Serratia marcescens strain, and use thereof in co-production of prodigiosin, chitinase and lipase

Abstract

Disclosed in the present invention are a Serratia marcescens strain, and the use thereof in co-production of prodigiosin, chitinase and lipase. The Serratia marcescens strain is classified and named Serratia marcescens LT-7 and has been preserved in China Center for Type Culture Collection with the accession number CCTCC NO: M20231646, the preservation date being September 07 2023. The strain synthesizes prodigiosin, chitinase and lipase by means of fermentation by using molasses as a carbon source and tryptone as a nitrogen source; and during a coproduction process, the concentration of prodigiosin accumulated in a culture medium reaches up to 20.70 g / L, the activity of chitinase is 49.78 U / mL and the activity of the lipase is 86.92 U / mL. The operation method disclosed by the present invention is simple, effective and low-cost, has a great industrial production prospect, and provides an innovative process for the biosynthesis of prodigiosin, chitinase and lipase.

Description

A strain of Serratia marcescens and its application in the co-production of prodigiosin, chitinase and lipase Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a strain of Serratia marcescens and application thereof in the co-production of prodigiosin, chitinase and lipase. Background Art

[0002] Prodigiosins (PGs) are a class of secondary metabolites produced by actinomycetes and bacteria. They are fat-soluble pigments. Their skeleton contains a common cyclic structure of pyrrole and bispyrromethene groups. They are virtually insoluble in water, soluble in alcohols and highly polar solvents such as acetone and pyridine, but insoluble in less polar solvents such as petroleum ether, ether, and ethyl acetate. Research has shown that PGs possess multiple physiological activities, including antibacterial, antitumor, and immunosuppressive properties, as well as nutritional benefits and as natural dyes, offering unique advantages for use in the pharmaceutical and textile industries.

[0003] Chitin, also known as chitin, is a polysaccharide extracted from the shells of marine crustaceans. Its natural abundance is second only to cellulose, and the physiological and biochemical functions of its degradation product, chito-oligosaccharides, are increasingly attracting attention. However, the industrial production of chito-oligosaccharides involves multiple steps and is costly. Therefore, using chitinase to biodegrade chitin to produce chito-oligosaccharides is a viable approach. Chitin is also a crucial component of the peritrophic membranes of insects, the eggshells of plant pathogenic nematodes, and the cell walls of plant fungi, primarily serving as a supporting skeleton and providing protection. Chitinase holds great promise for application in plant pest control.

[0004] Lipase, also known as triacylglycerol acylhydrolase, is a hydrolase that catalyzes the hydrolysis, alcoholysis, esterification, transesterification, and reverse synthesis of triacylglycerols and other water-insoluble esters. Lipase is widely available, found in some microorganisms, the pancreas of animals, and the seeds of oilseed crops. Lipase has broad application prospects in food processing, medicine, cosmetics, and biodiesel. In food processing, lipase can improve the taste and texture of pastries, meat products, and dairy products, enhancing food quality while reducing production costs and pollution. In other fields, lipase can be used to prepare pharmaceutical intermediates, cosmetic raw materials, and biodiesel production, demonstrating its high application value and market prospects.

[0005] Currently, the preparation of prodigiosin, chitinase, and lipase requires separate fermentation of different strains. S. marcescens SOCE 001, after fermentation optimization, obtained a prodigiosin concentration of 2.468 g / L, Bacillus thuringiensis Bt028, after fermentation optimization, obtained a chitinase activity of 10.48 U / mL, and Moesziomyces aphidis, after fermentation optimization, obtained a lipase activity of 83.14 U / mL. These strains produce low yields of single products, take a long time, are costly, and have low efficiency.

[0006] There are currently no reports on the co-production of prodigiosin, chitinase and lipase using Serratia marcescens fermentation. Summary of the Invention

[0007] Purpose of the invention: The problem to be solved by the present invention is to provide a strain of Serratia marcescens that has the ability to ferment prodigiosin, chitinase and lipase.

[0008] The technical problem that the present invention also aims to solve is to provide the use of the above-mentioned Serratia marcescens in the co-production of prodigiosin, chitinase and lipase.

