阅读说明：本技术 一种利用氧化还原电位调控甲基营养菌生产吡咯喹啉醌的方法 (Method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated by oxidation-reduction potential ) 是由 柯崇榕 杨欣伟 黄建忠 丁灵涛 任洋 刘孟粟 陶勇 于 2021-08-23 设计创作，主要内容包括：本发明公开一种利用氧化还原电位调控甲基营养菌生产吡咯喹啉醌的方法,包括菌种活化、种子培养和发酵生产；在甲基营养菌发酵生产吡咯喹啉醌的体系中引入氧化还原电位电极,通过控制体系中通气量和搅拌转速来调节发酵生产阶段的氧化还原电位在高电位氧化阶段和低电位还原阶段变化,进而保证菌体生长和吡咯喹啉醌的积累,本发明的方法能够缩短发酵时间,同时提高吡咯喹啉醌的产量和产率及发酵体系的稳定性。(The invention discloses a method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated by oxidation-reduction potential, which comprises the steps of strain activation, seed culture and fermentation production; an oxidation-reduction potential electrode is introduced into a system for producing pyrroloquinoline quinone by methylotrophic bacteria fermentation, and the oxidation-reduction potential in the fermentation production stage is adjusted to change in a high potential oxidation stage and a low potential reduction stage by controlling the ventilation quantity and the stirring speed in the system, so that the growth of bacteria and the accumulation of pyrroloquinoline quinone are ensured.)

1. A method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated by oxidation-reduction potential is characterized by comprising the following steps: comprises strain activation, seed culture and fermentation production; an oxidation-reduction potential electrode is introduced into a system for producing pyrroloquinoline quinone by methylotrophic bacteria fermentation, and the oxidation-reduction potential in the fermentation production stage is adjusted to change in a high potential oxidation stage and a low potential reduction stage by controlling the ventilation quantity and the stirring speed in the system, so that the growth of thalli and the accumulation of pyrroloquinoline quinone are ensured.

2. The method for producing pyrroloquinoline quinone by using methylotrophic bacteria under the action of oxidation-reduction potential according to claim 1, wherein: after an oxidation-reduction potential electrode is introduced into the fermentation production, three stages are set to control the fermentation process, the oxidation-reduction potential is maintained within the range of 190-250 mV, wherein the first stage is a high-potential strong oxidation stage within 35-60 hours from the beginning of fermentation; in 35-60 h to 70-100 h after the second stage fermentation is started, the second stage is a low-potential strong reduction stage; the third stage is a neutralization stage after 70-100 h to 150-200 h after the fermentation is started.

3. The method for producing pyrroloquinoline quinone by using methylotrophic bacteria under the action of oxidation-reduction potential according to claim 2, wherein: the methylotrophic bacteria are denitrogenated hyphomycetes or methylobacterium toruloides or mutant bacteria of the denitrogenated hyphomycetes or the methylotrophic bacilli after genetic engineering transformation.

4. The method for producing pyrroloquinoline quinone by using methylotrophic bacteria under the action of oxidation-reduction potential according to claim 3, wherein: after the redox potential electrode is introduced, when the redox potential is lower than a set value in the potential regulation process, firstly increasing the ventilation quantity and then increasing the stirring rotating speed; when the oxidation-reduction potential is higher than the set value, the stirring speed is firstly reduced and then the ventilation volume is reduced.

5. The method for producing pyrroloquinoline quinone by using methylotrophic bacteria according to any one of claims 1 to 4, comprising the steps of:

s1, strain activation: adopting a solid culture medium with the pH of 6.0-7.5, containing methanol and capable of providing a carbon source, a nitrogen source and inorganic salts, performing plate streaking on the strain in the solid culture medium during activation culture, and culturing for 3-4 days in a constant-temperature incubator at the temperature of 25-30 ℃;

