CN102732468A - Enterococcus faecalis and broad-spectrum antibacterial action thereof - Google Patents

Abstract

The invention discloses enterococcus faecalis and application of broad-spectrum antibacterial action thereof. The invention provides Enterococcus faecalis E15-CUI with the collection number of CGMCC NO. 6130. The invention also provides application of the Enterococcus faecalis E15-CUI CGMCC NO.6130 or a fermentation product thereof in preparation of antibacterial products. Experiments prove that enterococcus faecalis E15-CUI is quickly screened from gastrointestinal tracts of animals, the strain is subjected to fermentation culture, and the fermentation product can inhibit various bacteria such as Escherichia coli, salmonella pullorum, salmonella gallinarum, salmonella enteritidis, Klebsiella pneumoniae, proteus, Pasteurella multocida, Streptococcus equinus, Streptococcus suis, Staphylococcus aureus and Bacillus cereus.

Description

One strain enterococcus faecalis and broad-spectrum antibacterial effect thereof

Technical field

The present invention relates to biological technical field, relate in particular to a strain enterococcus faecalis and broad-spectrum antibacterial effect thereof.

Background technology

China is a livestock industry big country, also is livestock product consumption big country.Microbiotic is extensively used in prevention or treatment zoogenetic infection parasite and bacteriosis in a large number; It has been recognized that the harm of abuse of antibiotics; Be limited or forbid as animal growth promoter, microbiotic uses the drug residue problem that causes to become the serious hindrance of animal husbandry development.The animal-derived food microbiotic exceeds standard and the exit loss that causes and public health security problem have caused widely and pay close attention to.People press for and seek possible substitute, more hope to find related material seldom with human microbiotic to carry out prevention and treatment of diseases, to eliminate the threat of antibiotic resistance to human health.Bacteriocin is one type of natural antimicrobial substance with great potential beyond doubt, so bacteriocin becomes the focus of Recent study.

Bacteriocin (Bacteriocins) is that some bacterium is in metabolic process; Through one type of polypeptide, protein or protein complex that the rrna synthesis mechanism produces, have advantages such as efficient, nontoxic, high temperature resistant, noresidue, no resistance with anti-microbial activity.And traditional microbiotic such as penicillium mould are the meta-bolitess that is formed by the catalysis of cell multienzyme complex, do not have structure gene; And bacteriocin can be transformed through engineered means by genes encoding.Nutrition 21 Nisin is generally regarded as safe in the world sanitas, and existing 52 countries and regions use Nisin as food preservative, thereby has promoted the research of other kind bacteriocin and the development and application in field outside food preservatives.

In recent years, bacteriocin has been widely used as foodstuff additive and probiotics because of it has advantages such as nontoxic, efficient, high temperature resistant, noresidue, no resistance.Along with the popularization on the aquaculture model of the fast development of fodder industry and mass-producing, people pay close attention to the security level of animal-derived food more.If the bacterial strain of bacteriocinogeny is prepared into the probiotics active bacteria formulation or develops the bacteriocin fodder additives, to reduce the abuse of Antibiotic Additive in feed, the application potential of bacteriocin in livestock industry will be very huge so.

Summary of the invention

The invention provides a strain enterococcus faecalis (Enterococcus faecalis) E15-CUI.

Enterococcus faecalis provided by the invention (Enterococcus faecalis) E15-CUI, its preserving number is CGMCCNO.6130.

The application in preparing antibacterial and/or sterilised products of above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or its tunning also is the scope that the present invention protects.

In the above-mentioned application, said antibacterial for suppressing at least a in ETEC, white dysentery Salmonellas, fowl typhoid Salmonellas, Klebsiella pneumonia, pasteurella multocida, monocyte hyperplasia Listeria monocytogenes, strangles suis epizootic disease subspecies, streptococcus aureus, Bacillus anthracis, Bacillus cereus, faecium and the enterococcus faecalis;

Also can suppress resistance fowl typhoid Salmonellas, resistance Salmonella enteritidis, proteus vulgaris or swine streptococcus.

Said sterilization is for killing ETEC; The said ETEC of killing is specially and kills ETEC CVCC C83707.

In the above-mentioned application, said tunning prepares according to the method that comprises the steps: above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 is carried out liquid fermenting, collect product, be tunning.

In the above-mentioned application, the temperature of said liquid fermenting is 37 ℃, and the time of said liquid fermenting is 24h, and the rotating speed of said liquid fermenting is 200r/min, and the substratum of said liquid fermenting is the MRS liquid nutrient medium.

In the above-mentioned application, also comprise the steps: among the said tunning preparation method said product, collect supernatant, obtain tunning at 4 ℃, the centrifugal 10min of 9100 * g.

Another object of the present invention provides a kind of antibacterial product.

Antibacterial product provided by the invention, its activeconstituents are above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or the tunning in the above-mentioned application; Above-mentioned antibacterial being specially suppressed at least a in ETEC, white dysentery Salmonellas, fowl typhoid Salmonellas, Klebsiella pneumonia, pasteurella multocida, monocyte hyperplasia Listeria monocytogenes, strangles suis epizootic disease subspecies, streptococcus aureus, Bacillus anthracis, Bacillus cereus, faecium and the enterococcus faecalis.

The 3rd purpose of the present invention provides a kind of sterilised products.

Sterilised products provided by the invention, its activeconstituents are above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or the tunning in the above-mentioned application; Said sterilization is for killing ETEC; The said ETEC of killing is specially and kills ETEC CVCC C83707.

The 4th purpose of the present invention provides a kind of method for preparing antibacterial product or sterilised products.

Method provided by the invention comprises the steps: above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 is carried out liquid fermenting, collects product, promptly obtains antibacterial product or sterilised products.

In the aforesaid method, the temperature of said liquid fermenting is 37 ℃, and the time of said liquid fermenting is 24h, and the rotating speed of said liquid fermenting is 200r/min, and the substratum of said liquid fermenting is the MRS liquid nutrient medium.

Aforesaid method also comprises the steps: behind said collection product, also to comprise the steps: with said product at 4 ℃, the centrifugal 10min of 9100 * g, collects supernatant, obtains antibacterial product;

Above-mentioned antibacterial for suppressing at least a in ETEC, white dysentery Salmonellas, fowl typhoid Salmonellas, Klebsiella pneumonia, pasteurella multocida, monocyte hyperplasia Listeria monocytogenes, strangles suis epizootic disease subspecies, streptococcus aureus, Bacillus anthracis, Bacillus cereus, faecium and the enterococcus faecalis;

Said sterilization is for killing ETEC; The said ETEC of killing is specially and kills ETEC CVCC C83707.

Bacterial strain E15-CUI is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 23rd, 2012 and (is called for short CGMCC; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; Postcode 100101), preserving number is CGMCC No.6130, classification called after enterococcus faecalis Enterococcus faecalis.

Experiment of the present invention proves; The present invention goes out enterococcus faecalis E15-CUI from the animal gastrointestinal tract rapid screening; This bacterial strain is carried out fermentation culture; Tunning can suppress multiple bacterium such as ETEC, white dysentery Salmonellas, fowl typhoid Salmonellas, Salmonella enteritidis, Klebsiella pneumonia, proteus vulgaris, pasteurella multocida, strangles suis, swine streptococcus, streptococcus aureus, Bacillus cereus; And fungistatic effect judges that through preliminary antibacterial is because fermentation using bacteria has produced a kind of bacteriocin material with property of protein and caused greater than similar enterococcus faecalis.Therefore bacterial strain of the present invention and the application potential of tunning in livestock industry thereof will be very huge.

Description of drawings

Fig. 1 is the morphologic observation of E15-CUI

Fig. 2 is the E15-CUI cultural characters

Fig. 3 is the influence of transmission electron microscope observing E15-CUI fermented liquid to intestinal bacteria CVCC C83707 form

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

The separation of embodiment 1, E15-CUI and evaluation

1, the separation of E15-CUI

(1) processing of animal gastrointestinal tract

The aseptic whole digestive tube of getting health pig comprises stomach, duodenum, jejunum, ileum, caecum, colon, rectum; Get each position mucous membrane tissue 1g respectively; Place sterilization plate, shred, process suspension with the SPSS of 5mL, get in the 100 μ L suspension inoculation milk-acid bacteria enrichment medium; Put candle jar method cultivation 48h in 37 ℃ of incubators, obtain increasing the bacterium liquid after bacterium is cultivated.

(2) separation and purification

Get the bacterium liquid that increases after bacterium is cultivated and select the separation and Culture of ruling on the substratum (available from sky, Hangzhou and reagent ltd), cultivate 24h for 37 ℃ faecalis.Picking colonies typical, microscopy, the assorted bacterium of nothing is transplanted, purifying is cultivated, and is stored in 4 ℃ of refrigerators subsequent use.

(3) bacteriostatic experiment

The MRS liquid nutrient medium: Tryptones 10g, yeast soak powder 5g, glucose 10g, sucrose 5g, Diammonium citrate 2g, sodium-acetate 15g, KH ₂PO ₄6g, MnSO ₄4H ₂0MgSO ₄7H ₂0 0.58g, 0.25g, FeSO ₄7H ₂0,0.03g, tween-80 1g, zero(ppm) water 1000ml, pH7.0.

The PYG substratum: soy peptone 0.5g, yeast soak powder 0.5g, KH ₂PO ₄0.1g, MgSO ₄7H ₂00.02g, MnSO ₄4H ₂0 0.002g, glucose 0.1g, zero(ppm) water 100ml, pH6.5.

Fermentation: the microbionation of the separation and purification that above-mentioned (2) are obtained is to the MRS liquid nutrient medium, and 37 ℃, 200r/min oscillation and fermentation cultivation 24h obtain tunning; At 4 ℃, the centrifugal 10min of 9100 * g, it is subsequent use to get supernatant with tunning;

The cultivation of indicator (seeing the following form 1): respectively indicator is cultivated logarithmic phase, be diluted to 10 with sterile saline ⁷CFU/mL gets 100ul coating PYG culture medium flat plate; Adopt agar punching double-layer plate diffusion process, spread 1h in the rearmounted 4 ℃ of refrigerators of application of sample (above-mentioned supernatant), cultivate 24h for 37 ℃ and observe bacteriostatic activity, the result sees table 1:

Table 1 is the bacteriostatic test result

Filter out bacterial strain, called after E15-CUI with bacteriostatic activity.

2, the evaluation of E15-CUI

1) ne ar, cultural characters are identified

The E15-CUI that above-mentioned 1 screening is obtained carries out gramstaining, and is positive, microscopy, and the result is circle or oval, and single, paired or short chain shape is arranged, and no pod membrane, no gemma, atrichous coccus see Fig. 1.

Above-mentioned 1 E15-CUI that obtains of screening is selected substratum (available from sky, Hangzhou and reagent ltd) to upload to be commissioned to train foster faecalis, observe bacterium colony, form red-purple, smooth surface is protruding, periphery neatly, the bacterium colony of diameter 0.5-1.0mm is seen Fig. 2.

The E15-CUI that above-mentioned 1 screening is obtained goes up cultivation at ordinary nutrient agar substratum (available from sky, Hangzhou and reagent ltd), observes bacterium colony, forms circular pearl; Tip-like or the small colonies that reveals a shape; Moistening, translucent, the bacterium colony of neat in edge, the no hemolytic of growth on blood agar.

2) Physiology and biochemistry is identified

The E15-CUI that above-mentioned 1 screening is obtained carries out respectively like the test of the Characteristics Detection in the following table 2, and the result sees table 2:

Table 2 is the physiological and biochemical property of E15-CUI

3) molecular level is identified

The E15-CUI that above-mentioned 1 screening is obtained extracts genomic dna; Pcr amplification; Primer is P1:5 '-TCATCTGTCCCACCTTGCGC-3 ', and P2:5 '-GAGTTTGATCCTGGCTCAGG-3 ' obtains the 16SrDNA gene; Through order-checking, the nucleotides sequence of this 16SrDNA gene is classified the sequence 1 in the sequence table as.

Analyze above-mentioned form, Physiology and biochemistry result, and combine the classification position of 16SrDNA gene at GenBank, can obtain E15-CUI is enterococcus faecalis.

Bacterial strain E15-CUI is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 23rd, 2012 (is called for short CGMCC; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; Postcode 100101), preserving number is CGMCC No.6130, classification called after enterococcus faecalis Enterococcus faecalis.

Embodiment 2, the application of enterococcus faecalis E15-CUI CGMCC No.6130 in antibacterial

1, the antibacterial research of enterococcus faecalis E15-CUI CGMCC No.6130

With enterococcus faecalis E15-CUI CGMCC No.6130 according to the method for 1 (3) among the embodiment 1 ferment, centrifugal, and collect supernatant and carry out bacteriostatic experiment, table 1 is seen in the indicator source.With enterococcus faecalis E5 and faecium E9 is contrast.

The enterococcus faecalis E5 public can obtain from Beijing Agricultural College; Putting down in writing the article that this bacterial strain delivered is: produce enterocin E5 bacteriocin enterococcus faecalis Optimizing Conditions of Fermentation; Cui Defeng, cycle, Yang Guimei, Li Huanrong, Ruan Wenke, Wang Ming, Zhang Yonghong; 2011,45 (3): 13～17, Chinese veterinary drug magazine.

The faecium E9 public can obtain from Beijing Agricultural College; Putting down in writing the article that this bacterial strain delivered is: generation and the characteristic research of the plain E9 of faecium, cycle, Liu Fenghua, Cui Defeng, Nie Xiaohua, Wang Xueting, Bai Yan, Chinese veterinary drug magazine; 2011,45 (2): 16-19.

The result sees table 3, can find out that the fermented supernatant fluid of bacterial strain E15-CUI has shown broad-spectrum antibacterial activity, to 23 strain indicators inhibited (comprising 21 strain animal source pathogenic micro-organisms and 2 probiotics).The Gram-negative pathogenic bacteria that can extensively suppress enterobacteriaceae; The strongest to 7 strain pig source enterotoxin type intestinal bacteria and 1 strain chicken source pathogenic colon bacillus restraining effect; Antibacterial circle diameter reaches 17.7-24mm; White dysentery, fowl typhoid Salmonellas, Bacillus proteus and kerekou pneumonia uncle also there are stronger restraining effect, have tangible bacteriostatic activity to producing drug-fast fowl typhoid Salmonellas of wide spectrum and Salmonella enteritidis strain isolated especially; Can suppress pasteurella multocida (separation) from dying of illness pig and chicken; Suppress Gram-positive streptococcus aureus, strangles suis, swine streptococcus, 2 strain Bacillus anthracis low virulent strains and bacillus cereus are produced significant bacteriostatic activity; Can suppress to belong to together faecium, enterococcus faecalis; Only 1 strain monocyte hyperplasia Listeria monocytogenes is not had restraining effect, the fermented supernatant fluid fungistatic effect of E15-CUI is good than enterococcus faecalis E5 and faecium E9 obviously.

Table 3 is the comparison of E15-CUI and faecium E9 and enterococcus faecalis E5 fermented liquid bacteriostatic activity

2, enterococcus faecalis E15-CUI CGMCC No.6130 bacteriostatic action mechanism (antibacterial substance analysis)

(1) eliminating of hydrogen peroxide bacteriostatic action

Enterococcus faecalis E15-CUI CGMCC No.6130 according to the method fermentation of 1 among the embodiment 1 (3), centrifugal, collection supernatant, is got lmg/mL hydrogen peroxide enzyme aqueous solution, add the supernatant that centrifugal collection obtains, obtain handling product.Carry out bacteriostatic experiment with handling product according to the method for 1 among the embodiment 1 (3), indicator is intestinal bacteria CVCC C83707.With the supernatant handled without katalase as contrast.

The result is following: the supernatant of handling and handling without katalase through katalase all has restraining effect to indicator, and the diameter of inhibition zone is respectively 18.5mm and 18.4mm.Explain that enterococcus faecalis E15-CUI CGMCC No.6130 supernatant is not the effect of Hydrogen Peroxide Metabolism product to the restraining effect of indicator.

(2) discharge of acid bacteriostatic action

Enterococcus faecalis E15-CUI CGMCC No.6130 according to the method fermentation of 1 among the embodiment 1 (3), centrifugal, collection supernatant, is regulated the fermentation using bacteria supernatant to pH7.0 effect 30min with 6mol/L NaOH.

Carry out bacteriostatic experiment with handling product according to the method for 1 among the embodiment 1 (3), indicator is streptococcus aureus CVCC2086 and intestinal bacteria CVCC C83707, is that 4.5 lactic acid, hydrochloric acid are contrast not transfer pH fermented supernatant fluid and pH.

The result sees table 4, can be found out by table 4, and the fermented supernatant fluid of E15-CUI CGMCC No.6130 still has stronger anti-microbial activity to 2 strain indicators after discharging the acid interference.

Table 4 is the bacteriostatic action after acid is got rid of

(3) proteasome degradation experimental verification bacteriocinogeny milk-acid bacteria

With enterococcus faecalis E15-CUI CGMCC No.6130 according to the method for 1 among the embodiment 1 (3) fermentation, centrifugal, collect supernatant; Adding Proteinase K, trypsinase, papoid, lypase and glycase (Sigma) final concentration respectively is 5mg/mL; In 37 ℃ of water-baths, get 100ul behind the incubation 2h; The supernatant that will pass through after different enzymes are handled carries out bacteriostatic experiment (indicator is intestinal bacteria CVCC C83707) according to the method for 1 among the embodiment 1 (3), with the supernatant handled without any enzyme as contrast.

The result: the inhibition zone size of the supernatant of handling without any enzyme is 20mm;

The inhibition zone size of the bacterium supernatant after Proteinase K, trypsinase and papoid are handled is respectively 6.5mm, 6.8mm, 8.0mm;

The inhibition zone size of the bacterium supernatant after lypase and glycase processing is respectively 20mm and 19.9mm;

Can find out that the bacteriostatic activity of the bacterium supernatant after Proteinase K, trypsinase and papoid are handled obviously reduces, lypase and glycase are handled no change, can judge tentatively that fermentation using bacteria has produced a kind of bacteriocin material with property of protein.

(4) transmission electron microscope observing is to the germicidal action of intestinal bacteria CVCC C83707

With enterococcus faecalis E15-CUI CGMCC No.6130 according to the method for 1 among the embodiment 1 (3) fermentation, centrifugal, collect supernatant 5ml;

With 37 ℃ of intestinal bacteria CVCC C83707 that cultivate 3h, the centrifugal 10min of 9100 * g collects thalline; The 5ml supernatant of enterococcus faecalis E15-CUI CGMCC No.6130 is added in the above-mentioned intestinal bacteria CVCC C83707 thalline acts on 0.5-1h, PBS damping fluid (NaCl0.8g, KH are established in centrifugal, film-making simultaneously ₂PO ₄0.024g, KCl0.02g, Na ₂HPO ₄12H ₂O 0.363g, deionized water 100mL, (0.1mol/L pH7.2-7.3) is contrast, electron microscopic observation result.

The result sees Fig. 3, and wherein, the A:E15-CUI fermented liquid acts on 1h (8000X) to intestinal bacteria; The B:E15-CUI fermented liquid is to 0.5 hour (20000X) of intestinal bacteria effect; C:PBS control group effect 1h (8000X); D:PBS control group effect 0.5h (20000X)

Can find out that the PBS behind processing 0.5h and the 1h is hatched intestinal bacteria CVCC C83707 form homogeneous, full, the neat in edge of structure; The coli somatic of E15-CUI fermentation liquor treatment is along with the prolongation of action time, and intestinal bacteria CVCC C83707 form is destroyed gradually, and the lifetime dependency.When handling 30min, intestinal bacteria CVCCC83707 structure complete basically but begin to blur, form is uneven, edge shrinkage to some extent; After handling 1h, can't see the intestinal bacteria CVCC C83707 of structural integrity under the Electronic Speculum, all bacteriums are all broken, and dissipate.

Above result has further proved the truly have killing action of E15-CUI fermented liquid to intestinal bacteria CVCC C83707, and its influence to target bacteria mainly is to make the bacterium shrinkage, and cellularstructure is destroyed and kytoplasm leaks and death.

Claims (10)

1. enterococcus faecalis (Enterococcus faecalis) E15-CUI, its preserving number is CGMCC NO.6130.

2. the described enterococcus faecalis of claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or its tunning application in preparing antibacterial and/or sterilised products.

3. application according to claim 2 is characterized in that:

Said antibacterial for suppressing at least a in ETEC, white dysentery Salmonellas, fowl typhoid Salmonellas, Klebsiella pneumonia, pasteurella multocida, monocyte hyperplasia Listeria monocytogenes, strangles suis epizootic disease subspecies, streptococcus aureus, Bacillus anthracis, Bacillus cereus, faecium and the enterococcus faecalis;

Said sterilization is for killing ETEC.

4. according to claim 2 or 3 described application, it is characterized in that:

Said tunning prepares according to the method that comprises the steps: the described enterococcus faecalis of claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130 is carried out liquid fermenting, collect product, be tunning.

5. application according to claim 4 is characterized in that:

The temperature of said liquid fermenting is 37 ℃, and the time of said liquid fermenting is 24h, and the rotating speed of said liquid fermenting is 200r/min, and the substratum of said liquid fermenting is the MRS liquid nutrient medium.

6. according to claim 4 or 5 described application, it is characterized in that: also comprise the steps: among the said tunning preparation method said product at 4 ℃, the centrifugal 10min of 9100 * g, collect supernatant, obtain tunning.

7. antibacterial product, its activeconstituents is the tunning in arbitrary said application among the described enterococcus faecalis of claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or the claim 2-6; Said antibacterial being specially suppressed at least a in ETEC, white dysentery Salmonellas, fowl typhoid Salmonellas, Klebsiella pneumonia, pasteurella multocida, monocyte hyperplasia Listeria monocytogenes, strangles suis epizootic disease subspecies, streptococcus aureus, Bacillus anthracis, Bacillus cereus, faecium and the enterococcus faecalis.

8. sterilised products, its activeconstituents is the tunning in arbitrary said application among the described enterococcus faecalis of claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or the claim 2-6; Said sterilization is for killing ETEC.

9. method for preparing antibacterial product or sterilised products; Comprise the steps: the described enterococcus faecalis of claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130 is carried out liquid fermenting; Collect product, promptly obtain antibacterial product or sterilised products.

10. method according to claim 9 is characterized in that:

The temperature of said liquid fermenting is 37 ℃, and the time of said liquid fermenting is 24h, and the rotating speed of said liquid fermenting is 200r/min, and the substratum of said liquid fermenting is the MRS liquid nutrient medium;

Said method also comprises the steps: behind said collection product, also specifically to comprise the steps: with said product at 4 ℃, the centrifugal 10min of 9100 * g, collects supernatant, obtains antibacterial product;

Said antibacterial for suppressing at least a in ETEC, white dysentery Salmonellas, fowl typhoid Salmonellas, Klebsiella pneumonia, pasteurella multocida, monocyte hyperplasia Listeria monocytogenes, strangles suis epizootic disease subspecies, streptococcus aureus, Bacillus anthracis, Bacillus cereus, faecium and the enterococcus faecalis;

Said sterilization is for killing ETEC.

Images