A method for producing prodigiosin by fermentation using sugar cane molasses and sugar beet molasses

Abstract

The application discloses a method for producing prodigiosin by fermentation of sugarcane molasses and beet molasses. According to the Serratia marcescens PG-Zeno-001 strain developed by the company in the early stage, on the one hand, the by-product of the sugar industry, sugarcane molasses and beet molasses, is used as the carbon source for the production of prodigiosin by optimizing the formula of the fermentation medium; on the other hand, the method for producing prodigiosin by fermentation using the fermentation medium is provided, and in the large-scale fermentation of a 100L fermentation tank, the prodigiosin yield is up to 16.18 g / L at 48 h. The application can greatly reduce the cost of prodigiosin fermentation production, and is expected to build a new closed loop for the environment-friendly production of the sugar industry.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, specifically to a method for producing styraxin by fermenting sugarcane molasses and beet molasses. Background Technology

[0002] The chemical formula of prodigiosin is C20H25N3O, and its molecular weight is 323.1968 g·mol⁻¹. -1 With a melting point of 151-152℃, it has a methoxypyrrole skeleton structure with three pyrrole rings. Two pyrrole rings are linked by C-Cδ- bonds, and the third pyrrole ring is linked by a methylene group. It has a strong absorption peak at 535 nm in the UV-Vis spectrum and is a red microbial pigment with green reflectivity. Pyroglucinol was first isolated from a secondary metabolite produced by *Serratia marcescens*. In recent years, researchers have also tried using new chassis strains, such as *Streptomyces*, *Vibrio*, and *Pseudomonas putida*, but the results have been unsatisfactory.

[0003] Currently, the most widespread use of squalene is as a dye in the traditional textile printing and dyeing industry. Squalene possesses antibacterial, antimalarial, and antitumor effects, and can induce apoptosis of T and B lymphocytes, attracting the interest of researchers in medicine, pharmaceuticals, and various other industries. Its antibacterial activity against pathogenic microorganisms, regulation of cell motility in synthetic bacteria, and regulation of product transcriptional synthesis, especially its unique mechanism of action in inducing apoptosis in cancer cells, make it considered a potentially promising anticancer drug. Natural squalene exhibits photostability, and micro / nano encapsulation technology is a relatively effective method to improve the stability of natural pigments. Currently, the main encapsulation technologies include spray drying, freeze drying, emulsion, coagulation, liposomes, complexation, and nanoencapsulation. Furthermore, in the field of novel functional materials, due to the pH-responsive color-changing properties of squalene, recent reports in materials science have demonstrated its pH-responsive color-changing properties and biodegradable color-changing films, showing broad application prospects in labeling, packaging, and substrates. However, the yield of squalene from wild-type Serratia marcescens is low, hindering large-scale production. To truly utilize styraxone, breakthroughs are needed in the breeding of high-yielding strains. Literature reports that microwave mutagenesis of *S. marcescens* jx1 increased its styraxone yield from 3.1 g / L to 6.5 g / L. On the other hand, existing styraxone-producing strains generally exhibit poor substrate tolerance, with their biological activity decreasing sharply at high substrate concentrations, thus limiting further yield improvements.

[0004] Furthermore, studies have found that ultraviolet (UV) irradiation affects the stability of erythromycin, and this stability is also influenced by pH; the higher the pH, the greater the effect. The pigment is relatively stable at pH 3, retaining approximately 83% of its original content after 1 hour of UV irradiation, while the content significantly decreases under alkaline conditions. Additionally, it was found that the absorbance of the pigment drops sharply after exposure to direct outdoor light. After 7 hours of irradiation, the pigment loss rate reached as high as 92%, indicating that the pigment is unstable under direct light, possibly due to the destruction of the double bonds in the pyrrole ring of the erythromycin. *Serratia marcescens* is opportunistically pathogenic, and genetic manipulation tools are still immature, making gene modification a lengthy process with potentially limited effectiveness. Traditional mutagenesis screening offers advantages such as high mutation rates, low cost, and high strain stability, making it the most effective method for screening non-model microorganisms.

