Lactobacillus plantarum DH-3 and application thereof

Abstract

The invention discloses lactobacillus plantarum DH-3 and application thereof, and belongs to the technical field of microbial fermentation. The lactobacillus plantarum DH-3 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 11, 2025, and the preservation number is CGMCC No.35560. The lactobacillus plantarum DH-3 is preserved in the China General Microbiological Culture Collection Center (CGMCC). According to the method, lactobacillus plantarum DH-3 is used for carrying out microbial fermentation on roots and stems of asparagus cochinchinensis, a compound enzyme system generated in the growth process of the lactobacillus plantarum DH-3 is used for biologically converting saponin cytotoxic components and decomposing complex insoluble polysaccharide substances into oligosaccharides, so that active secondary metabolites are obtained, meanwhile, active components in the active secondary metabolites are effectively dissolved out, and the activity of asparagus cochinchinensis is improved. The obtained biological fermentation liquor is safe and effective, and has excellent performance in the aspects of resisting skin aging, reducing skin sensitivity and irritation and relieving skin pigmentation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to a strain of Lactobacillus plantarum DH-3 and its applications. Background Technology

[0002] Lactic acid bacteria are internationally recognized as safe microorganisms. The bacteria themselves and their various metabolites have nutritional and immune functions for the body, making them a valuable safe carrier. Using lactic acid bacteria as fermentation agents to increase the nutritional or medicinal value of fermentation substrates is highly significant. Lactic acid bacteria, which do not produce endotoxins, do not contain antibiotic resistance genes, and whose expressed active substances do not require purification, can be directly applied to skin care along with the bacteria.

[0003] Microbial fermentation technology, which represents additive-free and chemical-free residues, aligns with the current natural and environmentally friendly skincare philosophy. The establishment of a skin microecology gives it a greater advantage in the use of effective raw materials.

[0004] Plant secondary metabolites are a variety of small-molecule organic compounds produced by the secondary metabolism of plants, and are an important source of natural bioactive ingredients. Plant secondary metabolites are abundant and diverse in origin, and can be divided into three main categories: nitrogenous compounds, terpenes, and phenols.

[0005] Microbial transformation can synthesize many substances that are difficult to obtain through chemical synthesis. The microbial method for producing plant secondary metabolites is not only specific, fast, and under mild conditions, but also inexpensive and yields high amounts of secondary metabolites, providing a new approach for the large-scale production of plant secondary metabolites.

[0006] To date, scholars both at home and abroad have conducted extensive research on the chemical composition of plants in the genus Asparagus, and have isolated a variety of chemical components from these plants, mainly steroidal saponins and carboxylic lignans, as well as alkaloids, sterols, furfural, flavonoids, anthraquinones, cardiac glycosides, and other components.

[0007] The alkaloids and saponins contained in plant asparagus can be quite irritating to the skin. It is essential to use safe microorganisms to ferment the plant and obtain secondary metabolites, thereby reducing the irritation and toxicity of plant asparagus to the skin. The resulting fermented products are beneficial for skin care and repair. Summary of the Invention

[0008] This invention utilizes *Lactobacillus plantarum* DH-3 to ferment the rhizomes of *Asparagus cochinchinensis*. During the growth process, the complex enzyme system produced by *Lactobacillus plantarum* DH-3 biotransforms alkaloids and saponins into cytotoxic components, decomposes complex insoluble polysaccharides into oligosaccharides, thereby obtaining active secondary metabolites. At the same time, it effectively dissolves the active ingredients, resulting in a safe and effective bio-fermentation broth that exhibits excellent performance in combating skin aging, reducing skin sensitivity and irritation, and alleviating skin pigmentation.

[0009] This invention provides a strain of Lactobacillus plantarum DH-3, which has the accession number CGMCC No. 35560.

[0010] The present invention also provides the application of the above-mentioned Lactobacillus plantarum DH-3 in the preparation of asparagus fermentation broth, including fermentation of the above-mentioned Lactobacillus plantarum DH-3 in a culture medium containing asparagus aqueous extract.

[0011] Preferably, the culture medium containing asparagus aqueous extract contains 0.3-0.5 wt% yeast extract, 0.1-0.3 wt% soybean peptone, 0.05-0.15 wt% KH2PO4, 0.005-0.015 wt% MnSO4·H2O, 0.05-0.07 wt% MgSO4·7H2O, 0.04-0.06 wt% TW-80, and 1-3 wt% glucose, and is prepared using asparagus aqueous extract instead of water.

