Lactobacillus plantarum for preparing roxburgh rose fermentation liquor and application of lactobacillus plantarum

Abstract

The invention provides lactobacillus plantarum for preparing roxburgh rose fermentation liquor and application of the lactobacillus plantarum, lactobacillus plantarum GL-1 is obtained through self-screening, and the preservation number of the lactobacillus plantarum GL-1 is GDMCC NO: 66711. The metabolite of the strain can significantly improve the activity of SOD in HaCaT cells; the strain has better activity in roxburgh rose pulp at 15 DEG C, and the SOD activity, the flavonoid compound content and the phenolic compound content in a roxburgh rose fermented product obtained by fermenting the roxburgh rose pulp with the strain are remarkably improved; the obtained rosa roxburghii tratt leavening has a remarkable anti-oxidation effect and an inhibition effect on tyrosinase activity, and can be applied to products with anti-oxidation and whitening effects.

Description

Technical Field

[0001] This invention relates to a novel Lactobacillus plantarum GL-1, and also to the application of this Lactobacillus plantarum GL-1 in the preparation of fermented products, especially in the preparation of prickly pear fermentation products, belonging to the field of biofermentation technology. Background Technology

[0002] Rosa roxburghii is a wild fruit with extremely high nutritional and medicinal value, mainly distributed in southwestern China, especially the Guizhou Plateau. This Rosaceae plant's fruit is rich in various bioactive components, including superoxide dismutase (SOD), flavonoids, polyphenols, and vitamin C, making it a promising candidate for applications in health products, cosmetics, and pharmaceuticals.

[0003] Although the various bioactive components in prickly pear exhibit excellent physiological functions, their stability is significantly affected by a variety of environmental factors, which has become a key technical challenge restricting the processing and storage of prickly pear.

[0004] For example, superoxide dismutase (SOD) in prickly pear begins to undergo irreversible denaturation at temperatures exceeding 40°C, resulting in a rapid decrease in activity. Studies have shown that the optimal activity temperature range for SOD is 4-40°C. Within this range, the enzyme's molecular structure remains stable, maintaining high catalytic activity. When the temperature rises above 45°C, SOD begins to inactivate; while low-temperature environments (below 4°C) reduce enzyme activity but do not damage its structure, and activity can be partially restored after rewarming. This thermal instability mainly stems from the protein-like nature of SOD—high temperatures cause the protein's three-dimensional structure to unfold, disrupting the coordination environment of metal ions at the active site, thereby losing its catalytic function.

[0005] While prickly pear flavonoids exhibit relatively good temperature tolerance, prolonged exposure to high temperatures still leads to significant degradation. Studies have found that prickly pear flavonoids are relatively stable below 50°C, but their content decreases significantly with prolonged treatment time when the temperature rises above 80°C. This is mainly because high temperatures accelerate the hydrolysis of flavonoid glycosidic bonds and the oxidative decomposition of the parent nucleus structure. Particularly under aerobic conditions, high temperatures promote the auto-oxidation and polymerization of flavonoids, not only reducing their content but also potentially altering their biological activity.

[0006] The thermal stability of prickly pear polyphenols is similar to that of flavonoids; short periods of high temperature (such as a few minutes at 100°C) have little effect, but prolonged heating leads to significant losses. The degradation mechanisms of polyphenols include oxidation, polymerization, and hydrolysis. Notably, vitamin C in prickly pear is readily oxidized and decomposed at high temperatures, and the loss of vitamin C indirectly accelerates the oxidation of polyphenols.

[0007] For the reasons mentioned above, traditional extraction methods such as alcohol extraction and water extraction require prolonged extraction under specific temperature conditions, which can damage the active ingredients to some extent. Furthermore, both alcohol and water extraction yields relatively low amounts of active substances from prickly pear. Microbial fermentation extraction can significantly improve the yield of active substances in prickly pear. Microbial fermentation technology, due to its mild reaction conditions and efficient biotransformation capabilities, exhibits unique advantages in the field of plant active ingredient extraction. Compared to traditional thermal extraction methods, microbial fermentation can release bound active substances through enzymatic hydrolysis at room temperature or low temperature, while simultaneously generating new bioactive components. Low-temperature fermentation not only avoids SOD inactivation and flavonoid degradation during fermentation but also effectively disrupts plant cell wall structures through cellulase and pectinase produced by microbial metabolism, significantly improving the dissolution rate of active substances.

[0008] However, the key to achieving efficient low-temperature fermentation of prickly pear lies in obtaining microbial strains adapted to a specific fermentation system. Prickly pear pulp has three significant characteristics: firstly, a low pH (2.8-3.5); secondly, it contains abundant antibacterial components such as tannins; and thirdly, a low-temperature environment (10-20℃) inhibits the metabolic activity of most strains. Conventional fermentation strains such as yeast (Saccharomyces cerevisiae) and lactic acid bacteria (Lactobacillus spp.) often experience limited growth and insufficient enzyme secretion in this environment. Therefore, screening for a strain with fermentation activity in low-temperature prickly pear pulp is particularly important. This strain must simultaneously possess the following characteristics: (1) acid resistance, maintaining metabolic activity in an environment with pH < 3.5; (2) low-temperature adaptability, still efficiently secreting cell wall degrading enzymes at 10-20℃; (3) tolerance to the natural antibacterial components in prickly pear; and (4) non-decomposition of the target active ingredient. Obtaining such strains will provide key technical support for the high-value processing of prickly pear. Summary of the Invention

[0009] To address the aforementioned limitations of existing technologies, this invention has screened and obtained a high-performance *Lactobacillus plantarum* strain. This strain exhibits good activity, a short fermentation cycle, easy control of the fermentation process, and good batch-to-batch stability of the fermentation product. Using this *Lactobacillus plantarum* to ferment prickly pear yields stable fermentation products in a short time. Furthermore, the fermentation products contain high levels and high activity of flavonoids, superoxide dismutase (SOD), and phenolic active substances. The resulting fermentation products possess good antioxidant effects and significantly inhibit tyrosinase activity.

[0010] This invention utilizes a strain of *Lactobacillus plantarum* GL-1 with excellent fermentation performance. This strain was deposited on July 18, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 66711. The 16S sRNA gene sequence of *Lactobacillus plantarum* GL-1 is shown in SEQ ID NO: 1.

[0011] Furthermore, the present invention provides a microbial agent containing the aforementioned *Lactobacillus plantarum* GL-1.

[0012] This invention also provides the application of the aforementioned *Lactobacillus plantarum* GL-1 or the aforementioned inoculum in the preparation of fermented products. In this application, *Lactobacillus plantarum* GL-1 or the aforementioned inoculum is generally added to a substrate for fermentation to obtain the fermented product.

[0013] The fermentation uses of Lactobacillus plantarum in various fields are disclosed in the prior art. The Lactobacillus plantarum GL-1 of the present invention can be used in all of these disclosed fields to replace existing Lactobacillus plantarum in obtaining fermentation products.

[0014] Preferably, the *Lactobacillus plantarum* GL-1 or the above-mentioned bacterial agent is used to prepare prickly pear fermented products, wherein the prickly pear fermented products are obtained by fermenting prickly pear pulp with *Lactobacillus plantarum* or its bacterial agent.

[0015] This invention provides a method for preparing a fermentation product of prickly pear, the method comprising the following steps:

[0016] Step 1: Wash and remove impurities from the prickly pear, pulp it, and mix the resulting prickly pear pulp with water to obtain a suspension;

[0017] Step 2: Prepare the Lactobacillus plantarum GL-1 seed liquid into a bacterial culture using sterile deionized water, inoculate the bacterial culture into the suspension, and carry out anaerobic fermentation;

[0018] Step 3: After fermentation is complete, filter, sterilize, and freeze-dry the fermentation liquid to obtain the prickly pear fermentation product.

[0019] Preferably, the volume ratio of prickly pear pulp to water in step 1 is 1:0.7-1.2.

[0020] Furthermore, the prickly pear pulp is obtained by pulping whole prickly pears. Before pulping, the prickly pears can be washed, and the washed prickly pears can be pulped using conventional food processors, juicers, blenders, or other equipment.

