CN109628354A - The lactobacillus plantarum of one plant height bacteriostatic activity and application - Google Patents
The lactobacillus plantarum of one plant height bacteriostatic activity and application Download PDFInfo
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- CN109628354A CN109628354A CN201910089490.XA CN201910089490A CN109628354A CN 109628354 A CN109628354 A CN 109628354A CN 201910089490 A CN201910089490 A CN 201910089490A CN 109628354 A CN109628354 A CN 109628354A
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- lactobacillus plantarum
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 48
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 48
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 48
- 230000003385 bacteriostatic effect Effects 0.000 title abstract description 24
- 241000894006 Bacteria Species 0.000 claims abstract description 28
- 238000000855 fermentation Methods 0.000 claims abstract description 20
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 12
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 7
- 241000588724 Escherichia coli Species 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 12
- 244000052616 bacterial pathogen Species 0.000 claims description 9
- 241000196324 Embryophyta Species 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 6
- 241000607142 Salmonella Species 0.000 claims description 5
- 235000013305 food Nutrition 0.000 claims description 5
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 4
- 241000191967 Staphylococcus aureus Species 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 239000006071 cream Substances 0.000 claims description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 231100000350 mutagenesis Toxicity 0.000 abstract description 26
- 238000002703 mutagenesis Methods 0.000 abstract description 26
- 231100000219 mutagenic Toxicity 0.000 abstract description 20
- 230000003505 mutagenic effect Effects 0.000 abstract description 20
- 230000001580 bacterial effect Effects 0.000 abstract description 11
- 230000000843 anti-fungal effect Effects 0.000 abstract description 2
- 229940121375 antifungal agent Drugs 0.000 abstract description 2
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 238000001228 spectrum Methods 0.000 abstract description 2
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 15
- 230000035772 mutation Effects 0.000 description 11
- 231100000225 lethality Toxicity 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 239000000725 suspension Substances 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 108010053775 Nisin Proteins 0.000 description 5
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000004309 nisin Substances 0.000 description 5
- 235000010297 nisin Nutrition 0.000 description 5
- 230000001408 fungistatic effect Effects 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
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- 239000003755 preservative agent Substances 0.000 description 3
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- 238000012545 processing Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
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- 239000006916 nutrient agar Substances 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000010563 solid-state fermentation Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IDCPFAYURAQKDZ-UHFFFAOYSA-N 1-nitroguanidine Chemical compound NC(=N)N[N+]([O-])=O IDCPFAYURAQKDZ-UHFFFAOYSA-N 0.000 description 1
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 229930182843 D-Lactic acid Natural products 0.000 description 1
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 1
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000186605 Lactobacillus paracasei Species 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000896292 Odontothrips loti Species 0.000 description 1
- 241000191998 Pediococcus acidilactici Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 101150038956 cup-4 gene Proteins 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- BZDIAFGKSAYYFC-UHFFFAOYSA-N manganese;hydrate Chemical compound O.[Mn] BZDIAFGKSAYYFC-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- 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
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- C12N1/20—Bacteria; Culture media therefor
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- 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
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- C—CHEMISTRY; METALLURGY
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- 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
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Abstract
The invention discloses the lactobacillus plantarum of a plant height bacteriostatic activity and applications, belong to biological genetic breeding and fermentation engineering field.The present invention provides one plant of mutagenic obtained lactobacillus plantarum DY6-E1 (Lactobacillus plantarum), are preserved in China typical culture collection center on October 31st, 2018, and deposit number is CCTCC M 2018734.The bacterial strain has a broad antifungal spectrum, bacteriostatic activity is high, and mutagenesis technique can significantly improve the antibacterial energy energy of original strain, improves nearly 30%, and mutagenic strain can be applied to the prevention and treatment of harmful bacteria well, improve the quality of feed.
Description
Technical field
The present invention relates to the lactobacillus plantarum of a plant height bacteriostatic activity and applications, belong to biological genetic breeding and Fermentation Engineering
Technical field.
Background technique
Lactobacillus plantarum has inhibiting effect to Gram-positive and gram negative pathogenic bacteria.It by with pathogen to limit
The ability of property nutrient processed competes, and to inhibit the growth of pathogen, adjusts the composition of intestinal microecology, forms biological containment,
And the effects of the substances to other bacteriums such as generated lactic acid, bacteriocin and biacetyl are metabolized by it, it adjusts between flora
Relationship, maintain and guarantee flora optimal vigor combination, by the growth of antagonism pathogenic bacteria and its adherency of toxin, it is prevented to invade
Enter and is colonized.
