CN108611299B - Lactobacillus plantarum for producing antibacterial peptide and application thereof - Google Patents
Lactobacillus plantarum for producing antibacterial peptide and application thereof Download PDFInfo
- Publication number
- CN108611299B CN108611299B CN201810447104.5A CN201810447104A CN108611299B CN 108611299 B CN108611299 B CN 108611299B CN 201810447104 A CN201810447104 A CN 201810447104A CN 108611299 B CN108611299 B CN 108611299B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus plantarum
- yly
- antibacterial peptide
- bacteria
- food
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 51
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 51
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 51
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 31
- 241000894006 Bacteria Species 0.000 claims abstract description 46
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 7
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
- 235000013305 food Nutrition 0.000 claims description 21
- 230000003385 bacteriostatic effect Effects 0.000 claims description 17
- 230000005764 inhibitory process Effects 0.000 claims description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 7
- 241000192130 Leuconostoc mesenteroides Species 0.000 claims description 4
- 244000063299 Bacillus subtilis Species 0.000 claims description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 241000191967 Staphylococcus aureus Species 0.000 claims description 3
- 125000003003 spiro group Chemical group 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 17
- 241000237858 Gastropoda Species 0.000 abstract description 6
- 238000009920 food preservation Methods 0.000 abstract description 3
- 241000585116 Galba truncatula Species 0.000 abstract description 2
- 230000002378 acidificating effect Effects 0.000 abstract description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 34
- 238000000855 fermentation Methods 0.000 description 17
- 230000004151 fermentation Effects 0.000 description 17
- 239000004310 lactic acid Substances 0.000 description 17
- 235000014655 lactic acid Nutrition 0.000 description 17
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 12
- 230000009471 action Effects 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 241000186660 Lactobacillus Species 0.000 description 11
- 229940039696 lactobacillus Drugs 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 239000002253 acid Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 244000005700 microbiome Species 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 210000001035 gastrointestinal tract Anatomy 0.000 description 8
- 102000016938 Catalase Human genes 0.000 description 7
- 108010053835 Catalase Proteins 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 210000003608 fece Anatomy 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000003794 Gram staining Methods 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 235000019249 food preservative Nutrition 0.000 description 4
- 239000005452 food preservative Substances 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 235000019419 proteases Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000004080 punching Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- RPAJSBKBKSSMLJ-DFWYDOINSA-N (2s)-2-aminopentanedioic acid;hydrochloride Chemical class Cl.OC(=O)[C@@H](N)CCC(O)=O RPAJSBKBKSSMLJ-DFWYDOINSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 231100000956 nontoxicity Toxicity 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000593558 Bullacta Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 244000245214 Mentha canadensis Species 0.000 description 1
- 235000016278 Mentha canadensis Nutrition 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a Lactobacillus plantarum for producing antibacterial peptide, which is Lactobacillus plantarum (Lactobacillus plantarum) YLY-1, is preserved in China general microbiological culture Collection center (CGMCC) at 3-8.2018 with the preservation number of CGMCC NO. 15429. The invention also discloses application of the lactobacillus plantarum YLY-1 for producing the antibacterial peptide in inhibiting drunken snail putrefying bacteria. The lactobacillus plantarum YLY-1 has good tolerance and stability under an acidic condition, and the generated antibacterial peptide has a good antibacterial effect on mud snail putrefying bacteria and has a wide application prospect in food preservation.
Description
Technical Field
The invention relates to a lactobacillus plantarum, in particular to a lactobacillus plantarum for producing antibacterial peptide and application thereof in inhibiting drunken snail putrefying bacteria, and belongs to the technical field of food microorganisms.
