Microorganism as well as application and method thereof in preparation of 1, 3-propylene glycol and / or lactic acid

Abstract

The invention provides a microorganism as well as an application and a method of the microorganism in preparation of 1, 3-propylene glycol and lactic acid, the microorganism is lactobacillus reuteri, the microorganism is preserved in Guangdong Microbial Culture Collection Center on May 29, 2024, and the preservation number is GDMCC NO: 64700. The invention further provides a method for preparing 1, 3-propylene glycol and lactic acid, and the microorganism is lactobacillus reuteri and is preserved in Guangdong Microbial Culture Collection Center on May 29, 2024. The Lactobacillus reuteri can co-produce 1, 3-propylene glycol and lactic acid, has the advantages of high yield of 1, 3-propylene glycol and lactic acid, fast growth and metabolism, strong stability, high safety and the like, provides a new idea for green, low-cost and efficient production of 1, 3-propylene glycol and lactic acid, and has a wide application prospect.

Description

Technical Field

[0001] This application relates to the field of biochemical engineering. Specifically, this application relates to microorganisms and their application and methods in the preparation of 1,3-propanediol and lactic acid. Background Technology

[0002] 1,3-Propanediol (1,3-PDO) and lactic acid (LA) are widely used in cosmetics, pharmaceuticals, food, and chemicals. Production methods for 1,3-propanediol and lactic acid include chemical and biological methods. Chemical methods primarily use non-renewable petrochemical resources as raw materials, resulting in high energy consumption, numerous difficult-to-separate and purify byproducts, and immeasurable environmental pollution. Compared to chemical methods, biological methods for synthesizing 1,3-propanediol and lactic acid offer advantages such as simpler production steps, the use of renewable resources, and environmental friendliness. However, the suitable microorganisms for the biological synthesis of 1,3-propanediol and lactic acid still require further research. Summary of the Invention

[0003] This invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, this application proposes a *Limosilactobacillus reuteri* strain that can co-produce 1,3-propanediol and lactic acid, possessing advantages such as high yield of 1,3-propanediol and lactic acid, rapid growth and metabolism, strong stability, and high safety. This provides a new approach for the green, low-cost, and efficient production of 1,3-propanediol and lactic acid, with broad application prospects.

[0004] In a first aspect, this application discloses a microorganism. According to an embodiment of this application, the microorganism is *Limosilactobacillus reuteri*, deposited on May 29, 2024, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 64700. The microorganism according to the embodiment of this application can co-produce 1,3-propanediol and lactic acid, possessing advantages such as high yield of 1,3-propanediol and lactic acid, rapid growth and metabolism, strong stability, and high safety. It provides a new approach for the green, low-cost, and efficient production of 1,3-propanediol and lactic acid, with broad application prospects.

[0005] In a second aspect, this application provides a microbial inoculant. According to an embodiment of this application, the microbial inoculant includes the microorganisms described in the first aspect above.

[0006] In a third aspect of this application, a ferment is provided. According to embodiments of this application, the ferment is obtained by fermentation using at least the microorganisms described in the first aspect or the microbial agents described in the second aspect. As previously mentioned, the *Lactobacillus reuteri* of this application has the advantage of high production of 1,3-propanediol and lactic acid, and the ferment obtained by its fermentation has high contents of 1,3-propanediol and lactic acid.

[0007] According to an embodiment of this application, the fermentation product includes at least one of the following: fermentation broth, a concentrate of fermentation broth, a dried product of fermentation broth, a lysate of fermentation broth, an extract of the lysate, fermentation supernatant, an extract of fermentation supernatant, lysate, an extract of lysate, and a dried product of lysate.

[0008] In a fourth aspect, this application discloses a food product. According to an embodiment of this application, the food product comprises at least one of the microorganisms described in the first aspect, the microbial agent described in the second aspect, and the fermentation product described in the third aspect.

[0009] In a fifth aspect of this application, a pharmaceutical product is proposed. According to an embodiment of this application, the pharmaceutical product comprises at least one of the microorganisms described in the first aspect, the microbial agent described in the second aspect, and the fermentation product described in the third aspect.