The main means for extending open date at present are addition food preservative, but a large amount of uses of chemical preservative
More or less side effect can be brought, influence not merely will also result on environment on people.Therefore it researches and develops out efficiently, surely
Fixed, the preservative of natural safety is the inevitable development trend of food service industry.But the natural antiseptic agent of animals and plants is extracted from, by
Raw material production, extraction process, various restrictions such as cost are difficult to carry out the production of scale.And microbe fermentation method is natural
The suitable selection of one of preservative, and the lactic acid bacteria as probiotics can produce a variety of antibacterial materials such as organic acid, there is suppression
The protein or polypeptides matter of bacterium effect.These antibacterial substances can not only inhibit and kill some pathogens and spoilage organisms,
Enteron aisle can also be improved.
Summary of the invention
The present invention goes out one plant of lactobacillus plantarum with broad spectrum antibiotic activity by mutagenesis screening, to a variety of harmful bacterias have compared with
Good inhibiting effect.In addition mutagenesis is carried out to it, filters out the optimal mutant strain of fungistatic effect, improves its control efficiency, from
And it is made to have good application.
The first purpose of the invention is to provide a lactobacillus plantarum (Lactobacillus plantarum) DY6-
E1 is preserved in China typical culture collection center, classification naming Lactobacillus on October 31st, 2018
Plantarum DY6-E1, deposit number are CCTCC No:M 2018734, and preservation address is China, Wuhan, Wuhan University.It should
Bacterium is the lactobacillus plantarum DY6 bacterial strain that screens from dregs of beans as starting strain, and ultraviolet nitrosoguanidine (NTG) is carried out to it
Complex mutation, screen the high to Escherichia coli bacteriostatic activities of acquisition, pass on stable mutagenic fungi.
Second object of the present invention provides the composition for containing the lactobacillus plantarum.
In one embodiment of the invention, the composition contains the lactobacillus plantarum (Lactobacillus
Plantarum) acceptable carrier on DY6-E1 and food.
In one embodiment of the invention, in the composition lactobacillus plantarum concentration >=1 × 108CFU/g。
In one embodiment of the invention, in the composition lactobacillus plantarum concentration >=1 × 108CFU/mL。
In one embodiment of the invention, the composition contains the lactobacillus plantarum (Lactobacillus
Plantarum) DY6-E1 and cell-protecting.
In one embodiment of the invention, the composition also contains the lactobacillus plantarum (Lactobacillus
Plantarum) the metabolin of DY6-E1.
Third object of the present invention is to provide the lactobacillus plantarum DY6-E1 in food or feed antibacterial aspect
Using.
Fourth object of the present invention is to provide a kind of method for inhibiting pathogenic bacteria in fermentation process, and the method is by institute
Lactobacillus plantarum DY6-E1 cell is stated to be added into the fermentation system containing pathogenic bacteria.
In one embodiment of the invention, the pathogenic bacteria include but is not limited to Escherichia coli, it is salmonella, golden yellow
Color staphylococcus.
In one embodiment of the invention, the fermentation system contains dregs of beans.
The utility model has the advantages that 1, lactobacillus plantarum of the invention (Lactobacillus plantarum) DY6-E1 has a broad antifungal spectrum,
Bacteriostatic activity is high, all has good bacteriostatic activity to Escherichia coli, salmonella and staphylococcus aureus；2, of the invention
The bacteriostasis that mutagenesis technique can significantly improve original strain is compared with existing bacteriostatic agent, reaches nisin 570 μ g/mL's
Bacteriostasis property；3, mutagenic strain of the invention can be applied to the prevention and treatment of harmful bacteria well, improve the quality of feed.
Biomaterial preservation
One lactobacillus plantarum (Lactobacillus plantarum) DY6-E1, in preservation on October 31 in 2018
In China typical culture collection center, classification naming is Lactobacillus plantarum DY6-E1, and deposit number is
CCTCC No:M 2018734, preservation address are China, Wuhan, Wuhan University.
Detailed description of the invention
Fig. 1 is ultraviolet mutagenesis lethality and positive mutation rate curve；
Fig. 2 is NTG mutagenesis lethality and direct mutation curve；
Fig. 3 is mutant strain genetic stability result.
