CN108330077A - A kind of yellow dark-coloured streptomycete and its application - Google Patents

Abstract

The invention discloses a Streptomyces aureus and application thereof. The taxonomic name of the strain is Streptomyces xanthophaeus, and it was deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on March 8, 2017, and the preservation number is CGMCC No.13734. The Streptomyces xanthophaeus of the present invention has the effect of inhibiting the growth of Acetobacter pasteurianus, Agroococcus and Bacillus, can be used to prevent the deterioration of flavors and fragrances for tobacco, and has a positive effect on the improvement of the quality control technology of flavors and fragrances for tobacco. important value.

Description

A kind of Streptomyces aureus and application thereof

technical field

The invention belongs to the technical field of applied microorganisms, and in particular relates to a Streptomyces aureus that can inhibit the growth of bacteria. Simultaneously, the present invention also relates to the use of the Streptomyces aureus in tobacco production.

Background technique

Tobacco is one of the important pillars of our country's finances. In the past two decades, tobacco taxation accounts for about 10% of the country's fiscal revenue every year, and has made great contributions to my country's social and economic development. Tobacco flavors and fragrances, as important auxiliary materials, play a very important role in cigarette production, and the stability of their quality is crucial to maintaining the normal production of cigarettes and ensuring the quality of cigarettes. Through a large number of experiments, after searching and analyzing the reasons from multiple levels and angles, it was found that there are mainly four microorganisms including Bayer combined yeast, Acetobacter pasteurianus, Agrococcus and Bacillus in the spoiled tobacco flavors and fragrances. At present, there is very little research on the quality control technology of tobacco flavors and fragrances, especially for the problem of corruption of tobacco flavors and fragrances, there is no corresponding solution.

Actinomycetes are a class of microbial resources with a wide range of practical uses, and are closely related to humans. According to statistics, 61.7% of the discovered antibiotics are produced by actinomycetes. In the prior art, the research on Streptomyces as a bactericide has been recorded, but the application of Streptomyces in preventing and controlling the spoilage of flavors and fragrances for tobacco has not been reported yet.

Contents of the invention

The object of the present invention is to aim at the deficiencies in the prior art, and provide a kind of Streptomyces aureus that can inhibit the growth of Acetobacter pasteurianus, Agrococcus and Bacillus.

The object of the present invention is also to provide the application of the Streptomyces luteus in tobacco production.

The purpose of the present invention is achieved through the following technical solutions.

* Unless otherwise specified, the percentages used in the present invention are all mass percentages.

The present invention isolates a strain of dominant microorganism YNAU‐2 from the soil as a test strain, and performs 16S rDNA analysis on it. The identification result shows that the bacterium belongs to the actinomycetes Streptomyces genus, and its classification is named as Streptomyces spp. xanthophaeus), deposit number CGMCC No.13734.

The morphological characteristics of the described Streptomyces xanthophaeus bacterial strain on Gaoshi No. 1 solid medium are: the aerial hyphae are off-white to purple-gray, and the hyphae in the base are light yellow to yellowish brown, producing a small amount of light yellow Soluble pigments.

Described Streptomyces xanthophaeus (Streptomyces xanthophaeus) strain molecular biology identification: Utilize the universal primer PA (5'-GAGAGTTTGATCCTGGCTCAG-3') PB (5'-CTACGGCTACCTTGTTACGA-3') synthesized by Shanghai Sangong Bioengineering Co., Ltd., A PCR product of about 1500 bp was amplified using the genomic DNA of Streptomyces chrysogenum as a template, and the partial 16S rDNA gene sequence of the Streptomyces luteus strain obtained after sequencing and correction was recovered. The 16S rDNA sequence of the strain was amplified by PCR for molecular taxonomic identification of actinomycetes. Using 2×Power Taq PCRMaster Mix, 25 μL reaction system, actinomycete reaction conditions: 94°C pre-denaturation for 6 minutes, 94°C denaturation for 1 minute, 53°C annealing for 1 minute, 72°C extension for 2 minutes, 30 cycles; final 72°C extension for 10 minutes. The identification results showed that the bacterium belonged to the actinomycetes Streptomyces genus, and its taxonomic name was Streptomyces xantophaeus.

The Streptomyces xanthophaeus has definite bacteriostasis effect and can effectively inhibit the growth of Acetobacter pasteurii, Agrococcus and Bacillus in the flavors and fragrances for tobacco, thereby preventing the flavors and fragrances for tobacco from deteriorating.

The preferred method of using Streptomyces xanthophaeus to prevent deterioration of tobacco flavors and fragrances: adding 5 ug of Streptomyces xanthophaeus active substances per milliliter of tobacco flavors and fragrances can achieve the best antiseptic effect.

Compared with the prior art, the present invention has the following advantages:

1. The present invention successfully introduces biological control technology into the production of tobacco flavor and fragrance, which is an important breakthrough in tobacco production technology. Experimental data show that Streptomyces xanthophaeus has a significant inhibitory effect on three dominant bacteria that cause deterioration of flavors and fragrances used in tobacco: Acetobacter pasteurianus, Agrococcus and Bacillus.

2. The Streptomyces aureus and its application disclosed in the present invention are green and pollution-free, and harmless to the environment.

3. The process is simple, the operation is easy, the cost is low, it is suitable for popularization and application, and it has positive significance for promoting the healthy development of the cigarette industry.

Instructions for Depositing Biological Material

The bacterial strain of the present invention has been preserved in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee on March 8, 2017; the address of the center: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China, Chinese Academy of Sciences Institute of Microbiology. The taxonomic name of the strain is Streptomyces xanthophaeus; the preservation number is CGMCC No.13734.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below through examples and test data. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the technical solution of the present invention.

Embodiment 1: Isolation and identification of Streptomyces xanthophaeus

(1) Soil sample treatment

The soil samples collected from the back mountain of Yunnan Agricultural University in Panlong District, Kunming City, Yunnan Province, China were naturally air-dried, and 1g of soil samples were weighed in a triangular flask filled with 99mL sterile water and glass beads, and shaken for 20min to make the bacteria in the soil , spores or spores are evenly dispersed, this is the soil suspension with a concentration of 10 ^‐2 , let it stand for 30s, and take three test tubes filled with 9ml sterile water, numbered 10 ^‐3 , 10 ^‐4 , 10 ^‐5 . Use a sterile pipette to aseptically take 1mL of the soil suspension with a concentration of ^10-2 and add it to a sterile test tube with a number of ^10-3 , blow and suck 3 times, and mix it with 9mL of water to form a soil suspension with a concentration of ^10-3 solution, and so on until the dilution reaches 10 ^‐5 . (change a sterile pipette for each dilution), and the dilution process is carried out under sterile conditions.

(2) Strain purification and culture

Take 10 ^‐3 , 10 ^‐4 , 10 ^‐5 soil suspension 100 μL and evenly spread it on Gaoshi No. For colonies with different morphology, take a single colony and streak culture on a fresh medium, and repeat this until the newly grown bacteria have a single shape and color, and a purely cultured strain is obtained. Store in glycerol at -80°C.

(3) Strain identification

The isolated microorganisms were identified by a combination of morphological and molecular biology methods.

Molecular biological identification: the present invention isolated a strain of dominant microorganism YNAU‐2 from the soil as an experimental strain, and carried out PCR amplification of the 16S rDNA sequence of the strain for molecular taxonomic identification of actinomycetes. The primers for PCR amplification of actinomycetes were: PA(5'‐GAGAGTTTGATCCTGGCTCAG‐3')PB(5'‐CTACGGCTACCTTGTTACGA‐3'). Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. Using 2×Power Taq PCR Master Mix, 25 μL reaction system, actinomycete reaction conditions: 94°C pre-denaturation for 6 minutes, 94°C denaturation for 1 minute, 53°C annealing for 1 minute, 72°C extension for 2 minutes, 30 cycles; finally 72°C extension for 10 minutes. The identification results showed that the bacterium belonged to the actinomycetes Streptomyces genus, and its taxonomic name was Streptomyces xanthophaeus (Streptomyces xanthophaeus), and the preservation number was CGMCC No.13734. .

Morphological identification: According to the physiological and biochemical characteristics such as the shape and color of the colony, analyze and identify according to conventional methods. The morphological characteristics of the described Streptomyces xanthophaeus bacterial strain on Gaoshi No. 1 solid medium are: the aerial hyphae are off-white to purple-gray, and the hyphae in the base are light yellow to yellowish brown, producing a small amount of light yellow Soluble pigments.

Example 2 Bacteriostatic effect experiment of Streptomyces xanthophaeus CGMCC No.13734.

Through a large number of experimental studies in the early stage, the results show that: Zygomyces bayer, Acetobacter pasteurianus, Agrococcus, and Bacillus are the main microorganisms that cause the deterioration of the concentrated liquid of flavor and fragrance for tobacco.

1. Experiment preparation

(1) Sample: Tobacco Flavors and Fragrances: Ziyun and Ruanzhen

(2) Culture medium preparation: Gaoshi No. 1 medium was used for actinomycetes, and LB medium for bacteria and fungi.

LB plate medium, 1L formula is tryptone (10g), yeast powder (5g), agar (16g), sodium chloride (10g), supplemented with distilled water to 1000mL, adjusted to pH 7.0-7.2, sterilized by high pressure steam at 121℃ for 20min .

Gaoshi No. 1 medium: soluble starch (20g), KNO3 (1g), K ₂ HPO ₄ (0.5g), MgSO ₄ 7H ₂ O (0.5g), NaCl (0.5g), FeSO ₄ 7H ₂ O (0.01g), adjust the pH to 7.2-7.4, and sterilize by autoclaving at 21°C for 20min.

Preparation of Gaoshi No. 1 synthetic medium: first weigh the soluble starch, make a paste with 50-100ml of water in a small beaker, then add 900-950ml of hot water in another container, pour the starch in the small beaker Mix well. Then weigh other medicines separately, heat and stir to dissolve them, adjust the pH to 7.2-7.4, sub-package, and sterilize by high-pressure steam at 0.1Mpa (15lb/in2) for 15-30min.

2. Antibacterial experiment

(1) Take the four kinds of bacteria isolated and identified from flavors and fragrances (Bayer combined yeast, Acetobacter pasteurianus, Agrococcus, Bacillus) and put them into 2mL centrifuge tubes containing LB liquid medium respectively, and vibrate in a constant temperature oscillator 24h standby.

(2) Use a pipette gun to take 100uL of the above four bacterial suspensions and spread them evenly on Gao's No. 1 medium.

(3) Pick a small amount of purified Streptomyces xanthophaeus (Streptomyces xanthophaeus) with an inoculation needle and place it on the No. Combine Bayer yeast, Acetobacter pasteurianus, Agrococcus, and Bacillus) in 3 dishes, and observe the growth of bacteria after culturing for 3 to 4 days.

(4) Determine whether Streptomyces jaundice has an inhibitory effect on the microorganisms that cause the deterioration of flavors and fragrances by observing whether there is a bacteriostatic zone, and the value is subtracted from the average.

Table 1 Antagonistic effect of Streptomyces chrysogenum on 4 kinds of microorganisms isolated from spoiled flavors and fragrances

Note: The average value of 3 treatments in the table "-" indicates that there is no inhibition zone

Table 1 shows that Streptomyces xanthophaeus has obvious inhibitory effect on three kinds of bacteria (Acetobacter pasteurianus, Agrococcus, and Bacillus) isolated from spoiled flavors and fragrances, but has no inhibitory effect on the fungus Bayer combined yeast .

Example 3: Bacteriostasis experiment of Streptomyces xanthophaeus fermented liquid on spoiled flavors and fragrances.

(1) Inoculate the purified Streptomyces aureus in Gao's No. 1 liquid culture medium, and culture it on a shaker at 30° C. for 2 weeks to prepare a fermentation broth.

(2) Use a pipette gun to take 100uL of the spoiled Ziyun and Ruanzhen flavors and fragrances and spread them evenly on the LB medium.

(3) Use an inoculation needle to pick a small amount of Streptomyces fermented liquid and connect it to the LB medium coated with the bacterial liquid, repeat 3 to 6 times for each culture dish, design 3 treatments, and observe after 3 to 4 days of culture The growth of bacteria.

(4) Determine whether Streptomyces chrysogenum has an inhibitory effect on the microorganisms that cause the deterioration of flavors and fragrances by observing whether there is a bacteriostatic zone.

Table 2 Antagonism of Streptomyces japonica fermented liquid stock solution to microorganisms in flavors and fragrances

The results in Table 2 show that the Streptomyces fermented liquid stock solution has obvious inhibitory effect on the microorganisms in the deteriorating flavor and fragrance

Embodiment 4: utilize fermentation broth secondary metabolites to inhibit bacteria

(1) Inoculate the purified Streptomyces aureus in Gao's No. 1 liquid culture medium, and culture it on a shaker at 30° C. for 2 weeks to prepare a fermentation broth.

(2) The fermentation broth was extracted three times with 2 times the volume of ethyl acetate, and the ethyl acetate extract was evaporated to dryness.

(3) Use a pipette gun to take 100uL of the spoiled Ziyun and Ruanzhen flavors and fragrances and spread them evenly on the LB medium.

(4) Use an inoculation needle to pick a small amount of Streptomyces jaundice extract and connect it to the LB medium coated with the bacteria solution, repeat 3 to 6 times for each petri dish, and observe the growth of the bacteria after 3 to 4 days of cultivation.

(5) Determine whether Streptomyces chrysogenum has an inhibitory effect on the microorganisms that cause the deterioration of flavors and fragrances by observing whether there is a bacteriostatic zone.

Table 3 Antagonistic effect of Streptomyces fumarus extract on microorganisms in flavors and fragrances

The results in Table 3 show that the extract of Streptomyces aureus has obvious inhibitory effect on microorganisms in spoiled flavors and fragrances.

Example 5: Inhibition of bacteria and yeasts using active substances of Streptomyces luteus.

(1) Inoculate the purified Streptomyces aureus in Gao's No. 1 liquid culture medium, and culture it on a shaker at 30° C. for 2 weeks to prepare a fermentation broth.

(2) The fermentation broth was extracted three times with 2 times the volume of ethyl acetate, and the ethyl acetate extract was evaporated to dryness.

(3) Add the active substance of Streptomyces aureus to the flavor and fragrance at 1ug/mL, 3ug/mL, 5ug/mL.

(4) Put the samples of the experimental group and the control group into a 150ml Erlenmeyer flask, seal it, and place it in a constant temperature shaker at 30°C for 7 days. Under sterile conditions, adopt the flat plate coating culture method, take 10ul of RZ and ZY samples for each treatment, mix them with 990ul sterile water, and then take 100μL of mixed samples and spread them evenly on the LB medium, with 3 replicates , Cultivate at a constant temperature of 30°C for 3 to 5 days to observe and record the number of microbial colonies. The changes in the number of microorganisms were used to judge whether the active substances of Streptomyces aureus had an inhibitory effect on the microorganisms that caused the quality changes of flavors and fragrances.

Table 4 Bacteriostasis determination of active substances of Streptomyces chrysogenum

The results in Table 4 show that adding the active substance of Streptomyces aureus to the ratio of 5 μg Streptomyces aureus active substance per milliliter of flavors and fragrances for cigarettes, the maximum antiseptic effect can be obtained, and the bacteriostasis rate to Ziyun up to 75%, and the antibacterial rate of Ruanzhen reached 91.67%.

It has been proved by the above experiments that the Streptomyces xanthophaeus disclosed by the present invention has an obvious inhibition zone to the main microbial populations Acetobacter pasteurianus, Agrococcus, and Bacillus that cause the deterioration of flavors and fragrances used in tobacco, and the activity of Streptomyces xanthophaeus is The substance has an obvious inhibitory effect on microorganisms in flavors and fragrances. When the active substance of Streptomyces obscensus is added at a concentration of 5ug/ml, the bacteriostatic rate reaches 75%-91.67%. Experiments have proved that microorganisms are the main cause of deterioration of tobacco flavors and fragrances. Therefore, the Streptomyces xanthophaeus disclosed in this experiment has obvious antiseptic effects on tobacco flavors and fragrances, which provides a basis for the quality control technology of tobacco flavors and fragrances. A new direction for prevention and control.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (4)

1. A Streptomyces xantophaeus, characterized in that: the classification of the bacterial strain is called Streptomyces xantophaeus (Streptomyces xantophaeus), and it was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on March 8, 2017, and the preservation number is CGMCC No. 13734. 2. the application of Streptomyces xanthophaeus (Streptomyces xantophaeus) CGMCC No.13734 described in claim 1 in inhibiting Acetobacter pasteurianus, Agrococcus and Bacillus. 3. The application of Streptomyces xanthophaeus CGMCC No.13734 described in claim 1 in preventing the deterioration of flavors and fragrances for tobacco. 4. The application of Streptomyces xanthophaeus (Streptomyces xanthophaeus) CGMCC No.13734 according to claim 3 in preventing the deterioration of tobacco flavors and fragrances, characterized in that: 5 ug of Streptomyces xanthophaeus active substances are added per milliliter of tobacco flavors and fragrances .