Propionibacterium jensenii bred through aerospace superposition chemical mutation and method for mutagenizing, screening and fermenting propionic acid by propionibacterium jensenii

Abstract

The invention discloses propionibacterium jensenii bred by aerospace superposition chemical mutation and a method for mutagenizing, screening and fermenting propionic acid by the propionibacterium jensenii. The propionibacterium jensenii CICC22741 with high propionic acid yield is obtained by taking the propionibacterium jensenii CICC22741 as an original strain and through superposition of'practice No.19 'satellite space mutagenesis and ethyl methanesulfonate chemical mutagenesis and through two rounds of mutagenesis screening, the propionibacterium jensenii DJ04-16 with high propionic acid yield is obtained. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 16, 2025, and the preservation number is CGMCC No.34914. The invention further discloses a preparation method of the strain. Then, the propionic acid fermentation condition of the propionibacterium jensenii DJ04-16 is optimized, fermentation in a 1-ton tank verifies that the maximum propionic acid yield reaches 67.36 g / L and the fermentation intensity is 0.702 g / L.h after fermentation is performed for 96 h, and the strain has good industrial fermentation potential.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a strain of Propionibacterium jannaschii bred by space mutagenesis superimposed on ethyl methanesulfonate chemical mutagenesis, and methods for mutagenesis, screening and propionic acid fermentation thereof. Background Art

[0002] Propionic acid (PA) is an important three-carbon organic acid. As a building block, it participates in the synthesis of valuable products such as n-propanol, propylene, vitamin B12, and antimicrobial compounds (including diketopiperazines, linear and cyclic peptides, and 3-phenyllactic acid). Propionic acid and calcium propionate are important preservatives in food, chemical, and pharmaceutical synthesis. Propionic acid is also a key precursor for the production of cellulose propionate (CAP), herbicides, and nonsteroidal anti-inflammatory drugs.

[0003] Currently, the industrial production of propionic acid is still mainly based on the chemical synthesis of ethylene and carbon monoxide. However, this process faces problems such as dependence on petroleum resources, high energy consumption and environmental pollution. Microbial fermentation has become a research hotspot for replacing petrochemical routes due to its renewable raw materials and mild reaction conditions. However, the cost of the microbial fermentation route is relatively high due to the high cost of fermentation raw materials, low fermentation efficiency and high extraction energy consumption. For example, Liu et al. (Long L, Ningzi G, Gexin Z, et al. Pathway engineering of Propionibacterium jensenii for improved production of propionic acid.[J].Scientific Reports,2016,6(1):1-9.) Using glycerol as the sole carbon source, Propionibacterium jannaschii ATCC4868 was used for fermentation, and a propionic acid yield of 34.93 g / L and a production intensity of 0.265 g / Lh were obtained; Coral et al. (Jefferson C, Grace SK, Luciana VSDP, et al. Batch fermentation model of propionic acid production by Propionibacterium acidipropioniciUsing sodium lactate as the carbon source, the fermentation was carried out by Propionibacterium acidophilus ATCC 4965, and a propionic acid yield of 15.06 g / L and a production intensity of 0.113 g / Lh were obtained; Liu et al. (Yin L, Yong-Guang Z, Ru-Bing Z, et al. Glycerol / glucose co-fermentation: one more proficient process to produce propionic acid by Propionibacterium acidipropionici .[J].Current Microbiology,2011,62(1):152-8.) Using glucose and glycerol as mixed carbon sources, the same fermentation was carried out using Propionibacterium acidophilus ATCC 4965, and a propionic acid yield of 21.90 g / L and a production intensity of 0.152 g / Lh were obtained; Hoffman et al. (Bruna ZH, Bergmann LS, Rodrigo EMD, et al. Evolutionary engineering and chemical mutagenesis of Propionibacterium acidipropionici For improved propionic acid production from sugarcane-derived saccharides[J].Process Biochemistry,2023,130584-594.) Propionibacterium acidi was chemically mutagenized with ethyl methanesulfonate and combined with a propionic acid tolerance test to obtain a mutant strain M8, which achieved a propionic acid yield of 31.83 g / L and a production intensity of 0.42 g / Lh.

[0004] It can be seen from the above-cited literature that the current production of propionic acid by microbial fermentation has defects such as poor acid production level, low production efficiency, and long acid production cycle, which cannot meet the needs of propionic acid production. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a strain of Propionibacterium jannaschii bred by space-based superimposed chemical mutagenesis and a method for mutagenesis, screening and propionic acid fermentation thereof.

[0006] In a first aspect, the present invention provides a strain of Propionibacterium jannaschii bred by space-induced superposition of chemical mutagenesis, which is achieved through the following technical solutions.

[0007] A strain of Propionibacterium jannaschii bred by spaceflight superimposed chemical mutagenesis ( Propionibacterium jensenii )DJ04-16, the Propionibacterium jannaschii was deposited in the General Microbiology Center of China Culture Collection Administration on June 16, 2025, with the deposit number CGMCC No.34914.

[0008] In a second aspect, the present invention provides a method for mutagenesis screening of Propionibacterium jannaschii bred by spaceflight superimposed chemical mutagenesis, which is achieved through the following technical solutions.

[0009] A method for mutagenesis screening of Propionibacterium jannaschii comprises the following steps: S1. Using Propionibacterium janaschii CICC22741 from the China Industrial Culture Collection of Microorganisms as the starting strain, we subjected the strain to space mutagenesis. We then conducted primary screening, secondary screening, and fermentation screening of the induced mutants to identify strains with high propionic acid production. S2. Using the strain screened in step S1 as the starting strain, a bacterial suspension was prepared. The bacterial suspension was added to ethyl methanesulfonate at a final concentration of 1% (v / v). The mutagenesis was terminated after 30-70 min. The chemically mutagenized mutants were subjected to primary screening, secondary screening, and fermentation screening to obtain strains with further increased propionic acid production.

[0010] Preferably, in step S2, the chemical mutagenesis time is 50 min.

[0011] Specifically, the specific screening method for mutant strains after space-induced mutagenesis and / or chemical mutagenesis is as follows: a. Preliminary screening: Dilute the induced bacterial suspension and apply it to the screening medium. Incubate at 30°C under anaerobic conditions for 120 hours. Select strains with fast-growing colonies, large diameters, and clear zones around the colonies, indicating good growth. b. Rescreening: The strains initially screened were inoculated onto screening medium and cultured at 30°C under anaerobic conditions for 144 hours. The diameter of each colony and the diameter of its clear zone were measured. Strains with a clear zone diameter to colony diameter ratio higher than that of the starting strain and showing good growth were selected. c. Fermentation verification: The rescreened strains were anaerobically fermented at 30°C for 144 h to obtain a strain with high propionic acid production.

[0012] Furthermore, in steps a and b, the screening culture medium formula is: 3 wt % glucose, 1 wt % yeast powder, 1 wt % peptone, 0.2 wt % calcium carbonate, 2 wt % agar powder, and the balance is water, with a pH value of 7.0.

[0013] Furthermore, in step c, the fermentation screening medium formula is: 20g / L glucose, 25g / L glycerol, 6g / L peptone, 5g / L yeast powder, 20mg / L ferrous sulfate, 100mg / L magnesium sulfate, 1g / L dipotassium hydrogen phosphate, 0.5g / L potassium dihydrogen phosphate, 4g / L corn steep liquor, pH 6.5.

[0014] In a third aspect, the present invention provides a use of Propionibacterium jannaschii selected and bred by space flight superimposed chemical mutagenesis, which is achieved through the following technical solutions.

[0015] An application of the above-mentioned Propionibacterium janalyti in high production of propionic acid.

[0016] Furthermore, Propionibacterium jannaschii is used for industrial production of high-concentration propionic acid by ton-scale tank fermentation.

[0017] In a fourth aspect, the present invention provides a method for high-yield propionic acid, which is achieved through the following technical solutions.

[0018] A method for high-production of propionic acid using the above-mentioned Propionibacterium jannaschii, wherein the method is used for industrial production of high-concentration propionic acid by fermentation in ton-scale tanks; The fermentation medium formula is: 30g / L glucose, 35g / L glycerol, 5g / L-9g / L peptone, 5g / L-10g / L yeast extract, 80mg / L ferrous sulfate, 160mg / L magnesium sulfate, 2g / L dipotassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 5g / L-8g / L corn steep liquor, pH 6.5-7.0; Fermentation conditions: Calcium hydroxide was added during the fermentation process to adjust the pH, specifically as follows: the pH was maintained at 6.0-5.5 from 0h to 36h, and at 5.0-5.5 from 37h to 96h; 20g / L-30g / L glucose and 20g / L-35g / L glycerol were added from 36h to 48h of fermentation, N2 gas was passed through the fermentation tank to maintain an anaerobic environment, the fermentation temperature was 30℃-32℃, and the fermentation was carried out for 96h.

