• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
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    • 专利摘要:
    A method for quickly separating and screening lactic acid bacteria with bacteriostasis comprises steps as follows: 1) preparing plates by using a PCA and an MRS agar; 2) enriching the sample with MRS broth medium; 3) bacteria-screening: performing gradient dilution after enrichment and coating on the 5 MRS agar plate, pressing the plate onto the PCA plate for inoculation after colonies grow; then culturing the PCA plate at 30 °C, and pouring a PCA medium containing 106 cfu/g indicator bacteria onto the plate to form a double-layer agar for culturing; 4) observing the inhibition zone; marking a colony with bacteriostasis; and finding out a yellow colony at the corresponding position on the MRS agar plate; 5) picking up the single colony and further purifying it by a plate streaking method. The 10 present invention can quickly and conveniently screen out lactic acid bacteria with bacteriostasis from the sample.
    公开号:NL2024641A
    申请号:NL2024641
    申请日:2020-01-10
    公开日:2020-02-17
    发明作者:Xie Jing；Shen Yong；Wang Jinfeng；Liu Wenru；Mei Jun
    申请人:Univ Shanghai Ocean；
    IPC主号:
    专利说明:

    Method for Quickly Separating and Screening Lactic Acid Bacteria with Bacteriostasis CROSS-REFERENCE TO RELATED APPLICATIONS
    This application claims benefit of Chinese Patent Applcation No.201910336137.7, filed on April 24, 2019 and entitled Method for quickly separating and screening lactic acid bacteria with bacteriostasis , the entire contents of which are incorporated herein by reference.
    FIELD OF TECHNOLOGY
    The present invention relates to the field of microbial technology; in particular, to a method for quickly separating and screening lactic acid bacteria with bacteriostasis.
    BACKGROUND
    In recent years, issues about food safety have become a concern in the world. Aquatic products are popular worldwide due to the rich nutritional value, and further, since the nutrients contribute to the growth and reproduction of spoilage microorganisms and common food-borne pathogens, the safety and nutritional value of seafood decline faster than other foods such as meat during storage and transportation.
    At present, the chemical preservative is one of the main means for extending shelf-life, but a bio-preservation technology that develops and uses more natural preservatives to improve food safety is receiving more and more attention due to the concern of consumer against the chemical additives. The microorganism can produce metabolites with bacteriostasis that act as bio-preservatives to inhibit the growth of spoilage bacteria and pathogenic bacteria, thereby extending food shelf-life and improving food safety. Lactic acid bacteria are generally considered to be safe microorganisms (GRAS). And, in order to extend the shelf-life of aquatic products and improve their quality, selecting microbial populations of lactic acid bacteria present in aquatic products and their processing environments is advantageous because they have adapted to these environments. Therefore, using a convenient and quick method to separate lactic acid bacteria with bacteriostasis is of significance.
    SUMMARY
    One purpose of the present invention is to provide a method for conveniently and quickly separating and screening lactic acid bacteria with bacteriostasis, so as to reduce the workload and quickly screen out lactic acid bacteria with bacteriostasis.
    The purpose of the present invention is achieved by the following technical solutions: a method for quickly separating and screening lactic acid bacteria with bacteriostasis, comprising steps as follows:
    (1) after purchasing an aquatic product from an aquatic product market, transporting the aquatic product with a cryopreservation box at 4°C to the laboratory· within half an hour;
    (2) taking out intestines of the aquatic product 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;
    (3) drawing 1 mL of the stock solution for enrichment culturing with an MRS broth medium for 48-72 h; and diluting the cultured bacterial solution with a sterile physiological saline to a total of 6 dilution gradients of 1 O'3, IO'4, 10-3.. 10'6, IO’7 and IO'8;
    (4) pouring the sterilized plate count agar (PCA) medium into a petri dish of 150 mm, and pouring an MRS agar added with bromocresol purple into a petri dish of 90 mm to prepare an MRS agar plate;
    (5) spreading the diluted bacterial solution obtained in step (3) on the MRS agar plate and culturing for 48- 72 h at 30°C ;
    (6) after colonies grow, pressing the MRS agar plate on the PCA plate for inoculation and culturing for 48-72 h at 30°C.
    (7) after colonies on the PCA plate are formed, pouring a PCA medium containing 106 cfu/ml indicator bacteria on the surface to form a double-layer agar, placing and culturing for 18-24 h at 30°C, to observe whether there is an inhibition zone near the colonies;
    (8) picking a colony in the MRS agar plate, whose position is corresponding to the colony with the inhibition zone in the PCA plate in step (7) and having a yellow color;
    (9) further separating and purifying the colony obtained in step (8) by a plate streaking method, and after performing gram staining and catalase tests for each strain, selecting gram-positive and catalase-negative strains for preservation to obtain potential strains;
    (10) extracting DNA of the potential strains to perform PCR amplification and sequencing on 16S rDNA, and performing analysis of the homology on the potential strains.
    Further, in above step (3) a sterile physiological saline is used to dilute the bacterial solution.
    Further, the preparation of the PCA plate employs a petri dish of 150 mm, and the preparation of the MRS agar plate (added with bromocresol purple) employs a petri dish of 90 mm.
    Further, the amount of bromocresol purple added to the MRS agar is 0.01 %.
    Further, all of the operations are performed in a sterile workstation to maintain a sterile environment.
    The beneficial effects of the present invention are:
    With the method of the present invention, lactic acid bacteria with bacteriostasis may be conveniently and quickly separated and screened, so as to have the advantages of simple operation, reduced workload, improved efficiency and the like. The method may realize rapid screening of lactic acid bacteria with bacteriostasis in a general biochemical laboratory·.
    DETAILED DESCRIPTION OF THE EMBODIMENTS
    The technical solutions of the present invention are further illustrated by the following embodiments, but the scope of the present invention is not limited by any forms of the embodiments.
    After purchasing a lateolabrax from an aquatic product market in Luchaogang market, transporting to the laboratory at a low temperature. After the intestines are thoroughly cut under sterile conditions, adding them into a homogenized bag containing appropriate amount of physiological saline for homogenization, after shaking to be uniform, 1 ml of the stock solution is drawn into an MRS broth medium for culturing for 48-72 h at 30°C; the cultured bacterial solution is diluted with a sterile physiological saline to a total of 6 dilution gradients of ΙΟ'3, ΙΟ'4. ΙΟ'3, 10/ 10'7 and 10/ and coated onto a plate (a petri dish of 90 mm) of MRS agar (containing bromocresol purple) for culturing for 48-72 h at 30°C; after colonies grow on the MRS agar plate , the plate is pressed onto the PCA plate (a petri dish of 150 mm) for inoculation, and cultured at 30°C for 48-72 h; after colonies on the PCA plate are formed, a PCA medium containing 106cfu/ml E. coli is poured on the above PCA plate (the petri dish of 150 mm), and cultured for 18-24 h at 30 °C after solidification; then, observing the inhibition zone, and a colony with bacteriostasis is marked, and a yellow colony is found out at the corresponding position on the MRS agar (containing bromocresol puiplc) plate (the petri dish of 90mm), and lactic acid bacteria strain with bacteriostasis is obtained by a streaking method. After performing gram staining and catalase tests for each strain, gram-positive and catalase-negative strains are selected for preservation; DNA of the screened strains is extracted to perform PCR amplification and sequencing on 16S rDNA, and analysis of the homology on the screened strains is performed.
    In the numbered clauses below, specific embodiments are described:
    1. A method for quickly separating and screening lactic acid bacteria with bacteriostasis, comprising steps as follows:
    (1) after purchasing an aquatic product from an aquatic product market, transporting the aquatic product with a cryopreservation box at 4°C to the laboratory within half an hour;
    (2) taking out intestines of the aquatic product in a sterile environment, cutting up the intestines thoroughly with a pair of sterile surgical scissors and mixing to be unifonn, then adding the mixture into sterile physiological saline of 25 mL and repeating with sufficient shaking uniformly, to prepare a stock solution;
    (3) drawing 1 mL of the stock solution for enrichment culturing with an MRS broth medium for 48-72 h; and diluting the cultured bacterial solution with sterile physiological saline to a total of 6 dilution gradients of 103, 10’4, 10/ 10/ 10 and 10/ (4) pouring the sterilized PCA medium into a petri dish of 150 mm, and pouring an MRS agar added with bromocresol purple into a petri dish of 90 mm to prepare an MRS agar plate;
    (5) spreading the diluted bacterial solution obtained in step (3) on tire MRS agar plate and culturing for 48-72 h at 30°C;
    (6) after colonies grow, pressing the MRS agar plate on the PCA plate for inoculation and culturing for 48-72 h at 30°C;
    (7) after colonies on the PCA plate are formed, pouring a PCA medium containing 104 * 6 cfu/ml indicator bacteria on the surface to form a double-layer agar, and culturing for 18-24 h at 30°C, to observe whether there is an inhibition zone near the colonies;
    (8) picking a colony in the MRS agar plate, whose position is corresponding to tire colony with the inhibition zone in the PCA plate in step (7) and having a yellow color;
    (9) further separating and purifying the colony obtained in step (8) by a plate streaking method, and after performing gram staining and catalase tests for each strain, selecting gram-positive and catalase-negative strains for preservation to obtain potential strains;
    (10) extracting DNA of the potential strains to perform PCR amplification and sequencing on 16S rDNA, and perfbnning analysis of the homology on the potential strains.
    2. The method for quickly separating and screening lactic acid bacteria with bacteriostasis according to clause 1, wherein all operations are perfonned in a sterile workstation to maintain a sterile environment.
    3. The method for quickly separating and screening lactic acid bacteria with bacteriostasis according to clause 1, wherein the amount of bromocresol purple added to the MRS agar is 0.01%.
    4. The method for quickly separating and screening lactic acid bacteria with bacteriostasis according to clause 1, wherein screening the lactic acid bacteria with bacteriostasis from the sample by a convenient and quick way to reduce the workload and improve the efficiency in screening bacteria.
    权利要求:
    Claims (10)
    [1]
    Conclusions
    A method for the rapid separation and screening of lactic acid bacteria with bacteriostasis, comprising the following steps:
    (1) after purchasing an aquatic product from an aquatic products market, transport the aquatic product to the laboratory within half an hour using a cryopreservation box at a temperature of 4 ° C;
    [2]
    (2) Remove the intestines of the aquatic product in a sterile environment, cut the intestines thoroughly with a pair of sterile surgical scissors and mix until evenness, then add the mixture to 25 ml sterile saline and shake repeatedly to obtain a stock solution. to prepare;
    [3]
    (3) drawing 1 ml of the stock solution for enrichment culture with an MRS broth medium for 48-72 hours; diluting the cultured bacterial saline to a total of 6 dilution gradients of 10 ' 2 3 , ΙΟ' 4 , ΙΟ ' 5 , ΙΟ 6 , 10' 7 and 10 '8;
    [4]
    (4) pouring the sterilized PCA medium into a 150 mm petri dish and pouring an MRS agar to which bromocresol purple has been added into a 90 mm petri dish to prepare an MRS agar plate;
    [5]
    (5) spreading the diluted bacterial solution obtained in step (3) on the MRS agar plate and culturing at 30 ° C for 48-72 h;
    [6]
    (6) after colonies grow, press the MRS agar plate onto the PCA plate for inoculation and grow at 30 ° C for 48-72 hours;
    [7]
    (7) after the colonies have been formed on the PCA plate, pour a PCA medium containing 106 cfu / ml indicator bacteria on the surface to form a double layer agar; culture at 30 ° C for 18-24 h to observe if there is an inhibition zone near the colonies;
    [8]
    (8) selecting a colony in the MRS agar plate with added bromocresol purple, the position of which corresponds to the colony with the inhibition zone in the PCA plate in step (7) and which has a yellow color;
    [9]
    (9) further separating and purifying the colony in step (8) by a plate streak method, and after performing gram staining and catalase assays for each strain, selecting gram positive and catalase negative strains for conservation to obtain potential strains;
    [10]
    (10) extracting DNA from the potential strains to perform PCR amplification and sequencing on 16S rDNA and perform homology analysis on the potential strains.
    The method for quickly separating and screening lactic acid bacteria with bacteriostasis according to claim 1, wherein all operations are performed in a sterile workstation to maintain a sterile environment.
    The method for rapidly separating and screening lactic acid bacteria with bacteriostasis according to claim 1, wherein the amount of bromocresol purple added to the MRS agar is 0.01%.
    The method for rapidly separating and screening lactic acid bacteria with bacteriostasis according to claim 1, wherein screening the lactic acid bacteria with bacteriostasis from a sample by an easy and rapid way to reduce the workload and to improve the efficiency of screening bacteria. improve.
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    同族专利:
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    CN110016448A|2019-07-16|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2006119780A2|2005-05-11|2006-11-16|Chr. Hansen A/S|New antibiotic-sensitive lactic acid bacteria strains|
    WO2019027376A2|2017-08-03|2019-02-07|Chiang Mai University|A method for inducing microbial mutagenesis to produce lactic acd3|
    CN110527642A|2019-04-24|2019-12-03|上海海洋大学|A kind of enterococcus faecalis with biological antibiotic effect|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN201910336137.7A|CN110016448A|2019-04-24|2019-04-24|A kind of method of quick separation screening biocidal property lactic acid bacteria|
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