CN1940060B - Lactobacillus acidophilus and its application - Google Patents

Abstract

The present invention relates to a novel lactobacillus acidophilus LASW which has excellent resistance to animal digestive juices and has inhibitory effects against Escherichia coli, Salmonella, Shigella, Staphylococcus aureus and other staphylococci. In addition, it has good adsorbability to intestinal epithelial cells of human and animals, and can inhibit the invasion of bacteria into the intestinal epithelial cells of animals, thereby reducing infection caused by transfer of bacteria to liver and spleen. The lactobacillus acidophilus disclosed by the invention can be applied to yoghourt, microbial inoculum and feed additive products, and has an excellent effect of adjusting the gastrointestinal function of animals.

Description

Novel lactic acidophilus and its application

technical field

The present invention mainly relates to a novel Lactobacillus acidophilus and its application. In particular, the Lactobacillus acidophilus of the present invention has excellent resistance to animal digestive juices, can survive in the gastrointestinal tract of animals and inhibit the growth of pathogenic bacteria. The lactobacillus acidophilus of the invention can be applied to yogurt, probiotics and feed additive products, and has the excellent effect of adjusting the gastrointestinal function of animals.

Background technique

Lactic acid bacteria are the main intestinal flora of humans and animals, and are often used in probiotic products for humans and animals. One of the main ways to improve health is to inhibit the growth of intestinal pathogenic bacteria (such as Salmonella or Escherichia coli, etc.) (Lehto et al., 1997); there are also studies showing that lactic acid bacteria can prevent the invasion of pathogenic bacteria, such as preventing Salmonella typhoid fever Salmonella typhimurium invades epithelial cells (Jin et al., 1996). However, if lactic acid bacteria are to be able to perform the above functions in animals, two basic characteristics must be considered: the first is whether the lactic acid bacteria can tolerate the gastric acid and bile salts secreted by the digestive system of the animal's gastrointestinal tract, and reach the intestinal tract to survive; the second is the lactic acid bacteria Adsorption capacity to intestinal epithelial cells of animal hosts. The ability of lactic acid bacteria to adsorb to the mucosal surface is the condition for them to compete with pathogenic bacteria in the human gastrointestinal tract (Rammelsberg and Radler, 1990), because the prerequisite for pathogenic bacteria to cause infection is also to adsorb to intestinal cells (Chou and Weimer, 1999) . Generally, this adsorption characteristic is applied to the in vitro model to study the adsorption between bacteria and intestinal cell lines (Marteau et al., 1997), such as Int-407 (Leung and Finlay, 1991) and Caco-2 (Conway et al. , 1987; Marteau et al., 1997) cell lines for the study of lactic acid bacteria adsorption and competitive exclusion of pathogenic bacteria.

Salmonella, such as Salmonella typhi can cause food poisoning (Boonmar et al., 1998), and in recent years, the number of multidrug-resistant Salmonella typhi strains has increased year by year (Boonmar et al., 1998; Gross et al., 1998), and There have also been strains resistant to third-generation antimicrobial agents such as the new generation of fluoroquinolones and the third-generation broad-acting cephalosporins, which have become a major problem in clinical treatment of Salmonella infection ( Herikstad et al., 1997; Amyes and Gemmell, 1997; Piddock, 1998). Therefore, scholars have turned the treatment of pathogenic bacteria into the use of probiotics to strengthen the defense mechanism of the intestinal mucosa and protect the intestinal tract from infection by pathogenic bacteria such as Salmonella (Nird and Edlund.1990).

