A multi-strain probiotic composition for enhancing skin radiance

Abstract

This invention discloses a multi-strain probiotic composition for enhancing skin radiance, relating to the field of microbial technology. This invention utilizes a specific combination of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum. These active probiotic strains work synergistically to effectively improve skin condition and enhance skin radiance. By regulating the balance of intestinal flora, it promotes the absorption of nutrients and the excretion of metabolic waste, thereby indirectly improving skin health, making the skin smoother, more elastic, and radiant with a natural glow.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a multi-strain probiotic composition for enhancing skin radiance. Background Technology

[0002] In today's society, with the improvement of people's living standards and the increasing pursuit of beauty, skin health and radiance have become a focus of attention for consumers. As the largest organ in the human body, the condition of the skin is not only influenced by factors such as genetics and environment, but also closely related to the balance of the gut microbiota. The gut microbiota, as the body's "invisible organ," plays a crucial role in regulating nutrient absorption, metabolic waste excretion, and immune function. When the gut microbiota is imbalanced, it not only affects the overall health of the body, but may also indirectly affect the skin condition through complex physiological mechanisms, leading to problems such as dullness and lack of radiance.

[0003] Traditional skincare methods often focus on external care, such as applying and massaging various skincare products to improve skin condition and enhance radiance. However, these methods often only work on the skin's surface and are insufficient to address skin problems caused by gut microbiota imbalance. Furthermore, some skincare products may contain chemical ingredients that, with long-term use, may irritate or burden the skin, exacerbating existing problems. While some oral beauty products exist, their ingredients are mostly singular and insufficient to comprehensively regulate gut microbiota balance, thus limiting their effectiveness in improving skin radiance.

[0004] In view of the limitations of traditional skin care methods and oral beauty products in improving skin radiance, the present invention proposes a multi-strain probiotic composition to improve skin radiance, which is of particular importance. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multi-strain probiotic composition that enhances skin radiance. Through the synergistic effect of a specific ratio of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum, it can effectively regulate the balance of intestinal flora, promote the absorption of nutrients and the excretion of metabolic waste, thereby indirectly improving skin health, making the skin smoother, more elastic, and radiant with a natural glow.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a multi-strain probiotic composition for improving skin radiance, made from the following raw materials in parts by weight: The active probiotic strains in the composition consist of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum.

[0007] Furthermore, in the composition, the viable count of each single strain of Lactobacillus retardii, Lactobacillus rhamnosus, and Lactobacillus plantarum is not less than 1.0 × 10^8 CFU / g.

[0008] Furthermore, the total viable count of Lactobacillus retardans, Lactobacillus rhamnosus, and Lactobacillus plantarum in the composition is not less than 1.0 × 10^9 CFU / g.

[0009] Furthermore, the live count ratio of Lactobacillus retardans, Lactobacillus rhamnosus, and Lactobacillus plantarum is (1-5):(1-3):(1-3).

[0010] Furthermore, the live count ratio of Lactobacillus retardans, Lactobacillus rhamnosus, and Lactobacillus plantarum is 2:1:1.

[0011] Furthermore, the composition also includes a food- or pharmaceutically acceptable excipient selected from at least one of prebiotics, fillers, lubricants, flavoring agents, and freeze-drying protectants.

[0012] Furthermore, the excipients include prebiotics, which are selected from at least one of fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, inulin, and stachyose.

[0013] Furthermore, the composition is an oral solid dosage form, which is selected from any one of lyophilized powder, hard capsule, tablet, and granule.

[0014] Furthermore, the Lactobacillus wrasse, Lactobacillus rhamnosus, and Lactobacillus plantarum are all present in the composition in the form of freeze-dried bacterial powder.

[0015] Furthermore, the excipients include a freeze-drying protectant selected from at least one of skim milk powder, trehalose, mannitol, and glycerin.

