CN114717136A

CN114717136A - Lactobacillus acidophilus with acne treatment effect and application thereof

Info

Publication number: CN114717136A
Application number: CN202111400280.1A
Authority: CN
Inventors: 崔洪昌; 段治; 郭超群; 张景燕; 步欣萍; 吴松洁; 李凯玲
Original assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Current assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Priority date: 2021-11-24
Filing date: 2021-11-24
Publication date: 2022-07-08
Also published as: CN116515666A

Abstract

The invention relates to the technical field of probiotic screening and application, and particularly provides a novel lactobacillus acidophilus strainLactobacillus acidophilus) And applications thereof. The lactobacillus acidophilus is separated from pickle fermentation liquor, and the preservation number is CCTCC NO: m2021595 has effects of relieving skin inflammation, improving skin health, and preventing and relieving acne symptoms.

Description

Lactobacillus acidophilus with acne treatment effect and application thereof

Technical Field

The invention belongs to the technical field of probiotic screening and application, and particularly relates to lactobacillus acidophilus with an acne treatment effect and application thereof.

Background

Acne is a chronic inflammatory skin disease that mainly involves the facial pilosebaceous glands. At present, the pathogenesis of acne is not completely clarified, and the occurrence of acne is mainly related to factors such as hyperseborrhea, blockage of pilosebaceous ducts, bacterial infection and the like. Hyperseborrhea can lead to increased proliferation of propionibacterium acnes, with varying degrees of inflammatory response. Acne hyperseborrhea is largely related to genetic and hormonal abnormalities.

Intestinal microorganisms affect the immune system and inflammatory reactions caused by dysregulated intestinal flora are one of the causes of many skin diseases. Increased permeability of intestinal epithelial cells activates cytokine expression in T cells and regulatory T cell production, leading to systemic inflammation that disrupts skin homeostasis. Proinflammatory cytokines further enhance small intestinal epithelial cell permeability and establish a vicious cycle of systemic inflammation. In addition, harmful metabolites such as phenol and p-toluene produced by certain intestinal microorganisms enter various parts of the body through blood circulation, and when they are accumulated at skin sites, they adversely affect epidermal cell differentiation and skin barrier function.

The world health organization/united nations food and agriculture organization defines probiotics as: viable microorganisms, when administered in sufficient quantities, can provide health benefits to the host. Probiotics and their metabolites have been shown to interact with gut-associated lymphoid tissues, an interaction that is critical in helping the immune system to cope with pathogens or allergens or commensal bacteria. It has been reported that probiotics inhibit the cytokine IL-8 in epithelial cells and keratinocytes, and reduce inflammatory mediators produced by propionibacterium acnes and reduce vasodilation, edema, mast cell degranulation and TNF- α release.

In a prospective, randomized, double-blind placebo-controlled trial, lactobacillus rhamnosus GG group acne patients had a significant improvement in acne after the end of the 12-week trial, as compared to the control group, and a decrease in IGF-1 gene expression and an increase in FoxO1 gene expression, which studies indicated that probiotics could benefit acne patients. In addition, in recent years, researchers find that the post-growth factor formed after the probiotic fermentation is inactivated can still regulate the host immune response and exert health benefits. Seit et al, applied an emollient containing Vitreoscilla filiformis to atopic dermatitis patients, significantly reduced the SCORAD index. Park et al found that the use of lactobacillus emollients significantly reduced the transdermal water loss and visual simulation scores of atopic dermatitis patients. Gueniche et al found that Lactobacillus paracasei accelerated the recovery of damaged skin barriers. After a female with dry sensitive skin such as Puch uses the mixed fermented milk emulsion for 24 weeks, the index of skin percutaneous water loss is reduced, and the cutin barrier function is obviously improved. Thus, probiotic postnatal products may be administered orally or topically or may be used to treat acne.

Disclosure of Invention

The invention aims to provide a new Lactobacillus acidophilus (Lactobacillus acidophilus) and application thereof; the provided lactobacillus acidophilus is separated from the pickle fermentation liquor, and can relieve skin inflammation, improve skin health condition, and prevent and relieve acne symptoms.

The Lactobacillus acidophilus provided by the invention is Lactobacillus acidophilus VHProbi Y21(Lactobacillus acidophilus VHPribo Y21), which is preserved in China center for type culture Collection at 24 months 5 in 2021, and the preservation number is CCTCC NO: m2021595.

The 16s rDNA sequence of the Lactobacillus acidophilus VHProbi Y21 strain is SEQ ID NO. 1.

The MALDI-TOF ribosomal protein molecular weight spectrum of the Lactobacillus acidophilus VHProbi Y21 strain provided by the invention is shown in figure 2; the Riboprinter fingerprint is shown in figure 3; the RAPD fingerprint is shown in figure 4; the rep-PCR fingerprint is shown in FIG. 5.

The lactobacillus acidophilus provided by the invention can be used for preparing functional foods, health products, medicines or cosmetics.

The lactobacillus acidophilus provided by the invention can be used for preparing products with antioxidant function;

the lactobacillus acidophilus provided by the invention can be used for preparing products for preventing or treating acne.

The product is functional food or cosmetic.