s2, seed culture: adopting a liquid culture medium with the pH of 6.0-7.5, containing methanol and capable of providing a carbon source, a nitrogen source and inorganic salts, selecting 1-2 rings of activated bacteria after strain activation during culture, inoculating the activated bacteria into a conical flask filled with the liquid culture medium, wherein the culture temperature is 25-30 ℃, the rotating speed of a shaking table is 150-220 rpm, the culture time is 18-36 h, and the OD is OD₆₅₀1.2 to 1.6;

s3, fermentation production: inoculating the cultured seeds into a liquid culture medium in a fermentation tank by adopting the liquid culture medium, wherein the pH of the liquid culture medium is 6.0-7.5, the liquid culture medium contains methanol and can provide a carbon source, a nitrogen source and inorganic salts, the inoculation amount is 5-10% v/v, the culture temperature is 25-30 ℃, the dissolved oxygen is not less than 10%, and the fermentation culture time is 150-200 h; and (3) measuring and controlling the aeration ratio and the stirring speed in the fermentation process to adjust the oxidation-reduction potential in the fermentation tank, simultaneously, feeding 25% ammonia water in the fermentation production process to maintain the pH, and feeding methanol to maintain the concentration of methanol in the fermentation system to be not less than 1 g/L.

6. The method for producing pyrroloquinoline quinone by using methylotrophic bacteria according to claim 5, wherein: the solid culture medium for activating the strains comprises the following components: methanol, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate and agar powder.

7. The method for producing pyrroloquinoline quinone by using methylotrophic bacteria according to claim 5, wherein: the liquid culture medium for seed culture comprises the following components: methanol, ammonium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and vitamin auxiliary liquid.

8. The method for producing pyrroloquinoline quinone by using methylotrophic bacteria according to claim 5, wherein: the liquid culture medium for fermentation production comprises the following components: methanol, ammonium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, trace element liquid and vitamin auxiliary liquid.

9. The method for producing pyrroloquinoline quinone according to any one of claims 8 or 9, which comprises the steps of: the vitamin auxiliary liquid comprises the following components: riboflavin, pyridoxine hydrochloride, thiamine hydrochloride, inositol, folic acid, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, and biotin.

10. The method of claim 8, wherein the method comprises using an oxidation-reduction potential-regulated methylotrophic bacterium to produce pyrroloquinoline quinone: the trace element liquid comprises the following components: ferrous sulfate, zinc sulfate, manganese sulfate, copper sulfate, sodium chloride, sodium molybdate, potassium chloride, cobalt chloride, boric acid and calcium chloride.

Technical Field

The invention particularly relates to a method for producing pyrroloquinoline quinone by regulating and controlling methylotrophic bacteria through oxidation-reduction potential (ORP), belonging to the technical field of fermentation engineering.

Background

Pyrroloquinoline quinone (PQQ) is a secondary NAD⁺/NADP⁺And the coenzyme of the 3 rd oxidoreductase found after FMN/FAD, naturally occurring in nature, are the only biological factors found so far in all animal and plant tissues. PQQ is used as a unique physiological active substance, has great potential in the aspects of anti-aging, anti-fatigue, immunity improvement and treatment or adjuvant therapy of inflammation, liver disease, osteoporosis and other diseases, and has good medical and health care development prospects. PQQ is not synthesized by mammals per se, can only be taken from food, and has high content in celery, spinach, green pepper, kiwi fruit and pawpaw, and particularly has the content of 180 mu g/L in human breast milk as high as 140 mu g/L. The published paper by the japanese scientist Kasahara in Nature in 2003 suggests the classification of PQQ as a vitamin, and the united states cancer and anti-aging specialist Bruce n. Currently, PQQ is classified as a retinoid in the united states, japan, and the european union for sale as a dietary supplement.