Technical Field
Currently, a large number of food preservatives have been used in the food industry to prevent food spoilage and thereby extend the shelf life of food. The artificially synthesized chemical preservatives have a leading position in the food industry in China due to low cost, convenient use and good preservative effect, but the artificially synthesized preservatives can generate negative influence on the taste and flavor of food in use and can be absorbed by the body along with the food to cause certain toxic action on the body. The natural preservatives of animal, plant and microorganism sources attract the attention of numerous scholars due to the characteristics of safety, no toxic or side effect, strong antibacterial property and the like, wherein the microorganism source such as the lactobacillus preservative is wide in the variety of bacteria and the source, is not limited by raw materials and is paid more attention by the scholars. Therefore, the search for a natural food preservative and fresh-keeping agent with microbial sources, which is safe, non-toxic, wide in bacteriostatic spectrum and strong in antibacterial property, has become a focus of the development in the food field.
Lactic acid bacteria are dominant bacteria in normal flora of intestinal tracts of human beings and animals, have a plurality of physiological functions beneficial to the health of human beings and animals, are often applied to fermented foods and provide unexpected flavor and special texture structure for the foods; more importantly, antibacterial substances such as organic acid, hydrogen peroxide, lactein and the like can be generated in the metabolic process, wherein the lactein plays an important role in food preservation and fresh keeping. In view of the probiotics of the lactic acid bacteria, the lactic acid bacteria are the generation bacteria of the natural biological food preservative of the microorganism source with potential development value.
The lactobacillus bacteriostatic peptide is a protein or polypeptide substance which is generated by lactobacillus in the metabolic process and has the function of inhibiting the growth of other microorganisms. It can effectively inhibit the growth of pathogenic bacteria and putrefying bacteria in food. The antibacterial peptide produced by the lactobacillus has the following characteristics, so that the antibacterial peptide becomes a development object of a natural food preservative. (1) The bacteria grow and reproduce quickly, the production cycle is short, and the method is suitable for industrial production; (2) has better stability, is suitable for food processing and is convenient to use; (3) can be degraded by protease in human body, and can not accumulate in vivo, and has no influence on normal flora of intestinal tract; (4) it will not generate drug resistance, and will not induce resistance of microorganisms such as food pathogenic bacteria to therapeutic antibiotics.
At present, many reports are reported on the separation of lactic acid bacteria from dairy products, pickles, intestinal tracts of human beings and animals, excrement, soil and the like, however, the reports on the separation of lactic acid bacteria from aquatic animals such as fish and shellfish are few, and due to the special environment of the animals living in water for a long time, microorganisms in the intestinal tracts necessarily evolve to own unique metabolic pathways and generate a plurality of metabolites which are novel in structure, unique in action and different from terrestrial microorganisms.
Disclosure of Invention
The invention aims to solve the technical problem of providing a lactobacillus plantarum YLY-1 for producing antibacterial peptide, wherein the strain is screened from intestinal tracts of weever, and the antibacterial peptide produced by fermentation can effectively inhibit the growth of drunken snail putrefying bacteria, and has the characteristics of safety, no toxicity, strong antibacterial property and broad antibacterial spectrum.
The invention also aims to solve the technical problem of providing the application of the lactobacillus plantarum YLY-1 for producing the antibacterial peptide in inhibiting drunken snail putrefying bacteria.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention takes lactobacillus separated and screened from weever intestinal tracts collected from the market of Hakka food center in Nanjing as a hyperthyroidism object, and screens out the lactobacillus which can inhibit the activities of food-borne spoilage bacteria and pathogenic bacteria and can produce antibacterial peptide.
The strain is separated from intestinal tracts of weever, is named as Lactobacillus plantarum (Lactobacillus plantarum) YLY-1 after MRS inoculation culture, separation and purification, is classified and named as Lactobacillus plantarum (Lactobacillus plantarum), is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.15429 and the preservation date of 2018, 3 months and 8 days.
The biological properties of the strain are as follows: the Lactobacillus plantarum YLY-1 strain is derived from intestinal tracts of weever, grows well in MRS culture medium under anaerobic conditions at 37 ℃, is round in colony size, 0.8-1.5mm in diameter, white in colony, opaque, smooth and moist in surface, positive in gram stain, and short in cell shape under microscope observation.
The lactobacillus strain for producing the antibacterial peptide is subjected to 16s rDNA sequencing, and the 16s rDNA complete sequence of the lactobacillus plantarum YLY-1 is compared with the 16s rDNA gene sequence of the lactobacillus plantarum registered by NCBI by adopting a BLAST analysis method, wherein the homology is 100 percent, so that the lactobacillus plantarum is obtained.
The screening method of the lactobacillus plantarum comprises the following steps:
step 1, separating and purifying lactic acid bacteria: screening and separating lactobacillus strains from weever feces by using an MRS culture medium, purifying the lactobacillus strains by using a plate streaking method, performing gram staining and catalase experiments on the purified strains, and selecting the strains with gram staining as positive and catalase experiments as negative;
step 2, screening of lactic acid bacteria producing antibacterial peptide: inoculating the lactobacillus strain completely purified in the step 1 into an MRS liquid culture medium, standing and culturing for 24 hours at 37 ℃ to obtain strain fermentation liquor, centrifuging the fermentation liquor for 10 minutes at 4 ℃ and 10000r/min, retaining supernatant, performing an antibacterial experiment by using a puncher perforating method, and removing acid action and hydrogen peroxide action in the experimental process; finally, the antibacterial peptide lactic acid bacteria which still have antibacterial activity after eliminating the acid action and the hydrogen peroxide action are screened out.
In the step 1, screening and separating the lactic acid bacteria strain from the weever feces, coating the diluted sample bacterial suspension on an MRS solid plate added with bromocresol purple by adopting a plate dilution method, culturing for 48h at 37 ℃, and selecting a faint yellow bacterial colony on a culture medium plate to perform four-zone streaking.
The application of the lactobacillus plantarum YLY-1 for producing the antibacterial peptide in inhibiting food spoilage bacteria.
Wherein the food spoilage bacteria are Escherichia coli, Bacillus subtilis and Staphylococcus aureus.
The lactobacillus plantarum YLY-1 producing the antibacterial peptide is applied to inhibiting drunken snail putrefying bacteria.
Wherein the drunkenness putrefying bacteria is leuconostoc mesenteroides.
Has the advantages that: the lactobacillus plantarum YLY-1 has good tolerance and stability under an acidic condition, and the generated antibacterial peptide has a good antibacterial effect on mud snail putrefying bacteria and has a wide application prospect in food preservation.
Drawings
FIG. 1 shows the colony morphology of Lactobacillus plantarum YLY-1 of the present invention on MRS solid medium;
FIG. 2 is a gram stain of Lactobacillus plantarum YLY-1 of the invention, panel I;
FIG. 3 is a gram stain image II of Lactobacillus plantarum YLY-1 of the present invention;
FIG. 4 shows the comparison of the 16s rDNA full sequence of Lactobacillus plantarum YLY-1 of the present invention with the 16s rDNA gene sequence of Lactobacillus plantarum registered at NCBI;
FIG. 5 shows the bacteriostatic effect of Lactobacillus plantarum YLY-1 on bullacta putrefying bacteria.
Detailed Description
The technical solution of the present invention is further explained with reference to the accompanying drawings and specific embodiments.
Example 1: separation and purification of lactic acid bacteria
Taking out the weever feces, weighing 1g of the weever feces, placing the weever feces into a test tube containing 9mL of sterile normal saline, and uniformly mixing the weever feces with a vortex mixer; performing gradient dilution under aseptic conditions, respectively sucking 0.1mL of diluent from sample bacterial suspensions with three dilutions of 10-3, 10-4 and 10-5, respectively coating the diluent on MRS solid plates added with bromocresol purple, culturing for 48h at 37 ℃, selecting faint yellow bacterial colonies on a culture medium plate, performing four-zone streaking, culturing for 24-48 h at 37 ℃, and repeatedly streaking for 2-3 times to obtain purified strains; and performing gram staining and catalase experiments on the purified strains, and selecting the strains with gram staining as positive and catalase experiments as negative.
Example 2: screening of lactic acid bacteria producing antibacterial peptide
(I) preparation of fermentation supernatant
The completely purified strain obtained in example 1 was inoculated into MRS liquid medium, and cultured by standing at 37 ℃ for 24 hours to obtain strain fermentation broth, which was centrifuged at 4 ℃ at 10000r/min for 10 minutes to retain the supernatant.
