Application of phytobacterium plantarum in biological preparation of cinnamic acid

Abstract

The invention discloses an application of plant lactobacillus in biological preparation of cinnamic acid. The invention provides a method for biosynthesizing cinnamic acid, cinnamic acid is synthesized and prepared through lactobacillus plantarum for the first time, it is identified through high performance liquid chromatography that the content of cinnamic acid is 0.4108 mg / mL, the purity is 98.01%, the recovery rate of a sample is 99.85%, the sample is stable within 24 hours, the average peak area of the same sample is 33590254.6667, and RSD is 0.6206%. According to the invention, more microbial sources are provided for high-yield cinnamic acid, and compared with the existing biosynthesis method, the method has the advantages that fine metabolic engineering transformation of microorganisms is not needed, the strain stability in the fermentation process is higher, the preparation method is simple, convenient and rapid, the prepared cinnamic acid has higher concentration and purity, and the method is suitable for industrial production. And a more efficient method is provided for biosynthesis of cinnamic acid.

Description

Technical Field

[0001] The present application relates to the field of biosynthesis technology, and in particular to the application of Lactobacillus plantarum in the biological preparation of cinnamic acid. Background Art

[0002] Cinnamic acid is a plant-derived aromatic organic acid produced by the deamination of phenylalanine via phenylalanine ammonia lyase (PAL). In the past decade, its biological activity in chronic metabolic diseases, particularly type 2 diabetes, has garnered widespread attention. Numerous studies have demonstrated that cinnamic acid possesses antioxidant, anti-inflammatory, and lipid-modulating properties, potentially improving insulin resistance and hyperglycemia. In diabetic animal models, cinnamic acid supplementation improves glucose tolerance and insulin secretion. It also significantly enhances antioxidant defenses, such as by increasing the activities of catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione levels, while reducing oxidative stress markers such as malondialdehyde (MDA). Cinnamic acid also reduces chronic inflammation and improves insulin sensitivity by inhibiting inflammatory signaling pathways, including the nuclear transcription factor NF-κB, and downregulating the expression of the proinflammatory cytokines tumor necrosis factor-α and interleukin-6. Furthermore, cinnamon consumption has been shown to have a positive impact on associated dyslipidemia, such as lowering LDL cholesterol and triglyceride levels and mildly increasing HDL cholesterol levels in patients with type 2 diabetes. Existing research, including published experimental, clinical, and systematic reviews, provides ample evidence demonstrating the potential of cinnamic acid to improve glucose and lipid metabolism and reduce inflammatory and oxidative stress, providing scientific support for its use in chronic metabolic diseases such as type 2 diabetes.

[0003] Cinnamic acid is widely found in essential oils and resins of many plants and can be obtained through natural product extraction. The most famous sources are the bark and balsam resins of trees of the genus Cinnamon: for example, Cinnamon bark ( Cinnamon cassia The dried inner bark of cinnamon is a traditional spice. Its volatile oil contains small amounts of free cinnamic acid and cinnamate esters. The main component of cinnamon oil is cinnamaldehyde (about 55–75%), but cinnamic acid also contributes to the sweet aroma of honey. Another important natural source is Storax resin, which is obtained from trees of the genus Liquidambar (such as Oriental Liquidambar). Eastern Liquidambar The balsamic gum extracted from the trunk secretions of the storax tree is rich in cinnamic acid and its esters. Purified storax resin contains 5-15% free cinnamic acid, which, along with its benzoic acid and cinnamic acid esters, constitutes the main component of the resin.

