A multi-strain probiotic composition for enhancing skin radiance

Abstract

The present invention discloses a multi-strain probiotic composition for enhancing skin radiance, relating to the technical field of microbial technology. By means of a specific combination of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum, the active probiotic strains act synergistically to effectively improve skin condition and enhance skin radiance. By regulating the balance of the intestinal flora and promoting absorption of nutrients and excretion of metabolic wastes, the composition indirectly improves skin health, making the skin smoother, more elastic, and naturally radiant.

Description

[0001] DESCRIPTION

[0002] A multi-strain probiotic composition for enhancing skin radiance TECHNICAL FIELD

[0003] The present invention relates to the technical field of microbial technology, and in particular to a multi-strain probiotic composition for enhancing skin radiance. BACKGROUND ART

[0004] In today's society, with improvement of living standards and increasing pursuit of beauty, skin health and radiance have become focal concerns for consumers. As the largest organ of the human body, the condition of the skin is influenced not only by genetic and environmental factors, but also closely related to the balance of the intestinal flora. The intestinal flora, as an “invisible organ” in the human body, plays a vital role in regulation of nutrient absorption, excretion of metabolic wastes, and immune functions. When the intestinal flora is imbalanced, not only is overall human health affected, but the skin condition may also be indirectly affected through complex physiological mechanisms, resulting in dull and lackluster skin.

[0005] Traditional skin-care methods mostly focus on external care, such as application and massage with various skin-care products, in the hope of improving skin condition and enhancing radiance. However, such methods often act only on the skin surface and can hardly fundamentally solve skin problems caused by imbalance of the intestinal flora. In addition, certain skin-care products may contain chemical ingredients, and long-term use may irritate or burden the skin and even aggravate skin problems. Although some oral beauty products are available on the market, most of them contain only single ingredients and are therefore unable to comprehensively regulate the intestinal-flora balance, resulting in limited effects in enhancing skin radiance.

[0006] In view of the limitations of traditional skin-care methods and oral beauty products in enhancing skin radiance, it is particularly important to DESCRIPTION provide a multi-strain probiotic composition capable of enhancing skin radiance.

[0007] SUMMARY OF THE INVENTION

[0008] An object of the present invention is to overcome the shortcomings of the prior art by providing a multi-strain probiotic composition for enhancing skin radiance. By synergistic action of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum in specific ratios, the composition can effectively regulate the balance of the intestinal flora and promote absorption of nutrients and excretion of metabolic wastes, thereby indirectly improving skin health, making the skin smoother, more elastic, and naturally radiant.

[0009] In order to solve the above technical problems, the present invention provides the following technical solution: a multi-strain probiotic composition for enhancing skin radiance, prepared from the following raw materials in parts by weight: the active probiotic strains of the composition comprise Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum.

[0010] Further, in the composition, the viable count of each individual strain of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum is not less than 1.0><10A8 CFU / g.

[0011] Further, in the composition, the total viable count of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum is not less than 1.0xI0A9 CFU / g.

[0012] Further, the viable-count mass ratio of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum is (l-5):(l-3):(l-3). DESCRIPTION

[0013] Further, the viable-count mass ratio of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum is 2: 1 : 1.

[0014] Further, the composition further comprises food-acceptable or pharmaceutically acceptable excipients selected from at least one of prebiotics, fillers, lubricants, flavoring agents, and lyophilization protectants.

[0015] Further, the excipients comprise a prebiotic selected from at least one of fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, inulin, and stachyose.

[0016] Further, the composition is an oral solid preparation selected from any one of lyophilized powder, hard capsules, tablets, and granules.

[0017] Further, Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum are all present in the composition in the form of freeze-dried bacterial powder.

[0018] Further, the excipients comprise a lyophilization protectant selected from at least one of skim milk powder, trehalose, mannitol, and glycerol.

[0019] Compared with the prior art, the multi-strain probiotic composition for enhancing skin radiance of the present invention has the following beneficial effects:

[0020] I. By means of the specific combination of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum, the active probiotic strains act synergistically to effectively improve skin condition and enhance skin radiance. By regulating the balance of the intestinal flora and promoting absorption of nutrients and excretion of metabolic wastes, the composition indirectly improves skin health, making the skin smoother, more elastic, and DESCRIPTION naturally radiant.