The organisms capable of synthesizing PQQ have been mainly gram-negative bacteria, and among them, filamentous microsclera have a high level of PQQ biosynthesis, and have been the main mode of PQQ production instead of chemical synthesis. By 12 months in 2020, 6 enterprises perform NDI certification or GRAS registration to the US FDA, wherein 5 enterprises adopt a microbial fermentation method using Hyphomicrobium as a production strain to perform production and synthesis of PQQ. The specific biosynthetic pathway of PQQ is now basically well-defined, and this process requires balancing the oxygen content in the cytoplasm, satisfying the demand of PqqE and PqqC for anaerobic microenvironment and oxygen, completing the condensation and hydroxylation of PqqA and the cyclization and oxidation of AHQQ. In the industrial production of PQQ, fed-batch fermentation (CN106282044A, GRN 641, GRN 694, GRN 701 and GRN 709) or semi-continuous fermentation (CN109628509A) is adopted to control the dissolved oxygen at 5% or more. However, the above fermentation process has three outstanding problems: 1. the low dissolved oxygen in the early stage influences the growth of thalli, so that the fermentation period is long; 2. the intermediate with insufficient oxygen supply in the middle period is accumulated in a large amount, so that the conversion rate of the carbon source is low; 3. the late stage autolysis of the bacteria chelates PQQ, resulting in low cell yield per unit. Chinese patent with application number CN201510262476.7 discloses a preparation method of a filamentous microbe and pyrroloquinoline quinone, wherein the preparation method specifically discloses a filamentous microbe, the filamentous microbe is cultured in an 80T fermentation tank by adopting an oxygen control process (DO is maintained at 5%), pyrroloquinoline quinone is obtained from fermentation liquor, and the PQQ yield is 1.78g/L after 240 hours. Therefore, according to the strong redox characteristics of PQQ and the unique biosynthesis pathway thereof, the establishment of an efficient fermentation production process is the key for improving the industrial production of PQQ by using the raw silk micro-bacteria.

Disclosure of Invention

The invention aims to provide a method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated and controlled by oxidation-reduction potential (ORP), which regulates the fermentation process of the pyrroloquinoline quinone by a three-stage ORP control strategy, so that the yield and the yield of the pyrroloquinoline quinone and the stability of a fermentation system are improved, and the fermentation time is shortened.

The technical scheme of the invention is as follows:

the invention aims to provide a method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated by oxidation-reduction potential, which comprises the steps of strain activation, seed culture and fermentation production; an oxidation-reduction potential electrode is introduced into a system for producing pyrroloquinoline quinone by methylotrophic bacteria fermentation, and the oxidation-reduction potential in the fermentation production stage is adjusted to change in a high potential oxidation stage and a low potential reduction stage by controlling the ventilation quantity and the stirring speed in the system, so that the growth of bacteria and the accumulation of pyrroloquinoline quinone are ensured.

Furthermore, after an oxidation-reduction potential electrode is introduced into the fermentation production, three stages are set to control the fermentation process, wherein the oxidation-reduction potential is maintained within the range of 190-250 mV, and the first stage is a high-potential strong oxidation stage within 35-60 hours from the beginning of fermentation; in 35-60 h to 70-100 h after the second stage fermentation is started, the second stage is a low-potential strong reduction stage; the third stage is a neutralization stage after 70-100 h to 150-200 h after the fermentation is started.

Further, the methylotrophic bacteria are Microbacterium denitrificans or Methylobacterium toruloides or genetically engineered mutant bacteria thereof, specifically Microbacterium denitrificans FJNU-6 or Microbacterium denitrificans DSM 1869 or Methylobacterium toruloides ATCC 8457; wherein the Denitrification hyphomycete FJNU-6 (Denitrification hyphomycete)Hyphomicrobium denitrificans) Has been preserved in China general microbiological culture Collection center (CGMCC for short, address: western road No.1, north chen institute of microbiology, postal code 100101, north academy of sciences, tokyo, beijing), the collection number is CGMCC No. 1.12893; microbacterium denitrificans DSM 1869 and Methylobacterium torvum ATCC 8457 are model strains and can be purchased from DSM or ATCC official websites.

Furthermore, after the redox potential electrode is introduced, when the redox potential is lower than a set value in the potential regulation process, firstly increasing the ventilation quantity and then increasing the stirring rotating speed; when the oxidation-reduction potential is higher than the set value, the stirring speed is firstly reduced and then the ventilation volume is reduced.

