CN116731909B - Strain for high-yield prodigiosin and application thereof - Google Patents
Strain for high-yield prodigiosin and application thereof Download PDFInfo
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Abstract
The invention discloses a strain for high-yield prodigiosin and application thereof. Serratia marcescens NRRL B-1481 is taken as an initial strain, is subjected to composite mutagenesis by means of ARTP and ultraviolet mutagenesis, and is further screened by increasing substrate concentration, culture pH and illumination intensity, so as to obtain a high-yield strain of light-tolerant prodigiosin with high substrate concentration, namely Serratia marcescens PG-Zeno-001. Compared with the original strain, the prodigiosin production efficiency is improved by 219.3 percent. In another aspect, the invention also provides a method for producing prodigiosin using said strain for fermentation.
Description
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a strain for high-yield prodigiosin and application thereof.
Background
Prodigiosin (Prodigiosin) is a red substance, the structure of which is composed of three pyrrole rings, and has a strong absorption peak at 535nm of the ultraviolet visible spectrum, so that the Prodigiosin is red, and is a relatively common microbial pigment reported at present. Because of its relatively complex structure, the literature reports two main types of prodigiosins: first are compounds containing linear alkyl side chains, such as prodigiosin (prodigiosin) and undecyl prodigiosin (undecyl prodigiosin); and cyclic compounds such as cycloprodigiosin (cycloprodigiosin), cycloprodigiosin (metaciloproigiosin), prodigiosin R1 (Prodigiosin R1), and streptavidin B (streptorubin B). Prodigiosin was originally isolated from secondary metabolites produced by Serratia marcescens (Serratia marcescens).
Besides being used as a dyeing agent in the traditional textile printing industry, the prodigiosin also has various functions of resisting oxidation, inflammation, bacteria, viruses, cancers and the like, and can effectively inhibit the activity of free radicals, enhance the immunity of organisms, promote metabolism, improve blood circulation, reduce blood fat, prevent cardiovascular diseases, resist aging, resist cancers and the like. The induction mechanism of the prodigiosin is not relatively approved by scientific researches, the signal transmission related to cell survival and reproduction is the main focus of the research on the mechanism of inducing apoptosis, and the research shows that the prodigiosin can induce kinase closely related to cell survival to increase phosphate groups, thereby leading cells to execute the apoptosis program. Has obvious effect in inducing colorectal cancer cell apoptosis and has potential of being used as a novel anticancer drug for tumor treatment. In the field of novel functional materials, since prodigiosin has pH response color-changing characteristics, in the field of material science, recent reports prove that the pH response color-changing performance and the biodegradable color-changing film have wide application prospects in the aspects of marking, packaging, base materials and the like; in addition, recently, it has also been reported that prodigiosin has natural antibacterial properties, has a remarkable inhibitory effect on escherichia coli above a Minimum Inhibitory Concentration (MIC), and does not observe remarkable DNA damage and cytoplasmic membrane disintegration. However, wild-type Serratia marcescens has a lower prodigiosin yield and does not have the ability to be produced on a large scale. If prodigiosin is to be truly utilized, breakthrough must be made in the breeding of high-yield strains. It has been reported in the literature that the mutagenesis treatment of S.marcescens jx1 by microwave mutagenesis technique increases the prodigiosin yield of the bacterium from 3.1g/L to 6.5g/L. On the other hand, the existing prodigiosin producing strain generally has the characteristic of poor substrate tolerance, and the biological activity of the prodigiosin producing strain at high concentration is drastically reduced, so that the further improvement of the yield is restricted.
In addition, the research shows that the stability of the prodigiosin can be seriously influenced by ultraviolet irradiation, the stability is greatly influenced by pH, the higher the pH value is, the larger the influence is, the more stable the pigment is at the pH of 3, the pigment content can still be kept about 83% after 1h of ultraviolet irradiation, and the content is obviously reduced after ultraviolet irradiation under alkaline conditions. In addition, the absorbance of the pigment was found to decrease sharply after the pigment was irradiated with direct outdoor light. The loss rate of the pigment at 7h of irradiation was as high as 92%, which suggests that the pigment is unstable under direct light, probably because the double bonds in the pyrrole ring of the red pigment are destroyed. Serratia marcescens has conditional pathogenicity, genetic operation tools are not mature, the period of gene modification is long, the effect is probably not obvious, and the traditional mutation screening has the advantages of high mutation rate, low cost, high strain stability and the like, and is the most effective mode for screening non-model microorganisms at present.
In summary, to achieve commercial production of prodigiosin, there is still a need for prodigiosin producing strains having high yields, high substrate tolerance and light tolerance properties.
Disclosure of Invention
In order to solve the defects of poor substrate tolerance, low yield, unstable high pH and unstable light of the traditional prodigiosin producing strain, the invention provides a high-prodigiosin producing serratia marcescens Serratia marcescens PG-Zeno-001 which is preserved in China center for type culture collection (China university of Wuhan) at the 3 rd month of 2023 and has survived, and is classified and named as Serratia marcescens PG-Zeno-001, and the preservation number is CCTCC NO: m2023361.
According to the invention, serratia marcescens NRRL B-1481 (purchased from ATCC) is taken as an initial strain, the initial strain is subjected to composite mutagenesis by means of ARTP and ultraviolet mutagenesis, and the mutant strain is further screened by improving substrate concentration, culture pH and illumination intensity, so that a high-yield strain of the light-tolerant prodigiosin with high substrate concentration, namely Serratia marcescens PG-Zeno-001, is obtained.
In a second aspect, the present invention provides a method for producing prodigiosin by fermentation using Serratia marcescens Serratia marcescens PG-Zeno-001 as described above.
Preferably, the fermentation medium used in the fermentation production method comprises: 5 to 7.5g/L of tryptone, 5 to 7.5g/L of soybean cake powder, 5 to 7.5g/L, caCl of sucrose, 2.5 to 4.0g/L of glycine, 1.0 to 1.5g/L of defoamer and 1.0mL/L.
Preferably, the fermentation production method comprises: inoculating Serratia marcescens PG-Zeno-001 activated by inclined plane into a seed culture medium, and obtaining primary seed liquid through shake flask culture and primary seed culture; inoculating the primary seed liquid into the fermentation medium for fermentation culture to obtain Serratia marcescens fermentation liquid; the volume percentage of the primary seed liquid in the fermentation medium is 5-10%.
More preferably, the temperature of the fermentation culture is 25-30 ℃ and the time is 48-96 hours; the fermentation culture is carried out under the condition of aeration and stirring, and the stirring speed is 200-350 rpm.
More preferably, the fermentation production method further comprises: centrifuging the Serratia marcescens fermentation liquor, and removing the supernatant to obtain red thalli; and (3) re-suspending the red thalli with acetone, stirring the thalli at room temperature in a dark place until the thalli are colorless, centrifuging to remove the thalli, collecting acetone extract, and concentrating the acetone extract by using a rotary evaporator to obtain the prodigiosin.
Preferably, the fermentation production method further comprises a step of detecting the prodigiosin in the serratia marcescens fermentation liquid.
More preferably, the detecting step includes: taking 1mL of the uniformly mixed Serratia marcescens fermentation liquor, adding 4mL of acid methanol mother liquor, performing ultrasonic cell disruption for 15-20min, centrifuging at low temperature to remove precipitate, diluting the supernatant by 5 times, then diluting the supernatant by different times according to gradient, and respectively taking 200 mu L of the supernatant and adding the diluted supernatant into a 96-well plate to measure 535nm absorbance; the pH of the acidic methanol was 3.0.
Through the technical scheme, the invention has the following beneficial effects:
1. the method comprises the steps of transforming an original strain by means of ARTP and ultraviolet mutagenesis to obtain the high-yield prodigiosin, and obtaining the serratia marcescens strain with good substrate tolerance and light tolerance.
2. Provides a prodigiosin fermentation production method, prodigiosin is produced by utilizing the method, and the fermentation yield in a 5L tank can reach 8.59g/L in 48 hours.
Drawings
FIG. 1 is a screening bar graph of screening high substrate tolerant strains with high concentration sucrose mother liquor;
FIG. 2 is a screening bar graph of screening alkaline resistant strains at high pH;
FIG. 3 is a screening bar graph of a light stable strain screened with intense light;
FIG. 4 is a graph showing the course of prodigiosin production by fermentation of a starting strain with Serratia marcescens PG-Zeno-001 on a 5L tank;
FIG. 5 shows a comparison of colonies from the starting strain Serratia marcescens NRRL B-1481 (left) with Serratia marcescens PG-Zeno-001 (right) plates.
Detailed Description
The invention is further described below with reference to the drawings and examples.
EXAMPLE 1ARTP mutagenesis method
Sample slide preparation and treatment: in an ultra-clean workbench, placing the slide glass in an alcohol lamp for burning by using forceps for 30s, then placing the slide glass in a sterilized culture dish, taking 10 mu L of bacterial suspension by using a pipette after cooling, and uniformly covering the surface of the slide glass to finish the preparation of the slide glass of the sample to be treated.
Pre-sterilization of art mutagenesis breeding instrument operation room: in order to prevent contamination of the sample to be treated by other microorganisms in the operating room, it is necessary to sterilize the operating room before using the art mutation breeding instrument, and close the door of the operating room.
Treatment conditions for art mutagenesis: the high-purity helium is used as the working gas of ARTP, the distance between the sample to be treated and the jet outlet of the plasma generator is set to be 2mm, the working power is 100W, the air flow (liter/min) is 10SLM, and the pressure of the low-pressure output end of the pressure reducing valve is 0.10-0.15 MPa. The length of treatment is therefore taken as a function of the amount of mutagen.
EXAMPLE 2 UV mutagenesis method
Pre-sterilization of uv mutagenesis operating room: in order to prevent other microorganisms in the operating room from polluting the sample to be treated, sterilization is required in the operating room before ultraviolet mutation breeding is used, and the ultra-clean bench door is closed.
Ultraviolet mutagenesis treatment conditions: using an ultra-clean workbench as an experimental instrument, placing the bacterial suspension in a flat plate, turning on an ultraviolet lamp of the ultra-clean workbench, and using the treatment time as a variable of the mutagen quantity, wherein the flat plate is irradiated by an 8W ultraviolet lamp in the ultra-clean workbench (irradiation time is 0, 30, 60, 120 and 240s respectively). After completion, the bacterial suspension in the plate is drawn into a sterilized centrifuge tube and immediately placed into an ice-water mixture.
Example 3 detection method of prodigiosin in fermentation liquor
Taking 1mL of a uniformly mixed serratia marcescens fermentation liquid, adding 4mL of an acidic methanol mother solution, performing ultrasonic cell disruption for 15-20min, centrifuging at a low temperature to remove precipitate, diluting the supernatant by 5 times, then diluting the supernatant by different times according to a gradient, and adding 200 mu L of each solution into a 96-well plate to measure 535nm absorbance; the pH of the acidic methanol was 3.0.
Example 4 screening of high substrate tolerant strains with high concentration sucrose mother liquor
The Serratia marcescens starting strain of the invention was Serratia marcescens NRRL B-1481 (purchased from ATCC). Culturing the initial strain Serratia marcescens NRRL B-1481 on LB solid culture medium plate, standing at 25-30deg.C for 24 hr, collecting the full colony, inoculating to LB liquid culture medium tube containing 5mL, culturing at 200rpm in 30 deg.C table for 7 hr, centrifuging the bacterial liquid grown to logarithmic phase, washing with sterile physiological saline for 2-3 times, diluting with sterile physiological saline to obtain OD600 value of 0.6-0.8 or bacterial concentration of 10 6 About one/mL of the bacterial suspension to be mutagenized. The bacterial suspension to be mutagenized was treated sequentially using the ARTP mutagenesis method in example 1 and the ultraviolet mutagenesis method in example 2.
The bacterial suspension obtained after the mutagenesis treatment is uniformly coated on an LB solid medium plate containing 100g/L, 150g/L and 200g/L sucrose, and is cultured for 48 hours at 30 ℃. At this time, only Serratia marcescens which can endure high concentration sucrose after mutation can grow into colonies due to metabolic pressure caused by high concentration sucrose concentration. The single colonies are further inoculated into 7mL LB liquid culture medium test tubes containing 100g/L, 150g/L and 200g/L sucrose respectively for preliminary screening, and strains with higher biomass and faster pigment production are selected for subsequent shake flask rescreening.
Shake flask rescreening was performed using a 250mL conical flask containing 50mL of liquid LB medium, and strain with better performance of the primary screening was selected, inoculated in the rescreening shake flask at 1% of inoculum size, cultured at 200rpm at 30 ℃ for 48h, and then biomass and prodigiosin yield were detected, and the strain obtained in the process of this round of screening was named Serratia marcescens PG-Zeno-28-3.
Compared with the original strain, the yield of the prodigiosin obtained by shaking flask fermentation of Serratia marcescens PG-Zeno-28-3 is obviously improved.
Example 5 screening of alkali-resistant strains at high pH
Culturing the strain Serratia marcescens Zeno-PG-28-3 obtained in example 4 on LB solid medium plate, standing at 25-30deg.C for 24 hr, collecting colony, inoculating to LB medium tube containing 5mL, culturing at 30 deg.C in shaker at 200rpm for 7 hr, centrifuging to collect bacterial liquid, washing with sterile physiological saline for 2-3 times, diluting with appropriate amount of sterile physiological saline to obtain OD600 value of 0.6-0.8 or bacterial concentration of 10 6 About one/mL of the bacterial suspension to be mutagenized. The bacterial suspension to be mutagenized was treated sequentially using the ARTP mutagenesis method in example 1 and the UV mutagenesis method in example 2.
Uniformly coating the obtained bacterial suspension subjected to mutagenesis treatment on an LB culture medium with the pH value of 9.0, respectively inoculating the grown single bacterial colonies into an LB liquid culture medium test tube filled with 5mL, culturing to the late logarithmic growth phase, preparing into a mutagenesis bacterial suspension, carrying out the next round of ultraviolet and ARTP mutagenesis, and uniformly coating the treated bacterial suspension on an LB culture medium plate with the pH value of 10.0. Repeating the above operation, screening alkaline plates with pH of 11.0, pH of 12.0, pH of 13.0 and pH of 14.0, inoculating single colony growing on the plate with pH of 14.0 to a 7mL LB liquid culture medium test tube without sucrose, culturing at 30 ℃ for 48 hours at 200rpm, detecting biomass and prodigiosin yield, and obtaining the strain with higher prodigiosin yield. The process strain obtained by this round of screening was named Serratia marcescens PG-Zeno-45-2.
Compared with Serratia marcescens PG-Zeno-28-3, serratia marcescens PG-Zeno-45-2 shake flask fermentation results in significantly improved prodigiosin yield.
EXAMPLE 6 selection of photostable strains with high light
Culturing the strain Serratia marcescens PG-Zeno-45-2 obtained in example 5 on LB solid medium plate, standing at 25-30deg.C for 24 hr, collecting colony, inoculating to LB medium tube containing 5mL, culturing at 30 deg.C in shaker at 200rpm for 7 hr, centrifuging to collect bacterial liquid, washing with sterile physiological saline for 2-3 times, diluting with appropriate amount of sterile physiological saline to obtain OD600 value of 0.6-0.8 or bacterial concentration of 10 6 About one/mL of the bacterial suspension to be mutagenized. The bacterial suspension to be mutagenized was treated sequentially using the ARTP mutagenesis method in example 1 and the UV mutagenesis method in example 2.
Uniformly coating the obtained bacterial suspension subjected to mutagenesis treatment on an LB (liquid-phase) culture medium, placing the LB culture medium in an ultra-clean bench for 12 hours, 24 hours and 36 hours under the irradiation of a fluorescent lamp, inoculating the bacterial colony which is not discolored and full into a 7mL LB liquid culture medium test tube which does not contain sucrose, culturing for 48 hours at 30 ℃ at 200rpm, detecting the biomass and the prodigiosin yield, and obtaining the strain with higher prodigiosin yield.
The strain obtained by final screening is named as Serratia marcescens PG-Zeno-001, and compared with the original strain Serratia marcescens PG-Zeno-45-2 subjected to mutagenesis in the round, the shake flask final yield of Serratia marcescens PG-Zeno-001 is 3.31g/L, which is higher than that of the strain Serratia marcescens PG-Zeno-45-2 by 11.44%, and the strain has the advantages of higher growth speed and stronger pigment production capability. The Serratia marcescens PG-Zeno-001 high-yield prodigiosin is preserved in China center for type culture collection (CCTCC NO) at university of Wuhan at 20/3/2023: m2023361. As shown in FIG. 5, the plate morphology of Serratia marcescens PG-Zeno-001 showed a significantly higher rate of growth and pigment deposition than that of the starting strain at the same time.
EXAMPLE 7 production of prodigiosin by 5L in-tank fermentation
(1) Preparation of fermentation medium: tryptone 6.0g/L and soybean cake powder 6.0g/L, caCl 2 3.5g/L, 5.0g/L of sodium chloride, 7.5g/L of sucrose and 1.0g/L of glycine, 1.0mL/L of defoamer is dissolved in sterile water, and hydrochloric acid is used for adjusting the pH of the culture medium to 6.0; and sterilizing at 121 ℃ for 20 minutes in a wet heat way for later use.
(2) Preparation of feed medium: 50g/L of peanut cake powder, 300g/L of sucrose and 10g/L of proline are dissolved in sterile water, pH is not regulated, and the peanut cake powder is sterilized for 20 minutes at 121 ℃ in a wet heat way for later use.
(3) Preparing an inclined plane: PG-Zeno-001 is inoculated on LB inclined plane for activation culture, and the constant temperature culture is carried out for 24 to 48 hours at the temperature of 25 to 30 ℃ to prepare the inclined plane, and the inclined plane is refrigerated for standby.
(4) Preparing shake flask seeds: and (3) drawing the single colony on the inclined plane cultured in the step (3), inoculating the single colony on a sterilized 250mL shaking flask filled with 50mL of LB culture medium, and starting culture. Culture conditions: the temperature is 25-30 ℃, the rotation speed of the shaking table is 180-200 rpm, and the culture time is 8 hours.
(5) Primary seed culture: inoculating the shake flask seeds cultured in the step (4) into a sterilized 10L primary seed tank filled with 5L of LB culture medium according to a volume ratio of 1%, and starting to culture. Culture conditions: aeration rate is 0.3-1.5 VVM, tank pressure is 0.1Mpa, temperature is 25-30 ℃, saturation degree of dissolved oxygen is 1-80%, and culturing time is 10h.
(6) Fermenting in a fermentation tank: inoculating the first-level seeds cultured in the step (4) into a sterilized 5L fermentation tank filled with 2.5L fermentation medium according to the volume ratio of 5-10%, and starting to culture. Culture conditions: the initial aeration rate is 0.3VVM, the tank pressure is 0.1Mpa, the temperature is 25-30 ℃, and the culture time is 48-96 hours. The dissolved oxygen control scheme is as follows: the initial stirring rotating speed is 200rpm for 0-12h, the rotating speed is slowly increased to 350rpm along with the reduction of dissolved oxygen, and then the dissolved oxygen is controlled to be 30% by being related to the dissolved oxygen through stirring; controlling the dissolved oxygen to be 15% by associating the dissolved oxygen with the feed for 12-36 h, and controlling the dissolved oxygen to be 10% by associating the dissolved oxygen with the feed after 36 h-fermenting and finally reducing the stirring speed to 200 rpm. Through detection, the prodigiosin yield of the final 48h can reach 8.59g/L, the yield of the original strain fermented under the same condition is only 2.69g/L, and compared with the original strain, the yield under the same fermentation condition at the same time is improved by 219.3 percent. The final dry and wet bacterial body amounts of the fermentation are 21.6g/L and 86.5g/L respectively, which are respectively higher than 89.6% and 105.3% of the contemporaneous starting strain, and the tolerance capability and the light resistance capability of the low pH and high sucrose concentration are obviously improved.
Example 8 harvesting of prodigiosin from fermentation broths
Centrifuging the Serratia marcescens fermentation broth obtained in the example 7, and removing the supernatant to obtain red thalli; and (3) re-suspending the red thalli with acetone, stirring the thalli at room temperature in a dark place until the thalli are colorless, centrifuging to remove the thalli, collecting acetone extract, and concentrating the acetone extract by using a rotary evaporator to obtain a crude prodigiosin product.
The above detailed description of the embodiments should not be construed as limiting the scope of the invention, but it will be apparent to those skilled in the art from this disclosure that many insubstantial modifications and adaptations of the invention are possible within the scope of the invention.
Claims (8)
1. Serratia marcescens (Serratia marcescens) PG-Zeno-001 with high prodigiosin yield is preserved in China center for type culture Collection (China center for type culture collection) for 3 months of 2023, and the preservation number is CCTCC NO: M2023361.
2. A fermentation production method of prodigiosin, characterized in that serratia marcescens PG-Zeno-001 is used for fermentation to produce prodigiosin according to claim 1.
3. The prodigiosin fermentation production method according to claim 2, characterized in that: the fermentation medium used in the fermentation production method comprises the following components: 5 to 7.5g/L of tryptone, 5 to 7.5g/L of soybean cake powder, 5 to 7.5g/L, caCl of sucrose, 2.5 to 4.0g/L of glycine, 1.0 to 1.5g/L of defoamer and 1.0mL/L.
4. The prodigiosin fermentation production method according to claim 2, characterized in that: inoculating slope activated Serratia marcescens PG-Zeno-001 into a seed culture medium, and carrying out shake flask culture and primary seed culture to obtain primary seed liquid; inoculating the primary seed liquid into the fermentation medium of claim 3 for fermentation culture to obtain Serratia marcescens fermentation liquor; the volume percentage of the primary seed liquid in the fermentation medium is 5-10%.
5. The prodigiosin fermentation production method according to claim 4, characterized in that: the temperature of the fermentation culture is 25-30 ℃ and the time is 48-96 hours; the fermentation culture is carried out under the condition of aeration and stirring, and the stirring speed is 200-350 rpm.
6. The prodigiosin fermentation production method according to claim 4, characterized by further comprising: centrifuging the Serratia marcescens fermentation liquor, and removing the supernatant to obtain red thalli; and (3) re-suspending the red thalli with acetone, stirring the thalli at room temperature in a dark place until the thalli are colorless, centrifuging to remove the thalli, collecting acetone extract, and concentrating the acetone extract by using a rotary evaporator to obtain the prodigiosin.
7. The prodigiosin fermentation production method according to claim 4, characterized in that: the method also comprises the step of detecting the prodigiosin in the serratia marcescens fermentation liquid.
8. The prodigiosin fermentation production method according to claim 7, characterized in that: the detection step comprises the following steps: taking 1mL of the uniformly mixed Serratia marcescens fermentation liquor, adding 4mL of acid methanol mother liquor, performing ultrasonic cell disruption for 15-20min, centrifuging at low temperature to remove precipitate, diluting the supernatant by 5 times, then diluting the supernatant by different times according to gradient, and respectively taking 200 mu L of the supernatant and adding the diluted supernatant into a 96-well plate to measure 535nm absorbance; the pH of the acidic methanol was 3.0.
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|---|---|---|---|---|
| CN102888351A (en) * | 2012-09-03 | 2013-01-23 | 广东药学院 | Prodigiosin high-producing strain and production method thereof |
| CN105969702A (en) * | 2016-07-26 | 2016-09-28 | 南京师范大学 | Serratia marcescens RZ 21-C6 and application thereof |
| CN111154673A (en) * | 2020-01-08 | 2020-05-15 | 江苏师范大学 | Prodigiosin producing strain and production method and application thereof |
| CN111778298A (en) * | 2020-07-27 | 2020-10-16 | 中国热带农业科学院热带生物技术研究所 | Application of Serratia marcescens ITBB B5-1 in prodigiosin production |
| CN113025515A (en) * | 2020-11-23 | 2021-06-25 | 四川轻化工大学 | Serratia marcescens Ka3 strain with high prodigiosin yield and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888351A (en) * | 2012-09-03 | 2013-01-23 | 广东药学院 | Prodigiosin high-producing strain and production method thereof |
| CN105969702A (en) * | 2016-07-26 | 2016-09-28 | 南京师范大学 | Serratia marcescens RZ 21-C6 and application thereof |
| CN111154673A (en) * | 2020-01-08 | 2020-05-15 | 江苏师范大学 | Prodigiosin producing strain and production method and application thereof |
| CN111778298A (en) * | 2020-07-27 | 2020-10-16 | 中国热带农业科学院热带生物技术研究所 | Application of Serratia marcescens ITBB B5-1 in prodigiosin production |
| CN113025515A (en) * | 2020-11-23 | 2021-06-25 | 四川轻化工大学 | Serratia marcescens Ka3 strain with high prodigiosin yield and application thereof |
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