CN113564068B - Lactobacillus plantarum 1201 and application thereof in preparation of salmonella typhimurium inhibition products - Google Patents
Lactobacillus plantarum 1201 and application thereof in preparation of salmonella typhimurium inhibition products Download PDFInfo
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Abstract
The invention discloses a lactobacillus plantarum which is lactobacillus plantarum 1201 and is preserved in the China Center for Type Culture Collection (CCTCC) in Wuhan City in 1 month and 12 days, wherein the preservation number is CCTCC M2021050. The invention also discloses an effect of the lactobacillus plantarum 1201 on inhibiting salmonella typhimurium, which can be prepared into a probiotic bacterial agent for inhibiting salmonella typhimurium and can be used for treating enteritis caused by salmonella typhimurium. The lactobacillus plantarum 1201 regulates and controls the expression of inflammatory factors by regulating TLR4/NF- κB inflammatory pathways, thereby achieving the effect of effectively preventing and controlling intestinal inflammation induced by salmonella typhimurium infection.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum 1201 and application thereof in preparation of salmonella typhimurium inhibition products.
Background
The salmonella typhimurium infection-caused diseases have become a major health problem in society, and researches show that after the salmonella typhimurium enters the digestive tract orally, the salmonella typhimurium can increase the content of pathogenic bacteria in the intestinal tract, destroy the balance of intestinal flora, and colonise intestinal colonies through competition and occupation, thereby causing lamina propria inflammation. However, high-fat diets are becoming more common, and research shows that high-fat diets can promote the colonisation of salmonella typhimurium in the intestinal tract, so that salmonella typhimurium infection can be aggravated by high-fat diets. At present, antibiotics are mainly used for preventing and treating salmonella typhimurium infection, but excessive use of antibiotics easily causes drug resistance of pathogenic bacteria, so that the pathogenic bacteria are difficult to treat again. There is no method of inhibiting salmonella typhimurium without side effects.
Lactobacillus plantarum is used as an intestinal gram positive probiotic, can regulate the balance of intestinal flora, inhibit the growth of harmful bacteria, and plays an important role in intestinal immunity.
The lactobacillus plantarum 1201, 2021, is preserved in China center for type culture collection (China Center for Type Culture Collection, CCTCC M for short, address: eight paths 299 of university of Wuhan in Wuchang district of Wuhan, hubei province, post code: 430072) for 12 days in 1 month, and the preservation number is CCTCC M2021050.
Disclosure of Invention
Aiming at the defects and the problems in the prior art, the invention aims to provide the application of lactobacillus plantarum 1201 in preparing a salmonella typhimurium inhibiting product.
The invention is realized by the following technical scheme:
one of the purposes of the present invention is to provide a lactobacillus plantarum which is lactobacillus plantarum 1201, and is preserved in China center for type culture collection (China Center for Type Culture Collection, CCTCC M for short, address: eight-path 299 of Wuchang district of Wuhan, hubei province, in the university of Wuhan, post code 430072) of Wuhan, wherein the preservation number is CCTCC M2021050.
A second object of the present invention is to propose a probiotic bacterial agent for inhibiting salmonella typhimurium, the active ingredient of which comprises lactobacillus plantarum according to claim 1.
The probiotic bacterial agent can be prepared from lactobacillus plantarum 1201, exists in a form of a fresh live bacterial body in a stable period, and can be used for relieving intestinal inflammation induced by salmonella typhimurium infection.
The third object of the invention is to provide a preparation method of a probiotic bacterial agent for inhibiting salmonella typhimurium, which comprises the following steps:
(1) Anaerobic culture is carried out on the lactobacillus plantarum 1201MRS liquid culture medium for 24+/-2 hours at 37+/-1 ℃ to reach thalli in a stationary phase, and the preservation number of the lactobacillus plantarum 1201 is CCTCC M2014170;
(2) Centrifugally collecting the thalli in the step (1), and washing the thalli for 2 to 4 times by using sterile 1 XPBS;
(3) Adjusting the concentration of the cells obtained in the step (2) to 1-2X 10 by using sterile 1X PBS 8 cfu/mL。
In application, the obtained thallus is added with proper auxiliary materials to prepare medicine or food or health care product.
The fourth object of the invention is to propose the application of lactobacillus plantarum in preparing a product for inhibiting salmonella typhimurium, which is used for preparing a medicine or health care product or food for inhibiting salmonella typhimurium infection.
A fifth object of the present invention is to propose the use of said lactobacillus plantarum for the preparation of a medicament for the prevention or treatment of colitis caused by salmonella typhi.
Preferably, the product is health product, functional food or medicine
Further preferably, the food is a food which can survive various lactobacillus such as beverage, yoghurt, jelly and the like, so long as the food can survive lactobacillus plantarum, and the food is particularly according to actual needs.
Preferably, the lactobacillus plantarum 1201 is used in a1×10 dosage of the microbial agent 8 cfu/kg, namely, each kg of microbial inoculum used by animals and human bodies contains lactobacillus plantarum 1201 which reaches 10 8 cfu。
Compared with the prior art, the invention screens lactobacillus plantarum 1201 with the preservation number of CCTCC M2021050. The lactobacillus plantarum 1201 regulates and controls the expression of inflammatory factors by regulating TLR4/NF- κB inflammatory pathways, thereby achieving the effect of effectively preventing and controlling intestinal inflammation induced by salmonella typhimurium infection.
Drawings
FIG. 1 shows the change of Salmonella typhimurium content in the feces of different groups of mice;
FIG. 2 shows the changes in Salmonella typhimurium content in spleen, mesenteric lymph nodes, and jejunum tissues of different groups of mice;
FIG. 3 is a HE stained section of colon tissue from different groups of mice;
FIG. 4 shows the differences in TLR4/NF- κB inflammatory pathways in serum of different groups of mice;
FIG. 5 shows the difference in inflammatory factor expression in jejunal tissue in different groups of mice;
FIG. 6 shows the difference in inflammatory factor expression in colon tissue of different groups of mice.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1
Activation of the cells: lactobacillus plantarum 1201 stored in glycerol is uniformly mixed by vortexing, 10 mu L of lactobacillus plantarum 1201 is sucked and coated in an MRS solid culture medium, anaerobic culture is carried out for 24 hours at 37 ℃, single bacterial colony is selected and inoculated in 4mL of liquid MRS culture medium, anaerobic culture is carried out for 24 hours at 37 ℃, 1% inoculum size is inoculated in 10mL of MRS liquid culture medium, and anaerobic culture is carried out for 24 hours at 37 ℃ to reach a stationary phase.
Preparing MRS culture medium: 5g/L of peptone, 5g/L of beef extract, 10g/L of tryptone, 5g/L of yeast powder, 20g/L of glucose, 5g/L of anhydrous sodium acetate, 2g/L of diamine hydrogen citrate, 2g/L of dipotassium hydrogen phosphate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 1 ml/L of Tween-80, pH 6.3+/-0.1, 121 ℃ and 15 minutes.
Example 2
Preparation of lactobacillus plantarum microbial inoculum: centrifuging activated Lactobacillus plantarum 1201 at stationary phase, collecting fresh thallus, washing with sterile 1XPBS 3 times, and adjusting thallus concentration to 10 with sterile 1XPBS 8 cfu/mL, and preparing the microbial inoculum required by the experiment for standby.
Example 3 construction and experimental design of a mouse model infected with salmonella typhimurium:
after one week of adaptation in sterile mouse rooms, the MD, MD2, MD3 group (n=8) mice were given normal diet and normal drinking water, and the HFD, HFD2, HFD3 group (n=8) mice were given 60% high calorie diet and normal drinking water. Then, 200. Mu.L of 1xPBS was irrigated to MD and HFD groups (n=8), and 100. Mu.L of 1xPBS and 100. Mu.L of 1x10 were irrigated to MD2 and HFD2 groups (n=8), respectively 8 cfu/mL salmonella typhimurium ATCC13311, group md3 and HFD3 (n=8) were gavaged 100 μl 1x10 8 cfu/mL Lactobacillus plantarum A19 and 100. Mu.L 1X10 8 cfu/mL Salmonella typhimurium ATCC13311. Mouse faeces were collected 3h,6h,24h,48h,72h,96h after gavage. After 4d of lavage, mice were euthanized with diethyl ether and the mouse serum, spleen, mesenteric lymph nodes, colon, cecum, jejunum and colon contents were collected in a sterile environment for use. Taking out colonTissue was quickly rinsed with saline, and specimens were immersed in 10% formalin solution and HE stained.
Experimental results:
(1) Obviously reduce the salmonella typhimurium content in the mouse excrement
The salmonella typhimurium content in the high-fat diet group mouse feces is larger than that in the normal diet group, and after the intervention of the lactobacillus plantarum 1201 microbial inoculum for 96 hours, the salmonella typhimurium content in the intervention group mice is obviously reduced compared with that in the infected group mouse feces, as shown in figure 1. The invention can effectively reduce salmonella typhimurium in the mouse feces.
