CN116555369B - Method for producing prodigiosin by fermentation of waste corn steep liquor - Google Patents

Method for producing prodigiosin by fermentation of waste corn steep liquor Download PDF

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CN116555369B
CN116555369B CN202310519215.3A CN202310519215A CN116555369B CN 116555369 B CN116555369 B CN 116555369B CN 202310519215 A CN202310519215 A CN 202310519215A CN 116555369 B CN116555369 B CN 116555369B
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朱天择
季立豪
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Zeno Suzhou Biotechnology Co ltd
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Abstract

The invention discloses a method for producing prodigiosin by fermenting waste corn steep liquor. On one hand, by optimizing the formula of a fermentation medium, the byproduct corn steep liquor dry powder of the starch industry is used for partially replacing tryptone to be used as a nitrogen source; on the other hand, a method for producing the prodigiosin by using the fermentation medium is also provided, and in the large-scale fermentation of a 100L fermentation tank, the prodigiosin yield of 48 h can reach 10.68 g/L. The invention can greatly reduce the cost of prodigiosin fermentation production and is hopeful to construct a new closed loop for starch industrial environment-friendly production.

Description

Method for producing prodigiosin by fermentation of waste corn steep liquor
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a method for producing prodigiosin by utilizing waste corn steep liquor.
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 has multiple functions of resisting oxidation, inflammation, bacteria, viruses, cancers and the like, 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, cancers and the like (see background document 1: stankovic N, senroxic L, ilc-Tomic T, et al Properties and applications of undecylprodigiosin and other bacterial prodigiosins Appl Microbiol Biotechnol,2014, 98 (9): 3841-3858.; background document 2: nisha, kumar K, kumar V Prodigiosin alkaloids: recent advancements in total synthesis and their biological potential Rsc Adv,2015 (15): 10899-10920.). 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 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 a pH response color-changing characteristic, in the field of material science, recent reports have demonstrated pH response color-changing performance and a biodegradable color-changing film, which have wide application prospects in the aspects of labeling, packaging, base materials and the like (Amorim LFA, gomes AP, gouveia IC. Design and Preparation of a Biobased Colorimetric pH Indicator from Cellulose and Pigments of Bacterial Origin, for Potential Application as Smart Food packaging. Polymers (Basel). 2022 Sep 15;14 (18): 3869.); in addition, recently, it has also been reported that Prodigiosin has natural antibacterial properties, has a remarkable inhibitory effect on E.coli at a concentration higher than the Minimum Inhibitory Concentration (MIC), and has not been observed as remarkable DNA damage and cytoplasmatic membrane disintegration (Danev č i č T, bori ć Vezjak M, zorec M, stopar D. Prodigiosin-A Multifaceted Escherichia coli Antimicrobial agent. PLoS one. 2016 Sep 9;11 (9): e 0162412.). 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 that the mutagenesis treatment of S.marcescens jx1 by microwave mutagenesis technique increases the prodigiosin yield of the bacterium from 3.1 g/L to 6.5 g/L (Liu XX, wang YJ, sun SQ, et al Mutant breeding of Serratia marcescens strain for enhancing prodigiosin production and application to textiles Prep Biochem Biotechnol,2013, 43 (3): 271-284.). 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.
Through preliminary research, serratia marcescens NRRL B-1481 (purchased from ATCC) is taken as a starting strain, the starting strain is subjected to composite mutagenesis by means of ARTP and ultraviolet mutagenesis, and the mutated strain is further screened by improving substrate concentration, culture pH and illumination intensity, so that a high-yield strain of light-tolerant prodigiosin with high substrate concentration, namely Serratia marcescens ZN, is obtained. And a method for fermentation using the fermentation strain.
However, the fermentation medium used in the fermentation method uses tryptone, soybean cake powder and the like as nitrogen sources, so that the cost is high, and the production cost of the prodigiosin is not reduced.
Corn steep liquor is a byproduct of producing corn starch, and raw materials comprise corn grits, water and corn juice. The corn starch silk is produced by soaking corn kernels with sulfurous acid, concentrating the soaking solution to obtain a tan liquid, namely corn steep liquor, which contains abundant soluble proteins, auxin and some precursor substances, and contains about 40% -50% of solid substances. The taste is brackish, is an organic nitrogen source which is widely applied to microbial growth, and can promote the biosynthesis of antibiotics such as penicillin. Corn steep liquor is a high-quality fermentation raw material, is an acidic viscous liquid byproduct generated during the production of corn starch by a wet process, has low cost, is rich in various amino acids, proteins, soluble saccharides and the like, and is a cheap nitrogen source most commonly used in the amino acid industry at present.
However, the technical proposal of fermenting and producing corn steep liquor as a prodigiosin fermentation medium has not been reported yet. Effective ways to reduce the cost of prodigiosin production are still not clear.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a novel fermentation production process of prodigiosin, so as to improve the conversion rate of products and reduce the production cost of prodigiosin.
In a first aspect, the present invention provides a fermentation medium for producing prodigiosin comprising: nitrogen source, peanut cake powder 2.5-4 g/L, caCl 2.5-4.0 g/L, sodium chloride 5.0 g/L, sucrose 5-7.5 g/L, glycine 0.5-1 g/L, proline 0.5-1 g/L and defoamer 1 mL/L; the method is characterized in that: the nitrogen source consists of corn steep liquor dry powder and tryptone, and the ratio of the corn steep liquor dry powder to the tryptone is 16:4-8:12.
Corn steep liquor dry powder is conventionally obtained from commercial sources and is prepared by concentrating and drying waste steep liquor in the process of producing corn starch, so that the cost is extremely low.
Preferably, the sum of the concentrations of the corn steep liquor dry powder and the tryptone is in the range of 16-24 g/L.
Preferably, the concentration of the corn steep liquor dry powder is 8-16 g/L, and the concentration of the tryptone is 4-12 g/L.
Wherein, the concentration of the corn steep liquor dry powder can be independently 8 g/L, 9 g/L, 10g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L and 16 g/L, and the concentration of the tryptone can be independently 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10g/L, 11 g/L and 12 g/L.
In a second aspect, the present invention provides a fermentation production method of prodigiosin, characterized in that Serratia marcescens is fermented using the medium as described above to produce prodigiosin.
Preferably, the Serratia marcescens is Serratia marcescens PG-Zeno-001 strain which is preserved in China center for type culture Collection (CCTCC M2023361) in 2023 and 3 months and 20 days, and the preservation number is the preservation unit address: beijing, china.
As a further preferred aspect, the prodigiosin fermentation production method comprises the steps of:
(1) Selection of fermentation strains: the strain is Serratia marcescens (Serratia marcescens) PG-Zeno-001:
(2) Preparing a fermentation medium: corn steep liquor dry powder 16 g/L, tryptone 4 g/L, peanut cake powder 2.5-4 g/L, caCl 2.5-4.0 g/L, sodium chloride 5.0 g/L, sucrose 5-7.5 g/L, glycine 0.5-1 g/L, proline 0.5-1 g/L, defoamer 1 mL/L, and hydrochloric acid is used for regulating the pH value of the culture medium to 6.0; sterilizing at 121deg.C for 20 min;
(3) Preparing a feed medium: corn steep liquor dry powder 300 g/L, peanut cake powder 50 g/L, sucrose 300 g/L and proline 10g/L, and sterilizing at 121deg.C for 20 min without adjusting pH;
(4) Preparing an inclined plane: inoculating LB inclined plane with the fermentation strain glycerol tube for activation culture, and culturing at constant temperature of 25-30 ℃ for 24-48 h to obtain an inclined plane, and refrigerating the inclined plane for later use;
(5) Shake flask seed culture: inoculating the slant strain obtained in the step (4) into a sterilized shake flask filled with a shake flask LB seed medium, and culturing at about 8 h at 25-30 ℃ at 200 rpm:
(6) Primary seed culture: inoculating the shake flask seeds cultured in the step (5) into a sterilized 10L first-stage seed tank filled with 5L LB medium according to a volume ratio of 1%, and starting to culture, wherein the culture conditions are as follows: aeration rate is 0.3-1.5 VVM, tank pressure is 0.1Mpa, temperature is 25-30 ℃, saturation of dissolved oxygen is 1-80%, and culture time is 10 h;
(7) Fermenting in a fermentation tank: inoculating the first-level seeds cultured in the step (6) into a sterilized 100L fermentation tank filled with 50L fermentation medium according to the volume ratio of 5-10%, and starting to culture; culture conditions: initial ventilation of 0.3 VVM, tank pressure of 0.1Mpa, temperature of 25-30 ℃ and cultivation time of 48-96 hours; the dissolved oxygen control scheme is as follows: an initial rotation speed of 0-12 h is 200 rpm, the rotation speed is slowly increased to 350 rpm along with the reduction of dissolved oxygen, and then the dissolved oxygen is controlled by 30% in association with the dissolved oxygen through stirring; 12-36 h, controlling 15% of dissolved oxygen by associating the dissolved oxygen with the feed, 36-h, and finally reducing the stirring speed to 200 rpm, and controlling 10% of dissolved oxygen by associating the dissolved oxygen with the feed.
