Pediococcus acidilactici strain FSE3 with high phenyllactic acid yield and application thereof

Abstract

The invention provides a pediococcus acidilactici strain FSE3 with high phenyllactic acid yield and application of the pediococcus acidilactici strain FSE3, and belongs to the technical field of functional microorganisms. The pediococcus acidilactici strain FSE3 for producing phenyllactic acid is screened from a dreg vinegar fermentation system, and the content of phenyllactic acid produced by metabolism of the strain FSE3 is 2.1 g / L; when the strain is applied to fermentation of the dregs vinegar, the content of phenyllactic acid in the dregs vinegar is remarkably increased, the flavor and quality of the dregs vinegar can be greatly improved, and a new way is provided for development of vinegar or other fermentation products.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial technology, specifically relating to a high-producing Pediococcus lactis strain FSE3 and its applications. Background Technology

[0002] Zhaobo vinegar, also known as "Women's Vinegar," is a traditional flavored condiment from Hainan Province, possessing a unique flavor, texture, appearance, and functions. Besides organic acids, it is rich in beneficial components such as amino acids, polypeptides, trace elements, polyphenols, and flavonoids, which contribute to its various health benefits, including lowering blood pressure and cholesterol, anti-oxidation, and cardiovascular health. Research has discovered a functional component—phenyllactic acid—in Zhaobo vinegar and other fermented foods. Phenyllactic acid (also known as 2-hydroxy-3-phenylpropionic acid) is a structural analogue of tanshinone and possesses similar pharmacological effects, including inhibiting platelet aggregation, thrombus formation, and improving microcirculation; it also has certain medicinal value for ischemic heart disease. Phenyllactic acid (PLA), a phenolic acid, was first identified in honey and is naturally found in traditional Chinese medicine (such as iris, snow lotus, gastrodia elata, and rye) and lactic acid bacteria fermented foods. PLA is structurally stable under high temperatures and acidic environments, making it a potential natural food preservative. It has a broad antibacterial spectrum, inhibiting the growth of Listeria monocytogenes and the proliferation of foodborne spoilage bacteria and pathogens. It also exhibits good inhibitory effects against various foodborne pathogens such as Escherichia coli, Salmonella, and Bacillus cereus.

[0003] Studies have reported that many strains can produce phenyllactic acid, such as Lactobacillus, Geotrichum candida, Bacillus, Propionibacterium, and Pediococcus pentosaceus, but the yield varies considerably among different strains. Existing reports of phenyllactic acid-producing lactic acid bacteria show that their ability to synthesize phenyllactic acid is relatively weak, with most yielding less than 100 mg / L. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a high-producing Pediococcus lactis strain FSE3, which has a significantly increased content of phenyllactic acid produced by metabolism.

[0005] This invention provides a strain of Pediococcus acidilactici, FSE3, with accession number CGMCC No:66666.

[0006] The present invention provides a microbial preparation comprising the Pediococcus lactis strain FSE3.

[0007] Preferably, the viable bacterial concentration of the *Pediococcus lactis* strain FSE3 is 10. 7 CFU / mL or 10 7CFU / g or higher;

[0008] The microbial preparation also includes Saccharomyces cerevisiae and / or Lactobacillus plantarum.

[0009] This invention provides the application of the *Pediococcus lactis* strain FSE3 or the microbial preparation in the fermentation production of phenyllactic acid or products containing phenyllactic acid.

[0010] Preferably, the product containing phenyl lactic acid includes at least one of the following: vinegar, baijiu (Chinese white liquor), and huangjiu (yellow wine).

[0011] Preferably, the vinegar includes vinegar made from fermented grains.

[0012] This case provides a method for producing phenyl lactic acid by fermentation, including the following steps:

[0013] Using phenylalanine and / or phenylpyruvic acid as substrates, fermentation is carried out using the *Pediococcus lactis* strain FSE3 or the microbial preparation to obtain a fermentation broth rich in phenyllactic acid.

[0014] Preferably, the fermentation temperature is 32℃~37℃, and the fermentation time is 48~72h;

[0015] After obtaining the fermentation broth rich in phenyllactic acid, the process further includes removing the bacterial cells from the fermentation broth, extracting the phenyllactic acid with ethyl acetate, collecting the organic phase, removing the organic phase, and obtaining phenyllactic acid.

[0016] This invention provides a brewing method for vinegar rich in phenyllactic acid, comprising the following steps:

[0017] Fermentation agents are inoculated into the saccharification liquid prepared from grains;

[0018] The fermentation agent includes the *Pediococcus lactis* strain FSE3 described in the above technical solution or the microbial preparation described in the above technical solution.

[0019] Preferably, the Pediococcus lactis strain FSE3 is inoculated in the form of Pediococcus lactis strain FSE3 bacterial suspension;

[0020] The inoculum amount of the *Pediococcus lactis* strain FSE3 was 1%–3%, the fermentation temperature was 28°C–37°C, and the fermentation time was 13–17 days.

[0021] This invention provides a *Pediococcus acidilactici* strain FSE3, with accession number CGMCC No: 66666. This invention is the first to screen a *Pediococcus acidilactici* strain producing phenyllactic acid from a fermentation system for vinegar made from fermented grains, with the strain producing phenyllactic acid at a concentration of 2.1 g / L. When applied to the fermentation of vinegar made from fermented grains, the results show a significant increase in the content of phenyllactic acid and lactic acid in the vinegar system, resulting in a substantial improvement in the flavor and quality of the product, providing a new avenue for developing the health benefits of vinegar made from fermented grains. Attached Figure Description

[0022] Figure 1 The colony morphology of Pediococcus lactis strain FSE3 on agar plates;

[0023] Figure 2 The cell morphology of *Pediococcus lactis* strain FSE3 stained with crystal violet;

[0024] Figure 3 This is a phenyllactic acid content-chromatographic standard curve for the detection of phenyllactic acid by high performance liquid chromatography.

[0025] Information on the preservation of biological materials

[0026] Pediococcus acidilactici strain FSE3 was deposited on July 14, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number CGMCC No: 66666. Detailed Implementation

[0027] This invention provides a strain of Pediococcus acidilactici, FSE3, with accession number CGMCC No:66666.

[0028] In this invention, strain FSE3 was isolated from fermented vinegar residue and exhibits a significant advantage in phenyllactic acid (PLA) production compared to other strains isolated in the same batch. For example, after culturing at 37°C for 48 hours in a screening medium, the PLA concentration produced by strain FSE3 reached 298.03 mg / L, which is 2 to 3 times higher than that of other strains. This invention further optimizes the PLA production medium of strain FSE3. After culturing at 37°C for 60 hours in MRS medium containing 20 g / L phenylalanine, the PLA concentration produced by strain FSE3 reached over 2135 mg / L.

[0029] The present invention provides a microbial preparation comprising the Pediococcus lactis strain FSE3.

[0030] In this invention, the microbial preparation preferably comprises an aqueous solution or a powder. The viable cell concentration of the *Pediococcus lactis* strain FSE3 is preferably 10. 7 CFU / mL or 10 7 CFU / g or higher, can be 5×10 7 CFU / mL ~ 1×10 9 CFU / mL or 5×10 7 CFU / g ~ 1×10 9 CFU / g can also be 1×10 8 CFU / mL or 1×10 8 CFU / g. This invention does not impose any particular limitations on the preparation method of the microbial preparation; any method well-known in the art for preparing microbial preparations may be used.

[0031] This invention provides the application of the *Pediococcus lactis* strain FSE3 or the microbial preparation in the fermentation production of phenyllactic acid or products containing phenyllactic acid.

[0032] In this invention, the product containing phenyllactic acid preferably includes at least one of the following: vinegar, baijiu (Chinese white liquor), and huangjiu (yellow wine). The vinegar preferably includes vinegar made from fermented grains. When producing phenyllactic acid through fermentation, liquid fermentation is preferably carried out using an MRS medium containing phenylalanine and / or phenylpyruvic acid or phenylalanine and / or phenylpyruvic acid as the substrate. The working concentration of phenylalanine and / or phenylpyruvic acid is preferably 2–40 g / L, but can also be 3–30 g / L, 5–20 g / L, or more preferably 8–15 g / L. The temperature of the liquid fermentation is preferably 28–37°C, but can also be 30–35°C, 32–34°C, or 33°C. The time of the liquid fermentation is preferably 48–72 h, but can also be 50–70 h, 54–68 h, or 60 h. Compared with strains screened in the same batch, the *Pediococcus lactis* strain FSE3 has a significant advantage in producing phenyllactic acid, and under optimized substrate culture conditions, the phenyllactic acid content reaches 2135 mg / L.

[0033] In one embodiment of the present invention, the fermentation of the *Pediococcus lactis* strain FSE3 to prepare vinegar not only increases the concentration of phenyllactic acid in the vinegar to 470 mg / L, but also improves the sensory evaluation indicators (color, aroma, taste and state) of the vinegar, thus significantly improving the quality of the vinegar.

[0034] This invention provides a method for producing phenyllactic acid by fermentation, comprising the following steps:

[0035] Using phenylalanine and / or phenylpyruvic acid as substrates, fermentation is carried out using the *Pediococcus lactis* strain FSE3 or the microbial preparation to obtain a fermentation broth rich in phenyllactic acid.

[0036] In this invention, the fermentation temperature is preferably 32℃~37℃, but can be 32~36℃ or 35℃. The fermentation time is preferably 15 days.

[0037] In this invention, after obtaining the fermentation broth rich in phenyllactic acid, the process preferably further includes removing the bacterial cells from the fermentation broth, extracting the phenyllactic acid with ethyl acetate, collecting the organic phase, removing the organic phase, and obtaining phenyllactic acid. The method for removing the bacterial cells is preferably centrifugation. The centrifugation speed is preferably 10000 r / min to 14000 r / min, and can be 12000 r / min. The centrifugation time is 8 to 10 min, and can be 9 min. During the ethyl acetate extraction, the volume of ethyl acetate is 2 to 3 times, and can be 2.5 times, the volume of the fermentation broth from which the bacterial cells were removed. The method for removing the organic phase preferably includes nitrogen blowing evaporation. The temperature of the nitrogen blowing evaporation is preferably 60 to 85°C, and can be 65 to 80°C, and can be 70 to 75°C.

[0038] In this invention, the determination of phenyllactic acid is preferably performed using high-performance liquid chromatography (HPLC). An Xtimate Sugar-H column is used for the determination of phenyllactic acid. To avoid clogging before determination, membrane filtration is preferably performed. The pore size of the membrane is preferably 0.22 μm. The HPLC test conditions are as follows: mobile phase: 0.005 mol / L sulfuric acid; flow rate: 0.6 mL / min; injection volume: 20 μL; column temperature: 60 °C.

[0039] This invention provides a brewing method for vinegar made from phenyllactic acid-rich lees, comprising the following steps:

[0040] Fermentation agents are inoculated into the saccharification liquid prepared from grains;

[0041] The fermentation agent includes the *Pediococcus lactis* strain FSE3 described in the above technical solution or the microbial preparation described in the above technical solution.

[0042] In this invention, the Pediococcus lactis strain FSE3 is preferably inoculated in the form of a broth containing Pediococcus lactis strain FSE3. The inoculation amount of the broth containing Pediococcus lactis strain FSE3 is preferably 1% to 3%, and can be 2%.

[0043] The vinegar prepared by this invention has a pH of around 3 and a total acid concentration of 17.46, which is more than 36% higher than the blank control group.

[0044] The following detailed description, in conjunction with embodiments, illustrates a high-phenyllactic acid-producing Pediococcus lactis strain FSE3 and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Isolation, purification, and identification of bacterial strains

[0047] 1. Culture medium preparation

[0048] MRS liquid culture medium: purchased from Guangdong Huankai Microbial Technology Co., Ltd., 1000mL distilled water, pH 6.2-6.6, sterilized at 121℃ for 15min before use.

[0049] Agar: Purchased from Beijing Solarbio Technology Co., Ltd.

[0050] Phenylalanine: Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0051] MRS solid medium is made by adding 2% agar by weight / volume to MRS liquid medium and sterilizing at 121°C for 15 minutes before use.

[0052] The screening medium was prepared by adding 3g of phenylalanine to the MRS liquid medium and sterilizing it at 121℃ for 15min before use.

[0053] 2. Methods

[0054] (1) Isolation and purification of acid-producing strains

[0055] Take 10 ml of traditionally fermented lees vinegar sample (sample obtained from Ren'an Lees Vinegar Co., Ltd., Wenchang City, Hainan Province), and perform 10-fold serial dilutions on the sample. Select a dilution gradient of 10... -3 10 -4 10 -5 and 10 -6 For each dilution, 100 μL of each serial dilution was spread onto MRS solid agar plates and incubated upside down at 37°C for 48 h. Colonies with large clear zones were picked from the MRS solid agar plates and streaked three times to obtain pure cultures, which were then stored at 4°C for trial use.

[0056] (2) Screening of high-yield phenyllactic acid strains

[0057] The acid-producing bacteria obtained above were initially screened for high-phenyllactic acid producing strains (FSE3, LFMse11, LFMYU14, LHye10, LHye11, L-me8). The strains were inoculated into the screening medium and cultured at 37℃ for 48 h. The fermentation broth was centrifuged at 12000 r / min for 10 min to obtain a supernatant from which the bacterial cells were removed. The phenyllactic acid in the supernatant was then extracted three times with ethyl acetate. The volume ratio of ethyl acetate to supernatant was 2:1 for each extraction. The supernatant was then evaporated under nitrogen at 75℃ to obtain phenyllactic acid. The solution was reconstituted with sulfuric acid solution, shaken well, and the supernatant was filtered through a 0.22 μm filter membrane. The concentration of phenyllactic acid in the liquid was determined by high-performance liquid chromatography (HPLC) (differential refractive index). High-yielding phenyllactic acid-producing strains were screened. The HPLC was performed using an Ultimate Sugar-H column. The HPLC operating conditions were: mobile phase 0.005 mol / L sulfuric acid; flow rate 0.6 mL / min; injection volume 20 μL; column temperature 60℃. Phenyllactic acid standard was used as an external standard for quantitative analysis.

[0058] The results of liquid chromatography determination of phenyl lactic acid produced by some strains are shown in Table 1. Among them, the fermentation broth of strain FSE3 had the highest phenyl lactic acid content, reaching 298 mg / L.

[0059] Table 1. PLA production content (mg / L) of some strains

[0060]

[0061]

[0062] (3) Optimization of phenyllactic acid producing strains

[0063] Based on preliminary experiments, it was determined that 20 g / L phenylalanine should be added to MRS medium, and the mixture should be cultured at 37℃. The phenyllactic acid content of the fermentation broth was then determined using liquid chromatography. The relationship between fermentation time and phenyllactic acid content is shown in Table 2 below.

[0064] Table 2 Relationship between fermentation time and phenyllactic acid content

[0065] Incubation time (h) PLA content (mg / L) 0 0 48 1189±3.57 60 2135±1.02 72 2041±2.11

[0066] (4) Morphological identification of strains

[0067] After being cultured at 37℃ for 48 hours, strain FSE3 showed the following colony morphology on agar plates: milky white, round, raised, smooth, opaque, moist, and easily picked up. Under a microscope, the bacterial cells appeared spherical, arranged singly, in pairs, or in short chains. Based on the colony and cell morphology, it was preliminarily identified as a coccus.

[0068] (5) Molecular biological identification of strains

[0069] The bacteria obtained through purification and screening were collected by centrifugation of fresh bacterial culture in the exponential growth phase, and genomic DNA was extracted using a genomic DNA extraction kit. The full-length 16S rDNA sequence was amplified using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:2). Sequencing of the PCR amplification products was performed by Shanghai Paisenuo Biotechnology Co., Ltd. The obtained 16S rDNA sequence was then compared with the NCBI database using BLAST to determine its species. The full-length 16S rDNA sequence of the screened strain was 1479 bp, and the strain with the highest homology was *Pediococcus acidilactici*, with a similarity of 99.25%. This strain was named *Pediococcus acidilactici* FSE3.

[0070]

[0071] Example 2

[0072] A traditional method for fermenting vinegar made from fermented grains consists of the following steps:

[0073] 1. Select a 10L liquid fermentation tank, add 4L of water, and add 1kg of crushed rice flour;

[0074] 2. Add 20,000 U / mL of high-temperature α-amylase, in a volume of 5.0 mL, and liquefy at 90℃ for 30 min;

[0075] 3. Then, lower the temperature of the mash to 55°C; add 0.02% (v / v) of saccharifying enzyme (glucosidase, 100,000 U / g) and saccharify at 60°C for 2 hours to obtain the saccharified liquid;

[0076] 4. Cool to 30℃.

[0077] 5. Inoculate with a 5% inoculation rate of fermenting agent (the mass of the fermenting agent is 5% of the mass of the rice);

[0078] 6. Cover with a sealed lid and place in an incubator at 35°C for 15 days of fermentation.

[0079] Example 3

[0080] Application of Pediococcus lactis strain FSE3 in the brewing of vinegar from fermented grains

[0081] 1. Large-scale culture of the strain

[0082] Saccharomyces cerevisiae: Purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number 2.3886.

[0083] Saccharomyces cerevisiae: Purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number 1.12974.

[0084] (1) Inoculate the brewer's yeast into YPD medium at a mass-volume ratio of 2%, and culture it at 28℃ and 150 rpm for 12 h with shaking to obtain the initial culture of brewer's yeast.

[0085] (2) Inoculate the initial culture of *Saccharomyces cerevisiae* into YPD medium at a volume ratio of 2%, and culture at 28°C and 150 rpm for 12 h with shaking. Adjust the cell density of the culture to 1 × 10⁻⁶ cells / mL. 7 CFU / mL was used to obtain the brewing yeast broth.

[0086] (3) Inoculate Lactobacillus plantarum into MRS liquid medium at an inoculation rate of 2% by weight and culture at 37°C for 24 hours to obtain the initial bacterial culture of Lactobacillus plantarum.

[0087] (4) Inoculate the initial Lactobacillus plantarum culture at a volume ratio of 2% into MRS liquid medium, incubate at 37°C for 24 hours, and adjust the bacterial density of the culture to 1×10⁻⁶. 7 CFU / mL was used to obtain the fermentation broth of Lactobacillus plantarum.

[0088] (5) Fermentation agent for lees vinegar is obtained by mixing brewing yeast fermentation liquid and Lactobacillus plantarum fermentation liquid in a mass ratio of 1:1.

[0089] (6) Preparation of FSE3 seed culture: Purified *Pediococcus lactis* strain FSE3, in the logarithmic growth phase, was inoculated into MRS medium at an inoculation rate of 2% (v / v). The medium was incubated at 37°C for 24 hours, and the bacterial density was adjusted to 1×10⁻⁶. 7 CFU / mL was used to obtain the fermentation broth of Pediococcus lactis strain FSE3.

[0090] 2. Inoculation and Fermentation Test Group 1: 1% of the weight of the rice flour raw material of the saccharified liquid was inoculated with the fermentation liquid of Pediococcus lactis strain FSE3 and 5% of the fermentation agent. After being thoroughly mixed, the fermentation was carried out according to the fermentation process of the lees vinegar in Example 2 until the end of fermentation.

[0091] Control group: 5% fermentation agent by weight of raw materials was added to the saccharified liquid. After the bacterial liquid and the added raw materials were thoroughly mixed, fermentation was carried out according to the fermentation process of lees vinegar in Example 2 until the fermentation was completed.

[0092] 3. Determination of pH and total acid content

[0093] At the end of fermentation, the pH and total acid of the two fermentation broths were measured. pH was measured using a pH meter, and total acid was expressed as acetic acid and determined using acid-base titration.

[0094] The results are shown in Table 3. The total acid content in the group with added Pediococcus lactis was 17.46 g / L, while that in the control group was 12.834 g / L, representing a 36.04% increase in total acid content compared to the control group.

[0095] Table 3. pH and total acid content of fermentation broth (in g / L)

[0096] Group pH Total acid (g / L) Added Pediococcus lactis group 3.02±0.03 17.46±0.04 control group 3.44±0.02 12.834±0.01

[0097] 4. The content of phenyllactic acid in raw vinegar liquid

[0098] The content of phenyl lactic acid in raw vinegar was determined by high performance liquid chromatography. The test method was based on the screening of high phenyl lactic acid producing strains in Example 1 (2). The detection results of phenyl lactic acid in raw vinegar are shown in Table 4.

[0099] Table 4. Content of phenyllactic acid in raw vinegar liquid

[0100] Group Phenylated acid (mg / L) Added Pediococcus lactis group 470±0.35 control group 22.58±0.11

[0101] 5. Sensory index analysis of finished product vinegar made from lees

[0102] The sensory evaluation results of the fermented grain vinegar products from the group with added Pediococcus lactis and the control group are shown in Table 5. The aroma and taste of the finished product from the group with added Pediococcus lactis were significantly better than those from the control group, indicating that adding Pediococcus lactis during the initial fermentation stage significantly improved the product quality.

[0103] Table 5 Sensory Index Analysis of Finished Vinegar

[0104] Group Color (10 points) Aroma (10 points) Taste (10 points) Status (10 points) Added Pediococcus lactis group 8.9 9.2 9.3 7.4 control group 8.6 8.1 8.2 7

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Pediococcus acidilactici, FSE3, characterized in that, The accession number is CGMCCNo:66666.

2. A microbial preparation, characterized in that, Includes the *Pediococcus lactis* strain FSE3 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viable bacterial concentration of the *Pediococcus lactis* strain FSE3 was 10. 7 CFU / mL or 10 7 CFU / g or higher; The microbial preparation also includes Saccharomyces cerevisiae and / or Lactobacillus plantarum.

4. The use of the *Pediococcus lactis* strain FSE3 of claim 1 or the microbial preparation of claim 2 or 3 in the fermentation production of phenyllactic acid or products containing phenyllactic acid.

5. The application according to claim 4, characterized in that, The products containing phenyl lactic acid include at least one of the following: vinegar, baijiu (Chinese white liquor), and huangjiu (yellow wine).

6. The application according to claim 4, characterized in that, The vinegar mentioned includes vinegar made from fermented grains.

7. A method for producing phenyllactic acid by fermentation, characterized in that, Includes the following steps: Using phenylalanine and / or phenylpyruvic acid as substrates, fermentation is carried out using the *Pediococcus lactis* strain FSE3 as described in claim 1 or the microbial preparation as described in claim 2 or 3 to obtain a fermentation broth rich in phenyllactic acid.

8. The method according to claim 7, characterized in that, The fermentation temperature is 32℃~37℃, and the fermentation time is 48~72h; After obtaining the fermentation broth rich in phenyllactic acid, the process further includes removing the bacterial cells from the fermentation broth, extracting the phenyllactic acid with ethyl acetate, collecting the organic phase, removing the organic phase, and obtaining phenyllactic acid.

9. A method for brewing vinegar rich in phenyllactic acid, characterized in that, Includes the following steps: Fermentation agents are inoculated into the saccharification liquid prepared from grains; The fermentation agent includes the *Pediococcus lactis* strain FSE3 of claim 1 or the microbial preparation of claim 2 or 3.

10. The brewing method according to claim 9, characterized in that, When inoculating the *Pediococcus lactis* strain FSE3, inoculation is performed in the form of *Pediococcus lactis* strain FSE3 bacterial suspension. The inoculation amount of the *Pediococcus lactis* strain FSE3 was 1% to 3%. The fermentation temperature is 32℃~37℃, and the fermentation time is 13~17 days.

Images