[0004] Bioproduction of cinnamic acid through natural extraction has become an emerging technology in recent years and is considered a more environmentally friendly and efficient modern production method. In natural biosynthetic pathways, cinnamic acid is a key intermediate in many plant secondary metabolites (such as lignin and flavonoids). It is produced by the enzyme PAL, catalyzed by the enzyme. Therefore, it is theoretically possible to produce it using microorganisms or enzyme engineering. Currently, there are two main approaches to bioproduction of cinnamic acid: enzymatic or whole-cell catalysis, using plant or microbial PAL enzymes to directly deaminate the exogenous substrate L-phenylalanine to produce trans-cinnamic acid; and metabolic engineering fermentation, which involves constructing engineered strains that ferment L-phenylalanine from simple carbon sources and further convert it into cinnamic acid. For example, existing research has reported the use of recombinant strains to directly ferment inexpensive carbon sources such as glucose to cinnamic acid. This involves enhancing the aromatic amino acid synthesis pathway in Escherichia coli to produce high yields of D-phenyllactic acid from glucose under aerobic conditions. Subsequently, a cinnamic acid-producing strain was introduced to dehydrate D-phenyllactic acid to cinnamic acid under anaerobic conditions. Compared to chemical synthesis, biofermentation offers advantages in terms of renewable substrates (e.g., glucose and biomass hydrolyzate), mild process conditions (often conducted at moderate temperatures and atmospheric pressure), highly specific enzyme catalysts, minimal byproducts, and easier product purification. In particular, cinnamic acid derived from biofermentation can be classified as a "natural fermentation product," offering greater market acceptance and safety credentials compared to petrochemical-derived products.

[0005] With the advancement of synthetic biology and fermentation engineering, the biological production of cinnamic acid has shown good prospects for industrialization. The current common method is to express cinnamic acid in Escherichia coli through genetic modification. The resulting cinnamic acid has high purity and yield (95%-99% purity after purification and 1.5-2.5 g / L yield). However, this method requires sophisticated metabolic engineering of the microorganisms and the preparation method is cumbersome. The purity and yield of cinnamic acid synthesized in bacteria are often low, such as photosynthetic bacteria ( Rhodobacter sphaeroides Cinnamic acid is a chemical that is produced by a variety of microbial strains, but its purity is low (approximately 30-50%, requiring purification) and its yield is low (approximately 0.1-0.3 g / L). Currently, there are relatively few strains capable of biosynthesizing cinnamic acid. Furthermore, the yield and purity of cinnamic acid produced by these microbial strains are both low and need to be improved. Therefore, identifying more microbial strains capable of efficiently synthesizing cinnamic acid is of great significance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortage of existing microbial strain resources for efficiently synthesizing cinnamic acid and provide application of Lactobacillus plantarum in biological preparation of cinnamic acid.

[0007] The purpose of the present invention is to provide a method for biosynthesizing cinnamic acid.

[0008] Another object of the present invention is to provide the use of Lactobacillus plantarum in the biological preparation of cinnamic acid.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a method for biosynthesizing cinnamic acid, comprising the following steps: S1. Preparation of Lactobacillus plantarum fermentation broth: First, the probiotic Lactobacillus plantarum ( Lactiplantibacillus plantarum ) After activation culture, inoculate into fermentation medium, culture at 35-40°C for 12-36 hours, and centrifuge to obtain the fermentation supernatant; S2. Preparation of crude cinnamic acid: Extract the fermentation supernatant to remove excess water; then rotary evaporate at 50-60°C until the sample becomes a light yellow oil to obtain cinnamic acid.

[0010] The present invention shows that Lactobacillus plantarum ( L. plantarum ) The metabolites of the LY4 strain contain cinnamic acid. Cinnamic acid was prepared for the first time through biosynthesis by Lactobacillus plantarum. The results of HPLC determination confirmed that the content of cinnamic acid prepared from the fermentation supernatant of the LY4 strain was 0.4108 mg / mL, with a purity of 98.01% and a sample recovery rate of 99.85%. The sample was stable within 24 hours. The average peak area of ​​the same sample was 33590254.6667, and the RSD was 0.6206%. The present invention provides more microbial sources and preparation methods for high-yield cinnamic acid, reports for the first time that Lactobacillus plantarum can be used to prepare cinnamic acid, and the prepared product has high purity and high yield. Compared with existing microbial strain preparation methods, no further extraction is required after extraction, and the crude cinnamic acid product prepared by the present invention has high purity and high yield. Compared with the currently available biosynthesis method, the present invention has the advantages of not requiring sophisticated metabolic engineering transformation of microorganisms, and higher strain stability during the fermentation process. The preparation method is simple, fast, natural, non-toxic, low-cost, and the raw materials are easily available. The prepared cinnamic acid has high concentration and purity, and provides more efficient methods for biosynthesis of cinnamic acid.

[0011] Preferably, the Lactobacillus plantarum strain is the Lactobacillus plantarum LY4 strain, which was preserved in the Guangdong Provincial Microbial Culture Collection Center on April 19, 2023, with the culture collection number: GDMCC No: 63373.

