CN113913330B - Lactobacillus plantarum for regulating OVA-specific IgE and application thereof - Google Patents
Lactobacillus plantarum for regulating OVA-specific IgE and application thereof Download PDFInfo
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Abstract
The invention discloses lactobacillus plantarum for regulating OVA specific IgE and application thereof, and belongs to the technical field of microorganisms and medicines. The lactobacillus plantarum CCFM1189 disclosed by the invention is preserved in the microorganism strain preservation center of Guangdong province at the year 6 and the month 30 of 2021, and the preservation number is GDMCC No:61763. after the lactobacillus plantarum CCFM1189 provided by the invention is acted on a food allergy mouse, the anaphylactic reaction of the mouse can be obviously relieved, and the method is particularly embodied in that the weight reduction of the food allergy mouse is relieved, the jejunal tissue lesion degree of the food allergy mouse is relieved, the IL-4 level, the IL-5 level and the IL-13 level in jejunum of the food allergy mouse are obviously reduced, and the content of indoleacrylic acid in the mouse excrement is obviously improved, so that the lactobacillus plantarum CCFM1189 has great application prospect in preparing products for preventing and/or treating the food allergy.
Description
Technical Field
The invention discloses lactobacillus plantarum for regulating OVA specific IgE and application thereof, and belongs to the technical field of microorganisms and medicines.
Background
Food allergy refers to an immune response caused by food or food additives, which can lead to allergies in the digestive system or systemically. With the increasing incidence of food-borne allergic diseases, food allergy is one of the important public health problems of global concern, and the incidence of food allergy is high in north america, europe and asia. The clinical symptoms of food allergy are various, may have gastrointestinal symptoms, respiratory symptoms or skin symptoms, and have different attack time, and may be acute, subacute or chronic, so that the health of patients is greatly affected. Since food allergy is usually a hypersensitivity reaction caused by the fact that some proteins existing in the food allergy are considered as antigen substances by an organism when the food is ingested and absorbed by the gastrointestinal tract, the gastrointestinal tract is often the most severely damaged target organ in the food allergy, for example, for infants whose gastrointestinal functions are not fully developed, the food allergy is easy to cause noninfectious chronic diarrhea of the infants, the course of the disease is long and the disease is easy to repeatedly attack, and the growth and the physical health of the infants are greatly affected.
Food allergy can be classified into IgE-mediated food allergy and non-IgE-mediated food allergy according to whether or not the allergic reaction is mediated by immunoglobulin E (IgE), and mixed food allergy mediated by both IgE and immune cells. IgE-mediated food allergy belongs to type I hypersensitivity, and its clinical symptoms mainly include gastrointestinal symptoms (oral allergy syndrome, gastrointestinal allergy, etc.), skin (urticaria, measles, angioedema, rash, flushing, etc.), anaphylactic shock, etc., the onset is usually acute, and symptoms often occur within 2 hours after allergen intake. In IgE-mediated type I allergic reactions, proteins and the like in foods are regarded as antigenic substances, and after passing through intestinal mucosal barriers, they are taken up by dendritic cells and taken to regional lymph nodes, degraded into peptides, presented to primary CD4 positive T cells by class II molecules (MHC II) of the major histocompatibility complex on the surface of Dendritic Cells (DCs), activate helper T lymphocyte 2 (Th 2) cells to differentiate and expand, and secrete a large amount of Th 2-type cytokines such as interleukin 4 (IL-4) which induce proliferation and differentiation of specific B cells into plasma cells capable of IgE production. The Fc segment of IgE produced by plasma cells has a receptor Fc epsilon RI with high affinity to mast cells and basophils, and the receptor Fc epsilon RI is combined with mast cells or basophils to make the organism in a sensitized state.
The current methods for treating food allergy mainly avoid eating foods containing allergens, desensitization therapy and drug treatment strictly, but have some defects. Due to the imperfections of the food allergen tags, ways to strictly avoid eating food containing allergens are sometimes difficult to perform; the course of treatment, dosage and therapeutic effect of desensitization therapy varies from person to person and presents a certain risk; the medicines for treating allergy on the market only treat a specific symptom caused by allergy, and cannot change the progress of allergy. The drugs for treating allergy commonly used at present mainly comprise omalizumab. However, the omalizumab is mainly applied to respiratory tract allergy, and some researches have explored the therapeutic effect of the omalizumab on gastrointestinal allergy, and the results show that the omalizumab can improve symptoms of patients with severe food allergy, but can not change pathological damage of intestinal tissues caused by food allergy, and can not achieve the curative effect fundamentally. Thus, for food allergy, there is an urgent need to find a safe and effective way to alleviate its symptoms.
Numerous studies have shown that food allergy causes a change in the intestinal flora of patients or mice with a difference in the composition of the intestinal flora of healthy people or mice. On the other hand, the composition of the intestinal flora is different and affects the onset of food allergy. Some beneficial microorganisms in the intestinal tract can improve intestinal peristalsis, release beneficial substances, prevent pathogenic bacteria from colonizing the intestinal tract, and maintain the balance of intestinal flora. Whereas the intestinal flora of food allergy sufferers is often altered. The probiotics such as bifidobacteria and lactobacillus can maintain the regulation function of intestinal mucosa immune system through various mechanisms, regulate type 2 immunity, promote the barrier function of intestinal tracts and the like. Thus, probiotics may further modulate host immune system function by affecting the composition of the intestinal flora, host intestinal metabolism to enhance the intestinal barrier effect, to alleviate food allergy.