[0019] This application has the following beneficial effects.

[0020] The Propionibacterium janalysii DJ04-16 of the present invention is a high-propionic acid-producing strain obtained through space-based superimposed chemical mutagenesis screening. After optimizing its fermentation conditions, the strain can produce propionic acid in a ton-level fermenter within 96 hours, with a maximum propionic acid yield of 67.36 g / L and a fermentation intensity of 0.702 g / L / h. This greatly shortens the propionic acid fermentation cycle, accelerates the propionic acid production rate, and significantly reduces the cost of producing propionic acid, thus making it possible to achieve large-scale industrial production of propionic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the space-based superimposed chemical mutagenesis and screening process of the present invention; Figure 2 is the chemical mutagenesis lethality curve of the present invention; Figure 3 This is a diagram showing the verification results of the space-induced fermentation of the present invention; Figure 4 This is a diagram showing the verification results of aerospace superimposed chemical mutagenesis fermentation of the present invention; Figure 5 Morphological observation images of Propionibacterium janaschii DJ04-16 and its starting strain of the present invention (A. Colony and clear zone morphology of Propionibacterium janaschii DJ04-16 in screening culture medium; B. Microscopic observation image of Propionibacterium janaschii DJ04-16 bacteria; C. Colony and clear zone morphology of the starting strain of Propionibacterium janaschii CICC22741 in screening culture medium). DETAILED DESCRIPTION

[0022] The invention is further described below with reference to the accompanying drawings and examples. Unless otherwise specified, the experimental methods used in the present invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can be purchased from relevant material sales companies.

[0023] The present invention uses Propionibacterium jannaschii CICC22741 from the China Industrial Microbial Culture Collection as the starting strain, conducts space mutagenesis via the "Shijian 19" satellite, and then superimposes 1% ethyl methanesulfonate (EMS) chemical mutagenesis for 50 minutes, combined with a high propionic acid-producing strain screening method, and finally obtains a high-propionic acid-producing Propionibacterium jannaschii ( Propionibacterium jensenii ) DJ04-16, which was deposited with the China General Microbiological Culture Collection Centre (GCMCC) on June 16, 2025, under accession number CGMCC No. 34914. The address of the deposit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Propionibacterium jannaschii DJ04-16 was used for propionic acid fermentation. By optimizing the culture conditions for propionic acid fermentation, propionic acid fermentation was achieved in a single cubic tank for 96 hours, with a maximum propionic acid yield of 67.36 g / L and a fermentation intensity of 0.702 g / L / h. This strain has good potential for industrial propionic acid production. The specific experimental methods are as follows: The mutagenesis and screening process of a high-propionic acid-producing Propionibacterium jannaschii strain DJ04-16 is as follows: 1. Spaceflight Superimposed on Chemical Mutation Process Space superimposed chemical mutagenesis and its screening process Figure 1 shown.