[0012] More preferably, the culture medium containing asparagus aqueous extract contains 0.4 wt% yeast extract, 0.2 wt% soybean peptone, 0.1 wt% KH2PO4, 0.01 wt% MnSO4·H2O, 0.06 wt% MgSO4·7H2O, 0.05 wt% TW-80, and 2 wt% glucose, and is prepared using asparagus aqueous extract instead of water.

[0013] More preferably, the preparation method of the asparagus aqueous extract is as follows: add dried asparagus to deionized water at a mass ratio of 1:10, heat to 50°C and soak for 6 hours, crush the asparagus, add deionized water to a mass ratio of 1:14, stir slowly and heat to 80°C, keep warm for 2 hours, centrifuge with a filter bag to remove residue, and collect the filtrate to obtain the asparagus aqueous extract.

[0014] More preferably, it includes the following steps: (1) Seed culture Lactobacillus plantarum DH-3 was inoculated into MRS liquid medium, filling the bottle completely, sealing it, and incubating it statically at 37°C for 24 hours, then at 40°C for 6-8 hours. The OD of the seed culture was then measured. 600 =2~4, to obtain the primary culture seed solution; (2) Anaerobic fermentation The primary culture seed liquid from step (1) was inoculated into a culture medium containing asparagus aqueous extract, stirred, and then subjected to anaerobic fermentation. During the fermentation process, the pH of the fermentation system was maintained at 3-4, and the fermentation was carried out at a constant temperature of 38°C for 120 hours.

[0015] More preferably, in step (2), the primary culture seed solution is inoculated into the culture medium containing asparagus aqueous extract at an inoculation rate of 5 wt%.

[0016] More preferably, it also includes step (3) heating the fermentation broth to 80°C in place and holding it for 2 hours.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) Lactobacillus plantarum DH-3 has high acid resistance. This type of lactobacillus has a safe probiotic effect and belongs to the category of typical probiotics. It does not harm the human intestines and mucosa.

[0018] (2) The fermentation product of Lactobacillus plantarum DH-3 asparagus can regulate the microecological balance of skin and mucous membrane, inhibit the production of harmful free radicals in the body, reduce cellular oxidative stress and inflammatory response, thereby slowing down the aging process.

[0019] (3) Fermentation with Lactobacillus plantarum DH-3 can safely and effectively increase the active ingredients in asparagus plant cells. The resulting bio-fermentation liquid is safe and effective, and has excellent performance in combating skin aging, reducing skin sensitivity and irritation, and alleviating skin pigmentation.

[0020] (4) The fermentation of asparagus by Lactobacillus plantarum DH-3 strain can produce extracellular polysaccharides and flavonoid active substances with diverse structures. These compounds play an important role in regulating host health and treating diseases, and are expected to be developed into health food in the future.

[0021] (5) Bio-fermentation and extraction by Lactobacillus plantarum DH-3 strain can effectively avoid the use of organic reagents, make high-efficiency use of raw materials such as asparagus, fermentation starts from probiotics, and the active substances obtained by fermentation can be widely used in the food and daily chemical fields; the residue obtained by fermentation is rich in nutrients and can be used as organic feed and fertilizer in the breeding and planting industries.

[0022] (6) The complex enzyme system produced during the growth of Lactobacillus plantarum can effectively transform saponin cytotoxic components from asparagus tubers and decompose some complex insoluble heteropolysaccharides into oligosaccharides, thereby obtaining secondary metabolites.

[0023] Biological preservation instructions for Lactobacillus plantarum DH-3: Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; Accession number: CGMCC No. 35560; Deposit date: August 11, 2025; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Taxonomic name: Lactobacillus plantarum. Attached Figure Description

[0024] Figure 1 This is the phylogenetic tree of the DH-3 strain in Example 1.

[0025] Figure 2 The image shows a high-performance liquid chromatogram of the fermentation product of Lactobacillus plantarum DH-3 asparagus in Example 4.

[0026] Figure 3 This is a high-performance liquid chromatogram of the asparagus aqueous extract in Example 4.

[0027] Figure 4 The results are the determination results of DPPH scavenging rate in Example 5.

