CN108330077B - A kind of yellow dark streptomyces and its application - Google Patents

A kind of yellow dark streptomyces and its application Download PDF

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CN108330077B
CN108330077B CN201710276460.0A CN201710276460A CN108330077B CN 108330077 B CN108330077 B CN 108330077B CN 201710276460 A CN201710276460 A CN 201710276460A CN 108330077 B CN108330077 B CN 108330077B
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李源栋
刘秀明
段焰青
岳蕊
杨根华
董文汉
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China Tobacco Yunnan Industrial Co Ltd
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    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
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Abstract

The invention discloses a streptomyces flavochromogenes and application thereof. The strain is classified and named as Streptomyces xanthophaeus (Streptomyces xanthophaeus), and is preserved in China general microbiological culture collection center on 03-08 th month 2017 with the preservation number of CGMCC No. 13734. The Streptomyces xanthochrous (Streptomyces xanthophaeus) has the effect of inhibiting the growth of acetobacter pasteurianus, agrobacterium tumefaciens and bacillus, can be used for preventing the deterioration of tobacco essence and flavor, and has important value for improving the quality control technology of the tobacco essence and flavor.

Description

Streptomyces flavochromogenes and application thereof
Technical Field
The invention belongs to the technical field of applied microorganisms, and particularly relates to streptomyces flavochromogenes capable of inhibiting bacterial growth. Meanwhile, the invention also relates to the application of the streptomyces flavochromogenes in tobacco production.
Background
Tobacco is one of the important pillars of our country's finances. In the last two decades, tobacco tax accounts for about 10% of national financial income every year, and makes great contribution to the social and economic development of China. 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. Through a large number of experiments, after the reasons are searched and analyzed from multiple layers and multiple angles, the essence and flavor for the deteriorated tobacco mainly contain four microorganisms including Bayer combined yeast, acetobacter pasteurianus, agrobacterium tumefaciens and bacillus. At present, researches on quality control technologies of tobacco flavors and fragrances are few, and particularly, no corresponding measures are provided for solving 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. In the prior art, researches on streptomyces as bactericides are recorded, but the application of streptomyces in preventing and treating the spoilage of tobacco flavors and fragrances is not reported.
Disclosure of Invention
The invention aims to provide streptomyces flavochromogenes capable of inhibiting the growth of acetobacter pasteurianus, agrobacterium tumefaciens and bacillus aiming at overcoming the defects of the prior art.
The invention also aims to provide the application of the streptomyces luteologen 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-2 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 xanthophaeus (Streptomyces xanthophaeus) and has the collection number of CGMCC No. 13734.
The morphological characteristics of the Streptomyces xanthophaeus strain on a solid culture medium of a Gao-shi I are as follows: aerial hyphae are off-white to purple-gray, and intrabasal hyphae are light yellow to yellowish-brown and produce a small amount of light yellow soluble pigment.
The Streptomyces xanthophaeus strain is characterized by utilizing a general primer PA (5'-GAGAGTTTGATCCTGGCTCAG-3') PB (5'-CTACGGCTACCTTGTTACGA-3') synthesized by Shanghai biological engineering limited company, amplifying by taking genomic DNA of the Streptomyces xanthochrous strain as a template to obtain a PCR product of about 1500bp, and recovering, sequencing and correcting a partial 16S rDNA gene sequence of the Streptomyces xanthochrous strain. And carrying out PCR amplification on the 16S rDNA sequence of the strain for molecular taxonomic identification of actinomycetes. 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 actinomycete Streptomyces, and the bacterium is classified and named as Streptomyces xanthophaeus.
The Streptomyces xanthochrous (Streptomyces xanthophaeus) has exact bacteriostatic effect, and can effectively inhibit the growth of acetobacter pasteurianus, agrobacterium tumefaciens and bacillus in the tobacco flavor and fragrance, thereby preventing the tobacco flavor and fragrance from deteriorating.
A preferred method for preventing the deterioration of the tobacco flavor and fragrance by using Streptomyces xanthophaeus comprises the following steps: the best antiseptic effect can be obtained by adding 5ug of streptomyces flavochromogenes active substance into each milliliter of tobacco flavor and fragrance.
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. The experimental data show that Streptomyces xanthochrous (Streptomyces xanthophaeus) has 3 dominant bacteria causing deterioration of tobacco flavor and fragrance: acetobacter pasteurianus, Agrobacterium and Bacillus have obvious inhibiting effect.
2. The streptomyces flavochromogenes and the application thereof disclosed by the invention are green, pollution-free and 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 strain is classified and named as Streptomyces xanthophaeus; the preservation number is CGMCC No. 13734.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail by examples and experimental data below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of the invention.
Example 1: isolation and characterization of Streptomyces xanthophaeus
(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-2 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; and finally 72Extend at deg.C for 10 min. The identification result shows that the bacterium belongs to actinomycete Streptomyces, and the bacterium is classified and named as Streptomyces xanthophaeus (Streptomyces xanthophaeus) with the preservation number of CGMCC No. 13734.。
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 xanthophaeus strain on a solid culture medium of a Gao-shi I are as follows: aerial hyphae are off-white to purple-gray, and intrabasal hyphae are light yellow to yellowish-brown and produce a small amount of light yellow soluble pigment.
Example 2 Streptomyces xanthus (Streptomyces xanthophaeus) CGMCC No.13734 bacteriostatic effect experiment.
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: ziyun and Runzhen
(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) inoculating a small amount of purified Streptomyces luteovirens (Streptomyces xanthophaeus) on the culture medium I of Gao's type coated with the bacterial liquid by using an inoculating needle, repeating the step 3-6 times in each culture dish, culturing each bacterium (Bayer combined yeast, acetobacter pasteurianus, soil coccus and bacillus) in 3 dishes, and observing the growth condition of the bacteria after culturing for 3-4 days.
(4) Whether the streptomyces flavedo has an inhibiting effect on microorganisms causing the deterioration of essences and spices is judged by observing whether an inhibition zone is generated, and the numerical value is averaged.
TABLE 1 antagonistic action of Streptomyces flavedo against 4 microorganisms isolated from deteriorated flavors and fragrances
Figure BDA0001278448110000061
Note: the average value "-" of the 3 treatments in the table indicates that there is no zone of inhibition
Table 1 shows that Streptomyces xanthochrous (Streptomyces xanthophaeus) has obvious inhibition effect on 3 bacteria (Acetobacter pasteurianus, Agrobacterium, Bacillus) separated from deteriorated flavors and fragrances, but has no inhibition effect on Bayer-associated yeast.
Example 3: bacteriostatic experiments of Streptomyces xanthophaeus (Streptomyces xanthophaeus) fermentation liquor on deteriorated flavors and fragrances are carried out.
(1) Inoculating the purified streptomyces flavochromogenes into a Gao's I liquid culture medium, and performing shake culture at 30 ℃ for 2 weeks to prepare a fermentation liquid.
(2) Uniformly coating 100uL of deteriorated Ziyun and soft-pearl essence and flavor on LB culture medium by using a liquid-transfering gun.
(3) And (3) inoculating a small amount of streptomyces luteochromogenes 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.
(4) Whether the streptomyces flavedo has an inhibiting effect on microorganisms causing the deterioration of the essence and the spice is judged by observing whether an inhibition zone is generated.
TABLE 2 antagonistic action of yellow-dark color Streptomyces fermentation broth on microorganisms in flavors and fragrances
Figure BDA0001278448110000071
The results in Table 2 show that the Streptomyces flavedo fermentation broth stock solution has obvious inhibition effect on microorganisms in deteriorated flavors and fragrances
Example 4: inhibiting bacteria by using secondary metabolite of fermentation liquid
(1) Inoculating the purified streptomyces flavochromogenes into a Gao's I liquid culture medium, and performing shake culture at 30 ℃ for 2 weeks to prepare a fermentation liquid.
(2) Extracting fermentation liquor ethyl acetate 2 times by volume for 3 times, and evaporating ethyl acetate extract to dryness.
(3) Uniformly coating 100uL of deteriorated Ziyun and soft-pearl essence and flavor on LB culture medium by using a liquid-transfering gun.
(4) And (3) inoculating a small amount of streptomyces luteochromogenes extract 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, and observing the growth condition of the bacteria after culturing for 3-4 days.
(5) Whether the streptomyces flavedo has an inhibiting effect on microorganisms causing the deterioration of the essence and the spice is judged by observing whether an inhibition zone is generated.
TABLE 3 antagonistic action of Streptomyces flavedo extract on microorganisms in flavors and fragrances
Figure BDA0001278448110000072
Figure BDA0001278448110000081
The results in Table 3 show that the Streptomyces xanthus extract has obvious effect of inhibiting microorganisms in deteriorated flavors and fragrances.
Example 5: the Streptomyces xanthus active substance is used for inhibiting bacteria and yeast.
(1) Inoculating the purified streptomyces flavochromogenes into a Gao's I liquid culture medium, and performing shake culture at 30 ℃ for 2 weeks to prepare a fermentation liquid.
(2) Extracting fermentation liquor ethyl acetate 2 times by volume for 3 times, and evaporating ethyl acetate extract to dryness.
(3) Adding the Streptomyces xanthus active substances into the essence and flavor according to the ratio of 1ug/mL,3ug/mL and 5 ug/mL.
(4) 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 flavedo active substances have an inhibiting effect on microorganisms causing quality changes of the essences and the spices or not through the change of the number of the microorganisms.
TABLE 4 bacteriostatic assay for Streptomyces flavochromogenes active substances
Figure BDA0001278448110000082
Figure BDA0001278448110000091
The results in Table 4 show that the addition of the Streptomyces flavedo active substance into the tobacco flavor and fragrance according to the proportion of adding 5ug of the Streptomyces flavedo active substance into each ml of the tobacco flavor and fragrance can achieve the maximum antiseptic effect, the bacteriostasis rate to purple cloud reaches 75%, and the bacteriostasis rate to soft pearl reaches 91.67%.
The experiments prove that the Streptomyces flavochromogenes (Streptomyces xanthophaeus) disclosed by the invention has an obvious inhibition zone on main microorganism populations including acetobacter pasteurianus, soil coccus and bacillus which cause the deterioration of tobacco flavor and fragrance, the Streptomyces flavochromogenes active substance has an obvious inhibition effect on microorganisms in the flavor and fragrance, and when the Streptomyces flavochromogenes active substance with the concentration of 5ug/ml is added, the inhibition rate reaches 75% -91.67%. Experiments prove that microorganisms are the main cause of deterioration of the tobacco flavor and fragrance, so that the Streptomyces xanthochrous (Streptomyces xanthophaeus) disclosed by the experiment has an obvious preservative effect on the tobacco flavor and fragrance, and a new prevention and control direction is provided 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 (4)

1. A Streptomyces luteus strain is characterized in that: the strain is classified and named as Streptomyces xanthophaeus (Streptomyces xanthophaeus), and is preserved in China general microbiological culture collection center on 03-08 th month 2017 with the preservation number of CGMCC No. 13734.
2. Use of the strain of claim 1 for inhibiting Acetobacter pasteurianus, Agrobacterium and Bacillus.
3. The use of the strain of claim 1 for preventing the deterioration of flavors and fragrances for tobacco.
4. The application of the strain according to claim 3 in preventing tobacco flavor and fragrance from deteriorating is characterized in that: adding 5 mu g of streptomyces flavochromogenes active substance into each ml of tobacco flavor and fragrance.
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