[0012] Preferably, the fermentation medium is MRS broth medium or broth medium.

[0013] More preferably, the fermentation medium is MRS broth medium.

[0014] Preferably, the extraction method in S2 is: adjusting the pH of the supernatant to 2±0.1, adding NaCl to saturation; using ethyl acetate-methanol as the extractant for extraction, the volume ratio of the supernatant to the extractant is 1-2:1, vortexing and standing to clearly separate the organic phase, taking the aqueous phase and repeating the above steps twice, and combining the organic phases.

[0015] Preferably, the volume ratio of the supernatant to the extractant in S2 is 1-2:1.

[0016] Preferably, the volume ratio of the supernatant to the extractant in S2 is 2:1.

[0017] Preferably, the extraction conditions in S2 are: the extraction temperature is 25°C-30°C.

[0018] As the most preferred embodiment, the present invention provides a specific method for biosynthesizing cinnamic acid: (1) Preparation of fermentation supernatant of Lactobacillus plantarum LY4 strain: Use a pipette to draw 100 μL of the probiotic Lactobacillus plantarum LY4 bacterial solution, inoculate it into a test tube containing 5 mL of MRS broth medium, and culture it at 37°C for 24 hours; take 2 mL of the bacterial solution and inoculate it into a conical flask containing 100 mL of MRS broth medium, and culture it at 37°C for 24 hours; take 20 mL of the bacterial solution and inoculate it into a conical flask containing 1 L of MRS broth medium, and culture it at 37°C for 24 hours, remove it, and centrifuge it at 6500 r for 10 minutes to obtain the supernatant, and store the supernatant at 4°C for later use; (2) Preparation of crude cinnamic acid product from the fermentation supernatant of Lactobacillus plantarum LY4 strain: Take 200 mL of the prepared Lactobacillus plantarum LY4 strain fermentation supernatant into a separatory funnel, adjust the pH of the supernatant to pH = 2 with concentrated hydrochloric acid, and add 71.8 g of NaCl; add 100 mL of ethyl acetate-methanol mixed solution according to a volume ratio of 2:1, vortex at room temperature for 5 minutes, let it stand for 10 minutes, retain the organic phase after obvious stratification, and repeat the above steps twice for the aqueous phase; combine the organic phases, add an appropriate amount of anhydrous sodium sulfate, let it stand for 10 minutes to remove excess water; rotary evaporate at 55°C until the sample becomes a light yellow oil, and obtain 2 mL of crude cinnamic acid product.

[0019] The present invention provides cinnamic acid synthesized and prepared by the method.

[0020] The present invention uses ethyl acetate as an extraction solvent. By optimizing the extraction solvent ratio, extraction temperature, and extraction pH, multi-stage extraction is performed under optimal conditions to efficiently extract cinnamic acid from the fermentation supernatant of the Lactobacillus plantarum LY4 strain, obtaining a crude product with a purity of 98.01% and a sample recovery rate of 99.85%. The prepared sample is stable for 24 hours, and high-performance liquid chromatography analysis shows that the average peak area of ​​the same sample over 24 hours is 33,590,254.6667, with an RSD of 0.6206%.

[0021] Therefore, the present invention provides a method for efficiently extracting cinnamic acid from bacterial fermentation supernatant. The method comprises the following steps: extracting the bacterial fermentation supernatant with ethyl acetate-methanol as an extraction solvent, wherein the volume ratio of the supernatant to the extraction solvent is 1-2:1, the extraction temperature is 25°C-30°C, the extraction pH is 2±0.1, and multi-stage extraction is performed to obtain cinnamic acid.

[0022] Preferably, the bacterial fermentation supernatant is the fermentation supernatant of Lactobacillus plantarum LY4 strain.

[0023] The present invention also provides application of Lactobacillus plantarum in preparing cinnamic acid.

[0024] Preferably, the Lactobacillus plantarum strain is the Lactobacillus plantarum LY4 strain, which was preserved in the Guangdong Provincial Microbial Culture Collection Center on April 19, 2023, with the culture collection number: GDMCC No: 63373.