[0021] Other advantages, objectives, and features of the present invention will be set forth in part in the following description and in part will become apparent to those skilled in the art upon examination of the following disclosure, or may be learned from practice of the invention.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To describe the technical solutions in the embodiments of the present invention or in the prior art more clearly, the drawings required for describing the embodiments or the prior art are briefly introduced below. Obviously, the drawings in the following description merely illustrate certain embodiments of the present invention, and those of ordinary skill in the art can derive other drawings therefrom without inventive effort.

[0024] Figure 1 is a flow chart of the multi-strain probiotic composition for enhancing skin radiance.

[0025] DETAILED DESCRIPTION

[0026] In order to further illustrate the technical means and effects adopted by the present invention to achieve the intended objectives, the specific implementation, structure, features, and effects of the present invention are described in detail below in conjunction with the accompanying drawing and preferred embodiments.

[0027] Embodiment 1

[0028] This embodiment provides a multi-strain probiotic composition for enhancing skin radiance, specifically in the form of an oral lyophilized powder. Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum are compounded according to a viable-count mass ratio of 2: 1 : 1. The viable-count control standards of each strain and the complete preparation process of the composition are defined, and the regulatory effect of the DESCRIPTION composition on intestinal-flora balance and its improvement effect on skin radiance are verified, thereby providing an optimal implementation mode of the core technical solution of the present invention.

[0029] Strain and raw-material preparation. The active strains used in this embodiment are Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum. The excipients comprise a lyophilization protectant and a prebiotic, wherein the lyophilization protectant is skim milk powder and trehalose, and the prebiotic is fructooligosaccharide and inulin.

[0030] Preparation of freeze-dried bacterial powder of each individual strain. In the first step, strain activation is performed. Preserved strains of the three bacteria are separately inoculated into MRS liquid medium and cultured in an anaerobic incubator at 36 °C for 24 h to complete first-generation activation. The first-generation activated bacterial liquid is inoculated into fresh MRS liquid medium at an inoculation amount of 2%, and cultured under the same conditions for another 18 h to complete second-generation activation, thereby obtaining seed liquid with stable activity. In the second step, fermentation culture is performed. The seed liquid of each strain is separately inoculated into a fermentation medium at an inoculation amount of 3%. The fermentation medium comprises peptone, beef extract, yeast extract powder, glucose, anhydrous sodium acetate, diammonium hydrogen citrate, Tween 80, magnesium sulfate, and manganese sulfate. During fermentation, the temperature is controlled at 36 °C, the stirring speed is 80 rpm, the anaerobic environment is maintained throughout, and the fermentation period is 20 h. At the fermentation end point, the OD600 value of the fermentation broth is monitored to become stable and the bacterial concentration reaches a peak. In the third step, bacterial cells are collected and mixed with the protectant. After completion of fermentation, the fermentation broth is placed at 4 °C and centrifuged at 8000 rpm for 15 min. The supernatant is discarded and the bacterial-cell precipitate is collected. The bacterial-cell precipitate is washed DESCRIPTION twice with sterile physiological saline and centrifuged again to collect the bacterial cells, thereby obtaining wet bacterial cells. A lyophilization-protectant solution is added to the wet bacterial cells, the lyophilization-protectant solution comprising 10% skim milk powder and 5% trehalose, with a mass ratio of wet bacterial cells to lyophilization-protectant solution of 1:3. After full stirring and uniform mixing, a bacterial suspension is obtained. In the fourth step, vacuum freeze-drying is performed. The uniformly mixed bacterial suspension is spread flat in a sterile freeze-drying tray with a layer thickness of 8 mm and placed in a vacuum freeze dryer. Pre-freezing is first carried out at -40 °C for 4 h to ensure complete freezing of the bacterial suspension. After completion of pre-freezing, the vacuum system is turned on, the vacuum degree is controlled at 10 Pa, the first-stage sublimation drying temperature is controlled at -20 °C for 12 h, and the second-stage desorption drying temperature is controlled at 25 °C for 6 h, with the vacuum degree maintained stable throughout the process. After completion of drying, the product is crushed to obtain freeze-dried bacterial powder of each individual strain, which is sealed and stored at 4 °C in the dark.

