CN119530100A - A multi-lactobacillus composition for improving skin and its derivatives and applications - Google Patents
A multi-lactobacillus composition for improving skin and its derivatives and applications Download PDFInfo
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- CN119530100A CN119530100A CN202510084495.9A CN202510084495A CN119530100A CN 119530100 A CN119530100 A CN 119530100A CN 202510084495 A CN202510084495 A CN 202510084495A CN 119530100 A CN119530100 A CN 119530100A
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Abstract
本发明属于微生物技术领域，具体涉及一种改善皮肤的多乳杆菌组合物及其衍生物和应用。该组合物包含两歧双歧杆菌Bbifi‑1（CCTCC NO:M2023349）、青春双歧杆菌Badol‑1（CCTCC NO:M 20242593）、植物乳植杆菌Lplan‑4（CCTCC NO:M 20242594）和嗜酸乳杆菌（BA05）的菌株、纯培养物或灭活菌体。通过裂解、提取、发酵等处理方式制备的多乳杆菌衍生物组合物，可作为药物或化妆品活性成分，具有显著的抗氧化、抑菌，特别是针对痤疮丙酸杆菌及金黄色葡萄球菌、抗炎和抑制皮肤脂质堆积的作用，有助于消退皮肤红肿，避免毛囊堵塞，对痤疮具有显著效果。
The present invention belongs to the field of microbial technology, and specifically relates to a multi-lactobacillus composition for improving skin, its derivatives and applications. The composition comprises strains, pure cultures or inactivated bacteria of Bifidobacterium bifidum Bbifi‑1 (CCTCC NO: M2023349), Bifidobacterium adolescentis Badol‑1 (CCTCC NO: M 20242593), Lactobacillus plantarum Lplan‑4 (CCTCC NO: M 20242594) and Lactobacillus acidophilus (BA05). The multi-lactobacillus derivative composition prepared by lysis, extraction, fermentation and other treatment methods can be used as a drug or cosmetic active ingredient, and has significant antioxidant and antibacterial effects, especially against Propionibacterium acnes and Staphylococcus aureus, anti-inflammatory and inhibiting skin lipid accumulation, which helps to reduce skin redness and swelling, avoid hair follicle blockage, and has a significant effect on acne.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus plantarum composition for improving skin, a derivative thereof and application thereof.
Background
Acne is a common skin disorder that affects primarily teenagers and young people, but may also persist to adulthood. It is caused by the combined action of sebaceous gland hyperactivity, follicular keratinization abnormality, bacterial infection (e.g. Propionibacterium acnes, p. Acnes, propionibacterium acnes) and inflammatory reaction. Conventional treatment methods generally rely on antibiotics, retinoic acid-based drugs, hormone therapy, etc., but these methods may cause side effects, and long-term use may lead to the development of drug resistance.
In recent years, more and more research has focused on the use of microorganisms and their metabolites as alternative or complementary therapeutic means. Probiotics have been widely accepted as a beneficial viable bacteria in regulating intestinal microecological balance, however, their potential in the field of skin health has not been fully exploited. Studies have shown that certain probiotics can positively affect skin health by modulating the microbial community structure of the skin, enhancing skin barrier function, reducing inflammatory mediator release, etc. For example, the applicant has disclosed in CN118995463a that a derivative of bifidobacterium bifidum Bbifi-1 has some functions of improving the skin.
Nevertheless, there is currently no product on the market that effectively combines a variety of probiotics to fully address the complex etiology of acne. The prior art studies have focused on live bacterial preparations and mainly considered the effects of individual strains, neglecting the powerful biological activities of non-live bacterial components such as lysates and the synergistic effects exerted by the mutual supplementation between different strains. Therefore, it is of great importance to develop a composition based on a multi-probiotic.
Disclosure of Invention
The present invention aims to provide a more potential improvement to skin problems, in particular to provide an improvement to acne symptoms.
Based on the above, the present invention provides a Lactobacillus polymyxa composition comprising:
the bifidobacterium bifidum (Bifidobacterium bifidum) Bbifi-1 strain or a pure culture or an inactivated thallus thereof, wherein the preservation number of the Bbifi-1 strain is CCTCC NO: M2023349;
The bifidobacterium adolescentis (Bifidobacterium adolescentis) Badol-1 strain or a pure culture or an inactivated thallus thereof, wherein the preservation number of the Badol-1 strain is CCTCC NO: M20242593;
Lactobacillus plantarum (Lactiplantibacillus plantarum) Lplan-4 strain or pure culture or inactivated thallus thereof, wherein the Lplan-4 strain has a preservation number of CCTCC NO: M20242594, and
Lactobacillus acidophilus (Lactobacillus acidophilus) BA05 strain or pure culture or inactivated bacteria.
The above composition may be used for the preparation of a pharmaceutical or cosmetic active ingredient, which may be in the form of a lysate, extract, fermentation product lysate or a combination thereof.
Thus, the present invention also provides a composition of a Lactobacillus delbrueckii derivative, which is a lysate, extract, fermentation product lysate or combination thereof of Bbifi-1, badol-1, lplan-4 and BA05 strains or pure cultures or inactivated cells thereof. The composition of the lactobacillus polymorpha derivative can be prepared by respectively preparing lysate, extract and fermentation product lysate of each single bacterium, and then mixing the lysate, the extract and the fermentation product lysate of each single bacterium, or can be prepared by firstly mixing the single bacterium.
The ratio of each strain is not restricted, and the strains can be assembled in any proportion. Preferably, the components are mixed in equal proportions. For example, the lysate, extract, and lysate of fermentation product are prepared separately for each single fungus and then mixed, and an equal volume or equal mass of single fungus derivative may be selected and mixed. For the scheme that the single bacteria are firstly mixed in the preparation of lysate, extract and fermentation product lysate of the mixed bacteria, the single bacteria with equal colony number can be selected to be firstly mixed. Errors that do not significantly affect the effect of the final product are acceptable, for example, errors of + -10% are preferred, errors of + -5% are more preferred, and errors of + -1% are even more preferred.
Further, the invention also provides a cosmetic or medicine, and an active ingredient of the cosmetic or medicine contains the composition of the lactobacillus polymorpha derivative. The content of the composition of the Lactobacillus derivatives in the cosmetic or pharmaceutical is such that it can exert a pharmacological effect, which is easily determined by the person skilled in the art. The content is preferably 1 to 100%, more preferably 1 to 50%, still more preferably 5 to 15%, and further 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% may be selected.
Further, the invention provides the use of a probiotic, the active ingredient of which comprises a composition of the aforementioned lactobacillus derivatives, in a non-disease therapeutic use selected from one or more of antioxidant, bacteriostatic, anti-inflammatory, skin redness and swelling resolving, skin lipid accumulation inhibiting.
In some embodiments, the bacteriostatic means inhibiting one or both of propionibacterium acnes, staphylococcus aureus.
Furthermore, the invention also provides application of the microecological preparation in preparing a medicament for treating acne, wherein an active ingredient of the microecological preparation contains a composition of the lactobacillus polymorpha derivative.
The invention also provides a method for preparing the composition of the lactobacillus polymorpha derivative, which comprises the steps of respectively inactivating Bbifi-1, badol-1, lplan-4 and BA05 strains or pure cultures thereof, crushing thalli and filtering, or inactivating Bbifi-1, badol-1, lplan-4 and BA05 strains or pure cultures thereof together, crushing thalli and filtering.
The composition of the lactobacillus polymorpha derivative has remarkable antioxidant and anti-inflammatory effects, can inhibit acne and other common skin infection pathogenic bacteria such as propionibacterium acnes and staphylococcus aureus, can inhibit skin lipid accumulation, and can avoid hair follicle blockage. In the above aspects, the effect of the composition is similar to that of the optimal single bacterium Badol-1 derivative, but the composition of the lactobacillus derivatives has better anti-inflammatory effect and less scars/nodules compared with the single bacterium derivatives on the individual (such as rabbit model) level, and shows the synergistic effect of mutual complementation among different strains.
Drawings
FIG. 1 is a graph showing the results of detection of ear swelling rate of a rabbit acne model.
FIG. 2 shows the results of the total skin loss score test of the acne model of rabbits.
FIG. 3 shows the results of detection of total lesion number of the acne model of rabbits.
FIG. 4 shows the detection results of TNF-alpha in the rabbit acne model.
Fig. 5 is a representative picture of rabbit ear skin at the end of a rabbit acne model test.
Detailed Description
The meaning of a "strain" of a particular accession number as claimed in the present invention includes, but is not limited to:
(1) Strains deposited at said collection under a specific accession number;
(2) Strains having the same genome as the strain of (1), including cloned strains;
(3) The passaged strains without mutation based on the above (1) or (2), including progeny strains;
(4) The passaging strain based on the aforementioned (1), (2) or (3) which accumulates minute mutations in passaging but has no substantial change in toxicity, immunogenicity and biological activity.
As known in the art, strains inevitably introduce minor mutations by the use of progeny, and when mutations occur in non-coding sequence regions or synonymous mutations in coding regions or mutations that do not affect strain toxicity, immunogenicity and biological activity (e.g., residues that may be linked amino acid residues between two domains, or are located within the higher structure of the protein and do not affect toxicity, immunogenicity and biological activity by virtue of not contacting immune cells), it is reasonable to expect that these minor changes do not significantly affect toxicity, immunogenicity and biological activity of the progeny strain, and are derived from the strains contributed by the invention and therefore remain within the substantial technical contribution of the invention. These minor mutations remain insubstantial mutations and should be considered as mutant strains that have no alterations in toxicity, immunogenicity, and biological activity.
There is no substantial change in toxicity, immunogenicity, and biological activity, including, but not limited to, regarding toxicity, immunogenicity, and biological activity as being the same within the limitations and acceptable or unavoidable errors of detection techniques such as detection sensitivity, detection limits, and the like. The toxicity, immunogenicity and biological activity of the strain offspring are determined by cells, animals and the like, and the expected or unavoidable systematic errors are attributed to the insubstantial changes due to differences in cell lines, animal varieties, ages, sexes, health conditions, culture conditions and the like.
"Progeny strain" refers to progeny strains produced from the original strain or parent strain, including those strains that retain the key features and functional characteristics described in this patent application, although subjected to multiple passages.
By "clonal strain" is meant a genetically identical progeny strain derived directly from a single parent strain or its progeny by asexual propagation (e.g., cell division or gene replication).
"Pure culture" refers to a culture composed of a single species of microorganism. Pure culture means that all microorganisms grown on the medium are offspring from the same microorganism, which have the same genetic characteristics.
"Composition" refers to a product or collection of substances that is a mixture of two or more different components, which may be chemical substances, biological materials (e.g., cells, proteins, nucleic acids, etc.), pharmaceutical active ingredients, adjuvants, or other additives.
"Drug" refers to a formulation consisting of one or more active ingredients having therapeutic, prophylactic or diagnostic effects, together with pharmaceutically acceptable carriers, adjuvants or other additives. It is intended to access the organism by a suitable route of administration (e.g., oral, injectable, inhaled, topical application, etc.) to achieve the desired medical effect.
"Cosmetic" refers to a product designed specifically for personal care purposes such as beauty, skin care, etc., and generally consists of an active ingredient, a matrix material, auxiliary ingredients, and other additives, intended to clean, beautify, attract or alter the appearance of the human body, while maintaining the health of the skin, hair, etc.
