CN107541475A - One breeding oxen streptomycete and its application - Google Patents
One breeding oxen streptomycete and its application Download PDFInfo
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- CN107541475A CN107541475A CN201710276908.9A CN201710276908A CN107541475A CN 107541475 A CN107541475 A CN 107541475A CN 201710276908 A CN201710276908 A CN 201710276908A CN 107541475 A CN107541475 A CN 107541475A
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Abstract
The invention discloses a breeding oxen streptomycete and its application.The Classification And Nomenclature of the bacterial strain is bull streptomycete (streptomyces tauricus), and China Committee for Culture Collection of Microorganisms's common micro-organisms center was deposited on 03 08th, 2017, and deposit number is CGMCC No.13735.The present invention bull streptomycete (streptomyces tauricus) have the function that and meanwhile suppress bacterium and yeast-like fungi grow, it can be used for preventing the rotten of essence spice for cigarette, the lifting to essence spice for cigarette quality Control Technology has important value.
Description
Technical field
The invention belongs to microbe technical field, and in particular to it is a kind of can suppress simultaneously bacterium and fungi growth
Bull streptomycete.Meanwhile the purposes the invention further relates to described bull streptomycete in tobacco leaf production.
Background technology
Essence spice for cigarette has a very important role as important auxiliary material in production of cigarettes, the stabilization of its quality
For maintaining cigarette, normally production and guarantee cigarette quality are most important.But it is easy that essence spice for cigarette has during storage
The problem of putrid and deteriorated, by many experiments, reason is searched and analyzed from stage construction, multi-angle, the results showed that：Rotten microorganism
In be primarily present four kinds of microorganisms, be Bayer combining yeast, Pasteur's acetobacter, soil coccus, bacillus.Relevant cigarette is used at present
The research of essence and flavoring agent quality Control Technology is also considerably less, especially for essence spice for cigarette it is putrid and deteriorated the problem of do not have also
Effective solution.
Actinomyces are a kind of microbial resources for having extensive practical use, extremely close with the relation of the mankind.According to statistics,
In the antibiotic having found, have 61.7% produced by actinomyces.Screening application of the scientific research personnel to actinomyces has been carried out greatly
Quantity research, such as：A kind of bactericide product disclosed in Yunnan University and Yunnan Agriculatural Academy, it is using deposit number
CGMCC No.1682 bull streptomycete (Streptomyces tauricus) ECO 00008, through separation and Extraction fermented and cultured
Thing, will be merged after mycelium broken wall with fermented supernatant fluid with acetone or methanol, again through macroporous absorbent resin adsorbing separation, acetone or
Methanol elution, eluent concentration are further purified to obtain new phosphorus nitrogen mycin A.Newly the molecular formula of phosphorus nitrogen mycin A is
C31H51N2O10P, molecular weight are 642.72 dalton.Bactericide finished product is made after further adding carrier or auxiliary agent.The sterilization
Agent has good effect to preventing and treating rice blast, Alternaria alternate, graw mold of tomato and pepper anthracnose.With existing medicament phase
Than having the advantages that dosage is few, peasant's use cost is low, use range is wide.Yunnan Agriculatural Academy's agricultural environment resource is ground
A kind of application using new phosphorus nitrogen mycin A in anthracnose of tobacco is suppressed is studied carefully disclosed.New phosphorus nitrogen mycin A is as actinomyces
Secondary metabolite, its toxicity is low, fermentation yield is higher, raw material is easy to get, to the inhibitory action of Colletotrichumtabacum mycelial growth
By force, can emphasis carry out the application of new invention research and trituration for the target fungal disease based on anthracnose of tobacco and visit
Rope.
But in the prior art, relevant actinomyces can suppress bacterium simultaneously and the report of fungi is also considerably less, particularly close
It can suppress bacterium in essence spice for cigarette simultaneously in bull streptomycete and yeast-like fungi has not been reported.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide, one kind can suppress bacterium simultaneously and yeast class is true
The bull streptomycete of bacterium.
The present invention also aims to provide application of the bull streptomycete in tobacco leaf production.
The purpose of the present invention is achieved by the following technical programs.