[0009] Technical solution: To solve the above problems, the technical solution adopted by the present invention is as follows:

[0010] The present invention screened and obtained a strain of Serratia marcescens, which was classified and named Serratia marcescens LT-7. It has been deposited in the China Center for Type Culture Collection (CCTCC), with a deposit address of 430072, Wuhan City, Hubei Province, Bayi Road, Hongshan District, Wuhan University, Shandong Type Culture Collection, and a deposit number of CCTCC NO: M20231646. The deposit date is September 7, 2023. The following content uses this strain as the production strain.

[0011] The strain has the following properties:

[0012] The nucleotide sequence of the strain's 16S rDNA gene is 1471 bp long, and its gene sequence is shown in SEQ ID NO: 1. The sequence was compared for homology using the BLAST program from the Gene Bank database, and a phylogenetic tree based on the complete 16S rDNA sequence was constructed. The results showed that the strain shared 100% homology with Serratia marcescens strain JW-CZ2. Based on morphological observations and physiological and biochemical analysis of the strain, the strain used in the present invention was identified as Serratia marcescens, and designated Serratia marcescens LT-7.

[0013] The application of the above-mentioned Serratia marcescens LT-7 in the co-production of prodigiosin, chitinase and lipase also falls within the scope of protection of the present invention. Serratia marcescens LT-7 is inoculated into a fermentation medium and aerobic culture is performed to produce prodigiosin, chitinase and lipase.

[0014] The specific application method is to inoculate Serratia marcescens LT-7 into a slant solid culture medium, then transfer it to a seed culture medium, and finally inoculate it into a fermentation medium for aerobic culture. The fermentation broth is rich in prodigiosin, chitinase and lipase.

[0015] The Serratia marcescens LT-7 of the present invention and its use in preparing prodigiosin, chitinase and lipase comprise the following steps in sequence:

[0016] 1. Culture medium preparation:

[0017] (1a) The slant culture medium comprises: 10 g / L tryptone, 3 g / L yeast extract powder, 10 g / L sodium chloride, 20 g / L agar powder, water as the solvent, and a pH value of 6.0 to 7.0, preferably 6.8.

[0018] (1b) The liquid seed culture medium comprises: 10 g / L tryptone, 10 g / L sucrose, 6 g / L calcium chloride, the solvent is water, and the pH value is 6.0-7.0, preferably 6.8.

[0019] (1c) The solid seed culture medium comprises: 10 g / L tryptone, 10 g / L sucrose, 6 g / L calcium chloride, 20 g / L agar powder, water as solvent, and a pH value of 6.0 to 7.0, preferably 6.8.

[0020] (1d) The fermentation medium comprises the following components: 20-30 g / L carbon source, 10-15 g / L nitrogen source, 1-5 g / L metal salt, 1-3 g / L soybean powder, pH 6.0-7.0, preferably 6.8, and water as the solvent.

[0021] Wherein, the carbon source is any one or a combination of sucrose, glycerol, mannitol, maltose, glucose, and molasses; preferably molasses, and the addition amount of the molasses is preferably 20 g / L;

[0022] The nitrogen source is any one or a combination of beef extract, tryptone, yeast extract, corn steep liquor, soybean meal, peanut meal, soybean meal, wheat germ meal, yeast powder, and peptone, preferably tryptone, and the addition amount of the tryptone is preferably 12 g / L;

[0023] The metal salt is one or a combination of calcium chloride, zinc sulfate, ferrous sulfate, manganese chloride, magnesium sulfate, and sodium chloride, preferably calcium chloride, and the addition amount of the calcium chloride is preferably 3 g / L;

[0024] The addition amount of the soybean powder is preferably 3g / L;

[0025] The most preferred fermentation medium comprises the following components: 20 g / L molasses, 12 g / L tryptone, 3 g / L calcium chloride, 20 g / L molasses, 3 g / L soy flour, water as the solvent, and the initial pH of the fermentation broth is adjusted to 6.8 using sodium bicarbonate.

