CN114774314B - Lactobacillus rhamnosus strain LRa05, application and product thereof - Google Patents
Lactobacillus rhamnosus strain LRa05, application and product thereof Download PDFInfo
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- CN114774314B CN114774314B CN202210382570.6A CN202210382570A CN114774314B CN 114774314 B CN114774314 B CN 114774314B CN 202210382570 A CN202210382570 A CN 202210382570A CN 114774314 B CN114774314 B CN 114774314B
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Abstract
The invention relates to a lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05, application and a product thereof, belonging to the technical field of microorganisms. The preservation number of the lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 is CGMCC No.24377. The invention also provides application of the strain LRa05 in preparing the EPEC diarrhea relieving product, and the EPEC diarrhea relieving product based on the strain LRa05. The lactobacillus rhamnosus strain LRa05 can well inhibit EPEC in vitro, can greatly reduce the adhesion effect of EPEC on intestinal epithelial cells, and can greatly reduce the death rate, diarrhea score and weight loss of EPEC-challenged mice.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to a lactobacillus rhamnosus strain LRa05, and application and a product thereof.
Background
Diarrhea is a common symptom of the digestive tract, and refers to a clinical state in which the defecation frequency is significantly higher than that of the daily habit (more than 3 times per day), the feces are thin, the water content is increased, and the daily defecation amount is more than 200g, or undigested food or pus blood and mucus are contained. Diarrhea is often accompanied by symptoms such as urge to relieve constipation, anal discomfort, incontinence, etc. Diarrhea is classified into infectious diarrhea and non-infectious diarrhea according to the cause of the disease. Infectious diarrhea is caused by pathogens, common pathogens include bacteria, viruses, parasites, etc., with EPEC diarrhea being one of the infectious diarrhea. EPEC diarrhea refers to diarrhea caused by Enteropathogenic escherichia coli (EPEC). Currently, according to epidemiological investigation, EPEC is an important pathogenic bacterium causing acute and chronic diarrhea of infants and adult sporadic diarrhea worldwide, and this kind of bacterium can also cause infantile malnutrition. In the epidemic statistics of EPEC outbreaks in the middle and later stages of the 20 th century, the mortality rate reaches 25% -50%. EPEC is seen to be a major hazard. .
Currently, symptomatic treatments such as correction of water, electrolyte, acid-base imbalance and nutrient imbalance, supplementation of liquids, supplementation of vitamins, amino acids, fat emulsion and other nutrients as appropriate are generally selected for diarrhea. If necessary, selecting mucosa protectant such as montmorillonite powder, sucralfate, etc. For diarrhea caused by bacterial infection, antibiotic treatment is generally required, but different bacteria are selected from corresponding antibiotics. While antibiotics are used against pathogens, there are studies directed to host gut symbiotic bacteria that indicate that antibiotics can disrupt the composition of the gut microbiota, leading to dysbacteriosis and disease. In addition, the antibiotics are taken with the problems of large side effect, high cost, easy occurrence of drug resistance due to pathogenicity and the like. These drawbacks have limited the use of these antibiotics and a more safe and effective method for treating diarrhea is sought. Probiotics are a class of bacteria that are metabolized to produce beneficial metabolites, such as short chain fatty acids, by colonizing the human body, altering the composition of the host intestinal flora, to produce beneficial effects on the host. Compared with the common medicines, the probiotics has the advantages of high safety, no side effect, no drug resistance, low cost and the like.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) is one of probiotics, and although the lactobacillus rhamnosus (Lactobacillus rhamnosus) has been reported to have a relieving effect on diarrhea, the effect on EPEC diarrhea with higher mortality rate has not been reported in the field.
Disclosure of Invention
Based on the above, in order to effectively relieve the diarrhea symptom caused by EPEC and avoid drug resistance and adverse reaction caused by using drugs, the invention provides a lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with an effect of relieving the diarrhea caused by EPEC and application thereof. The invention aims to overcome the problems in the background technology and provide lactobacillus rhamnosus LRa05 capable of relieving EPEC diarrhea. The lactobacillus rhamnosus LRa05 can relieve diarrhea caused by EPEC. The invention also provides a preparation method of lactobacillus rhamnosus LRa05 capable of relieving EPEC diarrhea.
