Method for promoting synthesis of prodigiosin by utilizing peony seed oil and fermentation culture medium

Abstract

The invention discloses a method for promoting synthesis of prodigiosin by utilizing peony seed oil and a fermentation culture medium. A fermentation medium for high-yield synthesis of prodigiosin from serratia marcescens comprises 15.00 g / L of maltose, 13.39 g / L of tryptone, 10 g / L of NaCl and 3.12% (v / v) of peony seed oil. A fermentation medium is inoculated with a RedJ seed culture solution according to the volume ratio of 2%, fermentation culture is conducted for 60 h at the temperature of 20 DEG C and the speed of 200 r / min, and the yield of prodigiosin synthesized by the strain RedJ reaches 180.49 mg / L and is increased by 1.97 times compared with a fermentation medium without peony seed oil. The invention provides a fermentation method for improving the yield of prodigiosin by utilizing the peony seed oil, which not only can be applied to the aspect of large-scale biosynthesis of the prodigiosin, but also finds a new way for high-valued utilization of the peony seed oil.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a method for promoting the synthesis of prodigiosin by utilizing peony seed oil and a fermentation medium. Background Art

[0002] Prodigiosin is a natural pigment with a tripyrrole ring structure produced by microorganisms (Feng Miao et al. 2021). It is dark red and is often used as a natural dye due to its good color fastness and dye uptake (Tan Wenzhang et al. 2021). Studies have also shown that prodigiosin has numerous biological functions. For example, prodigiosin has significant inhibitory effects on bacteria, fungi, and malaria (Suryawanshi RK et al. 2017; Lapendada et al. 2015; Darshan et al. 2016); it has shown good effects on water pollution such as marine red tides and freshwater blooms caused by algae (Zhang et al. 2021); and it has excellent tolerance and removal effects on heavy metals, making it suitable for treating wastewater with excessive heavy metal content. Furthermore, studies have shown that prodigiosin can inhibit the proliferation of breast cancer, neuroblastoma, and lung cancer cells (Francisco et al. 2007; Tomás-Pérez et al. 2003; Soto-Cerrato et al. 2004; Liu Hancong et al. 2022). Therefore, prodigiosin has important research significance and wide application value.

[0003] Microorganisms capable of synthesizing prodigiosin primarily include strains of Serratia, Streptomyces, and Pseudomonas, with Serratia marcescens being the most widely studied (Zhang Wenming et al. 2024; Guo Di Ni et al. 2024). Numerous studies have been conducted on optimizing the fermentation conditions for prodigiosin synthesis using Serratia marcescens. Adding peanut powder (Shi Hongyu, 2012), different amino acids (proline, tryptophan, histidine, glycine, and methionine, among others) (Liu Qi, 2013), cassava flour, corn extract, palm oil (Manas et al., 2020), olive oil (Liu Sihang et al., 2018), Sophora flavescens seed oil (Ni Liang et al., 2020), soybean oil (Zhang Danfeng et al., 2015), peanut oil (Guo Di Ni et al., 2024), or sunflower oil (Buzozhiguli Kadier et al., 2015) to the culture medium can all increase the production of prodigiosin by Serratia marcescens to varying degrees. Further comparative analysis of the results of different studies on the promotion of prodigiosin synthesis by Serratia marcescens by adding different substances showed that plant oils significantly promoted the synthesis of prodigiosin by Serratia marcescens, and that the effect was positively correlated with the content of unsaturated fatty acids contained in the oils.

[0004] The present invention isolates and purifies a strain of Serratia marcescens capable of synthesizing prodigiosin from rice rhizosphere soil. Physiological and biochemical analyses and 16S rDNA sequence alignment identify it as Serratia marcescens and name it RedJ. To increase the yield of prodigiosin synthesized by RedJ and to verify the effect of unsaturated fatty acid content in plant oils and fats on the yield of prodigiosin synthesized by Serratia marcescens, the present invention utilizes response surface methodology to design a fermentation medium for high-yield prodigiosin synthesis by Serratia marcescens RedJ based on single-variable optimization of the addition of plant oils and fats with varying unsaturated fatty acid content, carbon sources, and nitrogen sources. This approach lays the foundation for the industrialization of large-scale biosynthesis of prodigiosin by Serratia marcescens. Summary of the Invention

[0005] The invention aims to optimize and improve the yield of prodigiosin synthesized by Serratia marcescens RedJ and its application, and to provide a fermentation medium for Serratia marcescens to synthesize prodigiosin with high yield.

[0006] Another object of the present invention is to provide a new fermentation method for increasing the yield of prodigiosin by using peony seed oil, which can not only be applied in the large-scale biosynthesis of prodigiosin, but also find a new way to increase the value of peony seed oil.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] The invention discloses a fermentation medium for Serratia marcescens to synthesize prodigiosin in high yield. The fermentation medium comprises 10-20 g / L maltose, 10-14 g / L tryptone, 10 g / L NaCl and 1-5% (v / v) peony seed oil.

[0009] As a preferred embodiment of the present invention, the Serratia marcescens is Serratia marcescens RedJ with a deposit number of CGMCC No.: 21691.

[0010] As a preferred embodiment of the present invention, the fermentation medium contains 15.00 g / L maltose, 13.39 g / L tryptone, 10 g / L NaCl, and 3.12% (v / v) peony seed oil.

[0011] The invention discloses an application of the fermentation medium in promoting Serratia marcescens to synthesize prodigiosin.

[0012] The invention discloses a method for promoting the synthesis of prodigiosin by utilizing peony seed oil, wherein the fermentation medium is used to ferment Serratia marcescens RedJ with a preservation number of CGMCC No.: 21691.