[0026] 2. Strain selection:

[0027] The deposited strain is Serratia marcescens LT-7.

[0028] 3. Bacteria activation:

[0029] The Serratia marcescens LT-7 strain was inoculated into a slant culture medium and cultured at 20-30°C for 16-36 hours. A single colony was streaked onto a common solid culture medium and cultured at 20-30°C for 16-36 hours to obtain an activated strain for use.

[0030] 4. Seed solution preparation:

[0031] Take the activated bacteria in step 3, inoculate 1 loop into the seed liquid shake flask under sterile conditions, place it on a shaker with a speed of 200 rpm, and culture it in the dark at a temperature of 24-30°C for 12 hours to obtain the fermentation seed liquid.

[0032] 5. Shake flask fermentation culture:

[0033] The fermentation seed liquid in step 3 is inoculated into a fermentation medium shake flask at a 3% (v / v) inoculation rate under sterile conditions, placed on a shaker at a speed of 200 rpm, and cultured at 24-32° C. in the dark for 36-48 hours; when the concentrations of prodigiosin, chitinase, and lipase in the fermentation broth substantially no longer increase, the fermentation is stopped.

[0034] 6. Fermentation culture in fermentation tank:

[0035] The fermentation seed liquid in step 4 is inoculated into a fermentation tank at an inoculum rate of 8% (v / v) under sterile conditions, with a liquid volume of 3L / 5L, a rotation speed of 200-500rpm, an aeration ratio of 0.8-1.0VVM, a culture temperature of 24-32°C, an initial pH of 6.0-7.0, and cultured for 24-72h. When the concentrations of prodigiosin, chitinase, and lipase in the fermentation broth basically no longer increase, the fermentation is stopped.

[0036] 7. Extraction of prodigiosin

[0037] (7a) Crude product preparation

[0038] The fermentation broth of Serratia marcescens LT-7 in step 5 or step 6 was centrifuged at 9000 rpm, the precipitated cells were extracted with methanol (pH 3) solution for 20 min and then extracted overnight to obtain a cell-treated liquid, which was concentrated to a paste, and ethyl acetate with a volume of 2 times that of the concentrate was added to dissolve it, the precipitate was removed by filtration, and the supernatant was concentrated and freeze-dried to obtain a crude prodigiosin.

[0039] (7b) Separation

[0040] The crude prodigiosin obtained in step (7a) was dissolved in 25 volumes of acetone for later use. Prodigiosin was separated and purified using a silica gel chromatography column. The mobile phase was petroleum ether:acetone (5:2), and the silica gel was 200-300 mesh. The components were collected and filtered through a 0.22 μm filter membrane and placed in a sample injection bottle for later use. The sample was detected by high performance liquid chromatography using a Sepax C18 column, mobile phase: acetonitrile:ammonium acetate (85:15), flow rate: 1.00 mL / min, injection volume: 10 μL, and column temperature: 30°C.

[0041] (7c) Purification

[0042] According to the analysis of the liquid chromatogram obtained in step (7b), the components containing prodigiosin are mixed, concentrated, and freeze-dried to obtain prodigiosin powder.

[0043] 8. Identification of Prodigiosin

[0044] High performance liquid chromatography, infrared spectrometry and nuclear magnetic resonance were used to identify the fermentation products of Serratia marcescens LT-7.

[0045] 100 mg of the prodigiosin powder obtained in step 7 was dissolved in 100 ml of methanol, and the pH was adjusted to 3 to obtain a prodigiosin methanol solution. The liquid chromatography results are shown in Figure 3. Under the same chromatographic conditions, this substance was compared with the prodigiosin standard. Both showed absorption peaks at the same location, indicating that the fermentation broth contained prodigiosin.

[0046] 0.2 mg of the prodigiosin powder obtained in step 7 was ground with a small amount of KBr and pressed into a tablet to prepare a sample. The infrared spectrum of the sample was then measured using an infrared spectrometer in the scanning range of 4000-400 cm~1. The results are shown in Figure 4. NMR1H and NMR13C detection of the purified product from Serratia marcescens LT-7 fermentation using CD3Cl as the solvent are shown in Figures 5 and 6.

[0047] 9. Determination of prodigiosin content

[0048] (9a) Drawing of the standard curve

[0049] Accurately weigh a constant weight of dry prodigiosin standard and prepare standard methanol solutions at concentrations of 0.05, 0.10, 0.15, 0.20, 0.25, and 0.30 mg / mL under acidic conditions (pH 3). The absorbance at 535 nm was measured using an enzyme-linked immunosorbent assay (ELISA). A standard curve was plotted with absorbance as the ordinate and concentration as the abscissa. The regression equation, shown in Figure (2A), was derived, and the prodigiosin content was calculated based on this standard curve.

[0050] (9b) Determination of prodigiosin content in fermentation broth

[0051] Under acidic conditions (pH 3), methanol was added to the fermentation broth for extraction for 20 minutes. The extract was centrifuged at 9000 rpm for 3 minutes. The absorbance of the centrifuged supernatant at 535 nm was measured using an enzyme-linked immunosorbent assay (ELISA). The measurement was repeated three times for each centrifuge tube, and the average value was taken. The data were derived and the prodigiosin content was calculated by referring to the prodigiosin standard curve.

[0052] 10. Chitinase Content Determination

[0053] (10a) Drawing of the standard curve

[0054] (10a-1) Preparation of standard solution

[0055] Accurately weigh N-acetyl-D-glucosamine powder and add a certain amount of distilled water to prepare 0.5, 1.0, 1.5, 2.0, and 2.5 μmol standard solutions.

[0056] (10a-2) Preparation of Reaction Solution

[0057] The standard solution in (10a-1) was added with an equal amount of dinitrosalicylic acid (DNS) reagent to prepare reaction solutions with different contents of N-acetyl-D-glucosamine.

[0058] (10a-3) Determination

[0059] After mixing, place in a boiling water bath for 10 minutes. After cooling, measure the absorbance at 535 nm. Use test tube No. 0 with only DNS added to set the value to zero. Repeat the measurement three times for each test tube and take the average value. Export the data and plot a standard curve with absorbance as the ordinate and concentration as the abscissa. Obtain the regression equation, as shown in Figure (2B). Calculate the N-acetyl-D-glucosamine content based on this standard curve.

[0060] (10b) Determination of chitinase in fermentation broth

[0061] (10b-1) Serratia marcescens LT-7 fermentation broth was centrifuged at 9000 rpm. The fermentation supernatant was divided into two groups for treatment: 1 ml of the supernatant from the first group was treated with boiling water for 15 min, and 1 ml of the fermentation broth from the second group was left untreated.

[0062] (10b-2) Take 1 mL of the fermentation supernatant from the first group after treatment in step (10b-2), add 500 μL of prepared 2% colloidal chitin (pH = 6) and 500 μL of PBS solution (50 mM, pH 7.0-7.4), and mix well; take the second group as a blank control for the fermentation broth from the first group;

[0063] (10b-3) After uniform mixing, the fermentation broth from step (10b-2) was reacted in a 37°C water bath for 1 hour and centrifuged at 10,000 rpm for 5 minutes. 1 ml of the supernatant was taken from each sample and added to the same volume of DNS solution in a boiling water bath for 10 minutes. After dilution 10-fold, the absorbance of the solution at a wavelength of 535 nm was measured. The chitinase activity was determined by reference to an N-acetyl-D-glucosamine standard curve. The chitinase activity (E) of the supernatant of the fermentation broth of the strain (the second fermentation group) = the activity (E2) of the supernatant of the fermentation group (the first fermentation group) - the activity (E1) of the supernatant of the fermentation group (the first fermentation group).

[0064] 11. Lipase enzyme content determination

[0065] (11a) Preparation of crude enzyme solution

[0066] A certain volume of fermentation broth after shake flask culture was taken and centrifuged at 1000 × 4 ° C for 5 min using a high-speed refrigerated centrifuge. The obtained supernatant containing enzyme was used as the crude enzyme solution for subsequent experiments.