[0016] Compared with existing technologies, this multi-strain probiotic composition for improving skin radiance has the following beneficial effects: I. This invention utilizes a specific combination of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum. These active probiotic strains work synergistically to effectively improve skin condition and enhance skin radiance. By regulating the balance of intestinal flora, it promotes the absorption of nutrients and the excretion of metabolic waste, thereby indirectly improving skin health, making the skin smoother, more elastic, and radiant with a natural glow.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 A flowchart of a multi-strain probiotic composition for improving skin radiance. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0021] Example 1 This embodiment provides a multi-strain probiotic composition for improving skin radiance, specifically an oral lyophilized powder formulation. It uses Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum in a live bacteria count ratio of 2:1:1. The control standards for the live bacteria count of each strain and the entire preparation process of the composition are clearly defined. The composition is verified to regulate the balance of intestinal flora and improve skin radiance. This is the optimal implementation of the core technical solution of this invention.

[0022] Strains and Raw Material Preparation: The active strains used in this embodiment are *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum*. The excipients used include a freeze-drying protectant and prebiotics. The freeze-drying protectant is skim milk powder and trehalose, and the prebiotics are fructooligosaccharides and inulin.

[0023] The first step in preparing single-strain freeze-dried bacterial powder is strain activation. Preserved strains of three different strains were inoculated into MRS liquid medium and cultured in an anaerobic incubator at 36°C for 24 hours to complete the first generation activation. The activated bacterial solution was then inoculated into fresh MRS liquid medium at a 2% inoculation rate and cultured for another 18 hours under the same conditions to complete the second generation activation, obtaining a stable seed culture. The second step is fermentation culture. The seed cultures of the three strains were inoculated into fermentation medium at a 3% inoculation rate. The fermentation medium consisted of peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, diammonium hydrogen citrate, Tween 80, magnesium sulfate, and manganese sulfate. The fermentation process was controlled at 36°C, with a stirring speed of 80 rpm, maintaining an anaerobic environment throughout. The fermentation period was 20 hours. At the end of fermentation, the OD600 value of the fermentation broth stabilized, and the cell concentration reached its peak. The third step involves cell collection and mixing with a protective agent. After fermentation, the fermentation broth was placed at 4 degrees Celsius and centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, and the bacterial precipitate was collected. The precipitate was washed twice with sterile physiological saline, and the cells were collected again by centrifugation to obtain wet cells. A lyophilization protectant solution consisting of 10% skim milk powder and 5% trehalose was added to the wet cells, with a mass ratio of wet cells to lyophilization protectant solution of 1:3. The mixture was thoroughly stirred to obtain a bacterial suspension. The fourth step was vacuum freeze-drying. The well-mixed bacterial suspension was spread evenly in a sterile freeze-drying tray to a thickness of 8 mm and placed in a vacuum freeze dryer. Pre-freezing was performed first, controlling the pre-freezing temperature at -40 degrees Celsius for 4 hours to ensure that the bacterial suspension was completely frozen. After pre-freezing, the vacuum system was activated and the vacuum level was controlled at 10 Pa. The first stage of sublimation drying was controlled at -20 degrees Celsius for 12 hours, and the second stage of desorption drying was controlled at 25 degrees Celsius for 6 hours, maintaining a stable vacuum level throughout the process. After drying, the single-strain freeze-dried bacterial powder was obtained by crushing, sealed, and stored in a light-protected environment at 4 degrees Celsius.

[0024] Preparation of the multi-strain probiotic composition: The viable count of the three single-strain lyophilized bacterial powders was tested using the lactic acid bacteria test method specified in GB4789.35-2016. The specific procedure included serially diluted samples, inoculated onto MRS agar medium, anaerobically cultured at 36°C for 48 hours, and colony count calculation. After testing, lyophilized Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum bacterial powders were weighed out at a viable count mass ratio of 2:1:1 and placed in a three-dimensional motion mixer. The mixing speed was controlled at 15 rpm for 20 minutes to obtain a multi-strain mixed bacterial powder. Prebiotic excipients were added to the multi-strain mixed bacterial powder. The prebiotics consisted of fructooligosaccharides and inulin in a 1:1 mass ratio. The mass ratio of the mixed bacterial powder to the prebiotics was 1:4. The mixture was continued to be mixed for 20 minutes under the same conditions. After mixing, the mixture was packaged in aluminum foil bags under sterile conditions. Each bag contained 2g of net weight. The bags were sealed and stored in a cool, dry place to obtain the multi-strain probiotic freeze-dried powder product.

