Lactobacillus reuteri Light-ɑ with whitening and anti-aging activities and its application

Abstract

The present invention provides a strain of Lactobacillus reuteri Light‑ɑ with whitening and anti-aging activities and related applications thereof, and relates to the field of microorganisms and their application technology. The Lactobacillus reuteri Light‑ɑ has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on January 2, 2024, with a deposit number of CGMCC No. 29488. The Lactobacillus reuteri Light‑ɑ provided by the present invention can effectively inhibit tyrosinase activity and reduce melanin synthesis; at the same time, the Light‑ɑ strain also has excellent free radical scavenging ability and high reducing power, exhibits strong antioxidant ability, and can tolerate acid, alkali and bile salt environments. Therefore, the Lactobacillus reuteri Light‑ɑ strain has broad market application prospects in the preparation of whitening, anti-oxidation, anti-aging and anti-aging products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and their application, in particular to Limosilactobacillus reuteri Light-ɑ with whitening and anti-aging activities and applications thereof. Background Art

[0002] Tyrosinase plays a major role in melanin synthesis. Excessive production and accumulation of melanin in specific parts of the skin can lead to hyperpigmentation such as chloasma, freckles and age spots and melanoma. In the food industry, tyrosinase is also one of the important causes of enzymatic browning of freshly cut fruits and vegetables. Therefore, the exploration and development of tyrosinase inhibitors has great application value. With the development of the economy and society, huge business opportunities have emerged in the skin whitening or freckle removal industry. Tyrosinase inhibitors produced by plants, fungi and bacteria are regarded as attractive potential skin whitening agents due to their low toxicity and bioavailability, especially for food and cosmetic applications.

[0003] The human body continuously produces free radicals in the body due to continuous contact with the outside world, including breathing, external pollution, ultraviolet radiation and other factors. At the same time, with the rapid development of society, the mental stress and negative emotions faced by people also promote the production of free radicals. Scientific research shows that the production of excessive free radicals is related to aging, cancer or other diseases. Among them, the most important and widely used application of antioxidants is anti-aging, especially skin aging. At the same time, antioxidants are also the basis and foundation of skin whitening. Taking enough antioxidants can slow down the body's degeneration, prevent skin aging, and always maintain youthful spirit. Therefore, antioxidants are one of the main research and development directions of health care products and cosmetics companies, and one of the most important functional demands of the market.

[0004] Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the bacterial flora in a certain part of the host. Studies have reported that some probiotics and their products have strong tyrosinase activity inhibition and antioxidant effects, reduce skin pigmentation and promote skin health; some clinical experiments have shown that taking Lactobacillus plantarum and lactic acid bacteria lysates can improve skin hydration, enhance skin barrier function, and resist photoaging. Considering the safety and effectiveness of probiotics, finding whitening and anti-aging probiotics that can be developed and utilized is an important direction that the market is currently focusing on research and development.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The present invention aims to provide a strain of Lactobacillus reuteri Light-ɑ with whitening and anti-aging activity and its application

[0007] The technical solution provided by the present invention is as follows:

[0008] In one aspect, the present invention provides a Lactobacillus reuteri Light-ɑ, the deposit number of the Lactobacillus reuteri is CGMCC No. 29488.

[0009] In another aspect, the present invention provides a bacterial agent, wherein the bacterial agent contains the Lactobacillus reuteri Light-ɑ. Preferably, the bacterial agent is a solid preparation or a liquid preparation. More preferably, the solid preparation is a powder prepared by freeze-drying.

[0010] The Lactobacillus reuteri strain Light-ɑ used in the present invention is isolated by the inventor and is classified and named as: Lactobacillus reuteri ( Limosilactobacillus reuteri ); it was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on January 2, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29488. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 It is the Gram staining picture of Lactobacillus reuteri Light-ɑ in the present invention.

[0013] Figure 2 A heat map of nucleotide identity (ANI) analysis of Light-ɑ in the present invention and other Lactobacillus reuteri is shown.

[0014] Figure 3 The effect of the culture supernatant of Lactobacillus reuteri Light-ɑ in the present invention on melanin synthesis. (A) In vitro tyrosinase activity inhibition rate, (B) In vivo tyrosinase activity inhibition rate, (C) Melanin content in B16F10 cells. Kojic acid was used as a positive control.

[0015] Figure 4 The antioxidant capacity of the culture supernatant of Lactobacillus reuteri Light-ɑ in the present invention is evaluated. (A) Anti-lipid peroxidation rate, (B) Hydroxyl radical scavenging rate, (C) DPPH radical scavenging rate, (D) Total reducing power. Vc was used as a positive control. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. 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.

[0017] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific examples. In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0018] The present invention separates and screens a Lactobacillus reuteri strain Light-ɑ from feces of centenarians living in Chengmai, Hainan Province for a long time. Experiments have shown that the Light-ɑ strain of the present invention not only has the ability to inhibit tyrosinase activity in vitro and in vivo and reduce melanin synthesis, but also has strong antioxidant activity.

[0019] Example 1 Identification and sample preparation of Lactobacillus reuteri Light-ɑ

[0020] (1) Strain identification

[0021] The Light-ɑ strain involved in the present invention comes from the centenarian population in Chengmai, Hainan, and is stored in the strain library built by Beijing Quantitative Health Co., Ltd. The Light-ɑ strain is blue-purple ( Figure 1 ), indicating that the strain is a Gram-positive bacterium (G+). The frozen glycerol bacteria were activated and the bacterial DNA was extracted and sent to a sequencing company. After sequencing, the genome was sorted and the genome information of Light-ɑ was compared with the genome information of 19 known Lactobacillus reuteri strains in the NCBI database (labeled as: GCF XXX). The FastANI (average nucleotide identity) value of Light-ɑ in the present invention was greater than 95% ( Figure 2 ), ANI = 95% is often used as the standard for species classification in identification analysis. Therefore, it is confirmed again that Light-ɑ in the present invention belongs to the species of Lactobacillus reuteri.

[0022] (2) Strain culture

[0023] Under aseptic operation conditions, Lactobacillus reuteri Light-ɑ stored in a -80℃ refrigerator was inoculated into the culture medium and cultured overnight in a 37℃ hypoxic workstation. The bacterial liquid after activation for 3 generations was used as the seed liquid for culture. The seed liquid was inoculated into the liquid culture medium with an inoculum volume fraction of 4%, and after culturing in a 37℃ constant temperature incubator for 18 h, it could be used as the strain working liquid. The obtained working strain mixture was then centrifuged at 4℃, 12000 r / min for 10 min, and the supernatant was collected to obtain the culture supernatant of Lactobacillus reuteri Light-ɑ for subsequent experiments. The samples were stored at 4℃ for later use.

[0024] Example 2 Effect of Lactobacillus reuteri Light-ɑ on melanin synthesis

[0025] This example is used to illustrate the inhibitory effect of Lactobacillus reuteri Light-ɑ on melanin synthesis.

[0026] Tyrosinase is a key rate-limiting enzyme in the process of melanin production. The ability of Lactobacillus reuteri Light-ɑ in the present invention to inhibit melanin production can be evaluated through in vitro and in vivo (cellular level) tyrosinase activity inhibition experiments.

[0027] (1) In vitro tyrosinase activity inhibition experiment

[0028] PBS and tyrosinase solution (250 μg / mL) were added to a 96-well plate, followed by the sample solution (A4). The 96-well plate was sealed and placed in a water bath at 37°C for 10 min. Thereafter, L-DOPA (10 mM) was added to the wells. After incubation in the dark for 10 min, the absorbance was measured at 490 nm using a microplate reader. A blank (A1) was set by adding an equal volume of PBS instead of the sample solution. A blank control (A2) and an experimental control (A3) were set by adding an equal volume of PBS instead of the tyrosinase solution. Kojic acid (100 μg / mL) was used as a positive control. The tyrosinase inhibition rate was calculated as follows: tyrosinase inhibition rate / %=1-(A4-A3 / A1-A2) ×100, where: A4 is the absorbance of PBS+tyrosinase+sample; A3 is the absorbance of PBS+sample+levodopa; A2 is the absorbance of PBS+tyrosinase+levodopa; A1 is the absorbance of PBS+levodopa. Figure 3 As shown in A, under the in vitro reaction conditions, the average inhibition rate of the culture supernatant of the Light-ɑ strain in the present invention is 83.62%, which is significantly higher than the positive control kojic acid (49.59%).