Further, the method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated by oxidation-reduction potential specifically comprises the following steps:

s1, strain activation: adopting a solid culture medium with the pH of 6.0-7.5, containing methanol and capable of providing a carbon source, a nitrogen source and inorganic salts, performing plate streaking on the strain in the solid culture medium during activation culture, and culturing in a constant-temperature incubator at 25-30 ℃ for 3-4 days;

s2, seed culture: adopting a liquid culture medium with pH of 6.0-7.5, containing methanol and capable of providing a carbon source, a nitrogen source and inorganic salts, and culturingThen 1-2 rings of activated bacteria after strain activation are selected and inoculated into a conical flask filled with a liquid culture medium, the culture temperature is 25-30 ℃, the rotating speed of a shaking table is 150-220 rpm, the culture time is 18-36 h, and the OD is OD₆₅₀1.2 to 1.6;

s3, fermentation production: inoculating the cultured seeds into a liquid culture medium in a fermentation tank by adopting the liquid culture medium, wherein the pH of the liquid culture medium is 6.0-7.5, the liquid culture medium contains methanol and can provide a carbon source, a nitrogen source and inorganic salts, the inoculation amount is 5-10% v/v, the culture temperature is 25-30 ℃, the pH is 6.0-7.5, the dissolved oxygen is not less than 10%, and the fermentation culture time is 150-200 h; in the fermentation production process, 25% ammonia water is fed back to maintain the pH, and methanol is fed back to maintain the concentration of methanol in the fermentation system to be not less than 1 g/L.

Further, the strain activated solid culture medium comprises the following components: methanol, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate and agar powder.

Further, the liquid culture medium for seed culture comprises the following components: methanol, ammonium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and vitamin auxiliary liquid.

Further, the liquid culture medium produced by fermentation comprises the following components: methanol, ammonium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, trace element liquid and vitamin auxiliary liquid.

Further, the vitamin auxiliary liquid comprises the following components: riboflavin, pyridoxine hydrochloride, thiamine hydrochloride, inositol, folic acid, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, and biotin.

Further, the trace element liquid comprises the following components: ferrous sulfate, zinc sulfate, manganese sulfate, copper sulfate, sodium chloride, sodium molybdate, potassium chloride, cobalt chloride, boric acid and calcium chloride.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, an oxidation-reduction potential electrode is introduced in the process of producing pyrroloquinoline quinone by methylotrophic bacteria fermentation, and a three-stage oxidation-reduction potential control strategy is set, wherein the first stage is a high-potential strong oxidation stage, which is beneficial to thallus growth and has high dissolved oxygen; the second stage is a low-potential strong reduction stage, can induce the synthesis of pyrroloquinoline quinone, and has low dissolved oxygen; the third stage is a neutralization stage, which is beneficial to both production and growth; on the other hand, the three-stage fermentation process overcomes the problem that the thalli are easy to autolyze when the dissolved oxygen is more than 5 percent in the prior art, so that the fermentation is unstable; therefore, the fermentation process of the pyrroloquinoline quinone is adjusted through three-stage fermentation control, the fermentation time is shortened, the stability of a fermentation system is improved, the rapid high-density growth of bacteria can be promoted, the efficient accumulation of the product pyrroloquinoline quinone can be promoted, and the yield of the pyrroloquinoline quinone can be further improved.

Reference numerals

FIG. 1 is a graph showing the effect of different oxidation-reduction potentials on PQQ production by Microbacterium denitrificans DSM 1869 in example 1 according to the present invention;

FIG. 2 is a graph showing the three-stage fermentation process for controlling PQQ production by the filamentous microbe denitrificans FJNU-6 according to example 4 of the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and preferred embodiments, which are given for illustrative purposes only and are not intended to limit the scope of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified; example 1

