CN104611251B - One plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application - Google Patents
One plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application Download PDFInfo
- Publication number
- CN104611251B CN104611251B CN201410752144.2A CN201410752144A CN104611251B CN 104611251 B CN104611251 B CN 104611251B CN 201410752144 A CN201410752144 A CN 201410752144A CN 104611251 B CN104611251 B CN 104611251B
- Authority
- CN
- China
- Prior art keywords
- gzph2
- lactic acid
- acid bacteria
- salivarius
- lactobacillus salivarius
- 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.)
- Expired - Fee Related
Links
Classifications
-
- 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
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to microorganism field, and in particular to one plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application.The described lactic acid bacteria with broad-spectrum antibacterial activity, it is named as Lactobacillus salivarius GZPH2 (Lactobacillus salivarius GZPH2), China typical culture collection center (CCTCC) is preserved on November 27th, 2014, deposit number is CCTCC M 2014598；The lactic acid bacteria Lactobacillus salivarius GZPH2 (Lactobacillus salivarius GZPH2) that the present invention is provided has good bacteriostasis to a variety of people and aquaculture organisms pathogenic bacteria；And with not producing histamine, the not characteristic such as haemolysis, acidproof, bile tolerance.
Description
Technical field
The invention belongs to microorganism field, and in particular to one plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application.
Background technology
Vibrios (Vibrio) is one of most commonly seen bacterial groups in marine environment, is widely present in seawater, seabed and sinks
In product thing and fish, shrimp, shellfish, crab, sea cucumber, sea-tangle etc. marine product.《Primary Jie Shi bacteriology handbook》8th edition describes 35 kinds of arcs
Bacterium, quite a few in them is the important pathogenic bacteria of aquaculture organisms.In aquatic products industry, by vibrio parahaemolytious, molten algae arc
The disease for the aquaculture organism that the various vibrios such as bacterium, Vibrio anguillarum trigger is referred to as vibriosis (vibriosis), and it gives aquatic products industry every year
Cause huge economic loss in boundary.At the same time, part vibrios is also the pathogenic bacteria of the mankind.They can not only cause human gastrointestinal
It is scorching, moreover it is possible to cause extra intestinal infection.In food, vibrios (Vibrio), salmonella (Salmonella), pathogenic large intestine angstrom
Uncommon bacterium (pathogenic Escherichia coli) and staphylococcus aureus (Staphylococcus aureus) etc. one
Rise, the main pathogeny bacterium as food posioning.Up to the present, it is known that such as comma bacillus (Vibrio
Cholerae), vibrio parahaemolytious (V.parahaemolyticus), vibrio fluvialis (V.fluvialis) etc. can cause the intestines of people
Gastritis, and Vibrio vulnificus (V.vulnificus), vibrio alginolyticus (V.alginolyticus) etc. cause wound infection and septicemia
Deng extra intestinal infection.In recent years, as the intestines problem caused by kinds of pathogenic vibrio, infectious disease and food poisoning generation scale and people
Group's exposure scale is in obvious ascendant trend, more than salmonella food poisoning, leaps to the first place of food posioning event, into
To influence the key factor of food security, cause extensive concern.
It is to use antibiotic to prevent and treat bacteriosis most common method, but antibiotic, which is used for a long time, to cause bacterium to produce
Drug resistance.Therefore, the research of effective substitute of antibiotic seems particularly urgent.Lactic acid bacteria (Lactic acid bacteria)
It is the general designation for the probiotics that a class produces lactic acid using carbohydrate.Although existing research shows that it has regulation machine at present
Body gut flora, keeps microecological balance, improves food digestion rate and biological value, strengthens immunity of organisms, improves body disease-resistant
The functions such as power, metabolite that is having or even can also producing tool bacteriostatic activity, such as lactobacillus peptide, bacteriocin, lactic acid, peroxidating
Hydrogen, acetic acid etc., have necessarily to some gram-positive bacterias such as listeria spp etc. and/or Gram-negative bacteria such as Escherichia coli etc.
Inhibitory action, but it is few for lactic acid bacteria to suppress the original scientific research that is carried out of vibrios, substantially only stops
Directly applied in transplanting this antibacterial concept of lactic acid bacteria in aquaculture.For can both suppress aquaculture can be caused to give birth to
The pathogenic vibrios of thing, and can suppress that the innovative research of the lactic acid bacteria of the vibrios of human foods poisoning and/or infection etc. can be caused
It is then less.