[0013] As a preferred embodiment of the present invention, the Serratia marcescens RedJ seed culture solution is inoculated into the fermentation medium of any one of claims 1-3 at a volume ratio of 1-3%, and the fermentation culture is carried out at 20-25° C. and 180-220 rpm for 50-72 hours.

[0014] As a further preferred embodiment of the present invention, the Serratia marcescens RedJ seed culture solution is inoculated into the fermentation medium of claim 3 at a volume ratio of 2%, and the culture is carried out at 20° C. and 200 rpm for 60 hours.

[0015] Detailed description of the invention

[0016] Activation and seed culture of RedJ

[0017] Remove the preserved RedJ strain (CGMCC No.: 21691) from the -80°C freezer and inoculate the strain onto LB solid medium using the plate streak method. Place in a 37°C constant temperature incubator and culture for 16 hours before transferring. After two consecutive transfers, the strain is activated. Use LB liquid medium as the seed medium. Use an inoculating loop to pick a single colony of the RedJ strain from the activated plate and inoculate it into 100mL / 250mL LB liquid medium. Incubate at 37°C, 200rpm, and shake for 16 hours until the logarithmic phase.

[0018] Extraction and Content Determination of Prodigiosin

[0019] Analytical grade methanol was selected as the extraction agent. 2 mL of the mixed bacterial solution and 8 mL of methanol were placed in a 50 mL centrifuge tube, and the cells were broken up by vortexing (the precipitate was white) to extract prodigiosin. The cell fragments were then placed in a high-speed refrigerated centrifuge, centrifuged at 10,000 rpm and 4°C for 10 minutes. The supernatant in the centrifuge tube was the prodigiosin extract. The maximum absorption wavelength of prodigiosin synthesized by Serratia marcescens RedJ was determined by full wavelength (200 nm-1000 nm) scanning using an ultraviolet spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd., UV-6100) to be 535 nm ( Figure 1 ). In the subsequent prodigiosin content determination test, the OD535 value of the prodigiosin extract was first measured with an ultraviolet spectrophotometer to calculate the concentration of prodigiosin.

[0020] LB liquid medium was used as the basic fermentation medium. The seed culture of RedJ was inoculated into a 250ml conical flask (50mL / 250mL) containing 50ml fermentation medium at an inoculation volume of 2% (v / v). The conical flasks were placed in incubators at 16°C, 20°C, 25°C, 28°C, 30°C and 37°C, respectively, and shaken at 200rpm for 60h. The OD535 value of prodigiosin in the fermentation broth was extracted and determined, and the content of prodigiosin was calculated. In order to ensure the reliability of the experimental results, three groups of parallel experiments were set for each temperature and three biological replicates were performed. The results showed that temperature had a greater effect on the yield of prodigiosin synthesized by Serratia marcescens RedJ ( Figure 2 ), among which the yield of prodigiosin synthesized by Serratia marcescens RedJ was the highest at 20°C (1.91 mg / L), and the lowest at 37°C (0.31 mg / L). The yields of prodigiosin at 16°C, 25°C, 28°C, and 30°C were 1.10 mg / L, 1.57 mg / L, 0.97 mg / L, and 0.68 mg / L, respectively. Therefore, the optimal temperature for the fermentation of prodigiosin by Serratia marcescens RedJ is 20°C.

[0021] Effects of vegetable oils with different unsaturated fatty acid contents on the production of prodigiosin synthesized by RedJ

[0022] Five kinds of plant oils with different unsaturated fatty acid contents were selected: peony seed oil, rapeseed oil, corn oil, soybean oil and peanut oil, and their average unsaturated fatty acid contents were 91.07%, 90.24%, 83.43%, 82.51% and 79.08%. Eight concentrations (v / v) were set for each plant oil, namely 0%, 0.1%, 0.2%, 0.5%, 1%, 3%, 5% and 10%, and added to LB basic fermentation medium respectively. In order to ensure the reliability of the experimental results, three groups of parallel experiments were set for each concentration of each oil and three biological replicates were performed. After high-pressure steam sterilization, the seed culture solution was transferred to LB fermentation basic medium (50mL / 250mL) at an inoculum size of 2% (v / v), and fermented at 20°C, 200rpm, and then prodigiosin was extracted after 60h of fermentation. By measuring the concentration of prodigiosin, the effect of plant oils with different unsaturated fatty acid contents on the yield of prodigiosin synthesized by RedJ was determined ( Figure 3 ).

[0023] Depend on Figure 3 It can be seen that the addition of five kinds of plant oils can promote the synthesis of prodigiosin by RedJ. When peony seed oil ( Figure 3 -A), rapeseed oil ( Figure 3 -B), corn oil ( Figure 3 -C) and soybean oil ( Figure 3 -D) was added at 3% (v / v), the corresponding concentrations of synthetic prodigiosin were the highest, namely 31.70 mg / L, 30.55 mg / L, 10.83 mg / L and 7.13 mg / L, which were 31.95 times, 31.99 times, 12.5 times and 5.47 times higher than those in the group without oil addition, respectively. When the addition amount was 5% and 10%, the yield of synthetic prodigiosin by RedJ decreased instead; however, in the experimental group with peanut oil addition ( Figure 3 -E), within the concentration range of 0%-10% (v / v), the yield of prodigiosin synthesized by Bacteria RedJ showed a continuous increasing trend. The concentration of prodigiosin synthesized by Bacteria RedJ with the addition of 10% (v / v) peanut oil was 12.96 mg / L, which was 14.65 times higher than that of the group without oil added. Among the five kinds of plant oils, peony seed oil and rapeseed oil had the highest unsaturated fatty acid content (>90%), while the unsaturated fatty acid content of corn oil and soybean oil was 83% on average, and the unsaturated fatty acid content of peanut oil was relatively low, averaging 79%. The results of the present invention show that plant oils can significantly increase the yield of prodigiosin synthesized by Serratia marcescens RedJ fermentation, and the higher the content of unsaturated fatty acids in plant oils, the higher the yield of prodigiosin synthesized by Bacteria RedJ after addition. Among the five kinds of oils, peony seed oil had the highest unsaturated fatty acid content (91.07%), and in the LB basic fermentation medium after its addition (3%, v / v), the yield of prodigiosin synthesized by Bacteria RedJ was the highest ( Figure 3 -A and Figure 4 ), with a concentration of 31.70 mg / L.

[0024] Optimization of carbon and nitrogen sources in the culture medium for the synthesis of prodigiosin by RedJ

[0025] Optimization of carbon source type and concentration for the synthesis of prodigiosin by RedJ

[0026] The yeast powder in the LB medium was used as a carbon source, and it was replaced with equal masses of glucose, maltose, soluble starch, mannitol, and citric acid in turn to prepare the fermentation medium for the synthesis of prodigiosin by the bacterium RedJ. After high-pressure steam sterilization, the bacterium RedJ seed culture solution was transferred to the fermentation medium (50mL / 250mL) at an inoculation volume of 2% (v / v). The prodigiosin was extracted after 60 hours of fermentation culture at 20°C and 200rpm. In order to ensure the reliability of the test results, three groups of parallel experiments were set up for each carbon source and three biological replicates were performed. The results of the synthesis of prodigiosin by the bacterium RedJ in fermentation broths with different carbon sources are shown in Figure 5 and Figure 6 . By further determining the concentration of prodigiosin, it was found that the optimal carbon source for Serratia marcescens RedJ to synthesize prodigiosin was maltose, and the concentration of synthesized prodigiosin at this time was 63.10 mg / L. The second was soluble starch, and the concentration of synthesized prodigiosin was 14.21 mg / L. Yeast powder was used as the carbon source, and the concentration of prodigiosin synthesized by RedJ was the lowest (2.41 mg / L). After determining that the optimal carbon source was maltose, the concentration of maltose was further optimized, and the concentration of maltose was set to 3g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L and 30g / L respectively. The concentration of prodigiosin in the fermentation broth was calculated using the same inoculation, culture and determination methods as mentioned above, thereby determining the optimal maltose concentration range. In order to ensure the reliability of the test results, three sets of parallel experiments were set for each maltose concentration and three biological replicates were performed. By Figure 7 The results showed that when the maltose concentration was between 3g / L and 15g / L, the production of prodigiosin increased with increasing maltose concentration; when the maltose concentration was between 15g / L and 30g / L, the production of prodigiosin decreased with increasing maltose concentration. When the maltose concentration was 15g / L, the concentration of prodigiosin synthesized by Serratia marcescens RedJ reached the highest level, 95.40mg / L.

[0027] Optimization of nitrogen source type and concentration for the synthesis of prodigiosin by RedJ

[0028] On the basis of determining the most suitable carbon source, the peptone in the LB medium was used as a nitrogen source and replaced with beef extract, trypsin, urea, ammonium acetate, sodium nitrate and ammonium chloride of equal mass. After high-pressure steam sterilization, the RedJ seed culture solution was transferred to the fermentation medium (50mL / 250mL) at an inoculation volume of 2% (v / v). The prodigiosin was extracted after 60 hours of fermentation culture at 20°C and 200rpm. In order to ensure the reliability of the test results, three parallel experiments were set up for each nitrogen source and three biological replicates were performed. The results of prodigiosin synthesis by RedJ in fermentation broths with different nitrogen sources are shown in Figure 8 and Figure 9 . By further determining the concentration of prodigiosin, it was found that the optimal nitrogen source for Serratia marcescens RedJ to synthesize prodigiosin was tryptone, with a synthesized prodigiosin concentration of 101.90 mg / L, followed by beef extract, with a prodigiosin concentration of 92.77 mg / L. When ammonium acetate was used as a nitrogen source, the concentration of prodigiosin synthesized by Serratia marcescens RedJ was the lowest (0.58 mg / L). After determining that the optimal nitrogen source was tryptone, the concentration of tryptone was further optimized, and the concentration of tryptone was set to 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L and 30 g / L, respectively. The concentration of prodigiosin in the fermentation broth was calculated using the same inoculation, culture and determination methods as mentioned above, thereby determining the optimal tryptone concentration range ( Figure 10 ).Depend on Figure 10 The results showed that when the tryptone concentration was between 5g / L and 10g / L, the production of prodigiosin increased with the increase in tryptone concentration; when the tryptone concentration was between 10g / L and 30g / L, the production of prodigiosin decreased with the increase in tryptone concentration. When the tryptone concentration was 10g / L, the concentration of prodigiosin synthesized by Serratia marcescens RedJ was the highest, at 95.01mg / L.

[0029] Response surface experiment design and analysis of the conditions for the synthesis of prodigiosin by RedJ

[0030] Based on a single-factor experiment, we determined the range of variation for the independent variable. Using Design Expert 11 software, we conducted a response surface design experiment (RDA) using the concentration of prodigiosin synthesized by Serratia marcescens RedJ fermentation as the response variable, and the concentrations of maltose and tryptone, as well as the amount of peony seed oil added, as the independent variables (Table 2). We conducted experiments based on the design (each experiment was performed in triplicate with three biological replicates), and the results are shown in Table 3. We then used Design Expert 11 software to analyze the model's significance, accuracy, and credibility, and identified the optimal experimental plan. The data in Table 3 were analyzed by Design Expert 11 software, and the quadratic multinomial regression model of A: maltose (g / L), B: tryptone (g / L), and C: peony seed oil (v / v, %) was obtained after fitting: Y = 831.87605-76.55966A+18.24080B-57.31205C+1.31348AB+9.57710AC+0.7967700BC+0.446156A 2 -1.51664B 2 -15.33152C 2 The regression model was subjected to variance analysis, and the results are shown in Table 4. The P value of the regression model is less than 0.0001, indicating that the regression model is extremely significant, while the P value of the lack of fit term is 0.4957, which is greater than 0.05, indicating that it is not significant. Therefore, the model is established. 2 and R 2 (Adj) The results are 0.9764 and 0.9460 respectively, which are close to each other, indicating that the model has high accuracy. CV = 4%, which is less than 10%, indicating that the model has a good fit with the experiment and high credibility. The model can be used to optimize the fermentation conditions of Serratia marcescens. Among them, A and B have significant effects, C has no significant effect, and AC has a significant effect in the interaction term, indicating that the effects of these three factors on the production of prodigiosin are not a simple linear relationship ( Figure 11 ). The optimal design and prediction results are obtained through software analysis ( Figure 12 ), the optimal conditioned medium was maltose 15.00 g / L, tryptone 13.39 g / L, and peony seed oil 3.12% (v / v). Under these predicted conditions, the concentration of prodigiosin synthesized by Serratia marcescens RedJ was 173.38 mg / L.

[0031] Experimental verification of the optimal fermentation conditions for the synthesis of prodigiosin by RedJ

[0032] According to the optimal culture medium components predicted by Design Except 11 software (carbon source: maltose 15.00g / L, nitrogen source: trypsin 13.39g / L, peony seed oil 3.12% (v / v)), the fermentation medium was prepared for experimental verification. In order to further verify the effect of peony seed oil on the synthesis of prodigiosin by bacteria RedJ, an experimental group without peony seed oil was set up. After high-pressure steam sterilization, the two culture media were inoculated with bacteria RedJ for fermentation and culture for 60 hours to extract and determine the concentration of prodigiosin, and calculate whether the experimental results are in line with expectations. The experiment set up three parallel experiments and performed three biological repetitions. After fermentation and culture, the color of the fermentation liquid of bacteria RedJ was seen Figure 13 The color of the fermentation broth with peony seed oil added was dark red (high prodigiosin content), while the color of the fermentation broth without peony seed oil added was orange-yellow (low prodigiosin content). Through further extraction and determination of the concentration of prodigiosin, the concentration of prodigiosin synthesized by RedJ in the culture medium with peony seed oil added was 180.49 mg / L (close to the concentration predicted by response surface design), which was 1.97 times higher than the fermentation medium without peony seed oil added (optimal carbon and nitrogen sources, yield of 60.81 mg / L). Figure 14 Therefore, the present invention uses response surface methodology to design a fermentation medium for high-yield prodigiosin synthesis by Serratia marcescens RedJ based on the optimization of single variables (carbon source, nitrogen source, and plant oil): 15.00 g / L maltose, 13.39 g / L tryptone, 10 g / L NaCl, and 3.12% (v / v) peony seed oil. The present invention provides a fermentation method for increasing prodigiosin production using peony seed oil. This method can not only be applied to the large-scale biosynthesis of prodigiosin, but also provides a new approach for the high-value utilization of peony seed oil.

[0033] Beneficial effects of the present invention:

[0034] 1. The method for promoting the synthesis of prodigiosin using peony seed oil provided by the present invention is a new method for promoting the production of prodigiosin by Serratia marcescens, which can significantly increase the production of prodigiosin;

[0035] 2. The method provided by the present invention for increasing the yield of prodigiosin synthesized by Serratia marcescens can be applied in the large-scale biosynthesis of prodigiosin, and also finds a new way to increase the value of peony seed oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the full slope scan result of the extract of prodigiosin synthesized by RedJ fermentation

[0037] The RedJ fermentation broth was extracted with methanol and centrifuged, and the supernatant was scanned at full wavelength (200nm-1000nm). The maximum absorption wavelength of prodigiosin synthesized by RedJ fermentation was found to be 535nm.

[0038] Figure 2 is the yield of prodigiosin synthesized by RedJ at different temperatures

[0039] The fermentation culture medium containing the inoculated strain RedJ was placed in an incubator at 16°C, 20°C, 25°C, 28°C, 30°C, and 37°C, with shaking at 200 rpm for 60 hours. The OD535 value of prodigiosin in the fermentation broth was extracted and measured, and the concentration of prodigiosin was calculated to determine the optimal temperature for the fermentation of prodigiosin by RedJ.

[0040] Figure 3 Effects of five kinds of plant oils on the production of prodigiosin synthesized by RedJ

[0041] Five kinds of plant oils with different unsaturated fatty acid contents were selected: peony seed oil (A), rapeseed oil (B), corn oil (C), soybean oil (D) and peanut oil (E). The effects of plant oils with different unsaturated fatty acid contents on the production of prodigiosin synthesized by RedJ were studied. Eight concentrations (v / v) of each plant oil were set, namely 0%, 0.1%, 0.2%, 0.5%, 1%, 3%, 5% and 10%, and added to the LB basic fermentation medium respectively. The results showed that the addition of five kinds of plant oils can increase the production of prodigiosin synthesized by RedJ. When the addition amount of peony seed oil (A), rapeseed oil (B), corn oil (C) and soybean oil (D) was 3% (v / v), the concentration of the corresponding synthetic prodigiosin was the highest, which were 31.70mg / L, 30.55mg / L, 10.83mg / L and 10.00mg / L, respectively. / L and 7.13 mg / L, which were increased by 31.95 times, 31.99 times, 12.5 times and 5.47 times respectively compared with the group without oil addition. When the addition amount was 5% and 10%, the production of prodigiosin synthesized by Bacteria RedJ decreased. However, in the experimental group (E) with the addition of peanut oil, the production of prodigiosin synthesized by Bacteria RedJ showed a continuous increasing trend within the concentration range of 0%-10%. The concentration of prodigiosin synthesized by Bacteria RedJ with the addition of 10% peanut oil was 12.96 mg / L, which was increased by 14.65 times compared with the group without oil addition.

[0042] Figure 4 The color of the fermentation liquid of RedJ in the fermentation medium with peony seed oil

[0043] Figure 5 The color of the fermentation broth of RedJ in different carbon source fermentation media

[0044] Yeast powder in LB medium was used as a carbon source and replaced with equal masses of soluble starch, maltose, mannitol, citric acid and glucose, respectively, to study the effects of different carbon sources on the fermentation and synthesis of prodigiosin by RedJ.

[0045] Figure 6 Effect of carbon source type on the yield of prodigiosin synthesized by RedJ

[0046] When yeast powder in LB medium was used as a carbon source, it was replaced with equal amounts of soluble starch, maltose, mannitol, citric acid, and glucose. Among them, the strain RedJ produced the highest concentration of prodigiosin, reaching 63.10 mg / L, in the fermentation medium with maltose as the carbon source.

[0047] Figure 7 Effect of maltose concentration on the yield of prodigiosin synthesized by RedJ

[0048] After determining that maltose was the optimal carbon source, the concentration of maltose was further optimized, and the concentrations of maltose were set to 3g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L and 30g / L, respectively. The results showed that when the maltose concentration was 15g / L, the amount of prodigiosin synthesized by Serratia marcescens RedJ was the largest, with a yield of 95.40mg / L.

[0049] Figure 8 The color of the fermentation broth of RedJ in different nitrogen source fermentation media

[0050] On the basis of determining the optimal carbon source, the peptone in LB medium was used as the nitrogen source and replaced with equal masses of beef extract, trypsin, urea, ammonium acetate, sodium nitrate and ammonium chloride, respectively, to study the effects of different nitrogen sources on the fermentation synthesis of prodigiosin by the fungus RedJ.

[0051] Figure 9 Effect of nitrogen source type on the yield of prodigiosin synthesized by RedJ

[0052] After determining the optimal carbon source, the peptone in LB medium was replaced with equal amounts of beef extract, tryptone, urea, ammonium acetate, sodium nitrate, and ammonium chloride as the nitrogen source. The strain RedJ produced the highest concentration of prodigiosin, reaching 101.90 mg / L, in the tryptone-based fermentation medium.

[0053] Figure 10 Effect of tryptone concentration on the production of prodigiosin synthesized by RedJ

[0054] After determining that the optimal nitrogen source was trypsin, the concentration of trypsin was further optimized, and the concentrations of trypsin were set to 5g / L, 10g / L, 15g / L, 20g / L, 25g / L and 30g / L, respectively. The results showed that when the trypsin concentration was 10g / L, the amount of prodigiosin synthesized by Serratia marcescens RedJ was the largest, with a yield of 95.01mg / L.

[0055] Figure 11 This is the response surface contour plot of the interaction between maltose, tryptone and peony seed oil.

[0056] The concentration of prodigiosin synthesized by Serratia marcescens RedJ was used as the response value, and the concentrations of maltose and tryptone and the amount of peony seed oil added were used as independent variables. The response surface experiment was designed using Design Expert 11 software. The experiment was carried out and the significance, accuracy and credibility of the experimental results were analyzed. The response surface contour map of the interaction between the three factors was obtained.

[0057] Figure 12 Response surface optimization design prediction diagram of RedJ fermentation synthesis of prodigiosin

[0058] The optimal culture medium formula and prediction results were obtained through analysis by Design Expert 11 software. The optimal culture medium formula was 15.00 g / L maltose, 13.39 g / L tryptone, and 3.12% (v / v) peony seed oil. Under these predicted conditions, the concentration of prodigiosin synthesized by Serratia marcescens RedJ was 173.38 mg / L.

[0059] Figure 13 The color of the prodigiosin fermentation liquid of RedJ in the optimal fermentation medium without peony seed oil and with peony seed oil

[0060] The color of the fermentation broth with peony seed oil added was dark red (high prodigiosin content), while that of the fermentation broth without peony seed oil added was orange-yellow (low prodigiosin content).

[0061] Figure 14 The concentration of prodigiosin synthesized by RedJ in the optimal fermentation medium without and with peony seed oil was

[0062] The concentration of prodigiosin synthesized by the fungus RedJ in the culture medium supplemented with peony seed oil was 180.49 mg / L (close to the concentration predicted by response surface design), which was 1.97 times higher than the yield in the fermentation medium without peony seed oil (yield of 60.81 mg / L).

[0063] Biomaterial deposit information

[0064] RedJ, classified as Serratia marcescens, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.21691 and the deposit date is January 20, 2021. DETAILED DESCRIPTION

[0065] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0066] Example 1

[0067] 1.1 Activation culture of RedJ

[0068] Prepare LB solid medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, 18 g / L agar powder, 1000 mL water), autoclave (121°C, 30 min), and pour onto plates. Remove the Serratia marcescens RedJ strain (CGMCC No. 21691) from a -80°C freezer and inoculate the LB solid medium using the streak method. Incubate in a 37°C incubator for 16 h before transferring the culture. Repeat this process twice to activate the strain and set aside.

[0069] 1.2 Seed culture of RedJ

[0070] Use LB liquid medium as the seed medium and fermentation basal medium. Use an inoculating loop to pick a single colony of RedJ from the activated plate and inoculate it into 100 mL / 250 mL of LB liquid medium. Incubate at 37°C, 200 rpm, and shake for 16 hours until the culture reaches the logarithmic phase.

[0071] 1.3 Extraction and content determination of prodigiosin

[0072] Analytical-grade methanol was used as the extraction solvent. First, 2 mL of bacterial culture (shake thoroughly before aspirating) and 8 mL of methanol (at a 1:4 ratio) were placed in a 50 mL centrifuge tube. A vortexer (QL-866, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.) was used to disrupt the cells for 2 minutes at 3,000 rpm to extract prodigiosin. The cell fragments were then placed in a high-speed refrigerated centrifuge and centrifuged at 10,000 rpm at 4°C for 10 minutes. After centrifugation, the supernatant in the centrifuge tube was the prodigiosin extract.

[0073] The maximum absorption wavelength of prodigiosin synthesized by Serratia marcescens RedJ was determined by ultraviolet spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd., UV-6100) at full wavelength (200nm-1000nm). Figure 1In the subsequent prodigiosin content determination test, the OD535 value of the extract was measured by UV-visible spectrophotometer, and the regression equation C = 3.944A + 0.007 (R 2 =0.9998) (Wang Chunling, 2007), and the concentration of prodigiosin was calculated.

[0074] Example 2

[0075] 2.1 Temperature optimization of RedJ fermentation for prodigiosin

[0076] LB liquid medium was used as the fermentation basal medium. A 2% (v / v) inoculum of RedJ seed culture was inoculated into a 250ml conical flask (50mL / 250mL) containing 50ml of fermentation medium. The conical flask was placed in an incubator at 16°C, 20°C, 25°C, 28°C, 30°C, and 37°C, respectively, with shaking at 200rpm for 60 hours. The OD535 value of prodigiosin in the fermentation broth was extracted and measured, and the prodigiosin content was calculated to determine the optimal temperature for the fermentation of prodigiosin by RedJ. To ensure the reliability of the experimental results, three parallel experiments were set for each temperature, with three biological replicates.

[0077] Depend on Figure 2 It can be seen that temperature has a great influence on the production of prodigiosin synthesized by Serratia marcescens RedJ. The production of prodigiosin synthesized by this strain at 20℃ is the largest, which is 1.91 mg / L, and at 37℃ the smallest (0.31 mg / L). The production of prodigiosin synthesized at 16℃, 25℃, 28℃ and 30℃ are 1.10 mg / L,

[0078] 1.57 mg / L, 0.97 mg / L and 0.68 mg / L. Therefore, the optimum temperature for the fermentation of prodigiosin by Serratia marcescens RedJ is 20℃.

[0079] 2.2 Effects of plant oils with different unsaturated fatty acid contents on the production of prodigiosin synthesized by RedJ

[0080] Five kinds of vegetable oils with different unsaturated fatty acid contents were selected: peanut oil, soybean oil, corn oil, rapeseed oil and peony seed oil, and their average unsaturated fatty acid contents were 79.08%, 82.51%, 83.43%, 90.24% and 91.07%. Eight concentrations (v / v) were set for each vegetable oil, namely 0%, 0.1%, 0.2%, 0.5%, 1%, 3%, 5% and 10%, and added to LB basic fermentation medium respectively. After high-pressure steam sterilization, the RedJ seed culture solution was transferred to LB fermentation basic medium (50mL / 250mL) at an inoculum size of 2% (v / v), and fermented at 20°C, 200rpm, and then prodigiosin was extracted after 60h of fermentation. By measuring the concentration of prodigiosin, the effect of vegetable oils with different unsaturated fatty acid contents on the yield of prodigiosin synthesized by RedJ was determined (see Figure 3 To ensure the reliability of the experimental results, three parallel experiments were set up for each concentration of each oil and three biological replicates were performed.

[0081] Depend on Figure 3 It can be seen that the addition of five kinds of plant oils can increase the production of prodigiosin synthesized by RedJ. When peony seed oil ( Figure 3 -A), rapeseed oil ( Figure 3 -B), corn oil ( Figure 3 -C) and soybean oil ( Figure 3 -D) was added at 3% (v / v), the corresponding concentrations of synthetic prodigiosin were the highest, namely 31.70 mg / L, 30.55 mg / L, 10.83 mg / L and 7.13 mg / L, which were 31.95 times, 31.99 times, 12.5 times and 5.47 times higher than those in the group without oil addition, respectively. When the addition amount was 5% and 10%, the yield of synthetic prodigiosin by RedJ decreased instead; however, in the experimental group with peanut oil addition ( Figure 3-E), in the concentration range of 0%-10%, the yield of prodigiosin synthesized by Bacteria RedJ showed a continuous increasing trend. The concentration of prodigiosin synthesized by Bacteria RedJ with the addition of 10% peanut oil was 12.96 mg / L, which was 14.65 times higher than that of the group without oil addition. Through extensive literature review (Wang Xiaohua et al., 2021; Hu Yan et al., 2014; Chen Jiangyan et al., 2024; Liu Yingsha et al., 2020; Qiao Shumin et al., 2023; Wang Yaping et al., 2024; Yu Shunbo et al., 2022; Gu Keren et al., 2012; Yang Xuqiang et al., 2022; Li Hongyan et al., 2021; Zhang Wenchao et al., 2021; Altan Tuya et al., 2019), it was found that among the five vegetable oils, peony seed oil and rapeseed oil had the highest unsaturated fatty acid content (>90%), while the unsaturated fatty acid content of corn oil and soybean oil averaged 83%, and the unsaturated fatty acid content of peanut oil was relatively low, averaging 79%. The results of the present invention show that vegetable oils can significantly increase the yield of prodigiosin synthesized by Serratia marcescens RedJ fermentation, and the higher the content of unsaturated fatty acids in the vegetable oil, the higher the yield of prodigiosin synthesized by RedJ after adding it. Among the five oils, peony seed oil had the highest content of unsaturated fatty acids (91.07%). After adding peony seed oil (3%, v / v) to the LB basic fermentation medium, the yield of prodigiosin synthesized by the bacterium RedJ was the highest ( Figure 3 A and Figure 4 ), with a concentration of 31.70 mg / L.

[0082] Optimization of carbon and nitrogen sources in the culture medium for the synthesis of prodigiosin by RedJ

[0083] In order to further increase the yield of prodigiosin synthesized by Serratia marcescens RedJ, the present invention uses LB liquid culture medium as a fermentation basal medium, optimizes the type of carbon source or nitrogen source in the culture medium for synthesizing prodigiosin by replacing the carbon source or nitrogen source, and after determining the type of carbon source or nitrogen source, further optimizes the optimal concentration of the carbon source or nitrogen source to increase the yield of prodigiosin synthesized by Serratia marcescens RedJ.

[0084] 2.4 Optimization of carbon source type and concentration for RedJ synthesis of prodigiosin

[0085] The yeast powder in the LB medium was used as a carbon source, and it was replaced with equal masses of glucose, maltose, soluble starch, mannitol, and citric acid to prepare the fermentation medium for the synthesis of prodigiosin by the bacterium RedJ. After high-pressure steam sterilization, the bacterium RedJ seed culture medium was transferred to the fermentation medium (50mL / 250mL) at an inoculation volume of 2% (v / v). The prodigiosin was extracted after 60 hours of fermentation culture at 20°C and 200rpm. In order to ensure the reliability of the test results, three parallel experiments were set up for each carbon source and three biological replicates were performed. The results of the synthesis of prodigiosin by the bacterium RedJ in fermentation broths with different carbon sources are shown in Figure 5 and Figure 6 Further analysis of prodigiosin concentration revealed that maltose was the optimal carbon source for Serratia marcescens RedJ to synthesize prodigiosin, producing a concentration of 63.10 mg / L. Soluble starch was the second most suitable carbon source, producing a concentration of 14.21 mg / L. Yeast extract, used as a carbon source, produced the lowest concentration of prodigiosin (2.41 mg / L).

[0086] After determining that the optimal carbon source was maltose, the concentration of maltose was further optimized and the concentrations of maltose were set to 3g / L, 5g / L, 10g / L, 15g / L, 20g / L, 25g / L and 30g / L respectively. The concentration of prodigiosin in the fermentation broth was calculated using the same inoculation, culture and determination methods as described above, thereby determining the optimal maltose concentration range. In order to ensure the reliability of the test results, three parallel experiments were set for each maltose concentration and three biological replicates were performed. Figure 7 The results showed that when the maltose concentration was between 3g / L and 15g / L, the production of prodigiosin increased with increasing maltose concentration; when the maltose concentration was between 15g / L and 30g / L, the production of prodigiosin decreased with increasing maltose concentration. At a maltose concentration of 15g / L, Serratia marcescens RedJ produced the highest amount of prodigiosin, with a yield of 95.40mg / L.

[0087] Optimization of nitrogen source type and concentration for prodigiosin synthesis by 2.5 bacterium RedJ

[0088] On the basis of determining the most suitable carbon source, the peptone in the LB medium was used as a nitrogen source and replaced with equal amounts of beef extract, trypsin, urea, ammonium acetate, sodium nitrate and ammonium chloride. After the medium was sterilized by high-pressure steam, the RedJ seed culture solution was transferred to the fermentation medium (50mL / 250mL) at an inoculation volume of 2% (v / v). After fermentation and cultivation at 20°C and 200rpm for 60h, prodigiosin was extracted. In order to ensure the reliability of the test results, three parallel experiments were set up for each nitrogen source and three biological replicates were performed. The results of prodigiosin synthesis by RedJ in fermentation broths with different nitrogen sources are shown in Figure 8 and Figure 9 Further determination of prodigiosin concentration revealed that the optimal nitrogen source for Serratia marcescens RedJ prodigiosin synthesis was tryptone, with a concentration of 101.90 mg / L, followed by beef extract, with a concentration of 92.77 mg / L. Using ammonium acetate as a nitrogen source, RedJ produced the lowest concentration of prodigiosin (0.58 mg / L).

[0089] After determining that the optimal nitrogen source was tryptone, the concentration of tryptone was further optimized and set to 5g / L, 10g / L, 15g / L, 20g / L, 25g / L and 30g / L. The concentration of prodigiosin in the fermentation broth was calculated using the same inoculation, culture and determination methods as above, thereby determining the optimal tryptone concentration range. Figure 10 The results showed that when the tryptone concentration was between 5g / L and 10g / L, the production of prodigiosin increased with the increase of tryptone concentration; when the tryptone concentration was between 10g / L and 30g / L, the production of prodigiosin decreased with the increase of tryptone concentration. When the tryptone concentration was 10g / L, the production of prodigiosin synthesized by Serratia marcescens RedJ was the highest, reaching 95.01mg / L.

[0090] Example 3 Response surface experiment design and analysis of conditions for the synthesis of prodigiosin by RedJ

[0091] The range of variation of the independent variable was determined according to the single-factor experiment in Example 2. A response surface experiment was performed using Design Expert 11 software, with the concentration of prodigiosin synthesized by Serratia marcescens RedJ fermentation as the response value, and the concentrations of maltose and tryptone and the amount of peony seed oil added as independent variables (Table 2).

[0092] Table 2 Experimental design

[0093]

[0094] Experiments were conducted according to the design provided by the software (see Table 3 for details). Each experiment was performed in triplicate with three biological replicates. The results were analyzed using Design Expert 11 software for model significance, accuracy, and credibility, and the optimal experimental plan was determined.