The lactobacillus acidophilus VHProbi Y21 provided by the invention has strong tolerance to artificial intestinal juice; the strain is sensitive to common antibiotics such as erythromycin, ampicillin and the like; does not produce hemolysin, can not dissolve blood cells and has good biological safety; the strain has antioxidant capacity, the lipid peroxidation inhibition rate of thalli is 9.7%, and the supernatant is 15.9%; DPPH clearance rate reaches 20.5 percent, HRS clearance rate reaches 45.75 percent; the degradation rate of cholesterol is 22.2%; in addition, the adsorption capacity of the strain aflatoxin B1 is 15.30%; the hydrophobicity of the cell surface is 11.78%; the skin cell adhesion was 3.78.

The lactobacillus acidophilus VHProbi Y21 and the lysate thereof can effectively relieve the red and swollen degree of rabbit ear acne, reduce the roughness and hardness of the skin of the rabbit ear acne part and improve the skin damage symptom of the rabbit ear acne part, and the rabbit ear skin has a relatively complete epidermal layer and a relatively complete dermal layer. The lactobacillus acidophilus VHProbi Y21 can also obviously reduce the level of proinflammatory cytokines in the white rabbits, regulate the immune response of the white rabbits and slow down the development process of acne inflammation. The lactobacillus acidophilus VHProbi Y21 has the effect of relieving the acne equivalent to that of a chemical agent.

The lactobacillus acidophilus VHProbi Y21 provided by the invention has no toxic action on organisms, can be added into cosmetics for treating acne, and has wide application prospect.

Drawings

FIG. 1 is a photograph of a crystal violet staining microscope and a photograph of a single colony of strain Y21;

FIG. 2 is MALDI-TOF ribosomal protein fingerprint of strain Y21;

FIG. 3 is a Riboprinter fingerprint of strain Y21;

FIG. 4 is RAPD fingerprint of strain Y21;

FIG. 5 is a rep-PCR fingerprint of strain Y21;

FIG. 6 is a comparison of acne symptoms in rabbit ears for each group;

FIG. 7 is a comparison of the results of cytokine detection in each group;

FIG. 8 shows the pathological section results of rabbit ears of each group.

Detailed Description

The lactobacillus acidophilus VHProbi Y21 provided by the invention meets the requirements of regulations, and the lactobacillus acidophilus VHProbi Y21 is a newly discovered strain through heterogeneous taxonomic identification. The lactobacillus acidophilus VHProbi Y21 provided by the invention can effectively prevent and relieve acne, and has a relieving effect on acne by singly using the strain without compounding with prebiotics and/or other probiotics; has important application value.

The lactobacillus acidophilus VHProbi Y21 is preserved in China center for type culture Collection of Wuhan university at 24/5/2021 by the applicant, and the preservation number is CCTCC NO: m2021595.

The screening method of the present invention is not limited to the examples, and any known method capable of achieving the screening purpose is possible, and the screening description of the examples is only illustrative of the present invention and is not limiting the scope of the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

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

Example 1 isolation screening of Lactobacillus acidophilus VHProbi Y21

1. Preliminary screening

Preparing MRS agar culture medium, adjusting pH to 6.2-6.5, and autoclaving at 121 deg.C for 15 min.

Taking 1g of fresh pickle fermentation liquor, diluting the fresh pickle fermentation liquor by using sterile normal saline, putting the diluted pickle fermentation liquor into a sterile sample bag, beating the sterile sample bag by using a homogenizer and uniformly mixing; and (3) taking 100 mu L of the uniformly mixed solution, diluting in a gradient manner, coating the uniformly mixed solution on an MRS agar culture medium, performing anaerobic culture at 37 ℃ for 48h, and performing microscopic examination on a single colony grown on a plate. According to microscopic results, the applicant screened 25 potential lactobacilli in total, which are named as Y01, Y02, … …, Y20, Y21, Y22, Y23, Y24 and Y25.

2. Double sieve

Preparing 1L MRS liquid culture medium, autoclaving at 121 deg.C for 15min, cooling, adding 3.2g pig mucosa pepsin, shaking for dissolving, and placing in 37 deg.C water bath shaker for 1 hr to obtain acid-resistant culture medium.

Respectively inoculating 25 screened lactobacillus strains Y01, Y02, … …, Y20, Y21, Y22, Y23, Y24 and Y25 into the acid-resistant culture medium according to 6 percent of inoculation amount, carrying out anaerobic standing culture at 37 ℃ for 48h, and taking and counting the bacterial amount of the fermentation liquor.

The result shows that the Y21 strain has the largest viable bacteria amount after being re-screened by the acid-resistant culture medium in the Log values of the viable bacteria amount in the 25 strains of lactobacillus fermentation liquor, and the Log value is as high as 9.50Log CFU/mL. Thus indicating that the Y21 strain has the highest acid resistance.

Example 2 Strain identification

1. Colony morphology identification

Inoculating Y21 strain on MRS agar medium, anaerobically culturing at 37 deg.C for 24 hr to obtain Y21 single colony which is milky white, has a diameter of about 2-3mm, and has a smooth and round surface, and is stained with crystal violet to obtain Brevibacterium under microscope.

2. Physiological and biochemical characteristic identification

The inoculation solution in this example was prepared as follows: under the aseptic condition, taking a proper amount of fresh Y21 bacterial liquid, centrifuging at 5000rpm/min for 5min, washing with PBS buffer for 2 times, and diluting by 50 times after thalli are weighed by the same volume of PBS buffer to be used as inoculation liquid.

2.1 temperature resistance test

Taking a proper amount of fresh bacterial liquid (24h, 37 ℃), centrifuging for 5min at 5000rpm, washing with the PSP solution once, resuspending with the same volume of the PSP solution, and diluting by 50 times to obtain inoculation liquid.

