A strain of Propionibacterium jannaschii bred by space-based superimposed chemical mutagenesis and methods for mutagenesis, screening, and propionic acid fermentation thereof

Abstract

This invention discloses a *Propionibacterium japonicum* strain selected through space-based combined chemical mutagenesis and its mutagenesis, screening, and propionic acid fermentation methods. Using *Propionibacterium japonicum* strain CICC22741 from the China Industrial Microbiological Culture Collection Center as the starting strain, this invention employs space-based mutagenesis via the "Shijian-19" satellite combined with ethyl methanesulfonate chemical mutagenesis. After two rounds of mutagenesis screening, a high-propionic acid-producing strain, *Propionibacterium japonicum* DJ04-16, was obtained. This strain was deposited at the China General Microbiological Culture Collection Center on June 16, 2025, with accession number CGMCC No. 34914. Subsequently, the propionic acid fermentation conditions of *Propionibacterium japonicum* DJ04-16 were optimized. Validation was performed in a 1-ton fermentation tank. After 96 hours of fermentation, the highest propionic acid yield reached 67.36 g / L, with a fermentation intensity of 0.702 g / L / h. This strain exhibits good industrial fermentation potential.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a strain of Propionibacterium japonicum bred through space mutagenesis combined with chemical mutagenesis with ethyl methanesulfonate, and methods for mutagenesis, screening and fermentation of propionic acid. Background Technology

[0002] Propionic acid (PA) is an important three-carbon organic acid that, as a modular molecule, participates in the synthesis of valuable products such as n-propanol, propylene, vitamin B12, and antibacterial compounds (including diketopiperazine, linear and cyclic peptides, and 3-phenyllactic acid). Propionic acid and calcium propionate, as preservatives, have significant applications in food, chemical, and pharmaceutical synthesis. Furthermore, propionic acid is a key precursor compound for the production of cellulose propionate (CAP), herbicides, and nonsteroidal anti-inflammatory drugs.

[0003] Currently, the industrial production of propionic acid still mainly relies on the chemical synthesis method using ethylene and carbon monoxide. However, this process faces problems such as dependence on petroleum resources, high energy consumption, and environmental pollution. Microbial fermentation, due to its renewable raw materials and mild reaction conditions, has become a research hotspot for alternatives to petrochemical routes. However, the microbial fermentation route is costly 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 japonicum ATCC4868 was used for fermentation, resulting in a propionic acid yield of 34.93 g / L and a production intensity of 0.265 g / Lh; 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, Propionibacterium acidogenetice ATCC 4965 was used for fermentation to obtain a propionic acid yield of 15.06 g / L and a production intensity of 0.113 g / Lh; 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 indifferent carbon sources.[J].Applied Biochemistry and Biotechnology,2008,151(2-3):333-41.) Propionibacterium acidipropionici [J]. Current Microbiology, 2011, 62(1): 152-8.) Using a mixed carbon source of glucose and glycerol, and fermented with Propionibacterium ATCC 4965, a propionic acid yield of 21.90 g / L and a production intensity of 0.152 g / Lh were obtained; Hoffmam et al. (Bruna ZH, Bergmann LS, Rodrigo EMD, et al. Evolutionary engineering and chemical mutationnesis of Propionibacterium acidipropionici For improved propionic acid production from sugarcane-derived saccharides[J]. Process Biochemistry, 2023, 130584-594.) By chemically mutagenesis of Propionibacterium acid-producing with ethyl methanesulfonate combined with propionic acid tolerance test, mutant strain M8 was obtained, with propionic acid production reaching a production intensity of 31.83 g / L and 0.42 g / Lh.

[0004] As can be seen from the above-cited literature, the current microbial fermentation production of propionic acid 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-mentioned technical problems, the present invention provides a strain of Propionibacterium japonicum selected by aerospace superimposed chemical mutagenesis and a method for mutagenesis, screening and fermentation of propionic acid.

