Lactobacillus polyactis composition for improving skin as well as derivative and application of lactobacillus polyactis composition

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus polyactis composition for improving skin as well as a derivative and application of the lactobacillus polyactis composition. The composition comprises a bacterial strain, a pure culture or an inactivated bacterium body of bifidobacterium bifidum Bbifid-1 (CCTCC NO: M2023349), bifidobacterium adolescentis Badol-1 (CCTCC NO: M 20242593), phytobacterium plantarum Lplan-4 (CCTCC NO: M 20242594) and lactobacillus acidophilus (BA05). The invention further relates to a preparation method of the composition. The lactobacillus plantarum derivative composition prepared through treatment modes of splitting decomposition, extraction, fermentation and the like can be used as an active ingredient of a medicine or a cosmetic, has remarkable effects of resisting oxidation and bacteria, especially resisting inflammation and inhibiting skin lipid accumulation aiming at propionibacterium acnes and staphylococcus aureus, is beneficial to fading skin red and swollen, avoids hair follicle blockage, and has a good application prospect. The obvious effect on the acne is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a multi-lactobacillus composition for improving skin and derivatives and applications thereof. Background Art

[0002] Acne is a common skin disease that primarily affects adolescents and young adults but may persist into adulthood. It is caused by overactive sebaceous glands, abnormal keratinization of hair follicles, bacterial infections (such as Propionibacterium acnes, P. acnes acnes) and inflammatory response. Traditional treatments usually rely on antibiotics, retinoids, hormone therapy, etc., but these methods may cause side effects, and long-term use may lead to drug resistance.

[0003] In recent years, more and more studies have focused on the use of microorganisms and their metabolites as alternative or complementary treatments. As a beneficial live bacteria, probiotics have been widely recognized in regulating the balance of intestinal microecology; however, their potential in the field of skin health has not been fully explored. Studies have shown that certain probiotics can have a positive impact on skin health by regulating the structure of skin microbial communities, enhancing skin barrier function, and reducing the release of inflammatory mediators. For example, the applicant has announced in CN118995463A that a derivative of Bifidobacterium bifidum Bbifi-1 has some functions of improving skin.

[0004] Despite this, there is a lack of products on the market that effectively combine multiple probiotics to comprehensively address the complex causes of acne. Existing technical research is mostly focused on live bacterial preparations and mainly considers the effects of a single strain, while ignoring the powerful biological activity contained in non-live bacterial components such as lysates and the synergistic effects of different strains complementing each other. Therefore, it is of great significance to develop a composition based on multiple probiotics. Summary of the invention

[0005] The purpose of the present invention is to provide a more potential improvement plan for skin problems, in particular to provide an improvement plan for acne symptoms.

[0006] Based on the above, the present invention first provides a polylactobacillus composition, which contains: Bifidobacterium bifidum ( Bifidobacterium bifidum ) Bbifi-1 strain or its pure culture or inactivated bacteria, the deposit number of the Bbifi-1 strain is CCTCC NO: M2023349; Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or inactivated bacteria, the deposit number of the Badol-1 strain is CCTCC NO: M 20242593; Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Lplan-4 strain or its pure culture or inactivated bacteria, the deposit number of the Lplan-4 strain is CCTCC NO: M 20242594; and Lactobacillus acidophilus ( Lactobacillus acidophilus ) BA05 strain or its pure culture or inactivated bacteria.

[0007] The above composition can be used to prepare a pharmaceutical active ingredient or a cosmetic active ingredient, and the active ingredient can be in the form of a lysate, an extract, a fermentation product lysate or a combination thereof.

[0008] Therefore, the present invention also provides a composition of polylactobacillus derivatives, wherein the derivatives are lysates, extracts, fermentation product lysates or combinations thereof of Bbifi-1, Badol-1, Lplan-4 and BA05 strains or their pure cultures or inactivated bacterial bodies. The composition of polylactobacillus derivatives described in the present invention can be prepared by mixing lysates, extracts, and fermentation product lysates prepared from each single bacterium, or by mixing each single bacterium before preparing a lysate, extract, or fermentation product lysate of a mixed bacterial body.

[0009] The ratio between the strains is not restricted and can be combined in any proportion. Preferably, they are combined in equal proportions. For example, for each single bacterium, a lysate, an extract, and a fermentation product lysate are prepared separately and then mixed, single bacterial derivatives of equal volume or mass can be selected for mixing. For each single bacterium, a lysate, an extract, and a fermentation product lysate are first mixed to prepare a mixed bacterial body, single bacteria with equal colony counts can be selected for mixing first. An error that does not significantly affect the effect of the final product is acceptable, for example, an error of ±10% is preferred, an error of ±5% is more preferred, and an error of ±1% is further preferred.

[0010] Further, the present invention also provides a cosmetic or medicine, wherein the active ingredient of the cosmetic or medicine contains the composition of the aforementioned lactobacillus derivatives. The content of the composition of the lactobacillus derivatives in the cosmetic or medicine is an amount that can play a pharmacological reaction role, which is easy to determine for those skilled in the art. The preferred content is 1-100%, more preferably 1-50%, further preferably 5-15%, and further 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% can be selected.

[0011] Furthermore, the present invention provides an application of a probiotic preparation for non-disease treatment purposes, wherein the active ingredient of the probiotic preparation contains a composition of the aforementioned polylactobacillus derivatives, and the non-disease treatment purposes are selected from one or more of anti-oxidation, antibacterial, anti-inflammatory, reduction of skin redness and swelling, and inhibition of skin lipid accumulation.

[0012] In some embodiments, the antibacterial activity refers to inhibiting one or both of Propionibacterium acnes and Staphylococcus aureus.

[0013] Furthermore, the present invention also provides a use of a probiotic preparation in the preparation of a drug for treating acne, wherein the active ingredient of the probiotic preparation contains the above-mentioned combination of lactobacillus derivatives.

[0014] The present invention also provides a method for preparing a composition of polylactobacillus derivatives, which comprises the steps of: inactivating, disrupting and filtering Bbifi-1, Badol-1, Lplan-4 and BA05 strains or their pure cultures respectively; or inactivating, disrupting and filtering Bbifi-1, Badol-1, Lplan-4 and BA05 strains or their pure cultures together.

[0015] The composition of the polylactobacillus derivatives of the present invention has significant antioxidant and anti-inflammatory effects, can inhibit pathogenic bacteria of acne and other common skin infections, such as Propionibacterium acnes and Staphylococcus aureus, and can also inhibit skin lipid accumulation and avoid clogging of hair follicles. In the above aspects, the effect of the composition is similar to that of the optimal single bacteria Badol-1 derivative, but at the individual level (such as rabbit model), the composition of polylactobacillus derivatives has better anti-inflammatory effects and fewer scars / nodules than each single bacteria derivative, showing a synergistic effect of mutual complementation between different strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the test result of ear swelling rate in rabbit acne model.

[0017] Figure 2 is the test result of the total score of skin lesions in the rabbit acne model.

[0018] FIG. 3 is the detection result of the total number of lesions in the rabbit acne model.

[0019] FIG. 4 is the detection result of TNF-α in rabbit acne model.

[0020] Figure 5 is a representative picture of the rabbit ear skin at the end point of the rabbit acne model test. DETAILED DESCRIPTION

[0021] The meaning of the "strain" with the specific deposit number claimed in the present invention includes but is not limited to: (1) A strain with a specific accession number deposited in the above-mentioned depository; (2) Strains with the same genome as the strain described in (1), including clonal strains; (3) A non-mutated strain based on the above (1) or (2), including progeny strains; (4) A bacterial strain based on (1), (2) or (3) above that accumulates minor mutations during subculture but has no substantial changes in toxicity, immunogenicity or biological activity.

[0022] As is known in the art, minor mutations are inevitably introduced into strains through passage and application. When mutations occur in non-coding sequence regions or synonymous mutations in coding regions or mutations that do not affect the toxicity, immunogenicity and biological activity of the strain (for example, it may be a connecting amino acid residue between two domains, or a minor mutation residue located inside the higher-level structure of a protein that does not contact immune cells and does not affect toxicity, immunogenicity and biological activity), it can be reasonably expected that when these minor changes do not significantly affect the toxicity, immunogenicity and biological activity of the offspring strains, the purpose of the present invention can still be achieved, and they are derived from the strains contributed by the present invention, and therefore are still within the scope of the substantial technical contribution of the present invention. These minor mutations are still non-substantial mutations and should be regarded as mutant strains with no changes in toxicity, immunogenicity and biological activity.

[0023] There is no substantial change in toxicity, immunogenicity and biological activity, including but not limited to, within the limits of detection technology such as detection sensitivity and detection limit and acceptable or unavoidable errors, the toxicity, immunogenicity and biological activity are considered to be the same. When the toxicity, immunogenicity and biological activity of the offspring of the strain are measured using cells and animals, there is no substantial change due to differences in cell strains, animal species, age, sex, health status, culture conditions, etc., as well as foreseeable or unavoidable systematic errors.

[0024] "Progeny strain" refers to a descendant strain produced by an original strain or parent strain, including those strains that have undergone multiple generations but still retain the key characteristics and functional properties described in this patent application.

[0025] "Clone strain" means a genetically identical offspring strain that is directly derived from a single parent strain or its progeny through asexual reproduction (such as cell division or gene duplication).

[0026] A "pure culture" is a culture that consists of a single species of microorganism. A pure culture means that all the microorganisms growing on the culture medium are descendants of the same microorganism and have the same genetic characteristics.

[0027] "Composition" refers to a product or substance collection composed of two or more different ingredients, which can be chemical substances, biological materials (such as cells, proteins, nucleic acids, etc.), active pharmaceutical ingredients, excipients or other additives.

[0028] "Drug" refers to a preparation composed of one or more active ingredients with therapeutic, preventive or diagnostic effects and pharmaceutically acceptable carriers, excipients or other additives. It is intended to enter the body through an appropriate route of administration (such as oral, injection, inhalation, topical application, etc.) to achieve the desired medical effect.

[0029] "Cosmetics" refer to products designed for personal care purposes such as beauty and skin care. They are usually composed of active ingredients, base materials, auxiliary ingredients and other additives. They are intended to clean, beautify, attract or change the appearance of the human body, while maintaining the health of the skin, hair and other parts.

[0030] "Inactivated bacteria" refers to microbial cells or their structurally intact cell walls and cell membranes that have lost their ability to reproduce after physical, chemical or other treatment methods. Although these cells are no longer viable (i.e., they cannot carry out metabolic activities or divide and proliferate), they still maintain a certain degree of structural integrity and can retain certain biologically active components.

[0031] "Derivative" in this patent refers to the product obtained by one or more steps of inactivation, crushing, separation, extraction, filtration, etc. of a strain, pure culture or inactivated bacteria through physical and chemical methods. The product contains the structural components, fermentation products, metabolites, etc. of the bacteria.

[0032] "Lysate" refers to a mixture formed by the destruction of the cell walls and cell membranes of microbial cells by physical, chemical or enzymatic methods, resulting in the release of cell contents.

[0033] "Extract" refers to a product obtained by physical, chemical or enzymatic treatment, in which the cell walls and cell membranes of microbial cells are destroyed, resulting in the release of cell contents, and then specific ingredients or components are selectively separated and concentrated by physical, chemical or biological methods.