A method for producing pyrroloquinoline quinone by using methylotrophic bacteria to regulate and control oxidation-reduction potential comprises introducing an oxidation-reduction potential electrode into a system for producing pyrroloquinoline quinone by fermenting methylotrophic bacteria, regulating the oxidation-reduction potential by controlling the gas ratio and the stirring speed in the system, realizing three-stage control fermentation of pyrroloquinoline quinone in fermentation production, and further ensuring the growth of thalli and the accumulation of pyrroloquinoline quinone; in this example, the methylotrophic bacterium is Micromyceliophthora denitrificans (Hyphomicrobium densificans) DSM 1869, which specifically comprises the following steps:

s1, strain activation: adopting a solid culture medium with the pH value of 6.5, taking 1-ring denitrogenation hyphomycete DSM 1869 during activation culture, carrying out plate streaking in the solid culture medium, and then culturing for 4 days in a constant temperature incubator at 30 ℃; wherein the solid culture medium for strain activation comprises the following components: 10g/L of methanol, 4g/L of ammonium sulfate, 3g/L of potassium dihydrogen phosphate, 2g/L of magnesium sulfate and 20g/L of agar powder;

s2, seed culture: adopting a liquid culture medium with the pH value of 6.5, selecting 1 ring of activated bacteria after strain activation during culture, inoculating the activated bacteria into a conical flask filled with the liquid culture medium, wherein the culture temperature is 30 ℃, the rotation speed of a shaking table is 220rpm, the culture time is 18-24 h, and the OD is OD₆₅₀Is 1.5; the liquid culture medium for seed culture comprises the following components: 5g/L of methanol, 2g/L of ammonium sulfate, 3g/L of monopotassium phosphate, 4g/L of disodium hydrogen phosphate, 1g/L of magnesium sulfate and 1mL/L of vitamin auxiliary liquid; wherein the vitamin auxiliary liquid contains 2g/L of riboflavin, 0.1g/L of pyridoxine hydrochloride, 0.2g/L of thiamine hydrochloride, 0.2g/L of inositol, 2mg/L of folic acid, 0.8g/L of nicotinic acid, 0.1g/L of p-aminobenzoic acid, 0.3g/L of calcium pantothenate and 6mg/L of biotin;

s3, fermentation production: inoculating the cultured seeds into a liquid culture medium in a 5L fermentation tank by adopting the liquid culture medium with the pH of 6.5, wherein the inoculation amount is 10% v/v, the culture temperature is 30 ℃, the dissolved oxygen is not less than 10%, and the fermentation culture time is 150-200 h; in the fermentation production process, 25% ammonia water is fed back to maintain the pH, and methanol is fed back to maintain the concentration of methanol in the fermentation system to be not lower than 1 g/L; the liquid culture medium for fermentation production comprises the following components: 10g/L of methanol, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 3g/L of disodium hydrogen phosphate, 1.6g/L of magnesium sulfate, 2mL/L of trace element liquid and 1mL/L of vitamin auxiliary liquid; wherein the microelements comprise 80g/L ferrous sulfate, 22.5g/L zinc sulfate, 40g/L manganese sulfate, 5g/L copper sulfate, 15g/L sodium chloride, 0.3g/L sodium molybdate, 0.3g/L potassium chloride, 0.03g/L cobalt chloride, 3g/L boric acid and 300g/L calcium chloride; the vitamin auxiliary liquid comprises 2g/L of riboflavin, 0.1g/L of pyridoxine hydrochloride, 0.2g/L of thiamine hydrochloride, 0.2g/L of inositol, 2mg/L of folic acid, 0.8g/L of nicotinic acid, 0.1g/L of p-aminobenzoic acid, 0.3g/L of calcium pantothenate and 6mg/L of biotin; measuring the oxidation-reduction potential of a fermentation system in a fermentation tank by an oxidation-reduction potential electrode, wherein the ventilation rate is 1.0vvm, the initial stirring speed is 200rpm, the oxidation-reduction potential is adjusted to be 190-250 mV by controlling the ventilation rate and the stirring rotating speed, the stirring rotating speed is adjusted to be 150-800 rpm, the ventilation rate is adjusted to be 0.8-2.0 vvm, for example, when the ORP is lower than a set value, the ventilation rate is increased firstly and then the stirring rotating speed is increased; when ORP is higher than the set value, the agitation speed is first reduced and then the aeration rate is decreased.