(2) Obviously inhibit the in vivo colonization of salmonella typhimurium
After the intervention of the lactobacillus plantarum 1201 microbial agent in the stomach is performed for 96 hours, the content of salmonella typhimurium in the jejunum, spleen and mesenteric lymph node of the mice in the intervention group is obviously reduced compared with that of the mice in the infection group, as shown in figure 2, the content of salmonella typhimurium in the organs of the mice in the intervention group is effectively reduced, and the lactobacillus plantarum 1201 microbial agent in the embodiment can inhibit the colonization of salmonella typhimurium in the mice.
(3) Significantly improve the pathological conditions of mice
The colon tissues of different groups of mice are made into pathological sections, and the colon tissues of the infected groups of mice are compared with the colon tissues of a natural control group, so that the colon of the infected group is obviously blurred in submucosa edge and infiltrated by inflammatory cells as can be seen from figure 3. The colon muscle layer of the group (lactobacillus plantarum microbial agent treatment group) is smooth, and the inflammation phenomenon is obviously reduced. The invention proves that the microbial inoculum can effectively relieve colon tissue inflammation of mice infected by salmonella typhimurium, and has the effect of repairing intestinal tract injury induced by pathogenic bacteria.
(4) The microbial inoculum obviously regulates TLR4/NF- κB signal path induced by salmonella typhimurium infection
Mouse serum is taken, and the expression level of TLR4, myD88, IKK-beta, IκB-alpha and NF- κB related proteins of TLR4/NF- κB signal path is detected.
As can be seen from FIG. 4, the protein expression level of the mice in the intervention group is obviously regulated compared with that of the mice in the infection group, the expression level of TLR4, myD88, IKK-beta and NF-kappa B is regulated down, and the expression level of Ikappa B-alpha is regulated up. The invention shows that the microbial inoculum can achieve the aim of protecting organism from inflammation by adjusting TLR4/NF- κB signal path related protein.
(5) The microbial inoculum obviously regulates the expression of colon tissue inflammatory factors induced by salmonella typhimurium infection
After RNA extraction and reverse transcription of colon tissue, the expression levels of inflammatory factors IFN-gamma, IL-1 beta, TNF-alpha, IL-6, IL-17A, IL-10, IL-22 and TGF-beta are detected.
As can be seen from FIGS. 5 and 6, the present invention significantly regulates the expression level of inflammatory factors in the mice in the intervening group compared with the mice in the infected group, down-regulates the expression level of pro-inflammatory factors IFN-gamma, IL-1β, TNF- α, IL-6, IL-17A, and down-regulates the expression level of anti-inflammatory factors IL-10, IL-22, TGF- β. The invention shows that the microbial inoculum can achieve the purpose of protecting organism from inflammation by regulating the expression level of cytokines.
The foregoing description of the preferred embodiments of the present invention has been presented only in terms of those specific and detailed descriptions, and is not, therefore, to be construed as limiting the scope of the invention. It should be noted that modifications, improvements and substitutions can be made by those skilled in the art without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (4)
1. A lactobacillus plantarum characterized in that: the lactobacillus plantarum is lactobacillus plantarum (Lactiplantibacillus plantarum) 1201 and is preserved in the China Center for Type Culture Collection (CCTCC) of Wuhan City in 2021, 1 month and 12 days, and the preservation number is CCTCC NO: m2021050.
2. A probiotic bacterial agent for inhibiting salmonella typhimurium, which is characterized in that: an active ingredient comprising the lactobacillus plantarum of claim 1.
3. The method for preparing the probiotic bacteria agent according to claim 2, which is characterized by comprising the following steps:
(1) Anaerobic culturing the lactobacillus plantarum 1201MRS liquid culture medium at 37+/-1 ℃ for 24+/-2 h until the lactobacillus plantarum 1201MRS liquid culture medium reaches a stable phase;
(2) Centrifugally collecting the thalli in the step (1), and washing the thalli with sterile 1XPBS for 2 to 4 times;
(3) Adjusting the concentration of the cells obtained in the step (2) to 1-2X 10 by using sterile 1X PBS 8 cfu/mL。
4. Use of lactobacillus plantarum according to claim 1 for the manufacture of a medicament for the prevention or treatment of colitis caused by salmonella typhi, characterized in that: the medicine can prevent or treat intestinal inflammation induced by Salmonella typhimurium infection by regulating TLR4/NF- κB inflammatory pathway and regulating and controlling the expression of inflammatory factors.
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