CN115786197B - Lactobacillus paracasei and preparation method and application of bacteriocin thereof, antibacterial peptide and identification method and application thereof - Google Patents
Lactobacillus paracasei and preparation method and application of bacteriocin thereof, antibacterial peptide and identification method and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The lactobacillus paracasei disclosed by the invention is named as lactobacillus paracasei LP-A1, and is preserved in China general microbiological culture Collection center (CGMCC) in the 9 th month 28 of 2022, and the registration number is CGMCC No.25792. The metabolite of the lactobacillus paracasei is separated and purified to obtain the bacteriocin crude extract LP-A1 and the refined extract LP-A1 S.LP-A1C, which have good broad-spectrum antibacterial capacity, antioxidation capacity, thermal stability, irradiation stability and enzymolysis stability, and can be applied to preparation of broad-spectrum bacteriostats, probiotic adjuvants in the sterilization process and food preservative. The LP-A1 S can be used for antagonizing vibrio parahaemolyticus with high efficiency and is applied to the preparation of vibrio parahaemolyticus antagonistic preparation. The mass spectrum identification of the captured 4 antibacterial peptides shows good antibacterial activity on vibrio parahaemolyticus, and can be applied to preparation of aquaculture disease prevention and control antibiotic substitutes or aquatic food preservative.
Description
Technical Field
The invention belongs to the fields of new microbial resources and biology and medicine, and in particular relates to a preparation method and application of lactobacillus paracasei and bacteriocin thereof, and an antibacterial peptide and an identification method and application thereof.
Background
Vibrio parahaemolyticus is common pathogenic vibrio in aquatic products, can cause explosive death of the aquatic products in a short time, and acute hepatopancreatic necrosis syndrome caused by the vibrio parahaemolyticus can cause halving of the yield of main-yield prawns of prawns in China, vietnam, thailand and the like, and becomes one of important aquatic pathogenic bacteria affecting the global prawn industry. In recent years, commercial aquatic products commonly have vibrio parahaemolyticus pollution, and isolated strains all show a certain degree of drug resistance, and searching for natural antibacterial drugs has become one of key links for reducing drug resistance obtained by aquatic pathogenic bacteria. After the use of antibiotic chemical drugs in aquaculture is forbidden in the 2008 part of countries, the prevention and treatment of probiotics are gradually promoted due to the advantages of environmental friendliness, biosafety and the like. There are relatively few reports of biological characteristics and safety evaluation of lactic acid bacteria, but research results show that part of lactic acid bacteria have drug resistance and transferable drug resistance. The screened lactobacillus strain with high safety, vigorous growth, stable metabolism and good adaptability to the peripheral environment is a key problem in the lactobacillus screening process, and determines the action effect and the application prospect of the lactobacillus and related preparations thereof.
Bacteriocins are usually macromolecular proteins or small peptide substances with good inhibition effect on related pathogenic bacteria, and bacteriocins in lactic acid bacteria metabolites are widely favored by researchers because of high safety, small side effect and long activity time. The existing research results show that the bacteriocin BM173 and the lactobacillus bacteriocin BM1300 can effectively inhibit the growth of escherichia coli and staphylococcus aureus, the enterococcus bacteriocin Durancin GL can effectively inhibit the growth of listeria monocytogenes, at present, more and more research reports of bacteriocin-producing lactobacillus are concentrated on streptococcus lactis and lactobacillus, but reports of the lactobacillus paracasei-derived bacteriocin are relatively less and not comprehensive, and the research on the bacteriocin on the antibacterial mode of vibrio parahaemolyticus is still limited at present, so that the development of new doors which can be applied to the starting strain of lactobacillus for preventing and controlling aquatic pathogenic bacteria and know the antibacterial mode of bacteriocin components in metabolic products of the lactobacillus bacteriocin is favorable for opening the research and development of aquatic medicines.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides A preparation method and application of lactobacillus paracasei (Lactobacillus paracasei) LP-A1 and bacteriocin thereof, antibacterial peptide and identification method and application thereof. The bacteriocin crude extract LP-A1 C of the lactobacillus paracasei has good broad-spectrum antibacterial capacity, oxidation resistance, thermal stability, irradiation stability and enzymolysis stability; the extract LP-A1 S can be used for antagonizing vibrio parahaemolyticus with high efficiency. The captured 4 antibacterial peptides (EVPCGKKPL, KQYVLT, TAKKAYQN, EVPPFKK) show good antibacterial activity on vibrio parahaemolyticus through LC-MS/MS mass spectrum identification of LP-A1 S.