Specific embodiment
Embodiment 1: the determination of starting strain
1 culture medium: MRS culture medium (g/L): peptone 10.0, beef extract 8.0, yeast powder 4.0, glucose 20.0, phosphoric acid
Hydrogen dipotassium 2.0, Triammonium citrate 2.0, sodium acetate 5.0, epsom salt 0.58, four water manganese sulfates 0.25, Tween 80 1ml steam
Distilled water 1L, 115 DEG C sterilize 20 minutes
The preparation of 2 bacterial strain seed liquors: laboratory preservation of bacteria strain is inoculated into MRS culture medium, 37 DEG C of work according to 1% inoculum concentration
Change for 24 hours, activates 2-3 generation in the same way, continue in MRS culture medium and cultivate, obtain the bacterium solution of lactic acid bacteria.
The preparation of 3 indicator bacteria bacterium solutions: three kinds of Escherichia coli, salmonella and Staphylococcus aureus indicator bacterias are inoculated in LB
Fluid nutrient medium, 37 DEG C of cultures are for 24 hours.
4 use Odontothrips loti: taking the plate of diameter about 90mm, pour into the nutrient agar 18- of heating and melting respectively
20mL makes it uniformly spread out cloth in plate, and place makes to solidify on level table, as bottom.Semisolid nutrient agar rouge is separately taken to train
After supporting base (agar content 1%) appropriate heating and melting, let cool to 48-50 DEG C, indicator bacteria bacterium is added in every 50-100mL culture medium
(dense bacterium is 10 to suspension 0.1-0.2mL8CFU/mL), it is separately added into 5mL in every 1 plate, it is made uniformly to spread out cloth on bottom,
As bacterium layer.It is spare with equidistant uniform placement Oxford cup 4 in every 1 plate, divide in the Oxford cup in each double-layer plate
It Di not fill 200 μ L lactic acid bacteria supernatants, after 37 DEG C of culture 18h, measure each antibacterial circle diameter (or area), the results are shown in Table 1,
It was found that lactobacillus plantarum DY6 (CCTCC 2017138) shows extremely strong bacteriostatic activity, it is selected as starting strain.
1 lactic acid bacteria bacteriostatic activity comparison result of table
Note: lactobacillus paracasei (CCTCC M 2017303) is disclosed in the patent application of Publication No. 107227280A；
Lactobacillus rhamnosus (CCTCC M 2017279) is disclosed in the patent application of Publication No. 107217022A；Pediococcus acidilactici
(CCTCC M 2017280) is disclosed in the patent application of Publication No. CN107227279A；Lactobacillus plantarum (CCTCC M
2017138) it is disclosed in the patent application of Publication No. CN107446852A.
Embodiment 2: ultraviolet mutagenesis condition determines
1, the preparation of bacteria suspension: lactobacillus plantarum DY6 is inoculated in MRS fluid nutrient medium with 2% inoculum concentration, 30 DEG C
Culture to logarithmic phase (12 ± 4) h, 6000rpm 10 minutes collection thallus of centrifugation is washed twice with the phosphate buffer of 1mol/L,
Thallus is suspended in phosphate buffer, is diluted to 107~108CFU/mL bacteria suspension, it is spare.
2, ultraviolet mutagenesis condition determines: the bacteria suspension for taking 10ml to dilute is put in the culture dish of 90mm, under dark condition
It is placed under 15W ultraviolet lamp at 15cm, irradiates 0,20,40,60,80,100,120s respectively under the action of magnetic stirrer, point
The bacterium solution of different irradiation times is not taken, carry out gradient dilution appropriate and carries out plate count, calculates the lethal of ultraviolet mutagenesis
Rate, every group is done three in parallel.Best mutation time is chosen, as a result such as Fig. 1
Lethality (%)=(viable count after control one mutagenesis of viable count)/control viable count × 100%
As shown in Figure 1, ultraviolet light improves the lethality of lactobacillus plantarum DY6 with the increase of irradiation time, works as photograph
Penetrate time lethality 88.11% when being 40s, upon irradiation between when being greater than 60s, the lethality of lactobacillus plantarum DY6 is close
100%, in addition irradiation time be 40s when positive mutation rate highest, so irradiation time 40s be best mutation time.
To strain mutagenesis 40s under the conditions of above-mentioned ultraviolet mutagenesis, the results are shown in Table 2, obtains bacteriostatic activity respectively and improves
10.14~17.10% mutagenic strain.
2 ultraviolet mutagenesis strain mutagenesis situation of table
| Bacterial strain | Bacteriostatic diameter (mm) | Bacteriostatic activity increase rate (%) |
| T1 | 20.28 | 17.10 |
| L3 | 19.16 | 10.63 |
| L18 | 19.07 | 10.14 |
| L20 | 19.19 | 10.78 |
| L21 | 19.90 | 14.91 |
| Z9 | 19.46 | 12.37 |
Embodiment 3:NTG mutagenic condition determines
1, the preparation of bacteria suspension: lactobacillus plantarum DY6 is inoculated in MRS fluid nutrient medium with 2% inoculum concentration, 30 DEG C
Culture to logarithmic phase (12 ± 4) h, 6000rpm 10 minutes collection thallus of centrifugation is washed twice with the phosphate buffer of 1mol/L,
Thallus is suspended in phosphate buffer, is diluted to 107~108CFU/mL bacteria suspension, it is spare.
2, NTG mutagenic condition determines: the nitrosoguanidine that 20mg is weighed in draught cupboard is dissolved in the acetone of 1ml, is made into
Then the nitroso guanidine solution of 20g/L is configured to the NTG solution of 2g/L with the phosphate buffer of 1mol/L (pH 7.0), draw
1ml bacteria suspension, is added suitable NTG solution and phosphate buffer makes the suspension of 0,0.2,0.4,0.6,0.8,1g/L respectively
Liquid, by mixed suspension, as 37 DEG C, the shaking table concussion of 200rpm processing 40 minutes, 6000rpm is centrifuged 10 minutes and abandons
Remove supernatant.It is cleaned thallus 2 times with phosphate buffer, isometric buffer is added and suspends, be coated with, apply after appropriate dilution
It is distributed on plate, calculates the lethality of nitrosoguanidine, every group is done three in parallel.Choose best mutagenesis concentration.As a result such as Fig. 2.
The lethality of lactobacillus plantarum DY6 increases as the concentration of NTG increases as shown in Figure 2, when the concentration of NTG is
Lethality has reached 80% when 0.4g/L, and when concentration is greater than 0.6g/L, in addition lethality is in mutagenesis concentration close to 100%
When 0.4g/L, positive mutation rate highest, so the best mutagenesis concentration of NTG is 0.4g/L.
Mutagenesis is carried out to bacterial strain using the NTG of 0.4g/L under the above conditions, the results are shown in Table 3, obtains respectively antibacterial
Activity improves 9.63~15.5% mutagenic strain.
Table 3NTG mutagenic strain mutagenesis situation
| Bacterial strain | Bacteriostatic diameter (mm) | Bacteriostatic activity increase rate (%) |
| S18 | 19.59 | 13.15 |
| S31 | 19.46 | 12.36 |
| S32 | 20.00 | 15.5 |
| S33 | 19.34 | 11.64 |
| S34 | 18.99 | 9.63 |
| S37 | 19.20 | 10.85 |
Embodiment 4: the complex mutation of bacterial strain
The seed liquor of lactobacillus plantarum DY6 is subjected to mutagenesis reaction with the optimum condition of ultraviolet mutagenesis, to plate after mutagenesis
On single colonie carry out the measurement to Escherichia coli bacteriostatic activity.Then the highest plant mutant bacterial strain of bacteriostatic activity is chosen with Asia
The optimum condition of nitroguanidine mutagenesis carries out mutagenesis reaction, and the measurement of bacteriostatic activity, knot are carried out to the single colonie on plate after mutagenesis
Fruit is as shown in table 4, using Mutation screening to one plant of bacterial strain E1 than initial strains bacteriostatic activity raising 30.3%.
4 complex mutation strain mutagenesis situation of table
| Bacterial strain | Bacteriostatic diameter (mm) | Bacteriostatic activity increase rate (%) |
| 1-21 | 21.25 | 22.7 |
| Y2 | 20.80 | 20.1 |
| Y3 | 20.84 | 20.3 |
| Y12 | 20.63 | 19.1 |
| E1 | 22.56 | 30.3 |
Embodiment 5: the bacteriostasis property of mutagenic strain E1
By mutagenic strain and starting strain after 37 DEG C of cultures for 24 hours, referring to the method for embodiment 1, compare the two to large intestine
The fungistatic effect of bacillus, salmonella and staphylococcus aureus, the results are shown in Table 5.
5 mutagenic strain of table is to pathogenic bacteria fungistatic effect
Embodiment 6: the inheritance stability Journal of Sex Research of mutagenic strain
The bacteriostatic activity obtained through one nitrosoguanidine complex mutation of ultraviolet light is higher than to 19.1% mutagenic strain, is transferred to
MRS culture medium is cultivated at 37 DEG C for 24 hours, is transferred 1 time every for 24 hours, after continuous 7 generations of transferring, is measured referring to the method for embodiment 2~4
Respectively for the bacteriostatic activity of bacterial strain.As a result Fig. 3 and table 6 are seen, it can be found that mutagenic strain inheritance stability.
6 mutagenic strain stability result of table
Note: data are bacteriostatic diameter (mm) of the mutagenic strain to Escherichia coli
Embodiment 7: control efficiency of the mutagenic strain to Escherichia coli
It takes 200g dregs of beans to be crushed, divides three groups, every group of 50g is added in 250mL triangular flask, in 105 DEG C of sterilizing 15min.
First group of addition concentration is 105The Escherichia coli 1mL of CFU/mL is added 50% water (by mass), 37 DEG C of fermentation 48h.Second
Group adds lactobacillus plantarum DY6-E1 bacterium solution 2mL (10 on the basis of first group8CFU/mL), basis of the third group at first group
Upper addition starting strain lactobacillus plantarum DY6 bacterium solution 2mL (108CFU/mL), Escherichia coli quantity reference after fermentation is measured
GB/T 18869-2002.Measurement result is shown in Table 5, it can be seen that after addition lactobacillus plantarum, can effectively inhibit Escherichia coli
Growth, adding mutagenic strain DY6-E1 than starting strain has better fungistatic effect.
Escherichia coli quantity after 7 solid state fermentation of table
| Group | Escherichia coli quantity (CFU/g) |
| Before fermentation (first group) | 2.1×104 |
| After fermentation (first group) | 7.9×107 |
| Before fermentation (second group) | 2.1×104 |
| After fermentation (second group) | 5.5×104 |
| Before fermentation (third group) | 2.1×104 |
| After fermentation (third group) | 8.3×104 |
Embodiment 8: the bacteriostasis property of mutagenic strain compares
With embodiment 5, difference is specific embodiment, replaces plant cream with the nisin that the concentration of 2mL is 570 μ g/mL
Bacillus DY6-E1 bacterium solution measures Escherichia coli quantity reference GB/T 18869-2002 after fermentation.The results show that by
The lower discovery of nisin effect is similar to DY6-E1 situation is added, and the ammonia benzyl which is equivalent to 100 μ g/mL is green
The effect of mycin.
Escherichia coli quantity after 8 solid state fermentation of table
| Group | Escherichia coli quantity (CFU/g) |
| Before fermentation (nisin processing) | 2.1×104 |
| After fermentation (nisin processing) | 4.8×104 |
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a lactobacillus plantarum (Lactobacillus plantarum) DY6-E1, is preserved on October 31st, 2018
China typical culture collection center, classification naming are Lactobacillus plantarum DY6-E1, and deposit number is
CCTCC No:M 2018734, preservation address are China, Wuhan, Wuhan University.
2. the composition containing lactobacillus plantarum DY6-E1 described in claim 1.
3. composition according to claim 2, which is characterized in that contain lactobacillus plantarum DY6- described in claim 1
Acceptable carrier on E1 and food.
4. composition according to claim 2 or 3, which is characterized in that concentration >=1 of lactobacillus plantarum DY6-E1 ×
108Concentration >=1 × 10 of CFU/g or lactobacillus plantarum DY6-E18CFU/mL。
5. according to any composition of claim 2~4, which is characterized in that contain plant cream bar described in claim 1
Bacterium DY6-E1 and cell-protecting.
6. according to any composition of claim 2~5, which is characterized in that also contain the lactobacillus plantarum DY6-E1
Metabolin.
7. lactobacillus plantarum DY6-E1 described in claim 1 is in the application of food or the antibacterial aspect of field of fodder.
8. a kind of method for inhibiting pathogenic bacteria in fermentation process, which is characterized in that the method is by the lactobacillus plantarum
DY6-E1 cell is added into the fermentation system containing pathogenic bacteria.
9. according to the method described in claim 8, it is characterized in that, the pathogenic bacteria include but is not limited to Escherichia coli, sramana
Salmonella, staphylococcus aureus.
10. according to the method described in claim 9, it is characterized in that, the fermentation system contains dregs of beans.
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