[0021] Preferably, the viable count of the inoculum solution in step 2 is 1×10⁻⁶. 7 -1×108 The inoculum concentration was CFU / mL, the inoculum volume in the suspension was 3-5 (v / v)%, the fermentation temperature was 15℃, the fermentation time was 48-72h, and the fermentation speed was 80-120 rpm.

[0022] The prickly pear fermented product prepared by the above method contains more active ingredients (flavonoids, polyphenols, superoxide dismutase, etc.) than the fermented products obtained by fermentation with other Lactobacillus plantarum in the prior art, and the prickly pear fermented product has better antioxidant and tyrosinase inhibition effects. It can be seen that the prickly pear fermented product prepared by the specific strain of the present invention has outstanding performance and has broad application prospects in products with antioxidant or whitening effects. Therefore, the prickly pear fermented product obtained by this method and its application in products with antioxidant or whitening effects are also within the protection scope of the present invention.

[0023] The present invention has the following advantages:

[0024] 1. This invention screened and obtained a strain of Lactobacillus plantarum GL-1, which has a good proliferation rate in prickly pear pulp at low temperature (15℃). In addition, the fermentation supernatant of this strain can significantly enhance the intracellular SOD activity of HaCaT cells.

[0025] 2. The fermented product of prickly pear obtained by fermenting prickly pear with Lactobacillus plantarum GL-1 as defined in this invention has a high content of active substances (flavonoids, polyphenols, and superoxide dismutase); and the obtained fermented product of prickly pear has good antioxidant and tyrosinase inhibition effects.

[0026] Preservation Information

[0027] The Lactobacillus plantarum GL-1 described in this invention was deposited on July 18, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66711, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0029] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0030] Screening and identification process of Lactobacillus plantarum GL-1

[0031] Using commercially available kimchi as raw material, the kimchi was ground into a homogenate. The homogenate was diluted 100 times with sterile water and evenly spread on MRS plates containing 0.04% bromomethylphenol purple using a spreader. The plates were incubated at 37°C for 2 days. Single colonies whose surrounding medium turned yellow were selected and inoculated onto MRS slant tubes. The plates were incubated at 37°C for 2 days. After the incubation period, the plates were stored at 4°C. A total of 6 strains were obtained and designated as strains 1-6.

[0032] Testing the activity of strains 1-6 in prickly pear pulp

[0033] Activation: The six strains obtained above were inoculated into MRS medium and cultured statically at 37°C until the logarithmic growth phase.

[0034] Seed culture preparation: The activated bacterial cultures of strains 1-6 were diluted with sterile water and inoculated into MRS broth medium, and incubated statically at 37°C until the viable count reached 1×10⁶. 8 CFU / mL, yielding seed solutions 1-6.

[0035] Preparation of suspension: Take fresh prickly pears without insect holes, wash and remove impurities, wipe off surface moisture, use a high-speed blender to make pulp, mix the obtained prickly pear pulp with water at a ratio of 1:0.7 (L / L) to obtain a suspension.

[0036] Experiment 1: Screening of strain proliferation activity in prickly pear pulp

[0037] The seed culture was inoculated into the suspension at a volume ratio of 1%, and the culture was incubated anaerobically at 37°C for 10 hours. OD was then measured after the incubation period. 600 The OD value is used to screen strains with higher values ​​for further screening.

[0038] Table 1. Screening of microbial strains using prickly pear pulp.

[0039] strains OD value strain 1 0.34 strain 2 ＜0.1 strain 3 0.16 strain 4 ＜0.1 strain 5 0.23 strain 6 0.31

[0040] As shown in Table 1, strains 1, 5, and 6 had higher OD values, indicating that they had better proliferation effects in the prickly pear suspension under anaerobic conditions at 37℃. They were selected as target strains for further screening.

[0041] Experiment 2: Screening of strain proliferation activity in prickly pear pulp at 15℃

[0042] Seed cultures of strains 1, 5, and 6 selected in Experiment 1 were inoculated into the suspension at a volume ratio of 1%, and then anaerobically cultured at 15°C for 12 hours. OD values ​​were then measured after the culture period. 600 The OD value was used to screen strains with higher values ​​as target strains.

[0043] Table 2 shows the screening of strains using specific temperatures.

[0044] strains OD value strain 1 0.15 strain 5 0.12 strain 6 0.28

[0045] As shown in Table 2, strain 6 has a high OD value, indicating that it adapts well to the prickly pear suspension at 15℃. It was selected as the target strain for identification and preservation.

[0046] Strain morphological identification

[0047] After Gram staining and microscopic observation, strain 6 was found to be a Gram-positive bacterium, appearing as long rods, arranged singly, in pairs, or in short chains.

[0048] Colony morphology characteristics

[0049] The colony morphology of strain 6 on MRS agar medium is milky white, smooth and moist, droplet-shaped, round, raised, with neat edges, and a colony diameter of 1-2 mm.

[0050] Molecular biological identification

[0051] Using the genomic DNA of strain 6 as a template, the 16S rRNA of this strain was amplified by PCR, and the sequence of its 16S rRNA was obtained by sequencing as shown in SEQ ID NO.1:

[0052] CATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACTACTATGCAATCTAAGAGATAAACGTCCCTTCGGGGAATGGATACAGGTG。

[0053] The sequencing results were compared with the NCBI database using BLAST and a phylogenetic tree was constructed. The results showed that the sequence was highly homologous to the gene sequence of Lactiplantibacillus plantarum (i.e., Lactobacillus plantarum), with a homology of 99.8%. Based on the morphological characteristics of the strain and the colony morphology, strain 6 was identified as belonging to Lactobacillus plantarum.

[0054] The strain was named *Lactobacillus plantarum* GL-1 and deposited on July 18, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. The deposit number is GDMCC No: 66711.

[0055] Example 1: Preparation of metabolites of Lactobacillus plantarum GL-1

[0056] Step 1: Prepare a 1×10⁻⁶ Lactobacillus plantarum GL-1 seed culture using sterile deionized water. 7 The CFU / mL bacterial culture was inoculated into MRS medium at 4 v / v% and anaerobic fermentation was carried out. The fermentation temperature was 15℃, the fermentation time was 60 h, the initial fermentation pH was 3.0, and the fermentation speed was 100 rpm.

[0057] Step 2: After fermentation is complete, filter the fermentation product to obtain the fermentation filtrate. Sterilize the fermentation filtrate by irradiation and freeze-dry it to obtain the metabolites of Lactobacillus plantarum.

[0058] Example 2: Preparation of Prickly Pear Fermentation Products

[0059] Step 1: Take fresh, insect-free prickly pears, wash and remove impurities, wipe off surface moisture, use a high-speed blender to make a pulp, mix the obtained prickly pear pulp with water at a ratio of 1:0.9 (L / L), sterilize by irradiation, and obtain a suspension.

[0060] Step 2: Prepare a 1×10⁻⁶ Lactobacillus plantarum GL-1 seed culture using sterile deionized water. 7 The CFU / mL bacterial culture was inoculated into the suspension at 4 v / v% and then subjected to anaerobic fermentation at a temperature of 15℃ for 60 h and a rotation speed of 100 rpm.

[0061] Step 3: After fermentation is complete, filter the fermentation product to obtain the fermentation filtrate, sterilize the fermentation filtrate by irradiation, freeze-dry it, and obtain the prickly pear fermentation product.

[0062] Example 3: Preparation of Prickly Pear Fermentation Products

[0063] Step 1: Take fresh, insect-free prickly pears, wash and remove impurities, wipe off surface moisture, use a high-speed blender to make a pulp, mix the obtained prickly pear pulp with water at a ratio of 1:0.7 (L / L), sterilize by irradiation, and obtain a suspension.

[0064] Step 2: Add 1×10⁻⁶ Lactobacillus plantarum GL-1 seed culture 8 CFU / mL was inoculated into the suspension at 3 v / v% and anaerobic fermentation was carried out; the fermentation temperature was 15℃, the fermentation time was 72 h, and the fermentation speed was 80 rpm.

[0065] Step 3: After fermentation is complete, filter the fermentation product to obtain the fermentation filtrate, sterilize the fermentation filtrate by irradiation, freeze-dry it, and obtain the prickly pear fermentation product.

[0066] Example 4: Preparation of Prickly Pear Fermentation Products

[0067] Step 1: Take fresh, insect-free prickly pears, wash and remove impurities, wipe off surface moisture, use a high-speed blender to make a pulp, mix the obtained prickly pear pulp with water at a ratio of 1:1.2 (L / L), sterilize by irradiation, and obtain a suspension.

[0068] Step 2: Prepare a 1×10⁻⁶ Lactobacillus plantarum GL-1 seed culture using sterile deionized water. 7 The CFU / mL bacterial culture was inoculated into the suspension at 5 v / v% and then subjected to anaerobic fermentation. The fermentation temperature was 15℃, the fermentation time was 48 h, and the fermentation speed was 120 rpm.

[0069] Step 3: After fermentation is complete, filter the fermentation product to obtain the fermentation filtrate, sterilize the fermentation filtrate by irradiation, freeze-dry it, and obtain the prickly pear fermentation product.

[0070] Comparative Example 1: Unlike Example 1, Lactobacillus plantarum seed culture with accession number GDMCC No. 61730 was used instead of Lactobacillus plantarum GL-1 seed culture. The rest of the preparation process and parameters were the same as in Example 1.

[0071] Comparative Example 2: Unlike Example 2, Lactobacillus plantarum seed culture with accession number CGMCC No.13121 was used instead of Lactobacillus plantarum GL-1 seed culture. The rest of the preparation process and parameters were the same as in Example 2.

[0072] Comparative Example 3: Unlike Example 2, Lactobacillus plantarum seed culture with accession number CGMCC No. 31277 was used instead of Lactobacillus plantarum GL-1 seed culture. The rest of the preparation process and parameters were the same as in Example 2.

[0073] Comparative Example A: Take fresh, insect-free prickly pears, wash and remove impurities, wipe off surface moisture, use a high-speed blender to make a pulp, mix the obtained prickly pear pulp with water at a ratio of 1:0.9 (L / L), sterilize by irradiation, obtain a suspension, and freeze-dry to obtain prickly pear powder.

[0074] Test Example 1: Investigating the effect of Lactobacillus plantarum metabolites on superoxide dismutase (SOD) activity in HaCaT cells. Test samples: Lactobacillus plantarum metabolites obtained in Example 1 and Comparative Example 1.

[0075] The complete culture medium was HaCaT complete culture medium produced by Shanghai Fuheng Company.

[0076] HaCaT cells were seeded in 6-well plates (cell count: 10). 5 After culturing for 24 h, cells in all groups except the normal control group were treated with complete medium containing 50 μM H2O2 for 1 h. The experimental samples (the *Lactobacillus plantarum* metabolites obtained in Example 1 and Comparative Example 1) were mixed with the complete medium to prepare 2.5 wt% complete medium containing the experimental samples. 100 μL of each of these groups was cultured in the H2O2-treated HaCaT cells for 24 h. The normal control group (HaCaT cells without H2O2 treatment) and the model group (HaCaT cells treated with H2O2) were supplemented with 100 μL of complete medium, and 100 μL of the H2O2-treated HaCaT cells was cultured in each group for 24 h. Six replicates were set up for each group. Cells were washed twice with PBS solution, and then scraped into PBS solution at 4°C. The cells were sonicated to fully disrupt them and release oxidized substances. The cells were centrifuged at 12000g for 5 min at 4°C, and the supernatant was stored at -80°C for analysis. The superoxide dismutase (SOD) test kit (Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number: ml076325) was used for the determination, and the results are shown in Table 3 below.

[0077] Table 3. Investigating the effects of Lactobacillus plantarum metabolites on SOD activity in HaCaT cells (n=3). )

[0078] Group SOD activity (U / L) normal control group 197.69±8.74* Model group 129.13±5.19* Example 1 716.04±14.47 Comparative Example 1 418.26±22.31*

[0079] Note: "*" indicates that compared with Example 1, p < 0.05.

[0080] As shown in Table 3, the Lactobacillus plantarum GL-1 metabolite provided by this invention can significantly enhance SOD activity in HaCat cells, with a marked improvement compared to Lactobacillus plantarum (GDMCC No. 61730).

[0081] Test Example 2: Investigating the effect of Lactobacillus plantarum GL-1 on the total phenol and total flavonoid content in the fermentation products of Prickly Pear.

[0082] The prickly pear fermentation products obtained in Examples 2-4 and Comparative Example 2, and the prickly pear powder obtained in Comparative Example A were prepared into 10 g / L test solutions using deionized water. The nutrient content in the test solutions was determined using the plant superoxide dismutase (SOD) ELISA kit (CB10021-Pt), plant total phenol (TP) ELISA kit (CB10189-Pt), and plant total flavonoid (flavone) ELISA kit (CB10424-Pt) produced by Shanghai Keaibo Biotechnology Co., Ltd. The change rates of SOD, total phenol, and total flavonoid in the test solutions were calculated. Change rate (%) = (AB) / B, where A is the SOD activity, total phenol content, or total flavonoid content in Examples 2-4 and Comparative Example 2, respectively, and B is the SOD activity, total phenol content, or total flavonoid content in Comparative Example A. The change rate results are shown in Table 4.

[0083] Table 4. Effects of *Lactobacillus plantarum* GL-1 on SOD activity, total phenolic and total flavonoid content in *Rhizopus rosae* pulp (n=3, ...). )

[0084] Group SOD activity change rate / % Change rate of total phenol content / % Change rate of total flavonoid content / % Example 2 11.52±0.19 18.87±0.63 64.12±1.22 Example 3 11.37±0.23 17.75±0.97 62.07±1.25 Example 4 10.96±0.38 18.24±0.59 61.66±1.68 Comparative Example 2 1.69±0.12* 7.94±0.78* 21.30±0.99*

[0085] Note: "*" indicates comparison with Example 2, p < 0.05

[0086] As can be seen from the results in Table 4, the use of *Lactobacillus plantarum* GL-1 selected by the present invention to ferment prickly pear pulp in Examples 2-4 can effectively increase the SOD activity, total phenolic content, and total flavonoid content in the prickly pear ferment. Comparing the results of Example 2 and Comparative Example 2, it can be seen that under the fermentation process defined by the present invention, the prickly pear ferment obtained by using *Lactobacillus plantarum* GL-1 selected by the present invention as the fermentation strain has a better increase rate in total phenolic content and total flavonoid content. The principle may be that fermentation is carried out in the original pH environment of prickly pear pulp, and the active substances in its fermentation products are more stable. Furthermore, the entire fermentation process is carried out at a lower temperature, and the heat-sensitive and easily decomposed flavonoids and polyphenols in its fermentation products can be more effectively retained.

[0087] Test Example 3: Experiment to investigate the detection of tyrosinase activity inhibition by prickly pear fermentation products.

[0088] Test samples: Prickly pear fermentation product prepared in Example 2, Prickly pear fermentation product prepared in Comparative Example 3, and Prickly pear powder prepared in Comparative Example A.

[0089] Solution preparation:

[0090] 1. Preparation of tyrosinase solution: Prepare tyrosinase to 500 U / ml using PBS buffer, aliquot, and store at -20℃ to avoid secondary freeze-thaw cycles.

[0091] 2. Preparation of L-tyrosine solution: Dissolve 0.025g of L-tyrosine in an appropriate amount of PBS buffer solution, sonicate to aid dissolution, and then bring the volume to 50mL with PBS to prepare a 0.5g / L L-tyrosine solution. Prepare and use immediately.

[0092] 3. Preparation of positive control solutions: Kojic acid, the positive control, was diluted with ultrapure water to prepare a series of mass concentration gradient solutions of 0.040 g / L, 0.020 g / L, 0.010 g / L, 0.008 g / L, 0.005 g / L, 0.002 g / L, and 0.001 g / L.

[0093] 4. Sample solution preparation: Prepare the test samples to 10% of the test concentration using PBS buffer, and use immediately after preparation.

[0094] Experimental Groups:

[0095] The experiment was divided into four groups: solvent background (Ta), solvent reaction wells (Tb), sample background (Tc), and sample reaction (Td). The solvent background group (Ta) consisted of PBS buffer and tyrosinase solution; the solvent reaction wells (Tb) consisted of L-tyrosine solution, PBS buffer, and tyrosinase solution; the sample background group (Tc) consisted of sample solution, PBS buffer, and tyrosinase solution; and the sample reaction group (Td) consisted of L-tyrosine solution, sample solution, PBS buffer, and tyrosinase solution. Three replicates were taken from each group, and the average value was recorded.

[0096] Experimental steps:

[0097] Following the reagent addition amounts in Table 5, L-tyrosine solution, sample solution, and PBS buffer were added sequentially to each well. After addition, the contents of each well were thoroughly mixed, and then incubated at 37°C for 10 min. At the end of the incubation, 20 μL of tyrosinase solution was added to each well sequentially, mixed, and the reaction was continued at 37°C, ensuring a reaction time of 5 min ± 5 s. The absorbance values ​​of different samples and positive controls were immediately measured at a wavelength of 475 nm.

[0098] Table 5. Inhibition capacity of tyrosinase activity (sample loading table)

[0099] name Ta (solvent background pores) Tb (solvent reaction well) Tc (sample bottom hole) Td (sample reaction well) L-tyrosine solution (μL) 0 40 0 40 Sample solution (μL) 0 0 40 40 PBS buffer (μL) 110 70 70 30 Tyrosinase solution (μL) 20 20 20 20 Total (μL) 130 130 130 130

[0100] Data processing:

[0101]

[0102] In the formula:

[0103] Aa—average absorbance of the solvent background pores;

[0104] Ab—average absorbance of the solvent reaction well;

[0105] Ac—Average absorbance of the sample's bottom wells;

[0106] Ad – Average absorbance of the sample reaction well.

[0107] Positive control half-maximal inhibitory concentration (IC50 value):

[0108] Plot a curve with the concentration of the positive control on the x-axis and the corresponding tyrosinase activity inhibition rate on the y-axis, and fit a linear regression equation (R0). 2 (≥0.900) and calculate the positive control concentration corresponding to a 50% inhibition rate of tyrosinase activity according to the equation. IC50 of positive control kojic acid. 50 If the concentration is between 0.005 g / L and 0.01 g / L, the experiment is considered valid.

[0109] The test results are shown in Table 6:

[0110] Table 6. Effects of Prickly Pear Fermentation Products on the Inhibition Rate of Tyrosinase

[0111]

[0112] The results are shown in Table 6. The fermentation product of prickly pear obtained by fermenting prickly pear with Lactobacillus plantarum GL-1 has a good inhibitory effect on tyrosinase activity, indicating that it has whitening effect and can be used to prepare whitening products, such as skin care products with whitening function.

[0113] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A Lactobacillus plantarum GL-1, characterized in that, The bacteria were deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 18, 2025, with accession number GDMCC NO: 66711.

2. A microbial agent, characterized in that, Includes Lactobacillus plantarum GL-1 as described in claim 1.

3. A fermentation product, characterized in that, It contains Lactobacillus plantarum GL-1 as described in claim 1 or the bacterial agent as described in claim 2.

4. A method for preparing a prickly pear fermentation product, characterized in that, Includes the following steps: Step 1: Wash and remove impurities from the prickly pear, pulp it, and mix the resulting prickly pear pulp with water to obtain a suspension; Step 2: Prepare the Lactobacillus plantarum GL-1 seed liquid into a bacterial culture using sterile deionized water, inoculate the bacterial culture into the suspension, and carry out anaerobic fermentation; Step 3: After fermentation is complete, filter, sterilize, and freeze-dry the fermentation liquid to obtain the prickly pear fermentation product.

5. The preparation method according to claim 4, characterized in that, The volume ratio of prickly pear pulp to water in step 1 is 1:0.7-1.

2.

6. The preparation method according to claim 4, characterized in that, The viable count of the inoculum solution in step 2 is 1 × 10⁻⁶. 7 -1×10 8 CFU / mL, the inoculum concentration in the prickly pear pulp is 3-5 (v / v)%.

7. The preparation method according to claim 4, characterized in that, The fermentation temperature in step 2 is 15℃, the fermentation time is 48-72h, and the fermentation speed is 80-120 rpm.

8. The application of a prickly pear fermentation product prepared by any one of the preparation methods described in claims 4-7 in the preparation of products with antioxidant and whitening effects.

Images