[0024] 1. Spaceflight Mutation Process Propionibacterium jannaschii CICC22741, purchased from the China Industrial Culture Collection of Microorganisms, was used as the starting strain. Streaks were made on basic solid medium plates for activation. Single colonies with good growth were selected and inoculated onto the slant of a basic solid medium test tube and incubated anaerobically at 30°C for 4 days. The strains were then transferred to sterile 2.0 mL cryovials (with screw-on caps) and incubated anaerobically at 30°C for 4 days. After observation for good growth, the strains were stored in a refrigerator at 4°C for future use. The "Shijian 19" satellite was launched on September 27, 2024, aboard a Long March 2 carrier rocket from the Jiuquan Satellite Launch Center. The orbital altitude was approximately 700 km (sun-synchronous circular orbit) at an inclination of approximately 98°. The strains underwent space mutagenesis treatment, experiencing the high vacuum pressure, microgravity, and strong radiation conditions of space. The strains were returned to Earth on October 11, 2024. Samples were stored in the satellite's return capsule throughout the mission. Upon return to Earth, the strains were retrieved and activated. The basic solid culture medium formula is: peptone 1wt%, yeast powder 1wt%, sodium lactate 1wt%, agar powder 2wt%, and the balance is water; 2. Chemical mutagenesis method and its mutagenic dose High-yielding strains selected by space mutagenesis were streaked onto basal solid medium plates for activation. A single colony was then inoculated into liquid basal medium (basal medium formulation: 1 wt% peptone, 1 wt% yeast extract, 1 wt% sodium lactate, balance water) and cultured at 30°C for 48 h. A 4% inoculum was then transferred to liquid basal medium and anaerobically cultured at 30°C for 25 h. After washing twice by centrifugation, the suspension was resuspended in 1 mL of 0.9% sterile saline and diluted 50-fold for later use. Five mL of the bacterial suspension was transferred to a 10-mL centrifuge tube and 50 μL of ethyl methanesulfonate (EMS) solution (final concentration 1% (v / v)) was added. The mutagenesis time was controlled (0, 20, 30, 50, 70, and 100 min). Afterwards, 2% sodium thiosulfate (mass fraction) was added at a 1:1 (v / v) ratio to terminate the mutagenesis. The supernatant was removed by centrifugation, washed twice with sterile saline, and resuspended in 5 mL of sterile saline, representing the post-mutagenized bacterial suspension. The strain after mutation treatment was diluted and spread on the basic culture medium and cultured under anaerobic conditions for 120 hours. The lethality curve of chemically mutated propionic acid bacteria was obtained with the number of colonies as an indicator. Figure 2 .from Figure 2 It can be concluded that the lethality of the strain increased rapidly within the 0-30 minute treatment period, remained at approximately 95% between 30 and 70 minutes, and reached 100% lethality after 100 minutes of treatment. Ultimately, a 50-minute ethyl methanesulfonate (EMS) treatment was selected as the chemical mutagenic dose.

[0025] 2. Screening of mutagenic strains 1. Space mutagenesis and strain screening The Propionibacterium jannaschii CICC22741 from the China Industrial Microorganism Culture Collection was used as the starting strain. After space mutagenesis, the mutant strain was aseptically scraped from the slant basal culture medium and inoculated into liquid basal culture medium at 30°C for 48 h of anaerobically activated culture. Then, the inoculum was transferred to liquid basal culture medium at a 4% inoculum volume and incubated at 30°C for 24 h of anaerobically activated culture. The mid-logarithmic bacterial liquid was taken and serially diluted 10 6 The CFU / mL was evenly spread on the screening medium plate and incubated at 30℃ for 120 h. Strains with fast growth and the appearance of colony transparent zone earlier than the starting strain were preliminarily screened. These strains were spotted on the screening medium and rescreened. They were incubated in an anaerobic box at 30℃ for 144 h, and the diameter of each colony and its transparent zone were measured. Strains with a ratio of transparent zone diameter to colony diameter higher than that of the starting strain and good growth (ratio ≥ 3.3) were selected. The results are shown in Table 1.

[0026] The screening medium formula is: glucose 3wt%, yeast powder 1 wt%, peptone 1 wt%, calcium carbonate 0.2wt%, agar powder 2 wt%, the balance is water, pH 7.0.

[0027] Table 1 Circular diameter ratio of Propionibacterium jannaschii induced by space flight

[0028] The circle diameter ratios of the mutagenic strains and the starting strains are shown in Table 1. The present application selected 17 strains with a circle diameter ratio greater than that of the CICC22741 strain and vigorous growth, namely: FN88-9, FN88-16, FN88-19, FN88-21, FN88-23, FN88-24, FN88-26, FN88-29, FN88-30, FN66-2, FN66-6, FN66-10, FN66-12, FN66-23, FN66-27, FN66-29, and FN66-30.

[0029] Seventeen strains were screened for propionic acid fermentation. The fermentation screening medium was as follows: 20 g / L glucose, 25 g / L glycerol, 6 g / L peptone, 5 g / L yeast powder, 20 mg / L ferrous sulfate, 100 mg / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 4 g / L corn steep liquor, pH 6.5. The strains were cultured in serum bottles at 30°C for 144 h, and the propionic acid content in the fermentation broth was determined.