The technical scheme adopted for solving the technical problems is as follows: lactobacillus paracasei, named lactobacillus paracasei (Lactobacillus paracasei) LP-A1, was deposited in the general microbiological center of the chinese microbiological bacterial culture management committee (No. 3 of the west way 1, the korean district beichen, the beijing city) at 9 months 28 of 2022, and the accession number was CGMCC No.25792, and the base sequence of the 16S rDNA thereof was shown in SEQ ID No. 1.
The preparation method of the bacteriocin of the lactobacillus paracasei, wherein the crude extract of the bacteriocin of the lactobacillus paracasei is named as LP-A1 C,LP-A1C, comprises the following steps: taking cell-free fermentation supernatant of lactobacillus paracasei LP-A1 as a crude extract, adding ammonium sulfate solid into the crude extract, magnetically stirring and dissolving until the saturation of ammonium sulfate is 70%, standing overnight at 4 ℃, centrifuging at 8000r/min for 10min to obtain precipitate, and re-dissolving the precipitate with sterile water accounting for 2% of the volume of the crude extract to obtain crude extract LP-A1 C.
Specifically, the preparation method of the crude extract original sample comprises the following steps: preparing seed liquid by taking single colony of lactobacillus paracasei LP-A1, inoculating the seed liquid into MRS liquid culture medium according to the inoculum size of 2% by volume, standing and fermenting for 30h at 37 ℃, and centrifuging for 10min at 8000r/min at the ambient temperature of 4 ℃ to obtain the crude extract original sample.
The preparation method of the bacteriocin of the lactobacillus paracasei, wherein the refined extract of the bacteriocin of the lactobacillus paracasei is named as LP-A1 S,LP-A1S, comprises the following steps of: mixing the refined original sample with n-butanol according to a volume ratio of 3:4 by taking the crude extract LP-A1 C as the refined original sample, magnetically stirring at 25 ℃ for 2 hours, and then evaporating the n-butanol at 55 ℃ in a rotary manner to obtain a solid matter, re-dissolving the solid matter to the volume of the refined original sample by using sterile water, and filtering with a 0.22 mu m filter membrane to obtain filtrate, namely the refined extract LP-A1 S.
The application of the crude extract LP-A1 C of the bacteriocin of the lactobacillus paracasei in a broad-spectrum bacteriostat, a probiotic adjuvant in a sterilization process and a food preservative. In the pH value range of 1-5, LP-A1 C is treated for 30min at 121 ℃, and has good thermal stability for the change of the antibacterial activity of vibrio parahaemolyticus is less than 5.70%. In addition, the LP-A1 C is subjected to irradiation energy treatment of 100-500J for 15 seconds, and the relative antibacterial activity is more than 80%, so that the irradiation stability is good; when the concentration of LP-A1 C is more than or equal to 300 mug/mL, the DPPH free radical clearance is higher than that of ascorbic acid with corresponding concentration, and the antioxidant capacity is good; after being treated by alpha-amylase, beta-amylase, lipase, hyaluronidase, pepsin, trypsin, alpha-chymotrypsin, catalase and protease, the relative antibacterial activity is more than 95%, and the enzymolysis stability is good.
Specifically, the pathogenic bacteria aimed by the broad-spectrum bacteriostat comprise one or more of candida albicans, listeria monocytogenes, staphylococcus aureus, enterobacter sakazakii, escherichia coli, salmonella typhimurium, rhodococcus equi, bacillus cereus, pseudomonas fluorescens, vibrio parahaemolyticus, vibrio harveyi, vibrio alginolyticus and aeromonas hydrophila. It can be seen that LP-A1 C shows good bacteriostatic activity against 13 listed pathogenic bacteria including gram-positive bacteria (Rhodococcus equi, listeria monocytogenes, bacillus cereus, staphylococcus aureus), gram-negative bacteria (Enterobacter sakazakii, escherichia coli, salmonella typhimurium, vibrio alginolyticus, vibrio harveyi, aeromonas hydrophila, vibrio parahaemolyticus, pseudomonas fluorescens) and fungi (Candida albicans).