Inoculating the inoculum solution into 10mL of MRS liquid culture medium according to the inoculation amount of 10%, using 5mL of MRS liquid culture medium without inoculation as a control, respectively culturing in a 15 ℃ constant-temperature incubator for 7 days, culturing in a 45 ℃ constant-temperature incubator for 2 days, and observing whether the bacterial solution becomes turbid.

The results showed that the Y21 strain did not grow at 15 ℃ and proliferated in large amounts at 45 ℃.

2.2 salinity tolerance test

Under sterile conditions, 190 μ L of BSM liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% was added to 96-well plates, 3 replicates per salt concentration, followed by 10 μ L of inoculum solution, and wells without inoculum were used as controls. 50 μ L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37 deg.C, and observing whether the culture medium turns turbid.

The results showed that the maximum salt concentration tolerated by the strain Y21 was 2%.

2.2 carbon source metabolism test

The basic medium formulation used in this example was as follows:

peptone 1.5 g; yeast extract 0.6 g; tween 800.1 g; 0.5mL of salt solution; 18mg of phenol red; 100mL of distilled water; pH 7.4. + -. 0.2. Salt solution composition: MgSO4 & 7H2O 11.5.5 g, MnSO4 & 4H2O2.8g and distilled water 100 mL.

A10 g/100mL solution of sugars, alcohols and carbohydrates of the glycosides was prepared and filtered through a 0.22 μm sterile filter. Under aseptic conditions, 20 μ L of sterilized carbohydrate solution, 4 replicates of each carbohydrate, was added to a 96-well plate, followed by 170 μ L of sterilized phenol red-containing basal medium, followed by 10 μ L of inoculum, and no-inoculum reaction wells served as controls. 50 μ L of liquid paraffin was added to each well to prevent evaporation of water during the culture. Anaerobic culture at 37 ℃, taking phenol red as an indicator, and observing the color change of the culture medium; specific results are shown in table 1.

TABLE 1 carbon source metabolism results of Y21 Strain

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

2.3 acid and gas evolution test for glucose

The media formulations used in this example were as follows:

peptone 0.5 g; yeast extract 0.3 g; tween 800.1 mL; salt solution A0.5mL; 0.5mL of saline solution B; 0.5g of sodium acetate; 2.5g of glucose; 0.05mL of 2% bromocresol green (w/v); 100mL of distilled water; the pH value is 6.8-7.0.

The prepared culture medium is subpackaged into large test tubes containing inverted small test tubes, 3 mL/tube, and autoclaved for 15min at 121 ℃.

Salt solution A composition: KH (Perkin Elmer)₂PO₄ 10g、K₂HPO₄1.0g, dissolved in distilled water and fixed to 100 mL.

Saline solution B composition: MgSO (MgSO)₄·7H₂O 11.5g、MnSO₄·2H₂O 2.4g、FeSO₄·7H₂0.68g of O, dissolving in distilled water, and fixing the volume to 100 mL.

Under aseptic conditions, inoculating the inoculum with 10% of inoculum size to culture medium, using non-inoculated culture medium as control, sealing the top with 2mL of sterile liquid paraffin, culturing at 37 deg.C for 24h, and observing whether the culture medium color changes.

The results show that: after culturing for 24h at 37 ℃, the culture medium changes from green to yellow, and no gas is in the small inverted tube, which indicates that the Y21 strain ferments glucose to produce acid and does not produce gas.

2.4 arginine Ammonia production test

Adding prepared arginine solution into PY basic culture solution.

The arginine solution comprises the following components:

1.5g of arginine; cysteine (1g/10mL H₂O)0.05 mL; 10mL of distilled water;

the pH was adjusted to 7.0, sterilized and 3 drops were added to 3mL of the medium.

The inoculum was inoculated at 10% inoculum size in 10mL arginine-containing medium, and simultaneously inoculated with arginine-free medium as a control. Culturing at a proper temperature for 1-3 days.

And (3) dropping 1 drop of the grown culture solution on the surface of a clean glass slide, adding 1-2 drops of Neisseria reagent, and if the yellow color is changed into brown color, performing positive reaction to produce ammonia.

The results show that: the detection result of the Neisseria reagent is negative, which indicates that the Y21 strain does not produce ammonia.

3. Molecular biological identification

3.116 s rDNA Gene sequence analysis

3.1.1 extraction of genomic DNA

Reference was made to the Tiangen bacterium genomic DNA extraction kit (catalog No.: DP 302).

3.1.2, 16s rDNA Gene amplification

The primer sequence is as follows:

27F：AGAGTTTGATCCTGGCTCA；

1492R：GGTTACCTTGTTACGACTT。

the 16s rDNA sequence SEQ ID NO 1 of the Y21 strain is obtained by sequencing and is compared in the NCBI database, and the Y21 strain is preliminarily determined to be lactobacillus acidophilus.

3.2 Riboprinter fingerprint

And (3) dipping the purified single colony from an agar culture medium plate by using a bacteria taking rod, putting the single colony into a sample tube with a buffer solution, stirring the single colony in the buffer solution by using a handheld stirrer to enable the single colony to be suspended in the buffer solution, putting a sample rack into a heater to inactivate, putting the sample rack into a Riboprinter system, and obtaining a bacteria identification result after DNA preparation, membrane conversion, imaging detection and data processing of the sample. The identification result shows that the Y21 strain is Lactobacillus acidophilus, and the Riboprinter fingerprint result is shown in figure 2.

3.3 MALDI-TOF-MS detection of ribosomal protein expression of strains

Inoculating fresh bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 0.1%, culturing at 37 ℃ and 150rpm for 48h, collecting the thalli, washing with sterile water for 4 times, and airing the surface water. Then taking a small amount of fresh thalli to be evenly coated on a target plate in a film form, adding 1 mu L of lysate to cover the sample, airing, then adding 1 mu L of matrix solution to cover the sample, airing, and then putting the sample target into a mass spectrometer for identification. Irradiating a cocrystallized film formed by the sample and the matrix with laser to ionize protein in the sample, accelerating the ions to fly through a flight pipeline under the action of a 10-20 KV electric field, and detecting the molecular weight of the protein according to different flight times of the ions reaching a detector. The protein fingerprint is obtained by using an Autof Analyzer v1.0 analysis software of Autofms 1000, and the ion peaks of the main ribosomal proteins of the Y21 strain are as follows: m/z2599.217, 3124.057, 3457.129, 4399.116, 5197.056, 5325.093, 6246.762, 6947.835, 7638.294. The results of the identification are shown in FIG. 3.

3.4 RAPD and rep-PCR fingerprinting

3.4.1 RAPD fingerprint identification

1) The primer sequence is as follows: GAGGGTGGCGGTTCT are provided.

2) RAPD reaction system

TABLE 2 RAPD reaction System

3) Electrophoresis

Preparing a 1.5% agarose gel plate, using DL2000 DNA Marker as result contrast, performing electrophoresis at 100V under a stable pressure for 80min, and finally detecting an electrophoretogram by using a gel imaging system. The RAPD fingerprint of the Y21 strain is shown in FIG. 4.

3.4.2 rep-PCR fingerprint

1) The primer sequence is as follows: CTACGGCAAGGCGACGCTGACG are provided.

2) rep-PCR reaction system

TABLE 3 reaction System for rep-PCR

3) Electrophoresis

DL2000 DNA Marker was used as a result control. The voltage is 100V, and the electrophoresis time is 80min to detect the amplification result. The rep-PCR fingerprint of the Y21 strain is shown in FIG. 5.

In conclusion, the results of colony morphology and physiological and biochemical characteristics of strain Y21 were uploaded to the website http:// www.tgw1916.net/bacterial _ logore _ desktop. htmL, and compared with the results published in De Clerck E, et al. By combining the identification results of molecular biology, the conclusion can be drawn that the strain Y21 is a new lactobacillus acidophilus strain which is named as lactobacillus acidophilus VHProbi Y21.

Example 3 tolerance test of Lactobacillus acidophilus VHProbi Y21 to artificial gastric and intestinal fluids

1. Preparation of artificial gastric juice

Separately, 5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl were weighed, 1000mL of distilled water was added, pH3.0 was adjusted with dilute hydrochloric acid, and then sterilization was carried out at 115 ℃ for 20 min. Then 3.2g of pig mucosa pepsin is added before use, shaken up and dissolved, and placed in a water bath shaker at 37 ℃ for a water bath for 1h to simulate the temperature of a human body.

2. Preparation of artificial intestinal juice

Separately weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH₂PO₄6.8g and 3.0g of ox-gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is adjusted to 1000mL, the pH value is adjusted to 6.8 +/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then 1g pancreatin is added before use, shaken up and dissolved, and put into a water bath shaker at 37 ℃ for water bath for 1h to simulate the temperature of a human body.

3. Test method

2mL of fresh bacterial liquid is taken, centrifuged at 5000rpm/min for 5min to collect thalli, the thalli are washed for 3 times by using normal saline, and then 2mL of normal saline is used for resuspension to serve as inoculation liquid. Taking 1mL of inoculation liquid, adding the inoculation liquid into 24mL of artificial intestinal juice, placing the artificial intestinal juice in a water bath shaker (200rpm/min) at 37 ℃ for 3h, sampling 1mL, and detecting the amount of live bacteria.

The viable bacteria counting method is used for measuring the bacterial quantity according to the national standard GB 4789.35-2016-food microorganism test lactobacillus test, and the viable bacteria quantity (Log CFU/mL) of the bacterial strain after being digested by artificial intestinal juice is shown in the table 4.

TABLE 4 viable cell count after digestion of artificial gastrointestinal fluids

As can be seen from Table 4, after the Lactobacillus acidophilus VHProbi Y21 screened by the method is digested by artificial gastric juice, the viable cell amount slightly increases, which indicates that the strain can tolerate the artificial gastric juice and germinate. After the artificial intestinal juice is reduced by 1.23Log value, the tolerance capability of the artificial intestinal juice is better.

Example 4 antibiotic resistance test of Lactobacillus acidophilus VHProbi Y21

1. Preparing antibiotics: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin were all prepared into 2048 μ g/mL stock solutions, and stored at-20 deg.C for further use. When in use, the stock solution is serially diluted into a use solution by 2 times of BMS liquid culture medium, and the gradient dilution concentration is 1-1024 mu g/mL for 11 gradients.

2. Preparing an inoculation solution: preparing inoculation liquid: taking a proper amount of fresh bacterial liquid (cultured for 24-48 h at 40 ℃), centrifuging for 5min at 5000rpm, washing once with sterile normal saline, and then diluting by 50 times after resuspending the bacteria with the same volume of normal saline to serve as inoculation liquid.