In the embodiment, the oxidation-reduction potential of a fermentation system in a fermentation tank is controlled to be 130mV and 160mV as a control group, the oxidation-reduction potential of the fermentation system in the fermentation tank is controlled to be 250mV, 220mV and 190mV as an experimental group, the oxidation-reduction potential of the experimental group and the control group is different only in the fermentation process when methylotrophic bacteria are used for producing pyrroloquinoline quinone, the rest conditions are the same, the final biological quantity and the yield of pyrroloquinoline quinone (PQQ) are compared, the specific result is shown in FIG. 1, the biomass is in direct proportion to the ORP potential, the dry weight of cells at 250mV is 51.89g/L, and the dry weight of cells at 130mV is only 17.15 g/L; the yield of PQQ is the highest when 190mV is taken in the first 80 hours, and the unit cell yield is 5.29 mg/g; after 80 hours, the yield of PQQ at 220mV is the highest and reaches 604.63mg/L, and the unit cell yield is 12.34 mg/g.

Example 2

In the embodiment, the methylotrophic bacteria adopts a hyphomycete denitrificans DSM 1869, and the method specifically comprises the following steps:

s1, strain activation: adopting a solid culture medium with the pH value of 7.5, taking 1-ring denitrogenation hyphomycete DSM 1869 during activation culture, carrying out plate streaking in the solid culture medium, and then culturing for 4 days in a constant temperature incubator at 25 ℃;

s2, seed culture: adopting a liquid culture medium with the pH of 7.5, selecting 2 rings of activated bacteria after strain activation during culture, inoculating the activated bacteria into a conical flask filled with the liquid culture medium, wherein the culture temperature is 25 ℃, the rotation speed of a shaking table is 200rpm, the culture time is 30-36 h, and the OD is OD₆₅₀1.2 to 1.5;

s3, fermentation production: inoculating the cultured seeds into a liquid culture medium in a 5L fermentation tank by adopting the liquid culture medium, wherein the inoculation amount is 5% v/v, the culture temperature is 25 ℃, the initial pH is 6.0, the dissolved oxygen is not lower than 10%, and the fermentation time is 200 h; adding 25% ammonia water in a flowing manner in the fermentation production process to maintain the pH, and adding methanol in a flowing manner to maintain the concentration of methanol in a fermentation system to be not less than 1 g/L; the oxidation-reduction potential of the fermentation system in the fermentation tank is measured by the oxidation-reduction potential electrode, the initial aeration is 1.0vvm, the initial stirring speed is 200rpm, in the embodiment, the oxidation-reduction potential electrode is introduced in the fermentation production and then three stages are set to control the fermentation process, and the first stage is set at 0-60h after the fermentation is started: adjusting ventilation volume and stirring speed, maintaining the oxidation-reduction potential in the fermentation system at 250 +/-5 mV and the pH value at 6.0-6.5; fermenting for 60-100h as a second stage, adjusting ventilation and stirring speed, maintaining oxidation-reduction potential in a fermentation system at 190 +/-5 mV and pH at 6.5-7.0; the third stage of fermentation is carried out for 200h after 100-fold, the aeration rate and the stirring rotation speed are adjusted, the oxidation-reduction potential in the fermentation system is maintained at 220 +/-5 mV, the pH value is maintained at 7.0-7.5, wherein the oxidation-reduction potential is adjusted by controlling the stirring rotation speed and the aeration rate, the stirring rotation speed is adjusted within the range of 150-800 rpm, and the aeration rate is adjusted within the range of 0.8-2.0 vvm;

wherein, the solid culture medium for activating the strains, the liquid culture medium for seed culture and the liquid culture medium for fermentation production adopted in the process are the same as the ones in the example 1;

in the embodiment, selecting that the oxidation-reduction potential is not controlled in the fermentation production process, only the dissolved oxygen in the fermentation system is maintained to be not less than 10% as a control group, controlling the oxidation-reduction potential and the dissolved oxygen to be not less than 10% according to three-stage fermentation as an experimental group, the rest conditions are the same, comparing the final biomass with the yield of pyrroloquinoline quinone (PQQ), and referring to Table 1 for specific results, the yield of PQQ per cell is improved by 64.67% and the synthesis rate of PQQ is improved by 49.41% by controlling ORP;

TABLE 1 comparison of ORP control and ORP non-control fermentation results of Micromyceliophthora denitrificans DSM 1869

Culture conditions	Biomass (g/L)	PQQ yield (mg/L)	Fermentation time (h)
				DO>10％	57.66	636.45	220
Control of ORP	47.56	864.44	200

Example 3

A method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated by oxidation-reduction potential, in the embodiment, methylotrophic bacteria adopts methylobacterium tornado ATCC 8457, and specifically comprises the following steps:

s1, strain activation: adopting a solid culture medium with the pH value of 7.0, taking 1-ring-torulopsis-methyl ATCC 8457 to perform plate streaking in the solid culture medium during activation culture, and then culturing in a constant-temperature incubator at 28 ℃ for 3 days;

s2, seed culture: adopting a liquid culture medium with the pH of 7.0, selecting 2 rings of activated bacteria after strain activation during culture, inoculating the activated bacteria into a conical flask filled with the liquid culture medium, wherein the culture temperature is 28 ℃, the rotation speed of a shaking table is 150rpm, the culture time is 24-30 h, and the OD is OD₆₅₀1.6 to 2.0;

s3, fermentation production: inoculating the cultured seeds into a liquid culture medium in a 5L fermentation tank by adopting the liquid culture medium with the initial pH of 7.5, wherein the inoculation amount is 8% v/v, the culture temperature is 28 ℃, the dissolved oxygen is not lower than 10%, and the fermentation time is 150 h; adding 25% ammonia water in a flowing manner in the fermentation production process to maintain the pH, and adding methanol in a flowing manner to maintain the concentration of methanol in a fermentation system to be not less than 1 g/L; the oxidation-reduction potential of the fermentation system in the fermentation tank is measured by the oxidation-reduction potential electrode, the initial aeration is 1.0vvm, the initial stirring speed is 200rpm, in the embodiment, the oxidation-reduction potential electrode is introduced in the fermentation production and then three stages are set to control the fermentation process, and the first stage is 0-35h after the fermentation is started: adjusting ventilation and stirring speed, maintaining the oxidation-reduction potential in the fermentation system at 250 +/-5 mV and the pH at 6.5; taking the fermentation for 35-70h as a second stage, adjusting the aeration quantity and the stirring speed, maintaining the oxidation-reduction potential in the fermentation system at 190 +/-5 mV and the pH value at 6.5-7.0; the third stage is that the fermentation is carried out for 70-150h, the aeration quantity and the stirring rotation speed are adjusted, the oxidation-reduction potential in a fermentation system is maintained at 220 +/-5 mV, the pH value is maintained at 7.5, wherein the oxidation-reduction potential is adjusted by controlling the stirring rotation speed and the aeration quantity, the stirring rotation speed is adjusted within the range of 150-800 rpm, and the aeration quantity is adjusted within the range of 0.8-2.0 vvm;

wherein, the solid culture medium for activating the strains, the liquid culture medium for seed culture and the liquid culture medium for fermentation production adopted in the process are the same as the ones in the example 1;

in this example, selecting a control group in which the oxidation-reduction potential is not controlled during the fermentation production process, and only maintaining dissolved oxygen in the fermentation system at not less than 10%, controlling the oxidation-reduction potential and controlling dissolved oxygen at not less than 10% according to three-stage fermentation as an experimental group, and the other conditions being the same, comparing the final biomass with the yield of pyrroloquinoline quinone (PQQ), and referring to table 2 as a specific result, by controlling ORP, the PQQ yield per cell is increased by 76.8%, and the PQQ synthesis rate is increased by 62.94%;

TABLE 2 comparison of ORP-controlled and ORP-uncontrolled fermentation results for Methylobacterium toruloides ATCC 8457

Culture conditions	Biomass (g/L)	PQQ yield (mg/L)	Fermentation time (h)
				DO>10％	62.37	227.84	160
Control of ORP	53.89	348.05	150

Example 4

A method for producing pyrroloquinoline quinone by using methylotrophic bacteria regulated by oxidation-reduction potential, in the embodiment, methylotrophic bacteria adopt denitrificated hyphomycete FJNU-6, and the method specifically comprises the following steps:

s1, strain activation: adopting a solid culture medium with the pH value of 6.5, taking 1-ring denitrogenation hyphomycete FJNU-6 during activation culture, carrying out plate streaking in the solid culture medium, and then culturing in a constant temperature incubator at 30 ℃ for 3 days;

s2, seed culture: adopting a liquid culture medium with the pH value of 6.5, selecting 1 ring of activated bacteria after strain activation during culture, inoculating the activated bacteria into a conical flask filled with the liquid culture medium, wherein the culture temperature is 30 ℃, the rotation speed of a shaking table is 220rpm, the culture time is 18-24 h, and the OD is OD₆₅₀1.5 to 2.0;

s3, fermentation production: inoculating the cultured seeds into a liquid culture medium in a 5L fermentation tank by adopting the liquid culture medium with the initial pH of 7.0, wherein the inoculation amount is 10% v/v, the culture temperature is 30 ℃, the dissolved oxygen is not lower than 10%, and the fermentation time is 192 h; in the fermentation production process, 25% ammonia water is fed back to maintain the pH, and methanol is fed back to maintain the concentration of methanol in the fermentation system to be not lower than 1 g/L; the oxidation-reduction potential of the fermentation system in the fermentation tank is measured by an oxidation-reduction potential electrode, the initial aeration is 1.0vvm, the initial stirring speed is 200rpm, in the embodiment, the oxidation-reduction potential electrode is introduced in the fermentation production and then three stages are arranged to control the fermentation process, and the first stage is the first stage after 0-40h from the beginning of the fermentation: adjusting ventilation and stirring speed, maintaining oxidation-reduction potential at 250 + -5 mV and pH at 6.5 in the fermentation system; fermenting for 40-70h as a second stage, adjusting the gas flow and the stirring speed, maintaining the oxidation-reduction potential in the fermentation system at 190 +/-5 mV and the pH value at 6.5-7.0; the third stage is that the fermentation is carried out for 70-192h, the aeration quantity and the stirring rotation speed are adjusted, the oxidation-reduction potential in a fermentation system is maintained at 220 +/-5 mV, and the pH value is maintained at 7.5, wherein the oxidation-reduction potential is adjusted by controlling the stirring rotation speed and the aeration quantity, the stirring rotation speed is adjusted within the range of 150-800 rpm, and the aeration quantity is adjusted within the range of 0.8-2.0 vvm;

wherein, the solid culture medium for activating the strains, the liquid culture medium for seed culture and the liquid culture medium for fermentation production adopted in the process are the same as the ones in the example 1;

in the embodiment, the oxidation-reduction potential is not controlled in the process of selecting fermentation production, only the dissolved oxygen in a fermentation system is maintained to be not less than 10%, the fermentation time is 192h and is taken as a control group, the oxidation-reduction potential is controlled according to three-stage fermentation while the dissolved oxygen is controlled to be not less than 10% and is taken as an experimental group, the rest conditions are the same, the biomass and the yield of pyrroloquinoline quinone (PQQ) in the fermentation solution are measured by sampling at regular time, the specific result is shown in figure 2, and the yield of PQQ reaches 2070.41mg/L (192h) by controlling ORP, and is improved by 45.77% (1420.29mg/L) compared with the yield of the uncontrolled PQQ.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications made by the equivalent structures or equivalent processes in the present specification, or directly or indirectly applied to other related technical fields are included in the scope of the present invention.