At present, there are many related studies on the inhibition of pathogenic bacteria by lactic acid bacteria, which are disclosed in patent documents or academic journals. For example, U.S. Patent No. 5,603,930 discloses that Lactobacillus johnsonii (Lactobacillus johnsonii) strain CNCM I-1225 can adsorb Caco-2 cells, and inhibits the adsorption of enterotoxins and intestinal invasive pathogens; Strain NRRL B-5628 can inhibit the growth of bacteria in other digestive systems (such as mouth or stomach), and is especially useful in the treatment of diarrhea or intestinal colic in piglets and other newborn animals, reducing Escherichia coli or Escherichia coli Group growth; U.S. Patent No. 6,491,956 disclosed that Lactobacillus acidophilus (Lactobacillus acidophilus) strain HY2177 and Lactobacillus casei (Lactobacillus casei) strain HY2743 can prevent and treat gastritis, duodenal and gastric ulcers caused by pylori infection; the United States Patent No. 4,874,704 discloses that Lactobacillus can produce an antibacterial substance, which can inhibit the growth of pathogenic bacteria and spoilage bacteria (such as Listeria and Salmonella) in food at refrigerated temperatures; U.S. Patent No. 5,340,577 and 5,604,127 disclose Lactobacillus, Lactococcus lactis, Citrobacter freundii, Enterococcus, Bifidobacterium and Propionibacterium can inhibit the growth of Salmonella; US Patent Publication No. 20020018770 Lactobacillus reuteri, Lactobacillus johnsonii and Bacillus subtilis can be applied to chicken feed to inhibit the growth of pathogenic bacteria, and promote the growth of poultry, thereby reducing the use of antibiotics or medicaments; US Patent Publication No. 20040028665 No. discloses that Lactobacillus and Propionibacterium freudenreichii (Propionibacterium freudenreichii) are used simultaneously, can inhibit the growth of Escherichia coli O157:H7 type and other pathogenic bacteria; U.S. Published Patent Application No. 20040029127 discloses that a kind of Lactobacillus casei can enhance immunity force, thereby inhibiting the growth of pathogenic bacteria; U.S. Published Patent Application No. 20040038379 discloses a kind of Lactobacillus salivarius (Lactobacillus salivarius), which has properties similar to bacteriostatin, and can inhibit Listeria and Staphylococcus, including resistance to methicillin Staphylococcus aureus (MRSA) and Bacillus, and can not inhibit other Lactobacillus; U.S. Published Patent Application No. 20040151708 discloses Lactobacillus fermentum (Lactobacillus acillus fermentum) strain ME-3, which is an antibacterial and antioxidant probiotic, can inhibit Escherichia coli, Shigella sonnei, Staphylococcus aureus, Salmonella typhi and Helicobacter pylori. pylori).

There are also many researches on lactic acid bacteria and their efficacy in academic journals. For example, Lactobacillus bifidus can prevent shiga toxin-producing E. coli O157:H7 infection in mice (Asahara et al., Infect .Immun.72:2240-2247); Lactobacillus rhamnosus (Lactobacillus rhamnosus) can reduce the infection of hemorrhagic Escherichia coli (Hirano, et al., Microbiol Immunol.47:405-409); Lactobacillus and lactose can inhibit Proliferation of Salmonella (Johannsen et al., Avian Dis.48:279-286); Lactobacillus can inhibit the growth of Listeria monocytogenes (Listeria monocytogenes) and Salmonella (Kuleasan et al., Nahrung., 46 : 408-410); Adsorbin produced by Bifidobacterium adolescentis strain 1027 can competitively inhibit the attachment of enterotoxic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cells Strain Lovo (Zhong et al., World J. Gastroenterol., 10: 1630-1633); Lactobacillus delbrueckii subsp. lactis can inhibit the growth of pathogenic bacteria and spoilage bacteria in meat products (Senne et al., J. Food Prot., 66:418-425); Lactococcus lactis subsp.lactis ATCC 11454 strain has antibacterial ability (Millette et al., J.Food Prot., 67:1184-1189); The supernatant significantly reduced the invasion of Salmonella to intestinal cells Caco-2/TC-7 (Coconnier et al., Appl. Environ. Microbiol., 66: 1152-1157); the bacterial fluid of Lactobacillus casei and its discarded culture The supernatant of the animals can inhibit Salmonella from invading the intestinal cell Caco-2, but loses its effect at pH 7, and the mice can maintain 100% survival rate after being infected with Salmonella after continuous feeding of L.casei GG for 7 days , and reduced the number of bacteria in the liver and spleen (Hudault et al., Appl.Enviro.Microbiol., 63:513-518); feeding Lactobacillus casei can reduce the invasion and infection of Salmonella in mice It provides protection, and it is believed that this protection is related to IgA secreted by the intestine (Perdigon et al., Journal of Dairy Research, 57:255-264); There are three periods. The first period is the period when Salmonella proliferates in organs such as the liver and spleen after the invasion. After one week, it enters the second period. The proliferation of Salmonella stops and reaches the peak of the highest number of bacteria. This period also lasts for a week. During the third period, the number of Salmonella in the liver and spleen gradually decreased (Nauciel et al., Infection and Immunity, 60: 450-454).

