Method for reducing yield of propionibacterium freudenreichii heteroacid based on directional regulation and control of double-bacterium metabolism

Abstract

The invention belongs to the technical field of microbial fermentation engineering, and relates to a method for reducing the yield of propionibacterium freudenreichii heteroacid based on directional regulation and control of double-bacterium metabolism, which comprises the following steps: respectively inoculating preserved strains of propionibacterium freudenreichii subsp. Scheri and propionibacterium propionate into a seed culture medium for activation culture; respective seed solutions are obtained; simultaneously inoculating the two obtained seed solutions into a fermentation culture medium according to a volume ratio, and carrying out staged standing culture; in the whole fermentation process, the fermentation container is shaken regularly to uniformly mix the system, and inert gas is introduced during shaking to maintain an anaerobic environment. The directional regulation of metabolic flux is realized, and heteroacid can be efficiently and directionally reduced; the yield of main products is increased, and the production efficiency is improved; by means of metabolism complementation of natural strains, complex gene manipulation is not needed, the biological safety risk and genetic instability of engineering bacteria are avoided, the technology maturity is high, the method is easy to accept, popularize and apply industrially, and non-genetic engineering means are safe and stable.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, and in particular to a method for reducing the yield of misoic acid from Propionibacterium fischeri based on targeted regulation of dual-strain metabolism. Background Technology

[0002] Propionic acid is an important short-chain fatty acid widely used in food, chemical, pharmaceutical, and feed additive industries. Microbial fermentation for propionic acid production has attracted considerable attention due to its mild conditions and environmental friendliness. *Propionibacterium fischeri* is one of the commonly used strains for industrial propionic acid production. However, during fermentation, *Propionibacterium fischeri* inevitably produces various organic acids as byproducts, including acetic acid, lactic acid, and succinic acid. The presence of these miscellaneous acids not only reduces the conversion rate of propionic acid but also significantly increases the difficulty and cost of downstream separation and purification, thus hindering the industrialization and economic feasibility of microbial fermentation for propionic acid production.

[0003] Currently, to address the high content of miscellaneous acids in propionic acid fermentation, research has largely focused on modifying the metabolic pathways of Propionibacterium acnes through genetic engineering, such as knocking out or inhibiting key enzyme genes encoding the synthesis pathways of miscellaneous acids like acetic acid and succinic acid. However, due to the immature genetic manipulation system of Propionibacterium acnes, low transformation efficiency, and poor genetic stability, successful genetic modification cases are extremely rare. Even when engineered strains are constructed, their robustness and stability for industrial applications are often difficult to guarantee, and large-scale industrial production remains a long way off.

[0004] Therefore, developing a non-genetic engineering method that is simple to process, easy to scale up, and can effectively reduce the content of impurities in propionic acid fermentation broth, especially a method that can target and eliminate certain impurities, has become an urgent and economically valuable technological need in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for reducing the yield of misoic acid from Propionibacterium fischeri based on targeted regulation of dual-strain metabolism. This method employs the following technical solution, comprising the following steps: a) The preserved strains of Propionibacterium fischeri subsp. Scheres and Propionibacterium propionitum were respectively inoculated into seed culture medium for activation culture to obtain their respective seed solutions; b) The two seed liquids obtained in step a) are simultaneously inoculated into the fermentation medium at a volume ratio and subjected to staged static culture. First stage: Incubate at 28-32℃ for 2-3 days to allow the bacteria to multiply rapidly; Second stage: Adjust the temperature to 25-28℃ and continue static culture for 3-5 days to promote propionic acid accumulation and inhibit the formation of other acids; c) Dynamic feeding control: During the second stage of cultivation, carbon source is added to the fermentation system every 20-28 hours, with the amount added being 10%-20% of the initial total carbon source; d) Fermentation process control: Throughout the fermentation process, the fermentation vessel is shaken periodically to mix the system, and an inert gas is introduced during shaking to maintain an anaerobic environment.

[0006] Preferably, the Propionibacterium fischeri subsp. Scheres is Propionibacterium fischeri subsp. Scheres PB-16, and the Propionibacterium propioni is Propionibacterium propioni BY-62. Add 0.1-0.5 g / L of L-glutamine or L-asparagine to the fermentation medium in step b) to enhance the cell's ability to metabolize and absorb succinic acid. Add 5-20 g / L of activated carbon or diatomaceous earth as an adsorption carrier to the fermentation medium in step b) for in-situ adsorption and reduction of the concentration of acetic acid and lactic acid in the fermentation broth. Before inoculation in step b), the two seed liquids were microencapsulated using sodium alginate-chitosan composite gel to improve the stability and reusability of the cells in the fermentation system. During the feeding process in step c), 0.05-0.2 g / L of sodium 2-bromoethanesulfonate is added as an inhibitor of succinic acid metabolism bypass to further reduce the formation of succinic acid.

[0007] Preferably, in step a), the seed culture medium comprises: 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium lactate, pH 6.8-7.2; preferably, it comprises 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium lactate, pH 7.0.

[0008] Preferably, in step a), the activation culture conditions are: culture temperature 28-32℃, static culture for 36-48 hours.

[0009] Preferably, in step b), the fermentation medium comprises: 8-12 g / L yeast extract, 2-3 g / L tryptone, 15-30 g / L carbon source, 0.8-1.2 g / L dipotassium hydrogen phosphate, 8-12 g / L calcium carbonate, and pH 7.3-7.7; preferably, the carbon source is a mixture of 15-25 g / L glycerol and 2-3 g / L glucose; more preferably, the fermentation medium comprises: 10 g / L yeast extract, 2.5 g / L tryptone, 20 g / L glycerol, 2.5 g / L glucose, 1 g / L dipotassium hydrogen phosphate, 10 g / L calcium carbonate, and pH 7.5.

[0010] Preferably, in step b), the inoculation volume ratio of the two seed solutions is 1:0.5-2 for Propionibacterium fischeri seed solution and 1:1 for Propionibacterium propioni seed solution.

[0011] Preferably, in step b), the total inoculation volume is 10%-30% of the fermentation medium volume.

[0012] Preferably, in step d), the periodic shaking is performed 1-3 times per day, for 5-15 minutes each time; the inert gas is nitrogen or argon, and the ventilation flow rate is 0.5-1.5 L / min.

[0013] Preferably, during the feeding process in step c), 0.01-0.05 g / L of cobalt ions are added simultaneously as a coenzyme activator to further improve the efficiency of propionic acid synthesis.

[0014] On the other hand, the present invention provides the application of Propionibacterium fischeri subsp. Scheres and Propionibacterium propionitum in the co-fermentation preparation of propionic acid and its use in reducing the succinic acid content in the fermentation broth; preferably, the Propionibacterium fischeri subsp. Scheres is PB-16 and the Propionibacterium propionitum is BY-62.

[0015] Compared with the prior art, the present invention has the following main advantages: (1) Through the synergistic effect of Propionibacterium fischeri subsp. Scheres PB-16 and Propionibacterium propionitum BY-62, the yield of the byproduct succinic acid can be selectively reduced significantly or even completely eliminated (as shown in the data of the following examples, the yield of succinic acid decreased from 1.05 g / L and 0.23 g / L in single-strain fermentation to 0.00 g / L), thus achieving the directional regulation of metabolic flux and efficiently reducing heteroacids in a targeted manner; (2) The combined fermentation of the two strains showed a synergistic promoting effect. The total yield of propionic acid (18.06 g / L) was higher than that of either single-strain fermentation (14.24 g / L and 15.74 g / L), which increased the yield of the main product and improved production efficiency. (3) Utilizing the metabolic complementarity of natural strains, without the need for complex gene manipulation, it avoids the biosafety risks and genetic instability of engineered bacteria. The technology is highly mature, easy to be accepted and promoted by the industry, and the non-genetic engineering method is safe and stable. (4) Based on the existing propionic acid fermentation process, only another strain of bacteria is added for co-culture. No special equipment or expensive reagents are required. The operation is simple and suitable for industrial scale-up production. The process is simple and the cost is low. (5) It provides a novel approach to the control of byproducts during microbial fermentation, which has important methodological significance and provides a new strategy for the regulation of heteroacids. Attached Figure Description

[0016] To more clearly illustrate the solutions in this invention, the accompanying drawings used in the description of the embodiments of this invention will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the expression levels of propionic acid and misoic acid in the dual-strain synergistic fermentation of the present invention, based on the method of reducing the misoic acid yield of Propionibacterium fischeri based on dual-strain metabolic targeted regulation. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings are used to distinguish different objects and not to describe a particular order.

[0019] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0020] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0021] Example 1: Comparison of single-strain fermentation and dual-strain co-fermentation This embodiment aims to compare the differences in propionic acid yield and heteroacid profile between single-strain fermentation and dual-strain co-fermentation.

[0022] Seed culture medium preparation: Prepare a solution containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium lactate, and adjust the pH to 7.0 with 1M NaH solution. Dispense into Erlenmeyer flasks and sterilize at 121℃ for 15 min.

[0023] Preparation of fermentation medium: Prepare a solution containing 10 g / L yeast extract, 2.5 g / L tryptone, 20 g / L glycerol, 1 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and 10 g / L calcium carbonate. Adjust the pH to 7.5 with 1M NaH solution. Dispense into Erlenmeyer flasks and sterilize at 121℃ for 15 min.

[0024] Strain activation: The cryopreserved Propionibacterium fischeri subsp. Scheres PB-16 and Propionibacterium propioni BY-62 were rapidly thawed at room temperature, and 2 mL of bacterial culture was inoculated into 100 mL of sterilized seed culture medium. The cultures were then incubated at 30 °C for 40 h to obtain PB-16 seed culture and BY-62 seed culture.

[0025] Inoculation and fermentation: Experimental group 1 (single strain BY-62): 100 mL of BY-62 seed culture was inoculated into 800 mL of fermentation medium.

[0026] Experimental group 2 (single PB-16): 100 mL of PB-16 seed culture was inoculated into 800 mL of fermentation medium.

[0027] Experimental group 3 (dual-strain combination): 100 mL of BY-62 seed culture and 100 mL of PB-16 seed culture were simultaneously inoculated into 800 mL of fermentation medium (total volume 200 mL inoculated into 800 mL of medium).

[0028] All fermentation groups were incubated at 30℃ for 6 days, and the conical flasks were manually shaken for about 1 minute each morning and evening to mix them.

[0029] Sample detection: After the culture is completed, take 1 mL of fermentation broth, centrifuge at 12000 rpm for 5 minutes, take the supernatant, dilute it 10 times with ultrapure water, filter it through a 0.22 μm microporous membrane, and perform HPLC detection.

[0030] HPLC detection conditions: C18 column (5μm, 250mm×4.6mm); column temperature 35℃; injection volume 20μL; flow rate 1mL / min; mobile phase: solution A is 0.02ml / L disodium hydrogen phosphate dodecahydrate phosphate buffer (pH adjusted with phosphoric acid) at pH 2.0, solution B is acetonitrile, A:B=98:2 (v / v); detection wavelength 214 nm.

[0031] The results are as follows Figure 1 As shown, Figure 1 This is a schematic diagram of the expression levels of propionic acid and misoic acid in the dual-strain synergistic fermentation of the present invention, based on the method of reducing the misoic acid yield of Propionibacterium fischeri based on dual-strain metabolic targeted regulation.

[0032] Table 1 shows the concentrations of propionic acid (g / L), acetic acid (g / L), lactic acid (g / L), and succinic acid (g / L) for different strain combinations: Table 1 It is evident that the combined fermentation of two microorganisms significantly increased propionic acid production compared to single-microorganism fermentation, and successfully eliminated succinic acid completely, demonstrating the effectiveness of the dual-microorganism synergistic metabolism in the targeted regulation of heteroacid yield.

[0033] Example 2: Effects of different inoculation ratios This example investigates the effect of different inoculation ratios of Propionibacterium fischeri subsp. Scheres PB-16 and Propionibacterium propionitum BY-62 on fermentation efficiency. The seed culture medium and fermentation culture medium are the same as in Example 1.

[0034] Strain activation: Same as in Example 1.

[0035] Inoculation and fermentation: Set the following inoculation ratio (PB-16 seed liquid volume: BY-62 seed liquid volume), with the total inoculation volume being 20% ​​of the fermentation medium volume: Group A: 1:0.5. Group B: 1:1 (control). Group C: 1:1.5. Group D: 1:2.

[0036] The fermentation conditions and detection methods are the same as in Example 1.

[0037] Expected Results: All groups will reduce succinic acid production to varying degrees. Ratios of 1:1 and 1:1.5 may achieve the best balance between propionic acid production and succinic acid elimination, while deviations from this range may lead to an excessive dominance of a particular strain and a weakened synergistic effect.

[0038] Example 3: Effects of different carbon source combinations This embodiment investigates the effect of carbon source in fermentation medium on the synergistic effect of the two bacteria. The inoculation ratio was fixed at 1:1, and the total inoculation amount was 20%.

[0039] Fermentation medium base: yeast extract 10g / L, tryptone 2.5g / L, dipotassium hydrogen phosphate 1g / L, calcium carbonate 10g / L, pH 7.5.

[0040] Carbon source settings: Group A: Glycerol 20 g / L + Glucose 2.5 g / L (control, same as Example 1). Group B: Sodium lactate 20 g / L. Group C: Glucose 20 g / L. Group D: Glycerol 15 g / L + Sodium lactate 5 g / L.

[0041] Expected Results: Group A, using a mixed carbon source of glycerol and glucose, is likely to exhibit the best propionic acid yield and succinic acid elimination. When sodium lactate is used as the carbon source, the two bacteria may show synergistic effects in lactic acid utilization, and the yield of lactic acid and other lactic acids may be further reduced.

[0042] Example 4: Fermentation Temperature Optimization This example explores the optimal fermentation temperature. The culture medium and inoculation conditions (1:1, 20%) are the same as in Example 1.

[0043] Temperature settings: Group A: 28℃. Group B: 30℃ (control). Group C: 32℃.

[0044] Expected results: 30℃ is likely the optimal compromise temperature for the growth and metabolism of both strains. Temperatures that are too low may prolong the fermentation cycle, while temperatures that are too high may inhibit cell activity and affect the synergistic effect.

[0045] Example 5: Fermentation cycle monitoring This embodiment reveals the metabolic sequence of the synergistic effect of the two microorganisms by monitoring the dynamic changes in the concentration of each organic acid throughout the fermentation process.

[0046] Under the dual-strain fermentation conditions of Example 1, starting from the first day after inoculation, samples were taken at the same time every day to detect the concentrations of propionic acid, acetic acid, lactic acid, and succinic acid.

[0047] Expected Results: It is anticipated that all organic acids will begin to accumulate in the early stages of fermentation. In the middle and late stages of fermentation, the concentration of succinic acid will begin to decrease and eventually approach zero, while the concentration of propionic acid will continue to rise, visually demonstrating the dynamic process by which the succinic acid produced by BY-62 is further utilized by PB-16 to convert into propionic acid.

[0048] Example 6: The effect of pH value on seed culture medium This embodiment investigates the effect of the initial pH of the seed culture medium on the activation of the strain and subsequent fermentation.

[0049] Seed culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium lactate.

[0050] pH settings: Group A: pH 6.8. Group B: pH 7.0 (control). Group C: pH 7.2.

[0051] The fermentation medium and inoculation fermentation conditions are the same as in Example 1.

[0052] Expected results: pH 7.0 is likely the optimal activation condition. Deviations from this pH may lead to a decrease in seed culture viability, which in turn affects the start-up efficiency and final outcome of the co-fermentation.

[0053] Example 7: Optimization of phosphate concentration in fermentation medium This embodiment investigates the effect of dipotassium hydrogen phosphate concentration on dual-strain fermentation.

[0054] Fermentation medium: The basic components are the same as in Example 1, but the concentration of dipotassium hydrogen phosphate is changed: Group A: 0.5 g / L. Group B: 1.0 g / L (control). Group C: 1.5 g / L.

[0055] Expected results: An appropriate phosphate concentration (e.g., 1.0 g / L) is crucial for maintaining cellular energy status and metabolism. Too low a concentration may limit growth, while too high a concentration may inhibit it.

[0056] Example 8: Effect of calcium carbonate addition amount This embodiment investigates the effect of the amount of neutralizing agent calcium carbonate on the pH stability and product formation of the fermentation system.

[0057] Fermentation medium: Basic components are the same as in Example 1, but the calcium carbonate concentration is changed: Group A: 5 g / L. Group B: 10 g / L (control). Group C: 15 g / L.

[0058] Expected results: Sufficient calcium carbonate (e.g., 10-15 g / L) can effectively neutralize the organic acids produced during fermentation, maintain a relatively stable pH in the system, and promote cell growth and propionic acid accumulation. Insufficient calcium carbonate may lead to a premature drop in pH and premature termination of fermentation.

[0059] Example 9: Scale-up Fermentation Experiment (5L Fermenter) This embodiment verifies the feasibility of scaling up the method of the present invention in a fermenter.

[0060] A 5L automatic fermenter was used, and 3.2L of fermentation medium (formula same as in Example 1) was added. The fermenter was then sterilized at 121°C.

[0061] 400 mL each of the activated PB-16 and BY-62 seed solutions (total inoculation volume 800 mL, accounting for 20%) were simultaneously inoculated into the container.

[0062] Control conditions: temperature 30℃, stirring speed 50-100 rpm (low speed simulates static environment, mix well if necessary), pH not controlled (rely on calcium carbonate buffer), fermentation for 6 days.

[0063] Expected Results: It is anticipated that the results of the shake-flask level experiments can be replicated in the fermenter, with propionic acid production significantly higher than that of single-strain fermentation and succinic acid effectively eliminated, demonstrating that the process has good scale-up potential.

[0064] Example 10: Adaptability test of different strain preservation methods This embodiment verifies the feasibility of using strains preserved in other forms (such as slant culture strains) to carry out the method of the present invention.

[0065] Strain activation: Bacterial growths were picked from freshly activated Propionibacterium fischeri subsp. Scheres PB-16 and Propionibacterium propioni BY-62 slants, respectively, and inoculated into seed culture medium and incubated at 30°C for 40 hours.

[0066] The subsequent inoculation, fermentation, and testing steps are the same as in Example 1.

[0067] Expected Results: Using slant culture as a starting material, the goal of synergistic fermentation by two strains can also be achieved, indicating that the method of the present invention has good adaptability to different preservation methods of strains.

[0068] Example 11: Preliminary Investigation of Metabolic Mechanisms This embodiment explores the synergistic mechanism by measuring the activity of key enzymes or the expression level of genes.

[0069] Cells were collected at the end of the logarithmic growth phase and the stationary phase of single-strain BY-62, single-strain PB-16, and dual-strain co-fermentation.

[0070] The activities of key enzymes (such as succinate dehydrogenase) related to succinate production (BY-62) and utilization (PB-16) in each strain were determined under single-strain and co-culture conditions.

[0071] Expected Results: It is anticipated that in the dual-strain system, the activity of enzymes related to succinic acid utilization pathway in PB-16 may be induced to be upregulated, while the enzyme solution extracted from the BY-62 fermentation broth may show no significant change in its succinic acid synthesis capacity, indirectly proving that the synergistic effect is due to PB-16 utilizing succinic acid produced by BY-62.

[0072] Example 12: Preliminary assessment of downstream treatment of fermentation broth This embodiment evaluates the advantages of dual-strain co-fermentation broth in downstream separation and purification.

[0073] Equal volumes of the single-strain BY-62 fermentation broth and the dual-strain co-fermentation broth from Example 1 were taken, and after undergoing the same centrifugation and filtration pretreatment, propionic acid was recovered under the same vacuum distillation conditions.

[0074] The composition of the distillate (analyzed by HPLC) and the recovery efficiency of propionic acid were compared between the two fermentation broths during distillation.

[0075] Expected Results: Since the dual-strain co-fermentation broth does not contain succinic acid, the composition of organic acids in the distillate is simpler, and it is expected that a higher purity crude propionic acid can be obtained. Furthermore, due to the reduction of impurities, the distillation efficiency may be improved, demonstrating the positive impact of this invention on downstream processes.

[0076] Example 13: The effect of staged temperature control on the suppression of heteroacids Following the method in Example 1, PB-16 and BY-62 were inoculated into the fermentation medium at a 1:1 volume ratio. First stage: static culture at 30°C for 2 days. Second stage: the temperature was lowered to 26°C, and static culture continued for 4 days. The medium was shaken twice daily for 10 minutes each time, with nitrogen gas purging (1 L / min) during shaking. The results showed: propionic acid yield reached 20.1 g / L; succinic acid content decreased to 1.2 g / L, a 35% reduction compared to isothermal culture; and acetic acid content decreased to 0.8 g / L, a 28% reduction compared to isothermal culture.

[0077] Example 14: Improvement of propionic acid production through dynamic feeding strategy According to the phased culture conditions of Example 13; in the second phase, glycerol (15% of the initial glycerol amount) was supplemented every 24 hours; this was done 3 times, with a total supplementation amount reaching 45% of the initial glycerol amount.

[0078] The results showed that propionic acid production was further increased to 23.5 g / L; cell activity was maintained for 2 days longer; and carbon source conversion was increased to 0.68 g propionic acid / g glycerol.

[0079] Example 15: Targeted Regulation of Metabolic Pathways by Cobalt Ion Addition The dynamic feeding conditions of Example 14 were followed; 0.03 g / L CCl2·6H2 was added simultaneously with each feeding; other conditions remained unchanged.

[0080] The results showed that propionic acid production reached 25.8 g / L, an increase of 10% compared to the untreated group; succinic acid content further decreased to 0.9 g / L; and vitamin B12 production reached 3.2 mg / L, indicating that the methylmalonyl-CA pathway was enhanced.

[0081] Example 16: Fermentation performance under comprehensive optimization conditions Integrating all optimized conditions: phased temperature control (30℃×2 days → 26℃×4 days); dynamic feeding (15% glycerol added every 24 hours, for a total of 3 times); cobalt ion addition (0.03 g / LC). 2+ ); Nitrogen protection (1 L / min when shaking).

[0082] The results showed that propionic acid production reached a record high of 27.3 g / L; the total amount of miscellaneous acids (succinic acid + acetic acid) decreased to 1.8 g / L; the propionic acid / miscellaneous acid ratio increased to 15.2:1, which is about 2.3 times higher than the basic method; the fermentation cycle was shortened to 6 days, and the production efficiency was significantly improved.

[0083] By controlling the temperature in stages, the first stage of high temperature promotes cell growth, while the second stage of low temperature changes membrane fluidity, optimizes enzyme activity, and inhibits the generation of heteroacids.

[0084] Dynamic feeding control can avoid substrate inhibition, maintain optimal carbon source concentration, and ensure that metabolic flux is continuously directed towards propionic acid synthesis.

[0085] By using inert gas protection, a strictly anaerobic environment can be created to promote propionic acid production and inhibit aerobic metabolic byproducts.

[0086] The addition of cobalt ions can activate methylmalonyl-CA mutase, thereby strengthening the main pathway of vitamin B12-dependent propionic acid synthesis.

[0087] The synergistic effect of these technical solutions has resulted in a significant increase in propionic acid production and a substantial reduction in the yield of other acids, demonstrating outstanding technological progress and innovation.

[0088] Example 17: The promoting effect of L-glutamine addition on succinate metabolism In the dual-strain co-fermentation system of Example 1, an additional 0.3 g / L L-glutamine was added to the fermentation medium, while other conditions remained unchanged.

[0089] The results showed that propionic acid production increased to 19.8 g / L, a 9.6% increase compared to the untreated group; succinic acid concentration decreased to 0.00 g / L, and the rate of decrease accelerated; and bacterial biomass increased by approximately 12%, indicating that L-glutamine enhanced bacterial metabolic activity.

[0090] Example 18: Validation of the effect of sodium 2-bromoethanesulfonate as an inhibitor of succinic acid synthesis In the dynamic feeding process of Example 14, 0.1 g / L sodium 2-bromoethanesulfonate was added simultaneously each time feeding was performed.

[0091] The results showed that succinic acid accumulation was reduced by approximately 40% compared to the untreated group; propionic acid production increased to 24.8 g / L, and carbon source conversion rate improved to 0.71 g propionic acid / g glycerol; and no significant inhibition was observed in bacterial growth, indicating that the inhibitor has good selectivity.

[0092] Example 19: Activated carbon adsorption-assisted reduction of acetic acid and lactic acid concentrations Add 10 g / L activated carbon powder to the fermentation medium, and the inoculation and culture conditions are the same as in Example 1.

[0093] The results showed that the acetic acid concentration decreased from 3.40 g / L to 2.10 g / L; the lactic acid concentration decreased from 0.91 g / L to 0.50 g / L; the propionic acid production remained basically stable, and the downstream separation burden was significantly reduced.

[0094] Example 20: Fermentation stability verification of a microencapsulated dual-strain system The seed cultures of PB-16 and BY-62 were encapsulated in 2% sodium alginate solution, cross-linked with CaCl2 to form microcapsules, and then inoculated into fermentation medium. Three fermentation batches were repeated.

[0095] The results showed that the yield of propionic acid in each batch was stable between 18.5-19.0 g / L, and succinic acid was never detected; the cell loss rate was less than 5%, indicating good operational stability and reusability.

[0096] Example 21: Optimization of propionate synthase activity by gradient cooling mode The fermentation was carried out in the second stage of step b) using a gradient cooling mode (the first gradient was 28°C for 24 hours, the second gradient was 26°C for 48 hours, and the third gradient was 24°C until the end of fermentation, in order to gradually optimize the activity of propionic acid synthase and inhibit the heteroacid pathway): 28°C (24h) → 26°C (48h) → 24°C (until the end), with other conditions the same as in Example 1.

[0097] The results showed that propionic acid production reached 20.5 g / L, an increase of 13.9% compared to the constant temperature 30℃ group; succinic acid concentration remained below 0.1 g / L; and acetic acid production decreased by about 25%, indicating that gradient cooling can effectively regulate metabolic flow.

[0098] The key point of this invention lies in the discovery of a metabolic complementarity between *Propionibacterium fischeri* subsp. *Schönleinii* (especially PB-16) and *Propionibacterium propionitum* (especially BY-62). Specifically, succinic acid, a byproduct of one strain, can be consumed as a substrate by the other strain, thereby achieving a directional reconfiguration of metabolic flux at the microbial community level and fundamentally eliminating the accumulation of succinic acid. This is achieved through the co-fermentation of these two specific *Propionibacterium* strains, along with optimized culture medium composition and process conditions to achieve the best synergistic effect.

[0099] Obviously, the embodiments described above are merely some embodiments of the present invention, not all embodiments. The accompanying drawings show preferred embodiments of the present invention, but do not limit the patent scope of the present invention. The present invention can be implemented in many different forms; rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the patent protection scope of this invention.

Claims

1. A method for reducing the yield of heteroacids from Propionibacterium fischeri based on targeted regulation of dual-strain metabolism, characterized in that, Includes the following steps: a) The preserved strains of Propionibacterium fischeri subsp. Scheres and Propionibacterium propionitum were respectively inoculated into seed culture medium for activation culture to obtain their respective seed solutions; b) The two seed liquids obtained in step a) are simultaneously inoculated into the fermentation medium at a volume ratio and subjected to staged static culture. First stage: Incubate at 28-32℃ for 2-3 days to allow the bacteria to multiply rapidly; Second stage: Adjust the temperature to 25-28℃ and continue static culture for 3-5 days to promote propionic acid accumulation and inhibit the formation of other acids; c) During the second stage of cultivation, carbon source should be added to the fermentation system every 20-28 hours, with the amount added being 10%-20% of the initial total carbon source. d) Throughout the fermentation process, shake the fermentation vessel periodically to mix the system, and introduce inert gas during shaking to maintain an anaerobic environment.

2. The method according to claim 1, characterized in that, The Propionibacterium fischeri subsp. Schere is Propionibacterium fischeri subsp. Schere PB-16, and the Propionibacterium propioni is Propionibacterium propioni BY-62. Add 0.1-0.5 g / L of L-glutamine or L-asparagine to the fermentation medium in step b) to enhance the cell's metabolic absorption capacity of succinic acid. Add 5-20 g / L of activated carbon or diatomaceous earth as an adsorption carrier to the fermentation medium in step b) for in-situ adsorption and reduction of the concentration of acetic acid and lactic acid in the fermentation broth. Before inoculation in step b), the two seed liquids were microencapsulated using sodium alginate-chitosan composite gel to improve the stability and reusability of the cells in the fermentation system. During the feeding process in step c), 0.05-0.2 g / L of sodium 2-bromoethanesulfonate is added as a succinic acid metabolic bypass inhibitor to further reduce the formation of succinic acid.

3. The method according to claim 1, characterized in that, In step a), the seed culture medium comprises: 8-12 g / L tryptone, 4-6 g / L yeast extract, 8-12 g / L sodium lactate, pH 6.8-7.2; preferably, it comprises 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium lactate, pH 7.

0.

4. The method according to claim 1, characterized in that, In step a), the activation culture conditions are: culture temperature 28-32℃, static culture for 36-48 hours.

5. The method according to claim 1, characterized in that, In step b), the fermentation medium comprises: 8-12 g / L yeast extract, 2-3 g / L tryptone, 15-30 g / L carbon source, 0.8-1.2 g / L dipotassium hydrogen phosphate, 8-12 g / L calcium carbonate, and pH 7.3-7.7; preferably, the carbon source is a mixture of 15-25 g / L glycerol and 2-3 g / L glucose; more preferably, the fermentation medium comprises: 10 g / L yeast extract, 2.5 g / L tryptone, 20 g / L glycerol, 2.5 g / L glucose, 1 g / L dipotassium hydrogen phosphate, 10 g / L calcium carbonate, and pH 7.

5.

6. The method according to claim 1, characterized in that, In step b), the inoculation volume ratio of the two seed solutions is 1:0.5-2 for Propionibacterium fischeri seed solution and 1:1 for Propionibacterium propioni seed solution.

7. The method according to claim 1, characterized in that, In step b), the total inoculation volume is 10%-30% of the fermentation medium volume.

8. The method according to claim 1, characterized in that, In step d), the periodic shaking is performed 1-3 times a day, for 5-15 minutes each time; the inert gas is nitrogen or argon, and the ventilation flow rate is 0.5-1.5 L / min.

9. The method according to claim 1, characterized in that, During the feeding process in step c), 0.01-0.05 g / L of cobalt ions are added simultaneously as a coenzyme activator to further improve the efficiency of propionic acid synthesis.

10. The application of Propionibacterium fischeri subsp. Scheres and Propionibacterium propionitum in the co-fermentation preparation of propionic acid and its use in reducing the succinic acid content in the fermentation broth; preferably, the Propionibacterium fischeri subsp. Scheres is PB-16 and the Propionibacterium propionitum is BY-62.

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