Method for rapidly screening antioxidant active bacteria in soil

Abstract

The invention relates to the technical field of microorganisms, and provides a method for rapidly screening antioxidant activity bacteria in soil. Comprising the following steps: 1, sieving soil with a 10-100-mesh sieve, mixing the soil with normal saline according to a ratio of 1: 9, stirring, and standing for 1-30 minutes; 2, centrifuging the supernate at 300 rpm for 0.5 min, centrifuging the supernate at 5000 rpm for 5 min, and suspending the precipitate with normal saline to the original volume; 3, screening bacteria of metabolic pathways related to DPPH and FRAP indexes, and treating S2 with 100mM H2O2 for 30 minutes; bacteria of metabolic pathways related to hydroxyl radical indexes are screened, and S2 is treated for 60 min with 100 mM H2O2. And 4, coating the product obtained in the step 3, separating and purifying to obtain the antioxidant active bacteria. According to the method, the screening efficiency of the antioxidant activity bacteria is improved, and reference is provided for screening of other functional microorganisms.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a rapid screening method for bacteria with antioxidant activity in soil. Background Art

[0002] Protecting human health requires solving the problems of oxidative stress and oxidative inflammation. Oxidative stress refers to the production of reactive oxygen species (ROS) that exceeds the clearance capacity of the cell's antioxidant defense system, causing inflammation and a variety of chronic diseases, such as diabetes and cancer.

[0003] Antioxidants can remove ROS and repair cell damage, but the body's endogenous defense is already insufficient, and the supplementation of exogenous antioxidants is particularly necessary. However, commonly used antioxidants such as vitamin C (VC) and vitamin E (VE) have side effects when used for a long time. Therefore, it is a global trend to find low-cost, green, and safe natural alternatives.

[0004] Since ancient times, medicinal plants and animals have been the main source of antioxidants. However, due to restrictions on seasons, geographical environments or protective properties, it has become difficult to extract large amounts of antioxidant active substances. Compared with plants and animals, microorganisms can grow rapidly under controlled conditions and have greater advantages in the production of antioxidant active species. In addition, compared with synthetic antioxidants, microbial antioxidants are non-toxic, have no carcinogenic risk, are biodegradable, safer and more environmentally friendly, and have great development and application value.

[0005] The premise of obtaining microbial products is to screen the target microorganisms, but there are two major problems with the traditional screening methods of microorganisms: first, low-abundance or slow-growing microorganisms are easily overlooked; second, the difference between the culture medium and the native environment may cause the microorganisms to enter a low metabolic state. Although single colonies can be screened using a culture medium containing an indicator, the low abundance problem is not fundamentally solved, and the process is time-consuming and labor-intensive. At the same time, there is currently no rapid screening method for soil antioxidant active strains. Summary of the invention

[0006] The invention provides a rapid screening method for anti-oxidative bacteria in soil. The method has high screening efficiency, low requirements on experimental equipment, simple operation, and economical saving, and is an ideal screening method.

[0007] The present invention provides a method for rapidly screening bacteria with antioxidant activity in soil, comprising the following steps:

[0008] S1. Explore the pre-treatment methods of soil leachate, aiming to retain as many bacteria in the soil as possible.

[0009] Furthermore, in step S1, it is necessary to eliminate the interference of soil particles and reducing metabolic components in the soil extract on the subsequent oxidative stress experiment.

[0010] Furthermore, in step S1, the pretreatment method is designed based on two common methods: centrifugal treatment and filtration treatment.

[0011] Furthermore, in step S1, the soil bacterial suspension of the pre-treatment group retaining the largest amount of soil bacteria is selected for subsequent oxidative stress experiments.

[0012] S2. Explore efficient oxidation methods, aiming to eliminate bacteria without antioxidant capacity to the maximum extent possible in a short period of time, leaving behind strains with high antioxidant activity.

[0013] Furthermore, in step S2, as many soil bacteria as possible are allowed to directly face oxidative stress.

[0014] Furthermore, in step S2, the most suitable oxidative stress treatment method is determined by counting the number of bacteria (500-2000 CFU / g) that can survive the soil bacterial suspension after being treated with different H2O2 oxidative stress concentrations and stress times.

