Rapid screening method for antioxidant actinomycetes in soil

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a rapid screening method for antioxidant actinomycetes in soil. The method comprises the following specific steps: S1, creating a treatment mode capable of retaining soil actinomycetes as many as possible and removing interference factors in soil: preparing a soil leachate according to a system in which V soil samples and V normal saline are in a ratio of 1: 9, centrifuging the soil leachate for 0.5 min at 300rpm, taking supernatant, centrifuging for 5 min at 5000rpm, and discarding the supernatant; and suspending the precipitate by using sterile normal saline according to the volume before centrifugation to obtain the soil bacterial suspension. And S2, creating a treatment mode capable of maximally and quickly removing actinomycetes without antioxidant activity, namely mixing the obtained soil suspension with 60mmol / L H2O2 according to the ratio of 1: 1, treating at 28 DEG C for 30 minutes, and coating a Gause No.1 culture medium with the obtained suspension to obtain the actinomycetes with antioxidant activity. The establishment of the method not only can greatly improve the screening efficiency of the antioxidant activity actinomycetes, serves for the development of natural antioxidants, but also can provide technical support for the rapid conversion and application of antioxidant actinomycetes resources.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology and more specifically, relates to a rapid screening method for soil antioxidant actinomycetes. Background Art

[0002] With the rapid development of economy and the improvement of human living standards, energy surplus and environmental pollution have come along with it, which lead to oxidative stress in the body. Bad eating habits, lifestyle, staying up late, smoking and other bad behaviors further disrupt the redox balance in the body and aggravate oxidative stress. Oxidative stress is closely related to chronic non-communicable diseases such as cardiovascular disease and cancer, which are the main causes of death in the global population. Therefore, controlling the level of oxidative stress is of great significance for maintaining health and preventing diseases.

[0003] Currently, antioxidants are mainly derived from plants, but they have problems such as long production cycles and high costs, making it difficult to meet the needs of large-scale production. Microorganisms, especially actinomycetes, have become a research hotspot due to their advantages such as variety, rapid reproduction, and low cost. Actinomycetes can produce a variety of bioactive substances, which have good antioxidant activity. Therefore, actinomycetes are very potential natural antioxidants.

[0004] To develop antioxidant actinomycetes, it is first necessary to screen and obtain high-quality actinomycete strains. The current screening methods rely on traditional isolation and culture, single bacterial activity determination and other steps, and the screening efficiency is low, which limits the development of antioxidant actinomycete resources. Based on this, it is necessary to find a method that can quickly screen and obtain antioxidant actinomycetes. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a new method for quickly screening antioxidant actinomycetes from soil.

[0006] The technical solution of the present invention is as follows:

[0007] A rapid screening method for soil antioxidant actinomycetes is carried out according to the following steps:

[0008] Step 1: Find a treatment method that can remove soil impurities while retaining the maximum amount of microorganisms;

[0009] Furthermore, in step 1, the treatment method includes two steps of removing soil impurities and collecting microorganisms;

[0010] Furthermore, the method for removing soil impurities includes centrifugation and filtration, and the method for collecting microorganisms includes centrifugation at 5000 rpm for 5 minutes and filtering with a 0.22 μm filter membrane to collect the precipitate;

[0011] Furthermore, the centrifugal removal of soil impurities conditions are set to 300, 500, 800 rpm for 1, 3, 5 min; the filtering method for removing soil impurities is to use eight layers of gauze, qualitative filter paper, and 0.45 μm filter membrane for filtration.

[0012] Step 2: Find a treatment method that can quickly remove non-antioxidant actinomycetes in the soil.

[0013] Furthermore, by statistically analyzing the soil bacterial suspension at different concentrations of H 2 O 2 The number of actinomycetes that can survive oxidative stress and treatment for different time periods (1000-10000 CFU / g) is used to determine the most suitable treatment method.

[0014] Furthermore, the H 2 O 2 The concentrations included 0, 0.1, 10, 20, 30, 40, 60, and 100 mmol / L, and the treatment times were 15, 30, and 60 min.

[0015] Furthermore, the precipitate collected in step 1 was resuspended in 0.85% saline to obtain a soil suspension. 2 O 2 ) was mixed with soil suspension at a ratio of 1:1 to obtain the sample solution. The sample solution was placed at 28°C for 15, 30, and 60 min, 0.2 mL was spread on Gao's solid medium No. 1, cultured at 28°C for 5 days, and the colonies were counted.

[0016] The specific method of the present invention is:

[0017] 200 g of soil sample was mixed with 1800 mL of sterile saline solution, stirred continuously for 5 min with a glass rod, and then allowed to stand for 5 min. The supernatant was taken as soil extract. The soil extract was centrifuged at 300 rpm for 0.5 min, the supernatant was taken, and centrifuged at 5000 rpm for 5 min. The supernatant was discarded. The precipitate was resuspended with the original volume of sterile saline solution to obtain the soil suspension. The soil suspension was mixed with 60 mmol / L H 2 O 2 Mix them in a ratio of 1:1, treat them at 28°C for 30 minutes, take 200 μL of each and spread them on Gao's No. 1 solid flat culture medium, culture them upside down at 28°C for 5 days, pick single colonies and streak them for isolation and culture, so as to obtain strains with high antioxidant activity.

[0018] The beneficial effects of the present invention are:

[0019] Compared with traditional methods, the method provided by the present invention can quickly identify strains with antioxidant activity, simplify the screening process, save costs, and can screen a large number of samples in a short time, thereby improving screening efficiency and helping to quickly find strains with potential application value.

[0020] The establishment of this method can provide technical support for the rapid transformation and application of actinomycete antioxidant resources, and also provide a reference for the screening and development of different active products of other groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Actinomycete content obtained by different pretreatment methods

[0022] Figure 2 Oxidative stress treatment pure culture diagram

[0023] Figure 3 Actinomycete content under oxidative stress treatment DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments. However, the content of the present invention is not limited to the scope described in the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0025] 1. Find a treatment method that can remove soil impurities while retaining the maximum amount of microorganisms

[0026] The surface soil (0-5 cm) was collected from Pinus yunnanensis near the tea garden of Dali University in Yunnan Province and immediately brought back to the laboratory. The soil was passed through a 10-mesh sieve, and 200 g of soil was weighed into a 5-L beaker. 1800 mL of sterile saline was added, stirred for 5 min, and allowed to stand for 5 min. The supernatant was taken as the soil extract.

[0027] The soil leachate was treated as shown in Table 1, with three replicates for each treatment. After the precipitate was collected, it was resuspended in sterile saline to obtain a soil suspension, which was marked as 10 -1 , dilute it to 10 -3 , take 0.1mL of soil suspension respectively, spread it on Gao's No. 1 solid culture medium, invert and culture at 28℃ for 5 days, use colony counter (XK97-A) to count colonies, and select the soil suspension of the pre-treatment group with the most soil bacteria for subsequent experiments.

[0028] Table 1 Soil leachate pretreatment method

[0029] The results showed that the LX1 group was able to retain the most actinomycetes. The specific treatment method was: centrifuge the soil extract at 300 rpm for 0.5 min, take the supernatant, centrifuge at 5000 rpm for 5 min, discard the supernatant, add a large volume of sterile saline to resuspend, and obtain the soil suspension. The specific results are shown in Figure 1 .

[0030] 2. Find a treatment method that can maximize the rapid removal of actinomycetes without antioxidant activity

[0031] The soil suspension prepared according to the method described in Example 1 was mixed with 0, 0.2, 2, 20, 40, 60, 80, 120, 200 mmol / L H 2 O 2 Mix in a 1:1 ratio to make H 2 O 2 The final working concentrations were 0.1, 1, 10, 20, 30, 40, 60, and 100 mmol / L, treated at 28°C for 15, 30, and 60 min, and spread on Gao's solid medium No. 1, with three replicates for each treatment. Incubate at 28°C for 5 days and count the colonies on the plates.

[0032] The results showed that the sample solution was treated with 30mmol / LH 2 O 2 The number of colonies on the plates obtained after treatment for 15, 30, and 60 minutes was between 20 and 200, and the content of actinomycetes was significantly reduced compared with the CK group, so 30mmol / L was considered to be the optimal treatment concentration.

[0033] 3. Verify the method for rapid screening of antioxidant actinomycete strains.

[0034] The plate colonies obtained by the 30 mmol / L treatment and the plate colonies obtained by the 0 mmol / L treatment described in Example 2 were picked for separation and purification, and a single colony was picked and placed in Gao's liquid culture medium No. 1, and cultured at 28°C and 140 rpm for 5 days.

[0035] The obtained bacterial suspension was centrifuged at 12000rpm for 1min to collect the bacteria, washed once with sterile saline, centrifuged again under the same conditions, the supernatant was discarded, 0.5mL sterile water was added, zirconium beads were added, and the mixture was ground at 6.5m / s and 4°C for 10min. The supernatant was collected and the DPPH, FRAP and hydroxyl free radical in vitro antioxidant indexes were determined. Blank sterile water was used as the blank control, and the changes in the absorbance values ​​were compared and analyzed to determine whether the strain had antioxidant activity.

[0036] The method for determining the DPPH free radical scavenging ability is as follows: take 100 μL of bacterial solution and 50 mg / L DPPH working solution in an equal volume in a 96-well plate, mix them, react in a 37°C water bath in the dark for 30 minutes, and measure the absorbance at 517 nm. At the same time, replace the sample solution with sterile water, repeat the above operation, and the absorbance value obtained will be used as the blank control data of the DPPH free radical of each strain. When the absorbance of the strain measured at 517 nm is lower than the blank control data, it is considered that the strain has antioxidant activity.

[0037] The hydroxyl radical scavenging rate was determined as follows: 1 mL of sample solution, 3 mmol / L FeSO 4 and 3mmoL / LH 2 O 2 Mix well and react in the dark for 10 minutes, then add 1mL 3mmoL / L salicylic acid and mix well and react in the dark for 30 minutes, and measure the absorbance at 517nm. At the same time, replace the sample solution with sterile water and repeat the above operation. The absorbance obtained will be used as the blank control data of hydroxyl free radicals of each strain. When the absorbance of the strain measured at 517nm is lower than the blank control data, it is considered that the strain has antioxidant activity.

[0038] The determination of iron ion reducing ability (FRAP) is as follows: 50 μL of sample solution is added to 1.2 mL of FRAP working solution and shaken, reacted at 37°C for 10 minutes, and the absorbance value is measured at 593 nm. At the same time, the sample solution is replaced with sterile water, and the above operation is repeated. The absorbance value obtained will be used as the blank control data of the iron redox ability of each strain. When the absorbance of the strain measured at 593 nm is higher than the blank control data, it is considered that the strain has antioxidant activity.

[0039] The results showed that when evaluating the scavenging ability of DPPH free radicals, 26.92% of the 15-min treatment group, 38.71% of the 30-min treatment group, and 17.95% of the 60-min treatment group were able to scaveng DPPH free radicals. Among them, the 60-min treatment group contained the strain with the strongest scavenging ability for DPPH free radicals, with an absorbance value of 0.164 (the blank was 0.217). When evaluating the scavenging ability for hydroxyl free radicals, 86.21% of the actinomycetes in the 15-min treatment group were able to scaveng hydroxyl free radicals, while the 30-min treatment group was able to scaveng hydroxyl free radicals. The 15-min treatment group accounted for 95.45%, the 30-min treatment group accounted for 92.31%, and the 15-min treatment group had the strongest ability to scavenge hydroxyl free radicals, with an absorbance value of 0.298 (blank was 0.620); finally, when evaluating the total iron reduction ability, all strains in the 15-min and 30-min treatment groups had reducing power, and 97.44% of the actinomycetes in the 60-min treatment group had reducing power, among which the 15-min treatment group contained the strain with the strongest ability to reduce iron ions, with an absorbance value of 0.204 (blank was 0.020). See Table 2 for details.

[0040] Table 2 Antioxidant activity test results

[0046] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A rapid screening method for antioxidant actinomycetes in soil, characterized in that: Follow these steps: S1. Find a treatment method that can remove soil impurities while retaining soil actinomycetes as much as possible; S2. Find a treatment method that can maximize the rapid removal of actinomycetes that do not have antioxidant activity.

2. The method according to claim 1, wherein S1 is characterized in that: soil impurities are removed by centrifugation at different speeds or filtration at different pore sizes; more preferably, after the first centrifugation or filtration, a second centrifugation or filtration is performed to collect the precipitate.

3. The method according to claim 2, characterized in that A soil leachate must first be prepared.

4. The method according to claim 3, characterized in that The preparation of the soil suspension comprises the following steps: Collect soil, pass it through a 10-100 mesh sieve, weigh 1-1000 parts of soil, add 9-9000 parts of sterile saline in a ratio of 1:9, stir continuously for 1-10 minutes, let it stand for 1-10 minutes, take the supernatant, and obtain the soil extract.

5. The method according to claim 2, characterized in that The filtration process includes eight-layer gauze filtration, 0.45 μm filter membrane filtration, 0.22 μm filter membrane filtration, and shaped filter paper filtration.

6. The method according to claim 2, characterized in that The centrifugation treatment includes centrifugation at 300, 500, and 800 rpm for 0.5, 1, 3, and 5 min, respectively; more preferably, the second centrifugation condition is centrifugation at 5000 rpm for 5 min.

7. The method according to claim 1, wherein S2 is characterized in that the treatment concentration that can maximize the removal of actinomycetes without antioxidant activity includes 0, 1, 10, 20, 30, 40, 60, 100 mmol / L H2O2.

8. The method according to claim 1, wherein S2 is characterized in that the processing time for finding the fastest method for screening out antioxidant actinomycetes includes 0, 15, 30, and 60 minutes.

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