CN1940060B - Acidophilous milk and its use - Google Patents
Acidophilous milk and its use Download PDFInfo
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Abstract
Acidophilic bacteria LASW and its use are disclosed. It has excellent animal digest-resisting characteristic and inhibition function for colibacillus, salmonella, shigella, staphylococcus aureus and other staphylococcus; it has good adsorptive ability, less infection and can adjust intestines and stomach functions and be used for sour milk and feed additives.
Description
Technical field
The present invention relates generally to a kind of Lactobacterium acidophilum (Lactobacillus acidophilus) of novelty and uses.Particularly, Lactobacterium acidophilum of the present invention has the characteristic of the good liquid of anti-the animal digestion, can survive in the animal intestine gastropore and suppresses growth of pathogenic bacteria.Lactobacterium acidophilum of the present invention can be applicable in sour milk, bacteria-promoting agent, the feedstuff additive product, has the excellent results of adjusting the animal functions of intestines and stomach.
Background technology
Milk-acid bacteria is human and the main flora of animal intestinal, is usually used in the humans and animals probiotic products.One of its salubrious main mode is for suppressing the growth (Lehto et al., 1997) of pathogenic bacteria (as Salmonellas or intestinal bacteria etc.) in the intestines; Also have to studies show that milk-acid bacteria can prevent the invasion of pathogenic bacteria, invade epithelial cell (Jinet al., 1996) as the Corynebacterium diphtheriae (Salmonella typhimurium) that can prevent salmonella.But, must consider two fundamental characteristics if milk-acid bacteria is wanted and can middlely in animal body bring into play above-mentioned functions: the firstth, can milk-acid bacteria stand animal intestine gastropore Digestive tract secreted hydrochloric acid in gastric juice and cholate, survives and arrive enteron aisle; Second is the adsorptive power of milk-acid bacteria to animal host's intestinal epithelial cell.The ability of milk-acid bacteria absorption mucous membrane surface, be that it can be in the condition (Rammelsberg and Radler, 1990) of human intestines and stomach and pathogenic bacteria competitive adsorption, because pathogenic bacteria is caused the prerequisite of infection, also be to be adsorbed in intestinal cell (Chou and Weimer, 1999).General this characterization of adsorption is the absorption situation of using between extracorporeal mode research thalline and the intestinal cell strain (Marteau et al., 1997), as Int-407 (Leung and Finlay, 1991) and Caco-2 (Conway et al., 1987; Marteau etal., 1997) cell strain, carry out the research of milk-acid bacteria absorption and competition excluding pathogenic bacteria.
Salmonella can cause food poisoning (Boonmar et al., 1998) as Corynebacterium diphtheriae, and in recent years, the Corynebacterium diphtheriae bacterial strain of tool multiple drug resistance increases (Boonmar et al., 1998 year by year; Gross et al., 1998), and also occurred the fluoroquinolones of new generation (fluoroquinolones) and the third generation are extensively imitated the head-germ spore element drug-fast bacterial strains of third generation biocide tool such as (cephalosporins), when becoming Salmonella infection, on the clinical treatment one big problem (Herikstad et al., 1997; Amyes and Gemmell, 1997; Piddock, 1998).Therefore, the scholar will treat pathogenic bacterial infection and transfer the use probiotic bacterium to, strengthen the intestinal mucosa defense mechanism, and the protection enteron aisle avoids the infection (Nird and Edlund.1990) of pathogenic bacterias such as Salmonellas.
At present existing many milk-acid bacterias can suppress the correlative study of pathogenic bacteria, are disclosed in patent documentation or the academic journal.For example United States Patent (USP) the 5th, 603, discloses the adsorbable Caco-2 cell of Lactobacillus johnsonii (Lactobacillusjohnsonii) bacterial strain CNCM I-1225 in No. 930, and suppress the absorption that enterotoxin and intestines are invaded pathogenic bacteria; United States Patent (USP) the 3rd, 953, disclose the growth that lactobacillus lactis (Lactobacilluslactis) bacterial strain NRRL B-5628 can suppress bacterium in other Digestive tract (as mouth or stomach) for No. 609, can be used on piggy and the diarrhoea of other new birth animal or the treatment of intestinal colic especially, reduce the growth of intestinal bacteria (Escherichia coli) or coliform; United States Patent (USP) the 6th, disclose Lactobacterium acidophilum (Lactobacillus acidophilus) bacterial strain HY2177 and lactobacterium casei (Lactobacilluscasei) bacterial strain HY2743 for 491, No. 956 and can prevent and treat gastritis and duodenum and the stomach ulcer that the Helicobacter pylori infection is caused; United States Patent (USP) the 4th, 874 discloses Bacterium lacticum No. 704 can produce a kind of antimicrobial substance, can be suppressed at the growth of the spoilage organism (as special bacterium of Li Shi and Salmonellas) in the pathogenic bacteria and food under the refrigerating temperature; United States Patent (USP) the 5th, 340,577 and 5, disclose the growth that Bacterium lacticum, Lactococcus lactis (Lactococcus lactis), Fo Shi citric acid bacillus (Citrobacter freundii), faecalis (Enterococcus), bifidus bacillus (Bifidobacterium) and bacterium acidi propionici (Propionibacterium) can suppress Salmonellas 604, No. 127; U.S. Patent Publication discloses Lip river moral Bacterium lacticum (Lactobacillus reuteri), Lactobacillus johnsonii and Bacillus subtilus (Bacillus subtilis) for No. 20020018770 can be applied to suppress in the chicken feed pathogenic bacteria growth, and the growth of promotion poultry, and then the usage quantity of minimizing microbiotic or medicament; U.S. Patent Publication discloses Bacterium lacticum No. 20040028665 and propionibacterium freudenreichii (Propionibacterium freudenreichii) uses simultaneously, can suppress Escherichia coli O 157: the growth of H7 type and other pathogenic bacteria; U.S.'s publication application discloses a kind of lactobacterium casei for No. 20040029127 can strengthening immunity, and then suppresses growth of pathogenic bacteria; U.S.'s publication application discloses a kind of lactobacillus salivarius (Lactobacillus salivarius) for No. 20040038379, it has the characteristic of similar ablastins, can suppress listeria bacteria and staphylococcus, comprise anti-methicillinum streptococcus aureus (MRSA) and bacillus, and can not suppress other Bacterium lacticum; U.S.'s publication application discloses lactobacillus fermentum (Lactobacillus fermentum) bacterial strain ME-3 No. 20040151708, be a kind of tool germ resistance and oxidation resistant probiotic bacterium, can suppress Shigellae (Shigella sonnei), streptococcus aureus, Corynebacterium diphtheriae and Hp (Helicobacter pylori) in intestinal bacteria, the Song.
Academic journal also is seen in many for correlative study and the effect thereof of milk-acid bacteria, for example lactobacillus bifidus can prevent to produce the Escherichia coli O 157 of shiga toxin (shiga toxin-producing) in the mouse test: and the infection of H7 type (Asahara et al., Infect.Immun.72:2240-2247); Lactobacillus rhamnosus (Lactobacillus rhamnosus) can reduce the infection (Hirano, et al., Microbiol Immunol.47:405-409) of Enterohemorrhagic E.coli; Bacterium lacticum and lactose can suppress Salmonellas propagation (Johannsen et al., Avian Dis.48:279-286); Lip river moral Bacterium lacticum can suppress listeria monocytogenes (Listeria monocytogenes) and Salmonellas growth (Kuleasan et al., Nahrung., 46:408-410); The absorption element that bifidobacterium adolescentis (Bifidobacterium adolescentis) bacterial strain 1027 is produced, can compete inhibition enterotoxigenic Escherichia coli, enteropathy originality intestinal bacteria and clostridium (Clostridium difficile) and be attached to intestinal epithelial cell strain Lovo (Zhonget al., World J.Gastroenterol., 10:1630-1633); De Shi milk-acid bacteria (Lactobacillusdelbrueckii subsp.lactis) in meat product, can suppress pathogenic bacterium and spoilage organism growth (Senne etal., J.Food Prot., 66:418-425); Lactococcus lactis (Lactococcus lactis subsp.lactis) ATCC 11454 strains have antibacterial ability (Millette et al., J.Food Prot., 67:1184-1189); The supernatant liquor of Bacterium lacticum LB obviously reduced Salmonellas to the intrusion of intestinal cell Caco-2/TC-7 (Coconnier et al., Appl.Environ.Microbiol., 66:1152-1157); The bacterium liquid of lactobacterium casei and discarded culture supernatants thereof all can suppress Salmonellas and invade intestinal cell Caco-2, but when pH 7, lose its effectiveness, and mouse was through continuous feeding L.casei GG 7 days, can after infecting Salmonellas, keep 100% survival rate, and reduced bacterium number (Hudault et al. in liver and the spleen, Appl.Enviro.Microbiol., 63:513-518); The feeding lactobacterium casei can be mouse and provides protection to intrusion and the infection of Salmonellas, and think the IgA of this provide protection and intestinal secretion relevant (Perdigon et al., Journal of Dairy Research, 57:255-264); In the Salmonella infection mouse test of sublethal dose, whole course of infection can be divided into three periods, and be to breed period at internal organs such as liver and spleens after Salmonellas is invaded first period, after a week, entered for second period, Salmonellas propagation stops, and reaches the peak period of the highest bacterium number, and also continue a week in this period, in the 3rd period, the bacterium number of Salmonellas in liver and spleen descend gradually (Nauciel et al., Infection and Immunity, 60:450-454).
In addition, also existing many report research milk-acid bacterias suppress the mechanism of Salmonellas.Fuller (1986) once proposed to utilize does not have a bacterial classification of adsorptive power, as dried suis (Streptococcus faecium), because the speed of its propagation surpasses the rate of discharge of chyme, and can survive in enteron aisle, therefore the bacterial classification that has this characteristic can stay in the large intestine mucous layer, and needn't be adsorbed on the epithelial cell. scholar (Hormaeche et al., 1980 are arranged in addition; Van Dissel et al., 1985) propose the propagation of the Salmonellas that infects in the host, the initial stage is the effectiveness that suppresses by the function performance after scavenger cell and the activation thereof.The mechanism that milk-acid bacteria suppresses the enteric microorganism infection is competitive the eliminating, comprises competition, secretion antibacterial substance (Hudault et al., Applied and EnvironmentalMicrobiology, 63:513-518 to nutrient and intestinal cell absorption position; Chauviere et al., FEMS Microbiology Letters, 91:213-218) and the activation intestinal tract immune system, can improve ability (Perdigonet al., Journal of Dairy Research, 57:255-264 that the host suppresses pathogenic bacteria; Schiffrin et al., AmericanJournal of Clinical Nutrition, 66:515S-520S).
Summary of the invention
The present invention is from chitling road screening and separate a strain Lactobacterium acidophilum, and it has the fundamental characteristics of the above-mentioned mankind and animal probiotic bacterium, and can resist pathogenic bacteria, invades and infect the effect of intestinal mucosa and liver, spleen organ as Salmonellas.Lactobacillus acidophilus strains of the present invention can be applicable in the fodder additives of sour milk and domestic animals and fowls, uses as bacteria-promoting agent, promotes the intestines and stomach function of animal.
Therefore, one aspect of the present invention provides a kind of Lactobacterium acidophilum LASW, and preserving number is CCTCCNO:M204083, is preserved in Chinese typical culture collection center.
Further aspect of the present invention provides a kind of probiotic composition, and it comprises above-mentioned Lactobacterium acidophilum LASW.
Another aspect of the invention provides a kind of feed composition, and it comprises above-mentioned Lactobacterium acidophilum LASW.
Another aspect of the invention provides a kind of medical composition that is used to prevent or treat pathogenic bacterial infection, and it comprises above-mentioned Lactobacterium acidophilum LASW.
Description of drawings
Fig. 1 shows that intestinal bacteria are subjected to the inhibition growth test result of Lactobacterium acidophilum supernatant liquor of the present invention (SCS).
Fig. 2 shows that Salmonella choleraesuls are subjected to Lactobacterium acidophilum of the present invention to suppress to invade test-results.
The mouse that Fig. 3 shows LASW and blank group general blood serum IL-6 of the 3rd and the 6th day behind the feeding Salmonellas changes.
Embodiment
The present invention relates generally to a kind of Lactobacterium acidophilum LASW of novelty, is preserved in Chinese typical culture collection center on November 17th, 2004, and preserving number is CCTCC NO:M204083.This Lactobacterium acidophilum has following characteristic:
1. filter out from chitling road, native country, Taiwan;
2. have an ability of excellent acid and bile tolerance, can be by surviving in the enteron aisle behind the gastro-intestinal digestion liquid in the mankind and animal body;
3. have the good mankind and animal intestinal epidermic cell adsorptive power;
Can with enteric bacterial pathogens competition absorption to intestinal cell, and then the inhibition enteric bacterial pathogens, as intestinal bacteria, Salmonellas, Shigellae, streptococcus aureus and other staphylococcic absorption growth with invade intestinal cell and intrusion and be transferred to the effect of liver and spleen.
Based on above-mentioned characteristic, lactobacillus acidophilus strains LASW of the present invention can be applicable to sour milk and domestic animals and fowls feed additive in, use as bacteria-promoting agent, promote the intestines and stomach function of animal.In addition, Lactobacterium acidophilum of the present invention also can be applicable in the medical composition, in order to prevention or treatment pathogenic bacterial infection.
The following example is in order to illustrating effect of the present invention, but not in order to limit category of the present invention.
Embodiment
One, acidproof and bile tolerance test
With reference to Conway et al., Journal of Dairy Science, the described method of 70:1-12 is carried out acid resistance test.With the LASW lactic acid bacterial liquid (10 of 100 μ l through cultivating
8～10
9The CFU/ milliliter), is added to through 0.1N HCl and is adjusted to pH2.0,2.5 and 3.2 phosphate buffer soln, or pig gastric juice (pH3.2), and cultivated 3 hours down in 37 ℃.Lactobacillus suspension adds the pH7.2 phosphate buffer soln of the same terms and organizes in contrast.Cultivate after behind the serial dilution, be incubated at MRS agar respectively, the milk-acid bacteria bacterium number of calculating survival.
With reference to Gilliland et al., Journal of Dairy Science, the method described in the 73:905-911 is carried out the bile tolerance test.With above-mentioned milk-acid bacteria through the acid resistance test survival, with 5, behind centrifugal 5 minutes of the 000rpm, clean with phosphate buffer soln (pH7.2), again milk-acid bacteria is moved in 9 milliliters the MRS nutrient solution and (contain and do not contain the cholate (Sigma) of 0.3%w/v respectively, cultivated 3,12 and 24 hours, and cultivated after serial dilution, tipping is incubated at the lactic acid bacteria number of MRS agar counting survival.
Test-results shows, Lactobacterium acidophilum LASW of the present invention, and its bacterium number reduces by 2 logarithmic value (reducing to 8.5 from 10.5) after the pH2.0 environment is cultivated 3 hours down; At pH2.5 and the next stable growth of 3.2 environment.After pig gastric juice (pH3.2) is cultivated 3 hours, only reduce by 0.6 logarithmic value.In containing the environment of cholate, cultivate, still can stabilized growth.Show that it has good acid and cholate tolerance.
Two, intestinal cell strain adsorption test
Get one 24 hole porous flat plates, put into a slice cover glass in every hole.The individual cells dihedral culture dish of Int-407 and Caco-2 completely will be cultivated, outwell old nutrient solution, outwell, add 1% trypsinase/EDTA and digest for about 1 milliliter with behind twice in 1 * PBS buffer solution for cleaning dihedral culture dish, pat under the dihedral culture dish number, make cell suspension.Add 40 milliliters of fresh BME (Int-407) or MEM (Caco-2) (all containing FBS and penicillin-Streptomycin sulphate) nutrient solution (promptly diluting 4 times), after shaking up, every hole adds 0.5 ml cells suspension, in 37 ℃, CO
2Cultivate in the gas, make cell adhere to cover glass by merisis.With old nutrient solution sucking-off, every hole adds 0.5 milliliter, 1 * PBS buffer solution for cleaning again, the cell that nutrient solution that eccysis is old and removal are not adhered to, twice of repeated washing.Add 0.5 milliliter of fresh nutrient solution (not containing penicillin-Streptomycin sulphate) that is fit to indivedual different cells, and 100 μ l lactic acid bacterial liquids, in 37 ℃, CO
2Cultivated 2 hours in the gas.
The old nutrient solution of sucking-off, 0.5 milliliter of every hole adding, 1 * PBS buffer solution for cleaning three times are rocked with the 100rpm rotating speed at every turn and were cleaned 5 minutes, with the dropper sucking-off.After adding 200 milliliters of 10% formalin, rock with the 100rpm rotating speed and to fix 30 minutes, make thalline and cell fixation in the hole on the cover glass, and with dropper sucking-off formalin.Add 0.5 milliliter, 1 * PBS buffer solution for cleaning three times, rock with the 100rpm rotating speed at every turn and cleaned 5 minutes.Adding at 24-hole porous culture dish outsourcing layer of aluminum foil paper, is rocked dyeing 5 minute with the 100rpm rotating speed through the Viola crystallina 200 μ l of filter paper coarse filtration dyeing, adds 0.5 milliliter, 1 * PBS buffer solution for cleaning and removes unnecessary stain.Drip 1 * PBS damping fluid on slide glass, take out cover glass with tweezers and be placed on it, avoid dry, observe down, and count the lactic acid bacteria number (Gopalet al., 2001) that adsorbs on each cell with inverted fluorescent microscope (Olympus IMT-2).
Test-results shows that Lactobacterium acidophilum of the present invention reaches the enteron aisle epidermic cell of pig, chicken, rabbit to human intestine's epidermic cell Int-407 and Caco-2 cell, and its adsorptive power all is at least each cell greater than 15 lactic acid bacteria numbers.
Three, the inhibition growth effect of various pathogenic bacteria is tested
Lactobacterium acidophilum of the present invention was cultivated in MRS 24 hours, the pH value of bacterium liquid reduces to 3.7, streptococcus aureus, other staphylococcus, intestinal bacteria and Shigellae are carried out sensitivity tests. the result shows, Lactobacterium acidophilum of the present invention, these pathogenic bacterias are all formed the inhibition circle that differs in size, really have the effect that suppresses the pathogenic bacteria growth. in addition, in this test with the SCS effect of LASW lactobacillus strain 4 hours; Remarkable to colibacillary inhibition growth action effect, can reduce bacterium several 10
2-10
4CFU/ milliliter (Fig. 1).
Four, suppress the test that Salmonella choleraesuls are invaded intestinal cell strain Int 407
Salmonella choleraesuls (Salmonella Choleraesuis) are the very strong infecting both domestic animals and human cause of disease bacterium of invasive, therefore, select its discussion as the inhibition bacterium intrusion of milk-acid bacteria LASW.This test method mainly is with reference to (1997) described methods such as Hudault.
(1) preparation of milk-acid bacteria
To test lactic bacterium strains takes out from freezing glycerine bottle, activate twice continuously with the MRS substratum, after cultivation overnight under 37 ℃, partly bacterium liquid in 4 ℃ with 6000rpm centrifugal 10 minutes, take out supernatant liquor (spent culture supernatant, SCS), utilize among the 1N NaOH of filtration sterilization and lactic acid bacterial liquid (bacterial culture) and supernatant liquor, prepare above-mentioned adjust with lactic acid bacterial liquid through being adjusted to pH neutral and supernatant liquor to test.
(2) preparation of Salmonella choleraesuls
Salmonella choleraesuls (bacterial strain SCV2a, SCV26a) are incubated in the LB substratum, collect thalline in centrifugal 10 minutes after re-activation is cultivated, and with after the cleaning of sterile phosphate buffered soln, are suspended in the phosphate buffer soln with volume.Bacterium liquid is cultivated in nutritional medium with colony counting method through serial dilution, carries out the counting of viable count.
(3) milk-acid bacteria suppresses Salmonella choleraesuls intrusion test in Int 407 intestinal cell strains
With the intestinal cell Int 407 in the 75T culturing bottle with trypsinase-EDTA digestion process of 1 milliliter after, pat culturing bottle cell taken, be sub-packed in 96-hole trace culture plate, add 1 * 10 in each hole
5Cells/ml, overnight through cultivating, make form monolayer cell after, remove old cell culture medium, add the cell culture medium of 90 μ l antibiotic-frees.Get the lactic acid bacterial liquid and the supernatant liquor (SCS) of preparation among the 10 μ l above-mentioned (1), add in each hole of cell cultures dish, place 37 ℃, 5%CO
2The middle cultivation 1 hour.With (2) described Salmonella choleraesuls suspension, make final concentration be about 10 through suitably taking out 10 μ l after the dilution, adding in each hole
6The CFU/ hole, made the bacterium sedimentation in 10 minutes with the 1000rpm low-speed centrifugal, the cell cultures dish moves to cell culture incubator and cultivates after 1 hour and 2 hours, clean each hole five times with phosphate buffer soln, after remaining in extracellular E.coli and Salmonella choleraesuls thalline with flush away, each hole adds the BME cell culture medium that 100 μ l contain ceftriaxone (ceftriaxone) (100 mcg/ml), place cell culture incubator to leave standstill 1 hour, to kill the outer Salmonella choleraesuls bacterium of Int 407 cell born of the same parents.Clean each hole 5 times with 0.01M PBS again, add after the aseptic triton X-100 of 100 μ l (1%) carry out 10 minutes lysis effect, with the mixed liquid in acutely mixed each hole of micropipet, take out mixed liquid and carry out serial dilution with 0.01M PBS, with the nutritional medium colony counting method, through cultivating 48 hours, counting is invaded Int 407 intracellular Salmonella choleraesuls bacterium numbers.
Can learn that by the result Lactobacterium acidophilum of the present invention after effect in a hour, can reach 10 for the inhibition intrusion of Salmonella choleraesuls
2～10
3The CFU/ milliliter; After 2 hours Salmonella choleraesuls intrusion, can suppress to surpass 10
3CFU/ milliliter (Fig. 2).
Five, measure TNF-α and IL-6 concentration with enzyme-linked immunosorbent assay (ELISA)
Mouse is invaded when infecting by Salmonellas, the cell of first reaction is a scavenger cell, scavenger cell is except that phagolysis, also can secrete some cytohormones, mainly contain TNF-α, IL-1, IL-6, IL-8 and IL-12, these cytohormones can be at local infection function of organization or systemic effect.Scavenger cell is not when being activated, few (the Beutler et al. of the output of TNF-α, 1986), Gram-negative bacteria stimulates scavenger cell (Balkwill et al. with lipopolysaccharide (LPS) usually, 2000), therefore, TNF-α can be as important indicator (the Nauciel and Espinasse-Maes that is subjected to the infection of Salmonellas initial stage, 1992), the TNF-α that produces can activate vascular endothelial cell in local action, increase vascular permeability, IgG, entering of complement and cell discharges TNF-α to whole body if cause liver and spleen scavenger cell, then can cause septicemia, this is that a kind of vasodilation of general causes a large amount of body fluid flow to tissue, cause normal blood to be unable to supply, cause organ failure, be called septic shock.IL-6 is when local infection, but activated lymphocyte and the manufacturing of increase antibody if be released into the manufacturing that acute phase protein then can be had a fever and induce to whole body, cause systematic inflammation (Hirano, 1992).In inflammatory response, TNF-α just increases the amount of its mRNA in 15-30 minute behind irriate, and the time that IL-6 produces is then later, and therefore, the output of measuring TNF-α and IL-6 can be understood the severity of whole inflammatory response.
(1) measures the concentration of TNF-α with enzyme-linked immunosorbent assay
According to Endogen (USA) standard step that company provided is carried out for Woburn, MA.:
The anti-ageing mouse TNF-alpha monoclonal antibodies (1.03 mcg/ml are in adsorption-buffering liquid) of getting 100 μ l adds in the ELISA flat chassis hole, at room temperature leaves standstill (about 14 to 16 hours) overnight and makes it adhere to (coating) to trying to get to the heart of a matter.After removing adsorption-buffering liquid, each hole adds the blocking-up damping fluid (with the analysis buffer filling) of 200 μ l, leave standstill 1 hour under the room temperature after, remove the blocking-up damping fluid, wash gently three times with cleaning buffer solution again.Monoclonal antibody (0.25 mcg/ml) the 50 μ l that get anti-ageing mouse TNF-α vitamin H-mark are in hole, the standard substance or the testing sample (serum after mouse liver grinds) that add 50 μ l again, after leaving standstill 2 hours under the room temperature, fall to remove the mixed liquid in the hole, after cleaning buffer solution cleaning three times, each hole adds the HRP--conjugation streptomycete avidin (streptavidin) (0.125 mcg/ml) of 100 μ l, effect is after 30 minutes under the room temperature, clean five times with cleaning buffer solution, above cleaning step all must be inhaled and be removed remaining liquid with 96 holes trace flat chassis back-off on paper handkerchief after finishing.Last each hole adds 3,3 ', 5 of 100 μ l, 5 '-tetramethyl benzidine (TMB) matrix solution, and the lucifuge effect made in 30 minutes under the room temperature carries out color reaction, adds the 0.18M sulphuric acid soln of 100 μ l at last, the color development stopping reaction.Read under wavelength 450nm, to read light absorption value in ELISA, and according to the concentration of typical curve conversion TNF-α.
2. the making of typical curve
Advise according to the specification sheets that Endogen company provides, with 400 μ l analysis buffer dissolvings TNF-α standard substance (mouse TNF-α ELISA standard substance) (TNF-α standard substance use in back 1 hour in redissolving), making its concentration is the 2450pg/ milliliter, carry out 2 times of serial dilutions with analysis buffer again, measure light absorption value under each concentration TNF-α standard substance wavelength 450nm according to above-mentioned analytical procedure, the result is made into typical curve.
(2) measure IL-6 concentration with enzyme-linked immunosorbent assay
According to Endogen (USA) standard step that provides of company is carried out for Woburn, MA.:
The anti-ageing mouse IL-6 monoclonal antibody (2.5 mcg/ml) of getting 100 μ l adds in the ELISA flat chassis hole, at room temperature leaves standstill (about 14 to 16 hours) overnight, primary antibody is adsorbed to tries to get to the heart of a matter.After removing adsorption-buffering liquid, each hole adds analysis (blocking-up) damping fluid (4%BSA is in PBS) of 200 μ l, after leaving standstill 1 hour under the room temperature, remove the blocking-up damping fluid, with 200 μ l cleaning buffer solution (50mM Tris, 0.2%Tween-20, pH8.0) wash three times gently. the biotin labeled monoclonal antibody of anti-ageing mouse IL-6 (0.25 mcg/ml is in analysis buffer) of getting 50 μ l is in hole, the standard substance or the testing sample (serum of mouse heart blood sampling) that add 50 μ l again, after leaving standstill 2 hours under the room temperature, fall to remove the mixed liquid in the hole, after cleaning buffer solution cleaning three times, each hole adds the HRP-conjugation streptomycete avidin (0.125 mcg/ml is in analysis buffer) of 100 μ l, effect is after 30 minutes under the room temperature, clean five times with cleaning buffer solution, above cleaning step all must be with 96 holes trace flat chassis back-off on paper handkerchief after finishing, remaining liquid is removed in suction. and each hole adds 3 of 100 μ l at last, 3 ', 5,5 '-tetramethyl benzidine (TMB) matrix solution, the lucifuge effect made it to carry out color reaction in 30 minutes under the room temperature, the 0.18M sulphuric acid soln that adds 100 μ l at last, color development stopping reaction. read under wavelength 490nm, to read light absorption value in ELISA, and according to the concentration of typical curve conversion IL-6.
2. the making of typical curve
Advise according to the specification sheets that Endogen company provides, with 400 μ l analysis buffer dissolving IL-6 standard substance (mouse IL-6ELISA standard substance), making its concentration is the 2450pg/ milliliter, carry out 2.5 times of serial dilutions to the 38.4pg/ milliliter with analysis buffer again, measure the light absorption value of each concentration IL-6 standard substance under wavelength 490nm according to above-mentioned analytical procedure, the result is made into typical curve.
This research suppresses the preferable animal-origin milk-acid bacteria of Salmonellas at mouse, the serum of the liver organization of infection after 3 and 6 days is detected the TNF-alpha content, the result shows the mouse that only feeds PBS, can't suppress the Salmonellas intrusion, so the TNF-α output of its inflammation index obviously increases after infecting 6 days.LASW suppresses Salmonellas infiltration capability the best, so its TNF-alpha content that infects after 6 days is minimum; The detection of IL-6 also has identical result (Fig. 3).
Through the foregoing description and with reference to the accompanying drawings, should understand that the present invention is not limited among above-mentioned these embodiment.The person of ordinary skill in the field changes and improves when carrying out difference under the situation departing from specification sheets of the present invention and defined scope of claim and spirit not, also belongs in the category 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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| CN102559539B (en) * | 2011-12-02 | 2015-04-15 | 北京大北农科技集团股份有限公司 | Lactobacillus acidophilus and application thereof and feed additive thereof and premix compound thereof |
| CN103283973A (en) * | 2013-03-20 | 2013-09-11 | 广州格拉姆生物科技有限公司 | A method for producing a probiotic agent of live Lactobacillus acidophilus |
| CN104415062A (en) * | 2013-08-27 | 2015-03-18 | 弘光科技大学 | Application of mixture containing four lactic acid bacteria strains in prevention and/or relieving of alcoholic liver diseases |
| CN104286408B (en) * | 2014-06-13 | 2017-10-24 | 杨雄 | Biological agent prepared by a kind of lactobacillus acidophilus XD13R is reducing the application process of antibiotic-resistant bacteria ratio in pet body |
| CN104877946B (en) * | 2015-03-13 | 2018-01-30 | 成都医学院 | Excrement lactobacillus FZB1 and its application |
| CN104673726B (en) * | 2015-03-13 | 2017-10-10 | 北京市农林科学院 | One boar source lactobacillus acidophilus freeze-drying preparation and its application |
| CN114395514B (en) * | 2022-02-28 | 2023-09-01 | 鲁东大学 | Lactobacillus acidophilus, microbial inoculum and application thereof |
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| US3953609A (en) * | 1974-02-06 | 1976-04-27 | Microlife Technics, Inc. | Method for the growth restriction of undesirable digestive system bacteria in animals and the establishment of lactobacillus lactis NRRL B-5628 |
| US6491956B2 (en) * | 1999-02-08 | 2002-12-10 | Korea Yakult Co. Ltd. | Food containing active strains for inhibiting infection and treating gastritis, gastric and duodenal ulcers |
| CN1641014A (en) * | 2004-01-18 | 2005-07-20 | 北京三元食品股份有限公司 | Acidophilic lactobacillus and its use |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3953609A (en) * | 1974-02-06 | 1976-04-27 | Microlife Technics, Inc. | Method for the growth restriction of undesirable digestive system bacteria in animals and the establishment of lactobacillus lactis NRRL B-5628 |
| US6491956B2 (en) * | 1999-02-08 | 2002-12-10 | Korea Yakult Co. Ltd. | Food containing active strains for inhibiting infection and treating gastritis, gastric and duodenal ulcers |
| CN1641014A (en) * | 2004-01-18 | 2005-07-20 | 北京三元食品股份有限公司 | Acidophilic lactobacillus and its use |
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