[0015] Further, in step S2, a soil bacterial suspension is prepared according to the optimal pretreatment method determined in S1 and concentrated to half, and H2O2 solution (0.2, 2, 20, 50, 100, 150, 200, 250, 300, 350, 400, 1000 mM) is added in a ratio of 1:1, so that the final concentration of H2O2 is 0.1, 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 500 mM, respectively, and the sample solution is obtained after oscillation and mixing. The sample solution is placed at 37°C for different oxidative stress times (15, 30, 60 min), and then sampled and coated, and the colonies on the plate are counted.

[0016] S3. All bacteria obtained by the most suitable oxidative stress treatment method are separated and purified to prepare bacterial liquid, and whether the strain has antioxidant activity is determined by measuring in vitro antioxidant indicators.

[0017] Furthermore, in step S3, the antioxidant activity of the bacterial solution is determined by DPPH free radical scavenging method, hydroxyl free radical scavenging method and iron ion reduction method, and beef extract peptone liquid culture medium is set as a blank control group. By comparing and analyzing the changes in the absorbance values ​​of the bacterial solution under different methods, it is determined whether the strain has antioxidant activity.

[0018] S4, the separation and purification of soil bacteria that have not been treated with oxidative stress, the preparation process of bacterial solution, and the determination method of antioxidant activity are the same as S3. By comparing the number of bacteria with antioxidant ability and antioxidant activity in S3 and S4, the success of the established rapid screening method for antioxidant active bacteria in soil is verified. The judgment criteria are: bacteria treated with oxidative stress have more bacteria with antioxidant ability and higher antioxidant activity.

[0019] Furthermore, in steps S3 and S4, the bacterial solution preparation process is as follows: the bacterial colonies are inoculated into beef extract peptone liquid culture medium, cultured at 37°C, and the bacterial solution OD is adjusted. 600 After the value reached 1, centrifuge at 5000 rpm for 5 min and collect the supernatant.

[0020] The specific method of the present invention is:

[0021] Mix 200g of soil sample with 1800mL of sterile saline, stir continuously for 5min with a glass rod, let stand for 5min, and take the supernatant as the soil extract; centrifuge the soil extract at 300rpm for 0.5min, take the supernatant, centrifuge at 5000rpm for 5min, and discard the supernatant; suspend the precipitate with 900mL of sterile saline to obtain the soil bacterial suspension; mix the soil bacterial suspension with 200mMH2O2 oxidant in a 1:1 ratio, and culture at 37℃ for 30min (to measure the DPPH free radical scavenging ability and iron ion reducing ability) or 60min (to measure the hydroxyl free radical scavenging rate), take 200μL and spread it on 90mm beef extract peptone solid flat culture medium, invert and culture at 37℃ for 36h, pick single colonies and streak for isolation and culture, so as to obtain pure culture of strains with high antioxidant activity.

[0022] The beneficial effects of the present invention are:

[0023] 1. This method has high screening efficiency, low requirements on experimental equipment, simple operation, and economical saving. It is an ideal screening method.

[0024] 2. The establishment of this method can not only greatly improve the screening efficiency of bacteria with antioxidant activity, serve the development of natural antioxidants, but also provide a reference for the screening of other microorganisms.

[0025] It is worth noting that different screening purposes require the determination of specific screening parameters, and the screening of functional microbial strains in the future will require more standard strain screening methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Bacterial content of soil leachate after pretreatment in different ways

[0027] Figure 2 Differential analysis of bacterial content after oxidative stress

[0028] Figure 3 Pure culture demonstration of the optimal concentration range for oxidative stress DETAILED DESCRIPTION

[0029] The following will be combined with the drawings and tables in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The embodiment of the present invention provides a method for rapid screening of bacteria with antioxidant activity in soil, comprising the following steps:

[0031] S1. Explore the pre-treatment methods of soil leachate, aiming to retain as many bacteria in the soil as possible.

[0032] S2. Explore efficient oxidation methods, aiming to eliminate bacteria without antioxidant capacity to the maximum extent possible in a short period of time, leaving behind strains with high antioxidant activity.

[0033] S3. All bacteria obtained by the most suitable oxidative stress treatment method are separated and purified to prepare bacterial liquid, and whether the strain has antioxidant activity is determined by measuring in vitro antioxidant indicators.

[0034] S4, the separation and purification of soil bacteria that have not been treated with oxidative stress, the preparation process of bacterial solution, and the determination method of antioxidant activity are the same as S3. By comparing the number of bacteria with antioxidant ability and antioxidant activity in S3 and S4, the success of the established rapid screening method for antioxidant active bacteria in soil is verified. The judgment criteria are: bacteria treated with oxidative stress have more bacteria with antioxidant ability and higher antioxidant activity.

[0035] In step S1, the specific method for preparing the soil leachate is as follows: 1000g of soil with a depth of 0-5cm was collected from the Yunnan pine forest near the tea garden of Dali University in Yunnan, and immediately brought back to the laboratory. After passing through a 10-mesh sieve to remove large particles of stones and tree roots, 200g of the soil sample was weighed and placed in a 5L glass beaker, and 1800mL of sterile saline was added to prepare a dilution system of V soil sample / V saline = 1 / 9. After continuous stirring with a glass rod for 5min, it was allowed to stand for 5min. The supernatant was taken as the soil leachate.

[0036] In step S1, the specific method of soil leaching solution pretreatment is as follows: The present invention is based on two common methods of centrifugation and filtration treatment, and designs 18 soil leaching solution pretreatment methods in order to find a pretreatment method that can retain the most soil bacteria. Prepare 54 50mL sterile centrifuge tubes, add 10mL of soil leaching solution to each centrifuge tube, and randomly divide them into 18 groups. Each group contains 3 centrifuge tubes as experimental duplications and parallel controls. The specific grouping and treatment methods are shown in Table 1. After the soil leaching solution is collected by different pretreatment methods, it is resuspended with 10mL of sterile saline to obtain a bacterial suspension, and 10% of the original solution is marked. -1 , repeat V 原液 / V 生理盐水 = 1 / 9 dilution method, all bacterial suspensions were diluted to 10 -3 100 μL of the prepared bacterial suspension was taken and spread on 90 mm beef extract peptone medium plates. Three parallel plates were spread for each treatment and each dilution gradient. The plates were incubated upside down at 37°C for 36 h. The colonies on the plates were counted using a colony counter (XK97-A). The bacterial suspension of the pre-treatment group with the largest amount of soil bacteria was selected for subsequent oxidative stress experiments.

[0037] Table 1 Soil leachate pre-treatment groups and treatment methods

[0038] In step S1, the treated supernatant is uniformly used to collect soil microorganisms.

[0039] S1 The results showed that the LX1 group retained the most soil bacteria. The specific treatment method was to centrifuge the soil extract at 300 rpm for 0.5 min, take the supernatant, centrifuge at 5000 rpm for 5 min, and discard the supernatant; the precipitate was suspended with sterile saline according to the volume before centrifugation, which was the soil bacteria suspension. Figure 1 .

[0040] In step S2, in order to explore how to maximize the killing of non-antioxidant bacteria in the soil while retaining strains with high antioxidant activity, the present invention determines the most suitable oxidative stress treatment method by counting the number of bacteria (500-2000 CFU / g) that can survive the bacterial suspension after treatment with different H2O2 oxidative stress concentrations and stress times. According to the optimal pretreatment method determined by S1, 80 mL of bacterial suspension was prepared, concentrated to 40 mL, mixed and added to the centrifuge tube according to the volume of 2 mL per tube, and then 2 mL of H2O2 solution (0.2, 2, 20, 50, 100, 150, 200, 250, 300, 350, 400, 1000 mM) was added to make the total volume of each tube 4 mL. The final concentrations of H2O2 were 0.1, 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 500 mM, respectively. After oscillation and mixing, the sample solution was obtained, and the sample solution was placed at 37°C for different oxidative stress times (15, 30, 60 min). After the oxidative stress ended, the sample solutions of different oxidative stress treatment groups were marked as 10% of the original solution. -1 , repeat V 原液 / V 生理盐水 = 1 / 9 dilution method, all samples were diluted to 10 -2 ; 200 μL of the sample solution from the oxidative stress treatment group with different dilution concentrations was taken and spread on a 90 mm beef extract peptone medium solid plate that had passed the sterility test. Three parallel plates were spread for each treatment, each repeat, and each dilution gradient. The plates were incubated at 37°C for 36 hours and the colonies on the plates were counted.

[0041] The results of S2 showed that treating the sample solution with 100 mM H2O2 for 15 min, 30 min, and 60 min respectively could be used as an ideal stress condition for further studying the antioxidant capacity of a single bacterium. Figure 2 , Figure 3 .

[0042] In step S3, the soil bacterial suspension is treated according to the most suitable oxidative stress treatment method determined in S2 and spread on a 90 mm beef extract peptone medium solid plate for culture. All bacteria on the plate are separated and purified to prepare a bacterial solution, and the antioxidant activity of the bacterial solution is determined by the DPPH free radical scavenging method, the hydroxyl free radical scavenging method and the iron ion reduction method.

[0043] In step S3, the isolation and purification of bacteria and the preparation of bacterial solution are as follows: According to the results of S2, all colonies on the plate of the most suitable oxidative stress treatment group are selected for antioxidant activity evaluation. The specific operation is: pick out all single colonies on the plate, separate and purify them by plate streak method, and after determining their purity by Gram staining, the selected colonies are resuspended in 10mL of normal saline. Then, take 1mL of the solution and inoculate it into 100mL of beef extract peptone liquid culture medium and culture it at 37℃ for 18h. Take 3mL of bacterial solution from the culture medium and adjust its OD 600 After the value reaches 1, centrifuge at 5000rpm for 5min and collect the supernatant. Transfer the supernatant to a new centrifuge tube and use DPPH free radical scavenging method, hydroxyl free radical (OH·) scavenging method and iron ion reduction method (FRAP) to determine its antioxidant activity. At the same time, beef extract peptone liquid culture medium is set as a blank control group. By comparing and analyzing the changes in the absorbance values ​​of the bacterial liquid under different methods, it is determined whether the strain has antioxidant activity.

[0044] In step S3, the determination method of DPPH free radicals is as follows: take 2 mL of bacterial solution and mix with 50 mg / LDPPH working solution in equal volumes, react in a 37°C water bath away from light for 30 minutes, and measure the absorbance at 517 nm. At the same time, replace the bacterial solution with beef extract peptone liquid culture medium, repeat the above operation, and the obtained absorbance value will be used as the blank control data of DPPH free radicals 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.

[0045] In step S3, the determination method of hydroxyl radical (OH·) is as follows: 1 mL of bacterial solution, 3 mmoL / L FeSO4 and 3 mmoL / L H2O2 are added in a ratio of 1:1:1, mixed and reacted in the dark for 10 minutes, and then 1 mL of 3 mmoL / L salicylic acid is added, mixed and reacted in the dark for 30 minutes, and the absorbance is measured at 517 nm. At the same time, the bacterial solution is replaced with beef extract peptone liquid culture medium, and the above operation is repeated. The absorbance value obtained will be used as the blank control data of hydroxyl radicals 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.

[0046] In step S3, the method for determining the iron ion reducing ability (FRAP) is as follows: 100 μL of bacterial solution is added to 2.4 mL of FRAP working solution and shaken well, reacted at 37°C for 10 minutes, and the absorbance value is measured at 593 nm. At the same time, the bacterial solution is replaced with beef extract peptone liquid culture medium, and the above operation is repeated. The absorbance value obtained will be used as the blank control data of the iron ion reducing 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.

[0047] S3 results showed that after the soil bacterial suspension was treated with a final concentration of 100mM H2O2 for 15min, 30min, and 60min, 112, 47, and 27 strains of bacteria were successfully purified from the plate of the coating culture, respectively. After these strains were tested by three antioxidant indicators, it was found that if it was necessary to develop bacteria in the metabolic pathways acted by the DPPH and FRAP indicators, 100mM H2O2 oxidative stress for 30min could be selected; if it was necessary to develop bacteria in the metabolic pathways acted by the hydroxyl free radical indicator, 100mMH2O2 oxidative stress for 60min could be selected. The results are shown in Table 2.

[0048] Table 2 Evaluation of antioxidant activity of strains treated with 100 mM H2O2 for different time periods

[0049] In step S3, when the strain is evaluated by the DPPH method and the hydroxyl radical scavenging method, the lower the absorbance value, the stronger the antioxidant activity of the strain; and when the iron ion reduction method is used for measurement, the higher the absorbance value, the better the antioxidant activity of the strain.

[0050] In step S4, 80 mL of bacterial suspension was prepared according to the optimal pretreatment method determined in S1, and after being cultured at 37°C for 15, 30, and 60 min, 200 μL was respectively applied to a 90 mm beef extract peptone medium solid plate that had been tested for sterility. Three parallel plates were applied to each treatment, each repetition, and each dilution gradient, and inverted culture was performed at 37°C for 36 h to obtain a soil bacterial plate that had not been treated with oxidative stress. The subsequent bacterial separation and purification, bacterial solution preparation process, and antioxidant activity determination method were the same as in S3. The rapid screening method for antioxidant active bacteria in soil established was verified to be successful by comparing the strain number and antioxidant activity with the bacteria treated with oxidative stress in S3. The judgment criteria are: bacteria treated with oxidative stress have more bacteria with antioxidant capacity and higher antioxidant activity.

[0051] The present invention selected 113 strains that were not subjected to oxidative stress for oxidative activity evaluation. The S4 results showed that the number of strains with antioxidant capacity and the activity of individual strains were lower than those of the rapid screening method. The results are shown in Table 3.

[0052] Table 3 Evaluation of antioxidant activity without oxidation treatment

[0053] In step S4, when the strain is evaluated by the DPPH method and the hydroxyl radical scavenging method, the lower the absorbance value, the stronger the antioxidant activity of the strain; and when the iron ion reduction method is used for measurement, the higher the absorbance value, the better the antioxidant activity of the strain.

[0054] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A rapid screening method for bacteria with antioxidant activity in soil, characterized in that: The steps include: S1. Preparation of soil leachate, dedicated to removing large particles from the soil; S2. Preparation of soil bacterial suspension, retaining as many bacteria as possible in the soil and removing interfering factors in the soil; S3. Treating the soil bacterial suspension with an oxidant is intended to remove bacteria without antioxidant capacity in the soil bacterial suspension and retain bacteria with antioxidant capacity. After separation and purification, bacterial strains with antioxidant capacity can be obtained.

2. The rapid screening method for anti-oxidative bacteria in soil according to claim 1, characterized in that: In step S1, the soil extract is prepared by collecting the surface soil (0-10 cm), filtering it through a 10-100 mesh sieve to remove large particles of impurities, weighing 1-1000 portions of soil samples, placing them in a beaker, and decanting the soil according to V. 土壤样本 :V 生理盐水 =1:9 system, add physiological saline, stir continuously for 1-10 minutes using a glass rod, let stand for 1-30 minutes, and take the supernatant to obtain the soil leachate.

3. The rapid screening method for anti-oxidative bacteria in soil according to claim 1, characterized in that: In step S2, the method for preparing the soil bacterial suspension is as follows: the soil extract is centrifuged at 300 rpm for 0.5 min, the supernatant is taken, and then centrifuged at 5000 rpm for 5 min, and the supernatant is discarded; the precipitate is suspended with sterile physiological saline according to the volume before centrifugation, which is the soil bacterial suspension.

4. The rapid screening method for anti-oxidative bacteria in soil according to claim 1, characterized in that: In step S3, the method of treating the soil bacteria suspension with an oxidant needs to be adjusted according to the characteristics of the antioxidant strain to be developed.

5. The method of treating soil bacterial suspension with an oxidant according to claim 4, wherein the specific operation is: bacteria that develop metabolic pathways acted upon by DPPH, hydroxyl radicals, and FRAP indicators are selected and subjected to oxidative stress of 100 mM H2O2 for 30 min; bacteria that develop metabolic pathways acted upon by hydroxyl radical indicators are selected and subjected to oxidative stress of 100 mM H2O2 for 60 min. The obtained suspension is coated and the bacteria are separated and purified to obtain bacteria with antioxidant activity.

Images