[0025] The quality indicators of the composition were tested. The results showed that the viable counts of single strains of Lactobacillus wrasse, Lactobacillus rhamnosus, and Lactobacillus plantarum in the finished product were not less than 1.0×10^8 CFU / g, the total viable count of the three strains was not less than 1.0×10^9 CFU / g, and the moisture content, total colony count, pathogenic bacteria and other indicators all met the relevant national standards.

[0026] The efficacy verification experiment of the composition involved 30 healthy subjects aged 20-45 years with dull and lackluster skin. Each subject took one sachet of the lyophilized powder prepared in this embodiment orally daily for 28 consecutive days. During the experiment, subjects maintained normal diet and lifestyle, and did not use other oral beauty products or functional skincare products. On day 0 and day 28 of the experiment, skin luster on the cheeks was measured using a skin luster meter under constant temperature and humidity conditions. Simultaneously, stool samples were collected, and gut microbiota diversity was detected using 16S rRNA high-throughput sequencing technology. The results showed that after 28 days of continuous use, the subjects' skin luster increased by an average of 14.2% compared to before the experiment, and the Shannon diversity index of gut microbiota increased by an average of 22.6% compared to before the experiment. Subjects generally reported improved skin smoothness and reduced dullness. No adverse reactions occurred during the experiment.

[0027] like Figure 1 As shown in the figure, the process flow chart for preparing multi-strain probiotic freeze-dried powder to enhance skin radiance in this embodiment is as follows: strain activation, seed liquid preparation, fermentation culture, cell centrifugation, collection, freeze-drying, protective agent mixing, vacuum freeze-drying, single-strain bacterial powder compounding, prebiotic excipient mixing, aseptic packaging, and finished product inspection.

[0028] This embodiment fully realizes the core content of the technical solution of the present invention. The prepared multi-strain probiotic composition meets the requirements for live bacteria count control. Through the synergistic effect of the three strains, the balance of intestinal flora is effectively regulated, and the skin luster is steadily improved, thus verifying the feasibility and beneficial effects of the technical solution of the present invention.

[0029] Example 2 This embodiment provides a multi-strain probiotic composition for improving skin radiance, specifically in the form of an oral hard capsule. It uses Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum in a live bacteria count ratio of 1:1:1. The entire preparation process and quality control standards of the composition are clearly defined, and the implementation effect of the lower limit parameter within the live bacteria count ratio range of the present invention is verified, covering the boundary protection scope of the technical solution of the present invention.

[0030] Strains and Raw Material Preparation: The active strains used in this embodiment are *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum*. The excipients used include a freeze-drying protectant, prebiotics, fillers, and lubricants. The freeze-drying protectant is skim milk powder and mannitol; the prebiotic is galactooligosaccharides; the filler is microcrystalline cellulose; and the lubricant is magnesium stearate.