[0095] A response surface experiment scheme of 17 groups was designed using Design Expert 11 software. Experiments were carried out according to the scheme, and the results are shown in Table 3. The data in the table were further analyzed using Design Expert 11 software, and the quadratic multinomial regression model for A: maltose (g / L), B: tryptone (g / L), and C: peony seed oil (v / v, %) was obtained after fitting:

[0096] Y=831.87605-76.55966A+18.24080B-57.31205C+1.31348AB+9.57710AC+0.7967700BC+0.446156A 2 -1.51664B 2 -15.33152C 2 .

[0097] Table 3 Response surface design scheme

[0098]

[0099] The regression model was subjected to variance analysis, and the results are shown in Table 4. The P value of the regression model is less than 0.0001, indicating that the regression model is extremely significant, while the P value of the lack of fit term is 0.4957, which is greater than 0.05, indicating that it is not significant. Therefore, the model is established. 2 and R 2 (Adj) The results are 0.9764 and 0.9460 respectively, which are close to each other, indicating that the model has high accuracy. CV = 4%, which is less than 10%, indicating that the model has a good fit with the experiment and high credibility. The model can be used to optimize the fermentation conditions of Serratia marcescens. Among them, A and B have significant effects, C has no significant effect, and AC has a significant effect in the interaction term, indicating that the effects of these three factors on the production of prodigiosin are not a simple linear relationship ( Figure 11 ).

[0100] Table 4 Analysis of variance

[0101]

[0102] The software analysis gives the optimal design and prediction results ( Figure 12 ), the optimal conditions were maltose 15.00 g / L, tryptone 13.39 g / L, and peony seed oil 3.12% (v / v). Under these predicted conditions, the concentration of prodigiosin synthesized by Serratia marcescens RedJ was 173.38 mg / L.

[0103] Example 4 Experimental Verification of the Optimal Fermentation Medium for Prodigiosin Synthesis by RedJ

[0104] According to the optimal fermentation conditions designed by Design Except 11 software (carbon source: maltose 15.00g / L, nitrogen source: trypsin 13.39g / L, added peony seed oil 3.12% (v / v)), the culture medium was prepared for experimental verification. In order to further verify the effect of peony seed oil on the synthesis of prodigiosin by bacteria RedJ, an experimental group without the addition of peony seed oil was set up. After the two culture media were sterilized with high-pressure steam, they were inoculated with bacteria RedJ for fermentation and culture for 60 hours, and then the prodigiosin was extracted and the concentration was measured to observe whether the experimental results were in line with expectations. The experiment set up three groups of parallel experiments and performed three biological repetitions. After fermentation and culture, the color of the fermentation liquid of bacteria RedJ was seen Figure 13The color of the fermentation broth with peony seed oil added was dark red (high prodigiosin content), while the color of the fermentation broth without peony seed oil added was orange-yellow (low prodigiosin content). Through further extraction and determination of the concentration of prodigiosin, the concentration of prodigiosin synthesized by RedJ in the culture medium with peony seed oil added was 180.49 mg / L (close to the concentration predicted by response surface design), which was 1.97 times higher than the fermentation medium without peony seed oil added (optimal carbon and nitrogen sources, yield of 60.81 mg / L). Figure 14 ). Therefore, the present invention uses response surface methodology to design a fermentation medium for Serratia marcescens RedJ to synthesize prodigiosin with high yield based on the optimization of single variables (carbon source, nitrogen source and vegetable oil): 15.00 g / L maltose, 13.39 g / L tryptone, 10 g / L NaCl, 3.12% (v / v) peony seed oil. Peony seed oil is a woody nut vegetable oil extracted from peony seeds. It was approved as a new resource food in 2011 and has broad application prospects in the edible vegetable oil, pharmaceutical excipients, health food and cosmetics industries. Since the peony seed oil industry faces the problems of being a new resource food, a short market promotion time, limited understanding of it by many consumers, unstable raw material output, uneven quality, high production costs, etc., it is difficult for peony seed oil to be recognized and promoted by the market in a short period of time. Therefore, the fermentation method for increasing the yield of prodigiosin by utilizing peony seed oil provided by the present invention can not only be applied in the large-scale biosynthesis of prodigiosin, but also finds a new way for the high-value utilization of peony seed oil.

Claims

1. A fermentation medium for high-yield prodigiosin synthesis by Serratia marcescens, characterized in that: The fermentation medium contains 10-20 g / L maltose, 10-14 g / L tryptone, 10 g / L NaCl, and 1-5% (v / v) peony seed oil.

2. The fermentation medium according to claim 1, characterized in that The Serratia marcescens is Serratia marcescens RedJ with a preservation number of CGMCC No.: 21691.

3. The fermentation medium according to claim 1, characterized in that The fermentation medium contains 15.00 g / L maltose, 13.39 g / L tryptone, 10 g / L NaCl, and 3.12% (v / v) peony seed oil.

4. Use of the fermentation medium according to any one of claims 1 to 3 in promoting the synthesis of prodigiosin by Serratia marcescens.

5. A method for promoting the synthesis of prodigiosin using peony seed oil, characterized in that: The Serratia marcescens RedJ with a preservation number of CGMCC No.: 21691 is fermented using the fermentation medium according to any one of claims 1 to 3.

6. The method according to claim 5, characterized in that The Serratia marcescens RedJ seed culture solution is inoculated into the fermentation medium according to any one of claims 1 to 3 at a volume ratio of 1-3%, and the fermentation culture is carried out at 20-25° C. and 180-220 rpm for 50-72 hours.

7. The method according to claim 6, characterized in that The Serratia marcescens RedJ seed culture solution was inoculated into the fermentation medium according to claim 3 at a volume ratio of 2%, and the culture was carried out at 20° C. and 200 rpm for 60 hours.

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