[0028] Figure 5 The results are the determination results of hydroxyl radical scavenging rate in Example 5.

[0029] Figure 6 The results are the determination results of ABTS free radical scavenging rate in Example 5.

[0030] Figure 7 The effect of different concentrations of Lactobacillus plantarum DH-3 asparagus fermentation products on hyaluronidase in Example 5 is shown.

[0031] Figure 8 This is the photoaging repair of HSF cells after 24 hours of treatment with different concentrations of Lactobacillus plantarum DH-3 fermentation products in Example 5. Detailed Implementation

[0032] Example 1 The inventor isolated a type of Lactobacillus plantarum from kimchi, and the specific process is as follows: (1) Selection of strains: Fresh asparagus was pulped with deionized water at 20wt%, and the filtered juice was adjusted to pH 2.0 with lactic acid and sterilized at 115℃ for 30min; Sichuan pickled vegetable juice from Qingcheng Mountain Taoist Canteen in Sichuan was inoculated at 10% (v / v), sealed and anaerobic at 37℃ for 72 hours, and the culture was inoculated into MRS agar medium (containing the following mass fractions: glucose 2%, peptone 2%, yeast extract 0.5%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, ammonium sulfate 0.2%, magnesium sulfate 0.1%, manganese sulfate 0.005%, Tween 80 0.1%, and agar powder 2%); double layer flat After anaerobic incubation at 37°C for 72 hours, five single colonies with good growth were collected and inoculated into MRS liquid medium (containing the following mass fractions: glucose 2%, peptone 2%, yeast extract 0.5%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, ammonium sulfate 0.2%, magnesium sulfate 0.1%, manganese sulfate 0.005%, and Tween 80 0.1%) in test tubes; the tubes were sealed with liquid paraffin and anaerobic incubated at 37°C for 24 hours. 30% glycerol cultures were then prepared and stored at -80°C. The strain with the best growth was ultimately selected as the strain for this patent.

[0033] (2) Strain identification: A single colony of DH-3 was streaked onto MRS agar medium. After the strain grew, Gram staining was performed to identify the strain. The LAB-specific biochemical tubes were used to perform the catalase experiment and to detect the 16S rRNA gene sequence and perform phylogenetic analysis on the strain. The 16S rRNA gene sequence was sequenced by PCR and its reaction products, and then aligned by BLAST. Phylogenetic analysis was performed using the aligned sequences of the 16S rRNA genes of *Lactobacillus plantarum* W9-1, *L. plantarum* FRT7, *Lacticaseibacillus zeae* 2985, *Lacticaseibacillus casei* JCM1134, *Pediococcus acidilactici* DSM20284, *Bacillus velezensis* K-XZ8, and *Pseudomonas aeruginosa* PAO1 strains. The results are as follows: Figure 1 As shown, by Figure 1 It can be seen that strain DH-3 is most closely related to strains L. plantarum W9-1 and L. plantarum FRT7, and the homology is as high as 100%, so strain DH-3 can be identified as L. plantarum.

[0034] Example 2 This embodiment utilizes Lactobacillus plantarum DH-3 isolated in Example 1 for fermentation. The specific process is as follows: 1. Raw material processing Asparagus (dried) is added to deionized water at a mass ratio of 1:10, heated to 50℃ and soaked for 6 hours, wet crushed, deionized water is added to bring the mass ratio to 1:14, slowly stirred and heated to 80℃ and kept warm for 2 hours, centrifuged with a filter bag to remove residue, and the filtrate is collected to obtain asparagus aqueous extract.

[0035] 2. Seed culture medium MRS liquid culture medium, sterilized at 115°C for 30 min.

[0036] 3. Fermentation medium The fermentation medium contains the following components by mass fraction: yeast extract 0.4%, soybean peptone 0.2%, KH2PO4 0.1%, MnSO4·H2O 0.01%, MgSO4·7H2O 0.06%, TW-80 0.05%, and glucose 2%. It is prepared using asparagus extract instead of water. The fermenter is sterilized in place at 115℃ for 30 minutes. Nitrogen gas is introduced during the sterilization cooling process, and the temperature is kept constant at 38℃ and pressure is maintained at 0.2MPa.

[0037] 4. Fermentation process control (1) Seed culture Inoculate MRS liquid medium with 0.1 wt% of *Lactobacillus plantarum* DH-3 glycerol seed culture, filling the bottle completely, seal, and incubate statically at 37°C for 24 hours, then incubate statically at 40°C for 6-8 hours. OD of the seed culture is then measured. 600 =2~4, no abnormalities were found under microscopic examination, and primary culture seed solution was obtained.

[0038] (2) Anaerobic culture in fermenter The primary culture seed solution from step (1) was inoculated into a fermenter containing fermentation medium at a 5 wt% inoculation rate. The mixture was stirred at 100 rpm, and nitrogen gas was continuously purged for 10 min. Gas purging was then stopped, and the pressure was maintained at 0.2 MPa. The pH of the fermentation system was adjusted using 4M NaOH to maintain it at pH 3.5. Fermentation was carried out at a constant temperature of 38℃ for 120 hours, after which fermentation was stopped. The fermentation broth was then heated to 80℃ and held for 2 hours before being transferred to a fermentation tank to obtain the sterilized asparagus fermentation broth.

[0039] 5. Purification process The asparagus fermentation sterilization broth obtained in step 4 was filtered at low temperature. The liquid portion was collected and sterilized by filtration through a 0.22µm cross-flow membrane. 20% by mass of butanediol was added to the filtrate to obtain the *Lactobacillus plantarum* DH-3 asparagus fermentation product.

[0040] Example 3 In this embodiment, Lactobacillus plantarum DH-3 isolated in Example 1 is used for fermentation. The specific process is carried out according to Example 2. Water is used instead of asparagus extract to prepare the fermentation culture medium. Fermentation and purification treatment yields the fermentation product of Lactobacillus plantarum DH-3.

[0041] Example 4 This embodiment analyzes the polysaccharides and flavonoids in the *Lactobacillus plantarum* DH-3 asparagus fermentation product, asparagus aqueous extract, and *Lactobacillus plantarum* DH-3 fermentation product obtained in Examples 2 and 3. The specific process is as follows: (1) Detection of total polysaccharide content To construct a standard curve: Accurately weigh 10 mg of D-anhydrous glucose standard, dissolve it in water, and then transfer it to a 25 mL volumetric flask and dilute to volume. This is the reference solution. Accurately pipette 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the anhydrous glucose reference solution into stoppered test tubes, add water to a final volume of 2 mL, slowly add 8 mL of 0.2% anthrone-sulfuric acid solution, mix well, and incubate in a boiling water bath for 10 min. Remove and cool in an ice-water bath for 10 min. The blank control is water. Measure the absorbance of the solution at 576 nm using UV-Vis spectrophotometry. Plot the standard curve with absorbance as the ordinate and the mass of anhydrous glucose as the abscissa to obtain the regression equation.

[0042] Sample solution detection: Accurately measure 0.5 mL of the sample to be tested, add water to 2 mL, and perform color development and determination according to the method in the standard curve plotting section. Calculate the total asparagus polysaccharide content in each sample using the reference standard. See the table below: Table 1

[0043] (2) Detection of total flavonoid content To construct a standard curve: Accurately weigh 50 mg of rutin standard, dissolve it in 60% ethanol, and dilute to 100 mL (concentration 0.5 mg / mL). Take six 25 mL colorimetric tubes and add 0, 0.5, 1.0, 2.0, 3.0, and 4.0 mL of rutin standard solution, respectively, and then add 60% ethanol to a final volume of 5 mL. Add 0.3 mL of 5% sodium nitrite solution sequentially, shake well, and let stand for 6 min; add 0.3 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min; add 4 mL of 4% sodium hydroxide solution, and dilute to the mark with 60% ethanol, shake well, and let stand for 15 min. Using a reagent blank as a reference, measure the absorbance at a wavelength of 510 nm. Plot a standard curve with absorbance as the ordinate and rutin mass as the abscissa to obtain the regression equation.

[0044] Sample solution detection: Accurately measure 0.5 mL of the sample to be tested, add water to 2 mL, and perform color development and determination according to the method in the standard curve plotting section. Calculate the content of total asparagus flavonoids in each sample using the reference standard. See the table below: Table 2

[0045] (3) High performance liquid chromatography detection Sample preparation: Accurately measure 1 mL of the sample to be tested, add methanol to 4 mL, incubate at 4℃ for 10 hours, centrifuge at 11000 rpm for 10 min, collect the supernatant and filter through a 0.22 µm microporous membrane, and perform HPLC analysis using the area normalization method. The detection conditions are as follows: Chromatographic column: Shimadzu C18 silica gel column (250mm×4.6mm, 5µm); Mobile phase: methanol: 0.1% phosphoric acid solution (30:70); Flow rate: 1.0 mL / min; column temperature: 30℃; injection volume: 10 μL; detection wavelength: 254 nm; isocratic elution.

[0046] The analysis results are shown in the figure. Figure 2 It is a fermentation product of Lactobacillus plantarum DH-3 asparagus. Figure 3 It is an aqueous extract of asparagus.

[0047] The above three tests clearly show that the content of polysaccharides and flavonoids in the asparagus water extract increased significantly after fermentation with Lactobacillus plantarum DH-3, and new flavonoids were produced.

[0048] Example 5 Efficacy test (1) DPPH scavenging rate test 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical. Its ethanol solution is deep purple and exhibits strong absorption around 517 nm. In the presence of free radical scavengers, the light absorption of the DPPH ethanol solution decreases due to the pairing of unpaired electrons with the free radical. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of a test sample to scavenge free radicals, i.e., the magnitude of its antioxidant activity.

[0049] The positive control was dissolved and diluted with 95% ethanol to prepare a series of concentration gradients of 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL to verify the test system.

[0050] Test substance preparation: Dilute the sample with deionized water to prepare samples of multiple concentrations.

[0051] Referring to Table 3, 10mL test tubes are used to set up sample tubes (T), sample background (T0), DPPH tubes (C) and solvent background (C0). For each sample, three parallel tubes are required for each test concentration of the sample tube (T), and three parallel tubes are also required for the DPPH tube (C).

[0052] Add 1 mL of sample solution of the same concentration to both the sample tube (T) and the sample background (T0).

[0053] Add 3 mL of deionized water to all test tubes (T, T0, C, C0) and mix well.

[0054] Add 1 mL of DPPH ethanol solution to the sample tube (T) and DPPH tube (C). Replace the sample background (T0) and solvent background (C0) with 95% ethanol. Shake gently and let stand at room temperature for 5 minutes.

[0055] Transfer each reaction solution into a 1 cm cuvette and measure the absorbance at 517 nm.

[0056] Table 3 Sample Addition Requirements

[0057] Calculate DPPH radical scavenging rate: ; Where: T—sample tube absorbance, i.e., the absorbance of the solution after the sample reacts with DPPH; T0—sample background absorbance; C—the average of three DPPH tube absorbance values, i.e., the absorbance of the DPPH solution without the sample; C0—solvent background absorbance.

[0058] Table 4 DPPH removal rate

[0059] DPPH scavenging rate test results: All three tested samples showed DPPH free radical scavenging activity with a dose-response effect (P<0.05); among them, the fermentation product of *Lactobacillus plantarum* DH-3 asparagus showed extremely strong DPPH free radical scavenging activity (see...). Figure 4 ).

[0060] (2) Hydroxyl radical scavenging test The Fenton reaction generates hydroxyl radicals, which react with salicylic acid to form the colored compound 2,3-dihydroxybenzoic acid. The amount of hydroxyl radicals is reflected by measuring the absorbance of this compound.

[0061] Reagent preparation: Prepare 1 mmol / L ferrous sulfate solution, 3 mmol / L H2O2 solution, and 3 mmol / L salicylic acid solution.

[0062] Experimental procedure: Add 5 mL of ferrous sulfate solution, 5 mL of H₂O₂, 1 mL of deionized water, and 1 mL of sample solution to a 25 mL colorimetric tube, and then bring the volume to 25 mL with salicylic acid solution. After mixing thoroughly, react in a 37℃ constant temperature water bath for 15 min, and then measure the absorbance at a wavelength of 510 nm using a spectrophotometer.

[0063] Calculation formula: Hydroxyl radical scavenging rate (%) = (A-(A-A0)) / A×100%, where A is the absorbance of the blank control, A0 is the absorbance of the sample, and A0 is the absorbance of the sample without the colorimetric reagent.

[0064] Table 5 Hydroxyl radical scavenging rate

[0065] Results of hydroxyl radical scavenging assay: All three tested samples showed hydroxyl radical scavenging activity with a dose-response effect (P<0.05). However, the fermentation product of *Lactobacillus plantarum* DH-3 asparagus exhibited significantly stronger scavenging ability at the same dose, demonstrating significant hydroxyl radical scavenging activity (see [link to relevant documentation]). Figure 5 ).

[0066] (3) ABTS free radical scavenging ability test The ABTS radical scavenging ability assay is a commonly used method for evaluating antioxidant activity. This method is based on the fact that ABTS radicals are reduced to alcohols under visible light, generating green ABTS+ ions. Antioxidants can scavenge ABTS radicals by inhibiting or slowing down the formation of ABTS+ ions, thus exhibiting antioxidant activity.

[0067] In this experiment, after ABTS free radicals are generated, the antioxidant sample reacts with them. By measuring the change in absorbance of ABTS before and after the reaction, the scavenging rate of the sample on ABTS free radicals can be calculated.

[0068] Experimental steps a. Reagent preparation: ABTS stock solution (7.4mM): Take 3 mg of ABTS and add 1.48 mL of distilled water.

[0069] K2S2O8 stock solution (2.6mM): Take 2 mg of K2S2O8 and add 2.8 mL of distilled water.

[0070] pH 7.4 Phosphate buffer: Prepared according to the requirements of the 2025 edition of the Chinese Pharmacopoeia.

[0071] ABTS working solution: Take 0.2 mL of ABTS stock solution and 0.2 mL of K2S2O8, mix them, and let them stand in the dark at room temperature for 12 hours. Dilute with pH 7.4 phosphate buffer 40-50 times, take 80 μL of this solution and mix it with 20 μL of 95% ethanol, and measure the absorbance at 734 nm. The absorbance should be 0.3 ± 0.01.

[0072] Sample solution: Dilute with deionized water to the concentration of the test sample and mix thoroughly.

[0073] ABTS free radical scavenging rate = (1-A / A0)*100%.

[0074] b. Measure absorbance: Blank control absorbance determination: Use a pipette to add 800 μL of ABTS working solution to the test tube, add 200 μL of 95% ethanol solution, shake for 10 seconds to mix thoroughly, let stand for 6 minutes, and then measure the A value at 734 nm to obtain the A0 value.

[0075] Sample absorbance measurement: Use a pipette to add 800 μL of ABTS working solution to the test tube, add 200 μL of sample solution, shake for 10 seconds to mix thoroughly, let stand for 6 minutes, and then measure the A value at 734 nm to obtain the A value.

[0076] c. Data processing: Calculate the scavenging rate of ABTS radicals by the sample based on the change in absorbance.

[0077] Results Analysis Record the clearance rate of each sample at different concentrations.

[0078] Table 6 ABTS free radical scavenging rate (%)

[0079] Results of ABTS free radical scavenging experiment: All three tested samples showed ABTS free radical scavenging activity with a dose-response effect (P<0.05), but the fermentation product of *Lactobacillus plantarum* DH-3 asparagus showed significantly stronger scavenging ability at the same dose, indicating significant ABTS free radical scavenging activity (see [link to relevant documentation]). Figure 6 ).

[0080] (4) Tyrosinase activity inhibition test Tyrosinase is widely found in microorganisms, animals, plants, and humans. It is a crucial rate-limiting enzyme involved in melanin production, and its expression level and activity determine the rate and yield of melanin synthesis. Inhibiting tyrosinase activity can improve tyrosinase metabolism in skin pigment cells and prevent the formation of pigmentation.

[0081] Add 1.5 mL of phosphate pH 6.8 buffer solution, 1 mL of sample (blank) solution, and 1 mL of tyrosine solution to a test tube in sequence, and incubate at 35 °C for 10 minutes. Then add 500 μL of tyrosinase solution, mix well, and incubate at 35 °C for 10 minutes. Quickly transfer the solution to a cuvette and measure the absorbance at 475 nm.

[0082] The absorbance value of the test group was A1, and the absorbance value of the blank control group was A1. 空 .

[0083] Inhibition rate = (A) 空 -A1) / A 空 ×100%.

[0084] Table 7 Tyrosinase activity inhibition rate

[0085] The test results of the tyrosinase inhibition ability of different concentrations of fermentation products of Lactobacillus plantarum DH-3 asparagus are shown in Table 7. It can be seen that the complex polyphenolic structure in the fermentation products of Lactobacillus plantarum DH-3 asparagus can significantly reduce the melanin intermediate product dopa and inhibit the oxidation process of melanin. Its antioxidant properties enable it to inhibit the synthesis of melanin by quenching free radicals and peroxides.

[0086] (6) Inflammatory factor inhibition test Experimental Methods: RAW264.7 macrophages were used as the research object. A cellular inflammation model was established by stimulating cells with lipopolysaccharide (LPS, bacterial endotoxin). Positive controls were obtained by adding 10 wt% *Lactobacillus plantarum* DH-3 asparagus fermentation product, 10 wt% asparagus aqueous extract, 10 wt% *Lactobacillus plantarum* DH-3 fermentation product, and 0.5 wt% quercetin. After culturing for 24 hours, cell supernatants were collected. Finally, the release level of the pro-inflammatory inflammatory factor TNF-α from RAW264.7 macrophages was analyzed using an ELISA kit.

[0087] Table 8

[0088] TNF-α is a pleiotropic pro-inflammatory cytokine belonging to the TNF ligand superfamily. TNF-α plays different roles in regulating various developmental and immune processes, including inflammation, differentiation, lipid metabolism, and apoptosis, and is associated with a variety of diseases. The fermentation product of *Lactobacillus plantarum* DH-3 asparagus significantly inhibited the release of TNF-α. The 10% fermentation product of *Lactobacillus plantarum* DH-3 asparagus showed a higher inhibitory effect on inflammatory factors than the 0.5% quercetin group, and this effect was concentration-dependent.

[0089] (7) Experiment on promoting the repair of damaged HaCaT cells Seed HaCaT cells (5×10) 4 Cells / well were added to 96-well plates and cultured overnight until cell adhesion occurred. 100 μl of 50 μg / ml SDS was added to each well, and the plates were incubated at 37°C for 8 h with 5% CO2. 10 wt% of *Lactobacillus plantarum* DH-3 asparagus fermentation product, asparagus aqueous extract, or *Lactobacillus plantarum* DH-3 fermentation product were added to each well, respectively. The control group was incubated with an equal volume of PBS for 24 h. 10 μl of CCK-8 solution was added to each well, and the plates were incubated for 4 h. The absorbance (A) at 450 nm was measured.

[0090] The formula for calculating cell proliferation rate is: Cell proliferation rate = (Experimental group A - Control group A) / Control group A * 100%.

[0091] Table 9

[0092] HaCaT cells are immortalized human epidermal cells, a non-tumor-derived immortalized keratinocyte line from normal human skin. They exhibit similar differentiation characteristics to normal human keratinocytes and can proliferate for over 150 generations. The fermentation product of *Lactobacillus plantarum* DH-3 asparagus significantly promoted the repair of SDS-induced HaCaT keratinocyte damage, increasing cell proliferation rate by 16.97%, thus demonstrating that the fermentation product of *Lactobacillus plantarum* DH-3 asparagus possesses excellent barrier repair function.

[0093] (8) Evaluation of anti-allergy and soothing effects Hyaluronidase is involved in type I hypersensitivity reactions. Hyaluronidase is strongly correlated with inflammation and allergies. Studies have reported that various drugs that release histamine from mast cells can regulate hyaluronidase activity. Some anti-allergy drugs have strong inhibitory effects on hyaluronidase activity. Therefore, inhibiting hyaluronidase activity is used as an indicator for studying soothing effects.

[0094] Assay method: Take 0.1 mL of 0.25 mM CaCl2 solution and 0.5 mL of hyaluronidase solution and incubate at 37℃ for 20 min; add 0.5 mL of each concentration of Lactobacillus plantarum DH-3 asparagus fermentation product sample solution and incubate at 37℃ for 20 min; add 0.5 mL of sodium hyaluronate solution and incubate at 37℃ for 30 min, then let stand at room temperature for 5 min; add 0.1 mL of 0.4 mol / L NaOH solution and 0.5 mL of acetylacetone solution, heat in a boiling water bath for 30 min, and immediately cool on ice for 5 min; add 1.0 mL of Ehrlich reagent and dilute with 3.0 mL of anhydrous ethanol, let stand for 20 min for color development; measure the absorbance value at 547 nm using a spectrophotometer. Formula for calculating hyaluronidase activity inhibition rate (%): Hyaluronidase activity inhibition rate (%) = [(AB) - (CD)] / (AB) × 100% In the formula: A—Absorbance value of the control solution; B—Absorbance value of blank control solution; C—Absorbance value of the sample solution; D—Absorbance value of the blank sample solution Table 10

[0095] Data Records Table 11

[0096] The fermentation product of *Lactobacillus plantarum* DH-3 asparagus exhibits a strong inhibitory effect on hyaluronidase, with the inhibitory effect increasing with increasing concentration. A 5.7% concentration (by weight) inhibits half of hyaluronidase activity, demonstrating a very significant soothing effect. A 15% concentration (by weight) completely inhibits hyaluronidase activity (see [link to product description]). Figure 7 ).

[0097] (9) Photoaging repair function This experiment used HSF cells as the research target and employed the MTT assay to detect 90 mJ / cm² cells. 2 The photoaging repair effect of HSF cells exposed to UVB irradiation after 24 h of treatment with samples of different concentrations of Lactobacillus plantarum DH-3 asparagus fermentation products (see [reference]). Figure 8 ).

[0098] Depend on Figure 8 The cell morphology results showed that HSF cells exhibited good repair effects on cellular senescence after 24 h of intervention with different concentrations of *Lactobacillus plantarum* DH-3 asparagus fermentation product samples. Compared with the blank irradiation group, the repair capacity of each concentration of samples was significantly demonstrated, and the repair level had a clear linear relationship with the sample concentration. The fibrous morphology of cells in the 50% *Lactobacillus plantarum* DH-3 asparagus fermentation product sample group was the most obvious.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Lactobacillus plantarum DH-3, characterized in that, The preservation number of the Lactobacillus plantarum DH-3 is CGMCC No. 35560.

2. The application of Lactobacillus plantarum DH-3 as described in claim 1 in the preparation of asparagus fermentation broth, characterized in that, This includes fermenting the Lactobacillus plantarum DH-3 in a culture medium containing an asparagus aqueous extract.

3. The application according to claim 2, characterized in that, The culture medium containing asparagus aqueous extract contains 0.3-0.5 wt% yeast extract, 0.1-0.3 wt% soybean peptone, 0.05-0.15 wt% KH2PO4, 0.005-0.015 wt% MnSO4·H2O, 0.05-0.07 wt% MgSO4·7H2O, 0.04-0.06 wt% TW-80, and 1-3 wt% glucose, and is prepared using asparagus aqueous extract instead of water.

4. The application according to claim 3, characterized in that, The culture medium containing asparagus aqueous extract contains 0.4 wt% yeast extract, 0.2 wt% soybean peptone, 0.1 wt% KH2PO4, 0.01 wt% MnSO4·H2O, 0.06 wt% MgSO4·7H2O, 0.05 wt% TW-80, and 2 wt% glucose, and is prepared using asparagus aqueous extract instead of water.

5. The application according to claim 4, characterized in that, The preparation method of the asparagus aqueous extract is as follows: add dried asparagus to deionized water at a mass ratio of 1:10, heat to 50°C and soak for 6 hours, crush the asparagus, add deionized water to a mass ratio of 1:14, stir slowly and heat to 80°C, keep warm for 2 hours, centrifuge with a filter bag to remove residue, and collect the filtrate to obtain the asparagus aqueous extract.

6. The application according to any one of claims 2 to 5, characterized in that, Includes the following steps: (1) Seed culture Lactobacillus plantarum DH-3 was inoculated into MRS liquid medium, filling the bottle completely, sealing it, and incubating it statically at 37°C for 24 hours, then at 40°C for 6-8 hours. The OD of the seed culture was then measured. 600 =2~4, to obtain the primary culture seed solution; (2) Anaerobic fermentation The primary culture seed liquid from step (1) was inoculated into a culture medium containing asparagus aqueous extract, stirred, and then subjected to anaerobic fermentation. During the fermentation process, the pH of the fermentation system was maintained at 3-4, and the fermentation was carried out at a constant temperature of 38°C for 120 hours.

7. The application according to claim 6, characterized in that, In step (2), the primary culture seed solution is inoculated into the culture medium containing asparagus aqueous extract at an inoculation rate of 5 wt%.

8. The application according to claim 7, characterized in that, It also includes step (3) heating the fermentation broth to 80°C and holding it for 2 hours.

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