[0005] Following preliminary research, we used *Serratia marcescens* NRRL B-1481 (purchased from ATCC) as the starting strain and performed combined mutagenesis using ARTP and UV mutagenesis. We further screened the mutant strain by increasing substrate concentration, culture pH, and light intensity, obtaining a high-yield strain of sparganum that is tolerant to high substrate concentrations and light, namely *Serratia marcescens* ZN86 (extraction name *Serratia marcescens* PG-Zeno-001 strain, deposited at the China Center for Type Culture Collection on March 20, 2023, accession number CCTCC NO: M 2023361). We also provided a method for fermentation using this strain.

[0006] According to reports, sucrose is mostly used as a carbon source in the production of sucrose, resulting in high costs and significant sucrose consumption during fermentation, which easily competes with the human food chain. For example, CN102002469B discloses a method for fermenting sucrose to produce sucrose, in which the carbon source of the fermentation medium is one or more of glycerol, mannitol, sucrose, glucose, fructose, soybean oil, and sesame oil, with an optimal sucrose yield of 9.10 g / L. CN103436476B discloses a sucrose-producing bacterium and its preparation method and application, in which the fermentation medium contains per liter: 0.5-1 g sodium chloride, 1 g potassium dihydrogen phosphate, 3 g dipotassium hydrogen phosphate, 1-1.2 g ammonium chloride, 1 g magnesium sulfate, 2 g ammonium nitrate, 20-50 g fructose or sucrose, 0.1-0.5 g yeast powder, with the remainder being water, and a pH of 7.0-8.0; the yield of sucrose can reach 120 mg / L after 24 hours of fermentation. CN111548977B discloses an engineered strain of Serratia marcescens and its application in the production of styraxone. The fermentation medium contains 15–25 g / L sucrose, 10–20 g / L beef extract, 5–15 g / L CaCl2, 5–10 g / L proline, 0.1–0.3 g / L MgSO4·7H2O, and 0.04–0.08 g / L FeSO4·7H2O. The yield of styraxone after 96 hours of fermentation is approximately 5.83 g / L. CN113549643A discloses a method for improving the synthesis of squalene by *Serratia marcescens* through overexpression of the gene *psrB*. The fermentation medium consists of 1.5–2.5% sucrose, 1.0–2.0% beef extract, 0.75–1.25% CaCl2, 0.5–1.0% L-proline, and 0.0025–0.0035% MgSO4·7H2O. The yield of squalene reaches 6.84 g / L after 72 hours of fermentation. The aforementioned patent literature all use sucrose or similar materials as carbon sources, resulting in high fermentation costs and low squalene yields (e.g., not exceeding 10 g / L), leading to persistently high production costs for squalene. Therefore, finding alternative carbon sources to reduce the production cost of squalene is of great significance.

[0007] Molasses is a byproduct of the sugar industry, and can be classified into sugarcane molasses, beet molasses, and soybean molasses, depending on the raw material used. The nutritional composition of different types of molasses varies. Carbohydrates account for about 60% of the total solids in molasses, and it contains a large amount of fermentable sugars (mainly sucrose). Its low cost makes it an excellent fermentation substrate, significantly reducing production costs. Due to the unique properties of molasses, it is currently only sold as animal feed, and there are few reports of its use in large-scale chemical production. Therefore, the development and utilization of molasses as a raw material is particularly important.

[0008] CN106754550A discloses the cultivation and application of the Serratia PFMP-5 strain for controlling root-knot nematode disease in vegetables. Claim 3 states that "the culture medium is NA medium, with a formula of 3% molasses, 10% ammonium sulfate, 5% peptone, 0.5% calcium carbonate, and the remainder being water." However, paragraphs 38-45 of the detailed embodiments section of the specification state that "the culture medium used in steps 1, 2, and 3 is NA medium. The culture medium used in step 4 has a formula of 3% molasses, 10% ammonium sulfate, 5% peptone, 0.5% calcium carbonate, and the remainder being water." Firstly, the content of this document is contradictory, making it impossible to determine whether steps 1-3 and step 4 use the same NA medium (according to the claims) or a different culture medium formula (according to the embodiments in the specification). Secondly, "NA medium" in this field generally refers specifically to "Nutrient Agar" (NA), which typically uses beef extract powder and peptone as nitrogen sources and agar as a solidifying agent (see the YY / T 0577-2005 standard "Nutrient Agar Medium" issued by the State Food and Drug Administration). It is clearly unsuitable for liquid seed culture and mixed fermentation culture. However, this document uses NA medium for both liquid seed culture (paragraph 44 of the specification) and fermentation culture (claims 1-3), leading those skilled in the art to reasonably doubt that it can achieve the intended purpose of the invention. Thirdly, the carbon source used in this document is glucose and glycerol (paragraph 46 of the specification), not molasses. This indicates that the document does not explicitly teach the use of molasses to replace glucose or other materials as a fermentation carbon source. Finally, molasses comes from various sources, and different sources can lead to significant differences in fermentation efficiency. This document does not disclose the specific source of its molasses. Therefore, based on the content disclosed in this document, those skilled in the art still do not understand how to substantially reduce the cost of styraxin fermentation production. Summary of the Invention

