CN107541475B - Streptomyces bulleyi and application thereof - Google Patents
Streptomyces bulleyi and application thereof Download PDFInfo
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Abstract
The invention discloses a streptomyces bulleyana and application thereof. The strain is classified and named as streptomyces tauricus (Streptomyces tauricus), and is preserved in China general microbiological culture Collection center on 03-08 month 2017 with the preservation number of CGMCC No. 13735. The streptomyces taurus (streptomyces taurus) has the effect of simultaneously inhibiting the growth of bacteria and yeast fungi, can be used for preventing the deterioration of tobacco essence and flavor, and has important value on the improvement of the quality control technology of the tobacco essence and flavor.
Description
Technical Field
The invention belongs to the technical field of applied microorganisms, and particularly relates to streptomyces bullae capable of inhibiting growth of bacteria and fungi simultaneously. Meanwhile, the invention also relates to the application of the streptomyces bullae in tobacco production.
Background
The essence and spice for cigarettes has very important function as an important auxiliary material in the production of cigarettes, and the stability of the quality of the essence and spice for cigarettes is vital to maintaining the normal production of the cigarettes and ensuring the quality of the cigarettes. But the essence spices for cigarette has the problem of easy spoilage in the storage process, through a large amount of experiments, look for and analyze the reason from multiple aspect, multi-angle, the result shows: four microorganisms mainly exist in the metamorphic microorganism, namely Bayer associative yeast, acetobacter pasteurianus, agrobacterium tumefaciens and bacillus. At present, researches on quality control technologies of tobacco flavors and fragrances are few, and particularly, no effective solution is provided for the problem of decay and deterioration of the tobacco flavors and fragrances.
Actinomycetes are a kind of microbial resource with wide practical use and have close relationship with human beings. Statistically, 61.7% of the antibiotics found are produced by actinomycetes. Researchers have conducted a great deal of research into screening applications for actinomycetes, such as: a bactericide product disclosed by Yunnan university and Yunnan province agricultural science institute is prepared by separating and extracting fermentation culture by using Streptomyces bulleyana (Streptomyces tauricus) ECO 00008 with the preservation number of CGMCC No.1682, breaking the wall of mycelium by using acetone or methanol, mixing with fermentation supernatant, adsorbing and separating by macroporous adsorption resin, eluting by acetone or methanol, and concentrating or further purifying eluent to obtain the neophosphazene A. The molecular formula of the neophosphazene A is C31H51N2O10P, and the molecular weight is 642.72 daltons. And further adding a carrier or an auxiliary agent to obtain a finished bactericide product. The bactericide has good effects of preventing and treating rice blast, tobacco brown spot, tomato gray mold and pepper anthracnose. Compared with the existing medicament, has the advantages of small dosage, low use cost for farmers, wide application range and the like. An application of neoazalomycin A in inhibiting tobacco anthracnose is disclosed by the research institute of agricultural environmental resources of agricultural academy of sciences in Yunnan province. The novel phosphazene A is used as a secondary metabolite of actinomycetes, has low toxicity, high fermentation yield and easily obtained raw materials, has strong inhibition effect on the growth of hyphae of tobacco anthracnose pathogen, and can be used for developing novel invention research and application exploration of preparation development aiming at target fungal diseases mainly including tobacco anthracnose.
However, in the prior art, reports about simultaneous inhibition of bacteria and fungi by actinomycetes are very few, and particularly, reports about simultaneous inhibition of bacteria and yeast fungi in tobacco flavors and fragrances by streptomyces bulleyana are not yet reported.
Disclosure of Invention
The invention aims to provide streptomyces bulleyi capable of simultaneously inhibiting bacteria and yeast fungi aiming at the defects of the prior art.
The invention also aims to provide the application of the streptomyces bullae in tobacco production.
The purpose of the invention is realized by the following technical scheme.
Unless otherwise stated, all percentages used in the present invention are mass percentages.
The invention separates a dominant microorganism YNAU-1 from soil as a test strain, carries out 16S rDNA analysis on the test strain, and the identification result shows that the bacterium belongs to actinomycete streptomyces, is classified and named as streptomyces bovis (streptomyces tauricus) with the collection number of CGMCC No. 13735.
According to the physiological and biochemical characteristics of colony, its color and others, the analysis and identification are carried out according to the conventional method. The morphological characteristics of the streptomyces taurus strain are as follows: the colony is pink, punctate, smooth in surface, round or oval.