[0029] (2) Intracellular tyrosinase activity inhibition experiment

[0030] Mouse melanoma cells B16F10 in the logarithmic growth phase were inoculated into 96-well plates and cultured in an incubator at 37°C and 5% CO2. When the cells grew to 70% to 80% confluence, the supernatant was discarded, and cell culture medium containing the sample solution (Light-ɑ strain culture supernatant or kojic acid or an equal volume of PBS solution) was added and incubated for 24 h. Then the culture medium was discarded, the cells were washed twice with PBS, 50 μL of 1% TritonX-100 solution was added, and the cells were shaken on a shaker for 5 min to lyse the cells. Then 50 μL of 10mM L-DOPA (10 mM) was added, and the cells were reacted at 37°C in the dark for 30 min, and the absorbance was measured at 490 nm by an enzyme reader. The positive control group was 1mM kojic acid solution. Figure 3 B shows that the average inhibition rate of the culture supernatant of Lactobacillus reuteri Light-ɑ on tyrosinase activity in B16F10 cells is about 88.21%, which is much higher than the inhibition rate of the positive control kojic acid of about 71.05%.

[0031] (3) Intracellular melanin content detection experiment

[0032] Mouse melanoma B16F10 cells in the logarithmic growth phase were inoculated into 6-well plates and treated with the sample solution for 24 h, referring to the method in (2). During the test, 1 mL of NaOH solution (1M) was added to each group for lysis for 1 h, and the color change of the cell suspension in the centrifuge tube was observed. Then, the suspension of each group was added to a 96-well plate and the absorbance at 490 nm was measured using an ELISA reader. Figure 3 As shown in CD, the supernatant sample of Lactobacillus reuteri Light-ɑ in the present invention can effectively reduce the production of melanin. Compared with the untreated control, the melanin content of the cells treated with Light-ɑ can be reduced to 32.11%, and the melanin content of the cells treated with kojic acid can be reduced to 39.40%.

[0033] In summary, according to the results of in vivo and in vitro tyrosinase activity inhibition and intracellular melanin content, Lactobacillus reuteri Light-ɑ in the present invention can effectively inhibit tyrosinase activity and reduce intracellular melanin production, and has the potential to be developed into whitening, freckle removal and other related products.

[0034] Example 3 Evaluation of the Antioxidant Capacity of Lactobacillus reuteri Light-ɑ

[0035] This example is used to illustrate the antioxidant capacity of Lactobacillus reuteri Light-ɑ, mainly involving the detection of anti-lipid peroxidation, free radical scavenging and total reducing power.

[0036] (1) Determination of anti-lipid peroxidation ability

[0037] Reactive oxygen species (ROS) generated after oxidative stress is enhanced can react with the side chains of polyunsaturated fatty acids in linoleic acid emulsion to form lipid peroxidation products, namely malondialdehyde (MDA). This substance is an important indicator for lipid peroxidation. Under high temperature and acidic environment, MDA can react with thiobarbituric acid (TBA) to produce purple-red compounds (532nm). During the experiment, 50 μL PBS, 100 μL linoleic acid emulsion, 100 μL 1% FeSO4 and 30 μL bacterial solution samples were added, and then placed in a 37°C water bath for 1.5 h after shaking. After taking out, vortex mix, add 20 μL 4% trichloroacetic acid and 200 μL 0.8% thiobarbituric acid (TBA), and place in a 100°C water bath for 30 min. After cooling in cold water, centrifuge at 5000 r / min for 5 min to collect the supernatant, and then measure its absorbance at a wavelength of 532 nm. The positive control was butylated hydroxyanisole (BHA, 0.01 mg / mL).

[0038] According to the analysis results ( Figure 4 A), the anti-lipid peroxidation rate of Lactobacillus reuteri Light-ɑ in the present invention is 18.90%, which is slightly lower than the anti-lipid peroxidation rate of the positive control BHA of 22.60%.

[0039] (2) Determination of hydroxyl radical scavenging ability

[0040] Hydroxyl free radical scavenging ability is an important indicator to measure the antioxidant capacity of a substance. Hydroxyl free radicals are generated by Fenton reaction: H2O2+Fe 2+ = OH+H2O+Fe 3+ Add salicylic acid to the reaction system, and the hydroxyl radical (·OH) generated by the Fenton reaction reacts with salicylic acid to generate 2,3-dihydroxybenzoic acid (purple) with special absorption at 510 nm. In a 1.5 mL EP tube, add 60 μL FeSO4 solution (9 mM), 60 μL H2O2 solution (8.8 mM), 60 μL salicylic acid (9 mM) and 10 μL sample solution, mix well, react in a 37°C water bath for 30 min, and then measure the absorbance at 510 nm.

[0041] according to Figure 4 As shown in Figure B, the supernatant sample of Lactobacillus reuteri Light-ɑ in the present invention can efficiently scavenge hydroxyl free radicals, with a scavenging rate of up to 85.81%, which is equivalent to the scavenging efficiency of vitamin C (Vc) in the positive group (87.07%).

[0042] (3) Determination of DPPH free radical scavenging ability

[0043] DPPH is a stable free radical in organic solvents. Its alcohol solution is purple and has maximum absorption at a wavelength of 517 nm. In the presence of free radical scavengers, the solution becomes lighter in color and the absorbance decreases, which can be used to indicate the antioxidant capacity of the sample. In the experiment, 10 μL of sample solution was added to 90 μL of DPPH solution (5 mM), oscillated to mix, and then incubated at room temperature in the dark for 30 min, then centrifuged and the supernatant was placed in a 96-well plate, and the absorbance was measured at 517 nm. According to Figure 4 As shown in Figure C, the supernatant of Lactobacillus reuteri Light-ɑ in the present invention can efficiently scavenge DPPH free radicals, with a scavenging rate of up to 82.01%, which is higher than the scavenging efficiency of vitamin C (Vc) in the positive group (76.87%).

[0044] (4) Determination of total reducing power

[0045] Antioxidants can reduce the trivalent iron of potassium ferrocyanide to divalent iron (yellow crystal potassium ferrocyanide), which can react with ferric chloride to generate a Prussian blue compound with a maximum absorbance at 700 nm. Based on this, the absorbance at 700 nm can indirectly reflect the reducing ability of the antioxidant. In the experiment, 30 μL of bacterial sample was added to a 1.5 mL centrifuge tube, and 60 μL of PBS buffer and 60 μL of 1% potassium ferrocyanide solution were added at the same time. The tube was placed in a 4°C water bath for 20 min, and then quickly cooled to room temperature. 60 μL of 10% trichloroacetic acid was added, mixed, and centrifuged at 3000 r / min in a centrifuge for 10 min. After taking 90 μL of the supernatant, 90 μL of deionized water and 20 μL of 0.1% ferric chloride solution were added, and the reaction was carried out at room temperature for 10 min. After the reaction, the absorbance at 700 nm was measured. The higher the value, the stronger the reducing ability of the sample. The positive control group was 0.2 mg / mL Vc solution.

[0046] According to the analysis results ( Figure 4 D), the absorbance reading of the blank control at 700 nm is 0.103, and the average absorbance value of the culture supernatant of Lactobacillus reuteri Light-ɑ in the present invention is 0.232, which is similar to the positive control group Vc solution, whose absorbance value is 0.258, indicating that the Light-ɑ strain has a strong reducing property.

[0047] During the metabolism of the body, free radicals with strong oxidative properties are generated. The resulting oxidative stress is also a factor that stimulates the production of melanin, which can lead to skin aging and dullness. The significant antioxidant capacity helps to reduce oxidative stress, help resist skin aging, and reduce melanin production. It can be seen that the Lactobacillus reuteri Light-ɑ of the present invention not only has the effect of directly inhibiting melanin production, but also can remove free radicals and resist oxidative stress by exerting antioxidant capacity.

[0048] Example 4 Analysis of acid-base and bile salt tolerance of Lactobacillus reuteri Light-ɑ

[0049] This example is used to evaluate the acid resistance, alkali resistance and bile salt resistance of Lactobacillus reuteri Light-ɑ.

[0050] (1) Artificial gastrointestinal fluid tolerance test

[0051] Preparation of artificial gastric and intestinal fluid: 1 M hydrochloric acid is added with water to adjust the pH to 2.5, 100 mL of hydrochloric acid solution with a pH of 2.5 is measured, 1 g of pepsin is added, and after it is fully dissolved, it is artificial gastric fluid. Weigh 6.8 g of KH2PO4, add 500 mL of distilled water to dissolve, adjust the pH to 6.8 with 0.01 M NaOH solution, transfer to a 1L volumetric flask, add water to the scale, add 10 g of trypsin, and after it is fully dissolved, it is artificial gastric fluid.

[0052] Take 0.5 mL of the culture solution of the Lactobacillus reuteri Light-ɑ strain in the present invention and add it to 4.5 mL of sterile artificial gastric juice and artificial intestinal juice, mix well, and incubate in a 37°C incubator for 3 hours. Take the culture solution at 0 h and 3 h for viable bacteria count, and calculate the survival rate according to the formula. The formula is as follows: survival rate %=logarithm of viable bacteria count at 3 h / logarithm of viable bacteria count at 0 h×100.

[0053] According to Table 1, after incubating Lactobacillus reuteri Light-ɑ strain in artificial gastric juice and artificial intestinal juice for 3 hours, the survival rates were as high as 75.83% and 63.68%, respectively, indicating that the Light-ɑ strain of the present invention has a high acid-base tolerance and can better resist the damage of gastric juice and intestinal juice when taken by the body.

[0054] Table 1 The results of artificial gastrointestinal fluid tolerance of Lactobacillus reuteri Light-ɑ

[0055]

[0056] (2) Bile salt tolerance test

[0057] Take 5 mL of Light-ɑ bacterial solution and mix it with 5 mL of bile salts with concentrations of 0% / 0.1% / 0.2% / 0.3% and 0.4%, respectively. Incubate at 37℃ for 3 h and then count the viable bacteria. Calculate the survival rate according to the formula: survival rate %=(number of viable bacteria after treatment with different concentrations of bile salts / number of viable bacteria without bile salt treatment)×100.

[0058] According to Table 2, after incubation for 3 hours at bile salt concentrations of 0.1% / 0.2% / 0.3% and 0.4%, the survival rates of Lactobacillus reuteri Light-ɑ in the present invention were 59.81%, 49.13% and 30.88%, respectively.

[0059] Table 2 Bile salt tolerance results of Lactobacillus reuteri Light-ɑ

[0060]

[0061] The oral antioxidant probiotic product developed based on the Lactobacillus reuteri Light-ɑ strain must be able to withstand the extreme environment of gastrointestinal fluid once it enters the body. According to the above tolerance experiment, the Lactobacillus reuteri Light-ɑ of the present invention can effectively withstand acid, alkali and bile salt environments, ensuring the survival of the strain in the body, laying a foundation for the development of the product.

[0062] Based on the results of all the above examples, the Lactobacillus reuteri Light-ɑ provided by the present invention can not only effectively inhibit the activity of tyrosinase, a key enzyme in melanin production, but also directly affect the production of melanin; it also has excellent antioxidant capacity, removes free radicals in the body, helps to resist oxidative stress, and reduces oxidative damage. It can be seen that the Light-ɑ strain has great development potential and broad application prospects in the preparation of new whitening and anti-aging probiotic products.

[0063] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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 Lactobacillus reuteri ( Limosilactobacillus reuteri )Light-ɑ, the preservation number of the Lactobacillus reuteri is CGMCC No. 29488.

2. A bacterial agent, comprising the Lactobacillus reuteri Light-ɑ according to claim 1.

3. The bacterial agent according to claim 2, wherein The bacterial agent is a solid preparation or a liquid preparation.

4. The bacterial agent according to claim 2, wherein The solid preparation is a powder prepared by freeze-drying.

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