Compound probiotics with effect of improving skin state and application of compound probiotics in solid beverage

Abstract

The invention relates to a compound probiotic with an effect of improving a skin state and application of the compound probiotic in a solid beverage. The compound probiotic comprises a combination of a bifidobacterium longum subsp. Longum SINE001 strain, a plant lactobacillus SINE002 strain, a lactobacillus reuteri SINE003 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 25664 and a bifidobacterium adolescentis BA-3 strain with the preservation number of CGMCC No. 27625. Potential interaction exists among the four strains, the four strains can cooperate with one another and achieve synergistic interaction, the skin state is remarkably improved by regulating and balancing intestinal flora, and the four strains are specifically expressed in the aspects of oxidation resistance, pigmentation resistance, water supplementing and moisturizing, whitening, aging resistance and the like. Meanwhile, the four bacteria are probiotics, so that the safety is high and dependence is not easy to generate when the four bacteria are used for preparing products with related effects.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and relates to a composite probiotic capable of improving skin condition and application thereof in a solid beverage. Background Art

[0002] The skin is the body's first line of defense. When it is exposed to the external environment and comes into contact with various external stimuli such as air, dust, and sunlight, its microecological homeostasis is easily destroyed, resulting in changes in skin structure and damage to barrier function. Specifically, it manifests as changes in skin surface acid-base imbalance, water loss, dullness and aging, pigmentation, dryness and roughness, fine lines, and even worse, skin cracking and difficulty in healing.

[0003] As the largest surface organ of a biological organism, the skin has a huge microbial ecosystem on its surface. Studies have shown that each square centimeter of skin is covered with about 1 million microorganisms, which is the second largest micro-ecosystem after the intestinal environment. The skin and the intestine have many common features in function and structure, and both are colonized with complex microbial systems. Studies have shown that the imbalance of intestinal flora homeostasis affects intestinal permeability, impairs the barrier function, causes metabolites to enter the blood circulation, and then causes skin inflammation, reduces keratin synthesis or affects epidermal cell differentiation. The balance between skin microbial flora can strengthen the skin barrier, promote skin health, and improve skin problems such as inflammation, allergies, acne, and dryness. Therefore, regulating intestinal flora can be used as one of the effective means to improve skin conditions. At present, there are relatively few probiotic products that improve the skin condition of the body by regulating intestinal flora. Therefore, if a probiotic product that can effectively improve the skin condition and enhance the health level of the skin microecological environment is provided, it has important application value. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a composite probiotic having the effect of improving skin condition and the application of the composite probiotic in a solid beverage.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a composite probiotic with the effect of improving skin condition, wherein the composite probiotic includes a combination of Bifidobacterium longum subsp. longum SINE001 strain, Lactobacillus plantarum SINE002 strain, Lactobacillus reuteri SINE003 strain with a preservation number of CGMCC No.25664, and Bifidobacterium adolescentis BA-3 strain with a preservation number of CGMCC No.27625.

[0007] Among them, the above-mentioned Bifidobacterium longum subspecies longum SINE001 strain is the CMCC P0001 strain preserved in the Chinese Medical Bacteria Collection Center, and is also included in the American Type Culture Collection with the number BAA-2753; the Lactobacillus plantarum SINE002 strain is the commercially available ATCC BAA-2819 strain; the Bifidobacterium adolescentis BA-3 strain is a strain numbered SINE YOUNG from Shanghai SPH Sinopharm Microecology Technology Co., Ltd.

[0008] The present invention develops a new probiotic compounding method and a new strategy for improving skin condition, namely compounding Bifidobacterium longum subspecies long SINE001 strain, Lactobacillus plantarum SINE002 strain, Lactobacillus reuteri SINE003 strain and Bifidobacterium adolescentis BA-3 strain, and finds that there is potential interaction between the four strains, which can cooperate with each other, synergize, and significantly improve skin condition by regulating and balancing intestinal flora, which is specifically manifested in anti-oxidation, anti-pigmentation, moisturizing, whitening, anti-aging and other aspects. At the same time, the four bacteria are all probiotics, so when they are used to prepare products with relevant efficacy, they are highly safe and not prone to dependence.

[0009] In accordance with the "List of Bacteria that Can Be Used in Food" issued by the National Health Commission of the People's Republic of China in 2022, the original Lactobacillus reuteri is now named Limosilactobacillus reuteri.

[0010] Preferably, the ratio of the viable cell counts of the SINE001 strain, the SINE002 strain, the SINE003 strain and the BA-3 strain is (0.5-2):(0.5-2):(0.5-2):(1-5), preferably 1:1:1:3.

[0011] The above “0.5-2” may be, for example, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc.;

[0012] The above-mentioned "1-5" can be, for example, 1, 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here;

[0013] Based on the potential interaction between the four strains, the present invention also found that when the four strains are compounded and used in the above-mentioned specific ratio of viable bacteria count, the effect in improving skin condition is more significant.

[0014] In a second aspect, the present invention provides a probiotic having the effect of improving skin condition, wherein the strain in the probiotic comprises the composite probiotic described in the first aspect.

[0015] Preferably, the total viable bacteria content in the probiotic is not less than 3×10 6 CFU / mL or 3×10 6 CFU / g, for example 3×10 6 CFU / mL (CFU / g), 4×10 6 CFU / mL (CFU / g), 5×10 6 CFU / mL (CFU / g), 8×10 6 CFU / mL (CFU / g), 1×10 7 CFU / mL (CFU / g), 5×10 7 CFU / mL (CFU / g), 1×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), etc. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0016] Preferably, the dosage form of the probiotic includes solution, lyophilized powder, capsule, tablet or granule.

[0017] Preferably, the probiotic further comprises a protective agent.

[0018] Preferably, the protective agent comprises any one of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinyl pyrrolidone, sorbitol, xylo-oligosaccharides, fructo-oligosaccharides or xylitol, or a combination of at least two thereof.

[0019] Preferably, the probiotic is in the form of a solution, which is prepared by the following method:

[0020] The four strains, namely, SINE001, SINE002, SINE003 and BA-3, are inoculated into culture medium respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain bacterial suspensions of the four strains; the bacterial suspensions of the four strains are mixed according to the ratio of live bacteria count to obtain the probiotic agent.

[0021] Preferably, the probiotic is in the form of a freeze-dried powder, which is prepared by the following method:

[0022] The four strains, namely, SINE001, SINE002, SINE003 and BA-3, are inoculated into culture medium respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and freeze-dried to obtain bacterial powders of the four strains; the bacterial powders of the four strains are mixed according to the ratio of live bacteria count to obtain the probiotic agent.

[0023] In a third aspect, the present invention provides a use of the composite probiotic according to the first aspect or the probiotic according to the second aspect in a skin care product.

[0024] In a fourth aspect, the present invention provides a solid beverage having the effect of improving skin condition, wherein the solid beverage comprises the composite probiotics described in the first aspect or the probiotic agent described in the second aspect.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention develops a new probiotic compounding method and a new strategy for improving skin condition, namely compounding Bifidobacterium longum subspecies long SINE001 strain, Lactobacillus plantarum SINE002 strain, Lactobacillus reuteri SINE003 strain and Bifidobacterium adolescentis BA-3 strain, and finds that there is potential interaction between the four strains, which can cooperate with each other, synergize and significantly improve skin condition, which is specifically manifested in multiple aspects such as anti-oxidation, anti-pigmentation, moisturizing, whitening, anti-aging and regulation and balanced intestinal flora. At the same time, the four bacteria are all probiotics, so when they are used to prepare products with relevant efficacy, they are highly safe and not easy to produce dependence.

[0027] The SINE003 strain involved in the present invention is classified and named as Lactobacillus reuteri, and the depository is the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 25664, a deposit date of September 7, 2022, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0028] The BA-3 strain involved in the present invention is classified and named Bifidobacterium adolescentis, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation number is CGMCC No.27625, the preservation date is June 14, 2023, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the yolk sac pigment signal diagram and the statistical result diagram of pigment signal intensity of each group of zebrafish samples;

[0030] Figure 2 It is the yolk sac fluorescence imaging and fluorescence intensity statistical result diagram of each group of zebrafish samples;