[0010] In a sixth aspect, this application discloses a cosmetic product. According to an embodiment of this application, the cosmetic product comprises at least one of the microorganisms described in the first aspect, the microbial agent described in the second aspect, and the fermentation product described in the third aspect.

[0011] In a seventh aspect of this application, the application proposes the use of the microorganisms described in the first aspect or the microbial agents described in the second aspect in the preparation of 1,3-propanediol and / or lactic acid.

[0012] In an eighth aspect of this application, a method for preparing 1,3-propanediol and / or lactic acid is provided. According to embodiments of this application, the method includes: fermenting the microorganisms described in the first aspect or the microbial inoculant described in the second aspect.

[0013] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0014] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0015] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0016] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this application.

[0017] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0018] The metabolic pathway for microbial production of 1,3-propanediol and lactic acid mainly includes: First, glycerol is converted to 3-hydroxypropanal (3-HPA) by glycerol dehydratase (GDHt) within the microorganism; then, 3-hydroxypropanal is reduced to 1,3-propanediol by 1,3-propanediol oxidoreductase (PDOR) under the action of the reducing coenzyme NADH, releasing NAD. + Meanwhile, glycerol dehydrogenases in microorganisms oxidize 3-hydroxypropanal to lactic acid. The pathways of 3-hydroxypropanal reduction to 1,3-propanediol and 3-hydroxypropanal oxidation to lactic acid are in competition with each other; when the yield of 1,3-propanediol increases, the yield of lactic acid tends to decrease.

[0019] Currently, existing technologies primarily produce 1,3-propanediol through microbial fermentation. Due to the competitive inhibition between the biosynthetic pathways of 1,3-propanediol and lactic acid, researchers typically increase 1,3-propanediol yield by inhibiting lactic acid growth through genetic modification or fermentation process optimization. Few researchers have attempted to co-produce 1,3-propanediol and lactic acid using biosynthesis, and even fewer have attempted to simultaneously increase the yields of both. This represents a significant technological barrier in the field. Furthermore, the microorganisms currently used for fermenting 1,3-propanediol mainly include Klebsiella pneumoniae and Escherichia coli. However, as pathogenic bacteria, Klebsiella pneumoniae and Escherichia coli face numerous limitations in strain transportation and preservation, fermentation processes, separation and purification processes, and applications in cosmetics and food. Moreover, their separation and purification processes suffer from complexity, high energy consumption, and high costs associated with waste treatment.

[0020] In view of this, this application selected multiple strains of Lactobacillus and Citrate Bacteria as initial strains, attempting to selectively breed strains that simultaneously produce high levels of 1,3-propanediol and lactic acid through mutagenesis. After a series of mutagenesis and screenings, a strain capable of simultaneously producing high levels of 1,3-propanediol and lactic acid—Limosilactobacillus reuteri PDO / LA-3—was successfully obtained. Furthermore, after multiple subcultures, it still exhibits good growth and metabolic capabilities, 1,3-propanediol and lactic acid production capacity, and strong stability.

[0021] Therefore, in a first aspect, this application proposes a microorganism. According to an embodiment of this application, the microorganism is *Limosilactobacillus reuteri*, deposited on May 29, 2024, at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO: 64700, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, postal code: 510070. The microorganism according to the embodiment of this application can co-produce 1,3-propanediol and lactic acid, possessing advantages such as high yield of 1,3-propanediol and lactic acid, rapid growth and metabolism, strong stability, and high safety. It provides a new approach for the green, low-cost, and efficient production of 1,3-propanediol and lactic acid, with broad application prospects. In this application, the terms "*Limosilactobacillus reuteri*" and "*Limosilactobacillus reuteri*" are used interchangeably.

[0022] The *Lactobacillus reuteri* PDO / LA-3 (hereinafter referred to as "PDO / LA-3") of this application was obtained through targeted selection of multiple strains of lactic acid bacteria and citric acid bacteria, specifically including ultraviolet-plasma combined mutagenesis. Exemplarily, the targeted selection steps include:

[0023] The activated bacterial strains were fermented and cultured. Strains in the logarithmic growth phase were collected, resuspended, and a bacterial suspension was obtained.