In addition, there have been many reports on the mechanism of lactic acid bacteria inhibiting Salmonella. Fuller (1986) once proposed to use non-absorbing strains, such as Streptococcus faecium, which can survive in the intestinal tract because their proliferation rate exceeds the excretion rate of chyme, so the strains with this characteristic can Stay in the mucosal layer of the large intestine without having to be adsorbed on the epithelial cells. In addition, some scholars (Hormaeche et al., 1980; Van Dissel et al., 1985) proposed that the proliferation of Salmonella infected in the host is initially through macrophages and their The activated function exerts an inhibitory effect. The mechanism by which lactic acid bacteria inhibit intestinal microbial infection is competitive exclusion, including competition for nutrients and intestinal cell adsorption sites, and secretion of antibacterial substances (Hudault et al., Applied and Environmental Microbiology, 63: 513-518; Chauviere et al., FEMS Microbiology Letters, 91:213-218) and activation of the intestinal immune system can improve the ability of the host to inhibit pathogenic bacteria (Perdigon et al., Journal of Dairy Research, 57:255-264; Schiffrin et al., American Journal of Clinical Nutrition, 66:515S-520S).

Contents of the invention

The present invention screens and isolates a strain of Lactobacillus acidophilus from the intestinal tract of pigs, which has the basic characteristics of the above-mentioned probiotics for humans and animals, and can resist pathogenic bacteria such as Salmonella invading and infecting intestinal mucosa, liver and spleen organs. The Lactobacillus acidophilus strain of the invention can be used in yogurt and feed additives for livestock and poultry, and can be used as a probiotic to improve the gastrointestinal tract function of animals.

Therefore, one aspect of the present invention is to provide a Lactobacillus acidophilus LASW, the preservation number is CCTCCNO: M204083, preserved in the China Center for Type Culture Collection.

Another aspect of the present invention is to provide a probiotic composition, which includes the above-mentioned Lactobacillus acidophilus LASW.

Another aspect of the present invention is to provide a feed composition, which includes the above-mentioned Lactobacillus acidophilus LASW.

Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating pathogenic bacteria infection, which includes the above-mentioned Lactobacillus acidophilus LASW.

Description of drawings

Figure 1 shows the results of the growth inhibition test of Escherichia coli by the Lactobacillus acidophilus supernatant (SCS) of the present invention.

Figure 2 shows the results of the invasion inhibition test of Salmonella choleraesuis by the Lactobacillus acidophilus of the present invention.

Figure 3 shows the change of systemic serum IL-6 in the mice of LASW and blank groups on the third and sixth day after being fed with Salmonella.

Detailed ways

The present invention mainly relates to a novel Lactobacillus acidophilus LASW, which was preserved in China Center for Type Culture Collection on November 17, 2004, and the preservation number is CCTCC NO: M204083. The Lactobacillus acidophilus has the following characteristics:

1. Screened from Taiwan's native pig intestine;

2. With excellent acid resistance and bile salt resistance, it can survive in the intestinal tract after passing through the gastrointestinal digestive juice in humans and animals;

3. Has excellent human and animal intestinal epidermal cell adsorption capacity;

4. Can compete with enteropathogenic bacteria for the adsorption of intestinal cells, thereby inhibiting the adsorption and growth of enteropathogenic bacteria, such as Escherichia coli, Salmonella, Shigella, Staphylococcus aureus and other staphylococci, and their invasion into intestinal cells And the effect of invasion and metastasis to the liver and spleen.