[0025] The present invention has the following beneficial effects: The present invention is the first to use a biosynthetic method in Lactobacillus plantarum ( L. plantarum ) was used to prepare cinnamic acid from the fermentation supernatant of a fermented product. High-performance liquid chromatography (HPLC) analysis revealed a cinnamic acid content of 0.4108 mg / mL, a purity of 98.01%, and a sample recovery of 99.85%. The sample was stable over 24 hours, with an average peak area of ​​33,590,254.6667 for the same sample and an RSD of 0.6206%. This invention provides a wider range of microbial sources for high-yield cinnamic acid production. Compared to existing biosynthesis methods, this invention offers advantages in that it does not require sophisticated metabolic engineering of the microorganisms, resulting in greater strain stability during the fermentation process. The preparation method is simple, rapid, natural, non-toxic, low-cost, and uses readily available raw materials. The resulting cinnamic acid exhibits high concentration and purity, providing a more efficient method for biosynthesis of cinnamic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the HPLC spectrum of cinnamic acid standard.

[0027] Figure 2 This is the standard curve of the relationship between cinnamic acid concentration and peak area.

[0028] Figure 3 This is the HPLC spectrum of the fermentation supernatant sample of Lactobacillus plantarum LY4 strain. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0031] The MRS culture medium used in the present invention has the following formula: 10 g of tryptone, 10 g of beef extract, 4 g of yeast extract powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate (anhydrous), 2 g of triammonium citrate (anhydrous), 5 g of sodium acetate trihydrate, 0.2 g of magnesium sulfate (containing heptahydrate), 0.05 g of manganese sulfate (containing tetrahydrate), 1 g of Tween-80, and a final pH of 6.8±0.2.

[0032] The standard grade cinnamic acid used in the following examples was analytically pure and purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0033] The plant lactobacillus used in the embodiment L. plantarum The LY4 strain is a research result of the applicant's research team and was deposited with the Guangdong Provincial Microbial Culture Collection on April 19, 2023. The strain is deposited with GDMCC No. 63373 at Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, and is recorded in the prior art as CN116478888A. The complete genome sequence of the strain has been uploaded to NCBI under accession number SRR24579381.

[0034] Example 1 Preparation of Crude Cinnamic Acid 1. Lactobacillus plantarum ( L. plantarum ) Preparation of LY4 strain fermentation supernatant Use a pipette to draw up the probiotic Lactobacillus plantarum ( L. plantarum ) Inoculate 100 μL of LY4 bacterial solution into a test tube containing 5 mL of MRS broth medium and culture at 37°C for 24 h; take 2 mL of bacterial solution and inoculate it into a conical flask containing 100 mL of MRS broth medium and culture at 37°C for 24 h; take 20 mL of bacterial solution and inoculate it into a conical flask containing 1 L of MRS broth medium and culture at 37°C for 24 h. Remove the culture and centrifuge at 6500 r for 10 min to obtain the supernatant. Store the supernatant at 4°C for later use.

[0035] 2. Preparation of Cinnamic Acid Crude Product from the Fermentation Supernatant of Lactobacillus plantarum LY4 Strain Take 200 mL of the prepared Lactobacillus plantarum LY4 strain fermentation supernatant into a separatory funnel, adjust the pH of the supernatant to pH = 2 with concentrated hydrochloric acid, and add NaCl to saturation; add 100 mL of ethyl acetate-methanol mixed solution according to a volume ratio of 2:1, vortex at room temperature for 5 minutes, let it stand for 10 minutes, retain the organic phase after obvious stratification, and repeat the above steps twice for the aqueous phase; combine the organic phases, add an appropriate amount of anhydrous sodium sulfate, let it stand for 10 minutes to remove excess water; rotary evaporate at 55°C until the sample becomes a light yellow oil, and obtain 2 mL of crude cinnamic acid product.

[0036] Example 2 Determination of cinnamic acid content 1. Preparation of mobile phase and chromatographic conditions Prepare 0.43w / v potassium dihydrogen phosphate aqueous solution, adjust the pH to pH = 2.5 with 85% concentrated phosphoric acid, filter through a 0.22μm filter membrane, sonicate for 15 minutes, and let it stand for later use; sonicate chromatographically pure acetonitrile for 10 minutes and let it stand for later use.

[0037] Chromatographic conditions: (1) Mobile phase: potassium dihydrogen phosphate aqueous solution: acetonitrile = 65:35; (2) Detection wavelength: 269 nm; (3) Column temperature: 30°C; (4) Flow rate: 1 mL / min; (5) Injection volume: 10 μL.

[0038] 2. Drawing of the standard curve of cinnamic acid Accurately weigh 10 mg of cinnamic acid and dissolve it in methanol to prepare a 1 mg / mL stock solution. Accurately pipette a certain amount of the stock solution to prepare standard solutions of 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, and 0.00625 mg / mL, respectively. Place these solutions in sample vials for later use. Draw a standard curve for cinnamic acid using the above conditions.

[0039] The peak diagram of cinnamic acid standard is as follows Figure 1 As shown in Figure 1, the response value is the largest at the retention time of 11.978 min, which is the retention time of cinnamic acid. The standard curve of cinnamic acid concentration and peak area is shown in Figure 1. Figure 2 As shown, the goodness of fit (R2) = 0.9999.

[0040] 3. Determination of cinnamic acid content in the fermentation supernatant of Lactobacillus plantarum LY4 strain Take 100 μL of the crude cinnamic acid product prepared above, add 9.9 mL of methanol, shake until completely dissolved, filter through a 0.22 μm filter membrane, and place in a sample bottle for later use. Determination is carried out according to the above conditions.

[0041] The peak diagram of the sample is as follows Figure 3As shown in the figure, the response value is the largest at the retention time of 12.001 min, which is consistent with the retention time of the cinnamic acid standard. Therefore, it is determined that the fermentation supernatant of Lactobacillus plantarum LY4 strain contains cinnamic acid. According to the standard curve, the cinnamic acid content in the sample is 0.4108 mg / mL; the peak area at this retention time accounts for 98.01% of the total area, that is, the purity of cinnamic acid in the sample is 98.01%.

[0042] 4. Precision test The prepared 0.4 mg / mL cinnamic acid standard was injected continuously 6 times, and the measured peak areas were 33811346, 33811445, 33811426, 33811399, 33811453, and 33811387, respectively. The average peak area was 33811409.3333, and the RSD was 0.0001%.

[0043] 5. Reproducibility test Six portions of fermentation supernatant of the same batch of Lactobacillus plantarum LY4 strain were taken and prepared according to the preparation method of the crude cinnamic acid product in the fermentation supernatant of the strain. The cinnamic acid contents were detected according to the above-mentioned determination method. The cinnamic acid contents were 0.4123, 0.4066, 0.3987, 0.4139, 0.4095, and 0.3958 mg / mL, respectively, with an average content of 0.4061 mg / mL and RSD=1.8158%.

[0044] 6. Stability test The same sample solution was taken and measured at 0, 2, 4, 8, 12, and 24 hours respectively. The peak areas were 33641238, 33215646, 33845231, 33652146, 33541285, and 33645982, respectively. The average peak area was 33590254.6667, and the RSD was 0.6206%.

[0045] 7. Sample recovery test Twelve portions of fermentation supernatant from the same batch of Lactobacillus plantarum LY4 strain were taken, of which 6 were blank experimental groups. Cinnamic acid was prepared according to the preparation method of the crude product in the fermentation supernatant of the strain, and then detected according to the method for determining the content of cinnamic acid in the fermentation supernatant of the strain; 6 were spiked experimental groups. Cinnamic acid standard (200 mL, cinnamic acid content 80 mg) with the same volume concentration of 0.4 mg / mL was added, and the product was prepared according to the preparation method of the crude product in the fermentation supernatant of the strain. After dilution twice, the product was detected according to the method for determining the content of cinnamic acid in the fermentation supernatant of the strain; the results are shown in Table 1, and the spiked recovery rate was 99.85%.

[0046] Table 1 Recovery rate of cinnamic acid

[0047] The cinnamic acid biosynthesized by Lactobacillus plantarum was detected by the above-mentioned high performance liquid chromatography, with high precision (average peak area of ​​33811409.3333, RSD=0.0001%) and good reproducibility (average content of 0.4061 mg / mL, RSD=1.8158%).

[0048] Example 3 A method for efficiently extracting cinnamic acid from bacterial fermentation supernatant Using an ethyl acetate-methanol mixed solution as the extraction solvent, by optimizing the extraction solvent ratio, extraction temperature, and extraction pH, and performing multi-stage extraction under optimal conditions, high-purity cinnamic acid can be efficiently synthesized in the bacterial fermentation supernatant. The specific method is as follows: Take 200 mL of the prepared Lactobacillus plantarum LY4 strain fermentation supernatant into a separatory funnel, adjust the pH of the supernatant to pH = 2 with concentrated hydrochloric acid, and add NaCl to saturation; add 100 mL of ethyl acetate-methanol mixed solution according to a volume ratio of 2:1, vortex at room temperature for 5 minutes, let it stand for 10 minutes, retain the organic phase after obvious stratification, and repeat the above steps twice for the aqueous phase; combine the organic phases, add an appropriate amount of anhydrous sodium sulfate, let it stand for 10 minutes to remove excess water; rotary evaporate at 55°C until the sample becomes a light yellow oil, and obtain 2 mL of crude cinnamic acid product.

[0049] The above extraction method can efficiently extract cinnamic acid from the fermentation supernatant of Lactobacillus plantarum LY4 strain and obtain a crude product with a purity of 98.01%. The product has high purity and does not require further purification. It is convenient and fast, providing more extraction methods and means for bacteria to synthesize high-purity cinnamic acid.

[0050] In summary, the present invention is the first to use a biosynthetic method in Lactobacillus plantarum ( L. plantarum ) was used to prepare a crude cinnamic acid product from the fermentation supernatant. High-performance liquid chromatography (HPLC) analysis revealed a cinnamic acid content of 0.4108 mg / mL, a purity of 98.01%, and a sample recovery of 99.85%. The sample was stable for 24 hours, with an average peak area of ​​33,590,254.6667 for the same sample and an RSD of 0.6206%. This invention provides a wider range of microbial sources for high-yield cinnamic acid production. Compared to existing biosynthetic methods, it offers advantages in that it does not require sophisticated metabolic engineering of the microorganisms, resulting in greater strain stability during the fermentation process. The preparation method is simple and rapid, and the cinnamic acid produced is of high concentration and purity, providing a more efficient method for the biosynthesis of cinnamic acid.

[0051] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for biosynthesizing cinnamic acid, characterized in that: The following steps are involved: S1. Preparation of Lactobacillus plantarum fermentation broth: First, the probiotic Lactobacillus plantarum ( Lactiplantibacillus plantarum ) After activation culture, inoculate into fermentation medium, culture at 35-40°C for 12-36 hours, and centrifuge to obtain the fermentation supernatant; S2. Preparation of crude cinnamic acid: Extract the fermentation supernatant to remove excess water, and then perform rotary evaporation at 50-60°C until the sample becomes a light yellow oil to obtain cinnamic acid.

2. The method according to claim 1, characterized in that The plant lactobacillus adopts the plant lactobacillus LY4 strain, which was preserved in the Guangdong Provincial Microbial Culture Collection Center on April 19, 2023, and the culture collection number is: GDMCC No: 63373.

3. The method according to claim 1, characterized in that The fermentation medium is MRS broth medium or broth medium.

4. The method according to claim 1, characterized in that The extraction method in S2 is as follows: adjust the pH of the supernatant to 2±0.1, add NaCl to saturation; use ethyl acetate-methanol as the extractant for extraction, the volume ratio of the supernatant to the extractant is 1-2:1, vortex and stand until obvious stratification occurs, retain the organic phase, take the aqueous phase and repeat the above steps twice, and combine the organic phases.

5. The method according to claim 1, characterized in that The extraction conditions in S2 are: extraction temperature is 25°C-30°C.

6. Cinnamic acid synthesized by the method according to any one of claims 1 to 5.

7. A method for efficiently extracting cinnamic acid from bacterial fermentation supernatant, characterized in that: The bacterial fermentation supernatant is extracted with ethyl acetate-methanol extraction solvent, the volume ratio of the supernatant to the extraction solvent is 1-2:1, the extraction temperature is 25°C-30°C, the extraction pH is 2±0.1, and multi-stage extraction is performed to obtain the product.

8. Application of Lactobacillus plantarum in the preparation of cinnamic acid.

9. The application according to claim 8, characterized in that: The plant lactobacillus adopts the plant lactobacillus LY4 strain, which was preserved in the Guangdong Provincial Microbial Culture Collection Center on April 19, 2023, and the culture collection number is: GDMCC No: 63373.

Images