Based on the technical scheme, the invention can obtain the following beneficial effects:
1. the novel process for large-scale production of prodigiosin by innovatively using the waste corn steep liquor as a nitrogen source for large-scale production of prodigiosin reshapes the novel process for low-cost large-scale production of prodigiosin, and surprisingly found that the waste corn steep liquor has the best substitution effect on tryptone compared with other nitrogen sources, and is expected to construct a novel closed loop for environmental-friendly production of starch industry.
2. The fermentation medium with optimized components is used for fermenting and producing the prodigiosin, and the prodigiosin yield at 48 h can reach 10.68 g/L in large-scale fermentation in a 100L fermentation tank.
Drawings
FIG. 1 shows the change in prodigiosin yield in a large scale fermentation production according to the invention;
description of the embodiments
The invention is further described below with reference to the drawings and examples.
Example 1 screening of Nitrogen Source produced by fermentation
In order to determine the most suitable nitrogen source for prodigiosin fermentation, we selected 20 g/L of corn steep liquor dry powder, soybean peptone, beef peptone, milk peptone, gelatin peptone, tryptone (the above alternative nitrogen sources were purchased from Shanghai chemical industry) as the alternative nitrogen source, respectively, and conducted shake flask experiments. The operation steps are as follows:
(1) Preparation of the fermentation strain: the strain is Serratia marcescens (Serratia marcescens) PG-Zeno-001 and is stored in a glycerol pipe;
(2) Preparing an inclined plane: inoculating the fermentation strain from a glycerol tube to an LB inclined plane for activation culture, culturing at a constant temperature of 25-30 ℃ for 24-48 hours to obtain an inclined plane, and refrigerating the inclined plane for later use;
(3) Culturing test tube seeds: inoculating the slant strain obtained in the step (2) into a sterilized test tube filled with 5 mL of LB seed culture medium, and culturing at 25-30 ℃ for about 8 h;
(4) Preparing a fermentation medium: 2.5 to 4. 4 g/L, caCl of nitrogen source mother liquor and peanut cake powder 2 2.5 to 4.0 g/L, 5.0 g/L sodium chloride, 5 to 7.5 g/L sucrose, 0.5 to 1 g/L glycine, 0.5 to 1 g/L proline, 1 mL/L defoamer, and adjusting the pH of the culture medium to 6.0 by using hydrochloric acid; the concentration of the nitrogen source mother liquor was varied according to the experimental requirements, and the experimental group nitrogen source mother liquor is shown in table 1 (groups 1 to 6).
(5) Fermentation culture: inoculating the test tube seeds cultured in the step (3) into a sterilized 500 mL shake flask containing 100 mL fermentation medium according to a volume ratio of 1%, and starting culturing, wherein the culturing conditions are as follows: after culturing at 25 to 130℃and 200 rpm for 48 hours, the final fermentation broth was examined for wet weight of cells and prodigiosin unit yield, and the results of each group are shown in Table 1.
TABLE 1 shaking flask experiment results for different Nitrogen sources
First, various kinds of single nitrogen sources, namely, 20 g/L corn steep liquor dry powder, soybean peptone, beef peptone, milk peptone, gelatin peptone, tryptone were compared, and the results are shown in Table 1. Finally, it is determined that tryptone is the single nitrogen source with the best production effect, the final thallus wet weight reaches 20.1 g/L at the maximum, and the prodigiosin content reaches 3.1 g/L at the maximum. Notably, the prodigiosin yield of 1.5 g/L was also achieved in the experimental group using almost zero cost corn steep liquor dry powder as the nitrogen source, and the wet weight of the cells was higher than that of the same concentration of soy peptone and milk peptone, indicating a very large prodigiosin production potential.
Although the fermentation yield of tryptone is highest, because of its high cost, large-scale production application is limited, so that the corn steep liquor dry powder is used for replacing the tryptone according to different proportions, the economy of production cost is maintained while the yield is ensured, and by changing the ratio of the corn steep liquor dry powder to the tryptone (as shown in groups 7-12 in Table 1), we surprisingly found that after the tryptone is partially replaced by the corn steep liquor dry powder, the yield of prodigiosin is not significantly reduced (for example, groups 9-12), and the thallus wet weight is even higher than that of the culture condition using the tryptone as a single nitrogen source when the corn steep liquor dry powder and the tryptone are added in a ratio of 1:1. The optimum production nitrogen source was thus determined to be a corn steep liquor dry powder to tryptone concentration ratio in the range of 16:4 to 8:12.
Although prodigiosin production can be increased at higher tryptone concentrations, the optimal medium formulation for the end determination is, for low carbon economy of production: corn steep liquor dry powder 16 g/L, tryptone 4 g/L, peanut cake powder 2.5-4 g/L, caCl 2 2.5 to 4.0 g/L, 5.0 g/L sodium chloride, 5 to 7.5 g/L sucrose, 0.5 to 1 g/L glycine, 0.5 to 1 g/L proline, 1 mL/L defoamer and hydrochloric acid are used for adjusting the pH value of the culture medium to 6.0.
Example 2 detection method of prodigiosin content in fermentation liquor
Taking Serratia marcescens fermentation liquor of which the concentration is 1 mL, adding 4 mL acid methanol mother liquor, performing ultrasonic cell disruption for 15-20 min, centrifuging at low temperature to remove precipitate, diluting supernatant by 5 times, then diluting the supernatant by different times according to gradient, and respectively taking 200 mu L of the diluted supernatant and adding the diluted supernatant into a 96-well plate to measure 535nm absorbance; the pH of the acidic methanol was about 3.0.
Example 3 Large Scale production of prodigiosin on 100L fermentors
The final fermentation medium ratio for prodigiosin production determined in example 1 was used to verify the prodigiosin yield in a 100L scale fermentor.
(1) Selection of fermentation strains: the strain is Serratia marcescens (Serratia marcescens) PG-Zeno-001:
(2) Preparing a fermentation medium: corn steep liquor dry powder 16 g/L, tryptone 4 g/L, peanut cake powder 2.5-4 g/L, caCl 2 2.5 to 4.0 percent g/L, 5.0 percent g/L sodium chloride, 5 to 7.5 percent g/L sucrose, 0.5 to 1 percent g/L glycine, 0.5 to 1 percent g/L proline, 1 percent mL/L defoamer and hydrochloric acid are used for cultivating the fishThe pH of the culture medium is adjusted to 6.0; sterilizing at 121deg.C for 20 min;
(3) Preparing a feed medium: corn steep liquor dry powder 300 g/L, peanut cake powder 50 g/L, sucrose 300 g/L and proline 10g/L, and sterilizing at 121deg.C for 20 min without adjusting pH;
(4) Preparing an inclined plane: inoculating LB inclined plane with the fermentation strain glycerol tube for activation culture, and culturing at constant temperature of 25-30 ℃ for 24-48 h to obtain an inclined plane, and refrigerating the inclined plane for later use;
(5) Shake flask seed culture: inoculating the slant strain obtained in the step (4) into a sterilized shake flask filled with a shake flask LB seed medium, and culturing at about 8 h at 25-30 ℃ at 200 rpm:
(6) Primary seed culture: inoculating the shake flask seeds cultured in the step (5) into a sterilized 10L first-stage seed tank filled with 5L LB medium according to a volume ratio of 1%, and starting to culture, wherein the culture conditions are as follows: aeration rate is 0.3-1.5 VVM, tank pressure is 0.1Mpa, temperature is 25-30 ℃, saturation of dissolved oxygen is 1-80%, and culture time is 10 h;
(7) Fermenting in a fermentation tank: inoculating the first-level seeds cultured in the step (6) into a sterilized 100L fermentation tank filled with 50L fermentation medium according to the volume ratio of 5-10%, and starting to culture; culture conditions: the initial ventilation is 0.3 VVM, 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: an initial rotation speed of 0-12 h is 200 rpm, the rotation speed is slowly increased to 350 rpm along with the reduction of dissolved oxygen, and then the dissolved oxygen is controlled by 30% in association with the dissolved oxygen through stirring; 12-36 h, controlling 15% of dissolved oxygen by associating the dissolved oxygen with the feed, 36-h, and finally reducing the stirring speed to 200 rpm, and controlling 10% of dissolved oxygen by associating the dissolved oxygen with the feed.
(8) Samples of fermentation broth were taken every 4 hours and the prodigiosin concentration was determined as in example 2 and the results are shown in figure 1. The prodigiosin yield of 48 th h of fermentation can reach 10.68 g/L.
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 (3)

1. A prodigiosin fermentation production method, which uses fermentation medium to ferment serratia marcescens to produce prodigiosin, is characterized in that:
the fermentation medium comprises: nitrogen source, peanut cake powder 2.5-4 g/L, calcium chloride 2.5-4.0 g/L, sodium chloride 5.0 g/L, sucrose 5-7.5 g/L, glycine 0.5-1 g/L, proline 0.5-1 g/L and defoamer 1 mL/L; the nitrogen source consists of corn steep liquor dry powder and tryptone, and the ratio of the corn steep liquor dry powder to the tryptone is 16:4-8:12; the sum of the concentration of the corn steep liquor dry powder and the concentration of the tryptone are in the range of 16-24 g/L;
the Serratia marcescens is Serratia marcescens PG-Zeno-001 strain which is preserved in China Center for Type Culture Collection (CCTCC) in the 3 rd month of 2023, and the preservation number is CCTCC NO: m2023361.
2. The prodigiosin fermentation production method according to claim 1, characterized in that: the concentration of the corn steep liquor dry powder is 8-16 g/L, and the concentration of the tryptone is 4-12 g/L.
3. The prodigiosin fermentation production method according to any one of claims 1-2, characterized by comprising the steps of:
(1) Selection of fermentation strains: the strain is Serratia marcescens (Serratia marcescens) PG-Zeno-001;
(2) Preparing a fermentation medium: corn steep liquor dry powder 16 g/L, tryptone 4 g/L, peanut cake powder 2.5-4 g/L, calcium chloride 2.5-4.0 g/L, sodium chloride 5.0 g/L, sucrose 5-7.5 g/L, glycine 0.5-1 g/L, proline 0.5-1 g/L, defoamer 1 mL/L, and hydrochloric acid is used for regulating the pH value of the culture medium to 6.0; sterilizing at 121deg.C for 20 min;
(3) Preparing a feed medium: corn steep liquor dry powder 300 g/L, peanut cake powder 50 g/L, sucrose 300 g/L and proline 10g/L, and sterilizing at 121deg.C for 20 min without adjusting pH;
(4) Preparing an inclined plane: inoculating LB inclined plane with the fermentation strain glycerol tube for activation culture, and culturing at constant temperature of 25-30 ℃ for 24-48 h to obtain an inclined plane, and refrigerating the inclined plane for later use;
(5) Shake flask seed culture: inoculating the slant strain obtained in the step (4) into a sterilized shake flask filled with a shake flask LB seed culture medium, and culturing at 200 rpm and 25-30 ℃ for about 8 h;
(6) Primary seed culture: inoculating the shake flask seeds cultured in the step (5) into a sterilized 10L first-stage seed tank filled with 5L LB medium according to a volume ratio of 1%, and starting to culture, wherein the culture conditions are as follows: aeration rate is 0.3-1.5 VVM, tank pressure is 0.1Mpa, temperature is 25-30 ℃, saturation of dissolved oxygen is 1-80%, and culture time is 10 h;
(7) Fermenting in a fermentation tank: inoculating the first-level seeds cultured in the step (6) into a sterilized 100L fermentation tank filled with 50L of fermentation medium according to the volume ratio of 5-10%, and starting to culture; culture conditions: initial ventilation of 0.3 VVM, tank pressure of 0.1Mpa, temperature of 25-30 ℃ and cultivation time of 48-96 hours; the dissolved oxygen control scheme is as follows: an initial rotation speed of 0-12 h is 200 rpm, the rotation speed is slowly increased to 350 rpm along with the reduction of dissolved oxygen, and then the dissolved oxygen is controlled by 30% in association with the dissolved oxygen through stirring; 12-36 h, controlling 15% of dissolved oxygen by associating the dissolved oxygen with the feed, 36-h, and finally reducing the stirring speed to 200 rpm, and controlling 10% of dissolved oxygen by associating the dissolved oxygen with the feed.
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CN102002469A (en) * 2010-09-28 2011-04-06 嘉兴学院 Bacterial strain for producing prodigiosin and method thereof
CN102311981A (en) * 2011-07-19 2012-01-11 东南大学 Method for preparing and purifying prodigiosin
CN103627747A (en) * 2013-08-15 2014-03-12 上海理工大学 Solid state fermentation production method for prodigiosin
CN111909864A (en) * 2020-06-29 2020-11-10 山东省食品发酵工业研究设计院 Method for one-bacterium multiple use of serratia marcescens strain

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