[0031] The first step in preparing the freeze-dried bacterial powder of a single strain is strain activation. Preserved strains of three different strains were inoculated into MRS liquid medium and cultured in an anaerobic incubator at 37°C for 22 hours to complete the first generation activation. The activated bacterial solution was then inoculated into fresh MRS liquid medium at a 2% inoculation rate and cultured for another 18 hours under the same conditions to complete the second generation activation, obtaining a stable seed culture. The second step is fermentation culture. The seed cultures of the three strains were inoculated into fermentation medium at a 3% inoculation rate. The composition of the fermentation medium was the same as in Example 1. The fermentation process was controlled at 37°C, with a stirring speed of 100 rpm, maintaining an anaerobic environment throughout. The fermentation period was 18 hours. At the end of fermentation, the OD600 value of the fermentation broth was monitored to be stable, and the cell concentration reached its peak. The third step is cell collection and mixing with the protective agent. After fermentation, the fermentation broth was placed at 4°C and centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, and the cell precipitate was collected. The bacterial cells were washed twice with sterile physiological saline, and then collected by centrifugation to obtain wet bacterial cells. A lyophilization protectant solution (12% skim milk powder and 4% mannitol) was added to the wet bacterial cells at a mass ratio of 1:3. The mixture was thoroughly stirred to obtain a bacterial suspension. The fourth step was vacuum freeze-drying. The well-mixed bacterial suspension was spread evenly in a sterile freeze-drying tray to a thickness of 10 mm and placed in a vacuum freeze dryer. Pre-freezing was performed at -45°C for 3.5 hours to ensure complete freezing. After pre-freezing, the vacuum system was activated and the vacuum level was controlled at 8 Pa. The first stage of sublimation drying was performed at -18°C for 10 hours, and the second stage of desorption drying was performed at 28°C for 5 hours, maintaining a stable vacuum level throughout the process. After drying, the bacteria were crushed to obtain single-strain freeze-dried powder, which was then sealed and stored in a light-protected environment at 4 degrees Celsius.

[0032] Preparation of the multi-strain probiotic composition: The viable count of the three single-strain lyophilized bacterial powders was tested using the same method as in Example 1. After the test, lyophilized Lactobacillus retardans, Lactobacillus rhamnosus, and Lactobacillus plantarum were weighed out in a viable count mass ratio of 1:1:1 and placed in a three-dimensional motion mixer. The speed was controlled at 12 rpm and the mixing time was 25 minutes to obtain a multi-strain mixed bacterial powder. Excipients were added to the multi-strain mixed bacterial powder. The excipient components were galacto-oligosaccharides, microcrystalline cellulose, and magnesium stearate. The mass ratio of each component was mixed bacterial powder: galacto-oligosaccharides: microcrystalline cellulose: magnesium stearate = 1:2:6.9:0.1. Mixing was continued under the same conditions for 30 minutes. After mixing, the final mixture was obtained. The mixed materials are fed into a fully automatic hard capsule filling machine, and No. 0 gelatin hollow capsules are used for filling. Each capsule is filled with 0.5g. After filling, the capsules are polished and unqualified capsules are removed. The capsules are then packaged in an aluminum-plastic blister pack under sterile conditions, sealed, and stored in a cool and dry place to obtain multi-strain probiotic hard capsules.

[0033] The quality indicators of the composition were tested. The test results showed that the viable counts of single strains of Lactobacillus retardii, Lactobacillus rhamnosus, and Lactobacillus plantarum in the finished product were not less than 1.0×10^8 CFU / g, the total viable count of the three strains was not less than 1.0×10^9 CFU / g, and the capsule weight, disintegration time, moisture content, pathogenic bacteria and other indicators all met the relevant national standards.

[0034] The efficacy verification test of the composition involved 30 healthy subjects aged 20-45 years with dull and lackluster skin. Each subject took four hard capsules prepared in this embodiment orally daily for 28 consecutive days. During the test, subjects maintained normal diet and lifestyle, and did not use other oral beauty products or functional skincare products. On day 0 and day 28 of the test, skin luster on the subjects' cheeks was measured using a skin luster meter under constant temperature and humidity conditions. Simultaneously, stool samples were collected, and gut microbiota diversity was detected using 16S rRNA high-throughput sequencing technology. The results showed that after 28 days of continuous use, the subjects' skin luster increased by an average of 10.8% compared to before the test, and the Shannon diversity index of gut microbiota increased by an average of 18.3% compared to before the test. Subjects generally reported an improvement in their dull skin condition, and no adverse reactions occurred during the test.