And (3) molecular biology identification, namely performing PCR amplification on a 16S rDNA sequence of the YNAU-1 strain for molecular taxonomic identification of actinomycetes. The primers for PCR amplification of the actinomycetes are as follows: PA (5'-GAGAGTTTGATCCTGGCTCAG-3') PB (5'-CTACGGCTACCTTGTTACGA-3'). The primers were synthesized by Shanghai Bioengineering Co., Ltd. 2 XPower TaqPCR Master Mix, 25. mu.L reaction system, actinomycetes reaction conditions: pre-denaturation at 94 deg.C for 6min, denaturation at 94 deg.C for 1min, annealing at 53 deg.C for 1min, extension at 72 deg.C for 2min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃. The identification result shows that the bacterium belongs to actinomycete streptomyces, and the bacterium is classified and named as streptomyces taurus (Streptomyces tauricus) with the preservation number of CGMCC No. 13735.
The streptomyces taurus has the function of simultaneously inhibiting the growth of bacteria and fungi, so that the streptomyces taurus can be used in the industrial production of tobacco flavor and fragrance, and can simultaneously and effectively inhibit the growth of bacteria and yeast fungi in the tobacco flavor and fragrance, thereby preventing the tobacco flavor and fragrance from deteriorating.
A preferable method for preventing the deterioration of the tobacco flavor and fragrance by using streptomyces taurus comprises the following steps: the best antiseptic effect can be obtained by adding 5ug of streptomyces bullae active substance into each milliliter of the essence and flavor for tobacco.
Compared with the prior art, the invention has the following advantages:
1. the invention successfully introduces the biological control technology into the production of tobacco flavors and fragrances, and is an important breakthrough of the tobacco production technology. Experimental data show that streptomyces taurus (streptomyces taurus) can simultaneously and effectively inhibit the growth of bacteria and yeast fungi which cause the deterioration of flavors and fragrances.
2. The streptomyces bulleyi disclosed by the invention is green and pollution-free and is harmless to the environment.
3. Simple process, easy operation, low cost, suitability for popularization and application and positive significance for promoting the healthy development of the cigarette industry.
Description of preservation of biological Material
The strain of the invention has been preserved in China general microbiological culture Collection center (CGMCC) in 2017, 03 and 08 months; the central address is: western road No.1 institute 3, institute of microbiology, china academy of sciences, north chen, chaoyang, china. The classification name of the strain is streptomyces taurus; the preservation number is CGMCC No. 13735.
Detailed Description
The present invention will be described in further detail with reference to examples, which are not intended to limit the scope of the present invention.
Example 1: isolation and characterization of Streptomyces taurinus
(1) Treatment of soil samples
Collecting soil sample from the back mountain of Yunnan agricultural university in Panlongdistrict of Kunming city, Yunnan province, China, air drying, weighing 1g of soil sample in a triangular flask containing 99mL of sterile water and glass beads, and oscillating for 20min to uniformly disperse thallus, spore or spore in soil to obtain 10‐2Standing the soil suspension for 30s, and taking three test tubes with 9ml of sterile water, wherein the number is 10‐3，10‐4，10‐5. Sterile-fetching 10 with sterile sucker‐21mL of the concentrated soil suspension was added to number 10‐3In the sterile test tube, 3 times of blowing and sucking are carried out to ensure that the sterile test tube is uniformly mixed with 9mL of water to obtain 10‐3Soil suspension of concentration, and so on until diluted to 10‐5. (sterile pipette is replaced for each dilution) and the dilution process is performed under sterile conditions.
(2) Purifying and culturing strain
Respectively take 10‐3，10‐4，10‐5Uniformly coating 100 mu L of soil suspension on a Gao's first culture medium, culturing at a constant temperature of 37 ℃, taking single colonies from the colonies with different forms on a fresh culture medium when the surface of the culture medium grows colonies with different forms and colors, and carrying out streak culture on the single colonies, repeating the steps until the newly grown colonies are single in form and color, thus obtaining the pure cultured strain. Storing at-80 deg.C with glycerol.
(3) Identification of strains
The isolated microorganisms are identified by a method combining morphology with molecular biology.
Molecular biology identification, the invention separates a dominant microorganism YNAU-1 from soil as an experimental strain, and performs PCR amplification on a 16S rDNA sequence of the strain for molecular taxonomic identification of actinomycetes. The primers for PCR amplification of the actinomycetes are as follows: PA (5'-GAGAGTTTGATCCTGGCTCAG-3') PB (5'-CTACGGCTACCTTGTTACGA-3'). The primers were synthesized by Shanghai Bioengineering Co., Ltd. 2 XPower Taq PCR Master Mix, 25. mu.L reaction system, actinomycetes reaction conditions: pre-denaturation at 94 deg.C for 6min, denaturation at 94 deg.C for 1min, annealing at 53 deg.C for 1min, extension at 72 deg.C for 2min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃. The identification result shows that the bacterium belongs to streptomyces, the classification name of the bacterium is streptomyces tarucicus, and the preservation number is CGMCC No. 13735.
And (3) morphological identification, namely analyzing and identifying according to physiological and biochemical characteristics such as the morphology, the color and the like of the colony by a conventional method. The morphological characteristics of the streptomyces taurus strain are as follows: the colony is pink, punctate, smooth in surface, round or oval.
Example 2 experiment of bacteriostatic effect of Streptomyces taurus.
Through a large number of experimental studies in the early stage, the results show that: zygosaccharomyces Bayer, Acetobacter pasteurianus, Agrobacterium and Bacillus are the main microorganisms causing the deterioration of the concentrated solution of the tobacco flavor and fragrance.
1. Preparation of the experiment
(1) Sample preparation: the tobacco essence and flavor: purple clouds and soft delicacies;
(2) preparing a culture medium:
the actinomycetes adopt a Gao's first culture medium, and the bacteria and the fungi adopt an LB culture medium.
LB plate culture medium, 1L formula is tryptone (10g), yeast powder (5g), agar (16g), sodium chloride (10g), supplement distilled water to 1000mL, adjust pH 7.0 ~ 7.2, 121 ℃ high pressure steam sterilization for 20 min.
Gao's first medium: soluble starch (20g), KNO3(1g), K2HPO4(0.5g),MgSO4·7H2O(0.5g),NaCl(0.5g),FeSO4·7H2O (0.01g), and autoclaving at pH7.2-7.4121 deg.C for 20 min.
Preparation of a Gao's I synthetic medium: weighing soluble starch, mixing the soluble starch into paste with 50-100 ml of water in a small beaker, adding 900-950 ml of hot water in another container, and pouring the starch in the small beaker into the small beaker and mixing the starch uniformly. Then weighing other medicines respectively, heating and stirring to dissolve the medicines, adjusting the pH value to 7.2-7.4, subpackaging, and sterilizing by high-pressure steam at 0.1Mpa (15lb/in2) for 15-30 min.
2. Experiment for inhibiting bacteria
(1) The four kinds of bacteria (Bayer associated yeast, Acetobacter pasteurianus, Agrobacterium and Bacillus) separated and identified from the essence and the spice are respectively put into a 2mL centrifuge tube filled with LB liquid culture medium and are shaken for 24h for standby application in a constant temperature oscillator.
(2) 100uL of the 4 bacterial suspensions are respectively and evenly coated on a Gao's No. I culture medium by using a pipette gun.
(3) And (3) picking a small amount of purified streptomyces taruis by using an inoculating needle, inoculating the purified streptomyces taruis on a Gao-shi culture medium coated with bacterial liquid, repeating the steps for 3-6 times in each culture dish, treating 3 bacteria, and observing the growth condition of the bacteria after culturing for 3-4 days.
(4) Whether the actinomycetes have the inhibiting effect on microorganisms causing the deterioration of the essence and the spice is judged by observing whether the inhibition zone is generated.
TABLE 1 antagonistic action of Streptomyces bullae against 4 microorganisms isolated from a flavor and fragrance which has been deteriorated
Note: the average value "-" of 6 replicates in the table indicates no zone of inhibition
Table 1 shows that the streptomyces bulleyi has obvious inhibiting effect on four microorganisms (Bayer zygosaccharomyces bailii, Acetobacter pasteurianus, Agrobacterium tumefaciens and bacillus) separated from deteriorated flavors and fragrances.
Example 3: bacteriostatic experiment of streptomyces bulleyi on deteriorated essence and spice
A. Inoculating the purified streptomyces bulleyi into a Gao's I liquid culture medium, and performing shake culture at 30 ℃ for 2 weeks to prepare a fermentation liquid.
B. Uniformly coating 100uL of deteriorated Ziyun and soft-pearl essence and flavor on LB culture medium by using a liquid-transfering gun.
C. And (3) inoculating a small amount of streptomyces bullae fermentation liquor on an LB (lysogeny broth) culture medium coated with a bacterial liquid by using an inoculating needle, repeating for 3-6 times in each culture dish, designing 3 treatments, and observing the growth condition of bacteria after culturing for 3-4 days.
D. Whether the actinomycetes have the inhibiting effect on microorganisms causing the deterioration of the essence and the spice is judged by observing whether the inhibition zone is generated.
TABLE 2 antagonistic Effect of stock solution of Bull-Streptomyces fermentation broth on microorganisms in deteriorated flavors and fragrances
The results in Table 2 show that the Streptomyces bullae fermentation broth stock solution has obvious inhibition effect on microorganisms in deteriorated flavors and fragrances.
Example 4: inhibiting bacteria and yeast by using secondary metabolite of fermentation liquid
A. Inoculating the purified streptomyces bulleyi into a Gao's I liquid culture medium, and performing shake culture at 30 ℃ for 2 weeks to prepare a fermentation liquid.
B. Extracting fermentation liquor ethyl acetate 2 times by volume for 3 times, and evaporating ethyl acetate extract to dryness.
C. Uniformly coating 100uL of deteriorated Ziyun and soft-pearl essence and flavor on LB culture medium by using a liquid-transfering gun.
D. And (3) picking a small amount of streptomyces bullae extract liquor by using an inoculating needle, inoculating the streptomyces bullae extract liquor on an LB (lysogeny broth) culture medium coated with a bacterial liquid, repeating the operation for 3-6 times in each culture dish, and observing the growth condition of the bacteria after culturing for 3-4 days.
E. Whether the actinomycetes have the inhibiting effect on microorganisms causing the deterioration of the essence and the spice is judged by observing whether the inhibition zone is generated.
TABLE 3 antagonistic action of Streptomyces bullae extract on microorganisms in flavors and fragrances
The results in Table 3 show that the streptomyces bullae extract has obvious inhibiting effect on microorganisms in deteriorated flavors and fragrances.
Example 5: inhibition of bacteria and yeast by streptomyces bullae active substances
And A, inoculating the purified streptomyces bulleyi into a Gao's I liquid culture medium, and performing shake culture at 30 ℃ for 2 weeks to prepare a fermentation liquid.
B. Extracting fermentation liquor ethyl acetate 2 times by volume for 3 times, and evaporating ethyl acetate extract to dryness.
C. Adding the streptomyces bullae extract into essence and spice according to the ratio of 1ug/mL,3ug/mL and 5 ug/mL.
D. The samples of the experimental group and the control group were filled in a 150ml triangular flask, sealed, and cultured in a 30 ℃ constant temperature shaker for 7 days. Under the aseptic condition, adopting a plate coating culture method, taking 10ul of each processed RZ and ZY sample, respectively, and uniformly mixing with 990ul of sterile water, then taking 100 mu L of the mixed sample, uniformly coating on an LB culture medium, repeating 3 times, culturing at the constant temperature of 30 ℃ for 3-5 days, observing and recording the number of microbial colonies. And judging whether the streptomyces bulleyi active substances have an inhibiting effect on microorganisms causing quality changes of the essences and the spices or not through the change of the microbial quantity.
TABLE 4 Streptomyces bullae active substance antibacterial assay
The results in Table 4 show that the maximum antiseptic effect can be achieved by adding the streptomyces bulleyi active substance into the tobacco flavor perfume according to the proportion of adding 5ug of the streptomyces bulleyi active substance into each milliliter of the tobacco flavor perfume, the bacteriostatic rate on the purple cloud reaches 83.33%, and the bacteriostatic rate on the soft pearl reaches 87.5%.
The experiments prove that the streptomyces tararicus disclosed by the invention has an obvious inhibition zone on main microorganism populations of Bayer-associated yeast, Acetobacter pasteurianus, soil coccus and Bacillus which cause the deterioration of tobacco flavors and fragrances, the active substances of the streptomyces tararicus have an obvious inhibition effect on microorganisms in the flavors and fragrances, and when the active substances of the streptomyces tararicus are added at a concentration of 5ug/ml, the inhibition rate reaches 83.33% -87.5%. Experiments prove that microorganisms are the main cause of deterioration of the tobacco flavor and fragrance, so that the streptomyces taurus (streptomyces taurus) disclosed in the experiments has an obvious anti-corrosion effect on the tobacco flavor and fragrance, and provides a new prevention and control direction for the quality prevention and control technology of the tobacco flavor and fragrance.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (3)
1. A streptomyces bulleyi is characterized in that: the strain is classified and named as streptomyces bovis (Streptomyces tauricus), and is preserved in China general microbiological culture Collection center on 03-08.2017 with the preservation number of CGMCC No. 13735.
2. The use of Streptomyces taurus (CGMCC No. 13735) as claimed in claim 1 for inhibiting both of Saccharomyces bayanus, Acetobacter pasteurianus, Agrobacterium and Bacillus.
3. The application of streptomyces taurus (streptomyces tauricus) CGMCC No.13735 in preventing tobacco flavor and fragrance from deteriorating as follows: adding 5ug of streptomyces bullae active substance into each ml of tobacco flavor and fragrance.
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| CN108587960A (en) * | 2018-04-28 | 2018-09-28 | 中山大学 | A kind of fragrant actinomycetes strain of production and its application |
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| 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 |
| 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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