The application of the refined extract LP-A1 S of the bacteriocin of the lactobacillus paracasei in the antagonistic preparation of vibrio parahaemolyticus. The minimum inhibitory concentration of LP-A1 S on Vibrio parahaemolyticus was 750 μg/mL.
An antibacterial peptide derived from the refined extract LP-A1 S of the bacteriocin of lactobacillus paracasei of claim 4, said antibacterial peptide comprising one or more of the peptide stretches of sequence EVPCGKKPL, KQYVLT, TAKKAYQN and EVPPFKK, respectively. EVPCGKKPL, KQYVLT, TAKKAYQN, EVPPFKK have molecular weights of 969.53Da, 750.43Da, 964.50Da and 843.49Da, respectively, and minimum inhibitory concentrations on Vibrio parahaemolyticus are 1.5mg/mL, 3mg/mL and 1.5mg/mL, respectively.
The identification method of the antibacterial peptide adopts an LC-MS/MS mass spectrum identification method, and comprises the following specific steps: the method comprises the step of using the refined extract LP-A1 S of the bacteriocin of Lactobacillus paracasei as a mass spectrometry sample, and performing pretreatment on the mass spectrometry sample, namely: desalting and concentrating the mass spectrum identification sample by a ZipTip C 18 column, dissolving by 20 mu L of a solution containing 0.1% formic acid and 5% acetonitrile, sufficiently vibrating and vortex, centrifuging for 20min at the ambient temperature of 4 ℃ at 10000r/min, and taking 8 mu L of supernatant for mass spectrum identification; wherein, the mass spectrum identification conditions are as follows: mobile phase A contained 0.1% formic acid, mobile phase B contained 0.1% formic acid and 80% acetonitrile, flow rate 500nL/min; mobile phase parameters are ：0～3min,95～90％A,3％B;3～4.5min,90～72％A,3～8％B;4.5～28min,72～62％A,8～32％B;28～31min,62～0％A,32～44％B;31～40min,99％B.
The antibacterial peptide is singly applied or compounded with other medicines to be applied to an antibacterial substitute for preventing and controlling diseases of aquaculture or an aquatic food preservative.
Compared with the prior art, the invention has the following advantages: the lactobacillus paracasei (Lactobacillus paracasei) LP-A1 disclosed by the invention is A novel lactobacillus paracasei obtained by separating excrement of A healthy infant, A metabolite of the novel lactobacillus paracasei is separated and purified to obtain A bacteriocin crude extract LP-A1 C, and the bacteriocin refined extract LP-A1 S.LP-A1C obtained by further purification has good broad-spectrum antibacterial capacity, antioxidant capacity, heat stability, irradiation stability and enzymolysis stability, and can be applied to preparation of A broad-spectrum antibacterial agent, A probiotic adjuvant in A sterilization process and A food preservative. The LP-A1 S can be used for antagonizing vibrio parahaemolyticus with high efficiency and is applied to the preparation of vibrio parahaemolyticus antagonistic preparation. The captured 4 antibacterial peptides (EVPCGKKPL, KQYVLT, TAKKAYQN, EVPPFKK) are identified by LC-MS/MS mass spectrum of LP-A1 S to show good antibacterial activity on vibrio parahaemolyticus, and can be applied to preparation of aquaculture disease prevention and control antibiotic substitutes or aquatic food preservative.
Drawings
FIG. 1 is a colony morphology of Lactobacillus paracasei LP-A1;
FIG. 2 is a gram-staining microscopic image of Lactobacillus paracasei LP-A1;
FIG. 3 is a thermal stability diagram of LP-A1 C;
FIG. 4 is a graph of the irradiation stability of LP-A1 C;
FIG. 5 is a graph of enzymatic stability of LP-A1 C;
FIG. 6 is a graph of DPPH radical scavenging for LP-A1 C;
FIG. 7 is a secondary mass spectrum of EVPCGKKPL peptide fragment;
FIG. 8 is a secondary mass spectrum of KQYVLT peptide fragment;
FIG. 9 is a secondary mass spectrum of TAKKAYQN peptide fragment;
FIG. 10 is a secondary mass spectrum of EVPPFKK peptide fragment.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
1. Isolation and identification of Lactobacillus paracasei (Lactobacillus paracasei) LP-A1
Isolation of LP-A1 from healthy infant faeces: coating the fecal diluent on an MRS agar plate, performing anaerobic culture at 37 ℃ for 48 hours, and picking single colony with strong typical characteristic of the colony, and performing continuous streak culture until the characteristic of the colony is consistent; the purified single colony was picked up in MRS broth at 37℃and cultured for 24 hours to give the LP-A1 strain, the colony morphology is shown in FIG. 1, and the gram staining microscopic image is shown in FIG. 2. The bacterial strain has positive gram staining, short rod shape, milky white circular colony, regular colony edge, bulges, easy picking and colony diameter of 1.0-1.5 mm.
16S rDNA amplification was performed using the DNA of the LP-A1 strain as a template, forward primer 27f:5'-AGAGTTTGATCCTGGCTC-3', reverse primer 1492r:5'-CTACGGCTACCTTGTTACGA-3'. PCR reaction conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, renaturation at 55℃for 1min, extension at 72℃for 90s,30 cycles; extending at 72℃for 10min. And (3) recovering and sequencing the PCR product, wherein the base sequence of the 16S rDNA is shown as SEQ ID NO. 1.
2. Preparation of crude extract of bacteriocin LP-A1 C of LP-A1 and test of broad-spectrum antibacterial ability
(1) Preparation of LP-A1 C
Taking cell-free fermentation supernatant of lactobacillus paracasei LP-A1 as a crude extract, adding ammonium sulfate solid into the crude extract, magnetically stirring and dissolving until the saturation of ammonium sulfate is 70%, standing overnight at 4 ℃, centrifuging at 8000r/min for 10min to obtain precipitate, and re-dissolving the precipitate with sterile water accounting for 2% of the volume of the crude extract to obtain crude extract LP-A1 C.
The preparation method of the cell-free fermentation supernatant of the lactobacillus paracasei LP-A1 comprises the following steps: preparing seed liquid by taking single colony of lactobacillus paracasei LP-A1, inoculating the seed liquid into MRS liquid culture medium according to the inoculum size of 2% by volume, standing and fermenting for 30h at 37 ℃, and centrifuging for 10min at 8000r/min at the ambient temperature of 4 ℃ to obtain cell-free fermentation supernatant of lactobacillus paracasei LP-A1.
(2) Broad-spectrum bacteriostatic ability test of LP-A1 C
13 Pathogenic bacteria including gram positive bacteria, gram negative bacteria and fungi listed in table 1 are activated and cultured to logarithmic phase, the concentration of bacterial liquid is regulated to be 1 multiplied by 10 7 CFU/mL, and an antibacterial spectrum of LP-A1 C is obtained through an antibacterial test, and is shown in table 1.
Table 1: LP-A1 C antibacterial spectrum
Note that: ++ represents that the diameter of the bacteriostasis ring is 15-20 mm; ++ represents that the diameter of the bacteriostasis ring is 12-15 mm; and the plus represents that the diameter of the inhibition zone is 7.8-12 mm.
As shown in Table 1, LP-A1 C has broad-spectrum antibacterial potential, shows antibacterial activity on 13 pathogenic bacteria including gram-positive bacteria, gram-negative bacteria and fungi, has particularly excellent antibacterial activity on 5 common aquatic pathogenic bacteria (vibrio alginolyticus, vibrio harveyi, aeromonas hydrophila, vibrio parahaemolyticus and pseudomonas fluorescens), has excellent antibacterial potential as a broad-spectrum antibacterial agent, and has the diameter of an antibacterial ring of more than 15 mm.
3. Thermal stability and irradiation stability test of LP-A1 C
(1) Thermal stability test of LP-A1 C
Taking LP-A1 C samples with pH values of 1.0, 2.0, 3.0, 4.0 and 5.0, heating the samples at 60 ℃, 70 ℃,80 ℃, 90 ℃, 100 ℃ and 121 ℃ for 30min respectively, quenching and stabilizing the samples in a refrigerator at 4 ℃ for 1h, taking untreated LP-A1 C as a blank control group, detecting the relative antibacterial activity of each sample through an oxford cup antibacterial test, and discussing and analyzing the thermal stability of the LP-A1 C under different pH values.
(2) Irradiation stability test of LP-A1 C
Equivalent LP-A1 C is taken to flash 15 times under the single pulse energy of 100J, 200J, 300J, 400J and 500J respectively at the flicker frequency of 1HZ, untreated LP-A1 C is taken as a blank control group, the relative antibacterial activity of each sample is detected through an oxford cup antibacterial test, and the irradiation stability of LP-A1 C is discussed and analyzed.
As shown in FIG. 3 and FIG. 4, in the pH value range of 1-5, the relative antibacterial activity of LP-A1 C is more than 80% after treatment at 60-121 ℃; LP-A1 C is treated at 121 ℃ for 30min, has antibacterial activity variation of <5.70% on vibrio parahaemolyticus, has good thermal stability, and can be used as probiotic adjuvant in thermal sterilization process. The LP-A1 C is treated by irradiation energy of 100-500J for 15s, the relative antibacterial activity is more than 80%, and the probiotic adjuvant has good irradiation stability and can be used in the cold sterilization process.
4. Enzymatic stability and antioxidant Capacity test of LP-A1 C
(1) Enzymatic stability test of LP-A1 C
And (3) respectively adding the equal amount of LP-A1 C into a catalase solution, a proteinase K solution, an alpha-chymotrypsin solution, pepsin solution and a trypsin solution with the final concentration of 1mg/mL, carrying out warm bath for 2h at the optimal temperature of each enzyme, carrying out water bath at 100 ℃ for 5min to inactivate the enzyme, taking the LP-A1 C which is not subjected to enzyme treatment as a blank control group, taking the enzyme solution with the same concentration as an interference control group, and detecting the relative antibacterial activity of each sample through an oxford cup antibacterial test to investigate and analyze the enzymolysis stability of the LP-A1 C.
(2) Antioxidant Capacity test of LP-A1 C
LP-A1 C mother liquor with the concentration of 12mg/mL is prepared, and the LP-A1 mother liquor is subjected to gradient dilution to obtain sample dilutions with the concentrations of 6000, 2400, 1200, 600, 300, 150, 75, 15, 7.5, 3, 1 and 0.25 mug/mL respectively. 2mL of each dilution sample solution is taken, 1mL of 0.2mmol/L DPPH-absolute ethanol solution is added, the mixture is uniformly mixed and then subjected to light-shielding reaction for 30min, the absorbance value is measured at an OD 517nm, the DPPH free radical scavenging capacity of each sample is calculated, and the same concentration of ascorbic acid is used as a control to discuss and analyze the antioxidant capacity of LP-A1 C.
The results are shown in fig. 5 and 6, and after the LP-A1 C is treated by alpha-amylase, beta-amylase, lipase, hyaluronidase, pepsin, trypsin, alpha-chymotrypsin, catalase and protease, the relative antibacterial activity is more than 95%, so that the enzymatic hydrolysis stability is good; when the concentration of LP-A1 C is more than 300 mug/mL, the DPPH free radical clearance is higher than that of ascorbic acid with corresponding concentration, and the compound has good antioxidant capacity and application potential as food preservative.
5. Preparation of crude extract of bacteriocin of LP-A1 S and test of antibacterial ability
(1) Preparation of LP-A1 S
Mixing the refined original sample with n-butanol according to a volume ratio of 3:4 by taking the crude extract LP-A1 C as the refined original sample, magnetically stirring at 25 ℃ for 2 hours, and then evaporating the n-butanol at 55 ℃ in a rotary manner to obtain a solid matter, re-dissolving the solid matter to the volume of the refined original sample by using sterile water, and filtering with a 0.22 mu m filter membrane to obtain filtrate, namely the refined extract LP-A1 S.
(2) Comparison of the purification conditions of LP-A1 C with LP-A1 S
The BCA kit is adopted to respectively measure the protein content of the cell-free fermentation supernatant of the lactobacillus paracasei LP-A1 and the protein content of the LP-A1 C、LP-A1S, the protein yield is calculated according to the formula 1, the specific activity is calculated according to the formula 2, and simultaneously, the minimum inhibitory concentration of the LP-A1 C and the LP-A1 S on the vibrio parahaemolyticus is measured by adopting a trace broth double dilution method.
Yield (%) = weight of dry matter before purification/weight of dry matter after purification×100 (formula 1)
Iu= [ Δa/(0.01×t) ]×d/m (formula 2)
Wherein:
IU-specific activity
Change in absorbance during delta A-reaction time
T-reaction time, min
D-extracting the total enzyme solution which is a multiple of the enzyme solution in the reaction system
Mass of m-reactive protein, mg
Table 2: comparison of purification effect
The results are shown in Table 2, and the LP-A1 C is subjected to 28 times of purification by ammonium sulfate precipitation, the specific activity of the protein is 770IU, and the minimum inhibitory concentration is 6mg/mL; through n-butanol extraction, LP-A1s is purified 64 times, the specific activity of protein is 1766IU, the minimum inhibitory concentration is 750 mug/mL, and the high-efficiency antagonistic capability to vibrio parahaemolyticus is presented, so that the method has great potential for preventing and controlling aquatic diseases.
6. Preparation of 4 antibacterial peptides and anti-Vibrio parahaemolyticus Activity
(1) Preparation of 4 antibacterial peptides
The 4 antibacterial peptides were captured by LC-MS/MS mass spectrometry identification from LP-A1 S. Desalting and concentrating the sample by a Ziptip C 18 column, dissolving by 20 mu L of a solution containing 0.1% formic acid and 5% acetonitrile, sufficiently vibrating and vortex, centrifuging for 20min at 10000r/min at the ambient temperature of 4 ℃, and taking 8 mu L of supernatant for mass spectrum identification; wherein, the mass spectrum identification conditions are as follows: mobile phase A contained 0.1% formic acid, mobile phase B contained 0.1% formic acid and 80% acetonitrile, flow rate 500nL/min; the mobile phase parameter is ：0～3min,95～90％A,3％B;3～4.5min,90～72％A,3～8％B;4.5～28min,72～62％A,8～32％B;28～31min,62～0％A,32～44％B;31～40min,99％B., the primary mass spectrum of 350-1550 m/z is obtained through single full scanning, and the secondary mass spectrum data is obtained through step normalization collision energy. The sequence adopts PEAKS software to search a database, and the antibacterial peptide sequence is screened according to the reliability of mass spectrum and the peptide property.
(2) Minimum inhibitory concentration determination of 4 antibacterial peptides
Reversely synthesizing the antibacterial peptide obtained by screening, and measuring the minimum antibacterial concentration by adopting a micro-broth double dilution method.
Table 3: minimum inhibitory concentration of antimicrobial peptides
As shown in Table 3 and FIGS. 7-10, 4 antibacterial peptides showing antibacterial effects on Vibrio parahaemolyticus are screened out, namely EVPCGKKPL (969.53 Da), KQYVLT (750.43 Da), TAKKAYQN (964.50 Da) and EVPPFKK (843.49 Da), and the minimum antibacterial concentrations on Vibrio parahaemolyticus are respectively 1.5mg/mL, 3mg/mL and 1.5mg/mL, so that the method has good potential for being applied to development of novel small molecular peptide antibacterial drugs.
Claims (10)
1. Lactobacillus paracasei, which is named as lactobacillus paracasei (Lactobacillus paracasei) LP-A1, and is preserved in China general microbiological culture Collection center (CGMCC) with the registration number of CGMCC No.25792 at 9 and 28 of 2022.
2. The bacteriocin of the lactobacillus paracasei is characterized in that the preparation method of the crude extract named as LP-A1 C,LP-A1C of the bacteriocin of the lactobacillus paracasei is as follows: taking the cell-free fermentation supernatant of the lactobacillus paracasei LP-A1 as a crude extract, adding ammonium sulfate solid into the crude extract, magnetically stirring and dissolving until the saturation of ammonium sulfate is 70%, standing overnight at 4 ℃, centrifuging at 8000r/min for 10min to obtain a precipitate, and re-dissolving the precipitate with sterile water accounting for 2% of the volume of the crude extract to obtain a crude extract LP-A1 C.
3. The bacteriocin of lactobacillus paracasei according to claim 2, characterized in that the preparation method of the crude extract raw sample is as follows: preparing seed liquid by taking single colony of lactobacillus paracasei LP-A1, inoculating the seed liquid into MRS liquid culture medium according to the inoculum size of 2% by volume, standing and fermenting for 30h at 37 ℃, and centrifuging for 10min at 8000r/min at the ambient temperature of 4 ℃ to obtain the crude extract original sample.
4. The bacteriocin of lactobacillus paracasei according to claim 2, characterized in that said refined extract of bacteriocin of lactobacillus paracasei is designated as LP-A1 S,LP-A1S, prepared by the following method: mixing the refined original sample with n-butanol according to a volume ratio of 3:4 by taking the crude extract LP-A1 C as the refined original sample, magnetically stirring at 25 ℃ for 2 hours, and then evaporating the n-butanol at 55 ℃ in a rotary manner to obtain a solid matter, re-dissolving the solid matter to the volume of the refined original sample by using sterile water, and filtering with a 0.22 mu m filter membrane to obtain filtrate, namely the refined extract LP-A1 S.
5. Use of a crude extract LP-A1 C of the bacteriocin of lactobacillus paracasei as defined in claim 2, for the preparation of a broad-spectrum bacteriostat.
6. The use according to claim 5, wherein the pathogenic bacteria for which the broad-spectrum antibacterial agent is directed are one or more of candida albicans, listeria monocytogenes, staphylococcus aureus, enterobacter sakazakii, escherichia coli, salmonella typhimurium, rhodococcus equi, bacillus cereus, pseudomonas fluorescens, vibrio parahaemolyticus, vibrio harveyi, vibrio alginolyticus and aeromonas hydrophila.
7. Use of the bacterial bacteriocin extract LP-A1 S of lactobacillus paracasei as defined in claim 4, for the preparation of a vibrio parahaemolyticus antagonistic preparation.
8. An antibacterial peptide derived from the bacterial bacteriocin extract LP-A1 S of lactobacillus paracasei of claim 4, wherein the antibacterial peptide is selected from one or more of the peptide fragments having the sequences EVPCGKKPL, KQYVLT, TAKKAYQN and EVPPFKK, respectively.
9. The method for identifying the antibacterial peptide according to claim 8, which is characterized by adopting an LC-MS/MS mass spectrum identification method, comprising the following specific steps: the method comprises the step of using the refined extract LP-A1 S of the bacteriocin of Lactobacillus paracasei as a mass spectrometry sample, and performing pretreatment on the mass spectrometry sample, namely: desalting and concentrating the mass spectrum identification sample by a ZipTip C 18 column, dissolving by 20 mu L of a solution containing 0.1% formic acid and 5% acetonitrile, sufficiently vibrating and vortex, centrifuging for 20min at the ambient temperature of 4 ℃ at 10000r/min, and taking 8 mu L of supernatant for mass spectrum identification; wherein, the mass spectrum identification conditions are as follows: mobile phase A contained 0.1% formic acid, mobile phase B contained 0.1% formic acid and 80% acetonitrile, flow rate 500nL/min; mobile phase parameters are ：0～3min,95～90％A,3％B;3～4.5min,90～72％A,3～8％B;4.5～28min,72～62％A,8～32％B;28～31min,62～0％A,32～44％B;31～40min,99％B.
10. The antibacterial peptide of claim 8 is applied to preparation of an aquaculture disease prevention and control antibiotic substitute or an aquatic food preservative independently or in combination with other medicines.
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| US8470583B1 (en) * | 2010-09-23 | 2013-06-25 | The United States Of America, As Represented By The Secretary Of Agriculture | Lactobacillus strain and bacteriocin |
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| US8470583B1 (en) * | 2010-09-23 | 2013-06-25 | The United States Of America, As Represented By The Secretary Of Agriculture | Lactobacillus strain and bacteriocin |
| KR20200056897A (en) * | 2018-11-15 | 2020-05-25 | 재단법인 발효미생물산업진흥원 | Lactobacillus paracasei SRCM102343 strain having antimicrobial activity and probiotics property and uses thereof |
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