CN114081901A - Probiotic composition, preparation method and application thereof - Google Patents
Probiotic composition, preparation method and application thereof Download PDFInfo
- Publication number
- CN114081901A CN114081901A CN202111585424.5A CN202111585424A CN114081901A CN 114081901 A CN114081901 A CN 114081901A CN 202111585424 A CN202111585424 A CN 202111585424A CN 114081901 A CN114081901 A CN 114081901A
- Authority
- CN
- China
- Prior art keywords
- seuneu
- lactobacillus
- probiotic composition
- plantarum
- lactobacillus plantarum
- 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.)
- Granted
Links
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Abstract
The invention 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 (A)Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentum (Lactobacillus fermentum) SEUNEU-102, Lactobacillus plantarum (A)Lactobacillus plantarum）SEUNEU101 and said lactobacillus fermentum (Lactobacillus fermentum) SEUNEU-102 is deposited in China center for type culture Collection, wherein Lactobacillus plantarum (A), (B), (C) and (C)Lactobacillu plantarum) SEUNEU-101 with the deposition number: CCTCC M20211279; lactobacillus fermentum (A)Lactobacillus fermentum) SEUNEU-102 with the accession number: CCTCC M20211280. The composition has effects of regulating human flora balance, improving skin condition, promoting skin repair, and relieving skin inflammation, especially autoinflammatory diseases in dermatology, and can be used in pharmaceuticals, foods, health products or cosmetics.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a probiotic composition, a preparation method and application thereof.
Background
The skin is a living area of a wide range of microorganisms as the largest organ of the human body, and the skin constitutes a habitat of various forms such as an entrapment part and a specialized space, and helps the survival of the widely distributed microorganisms. Microorganisms form a symbiotic relationship with a human as a host, and the skin microflora play an important role in the host. This is not only because of the ability of the skin microflora to resist the adhesion and development of skin pathogens, but also because of their ability to interact with and interact with the immune system. Moreover, in the event of dysbiosis at the skin level, probiotics may also act as a regulator to restore the balance of the skin microbiota.
The use of new technologies has facilitated taxonomic analysis of skin microbiota over the last decade, and it is known from this analysis that many skin diseases are associated with alterations in the skin microbiome. For example, acne, which is believed to be the bacterium associated primarily with propionibacterium acnes, is markedly elevated on the skin surface when acne occurs; in contrast, in patients with atopic dermatitis, the abundance of Staphylococcus epidermidis on the skin surface is reduced, and the abundance of Staphylococcus aureus is significantly increased.
The conventional approach to the above problem is to use antibacterial agents, i.e. topical disinfectants and antibiotics to inhibit the growth of pathogenic bacteria.
The use of antibiotics in the above methods is effective, but in addition to eliminating beneficial bacteria, they also carry the risk of eliminating beneficial bacteria, sensitization, and potential adverse events, which if used for a long period of time with broad spectrum antibiotics, may also cause an imbalance in skin flora, and make reconstitution difficult.
In addition, the skin barrier is damaged, 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 (hormone content in the body, release of enzymes in the body), physicochemical factors (powerful surfactants clean the skin), etc., all cause damage to the skin barrier. The damage of the skin barrier can not only cause the skin to be lack of water, dry, itchy and sensitive, but also cause the skin diseases such as eczema, psoriasis, acne and the like. In the modern large environment, the damage factors of the skin barrier are more and more, and 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 invention provides a probiotic composition, which can regulate the microbial flora of human skin, promote the proliferation and the repair of epidermal cells and has an anti-inflammatory effect, and the application of the probiotic composition and/or fermentation broth thereof in pharmaceuticals, foods, health care products or cosmetics.
The technical scheme of the invention is as follows:
the probiotic composition according to the embodiment of the invention comprises lactobacillus plantarum (lactobacillus plantarum) ((Lactobacillus plantarum）SEUNEU-101 and Lactobacillus fermentum: (Lactobacillus fermentum) SEUNEU-102, and/or, the Lactobacillus plantarum (A)Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) An evolutionary material of SEUNEU-102, two strains were isolated from natural pulp of sauerkraut. Said Lactobacillus plantarum: (Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) SEUNEU-102 is preserved in China center for type culture Collection with the address: wuhan university in Lojia mountain of Wuchang district, Wuhan city, wherein, the lactobacillus plantarum (Lactobacillus plantarum) SEUNEU-101 has the preservation number: CCTCC M20211279, preservation date is 2021, 10 months and 15 days; lactobacillus fermentum (Lactobacillus fermentum) SEUNEU-102 with a deposit number of: CCTCC M20211280, and the preservation date is 2021, 10 months and 15 days.
According to one embodiment of the present invention, the Lactobacillus plantarum (A)Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) The strains of SEUNEU-102 are live, dead or tyndallized.
According to one embodiment of the present invention, the Lactobacillus plantarum (A)Lactobacillus plantarum) The substances evolved from SEUNEU-101 include: any of lysate, extract, product, supernatant and derivative; the Lactobacillus fermentum (A), (B), (C)Lactobacillus fermentum) The evolved material of SEUNEU-102 includes any of lysate, extract, product, supernatant and derivatives.
According to one embodiment of the invention, the derivative is selected from one or more of metabolites, metabolic biological products, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components.
According to one embodiment of the present invention, the Lactobacillus plantarum (A)Lactobacillus plantarum) SEUNEU-101 or its evolved substance and said Lactobacillus fermentum (Lactobacillus fermentum: (L.))Lactobacillus fermentum) The mass ratio of SEUNEU-102 or its evolution material is 1: 1.
The invention also provides a preparation method of the probiotic composition, which comprises the following steps:
providing a composition containing live Lactobacillus plantarum (A)Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentum (Lactobacillus fermentum) A bacterial liquid of SEUNEU-102, Lactobacillus plantarum (L.)Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentum (Lactobacillus fermentum) SEUNEU-102 is deposited in China center for type culture Collection, wherein Lactobacillus plantarum (A), (B), (C) and (C)Lactobacillu plantarum) SEUNEU-101 with the deposition number: CCTCC M20211279; lactobacillus fermentum (A)Lactobacillus fermentum) SEUNEU-102 with the accession number: CCTCC M20211280;
adjusting the concentration of the bacterial liquid to OD600 of 0.1, and adding the lactobacillus plantarum (A), (B)Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) Inoculating SEUNEU-102 into MRS liquid culture medium, culturing at 37 deg.C, and fermenting to obtain mixed fermentation liquid;
and extracting the supernatant in the mixed fermentation liquor to obtain the probiotic composition.
According to the preparation method of the embodiment of the invention, in vitro culture experiments show that the lactobacillus plantarum (A), (B), (C) and (C)Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) The SEUNEU-102 is cultured together, and can reach the preset concentration more quickly than the culture of a single strain of any strain, so that the preparation efficiency of the bacterial liquid is improved.
According to one embodiment of the invention, the Lactobacillus plantarum is obtained: (Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) The mass ratio of SEUNEU-102 is 1: 1.
According to an embodiment of the present invention, the preparation method of the probiotic composition further comprises autoclaving the extracted supernatant at 121 ℃ for 30min to obtain the probiotic composition.
The invention also provides application of the probiotic composition in improving skin, inhibiting growth of staphylococcus aureus, promoting proliferation of staphylococcus epidermidis, inhibiting propionibacterium acnes, resisting inflammation, promoting epidermal cell repair, repairing skin barrier, up-regulating moisture retention related gene expression, removing free radicals and resisting oxidation.
The invention has at least the following beneficial effects:
in vitro culture experiments show that the culture of the SEUNEU-101 and the SEUNEU-102 together can reach the preset concentration faster than the culture of a single strain of any strain.
