Lactobacillus rhamnosus YYS-B2 with tyrosinase and elastase inhibitory activity and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a *Lactobacillus rhamnosus* YYS-B2 strain with tyrosinase and elastase inhibitory activity and its applications. *Lactobacillus rhamnosus* (… Lacticaseibacillusrhamnosus YYS-B2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28164. The *Lactobacillus rhamnosus* YYS-B2 provided by this invention exhibits excellent acid and bile salt resistance, enabling it to successfully reach the intestines and exert its probiotic effects. Furthermore, *Lactobacillus rhamnosus* YYS-B2 possesses inhibitory abilities against tyrosinase, elastase, and hyaluronidase, as well as high free radical scavenging capacity. When applied to probiotic products, it can effectively improve skin pigmentation, enhance skin's anti-wrinkle and moisturizing abilities, and help slow down skin aging.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of Lactobacillus rhamnosus YYS-B2 with tyrosinase and elastase inhibitory activity and its applications. Background Technology

[0002] As people's material lives become increasingly affluent, their attention to beauty and health is also rising, with skin anti-aging being a consistently hot research topic. Skin aging is caused by long-term exposure to ultraviolet radiation. Specifically, ultraviolet radiation accelerates the production of a large number of free radicals in the skin. The accumulation of free radicals damages skin cells, accelerates the degradation of collagen and elastin fibers, and consequently leads to sagging skin, wrinkles, age spots, and other signs of aging.

[0003] Meanwhile, skin aging is also influenced by endogenous factors, such as collagen degradation and hyaluronidase activity. Collagen is the main structural protein of the skin and is crucial for maintaining skin elasticity and firmness. However, with age and adverse effects from the external environment, collagen gradually degrades, causing the skin to lose elasticity and firmness, resulting in wrinkles and sagging. Hyaluronic acid is a natural polysaccharide that can absorb and retain a large amount of water, keeping the skin hydrated and moisturized. However, hyaluronidase degrades hyaluronic acid, leading to reduced skin moisture, loss of moisturizing ability, and causing dryness and aging.

[0004] Chinese patent document CN 110964673A discloses a strain of *Pediococcus pentosaceus* with whitening and antioxidant effects. Its extracellular supernatant exhibits a DPPH free radical scavenging rate of 81.89% and a tyrosinase inhibition rate of 64.82%. The resulting plant fermentation broth containing *Pediococcus pentosaceus* CCTCC M 2019323 effectively scavenges free radicals, promotes skin renewal, and reduces melanin synthesis. Experiments show that it exhibits high activity only in free radical scavenging and tyrosinase inhibition.

[0005] Therefore, it is necessary to develop a natural probiotic that can intervene in multiple skin aging factors to truly achieve comprehensive and integrated skin care effects. Summary of the Invention

[0006] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Lactobacillus rhamnosus* YYS-B2 with tyrosinase and elastase inhibitory activities. It has a highly efficient ability to scavenge free radicals and can inhibit the activities of elastase, tyrosinase, and hyaluronidase, achieving anti-wrinkle, anti-aging, moisturizing, and whitening effects, and has significant application value.

[0007] The *Lactobacillus rhamnosus* YYS-B2 provided by this invention is derived from breast milk and is classified as *Lactobacillus rhamnosus*. Its Latin scientific name is: Lacticaseibacillusrhamnosus It is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 28164, deposit date: August 14, 2023.

[0008] Among them, Lactobacillus rhamnosus YYS-B2 can inhibit tyrosinase, prevent the production of melanin, and has a whitening function.

[0009] Lactobacillus rhamnosus YYS-B2 can inhibit elastase, has anti-wrinkle function, and can increase skin elasticity.

[0010] Lactobacillus rhamnosus YYS-B2 can inhibit hyaluronidase and has moisturizing function.

[0011] Lactobacillus rhamnosus YYS-B2 can scavenge free radicals, has antioxidant function, and can prevent oxidative damage.

[0012] The present invention also provides the application of Lactobacillus rhamnosus YYS-B2 as described above in the preparation of probiotic products.

[0013] The present invention also provides the application of Lactobacillus rhamnosus YYS-B2 as described above in the preparation of whitening, anti-wrinkle, anti-aging and antioxidant products.

[0014] The present invention also provides a freeze-dried product, the components of which include Lactobacillus rhamnosus YYS-B2 as described above.

[0015] Furthermore, the viable count of the *Lactobacillus rhamnosus* YYS-B2 is not less than 1 × 10⁻⁶. 11 cfu / g.