"Inactivated bacteria" refers to microbial cells or their structurally intact cell walls and membranes that have been physically, chemically or otherwise rendered incapable of reproduction. Although these cells are no longer viable (i.e., are unable to perform metabolic activity or divide proliferation), they still maintain a degree of structural integrity that can retain certain bioactive components.
The term "derivative" in this patent refers to a product obtained by one or more steps of inactivation, crushing, separation, extraction, filtration, etc. of a strain, a pure culture or an inactivated strain by a physicochemical method, and the product contains a structural component of a strain, a fermentation product, a metabolite, etc.
"Lysate" refers to a mixture of microbial cells that, after physical, chemical or enzymatic treatment, has cell walls and membranes broken, resulting in the release of the cell contents.
"Extract" refers to a product obtained by physical, chemical or enzymatic treatment to disrupt the cell walls and membranes of microbial cells, resulting in the release of the cell contents, and then by physical, chemical or biological means to selectively separate and concentrate specific ingredients or components.
"Fermentation product lysate" refers to a mixture of cellular content and metabolites obtained from a microbial culture after a fermentation process by physical, chemical or enzymatic means. During this process, the microbial cell walls and membranes are destroyed, resulting in release of intracellular material into the external environment. The cell lysis process not only exposes and separates the components such as proteins, nucleic acids, enzymes, secondary metabolites and the like in the cells, but also retains extracellular metabolites generated in the fermentation process.
Reagent/culture medium formulation
Preparation of anaerobic MRS liquid culture medium MRS broth powder (027312, guangdong CycloKai Biotechnology Co., ltd.) 54.0. 54.0 g, cysteine hydrochloride monohydrate 0.5. 0.5g, dissolved in distilled water of 1L, N 2 substituted for oxygen removal, and sterilized at 121deg.C 15 min were weighed.
Preparation of anaerobic triple Mixed liquid Medium (BHI+MRS+modified GAM) A BHI broth powder (Qingdao sea Bo Biotechnology Co., ltd., HB 8297-5) 19.25 g, MRS broth powder (Guangdong Kai Biotechnology Co., ltd., 027312) 13.5 g, modified GAM broth powder (Qingdao sea Bo Biotechnology Co., ltd., HB 8518-3) 15 g were dissolved in distilled water of 1L, and N 2 was substituted for deoxidization and split charging, and subjected to high temperature wet heat sterilization at 121℃for 30 min, and stored in a cool and dry place.
Preparation of anaerobic and anaerobic Langerhans' three-mixed liquid culture medium (BHI+MRS+modified GAM) comprises weighing 19.25 g of BHI broth powder (Qingdao Highway Biotechnology Co., ltd., HB 8297-5), 13.5 g g of MRS broth powder (Guangdong CycloKai Biotechnology Co., ltd., 027312), 15 g g of modified GAM broth powder (HB 8518-3) dissolved in distilled water of 1L, boiling, cooling to room temperature, adding 0.5 g cysteine hydrochloride, stirring to dissolve, adjusting pH to 6.5-7.0, adding quantitative liquid separator and introducing N 2, heating to boil, maintaining 30 min in a micro-boiling state, cooling, sub-packaging, sterilizing at 121deg.C under wet heat and humidity 15min, and storing in shade and dry place.
1.5% Calcium carbonate MRS solid culture medium is prepared by weighing MRS finished product culture medium (OXOIDCM 1175) powder 52.0 g, agar powder 15.0 g, calcium carbonate 15.0 g, dissolving into distilled water 1L, heating to boil, adding cysteine hydrochloride 0.55 g after boiling, adjusting pH to 6.5,118 deg.C, sterilizing at 20. 20 min under high temperature and moist heat, storing in shade, and keeping away from light.
The preparation of the anaerobic PBS comprises the steps of weighing potassium dihydrogen phosphate 0.27 g, disodium hydrogen phosphate 1.42 g, sodium chloride 8 g and potassium chloride 0.2 g, dissolving in distilled water of 1L, heating and boiling, cooling to room temperature, adding 0.55 g cysteine hydrochloride, stirring and dissolving, adjusting the pH value to 6.5, installing a quantitative liquid dispenser, introducing N2, heating to boiling, boiling for 30 min in a micro-boiling state, cooling, split charging into a 10mL anaerobic tube, sterilizing at 121 ℃ at high temperature and humidity for 30 min, and storing in shade and in a dark place for later use.
Preparation of TSB (tryptone Soy Broth, qingdao sea Bo Biotechnology Co., ltd., HB 4114) Medium was weighed according to the instructions for dissolution, and storage in the shade and dry place at 121℃under high temperature humid heat sterilization 30 min.
Example 1 isolation and identification of strains
Fresh feces of healthy volunteers were collected, shaken, resuspended in an appropriate amount of anaerobic PBS, and filtered. The samples are subjected to gradient dilution under the protection of N 2 and then are subjected to anaerobic culture in a culture medium (such as an anaerobic three-mixed liquid culture medium). The cultures were subjected to MALDI-TOF mass spectrometry detection for de-duplication, followed by 16S rDNA gene amplification and sequencing (Beijing qing Biotechnology Co., ltd.) for identification. BLAST alignment was performed on the sequencing results.
The strain 1 has the highest sequence similarity with a strain of bifidobacterium bifidum, so that the strain is primarily identified as bifidobacterium bifidum (Bifidobacterium bifidum) and named as bifidobacterium bifidum (Bifidobacterium bifidum) Bbifi-1. The strain 2 has the highest sequence similarity with a strain of bifidobacterium adolescentis (Bifidobacterium adolescentis), and is identified as bifidobacterium adolescentis (Bifidobacterium adolescentis), and has been named as Latin Bifidobacterium stercoris, and has been named as bifidobacterium adolescentis (Bifidobacterium adolescentis) Badol-1, and has been named as Chinese excrement bifidobacterium. The strain 3 has the highest sequence similarity with a lactobacillus plantarum, and is identified as lactobacillus plantarum, also called lactobacillus plantarum, and named lactobacillus plantarum (Lactiplantibacillus plantarum) Lplan-4. Strain 4 is a strain of Lactobacillus acidophilus isolated from the commercial product VSL#3 (ALFASIGMA USA, INC, G2F 047) and known by the literature as Lactobacillus acidophilus (Lactobacillus acidophilus) BA05.
The separation method of the strain 4 comprises the steps of taking two VSL#3 capsules, opening the capsules in an anaerobic operation glove box, dissolving powder in the capsules in anaerobic PBS, and oscillating 2min to enable the capsule powder to be fully dissolved. One-time inoculating loop is used for dipping bacterial liquid, one loop is streaked on MRS solid culture medium containing 1.5% calcium carbonate, the MRS solid culture medium is placed in an anaerobic incubator for culturing at 37 ℃ for 48 h, single bacteria with calcium thawing loops are selected and cultured in anaerobic MRS liquid culture medium for 24 h, one part of bacterial liquid obtained after culturing is transferred for continuous culturing and preservation, and the other part of bacterial liquid is subjected to mass spectrum and sequencing detection, and is determined to be lactobacillus acidophilus (Lactobacillus acidophilus) BA05.
EXAMPLE 2 Whole genome analysis and identification
Strains 1, 2 and 3 obtained in example 1 were respectively cultured in anaerobic triple-mixed liquid medium to the late logarithmic growth phase, strain whole genome DNA was extracted, and whole genome sequencing was performed using Illumina high throughput sequencing platform NovaSeq 6000,6000. And after quality control is carried out on the off-machine data, assembling and annotating by a conventional method, comparing the obtained protein sequence with VFDB (Virulence Factor Databases) and CARD (The Comprehensive Antibiotic Resistance Database) databases, analyzing the development virulence factors and drug resistance genes, and evaluating the safety of the gene layer. Novel analyses of strains were performed using average nucleotide similarity (Average Nucleotide Identity, ANI) and Variant analysis based on single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNP).
The results of the strain 1 show that Bbifi-1 strain is safe in gene level. 180 published Bifidobacterium bifidum complete genomes were found by searching in Genbank, and by comparison fastANI (v 1.33), snippy (v 4.6.0), two strains closest to the complete genome of bifidobacterium bifidum Bbifi-1 of the present invention were found to be GCA_003466395.1 (ANI=99.01%) and GCA_003437945.1 (ANI=99.00%) respectively, so bifidobacterium bifidum Bbifi-1 could be considered as a new strain.
The results of the strain 2 show that Badol-1 strain is safe on the gene level. By searching in Genbank, 29 published Bifidobacterium adolescentis related whole genomes were found, and by comparison fastANI (v 1.33), snippy (v 4.6.0), the strain most closely resembling Badol-1 whole genome ANI was found to be GCA_003030905.1 (ANI=98.27%), so Badol-1 could be considered a new strain.
The result shows that Lplan-4 strain is safe on the gene level. By searching in Genbank, 854 of the public Lactiplantibacillus plantarum related whole genomes were found, and by comparison with fastANI (v 1.33), snippy (v 4.6.0), the strain most similar to Lplan-4 whole genome ANI was found to be GCA_002872355.1 (ANI=99.89%) so Lplan-4 could be considered to be a new strain.
Strains 1,2 and 3 have been deposited with China Center for Type Culture Collection (CCTCC) at the university of Wuhan, hubei province, post code 430072, telephone 027-68754052.
Strain 4 is commercially available.
The information for the preservation of strains 1,2 and 3 is given in the following table:
TABLE 1 microorganism preservation information
| Name of the Strain | Species information | Date of preservation | Deposit number | Check survival date |
| Bbifi-1 | Bifidobacterium bifidum (Bifidobacterium bifidum) | 2023, 3, 17 | CCTCC NO:M2023349 | 2023, 3, 24 |
| Badol-1 | Bifidobacterium adolescentis (Bifidobacterium adolescentis) | 2024 11, 18 | CCTCC NO:M 20242593 | 2024 11, 25 |
| Lplan-4 | Lactobacillus plantarum (Lactiplantibacillus plantarum) | 2024 11, 18 | CCTCC NO:M 20242594 | 2024 11, 25 |
Example 3 bacteriostatic Capacity against pathogenic bacteria
2 Common pathogenic bacteria causing acne are selected for bacteriostasis capacity detection, and the source information of pathogenic bacteria strains is as follows:
TABLE 2 pathogenic bead Source information
| Strain name | Strain deposit number | Strain preservation unit |
| Staphylococcus aureus | CMCC(B)26003 | Chinese food and drug testing institute |
| Propionibacterium acnes | BNCC330605 (ATCC 6919 second generation) | Henan province industrial microorganism strain engineering technology research center |
Preparation of Strain culture after 4 strains of single bacteria were activated, inoculated into 15mL of anaerobic MRS liquid medium at an inoculum size of 4% (v/v), and anaerobically cultured at 37℃for 24 hours. The culture was pasteurized at 65℃for 24h for 30min, then sonicated (working power 120W, ultrasound on for 3s, off for 4s, ultrasound under ice bath for 10 min) and filtered with a 0.22 μm filter to give a single fermentation product lysate stock solution.
And (3) assembling the stock solutions in an equal volume ratio to obtain a sample. The positive control was Saccharomyces cerevisiae (Shanghai Engineer Co., ltd.), the sample stock solution and the bifidus yeast are respectively diluted to 10% by using an anaerobic and resazurin-free three-mixed liquid culture medium to be used as a sample to be detected (hereinafter referred to as a probiotic composition).
The pathogenic bacteria are prepared by activating Staphylococcus aureus with TSB culture medium, diluting to OD 600 in TSB culture medium of 0.08-0.12, activating Propionibacterium acnes with anaerobic three-mixed liquid culture medium, and diluting to OD 600 of 0.16-0.24 in anaerobic three-mixed liquid culture medium without resazurin.
The experiment is divided into a probiotic composition to-be-detected sample group (probiotic composition), a positive control group of two-split yeast to-be-detected sample group (two-split yeast) and a blank control group, wherein the probiotic composition group and the two-split yeast group respectively take two pathogenic bacteria of 0.1mL and 0.1mL to-be-detected samples, the two pathogenic bacteria are placed in a 96-well plate, 3 groups of the two pathogenic bacteria are parallel, the blank control group respectively takes two pathogenic bacteria of 0.1mL and corresponding culture mediums of 0.1mL to be placed in the 96-well plate, and 3 groups of the two pathogenic bacteria are parallel. Aerobic culture of staphylococcus aureus for 24 hours, anaerobic culture of propionibacterium acnes for 24 hours, and detection of OD 600 of culture 0h and 24 h.
Calculating the bacteriostasis rate (R), wherein the formula is R= (1-DeltaOD 1/ΔOD0) multiplied by 100%, deltaOD 1 is the change of OD 600 before and after the culture of 24 h of a sample group to be tested, and DeltaOD 0 is the change of OD 600 before and after the culture of 24 h of a blank group of the corresponding same pathogenic bacteria.
Results are expressed as mean±sem of the bacteriostatic rate, and statistical analysis was performed using T-test. The bacteriostatic ability of the probiotic composition is compared with that of the saccharomyces cerevisiae, "" means that p <0.001, "" means that p <0.01, "" means that p <0.05 is compared with that of the saccharomyces cerevisiae.
TABLE 3 bacteriostasis test results of pathogenic bacteria
| Group of | Propionibacterium acnes antibacterial rate | Antibacterial rate of staphylococcus aureus |
| Saccharomyces bifidus | 31.21±1.78 | 27.14±1.67 |
| Probiotic compositions | 67.72±0.32 *** | 45.69±3.99 * |
As shown in Table 3, the probiotic composition has different degrees of inhibition on staphylococcus aureus and propionibacterium acnes, and the effect is similar to that of the derivatives of the single bacterium Badol-1, slightly better than that of other derivatives of the single bacterium (not shown in the table), and is obviously stronger than that of the saccharomyces cerevisiae.
Example 4 detection of Total antioxidant Capacity of probiotic compositions
Sample to be tested a stock solution of a probiotic composition was prepared according to the method of example 3. The positive control was Saccharomyces cerevisiae (Shanghai Engineer Co., ltd.).
And (3) adopting a BCA protein concentration determination kit (the kit is purchased from Beijing Soy Bao technology Co., ltd., PC 0020), drawing a standard curve according to the instruction operation of the kit, and detecting the protein concentration of the sample to be detected.
The total antioxidant capacity detection kit (the kit is purchased from Beijing Soy Bao technology Co., ltd., BC 1315) is adopted, and the antioxidant capacity of the sample to be detected is determined according to the specification operation of the kit and the standard curve. Total antioxidant capacity units μmol/mg prot.
Data processing 3 replicates per group were set, results were expressed as mean±sem, comparative analysis was performed using t-test, "x" indicated that p <0.05 compared to the saccharomyces cerevisiae group.
TABLE 4 Total antioxidant assay results
| Group of | Total antioxidant test results |
| Saccharomyces bifidus | 7.81±0.55 |
| Probiotic compositions | 10.20±0.23 * |
The experimental results show that the total antioxidant capacity of the probiotic composition reaches 10.20+/-0.23 mu mol/mg prot, which is obviously superior to that of the saccharomyces cerevisiae (p < 0.05), as shown in table 4, and the probiotic composition has stronger antioxidant capacity in vitro. The probiotic composition was similar to the derivatives of mono-fungus Badol-1, bbifi-1, slightly better than the other derivatives of mono-fungus (not shown in the table).
Example 5 probiotic compositions for DPPH radical scavenging Capacity detection
Sample to be tested a stock solution of a probiotic composition was prepared according to the method of example 3. The positive control was Saccharomyces cerevisiae (Shanghai Engineer Co., ltd.).
The DPPH radical scavenging ability of the sample to be tested was determined by using a DPPH radical scavenging ability detection kit (kit is available from Beijing Soy Bao technology Co., ltd., BC 4755) according to the instruction manual operation of the kit.
Data processing the experiment was performed in 3 replicates per group, the results are expressed as Mean ± SEM, and comparison analysis was performed using t-test, "×" indicates that p <0.001 compared to saccharomyces cerevisiae.
TABLE 5 DPPH free radical scavenging assay results
| Group of | DPPH free radical scavenging detection results |
| Saccharomyces bifidus | 1.60±0.73 |
| Probiotic compositions | 64.98±8.72 *** |
The experiment result shows that the DPPH free radical scavenging capacity of the probiotic composition reaches 64.98 +/-8.72 percent and is obviously superior to that of the saccharomyces cerevisiae (p < 0.0001), and the probiotic composition has stronger DPPH free radical scavenging capacity in vitro. The probiotic composition is slightly better than the single-bacterial derivative.
Example 6 probiotic compositions for detection of ABTS radical scavenging ability
Sample to be tested a stock solution of a probiotic composition was prepared according to the method of example 3. The positive control was Saccharomyces cerevisiae (Shanghai Engineer Co., ltd.).
The ABTS radical scavenging ability of the test samples was determined using ABTS radical scavenging ability assay kit (kit purchased from beijing solebao technologies, BC 4775) according to the instructions of the kit.
Data processing the experiment was performed in 3 replicates per group, the results are expressed as Mean ± SEM, and comparison analysis was performed using t-test, "×" indicates that p <0.001 compared to saccharomyces cerevisiae.
TABLE 6 ABTS free radical scavenging assay results
| Group of | ABTS radical scavenging assay |
| Saccharomyces bifidus | 19.71±0.47 |
| Probiotic compositions | 98.90±0.48 *** |
The experiment result shows that the ABTS free radical scavenging capacity of the probiotic composition reaches 98.90% +/-0.48%, which is obviously superior to that of the saccharomyces cerevisiae (p < 0.0001), and the probiotic composition has stronger ABTS free radical scavenging capacity in vitro. The probiotic composition effect is similar to that of each of the single bacterial derivatives.
Example 7 detection of lipid accumulation Capacity of probiotic compositions
Sample preparation sample treatment was performed as in example 3. Isotretinoin and Saccharomyces cerevisiae (Shanghai England) were used as positive controls.
The ability of the probiotic composition to clear lipid accumulation was evaluated by the amount of cellular lipid accumulation using linoleic acid (sigma aldrich (Shanghai) trade company, L1376) to induce lipid secretion by immortalized human sebaceous gland cell line sZ95 cells (Qingqi (Shanghai) Biotechnology development Co., ltd., BFN 60807569).
Linoleic acid was dissolved in a mixture of DMSO and ethanol (v/v=1/1) to prepare a 1M linoleic acid mother liquor, which was diluted 1000-fold to a linoleic acid working solution using serum-free DMEM medium (siemerfeier technology (china), C11065500 BT).
Isotretinoin was dissolved in DMSO to prepare 100 mM stock isotretinoin, which was diluted to 100 μm stock isotretinoin using linoleic acid.
The 5% (v/v) working solution of linoleic acid of the Saccharomyces cerevisiae is prepared by adding a stock solution of the Saccharomyces cerevisiae (Shanghai Bayan industries, ltd.) into the working solution of linoleic acid.
The 5% (v/v) probiotic composition linoleic acid working solution is prepared by adding the corresponding probiotic composition stock solution into the linoleic acid working solution.
SZ95 cells were inoculated in 96-well plates at 3X 10 4 cells/well, the supernatant was removed after incubation at 37℃and 5% CO 2 for 24 hours, and the cells were divided into 6 groups, 200. Mu.L of serum-free DMEM medium was added to the blank control group, 200. Mu.L of linoleic acid working solution was added to the model control group, 200. Mu.L of isotretinoin working solution was added to the isotretinoin group, and 200. Mu.L of the Saccharomyces linoleate working solution and the linoleic acid working solution were added to the Saccharomyces cerevisiae group and the probiotic composition, respectively.
After sample addition, the sample is cultured for 24 hours under the condition of 37 ℃ and 5% CO 2, 100 mu L of fluorescent dye is added for dyeing according to the requirements of a lipid drop Red fluorescent detection kit (Nile Red C2051M) of Biyun biotechnology Co., ltd, the sample is incubated for 10 minutes at 37 ℃ in a dark place for detection, excitation light is selected to be 485nm, and emission light is selected to be 535nm.
Data processing the experiment was performed in 6 replicates per group, results were expressed as Mean ± SEM, and multiple comparison analysis was performed using One-way ANOVA, "×" indicated that p <0.001 was compared to model control.
TABLE 7 detection of lipid accumulation removal Capacity
| Group of | Lipid accumulation clearance ability test results |
| Blank control | 100.00±6.07 *** |
| Model control | 176.53±3.72 |
| Isotretinoin A | 119.34±3.04 *** |
| Saccharomyces bifidus | 162.09±7.17 |
| Probiotic compositions | 141.26±6.37 *** |
The results of the test are shown in Table 7, the lipid secretion of cells after induction of linoleic acid is significantly increased, obvious lipid accumulation occurs, the lipid accumulation amount of the model control group is significantly higher than that of the blank control group (p < 0.001), the lipid accumulation amount after treatment of isotretinoin is significantly reduced compared with that of the model control group, no obvious effect is seen in the two-split yeast group, and the lipid accumulation amount is not significantly different from that of the model control group. The lipid accumulation of the probiotic composition group is obviously reduced compared with that of a model control group, and the probiotic composition effect is better than that of the saccharomyces cerevisiae.
Example 8 detection of the anti-IL-8 Effect of probiotic compositions
Sample preparation A sample stock solution was prepared as in example 3. Salicylic acid and Saccharomyces cerevisiae (Shanghai BayangLei Co., ltd.) were used as positive controls.
Human keratinocyte Hacat cells (cheng du gulon biotechnology limited, BNCC 339817) were stimulated by propionibacterium acnes (industrial microbial strain engineering center, BNCC330605 (ATCC 6919 second generation)) to produce cellular inflammatory factors, and the anti-inflammatory effects of the probiotic composition were evaluated by inflammatory factor levels.
The heat-inactivated Propionibacterium acnes working solution is prepared by selecting Propionibacterium acnes in logarithmic growth phase, heating at 80deg.C for 30min for inactivation, centrifuging at 12000rpm/min for 5min, removing supernatant, re-dissolving with serum-free DMEM medium (Cemer Feishl technologies (China) Co., C11065500 BT), and adjusting final concentration of Propionibacterium acnes to 5×10 8 CFU/ml.
Salicylic acid working solution preparation 200. Mu.g/ml salicylic acid working solution was prepared using heat inactivated Propionibacterium acnes working solution.
The 5% (v/v) working solution of Propionibacterium bifidum was prepared by adding a heat-inactivated Propionibacterium acnes working solution to a stock solution of Saccharomyces bifidus (Shanghai, england industries, ltd.).
The probiotic composition propionibacterium working solution with the concentration of 5% (v/v) is prepared by adding the heat-inactivated propionibacterium acnes working solution into the probiotic composition stock solution.
Hacat cells were inoculated in 96-well plates at 3×10 4 cells/well, incubated at 37 ℃ in 5% co 2 for 24 hours, the supernatant was aspirated, 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, and 200 μl of propionibacterium working solution of the diphyllotoxin and probiotic composition were added to the diphyllotoxin and probiotic composition respectively.
After sample addition, the cells were cultured at 37℃under 5% CO 2 for 24 hours, and the cell culture supernatants were collected, and inflammatory factor detection was performed according to the requirements of the human interleukin 8 (IL-8) ELISA kit (E-EL-H6008, inc., wohan Iretto Biotechnology Co., ltd.).
Data treatment the results are expressed in Mean ± SEM for multiple comparison analysis using One-way ANOVA, ", which means that p <0.001;" means that p <0.01, ", which means that p <0.05, which means that p <0.01, means that p <0.05, means that p <0.001, means that p <0.01, means that p <0.001, means that p is the same as the model.
TABLE 8 detection results of inflammatory factors IL-8
| Group of | Anti-inflammatory test results |
| Blank control | 11.13±0.12 *** |
| Model control | 36.69±3.45 |
| Salicylic acid | 15.72±0.80 *** |
| Saccharomyces bifidus | 28.05±2.02 * |
| Probiotic compositions | 21.85±2.36 ** |
The detection results are shown in Table 8, the heat-inactivated Propionibacterium acnes stimulates the secretion of inflammatory factors after induction, the IL-8 content of the model control group is obviously higher than that of the blank control group, the salicylic acid group has obvious anti-inflammatory effect, the IL-8 content after treatment is obviously reduced compared with the model control group, the two-split yeast group has no obvious effect, and the IL-8 content is not obviously different compared with the model control group. The probiotic composition has obvious anti-inflammatory effect, and the IL-8 content after treatment is obviously reduced compared with a model control group.
Example 9 detection of the anti-IL-6 Effect of probiotic compositions
Sample preparation stock sample treatment method was the same as in example 3, with dexamethasone (Shanghai Ala Biotechnology Co., ltd D754934) and Saccharomyces cerevisiae (Shanghai Qingdan England Co., ltd.) as positive controls.
Stimulation of human mononuclear leukocyte cell lines (THP-1 cells) derived from acute monocytic leukemia patients by lipopolysaccharide LPS induced production of cytokines by THP-1 cells (Bhanprinus life technologies Co., ltd., CL-0233) and evaluation of anti-inflammatory effects of probiotic compositions by inflammatory factor levels.
LPS (lipopolysaccharide, shanghai Ala Biotechnology Co., ltd L118716) working solution 1g/ml LPS and 20 ng/ml IFN-gamma (Interferon gamma receptor agonist, shanghai Ala Biotechnology Co., ltd rp 174266) molding working solution were prepared using serum-free 1640 medium (Simer Feishmanic technologies (China), C1187550 BT).
Dexamethasone working solution preparation 25. Mu.g/ml dexamethasone working solution was prepared using LPS working solution.
The 5% (v/v) working solution of the Saccharomyces cerevisiae LPS was prepared by adding an LPS working solution to a stock solution of Saccharomyces cerevisiae (Shanghai GmbH).
The 5% (v/v) probiotic composition LPS working solution is prepared by adding the probiotic composition stock solution into the LPS working solution.
THP-1 cells were inoculated in 96-well plates at 1X 10 5 cells/well, 100ng/ml PMA (Phorbol-myristate 13-acetate, medChemExpress (China) Limited) solution was added, the supernatant was aspirated after 24 hours of culture at 37℃under 5% CO 2 conditions, the experiment was divided into 5 groups, a blank control group was added with 200. Mu.l of serum-free 1640 medium, a model control group was added with 200. Mu.l of LPS working solution, a dexamethasone group was added with 200. Mu.l of dexamethasone working solution, and a two-split yeast group and a probiotic composition were added with 200. Mu.l of two-split yeast LPS working solution and probiotic composition LPS working solution, respectively.
After sample addition, the cells were cultured at 37℃under 5% CO 2 for 24 hours, and the cell culture supernatants were collected, and inflammatory factor detection was performed according to the requirements of the human interleukin 6 (IL-6) ELISA kit (E-EL-H6156, inc. of Wohan Iretto Biotechnology Co., ltd.).
Data treatment the results are expressed in Mean ± SEM for multiple comparison analysis using One-way ANOVA, ", which means that p <0.001;" means that p <0.01, ", which means that p <0.05, which means that p <0.01, means that p <0.05, means that p <0.001, means that p <0.01, means that p <0.001, means that p is the same as the model.
TABLE 9 detection results of inflammatory factors IL-6
| Group of | Anti-inflammatory test results |
| Blank control | 6.78±4.26 *** |
| Model control | 12611.65±983.76 |
| Dexamethasone | 128.62±15.24 *** |
| Saccharomyces bifidus | 3637.80±951.94 *** |
| Probiotic compositions | 7551.30±910.99 ** |
The test results are shown in Table 9, the IL-6 content of the model control group is obviously higher than that of the blank control group after LPS induction and the dexamethasone group and the schizosaccharomyces group have obvious anti-inflammatory effect, and the IL-6 content after treatment is obviously reduced compared with that of the model control group.
The probiotic composition has obvious anti-inflammatory effect, and the IL-6 content is obviously reduced compared with a model control group after use.
Example 10 probiotic compositions for improving acne in rabbit ears
Sample preparation the stock sample treatment method was the same as in example 3, with adapalene (Sichuan Ming's pharmaceutical Co., ltd.) and Saccharomyces cerevisiae (Repair Complex CLRTM PF, commercially available from CHEMISCHES LABORATORIUM Dr. Kurt Richter GmbH) as positive controls.
The preparation of the test sample comprises mixing the probiotic composition and 30ml of purchased positive control yeast stock solution respectively, adding 0.6g of hydroxyethyl cellulose (thickener, available from Shandong Heda Co., ltd.), mixing thoroughly, making into gel, packaging and preserving with 2ml sterile syringe at-20deg.C, and applying 1 piece per day.
The test group and administration of 25 clean grade male New Zealand white rabbits, 1.8-2.5 kg, were purchased from Pizhou Oriental culture Co. After 7 days of adaptive feeding, the animals were randomly divided into 5 groups of 5 probiotic compositions, each of which was a blank control group, a model control group, an adapalene group, a two-split yeast group, and a probiotic composition group. Except for the blank control group, the other groups are subjected to intradermal injection molding of propionibacterium acnes in the range of 3cm multiplied by 3cm at the inner side of the right ear of a rabbit at the injection position of 6 points, 25 mu l of each point is injected, 1 multiplied by 10 8 CFU is injected at each point, 0.2g of artificial sebum (Beijing Soulibao science and technology Co., ltd.) is uniformly smeared in the injection range immediately after the injection of the 1 st day, the continuous 14 days are carried out, the sebum smearing is carried out 1 time a day at the morning, and the blank control group is injected with the same amount of normal saline without smeared artificial sebum. The positive drug adapalene group is coated with adapalene according to 0.1 g/single, the positive drug mitomyces group and the probiotic composition group are both coated with the test product prepared above according to 0.4 ml/single, and the administration is carried out 1 time a day for 14 consecutive days in afternoon. The test cycle was 14 days, and on day 15, an appropriate amount of blood was drawn along the middle artery of the rabbit ear and serum was isolated for inflammatory factor detection.
Ear swelling ratio detection before the start of the test, the thickness of the right ear of each rabbit was measured by using a vernier caliper, three positions were measured, the skin thicknesses of the three positions which are approximately the same were measured again by using the vernier caliper after the end of the test, and the results were averaged to calculate a swelling ratio = (ear thickness at the end-ear thickness at the start)/ear thickness at the start × 100%. The test results are all expressed in mean+ -SEM and were analyzed by multiple comparisons using One-way ANOVA. P <0.05 and p <0.001 compared to model group.
And scoring the skin damage state of the rabbit ears according to the scoring rules of the following table, wherein the total skin damage score is obtained by adding the scores of all indexes of the following table. Test results are expressed as mean±sem, with non-parametric tests, kruskal-Wallis test for differences between groups, p <0.05 compared to model group, p <0.0001.
Table 10 skin loss evaluation chart
Counting the total focus number, namely recording the focus number of each rabbit ear modeling position, including papules, pustules, nodules, crumbs and crusts, and not counting if new skin tissues are obviously grown at skin lesions. Test results are expressed as mean±sem, with non-parametric tests, kruskal-Wallis test for differences between groups, p <0.01 and p <0.0001 compared to the model group.
Detection of inflammatory factor TNF-alpha according to the specification, the content of TNF-alpha in rabbit serum is detected by using a rabbit ELISA detection kit. The test results are all expressed in mean+ -SEM and were analyzed by multiple comparisons using One-way ANOVA. P <0.01 and p <0.001 compared to model group.
Table 11 detection results of rabbit acne model
| Group of | Ear swelling Rate (%) | Total score of skin lesions | Total focus number | TNF-alpha 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 ** |
| Two split yeast 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 *** |
Fig. 1 shows the results of detection of the rabbit acne model ear swelling rate, and compared with a blank control group, the model control group ear swelling rate is remarkably improved, and both the saccharomyces cerevisiae and the probiotic composition can remarkably reduce the rabbit ear swelling rate, and the probiotic composition has better improvement effect than adapalene and saccharomyces cerevisiae.
Fig. 2 shows the results of total skin loss score detection, wherein the total skin loss score of the model control group is significantly increased compared with the blank control group, and the total skin loss score of the probiotic composition is significantly reduced compared with the model control group.
Figure 3 is a statistical result of total lesion number, no lesions appear in the placebo group, and the probiotic composition significantly reduced the lesion number compared to the model control group. The improvement effect of the probiotic composition is better than that of adapalene and Saccharomyces cerevisiae.
FIG. 4 shows the results of serum inflammatory factor detection, wherein the serum TNF-alpha content of the model control group is significantly increased compared with the blank control group, and the serum TNF-alpha content of the adapalene Lin Heyi bacteria-producing composition is significantly reduced compared with the model control group. The improvement effect of the probiotic composition is better than that of the saccharomyces cerevisiae. The improvement of serum inflammatory factors by the probiotic composition is significantly better than that of the individual single bacterial derivatives (not shown).
Fig. 5 is a representative image of rabbit ear skin at the end of the test, with the model group forming a distinct acne lesion (primarily red papules at the beginning, then progressing to pustules, and finally forming cysts/nodules), with various degrees of improvement in each of the dosing groups, with a more pronounced improvement effect of the probiotic composition (significantly reduced nodule numbers, new skin tissue formation at pustule sites). The improvement effect of the probiotic composition is better than that of adapalene and Saccharomyces cerevisiae. And the probiotic composition is significantly better than the individual single bacterial derivatives (not shown) in terms of cysts/nodules.
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