Propionibacterium freudenreichii and application thereof in production of propionic acid

Abstract

This invention discloses a strain of *Propionibacterium freundii* and its application in propionic acid production. The strain was deposited at the China Center for Type Culture Collection on October 22, 2025, with accession number CCTCC M 20252289. This invention obtains a high-propionic acid-producing *Propionibacterium freundii* strain through screening from nature. Its 16S RNA was identified as *Propionibacterium freundii*. This strain is a safe edible strain approved by the Ministry of Health. Through optimization of its culture and fermentation conditions, the final propionic acid yield can reach over 50 g / L, the glucose conversion rate can reach 60%, and there is no lactic acid accumulation, meeting the requirements of industrial production. Furthermore, through fermentation optimization, the content of other acids can be effectively controlled, resulting in propionate products of different specifications that meet market demands.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a strain of Propionibacterium fischeri and its application in the production of propionic acid. Background Technology

[0002] Propionic acid is an important fine chemical product and basic chemical raw material. Propionic acid and its derivatives are widely used in food, feed, and pharmaceutical industries. The main production methods for propionic acid are chemical synthesis and biosynthesis. Chemical synthesis is less expensive, so it is the primary method used in industrial production of propionic acid. This mainly includes the oxidation of propionaldehyde, carboxylation of ethanol, and the recovery of propionic acid as a byproduct during the oxidation of light hydrocarbons to produce acetic acid. Compared to chemical synthesis, biosynthesis is more expensive due to its longer fermentation cycle, lower acid concentration, and higher proportion of other acids.

[0003] In recent years, due to the increasing demand for biologically derived products, propionic acid produced from renewable raw materials has garnered growing attention. Fermentation-based propionic acid production uses glucose as a carbon source, with host microorganisms utilizing their inherent metabolic pathways to produce propionic acid. The carbon in the resulting propionic acid is entirely derived from glucose, resulting in high naturalness and safety. Therefore, propionic acid produced through bio-fermentation is particularly favored in the food preservation field. However, natural Propionibacterium has drawbacks such as a long production cycle, low product concentration, and low conversion rate. Although there are reports in the literature on metabolic engineering of natural strains, the safety of genetically modified microorganisms used as food ingredients raises concerns. Therefore, continuously screening for high-yield natural propionic acid-producing strains and optimizing their fermentation processes is particularly important. Summary of the Invention

[0004] The technical problem to be solved is that existing technologies for producing natural Propionibacterium acnes using microbial fermentation suffer from long production cycles, low product concentrations, and low conversion rates. To address these problems, this invention provides a strain of Propionibacterium fischeri and its applications. The Propionibacterium fischeri provided by this invention produces propionic acid with a significantly higher efficiency than other products. This invention is mainly achieved through the following technical solutions: In a first aspect, the present invention provides a strain of Propionibacterium fischeri CR032, with the Latin name... Propionibacterium Freudenreichii CR032 ， It was deposited at the China Center for Type Culture Collection on October 22, 2025, with accession number CCTCC NO: M 20252289.

[0005] Furthermore, the Propionibacterium fischeri provided by this invention exhibits proliferative activity in an environment with a pH of 4.5-8.5 and a temperature of 28℃-37℃.

[0006] Furthermore, the *Propionibacterium freundii* provided by this invention has the ability to produce propionic acid through fermentation under conditions of pH 4.5-8.5 and temperature 28℃-37℃. Under these culture or fermentation conditions, the *Propionibacterium freundii* provided by this invention exhibits a higher propionic acid production capacity than other *Propionibacterium* species.

[0007] Secondly, the present invention provides a microbial preparation comprising one or more of the aforementioned Propionibacterium fischeri or its bacterial suspension or its culture medium or its fermentation product.

[0008] Thirdly, the present invention provides the application of Propionibacterium fischeri or a microbial preparation in the production or preparation of propionic acid.

[0009] Fourthly, the present invention provides the use of Propionibacterium fischeri or microbial preparations in the production or preparation of propionate products.

[0010] In this invention, a high-propionic acid-producing Propionibacterium strain was obtained by screening from nature. Its 16S RNA was identified as *Propionibacterium fischeri*, and its 16S RNA sequence is shown in SEQ ID NO.1. The screening method for Propionibacterium in this invention involves excavating deep soil samples from hillsides and dense forests in different regions to screen for anaerobic bacteria. Based on the results of propionic acid detection by high-performance liquid chromatography, high-propionic acid-producing strains were selected.

[0011] SEQ ID No.1 (1) Culture medium used for screening Screening medium (g / L): glucose 20, corn steep liquor 20, calcium carbonate 1, pH 6.9; Enrichment medium A (g / L): lactic acid 20, corn steep liquor 20, calcium carbonate 1, pH 6.9; Enrichment medium B (g / L): glycerol 20, corn steep liquor 20, calcium carbonate 1, antifoaming agent 0.2, pH 6.9; Solid plates for isolating culture medium (g / L): peptone 10, beef extract 10, yeast extract 5, diammonium citrate 2, glucose 20, Tween 80 1, sodium acetate 5, dipotassium hydrogen phosphate 2, magnesium sulfate 0.58, manganese sulfate 0.25, agar 18, bromophenol blue 0.04, pH 6.9; Liquid fermentation medium (g / L): glucose 20, peptone 5, yeast extract 10, dipotassium hydrogen phosphate 2.5, potassium dihydrogen phosphate 1.5, MgSO4 10.

[0012] (2) Specific method of screening culture: Take an appropriate amount of sample, dilute it with sterile water, inoculate it into a test tube containing screening culture medium, and anaerobically incubate it at 32℃ for 5 days.

[0013] (3) Specific method of enrichment culture: After centrifuging the selected bacterial solution, pick an appropriate amount of bacterial sludge and inoculate it into a test tube containing enrichment culture medium A and anaerobic culture at 32℃ for 4-5 days for the first enrichment culture; after the first enrichment culture, centrifuge the culture solution again, pick an appropriate amount of bacterial sludge and inoculate it into a test tube containing enrichment culture medium B and anaerobic culture at 32℃ for 4-5 days for the second enrichment culture.

[0014] (4) Specific method of isolation culture: Take an appropriate amount of the culture medium after enrichment culture, dilute it with sterile water, spread it on the solid plate of isolation culture medium, and anaerobic culture at 32℃ for 4-5 days. Select colonies with different shapes, such as milky white, neat edges, round and smooth, and light yellow to yellow back, and inoculate them into a 96-well deep plate containing liquid fermentation culture medium. After anaerobic culture at 32℃ for 4-5 days, take the fermentation broth and centrifuge it. Perform HPLC detection on the propionic acid content of the supernatant. Based on the detection results, screen out strains that produce high propionic acid.

[0015] The production of propionic acid by Propionibacterium fermentation includes the following steps: Propionibacterium seeds are inoculated into an activated seed liquid, and after growth, they are transferred to a fermentation seed liquid, and then inoculated into a fermentation medium for fermentation to obtain a fermentation broth.

[0016] Preferably, the fermentation seed liquid consists of 2-10 g / L peptone, 5-20 g / L yeast extract, 1-10 g / L KH2PO4, 1-10 g / L K2HPO4, 0.1-2 g / L magnesium sulfate, 10-20 g / L glucose or glycerol, pH 6.8-7.2, and is cultured anaerobically at 28-32℃ for 36-50 h.

[0017] Preferably, the liquid fermentation medium is: 2-10 g / L peptone, 5-30 g / L yeast extract, 1-10 g / L KH2PO4, 1-10 g / L K2HPO4, 2-4 g / L magnesium sulfate, 30-50 g / L glucose, pH 5.0-8.0.

[0018] Furthermore, the carbon source for the fermentation medium can be glycerol, glucose, or a mixture of glycerol and glucose.

[0019] Preferably, the seed culture is transferred into the fermentation medium at an inoculum rate of 1%-30% (V / V), and the fermentation conditions are 28-37℃, anaerobic culture for 96-240 h, and the pH is adjusted with calcium hydroxide solution or sodium hydroxide solution. The resulting propionate product is calcium propionate or sodium propionate.

[0020] The beneficial effects of this invention compared to the prior art are: This invention obtains a high-propionic acid-producing, GRAS-certified, food-grade Propionibacterium fischeri through screening, and optimizes its culture and fermentation conditions. The final propionic acid yield can reach more than 60 g / L, with a substrate conversion rate of 55%-60%, which meets the needs of industrial production. Through fermentation optimization, the content of miscellaneous acids can be effectively controlled, and there is no accumulation of lactic acid during the fermentation process. It can be used for the production of food preservatives containing natural propionate. Attached Figure Description

[0021] Figure 1 : Morphological image of a single colony of the screened Propionibacterium; Figure 2 Microscopic images of screened Propionibacterium acnes; Figure 3 : High-performance liquid chromatography (HPLC) chromatogram of the fermentation broth analyzed using a differential detector; Figure 4 High-performance liquid chromatography (HPLC) chromatogram of the fermentation broth analyzed using an ultraviolet detector. Detailed Implementation

[0022] The following examples are provided to better understand the present invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0023] Example 1: Screening for highly efficient Propionibacterium Soil samples from deep hills in different regions were collected, diluted with sterile water, and inoculated into test tubes containing screening medium. The samples were then incubated at 32°C for 5 days. The screening medium consisted of glucose (20 g / L), corn steep liquor (20 g / L), and calcium carbonate (1 g / L), with a pH of 6.9.

[0024] After centrifuging the fermentation broth, a suitable amount of bacterial sludge was transferred to a test tube containing enrichment medium A and incubated anaerobically at 32℃ for 4-5 days. After centrifuging the fermentation broth again, a suitable amount of bacterial sludge was transferred to a test tube containing enrichment medium B and incubated anaerobically at 32℃ for 4-5 days. A suitable amount of fermentation broth was diluted with sterile water and spread onto a solid agar plate for separation and incubated anaerobically at 32℃ for 4-5 days. The colony morphology on the plate was as follows. Figure 1 As shown in the figure. Colonies on the plate were inoculated into 96-well deep-well plates containing liquid fermentation medium and anaerobically cultured at 32°C for 4-5 days. The supernatant from fermentation centrifugation was then subjected to HPLC detection for propionic acid. Based on the detection results, strains producing high propionic acid were screened.

[0025] The culture medium used in this embodiment Screening medium (g / L): glucose 20, corn steep liquor 20, calcium carbonate 1, pH 6.9; Enrichment medium A (g / L): lactic acid 20, corn steep liquor 20, calcium carbonate 1, pH 6.9; Enrichment medium B (g / L): glycerol 20, corn steep liquor 20, calcium carbonate 1, antifoaming agent 0.2, pH 6.9; Solid plates for isolating culture medium (g / L): peptone 10, beef extract 10, yeast extract 5, diammonium citrate 2, glucose 20, Tween 80 1, sodium acetate 5, dipotassium hydrogen phosphate 2, magnesium sulfate 0.58, manganese sulfate 0.25, agar 18, bromophenol blue 0.04, pH 6.9; Liquid fermentation medium (g / L): glucose 20, peptone 5, yeast extract 10, dipotassium hydrogen phosphate 2.5, potassium dihydrogen phosphate 1.5, MgSO4 10.

[0026] Example 2: 16S RNA sequence amplification and sequencing Culture broth of *Propionibacterium fischeri* was collected, and the bacterial 16S RNA gene fragment was amplified using PCR technology. The amplified product was sequenced, and BLAST alignment confirmed that the strain was *Propionibacterium fischeri*. The 16S RNA sequence is shown in SEQ ID No. 1 of the sequence listing.

[0027] The above-mentioned Propionibacterium freundii is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20252289. Latin name Propionibacterium Freudenreichii The deposit date is October 22, 2025.

[0028] Example 3: Preparation of fermentation seed liquid from screened strains The selected *Propionibacterium fischeri* were streaked on solid culture medium and placed in an anaerobic bag for static incubation at 32°C for approximately 96 hours. Single colonies were then picked and inoculated into test tubes containing 10 mL of activated liquid culture medium and incubated at 32°C for 72-96 hours. Alternatively, seed cultured in glycerol tubes stored at -80°C was inoculated at a rate of 2%-10% into 10 mL of activated liquid culture medium and incubated at 32°C for 48-72 hours. The bacterial concentration (OD) was measured using a spectrophotometer. 600 Once the OD reaches approximately 1.5, transfer to a fermentation seed medium and incubate at 32°C for 36-50 hours. 600 When the count reaches 3 or higher, microscopic observation of the bacterial cell morphology shows that most cells appear as single rods, while a few appear as double rods. For example... Figure 2 As shown.

[0029] Example 4: Anaerobic fermentation of screened strains in a 5-L fermenter The seed culture was prepared in the same way as in Example 3. The fermentation seed culture was inoculated into the fermentation medium at 10% (V / V). The fermentation medium consisted of 5 g / L yeast extract, 5 g / L K2HPO4, 3 g / L KH2PO4, 3 g / L magnesium sulfate heptahydrate, and 60 g / L glucose. The culture was anaerobic at 32°C.

[0030] When the initial glucose concentration was below 5 g / L, a 70% glucose mother liquor was used for feeding, and the residual sugar concentration was controlled at 5-10 g / L throughout the process. The pH was maintained at 7.0 using a 20% calcium hydroxide solution. During fermentation, glucose, glycerol, propionic acid, succinic acid, acetic acid, and lactic acid were quantitatively analyzed using high-performance liquid chromatography (HPLC). An Agilent-HPLC 1200 HPLC system equipped with a BIO-RAD HPX-87H 300*7.8 mm column and differential detector was used. The mobile phase was 5 mM sulfuric acid, the column temperature was 65℃, and the flow rate was 0.65 mL / min. The fermentation broth samples were centrifuged at 12000 rpm for 2 min, diluted appropriately, filtered through a 0.22 μm membrane, and then analyzed by HPLC. After 150 h of fermentation, the chromatograms were analyzed by HPLC as shown below. Figure 3 As shown, the concentration of propionic acid was 58 g / L, the concentration of the byproduct acetic acid was 6.25 g / L, and the concentration of succinic acid was 3.25 g / L. No lactic acid accumulation was observed. Additionally, other impurities in the fermentation broth were observed using a UV detector. The detection conditions were as follows: Venusill MPC18 column (4.6 mm * 250 mm, 5 μm); mobile phase: 1.97% disodium hydrogen phosphate and 2% acetonitrile; pH adjusted to 2.0 with phosphoric acid; detection wavelength: 210 nm; column temperature: 35℃; flow rate: 1 mL / min; sample preparation was the same as described above. The HPLC results are as follows. Figure 4 As shown, the results indicate that the fermentation broth has fewer impurities under UV detection conditions.

[0031] Example 5: Anaerobic fermentation of screened strains in a 5-L fermenter The seed culture was prepared in the same manner as in Example 3. The fermentation seed culture was inoculated into the fermentation medium at a rate of 10% (V / V). The fermentation medium consisted of 20 g / L yeast extract, 5 g / L peptone, 5 g / L K₂HPO₄, 3 g / L KH₂PO₄, 3 g / L magnesium sulfate heptahydrate, and 40 g / L glycerol. Anaerobic fermentation was carried out at 32°C. When the initial glycerol concentration was below 5 g / L, 70% glycerol stock solution was used for feeding. The glycerol concentration in the fermentation broth was maintained at 5-10 g / L throughout the fermentation process, and the pH was controlled at 7.2 using a 20% calcium hydroxide solution. After 150 h of cultivation, high-performance liquid chromatography (HPLC) analysis showed a propionic acid concentration of 56 g / L, an acetic acid concentration of 4.68 g / L, and a succinic acid concentration of 3.25 g / L, with no lactic acid accumulation.

[0032] Example 6: Preparation of Fermentation Broth The seed culture was prepared in the same manner as in Example 2. The fermentation seed culture was inoculated into the fermentation medium at a rate of 10% (V / V). The fermentation medium consisted of 20 g / L yeast extract, 5 g / L peptone, 5 g / L K₂HPO₄, 3 g / L KH₂PO₄, 3 g / L magnesium sulfate heptahydrate, 40 g / L glucose, and 10 g / L glycerol. Anaerobic fermentation was carried out at 32°C. When the initial glucose concentration was below 5 g / L, a mixed mother liquor containing 70% glucose and 10% glycerol was used for feeding. The residual sugar concentration was controlled at 5-10 g / L throughout the process, and the pH was maintained at 7.0 using a 20% calcium hydroxide solution. After 140 h of cultivation, high-performance liquid chromatography (HPLC) analysis showed a propionic acid concentration of 59.32 g / L, an acetic acid concentration of 4.28 g / L, and a succinic acid concentration of 4.15 g / L, with no lactic acid accumulation.

[0033] It should be noted that, in this document, the terms “comprising,” “including,” or any other such terms are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0034] The above embodiments are merely illustrative of the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solutions based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A strain of Propionibacterium fischeri, Latin name Propionibacterium Freudenreichii It was deposited at the China Center for Type Culture Collection on October 22, 2025, with accession number CCTCC M 20252289.

2. The Propionibacterium fischeri according to claim 1, characterized in that, The Propionibacterium acnes exhibits proliferative activity and the ability to ferment and produce propionic acid or propionate products at a pH of 4.5-8.5 and a temperature of 28℃-37℃.

3. A microbial preparation, characterized in that, The microbial preparation comprises one or more of the following: Propionibacterium fischeri as described in claim 1, its bacterial suspension, its culture medium, or its fermentation product.

4. The use of Propionibacterium fischeri or a microbial preparation as described in any one of claims 1-3 in the production or preparation of propionic acid.

5. The use of Propionibacterium fischeri or a microbial preparation as described in any one of claims 1-3 in the production or preparation of propionate products.

6. The application according to claim 4 or 5, characterized in that, Select single clones or inoculate them from glycerol tubes into activation medium and culture anaerobically until OD. 600 Once the concentration reaches 0.8 or higher, inoculate it into liquid fermentation medium at a ratio of 1%-30% and culture it anaerobically for 4-10 days.

7. The application according to claim 6, characterized in that, The liquid fermentation medium includes one or more carbon sources, KH2PO4, K2HPO4 and MgSO4, wherein the carbon source is glucose or glycerol or a mixture of the two in any proportion.

8. The application according to claim 6, characterized in that, Liquid fermentation media include any one or a mixture of two of yeast extract or peptone.

9. The application according to claim 6, characterized in that, The anaerobic culture conditions are: pH 4.5-8.5 and temperature 28℃-37℃.

10. The application according to claim 9, characterized in that, Adjust the pH using a 10%-30% calcium hydroxide solution or sodium hydroxide solution.

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