(II) screening of bacteriostatic lactic acid bacteria
And selecting escherichia coli, bacillus subtilis and staphylococcus aureus aiming at common putrefying bacteria causing food putrefaction and deterioration. And (5) performing an antibacterial experiment by using the fermentation supernatant obtained in the step (I) through a puncher punching method, and screening out strains with antibacterial activity. The specific results of the bacterial strain YLY-1 in the screening process of the antibacterial lactic acid bacteria are shown in Table 1.
TABLE 1 in vitro bacteriostasis test preliminary screening results of Lactobacillus plantarum YLY-1 fermentation supernatant to three indicator bacteria
Elimination of acid inhibition
The inhibition of the fermentation supernatant to the indicator bacteria is probably the result of the action of lactic acid, acetic acid and other organic acids in metabolites, and in order to eliminate the possibility, the pH of the fermentation supernatant of the lactobacillus to be detected is adjusted to 5 by using 1.0mol/L NaOH and 1.0mol/L HCl, and an acid discharge experiment is carried out by using a hole puncher punching method, so that the strains with the inhibition zone after the acid discharge action are remained.
(IV) depletion of Hydrogen peroxide Effect
Taking 2.0mL of strain fermentation liquor which still has the bacteriostatic effect after the acid action is eliminated, adjusting the pH value to 7.0, adding 20mg of catalase, slightly shaking until the catalase is dissolved, placing the mixture at 37 ℃ for heat preservation for 2 hours, adjusting the pH value to be the control pH value determined when the acid action is eliminated, taking the acid-eliminating fermentation liquor which is not treated by the catalase as the control, and carrying out the bacteriostatic experiment by a puncher punching method. If the bacterial strain fermentation liquor still has an antibacterial zone after the action of eliminating acid and hydrogen peroxide, the antibacterial action is the action of the antibacterial peptide generated by the bacterial strain.
(V) protease enzymolysis test
Taking 4 parts of 2.0mL of strain fermentation liquor which still has bacteriostatic activity after acid and hydrogen peroxide are removed, adjusting the pH value to be the optimum pH value (respectively 7.4, 6.0 and 2.0) of the respective actions of trypsin, proteinase K, papain and pepsin, adding each enzyme solution according to the amount of 1.0mg/mL of final concentration, reacting for 4 hours at proper temperature, then adjusting the original pH value, and determining the bacteriostatic activity after enzyme treatment and before treatment.
Experimental results show that the lactobacillus plantarum YLY-1 can produce antibacterial peptide and has good antibacterial effect, and specific results are shown in Table 2.
Table 2 shows the results of in vitro bacteriostasis experiments of three indicator bacteria by lactobacillus plantarum YLY-1 fermentation supernatant
As can be seen from Table 2, the strains are subjected to protease enzymolysis, and the fermented supernatant of the strains subjected to the protease enzymolysis loses the bacteriostatic action on the three indicator bacteria.
Example 3: identification of lactic acid bacteria strain producing antibacterial peptide
Culturing the screened lactic acid bacteria strain YLY-1 for producing the antibacterial peptide to logarithmic growth phase, and extracting the genome DNA of the lactic acid bacteria strain YLY-1. A16 s rDNA fragment of the gene is amplified by using a primer (a forward primer AGAGAGTTTGATCCTGGCTCAG; a reverse primer AAGGAGGTGATCCAGCCGCA) and sent to a biological engineering (Shanghai) company Limited for sequencing, and the total sequence length is 1505bp (see a sequence table, SEQ ID NO, 1). The Lactobacillus plantarum is proved to be Lactobacillus plantarum through sequence alignment and is named as Lactobacillus plantarum (Lactobacillus plantarum) YLY-1.
Example 4: application of lactobacillus plantarum YLY-1 antibacterial peptide to intoxicated snail putrefying bacteria
Preparation of crude product of (I) antibacterial peptide
Inoculating lactobacillus plantarum YLY-1 into MRS liquid culture medium, standing and culturing at 37 deg.C for 24 hr to obtain strain fermentation liquid, centrifuging the fermentation liquid at 4 deg.C and 10000r/min for 10 min, and collecting supernatant. Freezing the fermented supernatant into powder to obtain powdered antibacterial peptide, and storing in a refrigerator at 4 deg.C.
(II) bacteriostatic effect of crude bacteriostatic peptide product on leuconostoc mesenteroides of drunkenness
With 10mL ddH2O-redissolving bacteriostatic peptide powder is used as a bacteriostatic agent, leuconostoc mesenteroides is used as an indicator bacterium, a puncher punching method is used for carrying out bacteriostatic test, and the diameter of a bacteriostatic ring is measured.
Experimental results show that the antibacterial peptide generated by the lactobacillus plantarum YLY-1 has a good inhibition effect on drunkenness putrefying bacteria, and the diameter of an inhibition zone is 17.36 mm. The specific results are shown in FIG. 5.
The antibacterial peptide produced by the Lactobacillus plantarum YLY-1 has good inhibition effect on the growth of gram-positive bacteria and gram-negative bacteria, has good stability, and can be hydrolyzed by various proteases. The lactobacillus plantarum YLY-1 fermentation supernatant can be frozen and dried to obtain a bacteriostatic peptide crude product; the antibacterial peptide generated by the lactobacillus plantarum YLY-1 has a good antibacterial effect, a wide antibacterial spectrum, safety and no toxicity, and can be widely used for preserving and refreshing food, so that the shelf life of the food is prolonged.
The preservation name of the lactobacillus plantarum strain of the invention: YLY-1;
the preservation number is as follows: CGMCC NO. 15429;
the preservation date is as follows: 2018-3-8;
the storage place: china general microbiological culture Collection center;
address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
Sequence listing
<110> university of Nanjing university
<120> lactobacillus plantarum for producing antibacterial peptide and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1505
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
aggcttagct ccttgttcga cttcacccta atcatctgtc ccaccttagg cggctggttc 60
ctaaaaggtt accccaccga ctttgggtgt tacaaactct catggtgtga cgggcggtgt 120
gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc cgcgattact agcgattccg 180
acttcatgta ggcgagttgc agcctacaat ccgaactgag aatggcttta agagattagc 240
ttactctcgc gagttcgcaa ctcgttgtac catccattgt agcacgtgtg tagcccaggt 300
cataaggggc atgatgattt gacgtcatcc ccaccttcct ccggtttgtc accggcagtc 360
tcaccagagt gcccaactta atgctggcaa ctgataataa gggttgcgct cgttgcggga 420
cttaacccaa catctcacga cacgagctga cgacaaccat gcaccacctg tatccatgtc 480
cccgaaggga acgtctaatc tcttagattt gcatagtatg tcaagacctg gtaaggttct 540
tcgcgtagct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc gtcaattcct 600
ttgagtttca gccttgcggc cgtactcccc aggcggaatg cttaatgcgt tagctgcagc 660
actgaagggc ggaaaccctc caacacttag cattcatcgt ttacggtatg gactaccagg 720
gtatctaatc ctgtttgcta cccatacttt cgagcctcag cgtcagttac agaccagaca 780
gccgccttcg ccactggtgt tcttccatat atctacgcat ttcaccgcta cacatggagt 840
tccactgtcc tcttctgcac tcaagtttcc cagtttccga tgcacttctt cggttgagcc 900
gaaggctttc acatcagact taaaaaaccg cctgcgctcg ctttacgccc aataaatccg 960
gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc cgtggctttc 1020
tggttaaata ccgtcaatac ctgaacagtt actctcagat atgttcttct ttaacaacag 1080
agttttacga gccgaaaccc ttcttcactc acgcggcgtt gctccatcag actttcgtcc 1140
attgtggaag attccctact gctgcctccc gtaggagttt gggccgtgtc tcagtcccaa 1200
tgtggccgat taccctctca ggtcggctac gtatcattgc catggtgagc cgttacccca 1260
ccatctagct aatacgccgc gggaccatcc aaaagtgata gccgaagcca tctttcaagc 1320
tcggaccatg cggtccaagt tgttatgcgg tattagcatc tgtttccagg tgttatcccc 1380
cgcttctggg caggtttccc acgtgttact caccagttcg ccactcactc aaatgtaaat 1440
catgatgcaa gcaccaatca ataccagagt tcgttcgact gcatgtatag cacccgccat 1500
gccaa 1505
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaggaggtga tccagccgca 20
Claims (3)
1. A lactobacillus plantarum for producing antibacterial peptide is characterized in that: the strain is lactobacillus plantarum (A)Lactobacillus plantarum) YLY-1, which is preserved in China general microbiological culture Collection center (CGMCC) at 3 and 8 months in 2018, and the preservation number is CGMCC NO. 15429.
2. Use of a lactobacillus plantarum producing a bacteriostatic peptide according to claim 1 for the inhibition of food spoilage bacteria in a food product, characterized by: the food spoilage bacteria are Escherichia coli, Bacillus subtilis and Staphylococcus aureus.
3. Use of a lactobacillus plantarum producing a bacteriostatic peptide according to claim 1 for inhibiting intoxicated spiro spoilage bacteria in food products, characterized by: the drunkenness putrefying bacteria is leuconostoc mesenteroides.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810447104.5A CN108611299B (en) | 2018-05-10 | 2018-05-10 | Lactobacillus plantarum for producing antibacterial peptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810447104.5A CN108611299B (en) | 2018-05-10 | 2018-05-10 | Lactobacillus plantarum for producing antibacterial peptide and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108611299A CN108611299A (en) | 2018-10-02 |
| CN108611299B true CN108611299B (en) | 2022-03-08 |
Family
ID=63662959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810447104.5A Active CN108611299B (en) | 2018-05-10 | 2018-05-10 | Lactobacillus plantarum for producing antibacterial peptide and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108611299B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055188A (en) * | 2019-03-07 | 2019-07-26 | 南京师范大学 | One plant of Paenibacillus polymyxa XW4 for producing bacteriostatic peptide and its separation screening and application |
| CN109971687B (en) * | 2019-05-05 | 2020-12-08 | 西南大学 | Lactobacillus sucicola and application thereof |
| CN112120140A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院大连化学物理研究所 | Engraulis japonicus Temminck et Schlegel antibacterial substance, its preparation method and its application in food preservation |
| CN113061550B (en) * | 2021-04-01 | 2023-03-24 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | Lactobacillus new strain Z6 and application thereof in food |
| CN114717150B (en) * | 2022-04-20 | 2023-01-17 | 河北农业大学 | Lactobacillus plantarum CRS33 and application thereof |
| CN116463248B (en) * | 2023-03-14 | 2024-02-23 | 华南农业大学 | Lactic acid bacteria capable of inhibiting growth of water mould and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974467A (en) * | 2010-10-26 | 2011-02-16 | 郑州大学 | Lactobacillus plantarum and application thereof |
| CN101974468A (en) * | 2010-10-26 | 2011-02-16 | 郑州大学 | Lactobacillus plantarum and application thereof |
| CN104232520A (en) * | 2014-08-29 | 2014-12-24 | 浙江工商大学 | Preparation method and application of lactobacillus plantarum and bacteriocin of lactobacillus plantarum |
| KR20150057636A (en) * | 2013-11-20 | 2015-05-28 | 서울대학교산학협력단 | Composition comprising cyclic dipeptides and DL-3-phenyllactic acid for antibacterial, antifungal and antiviral agents |
| CN104789511A (en) * | 2015-05-08 | 2015-07-22 | 内蒙古农业大学 | Lactobacillus plantarum AB-2 with broad-spectrum antifungal properties and application thereof |
| CN104830731A (en) * | 2015-05-08 | 2015-08-12 | 内蒙古农业大学 | Lactobacillus plantarum AB-1 with broad-spectrum bacteriostasis and application thereof |
-
2018
- 2018-05-10 CN CN201810447104.5A patent/CN108611299B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974467A (en) * | 2010-10-26 | 2011-02-16 | 郑州大学 | Lactobacillus plantarum and application thereof |
| CN101974468A (en) * | 2010-10-26 | 2011-02-16 | 郑州大学 | Lactobacillus plantarum and application thereof |
| KR20150057636A (en) * | 2013-11-20 | 2015-05-28 | 서울대학교산학협력단 | Composition comprising cyclic dipeptides and DL-3-phenyllactic acid for antibacterial, antifungal and antiviral agents |