The technical scheme of the invention is as follows:
lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 is characterized by having a preservation number of CGMCC No.24377.
Use of lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 in preparing a product for relieving EPEC diarrhea is characterized in that the lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 has a preservation number of CGMCC No.24377.
The relieving EPEC diarrhea comprises: inhibit EPEC growth, and/or reduce EPEC adhesion to cells, and/or reduce mortality due to EPEC infection, and/or reduce diarrhea score due to EPEC infection, and/or improve weight loss due to EPEC infection.
The cells are referred to as colonic epithelial cells.
The article is selected from a pharmaceutical or food product; preferably, the food is a health food.
An EPEC diarrhea relieving article comprising: an active ingredient; characterized in that the active ingredients comprise: lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with a collection number of CGMCC No.24377.
The relieving EPEC diarrhea comprises: inhibit EPEC growth, and/or reduce EPEC adhesion to cells, and/or reduce mortality due to EPEC infection, and/or reduce diarrhea score due to EPEC infection, and/or improve weight loss due to EPEC infection.
The article is selected from a pharmaceutical or food product; the food is preferably a health food.
The product for relieving EPEC diarrhea also comprises auxiliary materials.
The auxiliary materials are selected from pharmaceutical auxiliary materials or product additives.
The invention also claims the use of lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with a collection number of CGMCC No.24377 for alleviating or improving EPEC diarrhea, as allowed by some national or regional patent laws.
In another aspect, the invention also claims the use of lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with a collection number of CGMCC No.24377 for inhibiting EPEC growth, and/or for reducing EPEC adhesion to cells, and/or for reducing mortality due to EPEC infection, and/or for reducing diarrhea score due to EPEC infection, and/or for improving weight loss due to EPEC infection.
The invention provides lactobacillus rhamnosus LRa05 and application thereof. The lactobacillus rhamnosus (Lactobacillus rhamnosus) LRa05 strain has a preservation unit of China general microbiological culture Collection center, a preservation time of 2022, 1 month and 24 days, a preservation number of CGMCC No.24377, and an address of: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The application of lactobacillus rhamnosus LRa05 in preparing a product for relieving or treating EPEC diarrhea.
The lactobacillus rhamnosus powder and the preparation thereof are included, and the lactobacillus rhamnosus powder is prepared according to the following preparation method: inoculating lactobacillus rhamnosus LRa05 into a seed culture medium according to an inoculum size of 10% (V/V), and culturing at 37 ℃ for 6-8h to obtain first-stage seeds; inoculating the first-stage seeds into a seed culture medium according to an inoculum size of 5%, and standing and culturing at 37 ℃ for 5-6h to obtain second-stage seeds; inoculating the secondary seeds obtained by culture into a fermentation tank according to the inoculum size of 2% (v/v), culturing in the fermentation tank, and centrifuging the fermentation liquid to obtain bacterial sludge; resuspending the thalli with a lyoprotectant (the mass ratio of the lyoprotectant to the thalli is 1:1) to obtain a resuspension; and freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain freeze-dried powder.
The formula of the seed culture medium is as follows: 20g/L glucose, 10g/L beef extract, 5g/L yeast powder, 10g/L peptone, 5g/L sodium acetate, 2g/L sodium citrate, 3g/L dipotassium hydrogen phosphate, 0.5g/L magnesium acid, 0.1g/L manganese sulfate, 1g/L Tween 80,1g/L L-semi-gloss amino acid hydrochloride, and sterilizing at 115 ℃ for 20min.
The formula of the fermentation medium is as follows: 50g/L glucose, 15g/L yeast powder, 20g/L peptone, 5g/L beef liver extract powder, 5g/L sodium acetate, 3g/L sodium citrate, 3g/L dipotassium hydrogen phosphate, 0.5g/L magnesium sulfate, 0.1g/L manganese sulfate, 1g/L Tween 80,1g/L L-semi-optical amino acid hydrochloride, and sterilizing at 115 ℃ for 20min.
