CN114081901B - Probiotic composition, preparation method and application thereof - Google Patents
Probiotic composition, preparation method and application thereof Download PDFInfo
- Publication number
- CN114081901B CN114081901B CN202111585424.5A CN202111585424A CN114081901B CN 114081901 B CN114081901 B CN 114081901B CN 202111585424 A CN202111585424 A CN 202111585424A CN 114081901 B CN114081901 B CN 114081901B
- Authority
- CN
- China
- Prior art keywords
- seuneu
- lactobacillus
- lactobacillus plantarum
- probiotic composition
- fermentum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
- A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Birds (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The application provides a probiotic composition, a preparation method and application thereof, and belongs to the technical field of probiotic microorganisms. A probiotic composition comprising: lactobacillus plantarum (L.) KummerLactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, said Lactobacillus plantarumLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) SEUNEU-102 is preserved in China center for type culture Collection (SIG), wherein Lactobacillus plantarum is @)Lactobacillu plantarum) SEUNEU-101, accession number: cctccc M20211279; lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, accession number: cctccc M20211280. The composition has effects of regulating human body flora balance, improving skin state, promoting skin repair, and relieving skin inflammation, especially autoinflammatory disease in dermatology, and can be used in pharmaceuticals, foods, maintenance products or cosmetics.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a probiotic composition, a preparation method and application thereof.
Background
Skin, which is the largest organ of the human body, lives with a wide range of microorganisms, constitutes a habitat of various forms such as a trap, a specialized gap, etc., helping a wide range of microorganisms to survive. Microorganisms and persons as hosts form a symbiotic relationship, and the skin microbiota plays an important role in the host. This is not only because the skin microbiota have the ability to resist adhesion and development of skin pathogens, but also because they have the ability to interact with the immune system and to interact. Moreover, probiotics can also act as modulators to restore the balance of the skin microbiota when dysbiosis occurs at the skin level.
The use of new technologies has prompted a taxonomic analysis of skin microbiota over the last decade, from which it has been shown that many skin diseases are associated with alterations in the skin microbiome. For example, acne, propionibacterium acnes is considered to be a bacterium primarily associated therewith, and the number of propionibacterium acnes on the skin surface is markedly increased when acne occurs; whereas in the opposite patients with atopic dermatitis, the flora on the skin surface has a reduced abundance of staphylococcus epidermidis and a significantly increased abundance of staphylococcus aureus.
The conventional approach to solve the above problems is to use antibacterial agents, i.e., to inhibit the growth of pathogenic bacteria using topical disinfectants and antibiotics.
The use of antibiotics in the above methods is effective, but in addition to eliminating beneficial bacteria, they have the risk of eliminating beneficial bacteria, sensitization, and potential adverse events that can lead to an imbalance in the skin flora and difficult reconstitution if broad spectrum antibiotics are used for a long period of time.
In addition, there has been increasing attention in modern society to the damage of skin barrier, and the skin is directly contacted with the external environment and is easily damaged by various pressure conditions. Environmental factors (uv irradiation, environmental pollution, computer radiation, etc.), intrinsic regulatory factors (in vivo hormone content, in vivo enzyme release), physicochemical factors (powerful surfactants to clean the skin), etc. all lead to damage of the skin barrier. The damage of the skin barrier not only can lead to the lack of water, dryness, itching and sensitivity of the skin, but also can lead to the generation of skin diseases such as eczema, psoriasis, acne and the like. In the contemporary large environment, the factors of skin barrier damage are more and more increased, but people living in the environment are difficult to avoid, so that the development of an effective and safe method for repairing the skin barrier has very important significance.
Disclosure of Invention
In order to solve the problems, the application provides a probiotic composition which can regulate the microbial flora of human skin, promote the proliferation and repair of epidermal cells, and has anti-inflammatory effect, and the application of the probiotic composition and/or fermentation liquor thereof in medicines, foods, maintenance products or cosmetics.
The technical scheme of the application is as follows:
the probiotic composition according to the embodiment of the application comprises lactobacillus plantarumLactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, and/or said Lactobacillus plantarumLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) The evolution substance of SEUNEU-102 is obtained by separating natural pulp water of two strains selected from sauerkraut. The lactobacillus plantarum is [ ]Lactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) SEUNEU-102 was deposited at China center for type culture Collection, address: wu Changou university of Lojia mountain Wuhan in Wuhan, wherein Lactobacillus plantarum is @Lactobacillus plantarum) SEUNEU-101, accession number: cctccc M20211279, a preservation date of 2021, 10 months and 15 days; lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, accession number: CCTCC M20211280, the preservation date is 2021, 10, 15.