As can be seen here, study and then preferably there is the Lactic Acid Bacteria strain of excellent broad-spectrum antibacterial effect to suppress that water can be triggered
The vibrios of aquaculture organism and/or the disease of people is produced, the loss of aquatic products industry and infection and/or the hair of food poisoning of the mankind is reduced
It is raw, with important social effect and economic value.
Lactobacillus salivarius is one kind in lactic acid bacteria, is gram-positive bacteria, is located away from healthy mother's milk or baby face
Portion, can also separate from each age group human body intestinal canal, chicken intestinal mucous membrane, fermented food etc. and get.It is also the Ministry of Public Health in 2013
《New raw-food material security screening management method》Announce available for one of strain of food production.Study and be also shown that
Lactobacillus salivarius has immunoloregulation function, anti-inflammatory and the characteristic such as anti-infective.But as probiotics, the saliva reported at present
Lactobacillus exist or can not grow at a low ph, or not bile tolerance the shortcomings of.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, primary and foremost purpose of the invention is that providing one plant has broad-spectrum antibacterial
The lactic acid bacteria of activity.
Another object of the present invention is to provide the application of above-mentioned lactic acid bacteria.
The purpose of the present invention is achieved through the following technical solutions：
One plant has the active lactic acid bacteria of broad-spectrum antibacterial, entitled Lactobacillus salivarius (Lactobacillus
Salivarius) GZPH2, China typical culture collection center (CCTCC), address are preserved on November 27th, 2014：In
Wuhan University of Wuhan City of state, deposit number is CCTCC NO： M 2014598；
Described Lactobacillus salivarius (L.salivarius) GZPH2 is isolated and purified from pickles and obtained；
Described Lactobacillus salivarius (Lactobacillus salivarius) GZPH2 cultivation temperature is preferably 37 DEG C；
Described Lactobacillus salivarius (Lactobacillus salivarius) GZPH2 of culture culture medium is preferably MRS
Culture medium；
Described Lactobacillus salivarius (L.salivarius) GZPH2, with following morphological feature and physio-biochemical characteristics：
A, the Lactobacillus salivarius screened (L.salivarius) GZPH2 Gram's staining are positive, shaft-like, no gemma,
Atrichia；
B, the morphological feature of bacterium colony are：Described Lactobacillus salivarius (L.salivarius) GZPH2 is in MRS solid mediums
After 37 DEG C of culture 24h, bacterium colony is creamy white, projection, surface wettability, smooth glossy, neat in edge；
C, main physiological and biochemical property are as follows：Negative catalase, nitrate reduction negative, production gelatinase is negative,
Produce H2S is negative, produces the acid positive using glucose, utilizes glucose aerogenesis negative；
Described Lactobacillus salivarius (L.salivarius) GZPH2 16S rDNA sequences (5 ' -3 ') are as follows, with saliva
The homology of liquid lactobacillus type strain (L.salivarius ATCC 11741) is 99%；
ACTCTGTCACTTAGACGGCTGGCTCCTTGCGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGG
GCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCGATTACTAGCGATTCCGACTTCATGTA
GGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTAAACCTCGCGGTCTTGCGACTCGTTG
TACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGG
TTGGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAACTGACAACAAGGGTTGCGCTCGTAGCGGGAC
TTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAAGGGAAAACCTA
ATAACCTTAGGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTAGAATTAAACCACATGCTCCAC
CGCTTGTGCGGAGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTATTGCG
TTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCT
AATCCTGTTTGCTACCCACGCTTTCGAACCTCAGCGTCAGTTACAGACCAGAGAGCCGCTCTCGCCACTGGTGTTCT
TCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCCAA
TGCACTACTCCGGCTAAGCCGAAAGCTTTCACATCAGACTTAAAAGACCGCCTGCGTTCCCTTTACGCCCAATAAAT
CCGGACAACGCTTGCCACCTACGTATTAGCCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTGCTGGTTAGATACCG
TCATCGAATGAACAGTTACTCTCACTCGTGGTTCTTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCAC
TCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCC
GTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCACCTTGGTAGGCCGTTACCCCACC
AACTAGTTAATACGCCGCGGGTCCATCTAAAAGCGATAGCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTAG
AGATATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTTCTTTTAGGCAGG
TTACCCACGTGTTACTCACCCGTCCGCCACTCAACTTCTTACGATGAATGCAAGCATTCGGTGTAAGAAAGTTTCGT
TCGACTGCATGTATAGCACGCGCCCTGCA
Described Lactobacillus salivarius (L.salivarius) GZPH2 can suppress the arc that people can be triggered to infect and poison by food
Bacterium, can suppress that the vibrios of aquaculture organisms disease can be triggered again, while equally having to other food-borne pathogens good
Bacteriostasis, can be widely applied to microbial preparation field and food fermentation industry field, it can also be used to prepare bacteriostatic agent or anti-
Rotten agent.
The present invention has the following advantages and effect relative to prior art：
(1) the invention provides Lactobacillus salivarius (L.salivarius) GZPH2 that one plant has broad-spectrum antibacterial activity；
(2) Lactobacillus salivarius (L.salivarius) GZPH2 that the present invention is provided can suppress that people can be triggered to infect and eat
The vibrios of thing poisoning, can suppress that the vibrios of aquaculture organisms disease can be triggered, while also having other food-borne pathogens again
There is good bacteriostasis；
(3) Lactobacillus salivarius (L.salivarius) GZPH2 for providing of the present invention have do not produce histamine, not haemolysis, it is acidproof,
The characteristics such as bile tolerance.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
The culture medium being related in embodiment：
The formula (g/L) of MRS (de Man-Rogosa-Sharpe) fluid nutrient medium：Casease digest 10；Beef
Cream powder 10；Yeast extract powder 4；Triammonium citrate 2；Sodium acetate 5；Magnesium sulfate (MgSO4.7H2O)0.2；Manganese sulfate (MnSO4.4H2O)
0.05；Citric acid dipotassium 2；Glucose 20；Tween-80 1.08；Final pH 5.7 ± 0.2；
MRS solid culture based formulas：The agar powder that mass ratio is 1.5%, final pH 5.7 are added in MRS fluid nutrient mediums
±0.2；
The formula of blood plate：Blood plate finished product culture medium (sheep blood), finished product is purchased from the triumphant biological Co., Ltd of Guangdong ring.
Nutrient broth Liquid Culture based formulas (g/L)：Peptone 10；Beef extract powder 3；Sodium chloride 5, final pH 7.4 ±
0.2；
Nutrient broth solid culture based formulas：The agar powder that mass ratio is 1.5% is added in nutrient broth fluid nutrient medium,
Final pH 7.4 ± 0.2.
In the present embodiment in 39 plants of indicator bacterias, staphylococcus aureus (Staphylococcus aureus) AS
1.1865th, ETEC (Escherichia coli) AS 1.2420 and salmonella typhimurium (Salmonella
Typhimurium) CMCC 50115 is bought in China General Microbiological preservation administrative center；
Vibrio anguillarum (Vibrio anguillarum) ATCC 43305, vibrio fluvialis (Vibrio fluvialis) ATCC
33809th, vibrio parahaemolytious (V.parahaemolyticus) (tdh+) ATCC17802 is purchased from American Type Culture collection
(American Type Culture Collection)；
Vibrio alginolyticus (Vibrio alginolyticus) 1, vibrio alginolyticus (V.alginolyticus) 2, vibrio alginolyticus
(V.alginolyticus) 3, vibrio alginolyticus (V.alginolyticus) 4, comma bacillus (V.cholera) 6, vibrio parahaemolytious
(V.parahaemolyticus) 8, vibrio parahaemolytious (V.parahaemolyticus) 9, vibrio alginolyticus
(V.alginolyticus) 10, vibrio alginolyticus (V.alginolyticus) 11, Shewanella putrefaciens (Shewanella
Putrefaciens) 12, the husky thunder bacterium (Serratia ficaria) 15 of vibrio alginolyticus (V.alginolyticus) 13, smell, molten
Algae vibrios (V.alginolyticus) 16, pseudomonas aeruginosa (Pseudomonas aeruginosa) 17, pseudomonas aeruginosa
(P.aeruginosa) 18, the husky thunder bacterium (Serratia ficaria) 20 of vibrio alginolyticus (V.alginolyticus) 19, smell,
Vibrio parahaemolytious (V.parahaemolyticus) 21, pseudomonas aeruginosa (P.aeruginosa) 22, vibrio alginolyticus
(V.alginolyticus) 23, Shewanella putrefaciens (Shewanella putrefaciens) 24, vibrio parahaemolytious
(V.parahaemolyticus) 25, vibrio parahaemolytious (V.parahaemolyticus) 26, Shewanella putrefaciens
(Sh.putrefaciens) 27, Shewanella putrefaciens (Sh.putrefaciens) 28, pseudomonas aeruginosa
(P.aeruginosa) 29, pantoea agglomerans (Pantoea agglomerans) 30, acid-producing Klebsiella bacterium (Klebsiella
Oxytoca) 31, aeromonas salmonicida (Aeromonas salmonicida) 33, Shewanella putrefaciens
(Sh.putrefaciens) 34, pseudomonas aeruginosa (P.aeruginosa) 35 is by South China Science ＆ Engineering University's light industry and Foodstuffs Academy
(biological characterization of two marine bdellovibrio-and-like are provided
organisms isolated from Daya bay of Shenzhen,China and their application in
the elimination of Vibrio parahaemolyticus in oyster[J].Li H,Liu C,Chen L et
al.International Journal of Food Microbiology,2011,151:36–43.)；
Providencia rettgeri (Providencia rettgeri) 32 is by South China Science ＆ Engineering University's light industry and Foodstuffs Academy
(The protective effect of bdellovibrio-and-like organisms (BALO) on tilapia are provided
fish fillets against Salmonella enterica ssp.enterica serovar Typhimurium[J]
.Lu F,Cai J.Letters in Applied Microbiology,2010,51:625–631.)；
Vibrio anguillarum (Vibrio anguillarum) MVM425 is tested by East China University of Science's bioreactor state key
Room give (minimums of marine fishes pathogenic bacteria Vibrio anguillarum MVM425 virulence plasmid pEIB1 replication regions analysis [J] Wus sea is precious,
Zhang Huizhan, Li Gang, Ye Jiang, Ma Yue, Zhang Yuanxing food and medicine, 2007,04:1-4.)
The Lactobacillus salivarius of embodiment 1 (L.salivarius) GZPH2 screening, domestication and separation
From Guangzhou fairground stochastic buying pickles sample, the 50mL MRS liquid for taking sample liquids 1mL to add aseptic process (adds
Plus the CaCO that whole mass fraction is 0.05%3), 37 DEG C of enrichment culture 12h of 150r/min；Take pregnant solution sterile saline
It is diluted to 10-8, each dilution gradient applies flat board, 37 DEG C of culture 24h, the flat board that selection single bacterium colony number is 30~300 successively, and chooses
Periphery of bacterial colonies has the single bacterium colony of obvious dissolution of calcium carbonate circle, and the corresponding single bacterium of transfer needle picking drops down onto the friendship of the area of MRS solid mediums three
Fork line, until the single bacterium colony size occurred is homogeneous, selection Gram's staining is purple (positive), and dyeing thalli morphology size is equal
One single bacterium colony, tentatively confirms as lactic acid bacteria, and carry out strain idenfication；
The bacterium colony that purifying is obtained is identified that qualification result is as follows：
A, the lactic acid bacteria Gram's staining screened are the positive, shaft-like, no gemma, atrichia；
B, the morphological feature of bacterium colony are：After 37 DEG C of culture 24h of MRS solid mediums, bacterium colony is creamy white, projection, table
Face is moistened, smooth glossy, neat in edge；
C, main physiological and biochemical property are as follows：Negative catalase, nitrate reduction negative, production gelatinase is negative,
Produce H2S is negative, produces the acid positive using glucose, utilizes glucose aerogenesis negative；
Molecular biology identification result：
Take single bacterium colony to be inoculated in 10mL MRS fluid nutrient mediums to cultivate, 37 DEG C, 150r/min, 12h；Extract genome,
16S rDNA fragments are expanded, electrophoresis glue reclaim purpose fragment links PMD18-T (Takara) empty carrier plasmid, converts large intestine bar
Bacterium competence cell, amplification cultivation chooses monoclonal sequencing (Shanghai Sheng Gong bioengineering Co., Ltd)；Wherein, pcr amplification primer
Thing is：
27F：5’-AGAGTTTGATCMTGGCTCAG-3’；
1492R：5’-GGTTACCTTGTTACGACTT-3’；
PCR reaction conditions are：94 DEG C, pre-degeneration 4min；94 DEG C, it is denatured 30s；55 DEG C, anneal 30s；72 DEG C of extension 90s；
35 circulations；72 DEG C, extend 7min；The present invention uses monoclonal authentication method, through forward primer 27F and reverse primer 1492R
Amplification obtains two bar segments, and DNAStar splicing two sequences obtain 1469bp purpose fragments.The lactic acid bacteria 16S rDNA sequences
(5 ' -3 ') are as follows：
ACTCTGTCACTTAGACGGCTGGCTCCTTGCGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGG
GCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCGATTACTAGCGATTCCGACTTCATGTA
GGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTAAACCTCGCGGTCTTGCGACTCGTTG
TACCGTCCATTGTAGCACGTGT GTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCG
GTTGGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAACTGACAACAAGGGTTGCGCTCGTAGCGGGA
CTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAAGGGAAAACCT
AATAACCTTAGGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTAGAATTAAACCACATGCTCCA
CCGCTTGTGCGGAGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTATTGC
GTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATC
TAATCCTGTTTGCTACCCACGCTTTCGAACCTCAGCGTCAGTTACAGACCAGAGAGCCGCTCTCGCCACTGGTGTTC
TTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCCA
ATGCACTACTCCGGCTAAGCCGAAAGCTTTCACATCAGACTTAAAAGACCGCCTGCGTTCCCTTTACGCCCAATAAA
TCCGGACAACGCTTGCCACCTACGTATTAGCCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTGCTGGTTAGATACC
GTCATCGAATGAACAGTTACTCTCACTCGTGGTTCTTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCA
CTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGC
CGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCACCTTGGTAGGCCGTTACCCCAC
CAACTAGTTAATACGCCGCGGGTCCATCTAAAAGCGATAGCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTA
GAGATATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTTCTTTTAGGCAGGTTACCCACGTGTTACTCACCCG
TCCGCCACTCAACTTCTTACGATGAATGCAAGCATTCGGTGTAAGAAAGTTTCGTTCGACTGCATGTATAGCACGCG
CCCTGCA
16S rDNA gene sequencing result sequences are subjected to homologous comparison by NCBI BLAST in GenBank databases
Analyze and identify, RDP classifier classifications for search are lactobacillus (Lactobacillus), further NCBI BLAST analyses hair
Existing, the lactic acid bacteria and Lactobacillus salivarius type strain (L.salivarius strain ATCC 11741) homology are 99%；According to
According to homology>97% is same kind of principle, and therefore, bacterial strain GZPH2 is accredited as Lactobacillus salivarius (L.salivarius).
Summary is described, and the lactic acid bacteria that the present invention is screened belongs to lactobacillus (Lactobacillus), Lactobacillus salivarius
Plant (L.salivarius), Lactobacillus salivarius GZPH2 (Lactobacillus salivarius GZPH2) is named as, in 2014
On November 27, in is preserved in China typical culture collection center (CCTCC), address：Wuhan University of Wuhan, China city, preservation is compiled
Number be CCTCC NO： M 2014598.
The Lactobacillus salivarius of embodiment 2 (L.salivarius) GZPH2 broad-spectrum antibacterial activity
The preparation of (1) 39 plant of indicator bacteria
In 39 plants of indicator bacteria (table 1) single bacterium colony inoculation nutrient broth fluid nutrient mediums, 37 DEG C, 150r/min culture 8h are adjusted
Its concentration is saved to 1 × 106CFU/mL, 4 DEG C of preservations are standby；
The different experiment indicator strain of 1 39 plants of table
(2) preparation of lactic acid bacteria
Lactobacillus salivarius (L.salivarius) the GZPH2 single bacterium colonies identified in embodiment 1 are inoculated in 50mL MRS
Fluid nutrient medium, 37 DEG C, 150r/min cultivates 12h, cultivates its concentration to 1 × 108CFU/mL；10000RCF, 4 DEG C, 10min
Lactic acid bacteria culture solution is centrifuged, takes supernatant standby for bacteriostatic experiment, 4 DEG C of preservations are standby；
(3) (supernatant) Screening of Antibacterial Activities
Bacteriostatic experiment：The μ L of indicator bacteria 100 that 1. application step prepares are on nutrient broth solid medium, by Oxford
Cup is put down gently on the culture medium of coating indicator bacteria, and light pressure makes it fully contact, and adds 200 μ L of supernatant liquid, 37 DEG C of culture 24h, note
Record antibacterial circle diameter；
Interpretation of result：
Experiment is using 10 μ g/mL chloramphenicol as positive control, and MRS blank solutions coating culture medium is used as negative control.
Supernatant bacteriostatic activity result as shown in table 2, the pathogenic bacteria of 39 plants of separate sources as this experiment indicator bacteria, for lactic acid bacteria
For supernatant bacteriostatic activity, GZPH2 shows that positive ratio is 33/39 (84.6%) to 39 plants of indicator bacterias, and GZPH2 is to 39 plants
14 plants of indicator bacterias in indicator bacteria have extremely strong bacteriostatic activity (+++, antibacterial circle diameter be more than 15mm)；
Therefore, in the present invention, GZPH2 has broad-spectrum antibacterial activity, can suppress that the vibrios of people's enteritis can be triggered such as to contain
The vibrio parahaemolytious (Vibrio parahaemolyticus) of tdh toxin genes, can suppress that aquaculture organisms disease can be triggered again
Harmful vibrios, such as vibrio alginolyticus (Vibrio alginolyticus).
The GZPH2 supernatants of table 2 are analyzed the bacteriostatic activity of the pathogenic indicator bacteria of 39 plants of separate sources
Note：A, the diameter (mm) of bacteriostatic activity inhibition zone is represented.-, no bacteriostatic activity；+, 7<Antibacterial circle diameter<10mm；
++, 10mm<Antibacterial circle diameter<15mm；+++, antibacterial circle diameter>15mm.
The Lactobacillus salivarius of embodiment 3 (L.salivarius) GZPH2 other specificity analysises
(1) hemolytic analysis
Take 5 μ L seed liquors (final concentration of Lactobacillus salivarius (L.salivarius) GZPH2 identified in embodiment 1
8.0×108CFU/mL) the dibbling on blood plate (sheep blood, the triumphant biological Co., Ltd of Guangdong ring), 37 DEG C of culture 24h, observes bacterium colony
Whether surrounding has haemolysis.No. 8 vibrio parahaemolytious (Vibrio parahaemolyticus) of selection are used as positive control；
(2) histamine is detected
Nutrient broth fluid nutrient medium is prepared, containing kilnitamin (histidine 10.0g/L), regulation medium pH is 5.5,
Add bromocresol purple (0.06g/L), agar powder (20g/L, gel strength>1300g/cm2), 120 DEG C of sterilizing 15min are down flat plate；
Lactobacillus salivarius (L.salivarius) the GZPH2 bacterial strains identified in activation embodiment 1, after appropriate dilution, draw 100 μ L and apply
Cloth flat board, 48h is cultivated at 37 DEG C, while doing positive control [ETEC (E.coli) AS 1.2420] and feminine gender is right
According to (being not coated with any strain).Experiment do three times it is parallel.Because biogenic amine is alkaline matter, bromocresol purple meets the aobvious Huang of acidic materials
Color, meets alkaline matter and then shows purple or red.Therefore, yellow is feminine gender, and red or purple is the positive.
(3) acidproof detection
Lactobacillus salivarius (L.salivarius) the GZPH2 single bacteriums identified in inoculation embodiment 1 fall within 50mL MRS liquid
Cultivated in body culture, 37 DEG C, 12h, concentration is 1 × 108CFU/mL；10000RCF, 4 DEG C, 10min centrifugations abandon supernatant and take precipitation,
Correspondence pH MRS solution suspensions are taken, suspension is added into the 50mL MRS liquid that pH is respectively 6.5 (controls), 2.0,2.5 and 3.5
In body culture medium, apply flat board in 0h, 2h and 4h sampling dilution respectively and detect its concentration, each time point Concentration Testing does three and put down
OK, results averaged, is added standard deviation, is represented with logarithm Log10 values.
(4) bile tolerance is analyzed
Lactic acid bacteria single bacterium colony is inoculated in 50mL MRS fluid nutrient mediums and cultivated, 37 DEG C, 12h (exponential phase), concentration
For 1 × 108CFU/mL, 10000RCF, 4 DEG C, 10min centrifugations.Supernatant is abandoned, the MRS solution with the gallbladder salinity containing correspondence will be precipitated
Suspend, and it is respectively 0% (control group), 0.5%, 1%, 1.5% and 2% (w/v) containing cholate that precipitation suspension is added into 50mL
In MRS fluid nutrient mediums, 37 DEG C, 150r/min starts timing.100 μ L are taken to dilute and be coated in 0h, 2h, 4h and 6h respectively
In MRS solid mediums, 37 DEG C, 24h is cultivated, is counted, each bacterial concentration is detected.
(5) data analysis
In the present invention, each experimental group setting 3 is parallel, and lactic acid bacteria change in concentration is with average value ± standard deviation form table
Show.
Result of the test is as follows：
(1) hemolytic analysis
GZPH2 and indicator bacteria (positive control) liquid-transfering gun are respectively drawn into 5 μ L seed liquors (final concentration of 8.0 × 108CFU/
ML) dibbling is on blood plate (the triumphant biological Co., Ltd of Guangdong ring), 37 DEG C of culture 24h, No. 8 vibrio parahaemolytious (Vibrio in table 1
Parahaemolyticus) as positive control.As a result, the GZPH2 screened not haemolysis, positive control produces haemolysis.
(2) histamine is detected
Using bromocresol purple in sour environment (pH<5.8) yellow is shown, alkaline environment shows the principle of purple, qualitative analysis
Lactic acid bacteria produces histamine situation.ETEC (Escherichia coli) AS 1.2420 is used as positive control, negative control
For pH5.5 nutrient broth blank cultures.As a result show：ETEC (Escherichia coli) AS 1.2420
Plating medium shows purple, and GZPH2 plating mediums do not show purple, thus can distinguish that GZPH2 does not produce histamine.
(3) acidproof detection
As known from Table 3, compared with compareing (pH6.5) group, Lactobacillus salivarius (L.salivarius) GZPH2 of pH2.0 groups
There is downward trend in concentration, but pH2.5, pH3.5 group GZPH2 concentration then present fall before after the trend that rises.Thus disclose
GZPH2 at least has tolerance pH2.5 good acid-fast ability.
The resistance to acid analysis of the Lactobacillus salivarius of table 3 (L.salivarius) GZPH2 (concentration is represented with logarithm value)
Note：a:Average ± standard deviation, n=3；b:Incubation time
(4) bile tolerance is analyzed
The different gallbladder salinity of invention design, (concentration is 10 to 50mL GZPH2 seed liquors8CFU/mL), 1000 RCF/
After min, 10min, 4 DEG C of centrifugations, the MRS solution suspensions with correspondence gallbladder salinity will be precipitated, inoculation suspension is dense to different cholate
Spend in triangular flask, 37 DEG C, 150r/min shaking table cultures.Respectively MRS flat boards detection lactic acid is coated in 0h, 2h, 4h and 6h sampling
Bacteria concentration, as a result as shown in table 4.As known from Table 4, in control group (0% cholate), with the extension of time, GZPH2 concentration is presented
Ascendant trend.In 0.5% gallbladder salinity group, GZPH2 slightly now increases in 2h and 4h, is increased substantially during 6h (2.68log)；
1.0% and 1.5% gallbladder salinity group, GZPH2 concentration then occurs falling before the phenomenon that (2h) increases (4h, 6h) again；2.0% cholate
Concentration group, though GZPH2 concentration small size decline occurs in each experimental period point, its fall is also only within 0.71log.By
This is visible, and GZPH2 at least has the good bile tolerance ability of 1.5% gallbladder salinity of tolerance.
The Lactobacillus salivarius of table 4 (L.salivarius) GZPH2 bile tolerance analysis
Note：a:Average ± standard deviation, n=3；b:Incubation time
There is the active lactic acid bacteria of broad-spectrum antibacterial as one plant, Lactobacillus salivarius GZPH2 can suppress people can be triggered to infect
With the vibrios and other germs of food poisoning, it can suppress that the vibrios of aquaculture organisms disease can be triggered again, while having insoluble
Blood, histamine is not produced and good acidproof and bile tolerance ability.It is one plant of excellent lactic acid to be indicated above Lactobacillus salivarius GZPH2
Bacterium.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (3)
1. a strains of lactic acid bacteria, it is characterised in that：Entitled Lactobacillus salivarius (the Lactobacillus of described lactic acid bacteria
Salivarius) GZPH2, China typical culture collection center, address are preserved on November 27th, 2014：Wuhan, China city
Wuhan University, deposit number is CCTCC NO:M 2014598.
2. application of the lactic acid bacteria described in claim 1 in bacteriostatic agent is prepared.
3. application of the lactic acid bacteria described in claim 1 in preservative is prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410752144.2A CN104611251B (en) | 2014-12-09 | 2014-12-09 | One plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410752144.2A CN104611251B (en) | 2014-12-09 | 2014-12-09 | One plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104611251A CN104611251A (en) | 2015-05-13 |
| CN104611251B true CN104611251B (en) | 2017-10-20 |
Family
ID=53145903
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410752144.2A Expired - Fee Related CN104611251B (en) | 2014-12-09 | 2014-12-09 | One plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104611251B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109157546B (en) * | 2018-07-27 | 2021-05-28 | 江苏省海洋水产研究所 | Compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei boone and application thereof |
| KR102063544B1 (en) | 2018-09-12 | 2020-01-09 | (주)성운파마코피아 | Lactobacillus salivarius swpm101 which has antifungal activity or antibacterial activity |
| CN109759436B (en) * | 2019-01-07 | 2021-02-12 | 湖南工业大学 | Manganese-resistant providencia bacterial strain and method for repairing heavy metal contaminated soil |
| CN110157645B (en) * | 2019-05-29 | 2021-03-02 | 中国科学技术大学先进技术研究院 | Lactobacillus salivarius Y4 and application thereof |
| CN113151364A (en) * | 2021-05-26 | 2021-07-23 | 吉林大学 | Bacteriostatic active substance separated from chicken intestine source lactobacillus salivarius and application thereof |
| CN115053935A (en) * | 2022-05-12 | 2022-09-16 | 华南理工大学 | Application of lactobacillus salivarius in breeding of litopenaeus vannamei larvae |
| CN115216434B (en) * | 2022-09-14 | 2023-06-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Lactobacillus salivarius strain and application thereof |
| CN115948285A (en) * | 2022-11-23 | 2023-04-11 | 鲁东大学 | Biological disinfectant for lactobacillus salivarius BMC-06 and livestock breeding and application |
| CN116784421B (en) * | 2023-06-21 | 2024-05-31 | 华南理工大学 | Application of lactobacillus salivarius in bullfrog tadpole culture |
| CN117143783B (en) * | 2023-10-30 | 2024-03-22 | 杭州微致生物科技有限公司 | Saliva combined lactobacillus VB330 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538545A (en) * | 2009-04-08 | 2009-09-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | Lactobacillus salivarius and food composition thereof |
| CN103289926A (en) * | 2013-05-30 | 2013-09-11 | 中国农业科学院哈尔滨兽医研究所 | Lactobacillus salivarius strain and application thereof |
| CN103289927A (en) * | 2013-05-30 | 2013-09-11 | 中国农业科学院哈尔滨兽医研究所 | Lactobacillus salivarius strain and application thereof |
-
2014
- 2014-12-09 CN CN201410752144.2A patent/CN104611251B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538545A (en) * | 2009-04-08 | 2009-09-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | Lactobacillus salivarius and food composition thereof |
| CN103289926A (en) * | 2013-05-30 | 2013-09-11 | 中国农业科学院哈尔滨兽医研究所 | Lactobacillus salivarius strain and application thereof |
| CN103289927A (en) * | 2013-05-30 | 2013-09-11 | 中国农业科学院哈尔滨兽医研究所 | Lactobacillus salivarius strain and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 乳酸杆菌LH对几种水产养殖病原弧菌的抑制作用;周海平等;《台湾海峡》;20060831;第25卷(第3期);第389页第1段、第390页第3段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104611251A (en) | 2015-05-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104611251B (en) | One plant has the active lactic acid bacteria of broad-spectrum antibacterial and its application | |
| Arellano et al. | Safety evaluation and whole-genome annotation of Lactobacillus plantarum strains from different sources with special focus on isolates from green tea | |
| Buntin et al. | Screening of lactic acid bacteria from gastrointestinal tracts of marine fish for their potential use as probiotics. | |
| Hjelm et al. | Seasonal incidence of autochthonous antagonistic Roseobacter spp. and Vibrionaceae strains in a turbot larva (Scophthalmus maximus) rearing system | |
| Zhang et al. | Biological characteristics and probiotic effect of Leuconostoc lactis strain isolated from the intestine of black porgy fish | |
| Gupta et al. | Probiotic potential of Lactobacillus plantarum LD1 isolated from batter of Dosa, a South Indian fermented food | |
| CN105255752B (en) | High acid plant lactobacillus ZDY2013 | |
| CN105062921B (en) | It is a kind of inhibit fowl pathogenic salmonella Lactobacillus salivarius and its application | |
| CN108251335B (en) | Enterococcus faecalis HKF7 with lactic acid activity and screening culture method and application thereof | |
| CN105176874B (en) | Bacillus coagulans FM603 and its application | |
| Lamari et al. | Selection of lactic acid bacteria as candidate probiotics and in vivo test on Artemia nauplii | |
| CN113040390B (en) | Probiotic salt-tolerant lactobacillus johnsonii and application thereof in preventing and treating pathogenic bacteria in livestock and poultry aquiculture | |
| CN107858302B (en) | Bacillus subtilis 7K and application thereof | |
| CN107964524B (en) | Lactococcus lactis HKS2 with lactic acid activity and separation and screening method and application thereof | |
| CN112940982A (en) | Snakehead source Bacillus belezii and application thereof | |
| CN102559534A (en) | Bacillus cereus, and preparation and application of bacillus cereus | |
| Linh et al. | Screening of lactic acid bacteria isolated from fermented food as potential probiotics for aquacultured carp and amberjack | |
| CN104726359B (en) | One bacillus pumilus BP and application thereof | |
| CN105104712B (en) | A kind of additive for microbe feedstuff and preparation method thereof | |
| CN105062922B (en) | It is a kind of inhibit uropathogenic Escherichia coli Bacillus acidi lactici and its application | |
| CN111733117A (en) | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof | |
| 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 | |
| CN105624071A (en) | Lactobacillus salivarius XJP2 and application thereof | |
| CN116064324A (en) | Lactobacillus rhamnosus, culture method thereof and application thereof in preventing and treating diarrhea and enteritis | |
| CN102559533A (en) | Bacillus atrophaeus, and preparation and application of bacillus atrophaeus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171020 Termination date: 20211209 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |