CN102732468B - Enterococcus faecalis and broad-spectrum antibacterial action thereof - Google Patents
Enterococcus faecalis and broad-spectrum antibacterial action thereof Download PDFInfo
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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
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 Shi Yige livestock industry big country, Ye Shi Consumption of animal by-product big country.Microbiotic is extensively used in a large number in prevention or treatment zoogenetic infection parasite and bacteriosis, it has been recognized that the harm of abuse of antibiotics, as animal growth promoter, be limited or forbidding, microbiotic is used the drug residue problem causing to become the serious hindrance of animal husbandry development.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, in the urgent need to finding possible substitute, more wish to find material seldom associated with mankind's microbiotic to carry out prevention and treatment of diseases, to eliminate the threat of antibiotic resistance to human health.Bacteriocin is the natural antimicrobial substance with great potential of a class beyond doubt, so bacteriocin becomes the focus of Recent study.
Bacteriocin (Bacteriocins) is that some bacterium is in metabolic process, the class producing by Ribosome biogenesis mechanism has polypeptide, protein or the protein complex of anti-microbial activity, has efficient, nontoxic, high temperature resistant, noresidue, without advantages such as resistance.And traditional microbiotic such as penicillin are the meta-bolitess being formed by the catalysis of cell multienzyme complex, there is not structure gene; And bacteriocin is by genes encoding, can be transformed by engineered means.Nisin Nisin is generally regarded as safe sanitas in the world, and existing 52 countries and regions are used 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 because of its there is nontoxic, efficient, high temperature resistant, noresidue, without advantages such as resistance, be widely used as foodstuff additive and probiotics.Along with the popularization on the fast development of fodder industry and the aquaculture model of mass-producing, people pay close attention to more to the security level of animal-derived food.If the bacterial strain of bacteriocinogeny is prepared into probiotics active bacteria formulation or develops 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 is also the scope of protection of the invention.
In above-mentioned application, described antibacterial for suppressing at least one in colon bacillus, 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 enterococcus faecalis;
Also can suppress resistance fowl typhoid Salmonellas, resistance Salmonella enteritidis, proteus vulgaris or swine streptococcus.
Described sterilization is for killing colon bacillus; The described colon bacillus of killing is specially and kills colon bacillus CVCC C83707.
In above-mentioned application, described tunning is prepared according to the method comprising the steps: above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 is carried out to liquid fermenting, collect product, be tunning.
In above-mentioned application, the temperature of described liquid fermenting is 37 ℃, and the time of described liquid fermenting is 24h, and the rotating speed of described liquid fermenting is 200r/min, and the substratum of described liquid fermenting is MRS liquid nutrient medium.
In above-mentioned application, in described tunning preparation method, also comprise the steps: described product at 4 ℃, the centrifugal 10min of 9100 * g, collect supernatant liquor, obtain tunning.
Another object of the present invention is to provide a kind of antibacterial product.
Antibacterial product provided by the invention, its activeconstituents is above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or the tunning in above-mentioned application; Above-mentioned antibacterial at least one inhibition in colon bacillus, 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 enterococcus faecalis that be specially.
The 3rd object of the present invention is to provide a kind of sterilised products.
Sterilised products provided by the invention, its activeconstituents is above-mentioned enterococcus faecalis (Enterococcus faecalis) E15-CUI CGMCC NO.6130 or the tunning in above-mentioned application; Described sterilization is for killing colon bacillus; The described colon bacillus of killing is specially and kills colon bacillus CVCC C83707.
The 4th object of the present invention is to provide a kind of method of 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 to carry out liquid fermenting, collects product, obtains antibacterial product or sterilised products.
In aforesaid method, the temperature of described liquid fermenting is 37 ℃, and the time of described liquid fermenting is 24h, and the rotating speed of described liquid fermenting is 200r/min, and the substratum of described liquid fermenting is MRS liquid nutrient medium.
Aforesaid method also comprises the steps: also to comprise the steps: after described collection product by described product at 4 ℃, the centrifugal 10min of 9100 * g, collects supernatant liquor, obtains antibacterial product;
Above-mentioned antibacterial for suppressing at least one in colon bacillus, 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 enterococcus faecalis;
Described sterilization is for killing colon bacillus; The described colon bacillus of killing is specially and kills colon bacillus CVCC C83707.
Bacterial strain E15-CUI is preserved in China Committee for Culture Collection of Microorganisms's 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, and Classification And Nomenclature is enterococcus faecalis Enterococcus faecalis.
Of the present invention experimental results show that, the present invention goes out enterococcus faecalis E15-CUI from animal gastrointestinal tract rapid screening, this bacterial strain is carried out to fermentation culture, tunning can suppress colon bacillus, white dysentery Salmonellas, fowl typhoid Salmonellas, Salmonella enteritidis, Klebsiella pneumonia, proteus vulgaris, pasteurella multocida, strangles suis, swine streptococcus, streptococcus aureus, the multiple bacterium such as Bacillus cereus, and fungistatic effect is greater than similar enterococcus faecalis, antibacterial through preliminary judgement is to cause because fermentation using bacteria has produced a kind of bacteriocin material with property of protein.Therefore bacterial strain of the present invention and the application potential of tunning in livestock industry thereof will be very huge.
Accompanying drawing explanation
Fig. 1 is the morphologic observation of E15-CUI
Fig. 2 is E15-CUI cultural characters
Fig. 3 is the impact of transmission electron microscope observing E15-CUI fermented liquid on intestinal bacteria CVCC C83707 form
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The Isolation and Identification of embodiment 1, E15-CUI
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 respectively each position mucous membrane tissue 1g, be placed in sterilizing plate, shred, by the stroke-physiological saline solution of 5mL, make suspension, get in 100 μ L suspension inoculation milk-acid bacteria enrichment medium, put candle jar method in 37 ℃ of incubators and cultivate 48h, obtain increasing the bacterium liquid after bacterium is cultivated.
(2) separation and purification
Get the bacterium liquid that increases after bacterium is cultivated in the faecalis separation and Culture of selecting to rule on substratum (purchased from Hangzhou Tian He reagent company limited), cultivate 24h for 37 ℃.Picking colonies typical, microscopy, without miscellaneous bacteria transplant, purifying cultivates, and is stored in 4 ℃ of refrigerators standby.
(3) bacteriostatic experiment
MRS liquid nutrient medium: Tryptones 10g, yeast soaks 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, distilled water 1000ml, pH7.0.
PYG substratum: soy peptone 0.5g, yeast soaks powder 0.5g, KH
2pO
40.1g, MgSO
47H
200.02g, MnSO
44H
20 0.002g, glucose 0.1g, distilled water 100ml, pH6.5.
Fermentation: the microbionation of the separation and purification that above-mentioned (2) are obtained is to MRS liquid nutrient medium, and 37 ℃, 200r/min oscillation and fermentation cultivation 24h, obtain tunning; Tunning, at 4 ℃, the centrifugal 10min of 9100 * g, is got to supernatant liquor standby;
The cultivation of indicator (seeing the following form 1): respectively indicator is cultivated to 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, in the rearmounted 4 ℃ of refrigerators of application of sample (above-mentioned supernatant liquor), spread 1h, cultivate 24h for 37 ℃ and observe bacteriostatic activity, the results are shown in Table 1:
Table 1 is bacteriostatic test result
Filter out the bacterial strain with bacteriostatic activity, called after E15-CUI.
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, positive, microscopy, and result is circle or oval, single, paired or short catenation, without pod membrane, without gemma, atrichous coccus, is shown in Fig. 1.
The E15-CUI that above-mentioned 1 screening is obtained selects substratum (purchased from Hangzhou Tian He reagent company limited) to upload culture faecalis, observes bacterium colony, forms red-purple, smooth surface projection, and periphery is neat, and the bacterium colony of diameter 0.5-1.0mm, is shown in Fig. 2.
The E15-CUI that above-mentioned 1 screening is obtained, in the upper cultivation of ordinary nutrient agar substratum (purchased from Hangzhou Tian He reagent company limited), observes bacterium colony, forms circular canescence, tip-like or the small colonies that reveals a shape, moistening, translucent, the bacterium colony of neat in edge, on blood agar, growth is without hemolytic.
2) Physiology and biochemistry is identified
The E15-CUI that above-mentioned 1 screening is obtained carries out respectively, as the Characteristics Detection test in following table 2, the results are shown in 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 ', P2:5 '-GAGTTTGATCCTGGCTCAGG-3 ', obtain 16SrDNA gene, through order-checking, the nucleotides sequence of this 16SrDNA gene is classified the sequence 1 in sequence table as.
Analyze above-mentioned form, Physiology and biochemistry result, and in conjunction with 16SrDNA gene the classification position at GenBank, can obtain E15-CUI is enterococcus faecalis.
Bacterial strain E15-CUI is preserved in to China Committee for Culture Collection of Microorganisms's 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, and Classification And Nomenclature is 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
By enterococcus faecalis E15-CUI CGMCC No.6130 according to the method for 1 (3) in embodiment 1 ferment, centrifugal, and collect supernatant liquor and carry out bacteriostatic experiment, indicator is originated in Table 1.Take enterococcus faecalis E5 and faecium E9 is contrast.
Enterococcus faecalis E5 public Ke Cong Beijing Agricultural College obtains, recording the article that this bacterial strain delivered is: the optimization of producing enterocin E5 bacteriocin Enterococcus faecalis fermentation condition, Cui Defeng, cycle, Yang Guimei, Li Huanrong, Ruan Wenke, Wang Ming, Zhang Yonghong, 2011,45 (3): 13～17, Chinese veterinary drug magazine.
Faecium E9 public Ke Cong Beijing Agricultural College obtains, recording the article that this bacterial strain delivered is: generation and the characteristic research of faecium element E9, cycle, Liu Fenghua, Cui Defeng, Nie Xiaohua, Wang Xueting, Bai Yan, Chinese veterinary drug magazine, 2011,45 (2): 16-19.
The results are shown in Table 3, the fermented supernatant fluid that can find out bacterial strain E15-CUI has shown the anti-microbial activity of wide spectrum, 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 are also had to stronger restraining effect, to producing the drug-fast fowl typhoid Salmonellas of wide spectrum and Salmonella enteritidis strain isolated, there is obvious bacteriostatic activity especially; Can suppress pasteurella multocida (separated 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 to significant bacteriostatic activity; Can suppress to belong to together faecium, enterococcus faecalis; Only 1 strain monocyte hyperplasia Listeria monocytogenes is not had to 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 in embodiment 1 (3), centrifugal, collection supernatant liquor, is got to lmg/mL hydrogen peroxide enzyme aqueous solution, and the supernatant liquor that adds centrifugal collection to obtain, obtains processing product.By processing product, according to the method for 1 in embodiment 1 (3), carry out bacteriostatic experiment, indicator is intestinal bacteria CVCC C83707.With the supernatant liquor without catalase processing in contrast.
Result is as follows: the supernatant liquor of processing and processing without catalase through catalase all has restraining effect to indicator, and the diameter of inhibition zone is respectively 18.5mm and 18.4mm.Illustrate that enterococcus faecalis E15-CUI CGMCC No.6130 supernatant liquor is not the effect of Hydrogen Peroxide Metabolism product to the restraining effect of indicator.
(2) discharge of acid bacteriostatic action
According to the method fermentation of 1 in embodiment 1 (3), centrifugal, collection supernatant liquor, with 6mol/L NaOH, regulate fermentation using bacteria supernatant liquor to pH7.0 effect 30min enterococcus faecalis E15-CUI CGMCC No.6130.
By processing product, according to the method for 1 in embodiment 1 (3), carry out bacteriostatic experiment, indicator is streptococcus aureus CVCC2086 and intestinal bacteria CVCC C83707, take that not adjust lactic acid, the hydrochloric acid that pH fermented supernatant fluid and pH are 4.5 be contrast.
The results are shown in Table 4, as can be seen from Table 4, the fermented supernatant fluid of E15-CUI CGMCC No.6130 still has stronger anti-microbial activity to 2 strain indicators after discharging acid interference.
Table 4 is the bacteriostatic action after acid is got rid of
(3) proteasome degradation experimental verification bacteriocin-producing lactic acid bacteria
By enterococcus faecalis E15-CUI CGMCC No.6130 according to the method fermentation of 1 in embodiment 1 (3), centrifugal, collect supernatant liquor; Adding respectively Proteinase K, trypsinase, papoid, lipase and amylase (Sigma) final concentration is 5mg/mL, in 37 ℃ of water-baths, after incubation 2h, get 100ul, supernatant liquor after different enzymes are processed is carried out to bacteriostatic experiment (indicator is intestinal bacteria CVCC C83707) according to the method for 1 in embodiment 1 (3), with the supernatant liquor without any enzyme processing in contrast.
Result: the inhibition zone size of the supernatant liquor of processing without any enzyme is 20mm;
The inhibition zone size of the bacterium supernatant liquor after Proteinase K, trypsinase and papoid are processed is respectively 6.5mm, 6.8mm, 8.0mm;
The inhibition zone size of the bacterium supernatant liquor after lipase and amylase processing is respectively 20mm and 19.9mm;
Can find out, the bacteriostatic activity of the bacterium supernatant liquor after Proteinase K, trypsinase and papoid are processed obviously reduces, and lipase and amylase are processed unchanged, can tentatively judge that fermentation using bacteria has produced a kind of bacteriocin material with property of protein.
(4) germicidal action of transmission electron microscope observing to intestinal bacteria CVCC C83707
By enterococcus faecalis E15-CUI CGMCC No.6130 according to the method fermentation of 1 in embodiment 1 (3), centrifugal, collect supernatant liquor 5ml;
By 37 ℃ of intestinal bacteria CVCC C83707 that cultivate 3h, the centrifugal 10min of 9100 * g, collects thalline; The 5ml supernatant liquor of enterococcus faecalis E15-CUI CGMCC No.6130 is added in above-mentioned intestinal bacteria CVCC C83707 thalline and acts on 0.5-1h, and 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 results are shown in Figure 3, wherein, A:E15-CUI fermented liquid is to intestinal bacteria effect 1h(8000X); B:E15-CUI fermented liquid is to intestinal bacteria effect 0.5 hour (20000X); C:PBS control group effect 1h(8000X); D:PBS control group effect 0.5h(20000X)
Can find out, process PBS after 0.5h and 1h and hatch that intestinal bacteria CVCC C83707 form homogeneous, structure are full, neat in edge; 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 lifetime dependency.When processing 30min, intestinal bacteria CVCCC83707 structure substantially complete but start fuzzy, form is uneven, edge shrinkage to some extent; After processing 1h, under Electronic Speculum, can't see the intestinal bacteria CVCC C83707 of structural integrity, 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 it is mainly to make bacterium shrinkage on the impact of target bacteria, and cellularstructure is destroyed and kytoplasm leaks and death.
Claims (6)
1. enterococcus faecalis (Enterococcus faecalis) E15-CUI, its preserving number is CGMCC NO.6130.
2. the application of enterococcus faecalis claimed in claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130 in preparing antibacterial and/or sterilised products;
Described antibacterial for suppressing at least one in colon bacillus, 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 enterococcus faecalis;
Described sterilization is for killing colon bacillus.
3. an antibacterial product, its activeconstituents is enterococcus faecalis claimed in claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130; Described antibacterial at least one inhibition in colon bacillus, 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 enterococcus faecalis that be specially.
4. a sterilised products, its activeconstituents is enterococcus faecalis claimed in claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130; Described sterilization is for killing colon bacillus.
5. a method of preparing antibacterial product or sterilised products, comprise the steps: enterococcus faecalis claimed in claim 1 (Enterococcus faecalis) E15-CUI CGMCC NO.6130 to carry out liquid fermenting, collect product, obtain antibacterial product or sterilised products.
6. method according to claim 5, is characterized in that:
The temperature of described liquid fermenting is 37 ℃, and the time of described liquid fermenting is 24h, and the rotating speed of described liquid fermenting is 200r/min, and the substratum of described liquid fermenting is MRS liquid nutrient medium;
Described method also comprises the steps: also specifically to comprise the steps: after described collection product by described product at 4 ℃, the centrifugal 10min of 9100 * g, collects supernatant liquor, obtains antibacterial product;
Described antibacterial for suppressing at least one in colon bacillus, 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 enterococcus faecalis;
Described sterilization is for killing colon bacillus.
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| CN103409450A (en) * | 2013-01-10 | 2013-11-27 | 北京农学院 | Broad-spectrum heat-resistant bacteriocin-producing genetic engineering strain, as well as construction method and application thereof |
| CN103074281A (en) * | 2013-01-21 | 2013-05-01 | 黑龙江八一农垦大学 | Enterococcus faecalis FJL19 and application thereof |
| CN105779346B (en) * | 2016-03-25 | 2019-05-17 | 沈阳农业大学 | A kind of enterococcus faecium and its application of bacteriocinogeny |
| CN108048352B (en) * | 2017-12-22 | 2021-04-20 | 华中农业大学 | Enterococcus faecium XC2 capable of producing antibacterial substances and screening method and application thereof |
| CN109805167A (en) * | 2019-01-10 | 2019-05-28 | 河南爱猪人生物科技股份有限公司 | A kind of environment-friendly high-efficiency liquid feed additive |
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