[0034] "Fermentation product lysate" refers to a mixture of cell contents and metabolites obtained from a microbial culture after a fermentation process by physical, chemical or enzymatic methods. During this process, the cell wall and cell membrane of the microorganism are destroyed, resulting in the release of intracellular substances into the external environment. The lysis process not only exposes and separates intracellular components such as proteins, nucleic acids, enzymes, and secondary metabolites, but also retains the extracellular metabolites produced during the fermentation process.

[0035] Reagent / medium preparation Preparation of anaerobic MRS liquid culture medium: weigh 54.0 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312) and 0.5 g of cysteine ​​hydrochloride monohydrate, dissolve in 1 L of distilled water, deoxygenate with N2 replacement, and sterilize at 121°C for 15 min.

[0036] Preparation of anaerobic three-mixed liquid culture medium (BHI+MRS+modified GAM): weigh 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3), dissolve in 1 L of distilled water, deoxygenate by N2 replacement and package, sterilize at 121°C for 30 min, and store in a cool and dry place.

[0037] Preparation of anaerobic and resazurin-free three-mixed liquid culture medium (BHI+MRS+modified GAM): weigh 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3), dissolve in 1 L of distilled water, heat to boil, cool to room temperature, add 0.5 g of cysteine ​​hydrochloride, stir to dissolve, adjust the pH to 6.5-7.0, install a quantitative dispenser and pass N2, heat to boiling, keep at a slight boiling state for 30 min, cool and package, sterilize at 121°C for 15 min, and store in a cool and dry place.

[0038] Preparation of 1.5% calcium carbonate MRS solid culture medium: weigh 52.0 g of MRS finished culture medium (OXOIDCM1175) powder, 15.0 g of agar powder, and 15.0 g of calcium carbonate, dissolve in 1 L of distilled water, heat to boil, add 0.55 g of cysteine ​​hydrochloride after boiling, adjust the pH value to 6.5, sterilize at 118 °C for 20 min, store in a cool, dark place for later use.

[0039] Preparation of anaerobic PBS: weigh 0.27 g potassium dihydrogen phosphate, 1.42 g disodium hydrogen phosphate, 8 g sodium chloride, and 0.2 g potassium chloride, dissolve in 1 L of distilled water, heat to boil, cool to room temperature, add 0.55 g cysteine ​​hydrochloride, stir to dissolve, adjust the pH to 6.5, install a quantitative dispenser and pass N2, heat to boiling, cook at a slight boil for 30 min, cool and dispense into 10 mL anaerobic tubes, sterilize at 121°C for 30 min, store in a cool, dark place for later use.

[0040] TSB (Tryptone Soy Broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114) medium was prepared by weighing and dissolving according to the instructions, sterilizing with high temperature and moist heat at 121°C for 30 min, and storing in a cool and dry place.

[0041] Example 1 Isolation and identification of strains Fresh feces from healthy volunteers were collected, shaken, resuspended, and filtered in an appropriate amount of anaerobic PBS. Under N2 protection, the samples were gradient diluted and anaerobically cultured in a culture medium (such as anaerobic three-mixed liquid culture medium). The culture was subjected to MALDI-TOF mass spectrometry detection and deduplication, and then 16S rDNA gene amplification and sequencing (Beijing Qingke Biotechnology Co., Ltd. Chengdu Branch) was performed for identification. The sequencing results were compared by BLAST.

[0042] The sequence similarity between strain 1 and a strain of Bifidobacterium bifidum was the highest, so it was preliminarily identified as Bifidobacterium bifidum ( Bifidobacterium bifidum , named Bifidobacterium bifidum ( Bifidobacterium bifidum )Bbifi-1. Strain 2 had the highest sequence similarity with a strain of Bifidobacterium adolescentis and was identified as Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), formerly known by the Latin name: Bifidobacterium stercoris , formerly known as excremental bifidobacterium, was named adolescent bifidobacterium ( Bifidobacterium adolescentis ) Badol-1. Strain 3 had the highest sequence similarity with a strain of Lactobacillus plantarum and was identified as Lactobacillus plantarum, also known as Lactobacillus plantarum, and was named Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Lplan-4. Strain 4 is a strain of Lactobacillus acidophilus, which was isolated from the commercial product VSL#3 (ALFASIGMA USA, INC, G2F047). According to the literature, the strain name is Lactobacillus acidophilus ( Lactobacillus acidophilus )BA05.

[0043] Isolation method of strain 4: Take two VSL#3 capsules and open them in an anaerobic glove box, dissolve the powder in the capsule in anaerobic PBS, shake for 2 minutes, and fully dissolve the capsule powder. Use a disposable inoculation loop to dip a ring of bacterial solution and streak it on MRS solid medium containing 1.5% calcium carbonate. After culturing in an anaerobic incubator at 37°C for 48 hours, pick a single colony with a calcium melting circle and culture it in anaerobic MRS liquid medium for 24 hours. Transfer part of the bacterial solution obtained after culture for further culture and preservation, and perform mass spectrometry and sequencing on the other part of the bacterial solution, and it was determined to be Lactobacillus acidophilus ( Lactobacillus acidophilus )BA05.

[0044] Example 2 Whole genome analysis and identification Strains 1, 2 and 3 obtained in Example 1 were cultured in anaerobic three-mixed liquid medium, respectively, until the late logarithmic growth stage, and the whole genome DNA of the strains was extracted, and the whole genome sequencing was performed using the Illumina high-throughput sequencing platform NovaSeq 6000. After quality control of the offline data, it was assembled and annotated using conventional methods, and the obtained protein sequence was compared with the VFDB (Virulence Factor Databases) and CARD (The Comprehensive Antibiotic Resistance Database) databases to conduct virulence factor and drug resistance gene analysis and evaluate its genetic level safety. The novelty of the strains was analyzed using Average Nucleotide Identity (ANI) and Variant analysis based on single nucleotide polymorphism (SNP).

[0045] Strain 1: The results showed that the Bbifi-1 strain was safe at the genetic level. A search in Genbank found 180 published Bifidobacterium bifidum The whole genome was compared by fastANI (v1.33) and snippy (v4.6.0), and it was found that the two strains closest to the whole genome of Bifidobacterium bifidum Bbifi-1 of the present invention were GCA_003466395.1 (ANI=99.01%) and GCA_003437945.1 (ANI=99.00%), so Bifidobacterium bifidum Bbifi-1 can be considered as a new strain.

[0046] Strain 2: The results showed that the Badol-1 strain was safe at the genetic level. A search in Genbank found 29 published Bifidobacterium adolescentis The related whole genomes were compared by fastANI (v1.33) and snippy (v4.6.0), and it was found that the strain with the closest ANI to Badol-1 was GCA_003030905.1 (ANI= 98.27%), so Badol-1 can be considered as a new strain.

[0047] Strain 3: The results showed that the Lplan-4 strain was safe at the genetic level. A search in Genbank found 854 published Lactiplantibacillus plantarum The related whole genomes were compared by fastANI (v1.33) and snippy (v4.6.0), and it was found that the strain with the closest ANI to the Lplan-4 whole genome was GCA_002872355.1 (ANI= 99.89%), so Lplan-4 can be considered as a new strain.

[0048] Strains 1, 2, and 3 have been deposited in the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, Hubei Province, zip code: 430072, telephone: 027-68754052.

[0049] Strain 4 is commercially available.

[0050] The deposit information of strains 1, 2 and 3 is as follows: Table 1 Microbial deposit information Strain name Species information Date of deposit Accession number Test survival date Bbifi-1 Bifidobacterium bifidum ( March 17, 2023 CCTCC NO:M2023349 March 24, 2023 Badol-1 Bifidobacterium adolescentis( November 18, 2024 CCTCC NO:M 20242593 November 25, 2024 Lplan-4 Lactobacillus plantarum November 18, 2024 CCTCC NO:M 20242594 November 25, 2024 Example 3 Antibacterial ability against pathogenic bacteria Two common pathogens that cause acne were selected for antibacterial ability testing. The source information of the pathogenic bacteria strains is as follows: Table 2 Source information of pathogenic bacteria beads Strain name Strain accession number Strain collection unit Staphylococcus aureus CMCC(B)26003 China Food and Drug Inspection Institute Propionibacterium acnes BNCC330605 (ATCC6919 second generation) Henan Industrial Microbial Engineering Technology Research Center Preparation of strain culture: After activating the four strains, inoculate them into 15 mL of anaerobic MRS liquid culture medium at a 4% (v / v) inoculation rate and culture them anaerobically at 37°C for 24 hours. The culture cultured for 24 hours was pasteurized at 65°C for 30 minutes, and then treated with an ultrasonic disruptor (working power of 120W, ultrasound on for 3s, off for 4s, and ultrasound for a total of 10 minutes in an ice bath). After ultrasound, the fermentation product lysate of the single bacteria was filtered with a 0.22μm filter membrane to obtain the original solution.

[0051] The stock solutions were combined in equal volume ratios to obtain samples. Using bifid yeast (Shanghai Yanfu Industrial Co., Ltd.) as a positive control, the sample stock solution and bifid yeast were diluted to 10% using anaerobic resazurin-free three-mixed liquid culture medium as the test samples (hereinafter referred to as probiotic composition).

[0052] Preparation of pathogenic bacteria: Staphylococcus aureus was activated with TSB medium and diluted in TSB medium to OD 600 0.08-0.12; Propionibacterium acnes was activated by anaerobic three-mixed liquid medium and diluted to OD in anaerobic three-mixed liquid medium without resazurin. 600 It is 0.16-0.24.

[0053] Co-culture with pathogenic bacteria: The experiment was divided into a probiotic composition test sample group (probiotic composition), a positive control group of bifid yeast test sample group (bifid yeast) and a blank control group. The probiotic composition group and the bifid yeast group were respectively placed on a 96-well plate with 0.1 mL of two pathogenic bacteria and 0.1 mL of the test sample, with 3 parallel groups in each group; the blank control group was respectively placed on a 96-well plate with 0.1 mL of two pathogenic bacteria and 0.1 mL of the corresponding culture medium, with 3 parallel groups in each group. Staphylococcus aureus was cultured aerobically for 24 hours, and Propionibacterium acnes was cultured anaerobically for 24 hours, and the OD values ​​at 0 and 24 hours were detected. 600 .

[0054] Data processing: Calculate the inhibition rate (R) using the formula R=(1−ΔOD1 / ΔOD0) × 100%, where ΔOD1 is the OD value of the sample group before and after 24 h of culture. 600 ΔOD0 is the OD value of the blank control group of the same pathogen before and after 24 h of culture. 600 changes.

[0055] The results were expressed as Mean ± SEM of the inhibition rate, and statistical analysis was performed using T-test. The antibacterial ability of the probiotic composition was compared with that of bifid yeast; "***" means that compared with bifid yeast, p <0.001, "**" indicates comparison with Bifida Saccharomyces cerevisiae, p <0.01, "*" indicates comparison with Bifida Saccharomyces cerevisiae, p <0.05.

[0056] Table 3 Pathogenic bacteria inhibition test results Group Propionibacterium acnes inhibition rate Staphylococcus aureus inhibition rate Bifidobacterium 31.21±1.78 27.14±1.67 Probiotic composition 67.72±0.32 *** 45.69±3.99 * Experimental results: As shown in Table 3, the probiotic composition has different degrees of inhibitory effect on Staphylococcus aureus and Propionibacterium acnes, and the effect is similar to that of the single-bacteria Badol-1 derivative, slightly better than other single-bacteria derivatives (not shown in the table), and significantly stronger than Saccharomyces cerevisiae.

[0057] Example 4 Detection of total antioxidant capacity of probiotic composition Sample to be tested: The probiotic composition stock solution was prepared according to the method in Example 3. Bifidobacterium sp. (Shanghai Yanfu Industrial Co., Ltd.) was used as a positive control.

[0058] A BCA protein concentration determination kit (purchased from Beijing Solebow Technology Co., Ltd., PC0020) was used to draw a standard curve and detect the protein concentration of the sample to be tested according to the instructions of the kit.

[0059] The total antioxidant capacity detection kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC1315) was used to determine the antioxidant capacity of the sample according to the instructions of the kit and the standard curve. The total antioxidant capacity unit is μmol / mg prot.

[0060] Data processing: Three parallel groups were set up in this experiment, and the results were expressed as Mean ± SEM. t-test was used for comparative analysis. "*" indicates comparison with the bifid yeast group. p <0.05.

[0061] Table 4 Total antioxidant test results Group Total Antioxidant Test Results Bifidobacterium 7.81±0.55 Probiotic composition 10.20±0.23 * Experimental results: As shown in Table 4, the total antioxidant capacity of the probiotic composition reached 10.20 ± 0.23 μmol / mgprot, which was significantly better than that of Bifida yeast (p < 0.05), indicating that the probiotic composition has a strong antioxidant capacity in vitro. The effect of the probiotic composition is similar to that of the single bacteria Badol-1 and Bbifi-1 derivatives, and slightly better than other single bacteria derivatives (not shown in the table).

[0062] Example 5 Detection of the DPPH free radical scavenging ability of the probiotic composition Sample to be tested: The probiotic composition stock solution was prepared according to the method in Example 3. Bifidobacterium sp. (Shanghai Yanfu Industrial Co., Ltd.) was used as a positive control.

[0063] The DPPH free radical scavenging ability detection kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC4755) was used to determine the DPPH free radical scavenging ability of the sample to be tested according to the instructions of the kit.

[0064] Data processing: Three parallel groups were set up for each experiment. The results were expressed as Mean ± SEM and compared and analyzed by t-test. "***" means p < 0.001 compared with Bifida yeast.

[0065] Table 5 DPPH free radical scavenging test results Group DPPH free radical scavenging test results Bifidobacterium 1.60±0.73 Probiotic composition 64.98±8.72 *** Experimental results: As shown in Table 5, the DPPH free radical scavenging ability of the probiotic composition reached 64.98% ± 8.72%, which was significantly better than that of Bifida yeast (p < 0.0001), indicating that the probiotic composition has a strong DPPH free radical scavenging ability in vitro. The effect of the probiotic composition is slightly better than that of the single bacterial derivative.

[0066] Example 6 Detection of the ability of probiotic composition to scavenge ABTS free radicals Sample to be tested: The probiotic composition stock solution was prepared according to the method in Example 3. Bifidobacterium sp. (Shanghai Yanfu Industrial Co., Ltd.) was used as a positive control.

[0067] The ABTS free radical scavenging ability detection kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC4775) was used to determine the ABTS free radical scavenging ability of the sample to be tested according to the instructions of the kit.

[0068] Data processing: Three parallel groups were set up for each experiment. The results were expressed as Mean ± SEM and compared and analyzed by t-test. "***" means p < 0.001 compared with Bifida yeast.

[0069] Table 6 ABTS free radical scavenging test results Group ABTS free radical scavenging test results Bifidobacterium 19.71±0.47 Probiotic composition 98.90±0.48 *** Experimental results: As shown in Table 6, the ABTS free radical scavenging ability of the probiotic composition reached 98.90% ± 0.48%, which was significantly better than that of Bifida yeast (p < 0.0001), indicating that the probiotic composition has a strong ABTS free radical scavenging ability in vitro. The effect of the probiotic composition is similar to that of each single bacterial derivative.

[0070] Example 7 Detection of the ability of probiotic composition to remove lipid accumulation Sample preparation: The sample processing method was the same as in Example 3. Isotretinoin and bifid yeast (Shanghai Yanfu Industrial Co., Ltd.) were used as positive controls.

[0071] Linoleic acid (Sigma-Aldrich (Shanghai) Trading Co., Ltd., L1376) was used to induce lipid secretion in immortalized human sebaceous gland cell line SZ95 cells (Qingqi (Shanghai) Biotechnology Development Co., Ltd., BFN60807569) to induce cellular lipid accumulation. The ability of the probiotic composition to clear lipid accumulation was evaluated by the amount of cellular lipid accumulation.

[0072] Linoleic acid was dissolved in a mixture of DMSO and ethanol (v / v=1 / 1) to prepare a 1 M linoleic acid stock solution, which was then diluted 1000 times using serum-free DMEM culture medium (Thermo Fisher Scientific (China) Co., Ltd., C11065500BT) to prepare a linoleic acid working solution.

[0073] Isotretinoin was dissolved in DMSO to prepare a 100 mM isotretinoin stock solution, which was then diluted with linoleic acid working solution to prepare a 100 μM isotretinoin working solution.

[0074] 5% (v / v) bifid yeast linoleic acid working solution was prepared by adding bifid yeast stock solution (Shanghai Yanfu Industrial Co., Ltd.) to linoleic acid working solution.

[0075] The 5% (v / v) probiotic composition linoleic acid working solution is prepared by adding the corresponding probiotic composition stock solution to the linoleic acid working solution.

[0076] SZ95 cells were collected at 3×10 4 The cells were inoculated at a rate of 1000 μg / well in a 96-well plate and cultured at 37°C, 5% CO2 for 24 hours. The supernatant was removed and the plates were divided into 6 groups: 200 μL serum-free DMEM medium was added to the blank control group, 200 μL linoleic acid working solution was added to the model control group, 200 μL isotretinoin group was added to 200 μL isotretinoin working solution, 200 μL of bifid yeast linoleic acid working solution and 200 μL of probiotic composition linoleic acid working solution were added to the bifid yeast group and the probiotic composition group, respectively.

[0077] After adding the sample, the cells were cultured at 37°C and 5% CO2 for 24 hours. According to the requirements of the lipid droplet red fluorescence detection kit (Biyuntian Biotechnology Co., Ltd., Nile Red C2051M), 100 μL of fluorescent dye was added for staining, and the cells were incubated at 37°C in the dark for 10 minutes before detection. The excitation light was 485 nm and the emission light was 535 nm.

[0078] Data processing: Six parallel groups were set up in each group of this experiment. The results were expressed as Mean ± SEM. One-way ANOVA was used for multiple comparison analysis. "***" indicates comparison with the model control. p <0.001.

[0079] Table 7 Lipid accumulation removal ability test results Group Lipid accumulation clearance ability test results Blank control 100.00±6.07 *** Model comparison 176.53±3.72 Isotretinoin 119.34±3.04 *** Bifidobacterium 162.09±7.17 Probiotic composition 141.26±6.37 *** The test results are shown in Table 7. After linoleic acid induction, the cell lipid secretion increased significantly, and obvious lipid accumulation occurred. The lipid accumulation in the model control group was significantly higher than that in the blank control group ( p <0.001), the lipid accumulation after isotretinoin treatment was significantly reduced compared with the model control group, while no significant effect was seen in the bifid yeast group, and there was no significant difference in lipid accumulation compared with the model control group. The lipid accumulation in the probiotic combination group was significantly reduced compared with the model control group, and the probiotic combination was more effective than bifid yeast.

[0080] Example 8 Detection of the anti-IL-8 effect of the probiotic composition Sample preparation: The sample stock solution was prepared as in Example 3. Salicylic acid and bifid yeast (Shanghai Yanfu Industrial Co., Ltd.) were used as positive controls.

[0081] Human keratinocytes Hacat cells (Chengdu Gumeng Biotechnology Co., Ltd., BNCC339817) were stimulated with Propionibacterium acnes (Henan Industrial Microbial Strain Engineering Technology Research Center, BNCC330605 (ATCC6919 second generation)) to induce Hacat cells to produce cellular inflammatory factors, and the anti-inflammatory effect of the probiotic composition was evaluated by the level of inflammatory factors.

[0082] Preparation of heat-killed Propionibacterium acnes working solution: Select Propionibacterium acnes in the logarithmic growth phase, heat at 80°C for 30 min to inactivate, centrifuge at 12000 rpm / min for 5 min after inactivation, remove the supernatant, and use serum-free DMEM medium (Thermo Fisher Scientific (China) Co., Ltd., C11065500BT) to reconstitute, and adjust the final concentration of Propionibacterium acnes to 5×10 8 CFU / ml.

[0083] Preparation of salicylic acid working solution: Use heat-killed Propionibacterium acnes working solution to prepare 200 μg / ml salicylic acid working solution.

[0084] 5% (v / v) bifid yeast Propionibacterium working solution was prepared by adding heat-killed Propionibacterium acnes working solution to bifid yeast stock solution (Shanghai Yanfu Industrial Co., Ltd.).

[0085] The 5% (v / v) probiotic composition Propionibacterium working solution is prepared by adding the probiotic composition stock solution to the heat-inactivated Propionibacterium acnes working solution.

[0086] Hacat cells were collected at 3 × 10 4 The cells / well were inoculated in a 96-well plate and cultured at 37°C, 5% CO2 for 24 hours. The supernatant was removed and the experiment was divided into 5 groups: 200 μl of serum-free DMEM medium was added to the blank control group, 200 μl of Propionibacterium working solution was added to the model control group, 200 μl of salicylic acid working solution was added to the salicylic acid group, 200 μl of bifid yeast Propionibacterium working solution and 200 μl of probiotic composition Propionibacterium working solution were added to the bifid yeast group and the probiotic composition group, respectively.

[0087] After adding the sample, the cells were cultured at 37°C and 5% CO2 for 24 hours, and the cell culture supernatant was collected. Inflammatory factors were detected according to the instructions of the human interleukin 8 (IL-8) enzyme-linked immunosorbent assay kit (Wuhan Elaruite Biotechnology Co., Ltd., E-EL-H6008).

[0088] Data processing: Three parallel groups were set up in each group of this experiment. The results were expressed as Mean±SEM. One-way ANOVA was used for multiple comparison analysis. "***" means compared with the model control, p<0.001; "**" means compared with the model control, p<0.01; "*" means compared with the model control, p<0.05.

[0089] Table 8 Detection results of inflammatory factor IL-8 Group Anti-inflammatory test results Blank control 11.13±0.12 *** Model comparison 36.69±3.45 Salicylic acid 15.72±0.80 *** Bifidobacterium 28.05±2.02 * Probiotic composition 21.85±2.36 ** The test results are shown in Table 8. Heat-killed Propionibacterium acnes induces the secretion of inflammatory factors in cells. The IL-8 content in the model control group is significantly higher than that in the blank control group. The salicylic acid group has an obvious anti-inflammatory effect. After treatment, the IL-8 content is significantly reduced compared with the model control group. However, no obvious effect was observed in the bifid yeast group. There was no significant difference in IL-8 content compared with the model control group. The probiotic composition of the present invention has an obvious anti-inflammatory effect. After treatment, the IL-8 content is significantly reduced compared with the model control group.

[0090] Example 9 Detection of the anti-IL-6 effect of the probiotic composition Sample preparation: The stock solution sample treatment method was the same as in Example 3, with dexamethasone (D754934, Shanghai Aladdin Biochemical Technology Co., Ltd.) and bifida yeast (Shanghai Yanfu Industrial Co., Ltd.) as positive controls.

[0091] The human monocytic leukocyte cell line (THP-1 cells) derived from patients with acute monocytic leukemia was stimulated with lipopolysaccharide (LPS) to induce THP-1 cells (Wuhan Pronocell Life Science Technology Co., Ltd., CL-0233) to produce cellular inflammatory factors, and the anti-inflammatory effect of the probiotic composition was evaluated by the level of inflammatory factors.

[0092] Preparation of LPS (lipopolysaccharide, Shanghai Aladdin Biochemical Technology Co., Ltd. L118716) working solution: Use serum-free 1640 medium (Thermo Fisher Scientific (China) Co., Ltd., C1187550BT) to prepare 1g / ml LPS and 20ng / ml IFN-γ (interferon γ receptor agonist, Shanghai Aladdin Biochemical Technology Co., Ltd. rp174266) modeling working solution.

[0093] Preparation of dexamethasone working solution: Use LPS working solution to prepare 25 μg / ml dexamethasone working solution.

[0094] 5% (v / v) Bifida yeast LPS working solution was prepared by adding Bifida yeast stock solution (Shanghai Yanfu Industrial Co., Ltd.) to LPS working solution.

[0095] The 5% (v / v) probiotic composition LPS working solution is prepared by adding the probiotic composition stock solution into the LPS working solution.

[0096] THP-1 cells were collected at 1×10 5 The cells were inoculated in a 96-well plate at a concentration of 100 ng / ml PMA (Phorbol12-myristate 13-acetate, MedChemExpress (China) Co., Ltd. HY-18739) solution, and cultured at 37°C, 5% CO2 for 24 hours. The supernatant was removed and the experiment was divided into 5 groups. The blank control group was added with 200 μl of serum-free 1640 culture medium, the model control group was added with 200 μl of LPS working solution, the dexamethasone group was added with 200 μl of dexamethasone working solution, the bifid yeast group and the probiotic composition group were added with 200 μl of bifid yeast LPS working solution and probiotic composition LPS working solution, respectively.

[0097] After adding the sample, the cells were cultured at 37°C and 5% CO2 for 24 hours, and the cell culture supernatant was collected. Inflammatory factors were detected according to the instructions of the human interleukin 6 (IL-6) enzyme-linked immunosorbent assay kit (Wuhan Elaruite Biotechnology Co., Ltd., E-EL-H6156).

[0098] Data processing: Three parallel groups were set up in each group of this experiment. The results were expressed as Mean±SEM. One-way ANOVA was used for multiple comparison analysis. "***" means compared with the model control, p<0.001; "**" means compared with the model control, p<0.01; "*" means compared with the model control, p<0.05.

[0099] Table 9 Detection results of inflammatory factor IL-6 Group Anti-inflammatory test results Blank control 6.78±4.26 *** Model comparison 12611.65±983.76 Dexamethasone 128.62±15.24 *** Bifidobacterium 3637.80±951.94 *** Probiotic composition 7551.30±910.99 ** The test results are shown in Table 9. After LPS induction, the IL-6 content of the model control group was significantly higher than that of the blank control group. The dexamethasone group and the bifid yeast group had obvious anti-inflammatory effects. After treatment, the IL-6 content was significantly decreased compared with the model control group.

[0100] The probiotic composition has obvious anti-inflammatory effect, and the IL-6 content after use is significantly reduced compared with the model control group.

[0101] Example 10 Probiotic composition improves rabbit ear acne Sample preparation: The stock solution sample processing method was the same as in Example 3, with adapalene (Sichuan Mingxin Pharmaceutical Co., Ltd.) and bifida yeast (Repair Complex CLR™ PF, purchased from Chemisches Laboratorium Dr. KurtRichter GmbH) as positive controls.

[0102] Preparation of test samples: Take 30 ml of the above-mentioned probiotic composition and the purchased positive control bifid yeast stock solution, add 0.6 g of hydroxyethyl cellulose (thickener, purchased from Shandong Heda Co., Ltd.), mix well, and make gels respectively. Then use 2 ml sterile syringes to divide and store them separately. The storage condition is -20℃, and take one vial daily for application and administration.

[0103] Experimental grouping and administration: 25 clean-grade male New Zealand white rabbits, 3 months old and weighing 1.8-2.5 kg, were purchased from Pizhou Oriental Breeding Co., Ltd. After 7 days of adaptive feeding, they were randomly divided into 5 groups, namely blank control group, model control group, adapalene group, bifida yeast group, and probiotic composition group, with 5 rabbits in each group. Except for the blank control group, the other groups were injected with live Propionibacterium acnes intradermally between 9:00 and 10:00 am on the 1st, 3rd, and 5th day of the experiment. The injection site was 3 cm × 3 cm inside the right ear of the rabbit. Six points were injected each time, 25 μl was injected at each point, and 1×10 8 CFU, immediately after the injection on the first day, 0.2g of artificial sebum (Beijing Solebow Technology Co., Ltd.) was evenly applied within the injection range for 14 consecutive days, once a day, and the sebum application was performed in the morning. The blank control group was injected with the same amount of saline and no artificial sebum was applied. The positive drug adapalene group was applied with adapalene at 0.1g / piece, and the positive drug bifida yeast group and the probiotic composition group were both applied with the above-prepared test sample at 0.4ml / piece, and the drug was administered once a day for 14 consecutive days, and the administration was performed in the afternoon. The test cycle was 14 days. On the 15th day, an appropriate amount of blood was drawn along the rabbit middle ear artery, and the serum was separated for inflammatory factor detection.

[0104] Ear swelling rate detection: Before the experiment, the thickness of the right ear of each rabbit was measured with a vernier caliper at three locations. After the experiment, the skin thickness of the three roughly identical locations was measured again with a vernier caliper. The results were averaged and the swelling rate was calculated as (ear thickness at the end - ear thickness at the beginning) / ear thickness at the beginning * 100%. The experimental results were expressed as Mean ± SEM, and multiple comparisons were analyzed using One-way ANOVA. Compared with the model group, * p < 0.05; *** p < 0.001.

[0105] Skin lesion score: The skin lesion status of rabbit ears was scored according to the scoring criteria in the table below. The total skin lesion score was obtained by adding the scores of each index in the table below. The test results were expressed as Mean ± SEM. The differences between the groups were tested by non-parametric test, Kruskal-Wallis test. Compared with the model group, * p < 0.05; **** p < 0.0001.

[0106] Table 10 Skin lesion scoring table

[0107] Total lesion number statistics: The number of lesions at each rabbit ear modeling site was recorded, including papules, pustules, nodules, ulcers, and scabs. If new skin tissue obviously grew out of the lesions, it was not counted. The test results were expressed as Mean ± SEM, and the differences between the groups were tested by non-parametric tests, Kruskal-Wallis test. Compared with the model group, ** p < 0.01; **** p < 0.0001.

[0108] Detection of inflammatory factor TNF-α: According to the instructions, the TNF-α content in rabbit serum was detected using a rabbit ELISA detection kit. The test results were expressed as Mean±SEM, and one-way ANOVA was used for multiple comparison analysis. Compared with the model group, ** p<0.01; *** p<0.001.

[0109] Table 11 Rabbit acne model test results Group Ear swelling rate (%) Total skin lesion score Total number of lesions TNF-α content Normal control group 32.47±4.65 *** 0.00±0.00 *** 0.00±0.00 *** 6.55±1.00 *** Model control group 85.92±5.03 5.60±0.60 12.40±1.21 48.87±5.96 Adapalene group 71.20±7.71 4.60±0.24 12.00±1.30 13.15±2.77 ** Bifidobacterium group 66.89±7.18 * 4.80±0.37 14.00±0.84 42.08±12.16 Probiotic composition group 37.58±6.93 *** 4.25±0.25 * 9.50±1.26 * 8.71±2.34 *** Figure 1 The results are as follows: The ear swelling rate test results of the rabbit acne model. Compared with the blank control group, the ear swelling rate of the model control group was significantly increased. Both bifid yeast and the probiotic composition can significantly reduce the rabbit ear swelling rate. The improvement effect of the probiotic composition is better than that of adapalene and bifid yeast.

[0110] Figure 2 The total score of skin lesions was detected. Compared with the blank control group, the total score of skin lesions in the model control group was significantly increased. Compared with the model control group, the probiotic composition could significantly reduce the total score of skin lesions.

[0111] Figure 3 The total number of lesions is statistically shown in Table 1. There were no lesions in the blank control group. Compared with the model control group, the probiotic composition can significantly reduce the number of lesions. The probiotic composition has a better improvement effect than adapalene and bifid yeast.

[0112] Figure 4The results of serum inflammatory factor detection show that compared with the blank control group, the serum TNF-α content in the model control group was significantly increased. Compared with the model control group, adapalene and the probiotic composition can significantly reduce the serum TNF-α content. The probiotic composition has a better improvement effect than bifida yeast. The probiotic composition is significantly better than the single bacterial derivatives in improving serum inflammatory factors (not shown in the figure).

[0113] Figure 5 Representative pictures of rabbit ear skin at the end of the test. The model group formed obvious acne-like lesions (mainly red papules in the early stage, then developed into pustules, and finally formed cysts / nodules). Each drug-treated group had different degrees of improvement, and the improvement effect of the probiotic composition was more significant (the number of nodules was significantly reduced, and new skin tissue was generated at the pustule site). The improvement effect of the probiotic composition was better than that of adapalene and bifida yeast. Moreover, the effect of the probiotic composition on cysts / nodules was significantly better than that of the single bacterial derivatives (not shown in the figure).

Claims

1. A polylactobacillus composition, comprising: 1) Bifidobacterium bifidum ( Bifidobacterium bifidum ) Bbifi-1 strain or its pure culture or inactivated bacteria, the Bbifi-1 strain has a deposit number of CCTCCNO: M2023349; 2) Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) Badol-1 strain or its pure culture or inactivated bacteria, the Badol-1 strain has a deposit number of CCTCC NO: M 20242593; 3) Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Lplan-4 strain or its pure culture or inactivated bacteria, the Lplan-4 strain has a deposit number of CCTCC NO: M 20242594; and 4) Lactobacillus acidophilus ( Lactobacillus acidophilus ) BA05 strain or its pure culture or inactivated bacteria.

2. A composition of polylactobacillus derivatives, wherein the derivatives are the Bbifi-1, Badol-1, Lplan-4 and BA05 strains or their pure cultures or inactivated bacterial lysates, extracts, fermentation product lysates or combinations thereof as described in claim 1.

3. A cosmetic or a medicine, wherein the active ingredient of the cosmetic or the medicine contains the composition of the polylactobacillus derivative according to claim 2.

4. An application of a probiotic preparation in non-disease treatment purposes, wherein the active ingredient of the probiotic preparation contains the composition of the polylactobacillus derivatives according to claim 2, and the non-disease treatment purposes are selected from one or more of anti-oxidation, antibacterial, anti-inflammatory, reduction of skin redness and swelling, and inhibition of skin lipid accumulation.

5. The use according to claim 4, characterized in that: The antibacterial activity refers to inhibiting one or both of Propionibacterium acnes and Staphylococcus aureus.

6. Use of a microecological preparation in the preparation of a drug for treating acne, characterized in that: The active ingredients of the microecological preparation contain the composition of the lactobacillus derivatives according to claim 2.

7. A method for preparing the composition of polylactobacillus derivatives according to claim 2, the method comprising the steps of: inactivating, disrupting and filtering Bbifi-1, Badol-1, Lplan-4 and BA05 strains or their pure cultures respectively; or inactivating, disrupting and filtering Bbifi-1, Badol-1, Lplan-4 and BA05 strains or their pure cultures together.

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