CN112812989B - Bifidobacterium adolescentis capable of relieving psoriasis and application thereof - Google Patents
Bifidobacterium adolescentis capable of relieving psoriasis and application thereof Download PDFInfo
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- CN112812989B CN112812989B CN202011628281.7A CN202011628281A CN112812989B CN 112812989 B CN112812989 B CN 112812989B CN 202011628281 A CN202011628281 A CN 202011628281A CN 112812989 B CN112812989 B CN 112812989B
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Abstract
The invention discloses bifidobacterium adolescentis capable of relieving psoriasis and application thereof, belonging to the technical field of microorganisms and medicines. The Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 provided by the invention has the function of relieving psoriasis, relieving the skin condition of psoriasis-like mice lesion, inhibiting the thickening of the epidermis layer of the skin of the psoriasis-like mice, reducing the weight of the spleen of the psoriasis-like mice, reducing the IL-22 and IL-23 levels in the skin of the psoriasis-like mice by 30.2%, and has great application prospect in preparing products (such as food, medicine or health food and the like) for preventing and/or treating psoriasis.
Description
Technical Field
The invention relates to bifidobacterium adolescentis capable of relieving psoriasis and application thereof, belonging to the technical field of microorganisms and medicines.
Background
Psoriasis is a chronic, immune-mediated disease that affects primarily the skin and joints, has a complex inheritance, has a global prevalence of about 2-3%, and has increased year by year. Although onset can occur at any age, the incidence is highest between the ages of 18-39 years or 50-69 years. As a skin disease, psoriasis can manifest multiple phenotypes including plaque psoriasis (also known as psoriasis vulgaris), punctate psoriasis, inverse psoriasis, pustular psoriasis, palmoplantar psoriasis and erythrodermic psoriasis. The same individual may exhibit multiple phenotypes, with psoriasis vulgaris being the most common. Symptoms common to all phenotypes include itching, burning and soreness, among others. Psoriasis cannot endanger life, but seriously affects the physical health of a patient, and because the psoriasis can generate a plurality of papules and erythema with different sizes on the skin layer, and the surface of the psoriasis can be covered with silvery-white scales, and the psoriasis is mostly generated on the scalp and parts with outstretched sides of hands and feet which are easy to see by other people, the social life of the patient is affected, and the psychological stress is also brought to the patient.
Psoriasis is a systemic inflammatory disease, is a disease mediated by heredity, environment, immunity and the like, and the pathogenesis of the psoriasis is not completely clear. An increase in proinflammatory cytokines released by immune-related cells and chronic activation of the innate and adaptive immune systems are believed to be mechanisms that lead to long-term damage to multiple tissues and organs. Various psoriasis-associated abnormalities have been reported that promote the host immune response and infiltration of immune cells, including antigen presentation, activation of the NF-. kappa.B signaling pathway, differentiation of helper T cell populations, particularly TH17 cells, the major source of IL-17, and enhancement of the IL-17 response.
At present, no medical method and medicine for completely curing psoriasis exist, and the psoriasis is still a worldwide medical problem. The Chinese and western medicine are different in treatment method, along with the development of psoriasis genetics, the pathogenesis and the treatment method of the psoriasis are different and are continuously explored from the point of completely curing the psoriasis. The psoriasis is treated differentially according to different severity degrees, different medicines and treatment means are selected, and the disease course of the skin lesion treatment is divided into a progressive stage, a resting stage and a regressive stage in the medical field. At present, a plurality of Chinese and western medicines for treating psoriasis are provided, and a plurality of different treatment methods are tried, but the Chinese and western medicines can not completely cure psoriasis, can only relieve symptoms and reduce recurrence rate, and are mostly accompanied by different side effects.
Probiotics are active microorganisms which are beneficial to a host and change the composition of flora at a certain part of the host by colonizing in a human body. The health of the intestinal tract is kept by promoting the absorption of nutrients by regulating the immune function of the host mucous membrane and the system or by regulating the balance of flora in the intestinal tract, so that single microorganisms or mixed microorganisms with definite compositions which are beneficial to the health are generated. A large body of literature reports an imbalance in the intestinal flora of patients with psoriasis and a decrease in the relative abundance of various probiotics, such as Akkermansia (Akkermansia muciniphila), clostridium prarsonii (Faecalibacterium prausnitzii), etc., and thus it seems to be possible to alleviate psoriasis by supplementing probiotics. This possibility was also confirmed in foreign related studies, and Chen et al intervene in psoriasis-like mice with Lactobacillus pentosus (Lactobacillus pentosus) GMNL-77 and showed that this Lactobacillus pentosus alleviated the psoriasis condition. David et al found that the disease was alleviated in 26 patients with psoriasis after a continuous oral administration of Bifidobacterium infantis for 6 weeks. However, the strain only relieves the level of inflammation in the serum of a patient, and the relieving effect on the skin is not described.
Therefore, the current situation that psoriasis is difficult to treat, easy to repeat and great in side effect can be solved by developing probiotics capable of relieving psoriasis.
Disclosure of Invention
The technical problem is as follows: the invention aims to provide a probiotic strain capable of relieving psoriasis.
The first objective of the present invention is to provide a Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667, wherein the Bifidobacterium adolescentis CCFM667 has been deposited at the Guangdong province collection center in 12/7 of 2020 with a deposition number of GDMCC No: 61349, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
The sequence analysis of Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 of the present invention shows that the 16S rRNA sequence is shown in SEQ ID No.1, and the sequence is compared in NCBI to show that the strain is Bifidobacterium adolescentis (Bifidobacterium adolescentis) named Bifidobacterium adolescentis CCFM 667.
The microbial morphological characteristics of Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 of the invention are as follows: the cells were slightly irregular, rounded-ended Campylobacter, non-motile, non-sporulating.
It is a second object of the present invention to provide a composition comprising said bifidobacterium adolescentis CCFM 667.
In one embodiment, the number of Bifidobacterium adolescentis in the composition is greater than or equal to 1X 105CFU/g or 1X 105CFU/mL。
The third purpose of the invention is to provide the application of the Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 in preparing products for relieving psoriasis.
In one embodiment, the alleviating psoriasis comprises: relieving the symptoms of wrinkles, scales and/or erythema on the skin, and inhibiting the thickening of the skin cutin.
In one embodiment, the product includes, but is not limited to, a food product, a functional food product, a nutraceutical product, or a pharmaceutical.
In one embodiment, the viable count of Bifidobacterium adolescentis (CCFM 667) in the product is not less than 1 × 106CFU/mL。
In one embodiment, the medicament comprises Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.
In one embodiment, the pharmaceutical excipient comprises an excipient and an additive.
In one embodiment, the pharmaceutical excipient comprises an anti-adhesive agent, a penetration enhancer, a buffering agent, a plasticizer, a surfactant, an antifoaming agent, a thickener, an encapsulating agent, an absorbent, a humectant, a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a pH adjuster, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an integrating agent, an osmotic pressure adjuster, a stabilizer, a glidant, a flavoring agent, a preservative, a foaming agent, a suspending agent, a coating material, a fragrance, a diluent, a flocculating agent and a deflocculating agent, a filter aid, and a release retardant.
In one embodiment, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.
In one embodiment, the dosage form of the medicament comprises granules, capsules, tablets, pills or oral liquid.
In one embodiment, the food product comprises a dairy, soy or fruit and vegetable product produced using a starter culture comprising Bifidobacterium adolescentis (CCFM 667).
In one embodiment, the food product is a solid beverage comprising Bifidobacterium adolescentis (CCFM 667).
In one embodiment, the preparation method of the leavening agent is as follows: inoculating Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 into a culture medium according to an inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing at 37 ℃ for 30h to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing the thallus with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending with cryoprotectant (mass ratio of cryoprotectant to thallus is 2:1) to obtain re-suspension; freeze-drying the re-suspension by vacuum freezing method to obtain the ferment of Bifidobacterium adolescentis (CCFM 667).
In one embodiment, the medium comprises 87.7% water, 10% skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract solubles by total mass of the medium.
In one embodiment, the pH of the medium is 6.8.
In one embodiment, the skim milk powder: maltodextrin, 2: and the ratio of L-sodium glutamate is 8-10: 1.
The invention also claims the application of the bifidobacterium adolescentis CCFM667 in the field of food.
Has the advantages that: the invention provides a Bifidobacterium adolescentis (CCFM 667) with the effect of relieving psoriasis, which is specifically embodied in that:
(1) the skin condition of the psoriasis-like mice is relieved and improved;
(2) thickening of the epidermal layer of the skin of psoriasis-like mice is inhibited;
(3) spleen weight loss in psoriasis-like mice was 21.9%;
(4) a 30.2% reduction in IL-22 levels in the skin of psoriasis-like mice;
(5) a 24.6% reduction in IL-23 levels in the skin of psoriasis-like mice;
therefore, the Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 has a great application prospect in the preparation of products (such as food, medicine or health food and the like) for preventing and/or treating psoriasis.
Biological material preservation
Bifidobacterium adolescentis (CCFM 667), classified and named Bifidobacterium adolescentis, has been deposited in Guangdong province collection of microorganisms at 12 months and 7 days 2020 with the deposit number GDMCC No: 61349, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
Drawings
FIG. 1: different groups of experimental mice had pathological skin conditions.
FIG. 2: pathological sections of the skin of experimental mice of different groups.
FIG. 3: spleen weights of mice were tested in different groups.
FIG. 4: IL-22 content in the skin of different groups of experimental mice.
FIG. 5: IL-23 content in the skin of different groups of experimental mice.
Detailed Description
The invention is further illustrated with reference to specific examples.
The media involved in the following examples are as follows:
mMRS medium formula (1L): 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose and K2HPO42g of diammonium citrate, 2g of sodium acetate, 801 mL of Tween and MgSO4·7H20.5g of O, 0.5g of cysteine hydrochloride and MnSO4·4H20.25g of O, and the pH value is 7.2-7.4.
mMRS solid medium formula (1L): 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose and K2HPO42g of diammonium citrate, 2g of sodium acetate, 801 mL of Tween and MgSO4·7H20.5g of O, 0.5g of cysteine hydrochloride and MnSO40.25g of 4H2O 0.25 and 20g of agar, and the pH value is 7.2-7.4.
The detection methods referred to in the following examples are as follows:
the detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
And (3) an acidity detection method: the national standard GB 431334-.
Bifidobacterium adolescentis (Bifidobacterium adolescentis)1 is a strain isolated by the same method as in example 1, and its 16S rDNA sequence is shown in SEQ ID NO. 2.
Example 1: screening and identification of Bifidobacterium adolescentis
The method comprises the following specific steps:
taking human feces as a sample, pretreating the sample, storing the pretreated sample in 20% glycerol in a refrigerator at minus 80 ℃, taking out and unfreezing the sample, uniformly mixing the sample, sucking 0.5mL of the sample, adding the sample to 4.5mL of the sample, performing gradient dilution by using 9g/L of physiological saline containing 0.05g/L of cysteine, selecting a proper gradient dilution solution to coat the sample on an MRS solid culture medium, culturing the sample at 37 ℃ for 48 hours, selecting a typical bacterial colony of lactobacillus paracasei, streaking and purifying the typical bacterial colony on the MRS solid culture medium, selecting a single bacterial colony, transferring the single bacterial colony to an MRS liquid culture medium (containing 0.05g/L of cysteine) for enrichment, and preserving the single bacterial colony by using 30% glycerol to obtain a strain CCFM 667; wherein, the typical bacterial colony of the lactobacillus paracasei is milky white, irregular, round, convex and smooth.
The genome of CCFM667 was extracted, the 16S rDNA of CCFM667 was amplified and sequenced (shanghai bio-engineering gmbh), and the sequence was compared in GenBank, and the result showed that the strain was Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM 667.
Example 2: culture of Bifidobacterium adolescentis
The method comprises the following specific steps:
bifidobacterium adolescentis (CCFM 667) is inoculated into mMRS solid culture medium and cultured for 48h at 37 ℃, then its colony is observed and its thallus is observed under microscope, the colony is milk-white, irregular, round convex and smooth, its thallus shape is slightly irregular, and the campylobacter with round end usually exists in single, paired and small cluster.
Inoculating Bifidobacterium adolescentis (CCFM 667) into MRS liquid culture medium, culturing at 37 deg.C for 30h, transferring into fresh MRS liquid culture medium, culturing under the same conditions for 30h, centrifuging for 15min at 6000g, washing with 0.9% physiological saline, centrifuging again at 6000g for 10min to obtain thallus, resuspending with 30% sucrose solution to obtain sucrose suspension, and storing at-80 deg.C. When the mice are subjected to intragastric administration, the mice are taken out from the temperature of minus 80 ℃, centrifuged and supernatant is discarded, and physiological saline is used for resuspension to obtain the intragastric bacterial suspension.
Example 3: effect of bifidobacterium adolescentis on relieving skin with psoriasis lesions
The SPF-grade BALB/c female mice of 6-8 weeks are divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group 1 is intragastric bifidobacterium adolescentis CCFM667 (hereinafter, abbreviated as CCFM667 group), the experimental group 2 is intragastric bifidobacterium adolescentis B.adolescentis 1 (hereinafter, abbreviated as B.adolescentis 1 group), and each group comprises 6 mice, wherein the mice are bred in experimental animal center of south Jiangnan university, fed with common feed, kept at a constant temperature of 21-26 ℃, have a humidity of 40-70%, have noise of less than or equal to 60dB and have animal illumination of 15-20 (all animal experimental procedures are examined and approved by the university of animal welfare and ethical management committee of south Jiangnan).
The experimental period is 3 weeks in total, the molding is started at week 3, and the depilatory treatment is carried out on the back of the mouse one day before the molding, and the area is about 2.5cm multiplied by 2.5 cm. In the molding period, the ear and back depilatory areas of the model group and the experimental group were coated with imiquimod cream 10mg and 62.5mg daily, and the normal group was coated with vaseline in the same amount. During the experiment, the CCFM667 group gavage 0.2mL per day (viable count is 1X 10)9CFU/mL), and gavage 0.2mL per day (viable count 1X 10) in group B.adolescentis 19CFU/mL) b. adolescentis 1 bacterial suspension, normal and model groups were gavaged with only equal amounts of sterile saline as controls, and all groups were free to drink and ingest. The back of the mice was photographed daily during the molding period and the mice were sacrificed on day 1 of week 4. The results are shown in FIG. 1.
As can be seen from FIG. 1, the skin of the epilation area of the back of the normal group of mice is smooth, and the mice have no scales and erythema; the skin of the hairless area on the back of the model group mouse has wrinkle feeling and is covered with obvious scales accompanied with erythema; compared with the model group, the CCFM667 group has smooth skin in the epilation area of the back, and has no scales and erythema; the skin of the depilated area of the back of the b.adolescentis 1 mice had a wrinkled feeling, covered with visible scales, and had erythema as in the model group.
The above experimental results show that, compared with Bifidobacterium adolescentis b.adolescentis 1, Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 can alleviate the symptoms of psoriatic mouse lesions skin.
Example 4: inhibiting effect of bifidobacterium adolescentis on thickening of psoriasis mouse cutin
The SPF-grade BALB/c female mice of 6-8 weeks old are divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group 1 is intragastric bifidobacterium adolescentis CCFM667 (hereinafter, abbreviated as CCFM667 group), the experimental group 2 is intragastric bifidobacterium adolescentis B.adolescentis 1 (hereinafter, abbreviated as B.adolescentis 1 group), each group comprises 6 mice, the mice are bred in the experimental animal center of the university of south of the Yangtze river, fed with common feed, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illumination is 15-20LX (all animal experimental procedures are examined and approved by the university committee of animal welfare and ethical management in south of the Yangtze river).
The experimental period is 3 weeks in total, the 3 rd week is used for molding, the back of the mouse is depilated one day before molding, and the area is about 2.5cm multiplied by 2.5 cm. In the molding period, the ear and back depilatory areas of the model group and the experimental group were coated with imiquimod cream 10mg and 62.5mg daily, and the normal group was coated with vaseline in the same amount. During the experiment, the CCFM667 group gavage 0.2mL per day (viable count is 1X 10)9CFU/mL), and gavage 0.2mL per day (viable count 1X 10) in group B.adolescentis 19CFU/mL) b. adolescentis 1 bacterial suspension, normal and model groups were gavaged with only equal amounts of sterile saline as controls, and all groups were free to drink and ingest. Mice were sacrificed on day 1 at week 4, and pathological sections were prepared from the skin of the depilatory area of the back of the mice for histopathological analysis. The results are shown in FIG. 2.
As can be seen from FIG. 2, the epidermal layer of the skin of the mice in the blank group is only composed of one or two layers of cells, and inflammatory reaction is not seen in each layer of the epidermis; the cutin of the model group mice is obviously thickened and is accompanied with serious inflammation; compared with the model group, the CCFM667 group has lighter cutin thickening phenomenon and lighter inflammation phenomenon; the cutaneous cutin of mice in the adolescentis 1 group is obviously thickened, and the inflammation phenomenon is serious.
The experimental results show that Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 can inhibit the cutin thickening phenomenon of the pathological skin of the psoriasis mice better than Bifidobacterium adolescentis b.
Example 5: inhibition effect of bifidobacterium adolescentis on spleen weight gain of psoriasis mice
SPF-grade BALB/c female mice of 6-8 weeks old are divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group 1 is a gastric lavage bifidobacterium adolescentis CCFM667 (hereinafter, abbreviated as CCFM667 group), the experimental group 2 is a gastric lavage bifidobacterium adolescentis B.adolescentis 1 (hereinafter, abbreviated as B.adolescentis 1), each group comprises 6 mice, the mice are bred in experimental animal centers of south Jiangnan university, fed with common feed, kept at a constant temperature of 21-26 ℃, have a humidity of 40-70%, have noise of less than or equal to 60dB, and have animal illumination of 15-20LX (all animal experimental programs are reviewed and approved by the animal welfare and ethics administration committee in south Jiangnan).
The experimental period is 3 weeks in total, the 3 rd week is used for molding, the back of the mouse is depilated one day before molding, and the area is about 2.5cm multiplied by 2.5 cm. In the molding period, the ear and back depilatory areas of the model group and the experimental group were coated with imiquimod cream 10mg and 62.5mg daily, and the normal group was coated with vaseline in the same amount. During the experiment, the CCFM667 groups gavage 0.2mL per day (viable count is 1X 10)9CFU/mL), and gavage 0.2mL per day (viable count 1X 10) in group B.adolescentis 19CFU/mL) b. adolescentis 1 bacterial suspension, normal and model groups were gavaged with only equal amounts of sterile saline as controls, and all groups were free to drink and ingest. Mice were sacrificed on day 1 at week 4, spleens of the mice were immediately removed and weighed, and the results are shown in fig. 3.
As can be seen from FIG. 3, after the mice are gavaged with Bifidobacterium adolescentis CCFM667, the spleen weight is reduced by 21.9%, which is significantly reduced compared with the model group (p < 0.001), which indicates that the strain of the present invention can inhibit the spleen weight gain; after the mice are perfused with the bifidobacterium adolescentis B. adolescentis 1, the weight of the spleen is only reduced by 1.4 percent, and the weight of the spleen is not obviously different from that of a model group.
The experimental results show that Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 can inhibit the spleen weight increment phenomenon of psoriasis mice.
Example 6: effect of Bifidobacterium adolescentis on IL-22 in psoriasis mouse skin
The SPF-grade BALB/c female mice of 6-8 weeks are divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group 1 is intragastric bifidobacterium adolescentis CCFM667 (hereinafter, abbreviated as CCFM667 group), the experimental group 2 is intragastric bifidobacterium adolescentis B.adolescentis 1 (hereinafter, abbreviated as B.adolescentis 1 group), and each group comprises 6 mice, wherein the mice are bred in experimental animal center of south Jiangnan university, fed with common feed, kept at a constant temperature of 21-26 ℃, have a humidity of 40-70%, have noise of less than or equal to 60dB and have animal illumination of 15-20 (all animal experimental procedures are examined and approved by the university of animal welfare and ethical management committee of south Jiangnan).
The experimental period is 3 weeks in total, the 3 rd week is used for molding, the back of the mouse is depilated one day before molding, and the area is about 2.5cm multiplied by 2.5 cm. In the molding period, imiquimod cream is applied to ear hair removal areas and back hair removal areas of the model group and the experimental group every day, the ear cream is 10mg, the back cream is 62.5mg, and vaseline is only applied to a normal group in an equal amount. During the experiment, the CCFM667 group gavage 0.2mL per day (viable count is 1X 10)9CFU/mL), and gavage 0.2mL per day (viable count 1X 10) in group B.adolescentis 19CFU/mL) b. adolescentis 1 bacterial suspension, normal and model groups were gavaged with only equal amounts of sterile saline as controls, and all groups were free to drink and ingest. Mice were sacrificed on day 1 at week 4, and a portion of the skin in the depilatory area of the back of the mice was stored at-80 ℃ and the IL-22 content in the skin was measured by ELISA kit, and the results are shown in FIG. 4.
As can be seen from FIG. 4, after the bifidobacterium adolescentis CCFM667 is gavaged by the mouse, the content of IL-22 in the skin is reduced by 30.2%, which is obviously reduced compared with the model group (p is less than 0.01), which indicates that the strain CCFM667 in the invention can inhibit inflammatory reaction; after the mice are perfused with bifidobacterium adolescentis b.adolescentis 1, the content of IL-22 in the skin is even increased.
The experimental results show that Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 can significantly down-regulate typical up-regulated proinflammatory factors in psoriasis mice to normal levels, especially reduce IL-22 levels, and is significantly better than another Bifidobacterium adolescentis B.
Example 7: effect of Bifidobacterium adolescentis on IL-23 in psoriasis mouse skin
The SPF-grade BALB/c female mice of 6-8 weeks are divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group 1 is intragastric bifidobacterium adolescentis CCFM667 (hereinafter, abbreviated as CCFM667 group), the experimental group 2 is intragastric bifidobacterium adolescentis B.adolescentis 1 (hereinafter, abbreviated as B.adolescentis 1 group), and each group comprises 6 mice, wherein the mice are bred in experimental animal center of south Jiangnan university, fed with common feed, kept at a constant temperature of 21-26 ℃, have a humidity of 40-70%, have noise of less than or equal to 60dB and have animal illumination of 15-20 (all animal experimental procedures are examined and approved by the university of animal welfare and ethical management committee of south Jiangnan).
The experimental period is 3 weeks in total, the 3 rd week is used for molding, the back of the mouse is depilated one day before molding, and the area is about 2.5cm multiplied by 2.5 cm. In the molding period, the ear and back depilatory areas of the model group and the experimental group were coated with imiquimod cream 10mg and 62.5mg daily, and the normal group was coated with vaseline in the same amount. During the experiment, the CCFM667 group gavage 0.2mL per day (viable count is 1X 10)9CFU/mL), and gavage 0.2mL per day (viable count 1X 10) in group B.adolescentis 19CFU/mL) b. adolescentis 1 bacterial suspension, normal and model groups were gavaged with only equal amounts of sterile saline as controls, and all groups were free to drink and ingest. The mice were sacrificed on day 1 at week 4, and a portion of the skin in the depilated area of the back of the mice was stored at-80 ℃ and the content of IL-23 in the skin was measured by ELISA kit, and the results are shown in FIG. 5.
As can be seen from FIG. 5, after the bifidobacterium adolescentis CCFM667 is gavaged by the mouse, the content of IL-23 in the skin is reduced by 24.6 percent, which is obviously reduced compared with the model group (p is less than 0.001), which indicates that the strain of the invention can inhibit inflammatory reaction; after the mice are perfused with bifidobacterium adolescentis B.adolescentis 1, the content of IL-23 in the skin is not reduced, and the differences from a model group are not obvious.
The experimental results show that Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 can significantly down-regulate typical up-regulated proinflammatory factors in psoriasis mice to normal levels, especially reduce IL-23 levels, and is significantly better than another Bifidobacterium adolescentis B.
Example 8: preparation method of solid beverage containing bifidobacterium adolescentis CCFM667
Inoculating Bifidobacterium adolescentis (CCFM 667) preserved in a bacteria-preserving tube into a culture medium according to an inoculation amount accounting for 3% of the total mass of the culture medium, and culturing at 37 deg.C for 30h to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the thallus with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending with trehalose lyophilized protectant with trehalose concentration of 100g/L (mass ratio of lyophilized protectant to thallus is 2:1) to obtain re-suspension; freeze-drying the heavy suspension by vacuum freezing method to obtain Bifidobacterium adolescentis (CCFM 667 powder).
Will contain 109Mixing the powder of Bifidobacterium adolescentis (CCFM 667) of CFU with maltodextrin, wherein the total mass of the powder of CCFM667 and the maltodextrin is 1 g, and obtaining the solid beverage rich in Bifidobacterium adolescentis (CCFM 667).
Example 9: preparation method of fermented milk containing Bifidobacterium adolescentis CCFM667
The method comprises the steps of carrying out heat treatment on raw milk (fresh milk, skim milk and the like) at 95 ℃ for 20min, cooling to 37 ℃, inoculating Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 seed liquid stored in a bacteria preservation tube into the sterilized raw milk according to the inoculation amount accounting for 3% of the total volume, inoculating symbiotic commercial leavening agents (lactobacillus bulgaricus, streptococcus thermophilus and the like) into the raw milk according to the inoculation amount accounting for 3% of the total volume, and carrying out mixed fermentation on the raw milk at 37 ℃ until the titer acidity is 0.6% (calculated by lactic acid bacteria) to obtain the fermented milk rich in Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM 667.
Example 10: preparation of leaven of bifidobacterium adolescentis CCFM667
Inoculating a Bifidobacterium adolescentis (CCFM 667) seed solution preserved in a bacterium preservation tube into a fermentation culture medium according to an inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing at 37 ℃ for 30h to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing the thallus with phosphate buffer solution with pH of 7.2 for 3 times, then re-suspending with freeze-drying protective agent, and controlling the mass ratio of freeze-drying protective agent to thallus to be 2:1 to obtain re-suspension; freeze-drying the re-suspension by vacuum freezing method to obtain the ferment of Bifidobacterium adolescentis (CCFM 667).
The fermentation medium comprises water accounting for 87.7% of the total mass of the medium, skim milk accounting for 10%, glucose accounting for 0.5%, tryptone accounting for 1.5% and yeast extract accounting for 0.3%, and the pH value of the fermentation medium is 6.8.
Optionally, skimmed milk powder and sodium L-glutamate are also added into the fermentation medium, wherein the skimmed milk powder: maltodextrin (b): and the ratio of L-sodium glutamate is 8-10: 1.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (672)..(672)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (741)..(741)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (785)..(786)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (806)..(806)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (808)..(809)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (811)..(811)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (813)..(813)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (834)..(834)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (838)..(838)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (846)..(846)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (854)..(854)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (870)..(872)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (878)..(878)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (881)..(882)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (901)..(901)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (907)..(907)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (909)..(909)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (933)..(933)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (935)..(935)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (939)..(939)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (943)..(947)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (951)..(951)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (954)..(954)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (956)..(957)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (959)..(959)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (967)..(968)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (976)..(977)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (979)..(981)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (993)..(993)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1001)..(1002)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1005)..(1005)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1007)..(1009)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1016)..(1016)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1018)..(1019)
<223> n is a, c, g, or t
<400> 1
nnnnnnnnnn nnnnnnnnnn nnntgcagtc gacgggatcg gctggagctt gctccggccg 60
tgagagtggc gaacgggtga ntaatgcgtg accgacctgc cccatacacc ggaatagctc 120
ctggaaacgg gtggtaatgc cggatgctcc agttggatgc gtgtccttct gggaaagatt 180
ctatcggtat gggatggggt cgcgtcctat catcttgatg gcggggtaac ggcccaccat 240
ggcttcgacg ggtagccggc ctgagagggc gaccggccac attgggactg anatacggcc 300
canactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag 360
cgacgccgcg tgcgggatga cggccttcgg gttgtaaacc gcttttgact gggagcaagc 420
ccttcggggt gagtgtacct ttcgaataag caccggctaa ctacgtgcca gcagccgcgg 480
taatacgtag ggtgcaagcg ttatccggaa ttattgggcg taaagggctc gtaggcggtt 540
cgtcgcgtcc ggtgtgaaag tccatcgctt aacggaggat ccgcgccggg tacgggcggg 600
cttgagtgcg gnaggggana ctggaattcc cggtgtaacg gtgnaatgtg tagatatcgn 660
gaagaacacc gntggcgaag gcaggtctct gggccgtcac tgacgctgag gagcgaaagc 720
gtgaggagcg aacaggatta nataccctgg tagtccacgc cgtaaacgga ggatgctgga 780
tgtgnngacc attccacggt ctccgngnng nanccaacgc gttaatcatc ccgnctgngg 840
agtacngccg cgangctaaa actcaaagtn nnttgacngg nngcccgcac aagcggagga 900
ncatgcngna ttaattcgat gcaacgcgaa gancnttanc tgnnnnngac ntgntnncna 960
cagcccnnag agatgnngnn ntcccttcgg ggngggttca nnggngnnng catggncnn 1019
<210> 2
<211> 1172
<212> DNA
<213> Bifidobacterium adolescentis
<220>
<221> misc_feature
<222> (1)..(26)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (81)..(85)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (804)..(804)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (917)..(917)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (935)..(935)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (970)..(970)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (981)..(981)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1005)..(1005)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1008)..(1009)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1015)..(1016)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1022)..(1022)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1025)..(1025)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1029)..(1029)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1035)..(1035)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1043)..(1043)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1047)..(1047)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1055)..(1056)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1061)..(1061)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1068)..(1068)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1071)..(1071)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1075)..(1076)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1082)..(1085)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1088)..(1090)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1094)..(1101)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1103)..(1103)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1109)..(1109)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1111)..(1111)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1114)..(1117)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1122)..(1122)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1124)..(1124)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1127)..(1130)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1133)..(1133)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1136)..(1136)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1138)..(1138)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1140)..(1140)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1142)..(1142)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1147)..(1147)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1150)..(1151)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1155)..(1156)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1158)..(1163)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1167)..(1167)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1169)..(1172)
<223> n is a, c, g, or t
<400> 2
nnnnnnnnnn nnnnnnnnnn nnnnnntgca gtcgaacggg atcggctgga gcttgctccg 60
gccgtgagag tggcgaacgg nnnnntaatg cgtgaccgac ctgccccata caccggaata 120
gctcctggaa acgggtggta atgccggatg ctccagttgg atgcatgtcc ttctgggaaa 180
gattctatcg gtatgggatg gggtcgcgtc ctatcatctt gatggcgggg taacggccca 240
ccatggcttc gacgggtagc cggcctgaga gggcgaccgg ccacattggg actgagatac 300
ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat 360
gcagcgacgc cgcgtgcggg atgacggcct tcgggttgta aaccgctttt gactgggagc 420
aagcccttcg gggtgagtgt acctttcgaa taagcaccgg ctaactacgt gccagcagcc 480
gcggtaatac gtagggtgca agcgttatcc ggaattattg ggcgtaaagg gctcgtaggc 540
ggttcgtcgc gtccggtgtg aaagtccatc gcttaacggt ggatccgcgc cgggtacggg 600
cgggcttgag tgcggtaggg gagactggaa ttcccggtgt aacggtggaa tgtgtagata 660
tcgggaagaa caccaatggc gaaggcaggt ctctgggccg tcactgacgc tgaggagcga 720
aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa cggtggatgc 780
tggatgtggg gaccattcca cggnctccgt gtcggagcca acgcgttaag catcccgcct 840
ggggagtacg gccgcaaggc taaaactcaa agaaattgac gggggcccgc acaagcggcg 900
gagcatgcgg attaatncga tgcaacgcga agaancttac ctgggcttga catgttcccg 960
acagccccan agatggcggc ntcccttcgg ggcgtgttca caggnggnng catgnncgtc 1020
gncanctcnt gtcgngagat gtngggntaa gtccnncaac nagcgcancc ntccnncctg 1080
gnnnncannn cgtnnnnnnn ngnacctcnc nggnnnnacc gncnggnnnn aanctngnan 1140
gnagggnggn naatnncnnn nnnaatncnn nn 1172
Claims (8)
1. Bifidobacterium adolescentis (CCFM 667), which has been deposited at the Guangdong province Collection of microorganisms at 12/7/2020 with the deposit number GDMCC No: 61349.
2. a composition comprising bifidobacterium adolescentis CCFM667 according to claim 1.
3. The composition of claim 2, wherein the composition is a starter; the number of Bifidobacterium adolescentis in the leaven is more than or equal to 1 × 105CFU/g or 1X 105CFU/mL。
4. Use of a Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM667 according to claim 1, or a composition according to claim 2 or 3 in the manufacture of a product for the alleviation of psoriasis.
5. The use according to claim 4, wherein the psoriasis is: relieving the symptoms of wrinkles, scales and/or erythema, or inhibiting the thickening of skin cutin.
6. Use according to claim 4, wherein the product is a food product, a nutraceutical product or a pharmaceutical product.
7. A medicament comprising bifidobacterium adolescentis CCFM667 according to claim 1 further comprising a pharmaceutical carrier and/or a pharmaceutically acceptable adjuvant.
8. A solid beverage comprising bifidobacterium adolescentis CCFM667 according to claim 1.
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| CN202011628281.7A CN112812989B (en) | 2020-12-31 | 2020-12-31 | Bifidobacterium adolescentis capable of relieving psoriasis and application thereof |
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