CN117286043A - Lactobacillus salivarius capable of relieving symptoms of atopic dermatitis and application thereof - Google Patents
Lactobacillus salivarius capable of relieving symptoms of atopic dermatitis and application thereof Download PDFInfo
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- CN117286043A CN117286043A CN202210689815.XA CN202210689815A CN117286043A CN 117286043 A CN117286043 A CN 117286043A CN 202210689815 A CN202210689815 A CN 202210689815A CN 117286043 A CN117286043 A CN 117286043A
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- lactobacillus salivarius
- atopic dermatitis
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus salivarius (Lactobacillus salivarius) capable of relieving symptoms of atopic dermatitis and application thereof. The lactobacillus salivarius is separated from feces of healthy infants, and the preservation number is CCTCC NO: m2019940. The strain has strong tolerance to artificial intestinal juice, good biological safety and 6% of maximum tolerance salt concentration, and can effectively relieve the symptoms of atopic dermatitis (eczema). The strain has no toxic or harmful effect on organisms, can be added into foods, and can be prepared into functional foods with the effect of relieving atopic dermatitis, and has wide application prospects.
Description
Technical Field
The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus salivarius capable of relieving symptoms of atopic dermatitis and application of the lactobacillus salivarius.
Background
Atopic dermatitis (Atopic Dermatitis, AD) is an allergic skin disease with a genetic predisposition, also called atopic eczema, atopic dermatitis, generalized neurodermatitis, etc., and is mainly manifested by dry skin, severe itching, eczematoid changes. Atopic dermatitis has no known single causative agent, is currently thought to be related to genetic, environmental, immunological, and biological factors, and onset is mainly a combination of genetic and environmental factors. Most patients with atopic dermatitis are formed by recurrent episodes of infantile eczema and 70% of the patient families have a history of atopy such as allergy, asthma or allergic rhinitis.
Probiotics are a class of active microorganisms that act by improving the balance of the host intestinal microbial flora, and are capable of producing a definite health efficacy to improve the host's microecological balance, exerting beneficial effects. In 2001, the joint national Food and Agricultural Organization (FAO) and World Health Organization (WHO) have defined probiotics as follows: an active microorganism that produces a health benefit to the host when ingested in sufficient quantity. Most of the probiotics found at present belong to the traditional lactobacillus probiotics.
Research of the intestinal flora by the scientific community has shown explosive growth in the last two decades. The research shows that probiotics can help digestion and absorption, promote intestinal flora balance and maintain human health. There is increasing evidence that the health effects of probiotics on humans are not limited to the intestinal tract but also have a broader range of action, such as endocrine balance regulation, immune balance regulation, nervous system regulation, respiratory system regulation, etc. Meanwhile, intestinal flora disorder is related to a plurality of chronic diseases such as diabetes, obesity, depression and the like, and probiotics also have good treatment effect on atopic dermatitis and the multi-cause diseases related to immune regulation.
The invention patent with Chinese patent publication No. CN 110214014A discloses the use of probiotics in treating and/or preventing atopic dermatitis, and relates to a probiotic composition, namely bifidobacterium lactis CECT 8145, bifidobacterium longum CECE 7374 and/or lactobacillus casei CECT 9104, which are used for treating and/or preventing atopic dermatitis. The invention patent with the Chinese patent publication number of CN 110710627A discloses a functional probiotic solid beverage and a preparation method thereof, wherein the probiotic solid beverage comprises 40% of maltodextrin, 20% of microcrystalline cellulose, 10% of anhydrous glucose, 10% of fructo-oligosaccharide, 10% of stachyose, 5% of bifidobacterium longum, 2% of bifidobacterium lactis, 1% of lactobacillus acidophilus, 1% of lactobacillus casei and 1% of lactobacillus rhamnosus. However, the invention can achieve the effect of relieving atopic dermatitis by compounding a plurality of probiotics, and the effect of each strain is not clear, so that the specific strain or the compounded prebiotics can not be known, and the symptom of the atopic dermatitis can not be relieved. In addition, all previous studies focused on oral probiotics to modulate and alleviate atopic dermatitis by in vivo metabolism, and there was no precedent for topical application of probiotic lysates and metabolites.
Disclosure of Invention
The invention aims to provide a novel lactobacillus salivarius (Lactobacillus salivarius) and application thereof; the provided lactobacillus salivarius is separated from the feces of healthy infants, can regulate the immunity and improve the immunity of human bodies, thereby effectively preventing and relieving the occurrence of Jie Te atopic dermatitis (eczema).
The lactobacillus salivarius provided by the invention is a lactobacillus salivarius (Lactobacillus salivarius) VHProbi Y39 strain which is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in Wuhan, china on the 11 th month 18 days of 2019: m2019940.
The lactobacillus salivarius provided by the invention is applied to treating atopic dermatitis.
In another aspect, the invention also provides a bacterial preparation for treating atopic dermatitis, which comprises the lactobacillus salivarius VHProbi Y39 strain.
The lactobacillus salivarius provided by the invention can also be used for preparing functional foods with the effect of relieving atopic dermatitis.
The lactobacillus salivarius VHProbi Y39 provided by the invention has strong tolerance to artificial intestinal gastric juice, does not generate hemolysin, does not dissolve blood cells, is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety; can tolerate higher salinity with a maximum salt tolerance concentration of 6%.
The strain has strong antioxidant capacity, the lipid peroxidation inhibition rate of the supernatant fluid is above 54%, the lipid peroxidation inhibition rate of the thalli is above 71%, the DPPH clearance rate is above 43%, and the HRS clearance rate is above 54%. The strain can also effectively degrade cholesterol, and the degradation rate reaches more than 32%; in addition, the hydrophobicity of the cell surface of the strain is 70.61%, and the strain has certain adhesion capability.
The lactobacillus salivarius VHProbi Y39 has better effect of relieving atopic dermatitis. The lactobacillus salivarius VHProbi Y39 provided by the invention is used for lavaging or smearing the skin lesion part of a disease model mouse, and the dermatitis symptom is obviously reduced compared with a placebo group, which indicates that the strain can obviously improve the skin lesion progress of the atopic dermatitis model mouse no matter being taken orally or taken externally, and effectively relieve the atopic dermatitis.
The use of the lactobacillus salivarius VHProbi Y39 can effectively reduce IL-5, IL-6, IFN-gamma and total IgE in serum of a mouse with an atopic dermatitis model, so that the immune response of the mouse is TH1 type and the TH2 type immune response is inhibited.
From the results of the HE staining of the mouse skin, the skin keratinization and the skin thickening of the mouse are obviously improved compared with those of a placebo group after the treatment by lactobacillus salivarius VHProbi Y39.
The lactobacillus salivarius VHProbi Y39 provided by the invention has no toxic or harmful effect on organisms, can be added into food, can be prepared into functional food with the effect of relieving atopic dermatitis, and has wide application prospect.
Drawings
FIG. 1 is a Y39 strain Riboprinter fingerprint;
FIG. 2 is a RAPD fingerprint of Y39 strain;
FIG. 3 is a rep-PCR fingerprint of Y39 strain;
FIG. 4 is a graph showing daily observations of mice of each group;
FIG. 5 is a graph showing the results of cytokine measurement in serum of mice in each group;
FIG. 6 shows the results of HE staining of skin tissue of each group of mice (40X above, 200X below).
Detailed Description
Atopic dermatitis is a common chronic disease in the infant population, has the characteristics of recurrent attacks and genetic susceptibility, and can be triggered by a variety of risk factors. The influence of industrialized society has led to an increasing incidence of atopic dermatitis.
The lactobacillus salivarius VHProbi Y39 provided by the invention meets the requirements of regulations, can be used as a food raw material source, and has no side effect and excessive risk after long-term administration. The lactobacillus salivarius VPHrobi Y39 is a newly discovered strain through multiple taxonomic identification. The lactobacillus salivarius VHProbi Y39 provided by the invention can effectively relieve the symptoms of atopic dermatitis, and the strain can be singly used and has the effect of relieving the atopic dermatitis without being compounded with prebiotics and/or other probiotics; has important application value.
Applicant has deposited said lactobacillus salivarius VHProbi Y39 (l.salivarius VHProbi Y39) at the chinese collection of typical cultures at university of armed chinese at 11 and 18 days 2019 with the deposit number cctccc NO: m2019940.
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The present invention will be described in detail with reference to specific examples.
Example 1 isolation screening of Lactobacillus salivarius VHProbi Y39
1. Lactobacillus primary screening
Preparing MRS (Man Rogosa Sharpe) agar medium: 1000mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of citric acid diamine, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ for 15min.
Taking 1g of fresh feces of healthy infants which are from half a year of 1 and half a year and are not used with probiotic preparations, diluting the feces with sterile normal saline, putting the diluted feces into a sterile sample bag, and beating and uniformly mixing the feces with a homogenizer; and (3) taking 100 mu L of mixed solution for gradient dilution, coating the mixed solution on an MRS agar medium, and performing anaerobic culture at 37 ℃ for 48 hours, and performing microscopic examination on a single colony after the plate grows. According to the microscopic examination result, the applicant screens 40 potential lactobacillus strains, which are named as Y1, Y2, … … and Y40 respectively.
2. Lactobacillus re-screening
Preparing 1L MRS liquid culture medium, sterilizing at 121deg.C for 15min, cooling, adding 3.2g of pig mucosa pepsin, shaking for dissolving, and placing in a 37 deg.C water bath shaking table for 1 hr to obtain acid-resistant culture medium.
And respectively inoculating 40 strains of lactobacillus obtained by screening into the acid-resistant culture medium according to 6 percent of inoculum size, carrying out anaerobic static culture for 72 hours at 37 ℃, and taking fermentation liquor for bacterial count.
The result shows that the viable bacteria amount of the strain Y39 obtained by the primary screening is the largest after the strain Y39 is subjected to the secondary screening of the acid-resistant culture medium, and the logarithmic value of the bacterial amount is as high as 8.53Log CFU/mL. Thus demonstrating that the Y39 strain has the highest acid resistance.
Example 2 identification of strains
1. Colony morphology identification
After the Y39 strain is inoculated on MRS agar culture medium and anaerobically cultured for 48 hours at 37 ℃, the single colony of the Y39 is milky, the diameter of the colony is about 2mm, the surface is moist, the colony is short-rod-shaped under a microscope, and the two ends are round, and usually appear independently to be short-chain-shaped.
2. Identification of physiological and biochemical characteristics
The inoculum preparation in this example was as follows: under the aseptic condition, a proper amount of fresh Y39 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial cells are diluted by 50 times after the same volume of PBS buffer is used as an inoculation liquid.
2.1 salinity tolerance test
Under aseptic conditions, 190. Mu.L of BSM liquid medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, respectively, 3 replicates of each salt concentration, and then 10. Mu.L of inoculum was added thereto, and the wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid.
The results show that the Y39 strain grows at 1% -6% salt concentration, does not grow at 7% -8% salt concentration, and has a maximum tolerant salt concentration of 6%.
2.2 catalase experiments
The fresh bacterial liquid was taken and dropped onto a clean glass slide, and then a drop of 3% hydrogen peroxide solution was dropped thereon, and it was observed that the Y39 strain did not generate bubbles, which was a negative reaction.
2.3 carbon Source metabolism test
The basal medium formulation used in this example is as follows:
1.5g of peptone; 0.6g of yeast extract; tween 80.1 g; 0.5mL of saline solution; 18mg of phenol red; distilled water 100mL; pH 7.4.+ -. 0.2. Salt solution components: mgSO (MgSO) 4 ·7H 2 O 11.5g，MnSO 4 ·4H 2 O2.8 g, distilled water 100mL.
A10 g/100mL solution of sugar, alcohol and glycoside carbohydrate was prepared and filtered with a 0.22 μm sterile filter. Under aseptic conditions, 20. Mu.L of sterilized carbohydrate solution, 4 per carbohydrate, was added to the 96-well plate, then 170. Mu.L of sterilized phenol red-containing basal medium was added, and 10. Mu.L of inoculum was added, without inoculating the reaction well as a control. 50. Mu.L of liquid paraffin was added to each well to prevent evaporation of water during the culture. Anaerobic culture at 37deg.C, and observing color change of culture medium with phenol red as indicator. The specific results are shown in Table 1.
Table 1: carbon source metabolism results table of Y39 strain
Note that: a "+" positive response; "-" negative reaction.
3 molecular biological identification
3.1 16s rDNA Gene sequence analysis
3.1.1 genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
3.1.2, 16s rDNA Gene amplification
1) Primer sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
2) Reaction system (50. Mu.L)
Table 2:16s rDNA PCR amplification System Table
3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.
4) Sequencing of PCR products
The 16s rDNA sequence of the Y39 strain SEQ ID NO. 1 was obtained by sequencing and the sequences were aligned in NCBI database to preliminarily determine that the Y39 strain was Lactobacillus salivarius (Lactobacillus salivarius).
3.2Riboprinter finger print
The purified single colony is dipped from an agar culture medium plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then a sample frame is placed into a heater for inactivation and then placed into a Riboprinter system, and a bacterial identification result is obtained after DNA preparation, film transfer, imaging detection and data processing are carried out on the sample. The identification result shows that the Y39 strain is lactobacillus salivarius (Lactobacillus salivarius), and the result of the Riboprinter fingerprint is shown in figure 1.
3.3RAPD and rep-PCR fingerprinting identification
3.3.1 RAPD finger print identification
1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');
2) RAPD reaction system
Table 3: RAPD reaction System Table
3) Electrophoresis
1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and finally the electropherograms were detected using a gel imaging system. RAPD finger print of Y39 strain is shown in figure 2.
3.3.2 rep-PCR finger print
1) rep-PCR primer
CTACGGCAAGGCGACGCTGACG。
2) reaction system of rep-PCR
Table 4: table of the reaction System of rep-PCR
3) Electrophoresis
DL2000 DNA Marker served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of Y39 strain is shown in FIG. 3.
In summary, from the colony morphology, physiological and biochemical characteristics and molecular biology identification results of the Y39 strain, it can be concluded that the Y39 strain is a novel strain of Lactobacillus salivarius, which is designated as Lactobacillus salivarius VHProbi Y39 (Lactobacillus salivarius VHProbi Y39).
EXAMPLE 3 test of tolerance of Lactobacillus salivarius VHProbi Y39 to artificial gastric juice and artificial intestinal juice
1 preparation of artificial gastric juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.
2 preparation of artificial intestinal juice
Respectively weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH 2 PO 4 6.8g and 3.0g of ox gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.
3 test method
2mL of fresh bacterial liquid is taken, the bacterial liquid is collected by centrifugation at 5000rpm/min for 5min, the bacterial liquid is washed 3 times by physiological saline, and then 2mL of physiological saline is used as inoculation liquid for resuspension. 1mL of the inoculation liquid is taken and added into 24mL of artificial intestinal juice, and the mixture is placed on a water bath shaking table (200 rpm/min) at 37 ℃ for 3 hours, 1mL of sample is taken, and the viable bacteria amount is detected.
The viable bacteria counting method is used for measuring the bacterial amount according to national standard GB 4789.35-2016-lactobacillus test for food microorganism test, and the viable bacteria amount (Log CFU/mL) of the strain after artificial intestinal juice digestion is shown in Table 5.
Table 5: live bacterial load of Lactobacillus salivarius VHProbi Y39 digested by artificial gastric juice and artificial intestinal juice
As can be seen from Table 5, the live bacteria amount of the Lactobacillus salivarius VHProbi Y39 selected by the present invention was reduced only slightly after digestion with artificial gastric juice and artificial intestinal juice. Therefore, the strain has strong tolerance to artificial gastric juice and artificial intestinal juice.
EXAMPLE 4 haemolytic and antibiotic resistance test 1, haemolytic test of Lactobacillus salivarius VHProbi Y39
(1) Preparing an inoculation liquid: the frozen lactobacillus salivarius VHProbi Y39 strain is streaked and inoculated in MRS agar culture medium, cultured for 24-48 h at 37 ℃, then subcultured for 1 time by MRS liquid culture medium, and then the lactobacillus salivarius VHProbi Y39 is inoculated in fresh MRS liquid culture medium for 24-48 h at 37 ℃ with 5% of inoculation amount, so as to obtain fresh bacterial liquid as inoculation liquid.
(2) Preparation of blood cell culture medium: weighing the various components of TBS basic culture medium, dissolving, autoclaving at 121deg.C for 15min, cooling to 50deg.C, adding 5% sterilized defibrinated sheep blood, mixing, and plating.
(3) And (3) streaking culture: and streaking the test strain, inoculating the streaked strain to a prepared blood cell plate, culturing the strain in a 37 ℃ incubator, and observing whether the test strain has a hemolysis phenomenon or not in 24-48 hours.
The results show that: the inability of lactobacillus salivarius VHProbi Y39 to grow and the blood cell plates were unchanged, suggests that lactobacillus salivarius VHProbi Y39 did not produce hemolysin and was unable to lyse blood cells.
2. Antibiotic resistance test
(1) Preparing antibiotics: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline and vancomycin are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the stock solution is serially diluted by 2 times by using BSM liquid culture medium to form use solution, and the gradient dilution concentration is 1-1024 mu g/mL and total 11 gradients.
(2) Preparing an inoculation liquid: taking a proper amount of fresh bacterial liquid (24 h,37 ℃ for culture), centrifuging at 5000rpm for 5min, washing once with sterile physiological saline, and diluting 50 times after re-suspending bacterial cells with the same volume of physiological saline to obtain an inoculation liquid.
(3) Determination of minimum inhibitory concentration MIC value of antibiotic for Lactobacillus salivarius VHProbi Y39 by micro broth dilution method
an MRS liquid culture medium without antibiotics is added to the 1 st column of the 96-well plate as a negative control, 190 mu L of MRS liquid culture medium with antibiotics with different concentrations is sequentially added to the 2 nd to 12 th columns, 10 mu L of the inoculation liquid is inoculated respectively, 3 parallel wells are made, and 1 well of the non-added bacteria liquid is used as a blank.
b. 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.
c. The 96-well plate was incubated at 37℃for 24 hours, then removed, and OD was measured 600 Values, MIC values of antibiotics against strains were counted with 24h results, and specific results are shown in table 6.
Table 6: antibiotic MIC value of Lactobacillus salivarius VHProbi Y39
MIC units μg/mL.
From the results shown in Table 6, the lactobacillus salivarius VHProbi Y39 provided by the invention is sensitive to common antibiotics such as erythromycin and tetracycline, and has good biological safety.
EXAMPLE 5 measurement of antioxidant function of Lactobacillus salivarius VHProbi Y39
1. Determination of the ability of the Strain to scavenge DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) and Hydroxy Radical (HRS)
1) Preparation of PBS bacterial suspension
Single colony with excellent growth state is inoculated into 3mL of MRS liquid culture medium, and is cultured for 24h at 37 ℃, the culture solution is taken as an inoculating solution, and is inoculated into 50mL of MRS liquid culture medium according to the inoculating amount of 2 percent, and the culture solution of the strain is obtained by standing and culturing for 24 h. After 1mL of bacterial liquid is sucked up and bacterial cells are collected, the bacterial cells are washed by 1mL of buffer solution for 2 times, and then 2mL of buffer solution is added to resuspend the bacterial cells for standby.
2) Determination of DPPH free radical scavenging ability of Strain
1mL of PBS bacterial suspension of the strain to be detected is taken, 1mL of 0.4mM DPPH free radical solution is added, after uniform mixing, the mixture is placed at room temperature for shading reaction for 30min, then the absorbance A sample of the sample at the wavelength of 517nm is measured, and the sample is measured for 3 times of parallelism. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution and with equal volumes of PBS and ethanol mixed solution. The clearance is calculated according to the following formula: clearance% = [1- (a) Sample of -A Blank space )/A Control ]×100％。
The results are shown in Table 7 using Lactobacillus paracasei IMC-4 strain as a positive control.
Table 7: DPPH free radical scavenging rate table
As can be seen from the data in Table 7, the Lactobacillus salivarius VHProbi Y39 provided by the invention can effectively remove DPPH free radicals, and the removal rate reaches 43.08% which is obviously higher than that of Lactobacillus paracasei IMC-4 strain.
3) Determination of the ability of the Strain to clear HRS
100. Mu.L of 5mM sodium salicylate-ethanol solution, 100. Mu.L of 5mM ferrous sulfate, 500. Mu.L of deionized water and 200. Mu.L of lactic acid bacteria PBS bacterial suspension were mixed uniformly, 100. Mu.L of hydrogen peroxide solution (3 mM) was added thereto, and after 15min in a 37℃water bath, the absorbance of the sample was measured at a wavelength of 510 nm. The hydroxyl radical scavenging rate was calculated according to the following formula.
Clearance% = (a Sample of -A Control of )/(A Blank space -A Control of ) X 100%, where A Control of For deionized water to replace the sample, A Blank space Substitution of deionized water for sample and H 2 O 2 . The results are shown in Table 8 using Lactobacillus paracasei IMC-4 as a positive control.
Table 8: HRS free radical clearance table
From the data in Table 8, the lactobacillus salivarius VHProbi Y39 provided by the invention can effectively remove HRS free radicals, and the removal rate reaches 54.57%.
2. Identification of strains by anti-lipid peroxidation experiments
1) Preparation of culture and fermentation supernatant, cell and intracellular extract of lactic acid bacteria:
culturing lactobacillus in MRS liquid culture medium at 37deg.C for 24 hr, transferring for 3 generations, centrifuging at 6000rpm/min at 4deg.C for 10min, and collecting supernatant to obtain fermentation supernatant. The collected cells were centrifuged at 6000r/min for 10min with PBS buffer (pH 7.4), and washed 3 times. The bacterial cells were resuspended in PBS buffer to a bacterial cell concentration of 1.0X10 9 cells/mL to obtain a bacterial suspension.
2) Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
3) To 0.5mL of PBS solution (pH 7.4) was added 1mL of linoleic acid emulsion, 1mL of LFASO 4 (1%) and 0.5mL of sample are added, water bath is carried out at 37 ℃ for 1.5 hours, 0.2mL of TCA (4%) and 2mL of TBA (0.8%) are added into the mixed solution, water bath is carried out at 100 ℃ for 30 minutes, rapid cooling is carried out, centrifugation is carried out at 4000rpm/min for 15 minutes, and absorbance of the supernatant liquid is collected and measured at 532nm to obtain A; the control group was A with 0.5mL distilled water instead of the sample 0 . Inhibition rate/% = (a 0 －A)/A 0 ×100％
Note that: a isAbsorbance of the sample group; a is that 0 Absorbance was used as control. The results are shown in Table 9 using Lactobacillus paracasei IMC-4 as a positive control.
Table 9: anti-lipid peroxidation inhibition rate table
As can be seen from the data in table 9, the supernatant of lactobacillus salivarius VHProbi Y39 provided by the present invention has an anti-lipid peroxidation inhibition rate of 54.49%; the inhibition rate of the thallus to lipid peroxidation is 71.14 percent, which is far higher than that of lactobacillus paracasei IMC-4 strain
Example 6 Lactobacillus salivarius VHProbi Y39 in vitro cholesterol degradation assay
1. Preparing a cholesterol micelle solution: 1g of cholesterol was accurately weighed, dissolved in absolute ethanol, and fixed to 100mL, and sterilized by filtration through a 0.22 μm microporous filter under aseptic conditions.
2. 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of tween 80, 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, adjusting the pH value to 7.3, sterilizing at 115 ℃ for 30min, and then adding a cholesterol solution to ensure that the final concentration of cholesterol is 0.1%.
Inoculating fresh bacterial liquid according to 0.1% of inoculation amount, standing at 37 ℃ for 48 hours, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 r for 5 minutes, and taking supernatant for measuring cholesterol content. Cholesterol measurement method according to GB/T5009.128-2003 < measurement of cholesterol in food >.
The results show that: the degradation rate of the lactobacillus salivarius VHProbi Y39 provided by the invention on cholesterol reaches 32.22% (which is data without bile salts).
Example 7 hydrophobic cell surface test of Lactobacillus salivarius VHProbi Y39
1. Preparing a bacterial liquid to be tested: the purified lactobacillus salivarius VHProbi Y39 colony is picked and inoculated in a newly prepared MRS liquid culture medium, and is cultured for 24 to 48 hours at 37 ℃. Then according to 1% (V/V)Inoculating the seed amount into MRS liquid culture medium, continuously culturing at 37deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile physiological saline for 2 times, and sterilizing with 0.1M KNO 3 The bacterial cells were resuspended in 1mL of the solution and used as the bacterial liquid to be tested.
2. Surface hydrophobicity determination: mu.L of the above bacterial suspension was pipetted into 2450. Mu.L of 0.1M KNO 3 And record OD600 as A 0 1.5mL of the bacterial suspension was mixed with 500. Mu.L of xylene, and the mixture was allowed to stand at room temperature for 10 minutes (a two-phase system was formed). Vortex oscillating the two-phase system for 2min, standing for 20min, and reforming into water phase and organic phase. The aqueous phase (not to the organic phase) was carefully aspirated and absorbance A was measured at 600nm 1 . Cell Hydrophobicity = (a) according to the formula hydropathicity = (a) 0 -A 1 )/A 1 X% calculation, measurement of the average of three experiments.
The results show that: the hydrophobicity of the surface of the lactobacillus salivarius VHProbi Y39 cell provided by the invention is 70.61%, and the standard deviation is 1.89%.
Example 8 use of Lactobacillus salivarius VHProbi Y39 in the alleviation of atopic dermatitis in mice
1 Experimental materials
1.1 laboratory animals
30 male C57BL/6 mice of 5 weeks old, supplied by the Qinglong mountain animal breeding center, license number SCXK 2017-0001.
Conventional pellet feed (Jiangsu cooperative biological Co., ltd.) was fed to clean animal houses. And (3) 12h/12h of light/dark circulation, freely feeding drinking water, maintaining the temperature at 20-26 ℃ and the relative humidity at 40-70%, and using the obtained product for model construction after one week of adaptive growth.
1.2 reagent consumable
2 Experimental methods
2.1 preparation of solutions
Preparing bacterial liquid: dissolving lactobacillus salivarius VHProbi Y39 lyophilized powder in physiological saline to obtain a concentration of 0.1g/mL (10) 9 CFU/mL) bacteriaAnd (3) liquid.
10% chloral hydrate solution is prepared: accurately weighing 20g of chloral hydrate in a 500mL Schottky bottle, adding 180mL of Milli-Q water, fully mixing and dissolving, filtering by a 0.22 mu m filter membrane after dissolving, wrapping with tinfoil paper, avoiding light, and preserving at 4 ℃ for later use.
1% heparin sodium solution preparation: 200mg of heparin sodium is accurately weighed in a 50mL Corning centrifuge tube, 20mL of physiological saline solution is added, the solution is fully mixed and dissolved, the solution is filtered by a 0.22 mu m filter membrane after being dissolved, and the solution is wrapped by tinfoil paper and protected from light, and is preserved at 4 ℃ for standby.
4% glutaraldehyde fixative solution: 80mL of 50% glutaraldehyde solution (v/v) was measured, and 920mL of PBS buffer was added, i.e., 4% glutaraldehyde fixative (v/v).
1.5% Dinitrochlorobenzene (DNCB) solution: 150mg of dinitrochlorobenzene was weighed precisely in a small 10mL beaker and 3:1, fully stirring and dissolving the mixed solution of the acetone and the olive oil, then pouring the mixed solution into a 10mL volumetric flask, and adding the mixed solution of the acetone and the olive oil to 10mL for standby;
0.5% Dinitrochlorobenzene (DNCB) solution: 50mg of dinitrochlorobenzene was precisely weighed in a 10mL small beaker, and 3:1, fully stirring and dissolving the acetone-olive oil mixed solution, then pouring the solution into a 10mL volumetric flask, and adding the acetone-olive oil mixed solution to 10mL for later use.
2.2 model construction
After 3 days of adaptive growth of 5-week-old C57 mice, 30 mice were randomly picked out 6 for normal control, and the remaining 24 were used for model construction. The construction method comprises the following steps:
1) The day before the experiment, the razor shaved the hair of the mice with back areas of about 2cm by 3 cm;
2) Taking 250ul of 1.5% DNCB solution from mice in a model building group, uniformly smearing the solution on a dehairing area, and performing intensified stimulation once on the 2 nd day by using a blank control with an equivalent amount of physiological saline;
3) After that, skin irritation (250 ul of 0.5% DNCB solution) was added once again every 3 days, continuously for 4 times;
4) Mice were sacrificed 24h after stimulation 4 and the experiment was terminated.
2.3 grouping and intervention of laboratory animals
1) Gastric lavage pretreatment group (Pre-Gavage): the lactobacillus salivarius VHProbi Y39 bacterial liquid is infused 10 days before experimental modeling, 1mL each time is used for a day, and the process is continued until the experimental end
2) Post-lavage treatment group (Post-Gavage): on the day when DNCB solution is applied for the first time, the product post-treatment group starts to perfuse the lactobacillus salivarius VHProbi Y39 bacterial liquid (the dosage is the same as that of the product pre-treatment group), and the process is carried out once a day until the experiment is finished;
3) Smear group (Smear): the VHProbi Y39 bacterial liquid is smeared for 2 times every day to completely cover the shaved parts, and the shaved parts are respectively smeared once in the morning and evening (0.5 mL each time);
4) Placebo group (Placebo): modeling the 1mL of physiological saline for the stomach of the mice;
5) Normal control group (Normal): normal mice were perfused with 1mL of normal saline.
2.4 general observations in mice
The status of each group of mice was observed daily and recorded on day 7, 14, 18 post-modeling photographs.
2.5ELISA determination of cytokine expression levels in mouse serum
The final serum is used for measuring the content of IgE, IL-5, IL-6, IL-10, ECP, TNF-alpha and IFN-gamma, and the measuring steps are as follows:
1) The ELISA kit was removed from the refrigerator and equilibrated at room temperature for 20min.
2) And respectively setting blank, standard and to-be-detected sample holes, wherein a blank control hole is not added with a sample and an enzyme-labeled reagent, and the sample is added to the bottom of the enzyme-labeled plate in the sample adding process, and the liquid in the enzyme-labeled hole is gently shaken and mixed.
3) After sealing the film with a film sealing plate, the film is placed in an incubator at 37 ℃ for incubation for 30min.
4) The liquid in the wells was discarded, and the wells were gently dried, 50. Mu.L of biotin-labeled antibody working solution was added to each well, and incubated at 37℃for 30min.
5) Carefully uncovering the sealing plate film, pouring out the liquid in the holes, lightly spin-drying, adding the washing liquid into each hole, standing at room temperature for 30 seconds, pouring out, repeating the operation for 5 times, and lightly shooting to dry.
6) Each well was incubated with 50. Mu.L of horseradish peroxidase-labeled avidin working solution in an incubator at 37℃for 30min.
7) Pouring out the liquid in the holes, lightly spin-drying, adding the washing liquid into each hole, standing at room temperature for 30 seconds, pouring out, repeating the operation for 5 times, and lightly beating to dry.
8) And adding 100 mu L of a color development liquid into each hole in sequence, and developing for 15min in a dark place at 37 ℃.
9) The reaction was stopped by adding 50. Mu.L of stop solution to each well, and the absorbance (OD value) of each well was measured sequentially at a wavelength of Kong Diaoling, 450nm as a blank. The measurement is performed within 15min after adding the stop solution.
2.6 preparation of skin tissue sections
Skin tissue fixed in 4% glutaraldehyde solution was removed and 5 μm thick paraffin sections of tissue were prepared for HE staining.
Paraffin embedding
1) Dehydrating the fixed sample with 60%, 70%, 80% and 90% gradient alcohol for 8h respectively;
2) Dehydrating 100% alcohol I, II for 1 hr each;
3) Xylene I, II is transparent for 15min each;
4) Immersing the specimen in liquid paraffin, and placing the specimen in an oven at 65 ℃ for 2 hours;
5) Taking out the soaked paraffin specimen, and embedding the paraffin specimen;
6) The wax block was cut into 5 μm thick sections for HE staining.
HE staining
1) Xylene I, II for 15min each;
2) Gradient alcohol hydration to distilled water;
3) Hematoxylin staining for 5min;
4) Differentiating 1% hydrochloric acid alcohol for 1min;
5) Turning blue tap water for 30min;
6) Washing with tap water for 1min after eosin dyeing for 1min;
7) Gradient alcohol dehydration, xylene I, II for 15min each;
8) Sealing the tree glue;
9) And (5) photographing and analyzing.
3 data analysis
Data analysis was performed using SPSS 22.0 statistical software, and the results during this evaluation were expressed as mean ± standard error (x±sem). The comparison between two groups adopts independent sample t test, the comparison between multiple groups adopts single factor variance analysis, the comparison between two groups is further carried out by a minimum significant difference number method when the variances are uniform, and the 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 extremely significant. Mapping was performed using Oligo 8.0.
4 experimental results and analysis
4.1 mice treatment observations
After DNCB stimulation, the mice in the model building group begin to generate inflammatory reaction, oedema erythema is visible in the back skin preparation area, and the erythema is aggregated and aggravated along with repeated stimulation, and then exudation and crusting are generated, and then the oedema and the scars are converted. Normal mice had no obvious abnormalities and inflammation after dehairing.
The skin of mice in the treatment group had a significantly improved appearance compared to placebo group at the later stage of treatment with the lactobacillus salivarius VHProbi Y39 bacterial liquid intervention, suggesting that probiotic intervention could improve the progression of skin lesions, and that visual observations suggested that the difference between the prior administration and simultaneous administration after model construction was not significant, and that the effect of the application was slightly inferior to the results of intragastric administration (fig. 4).
4.2 results of cytokine ELISA assay in mouse serum
IgE, IL-5, IL-6, IL-10, ECP, TNF-. Alpha., IFN-. Gamma.in serum of the experimental end mice were assayed using ELISA method. The test results are shown in Table 10 and FIG. 5.
Table 10 results of cytokine measurements for each group
The serum IgE content of each model construction group is higher than that of the normal control group, and has extremely significant difference (p < 0.01), the serum IgE content of each group treated by probiotics falls back relative to the placebo group after the intervention treatment, but has extremely significant difference (p < 0.01) compared with the normal group, each group treated by probiotics has extremely significant difference (p < 0.01) compared with the placebo group, the difference (p > 0.05) compared with the treatment before and after the gastric lavage administration is not significant, and the difference (p < 0.05) between the smearing administration and the gastric lavage administration is significant.
The serum IL-5 content of each model construction group is higher than that of the normal control group, and has a significant difference (p < 0.05), the serum IL-5 content of each group treated by probiotics falls back relative to the placebo group after the intervention treatment, but has a significant difference (p < 0.05) compared with the normal group, the serum IL-5 content of each group treated by probiotics has a significant difference (p < 0.05) compared with the placebo group, the difference (p > 0.05) compared with the post-treatment before and after the gastric lavage administration is not significant, and the difference (p > 0.05) between the smearing administration and the gastric lavage administration is not significant.
The serum IL-6 content of each model construction group was higher than that of the normal control group, and had a significant difference (p < 0.05), the serum IL-6 content of each group treated with probiotics was returned relative to the placebo group after the intervention treatment, but still had a significant difference (p < 0.05) from the normal group, each group treated with probiotics had a significant difference (p < 0.05) from the placebo group, the difference was not significant (p > 0.05) from the pre-and post-lavage treatment, and the difference was not significant (p > 0.05) from the lavage administration.
The serum IL-10 content of each model construction group is higher than that of a normal control group, and has significant difference (p < 0.05), the serum IL-10 content of each group treated by probiotics is slightly higher than that of a placebo group after intervention treatment, the difference is significant (p < 0.05) compared with a normal group, the difference is not significant (p > 0.05) compared with treatment before and after gastric lavage, the difference between smearing administration and gastric lavage administration is not significant (p > 0.05), and the difference is not significant (p > 0.05) compared with a placebo group after smearing administration.
The serum ECP levels of mice in each model construction group were higher than that in the normal control group and had significant differences (p < 0.05), the serum ECP levels of each group treated with probiotics fell back relative to the placebo group after the intervention treatment, but were still significantly different (p < 0.05) from the normal group, each group treated with probiotics had significant differences (p < 0.05) from the placebo group, the differences between pre-and post-gavage treatments were not significant (p > 0.05), and the differences between the smear and gavage administration were not significant (p > 0.05).
The serum TNF- α content of each model construction group was higher than that of the normal control group and had a significant difference (p < 0.05), the serum TNF- α content of each group treated with probiotics was returned relative to the placebo group after the intervention treatment, but still significantly different (p < 0.05) from the normal group, each group treated with probiotics had a significant difference (p < 0.05) from the placebo group, the difference was not significant (p > 0.05) from the pre-and post-lavage treatment, and the difference was not significant (p > 0.05) from the lavage administration.
The serum IFN-gamma content of each group of mice of each model construction group is higher than that of a normal control group, and has a significant difference (p < 0.05), the serum IFN-gamma content of each group of the probiotics treatment group falls back relative to a placebo group after the intervention treatment, but has a significant difference (p < 0.05) compared with a normal group, the IFN-gamma content of each group of the probiotics treatment group is not significant (p > 0.05) compared with a placebo group, the difference between the pre-stomach administration treatment and the post-stomach administration treatment is not significant (p > 0.05), the difference between the smearing administration and the stomach administration is not significant (p > 0.05), and the difference between the probiotic treatment group and the stomach administration group is significant (p < 0.05) compared with the pretreatment.
4.3 results of HE staining of mouse skin
The results of HE staining of the skin of each group of mice are shown in fig. 6, the back epidermal cell structure of the normal group of mice is normal, the structures of all layers of the epidermis are clear, no obvious inflammatory reaction is seen, the surface thickening of the placebo group is obvious, the hyperkeratinization and the acanthosis cell hypertrophy are visible, the edema is visible in the layer, the large amount of lymphocytes in the shallow dermis infiltrate, and the chronic inflammatory hyperplasia change is obvious. After the intervention treatment by lactobacillus salivarius VHProbi Y39, the two groups of gavage administration groups only see inflammatory cell infiltration under high power mirror, and the skin keratinization and the epidermis thickening are obviously improved compared with the placebo group. The smeared administration group can be seen to thicken the epidermis under a high-power mirror, and is slightly obvious compared with the lavage administration group.
From the immune cytokine level and the skin pathological section test result, the lactobacillus salivarius VHProbi Y39 can effectively reduce the serum cytokines IL-5, IL-6, IFN-gamma and total serum IgE level in an atopic dermatitis mouse model, and has no obvious reduction effect on cytokines IL-10, ECP and TNF-alpha. And lactobacillus salivarius VHProbi Y39 can significantly improve the inflammatory status of skin tissue.
In conclusion, the lactobacillus salivarius VHProbi Y39 provided by the invention has strong tolerance to simulated artificial intestinal gastric juice, which lays a foundation for the probiotic strains to successfully pass through the gastrointestinal tract to perform the probiotic function by colonic colonisation. The hemolysis test proves that the lactobacillus salivarius VHProbi Y39 does not produce hemolysin, does not dissolve blood cells, and has good biological safety. Meanwhile, the lactobacillus salivarius VHProbi Y39 can remove DPPH and HRS free radicals, inhibit lipid peroxidation, has a certain antioxidant function activity, can degrade cholesterol, and has the probiotic property of reducing serum cholesterol. Animal experiments prove that the oral administration and smearing of the lactobacillus salivarius VHProbi Y39 can improve the inflammatory response of a atopic dermatitis model mouse, can inhibit TH2 type immune response while enhancing TH1 type cell immune response, reduce the inflammatory state of the organism and enhance the immunity, thus indicating that the lactobacillus salivarius VHProbi Y39 has potential application value in relieving the atopic dermatitis symptoms.
Sequence listing
<110> Qingdao blue biological Co., ltd
QINGDAO VLAND BIOTECH GROUP Co.,Ltd.
<120> Lactobacillus salivarius with effect of relieving symptoms of atopic dermatitis and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1396
<212> DNA
<213> Lactobacillus salivarius (Lactobacillus salivarius)
<400> 1
GCTTTGGGTG TTACAAACTC TCATGGTGTG ACGGGCGGTG TGTACAAGGC CCGGGAACGT 60
ATTCACCGCG ACATGCTGAT TCGCGATTAC TAGCGATTCC GACTTCATGT AGGCGAGTTG 120
CAGCCTACAA TCCGAACTGA GAACGGCTTT AAGAGATTAG CTAAACCTCG CGGTCTCGCG 180
ACTCGTTGTA CCGTCCATTG TAGCACGTGT GTAGCCCAGG TCATAAGGGG CATGATGACT 240
TGACGTCGTC CCCACCTTCC TCCGGTTTGT CACCGGCAGT CTCGCCAGAG TGCCCAACTT 300
AATGCTGGCA ACTGACAACA AGGGTTGCGC TCGTTGCGGG ACTTAACCCA ACATCTCACG 360
ACACGAGCTG ACGACAGCCA TGCACCACCT GTCACTTTGT CCCCGAAGGG AAAACCTAAT 420
CTCTTAGGTG GTCAAAGGAT GTCAAGACCT GGTAAGGTTC TTCGCGTTGC TTCGAATTAA 480
ACCACATGCT CCACCGCTTG TGCGGGCCCC CGTCAATTCC TTTGAGTTTC AACCTTGCGG 540
TCGTACTCCC CAGGCGGAAT GCTTATTGCG TTAGCTGCGG CACTGAAGGG CGGAAACCCT 600
CCAACACCTA GCATTCATCG TTTACGGCGT GGACTACCAG GGTATCTAAT CCTGTTTGCT 660
ACCCACGCTT TCGAACCTCA GCGTCAGTTA CAGACCAGAG AGCCGCTTTC GCCACTGGTG 720
TTCTTCCATA TATCTACGCA TTTCACCGCT ACACATGGAG TTCCACTCTC CTCTTCTGCA 780
CTCAAGTCTT CCAGTTTCCA ATGCACTACT CCGGTTAAGC CGAAGGCTTT CACATCAGAC 840
TTAAAAGACC GCCTGCGTTC CCTTTACGCC CAATAAATCC GGACAACGCT TGCCACCTAC 900
GTATTACCGC GGCTGCTGGC ACGTAGTTAG CCGTGACTTG CTGGTTAGAT ACCGTCATCG 960
AATGAACAGT TACTCTCACT CGTGTTCTTC TCTAACAACA GAGTTTTACG ATCCGAAGAC 1020
CTTCTTCACT CACGCGGCGT TGCTCCATCA GACTTGCGTC CATTGTGGAA GATTCCCTAC 1080
TGCTGCCTCC CGTAGGAGTT TGGGCCGTGT CTCAGTCCCA ATGTGGCCGA TCAACCTCTC 1140
AGTTCGGCTA CGTATCATCA CCTTGGTAGG CCGTTACCCC ACCAACTAGT TAATACGCCG 1200
CGGGTCCATC TAAAAGCGAT AGCAGAACCA TCTTTCATCT AAGGATCATG CGATCCTTAG 1260
AGATATACGG TATTAGCACC TGTTTCCAAG TGTTATCCCC TTCTTTTAGG CAGGTTACCC 1320
ACGTGTTACT CACCCGTCCG CCACTCAACT TCTTACGGTG AATGCAAGCA TTCGGTGTAA 1380
GAAAGTTTCG TTCGAC 1396
Claims (6)
1. The lactobacillus salivarius is characterized in that the collection number of the lactobacillus salivarius is CCTCC NO: m2019940.
2. Use of lactobacillus salivarius as claimed in claim 1 in the manufacture of a product for the treatment of atopic dermatitis.
3. A bacterial preparation comprising the lactobacillus salivarius of claim 1.
4. Use of the bacterial formulation of claim 3 in the preparation of a product for the treatment of atopic dermatitis.
5. The use according to claim 2 or claim 4, wherein the article is a functional food.
6. A functional food having an effect of relieving atopic dermatitis, characterized in that the Lactobacillus salivarius as claimed in claim 1 is used in the preparation process of the functional food.
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| CN202210689815.XA CN117286043A (en) | 2022-06-17 | 2022-06-17 | Lactobacillus salivarius capable of relieving symptoms of atopic dermatitis and application thereof |
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| Country | Link |
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| CN (1) | CN117286043A (en) |
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2022
- 2022-06-17 CN CN202210689815.XA patent/CN117286043A/en active Pending
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