[0024] The bacterial suspension was transferred into the processing chamber of the ultraviolet-plasma combined mutagenesis system for combined mutagenesis treatment.

[0025] The combined mutagenized lactic acid bacteria or citric acid bacteria were taken out and spread on a solid culture medium plate containing 1,3-propanediol and lactic acid. After cultivation, the cells with better growth were cultured in MRS broth and MRS broth containing glycerol and glucose in turn, and the fermentation broth was collected.

[0026] The fermentation broth collected in the previous step was centrifuged, and the supernatant was collected. The supernatant was analyzed by gas chromatography-mass spectrometry, and the strain with the highest yield of 1,3-propanediol and lactic acid was selected as the target strain, namely PDO / LA-3.

[0027] In a second aspect, this application proposes a microbial inoculant. According to embodiments of this application, the microbial inoculant includes the microorganisms described in the first aspect. The microbial inoculant of this application can co-produce 1,3-propanediol and lactic acid, possessing advantages such as high yield of 1,3-propanediol and lactic acid, rapid growth and metabolism, strong stability, and high safety. It provides a new approach for the green, low-cost, and efficient production of 1,3-propanediol and lactic acid, with broad application prospects.

[0028] It should be noted that the microorganisms contained in the microbial inoculant of this application are basically all alive. However, during actual production and storage, a small number of microorganisms may die due to various reasons. For example, the number of live bacteria in the microbial inoculant is not less than 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.9% of the total number of microorganisms.

[0029] According to embodiments of this application, the microbial agent further includes culture medium, solvent (physiological saline, sterile water), protectant (lyophilization protectant, antioxidant, desiccant, etc.), stabilizer, preservative, etc., thereby further facilitating the storage or use of Lactobacillus reuteri, improving fermentation performance, and increasing the yield of 1,3-propanediol and lactic acid.

[0030] This application does not impose strict requirements on the dosage form of microbial agents. For example, it can be a liquid (such as a suspension), powder, granules, etc., and can be flexibly selected according to actual needs.

[0031] It should be noted that the characteristics and advantages described above for microorganisms also apply to this microbial agent, and will not be repeated here.

[0032] In a third aspect of this application, a ferment is provided. According to embodiments of this application, the ferment is obtained by fermentation using at least the microorganisms described in the first aspect or the microbial agents described in the second aspect. As previously mentioned, the *Lactobacillus reuteri* PDO / LA-3 of this application can simultaneously produce high levels of 1,3-propanediol and lactic acid, and the ferment obtained by fermenting it is rich in 1,3-propanediol and lactic acid.

[0033] In this application, the term "fermentation product" refers to the substance obtained by fermentation of Lactobacillus reuteri PDO / LA-3. Fermentation products may contain bacterial cells, but do not necessarily need to contain bacterial cells.

[0034] It should be noted that the fermentation product of this application can be obtained in two ways: one is by directly fermenting using the aforementioned microorganisms or microbial agents; the other is by preparing the fermentation broth produced during the fermentation process through a treatment process.

[0035] According to embodiments of this application, the fermentation product includes at least one of the following: fermentation broth, a concentrate of fermentation broth, a dried product of fermentation broth, a lysate of fermentation broth, an extract of the lysate, a fermentation supernatant, an extract of the fermentation supernatant, a lysate, an extract of the lysate, and a dried product of the lysate.

[0036] In this application, the term "fermentation broth" refers to a liquid containing unconsumed culture medium components, microbial cells, and metabolic products during the fermentation process. The microbial cells in the fermentation broth may contain either live or dead microorganisms, for example, those that have undergone sterilization.

[0037] The fermentation broth can be concentrated using conventional concentration techniques in this field, such as distillation, to obtain a concentrate of the fermentation broth, the concentration factor of which can be adjusted according to actual needs.