[0031] Figure 3 It is the tail fin imaging and tail fin area statistical result diagram in the hydration and moisturizing effect test of each group of zebrafish samples;

[0032] Figure 4 This is a statistical result diagram of the melanin protein content and the relative expression of tyr gene in each group of zebrafish samples;

[0033] Figure 5 This is a statistical graph of telomerase activity and average telomere length in each group of zebrafish samples;

[0034] Figure 6 It is the statistical result graph of AGEs fluorescence intensity in each group of zebrafish samples;

[0035] Figure 7 The tail fin imaging and tail fin area statistical results of each group of zebrafish samples in the anti-photoaging effect test;

[0036] Figure 8 This is the statistical result of the content of type I collagen in each group of zebrafish samples;

[0037] Fig. 9 This is a test result of the ability of compound probiotics to improve skin condition;

[0038] Fig.10 This is the result of the alpha diversity analysis of compound probiotics to improve intestinal microbial flora;

[0039] Fig.11 This is the result of the β-diversity analysis of compound probiotics to improve intestinal microbial flora;

[0040] Fig.12 This is the result of the gate-level difference analysis of compound probiotics in improving intestinal microbial flora;

[0041] Fig.13This is the result of the genus-level difference analysis of compound probiotics in improving intestinal microbial flora;

[0042] Fig.14 This is the result of species-level difference analysis of compound probiotics in improving intestinal microbial flora. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0044] The following bacterial species information is:

[0045] ① The classification name of the SINE003 strain involved below is Lactobacillus reuteri, and the deposit number is CGMCC No.25664;

[0046] ② The BA-3 strain involved in the following classification is named Bifidobacterium adolescentis, with a preservation number of CGMCC No. 27625, and is from the strain numbered SINE YOUNG of Shanghai SINE Pharmaceuticals Microecology Technology Co., Ltd.;

[0047] ③ The SINE001 strain involved below is the commercially available BAA-2753 strain;

[0048] ④ The SINE002 strain mentioned below is the commercially available BAA-2819 strain.

[0049] The culture medium and its formula involved below are:

[0050] MRS medium: beef extract 10.0 g, glucose 20.0 g, peptone 10.0 g, yeast extract 5.0 g, sodium chloride 5.0 g, diammonium hydrogen citrate 2.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween-80 1.0 g, distilled water to 1000 mL, pH 6.2-6.4.

[0051] The hyaluronic acid mentioned below was purchased from By-Health Sodium Hyaluronate YEP Water Glow Bag Oral Product.

[0052] The water quality of the zebrafish farming water involved below is: 200 mg of instant sea salt is added to 1L of reverse osmosis water, the conductivity is 450-550 μS / cm; pH is 6.5-8.5; hardness is 50-100 mg / L CaCO3; the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458), IACUC ethics review number: IACUC-2024-10815-01.

[0053] The preparation method of bacterial suspension and bacterial powder involved below is: after activating the strain, inoculate it into culture medium for cultivation to obtain culture solution; centrifuge the culture solution, resuspend the bacteria to obtain bacterial suspension, or further add protective agent for freeze drying to obtain freeze-dried bacterial powder product.

[0054] The experimental results were statistically analyzed using SPSS26.0 software. Compared with the control group, *** represents p<0.001, ** represents p<0.01, and * represents p<0.05.

[0055] Example 1

[0056] This example explores the antioxidant and anti-pigmentation effects of composite probiotics

[0057] (1) Animal grouping, modeling, and intervention methods

[0058] Zebrafish (AL strain) 3 days after fertilization were randomly selected in 6-well plates, with 30 fish in each well and a volume of 3 mL per well. They were randomly divided into 6 groups: S1 group (blank control group, fish water), S2 group (model control group, fish water), S3 group (positive control group, water-soluble N-acetyl-L-cysteine ​​(fish water), water-soluble concentration of 62.5 μg / mL), S4 group (probiotic intervention group-1, water-soluble probiotics (fish water), the total concentration of water-soluble live bacteria of probiotics was 3.18×10 6 CFU / mL), S5 group (probiotic intervention group-2, water-soluble probiotics (water for fish farming), the total concentration of water-soluble live probiotics was 3.18×10 7 CFU / mL), S6 group (probiotic intervention group-3, water-soluble probiotics (water for fish farming), the total concentration of water-soluble live probiotics was 3.18×10 8 CFU / mL), where the probiotics used in groups S4-S6 were a combination of SINE001, SINE002, SINE003 and BA-3 with a live bacteria count ratio of 1:1:1:3. After 3 h of water treatment at 28°C, except for group S1, groups S2 to S6 were treated with water-soluble menadione (water-soluble concentration of 2.25 μM) to stimulate zebrafish to establish an oxidative stress model, and the test was performed after continuing to treat at 28°C for 21 h.

[0059] (2) Anti-pigmentation effect test

[0060] Ten fish were randomly selected from each group for observation, and the pigment level in the zebrafish yolk sac was analyzed to determine the degree of pigment deposition. Figure 1 shown.

[0061] Menadione can cause abnormal pigment deposition in zebrafish. As can be seen from the figure, compared with the pigment intensity signal test results of group S1 (blank control group), the pigment signal intensity in group S2 (model control group) increased significantly, proving that the zebrafish pigment deposition model was successfully established; while the pigment signal intensity in the zebrafish yolk sac in each probiotic intervention group S4-S6 showed a downward trend to varying degrees, and the anti-pigmentation effect of probiotic intervention in group S6 was close to the effect of the positive drug N-acetyl-L-cysteine. It can be seen that the composite probiotics provided by the present invention have excellent anti-pigmentation effects.

[0062] (3) Antioxidant effect test

[0063] The zebrafish in each group were fluorescently stained with Cell ROX Green reagent (2714479, Invitrogen). Ten fish were randomly selected from each group and photographed under a fluorescence microscope. The images were saved and analyzed using NIS-Elements D3.20 software to analyze the fluorescence intensity of the zebrafish yolk sac. The statistical analysis results of this index were used to evaluate the antioxidant efficacy of the samples. The results are shown in Table 1. Figure 2 shown.

[0064] Menadione can produce reactive oxygen species and cause oxidative stress in zebrafish, and the oxidation level is judged by the fluorescence intensity in the zebrafish yolk sac. The statistical results in the figure show that compared with the fluorescence imaging results of the S1 group (blank control group), the fluorescence intensity of zebrafish in the S2 group (model control group) increased significantly and was highly fluorescent throughout the yolk sac. The ROS fluorescence intensity in the zebrafish yolk sac in the S4-S6 probiotic intervention groups all showed varying degrees of decline, and the antioxidant effect was better than that of the S3 group (positive treatment group), indicating that the composite probiotics used in the present invention can scavenge free radicals and have excellent antioxidant effects.

[0065] Example 2

[0066] This example explores the hydrating and moisturizing ability of the composite probiotics

[0067] (1) Animal grouping, modeling, and intervention methods

[0068] Zebrafish (AL strain) 4 days after fertilization were randomly selected in a 6-well plate, with 30 fish in each well and a capacity of 3 mL in each well. They were randomly divided into 6 groups. Except for the positive control group S3, in which the water-soluble N-acetyl-L-cysteine ​​of the S3 group in Example 1 was replaced with water-soluble hyaluronic acid (water-soluble dosage concentration was 15.6 μL / mL), the grouping and intervention methods of the other 5 groups (S1-S2 and S4-S6 groups) were the same as those of the corresponding groups in Example 1.

[0069] After zebrafish samples in each group were treated at 28°C for 18 h, except for group S1, groups S2 to S6 were all treated with water-soluble sodium chloride (water-soluble concentration was 11 mg / mL) to stimulate zebrafish to establish a water deprivation model, and the treatment was continued at 28°C for 6 h before testing.

[0070] (2) Hydrating and moisturizing effect test

[0071] Ten fish were randomly selected from each group for dissection and photographed under a dissecting microscope. The tail area of ​​zebrafish was then analyzed using NIS-ElementsD 3.20 software. The results of this index were used to evaluate the moisturizing effect of the samples. The results are as follows: Figure 3 shown.