In summary, it is possible to solve the problem that the food allergy treatment is difficult to be carried out and easy to be repeated by developing probiotics capable of alleviating the pathological characteristics of the food allergy mice.
Disclosure of Invention
The invention provides a lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 which has been deposited with the Guangdong province microorganism strain collection at the year 6 and the month 30 of 2021, and the deposited number is GDMCC No:61763.
in one embodiment of the invention, the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 is isolated from a fermented fruit and vegetable product, the strain is subjected to sequencing analysis, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence alignment in Genbank, and the result shows that the similarity with the nucleic acid sequence of the lactobacillus plantarum is as high as 99.50 percent, and the sequence is named as lactobacillus plantarum (Lactobacillus plantarum) CCFM1189.
Lactobacillus plantarum CCFM1189 is inoculated into MRS solid culture medium, after being cultured for 24 hours at 37 ℃, bacterial colonies are observed and bacterial colonies are observed under a microscope, and the bacterial colonies are milky white, irregular, round convex and smooth, and the bacterial colonies are slightly irregular in shape and form campylobacter with round tail ends and usually exist singly, in pairs and in small clusters.
The invention also provides a microbial preparation containing the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189.
In one embodiment of the present invention, the number of Lactobacillus plantarum in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
The invention also provides a product, which contains the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189.
In one embodiment of the invention, the viable count of lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 in the product is at least: 1X 10 6 CFU/g。
In one embodiment of the invention, the product comprises a food or pharmaceutical product.
In one embodiment of the invention, the products include, but are not limited to, general foods, special foods, and pharmaceuticals.
In one embodiment of the invention, the food product is a food product comprising lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 or a fermented metabolite thereof.
In one embodiment of the invention, the food product is a dairy product, a soy product or a fruit and vegetable product produced using lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 or a starter culture comprising lactobacillus plantarum CCFM1189.
In one embodiment of the invention, the food product is a solid beverage containing lactobacillus plantarum (Lactobacillus plantarum) CCFM1189.
In one embodiment of the invention, the pharmaceutical product comprises lactobacillus plantarum (Lactobacillus plantarum) CCFM1189, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and additives.
In one embodiment of the invention, the pharmaceutical excipients comprise anti-adhesive, permeation enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH modifiers, adhesives, disintegrants, fillers, lubricants, wetting agents, integration agents, tonicity modifiers, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids, and release retarders.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.
In one embodiment of the invention, the dosage form of the pharmaceutical product comprises granules, capsules, tablets, pills or oral liquids.
The invention also provides application of the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 or the microbial preparation in preparing a product for relieving food allergy.
In one embodiment of the invention, the food allergy is an OVA-induced gastrointestinal allergy. Clinically, the indexes for judging food allergy, especially gastrointestinal allergy reaction, are mainly as follows: serum OVA-specific IgE.
In one embodiment of the invention, the alleviating food allergy comprises: relieving food allergy symptoms, relieving mouse serum OVA specific IgE, relieving mouse weight loss, relieving mouse jejunal lesions, and regulating Th1/Th2 reaction balance.
In one embodiment of the invention, the product includes, but is not limited to, a food product, a functional food product, a nutraceutical or a pharmaceutical.
In one embodiment of the invention, the products include, but are not limited to, general foods, special foods, and pharmaceuticals.
In one embodiment of the inventionIn an embodiment, the product has a viable count of lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 of at least: 1X 10 6 CFU/g。
In one embodiment of the invention, the pharmaceutical product comprises lactobacillus plantarum (Lactobacillus plantarum) CCFM1189, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and additives.
In one embodiment of the invention, the pharmaceutical excipients comprise anti-adhesive, permeation enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH modifiers, adhesives, disintegrants, fillers, lubricants, wetting agents, integration agents, tonicity modifiers, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids, and release retarders.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.
In one embodiment of the invention, the dosage form of the pharmaceutical product comprises granules, capsules, tablets, pills or oral liquids.
In one embodiment of the invention, the food product is a food product comprising lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 or a fermented metabolite thereof.
In one embodiment of the invention, the food product is a dairy product, a soy product or a fruit and vegetable product produced using lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 or a starter culture comprising lactobacillus plantarum CCFM1189.
In one embodiment of the invention, the food product is a solid beverage containing lactobacillus plantarum (Lactobacillus plantarum) CCFM1189.
In one embodiment of the invention, the preparation method of the starter is as follows: inoculating lactobacillus plantarum CCFM1189 into a culture medium according to the inoculum size accounting for 2-4% of the total mass of the culture medium, and culturing for 30 hours at 37 ℃ to obtain a culture solution; centrifuging the culture solution, and collecting thalli; washing thalli with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending with a freeze-drying protective agent to obtain re-suspension; lyophilizing the heavy suspension by vacuum freezing method to obtain lactobacillus plantarum CCFM1189 starter.
In one embodiment of the invention, the mass ratio of the lyoprotectant to the thallus is 2:1.
In one embodiment of the present invention, the lyoprotectant comprises skimmed milk powder, maltodextrin and sodium L-glutamate; wherein the skim milk powder: maltodextrin: the L-sodium glutamate is (8-10): 1.
In one embodiment of the present invention, the medium is prepared by dissolving 10% of skim milk, 0.5% of glucose, 1.5% of tryptone, and 0.3% of yeast extract in water, based on the total mass of the medium.
In one embodiment of the invention, the pH of the medium is 6.8.
The invention also provides application of the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 or the microbial preparation in producing fermented foods.
The invention also provides a solid beverage containing the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189.
Advantageous effects
The invention provides a lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 for relieving the pathological characteristics of food allergy mice, and further relieving the food allergy, and after the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 provided by the invention acts on the food allergy mice, the allergic reaction of the mice can be remarkably relieved, and the concrete expression is that compared with a modeling module:
(1) Weight loss in food-allergic mice was alleviated;
(2) Serum OVA-specific IgE levels were reduced by 68.1% in food allergy mice;
(3) The allergic symptoms of the food-allergic mice are relieved;
(4) The jejunum tissue lesion degree of the food allergy mice is relieved;
(5) IL-4 levels in jejunal tissues of food-sensitized mice were reduced by 50.5%;
(6) The IL-5 level in jejunal tissues of food-sensitized mice was reduced by 53.2%;
(7) The IL-13 level in jejunal tissues of food-sensitized mice was reduced by 52.6%;
(8) The content of indoleacrylic acid in the feces of the CCFM1189 mice is improved by 34.8 percent (log 2 peak area).
Therefore, the lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 has great application prospect in preparing products (such as foods, medicines or health-care foods) for preventing and/or treating food allergy.
Preservation of biological materials
Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189, classified under Lactobacillus plantarum, was deposited under the accession number GDMCC No:61763 the preservation address is building 5 of Guangdong national institute of microbiology, guangzhou Miao road 100, university 59.
Drawings
Fig. 1: the weight percentage of the mice in different groups was changed.
Fig. 2: serum OVA-specific IgE levels from mice in different groups.
Fig. 3: allergic symptoms score for different groups of experimental mice.
Fig. 4: jejunum histopathological sections of mice were tested in different groups.
Fig. 5: the mice were tested for IL-4 content in jejunal tissue in different groups.
Fig. 6: the mice were tested for IL-5 content in jejunal tissue in different groups.
Fig. 7: IL-13 content in jejunal tissues of mice was tested in different groups.
Fig. 8: the amount of indoleacrylic acid (log 2 peak area) in the feces of the mice was tested in different groups.
Fig. 9: animal experiment flow diagram.
Detailed Description
BALB/c female mice referred to in the examples below were purchased from Experimental animal technologies, inc., peking Vitrenlhua, and OVA referred to in the examples below was purchased from Sigma-Aldrich.
The preparation method of the OVA solution involved in the following examples is as follows:
sensitization solution: each mouse was intraperitoneally injected with 0.2mL of a sensitizing solution, and 0.2mL of the sensitizing solution was mixed with 0.1mL of a physiological saline solution containing 50. Mu.g of OVA and 0.1mL of an alum adjuvant containing 1mg of aluminum hydroxide.
Excitation liquid: each mouse was perfused with 0.2mL of challenge, and 0.2mL of challenge was physiological saline containing 50mg of OVA.
The allergy symptoms as referred to in the examples below are as follows:
"no allergic symptoms" is marked as "0"; the repeated ear grabbing and mouth grabbing are carried out, and the back foot is used for scratching the auditory canal and is marked as 1; "reduced activity, reddening and swelling of ear and eyes, reduced appetite" is recorded as "2"; the hair is kept still for a long time, the breathing rate is accelerated, and the hair is messy and has no luster, which is marked as 3; "the eyeball protrudes, conjunctiva is hyperemia, is stimulated and unresponsive, and has tremble and convulsion, and the bluish purple of lips is marked as" 4 "; "death" is noted as "5".
Detection of the content of indoleacrylic acid referred to in the following examples:
60mg of a mouse faecal sample is weighed, 4 times of the volume of the extracting solution (the volume ratio of methanol to acetonitrile to water=2:2:1) is added, and after vortex mixing, the tissue is ground until the faecal sample is mixed uniformly. Centrifuging at 12000r/min at 4deg.C for 15min, and concentrating the supernatant with vacuum concentrator (45deg.C, 1.0p,3-4 hr). After the concentration, 200. Mu.L of an aqueous methanol solution (methanol: water volume ratio=4:1) was added, and the mixture was centrifuged at 12000r/min at 4℃for 15 minutes, and the mixture was passed through a 0.22 μm filter membrane, and was subjected to on-line detection using Q exact. The off-line data were analyzed using Compound Discoverer 3.2.3.2, compared using mZ closed, KEGG, chemSpider databases, and metabolites were screened for VIP values (Variable important for the projection) > 1 and FC values (Fold change) > 2, and the peak areas were used to compare the levels.
The following examples relate to the following media:
LBS medium formulation (1L): 10g of peptone, 5g of yeast powder, 20g of glucose, 17g of anhydrous sodium acetate, 1mL of tween 80, 6g of monopotassium phosphate, 2g of ammonium citrate, 0.034g of ferrous sulfate, 0.575g of magnesium sulfate, 0.12g of manganese sulfate and pH of 5.4+/-0.2.
LBS solid medium formulation (1L): 10g of peptone, 5g of yeast powder, 20g of glucose, 17g of anhydrous sodium acetate, 1mL of tween 80, 6g of monopotassium phosphate, 2g of ammonium citrate, 0.034g of ferrous sulfate, 0.575g of magnesium sulfate, 0.12g of manganese sulfate, 15g of agar and pH of 5.4+/-0.2.
MRS Medium formulation (1L): 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose and K 2 HPO 4 2g, diammonium citrate 2g, sodium acetate 2g, tween 80 1mL, mgSO 4 ·7H 2 O 0.5g，MnSO 4 ·4H 2 O0.25 g, pH 7.2-7.4.
MRS solid Medium formulation (1L): 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose and K 2 HPO 4 2g, diammonium citrate 2g, sodium acetate 2g, tween 80 1mL, mgSO 4 ·7H 2 O 0.5g，MnSO 4 ·4H 2 O0.25 g, agar 20g, pH 7.2-7.4.
The detection method involved in the following examples is as follows:
the method for detecting the number of living bacteria comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus detection is adopted.
The acidity detection method comprises the following steps: the national standard GB 431334-2010 is adopted.
Lactobacillus plantarum 1 is another strain isolated from different fecal samples by the same method.
Example 1: acquisition of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189
(1) Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 screening:
the fermentation liquid sample (sample number is VJLHD 12) of the fermented fruit and vegetable is sucked by a pipetting gun, the sample is subjected to gradient dilution by using physiological saline, 100 mu L of dilution liquid is coated on a flat plate (LBS solid culture medium, sterile nystatin is added according to the volume of 0.5 per mill of the culture medium before the flat plate is poured into the flat plate), the flat plate is inverted and cultured for 48 hours by a 37 ℃ constant temperature incubator, single colonies are picked up and subjected to flat plate streaking according to colony morphology classification (the strain CCFM1189 is the picked up 4 th colony), the flat plate is inverted and cultured for 48 hours by the 37 ℃ constant temperature incubator, the single colonies are picked up and inoculated into 5mL of liquid LBS culture medium, after the flat plate is inoculated into the 37 ℃ constant temperature incubator for 16-18 hours, 1.5mL of thallus 6000r/min is taken, the supernatant is removed by centrifugation for 3min, 1mL of 30% sterile glycerin is added for preservation, and meanwhile, 1.5mL of thallus is left for centrifugation, and the supernatant is removed and then resuspended by sterile water for strain identification.
The sequence of the strain is shown as SEQ ID NO.1 by sequencing analysis, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in Genbank, and the result shows that the similarity with the nucleic acid sequence of lactobacillus plantarum is up to 99.50%, and the strain is named as lactobacillus plantarum (Lactobacillus plantarum) CCFM1189.
(2) Lactobacillus plantarum 1 screen:
performing gradient dilution on fermented vegetable samples, taking 100 mu L of diluent coating plates (LBS solid culture medium, adding sterile nystatin according to the volume of 0.5 per mill of the culture medium before pouring the plates, placing the plates in a constant temperature incubator for inversion culture for 48 hours at 37 ℃, classifying according to colony morphology, taking single colonies, placing the single colonies on the LBS solid culture medium for flat streaking, placing the plate in the constant temperature incubator for inversion culture for 48 hours at 37 ℃, taking single colonies, inoculating the single colonies into 5mL of liquid LBS culture medium, placing the plate in the constant temperature incubator for culture for 16-18 hours at 37 ℃, taking 1.5mL of bacteria 6000r/min for centrifugation for 3 minutes to remove supernatant, adding 1mL of 30% sterile glycerol for preservation, leaving a 1.5mL of bacteria for centrifugation, removing the supernatant, and then re-suspending the supernatant with sterile water for strain identification. The sequence obtained by sequencing is subjected to nucleic acid sequence comparison in Genbank, and the result shows that the similarity with the nucleic acid sequence of lactobacillus plantarum is up to 99.42%, and the sequence is named as lactobacillus plantarum L.plantarum 1.
Example 2: culture of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189
The method comprises the following specific steps:
inoculating lactobacillus plantarum CCFM1189 and lactobacillus plantarum L.plantarum 1 into an MRS liquid culture medium, culturing for 16 hours at 37 ℃, transferring the sucked bacterial liquid into a fresh MRS liquid culture medium according to an inoculum size of 4%, culturing for 12 hours under the same conditions, centrifuging the bacterial cells for 15 minutes at 8000r/min, washing the bacterial cells with 0.9% physiological saline, centrifuging for 10 minutes again at 8000r/min, collecting the bacterial cells, re-suspending the bacterial cells with 30% (m/v) sucrose solution, preparing heavy suspension, and freezing at-80 ℃ for later use.
Preparation of bacterial suspension for gastric lavage: when lactobacillus plantarum is used for lavaging mice, the prepared heavy suspension is taken out from the temperature of minus 80 ℃, centrifuged for 10min at the temperature of 4 ℃ and the pressure of 8000r/min, the supernatant is discarded, and the physiological saline with the concentration of 0.9% is used for resuspension, thus obtaining the bacterial suspension for lavaging the stomach.
Example 3: alleviation of weight loss in food-allergic mice by Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189
The method comprises the following specific steps:
SPF-class BALB/c female mice of 4-5 weeks of age are divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of lactobacillus curvulus CCFM1189 and an L.plantarum 1 group of lactobacillus curvulus L.plantarum 1;
6 animals are fed in the center of experimental animals of university of Jiangnan, and are 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 illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period is 7 weeks in total, the 1 st week is the adaptation period, the weight of the mice is weighed after the adaptation period is ended, and the mice are fed in the adaptation period: co60 irradiation laboratory mice grow and reproduce feed (Jiangsu province cooperative medical bioengineering Limited liability company); the culture process of each group at 2 to 7 weeks is shown in FIG. 9.
And (3) a molding process: molding is started at week 2, the weight of the mice is weighed at the same time every week, and the intraperitoneal injection of OVA solution is performed for sensitization on the third day of week 2 and the third day of week 4;
the third day of week 6 was followed by OVA solution lavage of 0.2mL, once every other day, each mouse was 0.2mL, until the last OVA solution lavage was performed on day 1 of week 8, the amount of lavage was 0.2mL, and the mice were weighed the next day of each OVA solution lavage. Mice were sacrificed 3 hours after the last challenge after the last weighing of the mice.
Model group: during the molding process, mice were perfused with 0.2mL of 0.9% physiological saline daily.
CCFM1189 group: in the molding process, 0.2mL of viable bacteria with the number of 1 multiplied by 10 is filled into the stomach of the mice of the experimental group every day 9 CFU/mL of CCFM1189 bacterial suspension was prepared as in example 2.
Group l.plantarum 1: in the molding process, 0.2mL of viable bacteria with the number of 1 multiplied by 10 is filled into the stomach of the mice of the experimental group every day 9 CFU/mL of L.plantarum 1 bacterial suspension was prepared in the same manner as in example 2.
Normal group: 0.2mL of 0.9% physiological saline was infused daily.
The results are shown in table 1 and fig. 1:
table 1: variation of weight percentage of mice of different groups (100% on day 0)
As can be seen from table 1 and fig. 1, the normal group effectively alleviated weight loss in mice caused by food allergy compared to the model group, and the alleviating effect was better compared to the l.plantarum 1 group.
The experimental results show that lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 can relieve the weight loss degree of the mice with food allergy more than lactobacillus plantarum L.plantarum 1.
Example 4: alleviation of serum specific IgE levels in food-allergic mice by Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189
The method comprises the following specific steps:
SPF-class BALB/c female mice of 4-5 weeks of age were divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of Lactobacillus curvatus CCFM1189 and an L.plantarum 1 group of Lactobacillus curvatus L.plantarum 1, 6 animals per group were fed to a university laboratory animal center in Jiangnan, and were fed with a normal feed at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and animal illuminance of 15-20LX (all animal laboratory procedures were examined and approved by the Committee for animal welfare and ethics in Jiangnan university).
The experimental period is 7 weeks in total, and week 1 is the adaptation period, and the adaptation period feeds: co60 irradiation laboratory mice grow and reproduce feed (Jiangsu province cooperative medical bioengineering Limited liability company); the culture process of each group at 2-7 weeks is shown in FIG. 9
And (3) a molding process: molding is started at week 2, and the intraperitoneal injection of OVA solution is performed for sensitization on the third day of week 2 and the third day of week 4;
the third day of week 6 was followed by an OVA solution lavage of 0.2mL, each time an OVA solution lavage was followed every other day, all of 0.2mL, until day 1 of week 8 was followed by a final OVA solution lavage of 0.2mL, and mice were sacrificed 3 hours after the final OVA solution lavage.
Model group: during the molding process, mice were perfused with 0.2mL of 0.9% physiological saline daily.
CCFM1189 group: in the molding process, 0.2mL of viable bacteria with the number of 1 multiplied by 10 is filled into the stomach of the mice of the experimental group every day 9 CFU/mL of CCFM1189 bacterial suspension was prepared as in example 2.
Group l.plantarum 1: in the molding process, 0.2mL of viable bacteria with the number of 1 multiplied by 10 is filled into the stomach of the mice of the experimental group every day 9 CFU/mL of L.plantarum 1 bacterial suspension was prepared in the same manner as in example 2.
Normal group: 0.2mL of 0.9% physiological saline was infused daily.
After taking blood from eyeballs and dying the mice, standing the blood of the mice for 2 hours, centrifuging at 3000r/min, taking serum of the mice, and measuring the content of OVA specific IgE in the serum of the mice by an ELISA kit, wherein the result is shown in figure 2.
As can be seen from fig. 2, after the mice were perfused with lactobacillus plantarum CCFM1189, the serum content of OVA-specific IgE was reduced, and the content was: 1.03ng/mL, which is significantly lower (p < 0.0005) than the model group (IgE content 3.23 ng/mL) by 68.1%; reduced by 27.5% compared with the normal group (content of IgE is 1.42 ng/mL); the strain lactobacillus plantarum CCFM1189 disclosed by the invention can inhibit the production of OVA-specific IgE; after mice were perfused with L.plantarum 1, the serum level of OVA-specific IgE was 2.09ng/mL, which also had a reduction but no significant differences.
The above experimental results show that lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 is more capable of alleviating OVA-specific IgE levels in serum of food-allergic mice than lactobacillus plantarum l.plantarum 1.
Example 5: alleviation of allergic symptoms in food-allergic mice by Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189
SPF-class BALB/c female mice of 4-5 weeks of age were divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of Lactobacillus curvatus CCFM1189 and an L.plantarum 1 group of Lactobacillus curvatus L.plantarum 1, 6 animals per group were fed to a university laboratory animal center in Jiangnan, and were fed with a normal feed at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and animal illuminance of 15-20LX (all animal laboratory procedures were examined and approved by the Committee for animal welfare and ethics in Jiangnan university).
The experimental period is 7 weeks in total, and week 1 is the adaptation period, and the adaptation period feeds: co60 irradiation laboratory mice grow and reproduce feed (Jiangsu province cooperative medical bioengineering Limited liability company); the culture process of each group in the 2 nd to 7 th weeks is as follows:
and (3) a molding process: molding is started at week 2, and the intraperitoneal injection of OVA solution is performed for sensitization on the third day of week 2 and the third day of week 4;
the third day of week 6 starts to perform the OVA solution lavage for 0.2mL, and the OVA solution lavage is performed once every other day, which is 0.2mL, until the last OVA solution lavage is performed on day 1 of week 8, and the lavage amount is 0.2mL.
Model group: during the molding process, mice were perfused with 0.2mL of 0.9% physiological saline daily.
CCFM1189 group: in the molding process, 0.2mL of viable bacteria with the number of 1 multiplied by 10 is filled into the stomach of the mice of the experimental group every day 9 CFU/mL of CCFM1189 bacterial suspension was prepared as in example 2.
Group l.plantarum 1: in the molding process, 0.2mL of viable bacteria with the number of 1 multiplied by 10 is filled into the stomach of the mice of the experimental group every day 9 CFU/mL of L.plantarum 1 bacterial suspension was prepared in the same manner as in example 2.
Normal group: 0.2mL of 0.9% physiological saline was infused daily.
Mice were scored for allergic symptoms according to the allergic symptoms scoring table after the last OVA solution lavage. Wherein, the "no allergic symptoms" is marked as 0 score, the "repeated ear and mouth grabbing occurs, the auditory canal is scratched by the hind legs, the tail part has scratching marks, the" hair is not smooth "is marked as 1 score, the" activity is reduced, the food intake is reduced, the hair is messy "is marked as 2 scores, the" long-time static, the respiration rate is accelerated, the hair is messy and not glossy, the hair is vertical, the "is marked as 3 scores, the" eyeballs are protruded, conjunctiva is hyperemia, no response is caused by stimulus, tremble and convulsion occur, the "bluish purple at the lips" is marked as 4 scores, the "death" is marked as 5 scores, and the scoring result is shown in figure 3.
As can be seen from fig. 3, the hair of the normal group mice was smooth and glossy, without allergic symptoms; the hair of the mice in the model group is messy and non-glossy, and the hair is vertical; compared with the L.plantarum 1 group, the CCFM1189 group of mice can relieve the allergic symptoms of the mice caused by food allergy.
The experimental results show that lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 can relieve allergic symptoms of food-allergic mice more than lactobacillus plantarum L.plantarum 1.
Example 6: alleviation of the extent of jejunal tissue lesions in food-allergic mice by Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189
SPF-class BALB/c female mice of 4-5 weeks of age were divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of Lactobacillus curvatus CCFM1189 and an L.plantarum 1 group of Lactobacillus curvatus L.plantarum 1, 6 animals per group were fed to a university laboratory animal center in Jiangnan, and were fed with a normal feed at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and animal illuminance of 15-20LX (all animal laboratory procedures were examined and approved by the Committee for animal welfare and ethics in Jiangnan university).
Specific treatments for model, CCFM1189, L.Plantarum 1 and normal groups were the same as in example 4, mice were sacrificed 3 hours after the last OVA solution gastric lavage. After the jejunum tissue of the mouse is fixed by 4% paraformaldehyde, washing, dehydrating, transparentizing, waxing, embedding, slicing, spreading, sticking, baking, staining by hematoxylin-eosin (HE), differentiating, rinsing, counterstaining, dehydrating, transparentizing and sealing are carried out, so that the jejunum HE staining slice is manufactured. The results of jejunum HE pathological sections are shown in FIG. 4.
As can be seen from fig. 4, the small intestinal villi of the jejunum of the normal group mice are complete and orderly arranged, and the lamina propria is compact and not loose;
small intestinal villi of jejunum of mice in the model group are broken, and are arranged in disorder, inflammatory cells infiltrate, and the lamina propria is loose;
compared with the model group, the integrity, the arrangement tightness, the inflammatory cell infiltration degree and the lamina propria loosening degree of the small intestinal villi of the jejunum of the CCFM1189 group mice are relieved, and the relieving effect is better than that of L.plantarum 1.
The experimental results show that compared with lactobacillus plantarum L.plantarum 1, lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 can relieve jejunal lesion degree of food allergy mice.
Example 7: effect of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 on IL-4 levels in jejunal tissues of food-sensitive mice
SPF-class BALB/c female mice of 4-5 weeks of age were divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of Lactobacillus curvatus CCFM1189 and an L.plantarum 1 group of Lactobacillus curvatus L.plantarum 1, 6 animals per group were fed to a university laboratory animal center in Jiangnan, and were fed with a normal feed at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and animal illuminance of 15-20LX (all animal laboratory procedures were examined and approved by the Committee for animal welfare and ethics in Jiangnan university).
The specific treatment procedures of the model group, the CCFM1189 group, the L.plantarum 1 group and the normal group are the same as in example 4, mice are sacrificed 3 hours after the last time of the stomach infusion of the OVA solution, 100mg of jejunum tissues are weighed after the jejunum tissues of the mice are quickly frozen by liquid nitrogen, 1mL of RIPA lysate is added to extract jejunum tissue supernatant, and the IL-4 content in the jejunum tissue supernatant of the mice is measured by ELISA kit, and the result is shown in FIG. 5.
As can be seen from FIG. 5, after the mice were perfused with Lactobacillus plantarum CCFM1189, the content of IL-4 in jejunum tissue (content 4.58pg/mg protein) was reduced, which was significantly reduced (p < 0.005) by 50.5% compared with the model group (content 9.26pg/mg protein of IL-4); reduced by 25.6% compared with the normal group (IL-4 content is 6.16pg/mg protein); the strain lactobacillus plantarum CCFM1189 can inhibit Th2 reaction; after the mice were perfused with Lactobacillus plantarum L.plantarum 1, the IL-4 content in jejunum tissue was also reduced (IL-4 content was 7.85pg/mg protein), but it was less significant than CCFM1189.
The above experimental results show that lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 is more capable of alleviating IL-4 levels in jejunal tissue in food-sensitive mice than lactobacillus plantarum L.plantarum 1.
Example 8: effect of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 on IL-5 levels in jejunal tissues of food-sensitive mice
SPF-class BALB/c female mice of 4-5 weeks of age were divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of Lactobacillus curvatus CCFM1189 and an L.plantarum 1 group of Lactobacillus curvatus L.plantarum 1, 6 animals per group were fed to a university laboratory animal center in Jiangnan, and were fed with a normal feed at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and animal illuminance of 15-20LX (all animal laboratory procedures were examined and approved by the Committee for animal welfare and ethics in Jiangnan university).
The specific treatment procedures of the model group, the CCFM1189 group, the L.plantarum 1 group and the normal group are the same as in example 4, mice are sacrificed 3 hours after the last time of the stomach infusion of the OVA solution, the jejunum tissues of the mice are quickly frozen by liquid nitrogen, 100mg jejunum tissues are weighed, 1mL of RIPA lysate is added to extract jejunum tissue supernatant, and the IL-5 content in the jejunum tissue supernatant of the mice is measured by ELISA kit, and the result is shown in FIG. 6.
As can be seen from FIG. 6, after the mice were perfused with Lactobacillus plantarum CCFM1189, the content of IL-5 in jejunum tissue (content 0.22pg/mg protein) was reduced, which was significantly reduced (p < 0.005) by 53.2% compared to the model group (content 0.47pg/mg protein of IL-5); compared with the normal group (the content of IL-5 is 0.35pg/mg protein), the content is reduced by 37.1 percent; the strain lactobacillus plantarum CCFM1189 can inhibit Th2 reaction; and after the mice are perfused with lactobacillus plantarum L.plantarum 1, the IL-5 content (content of 0.46pg/mg protein) in jejunal tissues has no significant difference.
The above experimental results show that lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 is more capable of alleviating IL-5 levels in jejunal tissue in food-sensitive mice than lactobacillus plantarum L.plantarum 1.
Example 9: effect of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 on IL-13 levels in jejunal tissues of food-sensitive mice
SPF-class BALB/c female mice of 4-5 weeks of age were divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of Lactobacillus curvatus CCFM1189 and an L.plantarum 1 group of Lactobacillus curvatus L.plantarum 1, 6 animals per group were fed to a university laboratory animal center in Jiangnan, and were fed with a normal feed at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and animal illuminance of 15-20LX (all animal laboratory procedures were examined and approved by the Committee for animal welfare and ethics in Jiangnan university).
The specific treatment procedures of the model group, the CCFM1189 group, the L.plantarum 1 group and the normal group are the same as in example 4, mice are sacrificed 3 hours after the last time of the stomach infusion of the OVA solution, the jejunum tissues of the mice are quickly frozen by liquid nitrogen, 100mg jejunum tissues are weighed, 1mL of RIPA lysate is added to extract jejunum tissue supernatant, and the IL-13 content in the jejunum tissue supernatant of the mice is measured by ELISA kit, and the result is shown in FIG. 7.
As can be seen from FIG. 7, after the mice were perfused with Lactobacillus plantarum CCFM1189, the content of IL-13 in jejunum tissue (content 0.90pg/mg protein) was reduced, which was significantly reduced (p < 0.005) by 52.6% compared to the model group (content 1.90pg/mg protein of IL-13); there was no significant decrease compared to the normal group (IL-13 content of 0.89pg/mg protein); the strain lactobacillus plantarum CCFM1189 can inhibit Th2 reaction; in contrast, after the mice were perfused with Lactobacillus plantarum L.plantarum 1, the IL-13 content (IL-13 content 1.81pg/mg protein) in jejunal tissue was not significantly reduced.
The above experimental results show that lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 is more capable of alleviating IL-13 levels in jejunal tissue in food-sensitive mice than lactobacillus plantarum L.plantarum 1.
Example 10: effect of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189 on the content of indoleacrylic acid in mouse intestinal metabolites
SPF-class BALB/c female mice of 4-5 weeks of age were divided into 4 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1189 group of Lactobacillus curvatus CCFM1189 and an L.plantarum 1 group of Lactobacillus curvatus L.plantarum 1, 6 animals per group were fed to a university laboratory animal center in Jiangnan, and were fed with a normal feed at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and animal illuminance of 15-20LX (all animal laboratory procedures were examined and approved by the Committee for animal welfare and ethics in Jiangnan university).
The specific treatment process of the model group, the CCFM1189 group, the L.plantarum 1 group and the normal group is the same as that of example 4, mice are killed 3 hours after the last time of the OVA solution is filled with stomach, the mouse manure is collected the day before the mice are killed, 50mg of the mouse manure is weighed for non-targeted metabonomics analysis, 4 times of volume of extracting solution (methanol: acetonitrile: water=2:2:1) is added, vortex mixing is carried out for 30 seconds, the tissue is ground until the manure sample is mixed evenly, centrifugation is carried out for 15 minutes at the temperature of 4 ℃ and the speed of 12000r/min, and the supernatant is taken and put into a vacuum concentrator to be concentrated until the liquid is evaporated to dryness. 200. Mu.L of aqueous methanol (methanol: water=4:1) was added, and the mixture was centrifuged at 12000r/min at 4℃for 15 minutes, passed through a 0.22 μm filter, and then subjected to detection by a machine. The log2 (peak area) results of indoleacrylic acid after Compound Discoverer comparison and analysis are shown in FIG. 8.
As shown in FIG. 8, after the mice were perfused with Lactobacillus plantarum CCFM1189, the content of indoleacrylic acid in the feces of the mice (log 2 peak area: 30.79) was significantly increased, compared with the model group (log 2 peak area: 22.84) (p < 0.05) and the L.plantarum 1 group (log 2 peak area: 23.31) (p < 0.0001), both the content of indoleacrylic acid was significantly increased, and the content of indoleacrylic acid in the feces of the mice was significantly increased by 34.81% and 32.09% of the peak area of the log2 indoleacrylic acid, respectively, which indicates that the Lactobacillus plantarum CCFM1189 strain of the present invention can effectively increase the content of indoleacrylic acid in the feces of the mice.
The experimental result shows that lactobacillus plantarum (Lacticaseibacillus plantraum) CCFM1189 can up-regulate the content of indoleacrylic acid in the mouse feces.
Example 11: preparation of lactobacillus plantarum (Lactobacillus plantarum) CCFM 1189-containing bacterial powder
The method comprises the following specific steps:
(1) Preparation of Lactobacillus plantarum CCFM1189 seed solution
Inoculating lactobacillus plantarum CCFM1189 and lactobacillus plantarum L.plantarum 1 into an MRS liquid culture medium, and culturing for 16 hours at 37 ℃ to prepare lactobacillus plantarum CCFM1189 seed liquid;
(2) Inoculating the prepared seed solution of lactobacillus plantarum CCFM1189 into an MRS culture medium according to the inoculum size accounting for 3 percent of the total mass of the culture medium, and culturing for 30 hours at 37 ℃ to obtain a culture solution;
centrifuging the culture solution, and collecting thalli; washing thalli for 3 times by using phosphate buffer solution with pH of 7.2, then re-suspending the thalli by using trehalose freeze-drying protective agent with the concentration of 100g/L, and controlling the mass ratio of the freeze-drying protective agent to the thalli to be 2:1 to obtain re-suspension;
and immediately transferring the re-suspension to a freeze dryer for drying for 24 hours after pre-cooling for 1.5 hours at the temperature of minus 80 ℃ to obtain lactobacillus plantarum CCFM1189 bacterial powder.
Example 12: preparation of yogurt containing Lactobacillus plantarum (Lactobacillus plantarum) CCFM1189
The method comprises the following specific steps:
(1) Milk powder, inulin, stevioside and water are mixed according to the weight ratio of 20:5:5:75, mixing, homogenizing,preparing fermentation raw materials; sterilizing at 121deg.C for 300s, cooling to 42deg.C, inoculating mixed powder of Lactobacillus bulgaricus and Streptococcus thermophilus, fermenting at 42deg.C for 12 hr, and controlling the thallus concentration of Lactobacillus bulgaricus and Streptococcus thermophilus to 10 5 CFU/g and 10 7 CFU/g, and then blending; cooling the fermentation product to 37 ℃;
(2) Adding lactobacillus plantarum CCFM1189 freeze-dried powder prepared according to the method of example 11 into the cooled fermentation product in the step (1), wherein the feeding amount of the lactobacillus plantarum CCFM1189 freeze-dried powder is 10 9 CFU lactobacillus plantarum CCFM1189/mL yoghurt is stirred, canned, stored for 2 days at 4 ℃ and naturally finished after-ripening, and then the probiotic yoghurt is prepared.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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
<110> university of Jiangnan
<120> Lactobacillus plantarum for regulating OVA-specific IgE and application thereof
<130> BAA211122A
<160> 1
<170> PatentIn version 3.3
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Claims (7)
1. Lactobacillus plantarum strainLactobacillus plantarum) Wherein the lactobacillus plantarum has been deposited at the microorganism strain collection of cantonese province at 30/6/2021 under the accession number GDMCC No:61763.
2. a microbial preparation comprising lactobacillus plantarum of claim 1.
3. The microbial preparation according to claim 2, wherein the number of lactobacillus plantarum in the microbial preparation is not less than 1 x 10 6 CFU/mL or 1X 10 6 CFU /g。
4. A product, characterized in that the lactobacillus plantarum according to claim 1 is contained in the product, and the product is common food, special food and medicine.
5. The product according to claim 4, wherein the viable count of lactobacillus plantarum in the product is at least: 1X 10 6 CFU /g。
6. Use of the lactobacillus plantarum of claim 1, or the microbial formulation of claim 2 or 3, for the manufacture of a medicament for alleviating food allergy, characterized in that the food allergy is an OVA-induced gastrointestinal allergy.
7. Use of the lactobacillus plantarum of claim 1, or the microbial preparation of claim 2 or 3, for the production of fermented food products.
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