[0038] Fermentation broth can be converted into a dried product using conventional drying techniques in the field, such as heating and drying or freeze drying. The water content can be selected as needed, and it can be completely water-free or contain a small amount of water, such as no more than 10%, no more than 8%, no more than 5%, no more than 3%, no more than 1%, no more than 0.8%, no more than 0.5%, no more than 0.1%, etc.

[0039] The fermentation broth is subjected to cell lysis, releasing all contents after cell rupture, including intracellular proteins, nucleic acids, lipids, and other organelles, as well as extracellular substances such as 1,3-propanediol and lactic acid, yielding a lysate of the fermentation broth. This lysate is then subjected to conventional extraction techniques in the art, such as centrifugation, sedimentation, ultrafiltration, nanofiltration, or chromatography, to obtain an extract.

[0040] By using conventional extraction techniques in this field, such as centrifugation, sedimentation, ultrafiltration, nanofiltration, or chromatography, the bacterial cells in the fermentation broth can be separated from the liquid. The liquid remaining after the bacterial cells are almost completely removed contains 1,3-propanediol and lactic acid, which is the fermentation supernatant.

[0041] "Lystosome" refers to all contents released after the fermentation broth is centrifuged, the precipitate is collected, and cells are disrupted. This includes intracellular proteins, nucleic acids, lipids, and other organelles. Using conventional drying techniques such as heat drying or freeze drying, the lystosome can be converted into a dried product. Its water content can be selected as needed, ranging from completely anhydrous to containing small amounts of water, such as no more than 10%, 8%, 5%, 3%, 1%, 0.8%, 0.5%, or 0.1%. Furthermore, using conventional extraction techniques such as centrifugation, sedimentation, ultrafiltration, nanofiltration, or chromatography, specific components, such as 1,3-propanediol and lactic acid, can be separated and extracted from the lystosome to obtain an extract.

[0042] In a fourth aspect, this application discloses a food product. According to embodiments of this application, the food product comprises at least one of the microorganisms described in the first aspect, the microbial inoculant described in the second aspect, and the fermentation product described in the third aspect. The food product of this application is rich in 1,3-propanediol and lactic acid, which can improve food quality. Specifically, 1,3-propanediol can act as a humectant, anti-caking agent, and thickener, helping to maintain the freshness of the food and improve its texture. Lactic acid can act as an acidulant and preservative, enhancing the flavor of the food and extending its shelf life. It also participates in the fermentation process of food as a fermenting agent, such as promoting the growth of specific microorganisms in dairy products and baked goods.

[0043] According to embodiments of this application, the food may further include other ingredients acceptable to the food, such as at least one of stabilizers, thickeners, emulsifiers, sweeteners, acidulants, flavorings, colorings, preservatives, and nutritional fortifiers, to improve the texture, taste, appearance, shelf life, and nutritional value of the food.

[0044] In a fifth aspect of this application, a pharmaceutical product is proposed. According to an embodiment of this application, the pharmaceutical product comprises at least one of the microorganisms described in the first aspect, the microbial agent described in the second aspect, and the fermentation product described in the third aspect.

[0045] The pharmaceutical product of this application is rich in 1,3-propanediol and lactic acid, which can improve the quality of the drug. Specifically, 1,3-propanediol can be used as a solvent, stabilizer, and wetting agent to improve the solubility, stability, and reduce irritation of the drug. Lactic acid has functions such as regulating pH and promoting drug absorption.

[0046] According to embodiments of this application, the pharmaceutical product may further include pharmaceutically acceptable excipients, including at least one of diluents, binders, disintegrants, lubricants, stabilizers, preservatives, colorants, and fragrances, to improve the processing performance, stability, absorption efficiency, and patient experience of the pharmaceutical product.

[0047] In a sixth aspect, this application discloses a cosmetic product. According to an embodiment of this application, the cosmetic product comprises at least one of the microorganisms described in the first aspect, the microbial agent described in the second aspect, and the fermentation product described in the third aspect.

[0048] The cosmetics described in this application are rich in 1,3-propanediol and lactic acid, which can enhance the quality of the cosmetics. Specifically, 1,3-propanediol can be used as a moisturizer and solvent to help other ingredients function better. Lactic acid has a gentle exfoliating effect, promotes skin renewal, improves skin texture, and acts as a moisturizer to help maintain the skin's moisture balance, thereby enhancing the product's skincare efficacy.

[0049] According to embodiments of this application, the cosmetic may further include at least one of emulsifiers, thickeners, preservatives, antioxidants, fragrances, pigments, moisturizers, active ingredients, sunscreens, and pH adjusters to improve the product's stability, user experience, efficacy, and safety.

[0050] It should be noted that the features and advantages described above for the microorganisms mentioned in the first aspect, the microbial agents mentioned in the second aspect, and the fermentation products mentioned in the third aspect also apply to this food, medicine, or cosmetic, and will not be repeated here.

[0051] In a fifth aspect of this application, the application of the microorganisms described in the first aspect or the microbial agents described in the second aspect in the preparation of 1,3-propanediol and / or lactic acid is proposed. The *Lactobacillus reuteri* strain of this application can produce high levels of 1,3-propanediol and lactic acid, exhibits rapid growth and metabolism, strong stability, and high safety, and can be used to prepare 1,3-propanediol, lactic acid, or a combination of both, making it suitable for industrial-scale application.

[0052] In a sixth aspect of this application, a method for preparing 1,3-propanediol and / or lactic acid is proposed. According to embodiments of this application, the method includes fermenting and culturing the microorganisms described in the first aspect or the microbial agent described in the second aspect. The *Lactobacillus reuteri* strain of this application can produce high yields of 1,3-propanediol and lactic acid, exhibits rapid growth and metabolism, strong stability, and high safety, and can be applied to the preparation of 1,3-propanediol, lactic acid, or a combination of both. Furthermore, this method is simple to operate, low in cost, and suitable for industrial-scale application.

[0053] It should be noted that this application does not strictly limit the fermentation conditions of the microorganisms or microbial agents. Fermentation conditions commonly used in the field for Lactobacillus reuteri can be used. For example, suitable culture media may include MRS medium or broth medium; anaerobic culture; and the culture temperature may be 30℃~40℃.

[0054] It should be noted that this application does not impose strict requirements on fermentation culture conditions. Commonly used methods in the art for culturing lactic acid bacteria to produce 1,3-propanediol and / or lactic acid can be employed, and the specific method can be flexibly selected according to actual needs. For example, the fermentation culture includes: inoculating *Lactobacillus reuteri* PDO / LA-3 into MRS medium and performing shaking culture to obtain a fermentation broth containing 1,3-propanediol and lactic acid. To obtain a product with higher purity, the fermentation broth can be further centrifuged and the supernatant collected. The supernatant can then undergo purification steps such as ultrafiltration and distillation to achieve effective collection and purification of 1,3-propanediol and lactic acid.

[0055] It should be noted that the features and advantages described above for the microorganisms in the first aspect and the microbial agents in the second aspect also apply to this application and method, and will not be repeated here.

[0056] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0057] Example 1

[0058] 1. Strains culture and mutagenesis

[0059] Purchase strains of Lactobacillus reuteri (ATCC55730, ATCC53608, ATCC23272) and Citrobacter freundii (ATCC8090).

[0060] 1.1 Strain reactivation

[0061] (1) Store Lactobacillus reuteri and Citrobacter freundi strains in freeze-dried tubes at 4°C;

[0062] (2) Disinfect the surface of the freeze-dried tube with 75% alcohol cotton, heat the top of the tube evenly over the outer flame of an alcohol lamp, drip 2-3 drops of sterile water onto the heated part, and knock off the freeze-dried tube head with tweezers.

[0063] (3) Add 0.5 mL of MRS broth to the Lactobacillus reuteri lyophilized tube, shake well, then pour into a 5 mL MRS broth test tube and shake well.

[0064] MRS broth: 10 g / L peptone, 8 g / L beef extract powder, 4 g / L yeast extract powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate (MgSO4·7H2O), 0.04 g / L manganese sulfate (MnSO4·4H2O), 1 mL / L Tween-80, with the remainder being water. After preparing the solution, adjust the pH to 5.5 using dilute hydrochloric acid and ammonia.

[0065] Add 0.5 mL of NB nutrient broth to the lyophilized tube of Citrobacter freundii, shake well, then pour into a 5 mL NB nutrient broth test tube and shake well.

[0066] NB Broth: 10g / L peptone, 3g / L beef extract powder, 5g / L sodium chloride, with the remainder being water. After preparing the solution, adjust the pH to 7.0 using dilute hydrochloric acid and ammonia.

[0067] (4) Place the MRS broth test tubes containing Lactobacillus reuteri in a 37°C incubator for static anaerobic culture for 48 h;

[0068] NB broth test tubes containing Citrobacter freundii were placed in a 37°C incubator for static aerobic culture for 24 hours.

[0069] 1.2 Ultraviolet-Plasma Composite Mutagenesis

[0070] (1) Transfer Lactobacillus reuteri MRS broth, cultured for 48 hours, to an Erlenmeyer flask containing MRS broth at a 10% inoculation concentration, and anaerobically culture at 37°C and 110 rpm for 16-24 hours until OD. 600 =0.6-0.8;

[0071] The *Citrobacter freundii* NB broth, cultured for 24 hours at a 10% inoculum concentration, was transferred to an Erlenmeyer flask containing NB broth and incubated aerobically at 37°C and 110 rpm for 6-10 hours until OD reached. 600 =0.6-0.8;

[0072] (2) During the logarithmic growth phase of the strain, transfer 10 mL of bacterial suspension to a 20 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, add 10 mL of phosphate buffer (0.1 M, pH 6.5), resuspend the bacterial cells, and centrifuge at 8000 rpm for 5 min. Repeat this step three times to obtain the final bacterial suspension with a cell concentration of 10. 7 CFU / mL;

[0073] (3) The bacterial suspension was transferred into the MPMS UV-plasma combined mutagenesis system. The UV lamp was turned on and nitrogen gas was injected to carry out UV-plasma combined mutagenesis. The plasma mutagenesis parameters were: nitrogen gas, gas flow rate of 12.05 slpm, mutagenesis distance of 20 mm, temperature of 25 ℃, and vertical irradiation. The UV mutagenesis parameters were: distance of 30 cm, wavelength of 254 nm, power of 300 W, irradiation at an angle of 45°, mutagenesis time of Lactobacillus reuteri of 75 s, and mutagenesis time of Citrobacter freundii of 60 s.

[0074] 2. Strain screening

[0075] 2.1 Lactobacillus reuteri

[0076] (1) Take out the Lactobacillus reuteri suspension after compound mutagenesis from the treatment room, spread it on MRS solid medium plates with 50 g / L of 1,3-propanediol and 50 g / L of lactic acid, and anaerobic culture in an incubator at 37°C for 48 h.

[0077] (2) The best-growing Lactobacillus reuteri colonies were transferred to test tubes containing 5 mL of MRS broth and statically anaerobically cultured at 37°C for 48 h.

[0078] (3) The Lactobacillus reuteri MRS broth, after being cultured for 48 hours, was transferred to five MRS broth conical flasks containing 20 g / L glycerol and 20 g / L glucose at a 10% inoculation concentration. The flasks were then shaken at 110 rpm at 37°C for 48 hours for anaerobic fermentation.

[0079] (4) After fermentation, take 10 mL of fermentation liquid into a 20 mL centrifuge tube and centrifuge at 8000 rpm for 5 min to separate the upper fermentation liquid and the cells.

[0080] (5) Place the upper fermentation broth into an ultrafiltration centrifuge tube (filter molecular weight <1000Da), centrifuge at 8000rpm for 20min, and take the filtrate for gas chromatography-mass spectrometry analysis.