According to one embodiment of the application, the lactobacillus plantarum isLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) The SEUNEU-102 strain is live or sterilized by batch sterilization.
According to one embodiment of the application, the lactobacillus plantarum isLactobacillus plantarum) The SEUNEU-101 evolving substance includes: any one of lysate, extract, product, supernatant and derivative; the lactobacillus fermentum is%Lactobacillus fermentum) The evolving substance of SEUNEU-102 includes any of lysate, extract, product, supernatant and derivative.
According to one embodiment of the application, the derivative is selected from one or more of metabolites, metabolic biological products, probiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components.
According to one embodiment of the application, the lactobacillus plantarum isLactobacillus plantarum) SEUNEU-101 or its evolution substance and lactobacillus fermentumLactobacillus fermentum) The mass ratio of SEUNEU-102 or its evolution substance is 1:1.
The application also provides a preparation method of the probiotic composition, which comprises the following steps:
providing a strain containing live Lactobacillus plantarumLactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentumLactobacillus fermentum) Bacterial liquid of SEUNEU-102, the lactobacillus plantarum is [ ]Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentumLactobacillus fermentum) SEUNEU-102 is preserved in China center for type culture Collection (SIG), wherein Lactobacillus plantarum is @)Lactobacillus plantarum) SEUNEU-101, accession number: cctccc M20211279; lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, accession number: cctccc M20211280;
the concentration of the bacterial liquid is regulated to be OD600 of 0.1, and the lactobacillus plantarum is preparedLactobacillus plantarum) SEUNEU-101 and hairLactobacillus fermentumLactobacillus fermentum) SEUNEU-102 is inoculated in MRS liquid culture medium, cultured at 37 ℃ and fermented to obtain mixed fermentation liquor;
and extracting the supernatant in the mixed fermentation broth to obtain the probiotic composition.
According to the preparation method of the embodiment of the application, in vitro culture experiments show that the lactobacillus plantarum is @ preparedLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) SEUNEU-102 co-culture can reach preset concentration faster than single strain culture of any strain, and preparation efficiency of bacterial liquid is improved.
According to one embodiment of the application, the lactobacillus plantarum is obtainedLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) The mass ratio of SEUNEU-102 is 1:1.
According to one embodiment of the present application, the method for preparing a probiotic composition further comprises autoclaving the extracted supernatant at 121 ℃ for 30min to obtain the probiotic composition.
The application also provides application of the probiotic composition in preparing medicines for inhibiting staphylococcus aureus growth, promoting staphylococcus epidermidis proliferation, inhibiting propionibacterium acnes, resisting inflammation, promoting epidermal cell repair, repairing skin barrier, up-regulating moisture-preserving related gene expression, and scavenging free radicals and resisting oxidation.
The application has at least the following beneficial effects:
in vitro culture experiments show that SEUNEU-101 and SEUNEU-102 can reach preset concentration faster than single strain culture of any strain.
The in vitro bacteriostasis test shows that the probiotic composition has the effect of inhibiting staphylococcus aureus, the bacteriostasis rate can reach 73.9 percent, and the bacteriostasis effect is higher than that of a single strain in the composition;
the probiotic composition has the effect of inhibiting Propionibacterium acnes, and after co-culture for 24 hours, the antibacterial rate of the composition for inhibiting Propionibacterium acnes can reach 39.4%;
the probiotic composition has the effect of promoting the proliferation of staphylococcus epidermidis, and after co-culture for 24 hours, the promoting rate of the composition for promoting staphylococcus epidermidis can reach 19.8%;
the probiotic composition has the effect of scavenging superoxide radicals, and the scavenging rate is 59.39%;
in vitro cell experiments show that the probiotic composition has anti-inflammatory effect, can reduce the NO release amount of Raw264.7 cells induced by LPS, and can be reduced by 24.14% compared with a control group;
in vitro cell experiments show that the probiotic composition has anti-inflammatory effect, and can reduce the expression level of the HaCaT cell inflammatory factor IL-1 alpha induced by staphylococcus aureus by 55%, reduce the expression level of IL-8 by 32.99% and reduce the expression level of COX-2 by 32.2%.
In vitro cell experiments show that the probiotic composition has the effect of promoting the repair of epidermal cells, the scratch healing rate is increased by 22% compared with a control group, the repair rate can reach 21% compared with the control group due to HaCaT cell damage caused by SDS.
In vitro cell experiments show that the probiotic composition has the effects of up-regulating the silk-polymer protein (filaggrin) FLG, the Involucrin (Involurin) IVL, the Ovo-like transcription factor 1 (Ovo Like Transcriptional Repressor 1) OVOL1 and the papilin (loricrin) LOR genes, and up-regulating by 1.3-7.8 times, so that the probiotic composition has the effect of promoting skin barrier repair.
In vitro cell experiments show that the probiotic composition has the effect of up-regulating the expression of Aquaporin 3 (Aquaporin 3) AQP3 and beta-glucocerebrosidase (beta-glucoerbrosidase) GBA, and the expression level can be up-regulated by 1.15-3.8 times.
Drawings
FIG. 1, a graph of the concentration change of a bacterial fluid obtained by culturing a probiotic composition and a single strain for 16 hours;
FIG. 2 is a graph of the rate of inhibition of Staphylococcus aureus by a probiotic composition;
FIG. 3 is a graph of the rate of inhibition of Propionibacterium acnes by probiotic compositions;
FIG. 4, graph of probiotic composition decreasing Raw264.7 cell NO release;
FIG. 5, a graph of probiotic composition down-regulating inflammatory-related factor expression;
FIG. 6 is a graph of a probiotic composition promoting HaCaT cell injury repair;
FIG. 7, a graph of a probiotic composition promoting HaCaT scratch repair;
FIG. 8, a graph of gene expression associated with upregulation of barrier repair by supernatant of probiotic compositions;
FIG. 9, a graph of gene expression associated with up-regulation of barrier repair by probiotic composition inactivated cells;
FIG. 10, graph of the expression of the moisturizing related factor gene on the supernatant of the probiotic composition;
FIG. 11 shows the expression pattern of the moisturizing-related factor gene on the inactivated cells of the probiotic composition.
Description of the embodiments
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. It should be noted that these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art, and the disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein.
Lactic acid bacteria have important roles in maintaining host organism steady state, activating immune system, maintaining organism immune balance and the like, and researches find that the lactic acid bacteria can prevent and treat various inflammatory diseases by regulating the immune function and phagocytic capacity of macrophages, can reduce skin inflammation and allergy by controlling the whole immune state including release of regulatory cytokines, and can regulate intestinal flora balance by eating lactic acid bacteria, and can regulate immune response. For the above reasons, the applicant has identified the use of probiotic compositions as a new solution for restoring the balance of the skin, intestinal tract and even the microbiota of the human body. The probiotic composition is separated and identified from natural syrup of pickled Chinese cabbage, comprises lactobacillus plantarum SEUNEU-101 and lactobacillus fermentum SEUNEU-102, has a synergistic effect between lactobacillus plantarum SEUNEU-101 and lactobacillus fermentum SEUNEU-102, can exert good antibacterial, anti-inflammatory and repairing effects after being compounded, and has great market potential in foods, medicines and cosmetics.
The probiotic compositions of the application and their use are described below in connection with specific examples.
It should be noted that, in the method mentioned in the present application, a conventional method or steps may be adopted without mention of specific steps.
Embodiment one: isolation and identification of strains
Preparing MC agar culture medium: 5g of soybean peptone, 5g of beef extract, 5g of yeast extract, 20g of glucose, 20g of lactose, 10g of calcium carbonate, 15g of agar, 5ml of 1% neutral red solution, 1000ml of distilled water, and sterilizing at 121 ℃ for 15min.
Preparing MRS liquid culture medium: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 80 lml of tween-80, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1000ml of distilled water, pH value of 6.4 and sterilizing at 121 ℃ for 15min.
The sample of this example was derived from natural syrup of sauerkraut, aseptically sampled in a sterilized coupon containing 5ml of glycerol, and frozen at-20 ℃ after retrieval. When separating, the sample is dissolved back, 1g is taken and mixed evenly by shaking in 9ml physiological saline, the supernatant is streaked on an MC flat plate, after the constant temperature culture at 37 ℃ for 48 hours, the red colony with a calcium dissolving ring is selected, and the inoculation and screening are repeated until a uniform single colony is obtained.
1. Primer:
according to the gene sequence of the lactobacillus 16S rRNA registered in GenBank, referring to 368-1049 locus gene fragments thereof, designing a primer:
F 5’-TCGGCTCGTAAAACTCTG-3’；
R 5’-GGACTTAACCCAACATCTCA-3’。
the primer is synthesized by Shanghai bioengineering Co-Ltd, the amplified fragment is V3-V7, 682bp long.
2. 16s rRNA gene sequence analysis:
single colony is selected and placed in an MRS centrifuge tube, after being cultured overnight at 37 ℃, thalli are collected by centrifugation, and the operation is carried out according to the step of DNA extraction kit. The PCR amplification system is a 50 mu L system, and the PCR amplification system is pre-denatured for 10min at 95 ℃;93℃1min,55℃1min,72℃1min,35 cycles; extending at 72℃for 7min.
3. Results:
the PCR product sequencing result is compared with the published standard sequence in GenBank to obtain SEUNEU-101 strain as lactobacillus plantarumLactobacillus plantarum) The method comprises the steps of carrying out a first treatment on the surface of the The SEUNEU-102 is Lactobacillus fermentumLactobacillus fermentum）。
Embodiment two: probiotics composition and single strain culture 16h bacterial liquid concentration change research
The activated probiotic composition, SEUNEU-101 and SEUNEU-102 bacterial solutions of the present application were adjusted to have a bacterial solution concentration of 0.1, 100. Mu.l were inoculated into 1ml MRS, cultured in an incubator at 37℃for 16 hours, and the bacterial count was measured by using a spectroluminometer OD600 nm.
Results:
after 16h of culture, as shown in FIG. 1, the concentration change results of the probiotic composition and the bacterial liquid of a single strain are shown as SEUNEU-101, SEUNEU-102 and OD600 detection results of the probiotic composition are respectively 3.0, 3.2 and 5.2, which indicate that the co-culture growth speed of the two strains is faster than that of the culture growth speed of the single strain, and the two strains have the effect of promoting proliferation mutually.
Embodiment III: probiotic composition staphylococcus aureus bacteriostasis research
1. Preparing a probiotic composition bacterial liquid and a single bacterial liquid:
the 1:1 probiotic composition of activated SEUNEU-101 and SEUNEU-102, SEUNEU-101 and SEUNEU-102 bacterial solutions were cultured in MRS in a 37℃incubator for 18 hours, and the bacterial count was measured by using a spectroluminometer OD600nm to adjust the concentration of the probiotic composition, SEUNEU-101 and SEUNEU-102 bacterial solutions to 2X 10 9 centrifuging cells/mL to obtain supernatant; sterilizing at 121deg.C for 30min to obtain inactivated supernatant.
2. Preparation of staphylococcus aureus bacterial liquid:
culturing in broth culture medium at 140r/min and 37deg.C overnight, measuring bacterial count with spectroluminometer OD600nm, and regulating golden yellow grape ballThe concentration of the bacterial liquid is 1 multiplied by 10 8 cells/mL。
3. Experiment for inhibiting staphylococcus aureus
The probiotic composition, SEUNEU-101, SEUNEU-102 supernatant and inactivated supernatant are added into staphylococcus aureus bacterial liquid at 5%, and the mixture is kept stand for 4 hours at 37 ℃, and the influence of the target strain on the staphylococcus aureus growth is evaluated according to the reduction percentage of the bacterial liquid concentration (OD 600).
4. Results
According to the result, the antibacterial rate of the probiotic composition is improved by 19.3-34.5% compared with that of a single strain in the composition, so that the probiotic composition has better antibacterial effect than that of the single strain in the composition. The results of the probiotic composition in inhibiting staphylococcus aureus bacteria inhibition rate are shown in figure 2.
Embodiment four: propionibacterium acnes assay-bacterial fluid concentration variation inhibition by probiotic compositions
1. Preparing a probiotic composition bacterial liquid:
culturing the activated probiotic composition bacteria liquid in a 37 deg.C incubator for 18 hours, measuring the bacteria number by using a spectro-luminance meter OD600nm, and adjusting the bacteria liquid concentration to 5×10 9 centrifuging at 4000rpm for 15min, and filtering with 0.22 μm filter membrane to obtain supernatant; and (3) inactivating the supernatant at 121 ℃ for 30min under high pressure to obtain an inactivated supernatant.
2. Preparing propionibacterium acnes bacterial liquid:
anaerobic culturing Propionibacterium acnes in BHI culture medium at 37deg.C for 48 hr, measuring bacterial count with spectroluminometer OD600nm, centrifuging at 4000rpm for 15min, discarding supernatant, sterilizing with 1xPBS buffer solution for two times, re-dissolving with 1xPBS phosphate buffer solution, and adjusting bacterial concentration of Propionibacterium acnes to 2.5X10 9 cells/mL。
3. Bacteriostasis experiment
Respectively adding the supernatant of the two probiotic compositions into the pathogenic bacteria bacterial liquid at 5%, standing at 37 ℃ for 24 hours, and evaluating the influence of the probiotic compositions on the growth of pathogenic bacteria according to the reduction percentage of the bacterial liquid concentration (OD 600).
4. Results
The probiotic composition has the effect of inhibiting Propionibacterium acnes, and after 24 hours of co-culture, the antibacterial rate of the supernatant of the probiotic composition for inhibiting Propionibacterium acnes is 39.4%, and the antibacterial rate of the supernatant of the probiotic composition for inactivating Propionibacterium acnes is 26.3%. The rate of inhibition of propionibacterium acnes by the probiotic composition is shown in figure 3.
Fifth embodiment: experiment of Probiotics composition to promote growth of Staphylococcus epidermidis
The probiotic composition was cultured overnight, OD600 = 1 was adjusted, the supernatant was centrifuged to 5% and added to the initial OD600 = 0.1 staphylococcus epidermidis broth, and the broth was allowed to stand at 37 ℃ for 4 hours, and the effect of the probiotic composition on staphylococcus epidermidis growth was evaluated as relative broth concentration (sample OD600 to control ratio).
Results: the relative bacterial liquid concentration of the supernatant added with the probiotic composition is 119.8%, and the promotion rate of the ratio of the supernatant to the control is 19.8%.
Example six: super oxygen radical scavenging capability of probiotic compositions
And (3) respectively picking up the monoclonal antibodies of SEUNEU-101 and SEUNEU-102, culturing in MRS liquid mixture for 24 hours, centrifuging to obtain supernatant, and measuring the superoxide radical scavenging capacity of the supernatant. The sample addition method is as follows:
each tube was allowed to stand for 5 minutes, the absorbance at 560nm was measured, and the clearance was calculated.
The clearance rate calculation formula: clearance% = [1- (sample 560-blank 560)/control 560 ]. Times.100.
Results: the supernatant clearance rate of the probiotic composition is 59.4 percent through detection and calculation.
Embodiment seven: probiotic composition for reducing LPS-induced Raw264.7 cell NO release
1. Preparation of probiotic composition samples
Use of probiotic compositions with MRSCulturing in culture medium overnight, detecting OD600, and adjusting bacterial liquid concentration to 1×10 8 After centrifugation, cells were autoclaved at 121℃for 30min to obtain cells, and the supernatant was filtered through a 0.22 μm filter membrane.
2. Cell preparation
Raw264.7 cells were digested and then treated with 2X 10 5 The wells were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator.
3. Probiotic composition addition and LPS stimulation
Different groups of 5% of supernatant and 10% of inactivated bacteria are added into Raw264.7 cells cultured overnight, after 2 hours, the Raw264.7 cells are induced to be inflamed by LPS with the concentration of 200ng, after 20 hours, the cell culture supernatant is taken, and the NO content detection kit is used for detecting the NO content, and three experiments are carried out every 3 compound holes.
Results:
in vitro cell experiments show that the probiotic composition has anti-inflammatory effect, can reduce the NO release amount of Raw264.7 cells induced by LPS, and compared with a LPS control group, the supernatant is reduced by 41.61%, and the inactivated thalli is reduced by 27.51%. The results of the probiotic composition reducing the NO release from raw264.7 cells are shown in figure 4.
Example eight: probiotic compositions for reducing staphylococcus aureus induced HaCaT inflammatory factorsIL-1α、IL-8、 COX-2Expression level
1. Preparation of probiotic composition samples
Culturing probiotic composition with MRS overnight, detecting OD600, and adjusting bacterial liquid concentration to 1×10 8 After centrifugation, cells were autoclaved at 121℃for 30min to obtain cells, and the supernatant was filtered through a 0.22 μm filter membrane.
2. HaCaT cell preparation
HaCaT cells were digested and then treated to 2X 10 5 The wells were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator.