[0067] (11b) Enzyme activity determination method

[0068] Lipase activity was determined using p-nitrophenyl fatty acid ester as a substrate. The standard assay reaction mixture contained 50 mmol / L phosphate buffer (280 μL, pH 7), 30 mmol / L p-NPA (10 μL), and crude enzyme solution (10 μL). The reaction was incubated at 40°C for 5 minutes, followed by the addition of 100 μL of 0.5 mol / L Na₂CO₃ (500 μL) to terminate the reaction. One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol (405 nm) per minute from p-NPA. Beneficial effects:

[0069] The present invention screened and obtained a strain that co-produces prodigiosin, chitinase, and lipase. This strain can ferment and synthesize prodigiosin using inexpensive molasses as a carbon source, and can also co-prodiginosin and chitinase. The maximum concentration of prodigiosin accumulated in the culture medium is 20.70 g / L, the maximum chitinase activity is 49.78 U / mL, and the maximum lipase activity is 86.92 U / mL. This method not only has low production costs but also is simple to operate, which is of great significance for the production and expanded application of prodigiosin, chitinase, and lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG1 is an agarose gel electrophoresis image of 16S rDNA PCR-purified Serratia marcescens LT-7 (A) and a phylogenetic tree of Serratia marcescens LT-7 (B).

[0071] FIG2 is a standard curve diagram of prodigiosin production (A) and chitinase production (B) by Serratia marcescens LT-7.

[0072] FIG3 is a high performance liquid chromatogram of prodigiosin produced by Serratia marcescens LT-7.

[0073] FIG4 is an infrared spectrum of prodigiosin produced by Serratia marcescens LT-7.

[0074] FIG5 is a nuclear magnetic resonance (NMR) 1H spectrum of prodigiosin produced by Serratia marcescens LT-7.

[0075] FIG6 is a nuclear magnetic resonance (NMR) 13C spectrum of prodigiosin produced by Serratia marcescens LT-7.

[0076] Figure 7 shows the effect of carbon source type on the synthesis of prodigiosin, chitinase and lipase by Serratia marcescens LT-7.

[0077] FIG8 shows the effect of carbon source concentration on the synthesis of prodigiosin, chitinase and lipase by Serratia marcescens LT-7.

[0078] Figure 9 shows the effect of nitrogen source type on the synthesis of prodigiosin, chitinase and lipase by Serratia marcescens LT-7.

[0079] FIG10 shows the effect of nitrogen source concentration on the synthesis of prodigiosin, chitinase and lipase by Serratia marcescens LT-7.

[0080] Figure 11 shows the process curve of the fed-batch synthesis of prodigiosin, chitinase and lipase in a 50 L fermenter.

[0081] Figure 12 shows the process curve of the fed-batch synthesis of prodigiosin, chitinase and lipase in a 1 t fermenter. DETAILED DESCRIPTION

[0082] The present invention can be better understood according to the following examples, and those skilled in the art will easily understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0083] Example 1: Isolation and screening of Serratia marcescens LT-7.

[0084] The composition of the culture medium used in this embodiment is as follows:

[0085] LB solid medium: peptone 10 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, agar 20 g / L, balance water, natural pH, sterilized at 121°C for 20 min.

[0086] The specific operation process of this embodiment is as follows:

[0087] The steps for screening pigment-producing bacteria are as follows: 1 g of each was added to a flask containing a sterile enrichment medium and cultured at 30°C on a shaker at 200 rpm for 48 hours. 1 mL of the culture was transferred to the same liquid enrichment medium and cultured under the same conditions for 48 hours. Under sterile conditions, the culture was diluted to 10 -6 and 10 -7 0.2 mL of each strain was spread onto LB solid medium and incubated at 30°C for 48 hours. Positive strains were screened based on colony surface color, isolated and purified, and then inoculated into fermentation medium. Incubated at 30°C, 200 rpm for 48 hours, the pigment production of the screened strains was measured, and the strain with the highest yield was identified. Subsequent physiological and biochemical assays revealed the presence of certain amounts of chitinase and lipase.

[0088] Example 2: Identification of Serratia marcescens LT-7.

[0089] Genomic DNA from Serratia marcescens LT-7 was extracted using a bacterial genomic DNA extraction kit. The 16S rDNA sequence was amplified by PCR using upstream primer 27F and downstream primer 1492R, as shown in Figure 1A. The PCR amplification product was gel-purified and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the 16S rDNA gene of the strain was measured to be 1471 bp in length, and its gene sequence is shown in SEQ ID NO: 1. The sequence was compared for homology using the BLAST program in the Gene Bank database, and a phylogenetic tree based on the complete 16S rDNA sequence was constructed (Figure 1B). The results showed that the strain shared 100% homology with Serratia marcescens JW-CZ2. Based on the results of strain morphological observation and physiological and biochemical experimental analysis, it was determined that the strain used in the present invention was Serratia marcescens, specifically Serratia marcescens LT-7.

[0090] Example 3: Identification of Serratia marcescens LT-7 fermentation products

[0091] High performance liquid chromatography, infrared spectrometry and nuclear magnetic resonance were used to identify the fermentation products of Serratia marcescens LT-7.

[0092] S. marcescens LT-7 fermentation broth was centrifuged at 9000 rpm. The precipitated cells were extracted with methanol (pH 3) for 20 minutes and then overnight. This precipitated cell solution was concentrated to a paste and dissolved in ethyl acetate (2 times the volume of the concentrate). The precipitate was removed by filtration, and the supernatant was concentrated and freeze-dried to obtain crude prodigiosin. The crude prodigiosin was dissolved in 25 volumes of acetone for later use. Prodigiosin was purified using a silica gel column chromatography column. The mobile phase consisted of petroleum ether:acetone (5:2) and silica gel (200-300 mesh). The purified solution was concentrated and freeze-dried at 50°C to obtain prodigiosin powder.

[0093] 100 mg of prodigiosin powder was dissolved in 100 ml of methanol and the pH was adjusted to 3 to obtain a prodigiosin methanol solution. The liquid chromatography results are shown in FIG3 .

[0094] 0.2 mg of prodigiosin powder was ground with a small amount of KBr and pressed into a pellet to prepare a sample. The infrared spectrum of the sample was then measured using an infrared spectrometer with a scanning range of 4000-400 cm-1. The results are shown in Figure 4. The product purified by fermentation of Serratia marcescens LT-7 was subjected to NMR 1 H and NMR 13 C analysis using CD3Cl as the solvent. The results are shown in Figures 5 and 6.

[0095] Example 4: Optimization of Carbon Source Types for Co-production of Prodigiosin, Chitinase, and Lipase by Serratia marcescens LT-7 Fermentation

[0096] This example illustrates the effects of different carbon sources on prodigiosin production, chitinase, and lipase activity in a strain. Seed culture fluid was inoculated at a 3% (v / v) inoculum into fermentation media containing sucrose, glycerol, mannitol, maltose, glucose, and molasses as the sole carbon source, respectively. The initial pH was 7.0, and the culture was shaken at 30°C, 200 rpm, and fermented for 36 hours. The strain produced significantly different levels of prodigiosin, chitinase, and lipase. When molasses was used as the carbon source for the fermentation medium, the pigment yield reached 1.03 g / L, with a production rate as high as 0.03 g / L / h; chitinase activity reached 17.62 U / mL; and lipase activity reached 35.37 U / mL. Therefore, molasses was selected as the optimal nitrogen source (Figure 7).

[0097] Note: The following examples were optimized with prodigiosin as the main product and chitinase and lipase as by-products.

[0098] Example 5: Optimization of carbon source concentration in the fermentation of Serratia marcescens LT-7 for the co-production of prodigiosin, chitinase and lipase.

[0099] This example illustrates the effects of different molasses concentrations on prodigiosin production, chitinase, and lipase activity. Seed culture fluid was inoculated at a 3% (v / v) inoculum into fermentation media containing molasses concentrations of 0, 10, 20, 30, 40, 50, and 60 g / L, respectively. The initial pH was 7.0, and the culture was shaken at 30°C, 200 rpm, and fermented for 36 hours. The strain produced significantly different levels of prodigiosin, chitinase, and lipase. When 20 g / L molasses was selected as the carbon source concentration in the fermentation medium, the pigment yield reached 3.57 g / L, with a production rate as high as 0.10 g / L / h; the chitinase activity reached 23.06 U / mL; and the lipase activity reached 42.96 U / mL. Therefore, a molasses concentration of 20 g / L was selected for subsequent fermentation (Figure 8).

[0100] Example 6: Optimization of nitrogen source for co-production of prodigiosin, chitinase and lipase by Serratia marcescens LT-7 fermentation.

[0101] This example illustrates the effects of different nitrogen sources on prodigiosin production, chitinase, and lipase activity in a strain. Seed culture broth was inoculated at a 3% (v / v) inoculum into fermentation media containing beef extract (A), tryptone (B), yeast extract (C), corn steep liquor (D), soybean meal (E), peanut meal (F), wheat germ meal (G), yeast extract (H), and peptone (I) as the sole nitrogen source. The initial pH was 7.0, and the culture was shaken at 30°C, 200 rpm, and fermented for 36 hours. The strains showed significant differences in prodigiosin, chitinase, and lipase production. When tryptone was used as the nitrogen source for the fermentation medium, pigment production reached 5.01 g / L, with a production rate as high as 0.14 g / L / h; chitinase activity reached 26.56 U / mL; and lipase activity reached 49.71 U / mL. Therefore, tryptone was selected as the optimal organic nitrogen source (Figure 9).

[0102] Example 7: Optimization of Tryptone Concentration for Co-production of Prodigiosin, Chitinase, and Lipase by Serratia marcescens LT-7

[0103] This example illustrates the effects of different tryptone concentrations on the prodigiosin production, chitinase, and lipase activities of the strain. Seed culture fluid was inoculated at a 3% (v / v) inoculum into fermentation media containing 0, 4, 8, 12, 16, 20, 24, and 28 g / L of tryptone, respectively. The initial pH was 7.0. The culture was shaken at 30°C, 200 rpm, and fermented for 36 h. The production of prodigiosin, chitinase, and lipase by the strain varied significantly. When 12 g / L of tryptone was selected as the nitrogen source concentration of the fermentation media, pigment production reached 6.94 g / L, with a production rate of up to 0.19 g / L / h; chitinase activity reached 30.81 U / mL; and lipase activity reached 59.72 U / mL. Therefore, 12 g / L of tryptone was selected for subsequent fermentation (Figure 10).

[0104] Example 8: Fed-batch co-production of prodigiosin, chitinase and lipase in a 50 L fermenter

[0105] The Serratia marcescens LT-7 strain is inoculated into a slant culture medium and statically cultured at 30°C for 24 hours. A single colony is again picked and streaked onto the slant culture medium and cultured at 30°C for 24 hours to obtain an activated strain for use. One loopful of the activated strain is aseptically inoculated into a shake flask containing a seed culture medium, and the flask is placed on a shaker at a speed of 200 rpm and cultured at a temperature of 30°C for 24 hours to obtain a fermentation seed liquid. The seed liquid is inoculated into a sterile fermentation medium at an inoculum volume of 8% by volume, and the total liquid volume of the fermenter is 30 L. The fermentation temperature is 30°C, the stirring speed is 200 rpm, and the ventilation volume is 0.8 VVM. The initial pH value of the fermentation is 7.0. During the fermentation process, an automatic pH control device is turned on and the pH value of the fermentation liquid is controlled at approximately 6.8 using sodium hydroxide or hydrochloric acid. 15 g / L of molasses is fed 38 hours after fermentation, and the total fermentation time is 72 hours. Samples were taken every four hours to measure the concentrations of prodigiosin, chitinase, and lipase in the fermentation broth. Analysis: The fermentation broth was centrifuged at 9,000 rpm for two minutes. The supernatant and precipitate were diluted with appropriate amounts of methanol and centrifuged at 12,000 rpm for two minutes. The absorbance of the supernatant at 535 nm was measured and compared to the prodigiosin standard curve. After a single feed, the prodigiosin yield reached 15.06 g / L, with a production rate of 0.42 g / L / h. The chitinase activity was 47.98 U / mL, and the lipase activity was 67.67 U / mL (Figure 11).

[0106] Example 9: Fed-batch co-production of prodigiosin, chitinase and lipase in a 1 t fermenter

[0107] Serratia marcescens LT-7 seed solution was inoculated at an 8% by volume into a sterile fermentation medium. The fermentation tank was filled with 700 L of liquid, maintained at a temperature of 30°C, a stirring speed of 220 rpm, and an aeration rate of 1.0 VVM. The initial pH of the fermentation was 6.8. During fermentation, an automatic pH control system was used to maintain the pH of the fermentation broth at approximately 6.8 using sodium hydroxide or hydrochloric acid. At 38 hours of fermentation, 15 g / L of molasses was added, and the total fermentation time was 72 hours. Samples were taken every four hours to measure the concentrations of prodigiosin, chitinase, and lipase in the fermentation broth. Determination and Analysis: The fermentation broth was centrifuged at 9000 rpm for 2 minutes. Appropriate amounts of methanol were added to dilute the supernatant and precipitate, respectively, and the mixture was centrifuged at 12000 rpm for 2 minutes. The absorbance of the supernatant at a wavelength of 535 nm was measured and compared with the prodigiosin standard curve. It was found that after one feeding, the prodigiosin yield reached 20.70 g / L, with a production rate of 0.58 g / L / h. The chitinase activity was 49.78 U / mL, and the lipase activity was 86.92 U / mL (Figure 12).

[0108] Note: The fermentation medium in this example is the optimized optimal medium, which is fed with a carbon source, thus obtaining a higher yield of prodigiosin, chitinase and lipase.

[0109] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the contents of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A Serratia marcescens, characterized in that, The classified name of the Serratia marcescens is Serratia marcescens LT-7, which has been deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC NO: M20231646, and the deposit date is September 7, 2023.

2. The application of the Serratia marcescens described in claim 1 in co-producing prodigiosin, chitinase and lipase.

3. The application according to claim 2, wherein Inoculate Serratia marcescens LT-7 into a fermentation medium for aerobic culture to prepare prodigiosin, chitinase and lipase.

4. The application according to claim 3, characterized in that The described fermentation medium comprises the following components: carbon source 20 - 30 g / L, nitrogen source 10 - 15 g / L, metal salt 1 - 5 g / L, pH value 6.0 - 7.0, and the solvent is water.

5. The application according to claim 4, characterized in that, The carbon source is any one or a combination of sucrose, glycerol, mannitol, maltose, glucose, molasses.

6. The application according to claim 4, wherein The nitrogen source is any one or a combination of beef extract, tryptone, yeast extract, corn steep liquor, soybean cake powder, peanut powder, soybean powder, wheat germ powder, yeast powder, peptone.

7. The application according to claim 4, wherein The metal salt is any one or a combination of calcium chloride, zinc sulfate, ferrous sulfate, manganese chloride, magnesium sulfate, sodium chloride.

8. The application according to claim 5, characterized in that, The carbon source is molasses.

9. The application according to claim 3, wherein The described fermentation medium contains the following components: molasses 20 - 30 g / L, tryptone 10 - 15 g / L, calcium chloride 1 - 5 g / L, soybean powder 1 - 3 g / L, the solvent is water, and the initial pH of the fermentation broth is adjusted to 6.0 - 7.0 with sodium bicarbonate.

10. The application according to claim 3, characterized in that, The described aerobic culture conditions are: the initial pH is 6.0 - 7.0, and the culture temperature is 24 - 32 °C; When the aerobic culture is flask shaking culture, the flask inoculation amount is 1 - 5% v / v, and the culture time is 24 - 48 h; When the aerobic culture is fermentor culture, the fermentor inoculation amount is 1 - 10% v / v, the fermentation method is fed-batch method, the aeration ratio is 0.8 - 1.0 VVM, and the culture time is 24 - 72 h.

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