[0081] 2.2 Citrobacter freundii

[0082] (1) Take out the Citrobacter freundii suspension after compound mutagenesis from the treatment room, spread it on NA solid culture medium plates with 1,3-propanediol concentration of 20-100 g / L and lactic acid concentration of 20-100 g / L, and incubate aerobically in an incubator at 37℃ for 24 h;

[0083] (2) The best-growing Citrobacter freundii colonies were transferred to test tubes containing 5 mL of NB broth and statically cultured aerobically at 37°C for 24 h.

[0084] (3) The NB broth of Citrobacter freundii after 48 hours of cultivation was transferred to five NB broth conical flasks containing 20 g / L glycerol and 20 g / L glucose at an inoculation concentration of 10% and then aerobically fermented at 37°C and 110 rpm for 24 hours.

[0085] (4) After fermentation, take 10 mL of fermentation liquid into a 20 mL centrifuge tube and centrifuge at 8000 rpm for 5 min to separate the upper fermentation liquid and the cells.

[0086] (5) Place the upper fermentation broth into an ultrafiltration centrifuge tube (filter molecular weight <1000Da), centrifuge at 8000rpm for 20min, and take the filtrate for gas chromatography-mass spectrometry analysis.

[0087] The results are shown in Table 1. PDO / LA-0 represents the initial strain ATCC53608, and PDO / LA-1, PDO / LA-2, PDO / LA-3, PDO / LA-4, and PDO / LA-5 are the mutagenized strains of PDO / LA-0 after mutagenesis. LR 1-0 represents the initial strain ATCC55730, and LR 1-1, LR 1-2, LR 1-3, LR 1-4, and LR 1-5 are the mutagenized strains of LR 1-0 after mutagenesis. LR2-0 represents the initial strain ATCC23272, and LR 2-1, LR 2-2, LR 2-3, LR 2-4, and LR 2-5 are the mutagenized strains of LR 2-0 after mutagenesis. CF 1-0 represents the initial strain ATCC8090, and CF 1-1, CF 1-2, CF 1-3, CF 1-4, and CF 1-5 are the mutagenic strains of CF 1-0 after mutagenesis treatment.

[0088] Table 1. Yields of 1,3-propanediol and lactic acid from different strains

[0089]

[0090]

[0091] As shown in Table 1, compared with the initial strains, after mutagenesis treatment, some strains showed increased production of 1,3-propanediol and lactic acid, such as PDO / LA-3, CF 1-4, and CF 1-5; some strains showed increased production of 1,3-propanediol but decreased production of lactic acid, such as PDO / LA-1, PDO / LA-4, LR1-1, LR 1-2, LR 1-5, LR 2-2, and CF 1-1; some strains showed decreased production of 1,3-propanediol but increased production of lactic acid, such as PDO / LA-2, PDO / LA-5, LR 1-3, LR 1-4, LR 2-4, and CF 1-2; and some strains showed decreased production of both 1,3-propanediol and lactic acid, such as LR 2-1, LR 2-3, LR 2-5, and CF 1-3. Overall, strain PDO / LA-3 produced the highest yields of 1,3-propanediol and lactic acid.

[0092] 3. Passage stability test

[0093] The strain PDO / LA-3 was inoculated into liquid MRS medium at an inoculum of 1% (v / v) and cultured at 37°C for 24 h. After inoculation, it was transferred to fresh liquid MRS medium at 1% (v / v). A 24 h passage cycle was used as one subculture cycle, and the number of generations in each cycle was log2100 = 6.64. The morphology of the strain was observed every 200 generations (30 days), and the strain was streaked onto solid MRS medium. The streaked plates were placed in an anaerobic incubator and cultured at 37°C for 48 h. The size, number, and morphology of the colonies were observed, and the stability of 1,3-propanediol and lactic acid yields was analyzed by glycerol fermentation.

[0094] The results are shown in Table 2. It can be seen that the *Lactobacillus reuteri* PDO / LA-3 strain of this application can still stably produce high levels of 1,3-propanediol lactic acid after multiple passages, and has a high survival rate.

[0095] Table 2. Passage stability experiment of PDO / LA-3 strain

[0096] Strain survival rate (%) 1,3-Propanediol yield (mol / mol) Lactic acid production (mol / mol) 2nd generation 100 0.48 0.28 200s 99.8 0.46 0.27 400s 99.6 0.47 0.27 600 generation 99.2 0.46 0.26 800s generation 99.1 0.45 0.25 1000s 98.3 0.45 0.23

[0097] Example 2

[0098] The strains PDO / LA-0 and PDO / LA-3 from Example 1 were subjected to fed-batch fermentation culture, and the specific steps are as follows:

[0099] 1. Fermentation of strains PDO / LA-0 and PDO / LA-3 was carried out in a 2.5L fermenter using MRS medium containing 30 g / L glycerol and 20 g / L glucose, at a temperature of 37°C, pH 5.5, and stirring at 300 rpm, in fed-batch fermentation mode. Nitrogen was purged before inoculation to establish anaerobic conditions. A separately sterilized MRS medium solution was added every 12 hours of fermentation to a concentration of 1 g / L. During fermentation, the pH was maintained at 5.5 using dilute hydrochloric acid and ammonia.

[0100] MRS medium contains the following components: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate (MgSO4·7H2O), 0.04 g / L manganese sulfate (MnSO4·4H2O), 1 mL / L Tween-80, and the remainder is water. After preparing the solution, the pH is adjusted to 5.5 using dilute hydrochloric acid and ammonia.

[0101] The feed consisted of 250 mM glycerol and 100 mM glucose aqueous solution.

[0102] The feeding start time for glycerol aqueous solution is 1 hour, and the feeding start time for glucose aqueous solution is 2 hours.

[0103] The feed rate for the glycerol aqueous solution was 10 ml / h, and the feed rate for the glucose aqueous solution was 10 ml / h.

[0104] 2. After 48 hours of fed-batch fermentation, the upper fermentation broth was placed into an ultrafiltration centrifuge tube (filtering molecular weight <1000Da), centrifuged at 8000 rpm for 20 min, and the filtrate was analyzed by gas chromatography-mass spectrometry.

[0105] Testing revealed that strain PDO / LA-0 produced 51.60 g / L of 1,3-propanediol and 13.91 g / L of lactic acid during fermentation. Strain PDO / LA-3 produced 90.26 g / L of 1,3-propanediol and 21.47 g / L of lactic acid, both superior to the pre-mutation strain PDO / LA-0. This further demonstrates that the mutagenic strains exhibit high yields of 1,3-propanediol and lactic acid, making them suitable for industrial production.

[0106] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0107] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A microorganism, characterized in that, The microorganism in question is *Limosilactobacillus reuteri*, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 29, 2024, with accession number GDMCC NO: 64700.

2. A microbial inoculant, characterized in that, include: The microorganism as described in claim 1.

3. A fermentation product, characterized in that, The fermentation product is obtained by fermentation of at least the microorganism described in claim 1 or the microbial agent described in claim 2.

4. The fermented product according to claim 3, characterized in that, The fermentation product includes at least one of the following: fermentation broth, concentrated fermentation broth, dried fermentation broth, lysate of fermentation broth, extract of the lysate, fermentation supernatant, extract of fermentation supernatant, lysate, extract of lysate, and dried lysate.

5. A food product, characterized in that, include: At least one of the microorganisms of claim 1, the microbial agent of claim 2, and the fermentation product of claim 3 or 4.

6. A medicine, characterized in that, include: At least one of the microorganisms of claim 1, the microbial agent of claim 2, and the fermentation product of claim 3 or 4.

7. A cosmetic product, characterized in that, include: At least one of the microorganisms of claim 1, the microbial agent of claim 2, and the fermentation product of claim 3 or 4.

8. The use of the microorganism of claim 1 or the microbial agent of claim 2 in the preparation of 1,3-propanediol and / or lactic acid.

9. A method for preparing 1,3-propanediol and / or lactic acid, characterized in that, include: The microorganism described in claim 1 or the microbial agent described in claim 2 is fermented and cultured.

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