Lactobacillus plantarum G4 with whitening effect and application thereof

Abstract

The invention provides lactobacillus plantarum G4 with a whitening effect and application of the lactobacillus plantarum G4, the lactobacillus plantarum G4 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.34672, and the preservation date is May 26, 2025. A fermentation supernatant of the lactobacillus plantarum G4 has a remarkable inhibition effect on tyrosinase activity, and meanwhile, the strain also shows excellent oxidation resistance. And the fermented supernate has a relatively high clearance rate on ABTS free radicals, DPPH free radicals and OH free radicals. Wherein the scavenging rate of ABTS free radicals can reach 60% or above, the scavenging rate of DPPH free radicals exceeds 90%, and the scavenging rate of OH free radicals is also 80% or above. The antioxidant activity can reduce the damage of active oxygen to the skin and inhibit the generation of melanin induced by oxidative stress, and the antioxidant activity and the tyrosinase inhibition effect cooperate to play a whitening effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a strain of Lactobacillus plantarum G4 with whitening effects and its applications. Background Technology

[0002] In today's society, the demand for skin whitening is booming in the cosmetics and health industries. Currently, common skin whitening methods on the market mainly rely on chemically synthesized whitening agents, such as hydroquinone and kojic acid. However, these chemically synthesized whitening agents have many problems. On the one hand, they may irritate the skin, leading to adverse reactions such as skin allergies and redness; on the other hand, the safety of some chemically synthesized whitening agents has been questioned, and long-term use may pose health risks. Therefore, developing safe and effective natural whitening ingredients has become a research hotspot in this field.

[0003] Tyrosinase is a key enzyme in melanin synthesis, catalyzing the oxidation of L-DOPA to dopaquinone, which in turn gradually forms melanin. Inhibiting tyrosinase activity can effectively reduce melanin production, thus achieving a skin-whitening effect. Simultaneously, oxidative stress also plays a crucial role in melanin production; excessive production of reactive oxygen species (ROS) promotes tyrosinase activity and accelerates melanin synthesis. Therefore, ingredients with antioxidant capabilities also contribute to skin-whitening effects.

[0004] Lactic acid bacteria, as an important class of probiotics, have been widely used in food fermentation and gut health. Recent studies have found that some lactic acid bacteria and their metabolites possess various biological activities. However, research on the application of lactic acid bacteria in skin whitening is relatively limited, especially regarding the screening and functional studies of lactic acid bacteria strains with highly effective tyrosinase inhibition and strong antioxidant capabilities, which still require further investigation. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a strain of Lactobacillus plantarum G4 with whitening effect and its application. This strain can significantly inhibit tyrosinase activity and has antioxidant function, and can be used to prepare whitening products.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The first aspect of this invention provides a strain of Lactobacillus plantarum G4 with whitening effects. The Lactobacillus plantarum G4 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34672 and deposit date of May 26, 2025.

[0008] The second aspect of the present invention provides a fermentation method for Lactobacillus plantarum G4 as described in the first aspect, comprising the following steps: inoculating Lactobacillus plantarum G4 into MRS medium, incubating it statically at 37°C for 24 h, subculturing it once with an inoculum of 2%, centrifuging it at 8000×g for 10 min at 4°C, and collecting the supernatant and bacterial cells respectively.

[0009] A third aspect of the present invention provides a microbial agent with whitening effect, wherein the agent contains Lactobacillus plantarum G4 and / or its fermentation products as described in the first aspect.

[0010] A fourth aspect of the present invention provides a skin whitening composition comprising the Lactobacillus plantarum G4 and / or its fermentation products as described in the first aspect.

[0011] The fifth aspect of this invention provides the application of Lactobacillus plantarum G4 as described in the first aspect, or the microbial agent as described in the third aspect, in the preparation of skin whitening products.

[0012] Furthermore, the product is a cosmetic, food, or medicine.

[0013] Furthermore, the product is one or more of the following:

[0014] (1) The product can inhibit tyrosinase activity;

[0015] (2) The product has antioxidant function.

[0016] Furthermore, the antioxidant function is manifested in the scavenging of ABTS free radicals, DPPH free radicals, or OH free radicals.