* unless otherwise indicated, the percentage employed in the present invention is mass percent.
The present invention isolates one plant of superior microorganism YNAU-1 as test strain from soil, and 16S rDNA are carried out to it
Analysis, qualification result show that the bacterium belongs to unwrapping wire strain streptomyces, and its Classification And Nomenclature is bull streptomycete
(streptomyces tauricus), preserving number CGMCC No.13735.
Conventionally analyzed and identified according to physio-biochemical characteristics such as the form of bacterium colony, colors.The public affairs of the present invention
The morphological feature of ox streptomycete (streptomyces tauricus) bacterial strain is：Bacterium colony pink, point-like, surface is smooth, circle
Shape or ellipse.
Molecular biology identification:Performing PCR amplification, the molecule for actinomyces are entered to the 16S rDNA sequences of YNAU-1 bacterial strains
Taxology is identified.Actinomyces PCR amplification primer be：PA(5'‐GAGAGTTTGATCCTGGCTCAG‐3')PB(5'‐
CTACGGCTACCTTGTTACGA‐3').Primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.Using 2 × Power Taq
PCR Master Mix, 25 μ L reaction systems, actinomyces reaction condition：94 DEG C of pre-degeneration 6min, 94 DEG C of denaturation 1min, 53 DEG C are moved back
Fiery 1min, 72 DEG C of extension 2min, 30 circulations；Last 72 DEG C of extensions 10min.Qualification result shows that the bacterium belongs to unwrapping wire strain
Streptomyces, its Classification And Nomenclature are bull streptomycete (streptomyces tauricus), preserving number CGMCC No.13735.
Described bull streptomycete (streptomyces tauricus) has the work(for suppressing bacterium and fungi growth simultaneously
Can, therefore can be used in the industrial production of essence spice for cigarette, can effectively suppress simultaneously in essence spice for cigarette bacterium and
The growth of yeast-like fungi, so as to prevent essence spice for cigarette from going bad.
Utilize the rotten method for optimizing of bull streptomycete (streptomyces tauricus) prevention essence spice for cigarette：
5ug bull streptomycete active materials are added by every milliliter of essence spice for cigarette, optimal antiseptic effect can be obtained.
Compared with prior art, the present invention has advantages below：
It is the one of tobacco leaf production technology 1. biological prevention is successfully introduced into essence spice for cigarette production by the present invention
Individual important breakthrough.Experimental data shows that bull streptomycete (streptomyces tauricus) can effectively suppress to cause simultaneously
The rotten bacterium of essence and flavoring agent and the growth of yeast-like fungi.
2. bull streptomycete disclosed by the invention and using green non-pollution, environmental sound.
3. technique is simple, operation is easy, and cost is low, suitable for popularization and application, has product to promoting cigarette industry to develop in a healthy way
Pole meaning.
The explanation of preservation biomaterial
The bacterial strain of the present invention, on 03 08th, 2017, it is general to be deposited in China Committee for Culture Collection of Microorganisms
Logical microorganism center (CGMCC)；The centre address：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3, the Chinese Academy of Sciences is micro-
Biological study institute.The Classification And Nomenclature of the bacterial strain is bull streptomycete (streptomyces tauricus)；Preserving number is CGMCC
No.13735。
Embodiment
Below by embodiment, the present invention is described in further detail, but embodiment is not to the technology of the present invention side
The restriction of case.
Embodiment 1：The separation and identification of bull streptomycete (streptomyces tauricus)
(1) soil sample is handled
Mountain gathers soil sample natural air drying after the Panlong District Yunnan Prov Agriculture University of Chinese yunnan province Kunming, weighs 1g soil samples in Sheng
There are 99mL sterilized waters and be equipped with the triangular flask of bead, vibrate 20min, make the thalline in soil, gemma or spore uniformly divide
Dissipate, this is 10‐2The Soil Slurry of concentration, 30s is stood, separately takes three, the test tube equipped with 9ml sterilized waters, numbering 10‐3, 10‐4, 10‐5.10 are taken with aseptic straw sterile working‐2The Soil Slurry 1mL of concentration simultaneously adds numbering 10‐3Sterile test tube in, blow
Inhale 3 times, make to mix with 9mL water, as 10‐3The Soil Slurry of concentration, by that analogy until being diluted to 10‐5.(each dilution
Degree changes an aseptic straw), dilution is aseptically carried out.
(2) bacterial strain purifying culture
10 are taken respectively‐3, 10‐4, 10‐5The μ L of Soil Slurry 100 are spread evenly across on Gause I culture medium, 37 DEG C of constant temperature
Culture, when media surface grow different shape, color bacterium colony when, by the bacterium colony of different shape, take single bacterium colony in fresh training
Culture of being rule on base is supported, untill so repeating when newly longer bacterium form and single color, that is, obtains the bacterium of pure culture
Strain.Using -80 DEG C of Cord bloods of glycerine.
(3) bacterial strain is identified
The method being combined using morphology with molecular biology is to separated microbial identification.
Molecular biology identification:The present invention has isolated one plant of superior microorganism YNAU-1 as experimental bacteria from soil
Kind, performing PCR amplification is entered to the 16S rDNA sequences of the bacterial strain, the chemotaxonomy for actinomyces is identified.Actinomyces PCR is expanded
Primer be：PA(5'‐GAGAGTTTGATCCTGGCTCAG‐3')PB(5'‐CTACGGCTACCTTGTTACGA‐3').Primer by
Shanghai Sheng Gong bioengineering Co., Ltd synthesizes.Using 2 × Power Taq PCR Master Mix, 25 μ L reaction systems, put
Line bacterium reaction condition：94 DEG C of pre-degeneration 6min, 94 DEG C of denaturation 1min, 53 DEG C of annealing 1min, 72 DEG C extend 2min, 30 circulations；
Last 72 DEG C of extensions 10min.Qualification result shows that the bacterium belongs to streptomyces, and its Classification And Nomenclature is bull streptomycete
(streptomyces tauricus), preserving number are CGMCC No.13735.
Morphological Identification:Conventionally analyzed and identified according to physio-biochemical characteristics such as the form of bacterium colony, colors.
The morphological feature of bull streptomycete (streptomyces tauricus) bacterial strain of the present invention is：Bacterium colony pink, point
Shape, surface is smooth, circular or ellipse.
The bull streptomycete of embodiment 2 (streptomyces tauricus) fungistatic effect is tested.
Pass through lot of experiments early stage, the results showed that：Bayer Zygosaccharomyces, Pasteur's acetobacter, soil coccus, gemma bar
Bacterium is the major microorganisms for causing essence spice for cigarette concentrate rotten.
1st, Preparatory work of experiment
(1) sample：Essence spice for cigarette：Purple cloud and soft treasure；
(2) prepared by culture medium：
Actinomyces use Gause I culture medium, and bacterium and fungi use LB culture mediums.
LB plating mediums, 1L formulas are tryptone (10g), dusty yeast (5g), agar (16g), sodium chloride (10g),
Distilled water is supplemented to 1000mL, 7.0~7.2,121 DEG C of high pressure steam sterilization 20min of regulation pH.
Gause I culture medium：Soluble starch (20g), KNO3 (1g), K2HPO4(0.5g),MgSO4·7H2O
(0.5g),NaCl(0.5g),FeSO4·7H2O (0.01g), adjust pH7.2-7.4121 DEG C of high pressure steam sterilization 20min.
The preparation of Gause I synthetic media：First soluble starch is weighed up, adjusted in small beaker with 50~100ml water
Into pasty state, then 900~950ml hot water is added in another container, starch in small beaker is poured into mixing.Weigh respectively again other
Medicine, and after heating stirring is allowed to dissolving, pH to 7.2~7.4, packing are adjusted, 0.1Mpa (15lb/in2) 15~30min high pressures are steamed
Vapour sterilizes.
2nd, bacteriostatic experiment
(1) separation from essence and flavoring agent is taken to identify four kinds of bacterium (Bayer combining yeast, Pasteur's acetobacter, the soil ball come
Bacterium, bacillus) the 2mL centrifuge tubes equipped with LB fluid nutrient mediums are respectively put into, it is standby in constant temperature oscillator vibration 24h.
(2) 100uL is taken to be evenly coated on Gause I culture medium respectively with liquid-transfering gun above-mentioned 4 kinds of bacteria suspensions.
(3) it is connected on and is scribbled with a small amount of bull streptomycete (streptomyces tauricus) after purification of transfer needle picking
On the Gause I culture medium of bacterium solution, each 3~6 repetitions of culture dish, each 3 processing of bacterium, culture is observed thin after 3~4 days
The growing state of bacterium.
(4) judge whether actinomyces have to the microorganism for causing essence and flavoring agent rotten by seeing whether to produce inhibition zone
Inhibition.
The bull streptomycete of table 1 is to the antagonism of the 4 kinds of microorganisms separated from the essence and flavoring agent that goes bad
Note：In table no inhibition zone is represented for 6 repetition average value "-"
Table 1 shows, bull streptomycete to four kinds of microorganisms isolated in the essence and flavoring agent that goes bad (Bayer Zygosaccharomyces,
Pasteur's acetobacter, soil coccus, bacillus) there is obvious inhibition.
Embodiment 3：Bacteriostatic experiment of the bull streptomycete to the essence and flavoring agent that goes bad
A. bull streptomycete after purification is inoculated in Gause I fluid nutrient medium, 30 DEG C of 2 weeks of shaking table culture,
Zymotic fluid is made.
B. rotten purple cloud and soft precious essence and flavoring agent with liquid-transfering gun 100uL will have been taken to be evenly coated on LB culture mediums respectively.
C. a small amount of bull streptomycete fermentation liquid of picking is connected on transfer needle and scribbles the LB of bacterium solution and accompany on foster base, each culture
3~6 repetitions of ware, design 3 processing, and the growing state of bacterium is observed in culture after 3~4 days.
D. judge whether actinomyces have to the microorganism for causing essence and flavoring agent rotten by seeing whether to produce inhibition zone
Inhibition.
Antagonism of the bull strepto- zymotic fluid stoste of table 2 to microorganism in the essence and flavoring agent that goes bad
The result of table 2 shows that bull streptomycete fermentation liquid stoste has obvious suppression effect to microorganism in rotten essence and flavoring agent
Fruit
Embodiment 4：Suppress bacterium and yeast using zymotic fluid secondary metabolite
A. bull streptomycete after purification is inoculated in Gause I fluid nutrient medium, 30 DEG C of 2 weeks of shaking table culture,
Zymotic fluid is made.
B. 2 times of volumes of zymotic fluid ethyl acetate are extracted 3 times, are evaporated acetic acid ethyl acetate extract.
C. rotten purple cloud and soft precious essence and flavoring agent with liquid-transfering gun 100uL will have been taken to be evenly coated on LB culture mediums respectively.
D. it is connected on a small amount of bull streptomycete extract of transfer needle picking and scribbles the LB of bacterium solution and accompany on foster base, each culture dish
The growing state of bacterium is observed in 3~6 repetitions, culture after 3~4 days.
E. judge whether actinomyces have to the microorganism for causing essence and flavoring agent rotten by seeing whether to produce inhibition zone
Inhibition.
Antagonism of the bull streptomycete extract of table 3 to microorganism in essence and flavoring agent
The result of table 3 shows that bull streptomycete extract has obvious inhibition to microorganism in rotten essence and flavoring agent.
Embodiment 5：Suppress bacterium and yeast using bull streptomycete active material
Bull streptomycete after purification is inoculated in Gause I fluid nutrient medium by A, 30 DEG C of 2 weeks of shaking table culture,
Zymotic fluid is made.
B. 2 times of volumes of zymotic fluid ethyl acetate are extracted 3 times, are evaporated acetic acid ethyl acetate extract.
C. bull streptomycete extract is pressed into 1ug/mL, 3ug/mL, 5ug/mL are added in essence and flavoring agent.
D. experimental group and control sample are loaded into 150ml triangular flasks, sealing, is placed in 30 DEG C of constant temperature oscillator cultures 7 days.
Aseptically, using spread plate method, take RZ and ZY samples each to handle each 10ul and mixed respectively with 990ul sterilized waters
Conjunction is uniform, then takes 100 μ L biased samples to be spread evenly across on LB culture mediums, each 3 repetitions, and 3~5d is cultivated under 30 DEG C of constant temperature
Observe and record microbe colony quantity.Judge bull streptomycete active material whether to causing by micro organism quantity change
The microorganism of essence and flavoring agent quality comparison has inhibitory action.
The bull streptomycete active material bacteria inhibition assay of table 4
The result of table 4 shows, adds 5ug bulls streptomycete to live by every milliliter of essence spice for cigarette bull streptomycete active material
Property material ratio, add in essence spice for cigarette, maximum antiseptic effect can be got, it is right to the bacteriostasis rate of purple cloud up to 83.33%
Soft precious bacteriostasis rate is up to 87.5%.
It is demonstrated experimentally that bull streptomycete disclosed by the invention (streptomyces tauricus) is to causing more than
The rotten primary Microbial Populations in Active Bayer combining yeast of essence spice for cigarette, Pasteur's acetobacter, soil coccus, bacillus has bright
Aobvious inhibition zone, bull streptomycete active material have obvious inhibition to microorganism in essence and flavoring agent, when addition concentration is
During 5ug/ml bull streptomycete active materials, bacteriostasis rate is up to 83.33%~87.5%.It is to cause cigarette to be used that experiment, which is proved microorganism,
The main reason for essence and flavoring agent goes bad, therefore the bull streptomycete (streptomyces tauricus) disclosed in this experiment is right
Essence spice for cigarette has obvious antiseptic effect, and new prevention and control direction is provided for essence spice for cigarette quality Control Technology.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (4)
- A 1. breeding oxen streptomycete, it is characterised in that：The Classification And Nomenclature of the bacterial strain is bull streptomycete (streptomyces Tauricus), China Committee for Culture Collection of Microorganisms's common micro-organisms center was deposited on 03 08th, 2017, protected It is CGMCC No.13735 to hide numbering.
- 2. bull streptomycete (streptomyces tauricus) CGMCC No.13735 described in claim 1 press down at the same time Application in bacterium processed and yeast-like fungi.
- 3. bull streptomycete (streptomyces tauricus) CGMCC No.13735 described in claim 1 are preventing cigarette With application of the essence and flavoring agent in rotten.
- 4. bull streptomycete (streptomyces tauricus) CGMCC No.13735 according to claim 3 are anti- Application during only essence spice for cigarette is rotten, it is characterised in that：5ug bulls streptomycete is added by every milliliter of essence spice for cigarette to live Property material.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108587960A (en) * | 2018-04-28 | 2018-09-28 | 中山大学 | A kind of fragrant actinomycetes strain of production and its application |
| CN109295157A (en) * | 2018-10-24 | 2019-02-01 | 云南中烟工业有限责任公司 | A kind of Brazil's aspergillus is used for the purposes and method of cigarette mould inhibitor fungistatic effect indicator bacteria |
| CN109303915A (en) * | 2018-08-01 | 2019-02-05 | 西北民族大学 | A kind of application prepared with D actinomycin D FGR for treating the drug of human cervical carcinoma cell Hela Xenografts in nude mice |
| CN109306331A (en) * | 2018-08-01 | 2019-02-05 | 西北民族大学 | The bull streptomycete bacterial strain screening method and identification method of one plant of production anti-cancer active matter |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108587960A (en) * | 2018-04-28 | 2018-09-28 | 中山大学 | A kind of fragrant actinomycetes strain of production and its application |
| CN109303915A (en) * | 2018-08-01 | 2019-02-05 | 西北民族大学 | A kind of application prepared with D actinomycin D FGR for treating the drug of human cervical carcinoma cell Hela Xenografts in nude mice |
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| CN109295157A (en) * | 2018-10-24 | 2019-02-01 | 云南中烟工业有限责任公司 | A kind of Brazil's aspergillus is used for the purposes and method of cigarette mould inhibitor fungistatic effect indicator bacteria |
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