[0009] To address the aforementioned problems in the existing technology, this invention provides a novel fermentation production process for squalene, thereby improving the product conversion rate and reducing the production cost of squalene.

[0010] In a first aspect, the present invention provides a fermentation medium for producing styraxone, comprising: 5–7.5 g / L tryptone, 2.5–4 g / L peanut meal, 2.5–4.0 g / L CaCl2, 4.8–5.2 g / L sodium chloride, 20–25 mL / L cane molasses or 25–30 mL / L beet molasses, 0.5–1 g / L glycine, 0.5–1 g / L proline, and 0.8–1.2 mL / L antifoaming agent. The pH of the fermentation medium is adjusted to 6.0 using hydrochloric acid, and the medium is sterilized by moist heat at 121°C for 20 minutes, then cooled before use.

[0011] Apart from the sucrose naturally present in molasses, this fermentation medium does not contain any additional commonly used carbon sources such as glycerol, mannitol, sucrose, glucose, fructose, vegetable oil, or animal oil, thereby reducing fermentation costs.

[0012] Molasses is readily available through commercial channels and is produced by concentrating the waste soaking liquid from the production of edible sugar, making it extremely inexpensive. Through extensive research, the inventors discovered that fermentation media prepared using sugarcane molasses or beet molasses instead of sucrose as the carbon source are most suitable for the company's self-developed sucralose-producing strain PG-Zeno-001, and determined the optimal concentrations to be 20 mL / L for sugarcane molasses and 25–30 mL / L for beet molasses.

[0013] In a second aspect, the present invention provides a method for producing squalene by fermentation, characterized in that *Serratia marcescens* is fermented using the culture medium described above to produce squalene. The *Serratia marcescens* strain is *Serratia marcescens* PG-Zeno-001, which was deposited at the China Center for Type Culture Collection on March 20, 2023, with accession number CCTCCNO: M 2023361.

[0014] Preferably, the fermentation production method of styrax erythrin includes the following steps:

[0015] (1) Selection of fermentation strain: The strain was Serratia marcescens PG-Zeno-001;

[0016] (2) Preparation of fermentation medium: tryptone 5-7.5 g / L, peanut cake powder 2.5-4 g / L, CaCl2 2.5-4.0 g / L, sodium chloride 5.0 g / L, sugarcane molasses 20 mL / L or beet molasses 30 mL / L, glycine 0.5-1 g / L, proline 0.5-1 g / L, defoamer 1 mL / L, adjust the pH of the medium to 6.0 with hydrochloric acid, sterilize at 121℃ for 20 minutes, and cool before use;

[0017] (3) Preparation of feed culture medium: 300 g / L tryptone, 50 g / L peanut cake powder, 400 mL / L sugarcane molasses or 600 mL / L beet molasses, 10 g / L proline, without adjusting pH, sterilize at 121℃ for 20 minutes, and then cool down for later use;

[0018] (4) Preparation of slant: The above-mentioned fermentation strain was inoculated into LB slant for activation culture and cultured at 25-30℃ for 24-48 h to obtain slant. The slant was then stored in the refrigerator for later use.

[0019] (5) Shake flask seed culture: The slant strain obtained in step (4) was inoculated into a sterilized shake flask containing shake flask LB seed medium and cultured at 25-30℃ for 8 h at 200 rpm.

[0020] (6) Primary seed culture: The shake flask seeds cultured in step (5) were inoculated at a volume ratio of 1% into a sterilized 10 L primary seed tank containing 5 L of LB medium and culture was started. The culture conditions were: aeration rate of 0.3 to 1.5 VVM, tank pressure of 0.1 MPa, temperature of 25 to 30°C, dissolved oxygen saturation of 1% to 80%, and culture time of 10 h.

[0021] (7) Fermentation in a fermenter: The primary seed cultured in step (6) is inoculated at a volume ratio of 5% to 10% into a sterilized 100 L fermenter containing 50 L of fermentation medium and culture begins. Culture conditions: initial aeration rate of 0.3 VVM, tank pressure of 0.1 MPa, temperature of 25 to 30°C, culture time of 48 to 96 hours. The dissolved oxygen control scheme is as follows: for 0-12 h, the initial stirring speed is 200 rpm, and as the dissolved oxygen decreases, the stirring speed is slowly increased to 350 rpm. Then, the dissolved oxygen is controlled at 30% by stirring and the dissolved oxygen is correlated. For 12 to 36 h, the dissolved oxygen is controlled at 15% by stirring and the feeding is correlated. For 36 h to the end of fermentation, the stirring speed is reduced to 200 rpm, and the dissolved oxygen is controlled at 10% by stirring and the feeding is correlated.

[0022] Unlike CN106754550A, the feed culture medium of this invention does not contain glucose and glycerol, but still uses sugarcane molasses or beet molasses as the fermentation carbon source, thereby greatly reducing the cost of fermentation production of styraxone.

[0023] Based on the above technical solutions, the present invention can achieve the following beneficial effects:

[0024] 1. In response to the national dual-carbon goals, this invention innovatively uses low-cost sugarcane molasses as the carbon source for the large-scale production of lecithin, reshaping the new process for the low-cost large-scale production of lecithin. In this process, it was surprisingly discovered that, compared with other carbon sources, sugarcane molasses and beet molasses have a better substitution effect on sucrose, which is expected to build a new closed loop for environmentally friendly production in the traditional sugar industry.

[0025] 2. The fermentation medium with optimized composition was used for the fermentation production of styraxone. In large-scale fermentation in a 100 L fermenter, the yield of styraxone could reach up to 16.18 g / L in 48 h. Attached Figure Description

[0026] Figure 1 Bar chart showing cell wet weight and pyruvic rubin content under different carbon source conditions during shake flask fermentation;

[0027] Figure 2 Bar chart showing the wet weight of mycelium and the content of lecithin under different concentrations of sugarcane molasses and beet molasses during shake-flask fermentation;

[0028] Figure 3 Fermentation progress curve for 100 L fermentation using sugarcane molasses;

[0029] Figure 4 Fermentation progress curve for 100 L fermentation using beet molasses. Detailed Implementation

[0030] Example 1: Screening of carbon sources for fermentation production

[0031] To reduce the carbon source cost of sucralose fermentation and determine the optimal carbon source, we selected sucrose (5 g / L), glycerol, glucose, cane molasses, beet molasses, soybean molasses, corn starch hydrolysate, wheat starch hydrolysate, and sweet potato starch hydrolysate as alternative carbon sources and conducted shake-flask experiments. The operating procedures are as follows:

[0032] (1) Sucrose, glycerol, glucose, sugarcane molasses, beet molasses and soybean molasses can all be obtained through commercial channels, while starchy raw materials such as corn starch hydrolysate, wheat starch hydrolysate and sweet potato starch hydrolysate are obtained by processing with starch hydrolyzing enzymes purchased from Novozymes (China) Investment Co., Ltd.

[0033] (2) Preparation of fermentation strain: The strain was Serratia marcescens PG-Zeno-001, which was stored in a glycerol tube;

[0034] (3) Preparation of slant: The above fermentation strain was inoculated from the glycerol tube into LB slant for activation culture. The culture was kept at 25-30℃ for 24-48 hours to obtain slant. The slant was then refrigerated for later use.

[0035] (4) Test tube seed culture: The slant strain obtained in step (2) is inoculated into a sterile test tube containing 5 mL of LB seed medium and cultured at 25-30℃ for about 8 h.