[0017] Information on strain preservation:

[0018] Lactobacillus plantarum G4, deposited at: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Deposit date: May 26, 2025; Accession number: CGMCC No. 34672; Classification: Lactobacillus plantarum.

[0019] The advantages of this invention compared to the prior art are as follows:

[0020] 1. Highly Effective Tyrosinase Inhibition: The fermentation supernatant of *Lactobacillus plantarum* G4 described in this invention exhibits a significant inhibitory effect on tyrosinase activity. In screening experiments, its inhibition rate reached as high as 97.3 ± 0.78%, significantly higher than many other lactic acid bacteria strains. This highly effective tyrosinase inhibition can block melanin synthesis at its source, providing a solid foundation for whitening effects.

[0021] 2. Significant Antioxidant Activity: This strain not only possesses strong tyrosinase inhibitory capabilities but also exhibits excellent antioxidant properties. Its fermentation supernatant shows high scavenging rates against ABTS, DPPH, and OH free radicals. Specifically, the scavenging rate against ABTS free radicals reaches over 60%, against DPPH free radicals exceeds 90%, and against OH free radicals is also over 80%. This antioxidant activity can reduce skin damage caused by reactive oxygen species, inhibit oxidative stress-induced melanin production, and synergistically exert a whitening effect with tyrosinase inhibition.

[0022] 3. Excellent biocompatibility: Experiments using a zebrafish model demonstrated that when the concentration of *Lactobacillus plantarum* G4 fermentation supernatant was less than or equal to 0.2%, it had no impact on the survival rate of zebrafish, indicating that this strain and its metabolites possess high biocompatibility. This characteristic gives it a significant advantage in applications in cosmetics and food, reducing safety risks during use.

[0023] 4. Inhibition of UVB-induced melanin production in zebrafish. The degree of pigmentation on the body surface of zebrafish treated with this fermentation supernatant was significantly reduced, with melanin mainly distributed in scattered dots. The zebrafish in the 0.2% concentration treatment group showed a very light body color. Simultaneously, this fermentation supernatant also regulated the ROS levels in zebrafish, further confirming its dual whitening and antioxidant effects.

[0024] 5. Potential multiple applications: Based on the whitening and antioxidant properties of Lactobacillus plantarum G4, it can be added to various skin care products as a natural whitening ingredient in the cosmetics field; in the food field, it can be used to develop functional foods with whitening effects; in addition, it may also have certain application potential in the pharmaceutical field, providing new ideas and methods for skin whitening and the prevention of related diseases. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0026] Figure 1 Comparative analysis of the inhibitory effects of different types of lactic acid bacteria on tyrosinase activity; where A: Lactobacillus plantarum, B: Lactobacillus paracasei, and C: Streptococcus thermophilus.

[0027] Figure 2 This study demonstrates the inhibitory effects of different types of lactic acid bacteria on tyrosinase at different concentrations.

[0028] Figure 3 The study demonstrates the scavenging ability of different lactic acid bacteria strains against ABTS free radicals.

[0029] Figure 4 The study demonstrates the scavenging ability of different lactic acid bacteria strains against DPPH free radicals.

[0030] Figure 5 The study demonstrates the scavenging ability of different lactic acid bacteria strains against OH free radicals.

[0031] Figure 6 The distribution of melanin in zebrafish is shown under a microscope;

[0032] Figure 7 The ROS distribution of zebrafish under a microscope is shown. Detailed Implementation

[0033] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0034] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0036] The detection methods involved in the following embodiments include:

[0037] 1. Strains culture and sample preparation

[0038] The lactic acid bacteria culture was removed from the -80℃ freezer and inoculated onto sterilized MRS solid medium. It was then incubated statically at 37℃ for 48 hours. Single colonies were picked and inoculated onto sterilized MRS liquid medium, incubated statically at 37℃ for 24 hours, and then passaged once more at a 2% inoculum level. The culture was then centrifuged at 8000×g for 10 minutes at 4℃, and the supernatant was collected for later use. After centrifugation, the bacterial cells were washed three times with sterilized PBS, and the bacterial cells were resuspended in PBS to obtain the bacterial cells.

