CN115851491A - Lactobacillus plantarum and application thereof - Google Patents
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Abstract
The invention relates to lactobacillus plantarum and application thereof. The invention provides a lactobacillus plantarum, which comprises the following bacteriocins; comprises the plant lactobacillus plantarum with an amino acid sequence shown as SEQ ID No. 4 or an amino acid sequence with at least 85 percent of sequence identity with the plant lactobacillus plantarum; and/or the amino acid sequence is shown in SEQ ID No. 5, or the amino acid sequence has at least 85% sequence identity with the plant lactobacillus.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to lactobacillus plantarum and application thereof.
Background
Lactobacillus plantarum (Lactplantibibacillus plantarum) is an anaerobic or facultative anaerobe of the genus Lactobacillus, the species being straight or bent rod-shaped, usually 0.9-1.2. Mu. M.times.3.0-8.0. Mu.m, single, paired or chained, generally lacking flagella, but capable of movement. Lactobacillus plantarum is a gram-positive bacterium, which does not produce spores. The colony surface is about 3mm in diameter, convex, round, smooth, fine, white, and occasionally light yellow or dark yellow. The lactobacillus chemoheterotrophic bacteria need a nutrient-rich culture medium for growth and can ferment pentose or gluconate, and more than 85% of the final products are lactic acid. Can grow in gluconate and produce CO 2 . Can grow at 15 ℃, and the optimal growth temperature is usually 30-35 ℃.
Lactobacillus plantarum (Lactobacillus plantarum) is a common probiotic in the food fermentation industry, is a member of GRAS (GRAS) microorganisms, and has been widely used in dairy products, meat products, and fruit and vegetable fermented foods. The lactobacillus plantarum producing the bacteriocin not only has probiotic properties, but also bacteriostatic properties.
Bacteriocins (bacteriocins) are proteins or antibacterial peptides synthesized by bacterial ribosomes that form channels in the cell membrane of target bacteria or directly inhibit certain functions of target bacterial cells to exert a bactericidal effect with high efficiency. Bacteriocins inhibit the proliferation of harmful bacterial flora. Bacteriocins can be classified into 4 types by molecular size: class I, having a molecular weight <5kD; class II, having a molecular weight of 5 to 10kD; class III, with a molecular weight >10kD; class IV is a macromolecular protein. Class I contains lanthionine amino acids and beta-methyllanthionine, and contains small-molecule modified peptides of 19-50 amino acid molecules. Class II contains no lanthionine amino acid bacteriocins.
Most of bacteriocins produced by the metabolism of lactobacillus plantarum are of Class I and Class II, have small molecular weight and stable structure, and are easily hydrolyzed by protease. The bacteriocins metabolized by Lactobacillus plantarum are generally Class I. The bacteriocins produced by the metabolism of the existing lactobacillus plantarum generally have narrow antibacterial spectrum and poor stability to enzymes and heat.
Therefore, there is a need to develop novel lactobacillus plantarum to broaden the antibacterial spectrum of the strain and even to improve the stability to enzymes and heat.
Disclosure of Invention
The invention aims to provide novel lactobacillus plantarum and application thereof, and aims to solve the problem that the conventional lactobacillus plantarum is narrow in antibacterial spectrum.
In a first aspect, the present disclosure provides a lactobacillus plantarum comprising the following bacteriocins:
comprises the plant lactobacillus which has an amino acid sequence shown as SEQ ID No. 4 or has at least 85 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 4; and/or
The amino acid sequence is shown as SEQ ID No. 5, or the amino acid sequence has at least 85% sequence identity with the amino acid sequence shown as SEQ ID No. 5.
In the present disclosure, the Lactobacillus plantarum is named Lactobacillus plantarum (Lactplantibibacillus plantarum) FJ6-1. The gene sequence of 16S rRNA of the lactobacillus plantarum FJ6-1 is shown as SEQ ID No. 3.
In some embodiments, the lactobacillus plantarum comprises the bacteriocins: comprises the plant lactobacillin with the amino acid sequence shown in SEQ ID No. 4 or the amino acid sequence with at least 90 percent of sequence identity with the amino acid sequence shown in SEQ ID No. 4.
In some embodiments, the lactobacillus plantarum comprises the following bacteriocins: comprises the plant lactobacillus having at least 95%, at least 98%, at least 99% or 100% sequence identity of the amino acid sequence as shown in SEQ ID No. 4.
In some embodiments, the lactobacillus plantarum comprises the following bacteriocins: comprises the plant lactobacillus with the amino acid sequence having at least 90 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 5.
In some embodiments, the lactobacillus plantarum comprises the bacteriocins: comprising a plant lactobacillus having an amino acid sequence with at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence as shown in SEQ ID No. 5.
In some embodiments, the lactobacillus plantarum comprises the bacteriocins: comprises the plant lactobacillus whose amino acid sequence is shown as SEQ ID No. 4 and/or the plant lactobacillus whose amino acid sequence is shown as SEQ ID No. 5.
In some embodiments, the lactobacillus plantarum comprises the following bacteriocins: comprises the plant lactobacillus whose amino acid sequence is shown as SEQ ID No. 4 and the plant lactobacillus whose amino acid sequence is shown as SEQ ID No. 5.
In some embodiments, the bacteriocin further comprises a plant lactobacillus EF.
In some embodiments, the amino acid sequence of Lactobacillus plantarum E in Lactobacillus plantarum EF is shown in SEQ ID No. 6, and the amino acid sequence of Lactobacillus plantarum F is shown in SEQ ID No. 7.
In some embodiments, the lactobacillus plantarum is deposited at the chinese type culture collection, deposit number CCTCC No. m20221260, 2022, 8/9/month.
In a second aspect, the present disclosure provides a bacteriocin produced by the lactobacillus plantarum provided in the first aspect of the present disclosure, comprising:
the amino acid sequence is shown as SEQ ID No. 4, or the amino acid sequence has at least 85 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 4; and/or
The amino acid sequence is shown as SEQ ID No. 5, or the amino acid sequence has at least 85 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 5.
In some embodiments, the bacteriocin comprises: the amino acid sequence is shown as SEQ ID No. 4, or the amino acid sequence has at least 90 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 4.
In some embodiments, the bacteriocin comprises: phytolactobacillin having an amino acid sequence with at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence as shown in SEQ ID No. 4.
In some embodiments, the bacteriocin comprises: the amino acid sequence is shown as SEQ ID No. 5, or the amino acid sequence has at least 90 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 5.
In some embodiments, the bacteriocin comprises: a plant lactocin having at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as set forth in SEQ ID No. 5.
In some embodiments, the bacteriocin comprises: the amino acid sequence of the plant lactobacillus is shown as SEQ ID No. 4, and/or the amino acid sequence of the plant lactobacillus is shown as SEQ ID No. 5.
In some embodiments, the bacteriocin comprises: the amino acid sequence of the plant lactobacillus is shown as SEQ ID No. 4, and the amino acid sequence of the plant lactobacillus is shown as SEQ ID No. 5.
In some embodiments, the bacteriocin further comprises a plant lactobacillus EF.
In some embodiments, the amino acid sequence of Lactobacillus plantarum E in Lactobacillus plantarum EF is shown in SEQ ID No. 6, and the amino acid sequence of Lactobacillus plantarum F is shown in SEQ ID No. 7.
In a third aspect, the present disclosure provides the use of a lactobacillus plantarum provided in the first aspect of the disclosure or a bacteriocin provided in the second aspect of the disclosure in an antibacterial or preservative.
In some embodiments, the antimicrobial agent or preservative is used to inhibit bacterial pathogens.
In some embodiments, the bacterial pathogenic bacteria comprise at least one of Escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas aeruginosa), salmonella (Salmonella), crohn's disease sakazakii (enterobacter sakazakii), listeria monocytogenes (Listeria monocytogenes), and Staphylococcus aureus (Staphylococcus aureus).
In a fourth aspect, the present disclosure provides a use of lactobacillus plantarum provided in the first aspect of the present disclosure in pickling pickles.
The antibacterial spectrum of the lactobacillus plantarum provided by the invention is wider. And the bacteriocin produced by the metabolism of the lactobacillus plantarum has higher stability to enzyme and heat.
Drawings
FIG. 1 shows a growth profile of Lactobacillus plantarum FJ6-1.
FIG. 2 shows the results of acid resistance assay of Lactobacillus plantarum FJ6-1.
FIG. 3 shows the bacteriostatic results of the supernatant after fermentation of Lactobacillus plantarum FJ6-1. FIG. 3A shows the results of the bacteriostasis of the supernatant on E.coli (ATCC 25922); FIG. 3B shows the results of the bacteriostatic action of the supernatant on Staphylococcus aureus (ATCC 25923).
FIG. 4 shows the results of the bacteriostasis of the bacteriocin extract of Lactobacillus plantarum FJ6-1. FIG. 3A shows the bacteriostatic results of bacteriocin extract against Staphylococcus aureus (ATCC 25923); FIG. 3B shows the results of bacteriostasis of the bacteriocin extract against E.coli (ATCC 25922).
FIG. 5 shows the results of the heat stability of the bacteriocin extract of Lactobacillus plantarum FJ6-1.
FIG. 6 shows the results of the enzyme stability of bacteriocin extracts of Lactobacillus plantarum FJ6-1.
Detailed Description
The present invention is further illustrated by the following examples, which should be construed as being merely illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced therein.
Definition of
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It is noted that the terms used herein should be interpreted as having a meaning that is consistent with the context of this specification and should not be interpreted in an idealized or overly formal sense.
The expression "about" as used herein is as understood by one of ordinary skill in the art and varies within certain limits depending on the context in which it is used. If one of ordinary skill in the art would not understand the use of this term based on the context of its use, then "about" would mean that the particular value is at most plus or minus 10%.
As used herein, "at least 85% sequence identity" to a sequence may include at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as compared to the sequence.
The term "contig" or "contig" as used herein refers to a group of clones which can be linked to each other via overlapping sequences at their ends to form a continuous long fragment of DNA.
The term "bacteriocin" or "bacteriocins" as used herein is a protein or antimicrobial peptide synthesized by bacterial ribosomes to form channels in the cell membrane of a target bacterium or to directly inhibit certain functions of a target bacterium cell to exert a bactericidal effect at a high efficiency. Bacteriocins inhibit the proliferation of harmful bacterial flora. Most of bacteriocins produced by the metabolism of lactobacillus plantarum are of Class I and Class II, have small molecular weight and stable structure, and are easily hydrolyzed by protease. The bacteriocins metabolized by Lactobacillus plantarum are generally Class I. The Class I contains lanthionine amino acid and beta-methyl lanthionine, and contains micromolecule modified peptide with 19-50 amino acid molecules. Class II contains no lanthionine amino acid bacteriocins.
The scheme of the invention will be explained with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. In the following description, descriptions of well-known technologies are omitted so as to avoid unnecessarily obscuring the concepts of the present disclosure. Such techniques are described in a number of publications, such as molecular cloning guidelines (fourth edition), cold spring harbor laboratory science publishers.
The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.
Examples
Example 1 screening and identification of Lactobacillus plantarum FJ6-1
(1) Screening of strains
Weighing a certain amount of pickle samples and liquid soup under aseptic condition, and enriching in aseptic normal saline and MRS liquid culture medium respectively. Adding 1mL of the mixed solution into 9mL of sterilized normal saline/MRS liquid culture medium to prepare 10 -1 Suspending the bacteria, and then gradually diluting. Each draw 100 μ L to spread the dilutions onto MRS solid medium and MRS + calcium carbonate solid medium plates. The bacterial suspension of the sterile physiological saline is selected to have a dilution gradient of 10 0 、10 -1 、10 -2 . The bacterial suspension selection gradient of the MRS liquid culture medium is 10 -5 、10 -6 、10 -7 . And (3) inversely placing the coated plate at the constant temperature of 30 ℃ for culturing for 48h, selecting 20 single colonies which grow well and are full and uniform, and culturing in a fresh MRS liquid culture medium at the constant temperature of 30 ℃ for 2 days. After the culture is finished, 7000r/min,4 ℃,10min centrifugation is carried out to remove the thalli, and fermentation supernatant is obtained. And (4) reserving the fermentation supernatant for determining the antibacterial activity.
The antibacterial activity is verified by adopting an oxford cup agar diffusion method, and escherichia coli (ATCC 25922) and staphylococcus aureus (ATCC 25923) are selected as indicator bacteria. The method comprises the following steps:
pouring 5-7mL of 1.5wt% sterile agar into a sterile plate to cover the bottom of the plate, and after solidification, placing the sterilized Oxford cups on an agar plate at equal intervals by using tweezers. Adding overnight activated indicator bacteria into LB culture medium which is cooled to about 45 ℃ and contains 0.7wt% of agar according to a ten-thousandth ratio, uniformly mixing, pouring 11-14 mL of the indicator bacteria into a sterile agar plate (not pouring the indicator bacteria into the holes of an Oxford cup), carefully clamping the Oxford cup by using sterile forceps after complete solidification, respectively adding 100 mu L of the fermentation supernatant into the small holes on the agar plate, placing the plate at 4 ℃ for 2h to fully diffuse the sample, transferring the plate to 37 ℃ for culture for 24h, observing bacteriostasis, and measuring the diameter of a bacteriostasis zone (see Table 1 for details). The strain FJ6-1 corresponding to the fermentation supernatant with the bacteriostatic activity is frozen and stored at-80 ℃ by a glycerol tube preservation method.
TABLE 1 screening results of bacteriostatic Activity of the strains
Remarking: -no bacteriostatic effect; + indicates that the bacteriostatic effect is slight (the diameter of the bacteriostatic circle is less than 9 mm); the + indicates that the bacteriostatic effect is obvious (9 m < the diameter of the bacteriostatic zone <13 mm); the bacteria inhibiting effect is obvious (the diameter of the bacteria inhibiting circle is larger than 13 mm).
The MRS liquid culture medium comprises the following components in percentage by mass: 1% of peptone, 0.8% of beef extract powder, 0.4% of yeast extract powder, 2% of glucose, 0.2% of dipotassium phosphate (anhydrous), 0.2% of triammonium citrate (anhydrous), 0.5% of sodium acetate (containing trihydrate), 0.22% of magnesium sulfate (containing heptahydrate), 0.05% of manganese sulfate (containing tetrahydrate), 800.1% of tween and distilled water.
The MRS solid culture medium comprises the following components in percentage by mass: 1% of peptone, 0.8% of beef extract powder, 0.4% of yeast extract powder, 2% of glucose, 0.2% of dipotassium phosphate (anhydrous), 0.2% of triammonium citrate (anhydrous), 0.5% of sodium acetate (containing trihydrate), 0.22% of magnesium sulfate (containing heptahydrate), 0.05% of manganese sulfate (containing tetrahydrate), 800.1% of tween, 2% of agar powder and distilled water.
MRS+CaCO 3 The solid culture medium comprises the following components in percentage by mass: peptone 1%, beef extract powder 0.8%, yeast extract powder 0.4%, glucose 2%, dipotassium hydrogen phosphate (anhydrous) 0.2%0.2% of triammonium citrate (anhydrous), 0.5% of sodium acetate (trihydrate), 0.22% of magnesium sulfate (heptahydrate), 0.05% of manganese sulfate (tetrahydrate), 800.1% of tween, 2% of agar powder, 2% of calcium carbonate and distilled water.
The LB liquid culture medium comprises the following components in percentage by mass: tryptone 1%, yeast extract 0.5%, naCl 1%, distilled water.
The LB solid medium comprises the following components in percentage by mass: tryptone 1%, yeast extract 0.5%, naCl 1%, agar powder 0.75%, and distilled water.
The formula of the sterile agar comprises the following components: 1.5 percent of agar powder and distilled water.
(2) Identification of the strains
Inoculating the strain FJ6-1 separated in the step (1) into an MRS liquid culture medium, and culturing for 12h at 30 ℃. Extracting the genome DNA of the strain according to the method of the Ezup column type bacteria genome DNA extraction kit specification. The genome DNA of the strain is taken as a template, 27F (SEQ ID No: 1) and 1492R (SEQ ID No: 2) are taken as primers to carry out 16s rDNA amplification, and the PCR amplification product is subjected to sequencing analysis.
The gene sequence of the obtained 16s rDNA of the strain is shown in SEQ ID No. 3. BLAST alignment analysis in NCBI database showed that Identity =99.51% (> 97%) of this strain to lactobacillus plantarum (lactentibacter plantarum). Thus, the strain is Lactobacillus plantarum
(Lactplantibacillus plantarum) is named as Lactobacillus plantarum FJ6-1 and is preserved in China center for type culture Collection (CCTCC for short, address: eight-path 299 th. In Wuchang district, wuhan city, hubei province) in 2022 years at 8-9 months, with the preservation number of CCTCC No. M20221260.
The Lactobacillus plantarum FJ6-1 is in the shape of a straight or curved rod, the diameter of the colony surface is about 3mm, and the colony surface is convex, round, smooth, fine, white, and occasionally light yellow or dark yellow. Gram-positive bacteria, no spore formation. The strain can not liquefy gelatin, the fermented glucose does not produce gas, and the optimal growth temperature is usually 30-35 ℃.
Example 2 cultivation of Lactobacillus plantarum FJ6-1 species:
(1) Growth curve of Lactobacillus plantarum FJ6-1
The lactobacillus plantarum FJ6-1 is inoculated into a fresh MRS liquid culture medium from a glycerol tube and is activated in a constant temperature incubator at 30 ℃ overnight to serve as a seed bacterium. The seed strain was inoculated into 100mL of a liquid medium at an inoculum size of 0.1%, added to a 96-well plate at an addition rate of 200. Mu.L per well, and cultured in a fully automatic growth apparatus at 30 ℃ for 72 hours. During the culture period, the OD of the bacterial suspension is measured every 1h 600 . The growth curve of Lactobacillus plantarum FJ6-1 is shown in FIG. 1. As can be seen from FIG. 1, lactobacillus plantarum FJ6-1 was in lag phase before 6h, in logarithmic growth phase at 6-16h, and in stationary phase after 16 h.
(2) Acid resistance of Lactobacillus plantarum FJ6-1
The deposited Lactobacillus plantarum FJ6-1 was inoculated from a glycerol tube into fresh MRS broth and activated overnight in a 30 ℃ incubator as a seed bacterium. The seed bacteria were inoculated into 100mL of liquid medium at different pH (pH =2.5, 4.5, 7.5) at an inoculum size of 0.1%. The cells were added to a 96-well plate at an amount of 200. Mu.L per well, and cultured in a fully automatic growth apparatus at 30 ℃ for 12 hours. During the culture period, the OD of the bacterial suspension is measured every 0.5h 600 . The acid resistance curve of Lactobacillus plantarum FJ6-1 is shown in FIG. 2. As can be seen from fig. 2, lactobacillus plantarum FJ6-1 was able to normally reach stationary phase in MRS liquid medium with initial pH =7.5, growth was slightly affected in MRS liquid medium with initial pH =4.5, and hardly grew in MRS liquid medium with initial pH = 2.5. As can be seen, the pH for the growth of Lactobacillus plantarum FJ6-1 is 4.5-7.5.
(3) Preparation of fermentation supernatant of lactobacillus plantarum FJ6-1 and antibacterial activity thereof.
Inoculating the preserved microbial strain FJ6-1 into a fresh MRS liquid culture medium from a glycerol tube, activating overnight in a constant temperature incubator at 30 ℃, then inoculating into the fresh MRS liquid culture medium by 1% of inoculation amount, and fermenting for 48h at 30 ℃ to obtain fermentation liquor. The fermentation liquor is centrifuged at 7000r/min at 4 ℃ for 10min to remove the thallus, and the fermentation supernatant is obtained. The bacteriostatic activity of the fermentation supernatant on staphylococcus aureus and escherichia coli was determined by an oxford cup method, as shown in fig. 3. As can be seen from FIG. 3, the fermentation supernatant has excellent bacteriostatic activity against Staphylococcus aureus and Escherichia coli.
The MRS liquid culture medium comprises the following components in percentage by mass: 1% of peptone, 0.8% of beef extract powder, 0.4% of yeast extract powder, 2% of glucose, 0.2% of dipotassium phosphate (anhydrous), 0.2% of triammonium citrate (anhydrous), 0.5% of sodium acetate (containing trihydrate), 0.22% of magnesium sulfate (containing heptahydrate), 0.05% of manganese sulfate (containing tetrahydrate), 800.1% of tween and distilled water.
EXAMPLE 3 preparation of bacteriocin extract of Lactobacillus plantarum FJ6-1 and antibacterial Activity thereof
(1) Preparation of bacteriocin extract
1L of the fermentation broth obtained in example 2 was centrifuged at 7500r/min at 4 ℃ for 15min to remove the cells, and the supernatant was obtained. Adding ethyl acetate into the supernatant according to the volume ratio of 1. Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were used as indicator bacteria, and the antimicrobial activity thereof was measured by Oxford cup method, as shown in FIG. 4.
(2) Antibacterial activity of crude bacteriocin extract
According to the method, oxford cup plates of different indicator bacteria (shown in Table 2) are respectively prepared, 100 mu L of crude bacteriocin extracts are respectively added into the holes, the mixture is cultured in a constant temperature incubator at 37 ℃ for 12h, and the diameter of the inhibition zone is counted. Do parallel 3 times.
TABLE 2 bacteriostasis profile of crude bacteriocin extract
As can be seen from the bacteriostatic results in Table 2, the Lactobacillus plantarum FJ6-1 of the present invention has a very good bacteriostatic effect on the strains in Table 2. Therefore, the antibacterial pedigree of the lactobacillus plantarum FJ6-1 is wide.
Example 4 stability of bacteriocin extracts
(1) Heat stability of bacteriocin extracts
The bacteriocin extract of Lactobacillus plantarum FJ6-1 was prepared according to the method for preparing the bacteriocin extract of example 3.
Treating the bacteriocin extract with water bath at 80 deg.C for 20min, 40min and 50min. Staphylococcus aureus was used as indicator, and 20. Mu.L of the heat-treated bacteriocin extract was subjected to determination of its bacteriostatic activity by Oxford cup method (see FIG. 5). As shown in FIG. 5, the bacteriostatic activity of the bacteriocin extract was not significantly reduced after the heat treatment at different times.
(2) Enzyme stability of bacteriocin extracts
The enzymes used in this experiment were Apain (papain), trypsin (Trypsin), pepsin (Pepsin) and Proteinase K (Proteinase K). The different enzymes were first dissolved in their respective optimal buffers (see Table 3 for details of buffers) at a concentration of 10mg/mL for each enzyme preparation.
TABLE 3 optimal buffers for different enzymes
A bacteriocin extract of Lactobacillus plantarum FJ6-1 was prepared as in example 3.
30. Mu.L of the bacteriocin extract was taken and the experiment was carried out at a final enzyme concentration of 1 mg/mL. After the enzyme solution was added to a fixed concentration, it was placed in a 37 ℃ water bath for 4h to react sufficiently. After the treatment, the sample was collected. The bacteriostatic activity of the bacteriocin extract was determined using the agar diffusion method using staphylococcus aureus as an indicator (see fig. 6). As shown in fig. 6, the bacteriocin extract still retained good bacteriostatic activity after papain, trypsin and pepsin treatment. After proteinase K treatment, the bacteriostatic activity of the bacteriocin extract was lost. As can be seen from the results, the bacteriocin produced by the Lactobacillus plantarum FJ6-1 of the invention has better tolerance to papain, trypsin and pepsin.
Example 5 identification of bacteriocin extract crude product
(1) Genomic DNA extraction
Taking 2mL of overnight culture liquid of lactobacillus plantarum FJ6-1, extracting the genomic DNA of a sample according to the method of a bacterial genomic DNA rapid extraction kit (B518225, biopsies, china) instruction, and detecting the purity and integrity of the DNA by agarose gel electrophoresis. Quantification was performed by using the Qubit, the DNA concentration was 215 ng/. Mu.L, OD 280 /OD 260 The value was 1.82, which met the sequencing requirements.
(2) Second-generation Illumina platform sequencing
DNA samples that were electrophoretically qualified were randomly fragmented into fragments of approximately 350bp in length using a Covaris sonicator. The DNA fragment after the completion of the treatment was used
Ultra TM The preparation of the whole Library is completed by the steps of end repair, A tail addition, sequencing joint addition, purification, PCR amplification and the like of a DNA Library Prep kit Illumina (NEB, USA) kit. After the library is constructed, firstly, using the Qubit 2.0 to carry out preliminary quantification, diluting the library to 2 ng/mu L, then using Agilent 2100 to detect the insert of the library, and after the insert size meets the expectation, using a Q-PCR method to accurately quantify the effective concentration of the library so as to ensure the quality of the library. Qualified libraries genomic DNA was subjected to Paired-End sequencing (2X 150 bp) by the Illumina Hiseq 4000 sequencing platform.
(3) Third generation Nanopore sequencing
The method comprises the steps of firstly adopting a BluePippin full-automatic nucleic acid fragment recovery system to recover large fragment DNA, then carrying out end repair, selecting a PCR-free method of an EXP-NBD104 kit of Oxford Nanopore Technologies, increasing Barcode, then using an AATI full-automatic capillary electrophoresis instrument to detect the size of the fragment, carrying out equimolar sample mixing on a sample, finally adopting an SQK-LSK109 connection kit of the Oxford Nanopore Technologies, carrying out connector linkage to construct a 10K library, and finally using a Nanopore platform to carry out sequencing.
(4) Genome assembly
The third generation Nanopore data and the second generation data were mixed and assembled using Unicycler software (v0.4.7). Third-generation assembly software: qminism + Racon, second generation assembly SPAdes, polising software Pilot. As a result: after assembly 8 contigs (contigs) were obtained with a total size of 3,556,373bp and a gc content of 44.13%.
(5) Identification of bacteriocins
The assembled genomic sequence of Lactobacillus plantarum FJ6-1 was loaded into the Bagel4 and anti SMASH databases for bacteriocin identification. In addition to the known plant lactobacillin EF (plantaricin EF), two novel bacteriocins were identified, the amino acid sequences of which are not identical to the amino acid sequence of any of the known bacteriocins. They were named as plantaricin Y61 (plantaricin Y61) and plantaricin LB61 (plantaricin LB 61), respectively, and the respective amino acid sequences were as follows:
the amino acid sequence of the plant lactobacillus Y61 is shown in SEQ ID No. 4.
The amino acid sequence of the plant lactobacillus LB61 is shown in SEQ ID No. 5.
The amino acid sequence of the plant lactobacillus E in the plant lactobacillus EF is shown as SEQ ID No. 6; the amino acid sequence of the plant lactobacillus F is shown as SEQ ID No. 7.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.
Claims (10)
1. A Lactobacillus plantarum strain comprising the bacteriocins:
comprises the amino acid sequence shown as SEQ ID No. 4, or the amino acid sequence has at least 85 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 4; and/or
The amino acid sequence is shown as SEQ ID No. 5, or the amino acid sequence has at least 85% sequence identity with the amino acid sequence shown as SEQ ID No. 5.
2. The lactobacillus plantarum of claim 2, wherein the bacteriocin further comprises a lactobacillus plantarum EF;
preferably, the amino acid sequence of the plant lactobacillus E in the plant lactobacillus EF is shown as SEQ ID No. 6, and the amino acid sequence of the plant lactobacillus F is shown as SEQ ID No. 7.
3. The lactobacillus plantarum of claim 1, wherein the gene sequence of 16S rRNA of the lactobacillus plantarum is shown as SEQ ID No: 3.
4. A Lactobacillus plantarum according to any of claims 1-3, characterized in that it was deposited in the China center for type culture Collection on 8/9/2022 with the accession number CCTCC No. M20221260.
5. A bacteriocin produced by the Lactobacillus plantarum of any one of claims 1-4, comprising:
the amino acid sequence is shown as SEQ ID No. 4, or the amino acid sequence has at least 85 percent of sequence identity with the amino acid sequence shown as SEQ ID No. 4; and/or
The amino acid sequence is shown as SEQ ID No. 5, or the amino acid sequence has at least 85% sequence identity with the amino acid sequence shown as SEQ ID No. 5.
6. The bacteriocin according to claim 5, further comprising a plant lactobacillin EF;
preferably, the amino acid sequence of the plant lactobacillus E in the plant lactobacillus EF is shown as SEQ ID No. 6, and the amino acid sequence of the plant lactobacillus F is shown as SEQ ID No. 7.
7. Use of a Lactobacillus plantarum strain according to any one of claims 1-4 or a bacteriocin according to claim 5 or 6 in an antibacterial or antiseptic agent.
8. Use according to claim 7, wherein the antibacterial or preservative is for the inhibition of bacterial pathogens.
9. The use according to claim 8, wherein the bacterial pathogenic bacteria comprise at least one of Escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas aeruginosa), salmonella (Salmonella), enterobacter sakazakii (Enterobacter sakazakii), listeria monocytogenes (Listeria monocytogenes), and Staphylococcus aureus (Staphylococcus aureus).
10. Use of lactobacillus plantarum as defined in any one of claims 1-4 for pickling pickles.
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