| CN104232520A (en) * | 2014-08-29 | 2014-12-24 | 浙江工商大学 | Preparation method and application of lactobacillus plantarum and bacteriocin of lactobacillus plantarum |
| CN104789511A (en) * | 2015-05-08 | 2015-07-22 | 内蒙古农业大学 | Lactobacillus plantarum AB-2 with broad-spectrum antifungal properties and application thereof |
| CN104830731A (en) * | 2015-05-08 | 2015-08-12 | 内蒙古农业大学 | Lactobacillus plantarum AB-1 with broad-spectrum bacteriostasis and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108611299A (en) | 2018-10-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108611299B (en) | Lactobacillus plantarum for producing antibacterial peptide and application thereof | |
| CN112358999B (en) | Lactobacillus reuteri and application thereof | |
| CN108148789B (en) | Lactobacillus rhamnosus and application thereof in preparation of bacteriocin | |
| CN110643544B (en) | Lactobacillus paracasei761, application thereof, silage additive and silage | |
| CN107981034B (en) | Lactobacillus buchneri HEW-A666 and application thereof | |
| CN114921385B (en) | Bacillus subtilis and application thereof in feed addition and antibiotic-free cultivation | |
| CN106754584A (en) | For the Lactobacillus plantarum SWUN5815 of bacteriocinogeny high and its application | |
| CN105176856A (en) | Screening and culture method of lactobacillus sakei LZ217 and application thereof | |
| CN116836830A (en) | Bacteriocin-producing lactobacillus plantarum and application thereof | |
| CN108432996B (en) | Hericium erinaceus lactobacillus fermented beverage and preparation method thereof | |
| CN111778178B (en) | Application of marine streptomyces griseoflavus HN60 in antibacterial aspect | |
| CN113980853A (en) | Lactococcus garvieae WBT0008 capable of producing lactic acid at high yield and application thereof | |
| CN110452857B (en) | Lactobacillus plantarum for producing non-protein micromolecule antibacterial metabolites and application thereof | |
| CN117551568A (en) | Lactobacillus plantarum TS with phenyllactic acid production capability | |
| CN116064324B (en) | Lactobacillus rhamnosus, culture method thereof and application thereof in preventing and treating diarrhea and enteritis | |
| CN113604387B (en) | Salt-tolerant and high-temperature-resistant lactobacillus reuteri and application thereof in prevention and treatment of pathogenic bacteria in livestock and poultry aquaculture | |
| CN113061550B (en) | Lactobacillus new strain Z6 and application thereof in food | |
| CN112592851B (en) | Lactobacillus acidophilus with broad-spectrum antagonistic effect on aquatic pathogenic bacteria and application thereof | |
| CN111154692B (en) | Lactobacillus plantarum strain capable of degrading nitrite and inhibiting ACE (angiotensin converting enzyme) | |
| CN113215067A (en) | VBNC (viable but non-viable) state lactobacillus brevis CSHRR5-3 strain and application thereof | |
| KR102272419B1 (en) | Pediococcus pentosaceus KCC-45 an composition containing the same | |
| CN114806944A (en) | Lactobacillus plantarum LP11, fermentation liquor thereof, preparation method and application | |
| CN108048341B (en) | Lactic acid bacteria and application thereof | |
| CN117106676B (en) | Bacillus subtilis and application thereof in feed production | |
| Maany et al. | Production of a polypeptide antimicrobial compound by lactic acid bacteria isolated from rhizospheric soil of Egyptian organic farms |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |