 FeN2O6、C 14 H 12 Cl6FeO4、C 12 H 14 Fe、C 36 H 36 Cl2FeN6O8、C 17 H 14 FeN4O 4S、C 24 H 30 FeN4O4、C 34 H 32 ClFeN4O6、C 12 H 12 Fe、Fe3H 14 O 12 P2 +6 、C 32 H 16 FeN8、FeS2、C 16 H 15 FeNO2 +2 、C 29 H 20 FeO6、C 23 H 28 FeO2、C 11 H 10 FeO2、C 13 H 14 FeO2、C12 H 12 FeO2、C 46 H 48 FeN4O6 +2 、C 47 H 59 FeN 13 O8 +2 、C 46 H 59 FeN 13 O8 +2 、C 48 H 62 FeN 12 O8S +2 、C 50 H 65 FeN 13 O8 +2 、C 48 H 63 FeN 13 O8 +2 、C 48 H 62 FeN 12 O8S +2 、C 55 H 76 FeN 14 O9 +2 、C 25 H 19 FeN3、C 15 H 17 FeN3OS +2 、C 22 H 23 FeN3OS +2 、C 26 H 28 ClFeN3、C 28 H 33 ClFeN4、C 27 H 31 ClFeN4、C 29 H 35 ClFeN4、C 30 H 37 ClFeN4、C 28 H 33 ClFeN4、C 27 H 30 ClFeN3、C 26 H 28 ClFeN3、C 29 H 35 ClFeN4、C 27 H 30 ClFeN5O +2 、C41 H 38 ClFeN5O3 +2 、C 42 H 41 FeN5O3 +2 、C 41 H 38 FFeN5O3 +2 、C 42 H 47 FeN5O3 +2 、C 43 H 49 FeN5O3 +2 、C 42 H 41 FeN5O3 +2 、C 42 H 40 ClFeN5O3 +2 、C 42 H 40 ClFeN5O3 +2 、C 42 H 40 FFeN5O3 +2 、C 41 H 45 FeN5O3 +2 、C 42 H 47 FeN5O3 +2 、C 41 H 39 FeN5O3 +2 、C 22 H 25 FeN5O5 +2 、C 24 H 23 ClFeN4O2 +2 、C 24 H 23 FFeN4O2 +2 、C 24 H 24 FeN4O2 +2 、C 15 H 21 FeN3S +2 、C 29 H 34 FeN4O2 +2 、C 28 H 31 ClFeN4O2 +2 、C 28 H 31 FFeN4O2 +2 、C 30 H 35ClFeN4O3 +2 、C 30 H 35 FFeN4O3 +2 、C 28 H 32 FeN4O2 +2 、C 27 H 30 FeN4O2 +2 、C 26 H 27 ClFeN4O2 +2 、C 30 H 36 FeN4O3 +2 、C 28 H 31 ClFeN4O3 +2 、C 28 H 31 FFeN4O3 +2 、C 28 H 32 FeN4O3 +2 、C 27 H 29 ClFeN4O3 +2 、C 26 H 27 FFeN4O2 +2 、C 26 H 28 FeN4O2 +2 、C 26 H 28 FeN4O2 +2 、C 27 H 29 FFeN4O3 +2 、C 27 H 30 FeN4O3 +2 、C 26 H 27 ClFeN4O3 +2 、C 26 H 27 FFeN4O3 +2 、C 26 H 28 FeN4O3 +2 、C 25 H 25 ClFeN4O3 +2 、C 25 H 25 FFeN4O3 +2 、C 25 H 26 FeN4O3 +2 、C24 H 23 ClFeN4O3 +2 、C 24 H 23 FFeN4O3 +2 、C 24 H 24 FeN4O3 +2 、C 25 H 25 ClFeN4O2 +2 、C 25 H 25 FFeN4O2 +2 、C 25 H 26 FeN4O2 +2 、C 25 H 26 FeN4O2 +2 、C 29 H 32 ClFeN7 +2 、C 33 H 32 ClFeN7 +2 、C 22 H 27 ClFeN3RuS + 、C 18 H 19 ClFeN3RuS + 、C 19 H 19 BFeO3 +2 、C 28 H 25 ClFeN4O +2 、C 31 H 38 FeN4O3、C 29 H 34 FeN4O3、C 31 H 41 FeN3O、C 28 H 32 FeN4O3、C 26 H 29 FeN3O2、C 26 H 30 FeN2O、C 31 H 36 FeN4O3、C 30 H 35 FeN5O4、C 29 H 35 FeN5O3、C 32 H 41 FeN5O3、C 35 H38 FeN4O3、C 32 H 40 FeN4O3、C 19 H 13 BBr2F2FeO2、C 19 H 14 BClF2FeO2、C 19 H 14 BBrF2FeO2、C 19 H 15 BF2FeO2、C 21 H 20 FeO4、C 20 H 18 FeO3、C 20 H 18 FeO3、C 20 H 18 FeO3、C 19 H 14 F2FeO2、C 19 H 14 Br2FeO2、C 19 H 15 BrFeO2、C 14 H 12 FeO3、C 21 H 19 BF2FeO4、C 20 H 17 BF2FeO3、C 20 H 17 BF2FeO3、C 20 H 17 BF2FeO3、C 19 H 13 BF4FeO2、C 19 H 13 BCl2F2FeO2、C 21 H 29 AuCl2FeN4S + 、C 30 H 24 Cl2FeN6 +2 、C 22 H 21 Cl2FeN3 +2 、C 23 H 22 FeN6 +2 、C 21 H 19 FeN7 +2 、C 23 H 24 FeN6O +2 、C 47 H64 FeN 14 O9、C 46 H 60 FeN 12 O 10 、C 41 H 53 FeN 11 O7、C 47 H 65 FeN 15 O8、C 45 H 59 FeN 13 O9、C 42 H 54 FeN 12 O7、C 43 H 67 FeN 15 O8、C 48 H 65 FeN 13 O8、C 47 H 64 FeN 12 O8、C 54 H 77 FeN 17 O9、C 51 H 71 FeN 15 O 10 、C 19 H 16 FeO2、C 44 H 48 FeN9O 17 P3、C 13 H9Cl2FeN3O6S、C 19 H 15 FeNO3、C 20 H 18 FeO2、C 20 H 18 FeO3、C 21 H 20 FeO3、C 17 H 20 FeN2O2、C 18 H 15 FeNO、C 17 H 14 FeOS、C 17 H 14 FeOS、C 17 H 14 FeO2、C 22 H 22 FeO4、C 20 H 18 FeO2、C20 H 18 FeO2, C 19 H 14 Cl2FeO, C 21 H 20 FeO3, C 48 H 28 FeN4O8, C 17 H 15 FeNS, C 34 H 30 FeN4O4 -2 , C 30 H 26 Br2FeN4O4, C 10 H 18 FeN2O7 +2 , C 14 H 12 FeO4, C 44 H 20 Cl8FeN4, C 64 H 64 FeN8O 12 S4, C 56 H 56 FeN8O8S4, C 56 H 44 Br8FeN4, C 56 H 52 FeN4, C 52 H 40 FeN8O 12 S4, C 44 H 32 FeN8O8S4, or C 44 H 28 FeN4. In some other cases, the source of iron has the following chemical formula: C a H b Fe c O d N e S f Br g Cl h P i Na j As k K l Al m C rn V o I p B q F r Te s W t , Here, a, b, c, d, e, f, g, h, I, j, kl, m, n, o, p, q, r, s, t are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any other integer from 21 to 1000000000000. The atoms C, H, Fe, O, N, S, Br, Cl, P, Na, As, K, Al, Cr, V, I, B, F, Te, and W preferentially occupy the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th positions, respectively. In some cases, at least one atom in a chemical formula can occupy any one of the 20 positions in the formula. In some other cases, the iron source can include chemical functional groups selected from the following groups: Hydrocarbons, alkanes (R(CH2)nH), alkenes (R2C = CR2), alkynes (RC≡CR'), benzene derivatives (RC6H5);Halogen-containing group, haloalkane (RX), oxygen-containing group, alcohol (ROH), carbonyl (RC or '), aldehyde (RCHO), acyl halide (RCOX), carbonate (ROCO or '), carboxylic acid (RCOO-), carboxylic acid (RCOOH), ester (RCO or '), methoxy (ROCH3), hydroperoxide (ROOH), peroxide (RO or '), ether (R or '), hemiacetal (RCH(or ')(OH)), hemiketal (RC(or ``(OH)R)), acetal (RCH(or ')(or ``)), ketal (RC(or ``(or '')R) '), orthoesters (RC(or')(or")(or'')), heterocyclic compounds (PhOCOPH), orthocarbonates (C(or)(or')(or")(or'')), nitrogen-containing groups, amides (RCONR2), amines (RNH2, R2NH, R3N, R4N+), imines (RC(=NH)R', RC(=NR'')R', RC(=NH)H, RC(=NR')H, imides ((RCO)2NR'), azides (RN3), azo compounds (RN2R'), cyanates (ROCN, RNCO), nitrates (RONO2), nitrates (RCN, RNC), nitrites (RONO), nitro compounds (RNO2), nitroso compounds (RNO), oximes (RCH = Groups including NOH), pyridine derivatives (RC5H4N), sulfur, thiols (RSH), sulfides (RSR'), disulfides (RSSR'), sulfoxides (RS or'), sulfones (RSO2R'), sulfinic acid (RSO2H), sulfonic acid (RSO3H), thiocyanates (RSCN, RNCS), thioketones (RCSR'), thiar (RCSH), phosphorus, phosphine (R3P), phosphonic acid (RP(=O)(OH)2), phosphates (ROP(=O)(OH)2), phosphodiesters (HOPO(or)2), boron, boronic acid (RB(OH)2), boronic acid esters (RB(or)2), boric acid (R2BOH), boric acid esters (R2B or), and several combinations from these groups; In some other cases, the iron source is an iron chelate agent.

[0210] In some cases, the amount of iron source is, preferentially, the amount or concentration of iron in the iron source or iron in the iron source, preferentially in the preliminary growth medium and / or growth medium.

[0211] The present invention also relates to a method of the present invention, wherein the source of iron or iron in the pre-growth medium is Fe 2+ and / or Fe 3+ Consists of or includes.

[0212] In some cases, if the chemical formula for iron includes Fe2, then the source of iron is Fe 2+ It contains. In some other cases, the source of iron is Fe if the chemical formula contains Fe3. 3+ Includes.

[0213] The present invention relates to a method for which the concentration of iron or an iron source in a preliminary growth medium is 20 μM or less. In some cases, the concentration of iron or an iron source in the preliminary growth medium is 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , or 20 μM or less. In other cases, the concentration of iron or iron source in the preliminary growth medium is 0, 10 -50 , 10 -20 , 10 -5 , 10 -1 , greater than 1, 5, 10 or 20 μM. Furthermore, in several other cases, the concentration of iron or iron source in the pre-growth medium is 10 -50 and 10 50 , 10 -10 and 10 10 , 10 -10 and 10 5 , 10 -10 and 10 3 , or between 10 -10 It is a μM between and 1.

[0214] The present invention relates to a method in which a growth medium comprises iron or at least one iron source, and the properties and amount of iron or the iron source enable the production of nanoparticles by nanoparticle-producing cells and / or cell proliferation.

[0215] The present invention relates to a method in which the iron source of the growth medium is the same as the iron source of the pre-growth medium.

[0216] The present invention relates to a method in which the concentration of iron or an iron source in the growth medium is equal to or greater than the concentration of iron or an iron source in the pre-growth medium.

[0217] In some cases, the concentration of iron or iron source in the growth medium is 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , or 20 μM or less. In other cases, the concentration of iron or iron source in the growth medium is 0, 10 -50 , 10 -20 , 10 -5 , 10 -1 , 5, 10 or greater than 20 μM. Furthermore, in some other cases, the concentration of iron or iron source in the growth medium is 10 -50 and 10 50 , 10 -10 and 10 10 , 10 -10 and 10 5 , 10 -10 and 10 3 , or 10 -10 It is a μM between and 1.

[0218] The present invention relates to a method for supplementing a growth medium with fed-batch medium.

[0219] In one embodiment of the present invention, the fed-batch medium includes at least one source common to the pre-growth and / or growth medium. In some cases, the concentration of this source in the fed-batch medium is 1.00001, 1.1, 2, 5, 10, 10 higher than that of the pre-growth and / or growth medium. 3or 10 5 Larger. In some other cases, the concentration of this source is at least 10 times higher in the fed-batch medium than in the pre-growth and / or growth medium. 5 , 10 3 , 10, 1, 1.1 or 1.00000001 lower.

[0220] This invention relates to a fed-batch medium, -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 20, 50, 100, 10 3 or 10 5 This relates to a method containing iron or an iron source at a concentration higher than μM. In some other cases, the fed-batch medium is 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10 -2 , 10 -10 or 10 -20 It contains iron or an iron source at a concentration lower than μM. Furthermore, in several other cases, the fed-batch medium contains 10 before adding the fed-batch medium to the growth medium. -50 and 10 50 , 10 -15 and 10 15 , 10 -10 and 10 5 , 10 -5 and 10 5 between 10 -3 and 10 3 The concentration is μM, or 0.5 nM to 50 M.

[0221] In one embodiment of the present invention, the fed-batch medium is acidic or has a pH lower than 7, 6, 5, 4, or 3, and preferably 2 or less. In some cases, the fed-batch medium has a pH greater than 0 or 1.

[0222] This invention relates to a fed-batch medium that is 10 per hour. -15 10 liters per hour 15 10 liters or per hour -15μM and 10 15 This relates to a method of introducing iron into growth medium at a rate of between μM. In some cases, the number of cell divisions in the growth medium is low, and 10 per second, hour, day, or month. 20 , 10 10 , 10 5 , 10 3 If cell division is 10 or less, feedbatch medium should be fed at a slow rate, preferentially 10 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10, 5, 1, 10 -2 , 10 -3 , 10 -5 or 10 -10 It is added to the growth medium at a rate lower than liters of fed-batch medium or μM iron per minute. In some other cases, the number of cell divisions in the growth medium is large, and it is 1, 2, 5, 10, 10 per second, hour, day, or month. 3 , 10 5 , 10 10 or 10 20 If the number of times is greater than 10, the culture medium is added to the growth medium at a high rate, preferentially 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 1, 5, 10, 10 2 , 10 3 , 10 5 or 10 10 It is added at a faster rate than liters of fed-batch medium / min or μM iron / min. In some cases, between two substeps of the growth step, a shortage of fed-batch medium is preferentially 1.0000001, 1.00001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 10 3 , 10 5 , 10 7 or 10 9 It decreases at coefficients exceeding 0. In some other cases, the shortage of fed-batch medium is preferentially 1.0000001, 1.00001, 1.0001, 1.01, 1.1, 1.2, 1.5, 2, 5, 10, 103 , 10 5 , 10 7 or 10 9 It increases by a coefficient of .

[0223] In one embodiment of the present invention, before being added to the growth medium, the fed-batch medium is enriched to contain at least 1.000001, 1.001, 1, 1.5, 2, 5, 10, and 10 iron concentrations relative to the growth medium. 2 , 10 3 or 10 5 It has a high iron concentration due to the coefficient.

[0224] In another embodiment of the present invention, the fed-batch medium becomes part of the growth medium after being preferentially added to the growth medium.

[0225] The present invention relates to a method by which a preliminary growth medium and / or growth medium comprises only one vitamin selected from the group consisting of biotin, calcium pantothenate, folic acid, inositol, nicotinic acid, p-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine HCl, and derivatives of these vitamins.

[0226] In some cases, the pre-growth and / or growth medium may be 10 50 , 10 20 , 10 10 , 10 5 , 10 3 It may contain 100, 75, 50, 25, 10, 5, 3, 2 or one or fewer different vitamins. In some cases, different vitamins may be at least 1, 2, 5, 10, 10 2 , 10 3 , 10 5 , 10 10 , 10 20 , or 10 50 It may be a vitamin containing different chemical elements. In some other cases, the preliminary growth medium and / or growth medium may be 1, 2, 5, 10, 10 2 , 10 3 , 10 5 or 10 10 It contains more than one different vitamin.

[0227] In some cases, vitamins may be water-soluble. In other cases, vitamins may be fat-soluble. In yet other cases, vitamins may be A, D, E, K, B1, B2, B3, B5, B6, B7, B9, B 12 It may belong to the vitamin C family. In several other cases, the vitamin is not produced by nanoparticle-producing cells. In several other cases, this vitamin is used to treat diseases such as those caused by or associated with vitamin deficiency.

[0228] In yet another embodiment of the present invention, the vitamin is selected from the following group: Adenosylcobalamin, aminobenzoic acid, ascorbic acid, biotin, calcium D-(+)-pantothenic acid, carotene thiamine, carotenoid beta, cholecalciferol (D3), cyanocobalamin, cyanacobalamin, ergocalciferol (D2), folate, folic acid, folic acid, hydroxocobalamin, inositol, menaquinone (K2), methylcobalamin, niacin, niatinalide, niacinamide, nicotinamide riboside, nicotinic acid, pantokinon, or one or more derivatives or combinations of these vitamins.

[0229] In yet another embodiment of the present invention, at least one vitamin contained in the growth medium is biotin, folic acid, riboflavin, nicotinic acid, or thiamine HCl.

[0230] In some cases, the vitamins in the preliminary growth medium are the same as those in the growth medium. In other cases, the vitamins in the preliminary growth medium are different from those in the growth medium.

[0231] The present invention also relates to a method in which the concentration of at least one vitamin contained in the preliminary growth medium and / or growth medium is 10 100 , 10 50 , 1020 , 10 10 , 10 5 , 10 3 , 10, 1, 10 -1 , 10 -3 , 10 -4 , 10 -6 , 10 -9 , 10 -20 , 10 -50 or 10 -100 This concerns methods that are lower than M, or preferentially 0.002 mol / L or less.

[0232] In another embodiment of the present invention, the pre-growth and / or growth medium contains at least one vitamin or one chemical element contained in at least one vitamin, 10 -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -4 , 10 -3 , or 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 It contains a concentration exceeding M.

[0233] The present invention also relates to a method thereof, wherein the pre-growth and / or growth medium contains at least one vitamin at concentrations higher than those of a source of carbon, nitrogen, sulfur, sulfate, phosphorus, phosphate, calcium, potassium, magnesium, oxygen, hydrogen, and / or iron, preferably 1.0001, 1.2, 1.5, 2, 5, 10, 10 3 or 10 5 The above coefficients are used to include the vitamins at low concentrations. In some cases, cells do not require high concentrations of vitamins to grow, divide, and / or synthesize nanoparticles.

[0234] The present invention also relates to a method according to the present invention, wherein the pre-growth and / or growth medium comprises the following amounts per gram or mL of growth or pre-growth medium: i) Yeast extract of 1 mg or less, ii) At least one component of 1 mg of yeast extract, iii) 1 mg of peptone, iv) 1 mg of at least one component of peptone, v) 1 mg of CMR agent, vi) At least one chelating agent in 1 mg, vii) 1 mg of at least one amino acid, viii) 1 mg of a toxic or cytotoxic compound, and / or ix) 1 mg of at least one heavy metal.

[0235] In one embodiment of the present invention, the pre-growth and / or growth medium contains yeast extract, peptone, CMR agent, chelating agent, amino acid, toxic or cytotoxic compound, and / or heavy metal at a concentration of 10 per liter or milliliter of the pre-growth / growth medium. 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10, 1, 10 -3 or 10 -5 Contains μg of yeast extract, peptone, CMR agent, chelating agent, amino acid, toxic or cytotoxic compound, and / or heavy metal. In some cases, this situation may occur if the yeast extract, peptone, CMR agent, chelating agent, amino acid, toxic or cytotoxic compound, and / or heavy metal have been removed from or are not present in the pre-growth or growth medium.

[0236] In one embodiment of the present invention, the first and / or second culture medium contains yeast extract, peptone, amino acids, and / or heavy metals at a concentration of 10 per liter or milliliter of the preliminary growth medium and / or growth medium. -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 10, 10 3 or 10 5Contains μg of yeast extract, peptone, amino acids, and / or heavy metals. In some cases, this situation may occur if yeast extract, peptone, CMR agents, chelating agents, amino acids, and / or heavy metals are preferentially and unintentionally added to the pre-growth and / or growth medium.

[0237] In one embodiment of the present invention, the yeast extract is a peptide, an amino acid, a purine base, a pyrimidine base, and / or a B group water-soluble vitamin, or contains them.

[0238] In one embodiment of the present invention, the amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and / or valine.

[0239] In one embodiment of the present invention, the heavy metals are arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb), selenium (Se), and / or zinc (Zn).

[0240] In one embodiment of the present invention, the agent that is carcinogenic, mutagenic, or reproductively toxic, and is also called a CMR agent, is nitriloacetate, trisodium salt, and / or boric acid.

[0241] In one embodiment of the present invention, toxic compounds or cytotoxic compounds are preferentially added to the pre-growth and / or growth medium, preferentially 10 -10 , 10 -5 , 10 -2 , 1, 5, 10, 10 3 , or 10 μM, preferably at a concentration comprised between 10 -10 and 10 10 For concentrations exceeding μM, 10 is preferred. -10 and 10 10This compound, when introduced at concentrations within the μM range, preferentially produces the death of nanoparticle-producing cells or organisms, or the death of nanoparticle-producing cells.

[0242] In one embodiment of the present invention, the pre-growth and / or growth medium does not contain minerals selected from the group selected from the following groups: C6H6NO6Na3, trisodium nitriloacetate, CoN2O66H2O, MnO4SH2O, manganese(II) sulfate monohydrate, NaCl, sodium chloride, CoN2O66H2O, cobalt(II) nitrate hexahydrate, O4SZn7H2O, zinc sulfate heptahydrate, CuO4S5H2O, copper(II) sulfate pentahydrate, AlKO8S212H2O, potassium sulfate dodecahydrate, H3BO3, boric acid, Na2MoO42H2O, sodium molybdate dihydrate, Cl2Ni6H2O, nickel(II) chloride hexahydrate, Na2SeO3, sodium selenite, and derivatives or combinations of these compounds.

[0243] In one embodiment of the present invention, the pre-growth and / or growth medium is 10 per liter or milliliter of growth medium. 100 , 10 50 , 10 20 , 10 10 , 10 5 , 10 2 , 10, 1, 10 -1 , 10 -3 or 10 -5 Contains mineral concentrations lower than μg. In some cases, this situation may occur when minerals are removed from the pre-growth and / or growth medium.

[0244] In one embodiment of the present invention, the first (pre-growth) and / or second growth medium is 10 per liter or milliliter of growth medium. -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 10, 10 3or 10 5 Contains mineral concentrations higher than μg. In some cases, this situation may occur when minerals are preferentially and unintentionally removed from the preliminary growth and / or growth medium.

[0245] The present invention also relates to a method comprising the additional step of purifying high-purity iron oxide nanoparticles by removing at least one impurity from the nanoparticles.

[0246] In one embodiment of the present invention, an additional step for purifying high-purity iron oxide nanoparticles comprises removing impurities from the nanoparticles and / or modifying and / or destroying impurities contained in the nanoparticles.

[0247] In one embodiment of the present invention, a preceding step of isolating or extracting nanoparticles from cells is performed before an additional step of purifying the nanoparticles. In some cases, this preceding step is a step of recovering the nanoparticles. In some cases, this preceding step is performed as follows: i) Cells preferentially obtained during the proliferation stage are mixed with surfactants such as KOH and NaOH. ii) Cells -270, -250, -200, -150, -100, -50, -30, -10, -5, 0, 5, 10, 20, 30, 50, 75, 100, 150, 200, 500, 10 3 , 10 5 or 10 10 ° Temperatures above C, or -270 and 10 10 -100 and 10 5 , or heat at a temperature between 0 and 100 °C, iii) 10 per hour, minute, or second -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 2, 5, 10, 10 3 , 10 5 or 10 10If the temperature exceeds °C, or if it is 10 per hour, minute, or second -50 and 10 10 Inducing a temperature gradient within °C, iii) For example, using a French press, preferentially 1, 10, 100, 500, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 Greater than atmospheric pressure or between 1 and 10 9 Applying pressure between atmospheric pressures to cells, and / or iv) Prioritize 10 -50 , 10 -20 , 10 -5 , 10 -1 , 1, 10, 10 2 , 10 3 , 10 5 or 10 10 Cells are sonicated with an output greater than watts.

[0248] In another embodiment of the present invention, an additional step of purifying nanoparticles allows for their removal as follows: i) In terms of mass percentage of impurities, 10 -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 25, 50, 75, 80 or more than 90%, or ii) 10 per gram of nanoparticles -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 1, 5, 10, 10 3 , 10 5 or 10 10 Impurities exceeding μg.

[0249] In one embodiment of the present invention, the impurities preferentially removed by the additional step of purifying the nanoparticles are carbon or carbonaceous material and / or not iron oxide. Preferably, these impurities are present in the coating of the nanoparticles.

[0250] In one embodiment of the present invention, impurities are removed from the core and / or coating of the nanoparticles, preferably from the coating of the nanoparticles. Preferably, the impurities removed are shallow impurities. In other cases, the impurities removed are deep impurities.

[0251] The present invention also relates to an additional step in a method for purifying high-purity iron oxide nanoparticles by removing at least one impurity from nanoparticles, comprising at least one heating step in which the temperature of the nanoparticles is increased, wherein the temperature of the nanoparticles is raised to T0, and maintained at T0 for a heating time preferably between 1 second and 1 minute, 1 second and 1 hour, 1 second and 12 hours, 1 second and 1 day, 1 second and 1 week, 1 second and 1 month, or 1 second and 1 year, wherein T0 is preferably between -200 and 10 5 -100 and 10 5 -50 and 10 5 -10 and 10 5 , 0 and 10 5 °C, 10 and 10 5 , 20 and 10 5 , 30 and 10 5 , 100 and 10 5 , 200 and 10 5 °C, 100 and 10 4 , 100 and 10 3 , or included between 100 and 500 °C.

[0252] The present invention also relates to an additional step in a method for purifying high-purity iron oxide nanoparticles by removing at least one impurity from the nanoparticles, which comprises at least two heating steps, in which: i) In step 1, the temperature of the nanoparticles rises to temperature T1 and is maintained at T1 during a heating time of 1 second to 20 years, wherein T1 is between 150°C and 250°C. ii) In step 2, the temperature of the nanoparticles rises to temperature T2 and is maintained at T2 for a heating time of 1 second to 20 years, wherein T2 is within the range of 350°C to 450°C.

[0253] In some cases, an additional step or heating step may be specified as part of the purification method for high-purity iron oxide nanoparticles.

[0254] The present invention relates to a method for removing at least one impurity from high-purity iron oxide nanoparticles, comprising an additional step between steps 1 and 2, wherein the temperature of the nanoparticles is raised to a temperature T3, and then maintained at T3 for a heating time ranging from 1 second to 20 years, wherein T3 is between 250°C and 350°C.

[0255] In some cases, the period during which the temperature of nanoparticles is maintained at temperatures T1, T2, and / or T3 may be shorter than 100 years, 50 years, 20 years, 10 years, 5 years, 2 years, 1 year, 11 months, 6 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 3 days, 1 day, 23 hours, 12 hours, 6 hours, 1 hour, 50 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 2 minutes, 1 minute, 50 seconds, 30 seconds, 10 seconds, 1 second, 1 millisecond, or 1 microsecond. In some other cases, the duration for which the nanoparticle temperature is maintained at temperatures T1, T2, and / or T3 is longer than 1 microsecond, 1 millisecond, 1 second, 10 seconds, 30 seconds, 50 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 1 hour, 6 hours, 12 hours, 23 hours, 1 day, 3 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 11 months, 1 year, 2 years, 5 years, 10 years, 20 years, 50 years, or 100 years. In some other cases, the duration for which the nanoparticle temperature is maintained at temperatures T1, T2, and / or T3 is between 1 microsecond and 100 years, 1 second and 20 years, 1 second and 1 year, 1 second and 1 month, 1 second and 1 day, 1 minute and 1 day, 5 minutes and 1 day, 10 minutes and 12 hours, 30 minutes and 6 hours, or 30 minutes and 3 hours.

[0256] In one embodiment of the present invention, the heating time is preferably 1.001, 1.1, 1.5, 2, 5, 10, 10 times longer than the time it takes for the temperature to rise to T1, T2, and / or T3. 3 , 10 5 or 10 10 The coefficients above indicate a long length.

[0257] In some cases, T1 falls between -273 °C and 250 °C, -200 °C and 250 °C, -100 and 250 °C, 0 and 250 °C, 50 and 250 °C, 150 and 250 °C, or 180 and 220 °C. Otherwise, T2 falls between 200 and 10 5 , 250 and 10 5 , 300 and 10 5 , 350 and 10 5 , 350 and 10 3 , 350 and 500, 350 and 450, or 360 and 400 °C. In some cases, T3 is between -273 and 10 5 -200 and 10 3 This includes temperatures between -100 and 500, -50 and 200, 0 and 500, 100 and 500, 200 and 500, 200 and 400, or between 250 and 350 °C.

[0258] In yet another embodiment of the present invention, T3 is included between T1 and T2. In some cases, T3 is preferred over T2 by 1.0001, 1.1, 1.2, 1.5, 2, 5, 10, 10 3 or 10 5 It shortens with coefficients exceeding this value. In other cases, T3 preferentially takes the values ​​1.0001, 1.1, 1.2, 1.5, 2, 5, 10, and 10 over T1. 3 or 10 5 It becomes longer with respect to the coefficient.

[0259] In one embodiment of the present invention, the temperature of the nanoparticles is the temperature of a heating device or furnace containing nanoparticles, used to heat the nanoparticles before, during, or after processing the nanoparticles by the purification method.

[0260] In one embodiment of the present invention, the temperature interval separating T1 and T2, specified as [TT1, T2], is: i) The nanoparticles exhibit the greatest variation or loss in weight or mass as a function of temperature, or ii) The derivative of the variation or loss of the weight or mass of the nanoparticles as a function of temperature is maximized.

[0261] In one embodiment of the present invention, the ratio [%W(T2)-%W(T1)] / (T2-T1), where %W(T2) and %W(T1) are percentages of the weight or mass of nanoparticles in T2 and T1, respectively, is 10 -50 , 10 -30 , 10 -20 , 10 -10 , 10 -5 , 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.5, 1, 10 or 10 5 Greater than % / °C. In some cases, this ratio is greater than 10% of the mass of carbon in the nanoparticles, preferably by purification or before processing the nanoparticles with purification. -20 , 10 -10 , 10 -5 , 10 -2 , 10 -1 It becomes larger when it is greater than 1, 5, 10, 20, 50, 75, 85, 95, or 100%.

[0262] In another embodiment of the present invention, the ratio [%W(T2) - %W(T1)] / (T2-T1) is 10 50 , 10 30 , 10 20 , 10 10 , 10 5 , 10, 5, 2, 1, 0.5, 0.05, 10 -3 , 10 -5 , 10 -10or 10 -20 Lower than % / °C. In some cases, the percentage of carbon mass in the nanoparticles is lower, preferably by purification or before processing the nanoparticles with purification, preferably 100, 95, 80, 70, 50, 30, 20, 10, 5, 2, 1, 10 -1 , 10 -3 , 10 -5 , 10 -10 or 10 -20 If it is less than a percent, this ratio will be low.

[0263] In one embodiment of the present invention, the temperature of the nanoparticles is such that T1, T2, and / or T3 are 10 5 , 10 3 , 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 3, 2, 1, 10 -5 , 10 -10 or 10 -20 If the change is less than %, it is maintained at T1, T2, and / or T3. In some cases, for each temperature T1, T2, and / or T3, this percentage is T maxi -T mini / T avi Equivalent to, T maxi , T mini , and T avi (i = 1, 2, 3) are the maximum, minimum, and average temperatures reached during the heating time or heating step after or at the time the temperature has been maintained at temperatures T1, T2, and / or T3. In some cases, this percentage will be lower if the furnace or heating device can maintain a stable temperature without large fluctuations and / or if the nanoparticles are less prone to endothermic and / or exothermic reactions. In some cases, an endothermic reaction is a reaction in which heat or energy is transferred from the medium surrounding the nanoparticles to the nanoparticles. In some other cases, an exothermic reaction is a reaction in which heat or energy is transferred from the nanoparticles to the medium surrounding the nanoparticles.

[0264] In one embodiment of the present invention, the temperature of the nanoparticles is 10 5 , 10 3 , 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 3, 2, 1, 10 -5, 10 -10 or 10 -20 If the change exceeds %, the nanoparticle temperature will not be maintained at T1, T2, and / or T3. In some cases, this percentage will be larger if the furnace or heating device cannot maintain a stable temperature without large fluctuations, and / or if the nanoparticles are prone to endothermic and / or exothermic reactions.

[0265] In one embodiment of the present invention, temperatures T1 and / or T2 are determined by: i) Nanoparticles at two temperatures T T <T1 and T T> T2 When heated between T, the change in the percentage of weight or mass of the nanoparticles is measured as a function of temperature. Here, T T <T1 teeth T1 Lower, T T> T2 It is higher than T2. ii) Measure, display, examine, investigate, or use at least one peak of the derivative of the variation in this rate as a function of temperature. iii) Estimate or estimate the temperature interval in which the variation in the percentage of weight or mass of nanoparticles as a function of temperature is maximum. The minimum and maximum temperatures in this interval are T1 and T2, respectively.

[0266] In some cases, the temperature at which the decreasing derivative of the variation in the weight or mass percentage of nanoparticles as a function of temperature shows a peak, or at which it begins, is the temperature at which the peak begins. In some other cases, the temperature at which the peak of the variation in the weight or mass percentage of nanoparticles as a function of temperature stops showing an increase, or at which it begins, is the temperature at which it ends.

[0267] In another embodiment of the present invention, the heat flux of nanoparticles is the heat flux generated by, emitted from, or originating from nanoparticles when the nanoparticles are heated, preferably in a heating device such as a furnace. Preferably, the heat flux can be measured using an instrument or thermal analysis method, or using differential thermal analysis (DTA) or differential scanning calorimetry (DSC).

[0268] In one embodiment of the present invention, the heating step i (where i is preferably a non-negative integer) of the purification method according to the present invention includes at least one of the following phases. • In the first phase, the temperature of the nanoparticles is time t i1P During the course of this, the temperature T i From temperature T iav It will rise to that point. • In the second phase, the temperature of the nanoparticles is time t i2P During the course of this, the temperature T iav It is maintained. • In the third phase, time t i3P During this process, the temperature of the nanoparticles reaches T iav From T f It decreases to [a certain level].

[0269] The present invention also relates to a purification method according to the present invention comprising at least one heating step i, wherein the heating step comprises at least one of the following first, second, and / or third phases, wherein: • In the initial stage, the temperature of the nanoparticles is time t i1P During the course of this, the temperature T i From temperature T iav It will rise to that point. • In the second stage, the temperature of the nanoparticles is controlled over time t i2P During the course of this, the temperature T iav It is maintained. • In the third stage, time t i3P During this process, the temperature of the nanoparticles reaches T iav From T f It decreases to [a certain level].

[0270] In some cases, T i and or t i1P is, T iav and or t i2P At least 1.0001, 1.1, 1.5, 2, 5, 10, or 100 times lower than. In some cases, T iav is equal to T1, T2, or T3, and or t i1P The temperature is T i It is equal to the time it takes to rise until it reaches [a certain value]. In several other cases, T fand or t i3 P is T i and or t i1P and 1.0001, 1.1, 1.5, 2, 5, 10, 10 2 or 10 5 The difference is not more than double.

[0271] The present invention also relates to a method according to the present invention, a method for preferential purification, wherein more than 10% by mass of carbon or carbonaceous material is removed from nanoparticles, and this percentage is (%C AT -%C BT ) / %C BT It is equal to %C. AT and %C BT These represent the proportion of carbon or carbonaceous material before and after treatment of the nanoparticles by the method.

[0272] In some cases, (%C AT -%C BT ) / %C BT is 10 -50 , 10 -20 , 10 -5 , 10 -2 , 10 -1 Greater than 1, 5, 10, 50, 75, 90, 95, or 99%. This is preferably lower than 99, 90, 70, 60, 50, 40, 30, 20, 10, or 1% if the purification method is efficient, or if the amount of carbon or carbonaceous material in the nanoparticles before processing the nanoparticles with the purification method is below a certain threshold.

[0273] In other cases, (%C AT -%C BT ) / %C is 99, 90, 70, 60, 50, 40, 30, 20, 10 or 1% or less. This is when the purification method is inefficient, or when the amount of carbon or carbonaceous material in the nanoparticles before processing the nanoparticles with the purification method is greater than a certain threshold, preferably 99, 90, 70, 60, 50, 40, 30, 20, 10 or 1%.

[0274] Furthermore, in some other cases, (%C AT -%C BT ) / %C BT This ranges from 0.1 to 100, 1 to 99, 10 to 99, 50 to 99, or 80 to 99%.

[0275] This invention relates to high-purity iron oxide-based nanoparticles obtained by the method of the present invention.

[0276] The present invention also relates to high-purity nanoparticles or high-purity nanoparticles that cannot be obtained by the present method.

[0277] In one embodiment of the present invention, high-purity nanoparticles, preferably coatings of these nanoparticles, consist of the following: i) Iron oxide between 0.8 and 0.999999999 g per gram of nanoparticles, and / or ii) 10 per gram of nanoparticles -40 and 10 5 Impurities between μg particles.

[0278] In one embodiment of the present invention, the high-purity nanoparticles contain, by mass percent, 90, 10, 5, 2, preferably 1, 0.5, 0.4, or 0.3% or less of carbon or carbonaceous material. In some cases, such a low percentage of carbon mass allows the nanoparticles to be coated with a coating that does not originate from the cells producing the nanoparticles.

[0279] In one embodiment of the present invention, the SAR (Specific Absorption Rate) of high-purity iron oxide nanoparticles is 10 per gram of nanoparticles. -100 , 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -3 , 10 -1 , 1, 10, 10 3 or 10 5 It is greater than watts. In some cases, the SAR of nanoparticles is highest when the amount of impurities in the nanoparticles is lowest. In some cases, the SAR of high-purity iron oxide nanoparticles is 10 per gram of nanoparticles.-100 and 10 100 , 10 -1 and 10 5 , or 0.1 and 10 3 It is composed of watts. In some cases, the SAR of nanoparticles is preferably proportional to the gradient of temperature change over time of nanoparticles surrounded by a medium such as water, biomaterials, or the body, preferably the initial gradient (DT / dt), where (DT / dt) is preferably estimated in °C / sec, and SAR = a(DT / dt). In some cases, a = C v / C nano And here C v This refers to the intrinsic heat capacity of water containing nanoparticles, biomaterials, body parts, or tissues, C nano SAR is the concentration or quantity or number of nanoparticles contained in water, biological materials, body parts, or tissues. In some cases, SAR is measured by exposing high-purity iron oxide nanoparticles to radiation, preferably heat-generating radiation, preferably lasers, magnetic fields, alternating magnetic fields, sound waves, ultrasound, or high-frequency radio waves.

[0280] In one embodiment of the present invention, high-purity iron oxide nanoparticles are 10 50 , 10 20 , 10 10 , 10 5 , 10 3 , 10 2 , 10, 1, 10 -1 , 10 -2 or 10 -5 It has a size distribution of less than nm. In some cases, when the method according to the present invention enables the production of nanoparticles having a low size distribution, the nanoparticle size distribution is low.

[0281] In another embodiment of the present invention, high-purity iron oxide nanoparticles are preferably in ml or mm 3 10 per or per cell -6 , 10 -3 , 10 -1 , at concentrations exceeding 1 or 10 mg of nanoparticles, 1, 10, 10 3 , 10 6 or 10 9It destroys more cells than a single individual.

[0282] The present invention also preferentially provides 10 per cell, preferably per liter of pre-growth and / or growth medium. -50 , 10 -30 , 10 -10 , 10 -5 , 10 -2 , 10 -1 , 1, 5, 10, 50, 10 2 , 10 3 or 10 5 This relates to high-purity iron oxide nanoparticles produced in yields exceeding mg, or to iron contained in nanoparticles.

[0283] The present invention also relates to nanoparticles obtained by the method of the present invention, wherein the yield of nanoparticle production is preferably 10 per cell, preferably 10 per liter of pre-growth and / or growth medium. 50 , 10 30 , 10 10 , 10 5 , 10 2 , 10, 5, 1, 10 -1 , 10 -2 , 10 -3 or 10 -5 This refers to nanoparticles of a few milligrams or less, or the amount of iron contained in the nanoparticles in milligrams.

[0284] The present invention also relates to high-purity iron oxide-based nanoparticles according to the present invention, wherein the high-purity iron oxide nanoparticles are magnetosomes.

[0285] In one embodiment of the present invention, the magnetosomes are nanoparticles produced by magnetotactic bacteria that are preferentially processed according to at least one of the following steps: i) Extract and / or isolate nanoparticles from bacteria to obtain magnetosomes containing crystallized minerals, preferably surrounded by a biological membrane. ii) Biological membranes are removed using the purification step preferentially. iii) Magnetosomes are coated with a non-nanoparticle-derived coating for stabilization, preferentially avoiding aggregation and / or deposition of the magnetosomes.

[0286] The present invention also relates to compositions, drugs, medical devices, diagnostic compositions, therapeutic compositions, or cosmetic compositions comprising high-purity iron oxide nanoparticles according to the present invention.

[0287] In another embodiment of the present invention, high-purity iron oxide nanoparticles result in the following: i) to provide medical or therapeutic activity, for example, by enabling the destruction of pathological cells, viruses, bacteria, or cancer cells, or by being less toxic to healthy tissue than pathological cells, viruses, bacteria, or cancer cells. ii) Diagnostic activities, such as enabling the detection of pathological cells, viruses, bacteria, or cancer cells, or having low toxicity to healthy tissue, and / or ii) Cosmetics: Activities that improve a person's appearance, for example.

[0288] In another embodiment of the present invention, the high-purity iron oxide nanoparticles are non-immunogenic or non-pyrogenic. In this case, they are preferred: i) A small number of immune cells, preferably 1, 5, 10, 10 3 , 10 10 , 10 50 or 10 100 To attract or bring forth the following immune cells, and / or ii) Generate temperature 10 5 , 10 3 , 10 2 An increase in the temperature of biological tissues to 50, 20, 10, 5, 2, 1, or 0.1 °C or less.

[0289] The present invention also relates to nanoparticles according to the present invention, preferably high-purity iron oxide-based nanoparticles, for use in the treatment of diseases selected from the following group: i) Diseases related to the proliferation of cells that differ from the cell proliferation of a healthy individual. ii) Diseases associated with the presence of pathological cells such as tumors or cancer cells in a body part or individual, iii) Diseases associated with the presence of pathological sites, i.e., sites containing pathological cells in an individual or body part. iv) Disease, disorder, or dysfunction of a body part, v) Diseases associated with the presence of radiation-resistant, acoustically resistant, laser-resistant, or magnetically resistant cells. vi) infectious diseases; vii) autoimmune diseases; viii) Neuropathology, ix) Cancer, x) Tumor, xi) A disease involving at least one cancer or tumor cell, xii) Skin condition, xiii) Endocrine disorders, xiv) Eye diseases or disorders, xv) Intestinal diseases, xvi) Communication disorder, xvii) Genetic disorders, xviii) Neurological disorders, xix) Voice disorder, xx) Vulvovaginal disorders, xxi) liver damage; xxii) Heart disorder, xxiii) Heat damage, xxiv) Mood disorders, xxv) Anemia, iron anemia is the preferred cause. xxvi) Personality disorder, xxvii) Adjuvants, especially Neuroaid, xxviii) Parkinson's, xxix) Alzheimer's disease, xxx) Bacterial and / or fungal infection or contamination, xxxi) Blood disorders due to, for example, a lack or absence of efficient clotting, and xxxii) Diseases due to a lack of immune function or immune disorders.

[0290] In one embodiment of the present invention, cancer or tumor is selected from the following group: Cancers of organs, blood cancers, cancers of the body's systems, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, heart cancer, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic and penile cancer, prostate cancer, reticular cancer Cancers such as membranocyte tumor, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, Castleman disease, Ewing tumor, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, myelodysplastic syndrome, pituitary tumors, and gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, malignant mesothelioma, and multiple myeloma.

[0291] In yet another embodiment of the present invention, the treatment of disease by nanoparticles according to the present invention occurs or is activated when the nanoparticles are exposed to radiation, and is preferentially not occurred or activated when the nanoparticles are not exposed to radiation.

[0292] In yet another embodiment, the radiation is a laser, an acoustic wave such as ultrasound, X-rays, gamma rays, and / or a magnetic field, preferably an alternating magnetic field.

[0293] In several other embodiments, the output or intensity of the radiation is 10 -50 , 10 -20 , 10 -10 , 10 -5 , 10 -1 , 0, 1 mT, T, W, W / cm, W / cm 2 or W / cm 3 and 1, 5, 10, 10 3 , 10 5 , 10 10 , 10 20 or 10 50 mT, T, W, W / cm, W / cm 2 or W / cm 3 That is the case.

[0294] In yet another embodiment of the present invention, a combination of properties or functions of nanoparticles or methods can be obtained by preferentially combining the properties or features of the nanoparticles or methods described in individual embodiments, sections, or sentences of this patent application.

[0295] In yet another embodiment of the present invention, if a compound such as a nanoparticle or chemical element has a property of condition 1 (P1) that is higher, longer, or greater by a coefficient than the property of condition 2 (P2), then P1 = a·P2 or P1 = a + P2, where a is preferably a number or integer greater than 1 or 0.

[0296] In yet another embodiment of the present invention, if a compound such as nanoparticles or chemical elements has property 1 (P1) which is smaller than property 2 (P2) or by a coefficient lower, this means P1 = P2 / a or P1 = P2 - a, where a is preferably a number or integer greater than 1 or 0. [Examples]

[0297] Materials and methods

[0298] Optical density measurement of fully magnetotactic bacterial suspensions to evaluate bacterial growth: Using a Secomam UviLine9400 spectrophotometer, OD 565nm The optical density of various suspensions of magnetostatic bacteria was measured at 565 nm, known as OD. 565nm The value is proportional to the concentration of bacteria in the suspension.

[0299] Measurement of the magnetic response of these living magnetotactic bacteria under magnetic field application using optical microscopy: 1 mL of the suspension of MSR-1 magnetotactic bacteria was centrifuged at 14500 rpm for 10 minutes. The growth medium was removed and replaced with PBS 0.1X until an OD of 0.5 at 565 nm was reached. 1 μL of this MSR-1 magnetotactic bacteria suspension was deposited onto a parallelepiped microscope slide (Menzel-Glaeser, 24 mm x 60 mm, 0.13-16 mm thick) for microscopic observation using a Zeiss Primo Vert optical microscope at 40x magnification. Four small cubic neodinium magnets (Supermagnet, N42 W-10-N 10x10x10 mm) with an intensity of 1.3 T were positioned on the microscope platform at a distance of approximately 2 cm from the bacterial suspension to generate a magnetic field. After 20 seconds, the percentage of bacteria aligned in the direction of the magnetic field was estimated, considering 200 magnetotactic bacteria. The magnet was placed at either position 1 or 2, parallel to the observer's position or the line between the two binoculars (position 1), or perpendicular to this position (position 2). Bacteria not aligned in the same direction as the magnetic field generated by the magnet were considered non-magnetic, and their number was n BNM It was designated as such. Bacteria aligned in the direction of the magnetic field generated by a magnet are considered magnetic, and their number is n BM It was designated as such. The proportion of magnetic bacteria is n BM / (n BM + n BNM The positive magnetic response of magnetobacteria is given by n BM / (n BM + n BNM )> This was equivalent to 0.5. The negative magnetic response of magnetobacteria is n BM / (n BM + n BNM This corresponds to a value of <0.5. In some cases, the proportion of magnetic bacteria can be measured by optical observation under a microscope in the presence of a magnetic field.

[0300] Measurement of intracellular iron concentration: The iron concentration inside magnetotactic bacteria was determined by destructive iron chemistry. For this purpose, 2 mL of MSR-1 magnetotactic bacteria were centrifuged at 14500 g for 10 minutes, and then the bacterial pellet was washed twice with 1X PBS and MilliQ water. After the second wash, the bacterial pellet was collected and 1 mL of 12N hydrochloric acid (HCl) was added to the pellet under a chemical hood. The sample was heated at 75°C for 2 hours with stirring at 300 rpm to convert intracellular iron to Fe³⁺ and Fe²⁺ ions. Next, the Fe²⁺ ions were oxidized to Fe³⁺ with 20% hydrogen peroxide (H₂O₂). Upon addition of potassium thiocyanate (KCN, 2 mol / L) to the acidic medium, the presence of Fe³⁺ ions was revealed, and a reddish-orange solution was formed that depended on the concentration of Fe³⁺ in the sample. Immediately after the addition of KCN, the absorbance of the solution was measured at 476 nm. Next, the iron concentration of the sample was estimated using the determined relationship between absorbance values ​​measured at 476 nm and the concentration of iron(III) chloride. This method makes it possible to estimate the total intracellular iron concentration.

[0301] Analysis of the elemental chemical composition of magnetosomes by ICP-AES: After fermentation, MSR-1 magnetotactic bacteria were concentrated in a 5 L volume by tangential filtration to an optical density of 25-30. The bacteria were then lysed for 1 hour. The bacterial lysate containing magnetosomes was placed on a neodymium magnet for 12 hours, and then the magnetosomes were separated from the bacterial lysate and resuspended in 10X PBS. This washing procedure was repeated twice with 10X PBS and three times with MilliQ water. The magnetosomes were then freeze-dried and heated in a muffle furnace under the conditions described below to obtain a magnetosome powder containing high-purity iron oxide crystals with a low carbon content. To analyze the elemental chemical composition, a solution of 500 μg of this powder was mixed with 200 μl of 12N HCl and 10 ml of 2% filtered HNO3. ICP-AES measurement of the powder allows us to determine the amount of each chemical element (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Si, Sn, Ti, Tl, W, Zn) per gram of iron contained in the magnetosome.

[0302] Chemicals used in the preparation of the growth medium: Aluminum sulfate dodecahydrate (AlK(SO4)212H2O, ref. NFG A6435, Merck); Ammonium hydroxide (NH4OH, ref. NFG 1336-21-6, Acros or ganics; ref. FG 105422, Merck); Ammonium chloride (NH4Cl, ref. NFG A9434, Merck; ref. FG 1011420001, Merck); Ammonium sulfate ((NH4)2SO4, ref. NFG A4418); Biotin (C10H16N2O3S, ref. NFG B4639, Merck; ref. FG B301, Merck); Boric acid (H3BO3, ref. NFG B6768, Merck); Calcium chloride (CaCl2, ref. NFG 223506, Merck; ref. FG 1.42002, Merck); Calcium pantothenate (HOCH2C(CH3)2CH(OH)CONHCH2CH2CO2·1 / 2Ca, ref. FG C0400000, Merck); Cobalt(II) nitrate hexahydrate (Cobalt(II) nitrate hexahydrate, ref. FG 239267, Merck); Copper(II) sulfate pentahydrate (CuO4S.5H2O) (ref. NFG C8027, Merck); DL-methionine (CH3SCH2CH2CH(NH2)COOH, ref. NFG M2768, Merck); DL-tryptophan (C11H12N2O2, ref. NFG T3300, Merck); EDTA ((HO2CCH2)2NCH2CH2N(CH2CO2H)2, ref. NFG E6758, Merck); Ferric citrate (C6H5FeO7, ref. NFG F3388, Merck; ref. FG B301, Merck); Folic acid (C19H19N7O6, ref. NFG F7876, Merck; ref. FG F0300000, Merck); Inositol (C6H12O6, ref. FG PHR1351, Merck); Iron(II) sulfate heptahydrate (FeO4S.7H2O, ref. NFG F8633, Merck; ref. FG 1.03963, Merck); Iron(III) oxalate hexahydrate (Fe2(C2O4)3.6H2O, ref. NFG 381446, Merck); L-histidine (C6H9N3O2, ref.FG PHR1108, Merck); Magnesium sulfate heptahydrate (MgSO4.7H2O, ref. NFG 63138, Merck; ref. FG 105882, Merck); Manganese(II) sulfate monohydrate (MnO4S.H2O, ref. NFG M7899, Merck); Nickel(II) chloride hexahydrate (Cl2Ni.6H2O, ref. NFG N6136, Merck); Nicotinic acid (C6H5NO2, ref. NFG N4126, Merck); Trisodium nitrilotriacetate (C6H6NO6Na3, ref. NFG N0253, Merck); p-aminobenzoic acid (H2NC6H4CO2H, ref. NFG A9878, Merck); Dibasic potassium phosphate (K2HPO4, ref. NFG P8281, Merck; ref. FG 105101, Merck); Monobasic potassium phosphate (KH2PO4, ref. NFG P9791, Merck); Protoporphyrin IX (C34H34N4O4, ref. NFG P8293, Merck); Pyridoxine HCl (C12H17ClN4OS.HCl, ref. NFG P9755, Merck); Riboflavin (C17H20N4O6, ref. NFG R9504, Merck; ref. FG PHR1054, Merck); Sodium chloride (NaCl, ref. NFG S7653, Merck); Sodium lactate (C3H5NaO3, ref. NFG L1375, Merck; ref. FG 106522, Merck); Sodium molybdate dihydrate (Na2Mo4.2H2O, ref. NFG M1003, Merck); Sodium selenite pentahydrate (Na2SeO3.5H2O, ref. FG 89771, Merck); Thiamine HCl (C12H17ClN4OS.HCl, ref. NFG 47858, Merck, ref. FG PHR1037, Merck); Yeast extract (ref. NFG Y1625, Merck); Zinc sulfate heptahydrate (O4Zn.7H2O, ref. NFG Z0251, Merck). NFG specifies non-pharmaceutical grade chemicals used in the preparation of the growth medium. FG specifies pharmaceutical grade chemicals used in the preparation of the growth medium. Deionized water (H2O) with a resistivity of 15 MΩ is also used.

[0303] Composition of various mineral elixirs: Table 6 shows the composition of various mineral elixirs (V0, CB1, V2, CB2, CB3, CB4, CB5, CB7, CB9, CB10, CB11, CB12, CB13). The amounts (in grams) of various chemicals used to prepare 1 liter of these elixirs are shown.

[0304] Composition of various yeast extracts: Various yeast extracts (Vi T1 X, Vit5X, Vi T1 Table 7 shows the composition of (0X, Vit0.5X, Vit0.1X). ​​It shows the amount (grams) of various chemicals used in the preparation of 1 liter of yeast. YNBWAA, YNBWoAA, and YNBWoAA.AS represent reduced yeast extract, while YE represents non-reduced yeast extract (see: Y0875, Sigma). YE consists of nitrogen compounds, carbon, sulfur, micronutrients, vitamin B complex, and other important growth factors.

[0305] Composition of various vitamin cocktails: Various vitamin cocktails (Vi T1 X, Vit5X, Vi T1 Table 8 shows the compositions of 0X, Vit0.5X, and Vit0.1X. The liters of these vitamin cocktails are also shown.

[0306] Composition of the preliminary growth medium for Condition 1 (Table 1): One liter of preliminary growth medium contains 1 liter of deionized water with 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, dibasic potassium phosphate, 0.1 g of yeast extract YE (Table 7), and 0.5 mL of any of the following mineral elixirs: V0, CB1, V2, CB2, CB3, CB4, CB5, CB7, CB9, CB10, CB11, CB12, or CB13 (Table 6).

[0307] Composition of the growth medium for Condition 1 (Table 1): 1 liter of growth medium consists of 1 liter of deionized water containing 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of dibasic potassium phosphate, 0.1 g of yeast extract YE (Table 7), 0.5 mL of any of the following mineral elixirs: V0, CB1, V2, CB2, CB3, CB4, CB5, CB7, CB9, CB10, CB11, CB12, or CB13 (Table 6), and 10 mL of ferric citrate (initial concentration 20 mM).

[0308] Composition of preliminary growth medium for Condition 2 (Table 2): 1 liter of preliminary growth medium is contained in 1 liter of deionized water: 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of dibasic potassium phosphate, 0.1 g of any of the following: yeast extract YE, YNBWAA, YNBWoAA, YNBWoAA.AS (Table 7), and 0.5 mL of mineral elixir CB3 (Table 6).

[0309] Composition of the growth medium for Condition 2 (Table 2): 1 liter of growth medium consists of 1 liter of deionized water containing 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of dibasic potassium phosphate, 0.1 g of any of the following: yeast extract YE, YNBWAA, YNBWoAA, or YNBWoAA.AS (Table 7), 0.5 mL of mineral elixir CB3 (Table 6), and 10 mL of ferric citrate (initial concentration of 20 mM).

[0310] Composition of the preliminary growth medium for Condition 3 (Table 3): One liter of preliminary growth medium contains 1 liter of deionized water, 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, dibasic potassium phosphate, and vitamin Vi. T1 X, Vit5X, Vi T1 0.1 mL of either 0X, Vit5X, Vit0.5X, or Vit0.1X (Table 8), or 0.5 mL of Mineral Elixir CB3 (Table 6).

[0311] Composition of growth medium for condition 3 (Table 3): 1 liter of growth medium consists of 1 liter of deionized water with 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of dibasic potassium phosphate, and vitamin cocktail Vi. T1 X, Vit5X, Vi T1 0.1 mL of either 0X, Vit5X, Vit0.5X, or Vit0.1X (Table 8), 0.5 mL of Mineral Elixir CB3 (Table 6), and 10 mL of ferric citrate (initial concentration of 20 mM).

[0312] Composition of the preliminary growth medium for condition 4 (Table 4): One liter of preliminary growth medium contains 1 liter of deionized water with 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, dibasic potassium phosphate, 0.1 mL of any of the individual vitamins Bt, CP, FA, I, NA, AA, P, R, or T (Table 9), and 0.5 mL of mineral elixir CB3 (Table 6).

[0313] Composition of growth medium for condition 4 (Table 4): 1 liter of growth medium consists of 1 liter of deionized water with 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of dibasic potassium phosphate, 0.1 mL of any of the individual vitamins Bt, CP, FA, I, NA, AA, P, R, or T (Table 9), 0.5 mL of mineral elixir CB3 (Table 6), and 10 mL of ferric citrate (initial concentration of 20 mM).

[0314] Table 5 shows the composition of the preliminary growth medium for Condition 5: the main components of the preliminary growth medium at various concentrations, namely sodium lactate, ammonium chloride, magnesium sulfate heptahydrate, and potassium phosphate. The dibasic (N, SL0, SL0.5X, SL0.2X, SL0.1X, AC0, AC0.5X, AC0.2X, AC0.1X, MG0, MG0.5X, MG0, MG0.5X, MG0.2X, MG0.1X, P, P0.5X, P0.2X, P0.1X) are shown in Table 5. Table 5 also shows the amounts (grams) of the various chemicals used to prepare 1 liter of these preliminary growth media.

[0315] Composition of growth medium for Condition 5 (Table 5): Table 5 shows the main components of preliminary growth mediums at various concentrations, namely sodium lactate, ammonium chloride, magnesium sulfate heptahydrate, and dipotassium phosphate (N, SL0, SL0.5X, SL0.2X, SL0.1X, AC0, AC0.5X, AC0.2X, AC0.1X, MG0, MG0.5X, MG0, MG0.5X, MG0.2X, MG0.1X, P, P0.5X, P0.2X, P0.1X). ​​Table 5 shows the amounts (grams) of various chemicals used to prepare 1 liter of these growth media.

[0316] Composition of preliminary growth medium, growth medium, and fed-batch medium under Condition 6 prepared using non-pharmaceutical grade chemicals (Table 14(a)): Preliminary growth mediums B1 and B4 consist of 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of dibasic potassium phosphate, 0.1 mL of vitamin cocktail Vit 0.1X (Table 8), and 0.5 mL of mineral elixir CB3 (Table 6) in 1 liter of deionized water. Growth mediums B1 and B4 contain 104 g of sodium lactate, 16 g of ammonium chloride, 1.2 g of magnesium sulfate heptahydrate, 2.8 g of dibasic potassium phosphate, 3.2 mL of vitamin cocktail Vit 0.1X (Table 8), and 2.8 mL of mineral elixir CB3 (Table 6) in 1 liter of deionized water. Fed-batch media B1 and B4 contain 100 g of lactic acid, 4.8 g of ammonia, 6 g of dibasic potassium phosphate, 2.4 g of magnesium sulfate heptahydrate, 1 mL of vitamin cocktail Vit0.1X in 1 liter of water (Table 8), 7 mL of mineral elixir CB3 (Table 6), and 1.8 g of ferric citrate (B1) or 2 g of iron III chloride (B4).

[0317] The composition of the preliminary growth medium, growth medium, and fed-batch medium prepared using pharmaceutical-grade chemicals under Condition 6 (Table 14(b)): Preliminary growth mediums B2 and B3 contain 2.6 g of sodium lactate, 0.4 g of ammonium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.5 g of dibasic potassium phosphate, 0.1 mL of vitamin cocktail Vit 0.1X (Table 8), and 0.5 mL of mineral elixir CB3 (Table 6) in 1 liter of deionized water. Growth mediums B1 and B4 contain: 104 g of sodium lactate, 16 g of ammonium chloride, 1.2 g of magnesium sulfate heptahydrate, 2.8 g of dibasic potassium phosphate, 3.2 mL of vitamin cocktail Vit 0.1X (Table 8), and 2.8 mL of mineral elixir CB3 (Table 6) in 1 liter of deionized water. The fed-batch media B1 and B4 contain: 100 g of lactic acid, 4.8 g of ammonia, 6 g of dibasic potassium phosphate, 2.4 g of magnesium sulfate heptahydrate, 1 mL of vitamin cocktail Vit0.1X (Table 8), 7 mL of mineral elixir CB3 (Table 6), and 1.8 g of ferric citrate (B2) or 2 g of iron III chloride (B3).

[0318] Stock of MSR-1 magnetotactic bacteria used in various cultures: MSR-1 magnetotactic bacteria are commercialized by DSMZ according to DSM 6361 standards. After acceptance, the MSR-1 bacterial suspension is OD 565nm The suspension of MSR-1 bacteria is stored in a -80°C freezer in 15 mL tubes (5 mL of bacterial suspension per tube) or 1.5 mL Eppendorf tubes (600 μL of bacterial suspension per tube) at 0.01 (565 nm). The MSR-1 bacterial suspension stored in a -80°C freezer constitutes the cell stock. In some cases, the culture and / or pre-culture growth medium may be the same as the growth and / or pre-growth medium.

[0319] Notation: The number X after the D in DX indicates the number of days since the start of the preliminary growth step. This is the day when the magnetotactic bacteria are first inserted into the preliminary growth medium, or during the first substep of the preliminary growth step.

[0320] Example 1: Determining the minimum mineral elixir that enables bacterial growth and magnetosome synthesis: This example describes an experimental protocol that minimizes the mineral elixir composition while still allowing for the growth of MSR-1 magnetotactic bacteria and the synthesis of magnetosomes by these bacteria. In this example, non-pharmaceutical grade chemicals are used for the preparation of the growth medium. Table 1 shows the composition of 1 liter of pre-growth medium and growth medium used in this example (Condition 1). On the first day of the experiment (D1), the first step is to collect 15 mL tubes containing 5 mL of MSR-1 cell stock tubes from a -80°C freezer. By thawing them in a hood at room temperature for 10 minutes, 5-10 mL tubes containing 5 mL of filtered pre-growth medium are inserted into 50 mL tubes. 6 100 μl containing MSR-1 magnetotactic bacteria was collected. In total, 13 different culture conditions were tested, corresponding to 13 different mineral elixirs tested. 50 ml tubes were cultured for 6 days between D1 and D6 in an incubator at 29.5°C under vibration conditions of 150 rpm. The next step was to add an iron source to the growth medium to enable magnetosome synthesis by MSR-1 bacteria. After 6 days of preliminary growth, 50 ml tubes were placed in a hood on D6, then 30 ml of filtered medium was added to the 50 ml tubes, and the bacteria grew between D6 and D13. A positive magnetic response was observed at D13, and the ratio of optical density at D13 to optical density at D6 was greater than 1 at V13 compared to V0, V2, CB2, CB3, CB4, CB5, CB7, CB10, CB11, CB12, and CB13 (Condition 1). In contrast, there is no magnetic response to conditions CB1 and CB9 where the concentrations of the chemical elements constituting the mineral elixir are 10⁻⁵ g / l or less. In conclusion, the minimum mineral elixir that enables the growth of MSR-1 bacteria (OD565nmD13 / OD565nmD6 greater than 4.8) and magnetosome synthesis (positive magnetic response) with a significant increase in optical density is CB13, which consists only of 1 g / l of ferrous sulfate heptahydrate and 20 g / l of calcium chloride.

[0321] Example 2: Determination of a Yeast Extract-Free Growth Medium Enabling Magnetotactic Bacteria Growth and Magnetosome Synthesis: This example describes an experimental protocol used to determine a reducing medium that replaces yeast extract, enabling the growth of MSR-1 magnetotactic bacteria and the synthesis of magnetosomes by these bacteria. In this example, non-pharmaceutical grade chemicals were used for the preparation of the growth medium. The compositions of the preliminary growth and growth medium in 1 liter of deionized water are shown in Table 2 (Condition 2), Table 3 (Condition 3), and Table 4 (Condition 4). On Day 1 of the experiment (D1), the first step is to collect 15 ml tubes containing 5 mL of MSR-1 cell stock tubes from a -80°C freezer and thaw the tubes at room temperature. In a hood, 100 μl of 5.106 MSR-1 magnetotactic bacteria were collected from these tubes, inserted into 50 mL tubes containing 8 mL of filtered preliminary growth medium of either Condition 2 (Table 2), Condition 3 (Table 3), or Condition 4 (Table 4). 50 ml tubes were cultured in an incubator at 29.5°C for 6 days between D1 and D6 under vibration conditions of 150 rpm. The next step was to add an iron source to the growth medium to enable magnetosome synthesis by MSR-1 bacteria. After 6 days of preliminary growth, the 50 ml tubes were placed in a hood on D6. Next, 30 ml of filtered growth medium was added to the 50 ml tubes (Condition 2, Table 2, Condition 3, Table 3, Condition 4, Table 4) on D6, and the bacteria grew between D6 and D13. Tables 11 and 12 show the yeast extracts YE, YNBWAA, YNBWAA, YNBWoAA, YNBWoAA, YNBWoAA.AS (Condition 2), Vi T1 The following conditions were observed: X, Vit0.5X, Vit0.1X (condition 3), biotin (Bt), folic acid (FA), nicotinic acid (NA), riboflavin (R), and thiamine HCl (T) (condition 4). These conditions showed a magnetic response of over 90% at D13, and a ratio of greater than 1 between the optical density measured at D13 and the optical density measured at D6 was observed. In contrast, the magnetic response was under the conditions Vit5X, Vi T1The values ​​were 0 in 0X (condition 3) and very low in conditions CP, I, AA, and P (condition 4). In conclusion, yeast extract can be replaced with a single vitamin, such as biotin, folic acid, riboflavin, nicotinic acid, or thiamine HCl. These vitamins have OD565nmD13 / OD565nmD6 values ​​of 9.8 (biotin), 2.9 (folic acid), 4.8 (riboflavin), 2.4 (nicotinic acid), 5.8 (thiamine HCl), and account for 90% of the magnetic response (Table 11).

[0322] Example 3: Determination of the minimum concentrations of the main components of the growth medium (sodium lactate, ammonium chloride, magnesium sulfate, potassium phosphate), which enables the growth of magnetotactic bacteria and the synthesis of magnetosomes by these bacteria. This example describes the experimental protocol used to determine a reduced growth medium that enables the growth of MSR-1 magnetotactic bacteria and the synthesis of magnetosomes by these bacteria. In this example, non-pharmaceutical grade chemicals were used to prepare the growth medium. Table 5 lists sodium lactate (conditions SL0, SL0.5X, SL0.2X, SL0.1X), ammonium chloride (AC0, AC0.5X, AC0.2X, AC0.1X), magnesium sulfate heptahydrate (MG0, MG0.5X, MG0.2X, MG0.1X), and dibasic potassium phosphate (P0, P0.5X, P0.2X, P0.1X). The chemical composition and concentrations of the preliminary and growth media N, SL0, SL0.5X, SL0.2X, SL0.1X, AC0, AC0.5X, AC0.2X, AC0.1X, MG0, MG0.5X, MG0.2X, MG0.1X, P0, P0.5X, P0.2X, and P0.1X are summarized per liter of growth medium. On day 1 of the experiment (D1), the first step was to collect 15 ml tubes containing 5 mL of MSR-1 cell stock tubes from a -80°C freezer and thaw the tubes at room temperature for 10 minutes. Under a hood, these tubes were collected containing 100 μl of 5.106 MSR-1 magnetotactic bacteria inserted into 50 mL tubes filled with 8 mL of preliminary growth medium (Condition 5, Table 5). The 50 ml tubes were cultured in an incubator at 29.5°C for 6 days between D1 and D6 under vibration conditions of 150 rpm. The next step was to add an iron source to the growth medium to enable magnetosome synthesis by MSR-1 bacteria. After 6 days of preliminary growth, 50 ml tubes were placed in a hood at D6. Next, 30 ml of filtered growth medium was added to the 50 ml tubes at D6, and the bacteria were grown between D6 and D13. Under conditions N, P0.5X, and P0.2X, the growth rate, or the ratio of optical density measured at D13 to optical density measured at D6, was greater than 1, and a positive magnetic response (magnetic response > 90%) was observed at D13. These conditions indicate that these conditions enabled bacterial growth and magnetosome generation. In contrast, under the conditions SL0, SL0.5X, SL0.2X, SL0.1X, AC0, AC0.5X, AC0.2X, AC0.1X, MG0, MG0.5X, MG0.2X, MG0.1X, P0.1X, and P0, magnetosome synthesis was very low (magnetic response <50%). In conclusion, the concentration of potassium phosphate in the growth medium can be reduced by a factor of 2 or 5 without affecting magnetosome growth and production. In fact, under these conditions, OD 565nmD13 / OD 565nmD6 The values ​​were 1.5 (condition P0.5X) and 2.1 (condition P0.2X), and the proportion of positive magnetic response between bacteria exceeded 90% (Table 13). In contrast, the concentrations of other chemicals in the culture medium (ammonium chloride, sodium lactate, magnesium phosphate) could not be reduced without significantly affecting the growth and / or magnetic response of MSR-1 magnetotactic bacteria.

[0323] Example 4: Determination of the iron source for a 1-liter fermenter that enables the growth of magnetotactic bacteria and the synthesis of magnetosomes by these bacteria, and reduction of impurities obtained by using high pharmaceutical-grade chemicals for the preparation of the preliminary growth medium and / or growth medium. This example describes the experimental protocol used to determine the iron source. This allows for the growth of MSR-1 magnetotactic bacteria, the synthesis of magnetosomes by these bacteria, and the reduction of impurities in the resulting magnetosomes using pharmaceutical-grade chemicals (Condition 6). In this example, B2 and B3 growth media (Table 14(b)) were prepared using pharmaceutical-grade chemicals, and B1 and B4 growth media (Table 14(a)) were prepared using non-pharmaceutical-grade chemicals. The compositions of the pre-growth medium, growth medium, and fed-batch medium are shown in Tables 14(a) and 14(b) for 1 liter of medium. On day 1 (D1), the first step of pre-growth was to collect 1.5 ml Eppendorf tubes containing 5 mL MSR-1 cell stock tubes from a -80°C freezer and thaw the tubes at room temperature for 10 minutes. Under a hood, these tubes containing 300 μl of 5.106 MSR-1 magnetotactic bacteria were collected and placed in 500 mL bottles filled with 250 mL of pre-growth medium. The 500 mL bottles were incubated in an incubator at 29.5°C for 7 days between D1 and D7 under vibration conditions of 150 rpm. The second step of preliminary growth was performed in a larger 2 L bottle. After 7 days of preliminary growth, the 500 mL bottle was placed in a hood on D8. The pre-growth medium containing MSR-1 bacteria was manually transferred to a 2 L sterile bottle filled with 1.5 L of filtered pre-growth medium for the second pre-growth stage. The 2 L bottle was incubated in an incubator at 29.5°C under shaking conditions of 150 rpm for 1 day between D8 and D9. On day 9 (D9), the growth stage began. To this end, four 1.5 L fermenters (conditions B1, B2, B3, B4) were filled with 780 mL of deionized water and sterilized under pressure. Next, 20 mL of filtered growth medium was added to each fermenter. Then, 200 mL of preliminary growth medium containing MSR-1 bacteria derived from the second step of preliminary growth was added to each of the four fermenters (conditions B1, B2, B3, B4). Between days D9 and D11, an acidic fuel-batch medium containing an iron source was added to the growth medium to maintain the pH of the growth medium at 6.9, enabling magnetosome synthesis by MSR-1 bacteria. During the growth phase, the temperature was maintained at 29.5°C, the airflow at 0.05 mL / min, and the stirring at 200 rpm. The optical density measured at 565 nm of bacterial suspensions on different days of the preliminary growth phases (D0 and D8) and growth phases (D9, D10, D11) is shown in Table 15 for conditions B1, B2, B3, and B4. After fermentation on D13, MSR-1 cells from fermenters B1, B2, B3, and B4 (conditions B1 to B4) were concentrated by centrifugation at 4000 rpm for 45 minutes. To lyse the bacteria, MSR-1 cells from fermenters B1, B2, B3, and B4 were resuspended in 15 mL of 1 M KOH solution and heated in 20 mL glass bottles in a 25 kHz sonication tank at 80°C for 2 hours. After bacterial lysation, the magnetosomes of the MSR-1 cells were separated from the organic material using a neodymium magnet overnight. On D14, the magnetosomes under conditions B1, B2, B3, and B4 were washed twice with 15 mL of 10 mL of phosphate-buffered saline and twice with 15 mL of deionized water using a neodymium magnet. Between each wash, the magnetosome suspension was left for 2 hours in front of a neodymium magnet attracting the magnetosomes. The supernatant containing organic debris was discarded and replaced with 15 mL of 10X phosphate-buffered saline or 15 mL of deionized water. On D16, after the final wash, the supernatant was discarded, and the magnetosomes under conditions B1, B2, B3, and B4 were transferred to ceramic cups and dried in front of a neodymium magnet for the entire day. On D17, the remaining liquid was discarded, the magnetosomes were placed in ceramic cups and placed in a muffle furnace, where they were heated at 200°C for 30 minutes, 300°C for 1 hour, and 380°C for 1 hour. On D17, approximately 1 mg of purified magnetosomes under conditions B1, B2, B3, and B4 were placed in 15 mL tubes filled with 200 μL of HCl 12N. The 15 mL tubes containing the magnetosomes were vortexed and incubated at room temperature for 2 hours, after which they were filled with 9.8 mL of 2% HNO3. Subsequently, the concentration of elemental impurities in μg per gram of nanoparticles was measured by ICP-AES. The results of these measurements are shown in Table 16 for conditions B1, B2, B3, and B4. The elemental impurities were Ag (silver), Al (aluminum), As (arsenic), Ba (barium), Cd (cadmium), Co (cobalt), Cr (chromium), Cu (copper), Mn (manganese), Mo (molybdenum), Ni (nickel), Pb (lead), Sb (antimony), Se (selenium), Si (silica), Sn (tin), Ti (titanium), Tl (thallium), W (tungstate), and Zn (zinc). In conclusion, condition B3 showed a maximum OD565nmD11 / OD565nmD9 value of 26.8 and a percentage of positive magnetic responses among bacteria (>90%) (Table 15), indicating that iron(III) chloride is the optimal iron source. Furthermore, in condition B3, where pharmaceutical-grade chemicals were used (except for Pb), the elemental impurity concentrations were reduced compared to condition B4, where non-pharmaceutical-grade chemicals were used (Table 16).

[0324] Example 5 (Purification Method): material and method:

[0325] Note: In this example, weight can be replaced with mass, and they will generally have the same meaning.

[0326] Equipment used for the analysis and heating of various samples:

[0327] TGA-DSC: Thermogravimetric analysis (TGA) combined with differential scanning calorimetry (DSC) is used to measure the heat flow (mW) or the percentage of mass loss (treated or untreated) of powders containing lyophilized magnetosomes. Alternatively, for the measurement of whole lyophilized bacteria or lyophilized SIGMA nanoparticles as a function of the heating temperature of these powders, the powders are heated at a rate of 6°C per minute between 20°C and 600°C. The TGA-DSC profile allows for the definition of the temperature at which the surface or material on the surface of the magnetosomes or nanoparticles, preferably organic materials, degrades. ATG and DSC analyses were performed using an SDT Q600 (TA Instrument), which consists of a sealed enclosure, a temperature-controlled furnace, a microbalance, and a thermocouple for measuring temperature. For TGA-DSC analysis, 3 mg of lyophilized bacteria, lyophilized magnetosomes, and SIGMA nanoparticle powders were used for CHNS measurements.

[0328] Furnace: A muffle furnace (Nabertherm L9 / 11 / B410) is used to heat 30 mg or 500 mg of magnetosomes extracted from magnetotactic bacteria, either without heat treatment above 200°C according to condition number 1 or 2, or with heat treatment above 200°C (according to condition numbers 3, 4, 5, 6, 7, 8, 9, 10, or 11). To do this, 30 or 500 mg of powder for each sample is placed in an uncovered porcelain cup and placed in the center of the furnace. Various heating conditions can be performed using the program. The furnace can maintain the temperature of the nanoparticles and / or the inside of the furnace at a predetermined temperature plus or minus 2°C, or the furnace can achieve a stable temperature of 20-380°C with a maximum variation of 2°C.

[0329] 完全走磁性 Sample containing bacteria (Sample 0): Magnetotactic bacteria obtained under Condition 1 (Mineral Elixir V2, Table 1) were collected and filtered using a tangential filtration system at 565 nm (OD 565nm The samples were concentrated to an optical density between 100 and 200 as measured by ). Sample 0 consists of condensed whole-body magnetotactic bacteria.

[0330] Samples containing magnetosomes extracted from magnetotactic bacteria without heat treatment above 200°C (Samples 1 and 2):

[0331] Dissolution condition 1 (Sample 1): OD 120 565nm A 100 ml concentrated sample was mixed with 400 ml of 5 M NaOH and heated at 60°C for 1 hour using an ultrasonic bath to lyse the bacteria. Next, the treated magnetosomes were isolated from the bacterial debris by placing a neodinum magnet on the wall of the container containing the lysed bacterial suspension overnight and replacing the supernatant containing the culture medium and bacterial debris with 1X PBS. The resulting suspension was then sonicated at 10 W for 20 seconds in the presence of 1X PBS, and then subjected to a neodinum magnet for 15 minutes to remove the supernatant, and the treated magnetosomes were resuspended in 1X PBS. This sequence of sonication and magnetic separation was repeated four times. This process was repeated 10 times for 10 different volumes for the entire fermenter. Thus, pyrogenic magnetosome chains extracted from MSR-1 magnetotactic bacteria were obtained, i.e., approximately 500 mg of iron in the magnetosomes contained in 1.7 ml of water. Sample 1 contains magnetosomes obtained under lysis condition 1.

[0332] Dissolution conditions 2 (Sample 2): Concentrated magnetotactic bacteria were frozen at -80°C for 48 hours. 30 OD 565nmTo obtain the concentrate, it was thawed and diluted with Milli-Q water, and then a certain amount of KOH was added to obtain the concentrated bacteria to obtain a final KOH concentration of 1 M. This solution was transferred to a polypropylene (PP) bottle and placed in an 80°C water bath, and stirred at 150 rpm for 30 minutes with a mechanical stirring pad (Fisher Scientific). Next, the contents of the bottle were transferred to four other 2 L glass bottles. Each bottle was subjected to an NdFeB magnet for 12 hours to magnetically separate the extracted magnetosomes from the bacterial debris. The magnetosomes were washed six times in a 500 mL bottle by magnetic selection until a clear supernatant was obtained. The first two washes were performed with 10X PBS, which allowed the pH to be returned to neutral. The other four washes were then performed with water. After lysis, the base pH of the lysate, which had been induced by KOH, was returned to neutral pH so as not to damage the magnetosomes. Thus, pyrogenic magnetosome chains extracted from the MSR-1 strain were obtained, i.e., approximately 500 mg of iron in 1.7 ml of magnetosomes. Sample 2 contains magnetosomes obtained after lysis condition 2.

[0333] Sample containing magnetosomes extracted from magnetotactic bacteria and treated with phenol-chloroform (Condition 3):

[0334] Processing Condition 3 (Sample 3): 100 μl of a suspension containing 30 mg of iron from magnetosomes, obtained according to Dissolution Condition 1, was mixed with 200 ml of a solution containing 1% Tri to n X-100 and 1% SDS. The mixture was heated overnight at 50°C, then subjected to a neodinium magnet, the supernatant was removed and replaced with 80 mL of phenol at pH 8. The resulting suspension was heated at 60°C for 2 hours with sonication and held overnight. Without sonication, the suspension was subjected to a magnet at 60°C, the supernatant was removed and replaced with 80 mL of chloroform. The suspension containing chloroform was placed against a neodinium magnet, the supernatant was removed, and residual chloroform adsorbed on the surface of the treated magnetosomes was removed by heating these magnetosomes under a hood for 2 hours. Finally, the cores of the thus obtained magnetosomes were desorbed from the glass wall of the tube containing them by adding 80 ml of 1 M NaOH heated in an ultrasonic bath at 60°C for 1 hour. A suspension containing the magnetosome core was placed against a neodinum magnet. The supernatant was removed and replaced with sterile MilliQ water. The suspension was sonicated at 10W for 20 seconds, and this washing sequence was repeated four times. Purified pyrogen-free magnetosomes were obtained with a small amount of pyrogen-free water. Sample 3 contains the magnetosomes obtained after treatment condition 3.

[0335] Samples containing magnetosomes extracted from magnetotactic bacteria and heated at temperatures exceeding 200°C (Samples 4 to 11):

[0336] Heat treatment condition 4 (Sample 4): After dissolution under condition 2, 100 μl of a suspension containing approximately 30 mg of iron from magnetosomes extracted from MSR-1 magnetotactic bacteria was freeze-dried, placed in a porcelain crucible, and baked in a Naberther mL9 / 11 / B410 furnace. The heating protocol was as follows: The furnace temperature was increased from 20°C to 200°C at a rate of 6°C / min until the furnace temperature reached 200°C, and the temperature inside the furnace was maintained at 200°C for 1 hour. The furnace temperature was then reduced from 200°C to 25°C over 12 hours. Sample 4 contains magnetosomes obtained after treatment condition 4.

[0337] Heat treatment condition 5 (Sample 5): After lysis under condition 2, 100 μl of a suspension containing approximately 30 mg of iron from magnetosomes extracted from MSR-1 magnetotactic bacteria was freeze-dried, placed in a porcelain crucible, and baked in a Naberther mL9 / 11 / B410 furnace. The heating protocol was as follows: The furnace temperature was increased from 20°C to 400°C at a rate of 6°C / min until the furnace temperature reached 400°C. The furnace temperature was maintained at 400°C for 1 hour. Subsequently, the furnace temperature was reduced from 400°C to 25°C over 20 hours. Sample 5 contains the magnetosomes obtained after treatment condition 5.

[0338] Heat treatment condition 6 (Sample 6): 100 μl of a suspension containing 30 mg of iron from magnetosomes, prepared according to dissolution condition 2, was freeze-dried and placed in a porcelain crucible. It was then heated in a Nabertherm L9 / 11 / B410 furnace. The heat treatment was carried out as follows: The furnace temperature was increased from 20°C to 200°C in 20 minutes at a rate of 9°C / min. The furnace temperature was then maintained at 200°C for 30 minutes. Next, the furnace temperature was increased from 200°C to 300°C in 10 minutes at a rate of 10°C / min. The furnace temperature was then maintained at 300°C for 1 hour. The furnace temperature was then decreased from 300°C to 25°C in 12 hours. Sample 6 contains the magnetosomes obtained after treatment condition 6.

[0339] Heat treatment condition 7 (Sample 7): 100 μl of a suspension containing 30 mg of iron from magnetosomes, prepared according to dissolution condition 2, was freeze-dried and placed in a porcelain crucible, then heated in a Nabertherm L9 / 11 / B410 furnace. The heat treatment was carried out as follows: The furnace temperature was increased from 20°C to 200°C in 20 minutes at a rate of 9°C / min. The furnace temperature was then maintained at 200°C for 30 minutes. Next, the furnace temperature was increased from 200°C to 300°C in 10 minutes at a rate of 10°C / min. The furnace temperature was then maintained at 300°C for 1 hour. Next, the furnace temperature was increased from 300°C to 380°C in 10 minutes at a rate of 8°C / min. The furnace temperature was then maintained at 380°C for 1 hour. Next, the furnace temperature was increased from 380°C to 550°C in 20 minutes at a rate of 8.5°C / min, and then maintained at 550°C for 1 hour. Subsequently, the furnace temperature was reduced from 550°C to 25°C over 20 hours. Sample 7 contains magnetosomes obtained after the treatment conditions.

[0340] Heat treatment condition 8 (Sample 8): 100 μl of a suspension containing 30 mg of iron from magnetosomes, prepared according to dissolution condition 2, was freeze-dried and placed in a porcelain crucible, then calcined in a Nabertherm L9 / 11 / B410 furnace. The heat treatment was carried out as follows: The furnace temperature was increased from 20°C to 200°C in 20 minutes at a rate of 9°C / min, and then maintained at 200°C for 30 minutes. Next, the furnace temperature was increased from 200°C to 300°C in 10 minutes at a rate of 10°C / min, and then maintained at 300°C for 1 hour. The furnace temperature was increased from 300°C to 380°C in 10 minutes at a rate of 8°C / min. Then, the furnace temperature was maintained at 380°C for 1 hour, and then decreased from 380°C to 25°C in 12 hours. Sample 8 contains magnetosomes obtained after treatment condition 8.

[0341] Heat treatment condition 9 (Sample 9): 100 μl of a suspension containing 30 mg of iron in magnetosomes, prepared according to dissolution condition 1, was freeze-dried and placed in a porcelain crucible, then heated in a Nabertherm L9 / 11 / B410 furnace. The heat treatment was carried out as follows: The furnace temperature was increased from 20°C to 200°C in 20 minutes at a rate of 9°C / min, and then maintained at 200°C for 30 minutes. Next, the furnace temperature was increased from 200°C to 300°C in 10 minutes at a rate of 10°C / min, and then maintained at 300°C for 1 hour. The furnace temperature was increased from 300°C to 380°C in 10 minutes at a rate of 8°C / min, and then maintained at 380°C for 1 hour. Subsequently, the furnace temperature was decreased from 380°C to 25°C in 12 hours. Sample 9 contains magnetosomes obtained after treatment condition 9.

[0342] Heat treatment condition 10 (Sample 10): 1.7 mL of a suspension containing 500 mg of iron from magnetosomes, prepared according to dissolution condition 2, was freeze-dried and placed in a porcelain crucible, then heated in a Nabertherm L9 / 11 / B410 furnace. The thermal protocol was as follows: The furnace temperature was increased from 20°C to 200°C in 2 hours and 30 minutes at a rate of 1.2°C / min. Next, the furnace temperature was maintained at 200°C for 1 hour, then increased from 200°C to 300°C in 1 hour and 20 minutes at a rate of 1.25°C / min. Next, the furnace temperature was maintained at 300°C for 2 hours, then increased from 300°C to 380°C in 1 hour and 20 minutes at a rate of 1°C / min. Subsequently, the furnace temperature was maintained at 380°C for 2 hours. Then, the furnace temperature was decreased from 380°C to 25°C in 12 hours. Sample 10 contains magnetosomes obtained after treatment condition 10.

[0343] Heat treatment conditions 11 (Sample 11): 1.7 mL of a suspension containing 500 mg of iron in magnetosomes, prepared according to dissolution conditions 1, was freeze-dried and placed in a porcelain crucible, then heated in a Nabertherm L9 / 11 / B410 furnace. The heat treatment was carried out as follows: The furnace temperature was increased from 20°C to 200°C at a rate of 1.2°C / min over 2 hours and 30 minutes. Next, the furnace temperature was maintained at 200°C for 1 hour, then increased from 200°C to 300°C at a rate of 1.25°C / min over 1 hour and 20 minutes. Next, the furnace temperature was maintained at 300°C for 2 hours, then increased from 300°C to 380°C at a rate of 1°C / min over 1 hour and 20 minutes. Subsequently, the furnace temperature was maintained at 380°C for 2 hours. After that, the furnace temperature was decreased from 380°C to 25°C over 12 hours. Sample 11 contains magnetosomes obtained after treatment condition 11.

[0344] Chemically synthesized nanoparticles (SIGMA, reference: 637106, batch number: MKBK2270V): The chemically synthesized nanoparticle powder is purchased from SIGMA. The size is 35 ± 13 nm and, in addition to iron oxide, contains 198 ppm aluminum (Al), 600 ppm calcium (Ca), 74 ppm chromium (Cr), 72 ppm magnesium (Mg), 642.5 ppm manganese (Mn), 30 ppm nickel (Ni), 128 ppm sodium (Na), 34 ppm titanium (Ti), 8.3 ppm vanadium (V), and 56.5 ppm zinc (Zn).

[0345] result:

[0346] Figure 1(a) shows the percentage of weight loss of a sample (Sample 0) containing 3 mg of lyophilized whole MSR-1 magnetotactic bacteria as the sample temperature increased from 20°C to 600°C, at a rate of 6°C per minute, and the first derivative of this percentage. These measurements were performed on a TGA-DTA / DSC combined instrument that measures both heat flow using differential scanning calorimetry of the material and weight change using thermogravimetric analysis as a function of temperature. CHNS measurements of 3 mg of lyophilized whole MSR-1 magnetotactic bacteria (Sample 0) show that it contains a large percentage of carbon, 44%, before heating (Table 19). The weight percentage of this sample decreased from 100% at 20°C to 5.5% at 600°C, indicating that the sample loses most of its weight between 20 and 600°C. More specifically, Figure 1(a) shows that the gradient of the weight percentage variation as a function of temperature is maximum in two temperature ranges: between 200 and 400°C (interval 1) and between 400 and 540°C (interval 2). Between 200 and 400°C, the variation of the weight percentage gradient as a function of temperature shows a double peak with maximums at 260°C and 315°C. This double peak may be due to loss by fully magnetotactic bacteria of organic materials, with the peak centered at 260°C being organic materials of type 1, and the peak centered at 315°C being of non-type 1 types, such as type 2, of which preferential. Between 400 and 540°C, the variation of the weight percentage gradient as a function of temperature shows a peak. This peak may be due to loss by nanoparticles of non-type 2 types of organic materials, such as type 1 or type 3, of which preferential.

[0347] Figure 1(b) shows the freeze-dried sample when its temperature increased from 20°C to 600°C at a rate of 6°C per minute. 完全走磁性The heat flow in milliwatts as a function of temperature for a sample containing 3 mg of bacteria is shown. These measurements were performed using a TGA-DSC instrument. Figure 1(b) shows two peaks where maximum heat flow was observed at temperatures of 330°C and 500°C. The peak centered at 330°C may be due to the combustion of nanoparticle mass. The peak at 500°C may be due to the combustion of nanoparticle clumps lost between 500 and 540°C.

[0348] Figure 1(c) shows the percentage change in weight of a sample containing a 3 mg lyophilized magnetosome chain prepared according to lysis condition 2, and the first derivative of this percentage as a function of sample temperature, when the sample temperature was increased from 20°C to 600°C at a rate of 6°C per minute. These measurements were performed using a TGA-DSC instrument. CHNS measurements of the lyophilized magnetosome chain (condition 2) showed that it contained 7% carbon before heating, which is much lower than the carbon percentage of fully magnetotactic bacteria (Table 19). The weight percentage of the sample containing the magnetosome chain decreased from 100% at 20°C to 91.4% at 600°C, and the loss of the sample containing the magnetosome chain was 8.6% less than that of the sample containing fully magnetotactic bacteria from 20°C to 600°C. More specifically, the gradient of the variation in weight percentage of magnetosome chains as a function of temperature is shown to be maximum in the temperature range of 200–400°C in Figure 1(c). The variation of the gradient of weight percentage of magnetosome chains as a function of temperature as a function of temperature shows a double peak between 260°C and 315°C, with maximums at positions similar to those observed in complete bacteria. This double peak may be due to loss of organic material, preferably type 1 organic material, at the peak centered at 260°C, and preferably type 2 organic material, at the peak centered at 315°C, in the magnetosome chain. This organic material likely originates from the organic membrane layer surrounding the inorganic iron oxide core of the magnetosome.

[0349] Figure 1(d) shows the heat flow in milliwatts as a function of sample temperature for a sample containing a 3 mg lyophilized chain of magnetosomes, as the sample temperature increased from 20°C to 600°C at a rate of 6°C per minute. These measurements were performed using a TGA-DSC instrument. Figure 1(d) shows three peaks where maximum heat flow was observed at temperatures of 250°C, 360°C, and 525°C. The peaks centered at 250°C and 360°C may be due to the combustion of nanoparticle clumps lost between 200 and 400°C. The peak at 525°C may be due to the combustion of nanoparticle clumps lost above 500°C, and / or the oxidation of magnetosomes from iron oxide to hematite, or from magnetite, maghemite, or an intermediate composition between magnetite and maghemite, and the transformation from maghemite to hematite, which may generate heat flow through an exothermic reaction.

[0350] Figure 2(a) shows the change in weight percentage of a sample (Sample 3) containing 3 mg of lyophilized magnetosomes prepared according to Condition 3, and the first derivative of this percentage as a function of sample temperature, as the sample temperature was increased from 20°C to 600°C at a rate of 6°C per minute. These measurements were performed using a TGA-DSC instrument. CHNS measurement of the lyophilized magnetosomes prepared according to Condition 3 shows that they contained a carbon percentage of 4% before heating. This is lower than the carbon percentage of the magnetosomes prepared according to Condition 2. The weight of the magnetosomes (Sample 3) decreased from 100% at 20°C to 95.1% at 600°C, which is less than that of Sample 2, i.e., a 4.9% weight loss for Sample 3. More specifically, Figure 2(a) appears to show that the slope of the change in weight percentage as a function of temperature is maximum in the temperature range of 200–400°C. Between 200 and 400°C, the variation of the weight percentage gradient as a function of temperature shows quadrupole peaks with maximums at 264°C, 286°C, 325°C, and 33...

Claims

1. A method for producing magnetosomes using magnetotactic bacteria, a) A preliminary growth step comprising growing the magnetotactic bacteria in a preliminary growth medium, b) A growth step comprising growing the magnetotactic bacteria derived from the preliminary growth step in a growth medium containing a fed-batch medium, c) A purification step for obtaining the magnetosomes produced in the growth step, the following steps: (1) Removal of at least one impurity from the magnetosome, (2) Denaturation of at least one impurity contained in the magnetosome, (3) Destruction of at least one impurity contained in the magnetosome, The process includes a purification step consisting of at least one of the following steps: The amount of the aforementioned preliminary growth medium and / or growth medium and / or fed-batch medium per kilogram or liter of preliminary growth medium and / or growth medium and / or fed-batch medium is: i) Yeast extract 0.005g or less, ii) Boric acid and nitrilotriacetic acid It contains a CMR agent of 0.001 grams or less selected from the group consisting of the following: When the aforementioned fed-batch medium is present, it is a supplementary medium to the growth medium, and in the growth step, more magnetosomes are produced than in the preliminary growth step, and The growth step differs from the preliminary growth step in that at least one characteristic selected from the group consisting of the following, i) A ratio greater than 1 C ＦｅＧＳ / C ＦｅＰＧＳ CFeGS and C ＦｅＰＧＳ These are the concentrations of iron or iron source in the growth medium and pre-growth medium, respectively. ii) A ratio greater than 1 C ＣＧＳ / C ＣＰＧＳ , C ＣＧＳ and C ＣＰＧＳ These are the concentrations of carbon or carbon source in the growth medium and pre-growth medium, respectively. iii) Ratio C greater than 1 ＮＧＳ / CNPGS, C ＮＧＳ and CNPGS are each the concentration of nitrogen or nitrogen source in said growth medium and pre-growth medium, respectively iv) Ratio less than 1 Δ p H GS / Δ p H PGS, Δ ｐ H ＧＳ and Δ ｐ H ＰＧＳ These represent the pH changes of the growth medium and the preliminary growth medium, respectively. v) A ratio greater than 1 Q ＧＧＳ / Q ＧＰＧＳ Q ＧＧＳ and Q ＧＰＧＳ These are the amounts of gas, oxygen, or air introduced or foamed into the growth medium and pre-growth medium, respectively, and vi) Ratio N less than 1 ＳＳＧＳ / N ＳＳＰＧＳ , N ＳＳＧＳ and N ＳＳＰＧＳ These are the number of substeps in the growth step and the number of substeps in the preliminary growth step, respectively, and the two substeps are separated by the movement of the magnetotactic bacteria from the first substep to the second substep. Here, the growth medium is replenished with fed-batch medium, but the preliminary growth medium is not replenished with such medium. The purification step includes removing at least one impurity from the magnetosomes generated in the growth step using at least one heating step, wherein in the heating step, the magnetosomes generated in the growth step are raised to a temperature T, and then maintained at T for a heating time ranging from 1 second to 20 years, where T is ranging from 50°C to 700°C, and prior to the purification step, (1) Isolating the magnetosomes from the magnetotactic bacteria, (2) Extracting the magnetosomes from the magnetotactic bacteria, (3) Recovering the magnetosomes. The preceding step is selected from the preceding steps, The fed-batch medium comprises a source of carbon, nitrogen, calcium, vitamins, oxygen, iron, phosphate, phosphorus, and / or magnesium. The fed-batch medium comprises lactic acid, ammonia, potassium phosphorylated, magnesium sulfate, ferric citrate, ferric quinate, ferric chloride, and / or ferric sulfate. The aforementioned magnetotactic bacterium is Magnetospirillum glutiswaldens strain MSR-1. method.

2. The method according to claim 1, wherein the purification step is initiated from magnetosomes isolated from the magnetotactic bacteria obtained at the end of the growth step.

3. The preceding step is, (1) Mix the magnetotactic bacteria with detergent, (2) Heat the magnetotactic bacteria to a temperature higher than 20°C, (3) 10 －３ Inducing a temperature gradient greater than °C / hour, minute, or second, (4) Apply a pressure greater than 1 atmosphere to the magnetotactic bacteria, and (5) Ultrasonically treat the magnetotactic bacteria with an output greater than 1W. The method according to claim 1, including the method described in claim 1.

4. The method according to claim 3, wherein the detergent in (1) is KOH or NaOH.

5. The method according to claim 3, wherein pressure is applied to the magnetotactic bacteria using a French press.

6. The removal of at least one impurity from the magnetosome in the purification step i) 10 －２ Removal of a mass percentage of impurities greater than % or ii) 10% per gram of magnetosomes －１ The method according to claim 1, comprising the removal of impurities larger than μg.

7. The method according to claim 1, wherein the at least one impurity removed by the purification step is carbon or a carbonaceous material.

8. The method according to claim 1, wherein the at least one impurity removed by the purification step is present in the coating of the magnetosome.

9. The purification step includes at least one heating step, wherein the heating step is at least one step selected from a first stage, a second stage, and a third stage: (1) During the first step, the temperature of the magnetosome is, time t ｉ１Ｐ Within the period, temperature T ｉ From temperature T ｉａｖ The stage where it is raised to this point, (2) During the second step, the temperature of the magnetosome is, time t ｉ２Ｐ Within the period, temperature T ｉａｖ The stage that is maintained (3) During the third step, the temperature of the magnetosome is, time t ｉ３Ｐ Within the period, T ｉａｖ From T ｆ The stage in which it decreases, The method according to claim 1, including the method described in claim 1.

10. The aforementioned growth step involves a gas selected from compressed air, air, oxygen, or more than 1% O ２ At least two of the following five substeps for introducing a gas containing into the growth medium: (1) 10 －３ ~10 ３ During the first substep, which lasts for a certain amount of time, the gas deficiency is 0 to 10 per liter of growth medium. １０ It is contained between mL / min and at the start of the first substep, 10 －１０ from 10 ３ From a value within that range to a value at the end of the first substep that is 1.0000001 times greater than the value at the start of the first substep, a substep that brings about an increase in the optical density of the magnetotactic bacteria, (2) 10 －３ ~10 ３ During the second substep, which lasts for a certain amount of time, the gas shortage increases by more than 1.0000001 times compared to the first substep, and is equal to or equal to the value obtained at the end of the first substep. －９ from 10 ４ From the initial value of the second substep included in between, to the final value of the second substep which is 1.0000001 times greater than the initial value of the second substep, a substep that brings about an increase in the optical density of the magnetotactic bacteria, (3) 10 －３ ~10 ３ During the third substep, which lasts for a certain amount of time, the gas shortage increases by more than 1.0000001 times compared to the second substep, and is equal to or equal to the value obtained at the end of the second substep. －９ from 10 ４ From the initial value of the third substep included in between, to the final value of the third substep which is 1.0000001 times greater than the initial value of the third substep, a substep that brings about an increase in the optical density of the magnetotactic bacteria, (4) 10 －３ ~10 ３ During the fourth substep, which lasts for a certain amount of time, the gas shortage increases by more than 1.0000001 times compared to the third substep, and is equal to or equal to the value obtained at the end of the third substep. －９ from 10 ４ From the initial value of the fourth substep included in the interval to the final value of the fourth substep which is 1.0000001 times greater than the initial value of the fourth substep, the substep that causes the increase in the optical density of the magnetotactic bacteria, (5) 10 －３ ~10 ３ During the fifth substep, which lasts for a certain amount of time, the gas shortage increases by more than 1.0000001 times compared to the fourth substep, and is equal to the value obtained at the end of the fourth substep, or 10 －９ from 10 ４ From the initial value of the fifth substep included in between, to the final value of the fourth substep which is 1.0000001 times greater than the initial value of the fifth substep, a substep that brings about an increase in the optical density of the magnetotactic bacteria, The method according to claim 1, including the method described in claim 1.

11. The method according to claim 10, wherein at least one of the following reaction conditions is set or observed: (1) Reaction conditions under which the percentage of oxygen is maintained at a level higher than 0.01% due to the aforementioned deficiency of the gas and lower than 0.9% due to the consumption of oxygen by the magnetotactic bacteria. (2) The growth medium is divided into 1 to 10 ５ Reaction conditions involving stirring at a speed of rotations per minute, (3) Reaction conditions under which the gas deficiency is reduced by increasing the stirring speed of the culture medium, (4) Reaction conditions under which the gas deficiency increases by reducing the stirring speed of the culture medium, (5) The optical density of the magnetotactic bacteria increases during any one of the substeps. (6) The pH of the growth medium is maintained under reaction conditions that fix, determine, or select a pH within the range of 0 to 14. (7) Reaction conditions under which the iron concentration in the growth medium increases during any one of the substeps.

12. The method according to claim 11, wherein the pH of the growth medium is maintained by adding an iron acid source contained in the fed-batch medium.

13. The aforementioned preliminary growth medium and / or growth medium is, per kilogram or liter, i) 5 × 10 －３ Mass% or volume% or 0.5 grams or 0.5 mL or 10 －８ mol or 10 －９ Vitamins in amounts less than mol or chemical components selected from the following group: folic acid, folate, pyridoxine, pyridoxine HCl, pyridoxamine, pyridoxal, riboflavin, biotin, thiamine, thiamine HCl, nicotinic acid, pantothenic acid, calcium pantothenate, inositol, p-aminobenzoic acid, aminobenzoic acid, thioacid, total trans-retinol, retinal, alternative provitamin A functional carotenoids including total trans-beta-carotene, niacin, niacinamide, Nicotinamide, riboside, cyanocobalamin, hydroxocobalamin, methylcobalcobamin, adenosylcobalamin, ascorbic acid, cholecalciferol, ergocalciferol, tocopherol, tocotrienol, phylloquinone, menaquinone, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin D2, vitamin D3, vitamin E, vitamin K, ii) 10 －２ Mass% or volume%, or 1 gram or 1 mL or 10 －７ mol or 10 －８ Minerals or chemical components selected from the following group in amounts less than mol: nitrilotriacetic acid, magnesium sulfate, sodium chloride, manganese sulfate, ferrous sulfate, ferrous sulfate heptahydrate, cobalt nitrate, calcium chloride, zinc sulfate, zinc sulfate heptahydrate, copper sulfate, copper sulfate hydrate, aluminum potassium sulfate, aluminum potassium sulfate dodecahydrate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, sodium tungstate dihydrate, nickel chloride, EDTA, MgSO4 ４ , MnSO ４ , NaCl, FeSO ４ CoSO ４ CaCl ２ ZnSO ４ , CdSO ４ , KAl(SO ４ ) ２ , H ３ BO ３ Na ２ MoO ４ NiCl ２ and Na ２ SeO ３ , iii) 0.005 grams or 10 －８ Yeast extract of at least one component less than M or at least one compound derived from yeast extract selected from the group consisting of: at least one protein, at least one nucleic acid, at least one functional peptide, glutathione, dextran, mannan, trehalose, flavor nucleotide, B vitamin, biotin, at least one volatile aromatic compound, calcium, phosphorus, zinc, iron, chromium, potassium, cobalt, manganese, strontium, and magnesium. iv) 0.01 grams or 10 －８ At least one component of peptone with a molecular weight less than M, or at least one compound derived from peptone selected from the group consisting of ash, protein, sucrose, stachyose, raffinose, neutral detergent fibers, and etheric extracts. v) EDTA less than 0.001 grams, vi) at least one amino acid in less than 0.001 grams, vii) At least one chemical element or heavy metal 10 selected from the group consisting of cadmium, lead, arsenic, mercury, cobalt, vanadium, nickel, lithium, antimony, and copper －５ Less than a gram, viiii) At least one CMR, toxic or cytotoxic compound 10 selected from the group consisting of nitrilotriacetic acid, manganese sulfate, cobalt nitrate, zinc sulfate, copper sulfate, potassium aluminum sulfate, boric acid, sodium molybdate, sodium selenite, sodium tungstate, and nickel chloride. －５ Less than a gram, and / or ix) Less than 0.01 grams of peptone The method according to claim 1, comprising a preliminary growth and / or growth and / or fed-batch medium.

14. At least one compound of the aforementioned pre-growth and / or growth medium is at concentration C ２ or concentration C ｔｏｔａｌ = C １ +C ２ It has, C １ This is a concentration in which at least one of the compounds in the preliminary growth medium and / or growth medium is not consumed by the magnetotactic bacteria. C ２ This is the concentration of the at least one compound in the pre-growth medium and / or growth medium consumed by the magnetotactic bacteria, C １ and C ２ The method according to claim 1, wherein is measured or considered at the start, during, or at the end of the preliminary growth and / or growth step.

15. Said preliminary growth medium and / or growth medium does not contain at least one compound at a concentration that affects said growth or magnetosome production of said magnetotactic bacteria, and / or said preliminary growth medium and / or growth medium does not contain said at least one compound, wherein said at least one compound is 1) Wolf vitamins, or a medium containing half or more of the total number of different components of Wolf vitamins, 2) one component of Wolf vitamins, 3) folic acid, 4) pyridoxine, 5) riboflavin, 6) biotin, 7) thiamine, 8) nicotinic acid, 9) pantothenic acid, 10) vitamin B12, 11) p-aminobenzoic acid, 12) thioic acid, 13) Wolf minerals, or a medium containing more than half of the total number of different components of Wolf minerals, 14) nitrilotriacetic acid, 15) magnesium sulfate, 16) sodium chloride, 17) manganese sulfate, 18) ferrous sulfate heptahydrate, 19) cobalt nitrate, 20) calcium chloride, 21) zinc sulfate heptahydrate, 22) copper sulfate hydrate, 23) potassium aluminum sulfate dodecahydrate, 24) boric acid, 25) sodium molybdate, 26) sodium selenite, 27) sodium tungstate dihydrate, 28) yeast extract, or a medium containing more than half of the total number of various components of yeast extract, 29) an equivalent of yeast extract or a medium containing more than half of the total number of various components of an equivalent of yeast extract, 30) one, two or five proteins derived from or contained in yeast extract, 31) one, two or five nucleic acids derived from or contained in yeast extract, 32) one, two or five peptides or functional peptides derived from or contained in yeast extract, 33) glutathione, 34) dextran, 35) mannan, 36) trehalose, 37) flavor nucleotides derived from or contained in yeast extract, 38) B vitamins, 39) biotin, 40) one, two or five volatile aromatic compounds derived from or contained in yeast extract, 41) chromium, 42) cobalt, 43) strontium, 44) nickel chloride, 45) a medium containing more than half of the total number of different components of mineral elixir, 46) MnSO ４ , 47) NaCl, 48) FeSO ４ , 49) CoSO ４ 、. ２ 、; ４ 、. ４ 、. ４ ) ２ 、!4)H ３ . ３ 、!!!! ２ *o﯁ ４ 、. ２ 、!?)!! ２ 3) ３ 58) Culture medium containing more than half the total number of different components of peptone, 59) One component of peptone, 60) One, two or five proteins derived from or contained in peptone, 61) Sugars derived from or contained in peptone, 62) One amino acid derived from or contained in peptone, 63) Ash derived from or contained in peptone, 64) One fiber derived from or contained in peptone, 65) One CMR agent, 66) Boric acid, 67) One amino acid, 68) Alanine, 69) Arginine, 70) Asparagine, 71) Aspartic acid, 72) Cyste 73) Glutamine, 74) Glutamic acid, 75) Glycine, 76) Histidine, 77) Isoleucine, 78) Leucine, 79) Lysine, 80) Methionine, 81) Phenylalanine, 82) Proline, 83) Serine, 84) Threonine, 85) Tryptophan, 86) Tyrosine, 87) Valine, 88) One cytotoxic or toxic compound, 89) Manganese sulfate, 90) Copper sulfate, 91) Aluminum potassium sulfate, 92) Boric acid, 93) Sodium tungstate, 94) One heavy metal other than iron, 95) Titanium, 96) Vanadium, 97) Manganese, 98) Nickel, 99) Copper, 100) Zinc, 101) Gallium, 102) Germanium, 103) Arsenic, 104) Zirconium, 105) Niobium, 106) Molybdenum, 107) Technetium, 108) Ruthenium, 109) Rhodium, 110) Palladium, 111) Silver, 112) Cadmium, 113) Indium, 114) Tin, 115) Tellurium, 116) Lutetium, 117) Hafnium, 118) Tantalum, 119) Tungsten, 120) Rhenium, 121) Osmium, 122) Iridium, 123) Platinum, 125) Gold, 126) Mercury, 127) Thallium, 128) Lead, 129) Bismuth, 130) Polonium, 131) Astatine, 132) Lanthanum, 133) Cerium, 134) Praseodyum, 135) Neodymium, 136) Promethium, 137) Samarium, 138) Europium, 139) Gadolinium, 140) Terbium, 141) Dysprosium, 142) Holmium, 143) Erbium, 144) Thulium, 145) Ytterbium, 146) Actinium, 147) Thorium, 148) Protactinium, 149) Uranium, 150) Neptunium, 151) Plutonium,152) Americium, 153) Curium, 154) Berkelium, 155) Carphonium, 156) Einsteinium, 157) Fermium, 158) Novelium, 159) Radium, 160) Lawrencium, 161) Lazafordium, 162) Dubnium, 163) Ceaborgium, 164) Volium, 165) Hasium, 166) Meitnerium, 167) Darmstadium, 168) Roentgen, 169) Copernicium, 170) Elements 113-118, 171) Helium, 172) Lithium, 173) Beryllium, 174) Bore, 175) Fluorine, 176) Aluminum, 177) Silicon, 178) Argon, 179) Scandium, 180) Chromium, 181) The method according to claim 1, comprising a compound selected from the group consisting of nickel, 182) copper, 183) selenium, 184) brom, 185) krypton, 186) rubidium, 187) yttrium, 188) Sn, 189) antimony, 190) iodine, 191) xenon, 192) cesium, 193) barium, 194) lutesium, 195) asterisk, 196) radon, 197) francium, 198) mendelevium, 199) morgan, 200) ununbium, 201) ununthrium, 202) ununquadium, 203) ununpentium, 204) ununhexium, 205) ununseptium, 206) unnunoctium, and 207) salts of these compounds 1) to 206).

16. The concentration of the at least one compound that affects the growth or magnetosome production of the magnetotactic bacteria is 1 pM, or 1 μM, or 1 mM, or 10 －３ The method according to claim 15, wherein the concentration in the pre-growth, growth and / or fed-batch medium is greater than ng of compound / liter, or 1 ng of compound / liter, or 103 ng of compound / liter.

17. i) The pH of the fed-batch medium is lower than the pH of the growth medium, and / or ii) a) Source of phosphorus or phosphate, b) Source of potassium, c) Source of magnesium, d) Source of iron, e) Source of vitamins, f) Source of calcium, g) KH ２ PO ４ , h) MgSO ４ i) FeCl ３ , j) thiamine, and k) CaCl ２ The method according to claim 1, wherein the concentration of at least one chemical element selected from the group consisting of is higher in the fed-batch medium than in the growth medium.

18. The aforementioned pre-growth and / or growth medium comprises a calcium source, a carbon source, a nitrogen source, a phosphate or phosphate source, a sulfur source, an iron source, a vitamin source, and a calcium source. The carbon source has a concentration in the pre-growth medium and / or growth medium that is higher than the concentration of at least one compound in the pre-growth medium and / or growth medium selected from the group consisting of phosphoric acid or phosphorus source, sulfur source, vitamin source, and calcium source, and / or The method according to claim 1, wherein the nitrogen source has a concentration in the pre-growth medium and / or growth medium that is higher than the concentration of at least one compound in the pre-growth medium and / or growth medium selected from the group consisting of the phosphate or phosphorus source, the sulfur source, the vitamin source, and the calcium source.

19. The method according to claim 1, further comprising the step of storing, growing, preparing or inserting the bank of magnetotactic bacteria into the pre-growth medium and / or growth medium and / or fed-batch medium, wherein the bank is stored, grown or prepared in a bank medium containing at least 1% of the same compound as the pre-growth medium and / or growth medium and / or fed-batch medium.

20. The method according to claim 1, wherein the yeast extract comprises at least one compound selected from the group consisting of peptides, amino acids, purine bases, pyrimidine bases, and water-soluble vitamins.

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