CN112553117B - Lactobacillus reuteri capable of inhibiting skin stratum corneum thickening and application thereof - Google Patents

Abstract

The invention discloses a strain of Lactobacillus reuteri capable of inhibiting skin stratum corneum thickening and its application, belonging to the technical field of microorganisms and the technical field of medicine. The invention provides a new use of Lactobacillus reuteri CCFM1132 or a starter containing Lactobacillus reuteri CCFM1132 in preparing a product for relieving psoriasis, and the strain can make psoriasis-like mice skin lesions The situation improved, and the thickening of the skin epidermis was inhibited; the level of IL-23 in the skin of psoriasis-like mice was reduced by 37.6%, and the level of IL-17 was reduced by 18.5%. , medicine or health food, etc.), has a huge application prospect.

Description

A strain of Lactobacillus reuteri that can inhibit skin stratum corneum thickening and its application

technical field

The invention relates to a Lactobacillus reuteri strain capable of inhibiting the thickening of skin stratum corneum and its application, belonging to the technical field of microorganisms and the technical field of medicine.

Background technique

The skin is an organ that is wrapped on the surface of the body and is in direct contact with the external environment. It has the functions of protecting, excreting, regulating body temperature and sensing external stimuli. It is the largest organ in the human body. The skin is divided into two layers, the epidermis and the dermis, and the epidermis is on the surface of the skin. The stratum corneum is the outermost part of the epidermis and consists mainly of 10 to 20 layers of flat, dead cells without nuclei. When these cells fall off, the underlying cells in the basal layer are pushed up to form a new stratum corneum. The primary role of the stratum corneum is to protect its subcutaneous tissue from infection, dehydration, and resistance to chemical and external stress. The stratum corneum generally ranges from 10 to 40 microns, depending on how much protection is required for its corresponding body part. Keratinocytes are the main constituent cells of the epidermis, accounting for more than 80% of epidermal cells, and produce keratin during differentiation. According to the differentiation stage and characteristics, it can be divided into five layers, from inside to outside, they are basal layer, spinous layer, granular layer, transparent layer and stratum corneum.

Psoriasis is a chronic relapsing inflammatory skin disease characterized by abnormal epidermal proliferation and erythematous scales. Its pathological changes mainly include abnormal proliferation and differentiation of epidermal keratinocytes, infiltration of dermal inflammatory cells and angiogenesis. The disease has a long course and is prone to recurrence, and even some patients cannot be cured in their lifetime. The disease is more common in young and middle-aged people. When the disease occurs, scales and erythema will appear on the body. It is more common in the scalp and limbs, and the situation will worsen in winter. It has seriously affected the patient's physical health and mental state. The cause of psoriasis is not fully understood. The medical community at home and abroad has done a lot of research on its pathogenesis and pathological mechanism, but there is still no complete conclusion. Western medicine believes that its pathogenesis is related to genetic factors, immune factors (mediated by immunity or inflammation), infectious factors (bacteria, fungi, viruses), endocrine factors (sex hormones), neuropsychiatric factors (psychological factors), living habits (drinking, smoking, etc.) etc.), drug factors, environmental factors (moisture, seasonal climate, etc.).

Psoriasis currently does not have a complete cure for medical methods and drugs, which is still a worldwide medical problem. Early treatment of psoriasis focuses on inhibiting the abnormally proliferating stratum corneum. Tar (Tar) has been used to treat skin diseases for 2000 years. At present, tar products have been widely used in the treatment of psoriasis, atopic dermatitis, seborrheic dermatitis and prurigo. It has inhibitory effects on sebaceous gland secretion, mitotic number of epidermal basal cells and the production of scales. Coal tar reduces sebaceous gland synthesis of sebum and inhibits its mitosis. In vitro experiments show that 0.351ag/ml of coal tar can inhibit the DNA synthesis of keratinocytes, thereby delaying the mitotic time of epidermal cell nuclei and exerting an anti-proliferative effect. But using tar products has side effects, both short-term and long-term. Short-term use can cause folliculitis due to blocked hair follicles, and some people may also be allergic to it. Long-term use can cause cancer due to polycyclic aromatic hydrocarbons (PAHs) in coal tar.

Probiotics are a class of active microorganisms that are beneficial to the host by colonizing the human body and changing the composition of the flora in a certain part of the host. Probiotics produce a variety of metabolites during growth to regulate human health. Rather et al. treated psoriasis-like mice with SEL001, an ethanolic extract of Lactobacillus sakei proBio-65, and found that the abnormal thickening of the mouse stratum corneum was inhibited and the disease was relieved. In addition, the effect of probiotics on the intestines can also be achieved. Chen et al. intervened psoriasis-like mice with Lactobacillus pentosus GMNL-77, and the results showed that the abnormal thickening of the mouse stratum corneum was inhibited and the disease was relieved.

Therefore, it is possible to solve the current situation of psoriasis that is difficult to treat, easy to repeat, and has large side effects by developing probiotics that can inhibit the thickening of the stratum corneum of the skin.

SUMMARY OF THE INVENTION

Technical problem: The technical problem to be solved by the present invention is to provide Lactobacillus reuteri

Novel use of CCFM1132 in the relief of psoriasis.

Technical solutions:

The first object of the present invention is to provide a new use of Lactobacillus reuteri CCFM1132 or a starter containing Lactobacillus reuteri CCFM1132 in the preparation of products for relieving psoriasis, the Lactobacillus reuteri CCFM1132 has been released in 2020 On July 22, 2009, it was deposited in the Guangdong Provincial Microbial Culture Collection Center, with the preservation number of GDMCC No.61093, and the preservation address is 5th Floor, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou.

In one embodiment, the alleviation of psoriasis comprises: alleviation of skin folds, scaling and/or erythema symptoms, or inhibition of skin keratinization.

In one embodiment, the product comprises a pharmaceutical product.

In one embodiment, the viable count of Lactobacillus reuteri CCFM1132 in the product is not less than 1×10 ⁶ CFU/mL.

In one embodiment, the medicine contains Lactobacillus reuteri CCFM1132, 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 excipients include excipients and additives.

In one embodiment, the pharmaceutical excipients comprise anti-adhesives, penetration enhancers, buffers, plasticizers, surfactants, antifoaming agents, thickeners, inclusion agents, absorbents, humectants, solvents , propellant, solubilizer, cosolvent, emulsifier, colorant, pH adjuster, binder, disintegrant, filler, lubricant, wetting agent, integration agent, osmotic pressure regulator, stabilizer, auxiliary Flow agents, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids and release retarders.

In one embodiment, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

In one embodiment, the dosage form of the medicine comprises granules, capsules, tablets, pills or oral liquids.

In one embodiment, the preparation method of the starter is as follows: inoculate the Lactobacillus reuteri CCFM1132 into the medium according to the inoculum amount of 2-4% of the total mass of the medium, and cultivate at 35-37° C. for 20 ~30h, the culture solution was obtained; the culture solution was centrifuged to collect the bacterial cells; the bacterial cells were washed with a phosphate buffer with a pH of 7.2, and then resuspended with a lyophilized protective agent to obtain a resuspended liquid; the resuspended liquid was frozen in vacuum Freeze-drying was carried out to obtain the starter of Lactobacillus reuteri CCFM1132.

In one embodiment, the mass ratio of the freeze-drying protective agent and the bacterial cells is 2:1.

In one embodiment, the freeze-drying protective agent contains skim milk powder, maltodextrin and sodium L-glutamate; wherein skim milk powder: maltodextrin: sodium L-glutamate=8～10:8～10 :1.

In one embodiment, the medium is prepared by dissolving 10% skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in 87.7% water based on the total mass of the medium.

In one embodiment, the pH of the medium is 6.8.

Beneficial effect: The present invention provides a strain of Lactobacillus reuteri CCFM1132 that can inhibit the thickening of skin stratum corneum and thereby alleviate psoriasis, which is embodied in:

(1) The skin condition of psoriasis-like mice improved;

(2) The thickening of skin epidermis in psoriasis-like mice was inhibited;

(3) The level of IL-23 in the skin of psoriasis-like mice decreased by 37.6%;

(4) The level of IL-17 in the skin of psoriasis-like mice decreased by 18.5%;

Therefore, Lactobacillus reuteri CCFM1132 has great application prospects in the preparation of products (such as medicines) for preventing and/or treating psoriasis.

biological material preservation

Lactobacillus reuteri CCFM1132, classified as Lactobacillus reuteri, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on July 22, 2020, and the deposit number is GDMCC No: 61093.

Description of drawings

Figure 1: Lesion skin conditions of different groups of experimental mice.

Figure 2: Pathological sections of the skin of different groups of experimental mice.

Figure 3: IL-23 content in the skin of different groups of experimental mice.

Figure 4: IL-17 content in the skin of different groups of experimental mice.

Detailed ways

The media involved in the following examples are as follows:

mMRS medium formula (1L): peptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, K ₂ HPO ₄ 2g, diammonium citrate 2g, sodium acetate 2g, Tween 80 1mL, MgSO ₄ 7H ₂ O 0.5 g, cysteine hydrochloride 0.5 g, MnSO ₄ ·4H ₂ O 0.25 g, pH 7.2-7.4.

mMRS solid medium formula (1L): peptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, K ₂ HPO ₄ 2g, diammonium citrate 2g, sodium acetate 2g, Tween 80 1mL, MgSO ₄ 7H ₂ O 0.5 g, cysteine hydrochloride 0.5 g, MnSO ₄ ·4H ₂ O 0.25 g, agar 20 g, pH 7.2-7.4.

The detection methods involved in the following examples are as follows:

The detection method of the number of viable bacteria: adopt the national standard "GB 4789.35-2016 National Food Safety Standard for Food Microbiology Detection of Lactic Acid Bacteria".

Acidity detection method: using the national standard GB 431334-2010.

L. reuteri 1 is a strain isolated from different stool samples using the same method.

Example 1: Culture of Lactobacillus reuteri and preparation of bacterial suspension

Lactobacillus reuteri CCFM1132 was inserted into mMRS solid medium and cultured at 37°C for 48 hours, and the colonies were observed and their cells were observed under a microscope. It was found that the colonies were milky white, irregular and round. Raised, smooth, slightly irregularly shaped, rounded-ended Campylobacter, usually present in singly, in pairs, and in small clusters.

Lactobacillus reuteri CCFM1132 was inserted into MRS liquid medium and cultured at 37°C for 30h, then transferred to fresh MRS liquid medium, cultured under the same conditions for 30h, centrifuged at 6000×g for 15min, 0.9% After washing the cells with physiological saline, the cells were centrifuged again at 6000×g for 10 min, and the cells were collected, resuspended with 30% (m/v) sucrose solution, and frozen at 80°C for use.

Optionally, when Lactobacillus reuteri is used for gavage in mice, it is taken out from -80°C, centrifuged to discard the supernatant, and resuspended with normal saline to obtain a bacterial suspension for gavage.

Example 2: Relief effect of Lactobacillus reuteri on skin lesions of mice with psoriasis

The 6-8-week-old SPF grade BALB/c female mice were divided into 4 groups, namely the normal group, the model group and the experimental group. L. reuteri 1 group of L. reuteri1, 6 animals in each group, were raised in the Experimental Animal Center of Jiangnan University, fed with common feed, constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, animals Illumination 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

The experimental period was 3 weeks in total, and the model was established in the third week. The back of the mice was depilated the day before the modeling, with an area of about 2.5cm×2.5cm. During the modeling period, imiquimod cream, 10 mg on the ear and 62.5 mg on the back, was applied to the ear and back hair removal areas of the mice in the model group and the experimental group every day, while the normal group only applied the same amount of Vaseline. During the experiment, the CCFM1132 group was given 0.2 mL of CCFM1132 bacterial suspension (the number of viable bacteria was 1×10 ⁹ CFU/mL) per day, and the L.reuteri1 group was given 0.2 mL (the number of viable bacteria was 1×10 ⁹ CFU mL) per day. The L. reuteri1 bacterial suspension, the normal group and the model group were only given the same amount of sterile normal saline as a control, and all groups were given free access to water and food. The back of the mice was photographed and recorded every day during the modeling period, and the mice were sacrificed on the first day of the fourth week. The results are shown in Figure 1.

It can be seen from Figure 1 that the skin of the dorsal depilation area of the mice in the normal group is smooth, without scales and erythema; while the skin of the dorsal depilation area of the mice in the model group is wrinkled, covered with obvious scales, and accompanied by erythema. group, the skin of the dorsal depilation area was smoother, with less scales and no erythema; while the skin of the dorsal depilation area of mice in the L. reuteri 1 group was wrinkled and covered with obvious scales like the model group.

The above experimental results showed that, compared with L. reuteri 1, L. reuteri CCFM1132 was more able to alleviate the symptoms of psoriatic skin lesions in mice.

Example 3: Inhibitory effect of Lactobacillus reuteri on keratin thickening in mice with psoriasis

The 6-8-week-old SPF grade BALB/c female mice were divided into 4 groups, namely the normal group, the model group and the experimental group. L. reuteri 1 group of L. reuteri1, 6 animals in each group, were raised in the Experimental Animal Center of Jiangnan University, fed with common feed, constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, animals Illumination 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

The experimental period was 3 weeks in total, and the model was established in the third week. The back of the mice was depilated the day before the modeling, with an area of about 2.5cm×2.5cm. During the modeling period, imiquimod cream, 10 mg on the ear and 62.5 mg on the back, was applied to the ear and back hair removal areas of the mice in the model group and the experimental group every day, while the normal group only applied the same amount of Vaseline. During the experiment, CCFM1132 group was given 0.2 mL of CCFM1132 bacterial suspension (viable count of 1×10 ⁹ CFU) per day, and L. reuteri group 1 was given 0.2 mL of L. reuteri (viable count of 1×10 ⁹ CFU) of L. .reuteri1 bacterial suspension, the normal group and the model group were given only the same amount of sterile saline as a control, and all groups were given free access to water and food. The mice were sacrificed on the first day of the 4th week, and part of the skin of the dorsal depilation area of the mice was taken to make pathological sections for histopathological analysis. The results are shown in Figure 2.

As can be seen from Figure 2, the skin epidermis of the mice in the blank group was composed of only one or two layers of cells, and no inflammatory reaction was seen in each layer of the epidermis. Compared with the model group, the phenomenon of keratin thickening was not obvious, while the skin keratin thickening of the L. reuteri 1 group was obvious.

The above experimental results showed that, compared with L. reuteri 1, L. reuteri CCFM1132 was more able to inhibit the keratin thickening phenomenon of lesional skin of psoriasis mice.

Example 4: Effect of Lactobacillus reuteri on IL-23 in the skin of psoriatic mice

The 6-8-week-old SPF grade BALB/c female mice were divided into 4 groups, namely the normal group, the model group and the experimental group. L. reuteri 1 group of L. reuteri1, 6 animals in each group, were raised in the Experimental Animal Center of Jiangnan University, fed with common feed, constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, animals Illumination 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

The experimental period was 3 weeks in total, and the model was established in the third week. The back of the mice was depilated the day before the modeling, with an area of about 2.5cm×2.5cm. During the modeling period, imiquimod cream, 10 mg on the ear and 62.5 mg on the back, was applied to the ear and back hair removal areas of the mice in the model group and the experimental group every day, while the normal group only applied the same amount of Vaseline. During the experiment, the CCFM1132 group was given 0.2 mL of CCFM1132 bacterial suspension (the number of viable bacteria was 1×10 ⁹ CFU/mL) every day, and the L.reuteri1 group was given 0.2 mL (the number of viable bacteria was 1×10 ⁹ CFU/mL) per day. ) of L. reuteri1 bacterial suspension, the normal group and the model group were given only the same amount of sterile normal saline as a control, and all groups were given free access to water and food. The mice were sacrificed on the first day of the 4th week, and part of the skin of the dorsal depilation area of the mice was taken and stored at -80°C, and the content of IL-23 in the skin was detected by ELISA kit. The results are shown in Figure 3.

It can be seen from Figure 3 that after the mice were given Lactobacillus reuteri CCFM1132, the content of IL-23 in the skin was reduced by 37.6%, which was significantly lower than that of the model group (p<0.0001), indicating that the strain in the present invention can inhibit inflammation. However, after the mice were given L. reuteri1, the content of IL-23 in the skin decreased by only 12.4%.

The above experimental results show that Lactobacillus reuteri CCFM1132 can significantly down-regulate the typical up-regulated pro-inflammatory factors in psoriatic mice to normal levels, especially the level of IL-23, which is significantly better than another strain of Reuteri Lactobacillus L. reuteri 1.

Example 5: Effect of Lactobacillus reuteri on IL-17 in the skin of psoriatic mice

The 6-8-week-old SPF grade BALB/c female mice were divided into 4 groups, namely the normal group, the model group and the experimental group. L. reuteri 1 group of L. reuteri1, 6 animals in each group, were raised in the Experimental Animal Center of Jiangnan University, fed with common feed, constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, animals Illumination 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

The experimental period was 3 weeks in total, and the model was established in the third week. The back of the mice was depilated the day before the modeling, with an area of about 2.5cm×2.5cm. During the modeling period, imiquimod cream, 10 mg on the ear and 62.5 mg on the back, was applied to the ear and back hair removal areas of the mice in the model group and the experimental group every day, while the normal group only applied the same amount of Vaseline. During the experiment, the CCFM1132 group was given 0.2 mL of CCFM1132 bacterial suspension (the number of viable bacteria was 1×10 ⁹ CFU/mL) every day, and the L.reuteri1 group was given 0.2 mL (the number of viable bacteria was 1×10 ⁹ CFU/mL) per day. ) of L. reuteri 1 bacterial suspension, the normal group and the model group were only given the same amount of sterile normal saline as a control, and all groups were given free access to water and food. The mice were sacrificed on the first day of the 4th week, and part of the skin of the dorsal depilation area of the mice was taken and stored at -80°C, and the content of IL-17 in the skin was detected by ELISA kit. The results are shown in Figure 4.

It can be seen from Figure 4 that after the mice were given Lactobacillus reuteri CCFM1132, the content of IL-17 in the skin was reduced by 18.5%, which was significantly lower than that of the model group (p<0.05), indicating that the strain of the present invention can inhibit inflammation. After the mice were given L. reuteri 1 by gavage, the content of IL-17 in the skin was only reduced by 9.7%, and there was no significant difference between the model group.

The above experimental results show that L. reuteri CCFM1132 can significantly down-regulate the typical up-regulated pro-inflammatory factors in psoriatic mice to normal levels, especially the level of IL-17, which is significantly better than another strain of L. reuteri L. reuteri 1.

Example 6: Preparation of bacteria powder containing Lactobacillus reuteri CCFM1132

The seed liquid of Lactobacillus reuteri CCFM1132 stored in the preservation tube was inoculated into the mMRS medium according to the inoculum amount of 3% of the total mass of the medium, and cultured at 37 ° C for 30 h to obtain a culture medium; the culture medium was centrifuged and collected. The thalline; the thalline is washed 3 times with a phosphate buffer with a pH of 7.2, and then resuspended with a trehalose freeze-drying protective agent with a trehalose concentration of 100 g/L, and the mass ratio of the lyophilized protective agent and the bacterial body is controlled to be 2:1 to obtain a re-suspension; the re-suspension was pre-cooled at -80°C for 1.5 h and then immediately transferred to a freeze dryer for drying for 24 h to obtain Lactobacillus reuteri CCFM1132 bacterial powder.

Example 7: Preparation of solid beverage containing Lactobacillus reuteri CCFM1132

The Lactobacillus reuteri CCFM1132 bacteria powder containing 10 ⁹ CFU prepared in Example 6 was mixed with maltodextrin in a ratio of 2:1 to obtain a solid beverage rich in Lactobacillus reuteri CCFM1132.

Example 8: Preparation of yogurt containing Lactobacillus reuteri CCFM1132

Mix milk powder, inulin, stevia, and water in a weight ratio of 20:5:5:75, and homogenize them to make fermentation raw materials; sterilize at 121°C for 300s at ultra-high temperature, then cool to 42°C for inoculation in Bulgaria The mixed bacterial powder of Lactobacillus and Streptococcus thermophilus was fermented at 42°C for 12 hours, and the bacterial concentration of Lactobacillus bulgaricus and Streptococcus thermophilus was controlled to be 10 ⁵ CFU/g and 10 ⁷ CFU/g, and then prepared; The fermented product was cooled to 37°C, added Lactobacillus reuteri CCFM1132 freeze-dried bacterial powder, the feeding amount was 10 ⁹ CFU/ml yogurt, stirred, canned, and stored at 4°C for 2 days after natural completion to make probiotics yogurt.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (6)

1. the application of Lactobacillus reuteri CCFM1132 or the starter containing Lactobacillus reuteri CCFM1132 in the preparation of medicines for relieving psoriasis, it is characterized in that, described Lactobacillus reuteri CCFM1132 has been in 2020 On July 22, 2008, it was deposited in the Guangdong Provincial Microbial Culture Collection Center, and the preservation number is GDMCC No.61093. 2 . The use according to claim 1 , wherein the alleviation of psoriasis comprises: alleviation of skin folds, scaling and/or erythema symptoms, or inhibition of skin keratinization. 3 . 3 . The application according to claim 1 , wherein the viable count of Lactobacillus reuteri CCFM1132 in the medicine is not less than 1×10 ⁶ CFU/mL or 1×10 ⁶ CFU/g. 4 . 4. The application according to claim 1, wherein the medicine contains Lactobacillus reuteri CCFM1132, a pharmaceutical carrier and/or a pharmaceutical excipient. 5 . The application according to claim 4 , wherein the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes; and the pharmaceutical excipients comprise excipients and additives. 6 . 6. The application according to claim 1, wherein the starter is prepared by the following method: inoculating Lactobacillus reuteri CCFM1132 into the culture medium, and culturing at 35～37°C for 20～30h to obtain culture solution; centrifuge the culture solution to collect the bacteria; wash the bacteria and resuspend them with a freeze-drying protective agent to obtain a resuspension; then freeze the resuspension by vacuum freezing to obtain Lactobacillus reuteri CCFM1132 of starter.

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