3. Preparation and addition of staphylococcus aureus
Staphylococcus aureus is inoculated into broth culture medium, shake cultured overnight at 37deg.C, and the concentration of bacterial solution is adjusted to 3×10 with MEM serum-free culture medium 9 cells/mL, 100 μl per well was added to the cultured overnight HaCaT cells, the cells were stimulated to produce inflammatory factors, the cell culture medium was discarded after 3 hours, PBS was washed 5 times, and 1mL of MEM serum-free medium was added again per well.
4. Probiotic composition sample addition
The supernatant was 5% added to staphylococcus aureus stimulated HaCaT cells, 3 duplicate wells per group, and incubated overnight.
5. Detection of inflammatory factorsIL-1α、IL-8、COX-2Expression level
After discarding the medium from the above cells, RNA was extracted with an RNA extraction kit, the concentration and purity of RNA were measured, all samples were adjusted to 1000ng, and reverse transcription and qPCR were performed with a reverse transcription kit, SYBRGreen qPCR kit.
Results:
probiotic compositions are capable of down-regulating staphylococcus aureus induced HaCaT inflammatory factorsIL-1α、IL-8、COX-2Expression, inflammatory factorsIL-1αThe expression level is reduced by 55.01 percent,IL-8the expression quantity is reduced by 32.98 percent,COX-2the expression level is reduced by 32.2%, so that the probiotic composition has anti-inflammatory effect. The probiotic composition down-regulates the expression of inflammation-related factors as shown in figure 5.
Example nine: probiotics composition for promoting scratch repair in HaCaT cell damage repair experiment
Human immortalized keratinocyte HaCaT cells (5×10) 5 ) To 6-well plate, overnight culture was performed until cells adhered, streaking was performed perpendicular to the bottom of 6-well plate using a 1ml gun head, 5% supernatant was added, photographing was performed, and recorded as D1, and culture was performed in a carbon dioxide incubator at 5% CO2 at 37 ℃. After 24h, a photograph is taken, designated D2. Data processing was performed using image J according to the formula: healing rate= (D1-D2)/D1. Data were plotted using GraphPad.
Results:
in vitro cell experiments show that the probiotic composition has the effect of promoting the repair of epidermal cells, and the scratch healing rate of the supernatant is increased by 22 percent compared with that of a control. See in particular fig. 6, 7.
Example ten: probiotics composition for promoting SDS damage repair of HaCaT cell damage repair experiment
Human immortalized keratinocyte HaCaT cells (5×10) 5 ) To 96-well plates, cells were cultured overnight to cell attachment. 50ug/ml SDS was prepared, 100ul was added per well, incubated for 8h, 5% supernatant of the probiotic composition was added, and incubated for 24h. 10ul of CCK-8 solution was added, incubated for 4h, and absorbance detected at 450 nm.
Cell viability= (a experimental group-a blank)/(a negative control group-a blank).
Results:
after detecting that HaCaT cells are damaged by SDS, the survival rate of the HaCaT cells is 121 percent and is increased by 21 percent compared with a control group, so that the probiotic composition has the effect of promoting the repair of the epidermal cells HaCaT cells. See in particular fig. 6.
Example eleven: experiment for up-regulating HaCaT barrier repair related gene expression by probiotics composition
Human immortalized keratinocyte HaCaT cells (5×10) 5 ) To 6-well plates, cells were cultured overnight to cell attachment. Adding 5% of supernatant of probiotic composition and 10% of inactivated thallus, respectively culturing for 24 hr, discarding culture medium supernatant, cleaning with PBS, adding RNA extraction lysate, extracting total RNA of cells, reverse transcribing into cDNA, and performing reverse transcription onFLG、IVL、OVOL1、LORThe expression level of the gene was qPCR-detected. And calculating the relative expression times according to a formula.
The formula: f=2 -ΔΔCT
Results:
the results show that barrier repair is relevant after HaCaT cells are stimulated with the probiotic compositionFLG、IVL、OVOL1、LORGene expression is up-regulated to different extents, so that the probiotics are combinedThe product has skin barrier repairing effect. The results of up-regulating the relative expression of repair related genes by the probiotic composition are shown in fig. 8 and 9.
Embodiment twelve: experiment for up-regulating HaCaT moisturizing related gene expression of probiotics composition
Human immortalized keratinocyte HaCaT cells (5×10) 5 ) To 6-well plates, cells were cultured overnight to cell attachment. Adding 5% of supernatant of probiotic composition and 10% of inactivated thallus, respectively culturing for 24 hr, discarding culture medium supernatant, cleaning with PBS, adding RNA extraction lysate, extracting total RNA of cells, reverse transcribing into cDNA, and qPCR detecting aquaporinAQP3、GBAIs expressed by (a). And calculating expression change times according to a formula.
The formula: f=2 -ΔΔCT
Results:
the results show that after HaCaT cells are stimulated by adding the probiotic composition, the moisturizing related genesAQP3、GBAGene expression is up-regulated, so that the probiotic composition has the effect of up-regulating the expression of the moisturizing factors. The relative expression results of the genes related to the up-regulation and the moisture preservation of the probiotic composition are shown in fig. 10 and 11. While the foregoing is directed to the preferred embodiments of the present application, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present application, and such modifications and adaptations are intended to be comprehended within the scope of the present application.
Claims (7)
1. A probiotic composition is characterized by comprising lactobacillus plantarumLactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, said Lactobacillus plantarumLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) SEUNEU-102 is preserved in China center for type culture Collection (SIG), wherein Lactobacillus plantarum is @)Lactobacillus plantarum) SEUNEU-101, accession number: cctccc M20211279; lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, accession number: cctccc M20211280.
2. The probiotic composition of claim 1, wherein said lactobacillus plantarum isLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) The SEUNEU-102 strain is live or sterilized by batch sterilization.
3. A probiotic composition according to any one of claims 1-2, characterized in that said lactobacillus plantarum is @ o @ mLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) The mass ratio of SEUNEU-102 is 1:1.
4. A method of preparing a probiotic composition comprising:
providing a strain containing live Lactobacillus plantarumLactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentumLactobacillus fermentum) Bacterial liquid of SEUNEU-102, the lactobacillus plantarum is [ ]Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentumLactobacillus fermentum) SEUNEU-102 is preserved in China center for type culture Collection (SIG), wherein Lactobacillus plantarum is @)Lactobacillus plantarum) SEUNEU-101, accession number: cctccc M20211279; lactobacillus fermentumLactobacillus fermentum) SEUNEU-102, accession number: cctccc M20211280;
the concentration of the bacterial liquid is regulated to be OD600 of 0.1, and the lactobacillus plantarum is preparedLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) SEUNEU-102 is inoculated in MRS liquid culture medium, cultured at 37 ℃ and fermented to obtain mixed fermentation liquor;
and extracting the supernatant in the mixed fermentation broth to obtain the probiotic composition.
5. The method for preparing a probiotic composition according to claim 4, wherein said lactobacillus plantarum is obtainedLactobacillus plantarum) SEUNEU-101 and lactobacillus fermentumLactobacillus fermentum) The mass ratio of SEUNEU-102 is 1:1.
6. The method of preparing a probiotic composition according to claim 4 or 5, further comprising autoclaving said extracted supernatant at 121 ℃ for 30min to obtain said probiotic composition.
7. Use of a probiotic composition according to any one of claims 1-2 for the preparation of a medicament for inhibiting staphylococcus aureus growth, promoting staphylococcus epidermidis proliferation, inhibiting propionibacterium acnes, anti-inflammatory, promoting epidermal cell repair, repairing skin barrier, up-regulating moisture-retention related gene expression, scavenging free radicals and anti-oxidation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111585424.5A CN114081901B (en) | 2021-12-23 | 2021-12-23 | Probiotic composition, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111585424.5A CN114081901B (en) | 2021-12-23 | 2021-12-23 | Probiotic composition, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114081901A CN114081901A (en) | 2022-02-25 |
| CN114081901B true CN114081901B (en) | 2023-08-29 |
Family
ID=80307631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111585424.5A Active CN114081901B (en) | 2021-12-23 | 2021-12-23 | Probiotic composition, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114081901B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115109714B (en) * | 2022-03-14 | 2023-10-13 | 山东锦鲤生物工程有限公司 | Pediococcus acidilactici SEUNEU-106 and application thereof in skin |
| CN114574408B (en) * | 2022-05-06 | 2022-07-22 | 山东锦鲤生物工程有限公司 | Probiotic SEUNEU-107 and application |
| CN114736831A (en) * | 2022-05-17 | 2022-07-12 | 廖梅香 | Lactobacillus fermentum and its application |
| CN114717162A (en) * | 2022-05-17 | 2022-07-08 | 廖梅香 | Lactobacillus fermentum and application thereof |
| CN114634899B (en) * | 2022-05-17 | 2022-08-16 | 山东锦鲤生物工程有限公司 | Lactobacillus fermentum and application thereof |
| CN115181698B (en) * | 2022-06-28 | 2023-09-08 | 广东粤港澳大湾区国家纳米科技创新研究院 | Probiotic composition and application thereof |
| CN117736942B (en) * | 2024-02-20 | 2024-05-31 | 山东中科嘉亿生物工程有限公司 | Fermented Lactobacillus mucilaginosus JYLF-315 for improving skin aging and its postbiotic preparation and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018174669A1 (en) * | 2017-03-24 | 2018-09-27 | 주식회사 팜스킨 | Fermented product of colostrum, and cosmetic composition using same |
| CN112940968A (en) * | 2021-02-07 | 2021-06-11 | 山东凤凰生物有限公司 | Lactobacillus fermentum, lactobacillus fermentum culture and preparation method thereof |
| KR102313770B1 (en) * | 2020-11-27 | 2021-10-18 | 한국식품연구원 | LACTOBACILLUS PLANTARUM WiKim0127 STRAIN DERIVED FROM JEJU PICKLED CABBAGE FOOD AND METHOD FOR PREPARING COMPOSITION USING SAME |
-
2021
- 2021-12-23 CN CN202111585424.5A patent/CN114081901B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018174669A1 (en) * | 2017-03-24 | 2018-09-27 | 주식회사 팜스킨 | Fermented product of colostrum, and cosmetic composition using same |
| KR102313770B1 (en) * | 2020-11-27 | 2021-10-18 | 한국식품연구원 | LACTOBACILLUS PLANTARUM WiKim0127 STRAIN DERIVED FROM JEJU PICKLED CABBAGE FOOD AND METHOD FOR PREPARING COMPOSITION USING SAME |
| CN112940968A (en) * | 2021-02-07 | 2021-06-11 | 山东凤凰生物有限公司 | Lactobacillus fermentum, lactobacillus fermentum culture and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Exploring the wound healing, anti-inflammatory, anti-pathogenic and proteomic effects of lactic acid bacteria on keratinocytes;Jessica Brandi 等;Sci Rep.;第10卷(第1期);文章号:11572 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114081901A (en) | 2022-02-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114081901B (en) | Probiotic composition, preparation method and application thereof | |
| CN115105452B (en) | Black tea fermentation filtrate and preparation method and application thereof | |
| US20250073281A1 (en) | Lactobacillus reuteri having improved anti-aging and hair health enhancement effects, and application thereof | |
| CN115960742B (en) | Lactobacillus fermentum XJC with functions of prolonging service life of dermal fibroblasts and resisting skin aging and application thereof | |
| CN111849836B (en) | Lactobacillus rhamnosus with antioxidant function and application thereof | |
| Sun et al. | Interaction of sulfated polysaccharides with intestinal Bacteroidales plays an important role in its biological activities | |
| CN115181695B (en) | Lactobacillus plantarum5b4m2 and application thereof | |
| CN114645001A (en) | Lactobacillus paracasei and application thereof in preparation of product for regulating skin microecological system | |
| CN115820498A (en) | Lactobacillus plantarum YJ2406 and application thereof | |
| CN114891683A (en) | Pediococcus pentosaceus and application thereof | |
| CN115094011A (en) | Lactobacillus gasseri and application thereof | |
| CN114990021A (en) | Lactobacillus salivarius and application thereof in preparation of skin care products | |
| CN114634899B (en) | Lactobacillus fermentum and application thereof | |
| CN115491334A (en) | Lactobacillus rhamnosus and application thereof | |
| CN115261273A (en) | Lactobacillus jensenii and application thereof | |
| CN115505550A (en) | Lactobacillus paracasei and application thereof | |
| CN114717220B (en) | Lactobacillus reuteri microcapsule and preparation method thereof | |
| CN114672442A (en) | ProfMIC-211 of probiotics and application | |
| CN114657106A (en) | A Lactobacillus plantarum and its application in the prevention and treatment of acne | |
| CN114645002A (en) | Lactobacillus paracasei for skin anti-inflammatory and acne treatment and application thereof | |
| CN119120268A (en) | Cat-derived animal bifidobacterium, postbiotics, pharmaceutical composition and application | |
| CN114574408B (en) | Probiotic SEUNEU-107 and application | |
| CN115044519B (en) | Lactobacillus amyloliquefaciens and application thereof | |
| CN116606781A (en) | Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof | |
| CN114621902A (en) | Lactobacillus paracasei and application thereof in preparation of products for preventing and treating skin diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |