CN114717134A - A strain of Bifidobacterium lactis and its application in the prevention and treatment of acne - Google Patents

Abstract

The invention relates to the technical field of probiotic screening and application, and particularly provides a novel Bifidobacterium lactis strain and application thereof. The preservation number of the bifidobacterium lactis is CCTCC NO: m2021598. The strain is isolated from fresh feces sample of breast-fed infant, has immunoregulatory activity, and can relieve skin inflammation, improve skin resistance, and effectively prevent and treat acne.

Description

A strain of Bifidobacterium lactis and its application in the prevention and treatment of acne

technical field

The invention belongs to the technical field of screening and application of probiotics, in particular to a strain of Bifidobacterium lactis with acne treatment efficacy and application thereof.

Background technique

The skin is the largest organ of the human body and constitutes a strong barrier between the outside world of the body, mainly composed of epidermis, dermis, subcutaneous tissue and skin appendages. The skin barrier in a broad sense includes physical barrier, immune barrier, pigment barrier and nerve barrier, etc., while the skin barrier in the narrow sense mostly refers to the physical or mechanical epidermal barrier structure. The epidermal structure prevents allergens or noxious substances from entering the body through skin moisture, temperature, pH and skin microbiome homeostasis. Therefore, skin inflammatory diseases such as acne are prone to occur when the integrity of the skin barrier is broken.

Acne is a chronic inflammatory skin disease that occurs in the pilosebaceous unit, mainly in adolescents. The occurrence of acne is mainly related to factors such as excessive sebum secretion, blockage of the pilosebaceous duct, bacterial infection and inflammatory response. Studies have shown that various microorganisms in hair follicles, especially Propionibacterium acnes, can activate the innate immune response, resulting in hyperkeratosis of the pilosebaceous glands. At present, the clinical treatment of acne mostly adopts retinoids, hormones and antibiotics, but has not received satisfactory results.

Probiotics are live microorganisms that, when administered in sufficient quantities, confer health benefits to the host. Common probiotic species include Lactobacillus, Bifidobacterium, Bacillus, and some Clostridium butyricum. Studies on probiotics have shown that they can be colonized in the human intestine to regulate the balance of the tract flora, and have an impact on the immune system, nervous system, digestive system, metabolic system and respiratory system of the human body. Therefore, it can alleviate or prevent IBS, IBD, Efficacy in diseases such as diabetes, depression and influenza. In recent years, studies on the gut-skin axis have shown that probiotics can regulate the composition of skin flora, inhibit inflammation in the body, and enhance skin barrier function. Changes in gut microbiota have been reported in almost half of mild acne patients.

Fabbrocini et al found that patients taking Lactobacillus rhamnosus SP1 fermentation broth restored the expression of genes related to insulin signaling and improved acne appearance compared with patients taking Lactobacillus rhamnosus SP1 fermentation broth. Rahmayani et al found that the anti-inflammatory cytokine interleukin IL-10 level in serum was significantly increased in patients with acne vulgaris after oral administration of the probiotic mixture for one month, indicating that oral probiotics can be used as an adjuvant therapy for acne vulgaris by regulating the inflammatory response. In addition, oral administration of Lactobacillus reuteri can also limit the major histocompatibility cells around the hair follicle, affect early inflammation, and prevent the occurrence of acne.

In addition to oral probiotics, there have also been many reports on topical probiotics in recent years, such as adding probiotic fermentation broth or its lysate to skin care products. Such postbiotics can affect the composition of skin microbes, such as inhibiting the colonization of Staphylococcus aureus, alleviating excessive inflammation, and ultimately improving skin health. In the future, the skin care products developed by Shengyuan as active ingredients have become a new way of preventing and treating acne.

The pathogenesis of acne is complex, and the probiotic properties of probiotics are different at the strain level. Therefore, it is still a difficulty and a hot spot in current research to screen and obtain probiotic strains with outstanding acne-prevention effect and clear mechanism of action.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new strain of Bifidobacterium lactis and its application. The strain, isolated from fresh fecal samples of breast-fed infants, has immunomodulatory activity, can reduce skin inflammation, improve skin resistance, and can effectively prevent and treat acne.

The Bifidobacterium lactis provided by the present invention is Bifidobacterium lactis VHProbi O20 (Bifidobacterium lactis VHProbi O20), which has been deposited in the Chinese Type Culture Collection Center of Wuhan University, Wuhan, China on May 24, 2021, and its deposit number is CCTCC NO: M2021598.

The Bifidobacterium lactis VHProbi O20 strain has a 16s rDNA sequence of SEQ ID NO: 1.

For the Bifidobacterium lactis VHP robi O20 strain, its MALDI-TOF ribosomal protein molecular weight map is shown in Figure 2; its Riboprinter fingerprint map is shown in Figure 3; its RAPD fingerprint map is shown in Figure 4; rep-PCR fingerprint The spectrum is shown in Figure 5.

The Bifidobacterium lactis provided by the present invention can be used for preparing functional food, health product, medicine or cosmetic.

The Bifidobacterium lactis provided by the present invention can be used to prepare products with antioxidant function;

The Bifidobacterium lactis provided by the present invention can be used to prepare a product for preventing or treating acne.

The product is functional food or cosmetic.

The Bifidobacterium lactis VHProbi O20 provided by the invention has strong tolerance to artificial gastrointestinal juice and can germinate in the artificial gastrointestinal juice; the strain is sensitive to common antibiotics such as erythromycin and ampicillin, and does not produce hemolysin , can not lyse blood cells, has good biological safety;

The strain has a certain antioxidant capacity, the inhibition rate of the cell against lipid peroxidation is 20.34%, the supernatant is 73.10%; the DPPH clearance rate is 20.21%, and the HRS clearance rate is 23.03%. In addition, the cell surface hydrophobicity of this strain was 64.7%.

Bifidobacterium lactis VHProbi O20 and its lysate can effectively relieve the redness and swelling of the rabbit ear acne site, reduce the roughness and hardness of the rabbit ear skin, and improve the skin lesion symptoms of the rabbit ear acne site. The rabbit ear skin has a relatively complete epidermis and Dermis.

Bifidobacterium lactis VHProbi O20 can also regulate the level of inflammatory factors in white rabbits, regulate its immune response, and slow down the development of acne inflammation.

The Bifidobacterium lactis VHProbi O20 provided by the invention has no toxic effect on the body, and can be added to food or skin care products for treating acne, and has broad application prospects.

Description of drawings

Fig. 1 is a photo of O20 bacterial strain colony;

Fig. 2 is O20 strain MALDI-TOF ribosomal protein peak map;

Fig. 3 is the fingerprint of O20 strain Riboprinter;

Fig. 4 is the RAPD fingerprint of O20 strain;

Fig. 5 is the rep-PCR fingerprint of O20 strain;

Fig. 6 is the comparison of acne symptoms in each group of rabbit ears;

Figure 7 is a comparison of cytokine detection results in each group;

Figure 8 shows the results of pathological sections of rabbit ears in each group.

Detailed ways

The Bifidobacterium lactis VHProbi O20 provided by the present invention complies with the requirements of laws and regulations, and is identified as a newly discovered strain by the multiphase taxonomy identification. The Bifidobacterium lactis VHProbi O20 provided by the invention can effectively treat and relieve acne, and can relieve acne by using the strain alone without compounding with prebiotics and/or other probiotics; it has important application value.

The applicant deposited the Bifidobacterium lactis VHProbi O20 in the China Center for Type Culture Collection of Wuhan University, Wuhan, China on May 24, 2021, and its deposit number is CCTCC NO: M2021598.

The screening method of the present invention is not limited to those described in the examples, and any known methods that can achieve the purpose of screening can be used. The screening descriptions in the examples are only for the description of the present invention, and do not limit the protection scope of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.

The present invention will be described in detail below with reference to specific embodiments.

Example 1 Isolation and screening of Bifidobacterium lactis VHProbi O20

1. Preliminary screening

Prepare BMS agar medium, adjust pH to 6.2-6.5, and sterilize by autoclaving at 121 °C for 15 min.

Take 1 g of fresh fecal samples of breastfed infants (the sampling process conforms to the ethical standards of biological sampling), dilute them with sterile saline, put them in sterile sample bags, and beat and mix with a homogenizer; take 100 μL of the mixed solution for gradient dilution , coated on BMS agar medium and cultured anaerobic at 37°C for 48h, until a single colony grew on the plate for microscopic examination. According to the microscopic examination results, the applicant screened a total of 25 potential lactobacilli strains, which were named as O01, O02, ..., O20, O21, O22, O23, O24, O25.

2. Re-screening

Prepare 1L of BMS liquid medium, autoclave at 121°C for 15min, add 3.2g of porcine mucosal pepsin after the medium is cooled, shake well to dissolve, and place it in a water bath shaker for 1h at 37°C to prepare an acid-resistant medium.

The 25 strains of Lactobacillus O01, O02, ..., O20, O21, O22, O23, O24, and O25 obtained by screening were inoculated into the above-mentioned acid-resistant medium at 6% inoculum, and anaerobic static culture was carried out at 37°C. 48h, take the fermentation broth for bacterial count.

The results showed that among the logarithm values of the viable bacteria in the fermentation broth of the 25 lactobacillus strains, the O20 strain had the largest amount of viable bacteria after being re-screened by the acid-resistant medium, and the logarithm value was as high as 8.70Log CFU/mL. This shows that the O20 strain has the highest acid resistance.

Example 2 Identification of strains

1. Identification of colony morphology

The O20 strain was inoculated on the BMS agar medium, and after anaerobic cultivation at 37°C for 24 hours, the single O20 colony was milky white, the colony diameter was about 1.5-2mm, the surface was smooth, and it was short rod-shaped under the microscope. The colonies and photos under the light microscope are shown in Figure 1.

2. Identification of physiological and biochemical characteristics

The preparation of the inoculum in this example is as follows: under aseptic conditions, take an appropriate amount of fresh O20 bacterial solution, centrifuge at 5000 rpm/min for 5 min, wash twice with PBS buffer, resuspend the bacterial cells with the same volume of PBS buffer and dilute 50 times as the inoculum.

2.1. Catalase experiment

Take a fresh bacterial solution, drop a drop on a clean glass slide, and then drop a drop of 3% hydrogen peroxide solution on it. It is observed that the O20 strain does not produce bubbles, which is a negative reaction.

2.2. Carbon source metabolism test

The basal medium formula used in the present embodiment is as follows:

Peptone 1.5g; yeast extract 0.6g; Tween 80 0.1g; saline solution 0.5mL; phenol red 18mg; distilled water 100mL; pH 7.4±0.2. Salt solution components: 11.5 g of MgSO ₄ ·7H ₂ O, 2.8 g of MnSO ₄ ·4H ₂ O, and 100 mL of distilled water.

A 10 g/100 mL solution of sugars, alcohols and glycosides carbohydrates was prepared and filtered through a 0.22 μm sterile filter. Under sterile conditions, add 20 μL of sterilized carbohydrate solution to the 96-well plate, 4 for each carbohydrate in parallel, then add 170 μL of sterilized phenol red-containing basal medium, and then add 10 μL of inoculum, without Inoculation wells served as controls. 50 μL of liquid paraffin was added to each well to prevent evaporation of water during incubation. Anaerobic culture at 37°C, using phenol red as an indicator, to observe the color change of the medium; the specific results are shown in Table 1.

Table 1: O20 strain carbon source metabolism result table

Note: "+" positive reaction; "-" negative reaction.

2.3 MALDI-TOF-MS detection of strain ribosomal protein expression

According to the inoculation amount of 0.1%, fresh bacterial liquid was inoculated in MRS liquid medium, and after culturing at 37 °C and 150 rpm for 48 h, the bacterial cells were collected, washed with sterile water for 4 times, and the surface moisture was dried. Then take a small amount of fresh cells and spread them evenly on the target plate in the form of a thin film, add 1 μL of lysis solution to cover the sample, and after drying, add 1 μL of matrix solution to cover the sample, after drying, put the sample target into the mass spectrometer for identification . The co-crystal film formed by the sample and the matrix is irradiated with a laser to ionize the protein in the sample. The ions are accelerated to fly through the flight pipeline under the action of an electric field of 10-20KV, and the molecular weight of the protein is detected according to the flight time to the detector. Using Autofms 1000 analysis software Autof Analyzer v1.0 to obtain protein fingerprints, the ion peaks of the main ribosomal proteins of O20 strain are: m/z 2658.901, 2648.948, 4002.334, 4376.813, 4561.980, 5316.587, 5713.658, 6871.363, 9003.998. The identification results are shown in Figure 2.

3. Molecular biological identification

3.1 16s rDNA gene sequence analysis

3.1.1. Genomic DNA extraction

The procedure was carried out with reference to the Genomex DNA Extraction Kit (Cat. No.: DP302).

3.1.2. 16s rDNA gene amplification

Primer sequence:

27F: AGAGTTTGATCCTGGCTCA;

1492R: GGTTACCTTGTTACGACTT.

The 16s rDNA sequence SEQ ID NO: 1 of the O20 strain was obtained by sequencing, and the sequence was compared in the NCBI database, and the O20 strain was preliminarily determined to be Bifidobacterium lactis.

3.2 Riboprinter fingerprint

Dip the purified single colony from the agar medium plate with a stick, put it into a sample tube with buffer, stir it with a hand stirrer to suspend it in the buffer, and then place the sample holder. After being inactivated in a heater, it was put into the Riboprinter system. After DNA preparation, membrane transfer, imaging detection and data processing, the bacterial identification results were obtained. The identification results showed that the O20 strain was Bifidobacterium lactis, and its Riboprinter fingerprint was shown in Figure 3.

3.3 RAPD and rep-PCR fingerprint identification

3.3.1. RAPD fingerprint identification

1) Primer sequence: GAGGGTGGCGGTTCT.

2) RAPD reaction system

Table 2 RAPD reaction system

3) Electrophoresis

A 1.5% agarose gel plate was prepared, DL2000DNA Marker was used as the result control, the voltage was regulated at 100V for 80min, and finally the electrophoresis was detected by a gel imaging system. The RAPD fingerprint of O20 strain is shown in Figure 4.

3.3.2. Fingerprint of rep-PCR

1) Primer sequence: CTACGGCAAGGCGACGCTGACG.

2) The reaction system of rep-PCR

Table 3 Reaction system of rep-PCR

3) Electrophoresis

DL2000 DNA Marker was used as a result control. The amplification results were detected at a voltage of 100 V and an electrophoresis time of 80 min. The rep-PCR fingerprint of the O20 strain is shown in Figure 5.

In summary, the results of the colony morphology and physiological and biochemical characteristics of the O20 strain were uploaded to the website http://www.tgw1916.net/bacteria_logare_desktop.html, and combined with the literature De Clerck E, et al. Systematic and applied microbiology, 2004, 27 (1 ) 50 published results for comparison. Based on the identification results of molecular biology, it can be concluded that the O20 strain is a new strain of Bifidobacterium lactis, which is named Bifidobacterium lactis VHProbi O20.

Embodiment 3 Tolerance test of Bifidobacterium lactis VHProbi O20 to artificial gastric juice and artificial intestinal juice

1. Preparation of artificial gastric juice

Weigh 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose and 2 g of NaCl respectively, add 1000 mL of distilled water, adjust pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115 °C for 20 min. Then add 3.2 g of porcine mucosal pepsin before use, shake well to dissolve, and place in a warm water bath for 1 hour in a 37°C water bath shaker to simulate human body temperature.

2. Preparation of artificial intestinal juice

Weigh 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose, 6.8 g of KH ₂ PO ₄ and 3.0 g of bovine bile salts, respectively, add 77 mL of 0.2 mol/L NaOH solution, make up to 1000 mL, and add dilute hydrochloric acid or sodium hydroxide. The solution was adjusted to pH 6.8±0.1 and sterilized at 115°C for 20min. Then add 1 g of pancreatin before use, shake well to dissolve, and place it in a warm water bath for 1 hour in a 37°C water bath shaker to simulate human body temperature.

3. Test method

Take 2 mL of fresh bacterial liquid, centrifuge at 5000 rpm/min for 5 min to collect the bacterial cells, wash the bacterial cells three times with physiological saline, and resuspend with 2 mL of physiological saline as the inoculum. Take 1 mL of inoculum, add it to 24 mL of artificial intestinal juice, place it on a water bath shaker (200 rpm/min) at 37°C for 3 h, and sample 1 mL to detect the amount of viable bacteria.

The count method of viable bacteria was determined according to the national standard "GB4789.35-2016 - Food Microbial Inspection Lactic Acid Bacteria Inspection", and the viable bacterial amount (Log CFU/mL) of this strain after being digested by artificial intestinal juice is shown in Table 4.

Table 4 Amount of viable bacteria after digestion of artificial gastrointestinal juice

As can be seen from Table 4, the Bifidobacterium lactis VHProbi O20 screened by the present invention has basically no change in the amount of viable bacteria after being digested by artificial gastric juice, and the amount of viable bacteria only decreases by 0.63 Log value after being digested by artificial intestinal juice. It shows that the strain has strong tolerance to artificial gastrointestinal fluid.

Example 4 Antibiotic tolerance test of Bifidobacterium lactis VHProbi O20

1. Antibiotic preparation: Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were all prepared into a stock solution of 2048 μg/mL, and stored at -20°C for later use. When using, the stock solution was serially diluted with BMS liquid medium by 2-fold serial gradient to obtain the use solution, and the gradient dilution concentration was 1-1024 μg/mL in a total of 11 gradients.

2. Preparation of inoculum: Preparation of inoculum: take an appropriate amount of fresh bacterial solution (24-48h, 40°C culture), centrifuge at 5000rpm for 5min, wash once with sterile saline, resuspend the bacteria with the same volume of saline and dilute 50 times as the inoculum.

3. The minimum inhibitory concentration (MIC) of antibiotics against Bifidobacterium lactis VHProbi O20 was determined by the micro-broth dilution method.

a. In the first column of the 96-well plate, add BMS liquid medium without antibiotics as a negative control. Add 190 μL of BMS liquid medium with different concentrations of antibiotics to columns 2 to 12 in turn, and then inoculate 10 μL of the above inoculum respectively. Make 3 parallel wells, and use 1 well without bacterial solution as a blank.

b. Add 50 μL of paraffin oil to cover to prevent water evaporation.

c. Take out the 96-well plate after shaking culture at 40°C for 48 hours, measure the OD600 value, and use the results of 48 hours to calculate the MIC value of antibiotics against the strain. The specific results are shown in Table 5.

Table 5 Antibiotic MIC value of Lactobacillus reuteri VHProbi O20

MIC unit μg/mL

It can be seen from the results in Table 5 that the Bifidobacterium lactis VHProbi O20 provided by the present invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biological safety.

Example 5 Determination of antioxidant function of Bifidobacterium lactis VHProbi O20

1. Determination of the ability of strains to scavenge DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) and hydroxyl radicals (HRS)

Determination of the ability of strains to scavenge DPPH free radicals

Take 1 mL of the PBS bacterial suspension of the strain to be tested, add 1 mL of 0.4 mM DPPH free radical solution, mix evenly, and then place it at room temperature for 30 min of shading reaction, and then measure the absorbance of the sample A sample at a wavelength of 517 nm. 3 parallels. The samples of the control group were zero-adjusted with equal volumes of PBS solution and DPPH·ethanol mixture, and blank-adjusted with equal volumes of PBS bacterial suspension and ethanol mixture. The clearance rate was calculated according to the following formula: clearance rate %=[1-(A _sample -A _blank )/A _control ]×100%. The results are shown in Table 6.

Table 6 DPPH free radical scavenging rate table

3) Determination of the ability of the strain to clear HRS

Mix 100 μL 5mM sodium salicylate-ethanol solution, 100 μL 5mM ferrous sulfate, 500 μL deionized water and 200 μL lactic acid bacteria PBS bacterial suspension, add 100 μL hydrogen peroxide solution (3 mM), water bath at 37 °C for 15 min at 510 nm Measure the sample absorbance at the wavelength. The hydroxyl radical scavenging rate was calculated according to the following formula.

Clearance %=(A _sample -A _control )/(A _blank -A _control )×100%, wherein A _control is deionized water substitute sample, A _blank is deionized water substitute sample and H ₂ O ₂ , the results are shown in the table 7.

Table 7 HRS free radical scavenging rate table

2. Identification of strains against lipid peroxidation

Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water. Add 1 mL of linoleic acid emulsion, 1 mL of FeSO ₄ (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample, water bath at 37°C for 1.5 h, add 0.2 mL of TCA (4%) to the mixture, 2 mL TBA (0.8%), 100°C water bath for 30min, rapidly cooled, centrifuged at 4000rpm/min for 15min, collected supernatant and measured the absorbance at 532nm as A; the control group was replaced by 0.5mL distilled water as A ₀ . Inhibition rate/%=(A ₀ -A)/A ₀ ×100%

Note: A is the absorbance of the sample group; _A0 is the absorbance of the control group, the results are shown in Table 8.

Table 8 Anti-lipid peroxidation inhibition rate table

Example 6 Adsorption capacity of aflatoxin B1 of Bifidobacterium lactis VHProbi O20

1. Prepare an AFB1-PBS solution with a concentration of 1 μg/mL.

2. Inoculation adsorption: Take 1mL of fresh bacterial solution (24h, 37℃), centrifuge at 8000rpm for 5min, discard the supernatant, wash the bacteria twice with the same volume of PBS buffer, centrifuge at 8000rpm for 5min, discard the supernatant, and then resuspend the bacteria Put 1 mL of the above AFB1-PBS solution in a 37°C constant temperature incubator, take it out after 1 h, centrifuge at 8000 rpm for 10 min, and take the supernatant for testing.

3. Determine the concentration of aflatoxin B1 in the supernatant according to the instructions of the aflatoxin B1 detection kit.

Before the assay, the supernatant was diluted 100-fold with methanol.

The results show that the aflatoxin B1 adsorption capacity of the Bifidobacterium lactis VHProbi O20 provided by the present invention is 15.90%, and the standard deviation is 0.01%.

Example 7 Hydrophobic cell surface test of Bifidobacterium lactis VHProbi O20

1. Preparation of the bacterial solution to be tested: Pick the purified Bifidobacterium lactis VHProbi O20 colony and inoculate it in the newly prepared MRS liquid medium, and cultivate at 37°C for 24-48h. The inoculum of 1% (V/V) was then added to MRS liquid medium at 37°C for 24-48 h, centrifuged at 6000 × g for 10 min, the cells were collected, washed twice with sterile saline, and then sterilized The bacteria were resuspended in 1mL solution of 0.1M KNO ₃ as the bacteria solution to be tested.

2. Determination of surface hydrophobicity: add 50 μL of the above bacterial suspension to 2450 μL of 0.1M KNO ₃ and record the OD600 as A ₀ , take 1.5 mL of the bacterial suspension and mix it with 500 μL of xylene, and then let it stand at room temperature for 10 min (the formation of two-phase system). The two-phase system was vortexed for 2 min and then allowed to stand for 20 min to re-form the aqueous and organic phases. Absorbance A ₁ was measured at 600 nm by carefully pipetting the aqueous phase (not the organic phase). The hydrophobicity of cells was calculated according to the formula Hydrophobicity%=(A ₀ -A ₁ )/A ₁ ×%, and the average value was obtained from three experiments.

The results show that the surface hydrophobicity of Bifidobacterium lactis VHProbi O20 cells provided by the present invention is 69.50%, and the standard deviation is 5.57%.

Example 8 Application of Bifidobacterium lactis VHProbi O20 and its lysate in relieving rabbit ear acne

1 Experimental animal handling and model construction

1.1 Experimental consumables

Table 9 Experimental consumables

1.2 Preparation of fresh bacterial solution and lysate

Bifidobacterium lactis VHProbi O20 was inoculated into freshly cultured MRS medium, and it was used for gavage after anaerobic culture at 37°C for 12 hours. The freshly cultured bacterial liquid was crushed by a high-pressure homogenizer at a pressure of 100 MPa, and the homogenization was repeated 3 times, and then placed in a 70°C water bath for complete inactivation treatment to prepare a lysate.

1.3 Handling of experimental animals

Clean-grade New Zealand white rabbits, male, 20, were randomly divided into 4 groups of 5 rabbits, provided by Qinglongshan Animal Breeding Center. The experimental rabbits were fed with special pellet feed and kept in a clean-grade animal room at a temperature of 18-20°C and a relative humidity of 40%.

The white rabbits were divided into blank group, modeling group, positive group and probiotic group after adaptive feeding. Among them, the blank group did not do any treatment, and the whole process was gavaged and smeared with normal saline; the modeling group, positive group and probiotic group were used to build acne models with Propionibacterium acnes and oleic acid. The patients were treated with normal saline and metronidazole gel, and the probiotic group was treated with Bifidobacterium lactis VHProbi O20 and its lysate. Animals were cared for and handled in strict accordance with animal ethics laws throughout the experimental period.

The specific processing method is as follows:

On the left and right external auditory canals of the rabbit ears, 0.2 mL of 50% oleic acid was evenly coated with a 0.25 mL applicator, once every other day, for 28 consecutive days. The cultured Propionibacterium acnes was counted, and the concentration was adjusted to 10 ⁷ /mL for use. On the 12th day of coating with oleic acid, 30ul/ear of Bacillus acnes was intradermally injected, and the injection site was marked for a total of one injection.

(1) Blank group: After adaptation, 10 mL of normal saline was administered to the stomach every day, and 1 mL of normal saline was applied to the external auditory canal, once in the morning, middle and evening until the end of the test on the 38th day;

(2) Modeling group: gavage 10 mL of normal saline every day, apply 1 mL of normal saline to the external auditory canal to cover the external auditory canal acne, once in the morning and once in the evening until the end of the experiment on the 38th day;

(3) Positive group: gavage 10 mL of normal saline every day, apply 1 g of metronidazole gel to cover the external auditory canal acne, once in the morning and evening until the end of the test on the 38th day;

(4) Probiotic group: 1×10 ⁹ CFU/mL Bifidobacterium lactis VHProbi O20 bacterial solution 10mL was administered by gavage every day, and 1 mL Bifidobacterium lactis VHProbi O20 lysate was applied to cover the external auditory canal acne, once in the morning and evening until the 38th day. Finish;

2 Indicator detection

On the 38th day after the test, the thickness of each rabbit was measured with a portable thickness gauge, and the swelling of the rabbit was observed. The acne skin lesion counting method was used to evaluate the therapeutic effect of probiotics application, and the skin condition of the rabbits was observed from the aspects of hardness, thickness and roughness of the rabbits.

Table 10 Observation and scoring criteria of acne model

The contents of IL-10, TNF-α and IFN-γ in serum of rabbits were detected by ELISA in venous blood.

Rabbit ear tissue was taken and made into 5 μm thick paraffin sections for HE staining. According to the experimental acne histopathological grading standard, according to the epidermal thickening of the model animal, the degree of hair follicle opening and the amount of keratin produced, the pathological changes of the specimen tissue were judged.

3 Data Analysis

SPSS 22.0 statistical software was used for data analysis, and the results were expressed as mean ± standard error. The independent sample t test was used for comparison between two groups, and the one-way analysis of variance was used for the comparison of differences among multiple groups. p<0.05 means the difference is significant, p<0.01 means the difference is extremely significant. Graphs were performed using GraphPad Prism 8.0.

4. Experimental results

4.1 Observation of the general state of the rabbits in each group at the end stage

On the 38th day after administration, the ears of the rabbits in the blank group were soft, with clear capillaries, and the hair follicles at the openings of the rabbit ears were neatly arranged; the ears of the rabbits in the modeling group were obviously red and swollen, with raised pustules; the ears of the rabbits in the positive group had horns The hair follicles become thicker, and the rabbit ears become thicker. In the probiotic group, the transmittance of the rabbit ears was improved, the degree of redness and swelling was reduced, no papules and pustules appeared, the phenomenon of blackheads was reduced, the rabbit ears became thinner, and the hardness and roughness were relieved. The observation results of typical rabbit ears in each group are shown in Figure 6, and the results of the appearance observation scores of rabbit ears in each group are shown in Table 11.

Table 11 Rabbit Ear Appearance Rating Scale

4.2 Serum ELISA test results of each group of white rabbits at the end-stage

IL-10 is a cytokine with pleiotropic effects in immune regulation and inflammation. It downregulates the expression of Th1 cytokines, MHC-II antigens and co-stimulatory molecules in macrophages, and also enhances the ability of B cells to survive, proliferate and produce antibodies. TNF-α is a cytokine involved in systemic inflammation and is one of many cytokines involved in acute responses. INF-γ is a water-soluble dimer cytokine, the only member of type II interferon, known as macrophage activating factor. After the experiment, the changes in the levels of the three cytokines in serum are shown in Figure 7.

As can be seen from the results in Figure 7, compared with the blank group, the serum levels of TNF-α and INF-γ cytokines in the white rabbits of the modeling group increased, and the difference was extremely significant (P<0.001). The level of IL-10 cytokines Compared with the modeling group, the serum levels of TNF-α and INF-γ cytokines in the positive group were decreased, and the difference was significant (P<0.001). IL-10 The cytokine levels were slightly higher, and the difference was not significant; compared with the model group, the serum TNF-α cytokine levels of the white rabbits in the probiotic group decreased, and the difference was extremely significant (P<0.001), and the IINF-γ cytokine levels Lower than the modeling group, L-10 cytokine level was slightly higher than the modeling group, the difference was not significant;

4.3 Pathological observation of rabbit ears in each group

The HE staining results of rabbit ears in each group were observed under an optical microscope, as shown in Figure 8.

In the blank group, the epidermis of the rabbit ears was thinner, the hair follicles and the junction between the dermis and the epidermis were clear, sparse mononuclear cell infiltration was seen in the dermis, and there was a sparse single structure in the dermis; in the modeling group, the dermis was hyperkeratinized under light microscope, and the hair follicle orifice and infundibulum were seen. There was accumulation of keratinized substances, the granular layer and acanthus layer of the hair follicle epithelium had obvious hyperplasia, a large number of inflammatory cells infiltrated in the dermis, and the epidermis and dermis were incomplete. The hair follicles are clear, mononuclear cell infiltration can be seen in the dermis, there is a sparse single structure in the dermis, and a small number of hair follicles can be seen dilated and filled with loose keratinized substances. Under the light microscope, the epidermis of the rabbit ears in the probiotic group was thinner, the hair follicles and the junction between the dermis and the epidermis were clear, a large number of mononuclear cells were infiltrated in the dermis, and keratin accumulation was visible in the dermis.

The results of the above animal experiments show that the simultaneous gavage of Bifidobacterium lactis VHProbi O20 and smearing of its lysate on the acne site can effectively relieve the redness and swelling of the acne site, reduce the roughness and hardness of the rabbit ear skin, and the skin lesions symptoms of the rabbit ear acne site. Improved, with clear epidermis and dermis. In addition, Bifidobacterium lactis VHProbi O20 can regulate the level of inflammatory factors in white rabbits, regulate the level of its immune response, and slow down the process of inflammation.

To sum up, the Bifidobacterium lactis VHProbi O20 provided by the present invention has strong tolerance to simulated artificial gastrointestinal fluid, and has strong cell surface hydrophobicity, which is good for probiotics to smoothly pass through the gastrointestinal tract and settle in the colon. Colonization and the development of probiotic functions laid the foundation. At the same time, the hemolytic experiment confirmed that Bifidobacterium lactis VHProbi O20 does not produce hemolysin, is sensitive to common antibiotics, and has good biological safety. Bifidobacterium lactis VHProbi O20 can scavenge free radicals such as DPPH and HRS, and has a certain antioxidant effect. In addition, Bifidobacterium lactis VHProbi O20 can adsorb aflatoxin B1 and has strong anti-lipid peroxidation ability. The rabbit ear acne experiment confirmed that the gavage of Bifidobacterium lactis VHProbi O20 and the application of its lysate at the same time can alleviate the severity of acne and regulate the level of its immune response. Bifidobacterium lactis is the normal intestinal flora of the human body, and its probiotic effect indicates that it can be developed into postbiotic products for use in food and skin care products. In addition, according to the latest taxonomic rules, Bifidobacterium lactis was renamed Bifidobacterium lactis subsp.

sequence listing

<110> Qingdao Weilan Biological Group Co., Ltd.

Qingdao Weilan Biological Co., Ltd.

<120> A strain of Bifidobacterium lactis and its application in the prevention and treatment of acne

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1319

<212> DNA

<213> Bifidobacterium lactis

<400> 1

gcttcgggtg ctacccactt tcatgacttg acgggcggtg tgtacaaggc ccgggaacgc 60

attcaccgcg gcgttgctga tccgcgatta ctagcgactc cgccttcacg cagtcgagtt 120

gcagactgcg atccgaactg agaccggttt tcagcgatcc gccccacgtc accgtgtcgc 180

accgcgttgt accggccatt gtagcatgcg tgaagccctg gacgtaaggg gcatgatgat 240

ctgacgtcat ccccaccttc ctccgagttg accccggcgg tcccacatga gttcccggca 300

tcacccgctg gcaacatgcg gcgagggttg cgctcgttgc gggacttaac ccaacatctc 360

acgacacgag ctgacgacga ccatgcacca cctgtgaacc ggccccgaag ggaaaccgtg 420

tctccacggc gatccggcac atgtcaagcc caggtaaggt tcttcgcgtt gcatcgaatt 480

aatccgcatg ctccgccgct tgtgcgggcc cccgtcaatt tctttgagtt ttagccttgc 540

ggccgtactc cccaggcggg atgcttaacg cgttggctcc gacacgggac ccgtggaaag 600

ggccccacat ccagcatcca ccgtttacgg cgtggactac cagggtatct aatcctgttc 660

gctccccacg ctttcgctcc tcagcgtcag tgacggccca gagacctgcc ttcgccattg 720

gtgttcttcc cgatatctac acattccacc gttacaccgg gaattccagt ctcccctacc 780

gcactccagc ccgcccgtac ccggcgcaga tccaccgtta ggcgatggac tttcacaccg 840

gacgcgacga accgcctacg agccctttac gcccaataaa tccggataac gctcgcaccc 900

tacgtattac cgcggctgct ggcacgtagt tagccggtgc ttattcgaac aatccactca 960

acacggccga aaccgtgcct tgcccttgaa caaaagcggt ttacaacccg aaggcctcca 1020

tcccgcacgc ggcgtcgctg catcaggctt gcgcccattg tgcaatattc cccactgctg 1080

cctcccgtag gagtctgggc cgtatctcag tcccaatgtg gccggtcacc ctctcaggcc 1140

ggctacccgt caacgccttg gtgggccatc accccgccaa caagctgata ggacgcgacc 1200

ccatcccatg ccgcaaaagc atttcccacc ccaccatgcg atggagcgga gcatccggta 1260

ttaccacccg tttccaggag ctattccggt gcacagggca ggttggtcac gcattactc 1319

Claims (10)

1. The bifidobacterium lactis is characterized in that the preservation number of the bifidobacterium lactis is CCTCC NO: m2021598.

2. The Bifidobacterium lactis according to claim 1, wherein the Bifidobacterium lactis has the 16s rDNA sequence of SEQ ID No. 1.

3. Bifidobacterium lactis according to claim 1, wherein the Bifidobacterium lactis has a MALDI-TOF ribosomal protein molecular weight profile as shown in FIG. 2, a Riboprinter fingerprint as shown in FIG. 3, an RAPD fingerprint as shown in FIG. 4 and a rep-PCR fingerprint as shown in FIG. 5.

4. Use of bifidobacterium lactis as claimed in claim 1 for the preparation of a food product, a health product, a pharmaceutical product or a cosmetic product.

5. Use of bifidobacterium lactis as claimed in claim 1 in the preparation of a product having antioxidant properties.

6. Use of bifidobacterium lactis as claimed in claim 1 in the manufacture of a product for use in the prevention or treatment of acne.

7. A probiotic preparation, characterized in that it comprises the live bacterium of Bifidobacterium lactis of claim 1 and/or a fermentation product thereof.

8. A probiotic preparation, characterized in that it comprises a lysate of bifidobacterium lactis according to claim 1.

9. The probiotic preparation according to claim 7 or 8, characterized in that it is a functional food.

10. The probiotic preparation according to claim 8, characterized in that it is a cosmetic preparation.

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