The freeze-drying protective agent comprises the following components in percentage by mass: 15% trehalose, 5% skimmed milk powder, 2% sucrose, 2% glycerol and 0.5% sorbitol.
(1) The lactobacillus rhamnosus (Lactobacillus rhamnosus) LRa05 strain has a preservation unit of China general microbiological culture Collection center, a preservation time of 2022, 1 month and 24 days, a preservation number of CGMCC No.24377 and an address of: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
(2) The invention also provides a preparation method of the bacterial powder of the lactobacillus rhamnosus LRa05 capable of relieving EPEC diarrhea
(3) The method for preparing lactobacillus rhamnosus LRa05 freeze-dried powder of claim 1 comprises the following steps: inoculating lactobacillus rhamnosus LRa05 into a seed culture medium according to an inoculum size of 10% (V/V), and culturing at 37 ℃ for 6-8h to obtain first-stage seeds; inoculating the first-stage seeds into a seed culture medium according to an inoculum size of 5%, and standing and culturing at 37 ℃ for 5-6h to obtain second-stage seeds; inoculating the secondary seeds obtained by culture into a fermentation tank according to the inoculum size of 2% (v/v), culturing in the fermentation tank, and centrifuging the fermentation liquid to obtain bacterial sludge; resuspending the thalli with a lyoprotectant (the mass ratio of the lyoprotectant to the thalli is 1:1) to obtain a resuspension; and freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain freeze-dried powder.
The lactobacillus rhamnosus LRa05 capable of relieving EPEC diarrhea is provided by the invention. The concrete steps are as follows:
(1) The lactobacillus rhamnosus LRa05 bacterial suspension and the metabolite thereof have good inhibition capability on EPEC in vitro.
(2) The lactobacillus rhamnosus can greatly reduce the adhesion effect of EPEC on intestinal epithelial cells.
(3) The lactobacillus rhamnosus can obviously reduce the death rate of EPEC virus-challenged mice.
(4) The lactobacillus rhamnosus can significantly reduce diarrhea scores of EPEC challenged mice.
(5) The lactobacillus rhamnosus can remarkably reduce the weight loss of EPEC virus-challenged mice.
Therefore, the lactobacillus rhamnosus LRa05 capable of relieving diarrhea has a great application prospect in preparing products (such as solid beverages) for relieving diarrhea.
The lactobacillus rhamnosus LRa05 provided by the invention can effectively relieve diarrhea of mice caused by EPEC, and is specifically characterized in that: (1) The lactobacillus rhamnosus LRa05 has good inhibition capability on EPEC in vitro; (2) The lactobacillus rhamnosus can greatly reduce the adhesion effect of EPEC on intestinal epithelial cells; (3) The lactobacillus rhamnosus can obviously reduce the death rate of EPEC virus-fighting mice; (4) The lactobacillus rhamnosus can obviously reduce diarrhea score of EPEC virus-challenged mice; (5) The lactobacillus rhamnosus can remarkably reduce the weight loss of EPEC virus-challenged mice. The lactobacillus rhamnosus LRa05 has a great application prospect in relieving diarrhea caused by EPEC.
The preservation information of lactobacillus rhamnosus strain LRa05 of the present invention is as follows:
strain name: LRa05;
deposit number: CGMCC No.24377;
classification naming: lactobacillus rhamnosus Lactobacillus rhamnosus;
preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);
deposit unit address: beijing, chaoyang area, north Chenxi Lu No. 1, 3;
preservation date: 2022, 1 month and 24 days.
Drawings
FIG. 1 is a graph showing the results of reducing the adhesion rate of EPEC to HT-29 cells by lactobacillus rhamnosus LRa05 of Experimental example 4; in the figure, the control group refers to the positive control group of experimental example 3.
FIG. 2 shows the effect of Lactobacillus rhamnosus LRa05 intervention in Experimental example 5 on the abdominal mortality of EPEC challenged mice.
FIG. 3 is the effect of Lactobacillus rhamnosus LRa05 intervention of Experimental example 6 of the present invention on diarrhea score of EPEC challenged mice.
FIG. 4 shows the effect of Lactobacillus rhamnosus LRa05 intervention in Experimental example 7 on the body weight of EPEC challenged mice.
The abscissa of fig. 2-4 is time: days.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the drawings and embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Sources of biological materials
The strain EPEC used in the experimental example of the invention is from China center for type culture Collection of Industrial microorganisms.
The mice used in the experimental examples of the present invention were purchased from Mikroot laboratories, inc. of Nannschluskida.
HT-29 cells used in Experimental example 3 of the present invention were purchased from China center for type culture Collection of microorganisms.
The experimental example of the invention uses MRS culture medium and TSA culture medium purchased from Qingdao sea Bo biotechnology Co.Ltd.1 group example, new application of strain LRa05
The present set of examples provides the use of lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 in the preparation of a product for alleviating diarrhea in EPEC. All embodiments of this group share the following common features: the preservation number of the lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 is CGMCC No.24377.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with a accession number of CGMCC No.24377 can be used by those skilled in the art to prepare any product having efficacy of alleviating diarrhea in EPEC in accordance with the teachings and teachings of the present invention. Any behavior of using, culturing, propagating, inoculating, preparing, producing, fermenting lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with the collection number of CGMCC No.24377 and using it for preparing any form of product for relieving EPEC diarrhea falls within the scope of the present invention.
In a specific embodiment, the alleviating EPEC diarrhea comprises: inhibit EPEC growth, and/or reduce EPEC adhesion to cells, and/or reduce mortality due to EPEC infection, and/or reduce diarrhea score due to EPEC infection, and/or improve weight loss due to EPEC infection.
In a more specific embodiment, the cells are colon epithelial cells.
In some embodiments, the article is selected from a pharmaceutical or food product.
In a preferred embodiment, the food product is a health food product.
According to the teachings of the present invention, the lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with the preservation number of CGMCC No.24377 can be prepared into medicines or foods by referring to the conventional technical means in the art (such as: biopharmaceutical preparation technology, "food microorganism", etc.) in combination with the conventional production process in the microorganism field, and by culturing, expanding propagation, inoculating, preparing, producing, fermenting, etc.
Group 2 example, EPEC diarrhea relieving article of the invention
This group of embodiments provides a product for alleviating EPEC diarrhea. All embodiments of this group share the following common features: the EPEC diarrhea relieving article comprises: an active ingredient; characterized in that the active ingredients comprise: lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 with a collection number of CGMCC No.24377.
In some embodiments, the alleviating EPEC diarrhea includes, but is not limited to: inhibit EPEC growth, and/or reduce EPEC adhesion to cells, and/or reduce mortality due to EPEC infection, and/or reduce diarrhea score due to EPEC infection, and/or improve weight loss due to EPEC infection.
In other embodiments, the article is selected from a pharmaceutical or food product; the food is preferably a health food.
In a further embodiment, the product for alleviating EPEC diarrhea further comprises an adjuvant.
In a specific embodiment, the adjuvant is selected from pharmaceutical adjuvants or food additives.
In more specific embodiments, the pharmaceutical excipients include, but are not limited to: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.
The pharmaceutical excipients can be selected and adjusted by a person skilled in the art according to the dosage form and specification of the medicament in actual production, so as to obtain a suitable medicament product which is suitable for preparing, producing, storing, transporting, taking, using, disintegrating and absorbing the medicament for relieving EPEC diarrhea and takes lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 as an active ingredient.
In other more specific embodiments, the food additives include, but are not limited to: antioxidants, enzyme preparations, flavoring agents, sweeteners, preservatives, leavening agents, acidulants, flavors, and the like.
According to the production requirements of the food field, the person skilled in the art can reasonably select and adjust the food additives according to factors such as the form, package, shelf life, taste and taste of the food on the premise of not affecting the activity of the lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05 and prepare the food meeting the food safety standard.
The technical effects achieved by the above embodiments of the present invention are demonstrated by the following experimental examples.
Experimental example 1, separation screening and identification of Lactobacillus rhamnosus
(1) Isolation and selection of strains
Collecting a healthy human body excrement sample, taking 1g to 9mL of sterile physiological saline, fully oscillating and dispersing the sample, taking 100 mu L of sample for gradient dilution, selecting a proper gradient to uniformly coat on an MRS agar plate, placing the sample on an anaerobic condition, culturing at 37 ℃ for 36 to 48 hours, selecting suspected single bacterial colonies, carrying out gram staining, observing the morphological characteristics of the bacterial colonies by microscopic examination, and primarily screening strains of suspected lactobacillus. The strain is repeatedly streaked and purified on MRS solid culture medium to obtain lactobacillus purified strain.
MRS Medium formulation (g/L): peptone 10, beef extract 5, yeast powder 5, glucose 20, anhydrous sodium acetate 5, diammonium hydrogen citrate 2, tween 80 1, K 2 HPO 4 2、MgSO 4 0.2、MnSO 4 0.05, pH6.5, and 121 ℃ for 20min, and adding 1.5% of agar powder into the solid MRS culture medium.
(2) Molecular biological identification
Liquid amplification culture is carried out on the screened target strain, thalli are collected, genome DNA is extracted, and universal primers 27F are adopted: agagttttgatcctggcttag and 1492R: GGTTACCTTGTTACGACTT amplifies 16S rDNA fragment, agarose gel electrophoresis detects PCR amplified product, and sequencing PCR product. Wherein the PCR reaction system comprises: 10 Xbuffer 10ul,10mM dNTP 2ul, 1ul of each of the upper and lower primers, 2ul of DNA template, 0.5ul of Taq enzyme, ddH 2 O34.5 ul. Pre-denaturation at 95℃for 10min; then, the temperature is 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min for 35 cycles, and the temperature is 72 ℃ for 5min after the completion of the cycle. The PCR products were detected by gel electrophoresis and sent to the Wohan Jin Kairui Bio-engineering Co., ltd for sequencing. The gene sequences to be identifiedThe strain is determined to be lactobacillus rhamnosus according to molecular biological identification results by comparing the strain with BLAST tools in NCBI database. The strain was named LRa05 and was deposited as follows:
strain name: LRa05;
deposit number: CGMCC No.24377;
classification naming: lactobacillus rhamnosus Lactobacillus rhamnosus;
preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);
deposit unit address: beijing, chaoyang area, north Chenxi Lu No. 1, 3;
preservation date: 2022, 1 month and 24 days.
Experimental example 2 inhibition of EPEC by Lactobacillus rhamnosus LRa05
Activating lactobacillus rhamnosus LRa05 stored in a glycerol tube on an MRS flat plate for 2-3 times, then picking single bacterial colony, inoculating the single bacterial colony into an MRS liquid culture medium, and carrying out anaerobic culture at 37 ℃ to obtain seed liquid; inoculating the seed solution into MRS liquid culture medium at a ratio of 2%, and culturing at 37deg.C for 17-24 hr to obtain culture solution. Collecting the centrifuged thallus, re-suspending with sterile physiological saline, and adjusting concentration to 10 8 -10 9 And obtaining the bacterial suspension by CFU/mL. Collecting supernatant after centrifugation, and filtering with a microporous membrane of 0.22 μm to obtain metabolite.
After the indicator strain EPEC is activated on a TSA solid plate culture medium, single bacterial colony is selected and inoculated to the TSA liquid culture medium, and after continuous transfer for two generations, the bacterial strain EPEC is centrifuged and collected. The thalli are subjected to resuspension washing by using sterile PBS, then are centrifugally collected, and the concentration of the bacterial suspension is adjusted to 10 by using the sterile PBS 8 CFU/mL。
Cooling MRS agar culture medium containing 1.5% agar to about 55deg.C, mixing with strain suspension at a certain ratio, and regulating concentration of indicator bacteria (10) 6 CFU/mL), then rapidly pouring the mixture into a plate in which an oxford cup is placed in advance, taking out the oxford cup after the culture medium is cooled and solidified, and adding 100 mu L of antagonistic bacteria liquid (the number of viable bacteria is 10) into each hole 7 CFU、10 8 CFU) and 5 are injected into each hole respectively0 mu and 100 mu L of metabolite, after standing for several minutes, put into a constant temperature incubator at 37 ℃, observe after proper time of culture, measure the diameter of the inhibition zone with vernier caliper, and the result is shown in Table 1. The results are shown in Table 1, from which it can be seen that Lactobacillus rhamnosus LRa05 shows good inhibition on EPEC.
TABLE 1 inhibition of EPEC by Lactobacillus rhamnosus LRa05
| Index (I) | Diameter of inhibition zone (mm) |
| Bacterial suspension (10) 7 CFU) | 17.3±1.1 |
| Bacterial suspension (10) 8 CFU) | 26.7±1.4 |
| Metabolite (50 mu L) | 11.8±1.2 |
| Metabolite (100 mu L) | 17.5±2.1 |
Experimental example 3 Lactobacillus rhamnosus LRa05 reduced the adhesion Rate of EPEC to colonic epithelial cells
HT-29 cells were cultured in DMEM complete medium (10% foetal calf serum, 1% penicillin and streptomycin solution) at 37℃with 5% CO 2 In a constant temperature cell incubator, the cell growth density is 80%, and the cells are digested and passaged to about 10 in a 24-well plate 5 After growing to a monolayer, the individual cells/wells were replaced with fresh culture without antibioticsA base.
Three experiments were used to study the adhesion inhibition of lactobacillus rhamnosus LRa05 to EPEC, the EPEC used in the experiments was 10 7 CFU/well, lactobacillus rhamnosus 10 8 CFU/well. Experiments were performed in four groups of 3 replicates each, 3 replicates were performed: (1) positive control group: adding EPEC and incubating for 3 hours; (2) competitive group: simultaneously adding EPEC and lactobacillus rhamnosus LRa05 to incubate for 3 hours; (3) exclusion group: firstly adding lactobacillus rhamnosus for incubation for 1.5 hours, and then adding EPEC for further incubation for 1.5 hours; (4) substitution set: the EPEC was added first for incubation for 1.5h, and then Lactobacillus rhamnosus LRa05 was added for further incubation for 1.5h. After incubation, the monolayer cells were washed 4 times with PBS, 0.5% Triton X-100.5 mL was added, and the cells were analyzed in an ice-water bath for 5min, and after gradient dilution of the cells analyzed from the cells, the EPEC numbers in the control and experimental groups were counted with LB plates. The extent of inhibition of EPEC by L.rhamnosus LRa05 on adherent HT-29 cells was expressed as relative adhesion rate (relative adhesion rate = number of treated colonies/number of control colonies x 100%).
The results of the experiment are shown in FIG. 1, and FIG. 1 shows the effect of different treatments on EPEC adhesion to HT-29 cells. The relative adhesion rate of the positive control is 100%, wherein the competition, rejection and replacement experiments reduce the adhesion rate of EPEC to HT-29 cells, the relative adhesion rate of the competition group is (61.2+/-5.66)%, the relative adhesion rate of the rejection group is (38.4+/-4.82)%, and the replacement group is (77.6+/-6.42)%, so that the inhibition effect of the rejection group is the best, and the EPEC adhesion can be inhibited by more than 60%, which indicates that lactobacillus rhamnosus can effectively inhibit the EPEC adhesion to intestinal long-skin cells through the occupation effect.
Experimental example 4 influence of Lactobacillus rhamnosus LRa05 intervention on mortality of EPEC-challenged mice
Preparation of lactobacillus rhamnosus LRa05 bacterial suspension: lactobacillus rhamnosus LRa05 is inoculated into an MRS liquid culture medium according to an inoculum size of 1% (v/v) from a glycerol tube, and is subjected to anaerobic culture at 37 ℃ for 24 hours to obtain first-stage seed liquid; inoculating the first-level seed solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and performing anaerobic culture at 37 ℃ for 16 hours to obtain a second-level seed solution; inoculating the second seed solution into MRS liquid culture medium according to 2% (v/v) inoculum size, and anaerobic culturing at 37deg.CCulturing for 16h to obtain bacterial liquid; centrifuging 8000g of bacterial liquid for 10min, and collecting bacterial cells; after washing the cells twice with sterile physiological saline, the cells were resuspended to a cell concentration of 5X 10 9 CFU/mL。
Preparation of EPEC bacterial suspension: after EPEC is activated on a TSA solid plate culture medium, single colony is selected and inoculated to a TSA liquid culture medium, and after continuous transfer for two generations, the bacteria are collected by centrifugation. Preparing bacterial suspension with sterile physiological saline, and regulating bacterial suspension concentration to 9×10 10 CFU/mL。
Establishment of EPEC diarrhea model mice: SPF grade (no specific pathogen) C57BL/6 male mice were tested for 60. The room temperature of animal feeding is 23+/-2 ℃, the humidity is 50% +/-10%, 12 hours/12 hours are alternated day and night, and the animal feeding is randomly divided into a normal control group (NC group), a modeling group (DC group) and an LRa05 intervention group (LRa 05 group) after infection after 1 week of feeding under the conditions of free feeding and drinking water, and each group is 20.
The test period was 20 days, starting from 10 days before molding and continuing until the end of the experiment, wherein the 3 days excluding antibiotic treatment, the control group and the molding group mice were filled with 200 μl of sterile physiological saline solution each day, and the LRa05 intervention group was filled with 200 μl of lactobacillus rhamnosus LRa05 bacterial suspension each day; the 11 th day after adaptation starts to be the molding period, the molding is carried out for 1-3 days, an antibiotic mixture of gentamicin (35 mg/L), vancomycin (45 mg/L), metronidazole (215 mg/L) and colistin (850U/mL) is added into drinking water of the mice so as to eliminate normal intestinal flora of the C57BL/6 mice, the molding is carried out for 4-10 days, sterile water without antibiotics is used for replacing water containing antibiotics as drinking water of the mice, and after the mice are fasted for 18 hours, 200 mu L of EPEC bacterial suspension is filled into each group of mice except a control group, the continuous gastric feeding is carried out for 7 days, the gastric feeding is carried out for 2 times in 1 day, and each time interval is 2 hours. Mice were observed daily for growth status, the number of deaths was recorded, and mortality was calculated. The mortality of mice is shown in figure 2.
Mice in the control group grow well in the dry expectation, ingest normally, have smooth and glossy hair, have a growing trend in body weight, and have no death by the end of the intervention period. The mice in the model group have symptoms of listlessness and diarrhea, and have death, and the death rate reaches 65% after 7 days. LRa 05-interfered mice also had symptoms of listlessness and diarrhea but were lighter than the model group, and also had death of mice, but overall mortality was 40% after the end of the intervention. The results indicate that LRa05 intervention can effectively reduce the lethality of EPEC infected mice.
Experimental example 5, lactobacillus rhamnosus LRa05 intervention improving diarrhea score in EPEC-challenged mice
Mouse modeling as in experimental example 3, the stool status of mice was recorded and scored at 1, 3, 5, and 7 days during the intervention period, and the average diarrhea score for each group of mice was calculated, and the mice were removed if death occurred. Diarrhea scoring criteria (0-4) were as follows: 0 to form good particles; 1-forming but softer, easily sticking to the EP tube wall; 2-thin or pasty stool; 3-diarrhea, watery feces; 4-is a thin watery sample and adheres to the anus. The diarrhea scoring criteria are common in the art and are described in Galcto-oligosaccharides protects against DSS-induced murine colitis through regulating intestinal flora and inhibiting NF- κB path. The diarrhea scores of the mice in each group are shown in fig. 3, and on days 3, 5 and 7 of the intervention, the diarrhea score of the LRa05 intervention group is lower than that of the mice in the model group, and statistical tests show that the diarrhea scores of the mice in the two groups are significantly different at the same time (p < 0.05), so that the diarrhea score of the EPEC infected mice can be effectively reduced by the probiotic intervention.
Experimental example 6 improvement of weight loss in EPEC-challenged mice by Lactobacillus rhamnosus LRa05 intervention
Mouse modeling the relative weights of the mice in each group were calculated by recording the weights of the mice at 1, 3, 5, and 7 days during the intervention period, respectively, and taking the weight value of the mice on the day of challenge as 100%. Calculation after rejection if death of mice occurred. As shown in fig. 4, during the intervention period, the weights of the mice in the control group steadily increase and finally increase to the initial weight 110, the weights of the mice in the model group and the LRa05 intervention group are in a decreasing trend on the 3 rd and 3 rd days, and the decreasing amplitude of the mice in the model group is larger, and after the intervention is finished, the weights of the mice in both groups are slightly increased, which indicates that the interference of lactobacillus rhamnosus LRa05 can weaken the weight decrease caused by EPEC infection to a certain extent, and is beneficial to the weight return of the mice. Statistical tests showed significant differences in body weight (p < 0.05) between the two groups of mice.
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
SEQUENCE LISTING
<110> micro Kang Yisheng (Suzhou) Co., ltd
<120> Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain LRa05, use and products thereof
<130> P220226/WKY
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Universal primer 27F
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agagtttgat cctggctcag 20
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<212> DNA
<213> Artificial Sequence
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Claims (9)
1. Lactobacillus rhamnosus strainLactobacillus rhamnosus) The strain LRa05 is characterized by having a preservation number of CGMCC No.24377; the strain LRa05 has a relieving effect on EPEC diarrhea and has good inhibition ability on EPEC.
2. Lactobacillus rhamnosus @Lactobacillus rhamnosus) The application of the strain LRa05 in preparing the product for relieving EPEC diarrhea is characterized in that the lactobacillus rhamnosus is ]Lactobacillus rhamnosus) The preservation number of the strain LRa05 is CGMCC No.24377.
3. Lactobacillus rhamnosus according to claim 2Lactobacillus rhamnosus) Use of strain LRa05 for preparing an EPEC diarrhea relieving product, wherein the EPEC diarrhea relieving product is: inhibit EPEC growth, and/or reduce EPEC adhesion to cells, and/or reduce mortality due to EPEC infection, and/or reduce diarrhea score due to EPEC infection, and/or improve weight loss due to EPEC infection.
4. Lactobacillus rhamnosus according to claim 3Lactobacillus rhamnosus) Use of strain LRa05 for the preparation of a product for alleviating diarrhea in EPEC, wherein said cells are colon epithelial cells.
5. Lactobacillus rhamnosus according to any of claims 2-4Lactobacillus rhamnosus) Use of strain LRa05 for the preparation of a product for alleviating EPEC diarrhea, characterized in that said product is selected from the group consisting of a medicament and a food.
6. An EPEC diarrhea relieving article comprising: an active ingredient; characterized in that the active ingredients comprise: lactobacillus rhamnosus with preservation number of CGMCC No.24377Lactobacillus rhamnosus) Strain LRa05.
7. A product for alleviating epac diarrhea according to claim 6, wherein said product is selected from the group consisting of a pharmaceutical or food product.
8. The product for alleviating diarrhea in EPEC according to claim 7, comprising an auxiliary material.
9. The product for relieving EPEC diarrhea according to claim 8, wherein said auxiliary material is selected from pharmaceutical auxiliary materials and product additives.
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| EP2693885A1 (en) * | 2011-04-08 | 2014-02-12 | Chr. Hansen A/S | Synergistic antimicrobial effect |
| CN107502575A (en) * | 2017-09-20 | 2017-12-22 | 中国农业科学院农产品加工研究所 | One plant of Lactobacillus plantarum with the high inhibitory activity of α glucuroides |
| CN112210507A (en) * | 2020-08-10 | 2021-01-12 | 江苏微康生物科技有限公司 | Shigella-antagonistic lactobacillus rhamnosus LRa05, screening method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2693885A1 (en) * | 2011-04-08 | 2014-02-12 | Chr. Hansen A/S | Synergistic antimicrobial effect |
| CN107502575A (en) * | 2017-09-20 | 2017-12-22 | 中国农业科学院农产品加工研究所 | One plant of Lactobacillus plantarum with the high inhibitory activity of α glucuroides |
| CN112210507A (en) * | 2020-08-10 | 2021-01-12 | 江苏微康生物科技有限公司 | Shigella-antagonistic lactobacillus rhamnosus LRa05, screening method and application thereof |
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| 潜在益生菌的筛选及抑菌功能特性评价;孙群;易华西;张冬;梁晶晶;蔡玉勇;张兰威;;食品科技(第12期);全文 * |
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