Application of quercetin in reducing number of resistance genes in soil, solid bacterial fertilizer and preparation method

Abstract

The invention discloses application of quercetin in reduction of the number of resistance genes in soil, solid bacterial fertilizer and a preparation method, and relates to the technical field of preparation of solid bacterial fertilizer and reduction of the number of resistance genes. Quercetin is introduced in the fermentation process of the solid bacterial fertilizer to inhibit propagation and diffusion of resistance genes, and the ecological safety of the bacterial fertilizer is improved on the premise that growth and proliferation of beneficial microorganisms are not affected. The concentration (0.01%-0.05%) of quercetin and the adding mode (added in the first-stage fermentation) are precisely designed, and the inhibition effect of quercetin on beneficial bacteria can be effectively avoided, so that the high efficiency and safety of the bacterial fertilizer are guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of solid bacterial fertilizer preparation and reduction of resistance gene quantity, and more specifically to application of quercetin in reducing the resistance gene quantity in soil, solid bacterial fertilizer and a preparation method thereof. Background Art

[0002] Antibiotic resistance (AMR) is a major challenge in the current global public health field. With the widespread use of antibiotics in medicine, agriculture and animal husbandry, a large number of bacteria carrying resistance genes and their resistance genes have entered the environment through waste discharge. Solid bacterial fertilizer, as an organic fertilizer widely used in agriculture, is mainly made from raw materials such as livestock and poultry manure, organic waste and fermented bacteria through fermentation. Solid bacterial fertilizer is not only rich in nutrients required by plants, but also contains a large number of active microorganisms, usually with a bacterial population of up to 10 8 CFU / g or more. This high concentration of microbial communities can not only effectively improve the soil during agricultural application, but also carry and spread resistance genes, constituting an important way for the spread of environmental resistance genes. When applied to the soil, these resistance genes may spread among soil microorganisms through horizontal gene transfer, thereby exacerbating the accumulation of resistance genes in the environment.

[0003] The high density of microorganisms in solid fertilizer greatly increases the risk of resistance gene transmission, and its potential for transmission and diffusion is far greater than that in a low-density bacterial community environment. The resistance genes carried in the fertilizer may be transferred to other microorganisms through horizontal gene transfer (HGT) in the soil environment, especially in a high-density bacterial community environment, where the gene transfer efficiency is higher. Once the resistance gene spreads to soil microorganisms, it will be difficult to remove and may further enter humans and animals through water sources, crops, and even the food chain.

[0004] At present, the main means to reduce resistance genes in solid bacterial fertilizers include high-temperature composting, chemical additives and physical treatment. However, these methods have certain shortcomings in practical applications: although high-temperature composting can kill some bacteria, some heat-resistant bacteria may still survive. In addition, the high-temperature composting process may be long and is not suitable for large-scale rapid production needs. Although chemical antimicrobial agents are effective, they have potential pollution to the environment and may have a negative impact on soil microbial communities, which does not meet environmental protection requirements. Although physical treatments such as radiation or ultraviolet treatment are effective, they have high application costs and are difficult to popularize in large-scale agricultural production.

[0005] Quercetin is a flavonoid compound naturally found in fruits, vegetables and tea leaves, and has multiple biological activities such as antioxidant, anti-inflammatory and antibacterial. Studies in recent years have shown that quercetin has significant effects in inhibiting bacterial growth, interfering with the expression of resistance genes and reducing the formation of bacterial biofilms. Generally speaking, the antibacterial effect of quercetin is more obvious at high concentrations, while at low concentrations it mainly manifests itself in inhibiting the activity of specific drug-resistant bacteria. At present, there is no technology for applying quercetin to solid bacterial fertilizers to reduce the number of resistance genes.

[0006] Therefore, how to apply quercetin to solid bacterial fertilizer to reduce the number of resistance genes is a technical problem that technicians in this field need to solve urgently. Summary of the invention

[0007] In view of this, the present invention aims to apply quercetin to solid bacterial fertilizer, which can reduce the survival ability of resistant bacteria by interfering with the expression pathway of resistance genes in bacteria, and can also significantly reduce the horizontal transfer efficiency of resistance genes between bacteria, thereby reducing the risk of resistance gene diffusion in bacterial fertilizer. At the same time, quercetin has good environmental degradability, is non-toxic and harmless to soil and plants, does not have a negative impact on the environment, and is suitable for large-scale agricultural applications. Quercetin can be extracted from a variety of plants, has low production costs, is suitable for additives in solid bacterial fertilizers, and greatly improves the economic feasibility of resistance gene control.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] In one aspect, an embodiment of the present invention provides the use of quercetin in reducing the number of resistance genes in soil.

[0010] A second aspect of an embodiment of the present invention provides a method for preparing a solid bacterial fertilizer, the process comprising:

[0011] Step 1, preparing a seed solution of growth-promoting bacteria;

[0012] Step 2: Evenly mix the coconut shell powder and soybean meal in a volume ratio of 6:4 to obtain a carrier material;

[0013] Step 3, spraying the quercetin solution evenly on the carrier material, adding 0.1-0.5 g of quercetin to 1 kg of the carrier material;

[0014] Step 4: Add 5-10% of the seed liquid to the carrier material and adjust the humidity to 55-60%;

[0015] Step 5: Yeast extract enhanced fermentation;

[0016] Step 6: Drying and molding of bacterial fertilizer.

[0017] In a preferred embodiment, in step 1, one loop of activated bacterial solution is inoculated into LB liquid culture medium, and cultured in a shaking incubator at 30° C. and 180 r / min for 24 hours to obtain a seed culture solution.

[0018] In a preferred embodiment, in step 5, the process of yeast extract enhanced fermentation is:

[0019] The first stage of fermentation: ferment at 30℃ for 48h, turn the pile regularly, keep ventilation, ensure that the bacteria get enough oxygen during the fermentation process, and promote the rapid proliferation of microorganisms;

[0020] Second stage fermentation: After 48 hours of fermentation, the yeast extract is diluted with a certain amount of water at a mass ratio of 1:30, and sprayed evenly in the fermentation matrix to adjust the humidity to 50-55%.

[0021] In a preferred embodiment, in step six, the drying and molding of the bacterial fertilizer is as follows: the bacterial fertilizer is low-temperature dried at 40-50°C for 4-6 hours until the moisture content of the fertilizer drops below 12% to ensure the survival of the microbial cells; then the dried bacterial fertilizer is pressed into 1-3 mm particles.

[0022] The third aspect of the embodiments of the present invention provides a solid bacterial fertilizer prepared by the method described above.

[0023] The fourth aspect of the embodiments of the present invention provides the use of the solid bacterial fertilizer in reducing the number of resistance genes in soil.

[0024] It can be seen from the above technical solution that, compared with the prior art, the present invention introduces quercetin during the fermentation process of solid bacterial fertilizer to inhibit the spread and diffusion of resistance genes, thereby improving the ecological safety of bacterial fertilizer without affecting the growth and proliferation of beneficial microorganisms. The concentration of quercetin (0.01%-0.05%) and the addition method (added in the first stage of fermentation) are precisely designed to effectively avoid its inhibitory effect on beneficial bacteria, thereby ensuring the efficiency and safety of bacterial fertilizer.

[0025] At the same time, in the process of preparing solid bacterial fertilizer, the present invention adopts a two-stage fermentation process, in which the expression of resistance genes is inhibited by adding quercetin in the first stage, and yeast extract (yeast extract powder or extract) is introduced as a fortifier in the second stage to promote the proliferation of beneficial bacteria, enhance the activity and fecundity of microorganisms, and reduce the potential inhibitory effect of quercetin on beneficial bacteria. The two-stage fermentation not only improves the bacterial concentration of microorganisms (reaching 10 10 ~10 12 CFU / g), and by precisely controlling temperature, humidity and the addition of yeast extract, the balance between efficient microbial proliferation and resistance gene inhibition during the fermentation process is ensured. This process improves the functionality and safety of solid bacterial fertilizer.

[0026] The present invention adopts coconut shell powder and soybean meal as carrier materials, which not only have good physical properties (such as air permeability and hygroscopicity), can provide an ideal growth environment for microorganisms, but also have a certain ability to inhibit the spread of resistance genes. By selecting natural materials such as coconut shell powder and soybean meal, the structural stability of the microorganism fertilizer is improved, and the diffusion of resistance genes in the fermentation process can be reduced, further improving the safety of the microorganism fertilizer. Technical details such as the mixing ratio of the carrier material and the pretreatment method help to enhance the uniform distribution and proliferation of microorganisms in the carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The attached figure is a distribution diagram of resistance gene composition under different treatments.

[0029] Figure 2 The attached figure shows the abundance of resistance genes under different treatments. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings 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.

[0031] The yeast extract was purchased from Oxoid yeast extract (LP0021B).

[0032] Example 1 Preparation of solid bacterial fertilizer

[0033] Step 1: Preparation of seed solution

[0034] In this experiment, Klebsiella sp. (CICC10702) from CICC was used as the bacterial strain. One loop of activated bacterial solution was inoculated into LB liquid culture medium and cultured in a shaking incubator at 30°C and 180 r / min for 24 h to obtain seed culture solution.

[0035] Step 2: Preparation of carrier material

[0036] The shell powder and soybean meal were uniformly mixed in a volume ratio of 6:4 to obtain a carrier material.

[0037] Step 3: Adding quercetin

[0038] Dissolve quercetin in an appropriate amount of deionized water to obtain a 0.5% quercetin solution, stir thoroughly and evenly spray it on the carrier material to ensure that each particle is evenly distributed. The initial concentration of quercetin is 0.01% (ie, 0.1 g of quercetin is added to 1 kg of carrier material).

[0039] Step 4: Microbial inoculation

[0040] Add the seed liquid obtained in step 1 to the carrier material at 7%, perform preliminary mixing, and adjust the humidity to 55-60%, ensuring that the bacteria are evenly distributed in the carrier and enhancing the subsequent fermentation effect of the bacterial fertilizer.

[0041] Step 5: Double-stage fermentation with yeast extract

[0042] The first stage of fermentation: Fermentation is carried out at 30℃ for 48 hours. Turn the pile regularly and keep it ventilated to ensure that the bacteria get enough oxygen during the fermentation process and promote the rapid proliferation of microorganisms.

[0043] Second stage fermentation: After 48 hours of fermentation, yeast extract was added. Yeast extract was diluted with a certain amount of water at a mass ratio of 1:30, and evenly sprayed on the fermentation matrix to adjust the humidity to 50-55%. The rich nutrients in yeast extract will significantly promote the secondary proliferation of microorganisms. The live bacteria count of the second stage fermentation was found to be 3.2×10 10 CFU / g.

[0044] Step 6: Drying and molding of bacterial fertilizer

[0045] After fermentation, the fertilizer is dried at 50°C for 4 hours until the moisture content of the fertilizer drops below 12% to ensure the viability of the microorganisms. After that, the fertilizer is granulated and pressed into 1-3mm particles to ensure easy use and even spread of fertilizer effect.

[0046] Example 2 Preparation of solid bacterial fertilizer

[0047] Step 1: Preparation of seed solution

[0048] In this experiment, Klebsiella sp. (CICC10702) was used as the bacterial species. One loop of activated bacterial solution was inoculated into LB liquid culture medium and cultured in a shaking incubator at 30°C and 180 r / min for 24 h to obtain seed culture solution.

[0049] Step 2: Preparation of carrier materials

[0050] The shell powder and soybean meal were uniformly mixed in a volume ratio of 6:4 to obtain a carrier material.

[0051] Step 3: Adding quercetin

[0052] Dissolve quercetin in an appropriate amount of deionized water, stir thoroughly, and evenly spray on the carrier material to ensure that each particle is evenly distributed. The initial concentration of quercetin is 0.01% (ie, 0.1 g of quercetin is added to 1 kg of carrier material).

[0053] Step 4: Microbial inoculation

[0054] Add the seed liquid obtained in step 1 at 5% of the carrier material, perform preliminary mixing, and adjust the humidity to 55-60%, ensuring that the bacteria are evenly distributed in the carrier and enhancing the subsequent fermentation effect of the bacterial fertilizer.

[0055] Step 5: Double-stage fermentation with yeast extract

[0056] The first stage of fermentation: Fermentation is carried out at 25℃ for 48 hours. Turn the pile regularly and keep it ventilated to ensure that the bacteria get enough oxygen during the fermentation process and promote the rapid proliferation of microorganisms.

[0057] Second stage fermentation: After 48 hours of fermentation, yeast extract was added. Yeast extract was diluted with a certain amount of water at a mass ratio of 1:30, and evenly sprayed on the fermentation matrix to adjust the humidity to 50-55%. The rich nutrients in yeast extract will significantly promote the secondary proliferation of microorganisms. The live bacteria count of the second stage fermentation was found to be 2.5×10 10 CFU / g.

[0058] Step 6: Drying and molding of bacterial fertilizer

[0059] After fermentation, the fertilizer is dried at 40°C for 6 hours until the moisture content of the fertilizer drops below 12% to ensure the survival of the microorganisms. After that, it is granulated and pressed into 1-3mm particles to ensure easy use and even spread of fertilizer effect.

[0060] Example 3 Preparation of solid bacterial fertilizer

[0061] Step 1: Preparation of seed solution

[0062] In this experiment, Klebsiella sp. (CICC10702) was used as the bacterial species. One loop of activated bacterial solution was inoculated into LB liquid culture medium and cultured in a shaking incubator at 30°C and 180 r / min for 24 h to obtain seed culture solution.

[0063] Step 2: Preparation of carrier material

[0064] The shell powder and soybean meal were uniformly mixed in a volume ratio of 6:4 to obtain a carrier material.

[0065] Step 3: Adding quercetin

[0066] Dissolve quercetin in an appropriate amount of deionized water, stir thoroughly, and evenly spray on the carrier material to ensure that each particle is evenly distributed. The initial concentration of quercetin is 0.03% (ie, 0.3 g of quercetin is added to 1 kg of carrier material).

[0067] Step 4: Microbial inoculation

[0068] Add the seed liquid obtained in step 1 at 5% of the carrier material, perform preliminary mixing, and adjust the humidity to 55-60%, ensuring that the bacteria are evenly distributed in the carrier and enhancing the subsequent fermentation effect of the bacterial fertilizer.

[0069] Step 5: Double-stage fermentation with yeast extract

[0070] The first stage of fermentation: Fermentation is carried out at 28℃ for 48 hours. Turn the pile regularly and keep it ventilated to ensure that the bacteria get enough oxygen during the fermentation process and promote the rapid proliferation of microorganisms.

[0071] Second stage fermentation: After 48 hours of fermentation, yeast extract was added. Yeast extract was diluted with a certain amount of water at a mass ratio of 1:30, and evenly sprayed on the fermentation matrix to adjust the humidity to 50-55%. The rich nutrients in yeast extract will significantly promote the secondary proliferation of microorganisms. The live bacteria count of the second stage fermentation product was found to be 2.1×10 10 CFU / g.

[0072] Step 6: Drying and molding of bacterial fertilizer

[0073] After fermentation, the fertilizer is dried at 45°C for 5 hours until the moisture content of the fertilizer drops below 12% to ensure the survival of the microorganisms. After that, it is granulated and pressed into 1-3mm particles to ensure easy use and even spread of fertilizer effect.

[0074] Example 4 Potted experiment to verify the effect of bacterial fertilizer (verified with the solid bacterial fertilizer of Example 1)

[0075] The potted experiment used a plastic flower pot with a height of 14 cm, a bottom diameter of 20.5 cm, and a diameter of 23.5 cm. Each pot was filled with 1.8 kg of air-dried soil. The experiment set up two treatments: ① The control group CK without any bacterial fertilizer; ② The bacterial fertilizer Kl was applied. Fertilizer amount: The bacterial fertilizer and soil were mixed evenly at a mass ratio of 1:100. Six pots were set up in each of the above four treatment groups. Thick and healthy tobacco seedlings were selected and transplanted into the pots designed by the experimental grouping. After growing at room temperature for 60 days, soil samples were collected and tobacco growth indicators were measured.

[0076] The effect of applying bacterial fertilizer Kl on tobacco growth was evaluated through potted experiments. As shown in Table 1, the plant height, aboveground fresh weight, aboveground dry weight and maximum leaf area of ​​the bacterial fertilizer Kl+quercetin group with quercetin added were significantly higher than those of the control group CK (p < 0.05). The plant height of the bacterial fertilizer Kl group increased by 22.44%, the aboveground dry weight increased by 173.27%, and the maximum leaf area increased to 160.94 cm 2 . Compared with the bacterial fertilizer without quercetin, the effect is equivalent. This shows that the addition of quercetin does not affect the growth of tobacco.

[0077] Table 1 Effect of applying bacterial fertilizer K1 on tobacco growth

[0078] Growth indicators CK Kl Kl+Quercetin Plant height(cm) 18.05±1.52c 19.05±7.23b 22.10±6.41ab Number of blades 9.50±1.87c 10.83±2.01bc 12.00±1.41ab Fresh weight of aboveground parts (g) 14.08±1.89c 36.70±1.17ab 38.28±8.14a Aboveground dry weight (g) 1.01±0.14c 1.60±0.89bc 2.76±0.66ab Fresh weight of underground part (g) 1.35±0.37a 1.34±0.11a 1.04±0.40ab Maximum leaf area (cm2) 88.99±6.79b 158.34±11.20a 160.94±20.96a

[0079] Example 5 Resistance gene determination

[0080] The test site is located in Daoer Science and Technology Park, Huayuan County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province. Random arrangement is adopted, and 3 plots are set up for each treatment. There are about 600 tobacco plants in each plot. The planting density of tobacco is 125cm in row and 60cm in plant. Protective rows are set up around, and the plots are randomly distributed. There are three treatments in the experiment: ① blank control CK without any bacterial fertilizer, ② bacterial fertilizer T1 with quercetin added, ③ bacterial fertilizer T2 without quercetin added, and ④ chemical tobacco disease agent kasugamycin KSG (KSG is 60g of kasugamycin diluted with 2L of water) T3. Solid bacterial fertilizer is applied by hole application when transplanting tobacco seedlings. The colony count of each tobacco seedling is controlled at 10×10 9 CFU / g. Other field management was carried out according to local standards. 15 days after application, soil samples were taken for metagenome analysis and resistance gene annotation. The results are shown in Figure 1 and Figure 2 , showing that the abundance of resistance genes decreased significantly after the application of solid bacterial fertilizer containing quercetin.

[0081] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0082] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of quercetin in reducing the number of resistance genes in soil.

2. A method for preparing solid bacterial fertilizer, characterized in that: The process includes: Step 1, preparing a seed solution of growth-promoting bacteria; Step 2: Evenly mix the coconut shell powder and soybean meal in a volume ratio of 6:4 to obtain a carrier material; Step 3, spraying the quercetin solution evenly on the carrier material, adding 0.1-0.5 g of quercetin to 1 kg of the carrier material; Step 4: Add 5-10% of the seed liquid to the carrier material and adjust the humidity to 55-60%; Step 5: Yeast extract enhanced fermentation; Step 6: Drying and molding of bacterial fertilizer.

3. The method for preparing a solid bacterial fertilizer according to claim 2, characterized in that: In step 1, one loop of activated bacterial solution was inoculated into LB liquid culture medium, and cultured in a shaking incubator at 30° C. and 180 r / min for 24 h to obtain seed culture solution.

4. The method for preparing a solid bacterial fertilizer according to claim 3, characterized in that: In step 5, the process of yeast extract enhanced fermentation is: The first stage of fermentation: ferment at 30℃ for 48h, turn the pile regularly, keep ventilation, ensure that the bacteria get enough oxygen during the fermentation process, and promote the rapid proliferation of microorganisms; Second stage fermentation: After 48 hours of fermentation, the yeast extract is diluted with a certain amount of water at a mass ratio of 1:30, and sprayed evenly in the fermentation matrix to adjust the humidity to 50-55%.

5. The method for preparing a solid bacterial fertilizer according to claim 4, characterized in that: In step six, the bacterial fertilizer is dried and formed as follows: the bacterial fertilizer is low-temperature dried at 40-50°C for 4-6 hours until the moisture content of the fertilizer drops below 12% to ensure the survival of the microbial cells; then the dried bacterial fertilizer is pressed into 1-3mm particles.

6. The solid bacterial fertilizer prepared by any one of the methods described in claims 2-5.

7. Use of the solid bacterial fertilizer according to claim 6 in reducing the number of resistance genes in soil.

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