[0036] (5) Preparation of fermentation medium: tryptone 5-7.5 g / L, peanut cake powder 2.5-4 g / L, CaCl2 2.5-4.0 g / L, sodium chloride 5.0 g / L, the concentration of the selected carbon source is adjusted to be basically the same as that of sucrose or glucose, glycine 0.5-1 g / L, proline 0.5-1 g / L, defoamer 1 mL / L; adjust the pH of the medium to 6.0 with hydrochloric acid; sterilize at 121℃ for 20 minutes, and then cool to room temperature for later use; change the concentration of carbon source mother liquor according to the experimental requirements. The carbon source mother liquor of the experimental group is shown in Table 1 (groups 1-9).

[0037] (6) Fermentation culture: The test tube seed cultured in step (3) was inoculated at a volume ratio of 1% into a sterilized 500 mL shake flask containing 100 mL of fermentation medium and culture was started. The culture conditions were: temperature 25-130℃, 200 rpm. After 48 hours of culture, the wet weight of the cell and the unit yield of styraxin were detected. The results of each group are shown in Table 1.

[0038] Table 1: Results of shake-flask experiments with different carbon sources

[0039]

[0040] First, different single carbon sources were compared, namely sucrose, glycerol, glucose, cane molasses, beet molasses, soybean molasses, corn starch hydrolysate, wheat starch hydrolysate, and sweet potato starch hydrolysate, and shake-flask fermentation was conducted for screening. The results are shown in Table 1. Among the three analytical-grade carbon sources (sucrose, glycerol, and glucose), sucrose showed the best fermentation effect, with a cell wet weight reaching 20.4 g / L and a final sucralose yield of 3.5 g / L, similar to the results reported in the literature. Surprisingly, glucose showed the worst fermentation effect, with a cell biomass only 44.1% of that in the sucrose-using experimental group, and a final sucralose yield of only 0.4 g / L. These results indicate that sucrose is the optimal pure carbon source for Serratia marcescens to produce and accumulate pigments. In the experimental group using molasses as raw material, both sugarcane molasses and beet molasses showed excellent substitution effects for sucrose, with the highest final cell wet weight reaching 25.6 g / L and the highest sucrose content reaching 4.9 g / L, exceeding the sucrose control group by 58%. This may be because molasses, as a complex carbon source, is rich in many nutrients besides sucrose, such as amino nitrogen, biotin, and some crude protein, which have a positive regulatory effect on the production and pigment accumulation of *Serratia marcescens*, thus leading to high sucrose production. In contrast, the growth of strains in the experimental group using starch hydrolysate was limited. This may be because the incomplete hydrolysis products of starch are starch, glucose, and some oligosaccharides. The glucose metabolism pathway in *Serratia marcescens* PG-Zeno-001 is relatively weak, leading to growth inhibition and consequently, lower pigment accumulation.

[0041] Based on the results of Example 1, the optimal carbon source for the large-scale production of lecithin was finally determined to be 25 mL / L sugarcane molasses and beet molasses, and this was used for subsequent optimization and fermentation production.

[0042] Example 2: Determination of the optimal concentration of sugarcane molasses and beet molasses

[0043] Based on the results of Example 1, we found that both 25 mL / L cane molasses and beet molasses could achieve good production results, so we further optimized the fermentation concentration.

[0044] Through shake-flask fermentation results at different concentrations, it was determined that when the concentration of cane molasses in the culture medium was increased from 5 mL / L to 20 mL / L, both the cell wet weight and the content of thiophanate-methyl reached their peak values. With further increases in concentration, both the cell wet weight and the thiophanate-methyl content in the fermentation broth showed a slight decrease. Therefore, the optimal concentration of cane molasses in the culture medium was determined to be 20 mL / L, and this concentration was used for subsequent large-scale fermentation of thiophanate-methyl. Similar results also determined the optimal concentration of beet molasses to be 30 mL / L.

[0045] Table 2: Results of shake-flask fermentation experiments with different carbon source concentrations

[0046]

[0047] Example 3: Method for detecting the content of pyruvic rubigin in fermentation broth

[0048] Take 1 mL of well-mixed Serratia marcescens fermentation broth, add 4 mL of acidic methanol stock solution, sonicate to disrupt cells for 15-20 min, centrifuge at low temperature to remove precipitate, dilute the supernatant 5 times, and then dilute it according to different gradients. Take 200 μL of each and add it to a 96-well plate to measure the absorbance at 535 nm. The pH of the acidic methanol is approximately 3.0.

[0049] Example 4: Large-scale production of styraxin in a 100L fermenter

[0050] The yield of styraxone produced in a 100L fermenter was verified according to the final fermentation medium ratio determined in Examples 1 and 2.

[0051] (1) Selection of fermentation strain: The strain was Serratia marcescens PG-Zeno-001;

[0052] (2) Preparation of fermentation medium: tryptone 5-7.5 g / L, peanut cake powder 2.5-4 g / L, CaCl2 2.5-4.0 g / L, sodium chloride 5.0 g / L, sugarcane molasses 20 mL / L or beet molasses 30 mL / L, glycine 0.5-1 g / L, proline 0.5-1 g / L, antifoaming agent 1 mL / L; adjust the pH of the medium to 6.0 with hydrochloric acid; sterilize by moist heat at 121℃ for 20 minutes, and then cool to room temperature for use.

[0053] (3) Preparation of supplemental culture medium: 300 g / L tryptone, 50 g / L peanut cake powder, 400 mL / L sugarcane molasses or 600 mL / L beet molasses, 10 g / L proline, without adjusting pH, sterilize at 121℃ for 20 minutes, and then cool to room temperature for later use;

[0054] (4) Preparation of slant: The above-mentioned fermentation strain was inoculated into LB slant for activation culture and cultured at 25-30℃ for 24-48 h to obtain slant. The slant was then stored in the refrigerator for later use.

[0055] (5) Shake flask seed culture: The slant strain obtained in step (4) is inoculated into a sterilized shake flask containing shake flask LB seed culture medium and cultured at 200 rpm at 25-30℃ for about 8 h.

[0056] (6) Primary seed culture: The shake flask seeds cultured in step (5) were inoculated at a volume ratio of 1% into a sterilized 10 L primary seed tank containing 5 L of LB medium and culture was started. The culture conditions were: aeration rate of 0.3 to 1.5 VVM, tank pressure of 0.1 MPa, temperature of 25 to 30°C, dissolved oxygen saturation of 1% to 80%, and culture time of 10 h.

[0057] (7) Fermentation in a fermenter: The primary seed cultured in step (6) is inoculated at a volume ratio of 5% to 10% into a sterilized 100 L fermenter containing 50 L of fermentation medium, and the culture begins. The culture conditions are: initial aeration rate of 0.3 VVM, tank pressure of 0.1 MPa, temperature of 25 to 30°C, and culture time of 48 to 96 hours. The dissolved oxygen control scheme is as follows: the initial stirring speed is 200 rpm for 0-12 h, and the stirring speed is slowly increased to 350 rpm as the dissolved oxygen decreases. Then, the dissolved oxygen is controlled at 30% by linking stirring with dissolved oxygen. From 12 to 36 h, the dissolved oxygen is controlled at 15% by linking dissolved oxygen with feeding. From 36 h to the end of fermentation, the stirring speed is reduced to 200 rpm, and the dissolved oxygen is controlled at 10% by linking dissolved oxygen with feeding.

[0058] (8) Fermentation broth samples were taken every 4 hours to determine the concentration of styraxone using the method in Example 2. The results are as follows: Figure 3 and Figure 4As shown, the yield of thiamethoxam using sugarcane molasses fermentation for 48 hours can reach 15.45 g / L; while the yield of thiamethoxam using beet molasses fermentation for 48 hours can reach 16.18 g / L.

[0059] The specific description of the above embodiments should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made to the present invention by those skilled in the art based on the above description shall fall within the scope of protection of the present invention.

Claims

1. A method for producing squalene by fermentation, comprising fermenting *Serratia marcescens* in a fermentation medium to produce squalene, characterized in that: The *Serratia marcescens* strain is *Serratia marcescens* PG-Zeno-001, which was deposited at the China Center for Type Culture Collection on March 20, 2023, with accession number CCTCC NO: M 2023361. The fermentation medium comprises: 5–7.5 g / L tryptone, 2.5–4 g / L peanut meal, 2.5–4.0 g / L CaCl2, 4.8–5.2 g / L sodium chloride, 20–25 mL / L cane molasses or 25–30 mL / L beet molasses, 0.5–1 g / L glycine, 0.5–1 g / L proline, and 0.8–1.2 mL / L defoamer.

2. The method for producing erythromycin by fermentation according to claim 1, characterized in that: The fermentation medium does not contain any of the following: glycerol, mannitol, sucrose, glucose, fructose, vegetable oil, or animal oil.

3. The method for producing erythromycin by fermentation according to claim 1, characterized in that: The pH of the fermentation medium was adjusted to 6.0 using hydrochloric acid, and then the fermentation medium was sterilized by moist heat at 121°C for 20 minutes. After cooling, it was ready for use.

4. The method for producing erythromycin by fermentation according to claim 1, characterized in that: During fermentation, a feed medium is added, which contains: 300 g / L tryptone, 50 g / L peanut meal, 400 mL / L sugarcane molasses or 600 mL / L beet molasses, and 10 g / L proline.

5. The method for producing erythromycin by fermentation according to claim 4, characterized in that: The feed medium does not contain any of the following: glycerol, mannitol, sucrose, glucose, fructose, vegetable oil, or animal oil.

6. The method for producing erythromycin by fermentation according to claim 5, characterized in that: After the feed culture medium is prepared, the pH is not adjusted. It is directly sterilized by moist heat at 121°C for 20 minutes and then cooled before use.

7. The method for producing styrax erythrin by fermentation according to any one of claims 1-6, characterized in that... Includes the following steps: (1) Selection of fermentation strain: The strain was Serratia marcescens PG-Zeno-001; (2) Preparation of fermentation medium: tryptone 5-7.5 g / L, peanut cake powder 2.5-4 g / L, CaCl2 2.5-4.0 g / L, sodium chloride 4.8-5.2 g / L, sugarcane molasses 20-25 mL / L or beet molasses 25-30 mL / L, glycine 0.5-1 g / L, proline 0.5-1 g / L, defoamer 0.8-1.2 mL / L; adjust the pH of the fermentation medium to 6.0 with hydrochloric acid, sterilize at 121℃ for 20 minutes, and cool before use; (3) Preparation of feed culture medium: 300 g / L tryptone, 50 g / L peanut cake powder, 400 mL / L sugarcane molasses or 600 mL / L beet molasses, 10 g / L proline, without adjusting pH, sterilize at 121℃ for 20 minutes, and then cool down for later use; (4) Preparation of slant: The above-mentioned fermentation strain was inoculated into LB slant for activation culture and cultured at 25-30℃ for 24-48 h to obtain slant. The slant was then stored in the refrigerator for later use. (5) Shake flask seed culture: The slant strain obtained in step (4) is inoculated into a sterilized shake flask containing shake flask LB seed culture medium and cultured at 200 rpm at 25-30℃ for 8 h. (6) Primary seed culture: The shake flask seeds cultured in step (5) were inoculated at a volume ratio of 1% into a sterilized 10 L primary seed tank containing 5 L of LB medium and culture was started. The culture conditions were: aeration rate of 0.3 to 1.5 VVM, tank pressure of 0.1 MPa, temperature of 25 to 30°C, dissolved oxygen saturation of 1% to 80%, and culture time of 10 h. (7) Fermentation in a fermenter: The primary seed cultured in step (6) is inoculated at a volume ratio of 5% to 10% into a sterilized 100 L fermenter containing 50 L of fermentation medium and culture begins. Culture conditions: initial aeration rate of 0.3 VVM, tank pressure of 0.1 MPa, temperature of 25 to 30°C, culture time of 48 to 96 hours. The dissolved oxygen control scheme is as follows: for 0-12 h, the initial stirring speed is 200 rpm, and as the dissolved oxygen decreases, the stirring speed is slowly increased to 350 rpm. Then, the dissolved oxygen is controlled at 30% by the linkage between stirring and dissolved oxygen. For 12 to 36 h, the dissolved oxygen is controlled at 15% by the linkage between dissolved oxygen and feeding. For 36 h to the end of fermentation, the stirring speed is reduced to 200 rpm, and the dissolved oxygen is controlled at 10% by the linkage between dissolved oxygen and feeding.

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