A composite microbial agent for degrading diazepam and its application

Abstract

The present invention discloses a composite microbial agent for degrading diazepam and its application. It belongs to the field of biodegradation technology. The composite microbial agent comprises the following components: Bacillus subtilis, yeast, Cladosporium cladosporium, Aspergillus oryzae and Aspergillus niger. The present invention prepares a composite microbial agent by screening suitable microbial strains and applies it to the degradation of DZP in aquaculture ponds. In terms of degradation rate index, the plant remediation method can reach up to about 70%, the nano zero-valent copper method can reach up to about 75%, and the degradation rate of the technical solution of the present invention can reach up to 100%. Comprehensive comparison shows that the technical solution of the present invention shows great superiority in degrading DZP residues in aquaculture water bodies and has significant promotion and demonstration prospects. At present, there is no report on the degradation of DZP in aquaculture pond water bodies by microbial methods.

Description

Technical Field

[0001] The present invention relates to the technical field of biodegradation, and more particularly to a composite microbial agent for degrading diazepam and application thereof. Background Art

[0002] Diazepam (DZP), also known as Valium, is a sedative drug, mainly used for anxiety, sedation, hypnosis, and can also be used for anti-epileptic and anticonvulsant. The "Maximum Residue Limits of Veterinary Drugs in Food" (GB 31650-2019) stipulates that DZP is allowed for therapeutic use, but it must not be detected in animal food. DZP can reduce the perception of fresh live fish to the outside world, reduce its metabolism, and ensure that the live fish remains fresh after transportation. DZP residues in fish are permanent and will be passed to humans through the food chain. DZP exceeding a certain dose may cause drowsiness, fatigue, movement disorders, mental confusion, etc. in humans. In severe cases, symptoms such as arrhythmia and coma may also occur.

[0003] Environmental water pollution is one of the main factors causing pollutant residues in aquatic organisms. Due to incomplete metabolism of DZP in humans or animals, drug abuse and other reasons, the drug can enter natural water bodies through aquaculture wastewater, domestic and medical wastewater, etc., causing environmental pollution. At present, DZP has been detected in natural water bodies in most countries and regions in the world. Most of the water used in fishery aquaculture is filtered and reused for aquaculture by using wastewater recycling technology. However, DZP in the recycled water cannot be effectively removed by traditional adsorption treatment, and it is easy to be lost, difficult to separate, and easy to cause secondary pollution during the adsorption-separation-circulation process, which poses a serious threat to the quality and safety of aquatic products and human health.

[0004] The existing methods for degrading DZP mainly include phytoremediation and chemical removal (nano zero-valent copper method). Wang Zhiwei et al. were able to accelerate the degradation of DZP in water bodies by planting water hyacinth and / or large hyacinth in water bodies where DZP pollution was detected. The results showed that planting water hyacinth could eliminate DZP in water bodies up to 70.55%; planting large hyacinth could eliminate DZP in water bodies up to 60.51%. Gong Zhiqiang took nano zero-valent copper as the research object and investigated the degradation characteristics and removal mechanism of DZP by nano zero-valent copper / persulfate oxidation system. The study showed that at 25°C and pH = 9, the degradation rate of DZP by this system can reach about 75%. The application of phytoremediation in the degradation of DZP in water bodies has the disadvantages of high cost, slow effect and long cycle. The nano-zero-valent copper method is also not suitable for aquaculture, mainly because the physical and chemical properties of nano-zero-valent copper and the environment in which it is located have a great impact on it. In addition, there will be great losses during use, and recycling and reuse are issues that need further consideration. At the same time, the toxicity of its degradation products needs to be further evaluated, especially for aquatic animals. Therefore, at this stage, there is a lack of effective, low-cost methods in actual aquaculture that do not pose risks to aquatic products and aquaculture ecosystems to degrade DZP residues in water.

[0005] In the practice of degradation of drug residues in water, bioremediation has attracted attention due to its low treatment cost and no secondary pollution. Microorganisms are diverse and have rich metabolic types, and can completely decompose pollutants into carbon dioxide, water, inorganic compounds and other substances that are harmless to the environment and human body. In recent years, the application of microorganisms in agriculture has been relatively widespread, and they also play an important role in the degradation of pesticide residues and other drugs. The bioremediation method that uses microorganisms themselves or microbial products to degrade pollutants has the advantages of being non-toxic and residue-free. It is a relatively cheap and effective method to eliminate pesticide residues or reduce residual toxicity.

[0006] In summary, how to provide a microbial agent that can degrade DZP residues in aquaculture water is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a composite microbial agent for degrading diazepam and application thereof.

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

[0009] A composite microbial agent for degrading diazepam, comprising the following components:

[0010] Bacillus subtilis, Saccharomyces cerevisiae, Cladosporium buddingum, Aspergillus oryzae, and Aspergillus niger;

[0011] The Bacillus subtilis is CCTCC AB 130001;

[0012] The yeast is CCTCC WY 2008323;

[0013] The Cladosporium cladosporium is BMZ103177;

[0014] The Aspergillus oryzae is GDMCC.NO: 3.31;

[0015] The Aspergillus niger is B71913;

[0016] The volume ratio of the bacterial liquid of each bacterial strain is 1-5:2-4:1-7:3-9:1-4.

[0017] Furthermore, the volume ratio of the bacterial liquid of each strain is 3:3:4:7:3.

[0018] Furthermore, the volume ratio of the bacterial liquid of each strain is 2:2:3:6:2.

[0019] Furthermore, the total effective viable count was 1.24×10 9 ~5.56×10 10 cfu / g.

[0020] Furthermore, the number of effective live bacteria in the bacterial solution of each strain was ≥3.0×10 7 cfu / g.

[0021] Furthermore, the number of effective live bacteria in the bacterial solution of each strain is as follows:

[0022] Bacillus subtilis: 5.2×10 8 ~3.0×10 9 cfu / g;

[0023] Yeast: 3.9×10 9 ~6.5×10 9 cfu / g;

[0024] Cladosporium cladodes: 4.2×10 8 ~2.5×10 9 cfu / g;

[0025] Aspergillus oryzae: 2.7×10 9 ~5.5×10 10 cfu / g;

[0026] Aspergillus niger: 3.6×10 9 ~2.1×10 10 cfu / g.

[0027] The preparation method of the composite microbial agent comprises the following steps:

[0028] (1) Activation of bacterial strains: The Bacillus subtilis strains preserved on the slant were inoculated on beef extract peptone medium plates and cultured at 28-32°C for 24-36 h; the yeast strains were inoculated on malt extract medium plates and cultured at 28-32°C for 24-48 h; the Cladosporium cladosporium strains were inoculated on basal salt medium plates and cultured at 26-32°C for 24-36 h; the Aspergillus oryzae strains were inoculated on potato dextrose broth medium plates and cultured at 28-32°C for 24-72 h; the Aspergillus niger strains were inoculated on potato dextrose broth medium plates and cultured at 28-32°C for 24-72 h.

[0029] (2) Seed liquid culture: Select activated strains and inoculate them into liquid beef extract peptone medium (with 5-10% phosphate) and culture them in shake flasks at 30-32°C for 24-36 h, with a rotation speed of 120-180 rpm and a ventilation volume of 0.5-1.0 V / V·min.

[0030] (3) Expanded culture: The seed solution is inoculated into a liquid fermentation medium at an inoculum rate of 2-6%, and cultured at 28-32° C. for 24-36 h, with a rotation speed of 150-200 rpm and a ventilation volume of 0.8-1.2 V / V·min.

[0031] (4) Preparation of composite microbial inoculants: Collect the bacterial cells of the required strains separately, prepare bacterial liquids, and mix the bacterial liquids of the different strains according to the volume ratio.

[0032] The application of the above-mentioned composite microbial agent in degrading diazepam residues in water bodies.

[0033] Furthermore, the composite microbial agent is evenly sprayed into the water body at a volume ratio of 100,000 to 8,000,000:1 between the water body and the composite microbial agent.

[0034] Furthermore, the specific operations in different application scenarios are as follows:

[0035] (1) Degradation of DZP residues before stocking: Sprinkle the composite microbial agent evenly into the water at a volume ratio of 1,000,000 to 8,000,000:1, and start the aeration pump at the same time. Repeat the operation twice after an interval of 48 to 72 hours.

[0036] (2) Degradation of DZP residues during daily aquaculture: When DZP residues are found in the water, the composite microbial agent can be evenly sprayed into the water at a volume ratio of 1,000,000 to 5,000,000:1, and the aeration pump can be turned on at the same time. The operation can be repeated after an interval of 72 to 96 hours. Alternatively, the composite microbial agent can be mixed into the feed at a mass ratio of 1,000 to 5,000:1 to 2, and the feed can enter the aquaculture water with the feed for 3 to 6 days.

[0037] (3) Degradation of DZP residues in aquaculture pond influent: Sprinkle the composite microbial agent evenly into the water at a volume ratio of 1,000,000 to 5,000,000:1. Repeat the operation three times after an interval of 24 to 48 hours.

[0038] (4) Degradation of DZP residues in tail water of aquaculture ponds: Sprinkle the composite microbial agent evenly into the water at a volume ratio of 100,000 to 400,000:1. Repeat the operation twice after an interval of 24 to 48 hours.

[0039] Furthermore, the water temperature is 15-25°C, and the water pH environment is 7.0-10.0.

[0040] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0041] The present invention prepares a composite microbial agent by screening suitable microbial strains and applies it to the degradation of DZP in aquaculture ponds. In terms of degradation rate index, the phytoremediation method can reach up to about 70%, the nano zero-valent copper method can reach up to about 75%, and the degradation rate of the technical solution of the present invention can reach up to 100%. Comprehensive comparison shows that the technical solution of the present invention shows great superiority in degrading DZP residues in aquaculture water bodies and has significant promotion and demonstration prospects. At present, there is no report on the degradation of DZP in aquaculture pond water bodies by microbial methods. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0043] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0044] Example 1

[0045] (1) Activation of bacterial strains: The Bacillus subtilis strains stored on the slant were inoculated on beef extract peptone medium plates and cultured at 30°C for 30 h; the yeast strains were inoculated on malt extract medium plates and cultured at 30°C for 36 h; the Cladosporium cladosporium strains were inoculated on basal salt medium plates and cultured at 28°C for 30 h; the Aspergillus oryzae strains were inoculated on potato dextrose broth medium plates and cultured at 30°C for 48 h; the Aspergillus niger strains were inoculated on potato dextrose broth medium plates and cultured at 30°C for 48 h.

[0046] The Bacillus subtilis is CCTCC AB 130001, the yeast is CCTCC WY 2008323, the Cladosporium cladosporium is BMZ103177, the Aspergillus oryzae is GDMCC.NO: 3.31, and the Aspergillus niger is B71913.

[0047] (2) Seed liquid culture: The activated strains were selected and inoculated into liquid beef extract peptone medium (added with 10% phosphate) and cultured in a shaking flask at 30°C for 30 h, with a rotation speed of 180 rpm and a ventilation volume of 0.8 V / V·min.

[0048] (3) Expansion culture: The seed solution was inoculated into a liquid fermentation medium (potato dextrose broth medium with 10% malt extract) at an inoculum rate of 4%, and cultured at 30°C for 30 h, with a rotation speed of 180 rpm and a ventilation volume of 1.0 V / V·min.

[0049] (4) Preparation of composite microbial agent: The required bacterial species were collected and prepared into bacterial liquid. The number of viable bacteria of each strain was as follows: Bacillus subtilis 6.21×10 8 cfu / g, yeast is 4.95×10 9 cfu / g, Cladosporium cladosporium was 1.05×10 9 cfu / g, Aspergillus oryzae is 3.75×10 9 cfu / g, Aspergillus niger was 5.03×10 9 cfu / g; after mixing the bacterial cultures of each strain at a volume ratio of 3:3:4:7:3, the total effective viable count was 3.11×10 9 cfu / g of composite microbial agent.

[0050] Test 1

[0051] Verification of degradation effect of high concentration DZP

[0052] This experiment sets the high DZP residual concentration at 4.8 μg / L to verify the degradation effect of the composite microbial agent prepared in Example 1. According to the volume ratio of water to composite microbial agent of 5000000:1, the composite microbial agent is evenly sprinkled into the water body, and the aeration pump is turned on at the same time. After an interval of 48 hours, the operation is repeated twice (experimental group); the control group is not sprinkled with microbial agent, and the operation is repeated twice, and the DZP content is sampled and detected every 24 hours. The detection method adopts liquid mass method (the same below). The water temperature is 20±2℃, and the water pH environment is 8.5±0.5. The results are shown in Table 1.

[0053] Table 1 Degradation effect of high concentration DZP

[0054]

[0055] It can be seen from Table 1 that under high concentration conditions, the degradation rates of the technical solution of the present invention at various time points from 24h to 192h after use were increased by 8.33%, 31.3%, 33.3%, 43.8%, 52.1%, 54.2%, 54.2%, and 56.3% respectively compared with the control group. By the 192th hour, the control group had naturally degraded 12.5%, while the present invention, by using a composite microbial agent, degraded 68.8%, and the degradation effect of DZP was significant.

[0056] Test 2

[0057] Verification of degradation effect of low concentration DZP

[0058] This experiment sets the low DZP residual concentration at 0.06 μg / L to verify the degradation effect of the composite microbial agent prepared in Example 1. According to the volume ratio of water to composite microbial agent of 5000000:1, the composite microbial agent is evenly sprinkled into the water body, and the aeration pump is turned on at the same time. After an interval of 48 hours, the operation is repeated twice (experimental group); the control group is not sprinkled with microbial agent, and the operation is repeated twice, and the DZP content is sampled and detected every 24 hours. The detection method adopts liquid mass method (the same below). The water temperature is 20±2℃, and the water pH environment is 8.5±0.5. The results are shown in Table 2.

[0059] Table 2 Degradation effect of low concentration DZP

[0060]

[0061] As can be seen from Table 2, under low concentration conditions, by the 192th hour, the control group was unable to degrade DZP through the natural degradation process, while the present invention, by using the composite microbial agent, had degraded 50% by the 96th hour, and by the 192th hour, had degraded 100%, with a significant effect in degrading DZP.

[0062] Test 3

[0063] Different microbial composition

[0064] The bacterial liquids were prepared according to the method of Example 1.

[0065] In this part, "Bacillus subtilis, yeast, Cladosporium buddingii, Aspergillus oryzae, and Aspergillus niger are mixed in a volume ratio of 3:3:4:7:3" is used as the demonstration group of the present invention, and different treatment groups are designed on this basis to verify the effects of different combinations. Table 3 shows the settings of different groups.

[0066] Table 3 Design of different treatment groups

[0067]

[0068]

[0069] In this experiment, the residual concentration of DZP was set at 4.8 μg / L to verify the degradation effect of the composite microbial agent of the present invention. According to the volume ratio of water to composite microbial agent of 5000000:1, different groups of composite microbial agents were evenly sprinkled into the water body, and the aeration pump was turned on at the same time. After an interval of 48 hours, the operation was repeated twice; the control group without spraying microbial agents was used as the control group. After repeating the operation twice, the DZP content was sampled and detected every 24 hours. The detection method adopts liquid mass method (the same below). The water temperature is 20±2℃, and the pH environment of the water is 8.5±0.5. Table 4 shows the verification effect of different treatment groups on the degradation of DZP.

[0070] Table 4 Effect of different microbial compositions on DZP degradation

[0071]

[0072]

[0073] It can be seen from the results in Table 4 that, compared with other treatment groups, the DZP concentration of the technical solution of the present invention has dropped to 1.5 μg / L at 192 hours after treatment, while in other treatment groups: the DZP concentration of 4 bacteria is 2.7-3.0 μg / L, the DZP concentration of 3 bacteria is 3.2-3.9 μg / L, the DZP concentration of 2 bacteria is 3.5-4.1 μg / L, and the DZP concentration of each bacteria is 4.2-4.3 μg / L. Therefore, only when 5 kinds of microorganisms are used in combination can the best effect be achieved, and the effect of removing one or several bacteria will be greatly affected.

[0074] Test 4

[0075] Different proportions of bacteria

[0076] The bacterial liquids were prepared according to the method of Example 1.

[0077] This part designs different proportions of bacterial species to verify the effects of different combinations.

[0078] In this experiment, the residual concentration of DZP was set at 4.8μg / L to verify the degradation effect of the composite microbial agent of the present invention. According to the volume ratio of water to composite microbial agent of 5000000:1, different groups of composite microbial agents were evenly sprinkled into the water body, and the aeration pump was turned on at the same time. After an interval of 48h, the operation was repeated twice; the control group was not sprinkled with microbial agents, and the operation was repeated twice, and the DZP content was sampled and detected every 24h. The detection method adopts liquid mass method (the same below). The water temperature is 20±2℃, and the pH environment of the water is 8.5±0.5. Table 5 shows the settings of different groups and the verification effect on DZP degradation.

[0079] Table 5 Design of different treatment groups and their effects on DZP degradation

[0080]

[0081]

[0082] Note: The proportions are Bacillus subtilis: yeast: Cladosporium cladosporium: Aspergillus oryzae: Aspergillus niger.

[0083] It can be seen from the results of Table 5 that, compared with other treatment groups, the optimal ratio treatment group (3:3:4:7:3) of the technical solution of the present invention has a DZP concentration of 1.5 μg / L at 192h after treatment, while in other treatment groups within the protection scope of the present invention, the DZP concentration is 2.2-3.3 μg / L, and the strain volume ratio of treatment group 5 is 2:2:3:6:2, and the DZP concentration is reduced to 2.2 μg / L, which is relatively close to the optimal embodiment of the present invention. According to the designed ratio group not within the protection scope of the present invention, it is found that the degradation effect of DZP is not significant, and the DZP concentration is 4.0-4.3 μg / L at 192h.

[0084] Test 5

[0085] Change the strain type to other commercially available strains

[0086] In order to verify that the strain model used in the present invention is the optimal strain, this experiment further verifies it by changing the strain model.

[0087] In this part, "Bacillus subtilis, yeast, Cladosporium buddingum, Aspergillus oryzae, and Aspergillus niger mixed at a ratio of 3:3:4:7:3" is used as the technical demonstration group of the present invention, and different treatment groups are designed on this basis to verify the effects of different combinations. Table 6 shows the settings of different groups.

[0088] Table 6 Design of treatment groups with different strains and models

[0089]

[0090]

[0091]

[0092] In this experiment, the residual concentration of DZP was set at 4.8μg / L to verify the degradation effect of the composite microbial agent of the present invention. According to the volume ratio of water body to composite microbial agent of 5000000:1, different groups of composite microbial agents were evenly sprinkled into the water body, and the aeration pump was turned on at the same time. After an interval of 48h, the operation was repeated twice; the control group without spraying microbial agents was used as the control group. After repeating the operation twice, the DZP content was sampled and detected every 24h. The detection method adopts liquid mass method (the same below). The water temperature is 20±2℃, and the water pH environment is 8.5±0.5. Table 7 shows the verification effect of different treatment groups on DZP degradation.

[0093] Table 7 Effect of different strains and types of microorganisms on DZP degradation

[0094]

[0095]

[0096] It can be seen from the results in Table 7 that, compared with other treatment groups, the DZP concentration of the technical solution of the present invention has dropped to 1.5 μg / L at 192 hours after treatment, while in other treatment groups: the DZP concentration of 2.6-3.0 μg / L when 1 bacterial type is changed, the DZP concentration of 3.0-3.5 μg / L when 2 bacterial types are changed, the DZP concentration of 3.4-3.8 μg / L when 4 bacterial types are changed, the DZP concentration of 3.9-4.1 μg / L when 5 bacterial types are changed, and the DZP concentration of 4.2-4.3 μg / L when 5 bacterial types are changed, indicating that the bacterial types specified in the technical solution of the present invention have the specificity of DZP degradation. When other types of bacterial types are replaced, the degradation effect of DZP becomes worse as the number of replaced types increases. Even when only one bacterial type is replaced, at 192 hours, the group with the highest DZP degradation rate (i.e., Group 3 in Table 7) is only 66.7% of the technical solution of the present invention.

[0097] Test 6

[0098] Change in viable count

[0099] The total effective viable count of the composite microbial agent of the present invention is 1.24×10 9 ~5.56×10 10 cfu / g. In order to verify that the number of viable bacteria in this range is the optimal range, this experiment further verifies it by changing the number of viable bacteria.

[0100] In this section, "Bacillus subtilis, yeast, Cladosporium cladosporium, Aspergillus oryzae, and Aspergillus niger mixed in a ratio of 3:3:4:7:3" is used as the technical demonstration group of the present invention, and different treatment groups are designed on this basis to verify the effects of different combinations.

[0101] In this experiment, the residual concentration of DZP was set at 4.8μg / L to verify the degradation effect of the composite microbial agent of the present invention. According to the volume ratio of water body to composite microbial agent of 5000000:1, different groups of composite microbial agents were evenly sprinkled into the water body, and the aeration pump was turned on at the same time. After an interval of 48h, the operation was repeated twice; the control group without spraying microbial agents was used as the control group. After repeating the operation twice, the DZP content was sampled and detected every 24h. The detection method adopts liquid mass method (the same below). The water temperature is 20±2℃, and the pH environment of the water is 8.5±0.5. Table 8 shows the verification effect of different treatment groups and different treatment groups on the degradation of DZP.

[0102] Table 8 Design of different treatment groups and their effects on DZP degradation

[0103]

[0104] It can be seen from the results in Table 8 that, compared with other treatment groups, the effective viable count range described in the technical solution of the present invention has a DZP concentration of 1.5-2.0 μg / L at 192 h after treatment, while in other treatment groups: the DZP concentration is 2.6-3.3 μg / L for those below the viable count range described in the technical solution of the present invention, and the DZP concentration is 2.4-3.0 μg / L for those above the viable count range described in the technical solution of the present invention (under high concentration, the concentration of the bacterial agent continues to increase. Due to the limited substrate concentration and nutrients, competitive inhibition occurs between different strains, resulting in the degradation effect reaching an inflection point, and the degradation efficiency of diazepam is reduced). It shows that within the effective viable count range described in the present invention, there is a good degradation effect on DZP, with the lowest degradation rate reaching 58.3%, while when it is not within the scope of the present invention, the highest degradation rate is 50.0% (i.e., the 10th treatment group in Table 8).

[0105] Test 7

[0106] Changing the pH range of water

[0107] This part of the experiment sets different water pH ranges to further verify the degradation effect of the technical solution of the present invention on DZP. The preparation method of the composite microbial agent is the same as that of Example 1.

[0108] The test set the residual concentration of DZP to 4.8μg / L to verify the degradation effect of the composite microbial agent of the present invention. According to the volume ratio of water to composite microbial agent of 5000000:1, different groups of composite microbial agents were evenly sprinkled into the water body, and the aeration pump was turned on at the same time. After an interval of 48h, the operation was repeated twice; the control group was not sprinkled with microbial agents, and the operation was repeated twice and the DZP content was sampled and detected every 24h. The detection method adopts liquid mass spectrometry (the same below). The water temperature is 20±2℃. The results are shown in Table 9.

[0109] Table 9 Different water pH and its effect on DZP degradation

[0110]

[0111] It can be seen from the results in Table 9 that, compared with other treatment groups, the pH range described in the technical solution of the present invention has reduced the DZP concentration to 1.5-1.6 μg / L at 192 h after treatment, while in other treatment groups: the DZP concentration is 2.5-3.9 μg / L for those below the pH range described in the technical solution of the present invention, and the DZP concentration is 3.5-4.2 μg / L for those above the pH range described in the technical solution of the present invention. It shows that the degradation effect of DZP is better within the pH range described in the present invention, and the degradation rate is as low as 66.7%, while when it is not within the scope of the present invention, the degradation rate is as high as 47.9% (i.e., the third treatment group in Table 9). At the same time, according to the results in Table 9, the composite microbial agent of the present invention is sensitive to pH, and an overly acidic (pH≤5) or overly alkaline (pH>10) environment may cause the microorganism to be inactivated, thereby failing to exert the performance of degrading DZP.

[0112] Test 8

[0113] According to research, most of the reports on positive detection of DZP in freshwater fish are related to fishing. In order to improve the fishing effect and the success rate of fishing, fishermen will mix fish attractants into the nesting materials and baits. It was found that 100% of the 10 bottles of fish attractants contained DZP, and the concentration reached the mg / kg level. Except for 4 bottles of unlabeled ingredients, the ingredients of the remaining fish attractants also contained attractants. It can be seen that attractants are likely to be an important source of DZP in nesting materials, baits and fish attractants. Throwing nesting materials or baits containing DZP into the water will form an environment with high concentration of DZP in a short time, prompting the surrounding fish to gather and eat, thereby achieving the effect of rapid fishing. The use of nesting materials and baits in leisure fishing makes the aquaculture water and aquatic products polluted by DZP. Long-term fishing outside and the long metabolic cycle of DZP itself have an impact on the aquaculture environment and the safety of aquatic products.

[0114] Based on this, the present invention focused on extracting water samples from 60 ponds specializing in providing fishing services in Jiangsu Province. After testing, it was found that DZP residues were detected in 100% of the water bodies, of which 25.5% of the water samples had diazepam residues of 1000ng / L~6500ng / L, 50.8% of the water samples had diazepam residues of 186ng / L~921ng / L, and the remaining water samples had diazepam residues of 17.5ng / L~93.4ng / L.

[0115] 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.

[0116] 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. A composite microbial agent for degrading diazepam, characterized in that: The components include: Bacillus subtilis, Saccharomyces cerevisiae, Cladosporium buddingum, Aspergillus oryzae, and Aspergillus niger; The Bacillus subtilis is CCTCC AB 130001; The yeast is CCTCC WY 2008323; The Cladosporium cladosporium is BMZ103177; The Aspergillus oryzae is GDMCC.NO: 3.31; The Aspergillus niger is B71913; The volume ratio of the bacterial liquid of each bacterial strain is 1-5:2-4:1-7:3-9:1-4.

2. The composite microbial agent according to claim 1, characterized in that: The volume ratio of the bacterial liquid of each strain is 3:3:4:7:

3.

3. The composite microbial agent according to claim 1, characterized in that The volume ratio of the bacterial liquid of each strain is 2:2:3:6:

2.

4. The composite microbial agent according to claim 1, characterized in that The total effective viable count was 1.24×10 9 ~5.56×10 10 cfu / g.

5. The composite microbial agent according to claim 1, characterized in that: The number of effective live bacteria in the bacterial solution of each strain is ≥3.0×10 7 cfu / g.

6. Use of the composite microbial agent according to any one of claims 1 to 5 in degrading diazepam residues in water.

7. The use according to claim 6, characterized in that Sprinkle the composite microbial agent evenly into the water body at a volume ratio of 100,000 to 8,000,000:1 between the water body and the composite microbial agent.

8. The use according to claim 6, characterized in that The water temperature is 15-25℃, and the water pH environment is 7.0-10.0.

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