Based on the above characteristics, the Lactobacillus acidophilus strain LASW of the present invention can be applied in feed additives for yogurt and livestock and poultry, and can be used as a probiotic to improve the gastrointestinal function of animals. In addition, the Lactobacillus acidophilus of the present invention can also be used in pharmaceutical compositions to prevent or treat pathogenic bacteria infections.

The following examples are used to illustrate the efficacy of the present invention, but not to limit the scope of the present invention.

Example

1. Acid resistance and bile salt resistance test

The acid resistance test was carried out according to the method described in Conway et al., Journal of Dairy Science, 70: 1-12. Add 100 μl of cultured LASW lactic acid bacteria solution (10 ⁸ ～10 ⁹ CFU/ml) to phosphate buffer solution adjusted to pH 2.0, 2.5 and 3.2 with 0.1N HCl, or pig gastric juice (pH 3.2), and incubated at 37°C for 3 hours. The lactic acid bacteria liquid was added to the pH7.2 phosphate buffer solution under the same conditions as the control group. After serial dilution after cultivation, they were cultured on MRS agar respectively, and the number of surviving lactic acid bacteria was calculated.

The bile salt tolerance test was carried out with reference to the method described in Gilliland et al., Journal of Dairy Science, 73:905-911. The above-mentioned lactic acid bacteria that survived the acid resistance test were centrifuged at 5,000rpm for 5 minutes, washed with phosphate buffer solution (pH7.2), and then the lactic acid bacteria were moved to 9 ml of MRS culture medium (with and without 0.3% w /v of bile salt (Sigma), cultivated for 3, 12 and 24 hours, serially diluted after cultivation, and cultured on MRS agar by pouring method to count the number of surviving lactic acid bacteria.

The test results show that the number of bacteria of the Lactobacillus acidophilus LASW of the present invention is reduced by 2 logarithms (from 10.5 to 8.5) after being cultivated for 3 hours under the environment of pH 2.0; it grows stably under the environment of pH 2.5 and 3.2 . After culturing in pig gastric juice (pH3.2) for 3 hours, it only decreased by 0.6 log. It can still grow stably when cultured in an environment containing bile salts. Shows that it has good acid and bile salt tolerance.

2. Intestinal cell line adsorption test

Take a 24-well multi-well plate and put a cover glass in each well. For the complete cultured Int-407 and Caco-2 individual cell angle culture dishes, discard the old culture medium, wash the angle culture dishes twice with 1×PBS buffer solution, and then pour it out, add 1% trypsin/EDTA for about 1 mL to digest, and tap the petri dish a few times to suspend the cells. Add 40 ml of fresh BME (Int-407) or MEM (Caco-2) (both containing FBS and penicillin-streptomycin) culture medium (diluted 4 times), shake well, add 0.5 ml of cell suspension to each well, Incubate at 37°C in CO ₂ gas to enable cells to divide and grow attached to the coverslip. Then suck out the old culture medium, add 0.5 ml of 1×PBS buffer solution to each well to wash, wash away the old culture medium and remove unattached cells, and repeat the washing twice. Add 0.5 ml of fresh culture medium suitable for individual different cells (without penicillin-streptomycin), and 100 μl of lactic acid bacteria broth, and incubate for 2 hours at 37°C in CO ₂ gas.

Aspirate the old culture solution, add 0.5 ml of 1×PBS buffer solution to each well to wash three times, shake and wash at 100 rpm for 5 minutes each time, and suck out with a dropper. After adding 200 ml of 10% formalin, shake and fix at 100 rpm for 30 minutes to fix the bacteria and cells on the coverslip in the well, and suck out the formalin with a dropper. Add 0.5 ml of 1×PBS buffer solution to wash three times, and shake and wash at 100 rpm for 5 minutes each time. Add 200 μl of crystal violet coarsely filtered through filter paper for staining, wrap a layer of aluminum foil paper in a 24-well porous culture dish, shake at 100 rpm for 5 minutes, add 0.5 ml, 1×PBS buffer to wash to remove excess dye. Drop a drop of 1 × PBS buffer solution on the glass slide, take out the cover glass with tweezers and place it on it to avoid drying, observe under an inverted fluorescent microscope (Olympus IMT-2), and count the number of lactic acid bacteria adsorbed on each cell ( Gopal et al., 2001).

The test results show that the Lactobacillus acidophilus of the present invention has an adsorption capacity of at least 15 per cell to human intestinal epidermal cells Int-407 and Caco-2 cells, and intestinal epidermal cells of pigs, chickens, and rabbits. The number of lactic acid bacteria.

3. The growth inhibition test of different pathogenic bacteria

Lactobacillus acidophilus of the present invention was cultivated in MRS for 24 hours, and the pH value of the bacterial solution was reduced to 3.7, and a sensitivity test was carried out to Staphylococcus aureus, other staphylococci, Escherichia coli and Shigella. The results showed that the The invented Lactobacillus acidophilus forms inhibition circles of different sizes to these pathogenic bacteria, and indeed has the effect of inhibiting the growth of pathogenic bacteria. In addition, in this test, the SCS of the LASW lactic acid bacteria strain was used for 4 hours; the inhibition of Escherichia coli The growth effect is remarkable, and the bacterial count can be reduced by 10 ² -10 ⁴ CFU/ml (Fig. 1).

4. Test of inhibiting Salmonella choleraesuis from invading intestinal cell line Int 407

Salmonella Choleraesuis (Salmonella Choleraesuis) is a highly invasive zoonotic pathogen, so it was selected as the study on the inhibition of bacterial invasion of lactic acid bacteria LASW. This test method mainly refers to the method described by Hudault et al. (1997).

(1) Preparation of lactic acid bacteria

The test lactic acid bacteria strains were taken out from the frozen glycerin bottle, activated twice with MRS medium, cultured overnight at 37°C, and centrifuged at 6000rpm for 10 minutes at 4°C, and the supernatant (spent culture supernatant, SCS), using filter-sterilized 1N NaOH to neutralize the lactic acid bacteria liquid (bacterial culture) and supernatant, prepare the above-mentioned unadjusted and adjusted to neutral pH value of the lactic acid bacteria liquid and supernatant for testing.

(2) Preparation of Salmonella choleraesuis

Salmonella choleraesuis (strains SCV2a, SCV26a) were cultured in LB medium, collected by centrifugation for 10 minutes after secondary activation culture, washed with sterile phosphate buffer solution, and suspended in the same volume of phosphate buffer solution . The bacterial solution was serially diluted, cultured in the nutrient medium by the plate counting method, and the number of viable bacteria was counted.

(3) Lactic acid bacteria inhibit the invasion of Salmonella choleraesuis in Int 407 intestinal cell line

Digest the intestinal cells Int 407 in the 75T culture flask with 1 ml of trypsin-EDTA, tap the culture flask to pat the cells, and distribute them in 96-well microculture plates, adding 1×10 ⁵ cells/ml, cultivated overnight to form a monolayer of cells, remove the old cell culture medium, and add 90 μl of antibiotic-free cell culture medium. Take 10 μl of the lactic acid bacteria liquid and supernatant (SCS) prepared in (1) above, add it to each hole of the cell culture plate, and place it at 37° C., 5% CO ₂ and incubate for 1 hour. Take out 10 μl of the Salmonella choleraesuis suspension described in (2) after appropriate dilution, add it into each well to make the final concentration about 10 ⁶ CFU/well, centrifuge at 1000rpm at low speed for 10 minutes to settle the bacteria, and move the cell culture plate to After culturing in the cell culture box for 1 hour and 2 hours, wash each hole five times with phosphate buffer solution to wash away the E.coli and Salmonella choleraesuis cells remaining in the extracellular space, and then add 100 μl of ceftriaxone to each hole (ceftriaxone) (100 μg/ml) BME cell culture medium was placed in a cell culture incubator for 1 hour to kill the extracellular Salmonella choleraesuis bacteria of the Int 407 cells. Then wash each hole 5 times with 0.01M PBS, add 100 μl sterile triton X-100 (1%) to lyse the cells for 10 minutes, then vigorously mix the mixture in each hole with a micropipette, take out the mixture and wash it with 0.01M The PBS was serially diluted, and counted the number of Salmonella choleraesuis invading Int 407 cells after culturing for 48 hours by nutrient medium plate counting method.

From the results, it can be known that the inhibition effect of Lactobacillus acidophilus of the present invention on the invasion of Salmonella choleraesuis can reach 10 ² to 10 ³ CFU/ml after one hour of action; , can inhibit more than 10 ³ CFU/ml (Figure 2).

5. Determination of TNF-α and IL-6 concentrations by enzyme-linked immunosorbent assay (ELISA)

When mice are invaded and infected by Salmonella, the first responding cells are macrophages. In addition to phagocytosis, macrophages also secrete some cytokines, mainly TNF-α, IL-1, IL-6, IL- 8 and IL-12, these cytokines can act locally in infected tissues or act systemically. When macrophages are not activated, TNF-α production is very small (Beutler et al., 1986), Gram-negative bacteria usually stimulate macrophages with lipopolysaccharide (LPS) (Balkwill et al., 2000), Therefore, TNF-α can be used as an important indicator of early Salmonella infection (Nauciel and Espinasse-Maes, 1992), and the produced TNF-α can activate vascular endothelial cells locally, increase vascular permeability, IgG, complement and cell secretion. Entering, if it causes liver and spleen macrophages to release TNF-α to the whole body, it will cause sepsis, which is a systemic dilation of blood vessels that causes a large amount of body fluid to flow to the tissue, causing normal blood to fail to supply, leading to organ failure, known as septic shock. IL-6 can activate lymphocytes and increase antibody production during local infection, and if released to the whole body, it will cause fever and induce acute phase protein production, causing systemic inflammation (Hirano, 1992). In an inflammatory response, TNF-α increases the amount of its mRNA within 15-30 minutes after being stimulated, while the production time of IL-6 is later. Therefore, measuring the production of TNF-α and IL-6 can understand the overall Severity of the inflammatory response.

(1) Determination of TNF-α concentration by enzyme-linked immunosorbent assay

1. According to the standard steps provided by Endogen (Woburn, MA., USA):

Take 100 μl of anti-mouse TNF-α monoclonal antibody (1.03 μg/ml in the adsorption buffer) and add it to the well of the ELISA flat bottom plate, and let it stand at room temperature overnight (about 14 to 16 hours) to allow it to attach (coat) to bottom of the plate. After removing the adsorption buffer, add 200 μl of blocking buffer (filled with assay buffer) to each hole, let stand at room temperature for 1 hour, remove the blocking buffer, and wash gently three times with washing buffer. Take 50 μl of anti-mouse TNF-α biotin-labeled monoclonal antibody (0.25 μg/ml) in the hole, and then add 50 μl of standard or sample to be tested (serum after grinding mouse liver), statically at room temperature After standing for 2 hours, pour off the mixed solution in the well, wash with washing buffer three times, add 100 μl of HRP-conjugated streptavidin (0.125 μg/ml) to each well, and keep at room temperature After 30 minutes of exposure, wash with washing buffer five times. After the above washing steps are completed, the 96-well microplate must be turned upside down on a paper towel to absorb the remaining liquid. Finally, add 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) matrix solution to each hole, and keep it in the dark for 30 minutes at room temperature to allow color reaction, and finally add 100 μl of 0.18M sulfuric acid solution , to terminate the color reaction. Read the absorbance value at a wavelength of 450nm with an ELISA reader, and convert the concentration of TNF-α according to the standard curve.

