CN113841737A - Application of lactobacillus reuteri strain derived from breast milk to prevention and treatment of atopic dermatitis - Google Patents
Application of lactobacillus reuteri strain derived from breast milk to prevention and treatment of atopic dermatitis Download PDFInfo
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- CN113841737A CN113841737A CN202111233636.7A CN202111233636A CN113841737A CN 113841737 A CN113841737 A CN 113841737A CN 202111233636 A CN202111233636 A CN 202111233636A CN 113841737 A CN113841737 A CN 113841737A
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- lactobacillus reuteri
- atopic dermatitis
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- treatment
- mice
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Abstract
The invention discloses an application of lactobacillus reuteri from breast milk in prevention and treatment of atopic dermatitis, and belongs to the technical field of microorganisms and medicines. The invention provides a new application of lactobacillus reuteri FN041 in preventing and/or treating atopic dermatitis, which can prevent the occurrence of the atopic dermatitis of mice and improve the ear swelling degree and the skin pathological symptoms of the mice; significantly reduces the score of SCORAD in children with mild atopic dermatitis. Therefore, the lactobacillus reuteri FN041 has huge application prospect in preparing products for preventing and/or treating children atopic dermatitis.
Description
Technical Field
The invention relates to an application of lactobacillus reuteri from breast milk in prevention and treatment of atopic dermatitis, and belongs to the technical field of microorganisms and medicines.
Background
Atopic Dermatitis (AD) is a chronic, recurrent, inflammatory skin disease in which more than about 60% of patients develop early infancy, mostly within 2 years of age, and persist into adolescence, even after adulthood. Severe itching of infant skin caused by AD seriously affects sleeping and growth development; with the development of the allergic process, children are also susceptible to asthma, allergic rhinitis and conjunctivitis. With the progress of urbanization and social development, in recent years, the incidence rate of skin and respiratory diseases caused by allergy in China is obviously increased, the prevalence rate of atopic dermatitis of urban children of 1 to 7 years old in China is up to 12.94%, and the prevalence rate of AD of infants in 1 to 12 months is up to 30.48%. The increasing prevalence severely impacts the quality of life for patients and families. The pathogenesis of atopic dermatitis has been established due to an immune imbalance between helper T cells 1(Th1) and helper T cells 2(Th2) in patients and the development of immune tolerance not established during the immune development window.
At the present stage, there is no therapeutic drug with definite therapeutic effect against AD. Therefore, at present, mainly: 1) anti-inflammatory effects are exerted by external drugs such as glucocorticoids and calcineurin inhibitors; 2) external zinc oxide oil (paste) preparation, black soybean distillate oil ointment, physiological sodium chloride solution and other wet-applied medicines or phosphodiesterase 4 for caring skin or preventing body fluid exudation; 3) systemic administration, including oral antihistamine, immunosuppressant, low-dose systemic glucocorticoid, biological preparation such as interleukin 4(IL-4)/13 receptor a chain fully human monoclonal antibody, Janus kinase inhibitor, etc.; 4) topically or systemically performing antibacterial, antiviral or antifungal treatment.
However, the above drugs have great adverse reactions, and most patients have concerns about hormone drugs, and are not suitable for direct application to infants, which all bring great difficulties to AD treatment. Meanwhile, the method brings great trouble to the life of patients and families. In addition, current therapeutic measures are mainly used for improving symptoms, but cannot regulate the immune balance of patients, and cannot fundamentally improve the allergic constitution of the patients. Thus, there is still a need for a medicament or treatment which is suitable for infant patients without side effects to the patient, which can be used to combat the onset of allergies and the development of skin inflammation, and which can be applied to various classes of patients.
Disclosure of Invention
The invention discovers that the lactobacillus reuteri FN041 has the effect of relieving atopic dermatitis, so the invention provides the application of the lactobacillus reuteri FN041 in preparing a product for preventing and/or treating the atopic dermatitis of a target population; the target population is pregnant women, lactating women and/or children.
In one embodiment, lactobacillus reuteri FN041 is disclosed in the patent publication with publication No. CN 110205261A.
In one embodiment, the prevention and/or treatment of atopic dermatitis comprises any one of (a) to (d):
(a) relieving inflammatory reaction and inflammatory hyperplasia;
(b) relieving the swelling degree of the affected part;
(c) reducing the number of mast cells and eosinophils;
(d) improve the infiltration degree of inflammatory cells of the mice.
In one embodiment, the gestational period is from week 14 of gestation until the infant is born.
In one embodiment, the lactation period is within 12 months from birth of the infant.
In one embodiment, the target child is no older than 12 years; preferably, the target child is no older than 8 years.
In one embodiment, the atopic dermatitis is mild atopic dermatitis, specifically atopic dermatitis having a skin lesion area of less than or equal to 40% of the body surface area.
In one embodiment, the product includes, but is not limited to, a food product, a pharmaceutical or pharmaceutical composition, and/or a nutraceutical.
In one embodiment, the product has a viable count of not less than 1 × 106CFU/day doses were given to children; preferably, the number of viable bacteria is 1X 109CFU/day doses were given to children.
In one embodiment, the form of lactobacillus reuteri comprises the form of viable cells, inactivated cells or fermentation products or metabolites of lactobacillus reuteri, or a mixture of any of the above forms.
In one embodiment, the lactobacillus reuteri is present in the product in an amount of not less than 1 x 104CFU/mL or 1X 104CFU/g。
In one embodiment, the dosage form of the product includes, but is not limited to: tablet, granule, capsule, powder, liquid or jelly.
In one embodiment, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.
In one embodiment, the food product comprises a dairy product, a soy product, a fruit and vegetable product or a beverage produced using a lactobacillus reuteri-containing starter; or the food comprises a solid beverage, tablet, soft candy and drops containing the lactobacillus reuteri.
In one embodiment, the food and health care product further comprises conventional auxiliary materials, wherein the auxiliary materials comprise one or more of fillers, flavoring agents, binders, disintegrating agents, lubricants, antacids and nutrition enhancers.
The invention has the beneficial effects that:
the invention provides a new application of lactobacillus reuteri FN041 in preventing and/or treating atopic dermatitis, has good effect and has great application prospect in preparing products for preventing and/or treating atopic dermatitis.
Animal experiments show that:
(1) the female mouse takes the lactobacillus reuteri FN041 in the gestation period and the lactation period to prevent the offspring from generating atopic dermatitis, which is shown as obviously improving the ear swelling degree and the skin pathological symptoms of the weaned offspring mouse;
(2) the female mouse takes the lactobacillus reuteri FN041 in the gestation period and the lactation period, and the offspring mouse also takes the lactobacillus reuteri FN041, so that the swelling degree and the skin pathological symptoms of the offspring mouse can be improved;
(3) administration of lactobacillus reuteri FN041 to young offspring mice significantly improved the degree of ear swelling and the skin pathology.
Human body experiments show that:
after the probiotic powder containing lactobacillus reuteri FN041 is taken by children suffering from mild atopic dermatitis, the SCORAD index of the children suffering from mild atopic dermatitis can be remarkably reduced.
Drawings
FIG. 1 is a design of an experiment; mice were treated with Lactobacillus reuteri FN041 or Lactobacillus rhamnosus GG (1X 10) daily gavage in late gestation and lactation9CFU/day), right ear of mice was swabbed with MC903 after weaning, inducing atopic dermatitis. GDMCC60546, lactobacillus reuteri FN 041; LGG, lactobacillus rhamnosus GG; MC903, calcipotriol.
FIG. 2 is a graph showing the effect of probiotic intake in the mother mice in preventing the offspring from developing atopic dermatitis; gdmcc60546 treatment significantly prevented the progeny mice from inducing ear redness by MC 903; daily ear thickness of mice after MC903 treatment; thickness of mouse ear relative to normal control at the end of mc903 treatment; GDMCC60546, lactobacillus reuteri FN 041; LGG, lactobacillus rhamnosus GG; MC903, calcipotriol.
FIG. 3 is a pathological feature of an ear section of an atopic dermatitis mouse; A. typical pathology pictures of hematoxylin-eosin stain, mast cell stain (tolylene olive blue stain), or eosinophil (Carbol 2R stain); B. the content of mast cells in unit area of the pathological section; C. eosinophil content per unit area of pathological section; GDMCC60546 is Lactobacillus reuteri FN 041; LGG is Lactobacillus rhamnosus GG; MC903 is calcipotriol.
FIG. 4 is a flow chart of the SCORAD index clinical trial for atopic dermatitis patients.
FIG. 5 is a graph comparing the effect of different groups on lowering the SCORAD index in patients with mild atopic dermatitis.
Detailed Description
The invention is further illustrated with reference to specific examples.
The enzyme-hydrolyzed skim milk, glucose, tryptone and yeast extract mentioned in the examples below were purchased from Shanghai pharmaceutical group, Inc.
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.
Lactobacillus reuteri FN041 used in the examples described below is disclosed in patent publication No. CN 110205261A.
Example i: effect of Lactobacillus reuteri FN041 on ear thickness in atopic dermatitis mice
Male and female BALB/C mice (2: 1 by number) 7 weeks old were housed in polypropylene cages, after female conception, the males were removed from the cages and the females were randomly divided into 4 groups, each: blank group (Control), MC903 dermatitis-inducing group (AD), MC903 dermatitis-inducing and gavage Lactobacillus reuteri FN041 group (AD + GDMCC60546), and MC903 dermatitis-inducing and gavage Lactobacillus rhamnosus GG group (AD + LGG), wherein the AD + GDMCC60546 group and the AD + LGG group are both treatment groups.
Gavage treatment was started one week before birth of pregnant mice: lavage PBS (1X 10) for blank group and MC903 induced dermatitis group9CFU/day, 1 time per day, 100 μ L each time), AD + GDMCC60546 group per day gavage Lactobacillus reuteri FN041(1 × 10)9CFU/day, 1 time per day, 100 μ L each time), AD + LGG group, and Lactobacillus rhamnosus GG (1 × 10) for intragastric administration9CFU/day, 1 time per day, 100 μ L each) until the end of the 3-week lactation period.
After young offspring is weaned, the offspring atopic dermatitis is induced by MC903 for 7 days, the right ear of the offspring mouse is smeared with MC 90320 mu L every day, the left ear is controlled by the same amount of absolute ethyl alcohol, a blank group is not treated, meanwhile, the stomach irrigation treatment of the same mother mouse is also carried out on each group of the offspring, and all the groups are free drinking water and food intake. The experimental procedure described above can be seen in FIG. 1. The ear thickness of 4 groups of mice was measured with an electronic vernier caliper before the start of molding (i.e., day 0), and then the change in ear thickness was measured every day, and after the end of molding (i.e., day 8), the mice were photographed and sacrificed.
As can be seen from FIG. 2, the treatment groups of Lactobacillus reuteri FN041 and Lactobacillus rhamnosus GG significantly reduced the thickness of the ears of the mice in the model group and the degree of redness of the ears of the mice after the experiment (P < 0.05). Compared with the AD + LGG group, the Lactobacillus reuteri FN041 has larger reduction degree on the thickness of the mouse ear and stronger relieving capacity on the swelling degree of the mouse ear. On day 5 of treatment, GDMCC60546 treatment inhibited ear swelling in mice by up to 50%, 25% more than LGG. At day seven, GDMCC60546 maintained a 50% inhibition, 30% higher than LGG.
Example 2: effect of Lactobacillus reuteri FN041 on skin inflammation in atopic dermatitis mice
Male and female BALB/C mice (2: 1 by number) 7 weeks old were housed in polypropylene cages, after female conception, the males were removed from the cages and the females were randomly divided into 4 groups, each: blank group (Control), MC903 dermatitis-inducing group (AD), MC903 dermatitis-inducing and gavage Lactobacillus reuteri FN041 group (AD + Fn041), MC903 dermatitis-inducing and gavage Lactobacillus rhamnosus GG group (AD + LGG), wherein the AD + Fn041 group and the AD + LGG group are both treatment groups.
The gavage treatment, the placebo group and the MC903 induced dermatitis group were started one week before the birth of pregnant mice, and the gavage PBS (1X 10)9CFU/day, 1 time per day, 100 μ L each time), AD + Fn041 group was administered daily Lactobacillus reuteri FN041(1 × 10)9CFU/day, 1 time per day, 100 μ L each time), AD + LGG group, and Lactobacillus rhamnosus GG (1 × 10) for intragastric administration9CFU/day, 1 time per day, 100 μ L each) until the end of the 3-week lactation period.
After weaning of young offspring, offspring atopic dermatitis was induced with MC903 for a total of 7 days, right ear of offspring mice was smeared with MC 90320 μ L each day, left ear was controlled with equal amount of absolute ethanol, blank group was left untreated, all groups were free to drink and eat. The specific experimental flow is shown in figure 1. After completion of molding (i.e., day 8), blood was taken and the mice were sacrificed to prepare ear sections, which were subjected to eosin staining, mast cell staining (toluidine blue staining) and eosinophil staining (Carbol 2R staining), respectively.
As can be seen from FIG. 3, the structure of epidermal cells of ear skin of the mice in the blank group is normal, and inflammatory reaction does not occur in each layer of the epidermis; the pathology of the ear skin of the mice in the dermatitis group shows obvious inflammatory hyperplastic change, meanwhile, the red and swollen degree of the ears is increased, and the number of mast cells and eosinophilic granulocytes is obviously increased; the degree of redness of ear skin of mice treated by lactobacillus reuteri FN041 and lactobacillus rhamnosus GG is reduced, the inflammatory hyperplasia is relieved, the number of eosinophilic granulocytes and mast cells is obviously reduced, and the degree of redness of ear of the mice treated by lactobacillus reuteri FN041 is lighter and the inflammatory cell infiltration is less. MC903 induction increased mouse ear mast cells by about 2.3 times. After the treatment of GDMCC60546, the number of mast cells per square millimeter of the ear section can be reduced by 62 percent, while the LGG treatment can be reduced by only 56 percent. MC903 induction increased eosinophils in the mouse ear by about 12-fold, MC903 induction decreased eosinophils in the mouse ear sections by 67% per square millimeter, while LGG decreased eosinophils by 58%.
The experiments show that the ingestion of lactobacillus reuteri FN041 and LGG in the maternal generation can prevent the offspring mice from generating AD, can obviously relieve the ear swelling degree of atopic dermatitis model mice, and improve the infiltration degree of inflammatory cells of the mice, thereby effectively relieving the atopic dermatitis symptoms of the mice, and the effect of GDMCC60546 is better than that of LGG.
Example 3: effect of Lactobacillus reuteri FN041 on SCORAD index of atopic dermatitis patients in children of 4-7 years old
80 children with atopic dermatitis were recruited in this clinical trial, and all patients were randomized into 3 groups of three groups
Group A: placebo group (20 persons) taking maltodextrin,
group B: treatment group with Lactobacillus rhamnosus GG (LGG group) (25 persons),
group C: treatment group (GDMCC 60546) with Lactobacillus reuteri FN041 was administered (35 patients).
Wherein the product administered to the placebo group is maltodextrin which does not contain a probiotic component; the GDMCC60546 group is administered with Lactobacillus reuteri FN041 (viable count 1 × 10)9CFU/bar) probiotic powder; LGG group is administered with Lactobacillus rhamnosus GG (viable count 1 × 10)9CFU/bar) probiotic powder.
The inclusion criteria of the children patients are as follows: 1) atopic dermatitis having a skin damage area of 40% or less; 2) the infant patient and the parents know the information, and the parents sign an informed consent; 3) the compliance is good; 4) patients who do not receive glucocorticoid, immunomodulator and antihistamine medicine treatment within 2 weeks before treatment.
Exclusion criteria were: 1) patients with organic, immunological, infectious, hematological diseases; 2) those who had been treated systemically with phototherapy, cytostatics, glucocorticoids, immunosuppressants within 2 weeks prior to observation; 3) no other trials were performed.
In the whole test process, the lactobacillus reuteri FN041 and the lactobacillus rhamnosus GG are taken as probiotic bacteria powder, and can be directly taken or taken with warm water (not more than 37 ℃) with the dosage of 1 strip/day.
The trial preparation period was 2 weeks, and the clinical trial period was 8 weeks, for a total of 10 weeks.
A preparation period: sick children were contacted 2 weeks prior to the trial, and the number of persons intentionally participating in the trial was counted, and they and parents or other guardians were explained about the nature and content of the trial, and informed consent was made while coordinating the time of the hospital and doctor, and the materials needed to prepare the trial.
And (3) a clinical trial period: the children who had been diagnosed by the doctor, voluntarily, and agreed to participate in the trial by the guardian were administered placebo and probiotic (the outer package was completely identical) for 8 weeks. Before the experiment, children and parents were educated in a centralized way, which shows that the reason for the AD and diet are measures for avoiding the AD in life, and hope that the children and the parents adhere to the corresponding principles during the experiment.
Diagnosis is performed after the end of the preparation period, at the beginning and at the end of the clinical trial. Recording the skin damage area, clinical characteristics, pruritus, sleep loss degree, score and other observation indexes, inquiring the compliance, and recording adverse reactions, wherein the specific experimental flow is shown in figure 4. The score index was used to assess the extent of mild AD, including:
1. area was assessed (rule 9), max score 100, and differences between infants under 2 years of age and adults were noted.
2. Six clinical features were evaluated: erythema/color deepening; edema/papules; oozing/crusting; denudation; lichenification/prurigo; and (5) drying.
The first 5 individual, average representative sites were evaluated and the uninvolved sites were evaluated dry.
Grading scale: 0-3, 0 ═ none; 1 is mild; 2 is moderate; with 3 being severe, the maximum score is 18 points.
3. Two visual analog scales: patients evaluated the degree of itching and sleep loss ties before the last 3 days (late).
The first two terms are objective SCORADs, which can be used independently, with a maximum score of 83 points, plus a maximum score of 103 points for term 3.
As shown in fig. 5, 5 final children were excluded and 75 completed the clinical study, and were administered probiotic powder and placebo for 8 weeks, respectively, and the score of lactobacillus reuteri FN041 was significantly reduced (P <0.05) after evaluation compared to that before administration of the probiotic, and the symptoms of the patients were significantly improved. Three groups of sodad significantly differed during the initial phase of intervention. Both probiotics at the end of the intervention period could alleviate the score, with LGG treatment reducing score by 16% per month, while after GDMCC60546 treatment score reduction by 28% was achieved.
Example 4: preparation method of fermented fruit and vegetable beverage containing lactobacillus reuteri FN041
The method comprises the following specific steps:
cleaning fresh vegetables and fruits, squeezing to obtain juice, sterilizing at 140 deg.C for 2 s, immediately cooling to 42 deg.C, and pressing at 10 deg.C6Inoculating Lactobacillus reuteri FN041 into CFU/mL fruit and vegetable juice, and fermenting the inoculated fruit and vegetable juice at 42 deg.C for 16 hr to obtain juice containing Lactobacillus reuteri FN041Fermented fruit and vegetable beverage containing live Lactobacillus reuteri FN041 is provided.
Through detection, the pH value of the fermented fruit and vegetable beverage containing the live lactobacillus reuteri FN041 is 3.6, and the number of the live bacteria can reach l.5 multiplied by 109CFU/mL。
The fermented fruit and vegetable beverage containing the live lactobacillus reuteri FN041 is continuously taken by children patients with atopic dermatitis for one week, so that the swelling of the affected parts of the dermatitis of the patients can be effectively relieved, the SCORAD index of the patients can be effectively reduced, and the symptoms and the life quality of the patients are obviously improved.
Example 5: preparation of fermented milk containing Lactobacillus reuteri FN041
The method comprises the following specific steps:
adding 2% casein peptide into raw milk (skim milk, fresh milk, reconstituted milk and the like), carrying out high-strength heat treatment at 95 ℃, 20min or 140 ℃ for 2 seconds, cooling to 40 ℃, inoculating 3-5% of lactobacillus reuteri FN041 in volume percentage to the sterilized raw milk, inoculating 3-5% of lactobacillus bulgaricus or streptococcus thermophilus which can be symbiotically fermented and used for preparing the commercial starter of the yoghourt in volume percentage to the raw milk inoculated with the lactobacillus reuteri FN041, and carrying out mixed fermentation on the inoculated raw milk at 42 ℃ for 4 hours to obtain the fermented milk containing the viable lactobacillus reuteri FN 041.
Through detection, the titration acidity of the fermented milk containing the live bacteria of the lactobacillus reuteri FN041 reaches 75 DEG T, and the number of the live bacteria reaches 1 × 109CFU/mL。
The fermented milk containing the live lactobacillus reuteri FN041 is continuously taken by atopic dermatitis patients for one week, so that the swelling of the dermatitis affected parts of the patients can be effectively relieved, the SCORAD index of the patients can be effectively reduced, and the symptoms and the life quality of the patients are obviously improved.
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 (10)
1. Use of lactobacillus reuteri (lactobacillus reuteri) FN041 in the manufacture of a product for the prevention and/or treatment of atopic dermatitis in a target population; the target population is pregnant women, lactating women and/or children.
2. The use according to claim 1, wherein the prevention and/or treatment of atopic dermatitis comprises any one of (a) to (d):
(a) relieving inflammatory reaction and inflammatory hyperplasia;
(b) relieving the swelling degree of the affected part;
(c) reducing the number of mast cells and eosinophils;
(d) improve the infiltration degree of inflammatory cells of the mice.
3. The use of claim 1 or 2, wherein the gestational period is from week 14 of gestation until birth of the infant; the lactation period is within 12 months from the birth of the infant; the target child is no older than 12 years.
4. Use according to any one of claims 1 to 3, wherein the product includes, but is not limited to, a food product, a pharmaceutical or pharmaceutical composition and/or a nutraceutical.
5. The use according to any one of claims 1 to 4, wherein the viable count of the product is not less than 1 x 106CFU/day doses were given to children.
6. The use according to any one of claims 1 to 5, wherein the form of Lactobacillus reuteri comprises viable cells, inactivated cells or fermentation products or metabolites of Lactobacillus reuteri, or a mixture of any of the above forms.
7. The use of any one of claims 1 to 6, wherein the product is in a dosage form including, but not limited to: tablet, granule, capsule, powder, liquid or jelly.
8. The use of any one of claims 1 to 7, wherein the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.
9. The use according to any one of claims 1 to 8, wherein the food product comprises a dairy product, a soy product, a fruit and vegetable product or a beverage produced using a fermentation broth comprising Lactobacillus reuteri; or the food comprises a solid beverage, tablet, soft candy and drops containing the lactobacillus reuteri.
10. The use according to any one of claims 1 to 9, wherein the food or health product further comprises conventional adjuvants including one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and fortifiers.
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CN113073066A (en) * | 2021-04-16 | 2021-07-06 | 北京华元生物技术研究院 | Lactobacillus reuteri and application, composition, medicine and food thereof |
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Effective date of registration: 20230322 Address after: Room 2505, Unit 2, Building 3, No. 186, Liaoning Road, Shibei District, Qingdao, Shandong Province, 266000 Applicant after: Shangpin Health Technology (Qingdao) Co.,Ltd. Applicant after: QINGDAO University Address before: 266071 Ningxia Road, Laoshan District, Qingdao, Shandong Province, No. 308 Applicant before: QINGDAO University |