[0006] In the first aspect, the present invention provides a strain of Propionibacterium japonicum bred through aerospace superimposed chemical mutagenesis, which is achieved through the following technical solution.

[0007] A strain of Propionibacterium japonicum bred through space-based superimposed chemical mutagenesis ( Propionibacterium jensenii DJ04-16, Propionibacterium japonicum was deposited at the China General Microbiological Culture Collection Center on June 16, 2025, with accession number CGMCC No. 34914.

[0008] Secondly, the present invention provides a mutagenesis screening method for Propionibacterium japonicum bred through aerospace superimposed chemical mutagenesis, which is achieved through the following technical solution.

[0009] A method for mutagenesis screening of the above-mentioned Propionibacterium japonicum includes the following steps:

[0010] S1. Using Propionibacterium japonicum CICC22741 from the China Industrial Microbial Culture Collection Center as the starting strain, space mutagenesis was carried out in the space environment. The mutant strains after space mutagenesis were screened first, second and fermented to obtain strains with high propionic acid production.

[0011] S2. Using the strains obtained in step S1 as the starting strains, prepare a bacterial suspension. Add the bacterial suspension to ethyl methanesulfonate at a final concentration of 1% (v / v). After mutagenesis for 30-70 min, terminate the mutagenesis. Perform primary screening, secondary screening, and fermentation screening on the chemically mutagenic mutants to obtain strains with further increased propionic acid production.

[0012] Preferably, in step S2, the chemical mutagenesis time is 50 min.

[0013] Specifically, the screening methods for mutant strains induced by space mutagenesis and / or chemical mutagenesis are as follows:

[0014] a. Preliminary screening: The mutagenized bacterial suspension was diluted and spread on the screening medium. It was cultured in an anaerobic environment at 30℃ for 120h. Strains with fast growth, large colony diameter, and clear zone around the colony were selected.

[0015] b. Secondary screening: The strains selected in the preliminary screening were inoculated on the screening medium and cultured in an anaerobic environment at 30℃ for 144h. 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 good growth were selected.

[0016] c. Fermentation verification: The re-screened strains were anaerobic fermented at 30℃ for 144 h to finally obtain strains with high propionic acid production.

[0017] Furthermore, in steps a and b, the screening culture medium formula is as follows: 3 wt% glucose, 1 wt% yeast extract, 1 wt% peptone, 0.2 wt% calcium carbonate, 2 wt% agar powder, with the remainder being water, and a pH of 7.0.

[0018] Furthermore, in step c, the fermentation screening medium formula is as follows: 20 g / L glucose, 25 g / L glycerol, 6 g / L peptone, 5 g / L yeast extract, 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.

[0019] Thirdly, the present invention provides a use for Propionibacterium japonicum bred through aerospace superimposed chemical mutagenesis, which is achieved through the following technical solution.

[0020] An application of the above-mentioned Propionibacterium japonicum in high-yield propionic acid production.

[0021] Furthermore, Propionibacterium japonicum is used in the industrial production of high-concentration propionic acid through ton-scale fermentation.

[0022] Fourthly, the present invention provides a method for high-yield propionic acid production, which is achieved through the following technical solutions.

[0023] A method for high-yield propionic acid using the above-mentioned Propionibacterium japonicum, which is used for industrial-scale production of high-concentration propionic acid in ton-scale tank fermentation;

[0024] The fermentation medium formula is as follows: 30 g / L glucose, 35 g / L glycerol, 5 g / L~9 g / L peptone, 5 g / L~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, 5 g / L~8 g / L corn steep liquor, pH 6.5~7.0;

[0025] Fermentation conditions: Calcium hydroxide was added during the fermentation process to adjust the pH, specifically as follows: pH was maintained at 6.0-5.5 from 0h to 36h, and at 5.0-5.5 from 37h to 96h; 20g / L to 30g / L glucose and 20g / L to 35g / L glycerol were added from 36h to 48h of fermentation. The fermenter was purged with N2 gas to maintain an anaerobic environment. The fermentation temperature was 30℃ to 32℃, and the fermentation time was 96h.

[0026] This application has the following beneficial effects.

[0027] The present invention relates to Propionibacterium japonicum DJ04-16, a high-propionic acid production strain obtained through aerospace superimposed chemical mutagenesis screening. After optimizing its fermentation conditions, this strain can produce propionic acid in a ton-scale fermenter in 96 hours, with a maximum propionic acid production of 67.36 g / L and a fermentation intensity of 0.702 g / L / h. This significantly shortens the propionic acid fermentation cycle, accelerates the propionic acid production rate, and substantially reduces the cost of propionic acid production, thus providing a possibility for large-scale industrial production of propionic acid. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the aerospace superimposed chemical mutagenesis and screening process of the present invention;

[0029] Figure 2 This is the chemical mutagenesis lethality curve of this invention;

[0030] Figure 3 This is a diagram showing the results of the space-induced mutagenesis fermentation verification of this invention;

[0031] Figure 4 This is a diagram showing the results of the aerospace superimposed chemical mutagenesis fermentation verification of this invention;

[0032] Figure 5 The images show morphological observations of Propionibacterium japonicum DJ04-16 and its starting strain in this invention (wherein, A. morphological images of colonies and clear zones of Propionibacterium japonicum DJ04-16 in the screening medium; B. microscopic observation of Propionibacterium japonicum DJ04-16 cells; C. morphological images of colonies and clear zones of the initial strain of Propionibacterium japonicum CICC22741 in the screening medium). Detailed Implementation

[0033] The invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can all be purchased from relevant material sales companies.

[0034] This invention uses *Propionibacterium japonicum* CICC22741 from the China Industrial Microbial Culture Collection Center as the starting strain. Space mutagenesis was induced via the "Shijian-19" satellite, followed by chemical mutagenesis with 1% ethyl methanesulfonate (EMS) for 50 minutes. Combined with a high-propionic acid-producing strain screening method, a high-propionic acid-producing *Propionibacterium japonicum* strain was finally obtained. Propionibacterium jensenii The strain DJ04-16 was deposited on June 16, 2025, at the China General Microbiological Cultuer Collection Centre (GCMCC), accession number CGMCC No. 34914, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. Propionibacterium japonicum DJ04-16 was used for propionic acid fermentation. Through optimization of fermentation conditions, propionic acid fermentation was successfully achieved in a 1 cubic meter 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 fermentation production of propionic acid. Specific experimental methods are as follows:

[0035] The mutagenesis and screening process of a high-propionic acid-producing Propionibacterium japonicum strain DJ04-16 is as follows:

[0036] I. Space-based superimposed chemical mutagenesis process

[0037] Space superimposed chemical mutagenesis and its screening process, such as Figure 1 As shown.

[0038] 1. Space-induced mutation process

[0039] Using *Propionibacterium japonicum* CICC22741, purchased from the China Industrial Microbial Culture Collection Center, as the starting strain, the bacteria were activated by streaking on basal solid medium plates. Single colonies with good growth were selected and inoculated onto slant agar plates in basal solid medium and anaerobically cultured at 30°C for 4 days. The colonies were then transferred to sterile 2.0 mL cryovials (with inner screw caps) and anaerobically cultured at 30°C for 4 days. After observing good growth, the colonies were stored at 4°C for later use. The bacteria were then launched on September 27, 2024, aboard a Long March 2 rocket from the Jiuquan Satellite Launch Center (orbit altitude: approximately 700 km, sun-synchronous circular orbit; inclination: approximately 98°). The strain underwent space mutagenesis under the unique environment of high vacuum pressure, microgravity, and strong radiation, and returned to Earth on October 11, 2024. During the entire mission, the samples were stored in the satellite's return capsule. Upon return to Earth, the bacteria were retrieved for activation. The basic solid culture medium formula is: 1 wt% peptone, 1 wt% yeast extract, 1 wt% sodium lactate, 2 wt% agar powder, and the remainder is water;

[0040] 2. Chemical mutagenesis methods and their mutagenic dosage

[0041] The high-yielding bacterial strains selected through space-induced mutagenesis were streaked onto basal solid medium plates for activation. Single colonies were then inoculated into liquid basal medium (basal liquid medium formulation: 1 wt% peptone, 1 wt% yeast extract, 1 wt% sodium lactate, balance water) and incubated at 30°C for 48 h. The inoculum was then transferred to liquid basal medium at a 4% inoculation rate and anaerobically cultured at 30°C for 25 h. After centrifugation and washing twice, the bacterial suspension was resuspended in 1 mL of 0.9% sterile physiological saline and diluted 50 times for later use. 5 mL of the bacterial suspension was placed in a 10 mL centrifuge tube, and 50 μL of ethyl methanesulfonate (EMS) solution (final concentration 1% (V / V)) was added. Mutagenesis was controlled at 0, 20, 30, 50, 70, and 100 min. Mutagenesis was then terminated by adding 2% sodium thiosulfate at a 1:1 (V / V) ratio. The supernatant was discarded by centrifugation, and the bacterial suspension was washed twice by centrifugation with sterile physiological saline and resuspended in 5 mL of sterile physiological saline to obtain the mutagenic bacterial suspension. The mutagenic strain was diluted and spread onto basal medium, and cultured under anaerobic conditions for 120 h. The lethality curve of the chemically mutagenic propionic acid bacteria was obtained using colony count as an indicator. Figure 2 .from Figure 2 It can be concluded that the lethality of the strain increases rapidly within the treatment period of 0-30 min, remains at approximately 95% within the treatment period of 30-70 min, and reaches 100% lethality after 100 min of treatment. Therefore, 50 min of ethyl methanesulfonate (EMS) treatment was ultimately selected as the chemical mutagenic dose.

[0042] II. Screening of Mutagenic Strains

[0043] 1. Space mutagenesis and strain screening

[0044] Using Propionibacterium japonicum CICC22741 from the China Industrial Microbial Culture Collection Center as the starting strain, after space mutagenesis, the space-mutated mutant strain was aseptically scraped from the slant basal medium and inoculated into liquid basal medium for anaerobic activation culture at 30℃ for 48 h; then, it was transferred to liquid basal medium at a 4% inoculum and anaerobic activated culture at 30℃ for 24 h. The mid-logarithmic bacterial culture was then collected and serially diluted 10⁻⁶ times. 6 CFU / mL was evenly spread on screening medium plates and incubated at 30℃ for 120 h. Strains with rapid growth and clear zones appearing earlier than the starting strain were initially screened. These strains were then inoculated onto screening medium for secondary screening and incubated in an anaerobic chamber at 30℃ for 144 h. 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 good growth (ratio ≥ 3.3) were selected. The results are shown in Table 1.

[0045] The screening medium formula is as follows: 3 wt% glucose, 1 wt% yeast extract, 1 wt% peptone, 0.2 wt% calcium carbonate, 2 wt% agar powder, with the balance being water, and pH 7.0.

[0046] Table 1. Space-induced mutagenesis of Propionibacterium japonicum. diameter ratio

[0047]

[0048] The circle diameter ratios of the mutagenized strains and the starting strains are shown in Table 1. From these, 17 strains with a circle diameter ratio greater than that of strain CICC22741 and exhibiting vigorous growth were selected: 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.

[0049] Seventeen strains were screened by propionic acid fermentation. The fermentation culture medium consisted of 20 g / L glucose, 25 g / L glycerol, 6 g / L peptone, 5 g / L yeast extract, 20 mg / L ferrous sulfate, 100 mg / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, and 4 g / L corn steep liquor, with a pH of 6.5. The cultures were incubated at 30°C in serum bottles for 144 h, and the propionic acid content in the fermentation broth was determined.

[0050] This application uses HPLC to detect propionic acid concentration: Detection conditions: Aminex HPX-87H column, 5 mmol sulfuric acid mobile phase, flow rate 0.8 mL / min, column temperature 30℃, sample loading volume 10 μL, UV detection wavelength 215 nm. Preparation of propionic acid standard solutions: 20 g / L propionic acid standard solution was diluted with 5 mmol sulfuric acid to 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, and 2.5 g / L, respectively. Propionic acid standard curve: A regression curve was established with propionic acid concentration as the abscissa and peak area as the ordinate. Within the detection concentration range, propionic acid showed good linearity. The regression equation was: y = 332.3x + 44.343, R0 2 =0.9998. Propionate fermentation intensity = propionate concentration (g / L) / propionate fermentation time (h).

[0051] The results are as follows Figure 3 As shown, all mutant strains exhibited significantly higher propionic acid yields than the starting strain CICC22741, achieving a 100% positive selection rate. Among these, 15 strains showed an improved propionic acid / total acid ratio compared to CICC22741, with a positive selection rate of 88.24%, indicating the good applicability of this plate clear zone screening method. The propionic acid yields of mutant strains FN88-21, FN88-30, and FN66-30 all increased by more than 7.06%. Strain FN88-30 showed a 7.52% increase in propionic acid yield compared to the starting strain *Propionibacterium japonicum* CICC22741. Ultimately, strain FN88-30, with the highest propionic acid yield, was selected for subsequent chemical mutagenesis to obtain a more superior strain.

[0052] 2. Obtaining high-yield propionic acid bacteria through superimposed chemical mutagenesis

[0053] Using strain FN88-30 as the starting strain, chemical mutagenesis with ethyl methanesulfonate (EMS) was performed for 50 min. The mutagenized strain was then diluted and plated onto basal medium. The mutagenized bacterial suspension was diluted 10... 6After CFU / mL concentration, 100 μL was spread onto screening medium and incubated at 30°C for 120 h. Initial screening was conducted on strains that grew rapidly and showed a clear zone earlier than the starting strain FN88-30. The preliminarily screened strains were then inoculated onto screening medium and incubated at 30°C in an anaerobic chamber for 144 h. Colony diameter and the diameter of the clear zone for the same colony were measured for secondary screening. Strains with a clear zone diameter to colony diameter ratio higher than the starting strain and exhibiting good growth (ratio ≥ 4) were selected. The results are shown in Table 2.

[0054] The screening medium formula is as follows: 3 wt% glucose, 1 wt% yeast extract, 1 wt% peptone, 0.2 wt% calcium carbonate, 2 wt% agar powder, with the balance being water, pH 7.0.

[0055] Table 2. Diameter ratio of chemically mutagenic strains

[0056]

[0057] Nineteen strains with a diameter-to-diameter ratio greater than that of the starting strain FN88-30 were selected for further fermentation screening. These strains are: 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.

[0058] Fermentation screening medium: 20 g / L glucose, 25 g / L glycerol, 6 g / L peptone, 5 g / L yeast extract, 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. Nineteen mutant strains obtained through secondary screening were cultured at 30℃ in serum bottles for 144 h. The propionic acid content in the fermentation broth was measured, and superior strains were screened using both propionic acid yield and its proportion of total organic acids. Results are as follows: Figure 4 As shown, the 144-hour fermentation endpoint assay revealed that all mutant strains exhibited significantly higher propionic acid yields than the starting strain FN88-30, with a 100% positive screening rate. Among these, 15 strains showed an improved propionic acid / total acid ratio compared to strain FN88-30, indicating the good applicability of this plate clear zone screening method. Mutant strains DJ03-2, DJ03-7, and DJ04-16 all showed propionic acid yield increases exceeding 20%, demonstrating a significant advantage in propionic acid production.

[0059] Among them, strain DJ04-16 showed a 22.11% increase in propionic acid production compared to the starting strain FN88-30 and a 31.27% increase compared to the original strain CICC22741, making it the highest-producing acid-producing strain. The stability of strain DJ04-16 was verified through subculturing. After 10 generations of slant culture, there was no difference in propionic acid production among the 0th, 2nd, 4th, 6th, 8th, and 10th generations. Therefore, strain DJ04-16, with the highest propionic acid production, was selected as the high-producing strain through superimposed mutagenesis screening and named *Propionibacterium japonicum* DJ04-16.

[0060] The physicochemical properties of Propionibacterium japonicum DJ04-16, which produces high levels of propionic acid, are as follows:

[0061] This strain belongs to the genus *Propionibacterium*, is a Gram-positive, non-motile, facultative anaerobic bacterium, generally appearing as short rods and not forming spores. It can utilize various carbon sources (glucose, fructose, mannose, glycerol, lactic acid, lactose, galactose) to produce propionic acid, but cannot grow using polysaccharides (cellulose, starch, hemicellulose, etc.). The colony morphology of this strain in the selection medium and its microscopic observation are shown below. Figure 5 The colony characteristics of this strain are as follows: the colonies are round, 1.5-2.6 mm, milky white, moist and smooth, opaque, and with neat edges; under microscopic observation, the bacterial cells are short rod-shaped, arranged in pairs or "V" shapes, and are uniform in size, 0.5-0.8×0.9-1.6 µm.

[0062] III. Optimization of propionic acid fermentation by Propionibacterium japonicum DJ04-16 with high propionic acid production

[0063] 1. Results of the orthogonal experiment using carbon and nitrogen sources

[0064] Carbon and nitrogen sources have an interactive effect on propionic acid fermentation. Therefore, orthogonal experiments were conducted with different concentrations of glucose, glycerol, peptone, and yeast extract. The results are shown in Table 3. The influence of each factor on the propionic acid fermentation result is represented by different R values, in the order D > C > B > A. The variance analysis is shown in Table 4. All four factors were significant (p < 0.05). The main and secondary factors affecting propionic acid fermentation were D > C > B > A, consistent with the results in Table 3. The optimal level combination is A1B2C3D3, namely 30 g / L glucose, 35 g / L glycerol, 9 g / L peptone, and 10 g / L yeast extract.

[0065] Table 3 Results and Analysis of Orthogonal Experiments for Carbon and Nitrogen Source Optimization

[0066]

[0067] Table 4. Variance Analysis of Orthogonal Experiments Using Carbon and Nitrogen Sources

[0068]

[0069] a:R2 = 0.934 (adjusted R) 2 = 0.876)

[0070] 2. Orthogonal experiment for optimal concentrations of phosphate and magnesium ions

[0071] Since magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and corn steep liquor are key influencing factors in propionic acid fermentation and have interactive effects, a four-factor, three-level orthogonal experiment was conducted. The results are shown in Table 5. The degree of influence of the four factors on propionic acid production during fermentation is C > D > A > B. From Table 5, the optimal level combination is A3B2C1D. 2； The analysis of variance is shown in Table 6. The results show that only B (dipotassium hydrogen phosphate) was an insignificant factor (p>0.05), while the other three factors were significant factors (p<0.05). The order of importance of the factors affecting the fermentation production of propionic acid was 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.

[0072] Therefore, the optimal fermentation formula for strain DJ04-16 was determined to be: 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, and 8 g / L corn steep liquor.

[0073] Table 5 Results and Analysis of Orthogonal Experiment for Optimal Fermentation Medium

[0074]

[0075] Table 6. Variance analysis of the orthogonal experiment for the optimal fermentation medium

[0076]

[0077] a:R 2 = 0.940 (adjusted R) 2 = 888)

[0078] Using the above-mentioned optimal fermentation formula, the DJ04-16 propionic acid fermentation experiment was carried out, and the final propionic acid production reached 33.05±0.08g / L after 144h of fermentation.

[0079] IV. Fermentation Test of Propionibacterium japonicum DJ04-16 with High Propionic Acid Production

[0080] A 1-ton fermentation test was conducted using Propionibacterium acnes DJ04-16, which produces high levels of propionic acid.

[0081] Fermentation medium: 30 g / L glucose, 35 g / L glycerol, 5 g / L~9 g / L peptone, 5 g / L~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, 5 g / L~8 g / L corn steep liquor, pH 6.5~7.0;

[0082] Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH. The pH was maintained at 6.0–5.5 from 0h to 36h, and at 5.0–5.5 from 37h to 96h. From 36h to 48h, 20g / L–30g / L glucose and 20g / L–35g / L glycerol were added. The fermenter was purged with N2 gas to maintain an anaerobic environment. After 96h of fermentation, the highest propionic acid production reached 67.36 g / L, with a fermentation intensity of 0.702 g / Lh. See the following examples for details.

[0083] Example 1

[0084] A 1-ton fermentation test was conducted using Propionibacterium acnes DJ04-16, which produces high levels of propionic acid.

[0085] 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;

[0086] Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH value, maintaining it at 6.0 from 0h to 36h and at 5.0 from 37h to 96h. After 40h of fermentation, 30g / L glucose and 35g / L glycerol were added, and the fermenter was purged with N2 gas to maintain an anaerobic environment. After 96h of fermentation, the highest propionic acid production reached 60.55 g / L, with a fermentation intensity of 0.63 g / Lh.

[0087] Example 2

[0088] A 1-ton fermentation test was conducted using Propionibacterium acnes DJ04-16, which produces high levels of propionic acid.

[0089] 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.

[0090] Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH value, maintaining it at 6.0 from 0h to 36h and at 5.5 from 37h to 96h. After 42h of fermentation, 30g / L glucose and 35g / L glycerol were added, and the fermenter was purged with N2 gas to maintain an anaerobic environment. After 96h of fermentation, propionic acid production reached 60.02 g / L, with a fermentation intensity of 0.625 g / Lh.

[0091] Example 3

[0092] A 1-ton fermentation test was conducted using Propionibacterium acnes DJ04-16, which produces high levels of propionic acid.

[0093] 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;

[0094] Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH value, maintaining it at 6.0 from 0h to 36h and at 5.5 from 37h to 96h. After 44h of fermentation, 20g / L glucose and 25g / L glycerol were added, and the fermenter was purged with N2 gas to maintain an anaerobic environment. After 96h of fermentation, propionic acid production reached 59.87g / L, with a fermentation intensity of 0.623g / Lh.

[0095] Example 4

[0096] A 1-ton fermentation test was conducted using Propionibacterium acnes DJ04-16, which produces high levels of propionic acid.

[0097] 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;

[0098] Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH value, maintaining it at 6.0 from 0h to 36h and at 5.5 from 37h to 96h. After 48h of fermentation, 30g / L glucose and 25g / L glycerol were added, and the fermenter was purged with N2 gas to maintain an anaerobic environment. After 96h of fermentation, propionic acid production reached 67.36g / L, with a fermentation intensity of 0.702g / Lh.

[0099] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A strain of Propionibacterium japonicum bred through space-based superimposed chemical mutagenesis ( Propionibacterium jensenii DJ04-16, Propionibacterium japonicum was deposited at the China General Microbiological Culture Collection Center on June 16, 2025, with accession number CGMCC No. 34914.

2. The application of Propionibacterium japonicum as described in claim 1 in high-yield propionic acid production.

3. The application according to claim 2, characterized in that: Propionibacterium japonicum is used in the industrial production of high-concentration propionic acid through fermentation in ton-scale tanks.

4. A method for high propionic acid production using Propionibacterium japonicum as described in claim 1, characterized in that: This method is used for the industrial production of high-concentration propionic acid using ton-scale tank fermentation. The fermentation medium formula is as follows: 30 g / L glucose, 35 g / L glycerol, 5 g / L~9 g / L peptone, 5 g / L~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, 5 g / L~8 g / 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: pH was maintained at 5.5-6.0 from 0h to 36h, and pH was maintained 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. Nitrogen gas was purged through the fermenter to maintain an anaerobic environment. The fermentation temperature was 30℃-32℃, and the fermentation time was 96h.

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