[0031] Preparation of the multi-strain probiotic composition. The three obtained individual-strain freeze-dried bacterial powders are subjected to viable-count testing according to the lactic-acid-bacteria testing method specified in GB4789. 35-2016, including gradient dilution of samples, spread plating on MRS agar medium, anaerobic culture at 36 °C for 48 h, colony counting, and calculation of results. After testing, freeze-dried bacterial powder of Lactobacillus reuteri, freeze-dried bacterial powder of Lactobacillus rhamnosus, and freeze-dried bacterial powder of Lactobacillus plantarum are separately weighed according to a viable-count mass ratio of 2:1: 1 and placed in a three-dimensional motion mixer, with a rotation speed of 15 rpm and a mixing time of 20 min, thereby obtaining a multi-strain mixed bacterial DESCRIPTION powder. A prebiotic excipient is then added to the multi-strain mixed bacterial powder, the prebiotic consisting of fructooligosaccharide and inulin in a mass ratio of 1:1, with a mass ratio of mixed bacterial powder to prebiotic of 1:4. Mixing is continued under the same conditions for 20 min. After completion of mixing, subpackaging is carried out in aluminum-foil bags under a sterile environment, each bag having a net content of 2 g. The bags are sealed and stored in a cool and dry place, thereby obtaining the finished multi-strain probiotic lyophilized powder.

[0032] Quality-index testing of the composition. The prepared finished product is subjected to quality-index testing. The test results show that the viable count of each individual strain of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum in the finished product is not less than 1.0xl0A8 CFU / g, the total viable count of the three strains is not less than 1.0x l0A9 CFU / g, and indices such as moisture content, total colony count, and pathogenic bacteria all comply with relevant national standards.

[0033] Efficacy verification test of the composition. Thirty healthy subjects aged 20-45 years with dull and lackluster skin were selected. Each subject orally took one bag of the lyophilized powder prepared in this embodiment every day for 28 consecutive days. During the test period, the subjects maintained normal diet and work-rest schedules and did not use any other oral beauty products or functional skin-care products. On day 0 and day 28 of the test, skin radiance at the cheek region of each subject was measured with a skin gloss meter under a constant-temperature and constant-humidity environment, and fecal samples of the subjects were collected. High-throughput 16S rRNA sequencing was used to determine intestinal-flora diversity. The test results showed that, after 28 consecutive days of administration, the average skin-radiance value of the subjects increased by 14.2% as compared with that before the test, and the Shannon diversity index of the intestinal flora increased by 22.6% on average as compared with that DESCRIPTION before the test. The subjects generally reported improvement in skin smoothness and reduction of dullness, and no adverse reactions occurred during the test period.

[0034] As shown in Figure 1, the preparation flow chart of the multi-strain probiotic lyophilized powder for enhancing skin radiance in this embodiment sequentially comprises strain activation, seed-liquid preparation, fermentation culture, centrifugal collection of bacterial cells, mixing with a lyophilization protectant, vacuum freeze-drying, blending of individual-strain bacterial powders, mixing with prebiotic excipients, aseptic subpackaging, and finished-product inspection.

[0035] This embodiment fully implements the core content of the technical solution of the present invention. The prepared multi-strain probiotic composition satisfies the viable-count control requirements, effectively regulates the balance of the intestinal flora through the synergistic action of the three strains, and at the same time achieves stable improvement of skin radiance, thereby verifying the practicability and beneficial effects of the technical solution of the present invention.

[0036] Embodiment 2

[0037] This embodiment provides a multi-strain probiotic composition for enhancing skin radiance, specifically in the form of oral hard capsules. Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum are compounded according to a viable-count mass ratio of 1 : 1 : 1 . The complete preparation process and quality-control standards of the composition are defined, thereby verifying the implementation effect of the lower-limit parameter within the viable-count ratio range of the present invention and covering the boundary protection scope of the technical solution of the present invention.

[0038] Strain and raw-material preparation. The active strains used in this embodiment are Lactobacillus reuteri, Lactobacillus rhamnosus, and DESCRIPTION

[0039] Lactobacillus plantarum. The excipients comprise a lyophilization protectant, a prebiotic, a filler, and a lubricant, wherein the lyophilization protectant is skim milk powder and mannitol, the prebiotic is galactooligosaccharide, the filler is microcrystalline cellulose, and the lubricant is magnesium stearate.

[0040] Preparation of freeze-dried bacterial powder of each individual strain. In the first step, strain activation is performed. Preserved strains of the three bacteria are separately inoculated into MRS liquid medium and cultured in an anaerobic incubator at 37 °C for 22 h to complete first-generation activation. The first-generation activated bacterial liquid is inoculated into fresh MRS liquid medium at an inoculation amount of 2%, and cultured under the same conditions for another 18 h to complete second-generation activation, thereby obtaining seed liquid with stable activity. In the second step, fermentation culture is performed. The seed liquid of each strain is separately inoculated into a fermentation medium at an inoculation amount of 3%. The composition of the fermentation medium is the same as that in Embodiment 1. During fermentation, the temperature is controlled at 37 °C, the stirring speed is 100 rpm, the anaerobic environment is maintained throughout, and the fermentation period is 18 h. At the fermentation end point, the OD600 value of the fermentation broth is monitored to become stable and the bacterial concentration reaches a peak. In the third step, bacterial cells are collected and mixed with the protectant. After completion of fermentation, the fermentation broth is placed at 4 °C and centrifuged at 8000 rpm for 15 min. The supernatant is discarded and the bacterial-cell precipitate is collected. The bacterial-cell precipitate is washed twice with sterile physiological saline and centrifuged again to collect the bacterial cells, thereby obtaining wet bacterial cells. A lyophilization-protectant solution is added to the wet bacterial cells, the lyophilization-protectant solution comprising 12% skim milk powder and 4% mannitol, with a mass ratio of wet bacterial cells to lyophilization-protectant solution of 1:3. After full stirring and uniform DESCRIPTION mixing, a bacterial suspension is obtained. In the fourth step, vacuum freeze-drying is performed. The uniformly mixed bacterial suspension is spread flat in a sterile freeze-drying tray with a layer thickness of 10 mm and placed in a vacuum freeze dryer. Pre-freezing is first carried out at -45 °C for 3.5 h to ensure complete freezing of the bacterial suspension. After completion of pre-freezing, the vacuum system is turned on, the vacuum degree is controlled at 8 Pa, the first-stage sublimation drying temperature is controlled at -18 °C for 10 h, and the second-stage desorption drying temperature is controlled at 28 °C for 5 h, with the vacuum degree maintained stable throughout the process. After completion of drying, the product is crushed to obtain freeze-dried bacterial powder of each individual strain, which is sealed and stored at 4 °C in the dark.

[0041] Preparation of the multi-strain probiotic composition. The three obtained individual-strain freeze-dried bacterial powders are subjected to viable-count testing by the same method as in Embodiment 1. After testing, freeze-dried bacterial powder of Lactobacillus reuteri, freeze-dried bacterial powder of Lactobacillus rhamnosus, and freeze-dried bacterial powder of Lactobacillus plantarum are separately weighed according to a viable-count mass ratio of 1:1: 1 and placed in a three-dimensional motion mixer, with a rotation speed of 12 rpm and a mixing time of 25 min, thereby obtaining a multi-strain mixed bacterial powder. Excipients are then added to the multi-strain mixed bacterial powder, the excipients comprising galactooligosaccharide, microcrystalline cellulose, and magnesium stearate, with a mass ratio of mixed bacterial powder: galactooligosaccharide: microcrystalline cellulose: magnesium stearate = 1:2:6.9:0.1. Mixing is continued under the same conditions for 30 min to obtain an overall mixed material. The overall mixed material is fed into a fully automatic hard-capsule filling machine, and No. 0 hollow gelatin capsules are selected for filling, each capsule having a fill weight of 0.5 g. After filling, capsule polishing is performed, unqualified capsules are io DESCRIPTION removed, and aluminum-plastic blister packaging is carried out under a sterile environment. The packaged capsules are sealed and stored in a cool and dry place, thereby obtaining the finished multi-strain probiotic hard capsules.

[0042] Quality-index testing of the composition. The prepared finished product is subjected to quality-index testing. The test results show that the viable count of each individual strain of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum in the finished product is not less than 1.0xl0A8 CFU / g, the total viable count of the three strains is not less than 1.0 10A9 CFU / g, and indices such as capsule fill-weight variation, disintegration time, moisture content, and pathogenic bacteria all comply with relevant national standards.

[0043] Efficacy verification test of the composition. Thirty healthy subjects aged 20-45 years with dull and lackluster skin were selected. Each subject orally took four hard capsules prepared in this embodiment every day for 28 consecutive days. During the test period, the subjects maintained normal diet and work-rest schedules and did not use any other oral beauty products or functional skin-care products. On day 0 and day 28 of the test, skin radiance at the cheek region of each subject was measured with a skin gloss meter under a constant-temperature and constant-humidity environment, and fecal samples of the subjects were collected. High-throughput 16S rRNA sequencing was used to determine intestinal-flora diversity. The test results showed that, after 28 consecutive days of administration, the average skin-radiance value of the subjects increased by 10.8% as compared with that before the test, and the Shannon diversity index of the intestinal flora increased by 18.3% on average as compared with that before the test. The subjects generally reported improvement of skin dullness, and no adverse reactions occurred during the test period.

[0044] As shown in Figure 1, the preparation flow chart of the multi-strain probiotic hard capsules for enhancing skin radiance in this embodiment ii DESCRIPTION sequentially comprises strain activation, seed-liquid preparation, fermentation culture, centrifugal collection of bacterial cells, mixing with a lyophilization protectant, vacuum freeze-drying, blending of individual-strain bacterial powders, mixing with excipients, capsule filling, polishing, inner packaging, and finished-product inspection.

[0045] This embodiment fully implements the boundary parameters of the technical solution of the present invention. The prepared multi-strain probiotic composition satisfies the viable-count control requirements and dosage-form standards, and exhibits stable effects in regulating the intestinal flora and improving skin radiance, thereby verifying the effectiveness and practicability of the protection scope of the technical solution of the present invention.

[0046] Embodiment 3

[0047] This embodiment provides a multi-strain probiotic composition for enhancing skin radiance, specifically in the form of oral tablets. Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum are compounded according to a viable-count mass ratio of 5:3:3. The complete preparation process and quality-control standards of the composition are defined, thereby verifying the implementation effect of the upper-limit parameter within the viable-count ratio range of the present invention and further covering the full-range protection requirements of the technical solution of the present invention.

[0048] Strain and raw-material preparation. The active strains used in this embodiment are Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum. The excipients comprise a lyophilization protectant, a prebiotic, a filler, a binder, a lubricant, and a flavoring agent, wherein the lyophilization protectant is trehalose and glycerol, the prebiotic is stachyose, the filler is lactose and microcrystalline cellulose, the binder is a 10% povidone K30 aqueous solution, the lubricant is magnesium stearate, and the flavoring agent is stevioside. DESCRIPTION

[0049] Preparation of freeze-dried bacterial powder of each individual strain. In the first step, strain activation is performed. Preserved strains of the three bacteria are separately inoculated into MRS liquid medium and cultured in an anaerobic incubator at 36 °C for 24 h to complete first-generation activation. The first-generation activated bacterial liquid is inoculated into fresh MRS liquid medium at an inoculation amount of 2%, and cultured under the same conditions for another 20 h to complete second-generation activation, thereby obtaining seed liquid with stable activity. In the second step, fermentation culture is performed. The seed liquid of each strain is separately inoculated into a fermentation medium at an inoculation amount of 3%. The composition of the fermentation medium is the same as that in Embodiment 1. During fermentation, the temperature is controlled at 36 °C, the stirring speed is 90 rpm, the anaerobic environment is maintained throughout, and the fermentation period is 22 h. At the fermentation end point, the OD600 value of the fermentation broth is monitored to become stable and the bacterial concentration reaches a peak. In the third step, bacterial cells are collected and mixed with the protectant. After completion of fermentation, the fermentation broth is placed at 4 °C and centrifuged at 8000 rpm for 15 min. The supernatant is discarded and the bacterial-cell precipitate is collected. The bacterial-cell precipitate is washed twice with sterile physiological saline and centrifuged again to collect the bacterial cells, thereby obtaining wet bacterial cells. A lyophilization-protectant solution is added to the wet bacterial cells, the lyophilization-protectant solution comprising 15% trehalose and 2% glycerol, with a mass ratio of wet bacterial cells to lyophilization-protectant solution of 1:2.5. After full stirring and uniform mixing, a bacterial suspension is obtained. In the fourth step, vacuum freeze-drying is performed. The uniformly mixed bacterial suspension is spread flat in a sterile freeze-drying tray with a layer thickness of 7 mm and placed in a vacuum freeze dryer. Pre-freezing is first carried out at -40 °C for 4.5 h to ensure DESCRIPTION complete freezing of the bacterial suspension. After completion of pre-freezing, the vacuum system is turned on, the vacuum degree is controlled at 10 Pa, the first-stage sublimation drying temperature is controlled at -20 °C for 13 h, and the second-stage desorption drying temperature is controlled at 25 °C for 6 h, with the vacuum degree maintained stable throughout the process. After completion of drying, the product is crushed to obtain freeze-dried bacterial powder of each individual strain, which is sealed and stored at 4 °C in the dark.

[0050] Preparation of the multi-strain probiotic composition. The three obtained individual-strain freeze-dried bacterial powders are subjected to viable-count testing by the same method as in Embodiment 1. After testing, freeze-dried bacterial powder of Lactobacillus reuteri, freeze-dried bacterial powder of Lactobacillus rhamnosus, and freeze-dried bacterial powder of Lactobacillus plantarum are separately weighed according to a viable-count mass ratio of 5:3:3 and placed in a three-dimensional motion mixer, with a rotation speed of 15 rpm and a mixing time of 20 min, thereby obtaining a multi-strain mixed bacterial powder. Fillers, a prebiotic, and a flavoring agent are then added to the multi-strain mixed bacterial powder, with a mass ratio of mixed bacterial powder: stachyose:lactose:microcrystalline cellulose: stevioside = 1:1.5:5:2.48:0.02. The materials are mixed under the same conditions for 20 min to obtain a mixed powder. A 10% povidone K30 aqueous solution is then added to the mixed powder, and soft material is prepared by a high-speed wet granulator. The soft material is granulated through a 20-mesh sieve. The wet granules are placed in a fluidized-bed dryer, the inlet-air temperature is controlled at 50 °C, and drying is continued until the moisture content of the granules is less than 3%. After drying, the granules are sized through a 20-mesh sieve and unqualified granules are removed. Magnesium stearate is added to the sized dry granules in an amount of 0.2% of the total mass of the dry granules, and mixing is carried out in a three-dimensional motion mixer DESCRIPTION for 10 min to obtain final mixed granules. The final mixed granules are then fed into a rotary tablet press for tableting, the tableting pressure is controlled at 6 kN, and the tablet weight is 0.5 g per tablet. After tableting, inner packaging is performed, and the packaged tablets are sealed and stored in a cool and dry place, thereby obtaining the finished multi-strain probiotic tablets.

[0051] Quality-index testing of the composition. The prepared finished product is subjected to quality-index testing. The test results show that the viable count of each individual strain of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum in the finished product is not less than 1.0><10A8 CFU / g, the total viable count of the three strains is not less than 1.0xl0A9 CFU / g, and indices such as tablet weight variation, disintegration time, hardness, friability, moisture content, and pathogenic bacteria all comply with relevant national standards.

[0052] Efficacy verification test of the composition. Thirty healthy subjects aged 20-45 years with dull and lackluster skin were selected. Each subject orally took four tablets prepared in this embodiment every day for 28 consecutive days. During the test period, the subjects maintained normal diet and work-rest schedules and did not use any other oral beauty products or functional skin-care products. On day 0 and day 28 of the test, skin radiance at the cheek region of each subject was measured with a skin gloss meter under a constant-temperature and constant-humidity environment, and fecal samples of the subjects were collected. High-throughput 16S rRNA sequencing was used to determine intestinal-flora diversity. The test results showed that, after 28 consecutive days of administration, the average skin-radiance value of the subjects increased by 11.5% as compared with that before the test, and the Shannon diversity index of the intestinal flora increased by 19.1% on average as compared with that before the test. The subjects generally reported improvement in skin condition, and no adverse reactions occurred during the DESCRIPTION test period.

[0053] As shown in Figure 1, the preparation flow chart of the multi-strain probiotic tablets for enhancing skin radiance in this embodiment sequentially comprises strain activation, seed-liquid preparation, fermentation culture, centrifugal collection of bacterial cells, mixing with a lyophilization protectant, vacuum freeze-drying, blending of individual-strain bacterial powders, mixing with excipients, wet granulation, drying, sizing, final mixing, tableting, inner packaging, and finished-product inspection.

[0054] This embodiment fully implements the upper-limit parameter of the technical solution of the present invention. The prepared multi-strain probiotic composition satisfies the viable-count control requirements and tablet dosage-form standards, and exhibits stable effects in regulating the intestinal flora and improving skin radiance, thereby further verifying the practicability and effectiveness of the full protection scope of the technical solution of the present invention.

[0055] Comparative Example

[0056] This comparative example provides a single-strain probiotic composition, specifically in the form of an oral lyophilized powder, in which only Lactobacillus reuteri is used as an active ingredient. The complete preparation process and quality-control standards of the composition are defined in order to verify the synergistic effect of the multi-strain compounding.

[0057] Strain and raw-material preparation. The active strain used in this comparative example is Lactobacillus reuteri. The excipients comprise a lyophilization protectant and a prebiotic, wherein the lyophilization protectant is skim milk powder and trehalose, and the prebiotic is fructooligosaccharide and inulin.

[0058] Preparation of freeze-dried bacterial powder of the individual strain. In the first step, strain activation is performed. A preserved strain of Lactobacillus reuteri is inoculated into MRS liquid medium and cultured in an DESCRIPTION anaerobic incubator at 36 °C for 24 h to complete first-generation activation. The first-generation activated bacterial liquid is inoculated into fresh MRS liquid medium at an inoculation amount of 2% and cultured under the same conditions for another 18 h to complete second-generation activation, thereby obtaining seed liquid with stable activity. In the second step, fermentation culture is performed. The seed liquid of Lactobacillus reuteri is inoculated into a fermentation medium at an inoculation amount of 3%. The fermentation medium comprises peptone, beef extract, yeast extract powder, glucose, anhydrous sodium acetate, diammonium hydrogen citrate, Tween 80, magnesium sulfate, and manganese sulfate. During fermentation, the temperature is controlled at 36 °C, the stirring speed is 80 rpm, the anaerobic environment is maintained throughout, and the fermentation period is 20 h. At the fermentation end point, the OD600 value of the fermentation broth is monitored to become stable and the bacterial concentration reaches a peak. In the third step, bacterial cells are collected and mixed with the protectant. After completion of fermentation, the fermentation broth is placed at 4 °C and centrifuged at 8000 rpm for 15 min. The supernatant is discarded and the bacterial-cell precipitate is collected. The bacterial-cell precipitate is washed twice with sterile physiological saline and centrifuged again to collect the bacterial cells, thereby obtaining wet bacterial cells. A lyophilization-protectant solution is added to the wet bacterial cells, the lyophilization-protectant solution comprising 10% skim milk powder and 5% trehalose, with a mass ratio of wet bacterial cells to lyophilization-protectant solution of 1:3. After full stirring and uniform mixing, a bacterial suspension is obtained. In the fourth step, vacuum freeze-drying is performed. The uniformly mixed bacterial suspension is spread flat in a sterile freeze-drying tray with a layer thickness of 8 mm and placed in a vacuum freeze dryer. Pre-freezing is first carried out at -40 °C for 4 h to ensure complete freezing of the bacterial suspension. After completion of pre-freezing, the vacuum DESCRIPTION system is turned on, the vacuum degree is controlled at 10 Pa, the first-stage sublimation drying temperature is controlled at -20 °C for 12 h, and the second-stage desorption drying temperature is controlled at 25 °C for 6 h, with the vacuum degree maintained stable throughout the process. After completion of drying, the product is crushed to obtain freeze-dried bacterial powder of Lactobacillus reuteri, which is sealed and stored at 4 °C in the dark.

[0059] Preparation of the single-strain probiotic composition. The obtained freeze-dried bacterial powder of Lactobacillus reuteri is subjected to viable-count testing according to the lactic-acid-bacteria testing method specified in GB4789.35-2016, including gradient dilution of samples, spread plating on MRS agar medium, anaerobic culture at 36 °C for 48 h, colony counting, and calculation of results. After testing, an appropriate amount of freeze-dried bacterial powder of Lactobacillus reuteri is weighed so that the viable count of the strain in the finished product is not less than 1.0><10A9 CFU / g, placed in a three-dimensional motion mixer, and mixed with a prebiotic excipient, the prebiotic consisting of fructooligosaccharide and inulin in a mass ratio of 1 : 1 , with a mass ratio of bacterial powder to prebiotic of 1:4. The rotation speed is controlled at 15 rpm and the mixing time is 40 min. After completion of mixing, subpackaging is carried out in aluminum-foil bags under a sterile environment, each bag having a net content of 2 g. The bags are sealed and stored in a cool and dry place, thereby obtaining the finished single-strain probiotic lyophilized powder.

[0060] Quality-index testing of the composition. The prepared finished product is subjected to quality-index testing. The test results show that the viable count of Lactobacillus reuteri in the finished product is not less than 1.0 10A9 CFU / g, and indices such as moisture content, total colony count, and pathogenic bacteria all comply with relevant national standards.

[0061] Efficacy verification test of the composition. Thirty healthy subjects aged 20-45 years with dull and lackluster skin were selected. Each subject DESCRIPTION orally took one bag of the lyophilized powder prepared in this comparative example every day for 28 consecutive days. During the test period, the subjects maintained normal diet and work-rest schedules and did not use any other oral beauty products or functional skin-care products. On day 0 and day 28 of the test, skin radiance at the cheek region of each subject was measured with a skin gloss meter under a constant-temperature and constant-humidity environment, and fecal samples of the subjects were collected. High-throughput 16S rRNA sequencing was used to determine intestinal-flora diversity. The test results showed that, after 28 consecutive days of administration, the average skin-radiance value of the subjects increased by 2.7% as compared with that before the test, and the Shannon diversity index of the intestinal flora increased by 5.2% on average as compared with that before the test. The subjects reported no obvious improvement in skin condition, and no adverse reactions occurred during the test period.

[0062] This comparative example adopts a standardized lyophilized-powder preparation process and controls the total viable count of the finished product to meet conventional quality-control requirements for oral probiotic products. Since only a single strain is used as the active ingredient, the effects of the composition on regulation of the intestinal flora and improvement of skin radiance are significantly inferior to those of the multi-strain compounded composition. This verifies that the combination of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum has a significant synergistic effect, thereby further proving the inventiveness and beneficial effects of the technical solution of the present invention.

[0063] The specific data are shown in the table below: DESCRIPTION

[0064] As can be seen from the above table, the multi-strain probiotic compositions prepared in the three embodiments of the present invention all satisfy the viable-count control requirements and exhibit significantly better effects than the comparative example in both improvement of skin radiance DESCRIPTION and regulation of the intestinal flora, wherein Embodiment 1 provides the best effect. By compounding the three strains in specific ratios, the present invention achieves a synergistic enhancement effect and stably improves skin radiance on the basis of regulating the intestinal-flora balance, thereby verifying the effectiveness, stability, and inventiveness of the technical solution of the present invention.

[0065] The foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form. 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 may make certain changes or modifications to equivalent embodiments by using the technical content disclosed above without departing from the scope of the technical solutions of the present invention. However, any simple modification, equivalent substitution, or improvement made to the above embodiments on the basis of the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the protection scope of the technical solutions of the present invention.

Claims

CLAIMS1. A multi-strain probiotic composition for enhancing skin radiance, characterized in that the composition is prepared from the following raw materials in parts by weight: the active probiotic strains of the composition comprise 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 individual strain of Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum is not less than 1.0><10A8 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 reuteri, Lactobacillus rhamnosus, and Lactobacillus plantarum is not less than 1.0xl0A9 CFU / g.

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

5. The multi-strain probiotic composition for enhancing skin radiance according to claim 4, characterized in that the viable-count mass ratio of Lactobacillus reuteri, 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 food-acceptable or pharmaceutically acceptable excipients selected from at least one of prebiotics, fillers, lubricants, flavoring agents, and lyophilization protectants.

7. The multi-strain probiotic composition for enhancing skin radiance according to claim 6, characterized in that the excipients comprise a prebiotic selected from at least one of fructooligosaccharide, galactooligosaccharide,CLAIMS isomaltooligosaccharide, 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 preparation selected from any one of lyophilized powder, hard capsules, tablets, and granules.

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

10. The multi-strain probiotic composition for enhancing skin radiance according to claim 6, characterized in that the excipients comprise a lyophilization protectant selected from at least one of skim milk powder, trehalose, mannitol, and glycerol.

Images