Lactiplantibacillus plantarum PIAS240228 and use thereof

Abstract

Provided are Lactiplantibacillus plantarum PIAS240228 and the use thereof. The provided Lactiplantibacillus plantarum PIAS240228 has been deposited in the China Center for Type Culture Collection on 09 April 2024, with the accession number being CCTCCNO: M2024647 and the deposit address being Wuhan University, Wuhan City, China.

Description

Lactobacillus plantarum PIAS240228 and its applications

[0001] Related applications

[0002] This application claims priority to Chinese Patent Application No. 202410795180.0, filed on June 19, 2024, entitled "Lactiplantibacillus plantarum PIAS240228 and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of microbial technology, specifically relating to Lactobacillus plantarum PIAS240228 and its application in the cosmetics field. Background Technology

[0004] *Lactobacillus plantarum* is a type of lactic acid bacteria, with an optimal growth temperature of 30℃~35℃. It is an anaerobic or facultative anaerobic bacterium, with straight or curved rod-shaped cells, sometimes in pairs or chains, and belongs to the homofermentative lactic acid bacteria category. Besides producing some active hydrolytic enzymes, lactic acid bacteria may also produce other active ingredients during their growth, such as extracellular polysaccharides, bacteriocins, and antimicrobial peptides. Extracellular polysaccharides from lactic acid bacteria possess moisturizing, antioxidant, and anti-aging properties, and have broad application prospects in the cosmetics industry.

[0005] Tanninase (EC 3.1.1.20), also known as tannic acidase, is a tannic acyl hydrolase. Tannins can be hydrolyzed by tanninase to produce gallic acid, ellagic acid, and other compounds with various biological activities. Microorganisms containing tanninase are mainly fungi and bacteria. Fungi mostly belong to the genera *Aspergillus*, *Penicillium*, *Trichoderma*, *Fusarium*, and yeast. Reports on bacterial tanninase are relatively few, with *Lactobacillus* and *Bacillus* being the most prevalent.

[0006] However, the tanninase in Lactobacillus plantarum has low enzyme activity and is mainly used in the fields of bio-agriculture and food. Summary of the Invention

[0007] According to various embodiments of this application, a Lactobacillus plantarum PIAS240228 and its applications are provided.

[0008] One embodiment of this application provides a Lactiplantibacillus plantarum PIAS240228, which has high tannin enzyme activity and high production of extracellular polysaccharides.

[0009] This application provides a strain of Lactiplantibacillus plantarum PIAS240228, which was deposited at the China Center for Type Culture Collection on April 9, 2024, with accession number CCTCC NO: M2024647, and deposit address: Wuhan University, Wuhan, China.

[0010] This application also provides a microbial agent comprising Lactiplantibacillus plantarum PIAS240228.

[0011] In one embodiment, the fermentation product includes the fermentation filtrate obtained by fermenting plant juice or plant extracts with Lactiplantibacillus plantarum PIAS240228.

[0012] In one embodiment, the plant juice or plant extract includes one or more of the following: pressed juice or extract of pomegranate, chestnut bark, persimmon fruit, rambutan fruit, and burnet bark; the pomegranate includes pomegranate seeds or pomegranate peel.

[0013] This application also provides a method for preparing a fermentation product, the method comprising the step of fermentation using the aforementioned *Lactiplantibacillus plantarum* PIAS240228, including: activating the *Lactiplantibacillus plantarum* PIAS240228; inoculating the activated *Lactiplantibacillus plantarum* PIAS240228 into a solution containing tannin plant extract for fermentation, collecting the fermentation filtrate, inactivating the fermentation broth, filtering, and taking the supernatant to obtain the fermentation product.

[0014] In one embodiment, the viable count of *Lactiplantibacillus plantarum* PIAS240228 inoculated into the tannin-containing plant extract was 2–4 × 10⁻⁶. 9 CFU / mL; the tannin-containing plant extract includes one or more of the following: pomegranate extract, chestnut extract, persimmon extract, rambutan extract, and burnet root extract.

[0015] In one embodiment, activating Lactobacillus plantarum PIAS240228 includes the following steps: inoculating Lactobacillus plantarum PIAS240228 into an MRS broth containing 0.8 w / w% to 1.2 w / w% tannins; the MRS broth comprises the following components: 8 g to 12 g casein digest, 1.8 g to 2.2 g triammonium citrate, 8 g to 12 g beef extract, 0.15 g to 0.25 g magnesium sulfate, 3 g to 5 g yeast extract, 0.04 g to 0.06 g manganese sulfate, 4 g to 6 g sodium acetate, 1.5 g to 2.5 g dipotassium hydrogen phosphate, 18 g to 22 g glucose, and 1.06 g to 1.12 g Tween 80; the pH of the MRS broth is 5.5 to 5.9.

[0016] In one embodiment, the fermentation conditions include: a temperature of 36°C to 38°C and a time of 8h to 12h; the filtration method of the fermentation filtrate includes centrifugation; the centrifugation conditions include: a temperature of 3°C to 5°C, a rotation speed of 8000×g to 12000×g, and a time of 18min to 22min.

[0017] Another aspect of this application provides the use of the fermentation product prepared by the method of preparing the fermentation product as a cosmetic ingredient.

[0018] This application also provides a cosmetic composition comprising a fermentation product prepared by the method described above, or a formulation of the fermentation product.

[0019] Details of one or more embodiments of this application are set forth in the following drawings and description. Other features, objects, and advantages of this application will become apparent from the specification, drawings, and claims. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 shows the colony morphology of Lactiplantibacillus plantarum PIAS240228.

[0022] Figure 2 shows the ability of the extracellular crude polysaccharide of Lactiplantibacillus plantarum to scavenge DPPH free radicals. Detailed Implementation

[0023] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0024] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] the term

[0026] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0027] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0028] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0029] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0030] The term "DPPH radical" refers to 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), a stable free radical that exists stably in organic solvents. DPPH alcoholic solutions are purple and require low-temperature, light-protected storage. Possessing a single electron, it can accept one electron or a hydrogen ion, exhibiting maximum absorption at 517 nm. In the presence of free radical scavengers, the single electron of DPPH is captured, causing its color to lighten and the absorbance at the maximum absorption wavelength to decrease linearly. This decrease in absorbance indicates increased antioxidant activity, thus evaluating the antioxidant capacity of the test sample. This antioxidant capacity is expressed as an inhibition rate; a higher inhibition rate indicates stronger antioxidant activity.

[0031] The term "tannic acid," also known as tannin, is an organic compound with the chemical formula C63-32 ... 76 H 52 O 46 It is a yellow or brownish-yellow powder. Its aqueous solution turns blue-black upon contact with iron salt solutions; the addition of sodium sulfite delays the color change. It is insoluble in ether, benzene, and chloroform, but readily soluble in water, ethanol, and acetone. Its aqueous solution has an astringent taste. It is not a single compound; its chemical composition is relatively complex, and it can be roughly divided into two types: one is condensed tannin, a flavanol derivative; the other is hydrolyzable tannin, which contains ester bonds in its molecule.

[0032] This application provides a strain of Lactiplantibacillus plantarum PIAS240228, which was deposited at the China Center for Type Culture Collection on April 9, 2024, with accession number CCTCC NO: M2024647, and deposit address: Wuhan University, Wuhan, China.

[0033] This application also provides a microbial agent comprising Lactiplantibacillus plantarum PIAS240228.

[0034] The *Lactobacillus plantarum* PIAS240228 provided in this application exhibits high tanninase activity and a high yield of crude extracellular polysaccharide (EPS). Lactic acid bacteria extracellular polysaccharides possess moisturizing, antioxidant, and anti-aging activities, and also demonstrate strong DPPH free radical scavenging ability. Therefore, the *Lactobacillus plantarum* PIAS240228 of this application is suitable for the cosmetics industry, and the fermentation product of *Lactobacillus plantarum* PIAS240228 can be used as a raw material for cosmetics.

[0035] This application also provides a fermentation product comprising sterilized fermentation filtrate obtained by fermenting plant juice or plant extracts with Lactiplantibacillus plantarum PIAS240228.

[0036] In a specific example, the plant juice or plant extract includes, but is not limited to, one or more of the following: pressed juice or extract of pomegranate, chestnut bark, persimmon fruit, rambutan fruit, and burnet bark;

[0037] Optionally, the pomegranate includes pomegranate seeds or pomegranate peel.

[0038] This application also provides a method for preparing a fermentation product, the method comprising the step of fermentation using the aforementioned Lactiplantibacillus plantarum PIAS240228.

[0039] In a specific example, the method includes: activating Lactiplantibacillus plantarum PIAS240228, inoculating the activated Lactiplantibacillus plantarum PIAS240228 into a solution containing tannin plant extract for fermentation, collecting the fermentation filtrate, inactivating the fermentation broth, filtering and taking the clear liquid to obtain the fermentation product.

[0040] Optionally, the inoculum size of *Lactiplantibacillus plantarum* PIAS240228 in the tannin-containing plant extract is 1.8% (v / v) to 2.2% (v / v). For example, inoculum sizes of 1.8% (v / v), 1.9% (v / v), 2.0% (v / v), 2.1% (v / v), and 2.2% (v / v) are used.

[0041] Optionally, the viable count of *Lactiplantibacillus plantarum* PIAS240228 inoculated into the tannin-containing plant extract is 2–4 × 10⁻⁶. 9 CFU / mL. For example, 2 × 10⁻⁶. 9 CFU / mL, 2.5×10 9 CFU / mL, 3×10 9 CFU / mL, 3.5×10 9 CFU / mL, 4×10 9 CFU / mL.

[0042] Further optionally, the tannin-containing plant extract includes one or more of pomegranate extract, chestnut extract, persimmon extract, rambutan extract, and burnet root extract.

[0043] In a specific example, activating Lactiplantibacillus plantarum PIAS240228 includes the following steps: inoculating Lactiplantibacillus plantarum PIAS240228 into an MRS broth containing 0.8%–1.2% tannic acid; the MRS broth comprises the following components: 8g–12g casein digest, 1.8g–2.2g triammonium citrate, 8g–12g beef extract, 0.15g–0.25g magnesium sulfate, 3g–5g yeast extract, 0.04g–0.06g manganese sulfate, 4g–6g sodium acetate, 1.5g–2.5g dipotassium hydrogen phosphate, 18g–22g glucose, and 1.06g–1.12g Tween 80; the pH of the MRS broth is 5.5–5.9.

[0044] The fermentation conditions include a temperature of 36℃ to 38℃ and a time of 8 to 12 hours. For example, temperatures of 36℃, 37℃, and 38℃, and times of 9 hours, 10 hours, 11 hours, and 12 hours, respectively.

[0045] Optionally, collecting the fermentation filtrate includes a centrifugation step; the centrifugation conditions include: a temperature of 3℃ to 5℃, a rotation speed of 8000×g to 12000×g, and a time of 18min to 22min. For example, temperatures of 3℃, 4℃, and 5℃; rotation speeds of 8000×g, 9000×g, 10000×g, 11000×g, and 12000×g; and times of 18min, 19min, 20min, 21min, and 22min, respectively.

[0046] Another aspect of this application provides the use of the fermentation product prepared by the method of preparing the fermentation product as a cosmetic ingredient.

[0047] This application also provides a cosmetic composition comprising a fermentation product prepared by the method described above, or a formulation of the fermentation product.

[0048] The preparations of the fermentation products include, but are not limited to, freeze-dried fermentation preparations and fermentation concentrates.

[0049] Understandably, in the cosmetics industry, fermented freeze-dried preparations can contain a variety of beneficial microbial metabolites, such as peptides, proteins, vitamins, and antioxidants, which have nourishing and repairing effects on the skin. The application of freeze-drying technology in cosmetics allows these fermented products to exist in the form of dry powder, which can be mixed with solvents (such as water or serum) before use to restore their original activity.

[0050] Fermentation concentrate is a liquid containing the target product produced during fermentation. Through specific techniques such as evaporation, filtration, and membrane separation, the water content is removed, thereby increasing the concentration of the target product. This concentrate can be used in the cosmetics industry.

[0051] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0052] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0053] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0054] Lactobacillus plantarum PIAS240228 has the following characteristics:

[0055] (1) Morphological characteristics: The bacteria are straight or curved rod-shaped, single or sometimes in pairs or chains.

[0056] (2) Colony characteristics: After culturing on MRS solid plates for 24h to 48h, the colonies are smooth, round, milky white or white.

[0057] Opaque, with colony diameters ranging from 0.5 mm to 2 mm.

[0058] The culture media involved in the following examples are as follows: MRS solid medium: 10.0g peptone, 2.0g triammonium citrate, 5.0g beef extract powder, 0.2g magnesium sulfate (MgSO4·7H2O), 4.0g yeast extract powder, 0.05g manganese sulfate (MnSO4·4H2O), 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 20.0g glucose, 1.0mL Tween 80, and 15.0g agar; the pH value of the MRS medium is 6.2±0.2.

[0059] MRS broth: 10.0g casein digest, 2.0g triammonium citrate, 10.0g beef extract, 0.2g magnesium sulfate (MgSO4·7H2O), 4.0g yeast extract, 0.05g manganese sulfate (MnSO4·4H2O), 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 20.0g glucose, 1.08g Tween 80; the pH of the MRS broth culture medium is 5.7±0.2.

[0060] Example 1

[0061] 1.1 Isolation, identification, and observation of Lactobacillus plantarum PIAS240228

[0062] Take 0.5g of samples from fermented foods including kimchi, cheese, butter, and pickled bamboo shoots, and add them to 4.5mL of 0.9% physiological saline for serial dilution. Select appropriate serial dilutions and spread them on MRS solid medium supplemented with 0.2% bromocresol purple. Incubate at 37℃ for 24-48 hours. Select single colonies with obvious yellow discoloration zones and inoculate them onto MRS solid medium for purification. Remove single colonies and transfer them to MRS broth for expansion. Preserve with 30% glycerol to obtain Lactobacillus plantarum strain PIAS240228.

[0063] The whole genome DNA of Lactobacillus plantarum PIAS240228 was extracted for 16S rDNA amplification. The amplified DNA fragments were collected for sequencing (completed by Sangon Biotech (Shanghai) Co., Ltd.). The sequence was compared with the nucleic acid sequence in NCBI. The results showed that the strain was Lactobacillus plantarum and named Lactobacillus plantarum PIAS240228.

[0064] The DNA sequence of Lactiplantibacillus plantarum PIAS240228 is shown below:

[0065] The colony morphology of *Lactiplantibacillus plantarum* PIAS240228 was observed. During the discovery phase, the colonies were round, white, and smooth, as shown in Figure 1. When *Lactiplantibacillus plantarum* PIAS240228 was cultured in MRS broth for 24–48 hours, the fermentation broth exhibited viscous and stringy characteristics.

[0066] 1.2 Comparison of Tanninase Activities among Strains

[0067] The strain was inoculated into MRS broth containing 1% tannins and incubated at 37°C for 24 hours to obtain a liquid culture medium containing tannins. The supernatant was collected by centrifugation, which was the crude enzyme solution for enzyme activity determination. Enzyme activity was determined using Bao Yuxin's tannin method. Three clean test tubes were labeled as blank, test, and control tubes, respectively. The substrate propyl gallate and the crude enzyme solution were preheated in a 30°C water bath for 5-10 minutes before the reaction began. Add 0.25 mL of propyl gallate solution to each of the three labeled test tubes. Then add 0.25 mL of citrate buffer to the blank tube. Add 0.25 mL of crude enzyme solution to the test tube. Incubate all three test tubes in a 30°C water bath for 5 minutes. Next, add 0.3 mL of methanol-to-rhotanine (0.05 mol / L) solution to each test tube and incubate at 30°C for 5 minutes. Afterward, add 0.2 mL of 0.5 mol / L KOH aqueous solution to each test tube and incubate at 30°C for 5 minutes. Then, add 0.25 mL of crude enzyme solution only to the reaction mixture in the control tube. Finally, dilute each test tube with 4 mL of distilled water and incubate at 30°C for 5-10 minutes. Measure the absorbance of the reaction mixture at 520 nm using distilled water as a blank.

[0068] Enzyme activity unit definition: The amount of enzyme required to produce 1 μmol of substrate per minute under the above reaction conditions is defined as one enzyme activity unit (U). Specific activity measurements are shown in Table 1.

[0069] Table 1. Determination of tanninase activity in different strains

[0070] As shown in Table 1, strain *Lactobacillus plantarum* PIAS240228 exhibits relatively high tanninase activity (584.26 U / mL), significantly higher than *Lactobacillus plantarum* PIAS41-3, *Lactobacillus bread-associated* PIAS140-2, *Lactobacillus brevis* PIAS2-1, and *Lactobacillus rhamnosus* CGMCC1.22. Furthermore, compared to other tannin-producing *Lactobacillus plantarum* strains in current related technologies, *Lactobacillus plantarum* PIAS240228 demonstrates significantly higher tanninase activity.

[0071] 1.3 Comparison of differences in tannin metabolism capacity among strains

[0072] Tannic acid can react with Fe 2+ The reaction has a maximum absorption peak at 760 nm, while the hydrolysis of tannic acid to gallic acid does not react with Fe. 2+A colorimetric reaction occurred. The strain was inoculated into MRS broth containing 1% tannic acid and incubated at 37℃ for 24 h. The supernatant was collected by centrifugation. The tannic acid content was relatively quantified by spectrophotometry. 200 μL of sample was diluted with 200 μL of water, and then 400 μL of 1 g / L FeSO4 solution was added for reaction. The absorbance was measured at 760 nm. Different concentrations were diluted using blank medium as a reference for relative quantification. The relative consumption of tannic acid by different strains is shown in Table 2.

[0073] Table 2: Determination of relative tannin consumption by different bacterial strains

[0074] As shown in Table 2, after fermentation in MRS broth containing 1% tannic acid for 24 hours, Lactobacillus plantarum PIAS240228 was able to hydrolyze 99.02% of the tannic acid, and its tannic acid consumption was significantly higher than that of other strains, which also indicates that Lactobacillus plantarum PIAS240228 has high tanninase activity.

[0075] 1.4 Extraction and preparation of crude extracellular polysaccharide (EPS) from Lactobacillus plantarum PIAS240228

[0076] Lactobacillus plantarum PIAS240228 was inoculated into MRS broth and fermented at 37°C for 24 hours. The fermentation broth was then sterilized and incubated with enzymes in a 95°C water bath for 15 minutes. After cooling, the broth was centrifuged at 10000×g for 20 minutes at 4°C to remove the precipitate, yielding the fermentation supernatant. Three volumes of anhydrous ethanol (or 95% ethanol) were added to the supernatant, and the mixture was allowed to stand overnight at 4°C. The supernatant was then centrifuged at 10000×g for 20 minutes at 4°C to obtain the precipitate. The precipitate was dried to constant weight in a vacuum drying oven, weighed, and the result was recorded.

[0077] The yield of crude extracellular polysaccharide EPS from Lactobacillus plantarum PIAS240228 obtained as described above was 1.15 g / L.

[0078] 1.5 Determination of total sugar and protein content of extracellular crude polysaccharide in Lactobacillus plantarum PIAS240228

[0079] The total sugar content of EPS was determined using the sulfuric acid-phenol method. An appropriate amount of *Lactobacillus plantarum* EPS sample was accurately weighed, 1 mL of sterile water was added, and the mixture was centrifuged at 10000 × g for 10 min. The solution was diluted to a specific volume with ddH₂O. 0.1 mL of the supernatant was transferred to a new test tube, mixed with phenol and concentrated sulfuric acid, allowed to react, and allowed to stand for 10 min. The absorbance was then measured at 490 nm.

[0080] The protein content of *Lactobacillus plantarum* EPS was determined using the BCA protein assay kit (Thermo Scientific). The EPS sample was accurately weighed.

[0081] The results showed that the total sugar content of Lactobacillus plantarum EPS was 848.49 mg / g and the protein content was 77.60 mg / g, as detailed in Table 3.

[0082] Table 3. Determination of total sugar and protein content of extracellular crude polysaccharides in Lactobacillus plantarum PIAS240228

[0083] 1.6 Determination of monosaccharide composition of extracellular crude polysaccharide of Lactobacillus plantarum PIAS240228

[0084] Take a clean chromatographic vial, weigh an appropriate amount of *Lactobacillus plantarum* EPS sample, add 1 mL of 2M TFA acid solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Wash with 99.99% methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water, transfer to a chromatographic vial, and proceed with the analysis.

[0085] Monosaccharide components were analyzed and detected using a Thermo ICS 5000+ ion chromatography system (ICS5000+, Thermo Fisher Scientific, USA) with an electrochemical detector. Dionex was used... TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume: 5μL.

[0086] Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5 mL / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0087] The monosaccharide composition of the extracellular crude polysaccharide of Lactobacillus plantarum PIAS240228 was determined and is shown in Table 4.

[0088] Table 4 Monosaccharide composition of extracellular crude polysaccharide of Lactobacillus plantarum PIAS240228

[0089] 1.7 Determination of the ability of extracellular crude polysaccharide from Lactobacillus plantarum PIAS240228 to scavenge DPPH free radicals

[0090] Accurately weigh 0.100 g of EPS sample, add 10 mL of sterile water, mix well, and prepare a 10 g / L EPS solution. Dilute isocratically with ddH₂O to 5 g / L, 2.5 g / L, 1.25 g / L, and 0.625 g / L, and determine the IC₀(t) of DPPH radical scavenging ability. 50 value.

[0091] The ability to scavenge DPPH free radicals was determined using a kit method, and the specific determination method is as follows:

[0092] 1. Preparation of DPPH solution: 2 × 10 -4 Preparation of DPPH ethanol solution (mol / L): Weigh 20 mg DPPH, dissolve it in anhydrous ethanol, and dilute to a volume of 250 mL in a volumetric flask.

[0093] 2. Preparation of test samples: Raw materials should be dissolved and diluted directly with distilled water or anhydrous ethanol; products should be dissolved and diluted with distilled water or anhydrous ethanol, with the concentration that can dissolve in the system reagents being considered the high concentration. The stock solution can be used. Set 3-5 concentration gradients for each test sample.

[0094] 3. Measurement: Mix an equal volume (750 mL) of sample solution with DPPH solution and name the tube A1; mix an equal volume (750 mL) of anhydrous ethanol (or the sample solution solvent) with DPPH solution and name the tube A2; mix an equal volume (750 mL) of sample solution with anhydrous ethanol and name the tube A3; perform three replicates for each sample. After reacting in the dark for 30 min, measure the absorbance of tubes A1, A2, and A3 at 517 nm using a microplate reader.

[0095] 4. Result calculation: Clearance rate (%) = [(A2+A3)-A1] / A2, and the specific results are shown in Figure 2.

[0096] 1.8 Application of Lactobacillus plantarum PIAS230228 in fermentation of tannin-containing plant extracts

[0097] Lactobacillus plantarum PIAS240228 was activated by inoculating it into MRS broth containing 1% tannins. After activation, it was inoculated into an aqueous solution containing tannin plant extract at a 2% (v / v) inoculation rate and fermented at 37°C for 8-12 hours. After fermentation, it was centrifuged at 10,000×g for 20 minutes at 4°C, and the supernatant was retained as the fermentation filtrate of Lactobacillus plantarum PIAS240228.

[0098] The aqueous solution of the plant extract containing tannins contains 10 g / L of plant extract, 15 g / L of fermentation nutrient salt NSO, and 0.05 mol / L of phosphate.

[0099] The fermented nutrient salts include ammonium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, glucose, and yeast extract. The tannin-containing plants include, but are not limited to, one or more combinations of pomegranate, chestnut, persimmon, rambutan, and burnet root.

[0100] Taking pomegranate as an example, relevant measurements were conducted: pomegranate contains a large amount of ellagic tannin, which can be hydrolyzed under the action of tanninase to produce ellagic acid. Therefore, pomegranate extract can be fermented using Lactobacillus plantarum PIAS240228 to prepare a fermentation filtrate rich in ellagic acid, which can be used as a cosmetic raw material.

[0101] Lactobacillus plantarum PIAS240228 was activated by inoculating MRS broth containing 1% tannic acid. After activation, it was inoculated into pomegranate extract aqueous solution at a 2% (v / v) inoculation rate and fermented at 37°C for 48 h. A certain amount of fermentation broth was collected at 0 h, 8 h, 24 h, and 48 h to determine pH, viable bacterial count, and ellagic acid yield.

[0102] The resulting fermentation filtrate can be used as a cosmetic ingredient and applied in the cosmetics industry.

[0103] Ellagic acid yield was determined by high-performance liquid chromatography (HPLC). A Thermo UltiMate 3000 HPLC system (Thermo Fisher Scientific, USA) with an Agilent ZORBAX Eclipse XDB-C18 column (250*4.6mm, 5μm) was used, with an injection volume of 10μL, and ellagic acid was measured at 254nm. Mobile phase A (0.1% trifluoroacetic acid in water), mobile phase B (methanol), flow rate 0.5 mL / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / (95:5, V / V), 10 min A phase / B phase / (80:20, V / V), 25 min A phase / B phase / (55:45, V / V), 35 min A phase / B phase / (35:65, V / V), 40 min A phase / B phase / (95:5, V / V), 45 min A phase / B phase / (95:5, V / V).

[0104] Table 5 shows the changes in pH, viable cell count, and ellagic acid content of pomegranate extract fermented by Lactobacillus plantarum PIAS240228 at different time points.

[0105] Table 5: pH, viable cell count and ellagic acid content of pomegranate extract fermented with Lactobacillus plantarum PIAS240228 at different times

[0106] Table 4 shows that the ellagic acid content increased by 293.82 mg / L from 0h to 8h, indicating that *Lactobacillus plantarum* PIAS240228 hydrolyzed the tannins in pomegranate extract into ellagic acid during fermentation. After 8h, the ellagic acid content gradually decreased, indicating that the strain further converted the ellagic acid into smaller molecules. Therefore, 8h–12h was considered the fermentation endpoint.

[0107] Example 2

[0108] The application of Lactobacillus plantarum PIAS230228 in fermenting tannin-containing plant extracts in cosmetics is understood to include other cosmetic fields, with examples of toners and lotions.

[0109] 2.1 Toner

[0110] Manufacturing process:

[0111] 1. Add A01 to the pot, sprinkle in A02 and disperse evenly, then add A03-A11 to the pot, stir and heat to 85℃, keep warm and stir for 20 minutes until completely dissolved, then vacuum and cool.

[0112] 2. Cool down to 55℃, add the pre-mixed and completely dissolved B-phase raw material, and stir evenly;

[0113] 3. Cool down to below 37℃, add the C phase raw material to the pot, homogenize at 1800rpm for 2 minutes, stir evenly, and degas under vacuum.

[0114] 4. After passing the inspection, the material is discharged through a 300-mesh filter.

[0115] The specific formula is shown in Table 6, wherein the fermentation broth of Lactobacillus plantarum PIAS230228 corresponding to the C01 phase is prepared by fermentation of Lactobacillus plantarum PIAS240228 in one or more embodiments of this application.

[0116] Table 6. Formulas for Toners

[0117] 2.2 Emulsion

[0118] Manufacturing process:

[0119] 1. Add A01 to the pot, sprinkle in A02 and disperse evenly, then add A03-A07 to the pot, stir and heat to 85℃, keep warm and stir for 20 minutes until completely dissolved;

[0120] 2. Accurately weigh the B-phase raw material, stir and heat to 85℃, and stir until uniform;

[0121] 3. Pour phase A into the emulsifying tank, then pour in phase B, homogenize at 85℃ and 2800rpm for 5 minutes, then cool down;

[0122] 4. Cool to 55℃, add the pre-mixed and completely dissolved C-phase raw material, and stir until homogeneous;

[0123] 5. Cool down to below 37℃, add D phase raw material, homogenize at 2500rpm for 2 minutes, stir evenly, and after passing the inspection, filter the material through an 80-mesh filter.

[0124] The specific formula is shown in Table 7, wherein the fermentation broth of Lactobacillus plantarum PIAS230228 corresponding to phase D01 is prepared by fermentation of Lactobacillus plantarum PIAS240228 in one or more embodiments of this application.

[0125] Table 7 Emulsion Formulation

[0126] The embodiments described above merely illustrate several implementation methods of this application to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

[0127] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A species of Lactiplantibacillus plantarum, PIAS240228, was deposited on April 9, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024647, at Wuhan University, Wuhan, China.

2. A microbial agent, characterized in that, The microbial agent includes Lactiplantibacillus plantarum PIAS240228 as described in claim 1.

3. A fermentation product, characterized in that, The fermentation product includes the fermentation filtrate obtained by fermenting plant juice or plant extracts with Lactiplantibacillus plantarum PIAS240228 as described in claim 1.

4. The fermentation product according to claim 3, wherein, The plant juice or plant extract includes one or more of the following: pomegranate, chestnut bark, persimmon fruit, rambutan fruit, and burnet bark. The pomegranate includes pomegranate seeds or pomegranate peel.

5. A method for preparing a fermentation product, characterized in that, The method includes the step of fermentation using Lactiplantibacillus plantarum PIAS240228 as described in claim 1, comprising: The *Lactiplantibacillus plantarum* PIAS240228 was activated; and the activated *Lactiplantibacillus plantarum* PIAS240228 was inoculated into a solution containing tannin plant extract for fermentation. The fermentation filtrate was collected, the fermentation broth was inactivated, filtered, and the clear liquid was collected to obtain the fermentation product.

6. The method for preparing the fermentation product according to claim 5, wherein, The viable count of *Lactiplantibacillus plantarum* PIAS240228 in the tannin-containing plant extract was 2–4 × 10⁻⁴. 9 CFU / mL; The tannin-containing plant extracts include one or more of the following: pomegranate extract, chestnut extract, persimmon extract, rambutan extract, and burnet root extract.

7. The method for preparing the fermentation product according to claim 5, wherein, Activating Lactobacillus plantarum PIAS240228 includes the following steps: Lactobacillus plantarum PIAS240228 was inoculated into MRS broth containing 0.8 w / w% to 1.2 w / w% tannins; The MRS broth comprises the following components: 8g-12g casein digest, 1.8g-2.2g triammonium citrate, 8g-12g beef extract, 0.15g-0.25g magnesium sulfate, 3g-5g yeast extract, 0.04g-0.06g manganese sulfate, 4g-6g sodium acetate, 1.5g-2.5g dipotassium hydrogen phosphate, 18g-22g glucose, and 1.06g-1.12g Tween 80; the pH of the MRS broth is 5.5-5.

9.

8. The method for preparing the fermentation product according to any one of claims 5 to 7, wherein, Fermentation conditions include: a temperature of 36℃~38℃ and a time of 8h~12h; The filtration methods for fermentation filtrate include centrifugation; The centrifugation conditions include: temperature of 3℃~5℃, rotation speed of 8000×g~12000×g, and time of 18min~22min.

9. The fermentation product prepared by the method according to any one of claims 5 to 8 is used as a cosmetic raw material.

10. A cosmetic composition, characterized in that, The composition comprises a fermentation product prepared by the method for preparing the fermentation product according to any one of claims 5 to 8, or a formulation of the fermentation product.

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