• 专利附录
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  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    The invention provides a strain of E. faecalis with biological antibacterial activity, belongs to the fields of a bio-preservation technology and an aquatic product preservation technology. The strain is capable of producing an antibacterial substance and preserved in China General Microbiological Culture Collection Center (CGMCC) on January 14, 2019 with a preservation number of CGMCC No. 17173. This strain has antibacterial activity on S. aureus (gram-positive bacteria) and E. coli (gram-negative bacteria). Therefore, the strain has great application potential and value in prolonging the shelf life of aquatic products and improving food safety.
    公开号:NL2024514A
    申请号:NL2024514
    申请日:2019-12-19
    公开日:2020-01-13
    发明作者:Xie Jing；Shen Yong；Liu Wenru；Mei Jun；Wang Jinfeng
    申请人:Univ Shanghai Ocean；
    IPC主号:
    专利说明:

    © 2024514 © Α OCTROOIAANVRAAG (2 ) Aanvraagnummer: 2024514 (22) Aanvraag ingediend: 19 december 2019 (51) Int. Cl.:
    C12N 1/20 (2020.01) C12Q 1/18 (2020.01) C12R
    1/46 (2020.01) (30) Voorrang:
    april 2019 CN 201910336138.1 © Aanvraag ingeschreven:
    januari 2020 © Aanvraag gepubliceerd:
    januari 2020 © Aanvrager(s):
    Shanghai Ocean University te Pudong District, China, CN (72) Uitvinder(s):
    Jing Xie te Pudong District (CN) Yong Shen te Pudong District (CN) Wenru Liu te Pudong District (CN) Jun Mei te Pudong District (CN) Jinfeng Wang te Pudong District (CN) © Gemachtigde:
    J. Hoekstra te Eindhoven (54) ENTEROCOCCUS FAECALIS WITH BIOLOGICAL ANTIBACTERIAL ACTIVITY (57) The invention provides a strain of E. faecalis with biological antibacterial activity, belongs to the fields of a bio-preservation technology and an aquatic product preservation technology. The strain is capable of producing an antibacterial substance and preserved in China General Microbiological Culture Collection Center (CGMCC) on January 14, 2019 with a preservation number of CGMCC No. 17173. This strain has antibacterial activity on S. aureus (gram-positive bacteria) and E. coli (gram-negative bacteria). Therefore, the strain has great application potential and value in prolonging the shelf life of aquatic products and improving food safety.
    NL A 2024514
    Deze publicatie komt overeen met de oorspronkelijk ingediende stukken.
    ENTEROCOCCUS FAECALIS WITH BIOLOGICAL ANTIBACTERIAL ACTIVITY CROSS-REFERENCE TO RELATED APPLICATIONS
    This application claims benefit of Chinese Patent Application No.201910336138.1. filed on April 24, 2019 and entitled “Enterococcus faecalis with biological antibacterial activity', the contents of which are hereby incorporated by reference in its entirely.
    FIELD OF TECHNOLOGY
    The present invention relates to fields of a bio-preservation technology and an aquatic product preservation technology, in particular, to a strain of Enterococcus faecalis which can generate antibacterial substance.
    BACKGROUND ART
    The consumption of aquatic products is large in China. Due to aquatic products is nutrient-rich, they are easy to cause spoilage during transportation and storage time. Escherichia coli (E. coli) and Staphyloccocus aureus (S. aureus) are common foodborne pathogenic microorganisms.
    At present, the research on preservatives mainly focused on the chemical preservatives such as sodium benzoate, potassium sorbate and so on. Food preservatives could reduce microbial activities to prolong the shelflife of aquatic products. Synthetic preservatives have adverse effect on the flavor and quality of aquatic products, and could be ingested by humans along with aquatic products. The demand for fresh aquatic products is increasing with the improvement of people's living standards, therefore, the demand for fresh and high quality aquatic products are increasing. It is necessary to find a safe, green and efficient preservative.
    Using bacteriocin as preservatives to inhibit the grow th of spoilage bacteria has been increasing interest in the research on natural preservatives. Compared with terrestrial organisms, marine organisms have many different physiological characteristics due to the differences in living environment. Exploring and discovering sources of antibacterial substances w ith unique roles and good effects of marine organisms for applying to the low-temperature storage and transportation of aquatic products have a broad market application prospect.
    SUMMARY
    One purpose of the present invention is to provide a strain of Enterococcus faecalis with antibacterial activity.
    The present invention provides a strain of E. faecalis, which was preserved in China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on January' 14, 2019 with a preservation number of CGMCC No. 17173.
    Further, the biological characteristics of the E. faecalis are: spherical gram-positive, catalasenegative, and the colony morphology is white smooth bulge, shiny, round,.
    Further, a screening method of the said E. faecalis is: taking the intestine of the lateolabrax as a source to screen the bacteria with biological antagonism by studying and comparing the antibacterial effect and the antibacterial spectrum. The specific steps are as follows :
    (1) after purchasing a lateolabrax from an aquatic product market, transporting with a cryopreservation box at 4oC to the laboratory' within half an hour;
    (2) taking out intestines of the lateolabrax in a sterile environment, cutting up the intestines thoroughly with a pair of sterile surgical scissors and mixing to be uniform, then adding the mixture into sterile physiological saline of 25 mL and repeating with sufficient shaking uniformly, to prepare a stock solution; drawing 1 mL of the stock solution for gradient dilution to obtain diluted liquids;
    (3) coating an MRS solid medium containing bromocresol purple with the stock solution obtained in step (2) and the diluted fluids treated with gradient dilution of 10-1, 10-2, 10-3, 10-4, 105, 10-6, and culturing for 48-84h at 30oC, then picking a single yellow colony' with an inoculation needle to obtain suspected strains, performing streaking separation on the MRS plate for the suspected strains, and subculturing for 2 to 4 times until purification; selecting gram-positive and catalasenegative strains for preservation to obtain potential strains after performing gram staining and catalase tests for each strain,;
    (4) after activating, inoculating the activated potential strains in an MRS broth medium at a dose of 1 to 5% for statically' culturing for 24-48 h at 30oC, then centrifuging the fermentation broth at 6000 to 12000 r/min for 10-30 min to obtain a cell-free supernatant, which is then lyophilized and preserved for future use;
    (5) inoculating respectively E. coli and S. aureus in a BHI liquid medium, and culturing for 12 h under a shaker with a temperature of 30oC and a rotating speed of 120 r/min to make E. coli and S. aureus in the logarithmic phase to obtain bacterial solutions of E. coli and S. Aureus, respectively;
    (6) adding the lyophilized cell-free supernatant into sterile ultrapure water for redissolution.
    (7) taking 1 mL of the bacterial solution of E. coli and S. aureus for gradient dilution to a dilution of 10-3 to perform antibacterial test, respectively: coating a PCA plate with 100 pL of the bacterial solution with a dilution of 10-3 uniformly, and placing an Oxford cup with an inner diameter of 6 mm and an outer diameter of 8 mm on the PCA plate under sterile condition, then drawing 200 pL of the redissolved cell-free supernatant into the cup for culturing for 12-24 h at 30oC to observe whether there is an inhibition zone, identify the antibacterial ability, and measure a size of the inhibition zone with a vernier caliper; performing physiological and biochemical experiments on the screened active strains;
    (8) extracting DNA of the active strains to perform PCR amplification and sequencing on 16S rDNA, and analyzing the homology on the active strains.
    Further, in step (3), coating an MRS solid medium containing bromocresol purple with the stock solution obtained in step (2) and the diluted fluids treated with gradient dilution of 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, and culturing for 72h at 30oC. then picking a single yellow colony turned from purple with an inoculation needle and streaking on a plate for separation and sub-culturing for 3 times.
    After activated, the preserved potential strains are inoculated in an MRS broth medium at a dose of 2% for static culturing for 32 h at 30 oC.
    In step (4), the fermentation broth is centrifuged at 8000 r/min for 20 min to collect a cell-free supernatant, which is then lyophilized and preserved.
    The method for measuring inhibition zone: the inhibition zone is measured by the Oxford Cup method. When measuring the inhibition zone by the Oxford Cup method, the redissolved cell-free supernatant is cultured for 14 hours at 30oC.
    The gram staining test and catalase test are performed for the suspected strains, selecting and preserving gram-positive and catalase-negative strains.
    Further, the lyophilized cell-free supernatant is redissolved during the antibacterial test. The concentration after redissolution is 10 times of the concentration before lyophilization.
    Further, the antibacterial test is performed on the suspected strains to select the strains having a significant inhibition effect on S. aureu and E. coli., wherein the size of the inhibition zone of one target strain reaches 26.98 mm and 27.67 mm, respectively.
    Further, after 16S rDNA identification for tire strain, tire target strain has 99% homology with E. faecalis.
    The invention protects, but is not limited to, the antibacterial activity' of this stain against S. aureus and E. coli.
    BRIEF DESCRIPTION OF THE DRAWINGS
    Fig. 1 is a photograph showing the antibacterial test of the E. faecalis supernatant using S. aureus as indicator;
    Fig. 2 is a photograph showing the antibacterial test of the E. faecalis supernatant using E. coli as indicator.
    DETAILED DESCRIPTION OF THE EMBODIMENTS
    To make the present invention easier to understand, the following further describes tire present invention in combination with specific embodiments.
    (1) After purchasing a lateolabrax from an aquatic product market in Luchaogang market, transporting the lateolabrax with a cryopreservation box at 4oC to the laboratory' within half an hour. Taking out intestines of the lateolabrax in a sterile environment, cutting up the intestines thoroughly with a pair of sterile surgical scissors and mixing to be uniform, then adding the mixture into sterile physiological saline of 25 mL and repeating with sufficient shaking uniformly, to prepare a stock solution; drawing 1 mL of the stock solution for gradient dilution to obtain diluted liquids.
    (2) Coating an MRS solid medium containing bromocresol purple with the stock solution mentioned in (1) and tire diluted liquids treated with gradient dilution of 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, and culturing for 72h at 30oC. then picking a single yellow colony with an inoculation needle. Treating the suspected colony by streaking separation on the MRS plate, and subculturing for 3 times until purification. After performing gram staining and catalase tests for each strain, selecting grampositive and catalase-negative strains to obtain suspected strains to obtain potential strains, and numbering and preserving the stains.
    (3) After activating the preserved potential strains, inoculating the activated potential strains in an MRS broth medium at a dose of 2% for statically culturing for 32 h at 30oC, then centrifuging the fermentation broth at 6000 to 8000 r/min for 20 min to obtain a cell-free supernatant, which is then lyophilized and preserved for future use. Redissolving the lyophilized cell-free supernatant to perform the antibacterial test, wherein the concentration after redissolution is 10 times of the concentration before lyophilization. Inoculating respectively E. coli and S. aureus in a BHI liquid medium, and culturing overnight under a shaker with a temperature of 30oC and a rotating speed of 120 r/min until a log phase of growth; taking 1 mL of culture solution respectively for gradient dilution to obtain a diluted culture solution of E. coli and a diluted culture solution of S. aureus. Coating a PC A plate with 100 pL of the diluted culture solution with a dilution of 10-3 uniformly, and placing an Oxford cup with an inner diameter of 6 mm and an outer diameter of 8 mm on the PCA plate under sterile conditions, then drawing 200 pL of the redissolved cell-free supernatant in the cup for culturing for 14 h at 30oC to observe whether there is an inhibition zone, identify the antibacterial ability, measure a size of the inhibition zone with a vernier caliper, and select the strains having a significant inhibition effect, wherein the size of the inhibition zone of one target strain against S. aureu and E. coli. reaches 26.98 and 27.67 mm, respectively.
    (4) Physiological and biochemical experiments are performed on the screened active strain having the best inhibition effects, its colony moiphology is white smooth bulge, shiny, round.
    (5) DNA of the screened strain is extracted to perform PCR amplification and sequencing on 16S rDNA, and analysis of the homology is performed on the active strains, finding that the strain has 99% homology with E. faecalis, so the strain is E. faecalis.
    The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in all the foregoing embodiments, or equivalently replace some of the technical features. Any modifications, equivalent substitutions, improvements, and the like within the spirit and principles of the invention are intended to be included within the scope of the present invention.
    In the numbered clauses below, specific embodiments are described:
    1. A strain of E. faecalis with biological antibacterial activity, wherein tire strain is capable of producing an antibacterial substance, and preserved in China General Microbiological Culture Collection Center (CGMCC) on January' 14, 2019 with a preservation number of CGMCC No. 17173.
    2. A method for screening the E. faecalis with biological antibacterial activity according to clause 1, wherein the strain with biological antagonism is screened from a lateolabrax by studying and comparing the antibacterial effect and the antibacterial spectrum; the method specifically comprises steps as follows:
    (1) after purchasing from an aquatic product market, transporting the lateolabrax with a cryoprcscrvation box at 4°C to the laboratory' within half an hour;
    (2) taking out intestines of the lateolabrax in a sterile environment, cutting up the intestines thoroughly with a pair of sterile surgical scissors and mixing to be uniform, then adding the mixture into sterile physiological saline of 25 mL and repeating with sufficient shaking uniformly, to prepare a stock solution; drawing 1 mL of the stock solution for gradient dilution to obtain diluted liquids;
    (3) coating an MRS solid medium containing bromocresol purple with the stock solution mentioned in step (2) and the diluted liquids treated with gradient dilution of IO’1, IO’2, 10”, 10'4, IO’5, 10*. and culturing for 48-8411 at 30°C, then picking a single y ellow' colony on tire MRS plate with an inoculation needle to obtain suspected strains; performing streaking separation on the MRS plate for the suspected strains, and subculturing for 2 to 4 times until purification; after performing gram staining and catalase tests for each strain, selecting gram-positive and catalase-negative strains for preservation to obtain potential strains;
    (4) after activating, inoculating the activated potential strains in an MRS broth medium at a dose of 1 to 5% for statically culturing for 24-48 h at 30°C, then centrifuging the fermentation broth at 6000 to 12000 r/min for 10-30 min to obtain a cell-free supernatant, w hich is then lyophilized and preserved for future use;
    (5) inoculating respectively E. coll and 5. aureus in a BHI liquid medium, and culturing for 12 hours under a shaker wdth a temperature of 30°C and a rotating speed of 120 r/min to make E. coll and 5. aureus in a log phase of growth, to obtain a bacterial solution of E. coll and a bacterial solution of 5.
    aureus:
    (6) adding the lyophilized cell-free supernatant into sterile ultrapure water for redissolution to perform the antibacterial test, wherein the concentration after redissolution is 10 times of the concentration before lyophilization;
    (7) taking 1 mL of the bacterial solution of E. coll and the bacterial solution of S. aureus respectively for gradient dilution to a dilution of 10”; coating a PCA plate with 100 pL of the diluted bacterial solution with a dilution of 10” uniformly, and placing an Oxford cup with an inner diameter of 6 mm and an outer diameter of 8 mm on the PCA plate under sterile conditions, then drawing 200 pL of the redissolved cell-free supernatant into the cup for culturing for 12-24 h at 30°C to observe whether there is an inhibition zone, identify the antibacterial ability, and measure a size of the inhibition zone with a vernier caliper;
    (8) extracting DNA of the strains to perform PCR amplification and sequencing on 16S rDNA, and perform analysis of the homology on the strains.
    3. The method for screening E. faecalis with biological antibacterial activity according to clause 2, wherein coating an MRS solid medium containing bromocresol purple with the stock solution mentioned in step (2) and the diluted liquids treated with gradient dilution of 10' , 10', 10', 10 , 10”, 10-6, and culturing for 72h at 30°C, then picking a single yellow colony on the MRS plate with an inoculation needle and streaking on a plate for separation, and subculturing for 3 times.
    4. The method for screening E. faecalis with biological antibacterial activity according to clause 2, wherein after activating, inoculating the activated potential strains in an MRS broth medium at a dose of 2%.
    5. The method for screening E. faecalis with biological antibacterial activity according to clause 2, wherein after activating, inoculating the activated potential strains in an MRS broth medium for static culturing for 32 h at 30 °C.
    6. The method for screening E. faecalis with biological antibacterial activity according to clause 2, wherein centrifuging the fermentation broth at 8000 r/min for 20 min.
    7. The method for screening E. faecalis with biological antibacterial activity according to clause 2, wherein when measuring the inhibition zone by the Oxford Cup method, the redissolved cell-free supernatant is cultured for 14 hours at 30°C.
    8. The method for screening E. faecalis with biological antibacterial activity according to clause 2, wherein the obtained suspected strains are subjected to gram staining test and catalase contact test to select gram-positive and catalase-negative strains for preservation.
    9. The method for screening E. faecalis with biological antibacterial activity· according to clause 2, wherein the preserved potential strains are tested for bacteriostasis against E. coll and S. aureus.
    10. The method for screening E. faecalis with biological antibacterial activity according to clause 2, wherein 16S rDNA identification for the strain with the best bacteriostasis is performed, tire strain has 99% homology with E. faecalis.
    权利要求:
    Claims (8)
    [1]
    Conclusions
    A strain of Enterococcus faecalis with biological antibacterial action, wherein the strain is capable of producing an antibacterial substance, and preserved at China General Microbiological Culture Collection Center (CGMCC) on January 14, 2019 with conservation number CGMCC No. 17173.
    A method for screening the Enterococcus faecalis with biological antibacterial action according to claim 1, wherein the strain with biological antagonism is screened from a lateolabrax by studying and comparing the antibacterial effect and the antibacterial spectrum; the method specifically comprising the following steps:
    (1) after purchasing a market for aquatic products, transporting the lateolabrax to the laboratory within half an hour with a cryopreservation box at 4 ° C;
    [2]
    (2) removing the intestines of the lateolabrax in a sterile environment, thoroughly cutting the intestines with a set of sterile surgical scissors and mixing into an even mix and then adding this mix to a sterile 25 ml physiological saline solution and repeat with sufficient even shaking , to prepare a stock solution; extracting 1 ml of the stock solution for gradient dilution to obtain diluted liquids;
    [3]
    (3) coating an MRS solid medium containing bromocresol purple with the stock solution mentioned in step (2) and the diluted liquids treated with gradient dilution of 10 ' 1 , 10' 2 , 10 ' 10' 4 , 10 * ', 10 *, and grow at 30 ° C for 48-84 hours; then picking a single yellow colony on the MRS plate with an inoculation needle to obtain suspected strains; perform smear separation on the MRS plate for the suspect strains, and subculture 2 to 4 times for purification; after performing gram staining and catalase testing for each strain, selecting gram positive and catalase negative strains for preservation to obtain potential strains;
    [4]
    (4) after activation, inoculating the activated potential strains in an MRS breeding medium at a dose of 1 to 5% for static culture for 24-48 hours at 30 ° C; then centrifuging the fermentation broth at 6,000 to 12,000 rpm for 10-30 minutes to obtain a cell-free supernatant, which is then freeze-dried and preserved for future use;
    [5]
    (5) inoculate Escherichia, coli and Staphylococcus aureus, respectively, in a liquid BHI medium and grow for 12 hours under a shaker with a temperature of 30 ° C and a rotation speed of 120 rpm to make Escherichia coli and Staphylococcus aureus in a exponential phase of growth, to obtain a bacterial solution of Escherichia coli and a bacterial solution of Staphylococcus aureus;
    [6]
    (6) adding the freeze-dried cell-free supernatant in sterile ultrapure water for redissolution to perform the bacteriostatic test, the concentration after redissolution being 10 times the concentration before freeze-drying;
    [7]
    (7) taking 1 ml of the bacterial solution of Escherichia coli and the bacterial solution of Staphylococcus aureus respectively for gradient dilution to a 10 ' 3 dilution; coating a PCA plate with 100 µL of the diluted bacterial solution with an even dilution of 10 ' 3 , and placing an Oxford beaker with an inner diameter of 6 mm and an outer diameter of 8 mm on the PCA plate under sterile conditions ; subsequently incorporating 200 μL of the resolved cell-free supernatant into the culture cup for 12-24 hours at 30 ° C to observe if there is an inhibition zone, determine antibacterial capacity and measure a size of the inhibition zone with a caliper;
    [8]
    (8) extracting DNA from the strains to perform PCR amplification and sequencing on 16S rDNA and perform homology analysis on the strains.
    Method for screening of Enterococcus faecalis with biological antibacterial action according to claim 2, comprising coating a solid MRS medium containing bromocresol purple with the stock solution mentioned in step 2 and the diluted liquids treated with gradient dilution of 10 '', 10 ' 2 , ΙΟ' 3 , 10 ' 4 , 10'',10' 6 , and grow for 72 hours at 30 ° C, then pick a single yellow colony on the MRS plate with an inoculation needle and spread on a plate for separation , and 3 times subcultures.
    A method for screening Enterococcus faecalis with biological antibacterial activity according to claim 2, wherein after activation, the activated potential strains are inoculated into an MRS breeding medium at a dose of 2%.
    A method for screening Enterococcus faecalis with biological antibacterial activity according to claim 2, wherein after activation the activated potential strains are inoculated into an MRS breeding medium for static culture for 32 hours at 30 ° C.
    A method for screening Enterococcus faecalis with a biological antibacterial effect according to claim 2, wherein the fermentation broth is centrifuged for 20 minutes at a rotation speed of 8000 rpm.
    The method for screening Enterococcus faecalis with biological antibacterial activity according to claim 2, wherein when the inhibition zone is measured by the Oxford Cup method, the redissolved cell-free supernatant is cultured for 14 hours at 30 ° C.
    A method for screening Enterococcus faecalis with biological antibacterial activity according to claim 2, wherein the suspect strains obtained are subjected to gram staining test and catalase contact test to select gram-positive and catalase-negative strains for preservation.
    The method for screening Enterococcus faecalis with biological antibacterial activity according to claim 2, wherein the preserved potential strains are tested for bacteriostasis against Escherichia coli and Staphylococcus aureus.
    ° A method for screening Enterococcus faecalis with biological antibacterial activity according to claim 2, wherein 16S rDNA identification is performed for the strain with the best bacteriostasis, wherein the strain has 99% homology to Enterococcus faecalis.
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    同族专利:
    公开号 | 公开日
    CN110527642A|2019-12-03|
    NL2024514B1|2020-10-30|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CN110016448A|2019-04-24|2019-07-16|上海海洋大学|A kind of method of quick separation screening biocidal property lactic acid bacteria|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN201910336138.1A|CN110527642A|2019-04-24|2019-04-24|A kind of enterococcus faecalis with biological antibiotic effect|
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