[0030] This application uses HPLC to detect the concentration of propionic acid: Detection conditions: chromatographic column AminexHPX-87H, mobile phase is 5mmol sulfuric acid, flow rate is 0.8 mL / min, column temperature is 30℃, sample volume is 10μL, and ultraviolet detection wavelength is 215nm. Preparation of propionic acid standard solution: 20 g / L of propionic acid standard solution is diluted with 5mmol sulfuric acid to 0.5g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, and 2.5 g / L respectively. Propionic acid standard curve: With the propionic acid concentration as the horizontal axis and the peak area as the vertical axis, a regression curve of the propionic acid concentration and the detection peak area is established. Within the detection concentration range, the linear range of propionic acid is good, and the regression equation is: y =332.3x+44.343, R 2 =0.9998. Propionic acid fermentation intensity = propionic acid concentration (g / L) / propionic acid fermentation time (h).

[0031] The results are as follows Figure 3 As shown, all mutant strains significantly outperformed the starting strain CICC22741 in propionic acid production, achieving a 100% positive screening rate. Fifteen strains exhibited improved propionic acid / total acid ratios compared to CICC22741, with a positive screening rate of 88.24%, demonstrating the applicability of this plate-clearing zone screening method. Mutants FN88-21, FN88-30, and FN66-30 all showed increases in propionic acid production exceeding 7.06%. Strain FN88-30 exhibited a 7.52% increase in propionic acid production compared to the starting strain, Propionibacterium jannaschii CICC22741. Ultimately, strain FN88-30, with the highest propionic acid production, was selected for subsequent chemical mutagenesis in the hope of obtaining a more dominant strain.

[0032] 2. Superimpose chemical mutagenesis to obtain high-yielding propionic acid bacteria The FN88-30 strain was used as the starting strain and subjected to chemical mutagenesis treatment with ethyl methanesulfonate (EMS) for 50 min. The strain after mutagenesis was diluted and spread on the basal culture medium, and the mutagenic bacterial suspension was diluted 10 6 After measuring the CFU / mL, 100 μL was spread onto screening medium and incubated at 30°C for 120 h. Initial screening was performed for strains that grew rapidly and appeared with a clearing zone earlier than the starting strain FN88-30. These strains were then inoculated onto screening medium and incubated at 30°C in an anaerobic chamber for 144 h. The colony diameters and clearing zone diameters of the same colonies were measured. Rescreening was performed to select strains with a clearing zone diameter to colony diameter ratio greater than that of the starting strain (ratio ≥ 4) and good growth. The results are shown in Table 2. The screening medium formula is: glucose 3wt%, yeast powder 1wt%, peptone 1 wt%, calcium carbonate 0.2 wt%, agar powder 2 wt%, and the balance is water, pH 7.0.

[0033] Table 2 Circle diameter ratio of chemically induced strains

[0034] Among them, 19 strains with a circle diameter ratio greater than that of the starting strain FN88-30 were selected for the next fermentation screening, namely: DJ01-11, DJ01-13, DJ01-15, DJ02-6, DJ02-12, DJ02-13, DJ02-16, DJ03-2, DJ03-5, DJ03-6, DJ03-7, DJ03-8, DJ03-10, DJ03-12, DJ03-13, DJ04-10, DJ04-11, DJ04-14, and DJ04-16.

[0035] Fermentation screening medium: 20g / L glucose, 25g / L glycerol, 6g / L peptone, 5g / L yeast extract, 20mg / L ferrous sulfate, 100mg / L magnesium sulfate, 1g / L dipotassium hydrogen phosphate, 0.5g / L potassium dihydrogen phosphate, 4g / L corn steep liquor, pH 6.5; 19 mutant strains obtained through multiple screening were cultured in serum bottles at 30°C for 144h, and the propionic acid content in the fermentation broth was determined. Excellent strains were selected based on the dual indicators of propionic acid production and its proportion in total organic acids. The results are as follows Figure 4 As shown, 144-h fermentation endpoint testing revealed that all mutant strains significantly outperformed the starting strain FN88-30 in propionic acid production, achieving a 100% positive screening rate. Fifteen of these strains exhibited improved propionic acid / total acid ratios compared to FN88-30, demonstrating the applicability of this plate-clearing zone screening method. Mutant strains DJ03-2, DJ03-7, and DJ04-16 all exhibited increases in propionic acid production exceeding 20%, demonstrating a significant advantage in propionic acid production.

[0036] Strain DJ04-16 produced 22.11% more propionic acid than the starting strain FN88-30 and 31.27% more propionic acid than the original strain CICC22741, making it the highest acid-producing strain. The stability of the DJ04-16 strain was verified by slant transfer for 10 generations. No difference in propionic acid fermentation was observed between the 0th, 2nd, 4th, 6th, 8th, and 10th generations of slant cultures. Therefore, the strain DJ04-16 with the highest acid production was selected as the high-yielding strain for the superimposed mutagenesis screening and named Propionibacterium jannaschii DJ04-16.

[0037] The physicochemical properties of the high-propionic acid-producing Propionibacterium jannaschii DJ04-16 are as follows: This strain belongs to the genus Propionibacterium, is a Gram-positive, non-motile, facultative anaerobic bacterium, generally short rod-shaped, non-spore-forming, and can produce propionic acid using a variety of carbon sources (glucose, fructose, mannose, glycerol, lactic acid, lactose, galactose), but cannot grow using polysaccharides (cellulose, starch, hemicellulose, etc.). The colony morphology of this strain in the screening medium and its microscopic observation are shown in Figure 2. Figure 5The colony characteristics of this strain are: round colonies, 1.5-2.6 mm, milky white, moist and smooth, opaque, with neat edges; microscopic observation shows that the bacterial cells are short rods, arranged individually in pairs or "V" shapes, and are uniform in size, 0.5-0.8×0.9-1.6 µm.

[0038] 3. Optimization of propionic acid fermentation by high-yielding Propionibacterium jannaschii DJ04-16 1. Orthogonal test results of carbon source and nitrogen source Carbon and nitrogen sources have interactive effects on propionic acid fermentation. Therefore, orthogonal experiments were carried out with different concentrations of glucose, glycerol, peptone and yeast powder. The results are shown in Table 3. Different R values ​​represent the degree of influence of each factor on the propionic acid fermentation results, which is D>C>B>A. After variance analysis, see Table 4, all four factors are significant factors (p<0.05). The primary and secondary factors affecting propionic acid fermentation are D>C>B>A, which is consistent with the results analysis in Table 3. The optimal level combination is A1B2C3D3; that is, 30g / L glucose, 35g / L glycerol, 9g / L peptone and 10g / L yeast powder.

[0039] Table 3 Results and analysis of orthogonal test for carbon and nitrogen source optimization

[0040] Table 4 Analysis of variance table of orthogonal test of carbon and nitrogen sources

[0041] a:R 2 = 0.934 (Adjusted R 2 = 0.876) 2. Orthogonal test of optimal concentrations of phosphate and magnesium ions Since magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and corn steep liquor are key influencing factors in the propionic acid fermentation process and have interactive effects, a four-factor three-level orthogonal experiment was conducted. The results are shown in Table 5. The influence of the four factors on the fermentation production of propionic acid is factor C>D>A>B. The optimal level combination is obtained from Table 5 as A3B2C1D 2； The analysis of variance is shown in Table 6. The results show that only factor B (dipotassium hydrogen phosphate) was insignificant (p>0.05), while the other three factors were significant (p<0.05). The primary and secondary factors affecting propionic acid fermentation production were C>D>A>B, consistent with the results in Table 5. The optimal fermentation combination was 160 mg / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, and 8 g / L corn steep liquor.

[0042] Therefore, the optimal fermentation formula of strain DJ04-16 was determined as: 30 g / L glucose, 35 g / L glycerol, 9 g / L peptone, 10 g / L yeast powder, 80 mg / L ferrous sulfate, 160 mg / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, and 8 g / L corn steep liquor.

[0043] Table 5 Results and analysis of orthogonal test of optimal fermentation medium

[0044] Table 6 Variance analysis of orthogonal test for optimal fermentation medium

[0045] a:R 2 = 0.940 (Adjusted R 2 = 888) The DJ04-16 propionic acid fermentation experiment was carried out using the above optimal fermentation formula, and the final propionic acid production reached 33.05±0.08g / L after 144h of fermentation.

[0046] 4. Fermentation test of high-propionic acid-producing Propionibacterium jannaschii DJ04-16 A 1-ton tank fermentation test was conducted using Propionibacterium DJ04-16, a high-propionic acid-producing strain.

[0047] Fermentation medium: 30g / L glucose, 35g / L glycerol, 5g / L-9g / L peptone, 5g / L-10g / L yeast extract, 80mg / L ferrous sulfate, 160mg / L magnesium sulfate, 2g / L dipotassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 5g / L-8g / L corn steep liquor, pH 6.5-7.0; Fermentation conditions: Calcium hydroxide was automatically added during fermentation to adjust the pH, maintaining it between 6.0 and 5.5 from 0 to 36 hours, and between 5.0 and 5.5 from 37 to 96 hours. Between 36 and 48 hours, 20 to 30 g / L glucose and 20 to 35 g / L glycerol were added, and the fermentor was purged with N2 to maintain an anaerobic environment. After 96 hours of fermentation, the maximum propionic acid yield reached 67.36 g / L, with a fermentation intensity of 0.702 g / L / h. See the examples below for details.

[0048] Example 1

[0049] A 1-ton tank fermentation test was conducted using Propionibacterium DJ04-16, a high-propionic acid-producing strain.

[0050] Optimized fermentation medium: 30 g / L glucose, 35 g / L glycerol, 9 g / L peptone, 10 g / L yeast extract, 80 mg / L ferrous sulfate, 160 mg / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 8 g / L corn steep liquor, pH 6.5; Fermentation conditions: Calcium hydroxide was automatically added during fermentation to adjust the pH, maintaining it at 6.0 from 0 to 36 hours and at 5.0 from 37 to 96 hours. After 40 hours of fermentation, 30 g / L of glucose and 35 g / L of glycerol were added, and the fermenter was purged with nitrogen to maintain an anaerobic environment. After 96 hours of fermentation, the maximum propionic acid yield reached 60.55 g / L, with a fermentation intensity of 0.63 g / L / h.

[0051] Example 2

[0052] A 1-ton tank fermentation test was conducted using Propionibacterium DJ04-16, a high-propionic acid-producing strain.

[0053] Optimized fermentation medium: 30 g / L glucose, 35 g / L glycerol, 7.5 g / L peptone, 8 g / L yeast extract, 80 mg / L ferrous sulfate, 160 mg / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 7 g / L corn steep liquor, pH 6.7; Fermentation conditions: Calcium hydroxide was automatically added during fermentation to adjust the pH, maintaining it at 6.0 from 0 to 36 hours and at 5.5 from 37 to 96 hours. After 42 hours of fermentation, 30 g / L of glucose and 35 g / L of glycerol were added, and the fermenter was vented with nitrogen to maintain an anaerobic environment. After 96 hours of fermentation, propionic acid production reached 60.02 g / L, with a fermentation intensity of 0.625 g / L / h.

[0054] Example 3

[0055] A 1-ton tank fermentation test was conducted using Propionibacterium DJ04-16, a high-propionic acid-producing strain.

[0056] Optimized fermentation medium: 30 g / L glucose, 35 g / L glycerol, 5 g / L peptone, 5 g / L yeast extract, 80 mg / L ferrous sulfate, 160 mg / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 5 g / L corn steep liquor, pH 7.0; Fermentation conditions: Calcium hydroxide was automatically added during fermentation to adjust the pH, maintaining it at 6.0 from 0 to 36 hours and at 5.5 from 37 to 96 hours. After 44 hours of fermentation, 20 g / L of glucose and 25 g / L of glycerol were added, and the fermenter was vented with nitrogen to maintain an anaerobic environment. After 96 hours of fermentation, propionic acid production reached 59.87 g / L, with a fermentation intensity of 0.623 g / L / h.

[0057] Example 4

[0058] A 1-ton tank fermentation test was conducted using Propionibacterium DJ04-16, a high-propionic acid-producing strain.

[0059] Optimized fermentation medium: 30 g / L glucose, 35 g / L glycerol, 9 g / L peptone, 10 g / L yeast extract, 80 mg / L ferrous sulfate, 160 mg / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 8 g / L corn steep liquor, pH 7.0; Fermentation conditions: Calcium hydroxide was automatically added during fermentation to adjust the pH, maintaining it at 6.0 from 0 to 36 hours and at 5.5 from 37 to 96 hours. After 48 hours, 30 g / L of glucose and 25 g / L of glycerol were added, and the fermenter was vented with nitrogen to maintain an anaerobic environment. After 96 hours of fermentation, propionic acid production reached 67.36 g / L, with a fermentation intensity of 0.702 g / L / h.

[0060] The embodiments of this specific implementation method are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Propionibacterium jannaschii bred by spaceflight superimposed chemical mutagenesis ( Propionibacterium jensenii )DJ04-16, the Propionibacterium jannaschii was deposited in the General Microbiology Center of China Culture Collection Administration on June 16, 2025, with the deposit number CGMCC No.34914.

2. A method for mutagenesis screening of Propionibacterium jannaschii according to claim 1, characterized in that: The following steps are involved: S1. Using Propionibacterium janaschii CICC22741 from the China Industrial Culture Collection of Microorganisms as the starting strain, we subjected the strain to space mutagenesis. We then conducted primary screening, secondary screening, and fermentation screening of the induced mutants to identify strains with high propionic acid production. S2. Using the strain screened in step S1 as the starting strain, a bacterial suspension is prepared. The bacterial suspension is added to ethyl methanesulfonate at a final concentration of 1% (v / v). The mutagenesis is terminated after 30 to 70 minutes. The chemically mutagenized mutants are subjected to primary screening, secondary screening, and fermentation screening to obtain strains with further increased propionic acid production.

3. The method for mutagenesis screening of Propionibacterium jannaschii according to claim 2, wherein: In step S2, the chemical mutagenesis time is 50 min.

4. The method for mutagenesis screening of Propionibacterium jannaschii according to claim 2, wherein: The specific screening method for mutant strains after space-induced mutagenesis and / or chemical mutagenesis is as follows: a. Preliminary screening: Dilute the induced bacterial suspension and apply it to the screening medium. Incubate at 30°C under anaerobic conditions for 120 hours. Select strains with fast-growing colonies, large diameters, and clear zones around the colonies, indicating good growth. b. Rescreening: The strains initially screened were inoculated onto screening medium and cultured at 30°C under anaerobic conditions for 144 hours. The diameter of each colony and the diameter of its clear zone were measured. Strains with a clear zone diameter to colony diameter ratio higher than that of the starting strain and showing good growth were selected. c. Fermentation verification: The rescreened strains were anaerobically fermented at 30°C for 144 h to obtain a strain with high propionic acid production.

5. The method for mutagenesis screening of Propionibacterium janalysii according to claim 4, wherein: In steps a and b, the screening medium formula is: 3 wt % glucose, 1 wt % yeast powder, 1 wt % peptone, 0.2 wt % calcium carbonate, 2 wt % agar powder, and the balance is water, with a pH value of 7.

0.

6. The method for mutagenesis screening of Propionibacterium jannaschii according to claim 4, wherein: In step c, the fermentation screening medium formula is: 20g / L glucose, 25g / L glycerol, 6g / L peptone, 5g / L yeast extract, 20mg / L ferrous sulfate, 100mg / L magnesium sulfate, 1g / L dipotassium hydrogen phosphate, 0.5g / L potassium dihydrogen phosphate, 4g / L corn steep liquor, pH 6.

5.

7. Use of the Propionibacterium janaphysonii according to claim 1 in high propionic acid production.

8. The application according to claim 7, characterized in that: Propionibacterium janalysii is used for industrial production of high-concentration propionic acid by ton-scale tank fermentation.

9. A method for high propionic acid production using the Propionibacterium janalysii according to claim 1, characterized in that: The method is used for industrial production of high-concentration propionic acid by fermentation in ton-scale tanks; The fermentation medium formula is: 30g / L glucose, 35g / L glycerol, 5g / L-9g / L peptone, 5g / L-10g / L yeast extract, 80mg / L ferrous sulfate, 160mg / L magnesium sulfate, 2g / L dipotassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 5g / L-8g / L corn steep liquor, pH 6.5-7.0; Fermentation conditions: Calcium hydroxide was added during the fermentation process to adjust the pH, specifically: the pH was maintained at 6.0-5.5 from 0h to 36h, and at 5.0-5.5 from 37h to 96h; 20g / L-30g / L glucose and 20g / L-35g / L glycerol were added from 36h to 48h of fermentation, N2 gas was passed through the fermentation tank to maintain an anaerobic environment, the fermentation temperature was 30℃-32℃, and the fermentation was carried out for 96h.

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