[0035] like Figure 1 As shown in the flowchart, this embodiment describes the preparation process of multi-strain probiotic hard capsules for improving skin radiance. The process includes: strain activation, seed liquid preparation, fermentation culture, cell centrifugation, collection, freeze drying, protective agent mixing, vacuum freeze drying, single-strain bacterial powder, compound excipient mixing, capsule filling, polishing, inner packaging, and finished product inspection.

[0036] This embodiment fully realizes the boundary parameters of the technical solution of the present invention. The prepared multi-strain probiotic composition meets the requirements for live bacteria count control and dosage form standards. It shows stable effects in regulating intestinal flora and improving skin luster, thus verifying the effectiveness and feasibility of the scope of protection of the technical solution of the present invention.

[0037] Example 3 This embodiment provides a multi-strain probiotic composition for improving skin radiance, specifically an oral tablet formulation. It uses Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum in a live bacteria ratio of 5:3:3. The entire preparation process and quality control standards of the composition are clearly defined, and the implementation effect of the upper limit parameter within the live bacteria ratio range of the present invention is verified, further covering the full scope of protection requirements of the technical solution of the present invention.

[0038] The strains and raw material preparation used in this example are *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum*. The excipients used include lyophilization protectants, prebiotics, fillers, binders, lubricants, and flavoring agents. Specifically, the lyophilization protectants are trehalose and glycerin; the prebiotic is stachyose; the fillers are lactose and microcrystalline cellulose; the binder is a 10% povidone K30 aqueous solution; the lubricant is magnesium stearate; and the flavoring agent is steviol glycosides.

[0039] The first step in preparing single-strain freeze-dried bacterial powder is strain activation. Preserved strains of three different strains were inoculated into MRS liquid medium and cultured in an anaerobic incubator at 36°C for 24 hours to complete the first generation activation. The activated bacterial solution was then inoculated into fresh MRS liquid medium at a 2% inoculation rate and cultured for another 20 hours under the same conditions to complete the second generation activation, obtaining a stable seed culture. The second step is fermentation culture. The seed cultures of the three strains were inoculated into fermentation medium at a 3% inoculation rate. The fermentation medium composition was the same as in Example 1. The fermentation process was controlled at 36°C, with a stirring speed of 90 rpm, maintaining an anaerobic environment throughout. The fermentation cycle was 22 hours. At the end of fermentation, the OD600 value of the fermentation broth stabilized, and the cell concentration reached its peak. The third step is cell collection and mixing with a protective agent. After fermentation, the fermentation broth was placed at 4°C and centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, and the cell precipitate was collected. The bacterial cells were washed twice with sterile physiological saline, and then collected by centrifugation to obtain wet bacterial cells. A lyophilization protectant solution (15% trehalose and 2% glycerol) was added to the wet bacterial cells at a mass ratio of 1:2.5. The mixture was thoroughly stirred to obtain a bacterial suspension. The fourth step was vacuum freeze-drying. The well-mixed bacterial suspension was spread evenly in a sterile freeze-drying tray to a thickness of 7 mm and placed in a vacuum freeze dryer. Pre-freezing was performed at -40°C for 4.5 hours to ensure complete freezing. After pre-freezing, the vacuum system was activated and the vacuum level was controlled at 10 Pa. The first stage of sublimation drying was performed at -20°C for 13 hours, and the second stage of desorption drying was performed at 25°C for 6 hours, maintaining a stable vacuum level throughout the process. After drying, the bacteria were crushed to obtain single-strain freeze-dried powder, which was then sealed and stored in a light-protected environment at 4 degrees Celsius.

[0040] Preparation of the multi-strain probiotic composition: The viable count of the three single-strain freeze-dried bacterial powders was tested using the same method as in Example 1. After the test, freeze-dried Lactobacillus retardans, Lactobacillus rhamnosus, and Lactobacillus plantarum were weighed out in a viable count mass ratio of 5:3:3 and placed in a three-dimensional motion mixer. The speed was controlled at 15 rpm and the mixing time was 20 minutes to obtain a multi-strain mixed bacterial powder. A prebiotic flavoring agent was added to the multi-strain mixed bacterial powder. The mass ratio of each component was mixed bacterial powder: stachyose: lactose: microcrystalline cellulose: steviol glycosides = 1:1.5:5:2.48:0.02. The mixture was mixed for 20 minutes under the same conditions to obtain a mixed powder. A 10% aqueous solution of povidone K30 was added to the mixed powder, and a soft mass was prepared using a wet high-speed granulator. The soft mass was granulated through a 20-mesh sieve. The wet granules were placed in a fluidized bed dryer, and the inlet air temperature was controlled at 50 degrees Celsius. The dry mass was dried until the moisture content of the granules was less than 3%. After drying, the granules were sizing through a 20-mesh sieve, and unqualified granules were removed. Magnesium stearate was added to the sizing dry granules at a rate of 0.2% of the total mass of the dry granules. The mixture was then placed in a three-dimensional motion mixer and mixed for 10 minutes to obtain a total mixed granule. The total mixed granules were then fed into a rotary tablet press for tableting, with the tableting pressure controlled at 6 kN. Each tablet weighed 0.5 g. After tableting, the tablets were inner-packaged, sealed, and stored in a cool, dry place to obtain multi-strain probiotic tablets.

[0041] The quality indicators of the composition were tested. The results showed that the viable counts of single strains of Lactobacillus retardii, Lactobacillus rhamnosus, and Lactobacillus plantarum in the finished product were not less than 1.0×10^8 CFU / g, and the total viable count of the three strains was not less than 1.0×10^9 CFU / g. The tablet weight difference, disintegration time, hardness, friability, moisture content, pathogenic bacteria and other indicators all met the relevant national standards.

[0042] The efficacy verification test of the composition involved 30 healthy subjects aged 20-45 years with dull and lackluster skin. Each subject took four tablets of the prepared tablets orally daily for 28 consecutive days. During the test, subjects maintained normal diet and lifestyle, and did not use other oral beauty products or functional skincare products. On day 0 and day 28 of the test, skin luster on the subjects' cheeks was measured using a skin luster meter under constant temperature and humidity conditions. Simultaneously, stool samples were collected, and gut microbiota diversity was detected using 16S rRNA high-throughput sequencing technology. The results showed that after 28 days of continuous use, the subjects' skin luster increased by an average of 11.5% compared to before the test, and the Shannon diversity index of gut microbiota increased by an average of 19.1% compared to before the test. Subjects generally reported improved skin condition, and no adverse reactions occurred during the test.

[0043] like Figure 1As shown in the flowchart, this embodiment describes the preparation process of multi-strain probiotic tablets that enhance skin radiance. The process includes: strain activation, seed liquid preparation, fermentation culture, cell centrifugation, collection, freeze drying, protective agent mixing, vacuum freeze drying, single-strain bacterial powder, compound excipient mixing, wet granulation, drying, granulation, total mixing, tableting, inner packaging, and finished product inspection.

[0044] This embodiment fully realizes the upper limit parameters of the technical solution of the present invention. The prepared multi-strain probiotic composition meets the requirements for live bacteria count control and tablet dosage form standards. It shows stable effects in regulating intestinal flora and improving skin luster, further verifying the feasibility and effectiveness of the full protection scope of the technical solution of the present invention.

[0045] Comparative Example This comparative example provides a single-strain probiotic composition, specifically an oral lyophilized powder formulation, using only Lactobacillus reuteri as the active ingredient. The entire preparation process and quality control standards of the composition are clearly defined to verify the synergistic effect of multiple strains combined.

[0046] The strain and raw material preparation used in this comparative example were *Lactobacillus reuteri*. The excipients included lyophilization protectants and prebiotics; the lyophilization protectants were skim milk powder and trehalose, and the prebiotics were fructooligosaccharides and inulin.

[0047] The first step in preparing single-strain freeze-dried bacterial powder is strain activation. Preserved *Lactobacillus reuteri* culture is inoculated into MRS liquid medium and cultured in an anaerobic incubator at 36°C for 24 hours to complete the first generation activation. The activated bacterial solution is then inoculated into fresh MRS liquid medium at a 2% inoculation rate, and cultured for another 18 hours under the same conditions to complete the second generation activation, obtaining a stable seed culture. The second step is fermentation culture. The *Lactobacillus reuteri* seed culture is inoculated into fermentation medium at a 3% inoculation rate. The fermentation medium consists of peptone, beef extract, yeast extract, glucose, anhydrous sodium acetate, diammonium hydrogen citrate, Tween 80, magnesium sulfate, and manganese sulfate. The fermentation process is controlled at 36°C, with a stirring speed of 80 rpm, maintaining an anaerobic environment throughout. The fermentation cycle is 20 hours. At the end of fermentation, the OD600 value of the fermentation broth is monitored to ensure stability, and the bacterial concentration reaches its peak. The third step involves bacterial collection and mixing with a protective agent. After fermentation, the fermentation broth was placed at 4 degrees Celsius and centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, and the bacterial precipitate was collected. The precipitate was washed twice with sterile physiological saline, and the cells were collected again by centrifugation to obtain wet cells. A lyophilization protectant solution consisting of 10% skim milk powder and 5% trehalose was added to the wet cells, with a mass ratio of wet cells to lyophilization protectant solution of 1:3. The mixture was thoroughly stirred to obtain a bacterial suspension. The fourth step was vacuum freeze-drying. The well-mixed bacterial suspension was spread evenly in a sterile freeze-drying tray to a thickness of 8 mm and placed in a vacuum freeze dryer. Pre-freezing was performed first, controlling the pre-freezing temperature at -40 degrees Celsius for 4 hours to ensure that the bacterial suspension was completely frozen. After pre-freezing, the vacuum system was activated and the vacuum level was controlled at 10 Pa. The first stage of sublimation drying was controlled at -20 degrees Celsius for 12 hours, and the second stage of desorption drying was controlled at 25 degrees Celsius for 6 hours, maintaining a stable vacuum level throughout the process. After drying, the Lactobacillus retardans lyophilized powder was obtained by crushing, sealed, and stored in a light-protected environment at 4 degrees Celsius.

[0048] Preparation of the single-strain probiotic composition: The viable count of the prepared *Lactobacillus reuteri* lyophilized powder was tested using the lactic acid bacteria test method specified in GB4789.35-2016. Specific procedures included serially diluting the sample, plating it onto MRS agar medium, anaerobically incubating at 36°C for 48 hours, and calculating the colony count. After testing, the corresponding mass of *Lactobacillus reuteri* lyophilized powder was weighed, ensuring the viable count in the finished product was not less than 1.0 × 10^9 CFU / g. The powder was placed in a three-dimensional motion mixer, and prebiotic excipients were added. The prebiotics consisted of fructooligosaccharides and inulin in a 1:1 mass ratio. The mass ratio of the bacterial powder to the prebiotics was 1:4. The mixing speed was controlled at 15 rpm for 40 minutes. After mixing, the mixture was aseptically packaged into aluminum foil bags, each containing 2g net content. The bags were sealed and stored in a cool, dry place to obtain the finished single-strain probiotic lyophilized powder.

[0049] The quality indicators of the composition were tested. The results showed that the viable count of Lactobacillus repens in the finished product was not less than 1.0×10^9 CFU / g, and the moisture content, total bacterial count, pathogenic bacteria and other indicators all met the relevant national standards.

[0050] The efficacy validation trial of the composition involved 30 healthy subjects aged 20-45 years with dull and lackluster skin. Each subject took one sachet of the lyophilized powder prepared in this comparative proportion orally daily for 28 consecutive days. During the trial, subjects maintained normal diet and lifestyle, and did not use other oral beauty products or functional skincare products. On day 0 and day 28 of the trial, skin luster was measured on the subjects' cheeks using a skin luster meter under constant temperature and humidity conditions. Simultaneously, stool samples were collected, and gut microbiota diversity was detected using 16S rRNA high-throughput sequencing technology. The results showed that after 28 days of continuous use, the subjects' skin luster improved by an average of 2.7% compared to before the trial, and the Shannon diversity index of gut microbiota improved by an average of 5.2% compared to before the trial. Subjects reported no significant improvement in their skin condition, and no adverse reactions occurred during the trial.

[0051] This comparative example uses a standardized freeze-drying powder preparation process to control the total number of live bacteria in the finished product to meet the routine quality control requirements for oral probiotic products. It uses only a single strain as the active ingredient, and its effect on intestinal flora regulation and skin luster improvement is significantly lower than that of a multi-strain compound composition. This verifies that the compound of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum has a significant synergistic effect, further demonstrating the inventiveness and beneficial effects of the technical solution of this invention.

[0052] The specific content is shown in the table below:

[0053] As can be seen from the table above, the multi-strain probiotic compositions prepared in the three embodiments of the present invention all meet the requirements for live bacteria count control, and all show significantly better effects than the comparative examples in terms of improving skin luster and regulating intestinal flora, with Example 1 showing the best effect. The present invention achieves a synergistic effect by combining three strains in a specific ratio, stably improving skin luster while regulating the balance of intestinal flora, thus verifying the effectiveness, stability, and inventiveness of the technical solution of the present invention.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A multi-strain probiotic composition for enhancing skin radiance, characterized in that, Made from the following raw materials in parts by weight: The active probiotic strains in the composition consist of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum.

2. The multi-strain probiotic composition for enhancing skin radiance according to claim 1, characterized in that, In the composition, the viable count of each single strain of Lactobacillus retardii, Lactobacillus rhamnosus, and Lactobacillus plantarum is not less than 1.0 × 10^8 CFU / g.

3. The multi-strain probiotic composition for enhancing skin radiance according to claim 1, characterized in that, In the composition, the total viable count of Lactobacillus retardans, Lactobacillus rhamnosus, and Lactobacillus plantarum is not less than 1.0 × 10^9 CFU / g.

4. The multi-strain probiotic composition for enhancing skin radiance according to claim 1, characterized in that, The viable count ratio of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum is (1-5):(1-3):(1-3).

5. The multi-strain probiotic composition for enhancing skin radiance according to claim 4, characterized in that, The viable count ratio of Lactobacillus retardans, Lactobacillus rhamnosus, and Lactobacillus plantarum is 2:1:

1.

6. The multi-strain probiotic composition for enhancing skin radiance according to claim 1, characterized in that, The composition further comprises a food- or pharmaceutically acceptable excipient selected from at least one of prebiotics, fillers, lubricants, flavoring agents, and freeze-drying protectants.

7. The multi-strain probiotic composition for enhancing skin radiance according to claim 6, characterized in that, The excipients include prebiotics, which are selected from at least one of fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, inulin, and stachyose.

8. The multi-strain probiotic composition for enhancing skin radiance according to claim 6, characterized in that, The composition is an oral solid dosage form, which is selected from any one of lyophilized powder, hard capsule, tablet, and granule.

9. The multi-strain probiotic composition for enhancing skin radiance according to claim 1, characterized in that, The Lactobacillus rubescens, Lactobacillus rhamnosus, and Lactobacillus plantarum are all present in the composition in the form of freeze-dried bacterial powder.

10. A multi-strain probiotic composition for enhancing skin radiance according to claim 6, characterized in that, The excipients include a freeze-drying protectant, which is selected from at least one of skim milk powder, trehalose, mannitol, and glycerin.

Images