In-vitro bacteriostatic tests show that the probiotic composition has the effect of inhibiting staphylococcus aureus, the bacteriostatic rate can reach 73.9 percent, and the bacteriostatic effect is higher than that of a single bacterial strain in the composition;
the probiotic composition has the effect of inhibiting propionibacterium acnes, and after the co-culture is carried out for 24 hours, the inhibition rate of the composition for inhibiting propionibacterium acnes can reach 39.4 percent;
the probiotic composition has the effect of promoting the proliferation of staphylococcus epidermidis, and after the co-culture is carried out for 24 hours, the promoting rate of the composition for promoting the staphylococcus epidermidis can reach 19.8 percent;
the probiotic composition has the function of eliminating superoxide radical, and the clearance rate is 59.39%;
in vitro cell experiments show that the probiotic composition has an 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 an anti-inflammatory effect, and can reduce the expression level of HaCaT cell inflammatory factor IL-1 alpha induced by staphylococcus aureus by 55%, the expression level of IL-8 by 32.99% and the expression level of COX-2 by 32.2%.
In vitro cell experiments show that the probiotic composition has the effect of promoting epidermal cell repair, the healing rate of scratches is increased by 22% compared with a control group, and the repair rate of HaCaT cell damage caused by SDS can reach 21% compared with a control group.
In vitro cell experiments show that the probiotic composition has the effects of up-regulating barrier repair-associated silk polymer protein (filaggrin) FLG, Involucrin (Involucrin) IVL, Ovo-Like transcription factor 1 (Ovo Like Transcriptional Reprossor 1) OVOL1 and lorin (loricrin) LOR genes, and is up-regulated 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 function of up-regulating the expression of Aquaporin 3 (Aquaporin 3) AQP3 and beta-glucocerebrosidase (beta-glucocerebrosidase) GBA, and the expression level can be up-regulated by 1.15-3.8 times.
Drawings
FIG. 1 is a graph showing changes in concentration of probiotic compositions and bacterial liquid cultured by a single strain for 16 h;
FIG. 2 is a graph of the bacteriostatic rate of probiotic compositions against Staphylococcus aureus;
fig. 3 is a graph of the bacteriostatic rate of probiotic compositions against propionibacterium acnes;
fig. 4, graph of the reduction of raw264.7 cell NO release by probiotic compositions;
FIG. 5, probiotic composition down-regulating inflammation-associated factor expression profile;
FIG. 6 is a graph of probiotic compositions promoting HaCaT cell damage repair;
FIG. 7 is a graph of probiotic composition promoting HaCaT scratch repair;
fig. 8, a graph of the expression of genes associated with barrier repair in the supernatant of a probiotic composition;
FIG. 9 shows a diagram of the expression of genes related to barrier repair, which are up-regulated by inactivated bacteria of probiotic compositions;
FIG. 10 is a graph of the gene expression of the probiotic composition supernatant up-regulated moisture retention-related factor;
FIG. 11 shows the expression diagram of probiotic composition inactivated bacteria for up-regulating the gene expression of moisturizing factors.
Detailed Description
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 can be more completely understood and fully conveyed to those skilled in the art, and the present disclosure may be implemented in various forms without being limited to the embodiments set forth herein.
The lactobacillus plays an important role in maintaining the steady state of a host body, activating an immune system, maintaining the immune balance of an organism and the like, researches find that the lactobacillus can prevent and treat various inflammatory diseases by regulating the immune function and phagocytic capacity of macrophages, probiotics can relieve skin inflammation and allergy by controlling the immune state of the whole body including the release of regulatory cytokines, and can regulate the balance of intestinal flora by eating the lactobacillus and regulate immune response. For the above reasons, the applicant has identified the use of probiotic compositions as a new solution to restore the balance of the skin, intestinal and even human microbiota. The probiotic composition disclosed by the invention is separated and identified from natural slurry of pickled Chinese cabbage, comprises the lactobacillus plantarum SEUNEU-101 and the lactobacillus fermentum SEUNEU-102, has a synergistic effect between the lactobacillus plantarum SEUNEU-101 and the lactobacillus fermentum SEUNEU-102, can exert good bacteriostatic, anti-inflammatory and repairing effects after being compounded, and has great market potential in foods, medicines and cosmetics.
The probiotic composition and its use of the present application are described below with reference to specific examples.
It should be noted that, the methods mentioned in the present application, and not the specific steps mentioned, may adopt conventional methods or steps.
The first embodiment is as follows: isolation and characterization of strains
Preparation of MC agar 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, pH value of 6 and sterilization at 121 ℃ for 15 min.
Preparing an 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 phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1000ml of distilled water, 6.4 of pH value and 15min of sterilization at 121 ℃.
The sample of this example was derived from natural serous fluid of sauerkraut, aseptically sampled in a sterile sampling tube containing 5ml of glycerol, and taken back and frozen at-20 ℃. During separation, the sample is redissolved, 1g of the sample is taken to be uniformly shaken in 9ml of normal saline, the supernatant is taken and streaked on an MC flat plate, after constant temperature culture for 48 hours at 37 ℃, the red bacterial colony with the calcium-dissolving ring is selected, and inoculation and screening are repeated until a uniform single bacterial colony is obtained.
1. Primer:
according to the gene sequence of lactobacillus 16S rRNA registered in GenBank, the 368-charge 1049 site gene fragment is referred to, and a primer is designed:
F 5’-TCGGCTCGTAAAACTCTG-3’；
R 5’-GGACTTAACCCAACATCTCA-3’。
the primer is synthesized by Shanghai bioengineering GmbH, and the amplified fragment is V3-V7 and is 682bp long.
2. 16s rRNA Gene sequence analysis:
and (3) picking a single colony, placing the single colony in an MRS centrifuge tube, culturing the single colony overnight at 37 ℃, centrifuging and collecting thalli, and operating according to the steps of the DNA extraction kit. The PCR amplification system is a 50 mu L system, and the pre-denaturation is carried out for 10min at 95 ℃; 35 cycles of 93 ℃ for 1min, 55 ℃ for 1min, 72 ℃ for 1 min; extension at 72 ℃ for 7 min.
3. As a result:
the SEUNEU-101 strain is lactobacillus plantarum after the homology comparison of the PCR product sequencing result and the published standard sequence in GenBank (L. plantarum) ((L. plantarum))Lactobacillu plantarum) (ii) a SEUNEU-102 is Lactobacillus fermentum (L.) (Lactobacillus fermentum）。
Example two: study on concentration change of probiotic composition and single strain cultured for 16h
The activated probiotic composition of the present invention, the bacterial liquid of SEUNEU-101 and SEUNEU-102, was adjusted to a bacterial liquid concentration of OD600 of 0.1, 100. mu.l was inoculated into 1ml of MRS, cultured in a 37 ℃ incubator for 16 hours by standing culture, and the number of bacteria was measured using a spectrophotometer OD600 nm.
As a result:
after the probiotic composition and the single strain are cultured for 16h, as shown in figure 1, the change results of the concentration of the bacterial liquid of the probiotic composition and the single strain after the bacterial liquid is cultured for 16h respectively show that the detection results of the SEUNEU-101, the SEUNEU-102 and the probiotic composition OD600 are 3.0, 3.2 and 5.2 respectively, which shows that the growth speed of the co-culture of the two strains is higher than that of the culture of the single strain, and the two strains have the function of mutually promoting the proliferation.
Example three: antibacterial study of probiotic composition staphylococcus aureus
1. Preparing probiotic composition bacteria liquid and single bacteria liquid:
culturing 1:1 probiotic composition, SEUNEU-101, and SEUNEU-102 bacteria solution with activated SEUNEU-101 and SEUNEU-102 in MRS at 37 deg.C, standing for 18 hr, measuring the number of bacteria with spectrophotometer OD600nm, and adjusting the concentration of probiotic composition, SEUNEU-101, and SEUNEU-102 bacteria solution to 2 × 109centrifuging the cells/mL to obtain a supernatant; sterilizing at 121 deg.C for 30min under high pressure to obtain inactivated supernatant.
2. Preparation of staphylococcus aureus liquid:
culturing in broth culture at 140r/min overnight at 37 deg.C, measuring the number of bacteria with spectrophotometer OD600nm, and adjusting the concentration of lactobacillus solution to 1 × 108 cells/mL。
3. Staphylococcus aureus inhibition experiment
Respectively adding the probiotic composition, the supernatant of SEUNEU-101, the supernatant of SEUNEU-102 and the inactivated supernatant into staphylococcus aureus bacteria liquid in an amount of 5%, standing for 4 hours at 37 ℃, and evaluating the influence of the target strain on the growth of staphylococcus aureus by the percentage reduction of the bacteria liquid concentration (OD 600).
4. Results
Bacteriostatic rate (%) of probiotic composition for inhibiting staphylococcus aureus
| Group of | SEUNEU-101 | SEUNEU-102 | SEUNEU-101+102 |
| Supernatant fluid | 45.3 | 51.1 | 70.4 |
| Inactivating the supernatant | 39.4 | 44.0 | 73.9 |
According to the results, the bacteriostatic 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 bacteriostatic effect than that of the single strain in the composition. The results of the probiotic composition inhibiting staphylococcus aureus bacteriostasis rate are shown in fig. 2.
Example four: probiotic composition staphylococcus aureus inhibition propionibacterium acnes experiment-bacterial liquid concentration change
1. Preparing probiotic composition bacteria liquid:
culturing the activated probiotic composition bacteria liquid in an incubator at 37 deg.C for 18 hr, measuring the bacteria number with a spectrophotometer OD600nm, adjusting the bacteria liquid concentration to 5 × 109cells/mL, centrifugation at 4000rpm for 15 minutes, and filtration through a 0.22 μm filter to obtain a supernatant; inactivating the supernatant at 121 deg.C for 30min under high pressure to obtain inactivated supernatant.
2. Preparing a propionibacterium acnes bacterial liquid:
culturing Propionibacterium acnes in BHI culture medium at 37 deg.C for 48h under anaerobic condition, measuring bacterial count with spectrophotometer OD600nm, centrifuging bacterial liquid at 4000rpm for 15min, discarding supernatant, washing bacterial cells with sterilized 1xPBS buffer solution twice, re-dissolving with 1xPBS phosphate buffer solution, and adjusting the concentration of Propionibacterium acnes to 2.5 × 109 cells/mL。
3. Experiment for inhibiting bacteria
Respectively adding the supernatant of the two probiotic compositions into the pathogenic bacteria liquid by 5 percent, standing for 24 hours at 37 ℃, and evaluating the influence of the probiotic compositions on the growth of the pathogenic bacteria by the percentage reduction of the bacteria liquid concentration (OD 600).
4. Results
The probiotic composition has the effect of inhibiting propionibacterium acnes, after 24 hours of co-culture, the inhibition rate of the supernatant of the probiotic composition on the propionibacterium acnes is 39.4%, and the inhibition rate of the inactivated supernatant of the probiotic composition on the propionibacterium acnes is 26.3%. The bacteriostatic rate of the probiotic composition against propionibacterium acnes is shown in fig. 3.
Example five: experiment for promoting growth of staphylococcus epidermidis by probiotic composition
The probiotic composition was cultured overnight, OD600=1 was adjusted, the supernatant was centrifuged to 5% and staphylococcus epidermidis broth with initial OD600=0.1 was added, left at 37 ℃ for 4h, and the effect of the probiotic composition on the growth of staphylococcus epidermidis was evaluated in terms of relative broth concentration (ratio of sample OD600 to control).
As a result: the relative bacteria liquid concentration of the supernatant added with the probiotic composition is 119.8 percent, and the promotion rate is 19.8 percent compared with the control.
Example six: superoxide radical scavenging capacity of probiotic composition
Respectively selecting a single clone from SEUNEU-101 and SEUNEU-102, performing mixed culture in MRS liquid, performing centrifugation after 24h of culture to obtain a supernatant, and determining the superoxide radical scavenging capacity of the supernatant. The sample addition method was as follows:
| name of solution | Sample tube | Blank tube | Control tube |
| Sample (supernatant) | 50 | 50 μl | - |
| 120 μ M Phenazine Methosulfate (PMS) | 250 μl | - | 250 μl |
| 936 μ M reduced form of beta-Nicotinamide Adenine Dinucleotide (NADH) | 250 μl | - | 250 μl |
| 300 μ M Nitro-tetrazolium chloride blue (NBT) | 250 μl | 250 μl | 250 μl |
| 0.1M phosphate buffer, pH 7.4 | - | 500 | 50 μl |
The tubes were allowed to stand for 5min, and the absorbance at 560nm was measured to calculate the clearance.
Clearance calculation formula: % clearance = [1- (sample 560-blank 560)/control 560 ]. 100.
As a result: the clearance rate of the supernatant of the probiotic composition is 59.4% by detection and calculation.
Example seven: probiotic composition for reducing LPS-induced NO release amount of Raw264.7 cells
1. Probiotic composition sample preparation
Culturing probiotic composition with MRS culture medium overnight, detecting OD600, and adjusting bacterial liquid concentration to 1 × 108cells/mL, after centrifugation, the thalli is sterilized for 30min at 121 ℃ under high pressure to obtain thalli, and the centrifugal supernatant is filtered by a 0.22 mu m filter membrane to obtain the supernatant.
2. Cell preparation
Raw264.7 cells were digested and then digested at 2X 105 Perwell was inoculated into 24-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. Probiotic composition addition and LPS stimulation
Adding 5% of the supernatant and 10% of inactivated thallus into Raw264.7 cells cultured overnight in different groups, inducing the Raw264.7 cells to be inflamed by LPS with the concentration of 200ng after 2 hours, taking the cell culture supernatant after 20 hours, carrying out NO content detection by using an NO content detection kit, and carrying out three experiments in total after 3 multiple holes each time.
As a result:
in vitro cell experiments show that the probiotic composition has an anti-inflammatory effect, can reduce NO release amount of Raw264.7 cells induced by LPS, and compared with an LPS control group, the content of the supernatant is reduced by 41.61%, and the content of inactivated bacteria is reduced by 27.51%. The results of the probiotic composition for reducing the amount of NO released from Raw264.7 cells are shown in FIG. 4.
Example eight: probiotic compositions for reducing staphylococcus aureus-induced HaCaT inflammatory factorsIL-1α、IL-8、 COX-2Amount of expression
1. Probiotic composition sample preparation
Culturing probiotic composition with MRS overnight, detecting OD600, and adjusting bacterial liquid concentration to 1 × 108cells/mL, after centrifugation, cellsSterilizing the strain at 121 deg.C under high pressure for 30min to obtain thallus, centrifuging, and filtering the supernatant with 0.22 μm filter membrane to obtain supernatant.
2. HaCaT cell preparation
HaCaT cells were digested and then treated at 2X 105Perwell was inoculated into 24-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. Preparation and addition of staphylococcus aureus
Inoculating Staphylococcus aureus into broth culture medium, shaking culturing at 37 deg.C overnight, and adjusting the concentration of the culture medium to 3 × 10 with MEM serum-free medium9cells/mL, 100. mu.l per well, were added to overnight cultured HaCaT cells to stimulate the production of inflammatory factors, after 3h the cell culture medium was discarded, washed 5 times with PBS, and 1mL MEM serum-free medium was added to each well.
4. Probiotic composition sample addition
5% of the supernatant was added to staphylococcus aureus-stimulated HaCaT cells, 3 replicates per group, and incubated overnight.
5. Detection of inflammatory factorsIL-1α、IL-8、COX-2Amount of expression
After the cells are discarded from the culture medium, RNA is extracted by using an RNA extraction kit, the concentration and purity of the RNA are detected, all samples are adjusted to 1000ng, and reverse transcription and qPCR are carried out by using a reverse transcription kit and a SYBRGreen qPCR kit.
As a result:
probiotic compositions down-regulate expression of inflammation-related factors
| Name of Gene | Relative fold expression of mRNA | Inhibition rate |
| IL-1α | 0.450 | 55.01% |
| IL-8 | 0.670 | 32.98% |
| COX-2 | 0.678 | 32.20% |
Probiotic compositions are capable of downregulating staphylococcus aureus-induced HaCaT inflammatory factorsIL-1α、IL-8、COX-2Expression of inflammatory factorsIL-1αThe expression level is reduced by 55.01 percent,IL-8the expression level is reduced by 32.98 percent,COX-2the expression level is reduced by 32.2%, therefore, the probiotic composition of the invention has anti-inflammatory effect. The probiotic composition down-regulates the expression of inflammation-related factors see figure 5.
Example nine: probiotic composition for promoting HaCaT cell damage repair experiment-scratch repair
Inoculation of human immortalized keratinocytes HaCaT cells (5X 10)5) Culturing in a 6-well plate overnight until the cells adhere to the wall, streaking the cells at the bottom of the 6-well plate by using a 1ml pipette tip, adding 5% of the supernatant, taking a picture as D1, and culturing in a carbon dioxide incubator at 37 ℃ and 5% of CO 2. The picture was taken after 24h and recorded as D2. Using image J to process data, according to the formula: healing rate = (D1-D2)/D1. Data were plotted using GraphPad.
As a result:
in vitro cell experiments show that the probiotic composition has the effect of promoting epidermal cell repair, and the healing rate of scratches of the supernatant is increased by 22 percent compared with that of a control. See fig. 6, 7 in particular.
Example ten: experiment for promoting HaCaT cell damage repair by probiotic composition, namely SDS damage repair
Inoculation of human immortalized keratinocytes HaCaT cells (5X 10)5) To 96-well plates, overnight culture until cells adhere. Preparing 50ug/ml SDS, adding 100ul of SDS into each well, incubating for 8h, adding 5% supernatant of probiotic composition, and incubating for 24 h. Adding 10ul of CCK-8 solution, incubating for 4h, and detecting the light absorption value at 450 nm.
Cell viability = (experimental group-a blank)/(negative control group a-a blank).
As a result:
after the HaCaT cells are damaged by SDS, the probiotic composition is added, the survival rate of the HaCaT cells is 121%, and compared with a control group, the survival rate is increased by 21%, so that the probiotic composition has the effect of promoting the repair of the HaCaT cells of epidermal cells. See in particular fig. 6.
Example eleven: probiotic composition up-regulation HaCaT barrier repair related gene expression experiment
Inoculation of human immortalized keratinocytes HaCaT cells (5X 10)5) To 6-well plates, culture overnight until cells adhere. Adding supernatant of probiotic composition 5% and inactivated thallus 10%, culturing for 24 hr, discarding culture medium supernatant, washing with PBS, adding RNA, extracting lysate, extracting total RNA from cells, reverse transcribing to cDNA, and treatingFLG、IVL、OVOL1、LORAnd (3) carrying out qPCR detection on the expression quantity of the gene. Relative fold expression calculations were performed according to the formula.
The formula: f =2-ΔΔCT
As a result:
probiotic composition up-regulation and modification of relative expression result of related genes
| Name of Gene | Supernatant fluid | Inactivated thallus |
| FLG | 7.60 | 1.45 |
| IVL | 3.81 | 1.50 |
| LOR | 1.85 | 1.34 |
| OVOL1 | - | 1.46 |
The results show that HaCaT cells are associated with barrier repair after stimulation by the addition of probiotic compositionsFLG、IVL、OVOL1、LORThe gene expression is up-regulated to different degrees, so the probiotic composition has the function of promoting the skin barrier repair. The relative expression results of the probiotic composition up-regulating the modified associated genes are shown in fig. 8 and 9.
Example twelve: probiotic composition up-regulation HaCaT moisturizing related gene expression experiment
Inoculation of human immortalized keratinocytes HaCaT cells (5X 10)5) To 6-well plates, culture overnight until cells adhere. Adding 5% of the supernatant of the probiotic composition and 10% of inactivated bacteria, respectively culturing for 24h, removing the supernatant of the culture medium, washing with PBS, adding RNA to extract lysate, extracting total RNA of cells, performing reverse transcription to obtain cDNA, and performing qPCR (quantitative polymerase chain reaction) to detect aquaporinAQP3、GBAExpression of (2). And calculating expression change times according to a formula.
The formula: f =2-ΔΔCT
As a result:
probiotics composition up-regulating relative expression of moisture retention related genes
| Relative fold expression of mRNA | Relative fold expression of mRNA | |
| Name of Gene | Probiotic composition supernatant | Probiotic composition inactivated thallus |
| AQP3 | 2.10 | 3.50 |
| GBA | 1.54 | 1.18 |
The results show that the moisturizing related genes after the stimulation of the HaCaT cells by adding the probiotic compositionAQP3、GBAThe gene expression is up-regulated, so that the probiotic composition has the function of up-regulating the expression of the moisturizing factor. The relative expression results of the probiotic composition up-regulating the moisturizing related genes are shown in fig. 10 and fig. 11. While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principles of the inventionThe decoration should also be regarded as the protection scope of the present invention.
Claims (7)
1. A probiotic composition, characterized in that it comprises Lactobacillus plantarum (Lactobacillus plantarum) ((R))Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentum (Lactobacillus fermentum) SEUNEU-102, Lactobacillus plantarum (A)Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) SEUNEU-102 is deposited in China center for type culture Collection, wherein Lactobacillus plantarum (A), (B), (C) and (C)Lactobacillu plantarum) SEUNEU-101 with the deposition number: CCTCC M20211279; lactobacillus fermentum (A)Lactobacillus fermentum) SEUNEU-102 with the accession number: CCTCC M20211280.
2. The probiotic composition according to claim 1, wherein said Lactobacillus plantarum (F: (A), (B), (CLactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) The strains of SEUNEU-102 are live, dead or tyndallized.
3. The probiotic composition according to any of claims 1 to 2, characterized in that said Lactobacillus plantarum (Lactobacillus plantarum) (II) ((III))Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (f)Lactobacillus fermentum) The mass ratio of SEUNEU-102 is 1: 1.
4. A method of preparing a probiotic composition, comprising:
providing a composition containing live Lactobacillus plantarum (A)Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentum (Lactobacillus fermentum) A bacterial liquid of SEUNEU-102, Lactobacillus plantarum (L.)Lactobacillus plantarum) SEUNEU-101 and Lactobacillus fermentum (Lactobacillus fermentum) SEUNEU-102 is deposited in China center for type culture Collection, wherein Lactobacillus plantarum (A), (B), (C) and (C)Lactobacillu plantarum) SEUNEU-101 with the deposition number: CCTCC M20211279; lactobacillus fermentum (A)Lactobacillus fermentum) SEUNEU-102 with the accession number: CCTCC M20211280;
adjusting the concentration of the bacterial liquid to OD600 of 0.1, and adding the lactobacillus plantarum (A), (B)Lactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) Inoculating SEUNEU-102 into MRS liquid culture medium, culturing at 37 deg.C, and fermenting to obtain mixed fermentation liquid;
and extracting the supernatant in the mixed fermentation liquor to obtain the probiotic composition.
5. Process for the preparation of the probiotic composition according to claim 4, characterized in that said Lactobacillus plantarum (F) (A) is obtainedLactobacillus plantarum) SEUNEU-101 and the Lactobacillus fermentum (A), (B), (C) and (C)Lactobacillus fermentum) The mass ratio of SEUNEU-102 is 1: 1.
6. The method for preparing the probiotic composition according to claim 4 or 5, further comprising autoclaving the extracted supernatant at 121 ℃ for 30min to obtain the probiotic composition.
7. Use of a probiotic composition according to any of claims 1-2 for improving skin, inhibiting the growth of staphylococcus aureus, promoting the proliferation of staphylococcus epidermidis, inhibiting propionibacterium acnes, anti-inflammatory, promoting epidermal cell repair, repairing skin barrier, up-regulating moisture retention related gene expression, scavenging free radicals and resisting oxidation.
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| CN115181698A (en) * | 2022-06-28 | 2022-10-14 | 广东粤港澳大湾区国家纳米科技创新研究院 | Probiotic composition and application |
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| CN117736942A (en) * | 2024-02-20 | 2024-03-22 | 山东中科嘉亿生物工程有限公司 | Fermented lactobacillus mucilaginosus JYLF-315 for improving skin aging and metagen preparation and application thereof |
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