[0072] By adding sodium chloride to the feeding environment of zebrafish, the environmental osmotic pressure can be changed to cause zebrafish to be dehydrated, so that the degree of dehydration can be effectively judged by observing the state of the zebrafish tail fin, to explore the water replenishment and moisturizing ability of the sample after the intervention of the composite probiotic sample. As can be seen from the figure, compared with the typical figure of the zebrafish tail fin area of ​​the S1 group (blank control group), the tail fin area of ​​the zebrafish in the S2 group (model control group) showed a significant downward trend, and wrinkling occurred; while the tail fin area of ​​the zebrafish samples in the S4-S6 probiotic intervention groups was reduced and the shrinkage was improved to varying degrees, and the S6 group effect was better than the treatment effect in the S3 positive control group, indicating that the composite probiotics provided by the present invention have excellent water replenishment and moisturizing effects.

[0073] Example 3

[0074] This example explores the whitening effect of composite probiotics

[0075] (1) Animal grouping, modeling, and intervention methods

[0076] Melanocyte stimulating hormone was microinjected into the yolk sac of wild-type zebrafish (AB strain) 3 days after fertilization to establish a zebrafish melanin increase model. Zebrafish in good condition were selected under a microscope and randomly assigned to 6-well plates, 30 per well, 3 mL per well, and randomly divided into 5 groups. Except for the positive control group of group S3, the water-soluble N-acetyl-L-cysteine ​​of group S3 in Example 1 was replaced with water-soluble whitening pills (POLA, water-soluble concentration of 25 μg / mL), and the grouping and intervention methods of the remaining 4 groups (groups S2 and S4-S6) were the same as those of the corresponding groups in Example 1. Group S1 was a blank control group, using wild-type zebrafish (AB strain) at 3dpf without modeling, and each group of zebrafish samples were tested after being treated at 28°C for 24h.

[0077] (2) Whitening effect test

[0078] After the treatment, the zebrafish samples from each group were collected, and the melanin protein content in the zebrafish was detected by the melanin biological kit; the total RNA of each group of zebrafish was extracted using an RNA extraction kit, and the total RNA concentration and purity were determined using a UV-visible spectrophotometer. Then, 2.00 μg of total RNA from the zebrafish samples was taken, and cDNA was synthesized according to the instructions of the cDNA first-chain synthesis kit. The relative expression of the tyrosinase (tyr) gene was detected and calculated by q-PCR. The oral whitening efficacy of the samples was evaluated based on the statistical analysis results of the zebrafish melanin protein content and the relative expression of the tyr gene. The results are as follows: Figure 4 shown.

[0079] As can be seen from the figure, the composite probiotics provided by the present invention can significantly reduce the melanin content and inhibit the expression of the rate-limiting enzyme in melanin synthesis, and has an excellent whitening effect.

[0080] Example 4

[0081] This example explores the anti-aging effect of compound probiotics

[0082] (1) Animal grouping, modeling, and intervention methods

[0083] Wild-type zebrafish (AB strain) 6 hours after fertilization were randomly selected in a 6-well plate, with 30 fish in each well and a capacity of 3 mL in each well. They were randomly divided into 6 groups. Except for the positive control group S3, in which the water-soluble N-acetyl-L-cysteine ​​of the S3 group in Example 1 was replaced with water-soluble resveratrol (water-soluble concentration of 20 μg / mL), the grouping and intervention methods of the other 5 groups (S1-S2 and S4-S6 groups) were the same as those of the corresponding groups in Example 1.

[0084] Except for group S1, groups S2 to S6 were stimulated with aqueous hydrogen peroxide solution (water-soluble concentration was 1000 μM) to establish an aging model in zebrafish. The solution was changed every day and the treatment was continued at 28°C for 6 days before testing.

[0085] (2) Anti-aging effect test

[0086] The total DNA of zebrafish in each group was extracted using a universal genomic DNA extraction kit, and the total DNA concentration and purity were measured using a UV-visible spectrophotometer. Using dio2 (deiodinase, iodothyronine, type II) as a single-copy gene, the ratio of telomere repeat copies (T) to single-copy (S) gene products was calculated, and the telomere length was expressed as a relative T / S ratio; zebrafish samples were collected according to the instructions of the Zebrafish TE Elisa Kit, and data were collected using a multifunctional microplate reader. The telomerase activity in zebrafish in each experimental group was analyzed, and the anti-aging effect of the samples was evaluated by the analysis results of the average telomere length and telomerase activity indicators. The results are shown in Figure 2. Figure 5 shown.

[0087] Each cell division in the organism is accompanied by DNA loss at the end of the chromosome, resulting in the gradual shortening of telomeres. When the telomere length reaches a critical length, the cell shows that it can no longer divide normally, which is the mechanism of aging. It can be seen that telomerase is a special reverse transcriptase, and telomerase activity refers to the ability of telomerase to repair and extend telomeres. Therefore, the detection of the average telomere length and telomerase activity can show the aging level in the organism. As can be seen from the figure, compared with the S1 group (blank control group), the average length of telomerase in the zebrafish in the S2 group (model control group) decreased significantly, and the activity was greatly reduced, indicating the successful construction of the zebrafish aging model; and the average length of telomeres and telomerase activity in the zebrafish in the S4-S6 probiotic intervention groups all showed varying degrees of increase, and the ability of the composite probiotics in the S6 group to repair the shortened telomere length caused by aging was better than the positive control drug resveratrol in the S3 group, indicating that the composite probiotics provided by the present invention can repair telomeres and improve telomerase activity, with excellent anti-aging effect.

[0088] Example 5

[0089] This example explores the anti-glycation effect of composite probiotics

[0090] (1) Animal grouping, modeling, and intervention methods

[0091] Wild-type zebrafish (AB strain) 5 days after fertilization were randomly selected in 1.5 mL centrifuge tubes, 10 in each well, and the capacity of each well was 150 μL. They were randomly divided into 6 groups. Except for the positive control group S3 group, in which the water-soluble N-acetyl-L-cysteine ​​of the S3 group in Example 1 was replaced with water-soluble POLA anti-sugar pills (water-soluble concentration of 125 μg / mL), the grouping and intervention methods of the other 5 groups (S1-S2 and S4-S6 groups) were the same as those of the corresponding groups in Example 1.

[0092] Except for group S1, groups S2 to S6 were treated with water-soluble glucose (water-soluble concentration was 0.4 M) to stimulate zebrafish to establish a saccharification model, and the test was performed after 24 h of shaking treatment at 60°C.

[0093] (2) Anti-glycation effect test

[0094] After the intervention breeding, the zebrafish in each group were treated at 60°C for 1 day, and then the zebrafish tissues in all groups were ground and centrifuged. The supernatant was taken at 80 μL / well, and the data were collected using a multifunctional microplate reader. The fluorescence value of advanced glycation end products (AGEs) in each group was analyzed. The anti-glycation efficacy of the samples was evaluated based on the statistical analysis results of this indicator. The results are as follows: Figure 6 shown.

[0095] AGEs are a class of complex compounds generated by the reaction of reducing sugars with the primary amino groups of macromolecules such as proteins, lipids or nucleic acids under non-enzymatic conditions. The level of sugar in the body can be indirectly reflected by detecting the level of AGEs. As can be seen from the figure, compared with the test results of AGEs levels in zebrafish in group S1 (blank control group), the level of AGEs in zebrafish in group S2 (model control group) increased significantly, indicating that the zebrafish glycation model was successfully constructed; and the level of AGEs in zebrafish samples in the probiotic intervention groups S4-S6 showed different degrees of reduction, indicating that the composite probiotics provided by the present invention have excellent anti-glycation effects.

[0096] Example 6

[0097] This example explores the anti-photoaging effect of composite probiotics

[0098] (1) Animal grouping, modeling, and intervention methods

[0099] Wild-type zebrafish (AB strain) 5 days after fertilization were randomly selected in a 6-well plate, with 30 fish in each well and a capacity of 3 mL in each well. They were randomly divided into 6 groups. Except for the positive control group S3 group, in which the water-soluble N-acetyl-L-cysteine ​​of the S3 group in Example 1 was replaced with water-soluble POLA anti-sugar pills (water-soluble concentration of 125 μg / mL), the grouping and intervention methods of the other 5 groups (S1-S2 and S4-S6 groups) were the same as those of the corresponding groups in Example 1.

[0100] Except for group S1, groups S2 to S6 used sunlight to stimulate zebrafish to establish a photoaging model, and the test was performed after 24 hours of irradiation at 28°C.

[0101] (2) Anti-light aging effect test

[0102] Ten zebrafish were randomly selected from each group and placed under a dissecting microscope to take pictures and save the pictures. NIS-Elements D3.20 advanced image processing software was used to analyze and collect data. The zebrafish tail fin area was analyzed. The results are as follows: Figure 7 shown.

[0103] At the same time, zebrafish samples from each group were collected according to the instructions of the ELISA kit, and data were collected using a multifunctional microplate reader to analyze the content of type I collagen in each group of zebrafish. The results are as follows: Figure 8 shown.

[0104] Photoaging is mainly caused by ultraviolet rays in sunlight, which induce abnormal changes in skin tissue, the most common manifestation of which is the loss of collagen and changes in tissue morphology. As can be seen from the figure, compared with the S1 group (blank control group), the zebrafish in the S2 group (model control group) were obviously wrinkled, morphologically changed, and the tail area was significantly reduced. Further detection of the type I collagen content in the zebrafish body was significantly reduced, indicating that the zebrafish photoaging model was successfully constructed; and the reduction in the tail fin area of ​​the zebrafish in the S4-S6 probiotic intervention groups was significantly improved, the wrinkled tail fin morphology was improved, and the collagen content levels in the body showed varying degrees of increase, indicating that the composite probiotics provided in the present invention have excellent anti-photoaging effects.

[0105] Example 7

[0106] This example explores the ability of composite probiotics to improve skin condition

[0107] (1) Chinese healthy subjects (19 females and 15 males) with no facial inflammation, scars, moles, hirsutism, etc., and no abnormal liver and kidney function in the past month were selected. The subjects had no allergic diseases, and their age range was 25-45 years old, with an average age of 33.26±5.54 years old. After arriving at the laboratory, the subjects sat quietly for 30 minutes and their facial skin was tested. The test environment was 20-22℃ and 40%-60% humidity.

[0108] (2) Test items include:

[0109] The facial skin color was tested using the DSMI Colormeter;

[0110] The moisture content of the facial stratum corneum was tested using a skin moisture meter, Corneometer CM825.

[0111] (3) Each subject took freeze-dried probiotic powder 30 minutes after dinner every day (the total number of viable bacteria in the powder was 3×10 10 CFU, the bacterial powder is composed of SINE001 powder, SINE002 powder, SINE003 powder and BA-3 powder with a ratio of 1:1:1:3 of viable bacteria count. After 30 days, the facial skin was tested again in the laboratory. The results are as follows Fig. 9 shown.

[0112] As can be seen from the figure, before taking the composite probiotic powder, the stratum corneum water content (mean) of the subjects was 61.18±12.32 (au); the skin erythema index E value (mean) was 15.55±2.52; the skin melanin index M value was 38.66±3.37; and the results of re-testing after oral probiotics for 30 days showed that compared with the above test results, the subjects' skin stratum corneum water content increased by 12.67%; the skin erythema index E value decreased by 2.44%; the skin melanin index M value decreased by 1.5%; it can be seen that the composite probiotics provided by the present invention can significantly increase the skin's stratum corneum water content and reduce the skin erythema index and skin melanin content after taking, and have an excellent effect on improving skin condition.

[0113] Example 8

[0114] This example explores the effect of compound probiotics on intestinal flora

[0115] The feces of the subjects in Example 7 were collected before (A1) and 30 days after taking the compound probiotics (A2) for intestinal flora detection. The raw data obtained by the detection were spliced ​​and filtered to obtain valid data, and then noise reduction was performed by DADA2 to obtain the final ASVs, and then Alpha diversity and Beta diversity analysis were performed; the T-test statistical analysis method was used to perform a significant difference test on the species composition and community structure of the grouped samples. The statistical analysis results are as follows Figures 10 to 14 shown.

[0116] Depend on Fig.10 As shown in the Alpha diversity analysis result graph of the subjects' intestinal microorganisms, the Alpha diversity of the subjects' intestinal microbial flora increased significantly after taking the compound probiotics for 30 days, indicating that the diversity of intestinal microorganisms can be effectively improved after oral administration of the compound probiotics provided by the present invention.

[0117] Depend on Fig.11From the Beta diversity analysis results of the intestinal microorganisms of the subjects, it can be seen that the Beta diversity of the intestinal microbial flora of the subjects increased significantly after taking the composite probiotics for 30 days, indicating that the abundance of intestinal microorganisms can be effectively improved after oral administration of the composite probiotics provided by the present invention.

[0118] Depend on Figure 12 to Figure 14 The results of the differential analysis of the intestinal microorganisms of the subjects at the phylum, genus and species levels show that oral administration of the composite probiotics provided by the present invention can significantly improve the diversity and abundance of the intestinal flora in the subjects, and further analysis found that the differences in the phylum level of the intestinal microorganisms of the subjects were mainly Bacteroidetes, Firmicutes, Actinobacteriota and Fusobacteriota; the differences in the genus level of the intestinal microorganisms of the subjects were mainly Bacteroides. , Prervotella_9, Faecalibacterium and Megamonas, among which Blautia_faecis was the microbial species with the most significant change, indicating that the composite probiotics composed of Bifidobacterium longum subsp. longum SINE001 strain, Lactobacillus plantarum SINE002 strain, Lactobacillus reuteri SINE003 strain and Bifidobacterium adolescentis BA-3 strain provided by the present invention can effectively regulate the diversity and richness of human intestinal flora, and improve the level of beneficial bacteria to give full play to the probiotic effect.

[0119] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0120] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0121] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A composite probiotic having the effect of improving skin condition, characterized in that: The composite probiotics include a combination of Bifidobacterium longum subsp. longum SINE001 strain, Lactobacillus plantarum SINE002 strain, Lactobacillus reuteri SINE003 strain with a preservation number of CGMCC No.25664, and Bifidobacterium adolescentis BA-3 strain with a preservation number of CGMCC No.27625.

2. The composite probiotic having the effect of improving skin condition according to claim 1, characterized in that: The ratio of the number of viable bacteria of the SINE001 strain, the SINE002 strain, the SINE003 strain and the BA-3 strain is (0.5-2):(0.5-2):(0.5-2):(1-5).

3. A probiotic agent having the effect of improving skin condition, characterized in that: The strains in the probiotics include the composite probiotics according to claim 1 or 2.

4. The probiotic agent having the effect of improving skin condition according to claim 3, characterized in that: The total live bacteria content in the probiotics is not less than 3×10 6 CFU / mL or 3×10 6 CFU / g.

5. The probiotic agent having the effect of improving skin condition according to claim 3, characterized in that: The dosage form of the probiotics includes solution, lyophilized powder, capsule, tablet or granule.

6. The probiotic agent having the effect of improving skin condition according to claim 3, characterized in that: The probiotics also include a protective agent; Preferably, the protective agent comprises any one of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinyl pyrrolidone, sorbitol, xylo-oligosaccharides, fructo-oligosaccharides or xylitol, or a combination of at least two thereof.

7. The probiotic agent having the effect of improving skin condition according to claim 5, characterized in that: The dosage form of the probiotic is a solution, which is prepared by the following method: The four strains, namely, SINE001, SINE002, SINE003 and BA-3, are inoculated into culture medium respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain bacterial suspensions of the four strains; the bacterial suspensions of the four strains are mixed according to the ratio of live bacteria count to obtain the probiotic agent.

8. The probiotic agent having the effect of improving skin condition according to claim 5, characterized in that: The dosage form of the probiotic is a freeze-dried powder, which is prepared by the following method: The four strains, namely, SINE001, SINE002, SINE003 and BA-3, are inoculated into culture medium respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and freeze-dried to obtain bacterial powders of the four strains; the bacterial powders of the four strains are mixed according to the ratio of live bacteria count to obtain the probiotic agent.

9. Use of the composite probiotic according to claim 1 or 2 or the probiotic according to any one of claims 3 to 8 in skin care products.

10. A solid beverage having the effect of improving skin condition, characterized in that: The solid beverage comprises the composite probiotics according to claim 1 or 2 or the probiotic agent according to any one of claims 3 to 8.

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