[0039] 2. Screening of lactic acid bacteria strains that inhibit tyrosinase

[0040] Referring to the Shanghai Daily Chemicals Industry Association Group Standard (T / SHRH 015-2018) "Cosmetics - Experimental Method for Inhibition of Tyrosinase Activity", based on the principle that tyrosinase catalyzes the conversion of L-DOPA to dopaquinone through diphenolase activity, this study investigated the inhibitory effect of lactic acid bacteria fermentation broth on tyrosinase activity using L-DOPA as a substrate.

[0041] According to Table 1, the 96-well plates were divided into four groups. The corresponding volumes of L-DOPA, PBS buffer, tyrosinase, and fermentation supernatant were added to each group, with three replicates per group. In each well, other solutions were added first, followed by the enzyme solution. The 96-well plates were shaken at 37°C for 10 min, then 125 U / mL tyrosinase solution was added. After reacting for 20 min, the absorbance was measured at 475 nm using a microplate reader.

[0042] The tyrosinase inhibition rate is calculated according to formula (1).

[0043]

[0044] Table 1. Dosage of Tyrosinase Activity Inhibitors

[0045]

[0046] By fixing the L-DOPA concentration and varying the tyrosinase concentration (final concentrations of 40, 80, 120, 160, and 200 U / mL), the absorbance of different lactic acid bacteria oxidizing L-DOPA under the catalysis of various concentrations of tyrosinase was measured.

[0047] 3. Determination of antioxidant capacity of lactic acid bacteria

[0048] (1) Determination of ABTS free radical scavenging ability

[0049] 7mM / LABTS: Accurately weigh 38.4mg ABTS, dissolve in distilled water, and bring the volume to 10mL to prepare a 7mM / LABTS solution. Store in the dark.

[0050] 2.45mM / L K2S2O8: Accurately weigh 66.2mg K2S2O8, dissolve it in distilled water, and make up to 100mL to prepare 2.45mM / L K2S2O8.

[0051] ABTS working solution: Mix 7 mM / LABTS and 2.45 mM / LK2S2O8 in equal volumes in a beaker, react in the dark for 12 hours, dilute the reacted solution with distilled water, and prepare the ABTS working solution with an absorbance of 0.7 ± 0.02 at a wavelength of 734 nm.

[0052] Mix 40 μL of sample with 800 μL of LABTS working solution in a 1.5 mL centrifuge tube, vortex for 20 s, and react in the dark for 6 min. Measure the absorbance of the sample at 734 nm. Perform triplicate for each sample. Replace the sample solution with an equal volume of distilled water for the blank control; this is the absorbance of the blank control.

[0053] ABTS clearance rate (%) = [(A blank - A sample) / A blank] × 100%

[0054] (2) DPPH free radical scavenging capacity determination

[0055] 0.1 mM / LDPPH: Accurately weigh 0.002 g of DPPH and dissolve it in 50 mL of ethanol. Store in the dark.

[0056] According to Table 2, the samples were divided into four groups. Each group was treated with the corresponding volume of sample, DPPH anhydrous ethanol solution, water or anhydrous ethanol, and reacted in the dark for 20 min. The bacterial cells were then centrifuged at 8000×g for 10 min at 4℃. The supernatant was collected and the absorbance was measured at 517 nm. Each group was tested in triplicate.

[0057] The DPPH free radical scavenging rate is calculated according to formula (2).

[0058]

[0059] Table 2. Dosage of DPPH Free Radical Scavenger Reagent

[0060]

[0061] (3) Determination of OH free radical scavenging ability

[0062] Add 0.5 mL of sample and 1 mL of PBS to 0.5 mL of O-phenanthroline (0.75 mM / L), mix thoroughly, then add 5 mL of FeSO4 (0.75 mM / L) and 0.5 mL of 0.01% H2O2. Incubate at 37℃ for 1 h, and measure absorbance at 536 nm. Centrifuge bacterial cells at 8000×g for 10 min at 4℃, and measure absorbance of the supernatant at 536 nm. Perform triplicate for each sample. Replace 0.5 mL of H2O2 with 0.5 mL of distilled water in the blank control group; this is the absorbance of the blank control group. Replace 0.5 mL of sample with 0.5 mL of distilled water in the control group; this is the absorbance of the control group.

[0063] OH removal rate (%) = [(Sample A - Control A) / (Blank A - Control A)] × 100%

[0064] 4. Analysis of the growth and probiotic characteristics of Lactobacillus plantarum G4

[0065] (1) Determination of the growth curve of Lactobacillus plantarum G4

[0066] Lactobacillus plantarum G4 culture preserved in glycerol was activated for three generations, inoculated into liquid MRS medium, and incubated in a constant temperature incubator at 37℃. The absorbance of the bacterial solution at OD600 was measured at 0, 2, 4, 6, 8, 10, 12, 14, 18, 24, 36 and 48 h, and growth curves were plotted.

[0067] (2) Analysis of the gastrointestinal digestive fluid tolerance of Lactobacillus plantarum G4

[0068] The activated Lactobacillus plantarum G4 was centrifuged at 8000×g for 10 min at 4℃. After centrifugation, the bacterial cells were washed three times with sterilized PBS. The bacterial cells were resuspended in PBS with pH 2.5, 3, and 4 and containing 3 g / L pepsin. The OD was adjusted to 1.0 and the cells were placed in a 37℃ incubator. At 0 and 3 h, 2 mL of the bacterial solution was taken and diluted with sterile PBS to the appropriate dilution and spread on MRS solid plates. After incubation at 37℃ for 36-48 h, plate counts were performed.

[0069] Pancreatic enzyme (1 g / L) suspension was placed in sterile PBS, and the pH was adjusted to 8.0 with sodium oxide. 100 μL of Lactobacillus plantarum G4 cells, which had been cultured in gastric juice for 3 h, was transferred to 900 μL of simulated artificial pancreatic juice and cultured at 37 °C for 4 h. At 0 and 4 h, 100 μL of the bacterial suspension was taken and diluted to the appropriate dilution with sterile physiological saline and plated on MRS solid plates. After incubation at 37 °C for 36-48 h, plate counts were performed.

[0070] (3) Hydrophobicity test of Lactobacillus plantarum G4

[0071] After activating the strain for 2-3 generations, wash the cells 2-3 times with sterile PBS (pH 6.5) buffer to adjust the bacterial count to 1.0 × 10⁻⁶. 9 The absorbance (A0) of the bacterial suspension at 600 nm was measured using a multi-functional microplate reader. 3 mL of the bacterial suspension was added to 1 mL of xylene, pre-cultured at room temperature for 10 min, then rapidly vortexed for 2 min, and allowed to stand at room temperature for 15 min. After the solution separated into layers, the absorbance (A0) of the lower aqueous phase at 600 nm was measured (the blank control was buffer). Each sample was tested in triplicate. The hydrophobicity of the bacterial cell surface was calculated using the following formula.

[0072] Cell surface hydrophobicity H% = (A0 - A) / A0 × 100%

[0073] (4) Self-aggregation ability of Lactobacillus plantarum G4

[0074] The strain was incubated in MRS liquid medium at 37°C for 18 h, and the bacterial count was adjusted to an equal volume of standard cell concentration (A600 = 1 ± 0.05, 1 × 10⁻⁶) using 0.1 mol / mL sterile phosphate-buffered saline. 8 CFU / mL). Bacterial cells were collected by centrifugation at 12,000 rpm for 10 min at 4 °C, washed twice with 0.1 mol / mL sterile phosphate-buffered saline (pH = 7.2 ± 0.2), resuspended, and incubated at room temperature. Absorbance was measured at 600 nm at 0 h, 2 h, 4 h, and 6 h of incubation. Each sample was tested in triplicate. The absorbance was calculated using the following formula:

[0075] Hydrophobicity (%) = (A0 - A) t ) / A0×100%

[0076] A0 represents the absorbance at 0h;

[0077] A t Absorbance at different times (0h, 2h, 4h and 6h).

[0078] (5) Determination of the antibacterial ability of Lactobacillus plantarum G4

[0079] The antibacterial activity was initially determined using the double-layer streak plating method. Single colonies were picked and streaked with two parallel lines of equal length on an MRS plate. The plates were incubated at 37°C for 48 hours. Indicator bacteria Staphylococcus aureus MRSA14-84 and Enterococcus faecalis 224-2 (OD2000) were then added. 600 =0.8) The volume of the medium is mixed at a ratio of 1:100. The medium containing the indicator bacteria is poured onto the MRS plate that has been grown with Lactobacillus plantarum G4 and incubated overnight at 37°C.

[0080] Example 1: Screening of lactic acid bacteria with whitening effects

[0081] This embodiment compares and analyzes the inhibition of tyrosinase activity in the fermentation supernatants of 82 strains of lactic acid bacteria, including *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Streptococcus thermophilus*. Lactic acid bacteria exhibiting the highest and lowest inhibition of tyrosinase activity were screened from each strain for further analysis.

[0082] The results are as follows Figure 1 As shown, at a tyrosinase concentration of 125 U / mL, the inhibition rate of tyrosinase in the control MRS medium was 33.2 ± 2.1%. *Lactobacillus paracasei* B20 showed the highest inhibition rate at 94.3 ± 0.7%, while M57-1 showed the lowest at 61.0 ± 1.3%. Among *Streptococcus thermophilus*, S56 showed the highest inhibition rate at 88.4 ± 1.1%, while strain 76 showed the lowest at 76.6 ± 0.5%. Among *Lactobacillus plantarum*, G4 showed the highest inhibition rate at 97.3 ± 0.78%, while 126-5 showed the lowest at 77.9 ± 4.3%.

[0083] Furthermore, to compare the differences in the inhibition of tyrosinase activity among the selected strains, such as... Figure 2As shown, this embodiment set different final tyrosinase concentrations (40–240 U / mL). It was found that as the tyrosinase concentration increased, the inhibitory effect of the fermentation supernatant of lactic acid bacteria on tyrosinase activity weakened. There were significant differences in the inhibitory effect of the fermentation supernatant on tyrosinase activity among different lactic acid bacteria strains, with *Lactobacillus plantarum* G4 and *Lactobacillus paracasei* B20 showing stronger inhibitory effects. At a final enzyme concentration of 40 U / mL, the inhibition rate of *Lactobacillus plantarum* G4 and *Lactobacillus paracasei* B20 reached over 95%; at a tyrosinase concentration of 200 U / mL, the inhibition rate of G4 on tyrosinase was approximately 75%. To save costs, a final tyrosinase concentration of 40 U / mL was selected for subsequent experiments.

[0084] Example 2: Analysis of the antioxidant capacity of lactic acid bacteria fermentation supernatant

[0085] This embodiment analyzes the scavenging ability of fermentation supernatants and bacterial cells of Lactobacillus paracasei B20, M57-1, Streptococcus thermophilus S56, 76 and Lactobacillus plantarum G4, 126-5 on ABTS free radicals.

[0086] like Figure 3 As shown, the scavenging ability of fermentation supernatant for ABTS free radicals was higher than that of bacterial cells. Except for Lactobacillus plantarum 126-5, which had a slightly lower scavenging rate for ABTS free radicals, the scavenging rates of the fermentation supernatants of the other five strains for ABTS free radicals were not significantly different, with a scavenging rate of up to 60%. There were significant differences in the scavenging of ABTS free radicals by bacterial cells of different strains. Among them, Lactobacillus paracasei B20 had the highest scavenging effect on ABTS free radicals, with a scavenging rate of 41.9 ± 0.02%.

[0087] Further analysis of the DPPH free radical scavenging ability of the fermentation supernatant and cell cells of the above six lactic acid bacteria strains showed that the fermentation supernatant of all six strains achieved a DPPH free radical scavenging rate of over 90%. Figure 4 Different bacterial cells showed significant differences in their ability to scavenge DPPH free radicals. Lactobacillus paracasei B20 exhibited the highest DPPH free radical scavenging rate, at 80.4 ± 0.02%.

[0088] Comparative analysis of the scavenging capacity of fermentation supernatants and bacterial cells of six lactic acid bacteria strains for OH free radicals revealed that the fermentation supernatants of *Lactobacillus plantarum* G4 and 126-5 exhibited high OH free radical scavenging capacity, exceeding 80%. The fermentation supernatants of *Lactobacillus plantarum* G4 and M57-1 showed moderate OH free radical scavenging capacity, approximately 70%. The fermentation supernatants of *Streptococcus thermophilus* S56 and 76 showed relatively low OH free radical scavenging capacity, approximately 60%. Figure 5Comparison of the OH free radical scavenging effects of different bacterial cells revealed that Streptococcus thermophilus S56 had the highest OH free radical scavenging rate, reaching 59.1±0.02%, followed by Lactobacillus paracasei B20 and Lactobacillus plantarum G4.

[0089] In summary, by comparing the tyrosinase activity and antioxidant capacity (including the ability to scavenge ABTS, DPPH, and OH free radicals) of multiple lactic acid bacteria strains, *Lactobacillus plantarum* G4 was selected as having higher inhibitory activity against tyrosinase and stronger antioxidant capacity.

[0090] Example 3: Whitening and antioxidant effects of Lactobacillus plantarum G4 fermentation broth on zebrafish models

[0091] To investigate the whitening and anti-aging effects of Lactobacillus plantarum G4 fermentation broth, this example uses a zebrafish model and conducts the following experiments:

[0092] 1. Screening of the concentration of fermentation supernatant from Lactobacillus plantarum G4

[0093] Using 12-well plates as containers, 15 embryos were placed in each well. Different concentrations of fermentation supernatant (0.05%, 0.1%, 0.2%, 0.3%, 0.4%) were prepared, along with a UV model group. Embryos without UV radiation served as a blank control. 24 hours after fertilization, different concentrations of fermentation supernatant were added to each well in the experimental groups. 3 mL of culture medium was added to each well. Zebrafish larvae were treated with different concentrations of fermentation supernatant for 24 hours, followed by UVB irradiation for 6 days, 5 times a day for 15 minutes each time, with a radiation power of 30 mJ / cm². The culture medium was changed every 24 hours, and dead embryos were removed. Mortality was recorded 7 days after embryo treatment. The mortality rate was calculated using the following formula (obtained by counting):

[0094]

[0095] 2. Determination of ROS in zebrafish after UVB induction

[0096] Zebrafish embryos aged 7-9 hpf post-fertilization were randomly divided into 5 groups of 15 embryos each and cultured in 6-well plates. Different concentrations of sample were added to each well, and after 1 hour of incubation, the liquid in all wells was discarded, retaining 1 mL of E3 medium. Except for the control group, the zebrafish embryos in each group were induced with UVB. After induction, an appropriate amount of E3 medium was added to each group for continued culture. Zebrafish embryos were induced daily until 72 hpf. They were then stained with DCFH-DA (20 μg / mL), with an appropriate amount of staining solution added to each well, and incubated at 28°C in the dark for 2 hours. The juvenile fish were anesthetized, observed under a fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ software.

[0097] 3. Observation of melanin production on the embryonic surface

[0098] Observe the melanin production of the embryos under a microscope and take pictures after 72 hours of treatment with the test substance. Carefully aspirate the embryos with a sterile pipette and place them vertically. When the zebrafish sinks to the mouth of the pipette, gently squeeze the pipette to drop the embryos into the groove of the concave glass slide. Discard the embryo culture medium and add 1-2 drops of 0.05 mg / mL MS-222 to anesthetize the zebrafish. Under the microscope, adjust the zebrafish so that both eyes and body segments are aligned and the tail is at the same level as the body. Take a clear top view of the zebrafish under the same magnification and light intensity.

[0099] 4. Determination of melanin content in zebrafish

[0100] The melanin content of zebrafish was determined using the NaOH pyrolysis method. The precipitate after centrifugation was resuspended in 600 μL of a 1 mol / L NaOH solution containing 10% DMSO and incubated at 80°C for 2 h. After cooling, the precipitate was transferred to a 96-well plate. Zebrafish without the test substance were used as a blank control, and the absorbance (A) at 405 nm was measured. 405 This refers to the melanin content in the zebrafish, which is calculated using the following formula:

[0101]

[0102] In the formula: A 空白 A represents the absorbance of the blank group. 样品 The absorbance value is the value after adding the sample.

[0103] The experimental results are as follows:

[0104] 1. Effects of different concentrations of fermentation supernatant on the survival rate of zebrafish

[0105] The results of the study on the effect of Lactobacillus plantarum G4 fermentation supernatant on zebrafish mortality showed (Table 3) that 0.3% Lactobacillus plantarum G4 fermentation supernatant resulted in a 30% mortality rate in zebrafish, 0.4% Lactobacillus plantarum G4 fermentation supernatant resulted in a 100% mortality rate, and 0.2% or less Lactobacillus plantarum G4 fermentation supernatant had no effect on zebrafish mortality. This indicates that 0.2% Lactobacillus plantarum G4 fermentation supernatant is the maximum safe dose for zebrafish.

[0106] Table 3. Effects of different concentrations of Lactobacillus plantarum G4 fermentation supernatant on zebrafish mortality.

[0107]

[0108] 2. Study on the inhibitory effect of Lactobacillus plantarum G4 fermentation supernatant on melanin production and antioxidant properties

[0109] The inhibitory effect of *Lactobacillus plantarum* G4 fermentation supernatant on melanin production in zebrafish was directly verified by the differences in melanin distribution on the body surface. The UVB model group of zebrafish showed significant melanin deposition on the skin, especially in the head region where it exhibited a typical patchy distribution. The experimental groups treated with *Lactobacillus plantarum* G4 fermentation supernatant showed different pigment distribution characteristics, with a significantly reduced degree of pigmentation on the body surface, and the melanin mainly exhibiting a scattered dotted distribution. The zebrafish treated with 0.2% *Lactobacillus plantarum* G4 fermentation supernatant had the lowest melanin content, with a very light body color; followed by the 0.1% *Lactobacillus plantarum* G4 fermentation supernatant treatment group (…). Figure 6 ).

[0110] 3. Determination of melanin and ROS content in zebrafish embryos

[0111] Reactive oxygen species (ROS), including hydrogen peroxide, superoxide, and hydroxyl radicals, are common byproducts of aerobic metabolism in all cells and important mediators of inflammation. The antioxidant stress effect of *Lactobacillus plantarum* G4 fermentation supernatant showed that, in terms of ROS scavenging, with increasing *Lactobacillus plantarum* G4 fermentation supernatant concentration, the green fluorescence in zebrafish initially darkened and then brightened, while ROS levels initially decreased and then increased. Figure 7 0.1% of Lactobacillus plantarum G4 fermentation supernatant reduced the fluorescence intensity in zebrafish to the lowest level, but 0.2% of Lactobacillus plantarum G4 fermentation supernatant significantly increased the fluorescence intensity.

[0112] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* G4 with whitening effects, characterized in that... The Lactobacillus plantarum G4 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 34672 and deposit date of May 26, 2025.

2. The fermentation method of *Lactobacillus plantarum* G4 as described in claim 1, characterized in that, Includes the following steps: Lactobacillus plantarum G4 was inoculated into MRS medium and cultured statically at 37°C for 24 h. It was then passaged once at an inoculum size of 2%, centrifuged at 8000×g for 10 min at 4°C, and the supernatant and bacterial cells were collected separately.

3. A microbial agent with whitening effects, characterized in that, The microbial agent contains Lactobacillus plantarum G4 as described in claim 1 and / or its fermentation products.

4. A whitening composition, characterized in that, The composition comprises Lactobacillus plantarum G4 as described in claim 1 and / or its fermentation products.

5. The application of Lactobacillus plantarum G4 as described in claim 1, or the microbial agent as described in claim 3, in the preparation of skin whitening products.

6. The application according to claim 5, characterized in that, The product in question is a cosmetic, food, or medicine.

7. The application according to claim 5 or 6, characterized in that, The product is one or more of the following: (1) The product can inhibit tyrosinase activity; (2) The product has antioxidant function.

8. The application according to claim 7, characterized in that, The antioxidant function is manifested in the scavenging of ABTS free radicals, DPPH free radicals, or OH free radicals.

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