2. Preparation of standard curve

According to the instructions provided by Endogen, dissolve the TNF-α standard (mouse TNF-α ELISA standard) with 400 μl of analysis buffer (the TNF-α standard should be used within 1 hour after reconstitution), so that its concentration is 2450 pg/ Then carry out 2-fold serial dilution with the analysis buffer, measure the absorbance value of each concentration TNF-α standard substance at a wavelength of 450nm according to the above analysis method, and make the result into a standard curve.

(2) Determination of IL-6 concentration by enzyme-linked immunosorbent assay

1. According to the standard steps provided by Endogen (Woburn, MA., USA):

Add 100 μl of anti-mouse IL-6 monoclonal antibody (2.5 μg/ml) into the wells of the ELISA flat bottom plate, and let it stand at room temperature overnight (about 14 to 16 hours) to allow the primary antibody to adsorb to the bottom of the plate. After removing the adsorption buffer, add 200 μl of analysis (blocking) buffer (4% BSA in PBS) to each well, let stand at room temperature for 1 hour, remove the blocking buffer, and replace with 200 μl of washing buffer (50 mM Tris , 0.2% Tween-20, pH8.0) gently washed three times. Take 50 μl of anti-mouse IL-6 biotin-labeled monoclonal antibody (0.25 μg/ml in assay buffer) in the hole, and then add 50 μl of Standard or sample to be tested (serum from mouse heart blood collection), after standing at room temperature for 2 hours, pour off the mixed solution in the hole, wash with washing buffer three times, add 100 μl of HRP-conjugated Anti-streptavidin (0.125 μg/ml in assay buffer), after 30 minutes at room temperature, wash five times with wash buffer. After the above wash steps, the 96-well microplate must be turned upside down On a paper towel, absorb the remaining liquid. Finally, add 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) matrix solution to each hole, and keep it in the dark for 30 minutes at room temperature for color reaction , and finally add 100 μl of 0.18M sulfuric acid solution to terminate the color reaction. Read the absorbance value at a wavelength of 490 nm with an ELISA reader, and convert the concentration of IL-6 according to the standard curve.

2. Preparation of standard curve

According to the instructions provided by Endogen, dissolve the IL-6 standard (mouse IL-6 ELISA standard) with 400 μl of analysis buffer to make its concentration 2450 pg/ml, and then perform 2.5-fold serial dilution with analysis buffer to 38.4 pg /ml, measure the absorbance value of each concentration IL-6 standard substance at a wavelength of 490nm according to the above analysis method, and make the result into a standard curve.

In this study, aiming at the animal source lactic acid bacteria that are better for inhibiting Salmonella in mice, the TNF-α content was detected in the serum of liver tissue after 3 and 6 days of infection. The results showed that mice fed only with PBS could not inhibit the invasion of Salmonella, so the inflammatory index The production of TNF-α increased obviously 6 days after infection. LASW has the best ability to inhibit the invasion of Salmonella, so its TNF-α content is the lowest 6 days after infection; the detection of IL-6 also has the same result (Figure 3).

After the above embodiments and referring to the drawings, it should be understood that the present invention is not limited to the above embodiments. Various changes and improvements may be made by those skilled in the art without departing from the scope and spirit defined in the specification and claims of the present invention, which also fall within the scope of the present invention.

Claims (4)

1. a Lactobacterium acidophilum (Lactobacillus acidophilus) LASW is characterized in that preserving number is CCTCC NO:M204083, is preserved in Chinese typical culture collection center.

2. a bacteria-promoting agent composition is characterized in that comprising the described Lactobacterium acidophilum LASW of claim 1.

3. a feed composition is characterized in that comprising the described Lactobacterium acidophilum LASW of claim 1.

4. a medical composition that prevents or treat Salmonellas, intestinal bacteria, Shigellae, staphylococcal infections is characterized in that comprising the described Lactobacterium acidophilum LASW of claim 1.

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