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

Lactobacillus reuteri capable of inhibiting skin stratum corneum thickening and application thereof Download PDF

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CN112553117B
CN112553117B CN202011548289.2A CN202011548289A CN112553117B CN 112553117 B CN112553117 B CN 112553117B CN 202011548289 A CN202011548289 A CN 202011548289A CN 112553117 B CN112553117 B CN 112553117B
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lactobacillus reuteri
ccfm1132
skin
psoriasis
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陈卫
陆文伟
邓雅丹
翟齐啸
田丰伟
崔树茂
王刚
赵建新
张灏
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Jiangnan University
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Abstract

The invention discloses lactobacillus reuteri capable of inhibiting skin cuticle thickening and application thereof, and belongs to the technical field of microorganisms and medicines. The invention provides a new application of Lactobacillus reuteri CCFM1132 or a leavening agent containing the Lactobacillus reuteri CCFM1132 in preparing a product for relieving psoriasis, wherein the strain can improve the pathological change skin condition of a psoriasis-like mouse and inhibit the thickening of the skin epidermal layer; the level of IL-23 in the skin of the psoriasis-like mouse is reduced by 37.6 percent, the level of IL-17 is reduced by 18.5 percent, and the preparation method has great application prospect in preparing products (such as food, medicine or health food and the like) for preventing and/or treating psoriasis.

Description

Lactobacillus reuteri capable of inhibiting skin stratum corneum thickening and application thereof
Technical Field
The invention relates to lactobacillus reuteri capable of inhibiting skin stratum corneum thickening and application thereof, and belongs to the technical field of microorganisms and medicines.
Background
The skin is an organ which is wrapped on the surface of the body, is directly contacted with the external environment, has the functions of protecting, excreting, regulating body temperature, sensing external stimulation and the like, and is the largest organ in human body organs. The skin is divided into two layers, namely epidermis and dermis, and the epidermis is arranged on the surface of the skin. The stratum corneum is the outermost part of the epidermis and is composed of mainly 10 to 20 flat, dead cells without nuclei. When these cells are shed, the cells in the underlying stratum basale are pushed up to form a new stratum corneum. The stratum corneum serves primarily to protect its underlying tissues from infection, dehydration, and stress from chemical and external forces. The stratum corneum typically varies from 10 to 40 microns depending on how much protection is required for its corresponding body part. Keratinocytes are the major constituent cells of the epidermis, accounting for more than 80% of the epidermal cells, and produce keratin during differentiation. The differentiation stage and characteristics of the traditional Chinese medicine composition can be divided into five layers, namely a basal layer, a spinous layer, a granular layer, a transparent layer and a cuticle from inside to outside.
Psoriasis is a chronic recurrent inflammatory skin disease which is clinically characterized by abnormal proliferation and erythema crumbs of epidermis, and the pathological changes of the psoriasis mainly comprise abnormal proliferation and differentiation of epidermal keratinocytes, infiltration of dermal inflammatory cells and angiogenesis. The disease has long course of disease and is easy to recur, and even some patients can not be cured for a long time. The disease is often seen in young and strong years, scales, erythema and the like appear on the body during the disease attack, the disease is often seen on the scalp and the limbs, and the condition is aggravated in winter. The physical health and mental state of the patient have been seriously affected. The cause of psoriasis is not completely clear. The medical circles at home and abroad make a great deal of research on the causes and the pathological mechanisms of the diseases, but the complete conclusion is not made up to now. 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), habits (drinking, smoking, etc.), drug factors, environmental factors (dampness, seasonal climate, etc.).
At present, no medical method and medicine for completely curing psoriasis exist, and the psoriasis is still a worldwide medical problem. Early treatment of psoriasis has focused primarily on inhibiting the abnormally proliferating stratum corneum. Tar (Tar) has been used for the treatment of skin diseases for 2000 years. At present, tar products are widely used for treating psoriasis, atopic dermatitis, seborrheic dermatitis, prurigo and the like. It has inhibitory effect on sebaceous gland secretion, mitotic number of epidermal basal cells and generation of scale. Coal tar can reduce sebum synthesis and inhibit mitosis of sebaceous glands. In vitro experiments show that 0.351ag/ml coal tar can inhibit DNA synthesis of keratin cells, thereby delaying mitosis time of epidermal cell nucleus and playing an anti-hyperplasia role. However, the use of tar products has side effects, including short-term and long-term side effects. Short term use may cause folliculitis due to the obstruction of the hair follicle, and some people may also be allergic to this condition. Polycyclic Aromatic Hydrocarbons (PAHs) in coal tar can cause carcinogenesis after long-term use.
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 probiotics generate various metabolites in the growth process to regulate the health of human bodies. Rather et al acted on psoriasis-like mice with ethanol extract SEL001 of Lactobacillus sakei (Lactobacillus sakei) proBio-65 and found that abnormal thickening of the horny layer was inhibited and the disease was alleviated in the mice. In addition, the probiotic bacteria act on the intestinal tract to achieve the same effect. Chen et al intervene in psoriasis-like mice with Lactobacillus pentosus (Lactobacillus pentosus) GMNL-77 and showed that abnormal thickening of the mouse stratum corneum was inhibited and the disease was alleviated.
Therefore, it is possible to solve the current situation that psoriasis is difficult to treat, easy to repeat and has a large side effect by developing probiotics capable of inhibiting the thickening of the horny layer of the skin.
Disclosure of Invention
The technical problem is as follows: the technical problem to be solved by the invention is to provide Lactobacillus reuteri (Lactobacillus reuteri)
Novel use of CCFM1132 in the relief of psoriasis.
The technical scheme is as follows:
the invention aims to provide a new application of lactobacillus reuteri CCFM1132 or a leavening agent containing the lactobacillus reuteri CCFM1132 in preparing a product for relieving psoriasis, wherein the lactobacillus reuteri CCFM1132 is preserved in Guangdong province microorganism culture collection center in 7-22.2020, and has the preservation number of GDMCC No.61093 and the preservation address of No. 5-building 59 of Mr. Zhou Lu 100 of Guangzhou city.
In one embodiment, the alleviating psoriasis comprises: relieving the symptoms of wrinkles, scales and/or erythema, or inhibiting the thickening of skin cutin.
In one embodiment, the product comprises a pharmaceutical product.
In one embodiment, the viable count of Lactobacillus reuteri CCFM1132 in said product is not less than 1 × 106CFU/mL。
In one embodiment, the pharmaceutical product comprises 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 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 pharmaceutical product is in a dosage form comprising granules, capsules, tablets, pills or oral liquid.
In one embodiment, the preparation method of the leavening agent is as follows: inoculating lactobacillus reuteri CCFM1132 into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing at 35-37 ℃ for 20-30 h to obtain a culture solution; centrifuging the culture solution, and collecting thalli; cleaning the bacteria with phosphate buffer solution with pH of 7.2, and then resuspending the bacteria with a freeze-drying protective agent to obtain a resuspension solution; and (3) freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the fermentation agent of the lactobacillus reuteri CCFM 1132.
In one embodiment, the mass ratio of the lyoprotectant to the microbial cells is 2: 1.
In one embodiment, the lyoprotectant comprises skim milk powder, maltodextrin, and sodium L-glutamate; wherein the skimmed milk powder comprises maltodextrin and L-sodium glutamate which are 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.
Has the advantages that: the invention provides a lactobacillus reuteri CCFM1132 capable of inhibiting skin cuticle thickening and further relieving psoriasis, which is specifically embodied in that:
(1) the skin condition of psoriasis-like mice is improved;
(2) thickening of the epidermal layer of the skin of psoriasis-like mice is inhibited;
(3) a 37.6% reduction in IL-23 levels in the skin of psoriasis-like mice;
(4) a 18.5% reduction in IL-17 levels in the skin of psoriasis-like mice;
therefore, the lactobacillus reuteri CCFM1132 has a great application prospect in preparing products (such as medicines) for preventing and/or treating psoriasis.
Biological material preservation
Lactobacillus reuteri (Lactobacillus reuteri) CCFM1132, classified and named as Lactobacillus reuteri, which is deposited in Guangdong province collection of microorganisms and strains in 7-22.2020, with the deposit number GDMCC No: 61093.
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: IL-23 content in the skin of different groups of experimental mice.
FIG. 4: IL-17 content in the skin of different groups of experimental mice.
Detailed Description
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 MnSO4·4H20.25g of O 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-.
Lactobacillus reuteri (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 (Lactobacillus reuteri) CCFM1132 is inoculated into an mMRS solid culture medium and cultured for 48 hours at 37 ℃, colonies are observed, thalli of the Lactobacillus reuteri are observed under a microscope, and the colonies are milky white, irregular, round and convex and smooth, the shape of the thalli of the Campylobacter with slightly irregular and round ends is usually single, paired and small cluster.
Inoculating Lactobacillus reuteri (Lactobacillus reuteri) CCFM1132 into an MRS liquid culture medium, culturing at 37 ℃ for 30h, transferring into a fresh MRS liquid culture medium, culturing under the same condition for 30h, centrifuging the strain at 6000 Xg for 15min, washing the strain at 6000 Xg with 0.9% physiological saline, centrifuging again at 6000 Xg for 10min, collecting the strain, resuspending the strain with 30% (m/v) sucrose solution, and freezing at 80 ℃ for later use.
Alternatively, when Lactobacillus reuteri is used for intragastric administration to mice, the mice are removed from the stomach at-80 ℃ and centrifuged to discard the supernatant, which is then resuspended in physiological saline to obtain a suspension for intragastric administration.
Example 2: alleviating effect of lactobacillus reuteri on psoriasis mouse lesion skin
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 comprises a CCFM1132 group of Lactobacillus gasseri CCFM1132 and an L.reuteri1 group of Lactobacillus gasseri L.reuteri1, 6 animals in each group are bred in the experimental animal center of south Jiangnan university, fed by common feed, have constant temperature of 21-26 ℃, humidity of 40-70%, noise of less than or equal to 60dB and animal illumination of 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethical administration committee of south Jiangnan university).
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. During the molding processThe ear and back depilatory areas of the model group and the experimental group mice were coated with imiquimod cream in 10mg on the ear and 62.5mg on the back, and the normal group was coated with vaseline in the same amount. During the experiment, the CCFM1132 group was gavaged with 0.2mL per day (viable count was 1X 10)9CFU/mL) of CCFM1132 bacterial suspension, and L.reuteri1 group gavage 0.2mL (viable count of 1 × 10)9CFU mL), 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 depilated area of the back of the normal group of mice is smooth, and the normal group of mice has 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 CCFM1132 group has smoother skin at the depilated area of the back, less scales and no erythema; the skin of the depilated area of the back of the l.reuteri1 mice had a wrinkled texture and was covered with noticeable scales, as in the model group.
The above experimental results show that compared with lactobacillus reuteri l.reuteri1, lactobacillus reuteri CCFM1132 is more capable of relieving the symptoms of psoriatic mouse lesion skin.
Example 3: inhibition of psoriasis mouse cutin thickening by lactobacillus reuteri
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 comprises a CCFM1132 group of Lactobacillus gasseri CCFM1132 and an L.reuteri1 group of Lactobacillus gasseri L.reuteri1, 6 animals in each group are bred in the experimental animal center of south Jiangnan university, fed by common feed, have constant temperature of 21-26 ℃, humidity of 40-70%, noise of less than or equal to 60dB and animal illumination of 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethical administration committee of south Jiangnan university).
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. The ear and back depilatory areas of the model group and experimental group mice are coated with imiquimod every day during the molding periodSpecial cream, 10mg on ear and 62.5mg on back, and normal group is only smeared with vaseline with equal amount. During the experiment, the CCFM1132 group was gavaged with 0.2mL per day (viable count was 1X 10)9CFU) of CCFM1132 bacterial suspension, and L.reuteri1 group gavage 0.2mL (viable count of 1 × 10)9CFU), 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 CCFM1132 group has no obvious cutin thickening phenomenon, and the L.reuteri1 group mice have obvious cutin thickening phenomenon.
The experimental results show that compared with lactobacillus reuteri1, lactobacillus reuteri CCFM1132 can inhibit the cutin thickening phenomenon of the psoriasis mouse lesion skin better.
Example 4: effect of Lactobacillus reuteri on IL-23 in psoriasis mouse skin
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 comprises a CCFM1132 group of Lactobacillus gasseri CCFM1132 and an L.reuteri1 group of Lactobacillus gasseri L.reuteri1, 6 animals in each group are bred in the experimental animal center of south Jiangnan university, fed by common feed, have constant temperature of 21-26 ℃, humidity of 40-70%, noise of less than or equal to 60dB and animal illumination of 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethical administration committee of south Jiangnan university).
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, CCFM1132 group drenched the stomach every day0.2mL (viable count 1X 10)9CFU/mL) of CCFM1132 bacterial suspension, and L.reuteri1 group gavage 0.2mL (viable count of 1 × 10)9CFU/mL), normal and model groups were gavaged with only equal amounts of sterile saline as controls, and all groups were free access to water and food. 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-23 content in the skin was measured by ELISA kit, and the results are shown in FIG. 3.
As can be seen from FIG. 3, after the Lactobacillus reuteri CCFM1132 is perfused to the mouse, the content of IL-23 in the skin is reduced by 37.6%, which is obviously reduced compared with the model group (p is less than 0.0001), and the strain can inhibit inflammatory reaction; after the mice are perfused with lactobacillus reuteri L.reuteri1, the content of IL-23 in the skin is only reduced by 12.4%.
The experimental results show that the Lactobacillus reuteri CCFM1132 can obviously reduce typical up-regulated proinflammatory factors in psoriasis mice to a normal level, especially reduce the IL-23 level, and is obviously superior to another Lactobacillus reuteri L.reuteri1 strain.
Example 5: effect of Lactobacillus reuteri on IL-17 in psoriasis mouse skin
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 comprises a CCFM1132 group of Lactobacillus gasseri CCFM1132 and an L.reuteri1 group of Lactobacillus gasseri L.reuteri1, 6 animals in each group are bred in the experimental animal center of south Jiangnan university, fed by common feed, have constant temperature of 21-26 ℃, humidity of 40-70%, noise of less than or equal to 60dB and animal illumination of 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethical administration committee of south Jiangnan university).
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 CCFM1132 group was gavaged with 0.2mL per day (viable count was 1X 10)9CFU/mL) of CCFM1132 bacterial suspension, and L.reuteri1 group gavage 0.2mL (viable count of 1 × 10)9CFU/mL), normal and model groups were gavaged with only the same amount of sterile saline as control, and all groups were free access to water and food. 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-17 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 Lactobacillus reuteri CCFM1132 is perfused to the mouse, the IL-17 content in the skin is reduced by 18.5%, which is obviously reduced compared with the model group (p is less than 0.05), and the strain can inhibit inflammatory reaction; after the mice are perfused with the lactobacillus reuteri1, the content of IL-17 in the skin is only reduced by 9.7 percent, and no obvious difference exists in a model group.
The experimental results show that the lactobacillus reuteri CCFM1132 can obviously reduce typical up-regulated proinflammatory factors in psoriasis mice to a normal level, especially reduce the IL-17 level, and is obviously superior to another lactobacillus reuteri L.reuteri 1.
Example 6: preparation method of powder containing lactobacillus reuteri CCFM1132 bacteria
Inoculating the seed solution of the lactobacillus reuteri CCFM1132 preserved in a bacterium-preserving tube into an mMRS culture medium according to the inoculation amount accounting for 3% of the total mass of the culture medium, and culturing at 37 ℃ for 30h to obtain a culture solution; centrifuging the culture solution, and collecting thalli; cleaning the thallus with phosphate buffer solution with pH of 7.2 for 3 times, then re-suspending with trehalose freeze-drying protective agent with trehalose concentration of 100g/L, and controlling the mass ratio of the freeze-drying protective agent to the thallus to be 2:1 to obtain re-suspension; precooling the resuspension at-80 ℃ for 1.5h, and immediately transferring to a freeze dryer for drying for 24h to obtain the Lactobacillus reuteri CCFM1132 powder.
Example 7: preparation method of solid beverage containing lactobacillus reuteri CCFM1132
Prepared from example 6 and containing 109CFU lactobacillus reuteri CCFM1132 powder mixed with maltodextrin at a ratio of 2:1 to obtain the solid beverage rich in lactobacillus reuteri CCFM 1132.
Example 8: preparation of yoghourt containing lactobacillus reuteri CCFM1132
Mixing milk powder, inulin, stevioside and water in a weight ratio of 20: 5: 5: 75, mixing and homogenizing to prepare a fermentation raw material; sterilizing at 121 deg.C for 300s, cooling to 42 deg.C, inoculating mixed powder of Lactobacillus bulgaricus and Streptococcus thermophilus, fermenting at 42 deg.C for 12 hr, and controlling thallus concentration of Lactobacillus bulgaricus and Streptococcus thermophilus to 105CFU/g and 107CFU/g, and then blending; cooling the fermentation product to 37 deg.C, adding lyophilized powder of Lactobacillus reuteri CCFM1132 with a feeding amount of 109And (3) stirring and canning the CFU/ml yoghourt, preserving at 4 ℃ for 2 days, naturally completing after-ripening, and preparing the probiotic yoghourt.
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.

Claims (6)

1. Use of Lactobacillus reuteri CCFM1132 or a fermentation broth comprising Lactobacillus reuteri CCFM1132 for the manufacture of a medicament for alleviating psoriasis, wherein the Lactobacillus reuteri CCFM1132 has been deposited with the cantonese culture collection in 7/22 of 2020 with the deposit number GDMCC No. 61093.
2. The use of claim 1, wherein the alleviating psoriasis comprises: relieving the symptoms of wrinkles, scales and/or erythema, or inhibiting the thickening of skin cutin.
3. The use according to claim 1, wherein the number of viable Lactobacillus reuteri CCFM1132 in the medicament is not less than 1 x 106CFU/mL or 1X 106CFU/g。
4. The use according to claim 1, wherein the medicament comprises lactobacillus reuteri CCFM1132, a pharmaceutical carrier and/or a pharmaceutical excipient.
5. The use of claim 4, wherein the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes; the pharmaceutic adjuvant comprises an excipient and an additive.
6. The use according to claim 1, wherein the starter is prepared as follows: inoculating lactobacillus reuteri CCFM1132 into a culture medium, and culturing at 35-37 ℃ for 20-30 h to obtain a culture solution; centrifuging the culture solution, and collecting thalli; washing the thalli, and then resuspending the thalli by using a freeze-drying protective agent to obtain a resuspension solution; and freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the fermentation agent containing the lactobacillus reuteri CCFM 1132.
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