[0016] This invention also provides a method for preparing a freeze-dried product, comprising the following preparation steps:

[0017] 1) Preparation of Lactobacillus rhamnosus seed culture YYS-B2;

[0018] 2) Seed culture expansion;

[0019] 3) Seed culture fermentation to obtain fermentation broth;

[0020] 4) Centrifuge the fermentation broth to obtain bacterial cells;

[0021] 5) After mixing the bacterial cells with the freeze-drying protectant, emulsify and embed them to obtain an emulsion;

[0022] 6) The emulsion is freeze-dried and pulverized to obtain freeze-dried Lactobacillus rhamnosus YYS-B2 bacterial powder.

[0023] The present invention also provides a microbial agent, wherein the components of the microbial agent comprise the freeze-dried product as described above, and / or, the components of the microbial agent comprise Lactobacillus rhamnosus YYS-B2 as described above.

[0024] Furthermore, the microbial agent also includes one or more of the following: prebiotics, fillers, acidulants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, and filter aids.

[0025] Based on the above characteristics, the Lactobacillus rhamnosus YYS-B2 provided by the present invention has the following beneficial effects:

[0026] The *Lactobacillus rhamnosus* YYS-B2 provided by this invention has a highly efficient ability to scavenge free radicals. It can inhibit the activity of elastase, tyrosinase, and hyaluronidase. Its tyrosinase inhibition rate can reach (79.2±1.69%), DPPH free radical scavenging rate can reach (83.1±1.28)%, ABTS free radical scavenging rate can reach (78.5±1.53)%, reducing power can reach (47.31±4.62)%, elastase inhibition rate can reach (58.60±2.82)%, and hyaluronidase activity inhibition can reach (73.0±1.20)%. At the same time, YYS-B2 has good acid and bile salt resistance, and can be applied to probiotic products and whitening products, providing new directions and more possibilities for the development of whitening products. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The colony morphology of Lactobacillus rhamnosus YYS-B2 provided in this embodiment of the invention;

[0029] Figure 2 A phylogenetic tree of Lactobacillus rhamnosus YYS-B2 based on the 16S rRNA sequence provided in this embodiment of the invention;

[0030] Figure 3The effect of Lactobacillus rhamnosus YYS-B2 on superoxide dismutase (SOD) activity in mouse serum and liver, as provided in this embodiment of the invention;

[0031] Figure 4 The effects of Lactobacillus rhamnosus YYS-B2 on the activity of glutathione peroxidase (GSH-PX) in mouse serum and liver provided in this embodiment of the invention;

[0032] Figure 5 The effects of Lactobacillus rhamnosus YYS-B2 on malondialdehyde (MDA) activity in mouse serum and liver, as provided in this embodiment of the invention;

[0033] Figure 6 The effects of Lactobacillus rhamnosus YYS-B2 and collagen on skin moisture content in mice, as provided in this embodiment of the invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] This invention provides a Lactobacillus rhamnosus ( Lacticaseibacillusrhamnosus YYS-B2 is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 28164.

[0036] Example 1: Isolation and Identification of Lactobacillus rhamnosus YYS-B2

[0037] 1.1 Separation

[0038] Aseptic sampling was performed on breast milk using the plate spread method. 5 mL of breast milk sample was placed in a sterile homogenizing bag, labeled, and 45 mL of 0.85% physiological saline was added. The mixture was then thoroughly agitated to obtain the sample. 100 μL of the sample was then serially diluted 10-fold. -4 10 -5 10 -6 100 μL of the sample was spread onto an MRS plate containing 2.5% CaCO3 and incubated upside down at 37°C for 24 h. Colonies with good growth and large calcium dissolution zones were selected and repeatedly isolated and purified using the streak plate method until single colonies were obtained (see [link to sample]). Figure 1 The isolated strain was named YYS-B2, numbered, and stored in a -80℃ culture medium with glycerol.

[0039] The colony morphology of the isolated and purified Lactobacillus rhamnosus YYS-B2 is as follows: the colony surface is raised, milky white, round, and smooth.

[0040] The formula for MRS liquid culture medium is as follows:

[0041] 20.0 g glucose, 10.0 g tryptone, 10.0 g beef extract, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate monohydrate, 1 L deionized water, pH=6.5 (with 1.5% agar added for MRS solid medium).

[0042] 1.2 Physiological and Biochemical Characteristics

[0043] The screened and purified strain YYS-B2 was subjected to Gram staining and catalase tests, and its physiological and biochemical indicators were measured. The results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the bacterial species. The tests showed that the screened strain YYS-B2 stained purple, indicating a Gram-positive result. Its cells were rod-shaped, catalase-negative, and did not form spores.

[0044] 1.3 Identification of 16S rRNA

[0045] DNA was extracted from YYS-B2 according to the instructions of the bacterial DNA extraction kit, and PCR amplification of the 16S rRNA gene was performed using the DNA as a template.

[0046] Universal primers are used for amplification:

[0047] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1).

[0048] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0049] PCR reaction system: DNA 2 μL, 27F 2 μL, 1492R 2 μL, Premix Ex Taq 25 μL, ddH2O 19 μL.

[0050] PCR reaction conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 5 min.

[0051] After extracting the DNA genome from YYS-B2, 16S rRNA fragments were amplified, and the PCR amplification products were then sent for DNA sequencing (Guangzhou Qingke Biotechnology Co., Ltd.). The sequencing results were used to search for similar sequences in the NCBI database using Blast software. The obtained sequences were compared with the 16S rRNA gene sequences of related species obtained from gene banks, and a phylogenetic tree was constructed using Mega11.0 software (see [link to documentation]). Figure 2 ).

[0052] The results of 16S rRNA gene sequencing are as follows:

[0053]

[0054] Based on a comprehensive analysis of the strain's colony and cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and with reference to Bergey's Manual of Systematic Bacteriology, 8th Edition, the strain was identified as *Lactobacillus rhamnosus*. Lacticaseibacillusrhamnosus ).

[0055] Example 2: Preparation of freeze-dried Lactobacillus rhamnosus YYS-B2 bacterial powder

[0056] S1. Inoculate Lactobacillus rhamnosus YYS-B2 into a test tube containing seed culture medium for activation, and incubate in a constant temperature incubator for 20 h to obtain the first-grade seed solution.

[0057] S2. Inoculate the primary seed culture into an Erlenmeyer flask containing seed culture medium at a volume of 3% (volume percentage), and incubate in a constant temperature incubator for 18 hours to obtain the secondary seed culture.

[0058] S3. Inoculate the secondary seed solution into an Erlenmeyer flask containing seed culture medium at a volume of 5% (volume percentage), and incubate in a constant temperature incubator for 16 hours to obtain the tertiary seed solution.

[0059] S4. Inoculate the tertiary seed culture into a fermenter containing fermentation medium at an inoculation amount of 5% (volume percentage), with a preferred stirring speed of 50 r / min, an initial fermentation pH of 6.0, and incubate at 37℃ for 8 h.

[0060] S5, viable cell count in the fermentation broth ≥ 3.0 × 10⁻⁶ 9 When the cfu / mL level is reached, stop fermentation and collect the cells by centrifugation (8000 r / min, 15 min).

[0061] S6. The bacterial cells and the freeze-drying protectant were emulsified and encapsulated at a mass ratio of 1:0.6. The emulsification temperature was 20℃ and the emulsification time was 20 min.

[0062] S7. Pre-freeze the emulsion for 6 hours, then freeze-dry it under vacuum at -30℃ for 30 hours at a vacuum degree of 0.2 mbar. Pulverize to obtain *Lactobacillus rhamnosus* YYS-B2 freeze-dried bacterial powder with a viable count of 3.0 × 10⁻⁶. 11 cfu / g.

[0063] The seed culture medium, by weight percentage, consists of: 5% glucose, 3% peptone, 1.5% yeast extract, 1% beef extract, 0.5% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.06% manganese sulfate, 0.1% Tween 80, and the balance being water, with a pH of 6.8.

[0064] The fermentation medium, by weight percentage, consists of: 3.6% glucose, 2.7% soybean peptone, 2% yeast extract, 1% beef extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% Tween 80, and the balance being water.

[0065] The freeze-drying protectant comprises, by weight percentage: 10% skim milk powder, 0.5% glycerin, and the balance being water.

[0066] Example 3: Experiment on the acid and bile salt tolerance of Lactobacillus rhamnosus YYS-B2

[0067] 3.1 Acid resistance test

[0068] Weigh 0.2 g NaCl and 0.35 g pepsin and dissolve them in an appropriate amount of distilled water to prepare three portions. Adjust the pH to 1.5, 2.5 and 3.5 respectively with 1.0 mol / L hydrochloric acid, and dilute to the mark with 100 mL volumetric flasks. Inoculate the freeze-dried Lactobacillus rhamnosus YYS-B2 bacterial powder obtained in Example 2 into simulated gastric acid solutions with different pH values ​​at an inoculation rate of 0.3% (w / v). After incubation at 37℃ for 0, 2 and 4 h, dilute 10-fold serially, shake well, and then take 100 μL of the bacterial solution, pour it onto a plate, and incubate at 37℃ for 24 h. Calculate the viable count of Lactobacillus rhamnosus YYS-B2. The results are shown in Table 1.

[0069] Table 1

[0070]

[0071] Table 1 shows that the viable count of *Lactobacillus rhamnosus* YYS-B2 varied in simulated gastric fluid at different pH values. As the pH increased, the viable count and survival rate continuously improved. However, in simulated gastric fluid at the same pH, the viable count decreased with increasing treatment time. Nevertheless, the viable count remained high after 2 hours of treatment in simulated gastric fluid at different pH values, indicating that *Lactobacillus rhamnosus* YYS-B2 maintains a high survival rate after digestion by human gastric fluid and exhibits excellent tolerance to gastric acid.

[0072] 3.2 Bile Salt Tolerance Test

[0073] The freeze-dried Lactobacillus rhamnosus YYS-B2 bacterial powder obtained in Example 2 was inoculated at an inoculum of 0.3% (w / v) into porcine bile salt solutions with mass concentrations of 0.03 g / mL, 0.3 g / mL, and 0.5 g / mL, respectively. After being treated in a water bath at 37°C for 0, 2, and 4 h, the solutions were serially diluted 10-fold, shaken thoroughly, and 100 μL of the bacterial solution was taken and poured onto a plate. After incubation at 37°C for 24 h, the viable count of Lactobacillus rhamnosus YYS-B2 was calculated, and the results are shown in Table 2.

[0074] Table 2

[0075]

[0076] Table 2 shows that the viable count and survival rate of *Lactobacillus rhamnosus* YYS-B2 varied under different initial concentrations of bile salts in simulated intestinal fluid. The viable count gradually decreased with increasing concentrations of bile salts and treatment time. However, after 2 hours of treatment at a bile salt concentration closest to that in humans (typically 0.03–0.3 g / mL in the human small intestine), a high number of viable bacteria remained, indicating that *Lactobacillus rhamnosus* YYS-B2 retains a significant number of viable bacteria after digestion in the human gastrointestinal tract.

[0077] In summary, the Lactobacillus rhamnosus YYS-B2 provided by this invention exhibits excellent tolerance to gastric acid and bile salts, enabling it to successfully reach the intestines, regulate intestinal flora, and exert probiotic effects.

[0078] Example 4: Experiment on the inhibition of tyrosinase by Lactobacillus rhamnosus YYS-B2

[0079] 4.1 Solution Preparation

[0080] L -Tyrosine solution: Accurately weigh 18.12 mg L -Tyrosine was dissolved in 50 mmol / L PBS buffer (pH 6.8) and brought to a final volume of 50 mL to obtain 2 mmol / L PBS. L -Tyrosine solution, store this solution at 4°C protected from light.

[0081] Tyrosinase solution: Accurately weigh 3.00 mg of tyrosinase, dissolve it in 50 mmol / L PBS buffer (pH 6.8), and bring the volume to 100 mL to obtain a 40 μg / mL tyrosinase solution. This solution should be stored at -20°C for later use and thawed at 4°C before use.

[0082] Test sample solution: Inoculate 3% (v / v) of Lactobacillus rhamnosus YYS-B2 seed culture into MRS liquid medium (formula as above), ferment at 37℃ for 24 h, centrifuge at 8000 r / min for 10 min, discard the supernatant, resuspend in PBS buffer, and quantify the bacterial concentration to 1x10⁻⁶. 8 cfu / mL.

[0083] 4.2 Detection and analysis of tyrosinase inhibition in samples

[0084] Add 80 μL of the sample solution to a 96-well microplate, then add 100 μL of preheated (37°C) microplate solution. L Add 2 mmol / L tyrosine solution, shake to mix, and incubate at 37°C for 10 minutes. Add 40 μL tyrosinase solution (40 μg / mL), mix well, and continue the reaction at 37°C for 10 minutes. The dosages of each reactant are shown in Table 3. The absorbance of the reaction mixture at 492 nm was rapidly measured using a microplate reader. The inhibition rate of tyrosinase by *Lactobacillus rhamnosus* YYS-B2 is calculated as follows:

[0085] Tyrosinase inhibition rate = [1 - (CD) / (AB)] × 100%

[0086] Where A is the absorbance value of the blank group, B is the absorbance value of the control blank group, C is the absorbance value of the control group, and D is the absorbance value of the sample group.

[0087] By wavelength OD 492nm The test results showed that Lactobacillus rhamnosus YYS-B2 inhibited tyrosinase by 79.2±1.69%.

[0088] The Lactobacillus rhamnosus YYS-B2 provided by this invention has a good inhibitory effect on tyrosinase and can be applied to the development of skin whitening products.

[0089] Table 3

[0090]

[0091] Example 5: Antioxidant capacity experiment of Lactobacillus rhamnosus YYS-B2

[0092] 5.1 DPPH Free Radical Scavenging Rate Determination Experiment

[0093] 5.1.1 Solution Preparation

[0094] DPPH solution: Dissolve 0.002 g of DPPH in 50 mL of ethanol each time, and store in the dark. Dilute with ethanol to an absorbance of about 0.7 before use, and prepare fresh each time.

[0095] 5.1.2 Detection and analysis of DPPH free radical scavenging in samples

[0096] Inoculate 3% (v / v) of Lactobacillus rhamnosus YYS-B2 seed culture into MRS liquid medium (formula as above) and ferment at 37°C for 24 h. Centrifuge at 8000 r / min for 10 min, discard the supernatant, resuspend in PBS buffer, and quantify the bacterial concentration to 1 x 10⁻⁶. 8The cfu / mL concentration was used to obtain the sample solution. The experiment was divided into three groups: A, B, and C. The dosage of each reactant is shown in Table 4. Each reactant was added sequentially to a standard 96-well plate, and after incubation at room temperature in the dark for 30 min, the absorbance of the reaction mixture at 517 nm was measured. The formula for calculating the DPPH free radical scavenging rate of *Lactobacillus rhamnosus* YYS-B2 is as follows:

[0097] DPPH free radical scavenging rate = [1 - (AC) / B] × 100%

[0098] Where A is the absorbance of the sample group, B is the absorbance of the blank group, and C is the absorbance of the control group.

[0099] By wavelength OD 517nm The test results showed that Lactobacillus rhamnosus YYS-B2 could scavenge DPPH free radicals at a rate of (83.1±1.28)%.

[0100] The Lactobacillus rhamnosus YYS-B2 provided by this invention has a good DPPH free radical scavenging effect and can be applied to the development of antioxidant products.

[0101] Table 4

[0102]

[0103] 5.2 ABTS Free Radical Scavenging Rate Determination Experiment

[0104] 5.2.1 Solution Preparation

[0105] ABTS stock solution (7 mmol / L): Weigh 0.0384 g of ABTS and dilute to 10 mL with distilled water.

[0106] K2S2O8 stock solution (2.45 mmol / L): Weigh 0.0066 g of K2S2O8 and dilute to 10 mL with distilled water.

[0107] ABTS working solution: Mix ABTS stock solution and K2S2O8 stock solution at a 1:1 ratio, let stand for 12-16 h, and prepare ABTS working solution.

[0108] 5.2.2 Detection and analysis of ABTS free radical scavenging in samples

[0109] 3% (v / v) of Lactobacillus rhamnosus YYS-B2 seed culture was inoculated into MRS liquid medium (formula as above) and fermented at 37°C for 24 h. After centrifugation at 8000 r / min for 10 min, the culture was resuspended and the cell concentration was quantified to 1 x 10⁻⁶. 8cfu / mL. Take an appropriate amount of ABTS working solution and dilute it with PBS until the absorbance at 734 nm is 0.7. The experiment was divided into three groups: A, B, and C. The dosage of each reactant is shown in Table 5. Add each reactant sequentially to a standard 96-well plate, incubate at room temperature in the dark for 6 min, and then measure the absorbance of the reaction mixture at 734 nm. The formula for calculating the ABTS free radical scavenging rate of Lactobacillus rhamnosus YYS-B2 is as follows:

[0110] ABTS radical scavenging rate = [1 - (AC) / B] × 100%

[0111] Where A is the absorbance of the sample group, B is the absorbance of the blank group, and C is the absorbance of the control group.

[0112] By wavelength OD 734nm The test results showed that Lactobacillus rhamnosus YYS-B2 could scavenge ABTS free radicals at a rate of (78.5±1.53)%.

[0113] The Lactobacillus rhamnosus YYS-B2 provided by this invention has a good ABTS free radical scavenging effect and can be applied to the development of antioxidant products.

[0114] Table 5

[0115]

[0116] 5.3 Experiment on the reducing ability of the strain

[0117] 3% (v / v) of Lactobacillus rhamnosus YYS-B2 seed culture was inoculated into MRS liquid medium (formula as above) and fermented at 37°C for 24 h. After centrifugation at 8000 r / min for 10 min, the culture was resuspended and the cell concentration was quantified to 1 x 10⁻⁶. 8 CFU / mL was used to obtain a suspension of *Lactobacillus rhamnosus* YYS-B2. 0.5 mL of the *Lactobacillus rhamnosus* YYS-B2 suspension was taken, and 0.5 mL of 0.2 mol / L, pH 6.6 PBS solution and 0.5 mL of 1% potassium ferricyanide were added. The mixture was incubated in a 50℃ water bath for 20 min, then rapidly cooled. 0.5 mL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 3500 rpm for 5 min. 1 mL of the supernatant was collected, and 1 mL of distilled water and 0.1% ferric chloride were added. The mixture was mixed thoroughly, and the absorbance was measured at 700 nm after 10 min. s The same treatment was performed using an equal volume of PBS instead of the bacterial suspension, and the absorbance value was measured as A. b .

[0118] Restoration capability = [(A s -A b ) / Ab ]×100%

[0119] By wavelength OD 700nm The test results showed that the reducing power of Lactobacillus rhamnosus YYS-B2 was (47.31±4.62)%.

[0120] The Lactobacillus rhamnosus YYS-B2 provided by this invention has good redox capabilities and can be applied to the development of antioxidant products.

[0121] Example 6: Experiment on the inhibition of elastase by Lactobacillus rhamnosus YYS-B2

[0122] 3% (v / v) of Lactobacillus rhamnosus YYS-B2 seed culture was inoculated into MRS liquid medium and fermented at 37°C for 24 h. After centrifugation at 8000 r / min for 10 min, the supernatant was discarded, and the culture was resuspended in PBS buffer to quantify the bacterial concentration to 1 x 10⁻⁶. 8 The sample solution was obtained by diluting cfu / mL. A 1.015 mmol / L N-methoxysuccinyl-propionyl-propionyl-prolyl-valine p-nitroaniline solution was prepared using 0.1 mol / L Tris-HCl buffer (pH = 8.0). The elastase inhibition was evaluated using the following steps: 10 μL of sample solution and 130 μL of N-methoxysuccinyl-propionyl-propionyl-prolyl-valine p-nitroaniline solution were added to an ELISA plate and incubated at 25 °C for 5 min. Then, 15 μL of elastase solution (0.5 U / mL) was added, and the plate was incubated for another 30 min. The absorbance at 410 nm was measured for each group and recorded as A0. An equal volume of Tris-HCl buffer was used to replace the sample solution, and the absorbance at 410 nm was measured using the same method and recorded as A1. Using an equal volume of Tris-HCl buffer instead of the elastase solution, the absorbance at 410 nm was measured according to the above method and denoted as A2, as shown in Table 6. The elastase inhibition rate was calculated using the following formula:

[0123] Elastase inhibition rate = [A1 - (A0 - A2)] / A1 × 100%

[0124] By wavelength OD 410nm The test results showed that Lactobacillus rhamnosus YYS-B2 inhibited elastase activity at a rate of (58.60±2.82)%.

[0125] The Lactobacillus rhamnosus YYS-B2 provided by this invention has a good anti-wrinkle effect and can be applied to the development of anti-wrinkle and anti-aging products.

[0126] Table 6

[0127]

[0128] Example 7: Experiment on the inhibition of hyaluronidase by Lactobacillus rhamnosus YYS-B2

[0129] 7.1 Solution Preparation

[0130] Bacterial suspension: Inoculate 3% (v / v) of Lactobacillus rhamnosus YYS-B2 seed culture into MRS liquid medium and ferment at 37℃ for 24 h. Centrifuge at 8000 r / min for 10 min, discard the supernatant, resuspend in PBS buffer, and quantify the bacterial concentration to 1 x 10⁻⁶. 8 CFU / mL was used to obtain a bacterial suspension.

[0131] Acetate buffer: 4.8 mL of solution A (0.2 mol / L acetic acid and 11.55 mL of glacial acetic acid dissolved in 1 L of distilled water) and 45.2 mL of solution B (0.2 mol / L acetic acid, 16.4 g of anhydrous sodium acetate or 27.2 g of sodium acetate trihydrate dissolved in 1 L of distilled water) were mixed and diluted to 100 mL to prepare an acetate buffer with pH=5.6.

[0132] Hyaluronidase: Concentration 600 U / mL, prepare fresh before use, using acetate buffer as solvent;

[0133] Sodium hyaluronate solution: concentration 0.5 mg / mL, prepared once, using acetate buffer as solvent;

[0134] Acetylacetone solution: Mix 50 mL of 1.0 mol / L sodium carbonate solution and 3.5 mL of acetylacetone solution thoroughly (prepare immediately before use).

[0135] P-DAB colorimetric reagent: Dissolve 0.8 g of p-dimethylaminobenzaldehyde in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol, mix thoroughly. (Prepare immediately before use)

[0136] 7.2 Detection and analysis of hyaluronidase inhibition in samples

[0137] Lactobacillus rhamnosus YYS-B2 strain was activated to the second generation according to the Elson-Morgan modified method. 0.1 mL of 2.5 mmol / L CaCl2 and 0.5 mL of hyaluronidase were added to the test tube, and the mixture was treated at 37°C for 20 min. Then, 0.5 mL of bacterial suspension was added, and the mixture was treated at 37°C for 20 min. Next, 0.5 mL of sodium hyaluronate solution was added, and the mixture was treated at 37°C for 30 min. After standing at room temperature for 5 min, 0.5 mL of acetylacetone solution and 0.1 mL of 0.4 mol / L NaOH solution were added, and the mixture was boiled in a water bath for 15 min, followed immediately by an ice bath for 5 min. Finally, 1 mL of P-DAB chromogenic reagent was added, and the mixture was incubated at room temperature for 20 min. The absorbance was measured at 530 nm after the reaction. The inhibition rate was calculated as follows:

[0138] Hyaluronidase inhibition rate = [(AB) - (CD)] / (AB) × 100%

[0139] Where A is the absorbance value of the control group (equal volume of acetate buffer replacing bacterial suspension), B is the absorbance value of the control blank group (equal volume of acetate buffer replacing enzyme solution and bacterial suspension), C is the absorbance value of the sample group, and D is the absorbance value of the sample blank group (equal volume of acetate buffer replacing enzyme solution).

[0140] By wavelength OD 530nm The test results showed that fermentation with Lactobacillus rhamnosus YYS-B2 significantly reduced the activity of hyaluronidase, with an inhibition rate of (73.0±1.20)%.

[0141] The Lactobacillus rhamnosus YYS-B2 provided by this invention has excellent ability to inhibit hyaluronidase and can be used to prepare moisturizing products.

[0142] Example 8: Experiment on the enhancement of antioxidant capacity in mice by Lactobacillus rhamnosus YYS-B2

[0143] Healthy male BALB / c mice were randomly divided into an experimental group and a control group, with 20 mice in each group. The lyophilized Lactobacillus rhamnosus YYS-B2 bacterial powder prepared in Example 2 was mixed with 0.9% physiological saline to prepare a 1% bacterial powder suspension with a concentration of 3.0 × 10⁻⁶. 9 Mice in the control group were given 0.2 mL of 0.9% saline solution at a concentration of cfu / mL. This treatment was repeated daily for 4 weeks. At the end of the experiment, serum and liver tissue samples were collected from the mice. The relevant indicators were measured using SOD, GSH-PX, and MDA activity assay kits.

[0144] Results of mouse serum oxidative stress activation assay as follows Figure 3 , Figure 4 and Figure 5As shown, compared with the control group, the experimental group mice showed significantly increased SOD activity and GSH-PX in serum and liver, and significantly decreased MDA content. This indicates that consuming freeze-dried Lactobacillus rhamnosus YYS-B2 bacterial powder can significantly improve SOD activity and GSH-PX activity, reduce MDA content, and enhance the body's antioxidant capacity.

[0145] Example 9: Effects of Lactobacillus rhamnosus YYS-B2 and collagen peptide fermentation broth on mouse skin

[0146] Healthy SKH-1 hairless mice were randomly divided into three groups: a normal group, a YYS-B2 group, and a YYS-B2 group + collagen peptide group, with 10 mice in each group. The lyophilized Lactobacillus rhamnosus YYS-B2 bacterial powder prepared in Example 2 was mixed with 0.9% physiological saline to prepare a 1% bacterial powder suspension with a concentration of 3.0 × 10⁻⁶. 9 CFU / mL; the normal control group received 0.2 mL of 0.9% saline daily, the YYS-B2 group received 0.2 mL of *Lactobacillus rhamnosus* YYS-B2 bacterial suspension, and the YYS-B2 group + collagen peptide group received 0.2 mL of *Lactobacillus rhamnosus* YYS-B2 bacterial suspension containing 5% collagen peptides. The experiment lasted for 4 weeks. In all experiments, food and water were freely available, and food intake and body weight were measured daily. Skin moisture content was measured weekly to assess the results.

[0147] Experimental results are presented (see below) Figure 6 Compared to the normal control group, mice fed only with Lactobacillus rhamnosus YYS-B2 showed increased skin moisture content, while mice fed with a mixture of collagen peptides and Lactobacillus rhamnosus YYS-B2 powder showed a significantly greater increase in skin moisture. This indicates that Lactobacillus rhamnosus YYS-B2 has a skin moisturizing effect, and its combination with collagen peptides enhances this moisturizing effect even more.

[0148] Example 10: Lactobacillus rhamnosus YYS-B2 and collagen peptide fermentation enzyme

[0149] Fresh fruits such as pineapple, kiwi, and lemon are added to a jar with 20% sugar and sealed for three weeks to allow the juices to be released. Then, Lactobacillus rhamnosus YYS-B2 is added and fermented for three months.

[0150] The composition by weight percentage is as follows: Lactobacillus rhamnosus YYS-B2 10%, xylitol 6%, sodium hyaluronate 0.15%, fish collagen 5%, GABA 0.3%, nicotinamide 0.02%, fermented pineapple liquid 0.5%, fermented kiwi liquid 0.5%, fermented lemon liquid 0.5%, vitamin E 0.025%, vitamin C 0.025%, malic acid 0.025%, citric acid 0.025%, sodium carboxymethyl cellulose 0.025%, potassium sorbate 0.015%, sucralose 0.03%, and the balance being water.

[0151] This formula helps promote gut and skin health, while providing more beauty and skincare nutrients.

[0152] Example 11: Preparation of probiotic products using Lactobacillus rhamnosus YYS-B2

[0153] The probiotic product is a combination of Lactobacillus rhamnosus YYS-B2 and other ingredients;

[0154] The viable count of *Lactobacillus rhamnosus* YYS-B2 in the product is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0155] The ingredients include one or more of the following: prebiotics, fillers, acidulants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, and filter aids.

[0156] The dosage form of the probiotic product can be a solid beverage, liquid beverage, compressed candy, granules, capsules, tablets, pills, or oral liquid.

[0157] In this embodiment, a probiotic product in the form of a solid beverage is provided, namely Lactobacillus rhamnosus YYS-B2 solid beverage, which, by weight, includes 10 parts of active bacteria powder, 24.8 parts of xylitol, 24 parts of whole milk powder, 12 parts of fermented mulberry powder, 23 parts of maltodextrin, 5.6 parts of galactooligosaccharides, 0.1 parts of fructooligosaccharides and 0.5 parts of anhydrous citric acid.

[0158] The preparation method of this solid beverage is as follows:

[0159] S1. The Lactobacillus rhamnosus YYS-B2 strain is cultured in liquid culture medium, the bacterial cells are collected and washed, excipients are added, and dried to prepare active bacterial powder.

[0160] S2. After thoroughly mixing the active bacteria powder, xylitol, whole milk powder, fermented mulberry powder, maltodextrin, galactooligosaccharides, fructooligosaccharides, and anhydrous citric acid, a viable count of *Lactobacillus rhamnosus* YYS-B2 is obtained, which is not less than 1 × 10⁻⁶. 8 Solid beverage with CFU / g.

[0161] Experimental procedures not explicitly stated in all embodiments are conventional experimental procedures in the art, and the reagents or instruments involved can be obtained from legitimate channels.

[0162] The significant difference between this probiotic and other products lies in its comprehensive functional performance. Compared to other probiotic whitening preparations on the market, Lactobacillus rhamnosus YYS-B2 alone can effectively achieve whitening effects. It possesses multiple functions including highly effective whitening, anti-oxidation, anti-wrinkle, and moisturizing. Lactobacillus rhamnosus YYS-B2 exhibits outstanding functions, including a tyrosinase inhibition rate of up to 79.2%, which can effectively prevent the formation of pigment; a DPPH of 83.1% and an ABTS free radical scavenging rate of 78.5% and a reducing capacity of 47.3%, which helps prevent and repair skin damage caused by oxidation; it has 56.8% elastase inhibitory activity, which helps maintain skin elasticity and reduce wrinkles and skin aging; this probiotic also shows a 73.0% inhibition of hyaluronidase activity, which helps maintain the skin's moisture balance and has a significant moisturizing effect. Mouse experiments have confirmed that the moisturizing ability is better when the probiotic combines with collagen, and it can effectively increase the skin's moisture content. Given the multiple benefits of this probiotic in beauty and skincare, developing it into corresponding probiotic products is feasible. This probiotic achieves whitening, anti-aging, anti-wrinkle, and moisturizing effects through natural biological pathways, with no side effects, making it more in line with modern consumers' demands for healthy beauty.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus YYS-B2, characterized in that, Its accession number is CGMCC No. 28164.

2. The application of Lactobacillus rhamnosus YYS-B2 as described in claim 1 in the preparation of probiotic products.

3. The application of Lactobacillus rhamnosus YYS-B2 as described in claim 1 in the preparation of whitening, anti-wrinkle, anti-aging and / or antioxidant products.

4. A freeze-dried product, characterized in that: Its components include Lactobacillus rhamnosus YYS-B2 as described in claim 1.

5. The freeze-dried product according to claim 4, characterized in that: The viable count of the *Lactobacillus rhamnosus* YYS-B2 is not less than 1×10⁻⁶. 11 cfu / g.

6. A microbial agent, characterized in that: The components of the microbial agent include the freeze-dried product as described in claim 4 or 5, or the components of the microbial agent include Lactobacillus rhamnosus YYS-B2 as described in claim 1.

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