3. Minimal inhibitory concentration MIC of antibiotics to Lactobacillus acidophilus VHProbi Y21 was determined by broth dilution.

and a.1, adding BMS liquid culture medium without antibiotics into the 1 st row of the 96 pore plate, using the BMS liquid culture medium as a negative control, sequentially adding 190 mu L of BMS liquid culture medium containing antibiotics with different concentrations into the 2 nd to 12 th rows, then respectively inoculating 10 mu L of the inoculation liquid, making 3 parallel pores, and using 1 pore without adding bacteria liquid as a blank.

b. Add 50. mu.L paraffin oil blanket to prevent water evaporation.

c. And taking out the 96-well plate after shaking culture at 40 ℃ for 48h, measuring the OD600 value, and counting the MIC value of the antibiotic to the strain by using the result of 48h, wherein the specific result is shown in Table 5.

TABLE 5 antibiotic MIC values of Lactobacillus acidophilus VHProbi Y21

MIC unit μ g/mL

The results in table 5 show that lactobacillus acidophilus VHProbi Y21 provided by the present invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biological safety.

Example 5 Lactobacillus acidophilus VHProbi Y21 assay for antioxidant function

1. Determination of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) and Hydroxyl Radical (HRS) removing capability of strain

Determination of DPPH free radical scavenging ability of strain

Taking 1mL of PBS bacterial suspension of the strain to be detected, adding 1mL of 0.4mM of ready-prepared DPPH free radical solution, uniformly mixing, then placing at room temperature for shading reaction for 30min, then measuring the absorbance A sample of the sample at the wavelength of 517nm, and measuring for 3 times of parallelism. The control group samples were blank-zeroed with equal volumes of PBS solution and DPPH-ethanol mixture, and equal volumes of PBS suspension and ethanol mixture. The clearance rate is calculated according to the following formula: clearance%_{Sample (I)}-A_{Blank space})/A_Control]X 100%. The results are shown in Table 6.

TABLE 6 DPPH radical scavenging Table

3) Determination of HRS-removing ability of Strain

mu.L of 5mM sodium salicylate-ethanol solution, 100. mu.L of 5mM ferrous sulfate, 500. mu.L deionized water and 200. mu.L of lactic acid bacteria PBS suspension were mixed, 100. mu.L of hydrogen peroxide solution (3mM) was added, and absorbance of the sample was measured at a wavelength of 510nm after water bath at 37 ℃ for 15 min. The hydroxyl radical clearance was calculated according to the following formula.

Clearance%_{Sample (I)}-A_{Control of})/(A_{Blank space}-A_{Control of}) X 100% where A_{Control of}To replace the sample with deionized water, A_{Blank space}Replacement of sample and H for deionized Water₂O₂The results are shown in Table 7.

TABLE 7 HRS radical scavenging Table

2. Experimental identification of bacterial strain for resisting lipid peroxidation

Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water. 0.5mL of PBS solution (pH 7.4) was added 1mL of an emulsion of linoleic acid, 1mL of LFeSO₄(1%), adding 0.5mL of sample, carrying out water bath at 37 ℃ for 1.5h, adding 0.2mL of TCA (4%), 2mL of TBA (0.8%), carrying out water bath at 100 ℃ for 30min, rapidly cooling, centrifuging at 4000rpm/min for 15min, collecting supernatant, and measuring the absorbance at 532nm to obtain A; the control group uses 0.5mL of distilled water to replace the sample to obtain A₀. Inhibition rate/% (A)₀－A)/A₀×100％

Note: a is the absorbance of the sample group; a. the₀The results are shown in Table 8, which is the absorbance of the control group.

TABLE 8 inhibition of lipid peroxidation

Example 6 Lactobacillus acidophilus VHProbi Y21 in vitro Cholesterol degradation experiment

1. Preparation of cholesterol micelle solution: accurately weighing 1g of cholesterol, dissolving in absolute ethyl alcohol, diluting to 100mL, and filtering and sterilizing with a 0.22-micron microporous filter membrane under aseptic condition.

2. 10.0g of peptone and 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 801.0 mL of Tween, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water are weighed, the pH value is adjusted to 7.3, sterilization is carried out at 115 ℃ for 30min, and then cholesterol solution is added to ensure that the final concentration of cholesterol is 0.1%. Inoculating fresh bacterial liquid according to the inoculation amount of 0.1%, performing static culture at 37 ℃ for 48h, then taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10min, centrifuging for 5min at 3000 rpm, and taking supernatant for measuring the cholesterol content. Method for measuring cholesterol GB/T5009.128-2003< determination of cholesterol in food >.

The results show that: the degradation rate of lactobacillus acidophilus VHProbi Y21 on cholesterol provided by the invention reaches 22.02% (this is data without bile salt).

Example 7 Aflatoxin B1 adsorption Capacity of Lactobacillus acidophilus VHProbi Y21

1. AFB1-PBS solution was prepared at a concentration of 1. mu.g/mL.

2. Inoculation and adsorption: taking 1mL of fresh bacterial liquid (24h, 37 ℃), centrifuging at 8000rpm for 5min, removing the supernatant, washing the thalli with PBS buffer solution with the same volume for 2 times, centrifuging at 8000rpm for 5min, removing the supernatant, then suspending the thalli in 1mL of AFB1-PBS solution, placing the thalli in a constant temperature incubator at 37 ℃, taking out the thalli after 1h, centrifuging at 8000rpm for 10min, and taking the supernatant to be tested.

3. The aflatoxin B1 concentration in the supernatant was determined according to the instructions of the aflatoxin B1 test kit.

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

The results show that: the adsorption capacity of aflatoxin B1 of lactobacillus acidophilus VHProbi Y21 provided by the invention is 15.30%, and the standard deviation is 0.02%.

Example 8 hydrophobic cell surface assay for Lactobacillus acidophilus VHProbi Y21

1. Preparing bacterial liquid to be detected: and (3) selecting a purified lactobacillus acidophilus VHProbi Y21 colony, inoculating the colony in a newly prepared MRS liquid culture medium, and culturing at 37 ℃ for 24-48 h. Inoculating to MRS liquid culture medium according to the inoculation amount of 1% (V/V), continuously culturing at 37 deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile normal saline for 2 times, and sterilizing with 0.1M KNO₃1mL of the solution was used as a suspension of the cells.

2. Surface hydrophobicity measurement: sucking 50 μ L of the above bacterial suspension, adding 2450 μ L of 0.1M KNO₃And record OD600 as A₀1.5mL of the suspension was mixed with 500. mu.L of xylene and allowed to stand at room temperature for 10min (a two-phase system was formed). And (3) vortexing and oscillating the two-phase system for 2min, and then standing for 20min to form a water phase and an organic phase again. The absorbance A was measured at 600nm by carefully taking up the aqueous phase (not taking up the organic phase)₁. Hydrophobicity of cells expressed by the formula Hydrophobicity%₀-A₁)/A₁Calculated as% x, three experiments were measured and averaged.

The results show that: the cell surface hydrophobicity of the lactobacillus acidophilus VHProbi Y21 provided by the invention is 11.78%, and the standard deviation is 0.57%.

Example 9 skin cell adhesion test of Lactobacillus acidophilus VHProbi Y21

Hacat cells at 2X 10⁶Inoculating the inoculation amount of cells/hole in a six-hole plate, and culturing for 24h in a carbon dioxide incubator for cell adhesion experiments;

the stationary phase strains were resuspended to 5X 10 with MRS medium⁷CFU/mL；

Adding 1mL of the strain into a six-hole plate with cells attached to the wall, and culturing for 2h in a carbon dioxide incubator;

washing with PBS for 3 times to remove non-adhered bacteria;

adding 500ul of pancreatin for digestion for 3 minutes, adding 1.5mL of cell culture solution to stop digestion, repeatedly blowing, collecting the obtained solution into a sterile EP tube, performing gradient dilution on the collected solution by 10 times, 100 times, 1000 times and 10000 times, and coating plates for counting. The cells of the blank group were counted simultaneously. The adhesive ability of the test strain was calculated according to the following formula:

capacity for attachment (CFU/cells) — total number of bacteria attached per culture well/total number of cells per culture well.

The results show that: the cell adhesion of the lactobacillus acidophilus VHProbi Y21 provided by the invention is 3.78, and the standard deviation is 0.20%.

Example 10 Lactobacillus acidophilus VHProbi Y21 and its lysate for alleviating acne in rabbit ears

1 Experimental animal treatment and model construction

1.1 Experimental consumables

TABLE 9 Experimental consumables

1.2 preparation of fresh bacterial liquid and lysate

Lactobacillus acidophilus VHProbi Y21 was inoculated into freshly cultured MRS medium and used for intragastric gavage after anaerobic culture at 37 ℃ for 12 h. Crushing fresh cultured bacteria liquid by a high-pressure homogenizer under the pressure of 100MPa, repeatedly homogenizing for 3 times, putting the mixture into a 70 ℃ water bath kettle, and completely inactivating the mixture to prepare lysate.

1.3 treatment of laboratory animals

Clean-grade New Zealand white rabbits, male rabbits and 24 rabbits, are randomly divided into 4 groups, 6 rabbits in each group, and are provided by the Qinglongshan animal breeding center. The special pellet feed for the experimental rabbits is used for feeding the experimental rabbits, the experimental rabbits are fed in clean animal rooms at the temperature of 18-20 ℃ and the relative humidity of 40 percent, the experimental rabbits are fed in cages, 1 animal is randomly placed in each cage, and the experimental rabbits can freely eat drinking water.

After the adaptive breeding of the white rabbits is finished, the white rabbits are respectively a blank group, a building group, a positive group and a probiotic group. Wherein, the blank group is not treated, and is perfused with stomach and smeared with normal saline in the whole process; the building module, the positive group and the probiotic group adopt propionibacterium acnes and oleic acid to build an acne model, the building module and the positive group are treated by normal saline and adapalene gel respectively after modeling, and the probiotic group adopts lactobacillus acidophilus VHProbi Y21 and lysate thereof. Animals were followed and treated strictly according to animal ethics throughout the experimental period.

The specific treatment method comprises the following steps:

on the left and right external auditory canals of rabbit ears, 0.1mL of 25% oleic acid was uniformly applied with a 0.25mL applicator for 1 time every other day for 50 consecutive days. Counting the cultured Propionibacterium acnes, and adjusting the concentration to 10⁶one/mL for use. On day 20 of oleic acid application, 50 ul/ear of acnes was injected intradermally and marked at the injection site for a total of one injection。

(1) Blank group: after the adaptability is finished, the stomach is irrigated with 10mL of normal saline every day, 1mL of normal saline is smeared on the external auditory meatus, and the test is finished from morning to evening to 50 th day. Oleic acid was not coated;

(2) building a module: the stomach is irrigated with 10mL of normal saline every day, the external auditory canal is smeared with 1mL of normal saline to cover the acne part of the external auditory canal, and the test is finished from morning to evening to 50 days. Coating oleic acid, and injecting propionibacterium acnes;

(3) positive group: the stomach is irrigated with 10mL of normal saline every day, 1g of adapalene gel is smeared to cover the acne part of the external auditory canal, and the test is finished from morning to night to 50 days. Coating oleic acid, and injecting propionibacterium acnes;

(4) probiotic group: gavage 1X 10 per day⁹10mL of the bacterial liquid of the CFU/mL lactobacillus acidophilus VHProbi Y21, and 1mL of the lysate of the lactobacillus acidophilus VHProbi Y21 are smeared to cover the acne part of the external auditory canal, and the test is finished from once in the morning to once in the evening to the 50 th day. Coating oleic acid, and injecting propionibacterium acnes;

2 index detection

After the test on the 50 th day is finished, the swelling condition of the rabbit is observed, the smearing treatment effect of the probiotics is evaluated by adopting an acne skin lesion counting method, and the skin condition of the rabbit is observed from the aspects of hardness, thickness, roughness and the like of the rabbit.

TABLE 10 evaluation criteria for acne model observation

And (3) extracting venous blood and detecting the contents of IL-6, TNF-alpha and IL-1 beta cytokines in the white rabbit serum by an ELISA method.

Rabbit ear tissue was sampled and made into paraffin sections of 5 μm thickness for HE staining. And judging the pathological change of the specimen tissue according to the histopathological judgment grading standard of the experimental acne and the epidermal thickening condition, the expansion degree of the hair follicle opening and the amount of the generated keratinized angle of the model animal.

3 data analysis

Data analysis was performed using SPSS 22.0 statistical software and results are presented as mean ± sem. The comparison between two groups adopts independent sample t test, the difference comparison between the groups adopts single-factor variance analysis, pairwise comparison is further carried out by a least significant difference method when the variances are uniform, and rank sum test is adopted when the variances are not uniform. p <0.05 indicates that the difference is significant, and p <0.01 indicates that the difference is very significant. Plotting was performed using GraphPad Prism 8.0.

4. Results of the experiment

4.1 general State Observation of Each group of white rabbits at the terminal stage

The typical rabbit ear observation results for each group are shown in FIG. 6, and the appearance observation scores for each group are shown in Table 11.

The results show that: after the administration on day 50, the rabbit ears in the blank group are smooth and soft in appearance, the rabbit ears are transparent, no angle plug is formed, and the capillary vessels are clearly visible; rabbit ears in a modeling group can be seen to have obvious red swelling and skin damage, the thickness is increased, and phenomena of scabbing, drying and peeling exist, which indicates that the modeling is successful; the rabbit ears in the positive group are cornified, the rabbit ears are thickened, hair follicles are thickened, and black heads appear. The transmittance of the probiotics group rabbit ears is improved, no skin damage or pustule appears, the red and swollen degree is reduced, the blackheads are reduced, and the hardness and roughness are relieved. Therefore, the lactobacillus acidophilus VHProbi Y21 and the lysate thereof provided by the invention can effectively relieve the severity of rabbit ear acne and improve the acne skin state.

TABLE 11 Rabbit ear appearance Scoring Scale

4.2 ELISA test results of serum of various groups of white rabbits at terminal stage

After the experiment was completed, the level of three proinflammatory cytokines in serum was varied as shown in FIG. 7.

The results show that the serum TNF-alpha, IL-6 and IL-1 beta cytokines of the white rabbits of the modeling group are increased compared with the blank group, and the difference is very significant (P <0.001), which indicates that the modeling of the model is successful. Compared with the modeling group, the serum TNF-alpha cytokine level of the white rabbits of the positive group is reduced, and the difference is significant (P < 0.05); IL-6 cytokine levels were reduced with insignificant differences; IL-1. beta. cytokine levels were reduced, with the difference being very significant (P < 0.001). Compared with the modeling group, the level of TNF-alpha cytokines in the serum of the white rabbits in the probiotic group is reduced, and the difference is significant (P < 0.05); IL-6 cytokine levels were reduced with insignificant differences; IL-1. beta. cytokine levels were reduced, with the difference being very significant (P < 0.001). Therefore, the lactobacillus acidophilus VHProbi Y21 provided by the invention can effectively reduce the level of proinflammatory cytokines in serum and slow down the inflammatory process of acne, and the effect is basically equivalent to that of chemical agent adapalene gel.

4.3 pathological observation of rabbit ears in each group

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

The ear epidermal layer of the blank group of rabbits is thin, hair follicles and junctions of dermis and epidermis are clear, sparse mononuclear cell infiltration can be seen in the dermis, and a sparse single structure is formed in the dermis; incomplete epidermis and dermis layers, hyperkeratosis of the dermis, enlargement of funnel parts and hyperkeratosis can be seen under a module building light mirror, inflammatory cells are infiltrated in the dermis, and the hyperplasia of a granular layer and a spinous layer of the hair follicle epithelium is obvious; the positive group of rabbits had thin ear epidermis, clear junction between dermis and epidermis and hair follicles, and mononuclear cell infiltration was observed in the dermis and was filled with loose keratinized substances. Under a light microscope, the ear epidermal layer of the probiotic group rabbit is thin, hair follicles and the junction of dermis and epidermis are clear, mononuclear cell infiltration can be seen sparsely in the dermis, and loose keratinized substances can be seen in the dermis. Therefore, the lactobacillus acidophilus VHProbi Y21 and the lysate thereof provided by the invention are beneficial to promoting the complete recovery of the structures of the epidermal layer and the dermal layer of rabbit ear skin, relieving the keratinization phenomenon and effectively relieving the inflammation degree, and the effect is equivalent to that of chemical agent adapalene gel.

The lactobacillus acidophilus VHProbi Y21 provided by the invention has strong tolerance to simulated artificial intestinal gastric juice and strong cell surface hydrophobicity, which lays a foundation for probiotics to successfully pass through gastrointestinal tract and colonize colon and exert probiotic function. Meanwhile, a hemolytic experiment proves that the lactobacillus acidophilus VHProbi Y21 does not produce hemolysin, is sensitive to common antibiotics and has good biological safety. Lactobacillus acidophilus VHProbi Y21 can remove DPPH and HRS free radicals, and has certain antioxidant effect. In addition, Lactobacillus acidophilus VHProbi Y21 can adsorb aflatoxin B1 and has strong anti-lipid peroxidation capability. The rabbit ear acne experiment proves that the severity of acne can be relieved by gavage lactobacillus acidophilus VHProbi Y21 and the lysate can be applied at the same time, and the immunoreaction level of the acne can be adjusted. Lactobacillus acidophilus belongs to the traditional lactobacillus probiotics, has a long use history, and the probiotic effect shows that the Lactobacillus acidophilus can be developed into a post-biotic product to be applied to food and skin care products.

Sequence listing

<110> Islands blue biological group Co Ltd

QINGDAO VLAND BIOTECH Inc.

<120> lactobacillus acidophilus with acne treatment effect and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1376

<212> DNA

<213> Lactobacillus acidophilus (Lactobacillus acidophilus)

<400> 1

tttgggcatt gcagacttcc atggtgtgac gggcggtgtg tacaaggccc gggaacgtat 60

tcaccgcggc gttctgatcc gcgattacta gcgattccag cttcgtgcag tcgagttgca 120

gactgcagtc cgaactgaga acagctttca gagattcgct tgccttcgca ggctcgcttc 180

tcgttgtact gcccattgta gcacgtgtgt agcccaggtc ataaggggca tgatgacttg 240

acgtcatccc caccttcctc cggtttgtca ccggcagtct cattagagtg cccaacttaa 300

tgctggcaac taataataag ggttgcgctc gttgcgggac ttaacccaac atctcacgac 360

acgagctgac gacagccatg caccacctgt cttagcgtcc ccgaagggaa ctcctaatct 420

cttaggatgg cactagatgt caagacctgg taaggttctt cgcgttgctt cgaattaaac 480

cacatgctcc accgcttgtg cgggcccccg tcaattcctt tgagtttcaa ccttgcggtc 540

gtactcccca ggcggagtgc ttaatgcgtt agctgcagca ctgagaggcg gaaacctccc 600

aacacttagc actcatcgtt tacggcatgg actaccaggg tatctaatcc tgttcgctac 660

ccatgctttc gagcctcagc gtcagttgca gaccagagag ccgccttcgc cactggtgtt 720

cttccatata tctacgcatt ccaccgctac acatggagtt ccactctcct cttctgcact 780

caagaaaaac agtttccgat gcaattcctc ggttaagccg agggctttca catcagactt 840

attcttccgc ctgcgctcgc tttacgccca ataaatccgg acaacgcttg ccacctacgt 900

attaccgcgg ctgctggcac gtagttagcc gtgactttct ggttgattac cgtcaaataa 960

aggccagtta ctacctctat ccttcttcac caacaacaga gctttacgat ccgaaaacct 1020

tcttcactca cgcggcgttg ctccatcaga cttgcgtcca ttgtggaaga ttccctactg 1080

ctgcctcccg taggagtttg ggccgtgtct cagtcccaat gtggccgttc agtctctcaa 1140

ctcggctatg catcattgcc ttggtaagcc gttaccttac caactagcta atgcaccgcg 1200

gggccatccc atagcgacag cttacgccgc cttttataag ctgatcatgc gatctgcttt 1260

attatccggt attagcacct gtttccaagt ggtatcctag actatggggc aggttcccca 1320

cgtgttactc acccatccgc cgctcgcgtc cccagcgtca ttaccgaagt aaatct 1376

Claims

1. The lactobacillus acidophilus is characterized in that the preservation number of the lactobacillus acidophilus is CCTCC NO: m2021595.

2. The lactobacillus acidophilus according to claim 1, characterized in that the lactobacillus acidophilus has the 16s rDNA sequence of SEQ ID No. 1.

3. The lactobacillus acidophilus according to claim 1, wherein the lactobacillus acidophilus has a MALDI-TOF ribosomal protein molecular weight profile as shown in fig. 2, a Riboprinter fingerprint as shown in fig. 3, a RAPD fingerprint as shown in fig. 4 and a rep-PCR fingerprint as shown in fig. 5.

4. Use of lactobacillus acidophilus according to claim 1 for the preparation of food products, health products, pharmaceutical products or cosmetics.

5. Use of lactobacillus acidophilus according to claim 1 for the preparation of a product with antioxidant function.

6. Use of lactobacillus acidophilus according to claim 1 for the preparation of a preparation intended for the prevention or treatment of acne.

7. A probiotic preparation, characterized in that it comprises the live Lactobacillus acidophilus strain according to claim 1 and/or its fermentation product.

8. A probiotic preparation, characterized in that it comprises a lysate of lactobacillus acidophilus 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.