Lactobacillus paracasei B20 derived from plateau region and capable of inhibiting melanin and application of lactobacillus paracasei B20

Abstract

The invention belongs to the technical field of microorganisms, and discloses a Lactobacillus paracasei B20 for inhibiting melanin from a plateau area and application of the Lactobacillus paracasei B20, the Lactobacillus paracasei B20 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32434, and the preservation date is October 31, 2024. Metabolite of the lactobacillus paracasei B20 comprises phenylalanine, p-hydroxycinnamic acid and the like, has strong tyrosinase inhibition effect and anti-ultraviolet ability, and can significantly reduce generation of melanin in skin. In addition, the lactobacillus paracasei B20 also has relatively strong oxidation resistance, has good tolerance in artificial gastric juice and artificial intestinal juice, and can smoothly reach the intestinal tract of a human body. In addition, the lactobacillus paracasei B20 has a relatively obvious growth inhibition effect on skin conditioned pathogens such as staphylococcus aureus and enterococcus faecalis.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a melanin-inhibiting Lactobacillus paracasei B20 originating from plateau areas and an application thereof. Background Art

[0002] Melanin is a pigment produced in specific parts of the skin. It absorbs ultraviolet rays and protects the skin. However, excessive melanin production and accumulation can lead to excessive pigmentation, forming chloasma, freckles, age spots and melanoma, affecting skin color. Melanin synthesis mainly occurs in melanocytes in the skin. Tyrosine undergoes a series of oxidation reactions and is converted into dopaquinone. Dopaquinone is further converted into a variety of intermediates in the cells, which eventually polymerize to form melanin.

[0003] Tyrosinase (EC 1.14.18.1, tyrosinase, TYR), a key core enzyme for melanin synthesis, can specifically catalyze the conversion of tyrosine into dopaquinone, thereby initiating a chain reaction of melanin synthesis. The activity of tyrosinase directly determines the rate and efficiency of melanin synthesis, which in turn affects the depth of skin color. When the skin is exposed to ultraviolet rays, the activity of tyrosinase will be significantly enhanced, prompting the production of a large amount of melanin to resist the damage of ultraviolet rays. This adaptive response is a self-protection mechanism of the body against the external environment. Therefore, the development of inhibitors against tyrosinase has become an important direction of whitening technology. However, most of the existing tyrosinase inhibitors are derived from chemical synthesis, which poses potential safety hazards, and long-term use may cause skin discomfort or allergic reactions.

[0004] In recent years, with the deepening of microbiological research, lactic acid bacteria have gradually emerged in the field of beauty and skin care due to their unique physiological functions and safety. As a kind of probiotics, lactic acid bacteria can not only indirectly affect the skin condition by regulating the balance of intestinal flora and improving the internal environment, but also its metabolites or active ingredients can directly act on the skin, inhibiting melanin synthesis or promoting melanin metabolism, which has unique advantages over traditional whitening products. For example, some lactic acid bacteria can produce substances with antioxidant and anti-inflammatory effects, which help reduce skin inflammation and pigmentation, thereby achieving a whitening effect.

[0005] At present, tyrosinase inhibitors can be divided into natural, semi-synthetic and synthetic ones. Among them, natural inhibitors are mainly isolated from bacteria, fungi and plants, and have broader application prospects due to their low toxicity. In recent years, lactic acid bacteria have also been proven to have inhibitory effects on tyrosinase through their metabolites, and it has been proven that the physiological activity of their fermented extracts is significantly higher than that of unfermented extracts, and their cytotoxic activity is significantly lower than that of unfermented extracts. Therefore, research can focus on the screening of lactic acid bacteria strains, in-depth exploration of the mechanism of action, and provide more efficient and safe solutions for inhibiting melanin synthesis.

[0006] For the relevant literature on the inhibitory effect of existing lactic acid bacteria on tyrosinase, most of them focus on antioxidant and tyrosinase inhibition rate, and some literature analyzes its main active substances through non-targeted metabolomics. For example, in patent CN110964673B "A kind of pentosaceae with high antioxidant and whitening effect and its application", the antioxidant capacity and tyrosinase inhibition activity of pentosaceae were explored. Its DPPH free radical scavenging rate was 81.89%, and its tyrosinase inhibition rate was 64.82%, indicating that the strain has a certain whitening effect. In the document "Screening of lactic acid bacteria with tyrosinase inhibition activity and its component analysis", the inhibition rate of Enterococcus faecium on tyrosinase was 68.9%, and its metabolites were analyzed to find that the main active substance was 5-aminovaleric acid.

[0007] However, the inhibition rate of existing lactic acid bacteria on tyrosinase is generally low, and the whitening effect is limited. Therefore, screening lactic acid bacteria strains with high tyrosinase inhibition ability has become a hot topic in current research. Summary of the invention

[0008] The invention aims to provide a Lactobacillus paracasei and application thereof. The Lactobacillus paracasei is a natural anti-ultraviolet bacteria species in the high-altitude area of ​​Tibet, has strong antioxidant capacity and tyrosinase inhibition capacity, has an inhibitory effect on some pathogenic bacteria, and can be widely used in whitening and skin care products.

[0009] To achieve the above purpose, the solution of the present invention is: a Lacticaseibacillus paracasei B20, which is deposited in the General Microbiology Center of China Microbiological Culture Treasure Management Committee, with a deposit number of CGMCC No.32434 and a deposit date of October 31, 2024.

[0010] Furthermore, the Lactobacillus paracasei B20 is obtained by separation and purification from homemade butter of farmers in Nagqu City, Tibet Autonomous Region.

[0011] Further, the separation and purification method of the Lactobacillus paracasei B20 is as follows: Aseptically sample the butter sample, use the plate spreading method, take 5 g of the sample and put it into a sterile homogenizing bag, add 45 mL of 0.85% saline and homogenize to obtain a sample dilution solution; After thorough mixing, 100 μL of the sample was pipetted in sequence for 10-fold gradient dilution. -3 , 10 -4 , 10 -5 200 μL of sample dilution was pipetted and applied to a plate containing 2.5% CaCO 3 MRS solid medium, inverted culture at 37°C for 48h; Different strains were preliminarily selected according to their morphology, color, and whether they produced calcium-dissolving circles. They were separated and purified repeatedly by the plate streak separation method until all colonies on the MRS solid culture medium maintained a single morphology. Single colonies were picked and placed in MRS liquid culture medium and cultured at 37°C for 12 h. The fermentation broth of the strains was mixed with 50% glycerol in equal proportions and stored in glycerol tubes, which were then stored in a -80°C bacteria bank.

[0012] Furthermore, the Lactobacillus paracasei B20 has the ability to resist ultraviolet rays and degrade melanin.

[0013] Furthermore, the phenylalanine produced by the metabolites of Lactobacillus paracasei B20 not only has the function of inhibiting melanin synthesis, but also can promote the health of the stratum corneum and make the skin smooth and elastic.

[0014] Furthermore, the Lactobacillus paracasei B20 has significant tyrosine activity inhibition ability.

[0015] A use of Lactobacillus paracasei in preparing a whitening product, wherein the whitening product comprises whitening capsules, whitening probiotic powder, whitening enzyme and after-sun repair powder; The invention discloses a use of Lactobacillus paracasei in preparing functional products in the fields of inhibiting pathogenic bacteria and anti-oxidation. The functional products include medicines, skin care products, cosmetics, food additives or antibacterial agents.

[0016] Furthermore, the pathogenic bacteria include Staphylococcus aureus and Enterobacter faecalis.

[0017] A method for detecting melanin degradation ability of lactic acid bacteria is as follows: zebrafish embryos at 7-9 hpf after fertilization are randomly divided into groups, an experimental group is induced to 72 hpf by UVB, fluorescence intensity is detected by fluorescence microscope and analyzed by Image J software after staining, and the melanin degradation ability of zebrafish in the experimental group is obtained by comparing with that of the control group; wherein the experimental group is added with the above-mentioned Lactobacillus paracasei B20.

[0018] After adopting the above scheme, the beneficial effects of the present invention are: The inhibition rate of the fermentation supernatant of Lactobacillus paracasei B20 on tyrosinase, the rate-limiting enzyme for melanin production, can reach 94.8±0.7%, which is significantly higher than that of existing strains, and has strong antioxidant capacity, and can resist oxidative damage to the skin caused by the external environment.

[0019] The present invention uses LC-MS non-targeted metabolomics analysis and DOPA oxidation test to find that the substances that inhibit tyrosinase in Lactobacillus paracasei B20 mainly include phenylalanine and p-hydroxycinnamic acid. These two substances can not only effectively inhibit the activity of tyrosinase and reduce the production of melanin, but phenylalanine also has multiple functions such as promoting cell differentiation, increasing skin smoothness and elasticity, regulating skin lipid metabolism and maintaining skin moisture balance, providing comprehensive help for improving skin condition.

[0020] Moreover, through zebrafish induction experiments, it was found that Lactobacillus paracasei B20 also has strong melanin degradation and UV resistance capabilities. It can not only reduce the production of melanin, but also promote the degradation of the melanin that has been produced, and effectively resist the damage of ultraviolet rays, providing all-round protection for the skin.

[0021] In addition, Lactobacillus paracasei B20 has inhibitory effects on the growth of Enterococcus faecalis and Staphylococcus aureus, which can reduce the occurrence of skin inflammation to a certain extent, further enhancing its application value in whitening skin care products.

[0022] In summary, the Lactobacillus paracasei B20 of the present invention can be widely used in whitening skin care products with its efficient whitening effect, multiple skin care effects and antibacterial ability, providing a safer and more effective whitening skin care solution for people who pursue beauty. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The growth morphology of Lactobacillus paracasei B20 on MRS medium; Figure 2 This is a graphic representation of the results of tyrosinase inhibition activity of different types of lactic acid bacteria; Figure 3 To test the inhibitory effect of the selected lactic acid bacteria on tyrosinase at different concentrations; Figure 4 This is an analysis chart of the scavenging ability of different lactic acid bacteria strains on ABTS free radicals; Figure 5 This is an analysis chart of the scavenging ability of different lactic acid bacteria strains on DPPH free radicals; Figure 6 This is an analysis chart of the scavenging ability of different lactic acid bacteria strains on OH free radicals; Figure 7It is a graph showing the gastric digestive juice tolerance of Lactobacillus paracasei B20 of the present invention; Figure 8 This is a graph showing the tolerance of Lactobacillus paracasei B20 to artificial pancreatic juice; Fig. 9 It is a screening diagram of compounds inhibiting tyrosinase activity in Lactobacillus paracasei B20 of the present invention; Fig.10 This is a diagram showing the production of melanin on the body surface of zebrafish; Fig.11 This is the ROS staining imaging result of 72 hpf zebrafish larvae; Fig.12 This is a graph showing the inhibitory effect of Lactobacillus paracasei B20 on Staphylococcus aureus and Enterococcus faecalis. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The invention provides a Lacticaseibacillus paracasei B20, which is deposited in the General Microbiology Center of China Microbiological Culture Treasure Management Committee, with a deposit number of CGMCC No.32434 and a deposit date of October 31, 2024.

[0026] Source of raw materials: The Lactobacillus paracasei was isolated from homemade butter of farmers in Nagqu City, Tibet Autonomous Region. The strain was sequenced and analyzed, and through Blast sequence alignment, it was found to be highly homologous to Lactobacillus paracasei, and was named Lactobacillus paracasei B20.

[0027] Colony morphology: In MRS solid medium, the colonies are milky white, round, with smooth and neat edges.

[0028] Function: It has antioxidant and tyrosinase inhibition capabilities; it has strong acid and bile salt resistance, is well tolerated in artificial gastric juice and artificial intestinal juice, and can smoothly reach the human intestine; it can inhibit the growth of skin conditional pathogens (Staphylococcus aureus and Enterococcus faecalis).

[0029] Example 1: Isolation and Identification of Lactobacillus paracasei B20 1. Separation: Aseptically sample the butter sample, use the plate spreading method, take 5 g of sample and put it into a sterile homogenizing bag, add 45 mL of 0.85% saline and mix it homogenously to obtain the sample dilution. After thorough mixing, take 100 μL of sample in turn to perform a 10-fold series of gradient dilutions, and select the dilution factor of 10 -3 , 10 -4 , 10 -5 200 μL of sample dilution was pipetted and applied to a plate containing 2.5% CaCO 3 The cells were cultured on MRS solid medium at 37°C for 48 h.

[0030] Different strains were preliminarily selected according to their morphology, color, and whether they produced calcium-dissolving circles. They were separated and purified repeatedly by the plate streak separation method until all colonies on the MRS solid culture medium maintained a single morphology. Single colonies were picked and placed in MRS liquid culture medium and cultured at 37°C for 12 h. The fermentation broth of the strains was mixed with 50% glycerol in equal proportions and stored in glycerol tubes, which were then stored in a -80°C bacteria bank.

[0031] The colony morphology of the isolated and purified Lactobacillus paracasei B20 is as follows: the colonies are milky white, round, and have smooth and neat edges in MRS solid culture medium.

[0032] The formula of MRS liquid culture medium is: beef powder 10.0 g, glucose 20.0 g, tryptone 10.0 g, yeast extract powder 5.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, anhydrous sodium acetate 5.0 g, magnesium sulfate 0.5 g, manganese sulfate 0.25 g, deionized water 1.0 L, pH 6.5 (add 2% agar for MRS solid culture medium).

[0033] 2. Identification of bacterial species: The screened and purified strains were subjected to Gram staining to detect whether the strain morphology was uniform.

[0034] The strain genome was extracted according to the instructions of the bacterial gene DNA extraction kit, and 16S rDNA was amplified. The amplified product was sent to Guangzhou Qingke Bioengineering Co., Ltd. for sequencing, and the isolated strain was identified as Lacticaseibacillus paracasei B20. Its gene sequence is shown in SEQ ID NO.1.

[0035] Example 2: Determination of the ability of bacterial strains to colonize the intestine The hydrophobicity and self-aggregation ability of lactic acid bacteria are considered to be important criteria for determining their ability to colonize in the intestine. Therefore, the experiment conducted tests on hydrophobicity and self-aggregation ability respectively.

[0036] 1. Hydrophobicity test After activating the strain for 2-3 generations, wash the bacteria 2-3 times with PBS (Phosphate Buffered Saline) to adjust the bacterial count to 1.0×10 9 cfu / mL, and measure its absorbance at 600 nm A 0 . Take 3 mL of bacterial suspension and add 1 mL of xylene. After pre-incubation at room temperature for 10 min, vortex and quickly mix for 2 min, and leave it at room temperature for 15 min. After the solution is separated, measure the absorbance value A of the lower aqueous phase at 600 nm. Each sample is repeated 3 times. The hydrophobicity of the strain cell surface is calculated according to formula (1). Among them, the hydrophobicity of Lactobacillus paracasei B20 is 3.45±0.01%.

[0037] Formula (1) 2. Self-aggregation test The strain was inoculated into MRS liquid medium and cultured for 12 h. The cells were collected by centrifugation at 12,000 rpm for 10 min, washed twice with PBS, and resuspended to adjust A 600 =1±0.05, incubate at room temperature, and measure OD at 0 h, 2 h, 4 h, and 6 h 600 nm, and each sample was repeated 3 times. 0 represents the absorbance at 0 h, A t represents the absorbance at different times, and the self-aggregation ability was calculated by formula (2). As shown in Table 1, the self-aggregation ability of Lactobacillus paracasei B20 was 66.22±0.04% at 12 h, indicating that Lactobacillus paracasei B20 has a certain adhesion ability to host tissue cells.

[0038] Formula (2) Table 1 Self-aggregation ability and hydrophobicity of Lactobacillus paracasei B20

[0039] The experimental exploration of lactic acid bacteria in whitening mainly includes three aspects: ① Tyrosinase inhibition experiment. The key to whitening is to reduce the production of melanin, and tyrosinase is an indispensable catalyst in this process. A large number of studies have shown that the fermentation supernatant of certain lactic acid bacteria strains can significantly inhibit the activity of tyrosinase, thereby reducing the production of melanin. The main components include organic acids, peptides, enzymatic hydrolysis products, etc.

[0040] ② Antioxidant capacity evaluation. Oxidative stress is one of the important factors leading to skin aging and pigmentation. In order to verify the antioxidant capacity of lactic acid bacteria, various antioxidant test methods such as DPPH free radical scavenging experiment and reducing power determination are usually used for determination.

[0041] ③ Animal experiments. In order to more intuitively evaluate the whitening effect of lactic acid bacteria, animal models are usually introduced in experiments to test the effect, such as zebrafish, mice, humans, etc., to further verify the actual effect of lactic acid bacteria in whitening, and further enhance consumers' confidence and interest in lactic acid bacteria whitening products.

[0042] Based on the above aspects, the present invention provides the following experiments to verify the whitening effect of Lactobacillus paracasei B20 of the present invention.

[0043] Example 3 Screening of tyrosinase-inhibiting lactic acid bacteria strains Take out the lactic acid bacteria glycerol tube from the -80℃ bacteria library, transfer it to MRS liquid medium for activation, and culture it at 37℃ for 12 hours. Pipette the bacterial solution and transfer it to MRS solid medium, culture it at 37℃ for 48 hours, select a single colony and inoculate it in MRS liquid medium, culture it at 37℃ for 12 hours, centrifuge the fermentation liquid at 8000×g for 10 minutes at 4℃, and collect the fermentation supernatant. Wash the centrifuged bacteria with PBS solution, resuspend it in PBS, and obtain bacterial cells.

[0044] With reference to the Shanghai Daily Chemical Industry Association Group Standard (T / SHRH 015-2018) "Cosmetics-Tyrosinase Activity Inhibition Test Method", based on the principle that tyrosinase catalyzes the conversion of L-DOPA into dopaquinone with diphenolase activity, L-DOPA was used as the substrate to explore the inhibitory effect of lactic acid bacteria fermentation broth on tyrosinase activity.

[0045] According to Table 2, the experiment was divided into four groups. Each group was added with the corresponding volume of L-DOPA (2 mM / L), PBS buffer (0.067 mol / L), and sample fermentation supernatant in the table, and three parallel experiments were performed. After shaking at 37°C for 10 min, 125 U / mL tyrosinase solution was added, and the mixture was allowed to stand at room temperature for 20 min. The absorbance at 475 nm was tested. The tyrosinase inhibition rate was calculated according to formula (3). By fixing the L-DOPA concentration and changing the tyrosinase concentration (final concentrations of 40, 80, 120, 160, and 200 U / mL), the absorbance produced by the oxidation of L-DOPA by different lactic acid bacteria under the catalysis of tyrosinase at various concentrations was measured.

[0046] Table 2 Tyrosinase activity inhibition reagent dosage table

[0047] The experiment compared and analyzed the inhibitory effects of tyrosinase activity of 85 lactic acid bacteria fermentation supernatants including Lactobacillus paracasei, Lactobacillus plantarum, and Streptococcus thermophilus. Figure 2As shown in the figure, when the tyrosinase concentration was 125 U / mL, the inhibition rate of Lactobacillus paracasei strain B20 on tyrosinase was high, reaching 94.8±0.7%.

[0048] From the selected strains of Lactobacillus paracasei, Lactobacillus plantarum, and Streptococcus thermophilus, the strains with the highest and lowest inhibition of tyrosinase activity of each genus of lactic acid bacteria were screened out, a total of 6 strains, and the inhibition rates of the 6 lactic acid bacteria to different tyrosine concentrations were tested. The results are as follows Figure 3 As shown in the figure, the results showed that the inhibitory effects of fermentation supernatants of different lactic acid bacteria strains on tyrosinase activity were significantly different, and the inhibitory effect of fermentation supernatants of lactic acid bacteria on tyrosinase weakened with the increase of tyrosinase concentration. Among them, Lactobacillus paracasei B20 had a stronger inhibitory effect on tyrosinase activity. At a final concentration of 40 U / mL enzyme concentration, the inhibition rate of Lactobacillus paracasei B20 could reach more than 95% ( Figure 3 ).

[0049] Formula (3) Example 4 Determination of Antioxidant Capacity of Lactobacillus paracasei B20 1. Ability to remove ABTS free radicals Pipette 40 μL of sample and 800 μL of ABTS working solution into a 1.5 mL centrifuge tube, shake for 20 seconds, react in the dark for 6 minutes, and measure the sample absorbance at 734 nm. Repeat 3 times for each sample. Replace the sample solution with an equal volume of distilled water in the blank group, which is the absorbance of the blank group.

[0050] The experiment tested the scavenging ability of several lactic acid bacteria on ABTS free radicals and found that the scavenging ability of the fermentation supernatant of the strains on ABTS free radicals was higher than that of the bacterial cells (such as Figure 4 As shown in the figure, the scavenging rate of the fermentation supernatant of Lactobacillus paracasei B20 on ABTS free radicals reached 70%, and its scavenging rate on ABTS free radicals was the highest among several strains, reaching 41.9±0.02%.

[0051] Formula (4) 2. Determination of DPPH free radical scavenging ability Mix 1 mL of DPPH ethanol solution (0.2 mmol / L) with 1 mL of the sample to be tested, and react for 30 min in a dark environment. Test the OD 517 nm, denoted as A s . Replace the DPPH solution with an equal volume of anhydrous ethanol solution and test the OD 517 nm, denoted as A b , the absorbance value of the polysaccharide sample replaced by an equal volume of distilled water is recorded as A c, calculate the scavenging rate of DPPH free radicals, the results are as follows Figure 5 shown.

[0052] Depend on Figure 5 It can be seen that the fermentation supernatants of the six lactic acid bacteria strains could scaveng more than 90% of DPPH free radicals, while the scavenging effects of different bacterial cells on DPPH free radicals varied greatly. The bacterial cells of Lactobacillus paracasei B20 had the highest scavenging rate of DPPH free radicals, which was 80.4±0.02%.

[0053] Formula (5) 3. Determination of OH radical scavenging ability Add 0.5 mL of L-phenanthroline (0.75 mM / L), 0.5 mL of the sample to be tested, and 1 mL of PBS solution to the test tube in sequence, mix thoroughly, and then add 5 mL of FeSO 4 (0.75 mM / L) and 0.5 mL of 0.01% H 2 O 2 , incubate in a 37℃ water bath for 1 h, and measure the absorbance at 536 nm. Centrifuge the bacterial cells at 8000×g for 10 min at 4℃, and measure the absorbance at 536 nm from the supernatant. Repeat 3 times for each sample. Replace 0.5 mL of H2O with 0.5 mL of distilled water in the blank group. 2 O 2 , which is the absorbance of the blank group. The control group used 0.5 mL of distilled water instead of 0.5 mL of sample, which is the absorbance of the control group. The calculation by formula (4) shows that the OH free radical scavenging rate of the fermentation supernatant of Lactobacillus paracasei B20 is about 70%. The results are as follows Figure 6 shown.

[0054] Formula (4) Example 5: Study on the acid and bile resistance of Lactobacillus paracasei B20 1. Tolerance in artificial gastric juice The activated Lactobacillus paracasei B20 was centrifuged at 8000×g for 10 min at 4°C, the supernatant was discarded, the bacteria were washed 3 times with PBS, resuspended in PBS with pH values ​​of 2.5, 3, and 4 and containing 3 g / L peptic ulcer, the OD was adjusted to 1.0, and placed in a 37°C constant temperature incubator. 2 mL of the bacterial solution was taken at 0 h and 3 h, respectively, diluted to a suitable dilution with sterile PBS and spread on an MRS solid plate. After incubation at 37°C for 36-48 h, the plate was counted to calculate the number of viable bacteria and the survival rate of Lactobacillus paracasei B20. The results are as follows: Figure 7 shown.

[0055] The results showed that the survival rate of Lactobacillus paracasei B20 after 3 h of culture at pH 2.5, pH 3, and pH 4 was 89.47%, 104.99%, and 90.91%, respectively, and the number of viable bacteria was about 10 8 CFU / mL, indicating that Lactobacillus paracasei B20 has strong tolerance to gastric juice.

[0056] 2. Tolerance in artificial pancreatic juice Pancreatin (1 g / L) was suspended in sterile PBS, and the pH was adjusted to 8.0 using sodium hydroxide. Lactobacillus paracasei B20 cultured in gastric juice for 3 h was added to simulated artificial pancreatic juice at a ratio of 10%, and cultured at 37°C for 4 h. 100 μL of the bacterial solution was taken at 0 h and 4 h for live bacterial count, and the live bacterial count and survival rate of Lactobacillus paracasei B20 were calculated. The results are shown in Figure 2. Figure 8 shown.

[0057] The results are as follows Figure 8 As shown in the figure, when Lactobacillus paracasei was cultured in an environment with pH 2.5, pH 3, and pH 4 for 4 h, the survival rates were 105.96%, 109.73%, and 105.96%, respectively, and the number of viable bacteria was about 10 8 CFU / mL, indicating that Lactobacillus paracasei B20 has strong tolerance to simulated pancreatic juice.

[0058] Example 6 Effect of the active ingredients of Lactobacillus paracasei B20 fermentation broth on tyrosinase activity Non-targeted metabolomics analysis of Lactobacillus paracasei B20 The fermentation broth of a single colony of Lactobacillus paracasei B20 cultured for 12 h was centrifuged at 8000 × g for 10 min at 4 °C.

[0059] Pipette 240 μL of the supernatant after centrifugation into each of the three tubes. Add 240 μL of L-DOPA solution (2 mM / L) and 240 μL of tyrosinase solution (40 u / mL) to M1. In the M2 group, 240 μL of L-DOPA solution (2 mM / L) and 240 μL of tyrosinase solution (200 u / mL) were added and reacted at 37°C for 20 min. Only 240 μL of supernatant was added to the S group, and 3 tubes were added to each group in parallel.

[0060] Take 100 μL of the sample to be tested and add 400 μL of the extract (methanol: acetonitrile = 1:1 (V / V)), vortex mix for 30 s, and ultrasonicate in an ice-water bath for 10 min; after standing at -40 ℃ for 1 hour, centrifuge at 12000 rpm for 15 min, take the supernatant into the injection bottle, and use Vanquish (Thermo Fisher Scientific) ultra-performance liquid chromatography to chromatographically separate the target compounds.

[0061] The fermentation supernatant of Lactobacillus paracasei B20 was analyzed by non-targeted LC-MS, and a total of 1641 chemical components of the fermentation supernatant were identified. The main metabolites are shown in Table 3. There are amino acids including: arginine, phenylalanine, leucine, tryptophan; Organic nitrogen compounds: L-carnitine; Carboxylic acids and their derivatives: L-2-hydroxyphenylalanine; Histidine alkaloids: creatinine; Fatty acids and derivatives: lactic acid; Tyrosine phosphorylation inhibitors: AG1295, hydroxyphenyllactic acid.

[0062] Table 3 Main components of the supernatant of Lactobacillus paracasei B20 fermentation

[0063] Analysis of the inhibitory effect of each fermentation component of Lactobacillus paracasei B20 on tyrosinase Based on the dominant metabolites detected by non-targeted metabolomics, standard products of each dominant component were purchased to compare the inhibitory effect of each component on tyrosinase activity. The inhibitory effect of each component compound on tyrosinase activity was compared using the DOPA oxidation method, and the method was the same as the screening of tyrosinase-inhibiting lactic acid strains. The results are shown in Fig. 9 As shown, Fig. 9 The letters in the table represent the following: A-phenylalanine; B-p-hydroxycinnamic acid; C-L-carnitine; D-leucine; E-L-2-hydroxyphenylalanine; F-lysine; G-arginine; H-lactic acid; J-creatinine; K-phenyllactic acid; L-kojic acid.

[0064] Depend on Fig. 9 The results show that the inhibition of tyrosinase activity by phenylalanine and p-hydroxycinnamic acid is dose-dependent. The inhibition rate of tyrosinase by 0.5 mg / mL p-hydroxycinnamic acid reaches 60%, and the inhibition rate of tyrosinase by 0.75 mg / mL phenylalanine reaches 50%.

[0065] Quantification of the phenylalanine content in the supernatant of Lactobacillus paracasei B20 fermentation Phenylalanine not only has the function of inhibiting tyrosinase activity and reducing the production of melanin, but also can promote cell differentiation, increase skin smoothness and elasticity, regulate skin lipid metabolism, and maintain skin moisture balance. It has excellent applications in balancing skin oil and strengthening the skin stratum corneum. Therefore, the experiment chose to determine the content of phenylalanine in the supernatant of Lactobacillus paracasei B20 fermentation broth.

[0066] Prepare phenylalanine standard solutions of 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1.0 g / L. Take 5.0 mL of Lactobacillus paracasei B20 fermentation broth, centrifuge at 10,000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the sample to be tested.

[0067] Chromatographic conditions Chromatographic column: Waters Symmetry C18 column (5μm, 4.6 × 250 mm); column temperature: 30℃; injection volume: 10.0μL; flow rate: 1.0mL / min; mobile phase: phase A is 0.05% trifluoroacetic acid methanol solution (V / V), phase B is 0.05% trifluoroacetic acid solution (V / V); elution program: 0-20 min, A:B linearly changes from 10% to 100%, 20-23 min is 100% phase A, 23-25 ​​min maintain A:B at 10%; detection wavelength: 210 nm.

[0068] According to HPLC (high performance liquid chromatography), the standard curve of phenylalanine is y=8481341.18x+2998599.22 R2=0.9992. MRS and Lactobacillus paracasei B20 were subjected to HPLC in the same manner, and the calculation results are shown in Table 4. The phenylalanine content in MRS medium is 1.04±0.02 g / L, and the phenylalanine content in Lactobacillus paracasei B20 is 1.73±0.01 g / L, which is significantly higher than the former.

[0069] Table 4 Phenylalanine content in the fermentation supernatant of Lactobacillus paracasei B20 and MRS

[0070] Example 7: Effects of the active ingredients of Lactobacillus paracasei B20 fermentation broth on the anti-ultraviolet and whitening effects of zebrafish 1. Determination of ROS in zebrafish after UVB induction Zebrafish embryos at 7-9 hpf (hours post fertilization) were randomly divided into 8 groups, with 15 zebrafish embryos in each group. They were blank group, model group, 0.05% lactic acid bacteria fermentation broth experimental group, 0.15% lactic acid bacteria fermentation broth experimental group, 0.25% lactic acid bacteria fermentation broth experimental group, 0.05% MRS medium experimental group, 0.15% MRS medium experimental group, and 0.25% MRS medium experimental group. They were raised in 6-well plates respectively. In the experimental group, samples of different concentrations were added to each well. After incubation for 1 h, the liquid in all wells was discarded, and 1 mL of E3 medium (trisaccharide iron medium) was retained.

[0071] Except for the blank group (control), different groups of zebrafish embryos were induced by UVB. After induction, an appropriate amount of E3 medium was added to each group for continued feeding. Zebrafish embryos were induced every day until 72 hpf. They were stained with DCFH-DA (20 μg / mL), and an appropriate amount of staining solution was added to each well, and incubated at 28°C in the dark for 2 h. The larvae were anesthetized, then observed with a fluorescence microscope, and the fluorescence intensity was analyzed with Image J software.

[0072] UVB radiation After the skin absorbs excessive UVB radiation, a large amount of ROS can be generated, which destroys the normal structure and function of biological macromolecules such as nucleic acids and proteins, induces a large number of cell apoptosis, and leads to the death of zebrafish. As shown in Table 5, compared with the MRS group, the fermentation supernatant of Lactobacillus paracasei B20 can significantly reduce the mortality of zebrafish.

[0073] Table 5 Effects of different amounts of fermentation supernatant added on zebrafish mortality

[0074] 2. Analysis of melanin production on embryo surface After the test substance (fermentation supernatant of Lactobacillus paracasei B20) was treated for 72 h, the melanin production of the embryos was observed under a microscope, the embryo culture medium was discarded with a sterile pipette, and 1 drop of 0.05 mg / mL MS-222 was added to anesthetize the zebrafish. The zebrafish was adjusted under a microscope so that the eyes and body segments on both sides overlapped and the tail was at the same level as the body. A clear side view of the zebrafish was taken at the same magnification and light intensity. Image J software was used for image analysis, the images were converted into 8-bit grayscale images, the background was eliminated, the threshold parameters were set, the total area of ​​zebrafish melanin spots was calculated, and the difference in the ratio of melanin area to total area in different concentration treatment groups was compared. The melanin area ratio of the blank group was marked as S 空白 The percentage of melanin area in the sample group is marked as S 样品 , the surface pigment content is calculated as shown in Formula 5.

[0075] Compared with the MRS group, the fermentation supernatant of Lactobacillus paracasei B20 could significantly reduce the melanin content on the zebrafish body surface. Fig.10 The addition amounts of Lactobacillus paracasei B20 fermentation supernatant were 0.05%, 0.15%, and 0.25%, respectively. As the concentration of fermentation supernatant increased, the effect on melanin removal showed a dose effect.

[0076] Formula (5) 3. Determination of melanin content in zebrafish The melanin content of zebrafish was determined by NaOH lysis method. The juvenile fish of 72 hpf treated with different concentrations of lactic acid bacteria supernatant were collected and placed in a 1.5 mL centrifuge tube, washed twice with ultrapure water, and the liquid was aspirated to obtain embryos. Triton X-100 containing 1 mM / L PMSF was added to the centrifuge tube, placed in a -80℃ refrigerator for 30 min, and placed at room temperature for 30 min, and repeated 3 times. The embryos were ultrasonicated for 35 s under ice bath conditions, then centrifuged at 10000 rpm and 4℃ for 10 min, and 600 μL of 1 mol / L NaOH solution containing 10% DMSO was added to the centrifuged precipitate to resuspend it and place it in an 80℃ water bath for 2 h. Zebrafish without test substance treatment were used as blank controls, and the ROS content in the body was analyzed by fluorescence staining. The results showed that as the amount of Lactobacillus paracasei B20 fermentation supernatant added increased, the green fluorescence in zebrafish first dimmed and then brightened, and the fluorescence intensity value showed a trend of first decreasing and then increasing. It is speculated that the 0.25% addition has a toxic effect on zebrafish, leading to an increase in ROS. Among the several treatment concentrations, 0.15% of the fermentation supernatant of Lactobacillus paracasei B20 can effectively reduce the ROS content in zebrafish ( Fig.11 ).

[0077] Example 8 Lactobacillus paracasei inhibits the functional activity of Staphylococcus aureus and Enterococcus faecalis Staphylococcus aureus and Enterococcus faecalis are two major opportunistic pathogens that are susceptible to skin infection. Therefore, the inhibitory effect of Lactobacillus paracasei on the two opportunistic pathogens was analyzed.

[0078] The double-layer plate streak method was used to conduct a preliminary determination of the antibacterial activity. A single colony was selected and two parallel lines of equal length were drawn on the MRS plate. The plates were cultured at 37°C for 24 hours. Staphylococcus aureus and Enterococcus faecalis (OD 600 =0.6) was added to the culture medium at a ratio of 1% and mixed evenly. The upper layer of the culture medium was poured onto the cultured lactic acid bacteria plate and cultured at 37°C for 12 h. Fig.12 As shown in the figure (Staphylococcus aureus on the left and Enterococcus faecalis on the right), Lactobacillus paracasei B20 has inhibitory effect on both strains of bacteria, and a relatively obvious transparent circle appears, indicating that in addition to inhibiting melanin production and anti-oxidation, Lactobacillus paracasei B20 also has relatively good resistance to skin pathogen infection.

[0079] According to the results of the above examples, the Lactobacillus paracasei B20 provided by the present invention has the following properties and effects: 1. It has strong acid and bile salt resistance, good tolerance in artificial gastric juice and artificial intestinal juice, and can smoothly reach the human intestine; 2. The inhibition rate of tyrosinase is high, up to 94.8±0.7%, and the main active substances are phenylalanine and p-hydroxycinnamic acid; 3. Has high antioxidant capacity; 4. The fermentation supernatant has good melanin removal and UV resistance capabilities; 5. The phenylalanine content in the metabolites is 1.73±0.01g / L, which has the function of reducing melanin production and protecting the skin; 6. The self-aggregation ability and hydrophobicity are strong, indicating that the strain has good intestinal adhesion and colonization ability; 7. This strain has a significant inhibitory effect on Staphylococcus aureus and Enterobacter faecalis, and can be used in the development of skin disease treatment and prevention products.

[0080] In summary, compared with the prior art, the Lactobacillus paracasei B20 provided by the present invention has the following beneficial effects: The Lactobacillus paracasei provided by the present invention has strong acid and bile salt resistance, good tolerance in artificial gastric juice and artificial intestinal juice, and can smoothly reach the human intestine; Lactobacillus paracasei B20 has good tyrosinase inhibition ability, can reduce melanin production, and can be used in the development of sunscreen and whitening products; At the same time, this strain has the ability to degrade melanin and resist UV degradation, and can be widely used in the development of after-sun repair products, which can significantly reduce the melanin content in the skin.

[0081] It should be noted that: (1) Definition: The term "food" as used herein is broad and includes food and drink for humans. In certain embodiments, the food product is suitable for and designed for human consumption. The application can be used to prepare solid preparations such as powders, tablets, gels, and also dispersed in liquids to prepare liquid preparations, including but not limited to the embodiment schemes.

[0082] (2) Relevant prior art means or prior art terms involved in this application: "OD" is the abbreviation of optical density, also known as absorbance. When light passes through the object, the energy difference before and after is the energy absorbed by the object. At a specific wavelength, there is a quantitative relationship between the concentration of the same object and the absorbed energy. This quantitative relationship can be used to determine the concentration of the object. 625 " is the optical density value measured when the wavelength is set to 625nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate bacterial cell density. Among them, the "OD" value determination method is a prior art, and its principles and methods will not be repeated here.

[0083] Strain application: Embodiments illustrate that Lactobacillus paracasei B20 can be applied to whitening products. According to the above design concept, the strain can be applied to whitening capsules, whitening probiotic powder, after-sun repair powder, etc.

[0084] The above description is only a preferred embodiment of the present invention and is not a limitation on the design of this case. Any equivalent changes made based on the design key of this case shall fall within the protection scope of this case.

[0085] Sequence Listing Sequence Listing <110> Xiamen Yuanzhidao Biotechnology Co., Ltd. <120> Lactobacillus paracasei B20 originating from plateau areas and inhibiting melanin and its application <160> 1 <170> WIPO Sequence 2.3.0 <210> 1 <211> 1447 <212> DNA <213> Lactobacillus paracasei B20 ( Lacticaseibacillus paracasei ) <400> 1 ctggtcagtc gaacgagttc tcgttgatgc atcggtgctt gcaccgagat tcaacatgga 60 acgagtggcg gacgggtgag taacacgtgg gtaacctgcc cttaagtggg ggataacatt 120 tggaaacaga tgctaatacc gcatagatcc aagaaccgca tggttcttgg ctgaaagatg 180 gcgtaagcta tcgcttttgg atggacccgc ggcgtattag ctagttggtg aggtaatggc 240 tcaccaaggc gatgatacgt agccgaactg agaggttgat cggccacatt gggactgaga 300 cacggcccaa actcctacgg gaggcagcag tagggaatct tccacaatgg acgcaagtct 360 gatggagcaa cgccgcgtga gtgaagaagg ctttcgggtc gtaaaactct gttgttggag 420 aagaatggtc ggcagagtaa ctgttgtcgg cgtgacggta tccaaccaga aagccacggc 480 taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gatttattgg 540 gcgtaaagcg agcgcaggcg gttttttaag tctgatgtga aagccctcgg cttaaccgag 600 gaagcgcatc ggaaactggg aaacttgagt gcaagaagagg acagtggaac tccatgtgta 660 gcggtgaaat gcgtagatat atggaagaac accagtggcg aaggcggctg tctggtctgt 720 aactgacgct gaggctcgaa agcatgggta gcgaacagga ttagataccc tggtagtcca 780 tgccgtaaac gatgaatgct aggtgttgga gggtttccgc ccttcagtgc cgcagctaac 840 gcattaagca ttccgcctgg ggagtacgac cgcaaggttg aaactcaaag gaattgacgg 900 gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960 ggtcttgaca tcttttgatc acctgagaga tcaggtttcc ccttcgggg caaaatgaca1020 ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag1080 cgcaaccctt atgactagtt gccagcattt agttgggcac tctagtaaga ctgccggtga1140 caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca1200 cacgtgctac aatggatggt acaacgagtt gcgagaccgc gaggtcaagc taatctctta1260 aagccattct cagttcggac tgtaggctgc aactcgccta cacgaagtcg gaatcgctag1320 taatcgcgga tcagcacgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc1380 acaccatgag agtttgtaac acccgaagcc ggtggcgtaa cccttttagg gagcgatcaa1440 cgattga 1447

Claims

1. A Lactobacillus paracasei, characterized in that: Lacticaseibacillus paracasei B20 is deposited in the General Microbiology Center of China Microbiological Culture Collection Management Committee, with the deposit number CGMCC No.32434 and the deposit date of October 31, 2024.

2. a kind of Lactobacillus paracasei as claimed in claim 1, is characterized in that: The Lactobacillus paracasei B20 is obtained by separation and purification from homemade butter of farmers in Nagqu City, Tibet Autonomous Region.

3. a kind of Lactobacillus paracasei as claimed in claim 1, is characterized in that: The separation and purification method of described Lactobacillus paracasei B20 is as follows: Aseptically sample the butter sample, use the plate spreading method, take 5 g of the sample and put it into a sterile homogenizing bag, add 45 mL of 0.85% saline and homogenize to obtain the sample dilution solution; After thorough mixing, 100 μL of the sample was pipetted in sequence for 10-fold gradient dilution. -3 , 10 -4 , 10 -5 200 μL of the sample dilution was pipetted and spread on MRS solid medium containing 2.5% CaCO3, and inverted cultured at 37°C for 48 h; Different strains were preliminarily selected according to their morphology, color, and whether they produced calcium-dissolving circles. They were separated and purified repeatedly by the plate streak separation method until all colonies on the MRS solid culture medium maintained a single morphology. Single colonies were picked and placed in MRS liquid culture medium and cultured at 37°C for 12 h. The fermentation broth of the strains was mixed with 50% glycerol in equal proportions and stored in glycerol tubes, which were then stored in a -80°C bacteria bank.

4. a kind of Lactobacillus paracasei as claimed in claim 1, is characterized in that: The Lactobacillus paracasei B20 has the ability to resist ultraviolet rays and degrade melanin.

5. a kind of Lactobacillus paracasei as claimed in claim 1, is characterized in that: The phenylalanine produced by the metabolites of Lactobacillus paracasei B20 not only has the function of inhibiting melanin synthesis, but also can promote the health of the stratum corneum and make the skin smooth and elastic.

6. A Lactobacillus paracasei as claimed in claim 1, characterized in that: The Lactobacillus paracasei B20 has significant tyrosine activity inhibition ability.

7. The use of a kind of Lactobacillus paracasei as claimed in claim 1 in preparing a whitening product, wherein the whitening product comprises whitening capsules, whitening probiotic powder, whitening enzyme and after-sun repair powder.

8. The purpose of a kind of Lactobacillus paracasei as claimed in claim 1 in preparing a functional product that inhibits pathogenic bacteria and antioxidant fields, wherein the functional product comprises medicine, skin care product, cosmetics, food additive or antibacterial agent.

9. The use of a kind of Lactobacillus paracasei as claimed in claim 8 in preparing a functional product in the field of inhibiting pathogenic bacteria and anti-oxidation, characterized in that: The pathogenic bacteria include Staphylococcus aureus and Enterobacter faecalis.

10. A method for detecting melanin degradation ability of lactic acid bacteria, characterized in that: Zebrafish embryos at 7-9 hpf after fertilization were randomly divided into groups, and the experimental group was induced to 72 hpf by UVB, and the fluorescence intensity was analyzed by fluorescence microscopy and ImageJ software after staining, and compared with the control group, the melanin degradation ability of the zebrafish in the experimental group was obtained; wherein, the experimental group was added with Lactobacillus paracasei B20 as described in any one of claims 1 to 9.

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